US010392630B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 , 392 ,630 B2 Aponte et al. (45 ) Date of Patent: Aug. 27 , 2019 (54 ) PLANTS HAVING INCREASED TOLERANCE (56 ) References Cited TO HERBICIDES U . S . PATENT DOCUMENTS ( 71 ) Applicant: BASF AGRO B . V. , EA Arnheim (NL ) 5 , 169 ,770 A 12 / 1992 Chee et al . 5 , 198 ,013 A 3 / 1993 Hirai et al. (72 ) Inventors : Raphael Aponte , Mannheim (DE ); 5 , 322 , 783 A 6 / 1994 Tomes et al. 5 , 366 , 892 A 11/ 1994 Foncerrada et al. Stefan Tresch , Kirchheim (DE ); 5 , 376 ,543 A 12 / 1994 Chee et al. Matthias Witschel, Bad Duerkheim 5 , 380 ,831 A 1 / 1995 Adang et al. (DE ); Jens Lerchl, Golm (DE ) ; Dario 5 ,436 , 391 A 7 / 1995 Fujimoto et al . Massa , Mannheim (DE ) ; Tobias Seiser , 5 , 485 , 192 A 1/ 1996 Nagahata et al . Mannheim (DE ) ; Thomas Mietzner , 5 , 593 , 881 A 1 / 1997 Thompson et al . 5 , 723 , 756 A 3 / 1998 Peferoen et al. Annweiler (DE ) ; Jill Marie Paulik , 5 , 737 ,514 A 4 / 1998 Stiffler Cary , NC (US ) ; Chad Brommer , 5 , 747 , 450 A 5 / 1998 Ohba et al . Raleigh , NC (US ) 5 , 767 , 373 A 6 / 1998 Ward et al. 5 , 773 ,702 A 6 / 1998 Penner et al. 5 , 859 , 348 A 1/ 1999 Penner et al. ( 73 ) Assignee: BASF AGRO B . V . , Arnhem (NL ) 5 , 939 , 360 A 8 / 1999 Adachi et al . 5 ,939 ,602 A 8 / 1999 Volrath et al . ( * ) Notice : Subject to any disclaimer, the term of this 5 , 948 , 917 A 9 / 1999 Adachi et al. patent is extended or adjusted under 35 5 , 990 , 387 A 11/ 1999 Tomes et al . 6 ,018 , 105 A 1 / 2000 Johnson et al. U . S .C . 154 (b ) by 0 days . 6 ,027 , 945 A 2 / 2000 Smith et al . 6 ,084 , 155 A 7 / 2000 Volrath et al . (21 ) Appl. No. : 14 /911 , 814 6 , 160 , 206 A 12 / 2000 Sato et al. 6 , 308 ,458 B1 10 / 2001 Volrath et al. Aug. 12 , 2014 6 , 368 , 800 B1 4 /2002 Smith et al. ( 22 ) PCT Filed : 6 ,653 , 529 B2 11/ 2003 Peng et al . ( 86 ) PCT No. : PCT/ IB2014 / 063877 (Continued ) $ 371 (c )( 1 ), FOREIGN PATENT DOCUMENTS ( 2 ) Date : Feb . 12 , 2016 CA 2382090 A1 2 / 2001 (87 ) PCT Pub. No. : W02015 /022640 CA 2807035 AL 2 / 2012 PCT Pub . Date : Feb . 19 , 2015 (Continued ) (65 ) Prior Publication Data OTHER PUBLICATIONS Maniatis et al. , 1982 , Molecular Cloning : A Laboratory Manual , US 2016 /0201078 A1 Jul. 14 , 2016 Cold Spring Harbor Laboratory , pp . 324 - 343 and 387 -389 . * Keskin et al . , 2004 , Protein Science 13 : 1043 - 1055 . * Guo et al. , 2004 , Proceedings of the National Academy of Sciences Related U . S . Application Data USA 101 : 9205 - 9210 . * Thornton et al. , 2000 , Nature Structural Biology , structural genomic ( 60 ) Provisional application No . 61/ 866 , 067, filed on Aug . supplement , Nov. 2000 : 991 - 994 . * 15 , 2013 , provisional application No . 61/ 864, 671 , Brachypodium distachyon protoporphyrinogen oxidase with UniProt filed on Aug. 12 , 2013 , provisional application No . accession No. I1IZ42 , published on Jun . 13 , 2012 . * 61/ 864, 672 , filed on Aug. 12 , 2013 . Koch et al. , 2004 , EMBO J . 23 : 1720 - 1728 . * (Continued ) (51 ) Int. Ci. C12N 15 / 82 ( 2006 . 01 ) Primary Examiner — Bratislav Stankovic AOIN 37 / 48 ( 2006 .01 ) (74 ) Attorney, Agent, or Firm — Marshall , Gerstein & AOIN 43 /54 ( 2006 .01 ) Borun LLP A01N 43 /653 ( 2006 .01 ) (57 ) ABSTRACT A01N 43 /84 ( 2006 .01 ) The invention refers to plants comprising wild -type or AOIN 57/ 16 ( 2006 . 01 ) mutated Alopecurus PPO enzymes, and methods of obtain C12N 9 / 02 ( 2006 .01 ) ing such plants . The present invention further refers to a (52 ) U .S . CI. method for controlling weeds at a plant cultivation site , the CPC ...... C12N 15 /8274 ( 2013 .01 ); A01N 37 / 48 method comprising the steps of providing , at said site , a ( 2013 .01 ) ; AOIN 43 / 54 (2013 .01 ) ; AOIN plant that comprises at least one nucleic acid comprising a 43 /653 (2013 .01 ) ; AOIN 43 /84 ( 2013 .01 ); nucleotide sequence encoding a wild - type or a mutated AOIN 57/ 16 ( 2013 .01 ); C12N 9 /001 Alopecurus PPO enzyme which is resistant or tolerant to a ( 2013 .01 ); C12N 9 /0071 ( 2013 .01 ); C12Y PPO - inhibiting herbicide by applying to said site an effec 103 /03004 ( 2013 .01 ) tive amount of said herbicide . (58 ) Field of Classification Search None 7 Claims, 6 Drawing Sheets See application file for complete search history. Specification includes a Sequence Listing . US 10 ,392 ,630 B2 Page 2

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Macias et al. , Optimization of benzoxazinones as natural herbicide Lewis et al. , Interactionsbetween redox partners in various cytochrome models by lipophilicity enhancement. J. Acric . Food Chem . 54 : P450 systems: functional and structural aspects , Biochim . Biophys. 9357 -65 (2006 ) . Acta , 1460 ( 2 - 3 ) : 353 - 74 ( 2000 ) . Mulwa et al. , Biotechnology approaches to developing herbicide Li et al. , Development of PPO inhibitor -resistant cultures and crops , tolerance / selectivity in crops . Afr. J. Biotechnol. 5 (5 ): 396 -404 Pest Management Science , 61( 3 ) : 277 - 85 ( 2005 ) . ( 2006 ) . Loppes, A new class of arginine- requiring mutants in Chlamydomonas Rousonelos, S ., Master ' s Thesis , University of Illinois , published reinhardi, Mol. Gen . Genet ., 104 ( 2 ) :172 - 7 (1969 ) . Aug. 2010 . Matringe et al ., Protoporphyrinogen oxidase as a molecular target Watanabe et al. , Molecular characterization of photomixotrophic for diphenyl ether herbicides , Biochem . J . , 260 ( 1 ) : 231 - 5 ( 1989 ) . tobacco cells resistant to protoporphyrinogen oxidase -inhibiting Matringe et al. , Protoporphyrinogen oxidase inhibition by three herbicides . Plant Physiol. 118 : 751 - 8 ( 1998 ) . peroxidizing herbicides: oxadiazon , LS 82 -556 and M & B 39279 , Supplemental Partial European Search Report , European patent FEBS Lett ., 245 ( 1 - 20 : 35 - 8 ( 1989 ). application No. EP 14836899 , dated Mar . 20 , 2017 . Murray et al. , Codon usage in plant genes , Nucleic Acids Res ., Extended European Search Report , issued in application No. EP 17 ( 2 ) :477 -98 ( 1989 ) . 14836729 . 5 , dated Jun . 6 , 2017 . Nandihalli et al. , Quantative structure -activity relationships of EBI Accession No. GSP : BBB23069 , Amaranthus tuberculatus PPO protoporphyrinogen oxidase - inhibiting diphenyl ether herbicides , variant R128A/ T208S / F420V # 1 (Feb . 13 , 2014 ). Pesticide Biochem Physiol ., 43 : 193 -211 ( 1992 ) . 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RNA /DNA oligonucleotide in a mammalian cell- free extract, Nucleic Randolph - Anderson et al. , Isolation and characterization of a mutant Acids Res ., 27 ( 5 ) : 1323 - 30 ( 1999 ) . protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii Dailey et al ., Expression of a cloned protoporphyrinogen oxidase , J. conferring resistance to porphyric herbicides, Plant Mol. Biol. , Biol . Chem ., 289 ( 2 ) : 813 - 15 ( 1994 ) . 38 (5 ): 839 -59 ( 1998 ) . Dayan et al. , Biochemical and structural consequences of a glycine Sasarman et al. , Mapping of a new hem gene in Escherichia coli deletion in the alpha -8 helix of protoporphyrinogen oxidase, Biochim . K12 , J . Gen . Microbiol. , 113 ( 2 ) : 297 - 303 ( 1979 ) . Biophys. Acta , 1804 (7 ): 1548 -56 (2010 ). Sasarman et al. , Nucleotide sequence of the hemG gene involved in Duke et al. , Protoporphyrinogen oxidase- inhibiting herbicides , Weed the protoporphyrinogen oxidase activity of Escherichia coli K12 , Sci ., 39 :465 -73 ( 1991) . Can . J. Microbiol. , 39( 12 ): 1155 -61 ( 1993 ). Geiser et al. , The hypervariable region in the genes coding for Shibata et al. , Isolation and characterization of a Chlamydomonas entomopathogenic crystal proteins of Bacillus thuringiensis : nucleo reinhardtii mutant resistant to an experimental herbicide S - 23142 , tide sequence of the kurhd1 gene of subsp . kurstaki HD1, Gene, which inhibits chlorophyll synthesis, in : Murata (ed .) , Research in 48 ( 1 ) : 109 - 18 ( 1986 ) . Photosynthesis , vol . III, pp . 567 - 570 ( 1992 ) . GenBank Accession No . 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XM _ 004975973 , Predicted : Setaria italica italica protoporphyrinogen oxidase , chloroplastic /mitochrondrial protoporphyrinogen oxidase ,mitochondrial (LOC101781148 ), mRNA like ( LOC101781148 ) , mRNA , Jun . 26 , 2013 . (Nov . 30 , 2015 ) . U . S . Appl. No. 14 /408 ,439 , filed Dec . 16 , 2014 . International Preliminary Report on Patentability, International Appli U . S . Appl. No. 13/ 918 ,212 , filed Jun . 14 , 2013 . cation No . PCT /IB2014 /063877 , dated Feb . 16 , 2016 . U . S . Appl. No . 13 / 994 , 374 , filed Oct . 9 , 2013 . International Search Report and Written Opinion , International Application No . PCT/ IB2014 /063877 , dated Feb . 10 , 2015 . * cited by examiner U . S . Patent Aug . 27, 2019 Sheet 1 of 6 US 10 ,392 ,630 B2

AMARE WC herb sus MVIOSITALSPNL?LPSPLSVSTKNYP ------VAVMGN - - - s me wow mo - ISEREEPTSAKRVAVVG AMARE AC herb Sus MIOSITELSPNL?LPSPISVSIKNY D - VAVMGN ------ISOREREOSAKRVAVVG AMARE AC her resis MVIQSITHLS PNL?LPSPLSVSTKNYP - - VAVMGN - - * * - - ISEREEPTSAKRVAVVG AMARE CC herb resis MIQSITELSPNL?LPSPISVSTKNY ? - -- - VAVMGN ------ISEREEPTSAKRVAVVG SEQ ID NO 002 ALOMY MLTSATTPSSSSASSRASTRFASSSRPRRTAYARGRRLRPVLAMAASDDPR - ARSVAVVG AMARE WC herb sus AGVSGLAAAYKIKSHGISVTLFEADSRAGGKLKTVKKDGFINDEGANTMIESEAEVSSLI AMARE _ AC herb sus AGVSGLAAAYKLKSHGLSVTIESADSRAGGKLKTVKKDGFIWDEGANTMTESEAEVSSLI AMARB AC herb _ resis AGVSGLAAAYKLKSHGLSVTLHEADSRAGGKLKTVKKDGFINDEGANTMIESEAEVSSLI AMARE CC herb resis AGVSGLAAAYKLKSHGLSVTLFEANSRAGGKLKTVKKDGFIWDEGANTMTESEAEVSSLI SEQ ID NO 002 ALOMY AGISGLVAAYRLSKSGVRVTVFEADDRAGGKIRTNSDSGFLWDEGANTMTESALEASRLI AMARE WC herb sus DDLGEREKQOLPISONKRYIARDGLPVLLESNPAALLISNILSAKSKLQIMLEPFLWRKH AMARE AC herb sus DOLGLREKOOLPISONKRYI ARAGLPVLLPSNPAÁLLTSNILSAKSKLQIMLEFFIWRKH AMARE AC herb tesis DOLGLREKQQL ? ISQNKRYIARDGLPVLLPSNPAALLTSNILSAKSKLQIMLEPFLWRKH AMARE CC herb resis DOLGLREKOOLPISQNKRYIARDGLPVILPSNPAALLTSNILSAKSKLQIMLEPFLWRKH SEQ ID NO 002 ALOMY DOLGLEDBLOYENSOHKRYTVKDGAPALIFSDPIALMKSSLISTKSKFKLFIEPFLYOKS AMARE WC herb Sus N - ~ ATELSDEHVQE?VGEFFERHFGKEFVDYVIDPFVAGTCGGDPOSLSMHHTFPEVWN AMARE 4C herb sus N -- .- -- ATELSDEHVOESVGEFFERHFGKEFVDYVIDPFVAGTCGGDPOSLSMHHIFPEVWN AMARE AC herb resis N - - - ATEISDEHVQESVGEEFERHF 'GKEFVDYVIOPEVAGTOG - OPOSLSMHIFPEVWN AMARE CC her resis N - - - ATEISDEHVQESVGEFEEREEGKEFVDYVIDOFVAGICG - DPOSLSMYRTFPEVWN SEO ID NO 002 ALOMY STKSSKKVSDEHISESVGSFFERHEGKEVVDYLIDPEVAGISAGOPESISIRHAFPGLWN AMARE WC herb sus IEKRFGSVFAGLIQSTLLSKKEKGGENASIKKPRVRGSFSFOGGMQTLVDTMCKQLGEDE AMARE AC herb sus IEKRFGSVFAGLIQSTLLSEKEKGGENASIKKPRVRGSFSFOGGMOTLVOTMCKQLGEDE AMARE AC herb resis IEKREGSVEAGLIOSTLI- SKKEKGGENASIKKORVRGSESFOGGMOTIVDTMCKQIGEDE AMARB CC herb resis IEKRFGSVEAGLIOSTLISKKEKGGENASIKKPRVRGSFSFOGGMOTLVOTMCKOLGEDE SEQ ID NO 002 ALOMY LEKKYGSIIVGAIMSKLTAKGDKKGSAVSGKGRNKRASFSFHGGMQTLVDALHKEVGDGN AMARE WC _ herb sus LKLOCEVLSLSYNOKGIPSLGNWSVSSMSNNTS - ~ - ~ EOQSYDAVVVTAPIRNVKEMKI AMARE AC _ herb _ sus LKLOCBVLSLSYNOKGIPSIGNWSVSSMSNNTS - ~ - ~ EDOSYDAVVVTAPIRNVKEMKI AMARE AC herb resis LKLOCEVLSLSYNOKGIPSLGNWSVSSMSNNTS - - - - - EDOSYDAVVVTAPIRNVKEMKI AMARE CC herb tesis LKLQCEVLSLSYNOKGIPSLGNWSVSSMSNNTS - - - - - 5DQSYDAVVVTAPIRNVKEMKI SEQ ID NO 002 ALOMY. VKIGAQVISLACICDGLSASDGWSISVDSKDASNKELTKNHSFDAVIMTAPLSNVQRMKF AMARE WC herb sus MKFGNPFSLDFIPEVTYVPI SVMI TAFKKDKVKRPLEGFGVLIPSKEQ - UNGIKTIGTIE AMARE AC herb sus MKFGNPFSLDFI PEVTYVPLSVMITAFKKDKVKRFLEGFGVIIPSKEQ - UNGLKTLGTLF AMARE AC herb resis MKFGNPFSLDEI PEVTYVPLSVMITAFKKDKVKAPLEGEGVLIPSKED - ANGLKTLGTIF AMARE CC herb resis MKFGNPFSLDFIPEVTYVPLSVMI TAFKKDKVKRPLEGFGVLIPSKRO - UNGLKTLGTLE SEO ID NO 002 . ALOMY IKGGAPFVIDFIPKVDYLP?SLMVTAFKKEDVKRPLEGFGVIIPYKEQOKHGIKTIGTIS AMARE WC herb sus SSMMFPDRAPSDMCLFTTFVGGSANRKLANASTDELKQIVSSDLOOLLGTEDEPSEVNHL AMARE AC herb sus SSMMFPDRAPSDMCLFITFVGGSRNRKLANASIDELKQIVSSOLQOLLGTEDEPSFVNHL AMARE AC herb resis SSMMFPDRAPSDMCLFTTFVGGSRNRKLANASTOELKOIVSSDLOQLIGTEDEPSEVNHL AMARE CC _ herb resis S .SMMFPDRAPSDMCLFTTEVGGSRNRKLANASIDEIKOIVSSDLQOLLGTEDEPSFVNHL SEQ ID NO 002 ALOMY SSMMEPORAPNDOHLEITFVGGSHNRDLAGAPISILKOLVISDLGKLLGVEGOPIEVKHI AMARE WC herb sus FWSNAFPLYGHNYDSVLRAIDKMEKDLPGFFYAGNHKGGLSVGKAMASGCKAAELVISYL AMARE AC herb sus FWSNAFPLYGHNYDSVLRAIDKMEKDLPGFFYAGNHKGGLSVGKAMASGCKAAELVISYL AMARE AC herbi tesis FWSNAFPLYGHNYDCVLRAIDKMEKDLPGFFYAGNHKGGLSVGKAMASGCKAAELVISYL AMARE CC herb resis TWSNAPPLYGINYDSVLRAIDKMEKDLPGF YAGNIKGGLSVGKAMASGCKAAELVISYL SEQ ID NO 002 ALOMY HWRNAFPLYGHDYDSALEAIGKMESDLPGFFYAGNNKDGLAVGNVIASGSKTADLVISYL AMARE WC herb sus DSHIYVKMDEKIA AMARE AC herb sus DSHIYVKMDEKTA AMARE AC herb tesis DSHIYVKMDEKTA AMARE C? herb resis DSHIYVKMDEKTA SEQ ID NO 002 ALOMY ESG - - IKODN - - - -- Figure 1a U . S . Patent Aug . 27, 2019 Sheet 2 of 6 US 10 ,392 ,630 B2

PPO1 ALOMY ww ww mw - MVGAIMAIATVTAALPLRVRVPGRSRR ~ - ~ ~ GQAR ~ ~ ~ ~ ~ ~ CAVASDATEAPAAPS PPOI AMARE MSAMALSSSILOCPPHSDISFRFFAHTRTQPPIFFGRPRKLSYIHCSTSSSSTANYONTI PPO1 ALOMY ARLS ------ADCVIVGGGISGLCTAQALATKYGVS - - DLLVTEARARPGGNITTVERPDE PPO1 AMARE TSOGEGDKVLDCVIVGAGISGLCIAOALSTKHIOSNLNFIVTEAKHRVGGNITTME - - SD PPOI ALOMY GYLWEEGPNSFQPSDPVLTMAVOSGLKDEIVEGDPNAPRFVLWEGKLRPVPSKPGDLPFE PPOI AMARE GYIWEEGPNSFQPSOPVLTMAVDSGLKDDLVLGDPNA PRFVLWNGKLRPVPSKPTDLPFE PPOI ALOMY DLMSIPGKIRAGLGALGIRPPPPGREESVEEFVRRNLGAEVFERLIEPFCSGVYAGOPSK PPOI AMARE DLMSFPGKIKAGLGALGLRPPPPSYEESVEEFVRRNLGDEVFERLIEPFCSGVYAGDPAK PPOI ALOMY LSMRAAFGKVWRLEENGGSIIGGTIKAIQDKGKNPKPPRDPRLPAPKGOTVASFRKGLAM PPOI AMARE LSMKAAFGKVWTLEQKGGSIIAGTIKTIOERKNNPPPPRDPRLPKPKGOTVGSFRKGLIM PPOI ALOMY LPNAIASRLGSKVKLSWKLISITKSENQGYVLGYETPEGVVSVOAKSVIMT IPSYIASDI PPOI AMARE LPTAIAARLGSKVKLSWTLSNIDKSINGEYNLIYOTPDGPVSVRTKAVVMIVPSYIASSI PPOI ALOMY LRPLSSDAADGLSKFYYPPVAAVIVSYPKEAIRKECLIDGELQGFGQLHPRSQGVETLGT PPOI AMARE LRPLSDVAADSLSKFYYPPVAAVSLSYPKEA IRPECLIDGELKGFGQLHPRSQGVETLGT PPOI ALOMY IYSSSLFPNRAPAGRVILLNYIGGATNTGIVSKTEŠDLVEAVDRDLRKMLINPRAADPLA - PP01 AMARE IYSSSLFPGRAPPGRTLILSYIGGATNLGILOKSEDELAETVDKDLRKILINPNAKGSRV PPOI ALOMY IGVRVWPQAIPOFLIGALDRLDAAKSALVRSGCSGLELGGNYVAGVALGRCIEGAYOSAS PPOI AMARE IGVRVWPKAIPQFI:VGHEDVLDAAKAGLANAGQKGLF ' LGGNYVSGVALGRCIEGAYOSAS PP01 ALOMY EVSDFLNKYAYK PPOI AMARE EVVDFLSQYKDK

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Figure 5 US 10 , 392 ,630 B2 PLANTS HAVING INCREASED TOLERANCE Application of PPO - inhibiting herbicides results in the TO HERBICIDES accumulation of protoporphyrinogen IX in the chloroplast and mitochondria , which is believed to leak into the cytosol This application is a National Stage application of Inter where it is oxidized by a peroxidase . When exposed to light, national Application No . PCT /IB2014 /063877 , filed Aug . 5 protoporphyrin IX causes formation of singlet oxygen in the 12 , 2014 , which claims the benefit of U . S . Provisional cytosol and the formation of other reactive oxygen species , which can cause lipid peroxidation and membrane disrup Application No. 61/ 864, 671 , filed Aug. 12 , 2013 , U . S . tion leading to rapid cell death (Lee et al . 1993 . Plant Provisional Application No . 61 / 864 ,672 filed Aug . 12 , 2013 , Physiol. 102 : 881 ) . and U . S . Provisional Application No. 61/ 866 , 067, filed Aug . 10 Not all PPO enzymes are sensitive to herbicides which 15 , 2013 ; the entire contents of which are hereby incorpo inhibit plant PPO enzymes . Both the Escherichia coli and rated herein by reference in their its entirety . Bacillus subtilis PPO enzymes (Sasarmen et al . 1993 . Can . J . Microbiol. 39 : 1155 ; Dailey et al. 1994 . J . Biol. Chem . REFERENCE TO SEQUENCE LISTING 269 : 813 ) are resistant to these herbicidal inhibitors . SUBMITTED VIA EFS -WEB 15 Mutants of the unicellular alga Chlamydomonas rein hardtii resistant to the phenylimide herbicide S - 23142 have This application was filed electronically via EFS - Web and been reported (Kataoka et al. 1990 . J . Pesticide Sci. 15 : 449; includes an electronically submitted sequence listing in .txt Shibata et al. 1992 . In Research in Photosynthesis , Vol. III, format . The .txt file contains a sequence listing entitled N . Murata , ed . Kluwer: Netherlands . pp . 567 - 70 ). At least “ 77455 _ Seqlisting .txt ” created on Feb . 8 , 2016 , and is 20 one of thesemutants appears to have an altered PPO activity 123 , 357 bytes in size . The sequence listing contained in this that is resistant not only to the herbicidal inhibitor on which . txt file is part of the specification and is hereby incorporated the mutant was selected , but also to other classes of protox by reference herein in its entirety . inhibitors (Oshio et al. 1993 . Z . Naturforsch . 48c : 339 ; Sato et al. 1994 . In ACS Symposium on Porphyric Pesticides, S . FIELD OF THE INVENTION 25 Duke , ed . ACS Press : Washington , D . C . ) . A mutant tobacco cell line has also been reported that is resistant to the The present invention relates in general to methods for inhibitor S -21432 (Che et al. 1993 . Z . Naturforsch . 48c : conferring on plants agricultural level tolerance to a herbi - 350 ) . Auxotrophic E . coli mutants have been used to confirm cide. Particularly , the invention refers to plants having an the herbicide resistance of cloned plant PPO - inhibiting increased tolerance to PPO - inhibiting herbicides . More spe - 30 herbicides . cifically , the present invention relates to methods and plants Three main strategies are available for making plants obtained by mutagenesis and cross -breeding and transfor- tolerant to herbicides, i. e . ( 1 ) detoxifying the herbicide with mation that have an increased tolerance to PPO - inhibiting an enzyme which transforms the herbicide, or its active herbicides . metabolite , into non - toxic products , such as, for example , the enzymes for tolerance to bromoxynil or to basta BACKGROUND OF THE INVENTION (EP242236 , EP337899 ) ; ( 2 ) mutating the target enzyme into a functional enzyme which is less sensitive to the herbicide , Herbicides that inhibit protoporphyrinogen oxidase (here or to its active metabolite , such as , for example , the enzymes inafter referred to as Protox or PPO ; EC : 1 . 3 . 3 . 4 ) , a key for tolerance to glyphosate ( EP293356 , Padgette S . R . et al. , enzyme in the biosynthesis of protoporphyrin IX , have been 40 J . Biol. Chem ., 266 , 33 , 1991 ) ; or ( 3 ) overexpressing the used for selective weed control since the 1960s . PPO cata - sensitive enzyme so as to produce quantities of the target lyzes the last common step in chlorophyll and heme bio enzyme in the plant which are sufficient in relation to the synthesis which is the oxidation of protoporphyrinogen IX herbicide , in view of the kinetic constants of this enzyme, so to protoporphyrin IX . (Matringe et al. 1989 . Biochem . 1 . as to have enough of the functional enzyme available despite 260 : 231 ). PPO - inhibiting herbicides include many different 45 the presence of its inhibitor. The third strategy was described structural classes of molecules (Duke et al. 1991 . Weed Sci . for successfully obtaining plants which were tolerant to PPO 39: 465 ; Nandihalli et al. 1992 . Pesticide Biochem . Physiol. inhibitors ( see e. g . U . S . Pat . No . 5 ,767 , 373 or 5 , 939 ,602 , and 43 : 193 ; Matringe et al . 1989 . FEBS Lett . 245 : 35 ; Yanase patent family members thereof. ) . In addition , US 2010 / and Andoh . 1989 . Pesticide Biochem . Physiol. 35 : 70 ) . 0100988 and WO 2007 /024739 discloses nucleotide These herbicidal compounds include the diphenylethers 50 sequences encoding amino acid sequences having enzymatic { e .g . lactofen , (+ - )- 2 -ethoxy - 1 -methyl - 2 - oxoethyl 5 - { 2 activity such that the amino acid sequences are resistant to chloro -4 -( trifluoromethyl ) phenoxy } - 2 -nitrobenzoate ; acif - PPO inhibitor herbicidal chemicals , in particular 3 -pheny luorfen , 5 - { 2 - chloro - 4 - ( trifluoromethyl) phenoxy } - 2 - nitrob - luracil inhibitor specific PPO mutants . enzoic acid ; its methyl ester , or oxyfluorfen , 2 - chloro - 1 - ( 3 - WO 2012/ 080975 discloses plants the tolerance of which ethoxy - 4 -nitrophenoxy ) - 4 - ( trifluorobenzene ) ) , oxidiazoles, 55 to a PPO - inhibiting herbicide named "benzoxazinone - de ( e . g . oxidiazon , 3 - { 2 , 4 -dichloro - 5 - ( 1 -methylethoxy )phe - rivative” herbicide ( 1 , 5 -dimethyl - 6 - thioxo - 3 - ( 2 , 2 , 7 -trif nyl ) - 5 - ( 1 , 1 -dimethylethyl ) - 1 , 3 ,4 -oxadiazol - 2 - (3H ) -one ) , luoro - 3 -oxo - 4 - (prop - 2 - ynyl) - 3 , 4 -dihydro - 2H -benzo [ b ] [ 1 , 4 ] cyclic imides ( e . g . S - 23142 , N - ( 4 -chloro - 2 - fluoro - 5 - propar- oxazin - 6 - yl) - 1 , 3 , 5 - triazinane - 2 , 4 -dione ; “ BAS 850H ” ) had gyloxyphenyl) - 3 , 4 , 5 ,6 - tetrahydrophthalimide ; chloroph - been increased by transforming said plants with nucleic thalim , N - ( 4 -chlorophenyl ) - 3 , 4 , 5 , 6 - tetrahydrophthalimide ), 60 acids encoding mutated PPO (mutated PPO ) enzymes . In phenyl pyrazoles (e . g . TNPP - ethyl, ethyl 2 - { 1 - ( 2 , 3 , 4 - particular, WO 2012 /080975 discloses that the introduction trichlorophenyl) - 4 -nitropyrazolyl - 5 - oxy } propionate ; M & B of nucleic acids which code for a mutated PPO of an 39279 ), pyridine derivatives (e . g. LS 82 -556 ), and phenopy - Amaranthus type II PPO in which the Arginine at position late and its O - phenylpyrrolidino - and piperidinocarbamate 128 had been replaced by a leucine , alanine , or valine, and analogs . Many of these compounds competitively inhibit the 65 the phenylalanine at position 420 had been replaced by a normal reaction catalyzed by the enzyme, apparently acting methionine, cysteine , isoleucine , leucine , or threonine , con as substrate analogs. fers increased tolerance/ resistance to a benzoxazinone -de US 10 , 392 ,630 B2 rivative herbicide . The inventors of the present invention (d ) a nucleic acid molecule having 30 % or more identity , have now surprisingly found that those types of double preferably 40 % , 50 % , 60 % , 70 % , 75 % , 80 % , 85 % , 90 % , mutants in the type II or type I PPO of Alopecurus myo 95 % , 96 % , 97 % , 98 % , 99 % , 99 .5 % , or more with the suroides, and , furthermore , novel substitutions which are not nucleic acid molecule sequence of a polynucleotide com disclosed in WO 2012 /080975 confer increased tolerance/ 5 prising the nucleic acid molecule of SEQ ID NO : 1 or 3 , resistance to a wide variety of PPO inhibitors . In addition , or a homolog thereof, and confers increased herbicide the inventors of the present invention have surprisingly tolerance or resistance , as compared to a corresponding, found , that the wildtype sequence of type II PPO of Alope e . g . non - transformed , wild type plant cell , a plant or a part thereof; curus myosuroides as well as single mutations introduced 10 ( e ) a nucleic acid molecule encoding a polypeptide having therein confers increased tolerance/ resistance to a wide 30 % or more identity , preferably at least 40 % , 50 % , 60 % , variety of PPO inhibitors . Thus, to date , the prior art has not 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , 99 % , described PPO - inhibiting herbicide tolerant plants contain 99. 5 % or more , with the amino acid sequence of the PPO ing a PPO nucleic acid according to the present invention , polypeptide sequence of SEQ ID NO : 2 or 4 , and confers which are tolerant/ resistant to a broad spectrum of PPO 15 . increased herbicide tolerance or resistance , as compared inhibitors . Therefore, what is needed in the art are crop to a corresponding , e . g . non - transformed , wild type plant plants and crop plants having increased tolerance to herbi cell , a plant or a part thereof; cides such as PPO - inhibiting herbicide and containing at (f ) nucleic acid molecule which hybridizes with a nucleic least one wildtype and /or mutated PPO nucleic acid accord acid molecule of ( a ), ( b ) , ( c ) , (d ) or ( e ) under stringent ing to the present invention . Also needed are methods for 20 hybridization conditions and confers increased herbicide controlling weed growth in the vicinity of such crop plants tolerance or resistance, as compared to a corresponding , or crop plants . These compositions and methods would e . g . non -transformed , wild type plant cell , a plant or a part allow for the use of spray over techniques when applying??ng thereof; herbicides to areas containing crop plants or crop plants. The invention further provides a method for increasing 25 herbicide tolerance or resistance of a crop plant , themethod SUMMARY OF THE INVENTION comprising the following steps: ( i) increasing or generating of the expression of at least one polynucleotide ; and/ or ( ii ) Accordingly, in one embodiment , the present invention increasing or generating the expression of an expression provides a method for producing a plant having an increased product encoded by at least one polynucleotide ; and /or ( iii ) herbicide tolerance or resistance as compared to a corre - 30 increasing or generating one or more activities of an expres sponding wild type plant whereby the method comprises at sion product encoded by at least one polynucleotide , least the following step : increasing or generating in a plant wherein the polynucleotide is selected from the group con the activity of a transgenic wildtype or mutant Alopecurus sisting of: myosuroides PPO polypeptide, or a homolog thereof. ( a ) an isolated polynucleotide encoding the PPO polypeptide Accordingly , the invention provides a transgenic plant 35 comprising the sequence of SEQ ID NO : 2 or 4 , or a that over - expresses an isolated PPO polynucleotide as homolog thereof ; defined herein , or a homolog thereof, in the sub -cellular ( b ) an isolated polynucleotide comprising the sequence of compartment and tissue as indicated herein . The transgenic SEQ ID NO : 1 or 3 , or a homolog thereof; plant of the invention demonstrates an improved or ( c ) an isolated polynucleotide, which , as a result of the increased herbicide tolerance or resistance as compared to a 40 degeneracy of the genetic code , can be derived from a wild type variety of the plant. PPO polypeptide comprising the sequence of SEQ ID Accordingly , the invention provides a method for produc NO : 2 or 4 , or a homolog thereof and confers an increased ing a plant with increased herbicide tolerance or resistance herbicide tolerance or resistance as compared to a corre as compared to a corresponding wild type plant comprising sponding , e . g . non - transformed , wild type plant cell , a at least one of the steps selected from the group consisting 45 transgenic plant or a part thereof; of: ( i ) increasing or generating the activity of a PPO poly - ( d ) an isolated polynucleotide having 30 % or more identity , peptide comprising the sequence of SEQ ID NO : 2 or 4 , or for example 50 % , 60 % , 70 % , 80 % , 85 % , 90 % , 95 % , a homolog thereof; or ( ii ) increasing or generating the 97 % , 98 % , or 99 % (percent ) or more identity with the activity of an expression product of one or more isolated sequence of a polynucleotide comprising the sequence of polynucleotide ( s ) comprising one or more polynucleotide ( s ) 50 SEQ ID NO : 1 or 3 , or a homolog thereof and conferring comprising the sequence of SEQ ID NO : 1 or 3 , or a an increased herbicide tolerance or resistance as com homolog thereof. pared to a corresponding , e . g. non - transformed , wild type The invention further provides an isolated and / or recom - plant cell , a transgenic plant or a part thereof; binant and / or synthetic nucleic acid molecule comprising a ( e ) an isolated polynucleotide encoding a polypeptide hav nucleic acid molecule selected from the group consisting of: 55 ing 30 % or more identity , for example 50 % , 60 % , 70 % , ( a ) a nucleic acid molecule encoding the PPO polypeptide 80 % , 85 % , 90 % , 95 % , 97 % , 98 % , or 99 % or more comprising the sequence of SEQ ID NO : 2 or 4 , or a identity with the amino acid sequence of the polypeptide homolog thereof; encoded by the isolated polynucleotide of ( a ) to ( c ) and ( b ) a nucleic acid molecule comprising the sequence of SEQ conferring an increased herbicide tolerance or resistance ID NO : 1 or 3 , or a homolog thereof, 60 as compared to a corresponding , e . g . non - transformed , ( c ) a nucleic acid molecule , which , as a result of the wild type plant cell, a transgenic plant or a part thereof; degeneracy of the genetic code , can be derived from a ( f ) an isolated polynucleotide which hybridizes with an PPO polypeptide sequence of SEQ ID NO : 2 or 4 , or a isolated polynucleotide of ( a ) to ( c ) under stringent homolog thereof, and confers increased herbicide toler - hybridization conditions and confers an increased herbi ance or resistance , as compared to a corresponding , e . g . 65 cide tolerance or resistance as compared to a correspond non -transformed , wild type plant cell, a plant or a part ing , e . g . non - transformed , wild type plant cell, a trans thereof; genic plant or a part thereof; US 10 , 392 ,630 B2

Furthermore , the invention relates to a method for pro - In another embodiment, the invention relates to a process ducing a transgenic plant with increased herbicide tolerance for preparing a combination useful for weed control com or resistance as compared to a corresponding , e . g . non - prising ( a ) providing a polynucleotide encoding a wildtype transformed , wild type plant, comprising transforming a or mutated PPO polypeptide according to the present inven plant cell or a plant cell nucleus or a plant tissue to produce 5 tion , which polynucleotide is capable of being expressed in such a plant, with an isolated polynucleotide selected from a plant to thereby provide to that plant tolerance to a PPO the group consisting of: inhibiting herbicide ; and (b ) providing a PPO inhibiting ( a ) an isolated polynucleotide encoding the PPO polypeptide herbicide . comprising the sequence of SEQ ID NO : 2 or 4 , or a In a preferred embodiment, said step of providing a homolog thereof; 10 polynucleotide comprises providing a plant containing the (b ) an isolated polynucleotide comprising the sequence of polynucleotide . SEQ ID NO : 1 or 3 , or a homolog thereof; In another preferred embodiment, said step of providing ( c ) an isolated polynucleotide , which , as a result of the a polynucleotide comprises providing a seed containing the degeneracy of the genetic code , can be derived from a polynucleotide . PPO polypeptide comprising the sequence of SEQ ID 15 In another preferred embodiment, said process further NO : 2 or 4 , or a homolog thereof and confers an increased comprises a step of applying the PPO inhibiting herbicide to herbicide tolerance or resistance as compared to a corre the seed . sponding , e. g. non -transformed , wild type plant cell, a In another embodiment, the invention relates to the use of transgenic plant or a part thereof; a combination useful for weed control, comprising ( a ) a ( d ) an isolated polynucleotide having 30 or more , for 20 polynucleotide encoding a wildtype or mutated PPO poly example 50 % , 60 % , 70 % , 80 % , 85 % , 90 % , 95 % , 97 % , peptide according to the present invention , which polynucle 98 % , or 99 % (percent ) or more identity with the sequence otide is capable of being expressed in a plant to thereby of a polynucleotide comprising the sequence of SEQ ID provide to that plant tolerance to a PPO inhibiting herbicide ; NO : 1 or 3 , or a homolog thereof and conferring an and ( b ) a PPO inhibiting herbicide , to control weeds at a increased herbicide tolerance or resistance as compared to 25 plant cultivation site . a corresponding , e . g . non - transformed , wild type plant cell , a transgenic plant or a part thereof; BRIEF DESCRIPTION OF THE DRAWINGS ( e ) an isolated polynucleotide encoding a polypeptide hav ing 30 or more , for example 50 % , 60 % , 70 % , 80 % , 85 % , FIG . 1 shows an amino acid sequence alignment of 90 % , 95 % , 97 % , 98 % , or 99 % or more identity with the 30 Amaranthus tuberculatus ( A . tuberculatus ) PPO sequences amino acid sequence of the PPO polypeptide encoded by with Alopecurus myosuroides sequence of the present inven the isolated polynucleotide of ( a ) to ( c ) and conferring an tion . FIG . la : AMARE _ WC _ herb _ sus ( SEQ ID NO : 27 ) ; increased herbicide tolerance or resistance as compared to AMARE _ AC _herb _ sus (SEQ ID NO : 28 ) ; AMA a corresponding, e . g . non - transformed , wild type plant RE _ AC _ herb _ resis (SEQ ID NO : 30 ) ; AMARE _ CC _ her cell , a transgenic plant or a part thereof; 35 b _ resis (SEQ ID NO : 29 ) ; and SEQ _ ID _ NO _ 002 _ ALOMY ( f ) an isolated polynucleotide which hybridizes with an (SEQ ID NO : 2 ) . FIG . 16 : PPO1_ ALOMY (SEQ ID NO : 4 ) ; isolated polynucleotide of ( a ) to ( c ) under stringent and PPO1 AMARE (SEO ID NO : 31 ) . hybridization conditions and confers an increased herbi FIG . 2 shows transgenic Arabidopsis plants transformed cide tolerance or resistance as compared to a correspond with nucleic acids encoding mutated Alopecurus (ALOMY ) ing, e .g . non -transformed , wild type plant cell , a trans - 40 PPO2 comprising a double mutation Am _ PPO2 _R137M _ genic plant or a part thereof; F438V , treated with various concentrations of BAS 850H Furthermore , the invention relates to a method for con - ( 1 , 5 - dimethyl- 6 - thioxo - 3 - ( 2 , 2 , 7 - trifluoro - 3 -oxo - 4 - ( prop - 2 trolling undesired vegetation at a plant cultivation site , the ynyl) - 3 , 4 -dihydro -2H -benzo [ b ] [ 1 , 4 ] oxazin - 6 -yl ) - 1 , 3 , 5 - tri method comprising the steps of: azinane - 2 , 4 - dione ) ; A , B , C , D , E , represent different events ; a ) providing, at said site , a plant that comprises at least one 45 2 - 8 represents different treatment concentrations . 1 repre nucleic acid comprising a nucleotide sequence encoding a sents mock treated control. 1 = DMSO , 2 = 1 . 00E - 09M ; PPO polypeptide comprising the sequence of SEQ ID 3 = 6 . 00E - 09M ; 4 = 1 . 00E - 08M ; 5 = 2 . 50E - 08M ; 6 = 5 . 00E NO : 2 or 4 , or a homolog thereof, which is resistant or 08M ; 7 = 1 .00E - 07M ; 8 = 3 .00E - 07M ; tolerant to a PPO - inhibiting herbicide and /or FIG . 3 shows T1 Arabidopsis plants ( 1 per pot ) trans b ) applying to said site an effective amount of said herbicide . 50 formed with nucleic acids encoding mutated Alopecurus In another embodiment, the invention refers to a method (ALOMY ) PPO1 comprising ( 1 ) Am _ PPO1 T299L _ for growing the plant according to the present invention Y420M ; or ( 2 ) Am _ PPO1_ T299L _ S300G _ Y420V . For while controlling weeds in the vicinity of said plant , said comparison , wildtype plants ( 3 per pot) always placed at the method comprising the steps of: bottom of each picture. a ) growing said plant; and 55 FIG . 4 shows clones of TO transformed soybean plants 7 b ) applying a herbicide composition comprising a PPO - days after treatment with the indicated herbicide + 1 % ( v / v ) inhibiting herbicide to the plant and weeds , wherein the MSO . Plants were sprayed at the V2 stage ; 1 = Wild type ; herbicide normally inhibits protoporphyrinogen oxi 2 = AlomyPPO2 _ R137L _ F438M ; 3 = AlomyPPO2 _ F438M ; dase, at a level of the herbicide that would inhibit the 4 = AlomyPPO2 _ R137L _ F438V ; 5 = AlomyPPO2 R137L growth of a corresponding wild - type plant. 60 F438L ; 6 = AlomyPPO2_ R137M _ F438M ; 7 = AlomyPPO2 _ In another embodiment , the invention relates to a com - R137A _ F438M ; 8 = AlomyPPO1_ R138A _ Y420V ; 9 = bination useful for weed control, comprising ( a ) a poly - Alomy - PPO1_ R138L _ Y420V ; 10 = AlomyPPO1_ R138L _ nucleotide encoding a wildtype or mutated PPO polypeptide Y420M ; concentrations of BAS800H (Saflufenacil ) and according to the present invention , which polynucleotide is BAS 850H ( 1 , 5 -dimethyl - 6 - thioxo - 3 - ( 2 , 2 , 7 -trifluoro - 3 - oxo capable of being expressed in a plant to thereby provide to 65 4 - (prop - 2 - ynyl) - 3 , 4 -dihydro - 2H -benzo [ b ] [ 1 , 4 ] oxazin - 6 - yl) that plant tolerance to a PPO inhibiting herbicide ; and ( b ) a 1 , 3 , 5 - triazinane - 2 , 4 - dione ) are given in g a . i . /ha . The con PPO inhibiting herbicide . centrations for constructs 1 , 2 , 3 , 4 , 5 , and 6 are 0 , 100 , 200 , US 10 , 392 ,630 B2 (BAS800H ; Saflufenacil ) ; and 25 , 50 , 75 BAS 850H ( 1 ,5 consecutively generations or BC1 - , BC2 - and consecutively dimethyl- 6 - thioxo - 3 - ( 2 , 2 , 7 - trifluoro - 3 - oxo - 4 - ( prop - 2 -yny generation as well as crossbreeds thereof with non - trans 1 ) - 3 , 4 - dihydro - 2H -benzo [ b ] [ 1 , 4 ]oxazin - 6 - yl) - 1 , 3 , 5 - triazin - genic or other transgenic plants . ane - 2 , 4 - dione ) g a . i. /ha , The concentrations for constructs 8 , A modification , i. e . an increase, can be caused by endog 9 , and 10 are 0 , 25 , 50 , 100 (BAS800H ; Saflufenacil) ; and 5 enous or exogenous factors . For example , an increase in 50 , 75 BAS 850H ( 1 , 5 - dimethyl- 6 -thioxo - 3 - ( 2 , 2 , 7 - trifluoro activity in an organism or a part thereof can be caused by 3 -oxo - 4 - (prop - 2 - ynyl) - 3 ,4 - dihydro - 2H -benzo [ b ][ 1 , 4 ]oxaz adding a gene product or a precursor or an activator or an in - 6 - yl) - 1, 3 , 5 - triazinane - 2 , 4 - dione) g a .i . / ha agonist to the media or nutrition or can be caused by FIG . 5 shows TO Transformed corn 3 days after treatment. introducing said subjects into a organism , transient or stable . Plants were sprayed at the V2 - V3 stage. Furthermore such an increase can be reached by the intro 1 = Wild type ; duction of the inventive nucleic acid sequence or the 2 = tp - SorbiPPO2 _ AlomyPPO2 _ R130L _ F431M (SEQ ID encoded protein in the correct cell compartment for example NO : 22 ) ; into the nucleus or cytoplasmic respectively or into plastids 3 = tp - ZeamaPPO2 _ AlomyPPO2 _ R130L ( SEQ ID NO : 14 ) ; 16 either by transformation and / or targeting . 4 = p - ZeamaPPO2 _ AlomyPPO2 _ R130L _ F431M (SEQ ID For the purposes of the description of the present inven NO : 17 ) ; tion , the terms" cytoplasmic ” and “ non - targeted ” shall indi 5 = p - ZeamaPPO2_ AlomyPPO2_ R130M _ F431M (SEQ ID cate , that the nucleic acid of the invention is expressed NO : 19 ) without the addition of a non - natural transit peptide encod 20 ing sequence . A non -natural transit peptide encoding KEY TO SEQUENCE LISTING sequence is a sequence which is not a natural part of a nucleic acid of the invention , e . g . of the nucleic acids depicted in SEQ ID NO : 1 or 3 , or a homolog thereof, but TABLE 1 is rather added by molecular manipulation steps which are 25 well known to the person skilled in the art or as for example SEQ . ID Accession described hereinafter . Therefore the terms " cytoplasmic ” NO : Description Organism Gene No : and “ non -targeted ” shall not exclude a targeted localization 1 PPO nucleic acid Alopecurus PPO2 to any cell compartment for the products of the inventive myosuroides nucleic acid sequences by their naturally occurring sequence 2 PPO amino acid Alopecurus PPO2 30 properties within the background of the transgenic organ myosuroides 3 PPO nucleic acid Alopecurus PPO1 ism . The sub - cellular location of the mature polypeptide myosuroides derived from the enclosed sequences can be predicted by a 4 PPO amino acid Alopecurus PPO1 skilled person for the organism (plant ) by using software myosuroides tools like TargetP (Emanuelsson et al ., ( 2000 ) , Predicting PPO amino acid Alopecurus PPO transit myosuroides peptide sub - cellular localization of proteins based on their N -termi 6 PPO nucleic acid Alopecurus PPO2 nal amino acid sequence . J . Mol. Biol. 300 , 1005 - 1016 ) , myosuroides codon ChloroP ( Emanuelsson et al. (1999 ) , Chlorop , a neural optimized 7 PPO nucleic acid Alopecurus PPO1 network -based method for predicting chloroplast transit myosuroides codon 40 peptides and their cleavage sites . Protein Science , 8 : 978 optimized 984 ) or other predictive software tools ( Emanuelsson et al. 8 PPO amino acid Zea mays PPO2 transit peptide (2007 ) , locating proteins in the cell using TargetP , SignalP , 9 PPO amino acid Sorghum bicolor PPO2 transit and related tools (Nature Protocols 2 , 953 -971 ) . peptide The term " organelle ” according to the invention shall 27 PPO amino acid Amaranthus PPO2 WC _ HS 45 mean for example “ mitochondria ” , “ plastid ” or endoplasmic 28 PPO amino acid Amaranthus PPO2 AC HS 29 PPO amino acid Amaranthus PPO2 CC HR reticulum ( ER ) . The term “ plastid ” according to the inven 30 PPO amino acid Amaranthus PPO2 AC _ HR tion is intended to include various forms of plastids includ 31 PPO amino acid Amaranthus PPO1 ing proplastids , chloroplasts , chromoplasts , gerontoplasts , leucoplasts, amyloplasts , elaioplasts and etioplasts , prefer 50 ably chloroplasts . They all have as a common ancestor the aforementioned proplasts . DETAILED DESCRIPTION The term “ introduced ” in the context of this specification shall mean the insertion of a nucleic acid sequence into the An " herbicide tolerance or resistance - increasing activity ” organism by means of a “ transfection ” , “ transduction ” or according to the invention refers to an activity of a PPO from 55 preferably by “ transformation ” . Alopecurus myosuroides comprising the sequence of SEQ A plastid , such as a chloroplast , has been “ transformed ” ID NO : 2 or 4 , or a homolog thereof. A polypeptide by an exogenous (preferably foreign ) nucleic acid sequence conferring a herbicide tolerance or resistance - increasing if nucleic acid sequence has been introduced into the plastid activity can be encoded by a nucleic acid sequence com - that means that this sequence has crossed the membrane or prising the sequence of SEQ ID NO : 1 or 3 , or a homolog 60 the membranes of the plastid . The foreign DNA may be thereof, and/ or comprises or consists of a polypeptide com - integrated (covalently linked ) into plastid DNA making up prising the sequence of SEQ ID NO : 2 or 4 , or a homolog the genome of the plastid , or it may remain not integrated thereof. (e . g. , by including a chloroplast origin of replication ). “ Sta A “ transgenic plant” , as used herein , refers to a plant bly ” integrated DNA sequences are those , which are inher which contains a foreign nucleotide sequence inserted into 65 ited through plastid replication , thereby transferring new either its nuclear genome or organelle genome. It encom - plastids , with the features of the integrated DNA sequence to passes further the offspring generations i .e . the T1 -, T2 - and the progeny. US 10 , 392 ,630 B2 10 As used herein , " plant " is meant to include not only a regulatory sequences. The boundaries of the coding whole plant but also a part thereof i. e ., one or more cells, and sequence are determined by a translation start codon at the tissues , including for example , leaves , stems, shoots , roots, 5 '- terminus and a translation stop codon at the 3 '- terminus . flowers , fruits and seeds. A coding sequence can include, but is not limited to mRNA , The term " herbicide tolerance or resistance " as used 5 cDNA , recombinant nucleotide sequences or genomic DNA , herein it is intended that a plant that is tolerant or resistant while introns may be present as well under certain circum to at least one herbicide at a level that would normally kill , stances or inhibit the growth of, a normal or wild - type plant . As used in the present context a nucleic acid molecule Any increase in herbicide tolerance or resistance is an may also encompass the untranslated sequence located at the improved herbicide tolerance or resistance in accordance 10 3 ' and at the 5 ' end of the coding gene region , for example with the invention . For example , the improvement in her - 2000 , preferably less , e . g . 500 , preferably 200 , especially bicide tolerance or resistance can comprise a 1 .5x , 2x , 2 .5x , preferable 100 , nucleotides of the sequence upstream of the 3x , 5x , 10x , 20x , 30x , 40x , 50x , 75x , 100x , 150x , 200x or 5 ' end of the coding region and for example 300 , preferably greater increase in any measurable parameter . less, e . g . 100 , preferably 50 , especially preferable 20 , Unless otherwise specified , the terms " polynucleotides” , 15 nucleotides of the sequence downstream of the 3 ' end of the “ nucleic acid ” and “ nucleic acid molecule ” are interchange - coding gene region . ably in the present context. Unless otherwise specified , the “ Polypeptide” refers to a polymer of amino acid (amino terms " peptide” , “ polypeptide ” and “ protein ” are inter- acid sequence ) and does not refer to a specific length of the changeably in the present context . The term “ sequence” may molecule . Thus, peptides and oligopeptides are included relate to polynucleotides , nucleic acids, nucleic acid mol - 20 within the definition of polypeptide. This term does also ecules, peptides , polypeptides and proteins , depending on refer to or include post- translational modifications of the the context in which the term “ sequence ” is used . The terms polypeptide, for example , glycosylations, acetylations, " gene ( s ) " , " polynucleotide” , “ nucleic acid sequence ” , phosphorylations and the like . Included within the definition “ nucleotide sequence ” , or “ nucleic acid molecule ( s ) ” as a re , for example, polypeptides containing one or more used herein refers to a polymeric form of nucleotides of any 25 analogs of an amino acid ( including , for example , unnatural length , either ribonucleotides or deoxyribonucleotides. The amino acids, etc . ) , polypeptides with substituted linkages, as terms " gene ( s )” , " polynucleotide” , “ nucleic acid sequence ” , well as other modifications known in the art , both naturally “ nucleotide sequence ” , or “ nucleic acid molecule ( s) ” as occurring and non -naturally occurring . An “ isolated ” poly used herein include known types of modifications, for nucleotide or nucleic acid molecule is separated from other example , methylation , " caps ” , substitutions of one or more 30 polynucleotides or nucleic acid molecules, which are present of the naturally occurring nucleotides with an analogue . in the natural source of the nucleic acid molecule . An Preferably , the DNA or RNA sequence comprises a coding isolated nucleic acid molecule may be a chromosomal sequence encoding the herein defined polypeptide . fragment of several kb , or preferably , a molecule only As also used herein , the terms “ nucleic acid ” and “ nucleic comprising the coding region of the gene . Accordingly, an acid molecule ” are intended to include DNA molecules (e . g . 35 isolated nucleic acid molecule of the invention may com cDNA or genomic DNA ) and RNA molecules ( e . g . mRNA ) prise chromosomal regions, which are adjacent 5 ' and 3 ' or and analogs of the DNA or RNA generated using nucleotide further adjacent chromosomal regions, but preferably com analogs . The nucleic acid molecule can be single - stranded or prises no such sequences which naturally flank the nucleic double - stranded . acid molecule sequence in the genomic or chromosomal An " isolated ” nucleic acid molecule is one that is sub - 40 context in the organism from which the nucleic acid mol stantially separated from other nucleic acid molecules , ecule originates ( for example sequences which are adjacent which are present in the natural source of the nucleic acid to the regions encoding the 5 '- and 3 ' -UTRs of the nucleic That means other nucleic acid molecules are present in an acid molecule ) . An “ isolated ” or “ purified ” polypeptide or amount less than 5 % based on weight of the amount of the biologically active portion thereof is free of some of the desired nucleic acid , preferably less than 2 % by weight, 45 cellular material when produced by recombinant DNA tech more preferably less than 1 % by weight, most preferably niques , or chemical precursors or other chemicals when less than 0 . 5 % by weight. Preferably , an " isolated ” nucleic chemically synthesized . The language " substantially free of acid is free of some of the sequences that naturally flank the cellular material” includes preparations of a protein in which nucleic acid ( i . e ., sequences located at the 5 ' and 3 ' ends of the polypeptide is separated from some of the cellular the nucleic acid ) in the genomic DNA of the organism from 50 components of the cells in which it is naturally or recom which the nucleic acid is derived . For example , in various binantly produced . embodiments , the isolated herbicide resistance and / or toler - The terms " comprise ” or “ comprising ” and grammatical ance related protein encoding nucleic acid molecule can variations thereof when used in this specification are to be contain less than about 5 kb , 4 kb , 3 kb , 2 kb , 1 kb , 0 . 5 kb taken to specify the presence of stated features , integers , or 0 . 1 kb of nucleotide sequences which naturally flank the 55 steps or components or groups thereof, but not to preclude nucleic acid molecule in genomic DNA of the cell from the presence or addition of one or more other features , which the nucleic acid is derived . Moreover, an “ isolated ” integers, steps, components or groups thereof . nucleic acid molecule , such as a cDNA molecule , can be free In accordance with the invention , a protein or polypeptide from some of the other cellular material with which it is has the " activity of a PPO protein if its de novo activity , or naturally associated , or culture medium when produced by 60 its increased expression directly or indirectly leads to and recombinant techniques , or chemical precursors or other confers increased herbicide tolerance or resistance , as com chemicals when chemically synthesized . pared to a corresponding , e . g . non - transformed , wild type A " coding sequence ” is a nucleotide sequence , which is plant and the protein has the above mentioned activity of a transcribed into an RNA , e . g . a regulatory RNA , such as a PPO . miRNA , a ta - sRNA , co - suppression molecule , an RNAi, a 65 Throughout the specification the activity or preferably the ribozyme, etc . or into a mRNA which is translated into a biological activity of such a protein or polypeptide or an polypeptide when placed under the control of appropriate nucleic acid molecule or sequence encoding such protein or US 10 , 392 ,630 B2 11 polypeptide is identical or similar if it still has the biological 30 % or more , especially preferably to 50 % , 70 % or more , or enzymatic activity of a protein comprising the sequence very especially preferably to 100 % ,most preferably to 500 % of SEQ ID NO : 2 or 4 , or a homolog thereof, or which has or more. However , a de novo expression is also regarded as 10 % or more of the original enzymatic activity , preferably subject of the present invention . 20 % , 30 % , 40 % , 50 % , particularly preferably 60 % , 70 % , 5 The terms " wild type ” , " control" or " reference ” are 80 % most particularly preferably 90 % , 95 % , 98 % , 99 % or exchangeable and can be a cell or a part of organisms such more in comparison to a protein comprising the sequence of as an organelle like a chloroplast or a tissue , or an organism , SEQ ID NO : 2 or 4 , or a homolog thereof. in particular a plant, which was not modified or treated In another embodiment the biological or enzymatic activ - according to the herein described process according to the ity of a protein comprising the sequence of SEQ ID NO : 2 10 invention . Accordingly, the cell or a part of organisms such or 4 , or a homolog thereof, has 100 % or more of the original as an organelle like a chloroplast or a tissue , or an organism , enzymatic activity, preferably 110 % , 120 % , 130 % , 150 % , in particular a plant used as wild type , control or reference particularly preferably 150 % , 200 % , 300 % or more in corresponds to the cell, organism , plant or part thereof as comparison to a protein comprising the sequence of SEQ ID much as possible and is in any other property but in the result NO : 2 or 4 , or a homolog thereof. 