US010294500B2 (12 ) United States Patent ( 10) Patent No. : US 10 ,294 , 500 B2 Sato et al. (45 ) Date of Patent: * May 21, 2019 (54 ) METHOD FOR PRODUCING (58 ) Field of Classification Search METHACRYLIC ACID AND /OR ESTER None THEREOF See application file for complete search history . (71 ) Applicant: Mitsubishi Chemical Corporation , ( 56 ) References Cited Tokyo ( JP ) U . S . PATENT DOCUMENTS (72 ) Inventors: Eiji Sato , Kanagawa (JP ) ; Michiko 8 ,241 , 877 B2 * 8 /2012 Burgard C12P 7 / 40 Yamazaki, Kanagawa ( JP ) ; Eiji 435 / 136 Nakajima, Kanagawa ( JP ) ; Fujio Yu , 8 , 501, 455 B2 8 / 2013 Gokarn et al. Kanagawa ( JP ); Toshio Fujita , 203014881 8 / 2003 DiCosimo et al. 2004 / 0076982 Al 4 / 2004 Gokarn et al . Kanagawa (JP ); Wataru Mizunashi, 2007 / 0184524 Al 8 / 2007 Gokarn et al. Kanagawa ( JP ) 2008 / 0076167 A1 3 / 2008 Gokarn et al. 2009 /0053783 Al 2 /2009 Gokarn et al. (73 ) Assignee : Mitsubishi Chemical Corporation , 2009 /0130729 A1 * 5 / 2009 Symes .. .. C12P 7 /62 Tokyo ( JP ) 435 / 135 2009 /0275096 AL 11 /2009 Burgard et al . 2010 / 0035314 A1 2 / 2010 Mueller et al. ( * ) Notice : Subject to any disclaimer, the term of this 2011/ 0165640 A17 / 2011 Mueller et al. patent is extended or adjusted under 35 2012 /0077236 AL 3 /2012 Gokarn et al. U . S . C . 154 (b ) by 252 days . 2012 /0276604 AL 11 / 2012 Burgard et al. 2012 /0276605 Al 11/ 2012 Burgard et al . This patent is subject to a terminal dis 2013/ 0065279 A1* 3/ 2013 Burk . . . C12P 19 / 32 claimer . 435 / 88 (21 ) Appl. No. : 14 /419 ,575 FOREIGN PATENT DOCUMENTS (22 ) PCT Filed : Sep. 10 , 2013 CA 2688292 A1 12 /2008 EP 0187680 A2 7 / 1986 0502476 A2 9 / 1992 ( 86 ) PCT No. : PCT/ JP2013 / 005359 2868648 A1 5 / 2015 $ 371 (c )( 1 ), 54 - 046887 4 / 1979 ( 2 ) Date : Feb . 4 , 2015 ??HE 61- 162191 A 7 / 1986 ( Continued ) ( 87) PCT Pub . No. : WO2014 /038216 PCT Pub. Date : Mar. 13 , 2014 OTHER PUBLICATIONS Office Action dated Jun . 28 , 2016 in Japanese Patent Application (65 ) Prior Publication Data No . 2013 -546110 . US 2015 /0191756 A1 Jul. 9 , 2015 Edwige J . F . Souleyre, et al ., " An alcohol acyl transferase from apple (cv . Royal Gala ) , MPAAT1 , produces esters involved in apple ( 30 ) Foreign Application Priority Data fruit flavor ” , FEBS Journal, vol. 272, No . 12 , 2005 , pp . 3132 -3144 . Sep . 10 , 2012 ( JP ) 2012 - 198840 (Continued ) Sep . 10 , 2012 ( JP ) . . . 2012 - 198841 Primary Examiner — Suzanne M Noakes Jan . 31 , 2013 ( JP ) 2013 -016947 ( 74 ) Attorney, Agent, or Firm — Oblon , McClelland , Jul. 30 , 2013 ( JP ) ...... 2013 - 157306 Aug . 1 , 2013 ( JP ) 2013 - 160300 Maier & Neustadt, L .L .P . Aug . 1 , 2013 ( JP ) ...... 2013 - 160301 (57 ) ABSTRACT Aug . 20 , 2013 ( JP ) ...... 2013 - 170404 To provide a method for directly and efficiently producing ( 51 ) Int. Cl. methacrylic acid in a single step from renewable raw mate C12P 7 /62 ( 2006 .01 ) rials and / or biomass arising from the utilization of the C12N 9 / 10 ( 2006 . 01 ) renewable raw materials. Further provided is a method for C12N 9 /00 ( 2006 . 01 ) producing methacrylic acid using microbes having the abil C07C 57/ 04 ( 2006 . 01 ) ity to produce methacrylic acid , from renewable raw mate C07C 69 /54 ( 2006 .01 ) rials and / or biomass arising from the utilization of the C12P 7740 ( 2006 . 01 ) renewable raw materials , as a carbon source and / or energy C12N 9 / 02 ( 2006 .01 ) source . The method for producing methacrylic acid enables (52 ) U . S . CI. methacrylic acid to be safely and easily produced from CPC ...... C12P 7762 (2013 .01 ); C07C 57 /04 biomass , without using petroleum - derived raw materials , by ( 2013 . 01 ); C07C 69 / 54 (2013 .01 ) ; C12N 9 /001 utilizing microbes having the ability to produce methacrylic ( 2013 .01 ) ; C12N 9 / 1029 (2013 . 01) ; C12P 7 /40 acid . (2013 .01 ) ; C12Y 103 /99 (2013 .01 ) ; C12Y 103 / 99003 (2013 .01 ) ; C12Y 203 /01084 5 Claims, 7 Drawing Sheets (2013 .01 ) Specification includes a Sequence Listing . US 10 ,294 ,500 B2 Page 2
References Cited Database UniProt [ online ] , Accession No . G2UBR1, Definition : ( 56 ) Probable acyl- CoA dehydrogenase, http : // www . uniprot . org/ uniprot/ GRUBR1, Nov . 16 , 2011 uploaded [retrieved Nov. 27 , 2013 ], pp . FOREIGN PATENT DOCUMENTS 1 - 3 Database UniProt [online ], Accession No. C3J12 , Definition : Isobutyryl 62 -014788 1187 COA dehydrogenase, http : // www .uniprot . org /uniprot / C3J112 , Jun . 01 - 132392 5 / 1989 16 , 2009 uploaded [retrieved on Nov . 27 , 2013 ], pp . 1 -3 . 5 -64589 3 / 1993 Database UniProt [ online ] , Accession No . M2XPW5, Definition : 10 - 337185 A 12 / 1998 Acyl- CoA dehydrogenase , http : / /www .uniprot .org / uniprot / 11 - 243957 A 9 / 1999 2004 - 514431 A 5 /2004 M2XPW5, May 1 , 2013 uploaded [ retrieved Nov . 27 , 2013 ] , pp . 2005 - 516622 A 6 / 2005 1 - 3 . 2007 - 54011 A 3 / 2007 Sang -Hyun Pyo , et al. , “ A new route for the synthesis of methacrylic 2009 - 538118 11 / 2009 acid from 2 -methyl - 1 , 3 - propanediol by integrating biotransforma 2010 - 528597 A 8 / 2010 tion and catalytic dehydration ," Green Chemistry , vol . 14 , 2012 , pp . 2011 -519561 A 7 / 2011 1942 - 1948 2011 / 200133 A 10 / 2011 Mark R . Smith , et al . , “ The Utilization of 3 -Mercapto - 2 wo 00 / 32789 A1 6 / 2000 Methylpropionate as Sulphur Source by a Phototropic Bacterium , " WO 2002 /42418 A2 5 /2002 Bioorganic & Medicinal Chemistry , vol. 2 , No . 7 , 1994 , pp . 589 WO 2003 /066872 A1 8 / 2003 593 . WO 2007 /039415 A1 4 / 2007 WO 2007 / 110394 A2 10 / 2007 Jerry Vockley , et al. , “ Mammalian Branched - Chain Acyl -COA WO 2008 / 145737 Al 12 / 2008 Dehydrogenases :Molecular Cloning and Characterization of Recom WO 2009 / 135074 A2 11 / 2009 binant Enzymes, ” Methods in Enzymology, vol. 324 , 2000 , pp . WO 2011 /031897 Al 3 / 2011 241 - 258 WO 2012 / 135789 A2 10 / 2012 Lori A . Maggio - Hall, et al. , “ A Single Acyl- CoA Dehydrogenase Is WO 2013 / 044076 Al 3 / 2013 Required for Catabolism of Isoleucine , Valine and Short - Chain WO WO 2014 / 071289 Al 5 / 2014 Fatty Acids in Aspergillus nidulans, " Fungal Genet Biology , vol . 45 (3 ), Mar. 2008 , pp . 180 - 189 . Ying -Xin Zhang , et al. , “ Genes encoding acyl- CoA dehydrogenase OTHER PUBLICATIONS ( AcdH ) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched J . Michael Shaw , et al. , “ Phosphatidylmonoglucosyl Diacylglycerol chain fatty acids and macrolide antibiotic production ,” Microbiol of Pseudomonas diminuta ATCC 11568” , Journal of Biological ogy, vol . 145 , 1999 , pp . 2323 -2334 . Chemistry , vol. 252 , No. 12 , 1977 , pp . 4395 -4401 . Linda K . Massey , et al. , “ Branched - Chain Amino Acid Catabolism Takashi Yamada, et al. , “ Biodegradation of 2 ,4 ,6 - Tribromophenol in Bacteria ,” Bacteriological Reviews, vol. 40 , No . 1 ., Mar. 1976 , by Ochrobactrum sp . Strain TB01 " , Biosci Biotechnol Biochem , pp. 42 -54 . vol. 72 , No. 5 , 2008 , p . 1264 - p . 1271 . Zuzana Swigonova, et al. , “ Acyl- CoA Dehydrogenases: Dynamic Database Uniprot [online ] , Accession No . Q91513 , < http : / /www . History of Protein Family Evolution , " J . Mol . Evol. , vol. 69 , 2009 , uniprot. org /uniprot / Q91513 .txt ?version = 72) , uploaded on May 29 , pp . 176 - 193 . 2013 , [ retrieved on Jun . 20 , 2016 ]. Kathryn L . Hester , et al . , " Purification of Pseudomonas putida Database Uniprot [online ], Accession No . G7EWVO,
FIG .1
SCOA SCOA OR 2 - oxoisovaleric acid isobutyryl -CoA methacrylyl - COA methacrylic ? ?????? acid ester atent May 21 , 2019 Sheet 2 of 7 US 10 ,294 ,500 B2
FIG . 2 . 0190100101
17.151 Itt
14.314
13.694 1AMY:1411111
112012101210011001000
wW.
0
0
D1
-W
Abundance 130000 110000 10000 10008 80000! Hoooo 0000 10000 imeH U . S . Patent May 21, 2019 Sheet 3 of 7 US 10 ,294 ,500 B2
.
.v
w
w
. w
.
FIG . 3 .
.
. 240220230
.
"
;?.
.-:7*?
.
W"YA.-
.' /3w." .
*1
.
V 145157167176185192205 210170180190200140150160 *99 .S
.
.
*.
.*
>
.wy-wwWw
. 130
I
110120130100. 13011911110497104111119 . xww. w
.
:
-w
S . 90
.
ww w .
of . . 79 80 .
.
. .
. - . . 69 70
Y .- 60
.
.!
W
W stlui) 5040
17-1W
30 : 29
.
. 20 4000 3000 2000 1000 Abundance m/z-> U . S . Patent May 21, 2019 Sheet 4 of 7 US 10 ,294 ,500 B2
FIG . 4
KmR LigD homolog downstream / h
PTJ002 8 . 8kbp sacB LigD homolog upstream rep _origin U . S . Patent May 21, 2019 Sheet 5 of 7 US 10 ,294 ,500 B2
FIG .5
upstream downstream sacB Km sequence of the sequence of the sacB Km target gene target gene In - Fusion _ 3 oriv oriv BamHI Bm gene - destroying plasmid plasmid PCR (pK19sacB1 ) target gene -
Fw1: sequence upstream from Bambi in the vector and sequence 1Kb PCR product 1 2 upstream from the target gene are contained (fragment 1 ) Rv1: surrounding sequences of 5 ' and 3 ' of the target gene are contained DCP productor Fw2 : surrounding sequences of 5 ' and 3 of the target gene are contained (fragment 2 ) Rv2 : sequence 1Kb -downstream from the target gene and sequence upstream from BamHI in the vector are contained U . S . Patent May 21, 2019 Sheet 6 of 7 US 10 ,294 , 500 B2
FIG .6
Sse83871 Sse83871 Nitrilase Nitrilase MPAATI PACDAAT1 promoter ACDI promoter ( native codon ) Sse83871 Sse83871 Nitrilase Nitrilase GST MpAAT1 PACDAAT2 promoter ACD1 promoter tag ( native codon ) Sse83871 Sse83871 Nitrilase Nitrilase MPAAT1 PACDAAT3 promoter ACD1 promoter (modified codon ) WMMMMMM Sse83871 Sse83871 Nitrilase Nitrilase GST MPAAT1 PACDAAT4 promoter ACD1 promoter tag (modified codon ) Sse83871 Nitrilase GST MPAATI PACDAATSPACDAAT6 - elementepromoter ACD1acolo tag (native spadre codon ) I Sse83871 Nitrilase GST MPAATI PACDAATS promoter ACD1 | tag (modified codon ) U . S . Patent May 21, 2019 Sheet 7 of 7 US 10 ,294 ,500 B2
FIG . 7
MH piros Pa_ add PA _ add ? Pl _ acds Ph _ addy cTreba RE _acdi FE acct 45kDa 45kDa
Play DET19b RE aodi RE_ 20d2 We . . RE1196 PA acdi PA acda PA acd3 På _acchi 45kDa
(C ) W pire93A ( pircaoft ) ET19b ac # 45 Date este US 10 , 294 , 500 B2
METHOD FOR PRODUCING an ester produced is basically dissociated due to hydrolysis METHACRYLIC ACID AND /OR ESTER activity , and thus it is difficult to be considered as an THEREOF effective production method . Meanwhile , alcohol acyl transferase (hereinbelow , TECHNICAL FIELD 5 “ ATT ” ) is known as an enzyme for synthesis of fruity flavor. In Patent Document 7 , a method for producing various esters s to as fruit flavor by identifying the gene of the same enzyme, methacrylic acid and /or an ester thereof. More specifically , which is contained in specific fruits , is suggested . However, it relates to a method for producing methacrylic acid using the possibility of synthesizing a methacrylic acid ester with microbes having the ability to produce methacrylic acid , the 10 the enzyme is not described and remains completely uncer microbes, methacrylic acid obtained by the production tain . As described above, although several suggestions or method , and the like . discussions have been made , there is no example showing BACKGROUND ART actual production of methacrylic acid derivatives using 15 microbes, and thus an effective production method needs to Methacrylic acid is useful as a raw material for paint or be established . resin modifying agent , and its esters are an industrially significantly important compound as a raw material for CITATION LIST acrylic resin . As a method for chemical production ofmethacrylic acid 20 Patent Document derivatives , the ACH method intermediated by acetone Patent Document 1 : WO 2007 / 110394 A cyanhydrin using cyanide and acetone as a raw material, the Patent Document 2 : WO 2008 / 145737 A C4 oxidation using isobutylene or tert -butanol as a raw Patent Document 3 : WO 2009 / 135074 A material, and the like are practically used . Those chemical 25 Patent Document 4 : WO 2011 /031897 A production methods rely on fossil fuels and also require a Patent Document 5 : WO 2012 / 135789 A large amount of energy . Patent Document 6 : WO 2007 /039415 A Recently , from the viewpoint of preventing global warm - Patent Document 7 : WO 00 / 32789 A ing and environmental protection , use of renewable biog - Patent Document 8 : JP 2011 -519561 A enous resources ( renewable raw materials ) as a carbon 30 Patent Document 9 : JP 2011 -200133 A source to be a substitute of fossil fuels of the related art Patent Document 10 : JP 5 -64589 A receives attention . For example , a method for producing Patent Document 11 : JP 10 - 337185 A 2 - hydroxyisobutyric acid and 3 - hydroxyisobutyric acid , which are a precursor of methacrylic acid , from a natural Non -Patent Document product such as sugar using microbes existing in nature has Non -Patent Document 1 : Green Chemistry , 2012 , 14 , 1942 been suggested ( see , Patent Documents 1 and 2 and Non 1948 Patent Document 1 ) . However , those methods still rely on a Non -Patent Document 2 : Bioorganic & Medicinal Chemis chemical method in terms of the process for producing try , 1994 , 7 , 589 -593 methacrylic acid by dehydration of a precursor. . 40 Non -Patent Document 3 : Methods in Enzymology, 2000 , Further, although a method for producing methacrylic 324 , 241- 258 acid from glucose using a recombinant microbe introduced Non - Patent Document 4 : Fungal Genet. Biol. , 2008 . 