15 of the process of the invention as identical to the subject The terms “ increased ” , “ raised ” , “ extended ” , “ enhanced ” , matter of the invention as possible . Thus, the wild type , " improved ” or “ amplified ” relate to a corresponding change control or reference is treated identically or as identical as of a property in a plant, an organism , a part of an organism possible , saying that only conditions or properties might be such as a tissue , seed , root, leave , flower etc . or in a cell and different which do not influence the quality of the tested are interchangeable . Preferably, the overall activity in the 20 property . volume is increased or enhanced in cases if the increase or Preferably , any comparison is carried out under analogous enhancement is related to the increase or enhancement of an conditions. The term “ analogous conditions” means that all activity of a gene product , independent whether the amount conditions such as , for example , culture or growing condi of gene product or the specific activity of the gene product tions , soil, nutrient, water content of the soil , temperature , or both is increased or enhanced or whether the amount, 25 humidity or surrounding air or soil , assay conditions ( such stability or translation efficacy of the nucleic acid sequence as buffer composition , temperature , substrates , pathogen or gene encoding for the gene product is increased or strain , concentrations and the like ) are kept identical enhanced . between the experiments to be compared . The terms “ increase ” include the change of said property The “ reference ” , " control” , or “ wild type” is preferably a in only parts of the subject of the present invention , for 30 subject, e . g . an organelle , a cell , a tissue , an organism , in example , the modification can be found in compartment of particular a plant, which was not modified or treated accord a cell , like a organelle , or in a part of a plant, like tissue , ing to the herein described process of the invention and is in seed , root , leave , flower etc . but is not detectable if the any other property as similar to the subject matter of the overall subject, i. e . complete cell or plant, is tested . Accord - invention as possible. The reference, control or wild type is ingly , the term “ increase ” means that the specific activity of 35 in its genome, transcriptome, proteome or metabolome as an enzyme as well as the amount of a compound or metabo - similar as possible to the subject of the present invention . lite , e . g . of a polypeptide, a nucleic acid molecule of the Preferably , the term " reference - ” " control- ” or “ wild type - " invention or an encoding mRNA or DNA , can be increased -organelle , - cell , -tissue or -organism , in particular plant , in a volume. The term “ increase ” includes, that a compound relates to an organelle , cell , tissue or organism , in particular or an activity , especially an activity , is introduced into a cell , 40 plant, which is nearly genetically identical to the organelle , the cytoplasm or a sub - cellular compartment or organelle de cell , tissue or organism , in particular plant, of the present novo or that the compound or the activity , especially an invention or a part thereof preferably 90 % or more , e . g . activity , has not been detected before , in other words it is 95 % , more preferred are 98 % , even more preferred are “ generated ” . Accordingly , in the following , the term 99. 00 % , in particular 99 . 10 % , 99. 30 % , 99 .50 % , 99 .70 % , " increasing” also comprises the term " generating ” or 45 99 . 90 % , 99 . 99 % , 99 . 999 % or more . Most preferable the " stimulating " . The increased activity manifests itself in " reference ” , " control" , or " wild type” is a subject, e . g . an increased herbicide tolerance or resistance , as compared to organelle, a cell, a tissue, an organism , in particular a plant, a corresponding , e . g . non -transformed , wild type plant cell, which is genetically identical to the organism , in particular plant or part thereof. plant, cell , a tissue or organelle used according to the process Under " change of a property ” it is understood that the 50 of the invention except that the responsible or activity activity , expression level or amount of a gene product or the conferring nucleic acid molecules or the gene product metabolite content is changed in a specific volume relative encoded by them are amended , manipulated , exchanged or to a corresponding volume of a control, reference or wild introduced according to the inventive process . In case , a type , including the de novo creation of the activity or control, reference or wild type differing from the subject of expression . 55 the present invention only by not being subject of the “ Amount of protein or mRNA” is understood as meaning process of the invention can not be provided , a control , the molecule number of polypeptides or mRNA molecules in reference or wild type can be an organism in which the cause an organism , especially a plant, a tissue, a cell or a cell for the modulation of an activity conferring the enhanced compartment . “ Increase” in the amount of a protein means tolerance or resistance to herbicides as compared to a the quantitative increase of the molecule number of said 60 corresponding , e . g . non - transformed , wild type plant cell, protein in an organism , especially a plant, a tissue , a cell or plant or part thereof or expression of the nucleic acid a cell compartment such as an organelle like a plastid or molecule of the invention as described herein has been mitochondria or part thereof- for example by one of the switched back or off , e . g . by knocking out the expression of methods described herein below - in comparison to a wild responsible gene product , e . g . by antisense or RNAi or type , control or reference . 65 miRNA inhibition , by inactivation of an activator or agonist, The increase in molecule number amounts preferably to by activation of an inhibitor or antagonist , by inhibition 1 % or more , preferably to 10 % ormore , more preferably to through adding inhibitory antibodies , by adding active com US 10 , 392 ,630 B2 13 14 pounds as e . g . hormones, by introducing negative dominant episomal mammalian vectors ) are integrated into the mutants , etc. A gene production can for example be knocked genome of a host cell or a organelle upon introduction into out by introducing inactivating point mutations, which lead the host cell, and thereby are replicated along with the host to an enzymatic activity inhibition or a destabilization or an or organelle genome. Moreover , certain vectors are capable inhibition of the ability to bind to cofactors etc . Accordingly , 5 of directing the expression of genes to which they are preferred reference subject is the starting subject of the operatively linked . Such vectors are referred to herein as present process of the invention . Preferably , the reference " expression vectors .” In general, expression vectors of util and the subject matter of the invention are compared after ity in recombinant DNA techniques are often in the form of standardization and normalization , e . g . to the amount of plasmids . In the present specification , " plasmid ” and “ vec total RNA , DNA , or protein or activity or expression of 10 tor ” can be used interchangeably as the plasmid is the most reference genes, like housekeeping genes , such as ubiquitin , commonly used form of vector. However , the invention is actin or ribosomal proteins . intended to include such other forms of expression vectors , The term " expression ” refers to the transcription and / or such as viral vectors ( e . g . , replication defective retroviruses , translation of a codogenic gene segment or gene . As a rule , adenoviruses, and adeno - associated viruses ), which serve the resulting product is an mRNA or a protein . 15 equivalent functions . The increase or modulation according to this invention As used herein , “ operatively linked ” is intended to mean can be constitutive , e . g . due to a stable permanent transgenic that the nucleotide sequence of interest is linked to the expression or to a stable mutation in the corresponding regulatory sequence ( s ) in a manner which allows for expres endogenous gene encoding the nucleic acid molecule of the sion of the nucleotide sequence ( e . g . in an in vitro transcrip invention or to a modulation of the expression or of the 20 tion / translation system or in a host cell when the vector is behavior of a gene conferring the expression of the poly - introduced into the host cell ) . The term “ regulatory peptide of the invention , or transient , e . g . due to an transient sequence ” is intended to include promoters , enhancers , and transformation or temporary addition of a modulator such as other expression control elements ( e . g . polyadenylation sig a agonist or antagonist or inducible , e . g . after transformation nals ) . with a inducible construct carrying the nucleic acid molecule 25 Such regulatory sequences are described , for example , in of the invention under control of a inducible promoter and Goeddel , Gene Expression Technology : Methods in Enzy adding the inducer , e . g. tetracycline or as described herein mology 185 , Academic Press, San Diego , Calif . ( 1990 ), and below . Gruber and Crosby , in : Methods in PlantMolecular Biology Less influence on the regulation of a gene or its gene and Biotechnology , eds. Glick and Thompson , Chapter 7 , product is understood as meaning a reduced regulation of the 30 89 - 108 , CRC Press ; Boca Raton , Fla . , including the refer enzymatic activity leading to an increased specific or cellu - ences therein . Regulatory sequences include those that direct lar activity of the gene or its product . An increase of the constitutive expression of a nucleotide sequence in many enzymatic activity is understood as meaning an enzymatic types of host cells and those that direct expression of the activity , which is increased by 10 % ormore , advantageously nucleotide sequence only in certain host cells or under 20 % , 30 % or 40 % or more , especially advantageously by 35 certain conditions. 50 % , 60 % or 70 % or more in comparison with the starting “ Transformation ” is defined herein as a process for intro organism . This leads to increased herbicide tolerance or ducing heterologous DNA into a plant cell, plant tissue , or resistance , as compared to a corresponding , e . g . non - trans - plant. It may occur under natural or artificial conditions formed , wild type plant or part thereof. using various methods well known in the art . Transforma The increase in activity of the polypeptide amounts in a 40 tion may rely on any known method for the insertion of cell, a tissue , an organelle , an organ or an organism , pref - foreign nucleic acid sequences into a prokaryotic or eukary erably a plant, or a part thereof preferably to 5 % or more , otic host cell . The method is selected based on the host cell preferably to 20 % or to 50 % , especially preferably to 70 % , being transformed and may include , but is not limited to , 80 % , 90 % or more , very especially preferably are to 100 % , viral infection , electroporation , lipofection , and particle 150 % or 200 % , most preferably are to 250 % or more in 45 bombardment. Such " transformed " cells include stably comparison to the control, reference or wild type . In one transformed cells in which the inserted DNA is capable of embodiment the term increase means the increase in amount replication either as an autonomously replicating plasmid or in relation to the weight of the organism or part thereof as part of the host chromosome. They also include cells ( w / w ) . which transiently express the inserted DNA or RNA for By “ vectors ” is meant with the exception of plasmids all 50 limited periods of time. Transformed plant cells , plant tissue , other vectors known to those skilled in the art such as by way or plants are understood to encompass not only the end of example phages , viruses such as SV40 , CMV , baculovi- product of a transformation process , but also transgenic rus , adenovirus , transposons , IS elements , phasmids , progeny thereof . phagemids, cosmids , linear or circular DNA. These vectors The terms “ transformed , ” “ transgenic, ” and “ recombi can be replicated autonomously in the host organism or be 55 nant” refer to a host organism such as a bacterium or a plant chromosomally replicated , chromosomal replication being into which a heterologous nucleic acid molecule has been preferred . As used herein , the term “ vector” refers to a introduced . The nucleic acid molecule can be stably inte nucleic acid molecule capable of transporting another grated into the genome of the host or the nucleic acid nucleic acid to which it has been linked . One type of vector molecule can also be present as an extra - chromosomal is a “ plasmid ” , which refers to a circular double stranded 60 molecule . Such an extra - chromosomal molecule can be DNA loop into which additional DNA segments can be auto - replicating . Transformed cells , tissues , or plants are ligated . Another type of vector is a viral vector, wherein understood to encompass not only the end product of a additional DNA segments can be ligated into the viral transformation process , but also transgenic progeny thereof. genome. Certain vectors are capable of autonomous repli - A “ non - transformed ” , “ non - transgenic " or " non -recombi cation in a host cell into which they are introduced (e . g . 65 nant” host refers to a wild -type organism , e . g . a bacterium or bacterial vectors having a bacterial origin of replication and plant, which does not contain the heterologous nucleic acid episomal mammalian vectors ). Other vectors ( e . g . non molecule . US 10 , 392 ,630 B2 15 16 The terms “ host organism ” , “ host cell” , “ recombinant formed plant obtained according to the invention can be (host ) organism ” and “ transgenic (host ) cell ” are used here used in a conventional breeding scheme or in in vitro plant interchangeably . Of course these terms relate not only to the propagation to produce more transformed plants with the particular host organism or the particular target cell but also same characteristics and /or can be used to introduce the to the descendants or potential descendants of these organ - 5 same characteristic in other varieties of the same or related isms or cells. Since , due to mutation or environmental effects species . Such plants are also part of the invention . Seeds certain modifications may arise in successive generations, obtained from the transformed plants genetically also con these descendants need not necessarily be identical with the tain the same characteristic and are part of the invention . As parental cell but nevertheless are still encompassed by the mentioned before , the present invention is in principle term as used here . 10 applicable to any plant and crop that can be transformed with For the purposes of the invention “ transgenic ” or “ recom - any of the transformation method known to those skilled in binant ” means with regard for example to a nucleic acid the art . sequence, an expression cassette ( = gene construct , nucleic The term “ homology ” means that the respective nucleic acid construct) or a vector containing the nucleic acid acid molecules or encoded proteins are functionally and /or sequence according to the invention or an organism trans - 15 structurally equivalent. The nucleic acid molecules that are formed by said nucleic acid sequences , expression cassette homologous to the nucleic acid molecules described above or vector according to the invention all those constructions and that are derivatives of said nucleic acid molecules are , produced by genetic engineering methods in which either for example , variations of said nucleic acid molecules which ( a ) the nucleic acid sequence comprising the sequence of represent modifications having the same biological function , SEQ ID NO : 1 or 3 , or a homolog thereof, or its 20 in particular encoding proteins with the same or substan derivatives or parts thereof; or tially the same biological function . They may be naturally ( b ) a genetic control sequence functionally linked to the occurring variations, such as sequences from other plant nucleic acid sequence described under ( a ) , for example a varieties or species, or mutations . These mutations may 3 ' - and /or 5 '- genetic control sequence such as a promoter occur naturally or may be obtained by mutagenesis tech or terminator, or 25 niques . The allelic variations may be naturally occurring ( c ) ( a ) and ( b ) ; allelic variants as well as synthetically produced or geneti are not found in their natural, genetic environment or have cally engineered variants . Structurally equivalents can , for been modified by genetic engineering methods, wherein the example , be identified by testing the binding of said poly modification may by way of example be a substitution , peptide to antibodies or computer based predictions. Struc addition , deletion , inversion or insertion of one or more 30 turally equivalent have the similar immunological charac nucleotide residues. teristic , e . g . comprise similar epitopes. “ Natural genetic environment” means the natural As used herein , the terms “ gene " and " recombinant gene ” genomic or chromosomal locus in the organism of origin or refer to nucleic acid molecules comprising an open reading inside the host organism or presence in a genomic library. In frame encoding the polypeptide of the invention or com the case of a genomic library the natural genetic environ - 35 prising the nucleic acid molecule of the invention or encod ment of the nucleic acid sequence is preferably retained at ing the polypeptide used in the process of the present least in part . The environment borders the nucleic acid invention . Such natural variations can typically result in 1 to sequence at least on one side and has a sequence length of 5 % variance in the nucleotide sequence of the gene . Any and at least 50 bp , preferably at least 500 bp , particularly all such nucleotide variations and resulting amino acid preferably at least 1 , 000 bp , most particularly preferably at 40 polymorphisms in genes encoding a polypeptide of the least 5 ,000 bp . A naturally occurring expression cassette invention or comprising a the nucleic acid molecule of the for example the naturally occurring combination of the invention that are the result of natural variation and that do natural promoter of the nucleic acid sequence according to not alter the functional activity as described are intended to the invention with the corresponding gene — turns into a be within the scope of the invention . transgenic expression cassette when the latter is modified by 45 unnatural, synthetic (" artificial" ) methods such as by way of Specific Embodiments example a mutagenation . Appropriate methods are described by way of example in U . S . Pat. No . 5 , 565 , 350 or WO Accordingly , this invention provides measures and meth 00 / 15815 . ods to produce plants with increased herbicide tolerance or The term “ transgenic plants ” used in accordance with the 50 resistance. invention also refers to the progeny of a transgenic plant, for Accordingly, the present invention provides transgenic example the T , T2, Tz and subsequent plant generations or plants showing increased tolerance or resistance to one or the BC ,, BC2 , BC3 and subsequent plant generations. Thus, more herbicides as compared to the corresponding origin or the transgenic plants according to the invention can be raised the wild type plant and methods for producing such trans and selfed or crossed with other individuals in order to 55 genic plants with increased herbicide tolerance or resistance . obtain further transgenic plants according to the invention . One or more enhanced herbicide tolerance - related pheno Transgenic plants may also be obtained by propagating types are increased in accordance with the invention by transgenic plant cells vegetatively . The present invention increasing or generating the activity of a PPO enzyme from also relates to transgenic plant material, which can be Alopecurus myosuroides . derived from a transgenic plant population according to the 60 The nucleic acid molecule of the present invention or used invention . Such material includes plant cells and certain in accordance with the present invention , encodes a protein tissues , organs and parts of plants in all their manifestations , conferring an activity of a PPO enzyme from Alopecurus such as seeds , leaves, anthers, fibers , tubers , roots , root hairs , myosuroides. stems, embryo , calli, cotelydons , petioles , harvested mate Accordingly , in one embodiment, the present invention rial, plant tissue, reproductive tissue and cell cultures, which 65 relates to a nucleic acid molecule that encodes a polypeptide are derived from the actual transgenic plant and /or can be with an herbicide tolerance or resistance - increasing activity used for bringing about the transgenic plant. Any trans which is encoded by a nucleic acid sequence comprising the US 10 , 392 ,630 B2 17 18 sequence of SEQ ID NO : 1 or 3 , or a homolog thereof, a plant or a part thereof, the activity of an PPO polypeptide and /or which is a protein comprising or consisting of a of the present invention , e . g . by the methods mentioned polypeptide comprising the sequence ofSEQ ID NO : 2 or 4 , herein ; and ( b ) cultivating or growing the plant cell , the plant or a homolog thereof. or the part thereof under conditions which permit the devel In a preferred embodiment, the homolog refers to a 5 opment of the plant cell , the plant or the part thereof; and ( c ) mutated PPO enzyme from Alopecurus myosuroides com recovering a plant from said plant cell nucleus , said plant prising the sequence of SEQ ID NO : 2 or 4 , in which the wildtype amino acid at or corresponding to position 137 of cell , or said plant part, which shows increased herbicide SEQ ID NO : 2 and / or the wildtype amino acid at or tolerance or resistance as compared to a corresponding , e . g . corresponding to position 415 of SEQ ID NO : 2 , and /or the 10 non - transformed , origin or wild type plant ; and ( d ) option wildtype amino acid at or corresponding to position 438 of ally , selecting the plant or a part thereof, showing increased SEQ ID NO : 2 is substituted by another amino acid , as herbicide tolerance or resistance , as compared to a corre described in greater detail hereinafter. sponding, e . g . non - transformed , wild type plant cell, e . g . The increase or generation of said “ activity ” is for which shows visual symptoms of deficiency and /or death . example conferred by the increase of activity or of amount 155 Furthermore , the present invention also relates to a in a cell or a part thereof of one or more expression products method for the identification of a plant with an increased of said nucleic acid molecule , e . g . proteins , or by de novo herbicide tolerance or resistance comprising screening a expression , i. e . by the generation of said “ activity ” in the population of one or more plant cell nuclei, plant cells , plant plant. tissues or plants or parts thereof for said “ activity ” , com In one embodiment, said herbicide tolerance or resis - 20 paring the level of activity with the activity level in a tance - increasing activity is increased by increasing the reference ; identifying one or more plant cell nuclei, plant amount and / or the specific activity of a PPO protein com - cells , plant tissues or plants or parts thereof with the activity prising the sequence of SEQ ID NO : 2 or 4 , or a homolog increased compared to the reference , optionally producing a thereof. plant from the identified plant cell nuclei , cell or tissue . Accordingly , in one embodiment, an increased herbicide 25 In one further embodiment, the present invention also tolerance or resistance as compared to a correspondingly relates to a method for the identification of a plant with an non -modified , e . g . a non - transformed , wild type plant is increased herbicide tolerance or resistance comprising conferred according to method of the invention , by increas screening a population of one or more plant cell nuclei, plant ing or generating the activity of a polypeptide comprising cells , plant tissues or plants or parts thereof for the expres the sequence of SEQ ID NO : 2 or 4 , or a homolog thereof , 30 sion level of an nucleic acid coding for an polypeptide or encoded by the nucleic acid molecule ( or gene ) the conferring said activity , comparing the level of expression sequence of SEQ ID NO : 1 or 3 , or a homolog of said nucleic with a reference ; identifying one or more plant cell nuclei , acid molecule or polypeptide. plant cells , plant tissues or plants or parts thereof with the Thus, in one embodiment, the present invention provides expression level increased compared to the reference , a method for producing a plant showing increased or 35 optionally producing a plant from the identified plant cell improved herbicide resistance or tolerance as compared to nuclei, cell or tissue. the corresponding origin or wild type plant, by increasing or Accordingly , in a preferred embodiment, the present generating the activity of an PPO enzyme, e . g . which is invention provides a method for producing a transgenic cell conferred by one or more polynucleotide ( s ) comprising the for the regeneration or production of a plant with increased sequence of SEQ ID NO : 1 or 3 , or a homolog thereof, or by 40 herbicide tolerance or resistance , as compared to a corre one or more protein ( s ), each comprising a polypeptide sponding , e . g . non - transformed , wild type cell by increasing encoded by one or more nucleic acid sequence ( s ) compris - or generating the activity of a PPO polypeptide of the ing the sequence of SEQ ID NO : 1 or 3 , or a homolog present invention . The cell can be for example a host cell , thereof, or by one or more protein ( s ) each comprising a e . g . a transgenic host cell . A host cell can be for example a polypeptide comprising the sequence of SEQ ID NO : 2 or 4 , 45 microorganism , e . g . derived from fungi or bacteria , or a or a homolog thereof, and ( b ) optionally , growing the plant plant cell particular useful for transformation . cell, plant or part thereof under conditions which permit the Thus , the present invention fulfills the need to identify development of the plant cell , the plant or the part thereof , new , unique genes capable of conferring increased herbicide and ( c ) regenerating a plant with increased herbicide toler- tolerance or resistance to plants , upon expression or over ance or resistance , as compared to a corresponding , e . g . 50 expression of exogenous genes . Accordingly , the present non - transformed , wild type plant or a part thereof. invention provides novel PPO enzymes comprising the Accordingly, in one further embodiment, the said method sequence of SEQ ID NO : 2 or 4 , or a homolog thereof. for producing a plant or a part thereof for the regeneration In one embodiment the increase in activity of the poly of said plant, the plant showing an increased herbicide peptide amounts in an organelle such as a plastid . In another tolerance or resistance , said method comprises ( i ) growing 55 embodiment the increase in activity of the polypeptide the plant or part thereof together with a , e . g . non - trans amounts in the cytoplasm . formed , wild type plant under conditions of herbicide treat The specific activity of a polypeptide encoded by a ment; and ( ii ) selecting a plant with increased herbicide nucleic acid molecule of the present invention or of the tolerance or resistance as compared to a corresponding, e . g . polypeptide of the present invention can be tested as non - transformed , wild type plant, for example after the , e . g . 60 described in the examples . In particular, the expression of a non - transformed , wild type plant shows visual symptoms of protein in question in a cell, e . g . a plant cell in comparison deficiency and / or death . to a control is an easy test and can be performed as described Further , the present invention relates to a method for in the state of the art. producing a plant with increased herbicide tolerance or Accordingly , in one embodiment, the process of the resistance as compared to a corresponding origin or wild 65 present invention for producing a plant with increased type plant, e . g . a transgenic plant, which comprises: ( a ) herbicide tolerance or resistance comprises increasing or increasing or generating , in a plant cell nucleus, a plant cell , generating the activity of a gene product conferring the US 10 , 392 ,630 B2 20 activity of a PPO enzyme from Alopecurus myosuroides or thereof or as encoded by the nucleic acid sequences its functional equivalent or its homolog , e . g . the increase of comprising the sequence of SEQ ID NO : 1 or 3 , or a ( a ) a gene product of a gene comprising the nucleic acid homolog thereof, and which is joined to a nucleic acid molecule comprising the sequence of SEQ ID NO : 1 or 3 , sequence encoding an organelle localization sequence , or a functional equivalent or a homologue thereof; or 5 especially a chloroplast localization sequence , in a plant ( b ) a polypeptide comprising a polypeptide, a consensus cell, sequence or a polypeptide motif comprising the sequence (a4 ) increasing or generating the activity of a protein com of SEQ ID NO : 2 or 4 or a functional equivalent or a prising the sequence of SEQ ID NO : 2 or 4 , or a homolog homologue thereof. thereof or as encoded by the nucleic acid sequences Accordingly, an activity of a PPO polypeptide from 10 comprising the sequence of SEQ ID NO : 1 or 3 , or a Alopecurus myosuroides is increased in one or more specific homolog thereof, and which is joined to a nucleic acid compartment ( s ) or organelle ( s ) of a cell or plant and confers sequence encoding an mitochondrion localization said increased herbicide tolerance or resistance. For sequence in a plant cell , example , said activity can be increased in plastids or mito - and chondria of a plant cell , thus conferring increase of herbicide 15 (b ) regererating a plant from said plant cell ; tolerance or resistance in a corresponding plant. ( c ) growing the plant under conditions which permit the In one embodiment , an activity conferred by an expres development of a plant with increased herbicide tolerance sion of a gene described herein or its expression product; i .e . or resistance as compared to a corresponding , e . g . non by a PPO polypeptide of the present invention is increased transformed , wild type plant. or generated in the plastid . 20 The skilled worker is able to link transit peptide nucleic In one embodiment, an activity conferred by the expres - acid sequences to the nucleic acid sequences comprising the sion of a gene described herein or its expression product ; i. e . sequence of SEQ ID NO : 1 or 3 , or a homolog thereof. by a PPO polypeptide of the present invention is increased Any transit peptide may be used in accordance with the or generated in the mitochondria . various embodiments of the present invention . For example , In one embodiment , an activity conferred by the expres - 25 specific nucleic acid sequences are encoding transit peptides sion of a gene described herein or its expression product; i. e . are disclosed by von Heijne et al. (Plant Molecular Biology by a PPO polypeptide of the present invention is increased Reporter, 9 ( 2 ), 104 , (1991 )) or other transit peptides are or generated in the cytoplasm . disclosed by Schmidt et al . ( J . Biol. Chem . 268 (36 ) , 27447 In one embodiment, an activity conferred by the expres (1993 ) ) , Della -Cioppa et al. (Plant . Physiol. 84 , 965 ( 1987) ) , sion of a gene described herein or its expression product; i. e . 30 de Castro Silva Filho et al. ( Plant Mol. Biol. 30 , 769 ( 1996 ) ) , by a PPO polypeptide of the present invention is increased Zhao et al. ( J. Biol. Chem . 270 (11 ) , 6081 ( 1995 )) , Römer et or generated in the endoplasmic reticulum . al. (Biochem . Biophys . Res . Commun . 196 ( 3 ) , 1414 As the terms " cytoplasmic ” and “ non - targeted ” shall not ( 1993 ) ) , Keegstra et al. (Annu . Rev . Plant Physiol. Plant exclude a targeted localisation to any cell compartment for Mol . Biol. 40 , 471 ( 1989 ) ) , Lubben et al. (Photosynthesis the products of the inventive nucleic acid sequences by their 35 Res . 17 , 173 ( 1988 ) ) and Lawrence et al . ( J . Biol. Chem . 272 naturally occurring sequence properties within the back (33 ) , 20357 (1997 ) ) , which are hereby incorporated by ground of the transgenic organism , in one embodiment, an reference . A general review about targeting is disclosed by activity as disclosed herein as being conferred by a poly - Kermode Allison R . in Critical Reviews in Plant Science 15 peptide shown in SEQ ID NO : 2 or 4 , or a homolog thereof (4 ), 285 ( 1996 ) under the title “ Mechanisms of Intracellular is increase or generated non - targeted . For the purposes of the 40 Protein Transport and Targeting in Plant Cells ” . description of the present invention , the term “ cytoplasmic " Additional nucleic acid sequences encoding a transit shall indicate , that the nucleic acid of the invention is peptide can be isolated from any organism such as micro expressed without the addition of a non -natural transit organisms, algae or plants containing plastids, preferably peptide encoding sequence . A non - natural transient peptide containing chloroplasts . A “ transit peptide ” is an amino acid encoding sequence is a sequence which is not a natural part 45 sequence , whose encoding nucleic acid sequence is trans of a nucleic acid of the invention but is rather added by lated together with the corresponding structural gene . That molecular manipulation steps which are well -known to the means the transit peptide is an integral part of the translated person skilled in the art . Therefore the term " cytoplasmic ” protein and forms an amino terminal extension of the shall not exclude a targeted localisation to any cell com - protein . Both are translated as so called “ pre -protein ” . In partment for the products of the inventive nucleic acid 50 general the transit peptide is cleaved off from the pre - protein sequences by their naturally occurring sequence properties . during or just after import of the protein into the correct cell In another embodiment the present invention is related to organelle such as a plastid to yield the mature protein . The a method for producing a , e . g . transgenic , plant with transit peptide ensures correct localization of the mature increased herbicide tolerance or resistance, or a part thereof, protein by facilitating the transport of proteins through as compared to a corresponding , e . g . non - transformed , wild 55 intracellular membranes . type plant, which comprises For example, such transit peptides , which are beneficially (al ) increasing or generating the activity of an PPO poly - used in the inventive process, are derived from the nucleic peptide , e . g . the activity of said gene or the gene product acid sequence encoding a protein selected from the group gene , in an organelle of a plant cell, or consisting of ribulose bisphosphate carboxylase /oxygenase , ( a2 ) increasing or generating the activity of a protein com - 60 5 - enolpyruvyl- shikimate - 3 - phosphate synthase , acetolactate prising the sequence of SEQ ID NO : 2 or 4 , or a homolog synthase , chloroplast ribosomal protein CS17 , Cs protein , thereof or as encoded by the nucleic acid sequences ferredoxin , plastocyanin , ribulose bisphosphate carboxylase comprising the sequence of SEQ ID NO : 1 or 3 , or a activase, tryptophan synthase , acyl carrier protein , plastid homolog thereof, and which is joined to a nucleic acid chaperonin -60 , cytochrome c552 , 22 -KDA heat shock pro sequence encoding a transit peptide in the plant cell ; or 65 tein , 33 -kDa Oxygen - evolving enhancer protein 1 , ATP (a3 ) increasing or generating the activity of a protein com - synthase y subunit , ATP synthase d subunit, chlorophyll - a / prising the sequence of SEQ ID NO : 2 or 4 , or a homolog b -binding proteinll - 1, Oxygen - evolving enhancer protein 2 , US 10 , 392 ,630 B2 21 22 Oxygen -evolving enhancer protein 3 , photosystem I: P21 , The skilled worker will recognize that various other photosystem I: P28 , photosystem I : P30 , photosystem I : nucleic acid sequences encoding transit or signal sequence P35 , photosystem I: P37 , glycerol- 3 - phosphate acyltrans peptides can easily isolated from plastid - localized , mito ferases, chlorophyll a / b binding protein , CAB2 protein , chondria - localized or endoplasmic reticulum - localized pro hydroxymethyl- bilane synthase , pyruvate - orthophosphate 5 teins , which are expressed from nuclear genes as precursors dikinase , CAB3 protein , plastid ferritin , ferritin , early light- and are then targeted to plastids, mitochondria or endoplas inducible protein , glutamate - 1 -semialdehyde aminotrans mic reticulum . Nucleic acid sequences encoding a transit or ferase , protochlorophyllide reductase , starch - granule -bound signal sequence peptide can be isolated from organelle amylase synthase, light- harvesting chlorophyll a / b - binding targeted proteins from any organism . Preferably , the transit protein of photosystem II , major pollen allergen Lol p 5a , 10 or signal sequence peptide is isolated from an organism plastid ClpB ATP - dependent protease , superoxide dis selected from the group consisting of the genera Acetabu mutase , ferredoxin NADP oxidoreductase , 28 -kDa ribo - laria , Arabidopsis , Brassica , Capsicum , Chlamydomonas, nucleoprotein , 31- kDa ribonucleoprotein , 33 -kDa ribo Cururbita , Dunaliella, Euglena , Flaveria , Glycine , Helian nucleoprotein , acetolactate synthase , ATP synthase CF . thus, Hordeum , Lemna, Lolium , Lycopersion , Malus, Med subunit 1 , ATP synthase CF , subunit 2 , ATP synthase CF , 15 icago , Mesembryanthemum , Nicotiana , Oenotherea , Oryza , subunit 3 , ATP synthase CF , subunit 4 , cytochrome f , Petunia , Phaseolus, Physcomitrella , Pinus, Pisum , Rapha ADP - glucose pyrophosphorylase, glutamine synthase , glu nus, Silene , Sinapis, Solanum , Spinacea , Stevia , Synechoc tamine synthase 2 , carbonic anhydrase , Gap , protein , heat occus , Synechocystis , Triticum and Zea . More preferably, the shock -protein hsp21 , phosphate translocator, plastid Clpa nucleic acid sequence encoding the transitor signal ATP -dependent protease , plastid ribosomal protein CL24 , 20 sequence peptide is isolated from an organism selected from plastid ribosomal protein CL9, plastid ribosomal protein the group consisting of the species Acetabularia mediterra PsCL18 , plastid ribosomal protein PsCL25 , DAHP syn nea , Arabidopsis thaliana , Brassica campestris , Brassica thase , starch phosphorylase , root acyl carrier protein II , napus, Capsicum annuum , Chlamydomonas reinhardtii, betaine - aldehyde dehydrogenase, GapB protein , glutamine Cururbita moschata , Dunaliella salina , Dunaliella tertio synthetase 2 , phosphoribulokinase , nitrite reductase , ribo - 25 lecta , Euglena gracilis , Flaveria trinervia , Glycine max , somal protein L12 , ribosomal protein L13 , ribosomal protein Helianthus annuus , Hordeum vulgare , Lemna gibba , Lolium L21 , ribosomal protein L35 , ribosomal protein L40 , triose perenne , Lycopersion esculentum , Malus domestica , Med phosphate -3 -phosphoglyerate -phosphate translocator, ferre icago falcata , Medicago sativa , Mesembryanthemum crys doxin -dependent glutamate synthase, glyceraldehyde - 3 - tallinum , Nicotiana plumbaginifolia , Nicotiana sylvestris , phosphate dehydrogenase , NADP - dependent malic enzyme 30 Nicotiana tabacum , Oenotherea hookeri, Oryza sativa , and NADP -malate dehydrogenase , chloroplast 30S ribo - Petunia hybrida, Phaseolus vulgaris , Physcomitrella patens, somal protein PSrp - 1 , and the like . Pinus tunbergii , Pisum sativum , Raphanus sativus , Silene In a preferred embodiment, the targeting sequence com - pratensis , Sinapis alba , Solanum tuberosum , Spinacea prises a nucleotide sequence that encodes a transit peptide oleracea , Stevia rebaudiana , Synechococcus, Synechocystis , comprising the amino acid sequence of SEQ ID NO : 5 , 8 , or 35 Triticum aestivum and Zea mays . Alternatively , nucleic acid 9 . Preferably , the transit peptide encoding nucleic acid is sequences coding for transit or signal sequence peptides may operably linked such that the transit peptide is fused to the be chemically synthesized either in part or wholly according valine at position 55 in SEQ ID NO : 2 or to the alanine at to structure of transit peptide sequences disclosed in the position 47 in SEQ ID NO : 4 . prior art. In a particularly preferred embodiment, the nucleic acid 40 Such transitor signal sequence peptides encoding sequences of the present invention are linked to a nucleic sequences can be used for the construction of other expres acid encoding a so -called " signal sequence peptide ” . For the sion constructs . The transit or signal sequence peptides purposes of the present invention , “ signal sequence peptide” advantageously used in the inventive process and which are refers to amino acid sequences of about 15 to about 50 part of the inventive nucleic acid sequences and proteins are amino acids in length which are known in the art to be 45 typically 20 to 120 amino acids , preferably 25 to 110 , 30 to generally located at the amino terminus of proteins and 100 or 35 to 90 amino acids, more preferably 40 to 85 amino which are capable of targeting said proteins to the endoplas - acids and most preferably 45 to 80 amino acids as for transit mic reticulum . The core of the signal peptide contains a long peptides , or about 15 to about 50 amino acids as for signal stretch of hydrophobic amino acids that has a tendency to sequence peptides in length and functions post- translational form a single alpha -helix . In addition , many signal peptides 50 to direct the protein to the plastid , preferably to the chloro begin with a short positively charged stretch of amino acids, plast, the mitochondrion or endoplasmic reticulum . The which may help to enforce proper topology of the polypep - nucleic acid sequences encoding such transit or signal tide during translocation by what is known as the positive sequence peptides are localized upstream of nucleic acid inside rule . At the end of the signal peptide there is typically sequence encoding the mature protein . For the correct a stretch of amino acids that is recognized and cleaved by 55 molecular joining of the transit or signal sequence peptide signal peptidase . However this cleavage site is absent from encoding nucleic acid and the nucleic acid encoding the transmembrane -domains that serve as signal peptides, which protein to be targeted it is sometimes necessary to introduce are sometimes referred to as signal anchor sequences. Signal additional base pairs at the joining position , which forms peptidase may cleave during, or after completion of, trans - restriction enzyme recognition sequences useful for the location to generate a free signal peptide and a mature 60 molecular joining of the different nucleic acid molecules . protein . The free signal peptides are then digested by spe - This procedure might lead to very few additional amino cific proteases . Those skilled in the art would readily appre - acids at the N -terminal of the mature imported protein , ciate that many signal sequence peptides are known (van which usually and preferably do not interfere with the Heijne , G ., J . Mol. Biol. 184 : 99 - 105 ( 1985 )) and that these protein function . In any case, the additional base pairs at the peptide sequences or analogues thereof can be easily sub - 65 joining position which forms restriction enzyme recognition stituted as long as they fulfill the requirements for a signal sequences have to be chosen with care , in order to avoid the peptide as described above . formation of stop codons or codons which encode amino US 10 , 392 ,630 B2 23 24 acids with a strong influence on protein folding, like e . g . In one embodiment, the process of the present invention proline . It is preferred that such additional codons encode comprises one or more of the following steps: small structural flexible amino acids such as glycine or ( a ) stabilizing a protein conferring the increased expression alanine . of a protein encoded by the nucleic acid molecule of the Asmentioned above , the nucleic acid sequence coding for 5 invention or of the polypeptide of the invention having the a protein comprising the sequence of SEQ ID NO : 2 or 4 , or herein -mentioned activity of an PPO and conferring a homolog thereof, can be joined to a nucleic acid sequence increased herbicide tolerance or resistance , as compared encoding a transit or a signal sequence peptide. The nucleic to a corresponding , e . g . non - transformed , wild type plant acid sequence of the gene to be expressed and the nucleic cell , plant or part thereof ; acid sequence encoding the transit or signal sequence pep - 10 ( b ) stabilizing an mRNA conferring the increased expression tide are operably linked . Therefore the transit or signal of a polynucleotide encoding a polypeptide as mentioned sequence peptide is fused in frame to the nucleic acid in ( a ); sequence coding for a protein comprising the sequence of ( c ) increasing the specific activity of a protein conferring the SEQ ID NO : 2 or 4 , or a homolog thereof. increased expression of a polypeptide as mentioned in ( a ); The proteins translated from said inventive nucleic acid 15 ( d ) generating or increasing the expression of an endogenous sequences are a kind of fusion proteins that means the or artificial transcription factor mediating the expression nucleic acid sequences encoding the transit or signal of a protein conferring the increased expression of a sequence peptide, are joint to a gene , e. g . the nucleic acid polypeptide as mentioned in ( a ) ; sequences comprising the sequence of SEQ ID NO : 1 or 3 , ( e ) stimulating activity of a protein conferring the increased or a homolog thereof. 20 expression of a polypeptide as mentioned in ( a ), by adding In a preferred embodiment, said fusion proteins refer to one or more exogenous inducing factors to the organism mutated PPO polypeptides comprising the sequence of SEQ or parts thereof; ID NO : 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , ( f ) expressing a transgenic gene encoding a protein confer 24 , 25, or 26 . It will be acknowledged by the person skilled ring the increased expression of a polypeptide as men in the art that by replacing the natural transit peptide of an 25 tioned in ( a ) ; and / or Alopecurus PPO2 comprising the sequence of SEQ ID NO : ( g ) increasing the copy number of a gene conferring the 2 , against a heterologous transit peptide from Zea mays increased expression of a nucleic acid molecule encoding (SEQ ID NO : 8 ) or Sorghum bicolor (SEQ ID NO : 9 ) , the a polypeptide as mentioned in ( a ) ; native N - terminal end of SEQ ID NO : 2 is shortened by 7 ( h ) increasing the expression of the endogenous gene encod amino acids. Therefore, preferred mutational sites at or 30 ing a polypeptide as mentioned in ( a ) by adding positive corresponding to positions R137 or F438 on SEQ ID NO : 2 , expression or removing negative expression elements , as described hereinafter in greater detail , are identical to e . g . homologous recombination can be used to either positions R130 or F431 in said fusion proteins . introduce positive regulatory elements like for plants the The person skilled in the art is able to join said sequences 35S enhancer into the promoter or to remove repressor in a functional manner . Advantageously the transit or signal 35 elements form regulatory regions. Further gene conver sequence peptide part is cleaved off from the protein part s ion methods can be used to disrupt repressor elements or during the transport preferably into the endoplasmic reticu to enhance to activity of positive elements -positive ele lum or plastids. The skilled worker knows that other short ments can be randomly introduced in plants by T -DNA or sequences are also useful in the expression of the PPO genes transposon mutagenesis and lines can be identified in of the present invention . Furthermore the skilled worker is 40 which the positive elements have been integrated near to aware of the fact that there is not a need for such short a gene of the invention , the expression ofwhich is thereby sequences in the expression of the genes . enhanced ; and /or Alternatively to the targeting of the gene , e . g . proteins ( i ) modulating growth conditions of the plant in such a having the sequences comprising the sequence of SEQ ID manner, that the expression or activity of the gene encod NO : 2 or 4 , or a homolog thereof, the nucleic acids of the 45 ing a polypeptide as mentioned in ( a ), or the protein itself invention can directly be introduced into the plastidic is enhanced ; genome. ( i) selecting of organisms with especially high activity of a By transforming the plastids the intraspecies specific polypeptide as mentioned in ( a ) from natural or from transgene flow is blocked , because a lot of species such as mutagenized resources and breeding them into the target corn , cotton and rice have a strict maternal inheritance of 50 organisms, e . g . the elite crops . plastids. By placing the gene e . g . the genes comprising the Preferably , said mRNA is encoded by the nucleic acid sequence of SEQ ID NO : 1 or 3 , or a homolog thereof, or molecule of the present invention and /or the protein confer active fragments thereof in the plastids of plants , these genes ring the increased expression of a protein encoded by the will not be present in the pollen of said plants . nucleic acid molecule of the present invention alone or In another embodiment of the invention the gene , e . g . the 55 linked to a transit nucleic acid sequence or transit peptide nucleic acid molecules comprising the sequence of SEQ ID encoding nucleic acid sequence or the polypeptide having NO : 1 or 3, or a homolog thereof, used in the inventive the herein mentioned activity , e . g. conferring with increased process are transformed into mitochondria , which are meta - herbicide tolerance or resistance , as compared to a corre bolic active. sponding , e . g . non - transformed , wild type plant cell , plant or For a good expression in the plastids the gene , e .g . the 60 part thereof after increasing the expression or activity of the nucleic acid sequences comprising the sequence of SEQ ID encoded polypeptide or having the activity of a polypeptide NO : 1 or 3 , or a homolog thereof, are introduced into an having an activity as the protein comprising the sequence of expression cassette using a preferably a promoter and ter - SEQ ID NO : 2 or 4 , or a homolog thereof minator, which are active in plastids , preferably a chloro - In general, the amount of mRNA or polypeptide in a cell plast promoter. Examples of such promoters include the 65 or a compartment of an organism correlates with the amount psbA promoter from the gene from spinach or pea , the rbcL of encoded protein and thus with the overall activity of the promoter, and the atpB promoter from corn . encoded protein in said volume. Said correlation is not US 10 , 392 ,630 B2 25 26 always linear , the activity in the volume is dependent on the skilled in the art willbe able to identify regulation domains stability of the molecules or the presence of activating or and binding sites of regulators by comparing the sequence of inhibiting cofactors . The activity of the abovementioned the nucleic acid molecule of the present invention or the proteins and /or polypeptides encoded by the nucleic acid expression product thereof with the state of the art by molecule of the present invention can be increased in 5 computer software means which comprise algorithms for the various ways . For example , the activity in an organism or in identifying of binding sites and regulation domains or by a part thereof, like a cell , is increased via increasing the gene introducing into a nucleic acid molecule or in a protein product number , e . g . by increasing the expression rate , like systematically mutations and assaying for those mutations introducing a stronger promoter, or by increasing the sta - which will lead to an increased specific activity or an bility of the mRNA expressed , thus increasing the transla - 10 increased activity per volume, in particular per cell . tion rate , and / or increasing the stability of the gene product, The mutation is introduced in such a way that increased thus reducing the proteins decayed . Further , the activity or herbicide tolerance or resistance, is not adversely affected . turnover of enzymes can be influenced in such a way that a It can therefore be advantageous to express in an organism reduction or increase of the reaction rate or a modification a nucleic acid molecule of the invention or a polypeptide of (reduction or increase ) of the affinity to the substrate results , 15 the invention derived from a evolutionary distantly related is reached . A mutation in the catalytic centre of an polypep organism , as e . g . using a prokaryotic gene in a eukaryotic tide of the invention , e .g . as enzyme, can modulate the turn host , as in these cases the regulation mechanism of the host over rate of the enzyme , e . g . a knock out of an essential cell may not weaken the activity ( cellular or specific ) of the amino acid can lead to a reduced or completely knock out gene or its expression product. activity of the enzyme, or the deletion or mutation of 20 The invention is not limited to specific nucleic acids , regulator binding sites can reduce a negative regulation like specific polypeptides , specific cell types , specific host cells , a feedback inhibition ( or a substrate inhibition , if the sub - specific conditions or specific methods etc . as such , but may strate level is also increased ) . The specific activity of an vary and numerousmodifications and variations therein will enzyme of the present invention can be increased such that be apparent to those skilled in the art. It is also to be the turn over rate is increased or the binding of a co - factor 25 understood that the terminology used herein is for the is improved . Improving the stability of the encoding mRNA purpose of describing specific embodiments only and is not or the protein can also increase the activity of a gene intended to be limiting . product . The stimulation of the activity is also under the Further, " proteins are generally composed of one or more scope of the term “ increased activity ” . functional regions , commonly termed domains . Different Moreover , the regulation of the abovementioned nucleic 30 combinations of domains give rise to the diverse range of acid sequences may be modified so that gene expression is proteins found in nature . The identification of domains that increased . This can be achieved advantageously by means of occur within proteins can therefore provide insights into heterologous regulatory sequences or by modifying , for their function . Pfam - A entries are high quality , manually example mutating , the natural regulatory sequences which curated families. The Pfam database is a large collection of are present . The advantageous methods may also be com - 35 protein families , each represented by multiple sequence bined with each other. alignments and hidden Markov models (HMMs ) . ” ( see : The In general, an activity of a gene product in an organism or Pfam protein families database : R . D . Finn , et al. , Nucleic part thereof , in particular in a plant cell or organelle of a Acids Research (2010 ), Database Issue 38 :D211 -222 ) . The plant cell , a plant, or a plant tissue or a part thereof or in a Pfam protein family database is a large collection of more microorganism can be increased by increasing the amount of 40 than ten thousand protein families and is available at pfam the specific encodingmRNA or the corresponding protein in . sanger .ac . uk / . Profile Hidden Markov Models (HMMs ) are said organism or part thereof. flexible, probabilistic models that can be used to describe the A modification , i. e . an increase, can be caused by endog - consensus patterns shared by sets of homologous protein / enous or exogenous factors . For example , an increase in domain sequences. HMMs in the Pfam database are con activity in an organism or a part thereof can be caused by 45 structed from an alignment of a representative set of adding a gene product or a precursor or an activator or an sequences for each protein domain , called a seed alignment. agonist to the media or nutrition or can be caused by Accordingly, the present invention relates to a nucleic introducing said subjects into a organism , transient or stable . acid molecule encoding a polypeptide which is 50 % or Furthermore such an increase can be reached by the intro - more , preferably 60 % , 70 % , or 75 % , more preferably 80 % , duction of the inventive nucleic acid sequence or the 50 85 % , 90 % , or 95 % , even more preferred 96 % , 97 % , 98 % , encoded protein in the correct cell compartment for example 99 % or more and most preferred 100 % identical to the into the nucleus or cytoplasm respectively or into plastids polypeptide of SEQ ID NO : 2 or 4 , and conferring the either by transformation and/ or targeting . increase of the herbicide tolerance or resistance of a plant as In one further embodiment of the process according to the described herein . The invention also relates to the polypep invention , organisms are used in which one of the above - 55 tide encoded by said polynucleotide . mentioned genes , or one of the abovementioned nucleic The present invention also relates to isolated nucleic acids acids, is mutated in a way that the activity of the encoded comprising a nucleic acid molecule selected from the group gene products is less influenced by cellular factors, or not at consisting of: all , in comparison with the not mutated proteins. For ( a ) a nucleic acid molecule encoding the polypeptide com example , well known regulation mechanisms of enzyme 60 prising the sequence of SEQ ID NO : 2 or 4 , or a homolog activity are substrate inhibition or feed back regulation thereof; mechanisms. Ways and techniques for the introduction of (b ) a nucleic acid molecule comprising the sequence of SEQ substitution , deletions and additions of one or more bases, ID NO : 1 or 3 , or a homolog thereof, nucleotides or amino acids of a corresponding sequence are ( c ) a nucleic acid molecule , which , as a result of the described herein below in the corresponding paragraphs and 65 degeneracy of the genetic code , can be derived from a the references listed there , e . g . in Sambrook et al. , Molecular polypeptide sequence of SEQ ID NO : 2 or 4 , or a homolog Cloning , Cold Spring Harbour, N Y , 1989. The person thereof, and confers increased herbicide tolerance or US 10 , 392 ,630 B2 27 28 resistance , as compared to a corresponding , e . g . non sp .; Mannheimia haemolytica ; Mesorhizobium loti; Methyl transformed , wild type plant cell, a plant or a part thereof; ophaga thalassica ; Microcystis aeruginosa ; Microscilla sp . ( d ) a nucleic acid molecule having 30 % or more identity , PRED; Moraxella sp . TA 144 ; Mycobacterium sp. ; Myco preferably 40 % , 50 % , 60 % , 70 % , 75 % , 80 % , 85 % , 90 % , plasma sp .; Neisseria sp .; Nitrosomonas sp .; Nostoc sp . PCC 95 % , 96 % , 97 % , 98 % , 99 % , 99. 