45 . with plural enzyme genes, which is not exist in nature, has 180 - 189 been suggested , it is a mere suggestion of simple combina - Non - Patent Document 5 : Microbiology , 1999 , 145 , 2323 tion between known enzyme reactions and imaginary 45 2334 enzyme reactions deduced therefrom , and has not been Non - Patent Document 6 : Bacteriol. Review , 1976 , 40 , 42 - 54 practically proved ( see, Patent Documents 3 to 5 ) . ( 1976 ) Meanwhile , as a naturally occurring microbe, a photo - Non -Patent Document 7: J. Mol. Evol. , 2009, 69, 176 - 193 synthetic microbe having the ability to produce methacrylic Non - Patent Document 8 : Methods in Enzymology , 2000 , acid is known (Non -Patent Document 2 ) . However, the 50 324, 129 - 138 photosynthetic microbe only converts 3 -mercaptoisobutyric acid , which is a special compound not existing in nature, to DISCLOSURE OF THE INVENTION methacrylic acid . Further, in Patent Document 6 , a method for producing an Problem to be Solved by the Invention acrylic acid ester according to an action of a hydrolase in the 55 presence of Acryloyl- CoA and an alcohol is disclosed . It is According to a chemical production method of the related suggested in the same literature that a methacrylic acid ester art, for example , the ACH method , an oxidation treatment of can be also similarly produced . However, considering diver- acetone cyanhydrin with an acid is required so that a waste sity and substrate specificity of an enzyme, it only demon - acid is generated in a large amount. Further , as separation or strates that an acrylic acid ester can be produced with few 60 purification is required for each step , there is a problem of limited hydrolases, and it remains uncertain whether or not having high energy consumption . For such reasons, a a methacrylic acid ester having a different structure can be method for directly and efficiently producing methacrylic also similarly produced by the hydrolase . Further, it remains acid in a single step using microbes capable of producing completely uncertain whether or not production can be made methacrylic acid from renewable raw materials and /or bio with a different kind of enzyme having a different reaction 65 mass arising from the utilization of the renewable raw mechanism . Further, when an ester is synthesized with the materials , as a carbon source and / or energy source , is hydrolase described in PatentDocument 6 , it is expected that needed . US 10 , 294 ,500 B2 Means for Solving Problem ( 2 ) The production method of ( 1) , in which methacrylic acid and / or an ester thereof is accumulated at 0 .04 ppm or more . To solve the problems described above , inventors of the ( 3 ) The production method of ( 1 ) or (2 ), in which meth invention conducted extensive searches for microbes having acrylic acid and / or an ester thereof is produced using the the ability to produce methacrylic acid derivatives, and as a 5 microbes having the ability to produce methacrylic acid result , found that methacrylic acid can be produced with from renewable raw materials and / or biomass arising from many microbes . the utilization of the renewable raw materials as a carbon Further , as described below , the inventors found that the source and / or energy source . microbes having the ability to produce methacrylic acid (4 ) The production method of any one of ( 1 ) to ( 3 ) , in which derivatives are a group of microbes having a certain specific 10 the microbes having the ability to produce methacrylic acid function and a constant common property , and they also are microbes capable of producing methacrylic acid from succeeded in finding a method for searching them . organic matters containing isobutyric acid or valine . First , the inventors of the invention tried to produce ( 5 ) The production method of any one of ( 1 ) to ( 4 ), in which methacrylic acid derivatives , which are an intermediate of the microbes having the ability to produce methacrylic acid the valine metabolism pathway, from 2 -oxoisovaleric acid 15 are microbes having 16S rRNA gene which consists of a based on a microbial reaction by producing recombinant E . nucleotide sequence showing an identity of 81 % or more to coli which has been introduced with a gene of the valine any one of nucleotide sequences described in SEQ ID NOS . metabolism pathway ( branched chain keto acid dehydroge - 1 to 41 . nase (hereinbelow , “ BCKAD ” ) and isobutyryl- CoA dehy - (6 ) The production method of any one of ( 1 ) to ( 4 ), in which drogenase (hereinbelow , “ ACD " ) ) . However, production of 20 the microbes having the ability to produce methacrylic acid the methacrylic acid derivatives as a target product was not are microbes having LSUrRNA gene which consists of a observed while production of isobutyric acid was observed . nucleotide sequence showing an identity of 81 % or more to Further, when a cell extract is prepared from the recombi- any one of nucleotide sequences described in SEQ ID NOS . nant and the in vitro activity of BCKAD and ACD is 42 to 49 . measured after adding a cofactor or the like , coactivity was 25 ( 7 ) The production method of any one of ( 1 ) to ( 6 ), in which observed . Based on such results , it was determined that the the microbes having the ability to produce methacrylic acid BCKAD reaction , but not the ACD reaction , has occurred are microbes belonging to the genus Pseudomonas, the under in vivo conditions. genus Bacillus , the genus Sphingobacterium , the genus In mammals , it is known that the dehydrogenase reaction Comamonas , the genus Brevundimonas, the genus Sphin known as metabolism of branched amino acid or ß -oxidation 30 gomonas, the genus Ochrobactrum , the genus Pedobacter , of fatty acid occurs in a mitochondria and liberated electrons the genus Paenibacillus , the genus Achromobacter , the are transferred to a respiratory chain via an electron trans - genus Acinetobacter , the genus Shewanella , the genus Listo ferring flavoprotein . In other words , it was assumed that the nella , the genus Agrobacterium , the genus Mesorhizobium , main reaction does not occur only by adding foreign ACD to the genus Rhizobium , the genus Paracoccus, the genus a microbe which does not have the same function as them . 35 Xanthobacter , the genus Streptomyces , the genus Geobacil The ACD reaction actually did not occur in the recombinant lus , the genus Rhodococcus , the genus Saccharomyces , the E . coli as described above . genus Candida , or the genus Aspergillus. To solve this new problem , the inventors of the invention (8 ) The production method of any one of (1 ) to (7 ) , in which conducted extensive searches for the microbes having the a methacrylic acid ester is produced by having an alcohol or ability to produce methacrylic acid derivatives by microbes, 40 a phenol to act on methacrylyl- CoA in the presence ofAAT . and found that the problem can be solved using microbes ( 9 ) The production method of ( 8 ) , in which AAT is derived capable of producing methacrylic acid and microbes capable from a plant. of metabolizing isobutyryl - CoA as a substrate of ACD . ( 10 ) The microbes having the ability to produce methacrylic Examples of the microbes capable of metabolizing isobu - acid of ( 7 ) . tyryl -CoA include valine assimilating viable microbes and 45 ( 11 ) An enzyme involved with synthesis ofmethacrylic acid , isobutyric acid assimilating viable microbes . Those which is derived from the microbes of ( 10 ) . microbes can be obtained from nature by growing them with (12 ) A gene encoding the enzyme of (11 ) . use of valine as a sole carbon source and /or a nitrogen source ( 13 ) Methacrylic acid and / or an ester thereof produced by or isobutyric acid as a sole carbon source . microbes having the ability to produce methacrylic acid The inventors of the invention further found that, by 50 from renewable raw materials and / or biomass arising from adding the action of AAT to the microbes having the ability the utilization of the renewable raw materials as a carbon to produce methacrylic acid , a methacrylic acid ester can be source and / or energy source . produced in the presence of an alcohol. ( 14 ) Culture product of microbes containing the microbes In addition , the inventors of the invention intensively having the ability to produce methacrylic acid and meth studied the enzymes involved with methacrylic acid synthe - 55 acrylic acid and / or an ester thereof. sis , which are derived from the aforementioned microbes , (15 ) The enzyme of ( 11 ) which consists of a protein selected and genes which encode the enzymes . As a result , they found from the group consisting of the following ( a ) to ( c ) : that there was an enzyme useful for production of meth - ( a ) a protein which consists of an amino acid sequence acrylic acid derivatives and succeeded in characterizing the represented by SEQ ID NO . 50 or 52 enzyme protein . They also found that, according to combi- 60 ( b ) a protein which consists an amino acid sequence show nation of those enzyme proteins , synthesis of methacrylic i ng an identity of 45 % or more to a protein consisting of an acid derivatives can be achieved . Accordingly, the invention amino acid sequence represented by SEQ ID NO . 50 or 52 was completed . Specifically , the invention is as described and which has the ACD activity , and below . ( c ) a protein which consists of an amino acid sequence with ( 1 ) A method for producing methacrylic acid and / or an ester 65 deletion , substitution , or addition of one or several amino thereof using microbes having the ability to produce meth - acids in an amino acid sequence represented by SEQ ID NO . acrylic acid . 50 or 52 and which has the ACD activity . US 10 ,294 , 500 B2 ( 16 ) A method for producing methacrylic acid and / or an from plants belonging to the genus Musaceae , the genus ester thereof including using the enzyme of ( 15 ) . Malus, the genus Pyrus, the genus Bower actinidia , the ( 17 ) The production method of any one of ( 1 ) to ( 7 ) , in genus Cucumis , the genus Carica , or the genus Persea . which the microbes having the ability to produce meth (13A ) The production method for producing a methacrylic acrylic acid are microbes belonging to the genus Sphingo - 5 acid ester of (5A ), in which the plant is any one selected bacterium , the genus Comamonas, the genus Brevundimo from the group consisting of banana, apple , Japanese apri nas, the genus Sphingomonas, the genus Ochrobactrum , the genus Pedobacter, the genus Paenibacillus, the genus Ach cot, pear, blueberry , kiwi, melon , papaya, and avocado . romobacter, the genus Acinetobacter , the genus Shewanella , ( 14A ) The production method for producing a methacrylic the genus Listonella , the genus Agrobacterium , the genus 10 acid ester of (5A ) , in which the plant is any one selected Mesorhizobium , the genus Paracoccus, the genus Xantho from the group consisting of banana , apple , pear , kiwi, bacter , the genus Geobacillus, the genus Rhodococcus , or melon , papaya , and avocado . the genus Candida . The inventors of the invention also found that a specific The inventors of the invention also found that the AAT has protein which is derived from a microbe and has a function an activity of synthesizing a methacrylic acid ester. Specifi- 1515 ofOf ACD has an ability of converting isobutyryl- CoA to cally , the invention is as follows according to another aspect. methacrylyl -CoA at high efficiency . It is also found that 1 ) ( 1A ) A method for producing a methacrylic acid ester according to combination with BCKAaanzyme for including a step of synthesizing a methacrylic acid ester by producing isobutyryl- CoA , methacrylyl- CoA is synthesized having an alcohol or a phenol to act on methacrylyl- CoA in from 2 -oxoisovaleric acid , 2 ) according to combination with the presence of AAT. 20 AAT, a methacrylic acid ester is synthesized from isobu (2A ) The production method for producing a methacrylic tyryl- CoA , and 3 ) according to combination with an enzyme acid ester of ( 1A ) , in which the methacrylic acid ester is having an activity of hydrolyzing methacrylyl- CoA , meth accumulated at 0 . 001 mM or more . acrylic acid is synthesized from isobutyryl- CoA . Specifi ( 3A ) The production method for producing a methacrylic cally , the invention is as follows according to still another acid ester of ( 1A ) or ( 2A ) , in which it further has a step of 25 aspect. producing methacrylyl- CoA from isobutyryl- CoA or 3 -hy - (1B ) A transformant which is introduced with ACD gene droxyisobutyryl- CoA . selected from the group consisting of the following ( a ) to (4A ) The production method for producing a methacrylic ( d ) : acid ester of ( 3A ) , in which the isobutyryl -CoA is produced (a ) a gene encoding a protein consisting of an amino acid from 2 - oxoisovaleric acid . 30 sequence represented by SEQ ID NO . 50 or 52 (5A ) The production method for producing a methacrylic (b ) a gene consisting of a nucleotide sequence represented acid ester of any one of ( 1A ) to (4A ) , in which AAT is by SEQ ID NO . 51 or 53 derived from a plant. ( c ) a gene encoding a protein which consists of an amino (6A ) The production method for producing a methacrylic acid sequence showing an identity of 45 % or more to a acid ester of (5A ) , in which the plant belongs to an order 35 protein consisting of an amino acid sequence represented by selected from the group consisting of the order Zingiberales , SEQ ID NO . 50 or 52 and which has the ACD activity , and the order Rosales, the order Ericales, the order Cucurbitales, (d ) a gene encoding a protein which consists of an amino the order Brassicales, and the order Laurales . acid sequence with deletion , substitution , or addition of one ( 7A ) The production method for producing a methacrylic or several amino acids in an amino acid sequence repre acid ester of (5A ) , in which the plant belongs to a family 40 sented by SEQ ID NO . 50 or 52 and which has the ACD selected from the group consisting of the family Musa , the activity . family Rosales, the family Ericales , the family Actinidia , the (2B ) The transformant of ( 1B ) , which is introduced with family Cucurbitales , the family Carica , and the family BCKAD complex gene in addition to ACD gene . Laurales . (3B ) The transformant of ( 2B ), in which the BCKAD (8A ) The production method for producing a methacrylic 45 complex gene is a gene selected from the group consisting acid ester of (5A ) , in which the plant belongs to genus of the following ( e ) to ( h ) : selected from the group consisting of the genus Musa , the ( e ) a gene encoding four polypeptides having an amino acid genus Fragaria , the genus Malus , the genus Armeniaca , the sequence represented by SEQ ID NOS . 54 , 56 , 58 and 60 genus Pyrus, the genus Vaccinium , the genus Actinidia , the ( f ) a gene consisting of a nucleotide sequence represented by genus Cucumis , the genus Carica , and the genus Persea . 50 SEQ ID NOS . 55 , 57, 59 and 61 ( 9A ) The production method for producing a methacrylic ( g ) a gene encoding a protein which hybridizes under a acid ester of (5A ), in which the plant is any one selected stringent condition to a complementary strand of a gene from the group consisting of banana , strawberry , apple , encoding four polypeptides having an amino acid sequence Japanese apricot, pear , blueberry, kiwi, melon , papaya, and represented by SEQ ID NOS. 54 , 56 , 58 and 60 and which avocado . 55 has the BCKAD activity , and ( 10A ) The production method for producing a methacrylic ( h ) a gene encoding a protein which consists of an amino acid ester of any one of ( 1A ) to ( 9A ) , in which a genetically acid sequence with deletion , substitution , or addition of one modified microbe with a gene introduced to express AAT is or several amino acids in an amino acid sequence repre used . sented by SEQ ID NOS . 54 , 56 , 58 and 60 and which has the ( 11A ) The production method for producing a methacrylic 60 BCKAD activity . acid ester of (5A ) , in which the plant is any one selected (4B ) The transformant of (2B ), in which the BCKAD from plants belonging to the genus Musaceae , the genus complex gene is a gene selected from the group consisting Malus, the genus Purunus, the genus Pyrus, the genus of the following ( i) to (1 ) : Vaccinium hirtum , the genus Bower actinidia , the genus ( i) a gene encoding four polypeptides having an amino acid Cucumis , the genus Carica , or the genus Persea . 65 sequence represented by SEQ ID NOS . 62, 64 , 66 and 68 ( 12A ) The production method for producing a methacrylic ( ) a gene consisting of a nucleotide sequence represented by acid ester of (5A ), in which the plant is any one selected SEQ ID NOS. 63 , 65 , 67 and 69 US 10 , 294 ,500 B2 ( k ) a gene encoding a protein which hybridizes under a carbon atoms. Examples of the particularly preferred include stringent condition to a complementary strand of a gene a n alkyl group with 1 to 8 carbon atoms such as a methyl encoding four polypeptides having an amino acid sequence group , an ethyl group , a n -propyl group , an isopropyl group , represented by SEQ ID NOS. 62, 64 , 66 and 68 and which a n -butyl group , an isobutyl group , a sec -butyl group , a has the BCKAD activity, and 5 tert -butyl group , a n - pentyl group , an isopentyl group , a ( 1 ) a gene encoding a protein which consists of an amino tert - pentyl group , a n - hexyl group , an isohexyl group , a acid sequence with deletion , substitution , or addition of one 2 -hexyl group , a dimethylbutyl group , an ethylbutyl group , or several amino acids in an amino acid sequence repre - a heptyl group , an octyl group , or a 2 - ethylhexyl group , a sented by SEQ ID NOS. 62 , 64 , 66 and 68 and which has the benzyl group , and a phenyl group . BCKAD activity . 10 (5B ) The transformant of ( 2B ), in which the BCKAD CH2= C (CH3 ) C00 — R (Formula 1) complex gene is a gene selected from the group consisting The “methacrylic acid derivatives” is a general name of of the following (m ) to ( p ): the compounds containing a methacryloyl group ( m ) a gene encoding three polypeptides having an amino (CH = CCH2C00 ) in addition to the aforementioned acid sequence represented by SEQ ID NOS. 70 , 72 and 74 15 methacrylic acid and methacrylic acid ester. Examples of the ( n ) a gene consisting of a nucleotide sequence represented compound containing a methacryloyl group include meth by SEQ ID NOS . 71 , 73 and 75 acrylyl- CoA . ( o ) a gene encoding a protein which hybridizes under a The “ ability to produce methacrylic acid ” means ability stringent condition to a complementary strand of a gene for producing methacrylic acid using renewable raw mate encoding three polypeptides having an amino acid sequence 20 rials as a carbon source and / or energy source . The " produce ” represented by SEQ ID NOS . 70 , 72 and 74 and which has means both the synthesis and decomposition of methacrylic the BCKAD activity , and acid in a living body (that is , balance ) , and it is not limited ( p ) a gene encoding a protein which consists of an amino to the synthesis . acid sequence with deletion , substitution , or addition of one The “ renewable raw materials ” indicate natural resources , or several amino acids in an amino acid sequence repre - 25 for example , resources derived from an animal, a plant, or a sented by SEQ ID NOS. 70 , 72 and 74 and which has the microbe , in which the resources can be reconstructed by a BCKAD activity . process of the nature at a rate which is faster than the rate (6B ) The transformant of any one of (1B ) to (5B ), in which consumed by human beings or the like . The resources should the transformant is genetically introduced into a host which be self- renewable at a rate which is the same as the rate of has a pathway of metabolizing glucose to 2 - oxoisovaleric 30 the consumption . For example , a plant material has an acid . advantage that it can be cultivated without consuming it to ( 7B ) The transformant of any one of ( 1B ) to (6B ) , in which cause a significant decrease in natural sources . a gene of enzyme having the AAT activity is additionally The " biomass” means general resources that can be introduced . utilized as an organic energy source derived from a living ( 8B ) The transformant of any one of ( 1B ) to (7B ) , in which 35 organism . Examples of the biomass include a plant material , a gene of an enzyme having an activity of hydrolyzing an animal material , and a biodegradable waste . methacrylyl- CoA is additionally introduced . The " carbon source ” means an organic compound which ( 9B ) A method for producing methacrylyl- CoA including can be assimilated by a microbe and used for producing a expressing a protein having the ACD activity using the new cell . transformant of ( 1B ) and using the protein . 40 The “ energy source ” is a general name of the compounds ( 10B ) A method for producing methacrylyl- CoA including for producing an energy molecule ( ATP ) , which is required expressing a protein having the ACD activity and 2 - oxois - for a carbon source to get assimilated into a cell constitu ovalerate dehydrogenase activity using the transformant of tional component via a metabolism intermediate . When an any one of (2B ) to (5B ) and using the protein . energy source required for metabolism to produce ATP , that ( 11B ) A method for producing methacrylyl- CoA including 45 is , a catabolism process , is needed for an oxidation reaction expressing a protein having the ACD activity and 2 -oxois - of various compounds in microbes, those compounds ovalerate dehydrogenase activity by introducing the ACD become the “ energy source .” gene selected from the group consisting of ( a ) to ( d ) of ( 1B ) The " utilization of the renewable raw materials” means a to a host and introducing the BCKAD complex gene which method for general utilization of the renewable raw mate is selected from ( e ) to ( h ) , (i ) to ( 1 ) , and ( m ) to ( p ) of 50 rials for obtaining fuel components and various organic ( 3B ) to ( 5B ) to the same host or a different host and using substances from the renewable raw materials , which the protein . includes at least one processing step like gasification , liq Hereinbelow , the terminology of the invention is uidification , saccharification , enzyme treatment, fermenta explained . tion , distillation of fermentation product , hydrolysis , dehy The " methacrylic acid ” ( IUPAC name : 2 -methyl - 2 - pro - 55 dration , concentration , and drying. penoic acid ) means a compound with a chemical formula The “ microbes ” indicate , according to general definition , CH2 = C (CH3 ) COOH , and it encompasses any salt or ion prokaryotes like bacteria of any type , arcahea , and cyano ized form thereof. Examples of the salt of methacrylic acid bacteria , eukaryotes like yeast, mold , acrasis , and protozoa , include a sodium salt , a potassium salt , a calcium salt, and and viruses. It further includes undifferentiated cells of an a magnesium salt . 60 animal or a plant, and also a tissue culture product. As Themethacrylic acideterma m pund repre = described herein , the microbes specifically means the sented by Formula 1 . In Formula 1 , R represents a linear or microbes having the ability to produce methacrylic acid . branched hydrocarbon group with 1 to 20 carbon atoms. The Further , with regard to those having the ability to produce hydrocarbon group can be either a saturated or unsaturated methacrylic acid , it is also used as a concept which includes noncyclic type , or a saturated or unsaturated cyclic type . 65 a culture product, a medium ( a culture product from which Preferably , it is a linear or branched and unsubstituted alkyl microbial cells are removed ) , microbial cells ( including both group , an aralkyl group , or an aryl group having 1 to 10 cultured microbial cells or washed microbial cells ), a pro US 10 , 294 , 500 B2 10 cessed product of microbial cells in which the microbial FIG . 5 is a drawing explaining the method for producing cells are disrupted or lysed , and a composition having an a plasmid for deleting a gene In Fusion method . activity of producing methacrylic acid which is obtained by FIG . 6 is a drawing illustrating the structure of a plasmid purifying and processing of the medium and /or cells ( crude for co - expressing ACD -AAT . enzyme solution and purified enzyme) . 5 FIG . 7 is a drawing illustrating the expression of the The “ derived strainstrain ” means a strainstrain which is recombinant protein based on SDS polyacrylamide gel elec derived from a certain microbe strainstrain either naturally , trophoresis . The arrow indicates the position of the target or by a chemical or physical treatment. In the invention , it protein . ( A ) Cell extract of the recombinant with the specifically means a strain which is derived from a microbe described gene introduced therein , in which E . coli JM109 strain having the ability to produce methacrylicorylic acid and 10 is used as a host and pTrc99A is used as a vector, ( B ) a cell maintains the ability to produce methacrylic acid from extract of the recombinant with the described gene intro renewable raw material as a carbon source and /or energy duced therein in which E . coli BL21 (DE3 ) is used as a host source . and PET19b is used as a vector , and ( C ) a cell extract of the The " contact ” means an exposure treatment of the 16 recombinant with the acdH gene introduced therein . microbes and materials ( renewable raw materials ) for a EMBODIMENTS FOR CARRYING OUT THE certain period of time. Specifically , it indicates that microbes INVENTION are added to an aqueous medium containing renewable raw materials followed by suspension mixing . At that time, it is Hereinbelow , preferred embodiments for carrying out the acceptable to have proliferation of the microbes. 20 invention are explained . Meanwhile , the embodiments that The " aqueousmedium ” means water , an aqueous solution are explained hereinbelow are to illustrate an example of the having water as a main component, and gel (agar ) , and those representative embodiment of the invention , and by no having undissolved liquid and solid dispersed therein are means the scope of the invention is construed narrowly by also included . them . The “ vapor phase ” means the portion taken by gas or 25 1 . Method for Producing Methacrylic Acid and /or Ester water vapor in a culture tank ( a vessel for culturing Thereof microbes ) or a reaction tank ( a vessel for performing the According to the method for producing methacrylic acid reaction ) , excluding the portion taken by the liquid (medium and /or an ester thereof of the invention (hereinbelow , also or the like ). simply referred to as the “ production method for methacrylic The “ culture” means a process proliferating cells of 30 acid ” ) , methacrylic acid is produced using microbes having microbes or a multicellular organisms in an artificial envi- the ability to produce methacrylic acid from renewable raw ronment. materials and /or biomass arising from the utilization of the The “ culture product” means those obtained by culture of renewable raw materials , as a carbon source and/ or energy microbial cells , a broth , a cell -free extract , or cell mem source . Specifically , according to a step of contacting the branes . The cell - free extract can be obtained by, after 35 microbes having the ability to produce methacrylic acid with physically disrupting microbial cells after culture with a renewable raw materials and /or biomass , methacrylic acid is homogenizer or the like with addition of a sodium phosphate produced . More specifically , by culturing the microbes in an buffer solution followed by centrifuge ( 15 ,000 rpm , 10 min , aqueous medium containing the renewable raw materials 4° C . ) , collecting the supernatant from which non - disrupted and /or biomass, methacrylic acid is obtained in the aqueous microbes or cell membranes are removed , 40 medium . The “ esterification ” indicates the reaction for producing ( 1 ) Microbes an ester from an alcohol and a fatty acid based on dehydra With regard to the microbes that are used for production tion . Further , it also includes the reaction for producing a method for methacrylic acid according to the invention , they new ester based on an alcohol exchange reaction with are not particularly limited as long as they are the microbes another alcohol or a partial ester of a polyhydric alcohol. 45 having the ability to produce methacrylic acid . As for the microbes, the microbes as follows can be used , for example . Effect of the Invention The microbes can be used either singly or as a combination of two or more types that are selected from the following According to the invention , a method for directly and microbes . efficiently producing methacrylic acid in a single step from 50 [Preferred Order of Prokaryotes ] renewable raw materials and / or biomass arising from the Specific examples of the prokaryotes that are suitable for utilization of the renewable raw materials , as a carbon the invention include microbes belonging to Rhodospirilla source and/ or energy source , is provided . les , Rhodobacterales , Sphingomonadales, Caulobacterales , Rhizobiales , Parvularculales , Burkholderiales , Hydrogeno BRIEF DESCRIPTION OF DRAWINGS 55 philales, Neisseriales, Nitrosomonadales, Rhodocyclales, Chromatiales, Acidithiobacillales, Xanthomonadales , Thio FIG . 1 is a drawing illustrating the step for producing a trichales , Oceanospirillales, Pseudomonadales , Altero methacrylic acid ester from 2 - oxoisovaleric acid . monadales, Vibrionales, Aeromonadales , Salinisphaerales, FIG . 2 is a total ion chromatograph illustrating the result Legionellales , Desulfobacterales, Desulfarculales , Desulfu of GC /MS analysis of methacrylic acid which has been 60 romonadales , Syntrophobacterales, Bdellovibrionales , produced in a broth ofmethacrylic acid -producing microbes Myxococcales, Acidobacteriales , Actinomycetales, Bifido ( Example 2 ) . bacteriales , Rubrobacterales, Solirubrobacterales , Bacilla FIG . 3 is a mass spectrum illustrating the result of GC /MS les , Lactobacillales , Clostridiales , Thermoanaerobacterales, analysis of methacrylic acid which has been produced in a Natranaerobiales , Sphingobacteriales , Bacteroidales , broth of methacrylic acid - producing microbes ( Example 2 ) . 65 Cytophagales , Flavobacteriales, Deinococcales , Thermales , FIG . 