5 % , or more with the 5 7120 ; Novosphingobium aromaticivorans; Denococcus nucleic acid molecule sequence of a polynucleotide com - oeni; Pantoea citrea ; Pasteurella multocida ; Pediococcus prising the nucleic acid molecule of SEQ ID NO : 1 or 3 , pentosaceus ; Phormidium foveolarum ; Phytoplasma sp . ; or a homolog thereof, and confers increased herbicide Plectonema boryanum ; Prevotella ruminicola ; Propioni tolerance or resistance , as compared to a corresponding , bacterium sp . ; Proteus vulgaris ; Pseudomonas sp . ; Ralsto e . g . non - transformed , wild type plant cell, a plant or a part 10 nia sp . , Rhizobium sp . ; Rhodococcus equi; Rhodothermus thereof; marinus; Rickettsia sp. ; Riemerella anatipestifer ; Rumi ( e ) a nucleic acid molecule encoding a polypeptide having nococcus flavefaciens; Salmonella sp . , Selenomonas rumi 30 % ormore identity , preferably at least 40 % , 50 % , 60 % , nantium ; Serratia entomophila ; Shigella sp . ; Sinorhizobium 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , 99 % , meliloti ; Staphylococcus sp . ; Streptococcus sp . , Streptomy 99 . 5 % or more , with the amino acid sequence of the 15 ces sp .; Synechococcus sp .; Synechocystis sp . PCC 6803 ; polypeptide encoded by the nucleic acid molecule of ( a ), Thermotoga maritima ; Treponema sp . ; Ureaplasma ure ( b ), (c ) or ( d ) and having the activity represented by a alyticum ; Vibrio cholerae ; Vibrio parahaemolyticus; Xylella nucleic acid molecule comprising a polynucleotide of fastidiosa ; Yersinia sp .; Zymomonas mobilis , preferably SEQ ID NO : 1 or 3 , or a homolog thereof, and confers Salmonella sp . or E . coli or plants , preferably from yeasts increased herbicide tolerance or resistance as compared to 20 such as from the genera Saccharomyces, Pichia , Candida , a corresponding, e . g . non - transformed , wild type plant Hansenula , Torulopsis or Schizosaccharomyces or plants cell , a plant or a part thereof; such as A . thaliana , maize , wheat , rye , oat , triticale , rice , ( f) nucleic acid molecule which hybridizes with a nucleic barley, soybean , peanut, cotton , borage , sunflower , linseed , acid molecule of (a ), (b ), ( c ), (d ) or ( e ) under stringent primrose , rapeseed , canola and turnip rape, manihot, pepper, hybridization conditions and confers increased herbicide 25 sunflower, tagetes, solanaceous plant such as potato , tolerance or resistance, as compared to a corresponding, tobacco , eggplant and tomato , Vicia species, pea , alfalfa , e . g . non - transformed , wild type plant cell , a plant or a part bushy plants such as coffee , cacao , tea , Salix species, trees thereof; such as oil palm , coconut, perennial grass , such as ryegrass (g ) a nucleic acid molecule encoding a polypeptide which and fescue , and forage crops, such as alfalfa and clover and can be isolated with the aid of monoclonal or polyclonal 30 from spruce , pine or fir for example . antibodies made against a polypeptide encoded by one of The proteins of the present invention are preferably the nucleic acid molecules of (a ) , ( b ) , ( c ), ( d ) , ( e ) or ( f ) and produced by recombinant DNA techniques . For example , a having the activity represented by the nucleic acid mol- nucleic acid molecule encoding the protein is cloned into an ecule comprising a polynucleotide as depicted in SEQ ID expression vector, for example in to a binary vector, the NO : 1 or 3 , or a homolog thereof; 35 expression vector is introduced into a host cell , for example ( h ) a nucleic acid molecule which is obtainable by screening the A . thaliana wild type NASC N906 or any other plant cell a suitable nucleic acid library, especially a cDNA library as described in the examples see below , and the protein is and / or a genomic library , under stringent hybridization expressed in said host cell. Examples for binary vectors are conditions with a probe comprising a complementary PBIN19 , pBI101, pBinAR (Hofgen and Willmitzer , Plant sequence of a nucleic acid molecule of ( a ) or (b ) or with 40 Science 66 , 221 ( 1990 ) ), PGPTV , PCAM BIA , PBIB - HYG , a fragment thereof, having 15 nt, preferably 20 nt, 30 nt, pBecks , pGreen or pPZP (Hajukiewicz , P . et al. , PlantMol . 50 nt, 100 nt, 200 nt, 500 nt, 750 nt or 1000 nt or more Biol. 25 , 989 ( 1994 ) , and Hellens et al , Trends in Plant of a nucleic acid molecule complementary to a nucleic Science 5 , 446 (2000 ) ) . acid molecule sequence characterized in ( a ) to ( e ) and In one embodiment as described in more detail SUPRA , encoding a polypeptide having the activity represented by 45 the protein of the present invention is preferably targeted to a protein comprising a polypeptide as depicted SEQ ID a compartment of the cell , e . g . to the endoplasmic reticulum NO : 2 or 4 , or a homolog thereof. or in the plastids. Ways of introducing nucleic acids into the In one embodiment the invention relates to homologs of endoplasmic reticulum or plastids and producing proteins in the aforementioned sequences, which can be isolated advan - this compartment are known to the person skilled in the art tageously from yeast, fungi , viruses , algae , bacteria , such as 50 have been also described in this application . In one embodi Acetobacter (subgen . Acetobacter ) aceti ; Acidithiobacillus ment, the polypeptide of the invention is a protein localized ferrooxidans; Acinetobacter sp . , Actinobacillus sp ; Aeromo - after expression e . g . non - targeted , mitochondrial or plas nas salmonicida ; Agrobacterium tumefaciens ; Aquifex tidic , for example it is fused to a transit or signal sequence aeolicus ; Arcanobacterium pyogenes; Aster yellows phyto peptide as described above for plastidic or endoplasmic plasma ; Bacillus sp . ; Bifidobacterium sp . ; Borrelia burgdor - 55 reticulum localisation . In another embodiment the protein of feri ; Brevibacterium linens ; Brucella melitensis ; Buchnera the present invention is produced without further targeting sp . ; Butyrivibrio fibrisolvens; Campylobacter jejuni; Cau signal (e . g. as mentioned herein ), e .g . in the cytoplasm of the lobacter crescentus, Chlamydia sp .; Chlamydophila sp . ; cell. Ways of producing proteins in the cytoplasm are known Chlorobium limicola ; Citrobacter rodentium ; Clostridium to the person skilled in the art . Ways of producing proteins sp . ; Comamonas testosteroni; Corynebacterium sp . , Cox - 60 without artificial targeting are known to the person skilled in iella burnetii ; Deinococcus radiodurans, Dichelobacter the art. nodosus ; Edwardsiella ictaluri; Enterobacter sp ., Erysip Advantageously , the nucleic acid sequences according to elothrix rhusiopathiae; E . coli ; Flavobacterium sp . ; Fran - the invention or the gene construct together with at least one cisella tularensis ; Frankia sp . Cpl 1 , Fusobacterium nuclea - reporter gene are cloned into an expression cassette , which tum ; Geobacillus stearothermophilus ; Gluconobacter 65 is introduced into the organism via a vector or directly into oxydans ; Haemophilus sp . ; Helicobacter pylori; Klebsiella the genome. This reporter gene should allow easy detection pneumoniae; Lactobacillus sp. ; Lactococcus lactis ; Listeria via a growth , fluorescence, chemical , bioluminescence or US 10 , 392 ,630 B2 29 30 tolerance assay or via a photometric measurement. linked to the intervening encoding sequence with one of the Examples of reporter genes which may be mentioned are nucleic acids of SEQ ID NO : 1 or 3 , or a homolog thereof. antibiotic - or herbicide - tolerance genes , hydrolase genes, By an operable linkage is meant the sequential arrangement fluorescence protein genes, bioluminescence genes, sugar or of promoter , encoding sequence , terminator and optionally nucleotide metabolic genes or biosynthesis genes such as the 5 other regulatory elements in such a way that each of the Ura3 gene , the Ilv2 gene , the luciferase gene , the B - galac - regulatory elements can fulfill its function in the expression tosidase gene , the gfp gene, the 2 -desoxyglucose - 6 -phos of the encoding sequence in due manner. In one embodiment phate phosphatase gene , the B - glucuronidase gene , B -lacta - the sequences preferred for operable linkage are targeting mase gene , the neomycin phosphotransferase gene , the sequences for ensuring subcellular localization in plastids. hygromycin phosphotransferase gene , a mutated acetohy - 10 However, targeting sequences for ensuring subcellular local droxyacid synthase (AHAS ) gene (also known as acetolac - ization in the mitochondrium , in the endoplasmic reticulum tate synthase (ALS ) gene ) , a gene for a D - amino acid ( ER ), in the nucleus, in oil corpuscles or other compart metabolizing enzyme or the BASTA (Egluphosinate - toler - ments may also be employed as well as translation promot ance ) gene . These genes permit easy measurement and ers such as the 5 ' lead sequence in tobacco mosaic virus quantification of the transcription activity and hence of the 15 (Gallie et al. , Nucl. Acids Res. 15 8693 ( 1987 ) . expression of the genes . In this way genome positions may A nucleic acid construct, for example an expression be identified which exhibit differing productivity . For cassette may , for example , contain a constitutive promoter or expression a person skilled in the art is familiar with a tissue- specific promoter (preferably the USP or napin different methods to introduce the nucleic acid sequences promoter ) the gene to be expressed and the ER retention into different organelles such as the preferred plastids . Such 20 signal. For the ER retention signal the KDEL amino acid methods are for example disclosed by Maiga P . (Annu . Rev. sequence (lysine , aspartic acid , glutamic acid , leucine ) or the Plant Biol. 55 , 289 (2004 ) ) , Evans T . (WO 2004 /040973 ) , KKX amino acid sequence ( lysine - lysine - X -stop , wherein X McBride K . E . et al . ( U . S . Pat. No. 5 , 455 ,818 ) , Daniell H . means every other known amino acid ) is preferably et al. ( U . S . Pat. Nos . 5 ,932 , 479 and 5 ,693 , 507 ) and Straub employed . J . M . et al . ( U . S . Pat . No . 6 ,781 , 033 ) . A preferred method is 25 For expression in a host organism , for example a plant, the the transformation ofmicrospore - derived hypocotyl or coty - expression cassette is advantageously inserted into a vector ledonary tissue (which are green and thus contain numerous such as by way of example a plasmid , a phage or other DNA plastids ) leaf tissue and afterwards the regeneration of which allows optimal expression of the genes in the host shoots from said transformed plant material on selective organism . Examples of suitable plasmids are : in E . coli medium . As methods for the transformation bombarding of 30 PLG338 , PACYC184 , PBR series such as e . g . PBR322 , PUC the plant material or the use of independently replicating series such as pUC18 or pUC19 , M113mp series , pKC30 , shuttle vectors are well known by the skilled worker . But pRep4 , PHS1, PHS2, PPLc236 , PMBL24, PLG200 , also a PEG -mediated transformation of the plastids or Agro - PUR290 , PIN - III113 - B1, Agt11 or pBdCl; in Streptomyces bacterium transformation with binary vectors is possible . p1J101, p1J364 , p1J702 or plJ361 ; in Bacillus pUB110 , Useful markers for the transformation of plastids are posi - 35 PC194 or pBD214 ; in Corynebacterium PSA77 or PAJ667 ; tive selection markers for example the chloramphenicol -, in fungi PALS1, PIL2 or pBB116 ; other advantageous streptomycin - , kanamycin - , neomycin - , amikamycin -, spec - fungal vectors are described by Romanos M . A . et al. , Yeast tinomycin - , triazine - and / or lincomycin - tolerance genes . As 8 , 423 ( 1992 ) and by van den Hondel, C . A . M . J . J . et al. additional markers named in the literature often as second - [ ( 1991) " Heterologous gene expression in filamentous ary markers , genes coding for the tolerance against herbi- 40 fungi” ) as well as in “More Gene Manipulations” in “ Fungi” cides such as phosphinothricin (= glufosinate , BASTATM in Bennet J . W . & Lasure L . L . , eds. , pp . 396 - 428 , Academic LibertyTM , encoded by the bar gene ), glyphosate (EN - Press , San Diego , and in “ Gene transfer systems and vector ( phosphonomethyl ) glycine, RoundupTM , encoded by the development for filamentous fungi” [ van den Hondel, C . A . 5 - enolpyruvylshikimate - 3 - phosphate synthase gene = epsps ), M . J . J . & Punt, P . J . ( 1991 ) in : Applied Molecular Genetics sulfonylureas ( like StapleTM , encoded by the acetolactate 45 of Fungi , Peberdy , J . F . et al. , eds. , pp . 1 - 28 , Cambridge synthase (ALS ) gene) , imidazolinones [ = IMI, like University Press : Cambridge ]. Examples of advantageous imazethapyr, imazamox , ClearfieldTM , encoded by the aceto - yeast promoters are 2 uM , PAG - 1, YEp6 , YEp13 or pEM hydroxyacid synthase ( AHAS ) gene , also known as aceto - BLYe23 . Examples of algal or plant promoters are pLGV23 , lactate synthase (ALS ) gene ] or bromoxynil ( = BuctrilTM PGHlac + , PBIN19 , PAK2004 , PVKH or PDH51 ( see encoded by the oxy gene ) or genes coding for antibiotics 50 Schmidt , R . and Willmitzer , L . , Plant Cell Rep . 7 , 583 such as hygromycin or G418 are useful for further selection . ( 1988 ) ) . The vectors identified above or derivatives of the Such secondary markers are useful in the case when most vectors identified above are a small selection of the possible genome copies are transformed . In addition negative selec - plasmids . Further plasmids are well known to those skilled tion markers such as the bacterial cytosine deaminase ( en - in the art and may be found , for example , in “ Cloning coded by the codA gene ) are also useful for the transforma - 55 Vectors ” ( Eds . Pouwels P . H . et al. Elsevier, Amsterdam tion of plastids . New York - Oxford , 1985 , ISBN 0 444 904018 ) . Suitable To increase the possibility of identification of transfor - plant vectors are described inter alia in " Methods in Plant mants it is also desirable to use reporter genes other then the Molecular Biology and Biotechnology ” (CRC Press , Ch . aforementioned tolerance genes or in addition to said genes. 6 / 7 , pp . 71 - 119 ) . Advantageous vectors are known as shuttle Reporter genes are for example B - galactosidase - , B - glu - 60 vectors or binary vectors which replicate in E . coli and curonidase - (GUS ) , alkaline phosphatase - and / or green - fluo - Agrobacterium . rescent protein - genes (GFP ). In a further embodiment of the vector the expression In a preferred embodiment a nucleic acid construct, for cassette according to the invention may also advantageously example an expression cassette , comprises upstream , i. e . at be introduced into the organisms in the form of a linear DNA the 5' end of the encoding sequence, a promoter and down - 65 and be integrated into the genome of the host organism by stream , i . e . at the 3 ' end , a polyadenylation signal and way of heterologous or homologous recombination . This optionally other regulatory elements which are operably linear DNA may be composed of a linearized plasmid or US 10 , 392 ,630 B2 31 32 only of the expression cassette as vector or the nucleic acid the art is the dipping of a flowering plant into an Agrobac sequences according to the invention . teria solution , wherein the Agrobacteria contains the nucleic A nucleic acid sequence can also be introduced into an acid of the invention , followed by breeding of the trans organism on its own . formed gametes . Other suitable methods for transforming or If in addition to the nucleic acid sequence according to the 5 transfecting host cells including plant cells can be found in invention further genes are to be introduced into the organ - Sambrook et al ., supra , and other laboratory manuals such as ism , all together with a reporter gene in a single vector or Methods in Molecular Biology , 1995 , Vol. 44 , Agrobacte each single gene with a reporter gene in a vector in each case rium protocols , ed : Gartland and Davey , Humana Press , can be introduced into the organism , whereby the different Totowa, N . J . vectors can be introduced simultaneously or successively. 10 In one embodiment of the present invention , transfection The vector advantageously contains at least one copy of of a nucleic acid molecule coding for a polypeptide com the nucleic acid sequences according to the invention and / or prising the sequence of SEQ ID NO : 2 or 4 , or a homolog the expression cassette ( = gene construct ) according to the thereof into a plant is achieved by Agrobacterium mediated invention . gene transfer . Agrobacterium mediated plant transformation The invention further provides an isolated recombinant 15 can be performed using for example the GV3101 (PM P90 ) expression vector comprising a nucleic acid encoding a (Koncz and Schell , Mol. Gen . Genet. 204 , 383 (1986 ) ) or polypeptide comprising the sequence of SEQ ID NO : 2 or 4 , LBA4404 (Clontech ) Agrobacterium tumefaciens strain . or a homolog thereof, wherein expression of the vector in a Transformation can be performed by standard transforma host cell results in increased herbicide tolerance or resis - tion and regeneration techniques (Deblaere et al. , Nucl. tance, as compared to a wild type variety of the host cell. 20 Acids Res . 13 , 4777 ( 1994 ) , Gelvin , Stanton B . and Schil The recombinant expression vectors of the invention peroort Robert A , Plant Molecular Biology Manual, 2nd comprise a nucleic acid of the invention in a form suitable Ed . - Dordrecht: Kluwer Academic Publ. , 1995 . — in Sect. , for expression of the nucleic acid in a host cell, which means Ringbuc Zentrale Signatur: BT11 - P ISBN 0 -7923 - 2731 - 4 ; that the recombinant expression vectors include one or more Glick Bernard R . , Thompson John E . , Methods in Plant regulatory sequences , selected on the basis of the host cells 25 Molecular Biology and Biotechnology , Boca Raton : CRC to be used for expression , which is operatively linked to the Press, 1993 360 S ., ISBN 0 - 8493 -5164 - 2 ) . For example , nucleic acid sequence to be expressed . It will be appreciated rapeseed can be transformed via cotyledon or hypocotyl by those skilled in the art that the design of the expression transformation (Moloney et al . , Plant Cell Report 8 , 238 vector can depend on such factors as the choice of the host (1989 ) ; De Block et al. , Plant Physiol. 91, 694 ( 1989 )) . Use cell to be transformed , the level of expression of polypeptide 30 of antibiotics for Agrobacterium and plant selection depends desired , etc . The expression vectors of the invention can be on the binary vector and the Agrobacterium strain used for introduced into host cells to thereby produce polypeptides or transformation . Rapeseed selection is normally performed peptides , including fusion polypeptides or peptides, encoded using kanamycin as selectable plant marker. Agrobacterium by nucleic acids as described herein . mediated gene transfer to flax can be performed using , for The recombinant expression vectors of the invention can 35 example , a technique described by Mlynarova et al. , Plant be designed for expression of the polypeptide of the inven - Cell Report 13 , 282 ( 1994 ) . Additionally , transformation of tion in plant cells . For example , nucleic acid molecules of soybean can be performed using for example a technique the present invention can be expressed in plant cells ( see described in European Patent No. 424 047 , U . S . Pat. No . Schmidt R . , and Willmitzer L ., Plant Cell Rep . 7 ( 1988 ) ; 5 , 322 ,783 , European Patent No . 397 687 , U . S . Pat. No . Plant Molecular Biology and Biotechnology, C Press , Boca 40 5 , 376 ,543 or 5 , 169 ,770 . Transformation of maize can be Raton , Fla ., Chapter 6 / 7 , p . 71 - 119 ( 1993 ) ; White F . F ., Jenes achieved by particle bombardment, polyethylene glycol B . et al. , Techniques for Gene Transfer , in : Transgenic mediated DNA uptake or via the silicon carbide fiber tech Plants , Vol. 1, Engineering and Utilization , eds. Kung and nique . ( see , for example , Freeling and Walbot “ The maize Wu R . , 128 -43 , Academic Press: 1993 ; Potrykus , Annu . Rev. handbook ” Springer Verlag : New York ( 1993 ) ISBN 3 - 540 Plant Physiol. Plant Molec . Biol . 42 , 205 ( 1991 ) and refer - 45 97826 - 7 ). A specific example of maize transformation is ences cited therein ) . Suitable host cells are discussed further found in U . S . Pat . No . 5 , 990 ,387 , and a specific example of in Goeddel, Gene Expression Technology :Methods in Enzy - wheat transformation can be found in PCT Application No . mology 185 , Academic Press: San Diego , Calif . (1990 ) . By WO 93 /07256 . way of example the plant expression cassette can be According to the present invention , the introduced nucleic installed in the PRT transformation vector ( ( a ) Toepfer et al ., 50 acid molecule coding for a polypeptides depicted in SEQ ID Methods Enzymol. 217 , 66 ( 1993 ) , ( b ) Toepfer et al ., Nucl. NO : 2 or 4 , or homologs thereof, may be maintained in the Acids . Res. 15 , 5890 ( 1987 ) ) . Alternatively , a recombinant plant cell stably if it is incorporated into a non - chromosomal vector ( = expression vector ) can also be transcribed and autonomous replicon or integrated into the plant chromo translated in vitro , e .g . by using the T7 promoter and the T7 somes or organelle genome. Alternatively , the introduced RNA polymerase . 55 nucleic acid molecule may be present on an extra - chromo In an further embodiment of the present invention , the somal non - replicating vector and be transiently expressed or nucleic acid molecules of the invention are expressed in transiently active . plants and plants cells such as unicellular plant cells ( e . g . In one embodiment, a homologous recombinant microor algae ) (see Falciatore et al. , Marine Biotechnology 1 ( 3 ) , 239 ganism can be created wherein the nucleic acid molecule is ( 1999 ) and references therein ) and plant cells from higher 60 integrated into a chromosome, a vector is prepared which plants ( e . g . , the spermatophytes , such as crop plants ), for contains at least a portion of a nucleic acid molecule coding example to regenerate plants from the plant cells . A nucleic for a protein depicted in SEQ ID NO : 2 or 4 , or a homolog acid molecule depicted in SEQ ID NO : 1 or 3 , or a homolog thereof into which a deletion , addition , or substitution has thereof may be “ introduced ” into a plant cell by any means, been introduced to thereby alter , e . g . , functionally disrupt, including transfection , transformation or transduction , elec - 65 the gene . For example , the gene is a yeast gene , like a gene troporation , particle bombardment , agroinfection , and the of S . cerevisiae , or of Synechocystis , or a bacterial gene , like like . One transformation method known to those of skill in an E . coli gene , but it can be a homolog from a related plant US 10 , 392 ,630 B2 33 34 or even from a mammalian or insect source. The vector can according to the invention and /or of the nucleic acid con be designed such that , upon homologous recombination , the struct according to the invention . endogenous nucleic acid molecule coding for a protein In principle all plants can be used as host organism . depicted in SEQ ID NO : 2 or 4 , or a homolog thereof is Preferred transgenic plants are , for example , selected from mutated or otherwise altered but still encodes a functional 5 the families Aceraceae , Anacardiaceae , Apiaceae , Aster polypeptide ( e . g ., the upstream regulatory region can be aceae, Brassicaceae, Cactaceae, Cucurbitaceae , Euphorbi altered to thereby alter the expression of the endogenous aceae , Fabaceae , Malvaceae , Nymphaeaceae , Papaveraceae , nucleic acid molecule ) . In a preferred embodiment the Rosaceae , Salicaceae , Solanaceae , Arecaceae , Bromeli biological activity of the protein of the invention is increased aceae , Cyperaceae, Iridaceae , Liliaceae , Orchidaceae , Gen upon homologous recombination . To create a point mutation 10 tianaceae , Labiaceae , Magnoliaceae , Ranunculaceae , Cari via homologous recombination , DNA -RNA hybrids can be folaceae , Rubiaceae , Scrophulariaceae , Caryophyllaceae , used in a technique known as chimeraplasty (Cole - Strauss et Ericaceae, Polygonaceae, Violaceae , Juncaceae or Poaceae al. , Nucleic Acids Research 27 (5 ) , 1323 ( 1999 ) and Kmiec , and preferably from a plant selected from the group of the Gene Therapy American Scientist . 87 ( 3 ) , 240 ( 1999 ) ) . families Apiaceae, Asteraceae , Brassicaceae, Cucurbitaceae , Homologous recombination procedures in Physcomitrella 15 Fabaceae , Papaveraceae, Rosaceae, Solanaceae , Liliaceae or patens are also well known in the art and are contemplated Poaceae . Preferred are crop plants such as plants advanta for use herein . geously selected from the group of the genus peanut, oilseed Whereas in the homologous recombination vector , the rape , canola , sunflower, safflower, olive , sesame, hazelnut, altered portion of the nucleic acid molecule coding for a almond , avocado , bay, pumpkin /squash , linseed , soya , pis protein depicted in SEQ ID NO : 2 or 4 , or a homolog thereof 20 tachio , borage , maize, wheat , rye, oats , sorghum and millet , is flanked at its 5 ' and 3 ' ends by an additional nucleic acid triticale , rice , barley , cassava , potato , sugarbeet , egg plant, molecule of the gene to allow for homologous recombina - alfalfa , and perennial grasses and forage plants , oil palm , tion to occur between the exogenous gene carried by the vegetables ( brassicas , root vegetables , tuber vegetables, pod vector and an endogenous gene, in a microorganism or plant. vegetables, fruiting vegetables , onion vegetables, leafy veg The additional flanking nucleic acid molecule is of sufficient 25 etables and stem vegetables ) , buckwheat, Jerusalem arti length for successful homologous recombination with the choke, broad bean , vetches , lentil, dwarf bean , lupin , clover endogenous gene. Typically , several hundreds of base pairs and Lucerne for mentioning only some of them . up to kilobases of flanking DNA (both at the 5 ' and 3 ' ends) In one embodiment of the invention transgenic plants are are included in the vector. See , e . g ., Thomas K . R . , and selected from the group comprising cereals , soybean , rape Capecchi M . R . , Cell 51 , 503 ( 1987 ) for a description of 30 seed ( including oil seed rape, especially canola and winter homologous recombination vectors or Strepp et al ., PNAS , oil seed rape ) , cotton , sugarcane , sugar beet and potato , 95 ( 8 ), 4368 (1998 ) for cDNA based recombination in especially corn , soy , rapeseed (including oil seed rape , Physcomitrella patens. The vector is introduced into a especially canola and winter oil seed rape ), cotton , wheat microorganism or plant cell ( e . g . via polyethylene glycol and rice . mediated DNA ) , and cells in which the introduced gene has 35 In another embodiment of the invention the transgenic homologously recombined with the endogenous gene are plant is a gymnosperm plant , especially a spruce , pine or fir. selected using art- known techniques . In one embodiment, the host plant is selected from the Whether present in an extra -chromosomal non - replicating families Aceraceae , Anacardiaceae , Apiaceae , Asteraceae , vector or a vector that is integrated into a chromosome, the Brassicaceae, Cactaceae , Cucurbitaceae, Euphorbiaceae , nucleic acid molecule coding for amino acid molecules 40 Fabaceae, Malvaceae , Nymphaeaceae , Papaveraceae, Rosa depicted in SEQ ID NO : 2 or 4 , or a homolog thereof ceae , Salicaceae , Solanaceae , Arecaceae , Bromeliaceae , preferably resides in a plant expression cassette . A plant Cyperaceae , Iridaceae , Liliaceae , Orchidaceae , Gentian expression cassette preferably contains regulatory sequences aceae , Labiaceae , Magnoliaceae , Ranunculaceae , Carifo capable of driving gene expression in plant cells that are laceae , Rubiaceae , Scrophulariaceae , Caryophyllaceae , Eri operatively linked so that each sequence can fulfill its 45 caceae , Polygonaceae, Violaceae , Juncaceae or Poaceae and function , for example , termination of transcription by poly - preferably from a plant selected from the group of the adenylation signals . Preferred polyadenylation signals are families Apiaceae , Asteraceae , Brassicaceae , Cucurbitaceae , those originating from Agrobacterium tumefaciens t- DNA Fabaceae , Papaveraceae , Rosaceae , Solanaceae , Liliaceae or such as the gene 3 known as octopine synthase of the Poaceae . Preferred are crop plants and in particular plants Ti- plasmid pTIACH5 (Gielen et al ., EMBO J . 3 , 835 ( 1984 ) ) 50 mentioned herein above as host plants such as the families or functional equivalents thereof but also all other termina - and genera mentioned above for example preferred the tors functionally active in plants are suitable . As plant gene species Anacardium occidentale , Calendula officinalis, expression is very often not limited on transcriptional levels , Carthamus tinctorius, Cichorium intybus, Cynara scolymus , a plant expression cassette preferably contains other opera - Helianthus annus , Tagetes lucida , Tagetes erecta , Tagetes tively linked sequences like translational enhancers such as 55 tenuifolia ; Daucus carota ; Corylus avellana , Corylus the overdrive - sequence containing the 5 ' - untranslated leader colurna , Borago officinalis ; Brassica napus, Brassica rapa sequence from tobacco mosaic virus enhancing the poly - ssp ., Sinapis arvensis Brassica juncea , Brassica juncea var. peptide per RNA ratio (Gallie et al. , Nucl. Acids Research juncea , Brassica juncea var. crispifolia , Brassica juncea var . 15 , 8693 ( 1987 ) ). Examples of plant expression vectors foliosa , Brassica nigra , Brassica sinapioides, Melanosina include those detailed in : Becker D . et al ., Plant Mol. Biol. 60 pis communis , Brassica oleracea , Arabidopsis thaliana , 20 , 1195 ( 1992 ); and Bevan M . W ., Nucl . Acid . Res . 12 , Anana comosus, Ananas ananas, Bromelia comosa , Carica 8711 (1984 ) ; and “ Vectors for Gene Transfer in Higher papaya , Cannabis sative , Ipomoea batatus, Ipomoea pan Plants ” in : Transgenic Plants , Vol. 1 , Engineering and Uti durata , Convolvulus batatas, Convolvulus tiliaceus, Ipom lization , eds. Kung and Wu R . , Academic Press, 1993 , S . oea fastigiata , Ipomoea tiliacea , Ipomoea triloba , Convol 15 -38 . 65 vulus panduratus, Beta vulgaris , Beta vulgaris var . The host organism transgenic organism ) advanta altissima , Beta vulgaris var. vulgaris , Beta maritima , Beta geously contains at least one copy of the nucleic acid vulgaris var. perennis , Beta vulgaris var . conditiva , Beta US 10 , 392 ,630 B2 35 36 vulgaris var . esculenta , Cucurbita maxima, Cucurbita mbda, Brassicaceae such as the genera Brassica , Melanosinapis , Cucurbita pepo , Cucurbita moschata , Olea europaea ,Mani Sinapis, Arabidopsis e. g . the species Brassica napus, Bras hot utilissima, Janipha manihot, Jatropha manihot, Manihot sica rapa ssp . [canola , oilseed rape , turnip rape ], Sinapis aipil, Manihot dulcis , Manihot manihot, Manihot melanoba arvensis Brassica juncea , Brassica juncea var. juncea , Bras sis , Manihot esculenta , Ricinus communis , Pisum sativum , 5 sica juncea var . crispifolia , Brassica juncea var. foliosa , Pisum arvense , Pisum humile , Medicago sativa , Medicago Brassica nigra , Brassica sinapioides, Melanosinapis com falcata , Medicago varia , Glycine max Dolichos soja , Gly - munis [mustard ], Brassica oleracea [ fodder beet] or Arabi cine gracilis , Glycine hispida , Phaseolus max, Soja hispida , dopsis thaliana ; Bromeliaceae such as the genera Anana , Soja max , Cocos nucifera , Pelargonium grossularioides , Bromelia e . g . the species Anana comosus, Ananas ananas or Oleum cocoas, Laurus nobilis , Persea americana , Arachis 10 Bromelia comosa [pineapple ) ; Caricaceae such as the genera hypogaea , Linum usitatissimum , Linum humile , Linum aus - Carica e . g . the species Carica papaya (papaya ) ; Canna triacum , Linum bienne , Linum angustifolium , Linum cathar - baceae such as the genera Cannabis e . g . the species Can ticum , Linum flavum , Linum grandiflorum , Adenolinum nabis sative [hemp ) , Convolvulaceae such as the genera grandiflorum , Linum lewisii , Linum narbonense , Linum Ipomea , Convolvulus e . g . the species Ipomoea batatus , perenne, Linum perenne var. lewisii, Linum pratense , Linum 15 Ipomoea pandurata , Convolvulus batatas, Convolvulus tili trigynum , Punica granatum , Gossypium hirsutum , Gossy aceus, Ipomoea fastigiata , Ipomoea tiliacea , Ipomoea tri pium arboreum , Gossypium barbadense , Gossypium herba loba or Convolvulus panduratus [sweet potato , Man of the ceum , Gossypium thurberi, Musa nana , Musa acuminata , Earth , wild potato ], Chenopodiaceae such as the genera Musa paradisiaca , Musa spp . , Elaeis guineensis , Papaver Beta , i .e . the species Beta vulgaris , Beta vulgaris var. orientate , Papaver rhoeas, Papaver dubium , Sesamum indi- 20 altissima , Beta vulgaris var. Vulgaris, Beta maritima , Beta cum , Piper aduncum , Piper amalago , Piper angustifolium , vulgaris var. perennis , Beta vulgaris var. conditiva or Beta Piper auritum , Piper betel, Piper cubeba , Piper longum , vulgaris var . esculenta ( sugar beet] ; Cucurbitaceae such as Piper nigrum , Piper retrofractum , Artanthe adunca , Artan - the genera Cucurbita e . g . the species Cucurbita maxima , the elongata , Peperomia elongata , Piper elongatum , Stef - Cucurbita mixta , Cucurbita pepo or Cucurbita moschata fensia elongata , Hordeum vulgare , Hordeum jubatum , Hor - 25 ?pumpkin , squash ] ; Elaeagnaceae such as the genera Elae deum murinum , Hordeum secalinum , Hordeum distichon agnus e . g . the species Olea europaea (olive ); Ericaceae such Hordeum aegiceras, Hordeum hexastichon ., Hordeum hex - as the genera Kalmia e . g . the species Kalmia latifolia , astichum , Hordeum irregulare , Hordeum sativum , Hordeum Kalmia angustifolia , Kalmia microphylla , Kalmia polifolia , secalinum , Avena sativa , Avena fatua , Avena byzantina , Kalmia occidentalis, Cistus chamaerhodendros or Kalmia Avena fatua var. sativa, Avena hybrida, Sorghum bicolor, 30 lucida [ American laurel, broad - leafed laurel, calico bush , Sorghum halepense , Sorghum saccharatum , Sorghum vul - spoon wood , sheep laurel, alpine laurel, bog laurel, western gare , Andropogon drummondii, Holcus bicolor , Holcus sor- bog - laurel, swamp- laurel ] ; Euphorbiaceae such as the gen ghum , Sorghum aethiopicum , Sorghum arundinaceum , Sor - era Manihot, Janipha , Jatropha , Ricinus e . g . the species ghum caffrorum , Sorghum cernuum , Sorghum dochna , Manihot utilissima, Janipha manihot, Jatropha manihot, Sorghum drummondii, Sorghum durra , Sorghum guineense , 35 Manihot aipil , Manihot dulcis , Manihot manihot, Manihot Sorghum lanceolatum , Sorghum nervosum , Sorghum sac melanobasis , Manihot esculenta (manihot , arrowroot, tapi charatum , Sorghum subglabrescens, Sorghum verticilliflo oca , cassava ] or Ricinus communis [ castor bean , Castor Oil rum , Sorghum vulgare , Holcus halepensis , Sorghum mili - Bush , Castor Oil Plant, Palma Christi , Wonder Tree ] ; aceum millet, Panicum militaceum , Zea mays, Triticum Fabaceae such as the genera Pisum , Albizia , Cathormion , aestivum , Triticum durum , Triticum turgidum , Triticum 40 Feuillea , Inga , Pithecolobium , Acacia , Mimosa , Medicajo , hybernum , Triticum macha , Triticum sativum or Triticum Glycine , Dolichos, Phaseolus, Soja e . g . the species Pisum vulgare , Coffea spp ., Coffea arabica , Coffea canephora , sativum , Pisum arvense , Pisum humile [ pea ] , Albizia berte Coffea liberica , Capsicum annuum , Capsicum annuum var . riana , Albizia julibrissin , Albizia lebbeck , Acacia berteri glabriusculum , Capsicum frutescens , Capsicum annuum , ana , Acacia littoralis , Albizia berteriana , Albizzia berteri Nicotiana tabacum , Solanum tuberosum , Solanum melon - 45 ana , Cathormion berteriana , Feuillea berteriana , Inga gena , Lycopersicon esculentum , Lycopersicon lycopersi- fragrans , Pithecellobium berterianum , Pithecellobium fra cum , Lycopersicon pyriforme, Solanum integrifolium , Solawala grans, Pithecolobium berterianum , Pseudalbizzia berteri num lycopersicum Theobroma cacao or Camellia sinensis. ana , Acacia julibrissin , Acacia nemu, Albizia nemu , Anacardiaceae such as the genera Pistacia , Mangifera , Feuilleea julibrissin , Mimosa julibrissin , Mimosa speciosa , Anacardium e . g . the species Pistacia vera [ pistachios , 50 Sericanrda julibrissin , Acacia lebbeck , Acacia macrophylla , Pistazie ] , Mangifer indica [Mango ] or Anacardium occiden Albizia lebbek , Feuilleea lebbeck , Mimosa lebbeck , Mimosa tale [Cashew ] ; Asteraceae such as the genera Calendula , speciosa [bastard logwood , silk tree , East Indian Walnut ), Carthamus, Centaurea , Cichorium , Cynara , Helianthus, Medicago sativa , Medicago falcata , Medicago varia [ al Lactuca , Locusta, Tagetes , Valeriana e . g. the species Calen falfa ] Glycine max Dolichos soja , Glycine gracilis, Glycine dula officinalis [Marigold ], Carthamus tinctorius [ saf- 55 hispida , Phaseolus max, Soja hispida or Soja max [ soy flower) , Centaurea cyanus [ cornflower ], Cichorium intybus bean ]; Geraniaceae such as the genera Pelargonium , Cocos , [blue daisy ], Cynara scolymus [Artichoke ), Helianthus Oleum e. g . the species Cocos nucifera , Pelargonium gros annus ( sunflower ) , Lactuca sativa , Lactuca crispa , Lactuca sularioides or Oleum cocois ( coconut ] ; Gramineae such as esculenta , Lactuca scariola L . ssp . sativa , Lactuca scariola the genera Saccharum e .g . the species Saccharum offici L . var. integrata , Lactuca scariola L . var. integrifolia , 60 narum ; Juglandaceae such as the genera Juglans, Wallia e . g . Lactuca sativa subsp . romana , Locusta communis , Valeri- the species Juglans regia , Juglans ailanthifolia , Juglans ana locusta [lettuce ] , Tagetes lucida , Tagetes erecta or sieboldiana , Juglans cinerea , Wallia cinerea , Juglans bix Tagetes tenuifolia [Marigold ); Apiaceae such as the genera byi , Juglans califomica , Juglans hindsii, Juglans interme Daucus e . g . the species Daucus carota [ carrot) ; Betulaceae dia , Juglans jamaicensis , Juglans major, Juglans micro such as the genera Corylus e . g . the species Corylus avellana 65 carpa , Juglans nigra or Wallia nigra [ walnut, black walnut, or Corylus colurna ?hazelnut] ; Boraginaceae such as the common walnut, persian walnut, white walnut, butternut, genera Borago e . g . the species Borago officinalis [ borage ]; black walnut) ; Lauraceae such as the genera Persea , Laurus US 10 , 392 ,630 B2 37 38 e . g . the species laurel Laurus nobilis [bay , laurel, bay laurel, white mullein , dark mullein , greek mullein , orange mullein , sweet bay ] , Persea americana Persea americana , Persea purple mullein , hoary mullein , great mullein ); Solanaceae gratissima or Persea persea ( avocado ) ; Leguminosae such such as the genera Capsicum , Nicotiana , Solanum , Lycoper as the genera Arachis e . g . the species Arachis hypogaea sicon e . g . the species Capsicum annuum , Capsicum annuum Speanut] ; Linaceae such as the genera Linum , Adenolinum 5 var . glabriusculum , Capsicum frutescens ( pepper ] , Capsi e . g . the species Linum usitatissimum , Linum humile, Linum cum annuum [ paprika ) , Nicotiana tabacum , Nicotiana alata , austriacum , Linum bienne , Linum angustifolium , Linum Nicotiana attenuata , Nicotiana glauca , Nicotiana langs catharticum , Linum flavum , Linum grandiflorum , Adenoli- dorffii, Nicotiana obtusifolia , Nicotiana quadrivalvis, Nico num grandiflorum , Linum lewisii , Linum narbonense, Linum tiana repanda , Nicotiana rustica , Nicotiana sylvestris [ to perenne , Linum perenne var. lewisii , Linum pratense or 10 baccol, Solanum tuberosum ?potato ) , Solanum melongena Linum trigynum [ flax , linseed ) ; Lythrarieae such as the [ egg - plant ] (Lycopersicon esculentum , Lycopersicon lyco genera Punica e . g . the species Pun /ca granatum [pomegran - persicum . , Lycopersicon pyriforme, Solanum integrifolium atel; Malvaceae such as the genera Gossypium e . g . the o r Solanum lycopersicum ?tomato ] ; Sterculiaceae such as the species Gossypium hirsutum , Gossypium arboreum , Gossy - genera Theobroma e . g . the species Theobroma cacao [ ca pium barbadense, Gossypium herbaceum or Gossypium 15 cao ] ; Theaceae such as the genera Camellia e .g . the species thurberi ?cotton ] ; Musaceae such as the genera Musa e . g . the Camellia sinensis ) ?tea ) . species Musa nana , Musa acuminata , Musa paradisiaca , The introduction of the nucleic acids according to the Musa spp . [ banana ] ; Onagraceae such as the genera Camis - invention , the expression cassette or the vector into organ sonia , Oenothera e . g . the species Oenothera biennis or isms, plants for example , can in principle be done by all of Camissonia brevipes (primrose , evening primrose ] ; Palmae 20 the methods known to those skilled in the art. The intro such as the genera Elacis e . g . the species Elacis guineensis duction of the nucleic acid sequences gives rise to recom [oil plam ]; Papaveraceae such as the genera Papaver e .g . the binant or transgenic organisms. species Papaver orientale , Papaver rhoeas, Papaver The transfer of foreign genes into the genome of a plant dubium (poppy , oriental poppy, corn poppy , field poppy, is called transformation . In doing this themethods described shirley poppies , field poppy , long - headed poppy , long - pod 25 for the transformation and regeneration of plants from plant poppy ); Pedaliaceae such as the genera Sesamum e . g . the tissues or plant cells are utilized for transient or stable species Sesamum indicum [sesame ]; Piperaceae such as the transformation . Suitable methods are protoplast transforma genera Piper, Artanthe , Peperomia , Steffensia e . g. the spe tion by poly ( ethylene glycol) - induced DNA uptake , the cies Piper aduncum , Piper amalago , Piper angustifolium , “ biolistic ” method using the gene cannon referred to as the Piper auritum , Piper betel , Piper cubeba , Piper longum , 30 particle bombardment method , electroporation , the incuba Piper nigrum , Piper retrofractum , Artanthe adunca , Artan - tion of dry embryos in DNA solution , microinjection and the elongata , Peperomia elongata , Piper elongatum , Stef- gene transfer mediated by Agrobacterium . Said methods are fensia elongata . Cayenne pepper, wild pepper ); Poaceae described by way of example in Jenes B . et al. , Techniques such as the genera Hordeum , Secale , Avena , Sorghum , for Gene Transfer, in : Transgenic Plants, Vol. 1 , Engineering Andropogon , Holcus, Panicum , Oryza , Zea , Triticum e . g . the 35 and Utilization , eds. Kung S . D and Wu R ., Academic Press species Hordeum vulgare , Hordeum jubatum , Hordeum (1993 ) 128 - 143 and in Potrykus , Annu . Rev . Plant Physiol. murinum , Hordeum secalinum , Hordeum distichon Hor - Plant Molec . Biol. 42, 205 ( 1991) . The nucleic acids or the deum aegiceras , Hordeum hexastichon . , Hordeum hexasti - construct to be expressed is preferably cloned into a vector chum , Hordeum irregulare, Hordeum sativum , Hordeum which is suitable for transforming Agrobacterium tumefa secalinum [barley , pearl barley, foxtail barley, wall barley , 40 ciens, for example pBin19 ( Bevan et al. , Nucl. Acids Res. meadow barley ] , Secale cereale [ rye) , Avena sativa , Avena 12 , 8711 ( 1984 ) ) . Agrobacteria transformed by such a vector fatua , Avena byzantina , Avena fatua var. sativa , Avena can then be used in known manner for the transformation of hybrida [oat ], Sorghum bicolor, Sorghum halepense, Sor plants , in particular of crop plants such as by way of example ghum saccharatum , Sorghum vulgare, Andropogon drum tobacco plants , for example by bathing bruised leaves or mondii, Holcus bicolor, Holcus sorghum , Sorghum aethio - 45 chopped leaves in an agrobacterial solution and then cultur picum , Sorghum arundinaceum , Sorghum caffrorum , ing them in suitable media . The transformation of plants by Sorghum cernuum , Sorghum dochna , Sorghum drummondii , means of Agrobacterium tumefaciens is described , for Sorghum durra , Sorghum guineense, Sorghum lanceolatum , example , by Hofgen and Willmitzer in Nucl . Acid Res . 16 , Sorghum nervosum , Sorghum saccharatum , Sorghum subg - 9877 ( 1988 ) or is known inter alia from White F . F ., Vectors labrescens, Sorghum verticilliftorum , Sorghum vulgare , Hol - 50 for Gene Transfer in Higher Plants ; in Transgenic Plants , cus halepensis , Sorghum miliaceum millet , Panicum milita - Vol. 1, Engineering and Utilization , eds . Kung S . D . and Wu ceum [ Sorghum , millet ] , Oryza sativa , Oryza latifolia [rice ], R ., Academic Press , 1993 , pp . 15- 38 . Zea mays ?corn , maize ] Triticum aestivum , Triticum durum , Agrobacteria transformed by an expression vector accord Triticum turgidum , Triticum hybernum , Triticum macha , ing to the invention may likewise be used in known manner Triticum sativum or Triticum vulgare (wheat , bread wheat, 55 for the transformation of plants such as test plants like common wheat] , Proteaceae such as the genera Macadamia Arabidopsis or crop plants such as cereal crops , corn , oats , e .g . the species Macadamia intergrifolia (macadamia ]; rye, barley, wheat, soybean , rice, cotton , sugar beet , canola , Rubiaceae such as the genera Coffea e . g . the species Coffea sunflower , flax , hemp , potatoes , tobacco , tomatoes, carrots , spp ., Coffea arabica , Coffea canephora or Coffea liberica paprika , oilseed rape , tapioca , cassava , arrowroot , tagetes , [ coffeel; Scrophulariaceae such as the genera Verbascum 60 alfalfa , lettuce and the various tree , nut and vine species , in e . g . the species Verbascum blattaria , Verbascum chabdi, particular oil - containing crop plants such as soybean , pea Verbascum densiflorum , Verbascum lagurus , Verbascum nut , castor oil plant, sunflower , corn , cotton , flax , oilseed longifolium , Verbascum lychnitis, Verbascum nigrum , Ver rape, coconut, oil palm , safflower (Carthamus tinctorius) or bascum olympicum , Verbascum phiomoides , Verbascum cocoa bean , or in particular corn , wheat, soybean , rice , phoenicum , Verbascum pulverulentum or Verbascum thap - 65 cotton and canola , e. g. by bathing bruised leaves or chopped sus ?mullein , white moth mullein , nettle - leaved mullein , leaves in an agrobacterial solution and then culturing them dense - flowered mullein , silver mullein , long- leaved mullein , in suitable media . US 10 , 392 ,630 B2 39 40 The genetically modified plant cells may be regenerated NO : 1 or 3 , or a homolog thereof can be determined , for by all of the methods known to those skilled in the art . example , in vitro as described in the Examples. In addition , Appropriate methods can be found in the publications a functional expression of the sequences of SEQ ID NO : 2 referred to above by Kung S . D . and Wu R ., Potrykus or or 4 , or a homolog thereof, encoded by the corresponding Hofgen and Willmitzer. 