4 is a drawing illustrating the structure of a plasmid Gemmatimonadales, Fusobacteriales , Chloroflexales , Her for deleting LigD homolog gene. petosiphonales, Thermomicrobiales , Thermotogales, Defer US 10 ,294 , 500 B2 11 ribacterales, Sulfolobales, Desulfurococcales , Thermopro phaceae, as those belonging to Bdellovibrionales , Bdellovi teales, Acidilobales , Halobacteriales , Thermoplasmatales , or brionaceae and Bacteriovoracaceae , as those belonging to Archaeoglobales . Myxococcales , Cystobacteraceae , Myxococcaceae , Polyan Among them , preferred microbes are the microbes giaceae and Kofleriaceae, as those belonging to Acidobac belonging to Rhodospirillales , Rhodobacterales , Sphin - 5 teriales , Acidobacteriaceae , as those belonging to Acidimi gomonadales , Caulobacterales , Rhizobiales , Parvularcula - crobiales, Acidimicrobiaceae , as those belonging to les, Burkholderiales, Hydrogenophilales, Neisseriales , Actinomycetales, Streptosporangiaceae , Nocardiopsaceae , Nitrosomonadales, Rhodocyclales , Chromatiales, Thermomonosporaceae, Pseudonocardiaceae , Actinosynne Xanthomonadales , Thiotrichales , Oceanospirillales , mataceae, Micromonosporaceae, Actinopolysporaceae , Pro Pseudomonadales , Alteromonadales , Vibrionales, Aero - 10 pionibacteriaceae , Nocardioidaceae , Corynebacteriaceae , monadales , Salinisphaerales, Legionellales , Desulfobacter - Nocardiaceae , Gordoniaceae, Dietziaceae , Mycobacteri ales, Desulfarculales , Desulfuromonadales , Syntropho - aceae, Tsukamurellaceae, Segniliparaceae , Microbacteri bacterales , Bdellovibrionales, Myxococcales , aceae , Micrococcaceae , Dermabacteraceae , Dermatophi Acidobacteriales, Actinomycetales, Rubrobacterales, Soli - laceae , Brevibacteriaceae , Cellulomonadaceae , rubrobacterales, Bacillales , Lactobacillales , Clostridiales, 15 Intrasporangiaceae , Jonesiaceae , Rarobacteraceae , Franki Thermoanaerobacterales , Natranaerobiales , Sphingobacteri aceae , Acidothermaceae , Nakamurellaceae, Cryptosporan ales , Bacteroidales , Cytophagales , Flavobacteriales, giaceae , Geodermatophilaceae , Glycomycetaceae, Actino Deinococcales , Thermales , Gemmatimonadales , Fusobacte - mycetaceae , Streptomycetaceae , Catenulisporaceae and riales, Chloroflexales, Herpetosiphonales, Thermomicrobi- Kineosporiaceae , as those belonging to Bifidobacteriales , ales , Thermotogales , Deferribacterales, Sulfolobales, Des - 20 Bifidobacteriaceae , as those belonging to Rubrobacterales, ulfurococcales , Thermoproteales, Acidilobales , Rubrobacteraceae , as those belonging to Solirubrobacter Halobacteriales , Thermoplasmatales , or Archaeoglobales . ales , Conexibacteraceae , as those belonging to Bacillales , Further , examples of the particularly preferred microbes Bacillaceae , Alicyclobacillaceae, Paenibacillaceae , Plano which allow experimental confirmation of the production of coccaceae , Sporolactobacillaceae and Thermoactinomyceta methacrylic acid derivatives include the microbes belonging 25 ceae , as those belonging to Lactobacillales, Lactobacil to Rhodobacterales , Sphingomonadales , Caulobacterales, laceae , Leuconostocaceae , Aerococcaceae , Rhizobiales , Burkholderiales , Pseudomonadales, Altero Carnobacteriaceae and Streptococcaceae , as those belonging monadales, Vibrionales, Actinomycetales, Bacillales, or to Clostridiales, Clostridiaceae , Syntrophomonadaceae , Sphingobacteriales . Eubacteriaceae , Peptococcaceae, Peptostreptococcaceae , [Preferred Family of Prokaryotes ] 30 Lachnospiraceae and Oscillospiraceae , as those belonging to Examples may include , as those belonging to Rho - Thermoanaerobacterales , Thermoanaerobacteraceae , as dospirillales , Rhodospirillaceae and Acetobacteraceae , as those belonging to Natranaerobiales, Natranaerobiaceae , as those belonging to Rhodobacterales, Rhodobacteraceae and those belonging to Sphingobacteriales , Sphingobacteriaceae Hyphomonadaceae , as those belonging to Sphin - and Saprospiraceae , as those belonging to Bacteroidales, gomonadales, Sphingomonadaceae and Erythrobacteraceae , 35 Bacteroidaceae , Marinilabiliaceae , Prevotellaceae and Por as those belonging to Caulobacterales , Caulobacteraceae , as phyromonadaceae , as those belonging to Cytophagales, those belonging to Rhizobiales , Rhizobiaceae , Xanthobacte Cytophagaceae and Flammeovirgaceae , as those belonging raceae , Brucellaceae , Phyllobacteriaceae, Auranti to Flavobacteriales, Flavobacteriaceae , as those belonging to monadaceae , Bradyrhizobiaceae, Methylobacteriaceae, Deinococcales, Deinococcaceae , as those belonging to Ther Hyphomicrobiaceae , Rhodobiaceae and Methylocystaceae , 40 males , Thermaceae , as those belonging to Gemmati as those belonging to Parvularculales , Parvularculaceae , as monadales , Gemmatimonadaceae , as those belonging to those belonging to Burkholderiales , Burkholderiaceae , Fusobacteriales , Fusobacteriaceae , as those belonging to Alcaligenaceae , Comamonadaceae and Oxalobacteraceae , Chloroflexales, Chloroflexaceae , as those belonging to Her as those belonging to Hydrogenophilales , Hydrogenophi petosiphonales , Herpetosiphonaceae , as those belonging to laceae , as those belonging to Neisseriales , Neisseriaceae , as 45 Thermomicrobiales , Thermomicrobiaceae , as those belong those belonging to Nitrosomonadales , Nitrosomonadaceae , ing to Thermotogales, Thermotogaceae , as those belonging as those belonging to Rhodocyclales, Rhodocyclaceae , as to Deferribacterales , Deferribacteraceae , as those belonging those belonging to Chromatiales , Chromatiaceae and Ecto - to Sulfolobales, Sulfolobaceae, as those belonging to Des thiorhodospiraceae , as those belonging to Acidithiobacilla - ulfurococcales, Desulfurococcaceae , as those belonging to les , Acidithiobacillaceae, as those belonging to 50 Thermoproteales, Thermoproteaceae , as those belonging to Xanthomonadales, Xanthomonadaceae , as those belonging Acidilobales, Acidilobaceae , as those belonging to Halobac to Thiotrichales, Francisellaceae and Piscirickettsiaceae, as t eriales , Halobacteriaceae , as those belonging to Thermo those belonging to Oceanospirillales , Oceanospirillaceae, plasmatales, Thermoplasmataceae , Picrophilaceae and Fer Hahellaceae , Halomonadaceae and Alcanivoracaceae , as roplasmaceae , and as those belonging to Archaeoglobales, those belonging to Pseudomonadales, Pseudomonadaceae 55 Archaeoglobaceae . and Moraxellaceae , as those belonging to Alteromonadales , Among them , preferred microbes are the microbes Alteromonadaceae , Shewanellaceae , Ferrimonadaceae , belonging to Rhodospirillaceae, Acetobacteraceae , Rhodo Idiomarinaceae , Colwelliaceae and Pseudoaltero - bacteraceae , Hyphomonadaceae, Sphingomonadaceae , monadaceae , as those belonging to Vibrionales , Vibrion - Erythrobacteraceae , Caulobacteraceae , Rhizobiaceae , Xan aceae , as those belonging to Aeromonadales , Aero - 60 thobacteraceae , Brucellaceae, Phyllobacteriaceae , Auranti monadaceae , as those belonging to Salinisphaerales , monadaceae , Bradyrhizobiaceae , Methylobacteriaceae , Salinisphaeraceae , as those belonging to Legionellales , Hyphomicrobiaceae, Rhodobiaceae , Methylocystaceae , Par Legionellaceae and Coxiellaceae , as those belonging to vularculaceae , Burkholderiaceae , Alcaligenaceae, Coma Desulfobacterales , Desulfobulbaceae , as those belonging to monadaceae , Oxalobacteraceae , Neisseriaceae , Desulfarculales , Desulfarculaceae , as those belonging to 65 Nitrosomonadaceae , Rhodocyclaceae, Chromatiaceae , Desulfuromonadales, Geobacteraceae , as those belonging to Ectothiorhodospiraceae , Xanthomonadaceae, Franci Syntrophobacterales, Syntrophobacteraceae and Syntro sellaceae, Piscirickettsiaceae , Oceanospirillaceae, Hahel US 10 ,294 ,500 B2 13 14 laceae , Halomonadaceae , Alcanivoracaceae , Aurantimonadaceae , the genus Aurantimonas, as those Pseudomonadaceae , Moraxellaceae , Alteromonadaceae , belonging to Bradyrhizobiaceae, the genus Bradyrhizobium , Shewanellaceae , Ferrimonadaceae, Idiomarinaceae , Col- the genus Agromonas, the genus Rhodopseudomonas and welliaceae, Pseudoalteromonadaceae , Vibrionaceae , Aero the genus Nitrobacter , as those belonging to Methylobacte monadaceae , Salinisphaeraceae , Legionellaceae , Coxiel- 5 riaceae , the genus Methylobacterium , as those belonging to laceae , Desulfobulbaceae , Desulfarculaceae , Hyphomicrobiaceae, the genus Hyphomicrobium , the genus Geobacteraceae , Syntrophobacteraceae , Syntrophaceae, Rhodomicrobium and the genus Pelagibacterium , as those Bdellovibrionaceae , Bacteriovoracaceae , Cystobacteraceae , belonging to Rhodobiaceae , the genus Parvibaculum , as Myxococcaceae , Polyangiaceae , Kofleriaceae , Acidobacte - those belonging to Methylocystaceae, the genus Methylo riaceae , Acidimicrobiaceae, Streptosporangiaceae , Nocardi- 10 cystis , as those belonging to Parvularculaceae , the genus opsaceae , Thermomonosporaceae , Pseudonocardiaceae , Parvularcula , as those belonging to Burkholderiaceae , the Actinosynnemataceae, Micromonosporaceae , Nocar - genus Burkholderia , the genus Ralstonia , the genus Cupria dioidaceae, Corynebacteriaceae , Nocardiaceae , Gordoni vidus and the genus Polynucleobacter , as those belonging to aceae, Dietziaceae , Mycobacteriaceae, Tsukamurellaceae Alcaligenaceae, the genus Achromobacter , the genus Alcali Segniliparaceae , Microbacteriaceae , Micrococcaceae , 15 genes , the genus Bordetella , the genus Taylorella , the genus Dermabacteraceae , Cellulomonadaceae , Intrasporangiaceae , Pusillimonas and the genus Oligella , as those belonging to Frankiaceae , Acidothermaceae , Nakamurellaceae , Geoder - Comamonadaceae, the genus Comamonas, the genus Alicy matophilaceae , Glycomycetaceae , Streptomycetaceae, cliphilus, the genus Delftia , the genus Ramlibacter , the Catenulisporaceae , Rubrobacteraceae , Conexibacteraceae , genus Rhodoferax , the genus Variovorax, the genus Pola Bacillaceae , Alicyclobacillaceae, Paenibacillaceae , Lactoba - 20 romonas, the genus Acidovorax and the genus Vermineph cillaceae , Carnobacteriaceae Clostridiaceae , Syntroph - robacter, as those belonging to Oxalobacteraceae , the genus omonadaceae , Eubacteriaceae , Peptococcaceae, Lachno - Herminiimonas , the genus Herbaspirillum and the genus spiraceae, Oscillospiraceae , Thermoanaerobacteraceae , Collimonas, as those belonging to Hydrogenophilaceae , the Natranaerobiaceae , Sphingobacteriaceae , Saprospiraceae , genus Hydrogenophilus and the genus Thiobacillus , as those Porphyromonadaceae, Cytophagaceae , Flavobacteriaceae , 25 belonging to Neisseriaceae , the genus Chromobacterium , Deinococcaceae , Thermaceae , Gemmatimonadaceae , Fuso - the genus Laribacter and the genus Pseudogulbenkianonas bacteriaceae , Chloroflexaceae, Herpetosiphonaceae, Ther- those belonging to Nitrosomonadaceae, the genus momicrobiaceae , Thermotogaceae , Deferribacteraceae , Sul- Nitrosomonas and the genus Nitrosospira , as those belong folobaceae, Desulfurococcaceae , Thermoproteaceae , ing to Rhodocyclaceae, the genus Aromatoleum , the genus Acidilobaceae , Halobacteriaceae , Thermoplasmataceae , 30 Azoarcus , the genus Dechloromonas, the genus Thauera and Picrophilaceae, Ferroplasmaceae , or Archaeoglobaceae . the genus Azospira (Dechlorosoma ), as those belonging to Further , examples of the particularly preferred microbes Chromatiaceae, the genus Rheinheimera , the genus Thio which allow experimental confirmation of the production of sphaera and the genus Nitrosococcus , as those belonging to methacrylic acid derivatives include the microbes belonging Ectothiorhodospiraceae , the genus Halorhodospira , as those to Rhodobacteraceae, Sphingomonadaceae , Caulobacter - 35 belonging to Acidithiobacillaceae , the genus Acidithiobacil aceae, Rhizobiaceae , Xanthobacteraceae , Brucellaceae , lus , as those belonging to Xanthomonas, the genus Phyllobacteriaceae , Alcaligenaceae, Comamonadaceae , Xanthomonas, the genus Stenotrophomonas, the genus Pseudomonadaceae , Moraxellaceae , Shewanellaceae , Vibri - Pseudoxanthomonas and the genus Rhodanobacter , as those onaceae, Nocardiaceae , Streptomycetaceae , Bacillaceae , belonging to Francisellaceae, the genus Francisella , as those Paenibacillaceae or Sphingobacteriaceae . 40 belonging to Piscirickettsiaceae , the genus Cycloclasticus, [Preferred Genus of Prokaryotes ] as those belonging to Oceanospirillaceae , the genus Oceano Examples may include , as those belonging to Rho - spirillum and the genus Marinospirillum , as those belonging dospirillaceae , the genus Magnetospirillum , the genus Rho - to Hahellaceae , the genus Hahella , as those belonging to dospirillum , the genus Azospirillum and the genus Tistrella , Halomonadaceae , the genus Halomonas, as those belonging as those belonging to Acetobacteraceae , the genus Aceto - 45 to Alcanivoracaceae , the genus Alcanivorax and the genus bacter , the genus Acidiphilium and the genus Gluconaceto - Kangiella , as those belonging to Pseudomonadaceae , the bacter , as those belonging to Rhodobacteraceae , the genus genus Pseudomonas and the genus Azotobacter , as those Rhodobacter , the genus Paracoccus, the genus Ruegeria , belonging to Moraxellaceae , the genus Acinetobacter and the genus Jannaschia , the genus Roseobacter , the genus the genus Psychrobacter , as those belonging to Altero Dinoroseobacter, the genus Pseudovibrio , the genus Phae - 50 monadaceae, the genus Alishewanella , the genus Alteromo obacter, the genus Octadecabacter and the genus Ahrensia , nas, the genus Glaciecola , the genus Marinobacter , the as those belonging to Hyphomonadaceae, the genus Hyph genus Marinobacterium and as those belonging to Saccha omonas , the genus Maricaulis , the genus Hirschia , as those rophagus , the genus Shewanellaceae , the genus Shewanella , belonging to Sphingomonadaceae , the genus Sphingomonas, as those belonging to Ferrimonadaceae , the genus Ferrimo the genus Novosphingobium , the genus Sphingopyxis , the 55 nas , as those belonging to Idiomarinaceae, the genus genus Sphingobium , the genus Lutibacterium and the genus Idiomarina , as those belonging to Colwelliaceae , the genus Zymomonas, as those belonging to Erythrobacteraceae, the Colwellia , as those belonging to Pseudoalteromonadaceae, genus Erythrobacter, as those belonging to Caulobacter the genus Pseudoalteromonas, as those belonging to Vibri aceae, the genus Brevundimonas, the genus Caulobacter, the onaceae, the genus Listonella , the genus Vibrio and the genus Phenylobacterium and the genus Asticcacaulis , as 60 genus Photobacterium , as those belonging to Aero those belonging to Rhizobiaceae, the genus Agrobacterium , monadaceae , the genus Aeromonas and the genus Oceani the genus Rhizobim and the genus Sinorhizobium , as those monas , as those belonging to Salinisphaeraceae , the genus belonging to Xanthobacteraceae, the genus Xanthobacter Salinisphaera , as those belonging to Legionellaceae , the and the genus Azorhizobium , as those belonging to Brucel - genus Legionella , as those belonging to Coxiellaceae , the laceae , the genus Brucella and the genus Ochrobactrum , as 65 genus Coxiella , as those belonging to Desulfobulbaceae , the those belonging to Phyllobacteriaceae , the genus Mesorhizo - genus Desulfococcus , the genus Desulfobacterium and the bium and the genus Chelativorans, as those belonging to genus Desulfatibacillum , as those belonging to Desulfarcu US 10 ,294 , 500 B2 15 16 laceae , the genus Desulfobulbus and the genus Desulfarcu mus, as those belonging to Nakamurellaceae , the genus lus, as those belonging to Geobacteraceae , the genus Geo Nakamurella , as those belonging to Cryptosporangiaceae , bacter , as those belonging to Syntrophobacteraceae , the the genus Cryptosporangium , as those belonging to Geo genus Syntrophobacter, as those belonging to Syntro dermatophilaceae , the genus Geodermatophilus, as those phaceae , the genus Syntrophus and the genus Desulfomonile , 5 belonging to Glycomycetaceae , the genus Glycomyces and as those belonging to Bdellovibrionaceae , the genus Bdell - the genus Stackebrandtia , as those belonging to Actinomy ovibrio , as those belonging to Bacteriovoracaceae , the genus cetaceae , the genus Arcanobacterium , as those belonging to Bacteriovorax , as those belonging to Cystobacteraceae, the Streptomycetaceae, the genus Streptomyces and the genus genus Stigmatella , as those belonging to Myxococcaceae, Kitasatospora , as those belonging to Catenulisporaceae , the the genus Myxococcus and the genus Anaeromyxobacter , as 10 genus Catenulispora , as those belonging to Kineospori those belonging to Polyangiaceae , the genus Sorangium , as aceae , the genus Kineosporia and the genus Kineococcus , as those belonging to Kofleriaceae , the genus Haliangium , as those belonging to Bifidobacteriaceae , the genus Bifidobac those belonging to Acidobacteriaceae, the genus Acidobac t erium , as those belonging to Rubrobacteraceae , the genus terium and the genus Granulicella , as those belonging to Rubrobacter, as those belonging to Conexibacteraceae , the Acidimicrobiaceae, the genus Ilumatobacter , as those 15 genus Conexibacter , as those belonging to Bacillaceae , the belonging to Streptosporangiaceae, the genus Streptospo genus Bacillus, the genus Geobacillus, the genus Oceano rangium , the genus Acrocarpospora , the genus