5 nucleic acid molecule as depicted in SEQ ID NO : 1 or 3 , Accordingly , a further aspect of the invention relates to and / or homologs modified in nature and level and its effect transgenic organisms transformed by at least one nucleic on herbicide tolerance or resistance , but also on the meta acid sequence , expression cassette or vector according to the bolic pathways performance can be tested on test plants in invention as well as cells , cell cultures , tissue , parts — such as, for example , leaves , roots, etc . in the case of plant 10 greenhouse trials ( see EXAMPLES ) . organisms— or reproductive material derived from such An additional object of the invention comprises trans organisms. genic organisms such as transgenic plants transformed by an In one embodiment of the invention host plants for the expression cassette containing sequences of as depicted in nucleic acid , expression cassette or vector according to the SEQ ID NO : 1 or 3 , or a homolog thereof according to the invention are selected from the group comprising corn , soy , 15 invention or DNA sequences hybridizing therewith , as well oil seed rape ( including canola and winter oil seed rape ) , as transgenic cells , tissue , parts and reproduction material of cotton , wheat and rice . such plants . Particular preference is given in this case to A further embodiment of the invention relates to the use transgenic crop plants such as by way of example barley, of a nucleic acid construct , e . g . an expression cassette , wheat , rye, oats , corn , soybean , rice , cotton , sugar beet , containing one or more DNA sequences encoding one or 20 oilseed rape and canola , sunflower, flax , hemp, thistle , more polypeptides shown in SEQ ID NO : 2 or 4 , or a potatoes, tobacco , tomatoes, tapioca , cassava , arrowroot, homolog thereof or comprising one or more nucleic acid alfalfa , lettuce and the various tree , nut and vine species . molecules as depicted in SEQ ID NO : 1 or 3 , or a homolog I n one embodiment of the invention transgenic plants thereof or encoding or DNA sequences hybridizing there - transformed by an expression cassette containing or com with for the transformation of plant cells , tissues or parts of 25 prising nucleic acid molecules or sequences as depicted in plants . SEQ ID NO : 1 or 3 , or a homolog thereof, according to the In doing so , depending on the choice of promoter , the invention or DNA sequences hybridizing therewith are nucleic acid molecules of the present invention can be selected from the group comprising corn , soy , oil seed rape expressed specifically in the leaves , in the seeds, the nod (including canola and winter oil seed rape ) , cotton , wheat ules , in roots , in the stem or other parts of the plant . Those 30 and rice . transgenic plants overproducing sequences , e . g . as depicted For the purposes of the invention plants are mono - and in SEQ ID NO : 1 or 3 , or a homolog thereof, the reproduc - dicotyledonous plants , mosses or algae , especially plants , tive material thereof , together with the plant cells , tissues or for example in one embodiment monocotyledonous plants , parts thereof are a further object of the present invention . or for example in another embodiment dicotyledonous The expression cassette or the nucleic acid sequences or 35 plants . A further refinement according to the invention are construct according to the invention containing nucleic acid transgenic plants as described above which contain a nucleic molecules or sequences as depicted in SEQ ID NO : 1 or 3 , acid sequence or construct according to the invention or a or a homolog thereof can , moreover, also be employed for expression cassette according to the invention . the transformation of the organisms identified by way of However, transgenic also means that the nucleic acids example above such as bacteria , yeasts , filamentous fungi 40 according to the invention are located at their natural posi and plants . tion in the genome of an organism , but that the sequence , Within the framework of the present invention , increased e . g . the coding sequence or a regulatory sequence , for herbicide tolerance or resistance, relates to , for example , the example the promoter sequence , has been modified in com artificially acquired trait of increased herbicide tolerance or parison with the natural sequence . Preferably, transgenic / resistance, by comparison with the non - genetically modified 45 recombinant is to be understood as meaning the transcription initial plants e . g . the trait acquired by genetic modification of one or more nucleic acids or molecules of the invention of the target organism , and due to functional over- expression and being shown in SEQ ID NO : 1 or 3 , or a homolog of one or more polypeptide (sequences ) of SEQ ID NO : 2 or thereof , occurs at a non - natural position in the genome. In 4 , or a homolog thereof, e. g . encoded by the corresponding one embodiment, the expression of the nucleic acids or nucleic acid molecules as depicted in SEQ ID NO : 1 or 3 , 50 molecules is homologous . In another embodiment, the and / or homologs, in the organisms according to the inven - expression of the nucleic acids or molecules is heterologous . tion , advantageously in the transgenic plant according to the This expression can be transiently or of a sequence inte invention or produced according to the method of the grated stably into the genome . Advantageous inducible plant invention , at least for the duration of at least one plant promoters are by way of example the PRP1 promoter (Ward generation . 55 et al. , Plant. Mol . Biol. 22361 ( 1993 ) ), a promoter inducible A constitutive expression of the polypeptide sequences of by benzenesulfonamide (EP 388 186 ) , a promoter inducible SEO ID NO : 2 or 4 , or a homolog thereof, encoded by the by tetracycline (Gatz et al. , Plant J . 2 , 397 ( 1992 ) ) , a corresponding nucleic acid molecule as depicted in SEQ ID promoter inducible by salicylic acid (WO 95 / 19443 ) , a NO : 1 or 3 , or a homolog thereof is , moreover , advanta - promoter inducible by abscisic acid ( EP 335 528 ) and a geous . On the other hand , however , an inducible expression 60 promoter inducible by ethanol or cyclohexanone (WO may also appear desirable . Expression of the polypeptide 93 / 21334 ) . Other examples of plant promoters which can sequences of the invention can be either direct to the advantageously be used are the promoter of cytoplasmic cytoplasm or the organelles, preferably the plastids of the FBPase from potato , the ST- LSI promoter from potato host cells , preferably the plant cells . (Stockhaus et al. , EMBO J. 8 , 2445 ( 1989 )) , the promoter of The activity of the protein encoded by the sequences of 65 phosphoribosyl pyrophosphate amidotransferase from Gly SEQ ID NO : 2 or 4 , or a homolog thereof, encoded by the cine max ( see also gene bank accession number U87999 ) or corresponding nucleic acid molecule as depicted in SEQ ID a nodiene - specific promoter as described in EP 249 676 . US 10 , 392 ,630 B2 41 42 Such promoters are known to the person skilled in the art tion can comprise only a portion of the coding region of one or can be isolated from genes which are induced under the of the sequences or molecules of a nucleic acid as shown in conditions mentioned above . In one embodiment, seed - SEQ ID NO : 1 or 3 , or a homolog thereof, for example, a specific promoters may be used for monocotylodonous or fragment which can be used as a probe or primer or a dicotylodonous plants . 5 fragment encoding a biologically active portion of a poly In principle all natural promoters with their regulation peptide -according to invention . sequences can be used like those named above for the Portions of proteins encoded by the polypeptide according expression cassette according to the invention and the to the invention or a polypeptide encoding nucleic acid method according to the invention . Over and above this , molecules of the invention are preferably biologically active synthetic promoters may also advantageously be used . In the 10 portions described herein . As used herein , the term “ bio preparation of an expression cassette various DNA frag - logically active portion of” a polypeptide is intended to ments can be manipulated in order to obtain a nucleotide include a portion , e . g . a domain /motif , of increased herbi sequence, which usefully reads in the correct direction and cide tolerance or resistance, in a plant. To determine whether is equipped with a correct reading frame. To connect the a polypeptide according to the invention , or a biologically DNA fragments (= nucleic acids according to the invention ) 15 active portion thereof, results in an increased herbicide to one another adaptors or linkers may be attached to the tolerance or resistance , an analysis of a plant comprising the fragments . The promoter and the terminator regions can polypeptide may be performed . Such analysis methods are usefully be provided in the transcription direction with a well known to those skilled in the art, as detailed in the linker or polylinker containing one or more restriction points Examples. More specifically , nucleic acid fragments encod for the insertion of this sequence . Generally , the linker has 20 ing biologically active portions of a polypeptide can be 1 to 10 , mostly 1 to 8 , preferably 2 to 6 , restriction points. prepared by isolating a portion of one of the sequences of the In general the size of the linker inside the regulatory region nucleic acid molecules listed in SEQ ID NO : 1 or 3 , or a is less than 100 bp , frequently less than 60 bp , but at least homolog thereof expressing the encoded portion of the 5 bp . The promoter may be both native or homologous as polypeptide or peptide thereof ( e. g ., by recombinant expres well as foreign or heterologous to the host organism , for 25 sion in vitro ) and assessing the activity of the encoded example to the host plant . In the 5 ' - 3 ' transcription direction portion . the expression cassette contains the promoter, a DNA Biologically active portions of the polypeptide according sequence which shown in SEQ ID NO : 1 or 3 , or a homolog to the invention are encompassed by the present invention thereof and a region for transcription termination . Different and include peptides comprising amino acid sequences termination regions can be exchanged for one another in any 30 derived from the amino acid sequence of the polypeptide desired fashion . encoding gene , or the amino acid sequence of a protein A nucleic acid molecule of the present invention , e. g. , a homologous to the polypeptide according to the invention , nucleic acid molecule encoding a polypeptide which confers which include fewer amino acids than a full length poly increased herbicide tolerance or resistance , in plants , can be peptide according to the invention or the full length protein isolated using standard molecular biological techniques and 35 which is homologous to the polypeptide according to the the sequence information provided herein . For example , a invention , and exhibits at least some enzymatic or biological microbial polypeptide encoding cDNA according to present activity of the polypeptide according to the invention . Typi invention can be isolated from a microbial c - DNA library cally , biologically active portions ( e . g . , peptides which are , using all or portion of one of the sequences shown in SEQ for example , 5 , 10 , 15 , 20 , 30 , 35 , 36 , 37 , 38 , 39 , 40 , 50 , 100 ID NO : 1 or 3 , or a homolog thereof. Moreover, a nucleic 40 or more amino acids in length ) comprise a domain or motif acid molecule encompassing all or a portion of one of the with at least one activity of the polypeptide according to the sequences of SEQ ID NO : 1 or 3 , or a homolog thereof can invention . Moreover, other biologically active portions in be isolated by the polymerase chain reaction using oligo - which other regions of the protein are deleted can be nucleotide primers designed based upon this sequence . For prepared by recombinant techniques and evaluated for one example , mRNA can be isolated from plant cells ( e . g . , by the 45 or more of the activities described herein . Preferably , the guanidinium - thiocyanate extraction procedure of Chirgwin biologically active portions of the polypeptide according to et al. , Biochemistry 18 , 5294 ( 1979 ) ) and cDNA can be the invention include one or more selected domains/motifs prepared using reverse transcriptase ( e . g . , Moloney MLV or portions thereof having biological activity . reverse transcriptase , available from Gibco / BRL , Bethesda , The term “ biological active portion ” or “ biological activ Md. ; or AMV reverse transcriptase , available from Seika - 50 ity ” means a polypeptide as depicted in SEQ ID NO : 2 or 4 , gaku America , Inc . , St. Petersburg , Fla . ). Synthetic oligo - or a homolog thereof or a portion of said polypeptide which nucleotide primers for polymerase chain reaction amplifi- still has at least 10 % or 20 % , preferably 30 % , 40 % , 50 % or cation can be designed based upon one of the nucleotide 60 % , especially preferably 70 % , 75 % , 80 % , 90 % or 95 % of sequences shown in SEQ ID NO : 1 or 3 , or a homolog the enzymatic or biological activity of the natural or starting thereof. A nucleic acid molecule of the invention can be 55 enzyme or protein . amplified using cDNA or, alternatively , genomic DNA , as a In the process according to the invention nucleic acid template and appropriate oligonucleotide primers according sequences or molecules can be used , which , if appropriate , to standard PCR amplification techniques. The nucleic acid contain synthetic , non -natural or modified nucleotide bases , molecule so amplified can be cloned into an appropriate which can be incorporated into DNA or RNA . Said syn vector and characterized by DNA sequence analysis . Fur - 60 thetic , non - natural or modified bases can for example thermore , the genes employed in the present invention can increase the stability of the nucleic acid molecule outside or be prepared by standard synthetic techniques , e . g . , using a inside a cell . The nucleic acid molecules of the invention can commercially available automated DNA synthesizer . contain the same modifications as aforementioned . In a embodiment, an isolated nucleic acid molecule of the As used in the present context the term “ nucleic acid invention comprises one of the nucleotide sequences or 65 molecule ” may also encompass the untranslated sequence or molecules as shown in SEQ ID NO : 1 or 3 , or a homolog molecule located at the 3 ' and at the 5 ' end of the coding gene thereof. Moreover , the nucleic acid molecule of the inven region , for example at least 500 , preferably 200 , especially US 10 , 392 ,630 B2 43 44 preferably 100 , nucleotides of the sequence upstream of the Conserved domains can be identified from all sequences 5 ' end of the coding region and at least 100 , preferably 50 , and are described using a subset of the standard Prosite especially preferably 20 , nucleotides of the sequence down notation , e .g . the pattern Y- x (21 ,23 ) -[ FW ] means that a stream of the 3 ' end of the coding gene region . It is often conserved tyrosine is separated by minimum 21 and maxi advantageous only to choose the coding region for cloning 5 mum 23 amino acid residues from either a phenylalanine or and expression purposes. tryptophane . Patterns can match at least 80 % of the inves tigated proteins . Conserved patterns can be identified with Preferably , the nucleic acid molecule used in the process the software tool MEME version 3 . 5 . 1 or manually . MEME according to the invention or the nucleic acid molecule of is described by Timothy L . Bailey and Charles Elkan ( Pro the invention is an isolated nucleic acid molecule . In oneir 10 ceedings of the Second International Conference on Intelli embodiment, the nucleic acid molecule of the invention is 10 gent Systems forMolecular Biology , pp . 28 -36 , AAAI Press , the nucleic acid molecule used in the process of the inven Menlo Park , Calif. , 1994 ). The source code for the stand tion . alone program is publicly available from the San Diego In various embodiments , the isolated nucleic acid mol Supercomputer centre . The Prosite patterns of the conserved ecule used in the process according to the inventiontion may , Torfor 15 domains can bebe used to search for protein sequences match example comprise less than approximately 5 kb , 4 kb , 3 kb , ing this pattern . Various established Bioinformatic centres 2 kb , 1 kb , 0 . 5 kb or 0 . 1 kb nucleotide sequences which provide public internet portals for using those patterns in naturally flank the nucleic acid molecule in the genomic database searches ( e. g . PIR (Protein Information Resource , DNA of the cell from which the nucleic acid molecule located at Georgetown University Medical Center ) or originates . 20 ExPASy (Expert Protein Analysis System ) ) . Alternatively , The nucleic acid molecules used in the process , for stand - alone software is available , like the program Fuzzpro , example the polynucleotide of the invention or of a part which is part of the EMBOSS software package . For thereof can be isolated using molecular- biological standard example , the program Fuzzpro not only allows searching for techniques and the sequence information provided herein . an exact pattern - protein match but also allows setting vari Also , for example a homologous sequence or homologous , 25 ous ambiguities in the performed search . conserved sequence regions at the DNA or amino acid level Degenerate primers can then be utilized by PCR for the can be identified with the aid of comparison algorithms. The amplification of fragments of novel proteins having above former can be used as hybridization probes under standard mentioned activity , e . g . conferring increased herbicide tol hybridization techniques ( for example those described in erance or resistance , as compared to a corresponding , e . g . Sambrook et al. , supra ) for isolating further nucleic acid 30 non - transformed , wild type plant cell, plant or part thereof sequences useful in this process. after increasing the expression or activity or having the A nucleic acid molecule encompassing a complete activity of a protein as shown in SEQ ID NO : 2 or 4 , or a sequence of the nucleic acid molecules used in the process , homolog thereof or further functional homologs of the for example the polynucleotide of the invention , or a part polypeptide of the invention from other organisms. thereof may additionally be isolated by polymerase chain 35 These fragments can then be utilized as hybridization reaction , oligonucleotide primers based on this sequence or probe for isolating the complete gene sequence . As an on parts thereof being used . For example , a nucleic acid alternative , the missing 5 ' and 3' sequences can be isolated molecule comprising the complete sequence or part thereof by means of RACE - PCR . A nucleic acid molecule according can be isolated by polymerase chain reaction using oligo - to the invention can be amplified using cDNA or, as an nucleotide primers which have been generated on the basis 40 alternative , genomic DNA as template and suitable oligo of this very sequence . For example , mRNA can be isolated nucleotide primers , following standard PCR amplification from cells ( for example by means of the guanidinium techniques . The nucleic acid molecule amplified thus can be thiocyanate extraction method of Chirgwin et al. , Biochem - cloned into a suitable vector and characterized by means of istry 18 , 5294 ( 1979 ) ) and cDNA can be generated by means DNA sequence analysis . Oligonucleotides , which corre of reverse transcriptase ( for example Moloney , MLV reverse 45 spond to one of the nucleic acid molecules used in the transcriptase , available from Gibco /BRL , Bethesda , Md. , or process can be generated by standard synthesis methods, for AMV reverse transcriptase , obtainable from Seikagaku example using an automatic DNA synthesizer. America , Inc . , St. Petersburg , Fla . ) . Nucleic acid molecules which are advantageously for the Synthetic oligonucleotide primers for the amplification by process according to the invention can be isolated based on means of polymerase chain reaction can be generated on the 50 their homology to the nucleic acid molecules disclosed basis of a sequence shown herein , using known methods . herein using the sequences or part thereof as or for the Moreover , it is possible to identify a conserved protein by generation of a hybridization probe and following standard carrying out protein sequence alignments with the polypep - hybridization techniques under stringent hybridization con tide encoded by the nucleic acid molecules of the present ditions. In this context, it is possible to use, for example , invention , in particular with the sequences encoded by the 55 isolated one or more nucleic acid molecules of at least 15 , nucleic acid molecule shown in SEQ ID NO : 1 or 3 , or a 20 , 25 , 30 , 35 , 40 , 50 , 60 or more nucleotides , preferably of homolog thereof, from which conserved regions, and in turn , at least 15 , 20 or 25 nucleotides in length which hybridize degenerate primers can be derived . Conserved regions are under stringent conditions with the above - described nucleic those , which show a very little variation in the amino acid acid molecules, in particular with those which encompass a in one particular position of severalhomologs from different 60 nucleotide sequence of the nucleic acid molecule used in the origin . Moreover , it is possible to identify conserved regions process of the invention or encoding a protein used in the from various organisms by carrying out protein sequence invention or of the nucleic acid molecule of the invention . alignments with the polypeptide encoded by the nucleic acid Nucleic acid molecules with 30 , 50 , 100 , 250 or more of the present invention , in particular with the sequences of nucleotides may also be used . the polypeptide molecule shown in SEQ ID NO : 2 or 4 , or 65 By “ hybridizing " it is meant that such nucleic acid a homolog thereof, from which conserved regions , and in molecules hybridize under conventional hybridization con turn , degenerate primers can be derived . ditions , preferably under stringent conditions such as US 10 , 392 ,630 B2 45 46 described by, e . g . , Sambrook (Molecular Cloning ; A Labo - eters can be kept constant while only the other is varied . ratory Manual, 2nd Edition , Cold Spring Harbor Laboratory Denaturants , for example formamide or SDS , may also be Press, Cold Spring Harbor, N . Y . ( 1989 )) or in Current employed during the hybridization . In the presence of 50 % Protocols in Molecular Biology , John Wiley & Sons, N . Y . formamide, hybridization is preferably effected at 42° C . ( 1989) , 6 . 3 . 1 - 6 . 3 . 6 . 5 Relevant factors like 1 ) length of treatment, 2 ) salt condi According to the invention , DNA as well as RNA mol- tions , 3 ) detergent conditions, 4 ) competitor DNAs, 5 ) ecules of the nucleic acid of the invention can be used as temperature and 6 ) probe selection can be combined case by probes . Further, as template for the identification of func - case so that not all possibilities can be mentioned herein . tional homologues Northern blot assays as well as Southern Thus , in a preferred embodiment , Northern blots are blot assays can be performed . The Northern blot assay 10 prehybridized with Rothi- Hybri - Quick buffer (Roth , advantageously provides further information about the Karlsruhe ) at 68° C . for 2 h . Hybridization with radioactive expressed gene product : e .g . expression pattern , occurrence labelled probe is done overnight at 68° C . Subsequent of processing steps, like splicing and capping , etc . The washing steps are performed at 68° C . with 1xSSC . For Southern blot assay provides additional information about Southern blot assays the membrane is prehybridized with the chromosomal localization and organization of the gene 15 Rothi- Hybri - Quick buffer (Roth , Karlsruhe ) at 68° C . for 2 encoding the nucleic acid molecule of the invention . h . The hybridization with radioactive labelled probe is A preferred , non - limiting example of stringent hybridiza conducted over night at 68° C . Subsequently the hybridiza tion conditions are hybridizations in 6x sodium chloride/ tion buffer is discarded and the filter shortly washed using sodium citrate ( SSC ) at approximately 45° C . , followed by 2xSSC ; 0 . 1 % SDS . After discarding the washing buffer new one or more wash steps in 0 . 2xSSC , 0 . 1 % SDS at 50 to 65° 20 2xSSC ; 0 . 1 % SDS buffer is added and incubated at 68° C . C ., for example at 50° C ., 55° C . or 60° C . The skilled for 15 minutes. This washing step is performed twice worker knows that these hybridization conditions differ as a followed by an additional washing step using 1xSSC ; 0 . 1 % function of the type of the nucleic acid and , for example SDS at 68° C . for 10 min . when organic solvents are present, with regard to the tem Some examples of conditions for DNA hybridization perature and concentration of the buffer . The temperature 25 ( Southern blot assays) and wash step are shown herein under " standard hybridization conditions” differs for below : example as a function of the type of the nucleic acid between (1 ) Hybridization conditions can be selected , for example , 42° C . and 58° C ., preferably between 45º C . and 50° C . in from the following conditions : an aqueous buffer with a concentration of 0 . 1x , 0 . 5x , 1x , 2x , ( a ) 4xSSC at 65° C ., 3x , 4x or 5xSSC (pH 7 . 2 ) . If organic solvent( s ) is / are 30 ( b ) 6xSSC at 45° C ., present in the abovementioned buffer, for example 50 % ( c ) 6xSSC , 100 mg/ ml denatured fragmented fish sperm formamide , the temperature under standard conditions is DNA at 68° C . , approximately 40° C . , 42° C . or 45° C . The hybridization ( d ) 6xSSC , 0 . 5 % SDS , 100 mg/ ml denatured salmon conditions for DNA :DNA hybrids are preferably for sperm DNA at 68° C ., example 0 .1xSSC and 20° C . , 25° C . , 30° C ., 35° C . , 40° C . 35 ( e ) 6xSSC , 0 . 5 % SDS, 100 mg/ ml denatured fragmented or 45° C ., preferably between 30° C . and 45° C . The salmon sperm DNA , 50 % hybridization conditions for DNA : RNA hybrids are prefer formamide at 42° C . , ably for example 0 . 1xSSC and 30° C ., 35° C . , 40° C ., 45° ( f ) 50 % formamide, 4xSSC at 42° C . , C ., 50° C . or 55° C ., preferably between 45º C . and 55° C . ( g ) 50 % ( v / v ) formamide , 0 . 1 % bovine serum albumin , The abovementioned hybridization temperatures are deter - 40 0 . 1 % Ficoll, 0 . 1 % polyvinylpyrrolidone , 50 mM mined for example for a nucleic acid approximately 100 bp sodium phosphate buffer pH 6 .5 , 750 mM NaCl, 75 Fbase pairs ) in length and a G + C content of 50 % in the mM sodium citrate at 42° C . , absence of formamide. The skilled worker knows to deter (h ) 2x or 4xSSC at 50° C . ( low - stringency condition ), or mine the hybridization conditions required with the aid of ( i ) 30 to 40 % formamide , 2x or 4xSSC at 42° C . ( low textbooks , for example the ones mentioned above , or from 45 stringency condition ) . the following textbooks : Sambrook et al ., “ Molecular Clon - ( 2 ) Wash steps can be selected , for example , from the ing ” , Cold Spring Harbor Laboratory, 1989 ; Hames and following conditions : Higgins ( Ed .) 1985 , “ Nucleic Acids Hybridization : A Prac (a ) 0 .015 M NaC1/0 .0015 M sodium citrate / 0 .1 % SDS at tical Approach ” , IRL Press at Oxford University Press , 50° C . Oxford ; Brown ( Ed . ) 1991 , “ Essential Molecular Biology : A 50 (b ) 0 . 1xSSC at 65° C . Practical Approach ” , IRL Press at Oxford University Press , ( c ) 0 . 1XSSC , 0 . 5 % SDS at 68° C . Oxford . ( d ) 0 . 1xSSC , 0 . 5 % SDS , 50 % formamide at 42° C . A further example of one such stringent hybridization ( e ) 0 .2xSSC , 0 . 1 % SDS at 42° C . condition is hybridization at 4xSSC at 65° C ., followed by (f ) 2xSSC at 65° C . ( low - stringency condition ) . a washing in 0 .1xSSC at 65° C . for one hour. Alternatively , 55 Polypeptides having above -mentioned activity , i. e. con an exemplary stringent hybridization condition is in 50 % ferring increased herbicide tolerance or resistance, as com formamide, 4xSSC at 42° C . Further, the conditions during pared to a corresponding , e . g . non - transformed , wild type the wash step can be selected from the range of conditions plant cell, plant or part thereof, derived from other organ delimited by low - stringency conditions (approximately isms, can be encoded by other DNA sequences which 2xSSC at 50° C . ) and high - stringency conditions (approxi - 60 hybridize to the sequences shown in SEQ ID NO : 1 or 3 , or mately 0 .2xSSC at 50° C ., preferably at 65° C . ) ( 20xSSC : a homolog thereof, under relaxed hybridization conditions 0 . 3 M sodium citrate , 3 M NaCl, pH 7 . 0 ) . In addition , the and which code on expression for peptides conferring the temperature during the wash step can be raised from low - increased herbicide tolerance or resistance , as compared to stringency conditions at room temperature , approximately a corresponding , e . g . non -transformed , wild type plant cell , 22° C . , to higher - stringency conditions at approximately 65° 65 plant or part thereof. C . Both of the parameters salt concentration and temperature Further, some applications have to be performed at low can be varied simultaneously , or else one of the two param - stringency hybridization conditions, without any conse US 10 , 392 ,630 B2 47 48 quences for the specificity of the hybridization . For example , increased herbicide tolerance or resistance as compared to a a Southern blot analysis of total DNA could be probed with corresponding , e . g . non - transformed , wild type plant cell, a nucleic acid molecule of the present invention and washed plant or part thereof. at low stringency (55º C . in 2xSSPE , 0 . 1 % SDS ) . The T he term “ one or several amino acids” relates to at least hybridization analysis could reveal a simple pattern of only 5 one amino acid but not more than that number of amino genes encoding polypeptides of the present invention or acids , which would result in a homology of below 50 % used in the process of the invention, e . g . having the herein identity . Preferably , the identity is more than 70 % or 80 % , mentioned activity of enhancing the increased herbicide more preferred are 85 % , 90 % , 91 % 92 % 93 % 94 % or tolerance or resistance , as compared to a corresponding , e . g . 95 % , even more preferred are 96 % , 97 % , 98 % , or 99 % non - transformed , wild type plant cell, plant or part thereof. 10 identity . A further example of such low - stringent hybridization con - Further , the nucleic acid molecule of the invention com ditions is 4xSSC at 50° C . or hybridization with 30 to 40 % prises a nucleic acid molecule , which is a complement of formamide at 42° C . Such molecules comprise those which one of the nucleotide sequences of above mentioned nucleic are fragments , analogues or derivatives of the polypeptide of acid molecules or a portion thereof. A nucleic acid molecule the invention or used in the process of the invention and 15 or its sequence which is complementary to one of the differ , for example , by way of amino acid and / or nucleotide nucleotide molecules or sequences shown in SEQ ID NO : 1 deletion ( s ), insertion ( s ) , substitution ( s ) , addition ( s ) and/ or or 3 , or a homolog thereof, is one which is sufficiently recombination ( s ) or any other modification ( s ) known in the complementary to one of the nucleotide molecules or art either alone or in combination from the above - described sequences shown in SEQ ID NO : 1 or 3 , or a homolog amino acid sequences or their underlying nucleotide 20 thereof such that it can hybridize to one of the nucleotide sequence( s ) . However , it is preferred to use high stringency sequences shown in SEQ ID NO : 1 or 3 , or a homolog hybridization conditions . thereof , thereby forming a stable duplex . Preferably , the Hybridization should advantageously be carried out with hybridization is performed under stringent hybridization fragments of at least 5 , 10 , 15 , 20 , 25 , 30 , 35 or 40 bp , conditions. However, a complement of one of the herein advantageously at least 50 , 60 , 70 or 80 bp , preferably at 25 disclosed sequences is preferably a sequence complement least 90 , 100 or 110 bp . Most preferably are fragments of at thereto according to the base pairing of nucleic acid mol least 15 , 20 , 25 or 30 bp . Preferably are also hybridizations ecules well known to the skilled person . For example, the with at least 100 bp or 200 , very especially preferably at bases A and G undergo base pairing with the bases T and U least 400 bp in length . In an especially preferred embodi- or C , resp . and visa versa . Modifications of the bases can ment, the hybridization should be carried out with the entire 30 influence the base - pairing partner. nucleic acid sequence with conditions described above . The nucleic acid molecule of the invention comprises a The terms “ fragment” , “ fragment of a sequence " or " part nucleotide sequence which is at least about 30 % , 35 % , 40 % of a sequence ” mean a truncated sequence of the original or 45 % , preferably at least about 50 % , 55 % , 60 % or 65 % , sequence referred to . The truncated sequence (nucleic acid more preferably at least about 70 % , 80 % , or 90 % , and even or protein sequence ) can vary widely in length ; the mini- 35 more preferably at least about 95 % , 97 % , 98 % , 99 % ormore mum size being a sequence of sufficient size to provide a homologous to a nucleotide sequence shown in SEQ ID NO : sequence with at least a comparable function and /or activity 1 or 3 , or a homolog thereof, or a portion thereof and of the original sequence or molecule referred to or hybrid - preferably has above mentioned activity , in particular having izing with the nucleic acid molecule of the invention or used a herbicide tolerance or resistance increasing activity after in the process of the invention under stringent conditions, 40 increasing the activity or an activity of a gene as shown in while the maximum size is not critical. In some applications , SEQ ID NO : 1 or 3 , or a homolog thereof or of a gene the maximum size usually is not substantially greater than product, by for example expression either in the cytosol or that required to provide the desired activity and /or func cytoplasm or in an organelle such as a plastid or mitochon tion ( s ) of the original sequence . dria or both , preferably in plastids . Typically , the truncated amino acid sequence or molecule 45 In one embodiment, the nucleic acid molecules compris will range from about 5 to about 310 amino acids in length . ing the sequence of SEQ ID NO : 1 or 3 , or a homolog thereof More typically , however, the sequence will be a maximum or gene products encoded by said nucleic acid molecules are of about 250 amino acids in length , preferably a maximum expressed in combination with a targeting signal as of about 200 or 100 amino acids . It is usually desirable to described herein . select sequences of at least about 10 , 12 or 15 amino acids , 50 The nucleic acid molecule of the invention comprises a up to a maximum of about 20 or 25 amino acids . nucleotide sequence or molecule which hybridizes, prefer The term " epitope ” relates to specific immunoreactive ably hybridizes under stringent conditions as defined herein , sites within an antigen , also known as antigenic determi- to one of the nucleotide sequences or molecule shown in nates . These epitopes can be a linear array of monomers in SEQ ID NO : 1 or 3 , or a homolog thereof, or a portion a polymeric composition such as amino acids in a pro - 55 thereof and encodes a protein having above -mentioned tein — or consist of or comprise a more complex secondary activity , e . g . conferring an increased herbicide tolerance or or tertiary structure . Those of skill will recognize that resistance , as compared to a corresponding , e . g . non - trans immunogens ( i. e . , substances capable of eliciting an formed , wild type plant cell, plant or part thereof by for immune response ) are antigens; however , some antigen , example expression either in the cytosol or in an organelle such as haptens, are not immunogens but may be made 60 such as a plastid or mitochondria or both , preferably in immunogenic by coupling to a carrier molecule . The term plastids , and optionally , having the activity of an PPO " antigen ” includes references to a substance to which an enzyme. antibody can be generated and / or to which the antibody is Moreover, the nucleic acid molecule of the invention can specifically immunoreactive . comprise only a portion of the coding region of one of the In one embodiment the present invention relates to a 65 sequences shown in SEQ ID NO : 1 or 3 , or a homolog epitope of the polypeptide of the present invention or used thereof , for example a fragment which can be used as a in the process of the present invention and confers an probe or primer or a fragment encoding a biologically active US 10 , 392 ,630 B2 49 50 portion of the polypeptide of the present invention or of a ance or resistance , as compared to a corresponding, e. g . polypeptide used in the process of the present invention , i. e . non -transformed , wild type plant cell , plant or part thereof. having above -mentioned activity , e . g . conferring an In one embodiment, the nucleic acid molecule of the increased herbicide tolerance or resistance , as compared to present invention comprises a nucleic acid that encodes a a corresponding , e . g . non -transformed , wild type plant cell, 5 portion of the protein of the present invention . The protein plant or part thereof fits activity is increased by for example is at least about 30 % , 35 % , 40 % , 45 % or 50 % , preferably at expression either in the cytosol or in an organelle such as a plastid or mitochondria or both , preferably in plastids. The least about 55 % , 60 % , 65 % or 70 % , and more preferably at nucleotide sequences determined from the cloning of the least about 75 % , 80 % , 85 % , 90 % , 91 % , 92 % , 93 % or 94 % present protein - according -to -the - invention -encoding gene 10 and most preferably at least about 95 % , 97 % , 98 % , 99 % or allows for the generation of probes and primers designed for more homologous to an entire amino acid sequence SEQ ID use in identifying and/ or cloning its homologues in other cell NO : 2 or 4 , and having above- mentioned activity , e . g . types and organisms. The probe/ primer typically comprises conferring an increased herbicide tolerance or resistance, as substantially purified oligonucleotide. The oligonucleotide compared to a corresponding , e . g . non -transformed , wild typically comprises a region of nucleotide sequence that 15 type plant cell, plant or part thereof by for example expres hybridizes under stringent conditions to at least about 12 , 15 sion either in the cytosol or in an organelle such as a plastid preferably about 20 or 25 , more preferably about 40 , 50 or or mitochondria or both , preferably in plastids. 75 consecutive nucleotides of a sense strand of one of the Portions of proteins encoded by the nucleic acid molecule sequences set forth , e . g ., in SEQ ID NO : 1 or 3 , or a of the invention are preferably biologically active , prefer homolog thereof, an anti -sense sequence of one of the 20 ably having above -mentioned annotated activity , e .g . con sequences, e . g ., set forth in SEQ ID NO : 1 or 3 , or a ferring an increased herbicide tolerance or resistance , as homolog thereof, or naturally occurring mutants thereof. compared to a corresponding , e . g . non -transformed , wild Primers based on a nucleotide of invention can be used in type plant cell, plant or part thereof after increase of activity . PCR reactions to clone homologues of the polypeptide of the As mentioned herein , the term “ biologically active por invention or of the polypeptide used in the process of the 25 tion ” is intended to include a portion , e . g . , a domain /motif , invention , e . g . as the primers described in the examples of that confers an increased herbicide tolerance or resistance , the present invention , e . g . as shown in the examples . A PCR e .g . an increased herbicide tolerance or resistance -related with primers based on SEQ ID NO : 1 or 3 will result in a trait , as compared to a corresponding , e . g . non - transformed , fragment of the gene product as shown SEQ ID NO : 2 or 4 , wild type plant cell , plant or part thereof or has an immu or a homolog thereof. 30 nological activity such that it is binds to an antibody binding Primer sets are interchangeable . The person skilled in the specifically to the polypeptide of the present invention or a art knows to combine said primers to result in the desired polypeptide used in the process of the present invention for product, e . g . in a full length clone or a partial sequence . increasing herbicide tolerance or resistance , as compared to Probes based on the sequences of the nucleic acid molecule a corresponding, e . g . non - transformed , wild type plant cell , of the invention or used in the process of the present 35 plant or part thereof. invention can be used to detect transcripts or genomic The invention further relates to nucleic acid molecules sequences encoding the same or homologous proteins. The that differ from one of the nucleotide sequences shown in probe can further comprise a label group attached thereto , SEQ ID NO : 1 or 3 , or a homolog thereof ( and portions e . g . the label group can be a radioisotope , a fluorescent thereof ) due to degeneracy of the genetic code and thus compound , an enzyme, or an enzyme co - factor. Such probes 40 encode a polypeptide of the present invention , in particular can be used as a part of a genomic marker test kit for a polypeptide having above mentioned activity , e . g . as that identifying cells which express an polypeptide of the inven - polypeptides depicted by the sequence shown in SEQ ID tion or used in the process of the present invention , such as NO : 2 or 4 , or the functional homologues . Advantageously , by measuring a level of an encoding nucleic acid molecule the nucleic acid molecule of the invention comprises , or in in a sample of cells , e . g . , detecting mRNA levels or deter - 45 an other embodiment has , a nucleotide sequence encoding a mining , whether a genomic gene comprising the sequence of protein comprising , or in an other embodiment having , an the polynucleotide of the invention or used in the processes amino acid sequence shown in SEQ ID NO : 2 or 4 , or the of the present invention has been mutated or deleted . functional homologues . In a still further embodiment, the The nucleic acid molecule of the invention encodes a nucleic acid molecule of the invention encodes a full length polypeptide or portion thereof which includes an amino acid 50 protein which is substantially homologous to an amino acid sequence which is sufficiently homologous to the amino acid sequence shown in SEQ ID NO : 2 or 4 , or the functional sequence shown in SEQ ID NO : 2 or 4 , or a homolog thereof homologues . such that the protein or portion thereof maintains the ability In addition , it will be appreciated by those skilled in the to participate in increasing herbicide tolerance or resistance , art that DNA sequence polymorphisms that lead to changes as compared to a corresponding , e . g . non -transformed , wild 55 in the amino acid sequences may exist within a population . type plant cell , plant or part thereof, in particular increasing Such genetic polymorphism in the gene encoding the poly the activity as mentioned above or as described in the peptide of the invention or comprising the nucleic acid examples in plants is comprised . molecule of the invention may exist among individuals As used herein , the language " sufficiently homologous ” within a population due to natural variation . refers to proteins or portions thereof which have amino acid 60 Nucleic acid molecules corresponding to natural variants sequences which include a minimum number of identical or homologues of a nucleic acid molecule of the invention , equivalent amino acid residues (e .g ., an amino acid residue which can also be a cDNA , can be isolated based on their which has a similar side chain as an amino acid residue in homology to the nucleic acid molecules disclosed herein one of the sequences of the polypeptide of the present using the nucleic acid molecule of the invention , or a portion invention to an amino acid sequence shown in SEQ ID NO : 65 thereof, as a hybridization probe according to standard 2 or 4 , or a homolog thereof such that the protein or portion hybridization techniques under stringent hybridization con thereof is able to participate in increasing herbicide toler - ditions . US 10 , 392 ,630 B2 51 52 Accordingly, in another embodiment, a nucleic acid mol present invention to the usage of the organism or the cell ecule of the invention is at least 15 , 20 , 25 or 30 nucleotides compartment for example of the plastid or mitochondria in in length . Preferably , it hybridizes under stringent conditions which the polynucleotide or polypeptide is expressed . In a to a nucleic acid molecule comprising a nucleotide sequence particular preferred embodiment, codon - adapted nucleic of the nucleic acid molecule of the present invention or used 5 acid molecules of the present invention comprise the in the process of the present invention , e . g . comprising the sequence of SEQ ID NO : 6 , or 7 , which represent codon sequence shown in SEQ ID NO : 1 or 3 , or a homolog adapted nucleic acid molecules corresponding to SEQ ID thereof. The nucleic acid molecule is preferably at least 20 , NO : 1 , or 3 . 30 , 50 , 100 , 250 or more nucleotides in length . Accordingly , the invention relates to nucleic acid mol The term " hybridizes under stringent conditions” is 10 ecules encoding a polypeptide having above -mentioned defined above . In one embodiment, the term “ hybridizes activity , in an organism or parts thereof by for example under stringent conditions” is intended to describe condi expression either in the cytosol or in an organelle such as a tions for hybridization and washing under which nucleotide plastid or mitochondria or both , preferably in plastids that sequences at least 30 % , 40 % , 50 % or 65 % identical to each contain changes in amino acid residues that are not essential other typically remain hybridized to each other . Preferably, 15 for said activity . Such polypeptides differ in amino acid the conditions are such that sequences at least about 70 % , sequence from a sequence contained in the sequences shown more preferably at least about 75 % or 80 % , and even more in SEQ ID NO : 2 or 4 , or a homolog thereof yet retain said preferably at least about 85 % , 90 % or 95 % or more identical activity described herein . The nucleic acid molecule can to each other typically remain hybridized to each other . comprise a nucleotide sequence encoding a polypeptide , Preferably , nucleic acid molecule of the invention that 20 wherein the polypeptide comprises an amino acid sequence hybridizes under stringent conditions to a sequence shown in at least about 50 % identical to an amino acid sequence SEQ ID NO : 1 or 3 , or a homolog thereof corresponds to a shown SEQ ID NO : 2 or 4 , or a homolog thereof and is naturally occurring nucleic acid molecule of the invention . capable of participation in increasing herbicide tolerance or As used herein , a “ naturally - occurring ” nucleic acid mol- resistance , as compared to a corresponding , e . g . non - trans ecule refers to an RNA or DNA molecule having a nucleo - 25 formed , wild type plant cell, plant or part thereof after tide sequence that occurs in nature ( e . g ., encodes a natural increasing its activity , e . g . its expression by for example protein ) . Preferably , the nucleic acid molecule encodes a expression either in the cytosol or in an organelle such as a natural protein having above -mentioned activity, e . g . con - plastid or mitochondria or both , preferably in plastids . ferring increasing herbicide tolerance or resistance , after Preferably , the protein encoded by the nucleic acid molecule increasing the expression or activity thereof or the activity of 30 is at least about 60 % identical to the sequence shown in SEQ a protein of the invention or used in the process of the ID NO : 2 or 4 , or a homolog thereof, more preferably at least invention by for example expression the nucleic acid about 70 % identical to one of the sequences shown in SEQ sequence of the gene product in the cytosol and / or in an ID NO : 2 or 4 , or a homolog thereof, even more preferably organelle such as a plastid or mitochondria , preferably in at least about 80 % , 90 % , 95 % homologous to the sequence plastids. 35 shown in SEQ ID NO : 2 or 4 , or a homolog thereof, and In addition to naturally -occurring variants of the most preferably at least about 96 % , 97 % , 98 % , or 99 % sequences of the polypeptide or nucleic acid molecule of the identical to the sequence shown in SEQ ID NO : 2 or 4 , or invention as well as of the polypeptide or nucleic acid a homolog thereof. molecule used in the process of the invention thatmay exist To determine the percentage homology ( = identity , herein in the population , the skilled artisan will further appreciate 40 used interchangeably ) of two amino acid sequences or of that changes can be introduced by mutation into a nucleotide two nucleic acid molecules, the sequences are written one sequence of the nucleic acid molecule encoding the poly - underneath the other for an optimal comparison ( for peptide of the invention or used in the process of the present example gapsmay be inserted into the sequence of a protein invention , thereby leading to changes in the amino acid or of a nucleic acid in order to generate an optimal alignment sequence of the encoded said polypeptide , without altering 45 with the other protein or the other nucleic acid ) . the functional ability of the polypeptide , preferably not The amino acid residues or nucleic acid molecules at the decreasing said activity . corresponding amino acid positions or nucleotide positions For example , nucleotide substitutions leading to amino are then compared . If a position in one sequence is occupied acid substitutions at “ non - essential ” amino acid residues can by the same amino acid residue or the same nucleic acid be made in a sequence of the nucleic acid molecule of the 50 molecule as the corresponding position in the other invention or used in the process of the invention , e . g . shown sequence , the molecules are homologous at this position ( i . e . in SEQ ID NO : 1 or 3 , or a homolog thereof. amino acid or nucleic acid “ homology ” as used in the A “ non - essential” amino acid residue is a residue that can present context corresponds to amino acid or nucleic acid be altered from the wild - type sequence of one without “ identity ” . The percentage homology between the two altering the activity of said polypeptide , whereas an “ essen - 55 sequences is a function of the number of identical positions tial” amino acid residue is required for an activity as shared by the sequences ( i . e . % homology = number of iden mentioned above , e .g . leading to increasing herbicide toler - tical positions/ total number of positionsx100 ) . The terms ance or resistance , as compared to a corresponding , e . g . “ homology ” and “ identity ” are thus to be considered as non - transformed , wild type plant cell, plant or part thereof in synonyms. an organism after an increase of activity of the polypeptide . 60 For the determination of the percentage homology ( = iden Other amino acid residues , however, ( e . g ., those that are not tity ) of two or more amino acids or of two or more conserved or only semi- conserved in the domain having said nucleotide sequences several computer software programs activity ) may not be essential for activity and thus are likely have been developed . The homology of two or more to be amenable to alteration without altering said activity . sequences can be calculated with for example the software Further, a person skilled in the art knows that the codon 65 fasta , which presently has been used in the version fasta 3 usage between organisms can differ. Therefore , he may ( W . R . Pearson and D . J . Lipman , PNAS 85 , 2444 ( 1988 ) ; W . adapt the codon usage in the nucleic acid molecule of the R . Pearson , Methods in Enzymology 183 , 63 ( 1990 ); W . R . US 10 , 392 ,630 B2 53 54 Pearson and D . J . Lipman , PNAS 85 , 2444 ( 1988 ) ; W . R . rithm for blastx ) [ Integer ); default = 0 ; -t Length of the largest Pearson , Enzymology 183 , 63 (1990 )) . Another useful pro intron allowed in tblastn for linking HSPs (o disables gram for the calculation of homologies of different linking ) [ Integer ); default = 0 . sequences is the standard BLAST® program , which is Results of high quality are reached by using the algorithm included in the Biomax pedant software (Biomax , Munich , 5 of Needleman and Wunsch or Smith and Waterman . There Federal Republic of Germany ) . This leads unfortunately fore programs based on said algorithms are preferred . sometimes to suboptimal results since BLAST® does not Advantageously the comparisons of sequences can be done always include complete sequences of the subject and the with the program PileUp ( J. Mol. Evolution ., 25, 351 (1987 ), querry . Nevertheless as this program is very efficient it can Higgins et al ., CABIOS 5 , 151 ( 1989 )) or preferably with the be used for the comparison of a huge number of sequences. " programs “ Gap ” and “ Needle ” , which are both based on the The following settings are typically used for such a com - algorithms ofNeedleman and Wunsch (J . Mol. Biol. 48 ; 443 parisons of sequences: - p Program Name [ String ] ; - d Data (1970 ) ) , and “ BestFit” , which is based on the algorithm of base [ String ] ; default = nr ; - i Query File [ File In ] ; Smith and Waterman ( Adv. Appl. Math . 2 ; 482 ( 1981 )) . default = stdin ; - e Expectation value ( E ) [Real ] ; default = 10 . 0 ; 15 . “ Gap ” and “ BestFit” are part of the GCG software- package - m alignment view options : ( = pairwise ; 1 = query -anchored (Genetics Computer Group , 575 Science Drive , Madison , showing identities ; 2 = query -anchored no identities; 3 = flat Wis . ,USA 53711 ( 1991) ; Altschul et al. , (Nucleic Acids Res . query - anchored , show identities ; 4 = flat query -anchored , no 25 , 3389 ( 1997) ) , “ Needle ” is part of the The European identities ; 5 = query - anchored no identities and blunt ends; Molecular Biology Open Software Suite (EMBOSS ) 6 = flat query - anchored , no identities and blunt ends ; 7 = XML 20 ( Trends in Genetics 16 (6 ), 276 (2000 )) . Therefore prefer Blast output; 8 = tabular ; 9 tabular with comment lines [ Inte - ably the calculations to determine the percentages of ger ); default = 0 ; - 0 BLAST® report Output File [ File Out ] sequence homology are done with the programs “Gap ” or Optional; default = stdout ; - F Filter query sequence ( DUST “ Needle ” over the whole range of the sequences. The with blastn , SEG with others ) [ String ) ; default = T ; - G Cost to following standard adjustments for the comparison of open a gap (zero invokes default behavior ) Integer ) ; 25 nucleic acid sequences were used for “ Needle ” : matrix : default = 0 ; - E Cost to extend a gap ( zero invokes default EDNAFULL , Gap _ penalty : 10 . 