Herbi bacillus, the genus Lysinibacillus and the genus Halobacil dospora , the genus Microbispora , the genus Nonomuraea , lus , as those belonging to Alicyclobacillaceae , the genus the genus Planobispora and the genus Planomonospora , as Alicyclobacillus and the genus Kyrpidia , as those belonging those belonging to Nocardiopsaceae , the genus Nocardiop - 20 to Paenibacillaceae , the genus Paenibacillus and the genus sis and the genus Thermobifida , as those belonging to Brevibacillus, as those belonging to Planococcaceae , the Thermomonosporaceae , the genus Actinocorallia , the genus genus Planococcus and the genus Kurthia , as those belong Actinomadura and the genus Thermomonospora , as those ing to Sporolactobacillaceae , the genus Sporolactobacillus, belonging to Pseudonocardiaceae , the genus Pseudonocar - as those belonging to Thermoactinomycetaceae , the genus dia , the genus Actinoalloteichus , the genus Amycolatopsis , 25 Thermoactinomyces , as those belonging to Lactobacillaceae , the genus Kibdelosporangium , the genus Saccharomono the genus Lactobacillus and the genus Pediococcus, as those spora , the genus Saccharopolyspora , the genus Saccharo - belonging to Leuconostocaceae , the genus Leuconostoc and thrix , the genus Streptoalloteichus , the genus Thermo - the genus Weissella , as those belonging to Aerococcaceae , bispora and the genus Thermocrispum , as those belonging to the genus Aerococcus and the genus Globicatella , as those Actinosynnemataceae, the genus Actinokineospora and the 30 belonging to Carnobacteriaceae , the genus Alloiococcus and genus Actinosynnema, as those belonging to Micromono the genus Carnobacterium , as those belonging to Strepto sporaceae , the genus Micromonospora , the genus Actino coccaceae , the genus Streptococcus, as those belonging to planes , the genus Catellatospora , the genus Couchioplanes , Clostridiaceae , the genus Clostridium and the genus Alka the genus Dactylosporangium , the genus Pilimelia , the liphilus, as those belonging to Syntrophomonadaceae , the genus Salinispora and the genus Verrucosispora , the genus 35 genus Syntrophomonas and the genus Syntrophothermus , as Actinopolysporaceae, the genus Actinopolyspora , as those those belonging to Eubacteriaceae, the genus Eubacterium , belonging to Propionibacteriaceae, the genus Propionibac - as those belonging to Peptococcaceae, the genus Peptococ terium and the genus Luteococcus , as those belonging to cus , the genus Desulfitobacterium , the genus Desulfoto Nocardioidaceae , the genus Nocardioides, the genus Aero - maculum and the genus Pelotomaculum , as those belonging microbium , the genus Kribbella and the genus Pimelobacter , 40 to Peptostreptococcaceae , the genus Peptostreptococcus , the as those belonging to Corynebacteriaceae , the genus genus Lachnospiraceae , the genus Butyrivibrio and the Corynebacterium , the genus Nocardiaceae , the genus genus Roseburia , as those belonging to Oscillospiraceae , the Nocardia and the genus Rhodococcus, as those belonging to genus Oscillibacter , as those belonging to Thermoanaero Gordoniaceae , the genus Gordonia , as those belonging to bacteraceae, the genus Thermoanaerobacter and the genus Dietziaceae , the genus Dietzia , as those belonging to Myco - 45 Carboxydothermus , as those belonging to Natranaerobi bacteriaceae , the genus Mycobacterium and the genus Amy - aceae , the genus Natranaerobius, as those belonging to colicicoccus, as those belonging to Tsukamurellaceae , the Sphingobacteriaceae , the genus Sphingobacterium , the genus Tsukamurella , as those belonging to Segniliparaceae , genus Pedobacter and the genus Mucilaginibacter , as those the genus Segniliparus, as those belonging to Microbacte - belonging to Saprospiraceae , the genus Haliscomenobacter, riaceae , the genus Microbacterium , the genus Agromyces , 50 as those belonging to Bacteroidaceae , the genus Bacte the genus Clavibacter , the genus Curtobacterium and the roides , as those belonging to Marinilabiliaceae , the genus genus Rathayibacter, as those belonging to Micrococcaceae , Marinilabilia , as those belonging to Prevotellaceae, the the genus Micrococcus, the genus Arthrobacter , the genus genus Prevotella , as those belonging to Porphy Citricoccus , the genus Renibacterium , the genus Kocuria romonadaceae , the genus Porphyromonas and the genus and the genus Rothia , as those belonging to Dermabacter- 55 Odoribacter , as those belonging to Cytophagaceae , the aceae, the genus Brachybacterium , the genus Dermacoccus , genus Flexibacter, the genus Spirosoma and the genus the genus Demetria and the genus Kytococcus, as those Runella , as those belonging to Flammeovirgaceae , the genus belonging to Dermatophilaceae, the genus Dermatophilus, Flammeovirga , as those belonging to Flavobacteriaceae, the as those belonging to Brevibacteriaceae , the genus Brevi- genus Flavobacterium , the genus Chryseobacterium and the bacterium , as those belonging to Cellulomonadaceae , the 60 genus Maribacter , as those belonging to Deinococcaceae , genus Cellulomonas and the genus Oerskovia , as those the genus Deinococcus , as those belonging to Thermaceae , belonging to Intrasporangiaceae , the genus Intrasporan - the genus Thermus, the genus Meiothermus , the genus gium , the genus Janibacter , the genus Terrabacter and the Oceanithermus and the genus Marinithermus , as those genus Serinicoccus, as those belonging to Jonesiaceae , the belonging to Gemmatimonadaceae , the genus Gemmatimo genus Jonesia , as those belonging to Rarobacteraceae , the 65 nas , as those belonging to Fusobacteriaceae, the genus genus Rarobacter , the genus Frankiaceae, the genus Fusobacterium and the genus Ilyobacter , as those belonging Frankia , the genus Acidothermaceae , the genus Acidother - to Chloroflexaceae , the genus Roseiflexus, as those belong US 10 ,294 , 500 B2 17 18 ing to Herpetosiphonaceae , the genus Herpetosiphon , as the genus Dechloromonas , the genus Thauera , the genus those belonging to Thermomicrobiaceae , the genus Ther Azospira (Dechlorosoma ), the genus Rheinheimera , the momicrobium , as those belonging to Thermotogaceae , the genus Nitrosococcus, the genus Halorhodospira , the genus genus Thermotoga , the genus Thermosipho and the genus Xanthomonas, the genus Stenotrophomonas, the genus Fervidobacterium , as those belonging to Deferribacteraceae , 5 Pseudoxanthomonas , the genus Rhodanobacter , the genus the genus Deferribacter , the genus Calditerrivibrio and the Francisella , the genus Cycloclasticus , the genus Oceano genus Flexistipes, the genus Sulfolobaceae, the genus Met spirillum , the genus Hahella , the genus Halomonas , the allosphaera and the genus Acidianus , as those belonging to genus Alcanivorax , the genus Kangiella , the genus Desulfurococcaceae , the genus Aeropyrum , as those belong . Pseudomonas , the genus Azotobacter , the genus Acineto ing to Thermoproteaceae , the genus Pyrobaculum , the genus 10 bacter , the genus Psychrobacter , the genus Alishewanella , Caldivirga and the genus Vulcanisaeta , as those belonging the genus Alteromonas , the genus Glaciecola , the genus to Acidilobaceae, the genus Acidilobus, as those belonging Marinobacter , the genus Marinobacterium , the genus Sac to Halobacteriaceae , the genus Halobacterium , the genus charophagus , the genus Shewanella , the genus Ferrimonas , Halococcus, the genus Haloarcula , the genus Haloquadra - the genus Idiomarina , the genus Colwellia , the genus Pseu tum , the genus Natronomonas , the genus Halorubrum , the 15 doalteromonas , the genus Listonella , the genus Vibrio , the genus Haloterrigena , the genus Natrialba , the genus Hala - genus Photobacterium , the genus Aeromonas, the genus lkalicoccus and the genus Halogeometricum , as those Oceanimonas, the genus Salinisphaera , the genus Legion belonging to Thermoplasmataceae, the genus Thermo - ella , the genus Coxiella , the genus Desulfococcus , the genus plasma , as those belonging to Picrophilaceae , the genus Desulfobacterium , the genus Desulfatibacillum , the genus Picrophilus, as those belonging to Ferroplasmaceae , the 20 Desulfobulbus, the genus Desulfarculus, the genus Geo genus Ferroplasma , and as those belonging to Archaeo - bacter , the genus Syntrophobacter , the genus Syntrophus , globaceae, the genus Archaeoglobus and the genus Ferro - the genus Desulfomonile , the genus Bdellovibrio , the genus globus. Further, examples of the microbes having no deter Bacteriovorax, the genus Stigmatella , the genus Myxococ mined order in terms of classification include the genus cus, the genus Anaeromyxobacter, the genus Sorangium , the Polymorphum , the genus Micavibrio , the genus Simiduia , 25 genus Haliangium , the genus Acidobacterium , the genus the genus Leptothrix , the genus Thiomonas , the genus Granulicella , the genus Ilumatobacter , the genus Strep Rubrivivax , and the genus Methylbum , examples of the toprangum , the genus Nocardiopsis , the genus Thermo microbes belonging to Bacillales but no determined family bifida , the genus Thermomonospora , the genus Pseudono in terms of classification include the genus Exiguobacte - cardia , the genus Amycolatopsis , the genus rium , examples of the microbes belonging to Clostridiales 30 Saccharomonospora , the genus Saccharopolyspora , the but no determined family in terms of classification include genus Thermobispora , the genus Actinosynnema , the genus the genus Clostridiales. Micromonospora , the genus Salinispora , the genus Verru Among them , preferred microbes are the microbes which cosispora , the genus Nocardioides, the genus Kribbella , the have been demonstrated to have an enzyme for the metabo genus Corynebacterium , the genus Nocardia , the genus lism pathway of branched amino acids , that is , microbes 35 Rhodococcus , the genus Gordonia , the genus Dietzia , the belonging to the genus Magnetospirillum , the genus Rho - genus Mycobacterium , the genus Amycolicicoccus, the dospirillum , the genus Azospirillum , the genus Tistrella , the genus Tsukamurella , the genus Segniliparus, the genus genus Acidiphilium , the genus Rhodobacter, the genus Microbacterium , the genus Micrococcus, the genus Arthro Paracoccus, the genus Ruegeria , the genus Jannaschia , the bacter , the genus Citricoccus, the genus Renibacterium , the genus Roseobacter , the genus Dinoroseobacter, the genus 40 genus Kocuria , the genus Kytococcus , the genus Cellulo Pseudovibrio , the genus Phaeobacter , the genus Octadeca - monas , the genus Intrasporangium , the genus Serinicoccus , bacter , the genus Hyphomonas, the genus Maricaulis , the the genus Frankia , the genus Acidothermus, the genus genus Hirschia , the genus Sphingomonas, the genus Nakamurella , the genus Geodermatophilus , the genus Stack Novosphingobium , the genus Sphingopyxis , the genus ebrandtia , the genus Streptomyces, the genus Catenulispora , Sphingobium , the genus Erythrobacter , the genus Brevundi- 45 the genus Rubrobacter , the genus Conexibacter , the genus monas , the genus Caulobacter , the genus Phenylobacterium , Bacillus , the genus Geobacillus , the genus Oceanobacillus , the genus Asticcacaulis , the genus Agrobacterium , the genus the genus Lysinibacillus, the genus Halobacillus, the genus Rhizobium , the genus Sinorhizobium , the genus Xantho - Alicyclobacillus, the genus Kyrpidia , the genus Paenibacil bacter , the genus Azorhizobium , the genus Brucella , the lus, the genus Lactobacillus , the genus Carnobacterium , the genus Ochrobactrum , the genus Mesorhizobium , the genus 50 genus Clostridium , the genus Alkaliphilus, the genus Syn Chelativorans, the genus Aurantimonas , the genus Bra - trophomonas , the genus Syntrophothermus, the genus dyrhizobium , the genus Agromonas, the genus Rho Eubacterium , the genus Desulfitobacterium , the genus Des dopseudomonas , the genus Nitrobacter , the genus Methyl - ulfotomaculum , the genus Pelotomaculum , the genus Butyr obacterium , the genus Rhodomicrobium , the genus ivibrio , the genus Roseburia , the genus Oscillibacter , the Pelagibacterium , the genus Parvibaculum , the genus Meth - 55 genus Thermoanaerobacter , the genus Carboxydothermus , ylocystis , the genus Parvularcula , the genus Burkholderia , the genus Natranaerobius, the genus Sphingobacterium , the the genus Ralstonia , the genus Cupriavidus, the genus genus Pedobacter , the genus Haliscomenobacter, the genus Polynucleobacter , the genus Achromobacter, the genus Bor - Porphyromonas, the genus Odoribacter, the genus detella , the genus Taylorella , the genus Pusillimonas, the Spirosoma, the genus Runella , the genus Maribacter , the genus Comamonas, the genus Alicycliphilus , the genus 60 genus Deinococcus, the genus Thermus, the genus Meio Delftia , the genus Ramlibacter , the genus Rhodoferax , the thermus, the genus Oceanithermus , the genus Marinither genus Variovorax , the genus Polaromonas, the genus Aci - mus, the genus Gemmatimonas , the genus Fusobacterium , dovorax , the genus Verminephrobacter , the genus Herminii - the genus Ilyobacter , the genus Roseiflexus , the genus Her monas, the genus Herbaspirillum , the genus Collimonas, the petosiphon , the genus Thermomicrobium , the genus Ther genus Chromobacterium , the genus Laribacter , the genus 65 motoga , the genus Thermosipho , the genus Fervidobacte Pseudogulbenkiania , the genus Nitrosomonas, the genus rium , the genus Deferribacter , the genus Calditerrivibrio , Nitrosospira , the genus Aromatoleum , the genus Azoarcus, the genus Flexistipes, the genus Metallosphaera , the genus US 10 ,294 , 500 B2 19 20 Aeropyrum , the genus Pyrobaculum , the genus Caldivirga , lus aeolius, Bacillus aerius, Bacillus aerophilus, Bacillus the genus Vulcanisaeta , the genus Acidilobus, the genus agaradhaerens, Bacillus akibai, Bacillus alcalophilus, Haloarcula , the genus Haloquadratum , the genus Natron Bacillus algicolaBacillus , Bacillus aerius, alkalidiazotrophicuslibai , Bacillushicus,, BacillusBacillus omonas, the genus Halorubrum , the genus Haloterrigena , alkalitelluriskalitellurisicolo , Bacillus: Bacillus altitudinisdikai ., Bacillus ilealveayuensisBacillus , the genus Natrialba , the genus Halalkalicoccus , the genus 5 Bacillus amyloliquefaciens, Bacillus anthracis , Bacillus Halogeometricum , the genus Thermoplasma, the genus aquimaris, Bacillus arenosi, Bacillus arseniciselenatis , Picrophilus, the genus Ferroplasma, the genus Archaeoglo Bacillus arsenicus , Bacillus arvi, Bacillus asahii , Bacillus bus, the genus Ferroglobus, the genus Polymorphum , the atrophaeus, Bacillus aurantiacus, Bacillus axarquiensis , genus Micavibrio , the genus Simiduia , the genus Leptothrix , Bacillus azotoformans, Bacillus badius, Bacillus barbari the genus Thiomonas , the genus Rubrivivax , the genus 10 cus, Bacillus bataviensis, Bacillus benzoevorans, Bacillus Methylibium , the genus Exiguobacteriumm , or the genus bogoriensis , Bacillus boroniphilus, Bacillus butanolivorans , Anaerococcus. Further , examples of the particularly preferred microbes Bacillus carboniphilus , Bacillus cecembensis , Bacillus cel which allow experimental confirmation of the production of lulosilyticus , Bacillus chagannorensis , Bacillus cibi, Bacil methacrylic acid derivatives include themicrobes belonging 15 luslus circulans, Bacillus clarkii , Bacillus clausii , Bacillus to the genus Paracoccus, the genus Sphingomonas , the coagulans, Bacillus coahuilensis , Bacillus cohnii, Bacillus genus Brevundimonas, the genus Agrobacterium , the genus decisifrondis , Bacillusdecolorationis , Bacillus drentensis , Rhizobium , the genus Xanthobacter, the genus Ochrobac Bacillus edaphicus, Bacillus endophyticus, Bacillus farragi trum , the genus Mesorhizobium , the genus Achromobacter , nis , Bacillus fastidiosus, Bacillus firmus, Bacillus flexus, the genus Comamonas , the genus Pseudomonas, the genus 20 Bacillus foraminis , Bacillus fordii, Bacillus fortis , Bacillus Acinetobacter , the genus Shewanella , the genus Listonella , fumarioli, Bacillus funiculus, Bacillus galactosidilyticus, the genus Rhodococcus , the genus Streptomyces , the genus Bacillus gelatini, Bacillus gibsonii , Bacillus halmapalus, Bacillus, the genus Geobacillus, the genus Paenibacillus, Bacillus halodurans, Bacillus halophilus, Bacillus hemicel the genus Sphingobacterium , or the genus Pedobacter. lulosilyticus, Bacillus herbersteinensis , Bacillus horikoshii , [ Preferred Species of Prokaryotes ] 25 Bacillus horti, Bacillus humi, Bacillus hwajinpoensis , Bacil [Genus Pseudomonas ] lus idriensis , Bacillus indicus, Bacillus infantis , Bacillus Examples of the microbes that are classified as the genus infernus, Bacillus insolitus, Bacillus isabeliae, Bacillus jeot Pseudomonas include Pseudomonas aeruginosa , gali , Bacillus koreensis , Bacillus krulwichiae , Bacillus Pseudomonas agarici, Pseudomonas alcaligenes, lehensis , Bacillus lentus, Bacillus licheniformis , Bacillus Pseudomonas amygdale , Pseudomonas anguiliseptica , 30 litoralis , Bacillus luciferensis , Bacillus macauensis , Bacillus Pseudomonas antimicrobica , Pseudomonas aspleni, macyae, Bacillus malacitensis , Bacillus mannanilyticus , Pseudomonas aurantiaca , Pseudomonas aureofaciens, Bacillusmarinus , Bacillusmarisflavi , Bacillusmassiliensis , Pseudomonas avellanae , Pseudomonas azotoformans , Bacillus megaterium , Bacillus methanolicus, Bacillus Pseudomonas balearica, Pseudomonas beijerinsckii , mojavensis, Bacillus mucilaginosus, Bacillus muralis , Pseudomonas beteli , Pseudomonas boreopolis , Pseudomo - 35 Bacillus murimartini, Bacillus mycoides , Bacillus nealsonii , nas carboxyhydrogena , Pseudomonas caricapapayae, Bacillus niabensis , Bacillus niacini , Bacillus novalis , Bacil Pseudomonas cichorii, Pseudomonas cissicola , Pseudomo lus odysseyi, Bacillus okhensis, Bacillus okuhidensis , Bacil nas citronellolis , Pseudomonas coronafaciens, Pseudomo lusoleronius, Bacillus oshimensis , Bacillus pallidus, Bacil nas corrugate , Pseudomonas doudoroffii , Pseudomonas lus panaciterrae, Bacillus patagoniensis , Bacillus echinoids, Pseudomonas elongate , Pseudomonas ficuserec - 40 plakortidis, Bacillus pocheonensis , Bacillus polygoni, Bacil tae , Pseudomonas flavescens, Pseudomonas flectens, lus pseudalcaliphilus, Bacillus pseudofirmus, Bacillus Pseudomonas fluorescens, Pseudomonas fragi, Pseudomo pseudomycoides , Bacillus psychrodurans, Bacillus psychro nas fulva , Pseudomonas fuscovaginae, Pseudomonas gelidi saccharolyticus, Bacillus psychrotolerans, Bacillus pumilus, cola , Pseudomonas geniculata , Pseudomonas glathei , Bacillus pycnus, Bacillus qingdaonensis , Bacillus ruris , Pseudomonas halophila , Pseudomonas hibiscicola , 45 Bacillus safensis , Bacillus salarius, Bacillus saliphilus, Pseudomonas huttiensis, Pseudomonas iners, Pseudomonas Bacillus schlegelii, Bacillus selenatarsenatis , Bacillus sel lancelota , Pseudomonas lemoignei , Pseudomonas lunden - enitireducens, Bacillus seohaeanensis , Bacillus shackle sis , Pseudomonas luteola , Pseudomonas marginalis , tonii, Bacillus silvestris , Bacillus simplex , Bacillus siralis , Pseudomonas meliae , Pseudomonas mendocina , Pseudomo - Bacillus smithii, Bacillus soli, Bacillus sonorensis , Bacillus nas mucidolens, Pseudomonas monteilli , Pseudomonas nau - 50 sphaericus, Bacillus sporothermodurans , Bacillus strato tica , Pseudomonas nitroreducens, Pseudomonas oleo - sphericus, Bacillus subterraneus, Bacillus taeanensis , vorans, Pseudomonas oryzihabitans , Pseudomonas Bacillus tequilensis , Bacillus thermantarcticus , Bacillus pertucinogena , Pseudomonas phenazinium , Pseudomonas thermoamylovorans, Bacillus thermocloacae , Bacillus thio pictorum , Pseudomonas pseudoalcaligenes , Pseudomonas parans , Bacillus tusciae, Bacillus vallismortis , Bacillus ved putida , Pseudomonas pyrrocinia , Pseudomonas resino - 55 deri , Bacillus velezensis , Bacillus vietnamensis , Bacillus vorans, Pseudomonas rhodesiae , Pseudomonas saccharo - vireti , Bacillus wakoensis , and Bacillus weihenstephanensis . phila , Pseudomonas savastanoi, Pseudomonas spinosa , [Genus Sphingobacterium ] Pseudomonas stanieri, Pseudomonas straminae, Pseudomo Examples of the microbes that are classified as the genus nas stutzeri, Pseudomonas synxantha , Pseudomonas syrin Sphingobacterium include Sphingobacterium multivorum , gae , Pseudomonas syzygii, Pseudomonas taetrolens , 60 Sphingobacterium spiritivorum , Sphingobacterium alimen Pseudomonas tolaasii , Pseudomonas veronii, Pseudomonas tarium , Sphingobacterium anhuiense , Sphingobacterium viridiflava , Pseudomonas vulgaris , and Pseudomonas wis antarcticum , Sphingobacterium bambusae , Sphingobacte consinensis . rum canadense , phigobacterium composti , Sphingobac [Genus Bacillus ] terium daejeonense , Sphingobacterium faecium , Sphingob Examples of the microbes that are classified as the genus 65 acterium heparinum , Sphingobacterium kitahiroshimense , Bacillus include Bacillus cereus, Bacillus subtilis , Bacillus Sphingobacterium lactis, Sphingobacterium mizutaii, Sphin thuringiensis , Bacillus acidiceler, Bacillus acidicola , Bacil gobacterium nematocida, Sphingobacterium piscium , US 10 ,294 , 500 B2 21 Sphingobacterium shayense, Sphingobacterium siyangense , bacter cryoconitis , Pedobacter daechungensis, Pedobacter Sphingobacterium thalpophilum , and Sphingobacterium duraquae , Pedobacter ginsengisoli , Pedobacter hartonius, wenxiniae . Pedobacter heparinus, Pedobacter himalayensis, Pedo [Genus Comamonas ] bacter j eongneungensis , Pedobacter koreensis , Pedobacter Examples of the microbes that are classified as the genus 5 lentus, Pedobacter metabolipauper, Pedobacter nyackensis , Comamonas include Comamonas acidovorans , Comamonas Pedobacter panaciterrae , Pedobacter piscium , Pedobacter aquatica , Comamonas badia , Comamonas composti, Coma roseus, Pedobacter saltans, Pedobacter steynii , Pedobacter monas denitrificans , Comamonas granuli , Comamonas ker suwonensis, and Pedobacter terricola . stersii , Comamonas koreensis , Comamonas nitrativorans, [Genus Paenibacillus ] Comamonas odontotermites , Comamonas terrae , Comamo - 10 Examples of the microbes that are classified as the genus nas terrigena , Comamonas testosteroni, Comamonas thio Paenibacillus include Paenibacillus aestuarii, Paenibacil oxydans , and Comamonas zonglianii. lus alginolyticus, Paenibacillus algorifonticola , Paenibacil [Genus Brevundimonas ] lus alvei , Paenibacillus amylolyticus , Paenibacillus api Examples of the microbes that are classified as the genus arius, Paenibacillus assamensis , Paenibacillus Brevundimonas include Brevundimonas alba , Brevundimo - 15 azoreducens, Paenibacillus azotofixans, Paenibacillus bar nas aurantiaca , Brevundimonas bacteroides , Brevundimo - cinonensis , Paenibacillus barengoltzii , Paenibacillus nas basaltis , Brevundimonas bullata , Brevundimonas campinasensis , Paenibacillus cellulosilyticus, Paenibacillus diminuta , Brevundimonas intermedia , Brevundimonas chibensis , Paenibacillus chitinolyticus , Paenibacillus chon kwangchunensis , Brevundimonas lenta , Brevundimonas droitinus , Paenibacillus curdlanolyticus, Paenibacillus dae mediterranea , Brevundimonas nasdae, Brevundimonas olei, 20 jeonensis , Paenibacillus durus , Paenibacillus ehimensis , Brevundimonas subvibrioides , Brevundimonas terrae , Bre - Paenibacillus elgii, Paenibacillus filicis, Paenibacillus vundimonas vancanneytii, Brevundimonas variabilis , Bre - frigoriresistens, Paenibacillus gansuensis , Paenibacillus vundimonas vesicularis , and Brevundimonas viscosa . ginsengihumi, Paenibacillus glucanolyticus, Paenibacillus Genus Sphingomonas ] glycanilyticus , Paenibacillus graminis , Paenibacillus Examples of the microbes that are classified as the genus 25 hodogayensis , Paenibacillus hordei, Paenibacillus humicus, Sphingomonas include Sphingomonas abaci, Sphingomonas Paenibacillus illinoisensis, Paenibacillus jamilae, Paeniba adhaesiva , Sphingomonas aerolata , Sphingomonas aquati cillus kobensis, Paenibacillus koleovorans, Paenibacillus lis , Sphingomonas asaccharolytica , Sphingomonas astaxan - konsidensis , Paenibacillus kribbensis, Paenibacillus larvae thinifaciens, Sphingomonas aurantiaca , Sphingomonas azo subsp . larvae , Paenibacillus larvae subsp . pulvifaciens , tifigens, Sphingomonas azotofornans, Sphingomonas 30 Paenibacillus lautus, Paenibacillus macerans, Paenibacil capsulata , Sphingomonas changbaiensis , Sphingomonas lus macquariensis subsp . defensor, Paenibacillus mendelii , chlorophenolica , Sphingomonas cynarae , Sphingomonas Paenibacillus motobuensis , Paenibacillus naphthaleno desiccabilis , Sphingomonas dokdonensis , Sphingomonas vorans, Paenibacillus nematophilus, Paenibacillus ocean echinoides, Sphingomonas endophytica , Sphingomonas isediminis , Paenibacillus odorifer , Paenibacillus pabuli , faeni, Sphingomonas fennica , Sphingomonas ginsenosi- 35 Paenibacillus pasadenensis, Paenibacillus peoriae, Paeni dimutans, Sphingomonas haloaromaticamans, Sphingomo bacillus phoenicis , Paenibacillus pini, Paenibacillus pini nas herbicidovorans, Sphingomonas humi, Sphingomonas humi, Paenibacillus polymyxa , Paenibacillus pueri , Paeni insulae , Sphingomonas japonica, Sphingomonas jaspsi, bacillus rigui, Paenibacillus stellifer, Paenibacillus Sphingomonas jejuensis, Sphingomonas koreensis , Sphin taiwanensis, Paenibacillus terrae , Paenibacillus terrigena , gomonas macrogoltabidus , Sphingomonas mali, Sphin - 40 Paenibacillus thermophilus, Paenibacillus thiaminolyticus, gomonas melonis , Sphingomonas molluscorum , Sphingomo- Paenibacillus tundrae , Paenibacillus validus, Paenibacil nas mucosissima, Sphingomonas natatoria , Sphingomonas lusvulneris, Paenibacillus wooponensis , and Paenibacillus oligophenolica , Sphingomonas panni, Sphingomonas para xylaniclasticus. paucimobilis , Sphingomonas paucimobilis, Sphingomonas [Genus Acgromobacter ] phyllosphaerae, Sphingomonas pituitosa , Sphingomonas 45 Examples of themicrobes that are classified as the genus polyaromaticivorans, Sphingomonas pruni, Sphingomonas Acgromobacter include Achromobacter arsenitoxydans , rosa , Sphingomonas rosei?lava , Sphingomonas rubra , Achromobacter cholinophagum , Achromobacter cyclo Sphingomonas sanguinis , Sphingomonas soli , Sphingomo - clastes, Achromobacter denitrificans, Achromobacter fisch nas suberifaciens, Sphingomonas subterranea , Sphingomo eri, Achromobacter hartlebii, Achromobacter immobilis , nas terrae , Sphingomonas trueperi , Sphingomonas ursin - 50 Achromobacter insolitus, Achromobacter lactolyticus, Ach cola , Sphingomonas wittichii , Sphingomonas yabuuchiae , romobacter lyticus, Achromobacter methanolophila , Achro Sphingomonas yanoikuyae , and Sphingomonas yunnanen mobacter pestifer, Achromobacter piechaudii, Achromo sis . bacter ruhlandii, Achromobacter spanios, Achromobacter [Genus Ochrobactrum ] viscosus , Achromobacter xerosis , Achromobacter xylosoxi Examples of the microbes that are classified as the genus 55 dans, Achromobacter xylosoxidans subsp . denitrificans, and Ochrobactrum include Ochrobactrum anthropi, Ochrobac- Achromobacter xylosoxidans subsp . xylosoxidans. trum cytisi , Ochrobactrum daejeonense , Ochrobactrum [Genus Acinetobacter ] gallinifaecis , Ochrobactrum grignonense, Ochrobactrum Examples of the microbes that are classified as the genus haemophilum , Ochrobactrum intermedium , Ochrobactrum Acinetobacter include Acinetobacter baumannii , Acineto lupini, Ochrobactrum oryzae , Ochrobactrum pseudinterme - 60 bacter aylyi, Acinetobacter beijerinckii , Acinetobacter ber dium , Ochrobactrum pseudogrignonense , Ochrobactrum eziniae, Acinetobacter boubetii, Acinetobacter calcoaceti thiophenivorans, and Ochrobactrum tritici. cus, Acinetobacter gerneri, Acinetobacter grimontii, [Genus Pedobacter ] Acinetobacter guilouiae, Acinetobacter gyllenbergii, Acine Examples of the microbes that are classified as the genus tobacter haemolyticus , Acinetobacter johnsonii , Acineto Pedobacter include Pedobacter africanus, Pedobacter agri, 65 bacter junii, Acinetobacter kyonggiensis , Acinetobacter Pedobacter alluvius, Pedobacter aquatilis , Pedobacter lwoffii, Acinetobacter oleivorans, Acinetobacter parvus , borealis , Pedobacter caeni, Pedobacter composti, Pedo - Acinetobacter psychrotolerans, Acinetobacter radioresist US 10 ,294 , 500 B2 23 24 ens , Acinetobacter schindleri, Acinetobacter soli , Acineto ense , Rhizobium multihospitium , Rhizobium nagarjuna bacter tandoii, Acinetobacter tartarogenes, Acinetobacter nagarensis , Rhizobium oryzae , Rhizobium phaseoli, Rhizo tjernbergiae , Acinetobacter towneri, Acinetobacterursingii, bium pisi , Rhizobium pusense , Rhizobium radiobacter, and Acinetobacter venetianus . Rhizobium rhizogenes, Rhizobium rubi, Rhizobium sel [Genus Shewanella ] 5 enitireducens, Rhizobium soli, Rhizobium sullae , Rhizobium Examples of the microbes that are classified as the genus tibeticum , Rhizobium trifolii, Rhizobium tropici, Rhizobium Shewanella include Shewanella piezotolerans, Shewanella tuxtlense , Rhizobium undicola , Rhizobium validum , and abyssi, Shewanella affinis, Shewanella algae, Shewanella Rhizobium vitis . algidipiscicola , Shewanella amazonensis, Shewanella [Genus Paracoccus ] aquimarina , Shewanella arctica , Shewanella atlantica , 10 Examples of the microbes that are classified as the genus Shewanella baltica , Shewanella basaltis , Shewanella ben Paracoccus include Paracoccus aestuarii, Paracoccus alca thica , Shewanella candadensis, Shewanella chilikensis, liphilus , Paracoccus alkenifer, Paracoccus aminophilus, Shewanella colwelliana , Shewanella corallii, Shewanella Paracoccus aminovorans, Paracoccus beibuensis, Paracoc decolorationis , Shewanella denitrificans , Shewanella dong cus bengalensis , Paracoccus chinensis , Paracoccus denitri haensis , Shewanella fidelis , Shewanella fodinae, Shewanella 15 ficans, Paracoccus halophilus, Paracoccus homiensis , frigidimarina , Shewanella gaetbuli , Shewanella gelidima Paracoccus kocurii , Paracoccus caeni, Paracoccus kon rina , Shewanella glacialipiscicola , Shewanella gopherii, dratievae, Paracoccus koreensis, Paracoccus marinus, Shewanella hafniensis , Shewanella halifaxensis, Shewanella Paracoccus methylutens, Paracoccus oceanense, Paracoc haliotis , Shewanella hanedai, Shewanella japonica , cus pantotrophus, Paracoccus seriniphilus, Paracoccus sol Shewanella kaireitica , Shewanella ivingstonensis , 20 ventivorans, Paracoccus sulfuroxidans, Paracoccus thio Shewanella loihica , Shewanella marina , Shewanella marin - cyanatus , Paracoccus versutus, Paracoccus