0 , Extend _ penalty : 0 . 5 . The behavior) [ Integer ) ; default = 0 ; - X X dropoff value for following standard adjustments for the comparison of gapped alignment ( in bits ) ( zero invokes default behavior) ; nucleic acid sequences were used for “ Gap ” : gap weight: 50 , blastn 30 , megablast 20 , tblastx 0 , all others 15 [ Integer ) ; length weight: 3 , average match : 10 . 000 , average mismatch : default = 0 ; - I Show GI' s in deflines [ TIF ] ; default = F ; - 9 30 0 .000 . Penalty for a nucleotide mismatch (blastn only ) [ Integer ]; For example a sequence , which has 80 % homology with default = - 3 ; - r Reward for a nucleotide match (blastn only sequence SEQ ID NO : 1 at the nucleic acid level is under [ Integer ) ; default = 1 ; -v Number of database sequences to stood as meaning a sequence which , upon comparison with show one- line descriptions for ( V ) [ Integer ) ; default = 500 ; - b the sequence SEQ ID NO : 1 by the above program “ Needle ” Number of database sequence to show alignments for ( B ) 35 with the above parameter set, has a 80 % homology . [ Integer) ; default = 250 ; - f Threshold for extending hits , Homology between two polypeptides is understood as default if zero ; blastp 11, blastn 0 , blastx 12 , tblastn 13 ; meaning the identity of the amino acid sequence over in each tblastx 13 , megablast 0 [ Integer) ; default = 0 ; - g Perfom case the entire sequence length which is calculated by gapped alignment (not available with tblastx ) [ T / F ] ; comparison with the aid of the above program “ Needle ” default = T ; - Q Query Genetic code to use [ Integer ); 40 using Matrix : EBLOSUM62, Gap _ penalty : 8 .0 , Extend _ default = 1; - D DB Genetic code ( for tblast[ nx ] only ) [ Inte penalty : 2 .0 . ger ) ; default = 1 ; - a Number of processors to use [ Integer ] ; For example a sequence which has a 80 % homology with default = 1 ; - O SeqAlign file [File Out] Optional; - J Believe sequence SEQ ID NO : 2 at the protein level is understood as the query defline [ T / F ]; default = F ; - M Matrix [String ]; meaning a sequence which , upon comparison with the default = BLOSUM62 ; - W Word size , default if zero (blastn 45 sequence SEQ ID NO : 2 by the above program " Needle " 11, megablast 28 , all others 3 ) [ Integer ); default = 0 ; - Z with the above parameter set , has a 80 % homology. Effective length of the database (use zero for the real size ) Functional equivalents derived from the nucleic acid [Real ] ; default = 0 ; - K Number of best hits from a region to sequence as shown in SEQ ID NO : 1 or 3 , or a homolog keep (off by default , if used a value of 100 is recommended ) thereof according to the invention by substitution , insertion [ Integer ) ; default = 0 ; - P O for multiple hit, 1 for single hit 50 or deletion have at least 30 % , 35 % , 40 % , 45 % or 50 % , [ Integer) ; default = 0 ; - Y Effective length of the search space preferably at least 55 % , 60 % , 65 % or 70 % by preference at ( use zero for the real size ) [Real ]; default = 0 ; - S Query least 80 % , especially preferably at least 85 % or 90 % , 91 % , strands to search against database ( for blast [nx ], and 92 % , 93 % or 94 % , very especially preferably at least 95 % , tblastx ) ; 3 is both , 1 is top , 2 is bottom [ Integer ); default = 3 ; 97 % , 98 % or 99 % homology with one of the polypeptides - T Produce HTML output [ T / F ] ; default = F ; - I Restrict 55 as shown in SEQ ID NO : 2 or 4 , or a homolog thereof search of database to list of GI' s [String ] Optional ; - U Use according to the invention and encode polypeptides having lower case filtering of FASTA sequence [ T / F ] Optional; essentially the same properties as the polypeptide as shown default = F ; -y X dropoff value for ungapped extensions in in SEQ ID NO : 2 or 4 , or a homolog thereof. bits ( 0 .0 invokes default behavior ) ; blastn 20 ,megablast 10 , Functional equivalents derived from one of the polypep all others 7 [Real ] ; default = 0 . 0 ; - Z X dropoff value for final 60 tides as shown in SEQ ID NO : 2 or 4 , or a homolog thereof gapped alignment in bits ( 0 . 0 invokes default behavior ) ; according to the invention by substitution , insertion or blastn /megablast 50 , tblastx 0 , all others 25 [ Integer ) ; deletion have at least 30 % , 35 % , 40 % , 45 % or 50 % , default = 0 ; - R PSI- TBLASTN checkpoint file [ File In ] preferably at least 55 % , 60 % , 65 % or 70 % by preference at Optional; - n MegaBlast search [ T / F ] ; default= F ; - L Location least 80 % , especially preferably at least 85 % or 90 % , 91 % , on query sequence [ String ] Optional ; - A Multiple Hits 65 92 % , 93 % or 94 % , very especially preferably at least 95 % , window size , default if zero (blastn /megablast 0 , all others 97 % , 98 % or 99 % homology with one of the polypeptides 40 [ Integer ); default = 0 ; - w Frame shift penalty (OOF algo - as shown in SEQ ID NO : 2 or 4 , or a homolog thereof US 10 , 392 ,630 B2 55 56 according to the invention and having essentially the same type PPO enzymes with SEQ ID NO : 2 , or 4 . Preferred properties as the polypeptide as shown in SEQ ID NO : 2 or substitutions of such a mutated PPO are those that increase 4 , or a homolog thereof . the herbicide tolerance of the plant, but leave the biological “ Essentially the same properties” of a functional equiva activity of the oxidase activity substantially unaffected . lent is above all understood as meaning that the functional 5 The term “ mutated PPO nucleic acid ” refers to a PPO equivalent has above mentioned activity , by for example nucleic acid having a sequence that is mutated from a expression either in the cytosol or in an organelle such as a wild - type PPO nucleic acid of SEQ ID NO : 1 or 3 , and that plastid or mitochondria or both , preferably in plastids while increasing the amount of protein , activity or function of said confers increased PPO -inhibiting herbicide tolerance to a functional equivalent in an organism , e . g . a microorganism , 10 plant in which it is expressed . Furthermore , the term a plant or plant tissue or animal tissue , plant or animal cells “ mutated protoporphyrinogen oxidase (mutated PPO )” or a part of the same. refers to the replacement of an amino acid of the wild - type A nucleic acid molecule encoding an homologous to a primary sequences SEQ ID NO : 2 , or 4 , or a variant, a protein sequence of SEQ ID NO : 2 or 4 , or a homolog derivative , a homologue , an orthologue , or paralogue thereof can be created by introducing one or more nucleotide 15 thereof, with another amino acid . The expression “ mutated substitutions, additions or deletions into a nucleotide amino acid ” will be used below to designate the amino acid sequence of the nucleic acid molecule of the present inven which is replaced by another amino acid , thereby designat tion , in particular of SEQ ID NO : 1 or 3 , or a homolog ing the site of the mutation in the primary sequence of the thereof such that one or more amino acid substitutions , protein . additions or deletions are introduced into the encoded pro - 20 Accordingly , in another object of the present invention the tein . Mutations can be introduced into the encoding key amino acid residues of a PPO enzyme, a variant, sequences of SEQ ID NO : 1 or 3 , or a homolog thereof by derivative , orthologue, paralogue or homologue thereof, is standard techniques, such as site -directed mutagenesis and substituted by any other amino acid . PCR -mediated mutagenesis . In one embodiment, the key amino acid residues of a PPO Preferably, conservative amino acid substitutions are 25 enzyme, a variant, derivative , orthologue, paralogue or made at one or more predicted non - essential amino acid homologue thereof, is substituted by a conserved amino acid residues. A “ conservative amino acid substitution ” is one in as depicted in Table 2 . which the amino acid residue is replaced with an amino acid residue having a similar side chain . Families of amino acid TABLE 2 residues having similar side chains have been defined in the 30 art. These families include amino acids with basic side Examples of conserved amino acid substitutions chains ( e . g . , lysine, arginine, histidine ) , acidic side chains Conservative Conservative ( e . g . , aspartic acid , glutamic acid ), uncharged polar side Residue Substitutions Residue Substitutions chains ( e . g . , glycine , asparagine , glutamine, serine , threo Ala Ser Leu Ile ; Val nine , tyrosine , cysteine ), nonpolar side chains ( e .g . , alanine , 35 Arg Lys Lys Arg ; Gln valine, leucine , isoleucine, proline, phenylalanine , methio Asn Gln ; His Met Leu ; Ile Asp Glu Phe Met ; Leu ; Tyr nine , tryptophane ), beta -branched side chains ( e . g . , threo Gln Asn Ser Thr; Gly nine, valine , isoleucine ) and aromatic side chains ( e . g . , Cys Ser Thr Ser; Val tyrosine, phenylalanine , tryptophane , histidine ) . Glu Asp Trp Tyr Thus , a predicted nonessential amino acid residue in a 40 Gly Pro Tyr Trp ; Phe polypeptide of the invention or a polypeptide used in the His Asn ; Gin Val Ile ; Leu process of the invention is preferably replaced with another Ile Leu , Val amino acid residue from the same family . Alternatively, in another embodiment, mutations can be introduced randomly It will be understood by the person skilled in the art that along all or part of a coding sequence of a nucleic acid 45 amino acids located in a close proximity to the positions of molecule of the invention or used in the process of the amino acids mentioned below may also be substituted . Thus , invention , such as by saturation mutagenesis , and the resul- in another embodiment the variant of SEQ ID NO : 2 , or 4 , tant mutants can be screened for activity described herein to a variant , derivative , orthologue , paralogue or homologue identify mutants that retain or even have increased above thereof comprises a mutated PPO , wherein an amino mentioned activity , e . g . conferring increased herbicide tol- 50 acid : 3 , + 2 or 1 amino acid positions from a key amino acid erance or resistance , as compared to a corresponding , e . g . is substituted by any other amino acid . non - transformed , wild type plant cell , plant or part thereof. Based on techniques well -known in the art , a highly The inventors of the present invention have surprisingly characteristic sequence pattern can be developed , by means found that by expressing in a plant a nucleic acid comprising ofwhich further of mutated PPO candidates with the desired a nucleotide sequence which comprises the sequence of SEQ 55 activity may be searched . ID NO : 1, or 3 , or a variant or derivative thereof, the Searching for further mutated PPO candidates by apply tolerance or resistance of said plant to particular PPO ing a suitable sequence pattern would also be encompassed inhibiting herbicides could be remarkably increased as com - by the present invention . It will be understood by a skilled pared to a control or wildtype plant, that lacks the herbicide - reader that the present sequence pattern is not limited by the resistance characteristics and / or particular PPO polynucle - 60 exact distances between two adjacent amino acid residues of otide of the invention that are disclosed herein . said pattern . Each of the distances between two neighbours Furthermore , the inventors of the present invention have in the above patterns may, for example , vary independently found that by substituting one or more of the key amino acid of each other by up to 110 , 15 , 13 , 12 or + 1 amino acid residues , employing e . g . one of the above described methods positions without substantially affecting the desired activity . to mutate the encoding nucleic acids , the tolerance or 65 Furthermore , by applying the method of site directed resistance to particular PPO - inhibiting herbicides could be mutagenesis , in particular saturation mutagenes ( see e . g . increased even more as compared to the activity of the wild Schenk et al. , Biospektrum 03 / 2006 , pages 277 - 279 ), the US 10 , 392 ,630 B2 57 58 inventors of the present invention have identified and gen - In another preferred embodiment, the mutated PPO com erated specific amino acid substitutions and combinations prises a sequence of SEQ ID NO : 2 a variant, derivative , thereof, which — when introduced into a plant by transform - orthologue , paralogue or homologue thereof , in which : ing and expressing the respective mutated PPO encoding the amino acid at or corresponding to position 137 is Ala , nucleic acid confer increased herbicide resistance or tol- 5 and the amino acid at or corresponding to position 438 is erance to a PPO inhibiting herbicide to said plant. Met. It is to be understood that any amino acid besides the ones In another preferred embodiment, the mutated PPO com mentioned hereinafter could be used as a substitutent. Assays to test for the functionality of such mutants are prises a sequence of SEQ ID NO : 2 a variant, derivative , readily available in the art, and respectively , described in the 10 orthologue , paralogue or homologue thereof, in which : Example section of the present invention . the amino acid at or corresponding to position 137 is Ala , In a preferred embodiment, the mutated PPO refers to a and the amino acid at or corresponding to position 438 is polypeptide comprising the sequence of SEQ ID NO : 2 or 4 , His . wherein the amino acid sequence differs from an amino acid In another preferred embodiment, the mutated PPO com sequence of SEO ID NO : 2 or 4 at or corresponding to 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , position 137 , 415 , and /or position 438 of SEO ID NO : 2 . orthologue , paralogue or homologue thereof, in which : Examples of differences at these amino acid positions the amino acid at or corresponding to position 137 is Ala , include , but are not limited to , one or more of the following : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is other Lys . than Arginine ; 20 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is other prises a sequence of SEQ ID NO : 2 a variant, derivative , than Leucine orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 438 is other the amino acid at or corresponding to position 137 is Ala , than Phenylalanine. and the amino acid at or corresponding to position 438 is In some embodiments , the mutated PPO enzyme of SEQ 25 Asp . ID NO : 2 comprises one or more of the following : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Leu , prises a sequence of SEO ID NO : 2 a variant, derivative , Ala , Val, Ile , Met , Tyr, Gly , Asn , Cys , Phe , Ser, Thr, Gln , or orthologue , paralogue or homologue thereof, in which : His ; the amino acid at or corresponding to position 415 is Ala , 30 the amino acid at or corresponding to position 137 is Ala , Arg , Val, Ile , Met, His , Lys , Asp , Glu , Ser , Thr, Asn, Gln , and the amino acid at or corresponding to position 438 is Cys, Gly , Pro , Phe, Tyr , or Trp Glu . the amino acid at or corresponding to position 438 is Ala , In another preferred embodiment, the mutated PPO com Leu , Val , Ile , Met, His , Lys, Asp , Glu , Ser, Thr, Asn , Gln , prises a sequence of SEQ ID NO : 2 a variant, derivative , Cys, Gly , Pro , Arg , Tyr, or Trp . 35 orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Ala , prises a sequence of SEQ ID NO : 2 a variant, derivative, and the amino acid at or corresponding to position 438 is Ser . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Ala , prises a sequence of SEQ ID NO : 2 a variant, derivative , Leu , Val , Ile , Met , His , Lys , Asp , Glu , Ser , Thr, Asn , Gln , 40 orthologue , paralogue or homologue thereof , in which : Cys, Gly , Pro , Phe, Tyr, or Trp , and the amino acid at or the amino acid at or corresponding to position 137 is Ala , corresponding to position 438 is Ala , Leu , Val , Ile , Met, His , and the amino acid at or corresponding to position 438 is Lys, Asp , Glu , Ser , Thr, Asn , Gin , Cys , Gly , Pro , Arg , Tyr, Thr. or Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Ala , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asn . Ala . 50 In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Ala , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 55 Gln . Leu . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Ala , 60 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. Val. In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 65 the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Ala , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . US 10 , 392 ,630 B2 59 60 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com - 20 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Ala , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Trp . 25 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 3 Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 35 the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gin . Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, 40 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . In another preferred embodiment, the mutated PPO com - 50 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met. 55 Pro . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is 60 and the amino acid at or corresponding to position 438 is His . Arg . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , 65 the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys. Tyr . US 10 , 392 ,630 B2 62 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 137 is Leu , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is 5 prises a sequence of SEO ID NO : 2 a variant, derivative , Trp . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 137 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val , 10 Asn . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Val , orthologue, paralogue or homologue thereof, in which : 1515 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 the amino acid at or corresponding to position 137 is Val, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Val, Cys . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 25 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Val, orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Val, Gly . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 30 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Met . 35 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereofof, in which :: the amino acid at or corresponding to position 137 is Val , the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 40 Arg . His . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Val, 45 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Trp . Asp . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 55 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof , in which the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 438 is Ala . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Glu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 60 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 438 is Leu . the amino acid at or corresponding to position 137 is Val, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Ser. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 65 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 438 is Val. US 10 , 392 ,630 B2 63 64 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 438 is Ile . 5 the amino acid at or corresponding to position 438 is Arg . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 438 is Met . the amino acid at or corresponding to position 438 is Tyr . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 438 is His . the amino acid at or corresponding to position 438 is Trp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ile , and 20 the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 438 is Lys . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Ile , and 25 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 438 is Asp . the amino acid at or corresponding to position 137 is Met , In another preferred embodiment, the mutated PPO com - and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Leu . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Ile , and 3 prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 438 is Glu . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 137 is Met , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : 35 Val . the amino acid at or corresponding to position 137 is Ile , and In another preferred embodiment, themutated PPO com the amino acid at or corresponding to position 438 is Ser. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Met , orthologue , paralogue or homologue thereof, in which : 40 and the amino acid at or corresponding to position 438 is Ile . the amino acid at or corresponding to position 137 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 438 is Thr. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Met , orthologue , paralogue or homologue thereof, in which : 45 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Ile , and Met. the amino acid at or corresponding to position 438 is Asn . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 137 is Ile , and and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 438 is Gln . His . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 438 is Cys . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Lys . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : 60 prises a sequence of SEQ ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 438 is Gly . the amino acid at or corresponding to position 137 is Met, In another preferred embodiment, the mutated PPO com - and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant , derivative , Asp . orthologue, paralogue or homologue thereof, in which : 65 In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 137 is Ile , and prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 438 is Pro . orthologue, paralogue or homologue thereof , in which : US 10 , 392 ,630 B2 65 66 the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . Ala . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 438 is Ser. and the amino acid at or corresponding to position 438 is Leu . In another preferred embodiment, the mutated PPO com 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. 15 Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEO ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 438 is 20 and the amino acid at or corresponding to position 438 is Ile. Asn . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Met, 25 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met. Gln . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 30 the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Met, His . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Cys . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, the mutated PPO com 35 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is His . orthologue , paralogue or homologue thereof, in which : and the amino acidacid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Met , Lys and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Gly . 40 prises a sequence of SEQ ID NO : 2 a variant , derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is His , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Met, Asp . and the amino acid at or corresponding to position 438 is 45 In another preferred embodiment , the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is His , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Met , 50 Glu . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is His , orthologue , paralogue or homologue thereof, in which : 55 and the amino acid at or corresponding to position 438 is Ser. the amino acid at or corresponding to position 137 is Met , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Tyr. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is His, prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 137 is Met, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Trp . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - 65 the amino acid at or corresponding to position 137 is His , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Asn . US 10 , 392 ,630 B2 68 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Gln . Met. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. His . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 16 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gly. Lys . In another preferred embodiment, the mutated PPO com - 20 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . 25 Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys, and the amino acid at or corresponding to position 438 is 3 and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 35 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser . Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, 40 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is Lys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asn . Ala . 50 In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is Lys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 55 Gln . Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys, the amino acid at or corresponding to position 137 is Lys, 60 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. Val. In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 65 the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . US 10 , 392 ,630 B2 69 70 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys, the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys, the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com - 20 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Trp . 25 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 3 Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 36 the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, 40 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . In another preferred embodiment, the mutated PPO com - 50 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met. 55 Pro . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is 60 and the amino acid at or corresponding to position 438 is His . Arg. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , 65 the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys. Tyr . US 10 , 392 ,630 B2 72 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Glu , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 137 is Asp , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is 5 prises a sequence of SEO ID NO : 2 a variant, derivative , Trp . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 137 is Glu , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glu , 10 Asn . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Glu , orthologue, paralogue or homologue thereof, in which : 15 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 438 is . In another preferred embodiment , the mutated PPO com Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 the amino acid at or corresponding to position 137 is Glu , orthologue, paralogue or homologue thereof , in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Glu , Cys. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 25 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 137 is Glu , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Glu , Gly . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 30 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Met . 35 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 40 Arg . His . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , 45 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Trp . Asp . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 55 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ala . Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Leu . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 73 74 the amino acid at or corresponding to position 137 is Ser , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Val. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is Ser, prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Gly . the amino acid at or corresponding to position 137 is Ser, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 10 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Ser , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Met. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser , the amino acid at or corresponding to position 137 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg. His . 20 In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 25 Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser , the amino acid at or corresponding to position 137 is Ser, 30 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , Orthologue. - paralogue or homologue thereof in which : 35 orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . Ala . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 438 is Ser . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , 45 In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ser, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Thr, Thr. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - 50 Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ser, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Thr, Asn . 55 and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 438 is 60 and the amino acid at or corresponding to position 438 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser , 65 the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. His . US 10 , 392 ,630 B2 75 76 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof , in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Lys. Tyr. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . Ala . In another preferred embodiment, the mutated PPO com - 20 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is Ser. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - 25 Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant , derivative, the amino acid at or corresponding to position 137 is Thr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Asn , Thr. U and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Val. prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Thr, as orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Asn , Asn . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 40 orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gin . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SER ID NO : 2 a variant , derivative , 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . His . In another preferred embodiment, the mutated PPO com - 50 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gly . 55 Lys . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is 60 and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, 65 the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . US 10 , 392 ,630 B2 77 78 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Gin , and the amino acid at or corresponding to position 438 is Ser. 5 and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Leu . In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , 10 the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Val . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 15 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid aat or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Asn . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Gln , the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met. Gln . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 25 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gin , the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is His . Cys . 30 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Gln , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Asn1 , 35 Lys . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Gly . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 137 is Gin , orthologue , paralogue or homologue thereof, in which : 40 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Asn , Asp . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , 45 the amino acid at or corresponding to position 137 is Gln , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Asn , Glu . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 50 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Gln , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ser . the amino acid at or corresponding to position 137 is Asn , In another preferred embodiment , the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Tyr. 55 orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Gin , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 137 is Asn , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is 60 prises a sequence of SEQ ID NO : 2 a variant , derivative , Trp . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is Gin , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 137 is Gln , 65 In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Ala . orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 79 80 the amino acid at or corresponding to position 137 is Gln , the amino acid at or corresponding to position 137 is Cys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gin , the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . His . In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gln , the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 438 is 15 and the amino acid at or corresponding to position 438 is Gly . Lys. In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gln , 20 the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 25 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gin , the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 30 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gln , the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Cys, the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 40 Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Cys, 45 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 55 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys, the amino acid at or corresponding to position 137 is Cys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Cys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 82 the amino acid at or corresponding to position 137 is Cys, the amino acid at or corresponding to position 137 is Gly, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys, the amino acid at or corresponding to position 137 is Gly, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 438 is 15 and the amino acid at or corresponding to position 438 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Cys, 20 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 25 the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , su orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Leu . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 40 Cys. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , 45 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gly , 50 the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met. Pro . In another preferred embodiment , the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is His . Arg . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SER ID NO : 2 a variant , derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys . Tyr. In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 83 84 the amino acid at or corresponding to position 137 is Gly , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Trp . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is Pro , prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 137 is Pro , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 10 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Pro , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Pro , Gln . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Leu . 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Pro , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Pro , Cys . and the amino acid at or corresponding to position 438 is 20 In another preferred embodiment, the mutated PPO com Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Pro , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Pro , 25 Gly . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , 30 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Met. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 35 the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg. His. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, 40 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Tyr. Lys . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Trp . Asp . 50 In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 55 Ala . Glu . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Pro , 60 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Leu . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , 65 the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Val . US 10 , 392 ,630 B2 85 86 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Phe , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Gly . the amino acid at or corresponding to position 137 is Phe , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Ile . 5 prises a sequence of SEO ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof , in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Phe , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 438 is 10 Pro . Met . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to nositionposition 137137 isis Phephe , 1515 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . His . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEO ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 20 the amino acid at or corresponding to position 137 is Phe, the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Tyr. Lys. In another preferred embodiment, themutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 25 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Phe, the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com - 30 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Tyr , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . 35 Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEO ID NO : 2 a variant. derivative . prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Tyr, and the amino acid ator corresponding to position 438 is Ser . 40 and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, themutated PPO com orthologue, paralogue or homologue thereof , in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Phe , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is 45 the amino acid at or corresponding to position 137 is Tyr, Thr. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Phe , 50 orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Tyr, Asn . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Tyr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Tyr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. His . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 87 88 the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Tyr , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys. Tyr . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Tyr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO comm 10 In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr , the amino acid at or corresponding to position 137 is Trp , and the amino acid at or corresponding to position 438 is 15 and the amino acid at or corresponding to position 438 is Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Trp , the amino acid at or corresponding to position 137 is Tyr, 20 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, 25 the amino acid at or corresponding to position 137 is Trp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Val. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 30 orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : 3 the amino acid at or corresponding to position 137 is Trp , the amino acid at or corresponding to position 137 is Tyr, and the amino acid at or corresponding to position 438 is Ile . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Asn . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com 35 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Tyr, Met and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Gln . 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Trp , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Tyr, His . and the amino acid at or corresponding to position 438 is 45 In another preferred embodiment , the mutated PPO com Cys. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof , in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Tyr, 50 Lys . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Gly . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : 55 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Tyr , Asp . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SER ID NO : 2 a variant , derivative , 60 the amino acid at or corresponding to position 137 is Trp , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Tyr , Glu . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 65 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ser . US 10 , 392 ,630 B2 89 90 In another preferred embodiment, themutated PPO com the amino acid at or corresponding to position 137 is Ala , prises a sequence of SEQ ID NO : 2 a variant, derivative, and the amino acid at or corresponding to position 415 is orthologue, paralogue or homologue thereof , in which : Ala . the amino acid at or corresponding to position 137 is Trp , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is 5 prises a sequence of SEQ ID NO : 2 a variant, derivative, Thr. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Ala , and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Arg . orthologue, paralogue or homologue thereof, in which : 10 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Trp , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 137 is Ala , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , 18 VAN orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Trp , prises a sequence of SEO ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Gln . the amino acid at or corresponding to position 137 is Ala , In another preferred embodiment, the mutated PPO com - 20 and the amino acid at or corresponding to position 415 is Ile . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Ala , Cys . 25 and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Met. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is 30 the amino acid at or corresponding to position 137 is Ala , Gly . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - His . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Trp , 35 orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Ala , Pro . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Lys . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Ala , Arg . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Asp . prises a sequence of SEQ ID NO : 2 a variant, derivative , 45 In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Ala , Tyr. and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - 50 Glu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Trp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Ala , Trp . 55 and the amino acid at or corresponding to position 415 is Ser. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Ala , Leu , Val, Ile , Met, His , Lys, Asp , Glu , Ser , Thr, Asn , Gln , 60 and the amino acid at or corresponding to position 415 is Cys , Gly , Pro , Phe , Tyr, or Trp , and the amino acid at or Thr. corresponding to position 415 is Ala , Arg , Val, Ile , Met ,His , In another preferred embodiment, themutated PPO com Lys, Asp , Glu , Ser , Thr, Asn , Gln , Cys , Gly , Pro , Phe , Tyr, prises a sequence of SEQ ID NO : 2 a variant, derivative , or Trp . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - 65 the amino acid at or corresponding to position 137 is Ala , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 415 is orthologue , paralogue or homologue thereof, in which : Asn . US 10 , 392 ,630 B2 91 92 In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is 5 and the amino acid at or corresponding to position 415 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys. His . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 16 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gly. Lys . In another preferred embodiment, the mutated PPO com - 20 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . 25 Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is 3 and the amino acid at or corresponding to position 415 is Phe . Glu . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ala , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, 40 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Ala , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asn . Ala . 50 In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 55 Gln . Arg . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , 60 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys . Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 65 the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ile . Gly . US 10 , 392 ,630 B2 93 94 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is 5 and the amino acid at or corresponding to position 415 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , " the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe . Glu . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 16 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Leu , the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 20 prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val , the amino acid at or corresponding to position 137 is Leu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. Trp . 25 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 30 Asn . Ala . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : as the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gln . Arg . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val , the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ile . Gly . In another preferred embodiment, the mutated PPO com - 50 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Met. 55 Pro . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val , the amino acid at or corresponding to position 137 is Val, and the amino acid at or corresponding to position 415 is 60 and the amino acid at or corresponding to position 415 is His . Phe . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val, 65 the amino acid at or corresponding to position 137 is Val , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Lys. Tyr . US 10 , 392 ,630 B2 95 96 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Val, the amino acid at or corresponding to position 137 is Ile , and and the amino acid at or corresponding to position 415 is 5 the amino acid at or corresponding to position 415 is Gln . Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue , paralogue or homologue thereof, in which : 10 the amino acid at or corresponding to position 415 is Cys. the amino acid at or corresponding to position 137 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue , paralogue or homologue thereof, in which : 15 the amino acid at or corresponding to position 415 is Gly . the amino acid at or corresponding to position 137 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Arg . prises a sequence of SEQ ID NO : 2 a variant , derivative, In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue, paralogue or homologue thereof, in which : 20 the amino acid at or corresponding to position 415 is Pro . the amino acid at or corresponding to position 137 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue , paralogue or homologue thereof, in which : 25 the amino acid at or corresponding to position 415 is Phe . the amino acid at or corresponding to position 137 is Ile , and in another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Ile . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEO ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue, paralogue or homologue thereof, in which : 30 the amino acid at or corresponding to position 415 is Tyr. the amino acid at or corresponding to position 137 is Ile , and In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 415 is Met. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile, and orthologue , paralogue or homologue thereof, in which : 35 the amino acid at or corresponding to position 415 is Trp . the amino acid at or corresponding to position 137 is Ile , and in another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is His . prises a sequence of SEQ ID NO : 2 a variant , derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Met , orthologue , paralogue or homologue thereof, in which : 40 and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Ile , and Ala . the amino acid at or corresponding to position 415 is Lys . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 45 the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 137 is Ile , and and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 415 is Asp . Arg . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 50 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ile , and the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 415 is Glu . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Val . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : 55 prises a sequence of SEQ ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ser. the amino acid at or corresponding to position 137 is Met, In another preferred embodiment, the mutated PPO com - and the amino acid at or corresponding to position 415 is Ile . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ile , and orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Thr. the amino acid at or corresponding to position 137 is Met, In another preferred embodiment, the mutated PPO com - and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Met. orthologue, paralogue or homologue thereof, in which : 65 In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 137 is Ile , and prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Asn . orthologue, paralogue or homologue thereof , in which : US 10 , 392 ,630 B2 97 98 the amino acid at or corresponding to position 137 is Met, the amino acid at or corresponding to position 137 is Met , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is His . Phe . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, the amino acid at or corresponding to position 137 is Met , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Lys. Tyr. In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, the amino acid at or corresponding to position 137 is Met, and the amino acid at or corresponding to position 415 is 15 and the amino acid at or corresponding to position 415 is Asp . Trp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, 20 the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Glu . Ala . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 25 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 415 is Ser . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : S prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Met, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is His , Thr. and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - 26 Val. prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, themutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Met, orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is His , Asn . 40 and the amino acid at or corresponding to position 415 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 415 is 45 and the amino acid at or corresponding to position 415 is Gln . Met. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met, 50 the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys . His . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gly. Lys . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Met , the amino acid at or corresponding to position 137 is His , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . Asp . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 99 100 the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Glu . Ala . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser. Arg . In another preferred embodiment, the mutated PPO com 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. 15 Val . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is His , the amino acid at or corresponding to position 137 is Lys , and the amino acid at or corresponding to position 415 is 20 and the amino acid at or corresponding to position 415 is Ile. Asn . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is His , 25 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Met . Gln . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , 30 the amino acid at or corresponding to position 137 is Lys , orthologue, paralogue or homologue thereof , in which : 3 and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is His , His . and the amino acid at or corresponding to position 415 is In another preferred embodiment , the mutated PPO com Cys . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com 35 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Lys , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is His , Lys. and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Gly . 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Lys , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is His , Asp . and the amino acid at or corresponding to position 415 is 45 In another preferred embodiment , the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 137 is Lys, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is His , 50 Glu . and the amino acid at or corresponding to position 415 is In another preferred embodiment , the mutated PPO com Phe . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Lys, orthologue , paralogue or homologue thereof, in which : 55 and the amino acid at or corresponding to position 415 is Ser. the amino acid at or corresponding to position 137 is His , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Tyr. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is Lys, prises a sequence of SER ID NO : 2 a variant , derivative , 60 and the amino acid at or corresponding to position 415 is orthologue, paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 137 is His , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Trp . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - 65 the amino acid at or corresponding to position 137 is Lys, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 415 is orthologue , paralogue or homologue thereof, in which : Asn . US 10 , 392 ,630 B2 101 102 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys, the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is 5 and the amino acid at or corresponding to position 415 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys, the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys. His . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 16 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gly. Lys . In another preferred embodiment, the mutated PPO com - 20 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . 25 Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is 3 and the amino acid at or corresponding to position 415 is Phe . Glu . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 28 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Lys, the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Lys, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asn . Ala . 50 In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 55 Gln . Arg . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , 60 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys . Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 65 the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ile . Gly . US 10 , 392 ,630 B2 103 104 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof , in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is 5 and the amino acid at or corresponding to position 415 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe . Glu . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 16 prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asp , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com - 20 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Asp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. Trp . 25 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof , in which : the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 30 Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : as the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gin . Arg . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys . Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ile . Gly . In another preferred embodiment, the mutated PPO com - 50 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Met. 55 Pro . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glu , the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is 60 and the amino acid at or corresponding to position 415 is His . Phe . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glu , 65 the amino acid at or corresponding to position 137 is Glu , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Lys . Tyr . US 10 , 392 ,630 B2 105 106 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Ser , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 415 is orthologue , paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 137 is Glu , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is 5 prises a sequence of SEO ID NO : 2 a variant, derivative , Trp . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 137 is Ser , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 415 is orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, 10 Asn . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ser, orthologue, paralogue or homologue thereof , in which : 15 and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Ser, Gln . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 the amino acid at or corresponding to position 137 is Ser , orthologue, paralogue or homologue thereof , in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Ser , Cys . and the amino acid at or corresponding to position 415 is In another preferred embodiment , the mutated PPO com Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 25 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Ser , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Ser, Gly . and the amino acid at or corresponding to position 415 is Ile . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 30 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Ser , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . Met . 35 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Ser , the amino acid at or corresponding to position 137 is Ser , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 40 Phe . His . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Ser , 45 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 137 is Ser, the amino acid at or corresponding to position 137 is Ser, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Trp . Asp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 55 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Ser , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ala . Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Ser, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser . Arg . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 107 108 the amino acid at or corresponding to position 137 is Thr, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Val. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is Thr, prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 and the amino acid at or corresponding to position 415 is orthologue , paralogue or homologue thereof, in which : Gly . the amino acid at or corresponding to position 137 is Thr, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is Ile . In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 10 orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . Met. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr , the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe. His . 20 In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Thr, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 25 Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Thr, 30 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asp . Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 35 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is theThr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Glu . Ala . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr , the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 415 is Ser . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , 45 In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Thr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Asn , Thr. and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - 50 Val . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Thr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Asn , Asn . 55 and the amino acid at or corresponding to position 415 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 415 is 60 and the amino acid at or corresponding to position 415 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Thr, 65 the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys. His . US 10 , 392 ,630 B2 109 110 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 415 is 5 and the amino acid at or corresponding to position 415 is Lys. Tyr. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , " the amino acid at or corresponding to position 137 is Asn , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asp . Trp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 16 prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Glu . Ala . In another preferred embodiment, the mutated PPO com - 20 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 415 is Ser. and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - 25 Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Asn , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Gin , Thr. and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Val . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Asn , zs orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Gln , Asn . and the amino acid at or corresponding to position 415 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 40 orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gln . Met. In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SER ID NO : 2 a variant , derivative , 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Gin , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys . His . In another preferred embodiment, the mutated PPO com - 50 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gly . 55 Lys . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 415 is 60 and the amino acid at or corresponding to position 415 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Asn , 65 the amino acid at or corresponding to position 137 is Gin , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe . Glu . US 10 , 392 ,630 B2 111 112 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gln , the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 415 is Ser. 5 and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Arg . In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Glnis , 10 the amino acid at or corresponding to position 137 is Cys, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. Val . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 15 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gln , thethe amino acid aat or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ile . Asn . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof , in which the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Gin , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Met. Gln . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 25 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys, the amino acid at or corresponding to position 137 is Gln , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is His . Cys . 30 In another preferred embodiment, themutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Cys, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Gln1 , 2535 LysIus . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Gly . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Cys , orthologue , paralogue or homologue thereof, in which : 40 and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Gin , Asp . and the amino acid at or corresponding to position 415 is In another preferred embodiment , the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SER ID NO : 2 a variant , derivative , 45 the amino acid at or corresponding to position 137 is Cys , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Gln , Glu . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Phe. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 50 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Cys , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is Ser . the amino acid at or corresponding to position 137 is Gin , In another preferred embodiment , the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Tyr. 55 orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Cys , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 415 is orthologue, paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 137 is Gln , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is 60 prises a sequence of SEQ ID NO : 2 a variant , derivative , Trp . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is Cys , prises a sequence of SEQ ID NO : 2 a variant, derivative, and the amino acid at or corresponding to position 415 is orthologue, paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 137 is Cys, 65 In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Ala . orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 113 114 the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys, the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys . His . In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is 15 and the amino acid at or corresponding to position 415 is Gly . Lys . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys, 20 the amino acid at or corresponding to position 137 is Gly, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . Asp . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 25 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe . Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , s prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Cys, the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Cys , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 40 Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , 45 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gln . Arg. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 55 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gly, the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 41515 is Cys. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Gly , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ile . Gly . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 115 116 the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gly, the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe . Glu . In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Gly , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is 15 and the amino acid at or corresponding to position 415 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Gly , 20 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 25 the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , su orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gln . Arg . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 40 Cys. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , 45 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ile . Gly . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , 50 the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Met. Pro . In another preferred embodiment , the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is His . Phe . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Pro , the amino acid at or corresponding to position 137 is Pro , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Lys. Tyr. In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 117 118 the amino acid at or corresponding to position 137 is Pro , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Trp . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 137 is Phe , prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 and the amino acid at or corresponding to position 415 is orthologue , paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 137 is Phe , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 10 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Phe , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Phe , Gln . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Arg . 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, themutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 137 is Phe , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Phe , Cys . and the amino acid at or corresponding to position 415 is 20 In another preferred embodiment, the mutated PPO com Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Phe , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Phe , 25 Gly . and the amino acid at or corresponding to position 415 is Ile . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Phe , 30 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Pro . Met . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 35 the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe . His . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, 40 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 137 is Phe , the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe, the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Trp . Asp . 50 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof , in which the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Phe , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is 55 Ala . Glu . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Phe , 60 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Ser. Arg . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Phe , 65 the amino acid at or corresponding to position 137 is Tyr , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Thr. Val . US 10 , 392 ,630 B2 119 120 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 137 is Tyr, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 415 is orthologue, paralogue or homologue thereof, in which : Gly . the amino acid at or corresponding to position 137 is Tyr, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is Ile . 5 prises a sequence of SEO ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Tyr, orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Tyr, and the amino acid at or corresponding to position 415 is 10 Pro . Met. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Tyr. 15 and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Phe . His . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEO ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 20 the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Tyr , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 25 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Tyr, and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asp . Trp . In another preferred embodiment, the mutated PPO com - 30 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Trp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Glu . 35 Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEO ID NO : 2 a variant. derivative . prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Trp , and the amino acid ator corresponding to position 415 is Ser . 40 and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, themutated PPO com orthologue, paralogue or homologue thereof , in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Tyr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is 45 the amino acid at or corresponding to position 137 is Trp , Thr. and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com - Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof , in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 137 is Tyr, 50 orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 415 is the amino acid at or corresponding to position 137 is Trp , Asn . and the amino acid at or corresponding to position 415 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr, the amino acid at or corresponding to position 137 is Trp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Gin . Met . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Tyr , the amino acid at or corresponding to position 137 is Trp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Cys. His . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 121 122 the amino acid at or corresponding to position 137 is Trp , the amino acid at or corresponding to position 137 is Trp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Lys . Tyr. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Trp , the amino acid at or corresponding to position 137 is Trp , and the amino acid at or corresponding to position 415 is and the amino acid at or corresponding to position 415 is Asp . Trp . In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 137 is Trp , the amino acid at or corresponding to position 415 is Ala , and the amino acid at or corresponding to position 415 is 16 Arg , Val, Ile, Met , His , Lys, Asp , Glu , Ser , Thr, Asn , Gln , Glu . Cys, Gly , Pro , Phe, Tyr, or Trp , and the amino acid at or In another preferred embodiment, the mutated PPO com - corresponding to position 438 is Ala , Leu , Val, Ile ,Met , His , prises a sequence of SEQ ID NO : 2 a variant, derivative , Lys , Asp , Glu , Ser, Thr, Asn , Gln , Cys, Gly, Pro , Arg , Tyr, orthologue, paralogue or homologue thereof, in which : or Trp . the amino acid at or corresponding to position 137 is Trp , 20 In another preferred embodiment , the mutated PPO com and the amino acid at or corresponding to position 415 is Ser. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ala , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Trp , 25 Ala . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Thr. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ala , orthologue , paralogue or homologue thereof, in which : S and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Trp , Leu . and the amino acid at or corresponding to position 415 is In another preferred embodiment, the mutated PPO com Asn . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com - 35 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ala , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 137 is Trp , Val. and the amino acid at or corresponding to position 415 is In another preferred embodiment , the mutated PPO com Gln . 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant , derivative, the amino acid at or corresponding to position 415 is Ala , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ile . the amino acid at or corresponding to position 137 is Trp , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , Cys. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Ala, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Met. the amino acid at or corresponding to position 137 is Trp , 50 In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Gly . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Ala , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : 55 His . the amino acid at or corresponding to position 137 is Trp , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Pro . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Ala , prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Lys . the amino acid at or corresponding to position 137 is Trp , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 415 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Phe. orthologue, paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com - 65 the amino acid at or corresponding to position 415 is Ala , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Asp . US 10 , 392 ,630 B2 123 124 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ala , the amino acid at or corresponding to position 415 is Arg , and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Glu . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 10 the amino acid at or corresponding to position 415 is Arg , the amino acid at or corresponding to position 415 is Ala , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 15 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ala , the aminoamino acid aat or corresponding to position 415 is Arg , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Val. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ala , the amino acid at or corresponding to position 415 is Arg , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Asn . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 25 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Arg , and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Ala , Met. and the amino acid at or corresponding to position 438 is 30 In another preferred embodiment, the mutated PPO com Gln . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Arg , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Ala , 26. His and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Cys. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Arg . orthologue, paralogue or homologue thereof, in which : 40 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Ala , Lys . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Gly . prises a sequence of SEQ ID NO : 2 a variant , derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SER ID NO : 2 a variant , derivative , 45 the amino acid at or corresponding to position 415 is Arg , orthologue, paralogue or homologue thereof , in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Ala , Asp . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com - 50 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Arg , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Ala , Glu . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Arg . 55 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Arg , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ser . the amino acid at or corresponding to position 415 is Ala , In another preferred embodiment , the mutated PPO com and the amino acid at or corresponding to position 438 is 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , Tyr. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Arg , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof , in which : Thr. the amino acid at or corresponding to position 415 is Ala , 65 In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Trp . orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 125 126 the amino acid at or corresponding to position 415 is Arg , the amino acid at or corresponding to position 415 is Val, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Asn . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Val, the amino acid at or corresponding to position 415 is Arg , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met. In another preferred embodiment, the mutated PPO com Gln . com . 10 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Val, orthologue, paralogue or homologue thereof, in which and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Arg , His . and the amino acid at or corresponding to position 438 is 15 In another preferred embodiment, the mutated PPO com Cys. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof , in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Val, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Arg , 20 Lys . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Gly. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Val , orthologue , paralogue or homologue thereof, in which : 25 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Arg , Asp . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , 30 the amino acid at or corresponding to position 415 is Val, orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Arg , Glu . and the amino acid at or corresponding to position 438 is In another preferred embodiment, themutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 35 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Val, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ser . the amino acid at or corresponding to position 415 is Arg , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Tyr. 40 orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof , in which : Thr. the amino acid at or corresponding to position 415 is Arg . In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is 45 prises a sequence of SEQ ID NO : 2 a variant , derivative , Trp . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 415 is Val, 50 In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Ala . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : 55 Gln . the amino acid at or corresponding to position 415 is Val, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Leu . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Cys . the amino acid at or corresponding to position 415 is Val, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Val. orthologue, paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com - 65 the amino acid at or corresponding to position 415 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Gly . US 10 , 392 ,630 B2 127 128 In another preferred embodiment , the mutated PPO com - the amino acid at or corresponding to position 415 is Ile , and prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 438 is Glu . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 415 is Val, prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is 5 orthologue , paralogue or homologue thereof, in which : Pro . the amino acid at or corresponding to position 415 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 438 is Ser. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Val, 10 orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Ile , and Arg . the amino acid at or corresponding to position 438 is Thr. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 15 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Val, the amino acid at or corresponding to position 415 is Ile , and and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 438 is Asn . Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ile , and the amino acid at or corresponding to position 415 is Val, the amino acid at or corresponding to position 438 is Gln . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Trp . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 25 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 415 is Ile , and orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 438 is Cys. the amino acid at or corresponding to position 415 is Ile , and in another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 438 is Ala . prises a sequence of SEO ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 30 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ile , and orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 438 is Gly . the amino acid at or corresponding to position 415 is Ile , and In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 438 is Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 35 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ile , and orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 438 is Pro . the amino acid at or corresponding to position 415 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 438 is Val . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 40 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ile , and orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 438 is Arg . the amino acid at or corresponding to position 415 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 438 is Ile. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 45 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ile , and orthologue , paralogue or homologue thereof , in which the amino acid at or corresponding to position 438 is Tyr . the amino acid at or corresponding to position 415 is Ile , and In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 438 is Met . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 50 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Ile , and orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 438 is Trp . the amino acid at or corresponding to position 415 is Ile , and In another preferred embodiment , the mutated PPO com the amino acid at or corresponding to position 438 is His prises a sequence of SEQ ID NO : 2 a variant , derivative , In another preferred embodiment, the mutated PPO com - 55 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Met, orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Ile , and Ala . the amino acid at or corresponding to position 438 is Lys . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 60 prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Met, the amino acid at or corresponding to position 415 is Ile , and and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 438 is Asp . Leu . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 129 130 the amino acid at or corresponding to position 415 is Met, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Val. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Met, prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Gly . the amino acid at or corresponding to position 415 is Met, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 10 orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Met , the amino acid at or corresponding to position 415 is Met, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Met . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Met , the amino acid at or corresponding to position 415 is Met, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . His . 20 In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Met , the amino acid at or corresponding to position 415 is Met , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 25 Tyr. Lys . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Met, 30 the amino acid at or corresponding to position 415 is Met , and the amino acid at or corresponding to position 438 is 30 and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : 35 orthologueof , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Met , the amino acid at or corresponding to position 415 is His , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . Ala . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Met, the amino acid at or corresponding to position 415 is His , and the amino acid at or corresponding to position 438 is Ser. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , 45 In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 415 is Met, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is His , Thr. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - 50 Val . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Met, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is His , Asn . 55 and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Met , the amino acid at or corresponding to position 415 is His , and the amino acid at or corresponding to position 438 is 60 and the amino acid at or corresponding to position 438 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Met, 65 the amino acid at or corresponding to position 415 is His , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. His . US 10 , 392 ,630 B2 131 132 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative, orthologue , paralogue or homologue thereof , in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is His , and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Lys. Tyr. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is His , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 15 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, inin whichwhich : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . Ala . In another preferred embodiment, the mutated PPO com - 20 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is Lys , and the amino acid at or corresponding to position 438 is Ser. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - 25 Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is His , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Lys, Thr. U and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is His , os orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Lys, Asn . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 40 orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Met. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SER ID NO : 2 a variant , derivative , 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is Lys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . His . In another preferred embodiment, the mutated PPO com - 50 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gly . 55 Lys . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , the amino acid at or corresponding to position 415 is Lys , and the amino acid at or corresponding to position 438 is 60 and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is His , 65 the amino acid at or corresponding to position 415 is Lys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . US 10 , 392 ,630 B2 133 134 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Lys, the amino acid at or corresponding to position 415 is Asp , and the amino acid at or corresponding to position 438 is Ser 5 and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Leu . prises a sequence of SEQ ID NO : 2 a variant , derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Lys, 10 the amino acid at or corresponding to position 415 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 15 orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Lys , the amino acid at or corresponding to position 415 is Asp . and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Asn . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 20 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof , in which the amino acid at or corresponding to position 415 is Asp , the amino acid at or corresponding to position 415 is Lys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gin . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 25 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asp , the amino acid at or corresponding to position 415 is Lys, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is His . Cys . 30 In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Asp , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Lys , 25 Ivo and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Gly . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Asp , orthologue , paralogue or homologue thereof, in which : 40 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Lys, Asp . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SER ID NO : 2 a variant , derivative , 45 the amino acid at or corresponding to position 415 is Asp , orthologue, paralogue or homologue thereof , in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Lys , Gu . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 50 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Asp , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ser. the amino acid at or corresponding to position 415 is Lys , In another preferred embodiment , the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Tyr. 55 orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Asp , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 415 is Lys , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is 60 prises a sequence of SEQ ID NO : 2 a variant , derivative , Trp . orthologue, paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Asp , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 415 is Asp , 65 In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Ala . orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 135 136 the amino acid at or corresponding to position 415 is Asp , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Met. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asp , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . His . In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asp , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is 15 and the amino acid at or corresponding to position 438 is Gly . Lys. In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asp , 20 the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 25 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asp , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 30 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asp , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Asp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 40 Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Glu , 45 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 55 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Glu , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 137 138 the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment, the mutated PPO com - " In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Glu , the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is 16 and the amino acid at or corresponding to position 438 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Ser, the amino acid at or corresponding to position 415 is Glu , 20 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 25 the amino acid at or corresponding to position 415 is Ser , the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , su orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Ser , the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Leu . 26 In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Ser, the amino acid at or corresponding to position 415 is Ser , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 40 Cys. Val . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ser, the amino acid at or corresponding to position 415 is Ser , 45 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ser, 50 the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met . Pro . In another preferred embodiment , the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ser, the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is His . Arg . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Ser, the amino acid at or corresponding to position 415 is Ser, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys . Tyr. In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 139 140 the amino acid at or corresponding to position 415 is Ser , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Trp . orthologue, paralogue or homologue thereof , in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Thr, prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 415 is Thr, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Ala . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 10 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Thr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Thr, Gln . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Leu . 1515 prisesP aa sequencequum VINYof SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 415 is Thr , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Thr, Cys . and the amino acid at or corresponding to position 438 is 20 In another preferred embodiment, the mutated PPO com Val. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Thr , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Thr, 25 Gly . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Thr, the amino acid at or corresponding to position 415 is Thr, 30 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Pro . Met. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 35 the amino acid at or corresponding to position 415 is Thr, the amino acid at or corresponding to position 415 is Thr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg. His. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Thr, the amino acid at or corresponding to position 415 is Thr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Tyr. Lys . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - 45 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Thr, the amino acid at or corresponding to position 415 is Thr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Trp . Asp . 50 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof , in which the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Thr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 55 Ala . Glu . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Thr, 60 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Leu . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Thr, 65 the amino acid at or corresponding to position 415 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Val . US 10 , 392 ,630 B2 141 142 In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Asn , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Gly . the amino acid at or corresponding to position 415 is Asn , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Ile . 5 prises a sequence of SEO ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 415 is Asn , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Asn , and the amino acid at or corresponding to position 438 is 10 Pro . Met . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Asn . 15 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . His . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEO ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 20 the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Tyr. Lys. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 25 orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Asn , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com - 30 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Gln , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . 35 Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEO ID NO : 2 a variant. derivative . prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Gln , and the amino acid ator corresponding to position 438 is Ser . 40 and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof , in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Asn , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is 45 the amino acid at or corresponding to position 415 is Gin , Thr. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com - Val. prises a sequence of SEQ ID NO : 2 a variant , derivative , In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Asn , 50 orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Gln , Asn . and the amino acid at or corresponding to position 438 is Ile . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Gln , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Met . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SER ID NO : 2 a variant , derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Asn , the amino acid at or corresponding to position 415 is Gin , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. His . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 143 144 the amino acid at or corresponding to position 415 is Gin , the amino acid at or corresponding to position 415 is Gln , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys . Tyr . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Gin , the amino acid at or corresponding to position 415 is Gln , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp. In another preferred embodiment, the mutated PPO comm 10 In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue, paralogue or homologue thereof, in which orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Gln , the amino acid at or corresponding to position 415 is Cys , and the amino acid at or corresponding to position 438 is 15 andAla the amino acid at or corresponding to position 438 is Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Cys , the amino acid at or corresponding to position 415 is Gln , 20 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Gin , 25 the amino acid at or corresponding to position 415 is Cys , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Val. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 30 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 30 the amino acid at or corresponding to position 415 is Cys, the amino acid at or corresponding to position 415 is Gln , and the amino acid at or corresponding to position 438 is Ile . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Asn . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com 35 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 415 is Cys, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Gln , Met . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Gln . 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 415 is Cys , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Gln , His . and the amino acid at or corresponding to position 438 is 45 In another preferred embodiment , the mutated PPO com Cys. prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant , derivative , the amino acid at or corresponding to position 415 is Cys , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Gin , 50 Lys . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Gly . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Cys , orthologue , paralogue or homologue thereof, in which : 55 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Gin , Asp . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment , the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 the amino acid at or corresponding to position 415 is Cys , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Gin , Glu . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 65 orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Cys , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ser. US 10 , 392 ,630 B2 145 146 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Cys, the amino acid at or corresponding to position 415 is Gly , and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Thr. Val. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : 10 the amino acid at or corresponding to position 415 is Gly , the amino acid at or corresponding to position 415 is Cys , and the amino acid at or corresponding to position 438 is Ile . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Asn . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivativederivative , 15 the amino acid at or corresponding to position 415 is Gly , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Cys, Met . and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Gln . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 20 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Gly , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Cys , His . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Cys . 25 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Gly , orthologue, paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Cys, Lys . and the amino acid at or corresponding to position 438 is 30 In another preferred embodiment, the mutated PPO com Gly . prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment, the mutated PPO com - orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Gly , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Cys , 35 Asp . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Pro . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Gly, orthologue , paralogue or homologue thereof, in which : 40 and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Cys , Glu . and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Arg . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SER ID NO : 2 a variant , derivative , 45 the amino acid at or corresponding to position 415 is Gly , orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is Ser. the amino acid at or corresponding to position 415 is Cys , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant , derivative, Tyr. orthologue , paralogue or homologue thereof, in which : In another preferred embodiment , the mutated PPO com - 50 the amino acid at or corresponding to position 415 is Gly, prises a sequence of SEQ ID NO : 2 a variant, derivative, and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Thr. the amino acid at or corresponding to position 415 is Cys , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Trp . 55 orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com the amino acid at or corresponding to position 415 is Gly , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Asn . the amino acid at or corresponding to position 415 is Gly , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , Ala . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Gly , prises a sequence of SEQ ID NO : 2 a variant, derivative , and the amino acid at or corresponding to position 438 is orthologue, paralogue or homologue thereof, in which : Gln . the amino acid at or corresponding to position 415 is Gly , 65 In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Leu . orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 147 148 the amino acid at or corresponding to position 415 is Gly , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . His . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Gly , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gly . Lys . In another preferred embodiment, the mutated PPO com - 10 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Gly , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is 15 and the amino acid at or corresponding to position 438 is Pro . Asp . In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Gly , 20 the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : 25 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Gly , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser. Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, 30 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Gly , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Thr. Trp . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 40 Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Pro , 45 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gln . Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 50 the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys . Val. In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , 55 orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ile . Gly . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Met . Pro . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 149 150 the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . Glu . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, 5 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Pro , the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Ser . Tyr. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - " prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Phe , the amino acid at or corresponding to position 415 is Pro , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 15 Thr . Trp . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant , derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative, orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Phe , the amino acid at or corresponding to position 415 is Phe , 20 and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asn . Ala . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant , derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 25 the amino acid at or corresponding to position 415 is Phe , the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Gin . Leu . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , su orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Phe , the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Cys. Val . 5 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com p rises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which the amino acid at or corresponding to position 415 is Phe, the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is lle . 40 Gly . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Phe , the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is 45 and the amino acid at or corresponding to position 438 is Met. Pro . In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Phe , 50 the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is His . Arg. In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : 55 orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Phe , the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys. Tyr . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SER ID NO : 2 a variant , derivative , 60 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Phe , the amino acid at or corresponding to position 415 is Phe , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com - 65 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : US 10 , 392 ,630 B2 151 152 the amino acid at or corresponding to position 415 is Tyr, In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is prises a sequence of SEQ ID NO : 2 a variant, derivative , Ala . orthologue , paralogue or homologue thereof, in which : In another preferred embodiment, the mutated PPO com - the amino acid at or corresponding to position 415 is Tyr, prises a sequence of SEQ ID NO : 2 a variant, derivative , 5 and the amino acid at or corresponding to position 438 is orthologue , paralogue or homologue thereof, in which : Gln . the amino acid at or corresponding to position 415 is Tyr , In another preferred embodiment, the mutated PPO com and the amino acid at or corresponding to position 438 is Leu . prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com - 10 orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Tyr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Tyr, Cys. and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Val. 16 prises a sequence of SEQ ID NO : 2 a variant, derivative , In another preferred embodiment, the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative, the amino acid at or corresponding to position 415 is Tyr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Tyr, Gly . and the amino acid at or corresponding to position 438 is Ile . 20 In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Tyr , the amino acid at or corresponding to position 415 is Tyr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is 25 Pro . Met. In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com - prises a sequence of SEO ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Tyr, 30 thea amino acid at or corresponding to position 415 is Tyr , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is His . Arg. In another preferred embodiment , the mutated PPO com In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , Orthologue. - paralogue or homologue thereof in which : 35 orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Tyr, the amino acid at or corresponding to position 415 is Tyr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Lys. Tyr. In another preferred embodiment, the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , 40 prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Tyr , the amino acid at or corresponding to position 415 is Tyr, and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Asp . Trp . In another preferred embodiment, the mutated PPO com - 45 In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Tyr, the amino acid at or corresponding to position 415 is Trp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Glu . 50 Ala . In another preferred embodiment, the mutated PPO com - In another preferred embodiment , the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Tyr, the amino acid at or corresponding to position 415 is Trp , and the amino acid at or corresponding to position 438 is Ser. 55 and the amino acid at or corresponding to position 438 is In another preferred embodiment, the mutated PPO com Leu . prises a sequence of SEQ ID NO : 2 a variant , derivative, In another preferred embodiment , the mutated PPO com orthologue, paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Tyr, orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is 60 the amino acid at or corresponding to position 415 is Trp , Thr. and the amino acid at or corresponding to position 438 is In another preferred embodiment , the mutated PPO com Val. prises a sequence of SEQ ID NO : 2 a variant, derivative, In another preferred embodiment , the mutated PPO com orthologue , paralogue or homologue thereof, in which : prises a sequence of SEQ ID NO : 2 a variant, derivative , the amino acid at or corresponding to position 415 is Tyr, 65 orthologue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 415 is Trp , Asn . and the amino acid at or corresponding to position 438 is Ile. US 10 , 392 ,630 B2 153 154 In another preferred embodiment , the mutated PPO com - In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof , in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Trp , the amino acid at or corresponding to position 415 is Trp , and the amino acid at or corresponding to position 438 is 5 and the amino acid at or corresponding to position 438 is Met. Pro . In another preferred embodiment, the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue , paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : 10 the amino acid at or corresponding to position 415 is Trp , the amino acid at or corresponding to position 415 is Trp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Arg . His . In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com prises a sequence of SEQ ID NO : 2 a variant, derivative, prises a sequence of SEQ ID NO : 2 a variant, derivativelive , 15 orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Trp , the amino acid at or corresponding to position 415 is Trp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Tyr. Lys. In another preferred embodiment , the mutated PPO com In another preferred embodiment, the mutated PPO com - 20 prises a sequence of SEQ ID NO : 2 a variant, derivative , prises a sequence of SEQ ID NO : 2 a variant, derivative , orthologue, paralogue or homologue thereof, in which : orthologue , paralogue or homologue thereof, in which : the amino acid at or corresponding to position 415 is Trp , the amino acid at or corresponding to position 415 is Trp , and the amino acid at or corresponding to position 438 is and the amino acid at or corresponding to position 438 is Trp . Asp . 25 In other embodiments , the mutated PPO comprises a In another preferred embodiment, the mutated PPO com - sequence of SEQ ID NO : 4 , a variant, derivative , ortho prises a sequence of SEQ ID NO : 2 a variant, derivative, logue , paralogue or homologue thereof, in which : orthologue, paralogue or homologue thereof, in which : the amino acid at or corresponding to position 299 is other the amino acid at or corresponding to position 415 is Trp , than Thr, and is preferably Leu , Ala , Pro , Lys, Arg , Val, Ile , and the amino acid at or corresponding to position 438 is 30 Met, lyr, Trp , Gly , Asn , Asp , Cys , Phe , Ser , Gin , Glu , or His ; and / or Glu . the amino acid at or corresponding to position 300 is other In another preferred embodiment, the mutated PPO com than Ser, and is preferably Leu , Ala , Arg , Val, Ile ,Met , His , prises a sequence of SEQ ID NO : 2 a variant, derivative , Lys , Asp , Glu , Thr, Asn , Gln , Cys , Gly , Pro, Phe , Tyr, or Trp orthologue , paralogue or homologue thereof, in which : 35 and / or the amino acid at or corresponding to position 415 is Trp , the amino acid at or corresponding to position 420 is other and the amino acid at or corresponding to position 438 is Ser. than Tyr and is preferably Ala , Leu , Val, Ile , Met . His . Lys . In another preferred embodiment, the mutated PPO com - Asp , Glu , Ser. Thr. Asn , Gln , Cys , Gly , Pro , Arg , Phe , or Trp . prises a sequence of SEQ ID NO : 2 a variant , derivative , In a preferred , the mutated PPO comprises a sequence of orthologue , paralogue or homologue thereof, in which : 40 SEQ ID NO : 4 , a variant, derivative , orthologue, paralogue the amino acid at or corresponding to position 415 is Trp , or homologue thereof , in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 299 is Leu , Thr. and the amino acid at or corresponding to position 420 is In another preferred embodiment, the mutated PPO com - Met. prises a sequence of SEQ ID NO : 2 a variant, derivative , 45 In another preferred , the mutated PPO comprises a orthologue, paralogue or homologue thereof , in which : sequence of SEQ ID NO : 4 , a variant, derivative , ortho the amino acid at or corresponding to position 415 is Trp , logue , paralogue or homologue thereof, in which : and the amino acid at or corresponding to position 438 is the amino acid at or corresponding to position 299 is Leu , Asn . and the amino acid at or corresponding to position 300 is In another preferred embodiment, the mutated PPO com - 50 Gly , and the amino acid at or corresponding to position 420 prises a sequence of SEQ ID NO : 2 a variant, derivative , is Val. orthologue, paralogue or homologue thereof, in which : It will be within the knowledge of the skilled artisan to the amino acid at or corresponding to position 415 is Trp , identify conserved regions and motifs shared between the and the amino acid at or corresponding to position 438 is homologues , orthologues and paralogues encoded by SEQ Gln . 55 ID NO : 1 , or 3 , such as those depicted in Table 1 . Having In another preferred embodiment, the mutated PPO com identified such conserved regions that may representsuitable prises a sequence of SEQ ID NO : 2 a variant, derivative , binding motifs , amino acids corresponding to amino acids at orthologue, paralogue or homologue thereof, in which : the preferred positions listed above , can be chosen to be the amino acid at or corresponding to position 415 is Trp , substituted by any other amino acid , for example by con and the amino acid at or corresponding to position 438 is 60 served amino acids as shown in table 2 , preferably by the Cys . amino acids listed above . In another preferred embodiment, the mutated PPO com - Furthermore , such conserved regions and motifs can be prises a sequence of SEQ ID NO : 2 a variant, derivative , identified by employing so -called amino acid substitution orthologue, paralogue or homologue thereof, in which : matrices which are well known in the art . See , e .g ., the PAM the amino acid at or corresponding to position 415 is Trp , 65 matrices ( Dayhoff et al. , 1978 ) and the BLOSUM matrices and the amino acid at or corresponding to position 438 is (Henikoff and Henikoff, 1992 .) Both substitution matrix Gly . families are parameterized by sequence divergence . The US 10 , 392 ,630 B2 155 156 PAM matrices are based on a formal Markov model of 1 . 0 , or 1 . 5 , or 2 . 0 , or 2 . 5 , or 3 . 0 , or 3 . 5 , or 4 , or 4 . 5 , or 5 ; sequence evolution . The BLOSUM matrices use an ad hoc calculated as the sum of the amino acids corresponding to approach . Both families were derived according to the position 126 , and the amino acid corresponding to position following general approach , although the details of each step 127 , and the amino acid corresponding to position 128, and differ between the two methods. 1 . Use a set of “ trustedmisteda ” 5 the aminoal acid corresponding to position 129 , and the amino multiple sequence alignments ( ungapped ) to infer model acid corresponding to position 130 . Preferably , the score is parameters ; 2 . Count observed amino acid pairs in the between 1 .69 and 4 .61 trusted alignments , correcting for sample bias ; 3 . Estimate substitution frequencies from amino acid pair counts ; 4 . 1397 : Construct a log odds scoring matrix from substitution fre - 10 quencies . Amino acid K395 T396 L397 G398 T399 In a preferred embodiment, the so -called Position Weight 0 .49 0 . 44 0 . 16 1 .03 0 . 36 Matrix (PWM ) is used (Stormo , et al. ( 1982 ) Nucleic Acids - 1 .47 - 1 .01 - 1 . 46 - 1. 78 - 0 . 06 Research 10 ( 9 ) : 2997 - 3011 ) . When the PWM elements are 0 . 14 0 . 28 0 . 48 - 0 . 16 - 1 . 07 - 0 . 53 - 0 .02 - 0 . 55 - 0 . 43 - 0 . 38 calculated using log likelihoods, the score of a sequence 13 - 1 . 10 0 . 52 - 0 . 08 - 1 . 16 0 . 07 given a particular PWM can be calculated be adding ( rather 0 . 39 - 0 . 76 - 0 . 47 1 .92 - 0 . 44 than multiplying ) the relevant values at each position in the - 0 . 30 - 1 . 70 - 1 . 06 - 0 . 92 - 0 . 95 PWM . The sequence score gives an indication of how - 1. 36 0 .96 0 . 46 - 1 .66 0 . 35 different the sequence is from a random sequence . The score - 0 . 24 - 0 .76 - 0 . 06 - 0 .69 - 1 .64 - 0 . 96 0 .62 1 . 42 - 0 . 82 0 . 38 is O if the sequence has the same probability of being a 2 - 1 .58 - 0 . 88 - 0 . 51 - 1 .66 - 0 . 38 functional site and of being a random site . The score is - 0 . 34 - 0 .66 - 0 .03 0 .26 - 0 . 47 greater than 0 if it is more likely to be a functional site than 1 . 29 - 0 .43 0 . 14 0 . 27 0 .07 a random site , and less than 0 if it is more likely to be a - 0 . 03 - 1 .08 - 1 . 20 - 0 . 37 0 . 05 0 .60 - 0 . 82 - 0 . 24 - 0 . 13 - 0 . 10 random site than a functional site . (Guigo , Roderic . " An 1 . 14 - 0 .04 - 0 . 13 0 .04 0 . 42 Introduction to Position Specific Scoring Matrices ". bioin - 4 0 . 14 0 . 32 0 . 42 - 0 . 57 0 . 64 formatica .upf . edu ) . The sequence score can also be inter - 0 .67 0 .62 - 0 .01 - 1 .03 0 . 94 preted in a physical framework as the binding energy for that - 2 . 54 0 . 37 - 2 . 36 - 1 .78 - 0 . 41 sequence . - 1. 47 - 0 .66 - 0 . 55 - 0 . 98 - 0 . 80 The values contributing to the matching score are calcu lated as the natural logarithm of the likelihood of the amino 30 worstScore : 2 .31021955717484 acid to occur at this position in the motif divided by the Thus, in another embodiment, the invention refers to a background probability of occurrence assuming a uniform nucleic acid encoding a polypeptide comprising a motif distribution . Hence, the value of 0 .42 for the occurrence of corresponding to position 395 , 396 , 397 , 398 , and 399 of A at position 126 is calculated as ln ( 0 . 076 / 0 .05 ) , showing SEQ ID : 27 with a matching score greater than 0 , or 0 . 5 , or that the likelihood of an A at this position is with 7 .6 % more » 1 .0 , or 1. 5 , or 2 .0 , or 2 .5 , or 3 .0 , or 4 . 5, or 5 , or 5. 5 , or 6 ; frequent than the expected random occurrence with 5 % . calculated as the sum of the amino acids corresponding to As an example , log odd values for positions 128, 397 , and position 395 , and the amino acid corresponding to position 420 of SEQ ID NO : 27 ( Amaranthus PPO2) , which is used 396 , and the amino acid corresponding to position 397 , and as reference sequence , are shown below . the amino acid corresponding to position 398 , and the amino acid corresponding to position 399 . Preferably , the score is between 2 .31 and 5 . 56 R128 :

Amino acid N126 K127 R128 Y129 1130 F420 : 0 .42 0 .22 0 .72 0 . 05 - 0 . 03 45 - 1. 45 - 0 .75 - 1 . 50 - 1 . 53 - 1 . 13 Amino acid T418 T419 F420 V421 422 0 .42 0 . 03 - 0 .27 - 1 .69 - 0 . 90 - 0 . 03 - 0 .05 - 0 . 85 - 1 .53 - 0 . 79 0 . 42 1. 26 - 1 .01 0 .82 0 . 52 - 0 .65 0 . 09 - 0 . 38 0 . 94 0 . 66 - 0 . 57 0 .32 - 2 .38 - 0 . 85 - 2 . 36 0 .69 0 . 43 - 0 . 01 - 0 .49 - 1 . 18 - 2 . 20 - 2 .07 - 1. 60 - 2 .03 - 0 . 99 - 0 .65 - 0 .58 - 0 .64 - 0 . 23 - 0 . 76 50 - 0 . 51 - 1 .62 - 0 . 64 - 1 . 68 - 0 . 94 - 0 .97 - 0 . 58 - 0 . 24 0 .08 1 . 29 - 0 . 29 - 0 . 26 1 .51 0 . 36 - 0 . 46 0 . 35 0 . 41 - 0 . 20 - 0 . 73 - 1 . 92 - 0 .71 1 . 11 - 3 . 27 - 0 .94 1 . 58 0 . 30 - 0 .05 - 0 . 12 0 .89 1 . 05 - 0 . 71 - 2 . 90 0 . 10 - 2 .03 - 0 . 55 - 0 .62 - 0 . 86 - 0 . 93 - 0 . 92 - 0 . 79 - 0 . 13 0 .40 - 0 . 42 1 . 10 - 0 . 85 0 . 17 0 . 20 - 0 . 33 - 1 . 00 - 1. 23 - 2 .04 - 2 .62 - 6 . 31 - 1 .68 - 1. 42 0 . 17 - 0 .42 - 0 .43 - 1 .45 - 0 . 46 55 0 . 40 0 .72 0 . 59 0 . 78 - 0 . 23 - 0 . 31 - 0 . 34 - 0 . 54 - 0 . 80 - 1 . 13 0 . 25 - 0 .66 0 . 35 0 . 27 - 1 .42 0 .73 - 1 .69 - 2 . 07 - 0 . 92 - 0 . 77 - 0 . 10 0 .34 1 . 32 - 0 .04 - 0 .63 COALOIXHEZAORE> - 1 .91 - 1 . 71 - 2 . 20 - 1 . 58 0 . 43 0 . 36 0 . 39 0 .79 - 0 . 36 - 0 . 54 - 0 .67 - 2 .62 - 0 . 32 - 1 . 34 - 0 . 99 0 .28 0 .06 0 . 38 - 0 . 27 - 0 .66 0 . 29 - 2 .62 - 2 . 38 - 2 . 18 - 0 . 77 - 0 . 44 0 . 11 0 . 32 0 .81 1 . 17 0 .57 0 . 25 0 . 25 0 . 12 0 . 92 - 1 .31 - 1 . 08 - 2 .27 - 0 . 34 - 0 . 86 60 1 . 39 - 0 . 15 - 0 . 04 0 .65 0 . 47 - 1 .62 0 .52 - 0 .54 1 .26 0 . 40 1 . 15 1 . 30 - 0 . 12 1 . 26 - 0 . 07 - 2 .04 - 2 .07 0 . 38 - 2 .86 - 0 . 73 worstScore: 1 .69444132814779 - 0 . 86 - 0 . 98 1 . 20 - 0 .43 - 0 . 12 Thus, in another embodiment, the invention refers to a nucleic acid encoding a polypeptide comprising a motif 65 worstScore : 3 .68475507992571 corresponding to position 126 , 127 , 128 , 129 , and 130 of Thus, in another embodiment, the invention refers to a SEQ ID : 27 with a matching score greater than 0 , or 0 . 5, or nucleic acid encoding a polypeptide comprising a motif US 10 , 392 ,630 B2 157 158 corresponding to position 418 , 419 , 420 , 421, 422 , and 423 deletion , insertion or substitution of nucleotides from the of SEQ ID : 27 with a matching score greater than 0 , or 0 . 5 , sequences shown, preferably from SEQ ID NO : 1 or 3 , or a or 1. 0 , or 1 .5 , or 2. 0 , or 2 .5 , or 3 .0 , or 4 . 5 , or 5, or 5 . 5 , or homolog thereof, or from the derived nucleic acid 6 ; or 6 . 5 , or 7 ; calculated as the sum of the amino acids sequences, the intention being , however , that the enzyme corresponding to position 418 , and the amino acid corre - 5 activity or the biological activity of the resulting proteins sponding to position 419 , and the amino acid corresponding synthesized is advantageously retained or increased . to position 420 , and the amino acid corresponding to posi In one embodiment of the present invention , the nucleic tion 421, and the amino acid corresponding to position 422 . acid molecule of the invention or used in the process of the Preferably , the score is between 3 .68 and 6 .73 invention comprises the sequences shown in any of the SEQ Table 2b shows an overview of preferred mutation sites 10 ID NO : 1 or 3 , or a homolog thereof. It is preferred that the that are shared between homologues , orthologues and par - nucleic acid molecule comprises as little as possible other alogues listed in Table 1. nucleotides not shown in any one of SEQ ID NO : 1 or 3 , or TABLE 2b SEQ Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 Pos 6 Pos 7 Pos 8 Pos 9 Pos 10 Pos 11 Pos 12 Pos 13 Pos 14 Pos 15 2 H135 K136 R137 Y138 T139V140 S158 L160 T163 P173 K178 E194 S195 E201 V208 4 A136 P137 R138 F139 V140 L141 F159 L161 1164 A174 P179 E186 S187 R193 V200 27 N126 K127 R128 Y129 1130 A131 S149 1151 A154 P164 K169 E182 S183 E189 F196 28 N126 K127 R128 Y129 1130 A131 S149 1151 A154 P164 K169 E182 S183 E189 F196 29 N126 K127 R128 Y129 1130 A131 S149 1151 A154 P164 K169 E182 S183 E189 F196 30 N126 K127 R128 Y129 1130 A131 S149 1151 A154 P164 K169 E182 S183 E189 F196 31 A155 P156 R157 F158 V159L160 F178 L180 F183 A193 P198 E205 S206R212 V219 SEQ Pos 16 Pos 17 Pos 18 Pos 19 Pos 20 Pos 21 Pos 22 Pos 23 Pos 24 Pos 25 Pos 26 Pos 27 Pos 28 Pos 29 Pos 30 2 D214 $ 221 A222 G223 L228 1230 R231 H232 N239 S246 A258 G271 R272 N273 L307 4 E206 Y213 A214 G215 L220 M222 R223 A224 R231 S238 K250 A264 P265 K266 T299 27 D202 C209G210 G211 L216 M218 H219 H220 N227 S234 S246 K259 P260 R261 L295 28 D202 C209 G210 G211 L216 M218 H219 H220 N227 S234 S246 K259 P260 R261 L295 29 D202 C209 G210 L215 M217 Y218 H219 N226 S233 S245 K258 P259 R260 L294 30 D202 C209 G210 L215 M217 H218 H219 N226 S233 S245 K258 P259 R260 L294 31 E225 Y232 233 234 239 M241 K242 A243 T250 S257 R269 K283 P284 K285 S318 SEQ Pos 31 Pos 32 Pos 33 Pos 34 Pos 35 Pos 36 Pos 37 Pos 38 Pos 39 Pos 40 Pos 41 Pos 42 Pos 43 Pos 44 Pos 45 2 S308 C313 D320 S341 S352 G363F366 L368 D369 D375 L401L415 F435 T436 T437 4 S300 E305 0307 S321 P332 L343 D346 A348 D349 Y355 L381 L397 L417 L418 N419 27 S296 Q301 G308 S324 R335 G346 F349 L351 D352 T358 L384 L397 F417 T418 T419 28 S296 Q301 G308 S324 R335 G346 F349 L351 D352 T358 L384 L397 F417 T418 T419 29 S295 Q300 G307 S323 R334 G345 F348 L350 D351 T357 L383 L396 F416 T417 T418 30 S295 0300 G307 S323 R334 G345 F348 1350 1351 1357 1383 1396 F416 T417 T418 31 N319 L324 G326 S340 P351L362 V365 367 D368 Y374 L400 L416 1436 L437 S438 SEQ Pos 46 Pos 47 Pos 48 Pos 49 Pos 50 Pos 51 Pos 52 Pos 53 Pos 54 Pos 55 Pos 56 Pos 57 Pos 58 Pos 59 Pos 60 2 F438 A450 T452 K456 L467 7469 H480 Y488 S494 A495 G500 Y511 K516 D533 K544 4 Y420 K432 E434 V438 L449 R453 V464 F472 D478 R479 K484 L497 V502 S519 27 F420 A432 T434 K438 L449 T451 F462Y470 S476 1477 D482Y493 K498 E515 K528 28 F420 A432 T434 K438 L449 T451 F462 Y470 S476 V477 D482 Y493 K498 E515 K528 29 F419 A431 T433 K437 L448 T450 F461 Y469 S475 V476 D481 Y492 K497 E514 K527 30 F419 A431 T433 K437 L448 T450 F461 Y469 C475 V476 D481 Y492 K497 E514 K527 31 Y439 K451 E453A457 1468 1472 1483 F491 D497 498 K503 L516 V521S538 -

Following mutagenesis of one of the sequences as shown a homolog thereof. In one embodiment , the nucleic acid herein , the encoded protein can be expressed recombinantly 50 molecule comprises less than 500 , 400 , 300 , 200 , 100 , 90 , and the activity of the protein can be determined using , for 80 , 70 , 60 , 50 or 40 further nucleotides . In a further example , assays described herein . embodiment, the nucleic acid molecule comprises less than The highest homology of the nucleic acid molecule used 30 , 20 or 10 further nucleotides . In one embodiment, the in the process according to the invention was found for the nucleic acid molecule use in the process of the invention is following database entries by Gap search . 55 identical to the sequences shown in SEQ ID NO : 1 or 3 , or Homologues of the nucleic acid sequences used , with the a homolog thereof. sequence shown in SEQ ID NO : 1 or 3, or a homolog A lso preferred is that the nucleic acid molecule used in the thereof, comprise also allelic variants with at least approxi- process of the invention encodes a polypeptide comprising mately 30 % , 35 % , 40 % or 45 % homology , by preference at the sequence shown in SEQ ID NO : 2 or 4 , or a homolog least approximately 50 % , 60 % or 70 % , more preferably at 60 thereof . In one embodiment, the nucleic acid molecule least approximately 90 % , 91 % , 92 % , 93 % , 94 % or 95 % and encodes less than 150 , 130 , 100 , 80 , 60 , 50 , 40 or 30 further even more preferably at least approximately 96 % , 97 % , amino acids . In a further embodiment, the encoded poly 98 % , 99 % or more homology with one of the nucleotide peptide comprises less than 20 , 15, 10 , 9 , 8 , 7 , 6 or 5 further sequences shown or the abovementioned derived nucleic amino acids . In one embodiment used in the inventive acid sequences or their homologues, derivatives or ana - 65 process , the encoded polypeptide is identical to the logues or parts of these . Allelic variants encompass in sequences shown in SEQ ID NO : 2 or 4 , or a homolog particular functional variants which can be obtained by thereof. US 10 , 392 ,630 B2 159 160 In one embodiment, the nucleic acid molecule of the 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 96 % , 97 % , 98 % , 99 % , invention or used in the process encodes a polypeptide 99 . 5 % or more, with the amino acid sequence of the comprising the sequence shown in SEQ ID NO : 2 or 4 , or polypeptide encoded by the nucleic acid molecule of ( a ) , a homolog thereof comprises less than 100 further nucleo ( b ), ( c ) or ( d ) and having the activity represented by a tides . In a further embodiment, said nucleic acid molecule 5 nucleic acid molecule comprising a polynucleotide of COcomprises less than 30 further nucleotides . In one embodi- SEQ ID NO : 1 or 3 , or a homolog thereof, and confers ment, the nucleic acid molecule used in the process is increased herbicide tolerance or resistance as compared to identical to a coding sequence of the sequences shown in a corresponding , e . g . non -transformed , wild type plant SEO ID NO : 1 or 3 , or a homolog thereof. cell , a plant or a part thereof; Polypeptides ( = proteins ), which still have the essential 10 ( f ) nucleic acid molecule which hybridizes with a nucleic biological or enzymatic activity of the polypeptide of the acid molecule of ( a ), ( b ) , ( c ) , ( d ) or ( e ) under stringent present invention conferring increased herbicide tolerance hybridization conditions and confers increased herbicide or resistance , as compared to a corresponding , e . g . non tolerance or resistance, as compared to a corresponding , transformed , wild type plant cell , plant or part thereof i . e . e . g . non - transformed , wild type plant cell, a plant or a part whose activity is essentially not reduced , are polypeptides 15 thereof; with at least 10 % or 20 % , by preference 30 % or 40 % , ( g ) a nucleic acid molecule encoding a polypeptide which especially preferably 50 % or 60 % , very especially prefer can be isolated with the aid of monoclonal or polyclonal ably 80 % or 90 or more of the wild type biological activity antibodies made against a polypeptide encoded by one of or enzyme activity , advantageously , the activity is essen the nucleic acid molecules of ( a ) , ( b ) , ( c ), ( d ) , ( e ) or ( f) and tially not reduced in comparison with the activity of a 20 having the activity represented by the nucleic acid mol polypeptide shown in SEQ ID NO : 2 or 4 , or a homolog ecule comprising a polynucleotide as depicted in SEQ ID thereof expressed under identical conditions. NO : 1 or 3 , or a homolog thereof; Homologues of SEQ ID NO : 1 or 3 , or of the derived ( h ) a nucleic acid molecule which is obtainable by screening sequences of SEQ ID NO : 2 or 4 , or a homolog thereof also a suitable nucleic acid library , especially a cDNA library mean truncated sequences , cDNA , single - stranded DNA or 25 and / or a genomic library , under stringent hybridization RNA of the coding and noncoding DNA sequence. Homo conditions with a probe comprising a complementary logues of said sequences are also understood as meaning sequence of a nucleic acid molecule of ( a ) or ( b ) or with derivatives , which comprise noncoding regions such as, for a fragment thereof, having 15 nt, preferably 20 nt, 30 nt, example , UTRs, terminators , enhancers or promoter vari 50 nt, 100 nt, 200 nt, 500 nt, 750 nt or 1000 nt or more ants . The promoters upstream of the nucleotide sequences 30 of a nucleic acid molecule complementary to a nucleic stated can be modified by one or more nucleotide substitu acid molecule sequence characterized in ( a ) to ( e ) and tion ( s ), insertion ( s ) and / or deletion ( s ) without, however, encoding a polypeptide having the activity represented by interfering with the functionality or activity either of the a protein comprising a polypeptide as depicted SEQ ID promoters , the open reading frame ( ORF ) or with the NO : 2 or 4 , or a homolog thereof. 3 ' -regulatory region such as terminators or other 3 '- regula - 35 The invention further provides an isolated recombinant tory regions , which are far away from the ORF. It is expression vector comprising the nucleic acid molecule of furthermore possible that the activity of the promoters is the invention , wherein expression of the vector or nucleic increased by modification of their sequence , or that they are acid molecule , respectively in a host cell results in an replaced completely by more active promoters , even pro increased herbicide tolerance or resistance , as compared to moters from heterologous organisms. Appropriate promoters 40 the corresponding , e . g . non - transformed , wild type of the are known to the person skilled in the art and are mentioned host cell . herein below . A plant expression cassette preferably contains regulatory Further , an embodiment of the invention is an expression sequences capable of driving gene expression in plant cells vector comprising a nucleic acid molecule comprising a and operably linked so that each sequence can fulfill its nucleic acid molecule selected from the group consisting of : 45 function , for example , termination of transcription by poly ( a ) a nucleic acid molecule encoding the polypeptide com - adenylation signals . Preferred polyadenylation signals are prising the sequence of SEQ ID NO : 2 or 4 , or a homolog those originating from Agrobacterium tumefaciens T -DNA thereof; such as the gene 3 known as octopine synthase of the ( b ) a nucleic acid molecule comprising the sequence of SEQ Ti- plasmid pTIACH5 (Gielen et al ., EMBO J . 3 , 835 1 ( 984 )) ID NO : 1 or 3, or a homolog thereof, 50 or functional equivalents thereof but also all other termina ( c ) a nucleic acid molecule , which , as a result of the tors functionally active in plants are suitable . As plant gene degeneracy of the genetic code , can be derived from a expression is very often not limited on transcriptional levels , polypeptide sequence of SEQ ID NO : 2 or 4 , or a homolog a plant expression cassette preferably contains other oper thereof, and confers increased herbicide tolerance or ably linked sequences like translational enhancers such as resistance , as compared to a corresponding , e . g . non - 55 the overdrive - sequence containing the 5 ' -untranslated leader transformed , wild type plant cell , a plant or a part thereof; sequence from tobacco mosaic virus enhancing the protein ( d ) a nucleic acid molecule having 30 % or more identity , per RNA ratio (Gallie et al ., Nucl . Acids Research 15 , 8693 preferably 40 % , 50 % , 60 % , 70 % , 75 % , 80 % , 85 % , 90 % , (1987 ) . 95 % , 96 % , 97 % , 98 % , 99 % , 99 . 5 % , or more with the Plant gene expression has to be operably linked to an nucleic acid molecule sequence of a polynucleotide com - 60 appropriate promoter conferring gene expression in a timely , prising the nucleic acid molecule of SEQ ID NO : 1 or 3 , cell or tissue specific manner. Preferred are promoters driv or a homolog thereof, and confers increased herbicide ing constitutive expression (Benfey et al. , EMBO J . 8 , 2195 tolerance or resistance , as compared to a corresponding , (1989 ) ) like those derived from plant viruses like the 35S e .g . non -transformed , wild type plant cell, a plant or a part CaMV (Franck et al ., Cell 21, 285 ( 1980 ) ), the 19S CaMV thereof ; 65 (see also U . S . Pat. No . 5 , 352 ,605 and PCT Application No . ( e ) a nucleic acid molecule encoding a polypeptide having WO 84 / 02913 ) or plant promoters like those from Rubisco 30 % or more identity , preferably at least 40 % , 50 % , 60 % , small subunit described in U . S . Pat. No . 4 , 962 ,028 . Other US 10 , 392 ,630 B2 161 162 promoters , e . g . super -promoter (Ni et al ., Plant Journal 7 , moters are useful in dicotyledones. The following promoters 661 ( 1995 ) ) , Ubiquitin promoter (Callis et al. , J . Biol. are useful for example in monocotyledones Ipt- 2 - or Ipt - 1 Chem ., 265 , 12486 ( 1990 ); U . S . Pat . Nos. 5 ,510 ,474 ; 6 , 020 , promoter from barley (WO 95 /15389 and WO 95 /23230 ) or 190 ; Kawalleck et al. , Plant. Molecular Biology, 21, 673 hordein promoter from barley. Other useful promoters are ( 1993 ) ) or 34S promoter (GenBank Accession numbers 5 M59930 and X16673 ) were similar useful for the present described in WO 99/ 16890 . It is possible in principle to use invention and are known to a person skilled in the art . all natural promoters with their regulatory sequences like Developmental stage -preferred promoters are preferentially those mentioned above for the novel process . It is also expressed at certain stages of development. Tissue and organ possible and advantageous in addition to use synthetic preferred promoters include those that are preferentially promoters . expressed in certain tissues or organs, such as leaves, roots , The gene construct may also comprise further genes seeds, or xylem . Examples of tissue preferred and organ preferred promoters include , but are not limited to fruit which are to be inserted into the organisms and which are for preferred , ovule - preferred , male tissue -preferred , seed -pre example involved in herbicide tolerance or resistance ferred , integument- preferred , tuber- preferred , stalk -pre 15 increase . It is possible and advantageous to insert and ferred , pericarp -preferred , and leaf- preferred , stigma express in host organisms regulatory genes such as genes for preferred , pollen -preferred , anther- preferred , a petal inducers , repressors or enzymes which intervene by their preferred , sepal -preferred , pedicel- preferred , silique enzymatic activity in the regulation , or one or more or all preferred , stem - preferred , root- preferred promoters , and the 20 genes of a biosynthetic pathway. These genes can be heter like . Seed preferred promoters are preferentially expressed during seed development and /or germination . For example , ologous or homologous in origin . The inserted genes may seed preferred promoters can be embryo -preferred , have their own promoter or else be under the control of same endosperm preferred , and seed coat -preferred . See Thomp promoter as the sequences of the nucleic acid of SEQ ID son et al ., BioEssays 10 , 108 (1989 ). Examples of seed , preferred promoters include , but are not limited to , cellulose synthase (celA ), Ciml, gamma- zein , globulin - 1, maize 19 The gene construct advantageously comprises, for expres kD zein ( cZ19B1) , and the like . sion of the other genes present, additionally 3 ' and / or 5 ' Other promoters useful in the expression cassettes of the terminal regulatory sequences to enhance expression , which invention include , but are not limited to , the major chloro - ze are selected for optimal expression depending on the phyll a / b binding protein promoter, histone promoters , the selected host organism and gene or genes. Ap3 promoter, the B -conglycin promoter, the napin pro These regulatory sequences are intended to make specific moter, the soybean lectin promoter , the maize 15 kD zein expression of the genes and protein expression possible as promoter, the 22 kD zein promoter, the 27 kD zein promoter , mentioned above . This may mean , depending on the host the g - zein promoter , the waxy , shrunken 1 , shrunken 2 and 35 organism , for example that the gene is expressed or over bronze promoters , the Zm13 promoter ( U . S . Pat . No. 5 ,086 , expressed only after induction , or that it is immediately 169 ) , the maize polygalacturonase promoters ( PG ) ( U . S . Pat. expressed and / or over -expressed . Nos. 5 ,412 ,085 and 5 ,545 ,546 ), and the SGB6 promoter ( U . S . Pat. No. 5 ,470 , 359 ) , as well as synthetic or other The regulatory sequences or factors may moreover pref natural promoters. 40 erably have a beneficial effect on expression of the intro Additional advantageous regulatory sequences are , for duced genes, and thus increase it . It is possible in this way example, included in the plant promoters such as CaMV /35S for the regulatory elements to be enhanced advantageously ( Franck et al. , Cell 21 285 ( 1980 ) ) , PRP1 (Ward et al ., Plant. at the transcription level by using strong transcription sig Mol. Biol. 22 . 361 ( 1993 ) ) . SSU , OCS . lib4 . usp . STLS1 . 45 nals such as promoters and /or enhancers . However , in addi B33 , LEB4 , nos , ubiquitin , napin or phaseolin promoter . tion , it is also possible to enhance translation by , for Also advantageous in this connection are inducible promot example , improving the stability of the mRNA . ers such as the promoters described in EP 388 186 (benzyl Other preferred sequences for use in plant gene expres sulfonamide inducible ), Gatz et al ., Plant J . 2 , 397 ( 1992 ) 50 sion cassettes are targeting -sequences necessary to direct the ( tetracyclin inducible ), EP - A - 0 335 528 (abscisic acid gene product in its appropriate cell compartment ( for review inducible ) or WO 93 /21334 ( ethanol or cyclohexenol induc ible ). Additional useful plant promoters are the cytoplasmic see Kermode, Crit . Rev . Plant Sci. 15 (4 ), 285 ( 1996 ) and FBPase promoter or ST -LSI promoter of potato (Stockhaus references cited therein ) such as the vacuole , the nucleus, all et al. , EMBO J. 8 , 2445 ( 1989 )) , the phosphorybosyl phy - 55 types of plastids like amyloplasts , chloroplasts , chro rophoshate amido transferase promoter of Glycine max moplasts , the extracellular space , mitochondria , the endo ( gene bank accession No. U87999 ) or the noden specific plasmic reticulum , oil bodies , peroxisomes and other com promoter described in EP - A -0 249 676 . Additional particu partments of plant cells . larly advantageous promoters are seed specific promoters Plant gene expression can also be facilitated via an which can be used for monocotyledones or dicotyledones 60 inducible promoter (for review see Gatz , Annu . Rev . Plant and are described in U . S . Pat. No. 5 ,608 , 152 (napin pro Physiol. Plant Mol. Biol. 48 , 89 ( 1997 ) ) . Chemically induc moter from rapeseed ), WO 98 /45461 (phaseolin promoter ible promoters are especially suitable if gene expression is from Arabidopsis ) , U . S . Pat . No. 5 , 504 , 200 (phaseolin pro wanted to occur in a time specific manner. moter from Phaseolus vulgaris ), WO 91/ 13980 (Bce4 pro - 65 Table xx lists several examples of promoters that may be moter from Brassica ) and Baeumlein et al. , Plant J. , 2 (2 ), used to regulate transcription of the nucleic acid coding 233 (1992 ) (LEB4 promoter from leguminosa ). Said pro sequences of the present invention . US 10 ,392 ,630 B2 163 164 TABLE xx Examples of tissue -specific and inducible promoters in plants Expression Reference Cor78 - Cold , drought, salt, Ishitani, et al. , Plant Cell 9 , 1935 ( 1997 ) , ABA , wounding - inducible Yamaguchi- Shinozaki and Shinozaki, Plant Cell 6 , 251 ( 1994 ) Rci2A - Cold , dehydration Capel et al. , Plant Physiol 115 , 569 ( 1997 ) inducible Rd22 - Drought, salt Yamaguchi - Shinozaki and Shinozaki, Mol . Gen . Genet . 238 , 17 ( 1993 ) Cor15A - Cold , dehydration , Baker et al ., Plant Mol. Biol. 24 , 701 ( 1994 ) ??? GH3 - Auxin inducible Liu et al. , Plant Cell 6 , 645 ( 1994 ) ARSK1- Root , salt inducible Hwang and Goodman , Plant J. 8 , 37 ( 1995 ) PtxA - Root , salt inducible GenBank accession X67427 SEHRGP3 - Root specific Ahn et al. , Plant Cell 8 , 1477 ( 1998 ). KST1 - Guard cell specific Plesch et al. , Plant Journal . 28 ( 4 ) , 455 - (2001 ) KAT1 - Guard cell specific Plesch et al. , Gene 249 , 83 (2000 ), Nakamura et al. , Plant Physiol. 109 , 371 ( 1995 ) salicylic acid inducible PCT Application No . WO 95 / 19443 tetracycline inducible Gatz et al. , Plant J . 2 , 397 ( 1992 ) Ethanol inducible PCT Application No . WO 93 / 21334 Pathogen inducible PRP1 Ward et al. , Plant . Mol. Biol . 22 , 361 - ( 1993 ) Heat inducible hsp80 U . S . Pat . No . 5 ,187 ,267 Cold inducible alpha -amylase PCT Application No . WO 96 / 12814 Wound -inducible pinII European Patent No . 375 091 RD29A - salt - inducible Yamaguchi -Shinozalei et al. Mol . Gen . Genet. 236 , 331 ( 1993 ) Plastid -specific viral RNA PCT Application No . WO 95 / 16783 , PCT Application WO polymerase 97 /06250

Additional flexibility in controlling heterologous gene 30 be assessed . Similarly , such a comparison permits an assess expression in plants may be obtained by using DNA binding ment of which regions of the sequence are conserved and domains and response elements from heterologous sources which are not, which may aid in determining those regions ( i . e . , DNA binding domains from non -plant sources ) . An of the polypeptide that are essential for the functioning of the example of such a heterologous DNA binding domain is the enzyme. This type of determination is of value for polypep LexA DNA binding domain (Brent and Ptashne , Cell 43 , 729 35 tide engineering studies and may give an indication ofwhat ( 1985 ) ) . the polypeptide can tolerate in terms of mutagenesis without In one embodiment, the language " substantially free of losing function . cellular material ” includes preparations of a protein having There are a number of mechanisms by which the altera less than about 30 % (by dry weight) of contaminating tion of the polypeptide of the invention may directly affect material ( also referred to herein as a " contaminating poly - 40 herbicide tolerance or resistance . peptide” ) ,more preferably less than about 20 % of contami- The effect of the genetic modification in plants regarding nating material , still more preferably less than about 10 % of herbicide tolerance or resistance can be assessed by treating contaminating material, and most preferably less than about the modified plant with respective herbicides as , e . g . , 5 % contaminating material . described in the EXAMPLES , and then analyzing the The nucleic acid molecules, polypeptides , polypeptide 45 growth characteristics and /or metabolism of the plant in homologs, fusion polypeptides, primers , vectors , and host comparison to non -modified plants . Such analysis tech cells described herein can be used in one or more of the niques are well known to one skilled in the art , and include following methods: identification of S. cerevisiae, E . coli or evaluation of the plant phenotype , dry weight, fresh weight, Brassica napus, Glycine max , Zea mays or Oryza sativa and polypeptide synthesis , carbohydrate synthesis , lipid synthe related organisms; mapping of genomes of organisms 50 sis , evapotranspiration rates , general plant and / or crop yield , related to S . cerevisiae , E . coli ; identification and localiza - flowering , reproduction , seed setting , root growth , respira tion of S . cerevisiae , E . coli or Brassica napus, Glycine max , tion rates , photosynthesis rates , etc . ( Applications of HPLC Zea mays or Oryza sativa sequences of interest ; evolutionary in Biochemistry in : Laboratory Techniques in Biochemistry studies; determination of polypeptide regions required for and Molecular Biology , Vol. 17 ; Rehm et al. , 1993 Biotech function ; modulation of a polypeptide activity ; modulation 55 nology , Vol. 3 , Chapter III: Product recovery and purifica of the metabolism of one or more cell functions; modulation tion , page 469- 714 , VCH : Weinheim ; Belter P . A . et al. , of the transmembrane transport of one or more compounds ; 1988 , Bioseparations : downstream processing for biotech modulation of herbicide tolerance or resistance , and modu nology, John Wiley and Sons; Kennedy J . F ., and Cabral J . lation of expression of polypeptide nucleic acids. M . S ., 1992 , Recovery processes for biological materials , The nucleic acid molecules of the invention are also 60 John Wiley and Sons; Shaeiwitz J . A . and Henry J. D ., 1988 , useful for evolutionary and polypeptide structural studies. Biochemical separations, in Ulmann ' s Encyclopedia of The metabolic and transport processes in which the mol- Industrial Chemistry , Vol. B3 , Chapter 11 , page 1 - 27 , VCH : ecules of the invention participate are utilized by a wide Weinheim ; and Dechow F . J ., 1989 , Separation and purifi variety of prokaryotic and eukaryotic cells ; by comparing cation techniques in biotechnology , Noyes Publications ) . the sequences of the nucleic acid molecules of the present 65 For example , plant expression vectors comprising the invention to those encoding similar enzymes from other nucleic acids disclosed herein , or fragments thereof, can be organisms, the evolutionary relatedness of the organisms can constructed and transformed into an appropriate plant cell US 10 , 392 ,630 B2 165 166 such as rape , maize , cotton , rice , wheat , sugar cane , sugar the genome of a plant cell and to design zinc finger tran beet, soy bean , Arabidopsis thaliana , potato , Medicago scription factors linked to a functional domain that will truncatula , etc . , using standard protocols . The resulting interact with the regulatory region of the gene . The interac transgenic cells and /or plants derived therefrom can then be tion of the zinc finger protein with the plant gene can be assayed for generation or alteration of their herbicide toler - 5 designed in such a manner as to alter expression of the gene ance or resistance . and preferably thereby to confer increasing herbicide toler The present invention also provides antibodies that spe - ance or resistance . cifically bind to the polypeptide according to the invention , In particular , the invention provides a method of produc or a portion thereof, as encoded by a nucleic acid described ing a transgenic plant with a coding nucleic acid , wherein herein . Antibodies can be made by many well -known meth - 10 expression of the nucleic acid (s ) in the plant results in ods ( see , e . g . Harlow and Lane , “ Antibodies ; A Laboratory increasing herbicide tolerance or resistance , as compared to Manual” , Cold Spring Harbor Laboratory , Cold Spring a wild type plant comprising : ( a ) transforming a plant cell Harbor , N . Y ., ( 1988 ) ) . Briefly , purified antigen can be with an expression vector comprising a encoding nucleic injected into an animal in an amount and in intervals acid , and (b ) generating from the plant cell a transgenic plant sufficient to elicit an immune response . Antibodies can either 15 with enhanced increased herbicide tolerance or resistance as be purified directly , or spleen cells can be obtained from the compared to a wild type plant. For such plant transforma animal . The cells can then fused with an immortal cell line tion , binary vectors such as pBinAR can be used (Hofgen and screened for antibody secretion . The antibodies can be and Willmitzer , Plant Science 66 , 221 (1990 )) . Moreover used to screen nucleic acid clone libraries for cells secreting suitable binary vectors are for example pBIN19 , pBI101 , the antigen . Those positive clones can then be sequenced . 20 PGPTV or pPZP (Hajukiewicz P . et al ., Plant Mol. Biol. , 25 , See , for example , Kelly et al. , Bio / Technology 10 , 163 989 ( 1994 ) ) . ( 1992 ) ; Bebbington et al. , Bio / Technology 10 , 169 ( 1992 ) . Alternate methods of transfection include the direct trans Gene expression in plants is regulated by the interaction fer of DNA into developing flowers via electroporation or of protein transcription factors with specific nucleotide Agrobacterium mediated gene transfer. Agrobacterium sequences within the regulatory region of a gene . One 25 mediated plant transformation can be performed using for example of transcription factors are polypeptides that con - example the GV3101 ( PM P90 ) (Koncz and Schell, Mol. tain zinc finger (ZF ) motifs. Each ZF module is approxi- Gen . Genet. 204 , 383 ( 1986 ) or LBA4404 (Ooms et al. , mately 30 amino acids long folded around a zinc ion . The Plasmid , 7 , 15 ( 1982 ) ; Hoekema et al ., Nature, 303 , 179 DNA recognition domain of a ZF protein is a a - helical ( 1983 ) ) Agrobacterium tumefaciens strain . Transformation structure that inserts into the major grove of the DNA double 30 can be performed by standard transformation and regenera helix . The module contains three amino acids that bind to the tion techniques (Deblaere et al. , Nucl . Acids. Res . 13 , 4777 DNA with each amino acid contacting a single base pair in ( 1994 ); Gelvin and Schilperoort , Plant Molecular Biology the target DNA sequence . ZF motifs are arranged in a Manual, 2nd Ed . - Dordrecht : Kluwer Academic Publ. , modular repeating fashion to form a set of fingers that 1995 . - in Sect . , Ringbuc Zentrale Signatur : BT11 - P ISBN recognize a contiguous DNA sequence. For example , a 35 0 -7923 -2731 - 4 ; Glick B . R . and Thompson J. E . , Methods in three - fingered ZF motif will recognize 9 bp of DNA . Hun - Plant Molecular Biology and Biotechnology, Boca Raton : dreds of proteins have been shown to contain ZF motifs with CRC Press , 1993 . - 360 S . , ISBN 0 - 8493 -5164 - 2 ). For between 2 and 37 ZF modules in each protein ( Isalan M . et example , rapeseed can be transformed via cotyledon or al. , Biochemistry 37 (35 ) , 12026 ( 1998 ) ; Moore M . et al. , hypocotyl transformation (Moloney et al. , Plant Cell Reports Proc . Natl . Acad . Sci. USA 98 ( 4 ) , 1432 ( 2001) and Moore 40 8 , 238 ( 1989 ) ; De Block et al. , Plant Physiol. 91 , 694 M . et al. , Proc . Natl. Acad . Sci . USA 98 ( 4 ), 1437 ( 2001) ; ( 1989 ) ). Use of antibiotics for Agrobacterium and plant U . S . Pat . Nos . 6 , 007 , 988 and 6 , 013 , 453 ) . selection depends on the binary vector and the Agrobacte The regulatory region of a plant gene contains many short rium strain used for transformation . Rapeseed selection is DNA sequences ( cis -acting elements ) that serve as recogni- normally performed using kanamycin as selectable plant tion domains for transcription factors , including ZF proteins . 45 marker . Agrobacterium mediated gene transfer to flax can be Similar recognition domains in different genes allow the performed using , for example , a technique described by coordinate expression of several genes encoding enzymes in Mlynarova et al. , Plant Cell Report 13 , 282 ( 1994 ) ) . Addi a metabolic pathway by common transcription factors . tionally , transformation of soybean can be performed using Variation in the recognition domains among members of a for example a technique described in European Patent No . gene family facilitates differences in gene expression within 50 424 047, U . S . Pat. No. 5 , 322, 783, European Patent No . 397 the same gene family , for example , among tissues and stages 687 , U . S . Pat. No. 5 , 376 , 543 or 5 , 169 , 770 . Transformation of development and in response to environmental condi - of maize can be achieved by particle bombardment, poly tions. ethylene glycol mediated DNA uptake or via the silicon Typical ZF proteins contain not only a DNA recognition carbide fiber technique (see , for example , Freeling and domain but also a functional domain that enables the ZF 55 Walbot “ The maize handbook ” Springer Verlag : New York protein to activate or repress transcription of a specific gene . (1993 ) ISBN 3 - 540 - 97826 - 7 ) . A specific example of maize Experimentally , an activation domain has been used to transformation is found in U . S . Pat. No . 5 , 990 , 387 and a activate transcription of the target gene (U . S . Pat. No . specific example of wheat transformation can be found in 5 , 789 ,538 and patent application WO 95 / 19431 ) , but it is PCT Application No . WO 93 /07256 . also possible to link a transcription repressor domain to the 60 In one embodiment, the present invention relates to a ZF and thereby inhibit transcription (patent applications WO method for the identification of a gene product conferring in 00 /47754 and WO 01/ 002019 ). It has been reported that an increasing herbicide tolerance or resistance, as compared to enzymatic function such as nucleic acid cleavage can be a corresponding , e . g . non - transformed , wild type cell in a linked to the ZF ( patent application WO 00 /20622 ) . cell of an organism for example plant, comprising the The invention provides a method that allows one skilled 65 following steps : in the art to isolate the regulatory region of one or more ( a ) contacting , e . g . hybridizing , some or all nucleic acid polypeptide according to the invention -encoding genes from molecules of a sample , e. g . cells , tissues , plants or micro US 10 , 392 ,630 B2 167 168 organisms or a nucleic acid library , which can contain a disclosed herein , in particular the proteins comprising poly candidate gene encoding a gene product conferring peptides as shown in SEQ ID NO : 2 or 4 , and their increased herbicide tolerance or resistance with a nucleic homolgous and in consequence in a natural variation of an acid molecule as shown in SEQ ID NO : 1 or 3 , or a increased herbicide tolerance or resistance . functional homologue thereof; 5 In consequence natural variation eventually also exists in ( b ) identifying the nucleic acid molecules , which hybridize form of more active allelic variants leading already to a under relaxed or stringent conditions with said nucleic relative increase in herbicide tolerance or resistance . Differ acid molecule , in particular to the nucleic acid molecule ent variants of the nucleic acids molecule disclosed herein , sequence shown in SEQ ID NO : 1 or 3 , or a homolog in particular the nucleic acid comprising the nucleic acid thereof, and , optionally , isolating the full length cDNA 10 molecule as shown SEQ ID NO : 1 or 3 , or a homolog clone or complete genomic clone ; thereof, which corresponds to different levels of increased ( c ) identifying the candidate nucleic acid molecules or a herbicide tolerance or resistance can be identified and used fragment thereof in host cells , preferably in a plant cell ; for marker assisted breeding for an increased herbicide ( d ) increasing the expression of the identified nucleic acid tolerance or resistance , molecules in the host cells for which increased herbicide 15 Accordingly , the present invention relates to a method for tolerance or resistance are desired ; breeding plants with an increased herbicide tolerance or ( e ) assaying the level of increased herbicide tolerance or resistance comprising resistance of the host cells ; and ( a ) selecting a first plant variety with an increased herbicide ( f ) identifying the nucleic acid molecule and its gene product tolerance or resistance , based on increased expression of which confers increased herbicide tolerance or resistance , 20 a nucleic acid of the invention as disclosed herein , in in the host cell compared to the wild type . particular of a nucleic acid molecule comprising a nucleic Relaxed hybridization conditions are : After standard acid molecule as shown in SEQ ID NO : 1 or 3 , or a hybridization procedures washing steps can be performed at homolog thereof, or a polypeptide comprising a polypep low to medium stringency conditions usually with washing tide as shown in SEQ ID NO : 2 or 4 , or a homolog thereof, conditions of 40° - 55° C . and salt conditions between 25 or a homologue thereof as described herein ; 2xSSC and 0 . 2xSSC with 0 . 1 % SDS in comparison to (b ) associating the level of increased herbicide tolerance or stringent washing conditions as e . g . 60° to 68° C . with 0 . 1 % resistance with the expression level or the genomic struc SDS . Further examples can be found in the references listed ture of a gene encoding said polypeptide or said nucleic above for the string end hybridization conditions . Usually acid molecule ; washing steps are repeated with increasing stringency and 30 (c ) crossing the first plant variety with a second plant variety , length until a useful signal to noise ratio is detected and which significantly differs in its level of increased herbi depend on many factors as the target, e . g . its purity , GC cide tolerance or resistance ; and content, size etc , the probe, e . g . its length , is it a RNA or a ( d ) identifying , which of the offspring varieties has got DNA probe , salt conditions, washing or hybridization tem - increased levels of herbicide tolerance or resistance , perature , washing or hybridization time etc . 35 As described SUPRA , the present invention provides In another embodiment, the present invention relates to a plants , plant tissues, plant cells , and host cells that are method for the identification of a gene product the expres - resistant or tolerant of at least one PPO - inhibiting herbicide. sion of which confers increased herbicide tolerance or In some embodiments , the plants , plant tissues , plant cells , resistance , in a cell, comprising the following steps : and host cells demonstrate enhanced resistance or enhanced ( a ) identifying a nucleic acid molecule in an organism , 40 tolerance to at least one PPO - inhibiting herbicide . The term which is at least 20 % , preferably 25 % , more preferably enhanced ' refers to an increase in the amount of resistance 30 % , even more preferred are 35 % . 40 % or 50 % , even or tolerance above that which is expected . The preferred more preferred are 60 % , 70 % or 80 % , most preferred are amount or concentration of the herbicide is an “ effective 90 % or 95 % or more homolog to the nucleic acid mol- amount” or “ effective concentration ." By “ effective amount" ecule encoding a protein comprising the polypeptide 45 and " effective concentration ” is intended an amount and molecule as shown in SEQ ID NO : 2 or 4 , or a homolog concentration , respectively, that is sufficient to kill or inhibit therefor being encoded by a nucleic acid molecule com - the growth of a similar, wild - type , plant, plant tissue , plant prising a polynucleotide as shown in SEQ ID NO : 1 or 3 , cell , microspore , or host cell , but that said amount does not or a homologue thereof as described herein , for example kill or inhibit as severely the growth of the herbicide via homology search in a data bank ; 50 resistant plants , plant tissues , plant cells , microspores, and ( b ) enhancing the expression of the identified nucleic acid host cells of the present invention . Typically , the effective molecules in the host cells ; amount of an herbicide is an amount that is routinely used ( c ) assaying the level of enhancement in increasing herbi - in agricultural production systems to kill weeds of interest. cide tolerance or resistance , in the host cells ; and Such an amount is known to those of ordinary skill in the art , ( d ) identifying the host cell , in which the enhanced expres - 55 or can be easily determined using methods known in the art . sion confers the increasing herbicide tolerance or resis - Furthermore , it is recognized that the effective amount of an tance in the host cell compared to a wild type . herbicide in an agricultural production system might be Further , the nucleic acid molecule disclosed herein , in substantially different than an effective amount of an herbi particular the nucleic acid molecule shown in SEQ ID NO : cide for a plant culture system such as, for example , the 1 or 3 , may be sufficiently homologous to the sequences of 60 microspore culture system . related species such that these nucleic acid molecules may The PPO enzymes of the present invention and PPO serve as markers for the construction of a genomic map in inhibiting herbicide resistant plants of the invention find use related organism or for association mapping. Furthermore in methods for controlling weeds. natural variation in the genomic regions corresponding to Thus, the present invention further provides a method for nucleic acids disclosed herein , in particular the nucleic acid 65 controlling undesired vegetation at a plant cultivation site in molecule shown in SEQ ID NO : 1 or 3 , or homologous the vicinity of an herbicide - resistant plant, such as a plant thereof may lead to variation in the activity of the proteins comprising a PPO nucleic acid molecule of the invention , US 10 , 392 ,630 B2 169 170 i. e . a nucleotide sequence encoding a polypeptide compris - Digitaria , Phleum , Poa , Festuca, Eleusine , Brachiaria , ing the sequence of SEQ ID NO : 2 or 4 , or a homolog Lolium , Bromus, Avena , Cyperus, Sorghum , Agropyron , thereof. Themethod comprises applying an effective amount Cynodon , Monochoria , Fimbristyslis , Sagittaria , of a PPO - inhibiting herbicide to the weeds and to the Eleocharis , Scirpus, Paspalum , Ischaemum , Sphenoclea , herbicide - resistant plant, wherein the plant has resistance to 5 Dactyloctenium , Agrostis , Alopecurus, and Apera . In addi at least one PPO - inhibiting herbicide , when compared to a tion , the weeds of the present invention can include , for wild -type plant . example , crop plants that are growing in an undesired Furthermore, the present invention provides method for location . For example , a volunteer maize plant that is in a growing the plant according to the present invention while field that predominantly comprises soybean plants can be controlling weeds in the vicinity of said plant, said method 10 considered a weed , if the maize plant is undesired in the field comprising the steps of: of soybean plants . c) growing said plant ; and Generally , the term “ herbicide” is used herein to mean an applying a herbicide composition comprising a PPO - inhib - active ingredient that kills , controls or otherwise adversely iting herbicide to the plant and weeds, wherein the herbicide modifies the growth of plants . The preferred amount or normally inhibits protoporphyrinogen oxidase , at a level of 15 concentration of the herbicide is an " effective amount" or the herbicide that would inhibit the growth of a correspond - " effective concentration .” By " effective amount” and “ effec ing wild - type plant. tive concentration ” is intended an amount and concentration , In another embodiment, the invention relates to a com respectively , that is sufficient to kill or inhibit the growth of bination useful for weed control, comprising ( a ) a poly - a similar , wild - type , plant, plant tissue , plant cell , or host nucleotide encoding a wildtype or mutated PPO polypeptide 20 cell, but that said amount does not kill or inhibit as severely according to the present invention , which polynucleotide is the growth of the herbicide -resistant plants , plant tissues , capable of being expressed in a plant to thereby provide to plant cells , and host cells of the present invention . Typically , that plant tolerance to a PPO inhibiting herbicide ; and (b ) a the effective amount of a herbicide is an amount that is PPO inhibiting herbicide. routinely used in agricultural production systems to kill In another embodiment, the invention relates to a process 25 weeds of interest . Such an amount is known to those of for preparing a combination useful for weed control com ordinary skill in the art. Herbicidal activity is exhibited by prising ( a ) providing a polynucleotide encoding a wildtype herbicides useful for the present invention when they are or mutated PPO polypeptide according to the present inven - applied directly to the plant or to the locus of the plant at any tion , which polynucleotide is capable of being expressed in stage of growth or before planting or emergence . The effect a plant to thereby provide to that plant tolerance to a PPO 30 observed depends upon the plant species to be controlled , inhibiting herbicide; and ( b ) providing a PPO inhibiting the stage of growth of the plant , the application parameters herbicide . of dilution and spray drop size , the particle size of solid In a preferred embodiment, said step of providing a components , the environmental conditions at the time of use , polynucleotide comprises providing a plant containing the the specific compound employed , the specific adjuvants and polynucleotide. 35 carriers employed , the soil type , and the like, as well as the In another preferred embodiment, said step of providing amount of chemical applied . These and other factors can be a polynucleotide comprises providing a seed containing the adjusted as is known in the art to promote non - selective or polynucleotide . selective herbicidal action . Generally , it is preferred to apply In another preferred embodiment, said process further the herbicide post emergence to relatively immature unde comprises a step of applying the PPO inhibiting herbicide to 40 sirable vegetation to achieve the maximum control of weeds . the seed . The herbicides useful for the present invention are those In another embodiment, the invention relates to the use of that interfere with the activity of the PPO enzyme such that a combination useful for weed control, comprising ( a ) a PPO activity is reduced in the presence of the herbicide . polynucleotide encoding a wildtype or mutated PPO poly - Such herbicides may also be referred to herein as “ PPO peptide according to the present invention , which polynucle - 45 inhibiting herbicides” or simply “ PPO inhibitors .” As used otide is capable of being expressed in a plant to thereby herein , an “ PPO - inhibiting herbicide ” or an “ PPO inhibitor” provide to that plant tolerance to a PPO inhibiting herbicide ; is not meant to be limited to single herbicide that interferes and ( b ) a PPO inhibiting herbicide , to control weeds at a with the activity of the PPO enzyme. Thus , unless otherwise plant cultivation site . stated or evident from the context, an “ PPO - inhibiting The term " control of undesired vegetation ” is to be 50 herbicide” or an “ PPO inhibitor” can be a one herbicide or understood as meaning the controlling , particularly killing a mixture of two, three , four, or more herbicides, each of of weeds and / or otherwise retarding or inhibiting the normal which interferes with the activity of the PPO enzyme. The growth of the weeds . Weeds, in the broadest sense , are PPO - inhibiting herbicide for use in the methods provided understood as meaning all those plants which grow in herein can be applied by any method known in the art locations where they are undesired , e . g . at a plant cultivation 55 including, but not limited to , seed treatment , soil treatment, site . The weeds of the present invention include, for and foliar treatment. example , dicotyledonous and monocotyledonous weeds. By a " herbicide -tolerant ” or “ herbicide -resistant ” plant, it Dicotyledonousweeds include, but are not limited to , weeds is intended that a plant that is tolerant or resistant to at least of the genera : Sinapis , Lepidium , Galium , Stellaria , Matri - one herbicide at a level that would normally kill , or inhibit caria , Anthemis , Galinsoga , Chenopodium , Urtica , Senecio , 60 the growth of, a normal or wild - type plant. By " herbicide Amaranthus, Portulaca , Xanthium , Convolvulus, Ipomoea , tolerant wildtype or mutated PPO protein ” or “ herbicide Polygonum , Sesbania , Ambrosia , Cirsium , Carduus, Son resistant wildtype or mutated PPO protein " , it is intended chus, Solanum , Rorippa , Rotala , Lindernia , Lamium , that such a PPO protein displays higher PPO activity , Veronica , Abutilon , Emex , Datura , Viola , Galeopsis , relative to the PPO activity of a wild -type PPO protein , Papaver , Centaurea , Trifolium , Ranunculus, and Taraxa - 65 when in the presence of at least one herbicide that is known cum . Monocotyledonous weeds include , but are not limited to interfere with PPO activity and at a concentration or level to , weeds of the genera : Echinochloa , Setaria , Panicum , of the herbicide that is known to inhibit the PPO activity of US 10 , 392 ,630 B2 171 172 the wild - type mutated PPO protein . Furthermore , the PPO mexyl ( 1 -methylhexyl ), meptyl ( 1 -methylheptyl ) , heptyl, activity of such a herbicide - tolerant or herbicide- resistant octyl or isooctyl ( 2 - ethylhexyl) esters . Preferred C , -C4 mutated PPO protein may be referred to herein as “ herbi alkoxy - C , -C4 - alkyl esters are the straight- chain or branched cide - tolerant" or "herbicide - resistant” PPO activity . C2 -C4 - alkoxy ethyl esters, for example the 2 -methoxyethyl , Generally , if the PPO - inhibiting herbicides ( also referred 5 2 -ethoxyethyl , 2 -butoxyethyl (butotyl ) , 2 -butoxypropyl or to as compounds A ) and /or the herbicidal compounds B as 3 -butoxypropyl ester. An example of a straight- chain or described herein , which can be employed in the context of branched C -C10 - alkylthio ester is the ethylthio ester. the present invention , are capable of forming geometrical Examples of PPO inhibiting herbicides which can be used isomers , for example E / Z isomers, it is possible to use both , according to the present invention are acifluorfen , acifluo the pure isomers and mixtures thereof, in the compositions 10 rfen - sodium , aclonifen , azafenidin , bencarbazone , benzfen useful for the present the invention . If the PPO - inhibiting dizone , bifenox , butafenacil, carfentrazone , carfentrazone herbicides A and /or the herbicidal compounds B as ethyl, chlomethoxyfen , cinidon - ethyl, fluazolate , flufenpyr, described herein have one or more centers of chirality and , flufenpyr - ethyl , flumiclorac , flumiclorac - pentyl, flumiox asers a, consequenceit is possible, areto presentuse both as enantiomersthe pure enantiomers or diastereom and 15 azin , fluoroglycofen , fluoroglycofen - ethyl, fluthiacet , fluthi diastereomers and their mixtures , in the compositions acet- methyl , fomesafen , halosafen , lactofen , oxadiargyl, according to the invention . If the PPO - inhibiting herbicides oxadiazon , oxyfluorfen , pentoxazone , profluazol, pyraclonil , A and /or the herbicidal compounds B as described herein pyraflufen , pyraflufen -ethyl , saflufenacil , sulfentrazone , have ionizable functional groups, they can also be employed thidiazimin , tiafenacil, chlornitrofen , flumipropyn , fluoroni in the form of their agriculturally acceptable salts . Suitable 20 trofen , flupropacil , furyloxyfen , nitrofluorfen , ethyl [ 3 - [ 2 are, in general, the salts of those cations and the acid chloro - 4 - fluoro - 5 - ( 1 -methyl - 6 - trifluoromethyl- 2 ,4 - dioxo - 1 , addition salts of those acids whose cations and anions , 2 , 3 , 4 -tetrahydropyrimidin - 3 - yl) phenoxy ) - 2 - pyridyloxy ] respectively , have no adverse effect on the activity of the acetate (CAS 353292 - 31 - 6 ; S - 3100 ), N - ethyl- 3 - 2 ,6 active compounds. Preferred cations are the ions of the alkali dichloro - 4 - trifluoromethylphenoxy ) - 5 -methyl - 1H - pyrazole metals , preferably of lithium , sodium and potassium , of the 25 l -carboxamide (CAS 452098 - 92 - 9 ), N - tetrahydrofurfuryl alkaline earth metals , preferably of calcium and magnesium , 3 - ( 2 , 6 -dichloro - 4 - trifluoromethylphenoxy ) - 5 -methyl - 1H and of the transition metals , preferably of manganese , cop pyrazole - 1 -carboxamide (CAS 915396 -43 - 9 ) , N - ethyl - 3 - ( 2 per , zinc and iron , further ammonium and substituted ammo chloro -6 - fluoro - 4 - trifluoromethylphenoxy )- 5 -methyl - 1H nium in which one to four hydrogen atoms are replaced by pyrazole - 1 - carboxamide (CAS 452099 - 05 - 7 ) , N - tetrahydro C , -C2 - alkyl, hydroxy - C , - C4 - alkyl , C1 -C4 - alkoxy -C1 - C4 - 30 furfurv1- 3 - ( 2 - chloro - 6 - fluoro - 4 - trifluoromethylphenoxv ) - 5 alkyl, hydroxy - C , -C4 - alkoxy - C1 -C4 - alkyl, phenyl or ben zyl, preferably ammonium , methylammonium , isopropy methyl - 1H - pyrazole - 1 - carboxamide (CAS 452100 - 03 - 7 ) , lammonium , dimethylammonium , diisopropylammonium , 3 - [ 7 - fluoro - 3 - oxo - 4 - ( prop - 2 -ynyl ) - 3 , 4 - dihydro - 2H - benzo trimethylammonium , heptylammonium , dodecylammo [ 1, 4 ]oxazin -6 -yl ) - 1, 5 - dimethyl- 6 - thioxo -[ 1, 3 ,5 ] triazinan - 2 , nium , tetradecylammonium , tetramethylammonium , tetra - 35 4 - dione (CAS 451484 - 50 - 7 ) , 1 , 5 -dimethyl - 6 - thioxo - 3 - ( 2 , 2 , ethylammonium , tetrabutylammonium , 2 - hydroxyethylam 7 - trifluoro - 3 -oxo -4 - (prop -2 -ynyl ) -3 , 4 - dihydro -2H -benzo [b ] monium (olamine salt ). 2 - ( 2 -hydroxyeth - 1 - oxV )eth - 1 - [ 1 , 4 ] oxazin - 6 - yl) - 1 , 3 , 5 -triazinane -2 , 4 - dione (CAS 1258 ylammonium (diglycolamine salt ) , di( 2 -hydroxyeth - 1 -yl ) 836 - 72 - 4 ) , 2 - ( 2 , 2 , 7 - Trifluoro - 3 -oxo - 4 -prop - 2 -ynyl - 3 ,4 - di ammonium (diolamine salt ), tris ( 2 -hydroxyethyl ) amm hydro - 2H -benzo [ 1, 4 ]oxazin -6 -yl ) -4 ,5 ,6 , 7 - tetrahydro - isoin onium (trolamine salt ), tris ( 2 - hydroxypropyl) ammonium , 40 dole - 1 , 3 - dione ( CAS 1300118 - 96 - 0 ) , 1 -Methyl - 6 -trifluo benzyltrimethylammonium , benzyltriethylammonium , NN , romethyl- 3 - ( 2 , 2 ,7 -trifluoro - 3 -oxo - 4 - prop - 2 - ynyl- 3 , 4 - dih N - trimethylethanolammonium ( choline salt ), furthermore ydro - 2H -benzo [ 1 , 4 ]oxazin -6 - yl) - 1H - pyrimidine - 2 , 4 - dione , phosphonium ions, sulfonium ions, preferably tri ( C , -C4 - methyl (E )- 4 - [ 2 -chloro -5 - [4 -chloro -5 -( difluoromethoxy ) alkyl) sulfonium , such as trimethylsulfonium , and sulfoxo - 1H -methyl - pyrazol - 3 -yl ) - 4 - fluoro -phenoxy ]- 3 -methoxy nium ions, preferably tri( C1 - C4 - alkyl ) sulfoxonium , and 45 but - 2 - enoate [CAS 948893 -00 - 3 ], 3- [7 -Chloro - 5 - fluoro - 2 finally the salts of polybasic amines such as N , N - bis - ( 3 - (trifluoromethyl ) - 1H -benzimidazol - 4 -yl ) - 1 -methyl - 6 - ( trifl aminopropyl) methylamine and diethylenetriamine . Anions uoromethyl) - 1H -pyrimidine - 2 , 4 - dione (CAS 212754 - 02 - 4 ) . of useful acid addition salts are primarily chloride , bromide , and fluoride, iodide , hydrogensulfate , methylsulfate , sulfate , dihydrogenphosphate , hydrogenphosphate , nitrate , bicarlate - , 50 uracils of formula III bonate , carbonate, hexafluorosilicate, hexafluorophosphate , benzoate and also the anions of C , -C4 - alkanoic acids, pref III erably formate , acetate , propionate and butyrate. The PPO - inhibiting herbicides A and/ or the herbicidal CH3 R36 compounds B as described herein having a carboxyl group 55 can be employed in the form of the acid , in the form of an R37, agriculturally suitable salt as mentioned above or else in the form of an agriculturally acceptable derivative , for example as amides, such as mono - and di- C1- C6 - alkylamides or arylamides, as esters, for example as allyl esters , propargyl 60 esters , C -C10 - alkyl esters , alkoxyalkyl esters , tefuryl ( tet R30 R31 rahydrofuran - 2 -yl ) methyl ) esters and also as thioesters , for R33 example as C , - C10 - alkylthio esters. Preferred mono - and di- C , -Co - alkylamides are the methyl and the dimethylam wherein ides. Preferred arylamides are , for example , the anilides and 65 R3° and R31 independently of one another are F , C1 or CN ; the 2 - chloroanilides . Preferred alkyl esters are , for example , R32 is O or S ; the methyl, ethyl, propyl, isopropyl , butyl, isobutyl, pentyl, R33 is H , F, C1, CHz or OCHz; US 10 , 392 ,630 B2 173 174 R34 is CH or N ; ethyl) - 1H -benzimidazol - 4 -yl ] - 1 -methyl - 6 - ( trifluorom R ” ) is O or S ; ethyl) - 1H -pyrimidine - 2 , 4 -dione (CAS 212754 - 02 - 4 ) R36 is H , CN , CH3, CF3, OCH3, OC2H5, SCHZ, SC2H5, uracils of formula III . 1 (corresponding to uracils of formula ( COOC , H , or CH , R38 III, wherein R3° is F , R31 is C1, R32 is O ; R33 is H ; R34 is CH ; wherein R38 is F , C1, OCHZ, SCH3, SC2H5, CH2F , 5 R35 is O and R37 is OR40 ) CH Br or CH2OH ; and R37 is (C / -Co - alkyl ) amino , (C ,- Co- dialkyl ) amino , (NH ) III . 1 OR39 , OH , OR40 or SR40 wherein R39 is CH3, C , H , or phenyl; and 10 R40 is independently of one another C2 -Co - alkyl , C2- C6- alkenyl , Cz -Co - alkynyl, C , -Co - haloalkyl , F3CN OR 40 C2 -Co - alkoxy - C , -Co - alkyl, C . - C . - alkoxy -C1 - C6 alkoxy -C , - C / -alkyl , C2 -C6 -cyanoalkyl , C , -C4 alkoxy - carbonyl - C , -C4 - alkyl, C7 -C4 - alkyl- carbo - 15 nyl- amino , C . - C . - alkylsulfinyl - C , -Co - alkyl , C , - C6 - alkyl- sulfonyl - C1- C6- alkyl, C7 -Co - di alkoxy -C2 - Cg- alkyl , C -Co - alkyl- carbonyloxy C1- C6- alkyl , phenyl - carbonyl- C , - CG- alkyl, tri (C1 Cz- alkyl ) -silyl - C , - Co -alkyl , tri ( C - Cz -alkyl ) - 20 wherein silyl - C , - Co- alkenyl, tri (C1 - Cz- alkyl) - silyl- C1 - C6 R36 is OCH3, OC2H5, SCH , or SC2H5; alkynyl, tri( C1 - Cz- alkyl ) -silyl - C , -Co - alkoxy - C1 and Co- alkyl , dimethylamino , tetrahydropyranyl, R40 is C , -Co - alkyl , Cz- Co - alkenyl , Cz -Co - alkynyl, C ,- Co tetrahydrofuranyl- C , -Cz - alkyl, phenyl- C , - C6 haloalkyl, C . -Co - alkoxy - C - C / - alkyl, C1- Co- alkoxy alkoxy -C1 - C6 - alkyl , phenyl- C1 - Cz- alkyl , pyridyl - 25 C - C . - alkoxy - C , - C6- alkyl, C . -Cz - cyanoalkyl, phenyl C -Cz - alkyl, pyridyl, phenyl, C -Cz - alkyl , pyridyl- C , -Cz - alkyl , Cz- Co - cycloalkyl or which pyridyls and phenyls independently of one C3 -C6 - cycloalkyl -C1 - C4 -alkyl , another are substituted by one to five substitu which cycloalkyls are unsubstituted or substituted by ents selected from the group consisting ofhalo one to five substituents selected from the group gen , C , -Cz - alkyl or C , - Cz- haloalkyl ; 30 consisting of halogen , C1- Cz- alkyl and C1- C2- ha Cz -Co -cycloalkyl or C3 -Co -cycloalkyl - C , -C4 -alkyl , loalkyl; which cycloalkyls independently of one another and are unsubstituted or substituted by one to five substituents selected from the group consisting uracils of formula III. 2 ( corresponding to uracils of formula of halogen , C , -Cz -alkyl and C - Cz- haloalkyl ; 35 III , wherein R30 is F ; R31 is Cl; R32 is O ; R33 is H ; R34 is N ; including their agriculturally acceptable alkali metal salts R35 is O and R37 is OR40 with R40 is C . -Co - alkyl ) or ammonium salts . Preferred PPO - inhibiting herbicides that can be used according to the present invention are : III. 2 Acifluorfen , acifluorfen - sodium , azafenidin , bencarbazone , 40 CH3 benzfendizone , butafenacil , carfentrazone - ethyl, cinidon 1 F?C . N . ethyl, flufenpyr- ethyl, flumiclorac -pentyl , flumioxazin , fluo C1- C6- alkyl , roglycofen - ethyl , fluthiacet- methyl , fomesafen , lactofen , oxadiargyl, oxadiazon , oxyfluorfen , pentoxazone , pyra flufen - ethyl, saflufenacil , sulfentrazone, ethyl [3 - [ 2 -chloro - 45 4 - fluoro - 5 -( 1 -methyl - 6 -trifluoromethyl - 2, 4 - dioxo - 1, 2 ,3 , 4 tetrahydropyrimidin - 3 - yl) phenoxy ) - 2 - pyridyloxy ] - acetate (CAS 353292 - 31 - 6 ; S - 3100 ), N - ethyl - 3 - ( 2 ,6 -dichloro - 4 - tri fluoromethylphenoxy ) - 5 -methyl - 1H -pyrazole - 1 - carboxam Particularly preferred PPO - inhibiting herbicides that can ide (CAS 452098 -92 - 9 ) , N -tetrahydrofurfuryl - 3 - ( 2 , 6 -di - 50 be used according to the present invention are : chloro - 4 - trifluoromethylphenoxy ) - 5 -methyl - 1H -pyrazole - acifluorfen , acifluorfen -sodium , butafenacil , carfentrazone 1 - carboxamide (CAS 915396 - 43 - 9 ) , N - ethyl- 3 - ( 2 - chloro - 6 - ethyl, cinidon - ethyl, flumioxazin , fluthiacet- methyl , fome fluoro - 4 - trifluoromethylphenoxy ) -5 -methyl - 1H -pyrazole - 1 - safen , lactofen , oxadiargyl, oxyfluorfen , saflufenacil, sulfen carboxamide (CAS 452099 -05 - 7 ) , N - tetrahydrofurfuryl- 3 - trazone , ethyl [ 3 -12 - chloro - 4 - fluoro - 5 - ( 1 -methyl - 6 - trifluo ( 2 -chloro - 6 - fluoro - 4 - trifluoromethylphenoxy) -5 -methyl - 55 romethyl- 2 , 4 - dioxo - 1 , 2 , 3 , 4 - tetrahydropyrimidin - 3 - yl) - phe 1H -pyrazole - 1 - carboxamide (CAS 452100 -03 - 7 ), 3- [ 7 - noxyl- 2 - pyridyloxy ]acetate (CAS 353292 - 31 - 6 ; S - 3100 ), fluoro - 3 -oxo - 4 - (prop - 2 -ynyl ) - 3 , 4 - dihydro -2H -benzo [ 1 , 4 ] 3 - [ 7 - fluoro - 3 - oxo - 4 - ( prop - 2 - ynyl) - 3 , 4 - dihydro - 2H - benzo oxazin - 6 -yl ] - 1 , 5 -dimethyl - 6 - thioxo - [ 1 , 3 , 5 ]triazinan - 2 , 4 [ 1 , 4 ]oxazin - 6 - yl] - 1 , 5 - dimethyl- 6 -thioxo - [ 1 , 3 , 5 ] triazinan - 2 , dione (CAS 451484 -50 - 7 ) , 1 , 5 - dimethyl- 6 -thioxo - 3 - ( 2 , 2 , 7 - 4 - dione (CAS 451484 - 50 - 7 ) , 1 , 5 - dimethyl- 6 - thioxo - 3 - ( 2 , 2 , trifluoro - 3 - oxo - 4 - (prop - 2 -ynyl ) - 3 , 4 - dihydro -2H - benzo [b ] 60 7 - trifluoro - 3 -oxo - 4 - ( prop - 2 - ynyl) - 3 , 4 - dihydro - 2H -benzo [ b ] [ 1 , 4 ] oxazin - 6 - yl) - 1 , 3 , 5 - triazinane - 2 , 4 - dione (CAS 1258 - [ 1 , 4 ] oxazin - 6 -yl ) - 1 , 3 , 5 - triazinane - 2 ,4 - dione (CAS 12588 836 -72 - 4 ), 2 - ( 2 , 2 , 7 - Trifluoro - 3 - oxo - 4 -prop - 2 - ynyl- 3 ,4 - di- 36 -72 - 4 ), and 2 - (2 , 2 , 7 - Trifluoro -3 -oxo - 4 -prop - 2 -ynyl - 3 ,4 hydro - 2H -benzo [ 1 ,4 ] oxazin -6 - yl) - 4 , 5 ,6 , 7 - tetrahydro - isoin dihydro -2H -benzo [ 1 , 4 ]oxazin -6 -yl ) - 4 , 5 , 6 , 7 -tetrahydroiso dole - 1 , 3 -dione ( CAS 1300118 - 96 - 0 ) ; 1 -Methyl - 6 - trifluo indole - 1 , 3 -dione (CAS 1300118 - 96 - 0 ) , 1 -Methyl -6 - trifluo romethyl- 3 -( 2 ,2 ,7 - trifluoro -3 - oxo -4 -prop - 2 - ynyl- 3 ,4 -di - 65 romethyl -3 - ( 2, 2 ,7 - trifluoro - 3 -oxo - 4 -prop - 2 -ynyl - 3 ,4 - di hydro -2H -benzo [ 1 , 4 ]oxazin -6 - yl) - 1H - pyrimidine - 2 , 4 -dione hydro - 2H -benzo [ 1 , 4 ]oxazin - 6 - yl) - 1H -pyrimidine - 2 , 4 - dione (CAS 1304113 -05 - 0 ), 3 - [ 7 - Chloro - 5 - fluoro - 2 - ( trifluorom - (CAS 1304113 - 05 - 0 ), uracils of formula III. 1 . 1 ( correspond US 10, 392 ,630 B2 175 176 ing to uracils of formula III, wherein R30 is F , R31 is C1, R32 TABLE A is O ; R33 is H ; R34 is CH ; R35 is O , R36 is OCHZ and R37 is A . 1 acifluorfen OR49 ) A . 2 butafenacil 4 . 3 carfentrazone - ethyl A . 4 cinidon -et ethyl III. 1 . 1 A . 5 flumioxazin A . 6 fluthiacet -methyl CH3 H3CO A . 7 fomesafen A . 8 lactofen FzCN . OR40 A . 9 oxadiargyl 10 A . 10 oxyfluorfen A . 11 saflufenacil A . 12 sulfentrazone A . 13 ethyl [ 3 - [ 2 - chloro - 4 - fluoro - 5 - ( 1 -methyl - 6 - trifluoromethyl- 2 , 4 dioxo - 1 , 2 , 3 , 4 - tetrahydropyrimidin - 3 -yl )phenoxy ] - 2 pyridyloxylacetate (CAS 353292- 31 - 6 ) 15 . A .14 1 , 5 - dimethyl- 6 -thioxo - 3 - (2 , 2 , 7 -trifluoro - 3 - oxo - 4 - (prop - 2 -ynyl ) - 3 , 4 dihydro -2H -benzo [b ] [ 1 , 4 ]oxazin - 6 - yl) - 1 , 3 ,5 -triazinane - 2 , 4 - dione (CAS 1258836 -72 - 4 ) wherein R40 is C , -Co - alkyl, C2- Cd -alkenyl , C3 -C6 - alkynyl, C . -C6 - The PPO - inhibiting herbicides described above that are haloalkyl, C , - Co- alkoxy -C2 - C6- alkyl, C , -Co - alkoxy - 20 useful to carry out the present invention are often best C -Co - alkoxy - C , -C6 - alkyl, C - Cz- cyanoalkyl, phenyl applied in conjunction with one or more other herbicides to C -Cz - alkyl, pyridyl- C / -Cz - alkyl, Cz- Co - cycloalkyl or obtain control of a wider variety of undesirable vegetation . C3- C6 - cycloalkyl -C , -C4 - alkyl, For example , PPO - inhibiting herbicides may further be used which cycloalkyls are unsubstituted or substituted by in conjunction with additional herbicides to which the crop one to five substituents selected from the group 25 plant is naturally tolerant, or to which it is resistant via consisting of halogen , C1- Cz - alkyl and C1- C2 - ha expression of one or more additional transgenes, e . g . those loalkyl; as mentioned hereinafter in greater detail, or to which it is is preferably CH3, CH , CH ,OC2H5 , CH2CHF2, cyclo resistant via mutagenesis and breeding methods as described hexyl, ( 1 -methylcyclopropyl ) methyl or CH2( pyridine hereinafter. 4 - yl) ; 30 When used in conjunction with other targeting herbicides , the PPO - inhibiting herbicides, to which the plant of the uracils of formula III . 2 . 1 (corresponding to uracils of for present invention had been made resistant or tolerant, can be mula III , wherein R30 is F ; R31 is Cl; R32 is O ; R33 is H ; R34 formulated with the other herbicide or herbicides , tank is N ; R35 is O and R37 is OR40 with R40 is CHz) mixed with the other herbicide or herbicides , or applied 35 sequentially with the other herbicide or herbicides . Suitable components for mixtures are, for example , III. 2 . 1 selected from the herbicides of class bl ) to b15 ) CH3 B ) herbicides of class bl ) to b15 ) : b1 ) lipid biosynthesis inhibitors ; F3C N 0 OCH3, 40 b2 ) acetolactate synthase inhibitors ( ALS inhibitors ) ; b3 ) photosynthesis inhibitors ; b4 ) protoporphyrinogen - IX oxidase inhibitors , b5 ) bleacher herbicides; b6 ) enolpyruvyl shikimate 3 - phosphate synthase inhibi 45 tors ( EPSP inhibitors ); b7 ) glutamine synthetase inhibitors ; b8 ) 7 ,8 -dihydropteroate synthase inhibitors ( DHP inhibi and tors ); b9 ) mitosis inhibitors ; uracils of formula III . 2 . 2 ( corresponding to uracils of for b10 ) inhibitors of the synthesis of very long chain fatty mula III, wherein R30 is F ; R31 is Cl; R32 is O ; R33 is H ; R34 30 acids ( VLCFA inhibitors ) ; is N ; R35 is O and R37 is OR 40 with R40 is C H5) b11 ) cellulose biosynthesis inhibitors ; b12) decoupler herbicides; III. 2 . 2 b13 ) auxinic herbicides ; 55 b14 ) auxin transport inhibitors ; and CH ; b15 ) other herbicides selected from the group consisting of bromobutide , chloroflurenol, chloroflurenol- methyl , F?C . N LOC2H5, cinmethylin , cumyluron , dalapon , dazomet, difenzo quat , difenzoquat -metilsulfate , dimethipin , DSMA, 60 dymron , endothal and its salts , etobenzanid , flamprop , flamprop - isopropyl , flamprop -methyl , flamprop - M isopropyl, flamprop - M -methyl , fluorenol, fluorenol butyl, flurprimidol, fosamine , fosamine - ammonium , indanofan , indaziflam , maleic hydrazide , mefluidide, 65 metam , methiozolin (CAS 403640 - 27 - 7 ) , methyl azide , Especially preferred PPO - inhibiting herbicides are the methyl bromide , methyl- dymron , methyl iodide , PPO - inhibiting herbicides . 1 to A . 14 listed below in table A : MSMA , oleic acid , oxaziclomefone , pelargonic acid , US 10 , 392 ,630 B2 177 178 pyributicarb , quinoclamine, triaziflam , tridiphane and ron - methyl, triasulfuron , tribenuron , tribenuron -methyl , tri 6 - chloro - 3 - ( 2 - cyclopropyl- 6 -methylphenoxy ) - 4 floxysulfuron , triflusulfuron , triflusulfuron -methyl and pyridazinol (CAS 499223 -49 - 3 ) and its salts and esters; tritosulfuron , including their agriculturally acceptable salts or derivatives . imidazolinones such as imazamethabenz , imazamethabenz Examples of herbicides B which can be used in combi- 5 methyl, imazamox , imazapic , imazapyr, imazaquin and nation with the PPO -inhibiting herbicides according to the imazethapyr , triazolopyrimidine herbicides and sulfo nanilides such as cloransulam , cloransulam -methyl , diclo present invention are : sulam , flumetsulam , florasulam , metosulam , penoxsulam , b1) from the group of the lipid biosynthesis inhibitors : pyrimisulfan and pyroxsulam , ACC -herbicides such as alloxydim , alloxydim -sodium , 10 pyrimidinylbenzoates such as bispyribac, bispyribac -so butroxydim , clethodim , clodinafop , clodinafop - propargyl, dium , pyribenzoxim , pyriftalid , pyriminobac , pyriminobac cycloxydim , cyhalofop , cyhalofop -butyl , diclofop , diclofop methyl, pyrithiobac, pyrithiobac - sodium , 4 -[ [ [ 2 - [ ( 4 , 6 - dime methyl, fenoxaprop , fenoxaprop - ethyl , fenoxaprop - P , fenox thoxy - 2 -pyrimidinyl ) oxylphenyl] methyljamino ] - benzoic aprop -P -ethyl , fluazifop , fluazifop -butyl , fluazifop -P , fluazi acid - 1 -methylethyl ester (CAS 420138 -41 - 6 ) , 4 - [ [ { 2 - [ ( 4 ,6 fop - P -butyl , haloxyfop , haloxyfop -methyl , haloxyfop - P , 15 dimethoxy - 2 - pyrimidinyloxylphenyllmethyllaminol- ben haloxyfop - P -methyl , metamifop , pinoxaden , profoxydim , zoic acid propyl ester (CAS 420138 -40 - 5 ) , N - ( 4 -bromophe propaquizafop , quizalofop , quizalofop - ethyl, quizalofop nyl) - 2 - [ ( 4 , 6 - dimethoxy - 2 - pyrimidinyl) oxy ]benzenemetha tefuryl, quizalofop - P , quizalofop - P - ethyl, quizalofop - P -tefu - namine (CAS 420138 - 01 - 8 ) . ryl, sethoxydim , tepraloxydim , tralkoxydim , sulfonylaminocarbonyl- triazolinone herbicides such as flu 4 -( 4 -Chloro -4 -cyclopropyl - 2 - fluoro [ 1, 1' - biphenyl ] - 3 -yl ) - 20 carbazone , flucarbazone -sodium , propoxycarbazone , prop 5 -hydroxy - 2 ,2 ,6 ,6 -tetramethyl - 2H - pyran - 3 (6H ) -one ( CAS oxycarbazone -sodium , thiencarbazone and thiencarbazone 1312337 - 72 -6 ) ; 4 - ( 2 , 4 '- Dichloro - 4 - cyclopropyl[ 1 , 1 '- biphe - methyl ; and triafamone; nyl] - 3 - yl) - 5 -hydroxy - 2 ,2 , 6 , 6 - tetramethyl- 2H -pyran - 3 (6H ) among these, a preferred embodiment of the invention one (CAS 1312337 -45 - 3 ) ; 4 - ( 4 ' -Chloro - 4 - ethyl- 2 ' - fluoro [ 1 relates to those compositions comprising at least one imi 1 '- biphenyl ] - 3 -yl ) - 5 -hydroxy - 2 , 2 , 6 ,6 - tetramethyl- 2H - 25 dazolinone herbicide; pyran - 3 (6H ) -one (CAS 1033757 - 93 - 5 ) ; 4 - ( 2 , 4 '- Dichloro - 4 - b3 ) from the group of the photosynthesis inhibitors : ethyl[ 1 , 1 ' -biphenyl ] - 3 - yl ) - 2 . 2 ,6 ,6 - tetramethyl- 2H -pyran - 3 , 5 amicarbazone , inhibitors of the photosystem II , e . g . triazine (4H ,6H ) -dione (CAS 1312340 -84 -3 ); 5 - ( Acetyloxy )- 4 -( 4 ' herbicides, including of chlorotriazine , triazinones, triazin chloro - 4 - cyclopropyl- 2' - fluoro [ 1 , 1 '- biphenyl] - 3 - yl) - 3 ,6 diones , methylthiotriazines and pyridazinones such as dihydro - 2 , 2 ,6 , 6 - tetramethyl- 2H - pyran - 3 - one (CAS 1312 -. 3030 ametryn , atrazine , chloridazone, cyanazine , desmetryn , 337 -48 -6 ) ; 5 -( Acetyloxy )- 4 -( 2 , 4 -dichloro -4 -cyclopropyl dimethametryn , hexazinone, metribuzin , prometon , prom [ 1, 1' - biphenyl ]- 3 -yl ) - 3 ,6 - dihydro - 2 ,2 ,6 ,6 - tetramethyl- 2H etryn , propazine , simazine , simetryn , terbumeton , terbuth pyran - 3 -one ; 5 - ( Acetyloxy ) -4 - ( 4 - chloro - 4 - ethyl - 2 ' - fluoro ylazin , terbutryn and trietazin , aryl urea such as chlorobro [ 1 , 1' - biphenyl ] - 3 - yl) - 3 , 6 -dihydro - 2 , 2 ,6 , 6 - tetramethyl - 2H muron , chlorotoluron , chloroxuron , dimefuron , diuron , pyran - 3 - one (CAS 1312340 - 82 - 1 ) ; 5 - ( Acetyloxy ) - 4 - ( 2 , 4 ' 35 fluometuron , isoproturon , isouron , linuron , metamitron , dichloro - 4 -ethyl [ 1 , 1 '- biphenyl] - 3 - yl ) - 3 ,6 - dihydro - 2 , 2 , 6 ,6 methabenzthiazuron , metobenzuron , metoxuron , monolin tetramethyl -2H -pyran - 3 -one (CAS 1033760 -55 - 2 ); 4 -( 4 '- uronnyl carbamates , neburon , siduronsuch as , tebuthiurondesmedipham and . karbutilat thiadiazuron , phenme , phe Chloro -4 -cyclopropyl - 2' - fluoro [1 , 1' - biphenyl ] - 3 - yl) - 5 ,6 dipham , phenmedipham -ethyl , nitrile herbicides such as dihydro - 2 , 2 , 6 , 6 - tetramethyl- 5 -oxo - 2H -pyran - 3 -yl carbonic 40 bromofenoxim , bromoxynil and its salts and esters , ioxynil acid methyl ester (CAS 1312337 -51 - 1 ) ; 4 - ( 2' , 4 ' -Dichloro - 4 - and its salts and esters , uraciles such as bromacil , lenacil and cyclopropyl- [ 1, 1' - biphenyl ] - 3 - yl) -5 ,6 -dihydro - 2 ,2 ,6 ,6 -te - terbacil , and bentazon and bentazon -sodium , pyridate , tramethyl - 5 -oxo -2H - pyran - 3 -yl carbonic acid methyl ester; pyridafol, pentanochlor and propanil and inhibitors of the 4 - ( 4 - Chloro - 4 - ethyl- 2 ' - fluoro [ 1 , 1 ' -biphenyl ] - 3 -yl ) - 5 , 6 - di- photosystem I such as diquat , diquat -dibromide , paraquat, hydro - 2 , 2 ,6 , 6 -tetramethyl - 5 -oxo - 2H -pyran - 3 - yl carbonic 45 paraquat- dichloride and paraquat- dimetilsulfate . Among acid methyl ester (CAS 1312340 - 83 - 2 ) ; 4 - ( 2 , 4 '- Dichloro - 4 - these , a preferred embodiment of the invention relates to ethyl[ 1 , 1 ' -biphenyl ] - 3 -yl ) - 5 , 6 -dihydro - 2 , 2 ,6 , 6 - tetramethyl- those compositions comprising at least one aryl urea herbi 5 -oxo -2H -pyran - 3 -yl carbonic acid methyl ester ( CAS cide . Among these , likewise a preferred embodiment of the 1033760 - 58 - 5 ) ; and non ACC herbicides such as benfure - invention relates to those compositions comprising at least sate , butylate , cycloate , dalapon , dimepiperate , EPTC , 50 one triazine herbicide. Among these , likewise a preferred esprocarb , ethofumesate, flupropanate , molinate, orbencarb , embodiment of the invention relates to those compositions pebulate , prosulfocarb , TCA , thiobencarb , tiocarbazil , trial comprising at least one nitrile herbicide ; late and vernolate ; b4 ) from the group of the protoporphyrinogen - IX oxidase b2) from the group of the ALS inhibitors : inhibitors : sulfonylureas such as amidosulfuron , azimsulfuron , bensul- 55 acifluorfen , acifluorfen -sodium , azafenidin , bencarbazone , furon , bensulfuron -methyl , chlorimuron , chlorimuron - ethyl, benzfendizone , bifenox , butafenacil, carfentrazone , carfen chlorsulfuron , cinosulfuron , cyclosulfamuron , ethametsul- trazone - ethyl, chlomethoxyfen , cinidon - ethyl, fluazolate , furon , ethametsulfuron -methyl , ethoxysulfuron , flazasulfu flufenpyr, flufenpyr- ethyl, flumiclorac , flumiclorac -pentyl , ron , flucetosulfuron , flupyrsulfuron , flupyrsulfuron -methyl - flumioxazin , fluoroglycofen , fluoroglycofen - ethyl, fluthi sodium , foramsulfuron , halosulfuron , halosulfuron -methyl , 60 acet, fluthiacet -methyl , fomesafen , halosafen , lactofen , imazosulfuron , iodosulfuron , iodosulfuron -methyl - sodium , oxadiargyl, oxadiazon , oxyfluorfen , pentoxazone , proflua iofensulfuron , iofensulfuron - sodium , mesosulfuron , meta - zol, pyraclonil, pyraflufen , pyraflufen - ethyl , saflufenacil, zosulfuron , metsulfuron , metsulfuron -methyl , nicosulfuron , sulfentrazone , thidiazimin , tiafenacil, ethyl [3 - [2 -chloro -4 orthosulfamuron , oxasulfuron , primisulfuron , primisulfu fluoro - 5 - ( 1 -methyl - 6 - trifluoromethyl- 2 , 4 - dioxo - 1 ,2 , 3 , 4 - tet ron -methyl , propyrisulfuron , prosulfuron , pyrazosulfuron , 65 rahydropyrimidin - 3 - yl) phenoxy ] - 2 -pyridyloxy ]acetate pyrazosulfuron - ethyl, rimsulfuron , sulfometuron , sulfo (CAS 353292 - 31 - 6 ; S - 3100 , N - ethyl- 3 - ( 2 ,6 -dichloro - 4 - trif meturon -methyl , sulfosulfuron , thifensulfuron , thifensulfu - luoromethylphenoxy )- 5 -methyl - 1H -pyrazole - 1 -carboxam US 10 , 392 ,630 B2 179 180 ide (CAS 452098 -92 - 9 ), N -tetrahydrofurfuryl -3 -( 2 ,6 -di chloro - 4 - trifluoromethylphenoxy ) -5 -methyl - 1H -pyrazole II. 1 1 -carboxamide (CAS 915396 -43 -9 ), N - ethyl- 3 -( 2 -chloro -6 F3CFzCN fluoro - 4 - trifluoromethylphenoxy ) - 5 -methyl - 1H -pyrazole - 1 F carboxamide (CAS 452099 -05 -7 ), N - tetrahydrofurfuryl- 3 - 5 9 N – CH, ( 2 - chloro -6 - fluoro - 4 -trifluoromethylphenoxy ) - 5 - methyl H3C / 1H -pyrazole - 1 - carboxamide (CAS 452100 -03 - 7) , 3- [ 7 OCHF2 fluoro - 3 - oxo - 4 - ( prop - 2 -ynyl ) - 3 , 4 - dihydro - 2H -benzo [ 1 , 4 ] HC WN oxazin -6 - yl) - 1 , 5 - dimethyl - 6 - thioxo - [ 1 , 3 , 5 ] triazinan - 2 , 4 II. 2 dione, 1 , 5 - dimethyl- 6 - thioxo - 3 - ( 2 , 2 , 7 - trifluoro - 3 -oxo - 4 - (pr - 10 F3C op - 2 - ynyl) - 3 , 4 -dihydro - 2H - benzo [ b ][ 1 , 4 ] oxazin - 6 - yl) - 1 , 3 , N - CH3 5 - triazinane - 2 , 4 - dione (CAS 1258836 - 72 - 4 ) , 2 - ( 2 , 2 , 7 - Trif luoro - 3 -oxo -4 -prop - 2 -ynyl - 3 ,4 - dihydro -2H -benzo [ 1, 4 ]ox azin - 6 - yl) - 4 , 5 , 6 , 7 - tetrahydro - isoindole - 1 , 3 - dione , 1 -Met - 16 OCHF2 hyl- 6 - trifluoromethyl- 3 - ( 2 , 2 , 7 - trifluoro - 3 -oxo - 4 -prop - 2 HzCb - N ynyl - 3 , 4 - dihydro -2H -benzo [ 1 , 4 ]oxazin - 6 -yl ) - 1H - pyrimi FC. II. 3 dine -2 ,4 - dione ( CAS 1304113 -05 -0 ), methyl (E )- 4 - [ 2 0 chloro -5 -[ 4 -chloro - 5 -( difluoromethoxy )- 1H -methyl - pyra F N - CH3 zol- 3 -yl ) -4 - fluoro -phenoxy ] -3 -methoxy -but - 2 -enoate [ CAS 20 948893 - 00 - 3 ] , and 3 - [ 7 -Chloro - 5 - fluoro - 2 - ( trifluorometh H3C yl) - 1H -benzimidazol - 4 -yl ] - 1 -methyl -6 - ( trifluoromethyl) H3Cb 1H -pyrimidine - 2 , 4 - dione (CAS 212754 - 02 - 4 ); II . 4 F C b5 ) from the group of the bleacher herbicides: 25 N PDS inhibitors : beflubutamid , diflufenican , fluridone , fluro N – CH3 chloridone , flurtamone , norflurazon , picolinafen , and 4 -( 3 trifluoromethylphenoxy ) - 2 - ( 4 - trifluoromethylphenyl) pyri H3C midine (CAS 180608 - 33 - 7 ) , HPPD inhibitors : benzobicy H3C clon , benzofenap , clomazone, isoxaflutole , mesotrione , 30 pyrasulfotole , pyrazolynate , pyrazoxyfen , sulcotrione , tefu 11. 5 ryltrione , tembotrione , topramezone and bicyclopyrone , F3C . bleacher , unknown target : aclonifen , amitrole and flu N - CH3 meturon ; b6 ) from the group of the EPSP synthase inhibitors : 35 H3C glyphosate, glyphosate - isopropylammonium , glyphosate HzCV potassium and glyphosate - trimesium ( sulfosate ) ; II. 6 b7 ) from the group of the glutamine synthase inhibitors : F3C . bilanaphos ( bialaphos ), bilanaphos- sodium , glufosinate , 40 N - CH3 glufosinate - P and glufosinate - ammonium ; H3C b8) from the group of the DHP synthase inhibitors : N F F OCHF2 asulam ; H3C b9 ) from the group of the mitosis inhibitors : 45 Fac, II. 7 compounds of group K1: dinitroanilines such as benfluralin , 0 0 butralin , dinitramine , ethalfluralin , fluchloralin , oryzalin , N - CH3 pendimethalin , prodiamine and trifluralin , phosphorami ?? dates such as amiprophos, amiprophos -methyl , and butami H3C phos , benzoic acid herbicides such as chlorthal , chlorthal- > H3C N F F dimethyl , pyridines such as dithiopyr and thiazopyr, benzamides such as propyzamide and tebutam ; compounds II. 8 of group K2 : chlorpropham , propham and carbetamide , F3C N among these, compounds of group K1, in particular dini F N - CH3 troanilines are preferred ; 55 H ;C b10 ) from the group of the VLCFA inhibitors : T F OCHF2 chloroacetamides such as acetochlor, alachlor, butachlor, H3C ' N F OCHF2 dimethachlor, dimethenamid , dimethenamid - P , metazachlor , II . 9 metolachlor, metolachlor - S , pethoxamid , pretilachlor , 60 F3C propachlor, propisochlor and thenylchlor, oxyacetanilides such as flufenacet and mefenacet, acetanilides such as diphe N – CH3 namid , naproanilide and napropamide , tetrazolinones such H3C fentrazamide , and other herbicides such as anilofos , cafen strole , fenoxasulfone , ipfencarbazone , piperophos , pyroxa - 65 H3C N F F sulfone and isoxazoline compounds of the formulae II . 1 , II . 2 , II. 3 , II . 4 . II . 5 , II. 6 , II . 7 , II . 8 and II . 9 US 10 , 392 ,630 B2 181 182 the isoxazoline compounds of the formula (I ) I are known in 2H -pyran -3 -one (CAS 1312340 -82 - 1 ) ; 5 - ( Acetyloxy ) -4 - ( 2 , the art , e . g . from WO 2006 / 024820 , WO 2006 / 037945 , WO 4 ' -dichloro - 4 -ethyl [ 1 , 1 '- biphenyl ] - 3 - yl) - 3 ,6 - dihydro - 2 , 2 , 6 , 2007 /071900 and WO 2007/ 096576 ; 6 - tetramethyl- 2H -pyran -3 -one (CAS 1033760 -55 - 2 ) ; 4 - (4 ' among the VLCFA inhibitors , preference is given to chlo Chloro - 4 -cyclopropyl - 2 - fluoro [ 1 , 1 ' - biphenyl] - 3 - yl ) - 5 , 6 roacetamides and oxyacetamides ; dihydro - 2 , 2 , 6 ,6 -tetramethyl - 5 -oxo - 2H -pyran - 3 -yl carbonic b11 ) from the group of the cellulose biosynthesis inhibitors: acid methyl ester (CAS 1312337 -51 - 1 ) ; 4 - ( 2 , 4 - Dichloro - 4 chlorthiamid , dichlobenil, flupoxam , indaziflam , triaziflam , cyclopropyl- [ 1 , 1' - biphenyl ]- 3 - yl ) - 5 ,6 -dihydro -2 , 2, 6 ,6 - te isoxaben and 1 -Cyclohexyl -5 -pentafluorphenyloxy - 14- [ 1, 2 , tramethyl- 5 -oxo - 2H -pyran - 3 - yl carbonic acid methyl ester ; 4 ,6 ] thiatriazin - 3 -ylamine ; 4 - (4 '- Chloro -4 -ethyl - 2 '- fluoro [ 1, 1 '- biphenyl ] - 3 -yl ) -5 , 6 - di b12 ) from the group of the decoupler herbicides: 10 hydro - 2 , 2 ,6 ,6 - tetramethyl - 5 -oxo - 2H - pyran - 3 - yl carbonic dinoseb , dinoterb and DNOC and its salts ; acid methyl ester (CAS 1312340 - 83 - 2 ) ; 4 - ( 2 , 4 ' - Dichloro - 4 b13 ) from the group of the auxinic herbicides : ethyl[ 1 , 1 '- biphenyl ] - 3 - yl) - 5 ,6 - dihydro - 2 , 2 , 6 , 6 - tetramethyl 2 ,4 - D and its salts and esters such as clacyfos , 2 , 4 -DB and 5 -oxo - 2H -pyran - 3 -yl carbonic acid methyl ester (CAS its salts and esters , aminocyclopyrachlor and its salts and 1033760 -58 - 5 ) ; benfuresate , dimepiperate , EPTC , espro esters , aminopyralid and its salts such as aminopyralid - tris 15 carb , ethofumesate , molinate , orbencarb , prosulfocarb , thio ( 2 - hydroxypropyl) ammonium and its esters, benazolin , bencarb and triallate ; benazolin -ethyl , chloramben and its salts and esters , clom - b2 ) from the group of the ALS inhibitors: eprop , clopyralid and its salts and esters , dicamba and its amidosulfuron , azimsulfuron , bensulfuron -methyl , bispyri salts and esters , dichlorprop and its salts and esters , dichlo - bac - sodium , chlorimuron - ethyl, chlorsulfuron , cloransulam rprop - P and its salts and esters , fluroxypyr , fluroxypyr - 20 methyl, cyclosulfamuron , diclosulam , ethametsulfuron butometyl, fluroxypyr- meptyl , halauxifen and its salts and methyl , ethoxysulfuron , flazasulfuron, florasulam , esters (CAS 943832 - 60 - 8 ) ; MCPA and its salts and esters , flucarbazone -sodium , flucetosulfuron , flumetsulam , flupyr MCPA - thioethyl, MCPB and its salts and esters , mecoprop sulfuron -methyl - sodium , foramsulfuron , halosulfuron and its salts and esters , mecoprop - P and its salts and esters , methyl, imazamethabenz - methyl, imazamox , imazapic , picloram and its salts and esters , quinclorac , quinmerac , 25 imazapyr, imazaquin , imazethapyr, imazosulfuron , iodosul TBA ( 2 , 3 , 6 ) and its salts and esters and triclopyr and its salts furon , iodosulfuron -methyl - sodium , iofensulfuron , iofensul and esters; furon -sodium , mesosulfuron , metazosulfuron , metosulam , b14 ) from the group of the auxin transport inhibitors : metsulfuron -methyl , nicosulfuron , orthosulfamuron , oxasul diflufenzopyr, diflufenzopyr -sodium , naptalam and napta - furon , penoxsulam , primisulfuron -methyl , propoxycarba lam - sodium ; 30 zone - sodium , propyrisulfuron , prosulfuron , pyrazosulfuron b15 ) from the group of the other herbicides : bromobutide, ethyl, pyribenzoxim , pyrimisulfan , pyriftalid , pyriminobac chloroflurenol, chloroflurenol- methyl , cinmethylin , cumy methyl, pyrithiobac - sodium , pyroxsulam , rimsulfuron , luron , cyclopyrimorate (CAS 499223 - 49 - 3 ) and its salts and sulfometuron -methyl , sulfosulfuron , thiencarbazone esters , dalapon , dazomet, difenzoquat, difenzoquat- metilsul - methyl, thifensulfuron - methyl, triasulfuron , tribenuron fate, dimethipin , DSMA, dymron , endothal and its salts , 35 methyl, trifloxysulfuron , triflusulfuron -methyl , tritosulfuron etobenzanid , flamprop , flamprop - isopropyl, flamprop and triafamone; methyl, flamprop - M - isopropyl , flamprop - M -methyl , fluore - b3) from the group of the photosynthesis inhibitors: nol, fluorenol- butyl , flurprimidol, fosamine , fosamine -am - ametryn , amicarbazone, atrazine, bentazone , bentazone - so monium , indanofan , indaziflam , maleic hydrazide , dium , bromoxynil and its salts and esters, chloridazone , mefluidide, metam , methiozolin ( CAS 403640 - 27 - 7 ) , 40 chlorotoluron , cyanazine , desmedipham , diquat- dibromide , methyl azide , methyl bromide , methyl -dymron , methyl diuron , fluometuron , hexazinone , ioxynil and its salts and iodide, MSMA , oleic acid , oxaziclomefone , pelargonic acid , esters, isoproturon , lenacil, linuron , metamitron , methaben pyributicarb , quinoclamine , triaziflam and tridiphane. zthiazuron , metribuzin , paraquat, paraquat- dichloride , phen Preferred herbicides B that can be used in combination medipham , propanil , pyridate , simazine , terbutryn , terbuth with the PPO - inhibiting herbicides according to the present 45 ylazine and thidiazuron ; invention are : b4 ) from the group of the protoporphyrinogen - IX oxidase bl) from the group of the lipid biosynthesis inhibitors : inhibitors : clethodim , clodinafop -propargyl , cycloxydim , cyhalofop - acifluorfen , acifluorfen -sodium , azafenidin , bencarbazone , butyl, diclofop -methyl , fenoxaprop - P - ethyl, fluazifop - P -bu - benzfendizone , butafenacil, carfentrazone- ethyl, cinidon tyl , haloxyfop - P -methyl , metamifop , pinoxaden , profoxy - 50 ethyl, flufenpyr- ethyl, flumiclorac - pentyl, flumioxazin , fluo dim , propaquizafop , quizalofop - P - ethyl, quizalofop - P - roglycofen - ethyl, fluthiacet -methyl , fomesafen , lactofen , tefuryl, sethoxydim , tepraloxydim , tralkoxydim , 4 - 4 '- oxadiargyl, oxadiazon , oxyfluorfen , pentoxazone , pyra Chloro -4 -cyclopropyl - 2' - fluoro [ 1, 1' - biphenyl] - 3 -yl ) - 5 . flufen - ethyl, saflufenacil , sulfentrazone, ethyl [3 - [ 2 -chloro hydroxy - 2 , 2 ,6 , 6 - tetramethyl- 2H - pyran - 3 (6H ) -one (CAS 4 - fluoro - 5 - ( 1 - methyl- 6 - trifluoromethyl- 2 , 4 -dioxo - 1 , 2 , 3 , 4 1312337 -72 -6 ); 4 -( 2 ,4 -Dichloro -4 -cyclopropyl [ 1 , 1' - biphe - 55 tetrahydropyrimidin -3 - yl) phenoxy ]- 2 -pyridyloxy ]- acetate nyl] - 3 -yl ) - 5 - hydroxy - 2 , 2 , 6 , 6 - tetramethyl - 2H -pyran - 3 (6H ) - (CAS 353292 -31 - 6 ; S - 3100 ) , N - ethyl - 3 - ( 2 ,6 - dichloro - 4 - tri one ( CAS 1312337 -45 - 3 ); 4 -( 4 -Chloro -4 - ethyl- 2 '- fluoro [ 1 , fluoromethylphenoxy )- 5 -methyl - 1H -pyrazole - 1 -carboxam 1 '- biphenyl ] - 3 - yl) - 5 -hydroxy - 2 , 2 , 6 , 6 - tetramethyl- 2H ide (CAS 452098 - 92 - 9 ), N -tetrahydrofurfuryl -3 - (2 , 6 - di pyran - 3 (6H ) - one (CAS 1033757 - 93 - 5 ) ; 4 -( 2 , 4 '- Dichloro -4 - chloro - 4 - trifluoromethylphenoxy ) - 5 -methyl - 1H -pyrazole ethyl[ 1 , 1 ' -biphenyl ] - 3 -yl ) - 2 , 2 , 6 ,6 - tetramethyl- 2H -pyran - 3 , 5 60 1 - carboxamide (CAS 915396 - 43 - 9 ) , N - ethyl- 3 - ( 2 - chloro -6 (4H ,6H ) -dione (CAS 1312340 - 84 - 3 ) ; 5 - Acetyloxy ) - 4 - ( 4 fluoro - 4 - trifluoromethylphenoxy ) - 5 -methyl - 1H -pyrazole - 1 chloro - 4 - cyclopropyl- 2 '- fluoro [ 1 , 1' -biphenyl ] - 3 -yl ) - 3 , 6 - carboxamide (CAS 452099 - 05 - 7 ), N - tetrahydrofurfuryl- 3 dihydro - 2 ,2 ,6 , 6 -tetramethyl - 2H -pyran - 3 -one (CAS (2 - chloro -6 - fluoro -4 - trifluoromethylphenoxy )- 5 -methyl 1312337 - 48 - 6 ) ; 5 - ( Acetyloxy ) - 4 - ( 2 , 4 ' - dichloro - 4 -cyclopro - 1H -pyrazole - 1 - carboxamide (CAS 452100 -03 - 7 ) , 3 - [ 7 pyl- [ 1 , 1' - biphenyl ] - 3 - yl) - 3 ,6 -dihydro -2 ,2 ,6 ,6 -tetramethyl - 65 fluoro - 3 -oxo -4 - (prop - 2 -ynyl ) - 3 ,4 -dihydro - 2H -benzo [ 1, 4 ] 2H -pyran - 3 - one; 5 - ( Acetyloxy ) - 4 - (4 - chloro - 4 - ethyl- 2 ' oxazin - 6 - yl] - 1 , 5 - dimethyl- 6 -thioxo - [ 1 , 3 , 5 ] triazinan - 2 , 4 fluoro [ 1 , 1' - biphenyl ] - 3 - yl ) - 3 ,6 - dihydro - 2 , 2 ,6 , 6 - tetramethyl dione, 1 , 5 -dimethyl - 6 - thioxo - 3 - ( 2 , 2 ,7 -trifluoro - 3 -oxo - 4