yeei, and intestina , Shewanella marisflavi, Shewanella morhuae , Paracoccus zeaxanthinifaciens. Shewanella olleyana , Shewanella oneidensis , Shewanella [Genus Xanthobacter ] pacifica , Shewanella pealeana , Shewanella pneumatophori, Examples of the microbes that are classified as the genus Shewanella profunda , Shewanella putrefaciens, Shewanella 25 Xanthobacter include Xanthobacter agilis , Xanthobacter sairae , Shewanella schlegeliana , Shewanella sediminis, aminoxidans, Xanthobacter autotrophicus, Xanthobacter Shewanella surugensis, Shewanella vesiculosa , Shewanella flavus, Xanthobacter tagetidis , and Xanthobacter viscosus. violacea , Shewanella waksmanii, Shewanella woodyi , and [Genus Streptomyces ] Shewanella xiamenensis. Examples of the microbes that are classified as the genus Genus Listonella 30 Streptomyces include Streptomyces abikoensis , Streptomy Examples of the microbes that are classified as the genus ces aburaviensis , Streptomyces aburaviensis subsp . tuftfor Listonella include Listonella anguillara , Listonella anguil - mis , Streptomyces achromogenes, Streptomyces achromo larum , and Listonella pelagia . genes subsp . achromogenes, Streptomyces achromogenes [Genus Agrobacterium ] subsp . rubradiris, Streptomyces achromogenes subsp . strep Examples of the microbes that are classified as the genus 35 tozoticus, Streptomyces achromogenes subsp . tomaymyceti Agrobacterium include Agrobacterium agile , Agrobacte - cus, Streptomyces acidiscabies , Streptomyces acidoresis rium aureum , Agrobacterium azotophilum , Agrobacterium tans, Streptomyces acrimycini, Streptomyces actuosus, gypsophilae , Agrobacterium luteum , Agrobacterium Streptomyces aculeolatus , Streptomyces adephospholyticus, pseudotsugae, Agrobacterium rhizogenes, Agrobacterium Streptomyces afghaniensis , Streptomyces africanus, Strep ferrugineum , Agrobacterium sanguineum , Agrobacterium 40 tomyces agglomeratus, Streptomyces ahygroscopicus, Strep tumefaciens , Agrobacterium viscosum , and Agrobacterium tomyces akiyoshiensis , Streptomyces alanosinicus, Strepto vitis . myces albaduncus, Streptomyces albiaxialis , Streptomyces [Genus Mesorhizobium ] albidochromogenes, Streptomyces albidoflavus, Streptomy Examples of the microbes that are classified as the genus ces albidus, Streptomyces albireticuli , Streptomyces albo Mesorhizobium include Mesorhizobium albiziae , 45 chromogenes, Streptomyces albocinerescens, Streptomyces Mesorhizobium amorphae , Mesorhizobium australicum , albofaciens, Streptomyces alboflavus, Streptomyces Mesorhizobium caraganae , Mesorhizobium chacoense , albogriseolus, Streptomyces albohelvatus, Streptomyces Mesorhizobium ciceri, Mesorhizobium gobiense , albolongus, Streptomyces alboniger, Streptomyces albospi Mesorhizobium loti , Mesorhizobium mediterraneum , nus, Streptomyces albosporeus, Streptomyces albosporeus Mesorhizobium metallidurans, Mesorhizobium opportunis - 50 subsp . labilomyceticus, Streptomyces albovinaceus, Strep tum , Mesorhizobium plurifarium , Mesorhizobium huakuii, tomyces alboviridis , Streptomyces albulus, Streptomyces Mesorhizobium septentrionale , Mesorhizobium shan - albus , Streptomyces albus subsp . albus, Streptomyces albus grilense , Mesorhizobium tarimense , Mesorhizobium tem - subsp . coleimyceticus, Streptomyces albus subsp . pathocidi peratum , Mesorhizobium thiogangeticum , and Mesorhizo cus, Streptomyces alcalophilus, Streptomyces almquistii, bium tianshanense. 55 Streptomyces alni, Streptomyces althioticus, Streptomyces [Genus Rhizobium ] amagasakensis, Streptomyces amakusaensis, Streptomyces Examples of the microbes that are classified as the genus ambofaciens, Streptomyces aminophilus, Streptomyces Rhizobium include Rhizobium alamii, Rhizobium alkalisoli, anandii , Streptomyces angustmyceticus, Streptomyces Rhizobium cellulosilyticum , Rhizobium daejeonense , Rhizo - ansochromogenes subsp . ansochromogenes , Streptomyces bium endophyticum , Rhizobium etli , Rhizobium fabae , 60 anthocyanicus , Streptomyces antibioticus, Streptomyces Rhizobium gallicum , Rhizobium giardinii , Rhizobium gra - antifibrinolyticus , Streptomyces antimycoticus, Streptomy hamii , Rhizobium hainanense , Rhizobium huautlense , ces anulatus, Streptomyces aomiensis, Streptomyces arabi Rhizobium galegae , Rhizobium indica, Rhizobium indicus, cus, Streptomyces ardus, Streptomyces arenae , Streptomyces Rhizobium indigoferae, Rhizobium larrymoorei, Rhizobium argenteolus, Streptomyces argenteolus subsp . toyonakensis , leguminosarum , Rhizobium leucaenae , Rhizobium loes - 65 Streptomyces argillaceus, Streptomyces armeniacus , Strep sense , Rhizobium lupini, Rhizobium lusitanum , Rhizobium tomyces armentosus , Streptomyces arsitiensis , Streptomyces mesosinicum , Rhizobium miluonense , Rhizobium mongol ascomycinicus, Streptomyces asiaticus, Streptomyces US 10 , 294 ,500 B2 25 26 asterosporus, Streptomyces atratus, Streptomyces atroau colombiensis , Streptomyces coralus, Streptomyces corcho rantiacus , Streptomyces atrocyaneus, Streptomyces atrofa rusii, Streptomyces coriofaciens, Streptomyces costarica ciens, Streptomyces atrolaccus, Streptomyces atroolivaceus, nus, Streptomyces craterifer, Streptomyces cremeus , Strep Streptomyces atroviolaceus, Streptomyces atrovirens, Strep tomyces croceus, Streptomyces crystallinus, Streptomyces tomyces aurantiacogriseus , Streptomyces aurantiacus, 5 curacoi, Streptomyces cuspidosporus, Streptomyces cyaneo Streptomyces aurantiogriseus, Streptomyces auratus, Strep - fuscatus, Streptomyces cyaneogriseus, Streptomyces cya tomyces aureocirculatus, Streptomyces aureofaciens, Strep neus , Streptomyces cyanoalbus , Streptomyces cyanocolor, tomyces aureomonopodiales , Streptomyces aureorectus, Streptomyces cyanothe genus, Streptomyces cyanogriseus, Streptomyces aureoverticillatus, Streptomyces aureus, Streptomycesre cylindosporus, Streptomyces daghestanicus , Streptomyces aurigineus , Streptomyces avellaneus, Strepto - 10 Streptomyces davawensis , Streptomyces decovicus , Strepto myces avermectinius, Streptomyces avermitilis , Streptomy- myces demainii, Streptomyces diastaticus, Streptomyces dia ces avicenniae, Streptomyces avidinii , Streptomyces axinel staticus subsp . ardesiacus, Streptomyces diastaticus subsp . lae , Streptomyces azureus, Streptomyces baarnensis , diastaticus, Streptomyces diastatochromogenes, Streptomy Streptomyces bacillaris , Streptomyces badius, Streptomyces ces diastatochromogenes subsp . luteus, Streptomyces distal baliensis , Streptomyces bambergiensis, Streptomyces ban - 15 licus, Streptomyces djakartensis, Streptomyces drozdowiczii, gladeshensis , Streptomyces bangladeshiensis , Streptomyces Streptomyces durhamensis , Streptomyces durmitorensis, beijiangensis, Streptomyces bellus, Streptomyces bellus Streptomyces eburosporeus, Streptomyces echinatus, Strep subsp . cirolerosus , Streptomyces bernensis , Streptomyces tomyces echinoruber, Streptomycesederensis , Streptomyces bicolor, Streptomyces bifurcus, Streptomyces bikiniensis, elgreteus, Streptomyces elizabethii , Streptomyces emeiensis , Streptomyces bikiniensis subsp . zorbonensis, Streptomyces 20 Streptomyces endus , Streptomyces enissocaesilis, Strepto blastmyceticus, Streptomyces bluensis , Streptomyces bobili, myces eridani, Streptomyces erumpens, Streptomyces eryth Streptomyces bottropensis , Streptomyces brasiliensis , Strep - rochromogenes, Streptomyces erythrogriseus, Streptomyces tomyces brevisprorus, Streptomyces brunneogriseus , Strep - espinosus, Streptomyces eurocidicus , Streptomyces euro tomyces bungoensis , Streptomyces cacaoi, Streptomyces paeiscabiei, Streptomyces eurythermus, Streptomyces exfo cacaoi subsp . asoensis , Streptomyces cacaoi subsp . cacaoi, 25 liatus , Streptomyces fasiculatus, Streptomyces felleus , Strep Streptomyces caelestis , Streptomyces caelicus , Streptomyces tomyces ferralitis , Streptomyces fervens subsp . melrosporus , caeruleus, Streptomyces caesius, Streptomyces californicus, Streptomyces ficellus , Streptomyces filamentosus, Strepto Streptomyces calvus , Streptomyces canadiensis, Streptomy - myces filipinensis , Streptomyces fimbriatus, Streptomyces ces canarius, Streptomyces candidus, Streptomyces cane - fimicarius, Streptomyces finlayi, Streptomyces flaveolus , scens , Streptomyces cangkringensis, Streptomyces 30 Streptomyces flaveus , Streptomyces flavidofuscus, Strepto caniferus, Streptomyces canus, Streptomyces capillispiralis , myces flavidovirens, Streptomyces flavidovirens subsp . fus Streptomyces capoamus, Streptomyces capuensis , Strepto - cus, Streptomyces flaviscleroticus, Streptomyces flavochro myces carnosus , Streptomyces carpaticus, Streptomyces mogenes , Streptomyces flavofungini, Streptomyces carpinensis , Streptomyces castaneoglobisporus , Streptomy- flavofuscus, Streptomyces flavogriseus, Streptomyces flavo ces castaneus, Streptomyces castelarensis , Streptomyces 35 macrosporus , Streptomyces flavorectus, Streptomyces fla catenulae, Streptomyces cattleya , Streptomyces cavourensis , votricini, Streptomyces flavotricini subsp . pseudochromo Streptomyces cavourensis subsp . cavourensis , Streptomyces genes, Streptomyces flavovariabilis, Streptomyces cebimarensis , Streptomyces cellostaticus, Streptomyces cel- flavovirens , Streptomyces flavoviridis , Streptomyces flavus, luloflavus, Streptomyces cellulosae, Streptomyces champa Streptomyces flocculus, Streptomyces floridae , Streptomyces vatii , Streptomyces chartreusis , Streptomyces chattanoogen - 40 fluorescens, Streptomyces fradiae , Streptomyces fradiae sis , Streptomyces cheonanensis , Streptomyces subsp . acinicolor, Streptomyces fradiae subsp . italicus , chiangmaiensis , Streptomyces chibaensis, Streptomyces Streptomyces fragilis , Streptomyces fragmentans, Strepto chlorochromogenes, Streptomyces chrestomyceticus, Strep - myces fragmentans subsp . aquatica , Streptomyces fulvissi tomyces chrestomyceticus subsp . rubescens, Streptomyces mus, Streptomyces fulvorobeus , Streptomyces fulvoviola chromofuscus , Streptomyces chromothe genus , Streptomyces 45 ceus , Streptomyces fulvoviridis , Streptomyces fumanus, chryseus, Streptomyces chrysomallus, Streptomyces chryso Streptomyces fumigatiscleroticus, Streptomyces fungicidi mallus subsp . fumigatus, Streptomyces chungwhensis , Strep cus, Streptomyces furlongus, Streptomyces furlongus subsp . tomyces cinereorectus , Streptomyces cinereoruber, Strepto - furlongus , Streptomyces fuscoatrus, Streptomyces gabonae , myces cinereoruber subsp . cinereoruber, Streptomyces Streptomyces galbus , Streptomyces galilaeus, Streptomyces cinereoruber subsp . fructofermentans, Streptomyces 50 gallinarius, Streptomyces gancidicus, Streptomyces gangto cinereospinus, Streptomyces cinereus, Streptomyces cinero kensis , Streptomyces gannmycicus, Streptomyces garden , chromogenes, Streptomyces cinnabarinus, Streptomyces Streptomyces gardneri, Streptomyces gedanensis , Strepto cinnamocastaneus, Streptomyces cinnamonensis , Strepto - myces gelaticus, Streptomyces geldanamycininus, Strepto myces cinnamoneus, Streptomyces cinnamoneus subsp . myces geysiriensis, Streptomyces ghanaensis , Streptomyces albosporus, Streptomyces cinnamoneus subsp . forma aza - 55 gibsonii, Streptomyces ginsengisoli, Streptomyces glauce coluta , Streptomyces cinnamoneus subsp . lanosus, Strepto scens , Streptomyces glauciniger, Streptomyces glaucospo myces cinnamoneus subsp . sparsus, Streptomyces circula rus, Streptomyces glaucus, Streptomyces globifer, Strepto tus, Streptomyces cirratus , Streptomyces ciscaucasicus, myces globisporus , Streptomyces globisporus subsp . Streptomyces citrofurescens , Streptomyces citrus, Strep caucasicus, Streptomyces globisporus subsp . globisporus, tomyces citricolor, Streptomyces clavifer , Streptomyces cla - 60 Streptomyces globosus, Streptomyces glomeratus, Strepto vuligerus, Streptomyces coacervatus , Streptomyces cocklen - myces glomeroaurantiacus, Streptomyces glomerochromo sis , Streptomyces coelescens , Streptomyces coeliatus, genes, Streptomyces gobitricini, Streptomyces goshikiensis , Streptomyces coelicoflavus, Streptomyces coelicolor, Strep - Streptomyces gougerotii , Streptomyces graminearus, Strep tomyces coeruleoaurantiacus, Streptomyces coeruleofuscus, tomyces gramineus, Streptomyces graminofaciens, Strepto Streptomyces coeruleoprunus, Streptomyces coeruleorubi- 65 myces griseiniger, Streptomyces griseinus, Streptomyces dus , Streptomyces coerulescens, Streptomyces collinus, griseoaurantiacus, Streptomyces griseobrunneus, Strepto Streptomyces collinus subsp . albescens, Streptomyces myces griseocarneus, Streptomyces griseochromogenes , US 10 , 294 , 500 B2 27 28 Streptomyces griseochromogenes subsp . suitaensis, Strepto color, Streptomyces lazureus, Streptomyces levis , Streptomy myces griseofaciens, Streptomyces griseoflavus, Streptomy ces levoris , Streptomyces libani, Streptomyces libani subsp . ces griseoflavus subsp . pyrindicus, Streptomyces griseofus libani, Streptomyces libani subsp . rufus , Streptomyces lieno cus, Streptomyces griseoincarnatus, Streptomyces mycini , Streptomyces lieskei, Streptomyces lilaceus, Strep griseoloalbus , Streptomyces griseolosuffuscus, Streptomy- 5 tomyces lilacinofulvus, Streptomyces lilacinus, Streptomyces ces griseolus, Streptomyces griseoluteus, Streptomyces gris - limosus, Streptomyces lincolnensis, Streptomyces lipmanii, eomycini, Streptomyces griseoplanus, Streptomyces grise - Streptomyces lisandri, Streptomyces litmocidini, Streptomy orubens , Streptomyces griseoruber, Streptomyces ces lividans , Streptomyces lividoclavatus , Streptomyces livi griseorubiginosus, Streptomyces griseospiralis , Streptomy- dus, Streptomyces loidensis , Streptomyces lomondensis , ces griseosporeus, Streptomyces griseostramineus , Strepto - 10 Streptomyces longisporoflavus , Streptomyces longispororu myces griseoverticillatus, Streptomyces griseoviridis , Strep ber, Streptomyces longisporus, Streptomyces longissimus , tomyces griseus, Streptomyces griseus subsp . bruneus, Streptomyces longwoodensis , Streptomyces lucensis , Strep Streptomyces griseus subsp . desideus, Streptomyces griseus tomyces lunalinharensis , Streptomyces lunalinharesii , Strep subsp . erizensis, Streptomyces griseus subsp . formicus, tomyces luridiscabiei , Streptomyces luridus, Streptomyces Streptomyces griseus subsp . griseus, Streptomyces griseus 15 lusitanus, Streptomyces lusitanus var. tetracyclini, Strepto subsp . purpureus, Streptomyces griseus subsp . rhodochrous, myces luteireticuli, Streptomyces luteocolor, Streptomyces Streptomyces griseus subsp . solvifaciens, Streptomyces luteogriseus, Streptomyces luteolutescens, Streptomyces guanduensis , Streptomyces gulbargensis , Streptomyces gyp - luteosporeus, Streptomyces luteoverticillatus, Streptomyces seus, Streptomyces hainanensis , Streptomyceshaliclonae, lydicus , Streptomyces macromomyceticus, Streptomyces Streptomyces halotolerans , Streptomyces halstedii , Strepto - 20 macrosporeus , Streptomyces macrosporus, Streptomyces myces hawaiiensis , Streptomyces hazeliensis , Streptomy - maizeus, Streptomyces malachiticus , Streptomyces mala ceshebeiensis , Streptomyces heimi, Streptomyces heliomy - chiticus subsp . griseospinosus, Streptomyces malachitofus cini, Streptomyces helvaticus, Streptomyces henetus, cus, Streptomyces malachitorectus, Streptomyces malachi Streptomyces herbaricolor, Streptomyces heteromorphus, tospinus, Streptomycesm alayensis, Streptomyces Streptomyces himgriensis , Streptomyces hiroshimensis , 25 malaysiensis, Streptomyces manipurensis , Streptomyces Streptomyces hirsutus, Streptomyces horton , Streptomyces marinus , Streptomyces maritimus , Streptomyces marok humidus, Streptomyces humidus subsp . antitumoris , Strep - konensis, Streptomyces mashuensis , Streptomyces massas tomyces humifer , Streptomyces humiferus, Streptomyces poreus , Streptomyces matensis , Streptomyces mauvecolor, hundungensis , Streptomyces hyalinus, Streptomyces hydera - Streptomyces mayteni, Streptomyces mediocidicus, Strepto badensis , Streptomyces hydrogenans , Streptomyces hygro - 30 myces mediolani, Streptomyces megasporus , Streptomyces scopicus, Streptomyces hygroscopicus subsp . aabomyceti - melanogenes, Streptomyces melanosporofaciens, Streptomy cus , Streptomyces hygroscopicus subsp . angustmyceticus , ces mentougouensis , Streptomyces mexicanus, Streptomyces Streptomyces hygroscopicus subsp . azalomyceticus, Strep - michiganensi, Streptomyces michiganensis subsp . amylolyti tomyces hygroscopicus subsp . crystallogenes, Streptomyces cus, Streptomyces microflavus, Streptomyces microsporus, hygroscopicus subsp . decoyicus, Streptomyces hygroscopi- 35 Streptomyces miharaensis , Streptomyces minoensis , Strep cus subsp . duamyceticus, Streptomyces hygroscopicus tomyces minutiscleroticus, Streptomyces mirabilis , Strepto subsp . geldanus, Streptomyces hygroscopicus subsp . glebo myces misakiensis , Streptomyces misawanensis , Streptomy sus, Streptomyces hygroscopicus subsp . hialomyceticus , ces misionensis , Streptomyces mobaraensis , Streptomyces Streptomyces hygroscopicus subsp . hygroscopicus, Strepto - moderatus, Streptomyces monomycini, Streptomyces mord myces hygroscopicus subsp . hygroscopius , Streptomyces 40 arskii, Streptomyces morookaense , Streptomyces moroo hygroscopicus subsp . limoneus, Streptomyces hygroscopicus kaensis , Streptomyces mucoflavus, Streptomyces multispira subsp . ossamyceticus, Streptomyces hygrospinosus, Strepto - lis , Streptomyces murinus, Streptomycesmutabilis , myces iakyrus, Streptomyces inaequalis , Streptomyces indi- Streptomyces mutomycini, Streptomyces mycarofaciens , aensis, Streptomyces indicus, Streptomyces indigocolor, Streptomyces myxogenes, Streptomyces naganishii, Strepto Streptomycesindigteru trepmesindonesiensis5mycesanningensis, Streptomyceanhensis, Streptomyces Streptomyces insignis , Streptomyces intermedius, Strepto - naraensis , Streptomyces narbonensis , Streptomyces nashvil myces inusitatus, Streptomyces ipomoeae , Streptomyces lensis , Streptomyces natalensis , Streptomyces neburosus , iranensis, Streptomyces ishigakiensis , Streptomyces jamai- Streptomyces neocaliberis , Streptomyces netropsis , Strepto censis , Streptomyces janthinus, Streptomycesjavensis, Strep myces neyagawaensis, treptomyces nigellus, Streptomyces tomyces jietaisiensis , Streptomyces jujuy, Streptomyces 50 niger, Streptomyces nigrescens , Streptomyces nigrifaciens, jumonjinensis , Streptomyces kagawaensis , Streptomyces Streptomyces nigrogriseolus, Streptomyces nigroviolens, kagoshimanus , Streptomyces kanamyceticus , Streptomyces Streptomyces nitrosporeus , Streptomyces niveoruber , Strep kaniharaensis , Streptomyces karnatakensis , Streptomyces tomyces niveus , Streptomyces nobilis, Streptomyces nobori kasugaensis , Streptomyces kasugaspinus, Streptomyces toensis , Streptomyces nodosus, Streptomyces nodosus subsp . katrae , Streptomyces kentuckensis , Streptomyces khandal- 55 asukaensis , Streptomyces nogalater, Streptomyces nojirien ensis , Streptomyces kitasatoensis , Streptomyces kobenensis , sis , Streptomyces noursei, Streptomyces novaecaesareae , Streptomyces koyangensis , Streptomyces krainskii, Strepto Streptomyces novoverticillus, Streptomyces ochraceiscle myces kunmingensis , Streptomyces kurssanovii, Streptomy - roticus , Streptomyces ochrosporus , Streptomyces odorifer, ces kuwaitiensis, Streptomyces labedae, Streptomyces lac - Streptomyces ogaensis , Streptomyces oidiosporus, Strepto eyi, Streptomyces laculatusporus, Streptomyces 60 myces olivaceiscleroticus , Streptomyces olivaceoviridis , laeteviolaceus , Streptomyces lanatus, Streptomyces lannen - Streptomyces olivaceus, Streptomyces olivochromogenes , sis , Streptomyces lasaliensis , Streptomyces lateritius, Strep - Streptomyces olivochromogenes subsp . cytovirinus , Strepto tomyces laurentii, Streptomyces lavendofoliae , Streptomyces myces olivogriseus, Streptomyces olivoverticillatus , Strep lavendulae, Streptomyces lavendulae subsp . fuscus, Strep tomyces olivoviridis , Streptomyces omiyaensis , Streptomy tomyces lavendulae subsp . grasserius, Streptomyces laven - 65 ces orinoci, Streptomyces ornatus, Streptomyces dulae subsp . lavendulae , Streptomyces lavendularectus, osmaniensis , Streptomyces ostreogriseus, Streptomyces Streptomyces lavenduligriseus, Streptomyces lavendulo owasiensis, Streptomyces pactum , Streptomyces padanus, US 10 , 294 ,500 B2 29 30 Streptomyces pallidus, Streptomyces panacagri , Streptomy ces rubrus, Streptomyces rufochromogenes, Streptomyces ces panaciterrae , Streptomyces panayensis , Streptomyces rutgersensis , Streptomyces ryensis , Streptomyces sahachi paradoxus , Streptomyces paraguayensis, Streptomyces par roi, Streptomyces sakaiensis , Streptomyces salinarum , vulus, Streptomyces parvus, Streptomyces paucidiastaticus, Streptomyces sampsonii , Streptomyces sanglieri, Streptomy Streptomyces paucisporeus, Streptomyces paucisporogenes , 5 ces sannanensis , Streptomyces sannurensis , Streptomyces Streptomyces paulus, Streptomyces peruviensis , Streptomy sanyensis , Streptomyces saprophyticus , Streptomyces sara ces peucetius , Streptomyces peucetius subsp . caesius, Strep ceticus , Streptomyces sayamaensis , Streptomyces scabiei, tomyces peucetius subsp . carneus, Streptomyces phaeochro - Streptomyces scabrisporus , Streptomyces sclerogranulatus, mogenes, Streptomyces phaeofaciens, Streptomyces Streptomyces sclerotialus, Streptomyces scopiformis , Strep phaeoluteichromatogenes, Streptomyces phaeoluteigriseus, 10 tomyces sedi, Streptomyces senoensis , Streptomyces seou Streptomyces phaeopurpureus, Streptomyces phaeoverticil lensis , Streptomyces septatus, Streptomyces serianimatus , latus subsp . takatsukiensis , Streptomyces phaeoviridis , Streptomyces setae , Streptomyces setonensis , Streptomyces Streptomyces pharetrae , Streptomyces phemorphus , Strep - setonii , Streptomyces shaanxiensis , Streptomyces shiodaen tomyces phytohabitans, Streptomyces pilosus, Streptomyces sis , Streptomyces showdoensis , Streptomyces sindenensis , piloviolofuscus, Streptomyces piomothe genus , Streptomyces 15 Streptomyces sioyaensis , Streptomyces sodiiphilus, Strepto platensis , Streptomyces platensis subsp . clarensis , Strepto m yces somaliensis , Streptomyces spadicis , Streptomyces myces platensis subsp . malvinus, Streptomyces platensis sparsogenes , Streptomyces sparsus, Streptomyces specialis , subsp . robigocidicus, Streptomyces plicatus , Streptomyces Streptomyces spectabilis , Streptomyces speibonae , Strepto plumbeus , Streptomyces plumbiresistens , Streptomyces plu myces spheroides , Streptomyces spinichromogenes , Strepto ricolorescens, Streptomyces polyantibioticus , Streptomyces 20 myces spinicoumarensis , Streptomyces spinoverrucosus, polychromogenes , Streptomyces polychromogenes subsp . Streptomyces spiralis , Streptomyces spiroverticillatus, arenicolus , Streptomyces poonensis , Streptomyces praecox , Streptomyces spongiae, Streptomyces sporocinereus, Strep Streptomyces prasinopilosus, Streptomyces prasinosporus, tomyces sporoclivatus, Streptomyces spororaveus, Strepto Streptomyces prasinus, Streptomyces pristinaespiralis , myces sporoverrucosus, Streptomyces steffisburgensis , Streptomyces propurpuratus , Streptomyces prunicolor, 25 Streptomyces steffisburgensis subsp . steffisburgensis , Strep Streptomyces psammoticus , Streptomyces pseudoechino - tomyces stelliscabiei, Streptomyces stramineus, Streptomy sporeus, Streptomyces pseudogriseolus, Streptomyces ces subrutilus, Streptomyces sudanensis , Streptomyces sul pseudogriseolus subsp . glucofermentans, Streptomyces fonofaciens, Streptomyces sulphureus , Streptomyces sviceus , pseudovenezuelae, Streptomyces pulcher, Streptomyces pul Streptomyces synnematoformans, Streptomyces tanashien veraceus , Streptomyces puniceus, Streptomyces puniceus 30 sis , Streptomyces tanashiensis subsp . cephalomyceticus, subsp . doliceus, Streptomyces puniciscabiei, Streptomyces Streptomyces tateyamensis, Streptomyces tauricus, Strepto purpeochromogenes, Streptomyces purpeofuscus, Strepto myces tendae , Streptomyces tenebrarius, Streptomyces ten myces purpurascens , Streptomyces purpureofuscus, Strep jimariensis , Streptomyces termitum , Streptomyces testaceus , tomyces purpureofuscus subsp . acoagulans , Streptomyces Streptomyces tetanusemus, Streptomyces thermoalcalitoler purpureus , Streptomyces purpurogeneiscleroticus , Strepto - 35 ans, Streptomyces thermoatroviridis , Streptomyces thermo myces pyridomyceticus, Streptomyces racemochromogenes , carboxydovorans, Streptomyces thermocarboxydus, Strepto Streptomyces racemosus, Streptomyces radiopugnans, myces thermocastaneus, Streptomyces thermocoerulescens , Streptomyces raffinosus, Streptomyces rameus, Streptomy . Streptomyces thermocoprophilus, Streptomyces thermocya ces ramosissimus, Streptomyces ramulosus , Streptomyces neomaculatus , Streptomyces thermocyaneoviolaceus, Strep rangoon , Streptomyces rangoonensis, Streptomyces 40 tomyces thermodiastaticus , Streptomyces thermoflavus , rapamycincus, Streptomyces recifensis , Streptomyces rec Streptomyces thermogriseoviolaceus, Streptomyces ther tiviolaceus, Streptomyces refuineus subsp . thermotolerans, mogriseus , Streptomyces thermohygroscopicus subsp . Streptomyces regalis , Streptomyces regensis , Streptomyces rubiginosus , Streptomyces thermolilacinus , Streptomyces resistomycificus, Streptomyces reticuli subsp . protomycicus, thermolineatus, Streptomyces thermoluteus, Streptomyces Streptomyces reticuliscabiei , Streptomyces rhizosphaericus , 45 thermoluteus subsp . fuscus , Streptomyces thermonitrificans, Streptomyces ribosidificus, Streptomyces rimofaciens, Strep Streptomyces thermoolivaceus subsp . fuscus, Streptomyces tomyces rimosus, Streptomyces rimosus subsp . paromomy - thermoolivaceus subsp . thermoolivaceus, Streptomyces cinus, Streptomyces rimosus subsp . rimosus, Streptomyces thermophilus, Streptomyces thermospinosisporus, Strepto rishiriensis , Streptomyces robefuscus, Streptomyces rochei, myces thermotolerans, Streptomyces thermoviolaceus , Streptomyces rosa , Streptomyces rosa subsp . notoensis , 50 Streptomyces thermoviolaceus subsp . apingens, Streptomy Streptomyces roseiscleroticus, Streptomyces roseoalbus, ces thermoviolaceus subsp . thermoviolaceus , Streptomyces Streptomyces roseoaurantius, Streptomyces roseochromo- thermovulgaris , Streptomyces thinghirensis , Streptomyces genes , Streptomyces roseochromothe genus , Streptomyces thioluteus , Streptomyces threomyceticus, Streptomyces toru roseochromothe genus subsp . albocyclini, Streptomyces losus, Streptomyces tosaensis , Streptomyces toxifertilis , roseocinereus, Streptomyces roseodiastaticus , Streptomyces 55 Streptomyces toxytricini, Streptomyces toyocaensis, Strep roseoflavus, Streptomyces roseofulvus, Streptomyces tomyces triangulatus, Streptomyces tricolor, Streptomyces roseogriseus, Streptomyces roseolilacinus, Streptomyces triculaminicus, Streptomyces triostinicus, Streptomyces tri roseolus, Streptomyces roseoluteus, Streptomyces roseopla - tolerans , Streptomyces tsukiyonensis , Streptomyces tsusi tus , Streptomyces roseorubens , Streptomyces roseosporus, maensis , Streptomyces tubercidicus , Streptomyces tuirus , Streptomyces roseoverticillatus, Streptomyces roseoviola - 60 Streptomyces tumemacerans , Streptomyces tumuli , Strepto ceus , Streptomyces roseoviolascens, Streptomyces roseo - myces turgidiscabies, Streptomyces umbrinus , Streptomyces viridis, Streptomyces roseus, Streptomyces ruber, Strepto - umbrosus, Streptomyces variabilis , Streptomyces variega myces rubicolor, Streptomyces rubidus, Streptomyces tus , Streptomyces varius, Streptomyces varsoviensis, Strep rubiginosohelvolus, Streptomyces rubiginosus, Streptomy tomyces vastus, Streptomyces vellosus, Streptomyces ven ces rubrireticuli , Streptomyces rubrocyanodiastaticus 65 dargensis , Streptomyces venezuelae , Streptomyces verne, subsp . piger, Streptomyces rubrogriseus, Streptomyces Streptomyces versipellis , Streptomyces verticillatus, Strep rubrolavendulae , Streptomyces rubroverrucosus , Streptomy tomyces verticillus, Streptomyces vietnamensis , Streptomy US 10 ,294 ,500 B2 31 ces vinaceus, Streptomyces vinaceusdrappus, Streptomyces Dinoroseobacter shibae , as the microbes that are classified violaceochromogenes , Streptomyces violaceolatus, Strepto as the genus Phaeobacter, Phaeobacter gallaeciensis , as the myces violaceorectus, Streptomyces violaceoruber, Strepto - microbes that are classified as the genus Octadecabacter , myces violaceorubidus, Streptomyces violaceus, Streptomy Octadecabacter antarcticus and Octadecabacter arcticus, as ces violaceusniger, Streptomyces violarus , Streptomyces 5 the microbes that are classified as the genus Hyphomonas, violascens, Streptomyces violens, Streptomyces violorubens, Hyphomonas neptunium , as the microbes that are classified Streptomyces virens , Streptomyces virginiae , Streptomyces as the genus Maricaulis , Maricaulis maris, as the microbes virginiae subsp . lipoxae , Streptomyces viridans , Streptomy - that are classified as the genus Hirschia , Hirschia baltica , ces viridifaciens, Streptomyces viridiflavus, Streptomyces Novosphingobium , Novosphingobium aromaticivorans , viridis, Streptomyces virdiolaceus, Streptomyces viridob phingopyxis , Sphingopyxis alskensis , as the microbes that runneus, Streptomyces viridochromogenes, Streptomyces are classified as the genus Sphingobium , Sphingobium viridochromogenes subsp . komabensis, Streptomyces viri - japonicum and Sphingobium chlorophenolicum , as the dochromogenes subsp . sulfomycini, Streptomyces viridodia - microbes that are classified as the genus Erythrobacter, staticus, Streptomyces viridogenes, Streptomyces viridospo Erythrobacter litoralis, as the microbes that are classified as rus , Streptomyces viridoverrucosus, Streptomyces 15 the genus Caulobacter, Caulobacter crescentus and Cau vitaminophilus , Streptomyces vulgaris, Streptomyces wed - lobacter segnis , as the microbes that are classified as the morensis , Streptomyces wellingtoniae , Streptomyces wer - genus Phenylobacterium , Phenylobacterium zucineum , as raensis , Streptomyces willmorei, Streptomyces wistariopsis , the microbes that are classified as the genus Asticcacaulis , Streptomyces woolensis , Streptomyces xanthochromogenes , Asticcacaulis excentricus, as the microbes that are classified Streptomyces xanthocidicus, Streptomyces xantholiticus, 20 as the genus Sinorhizobium , Sinorhizobium meliloti, Streptomyces xanthophaeus, Streptomyces xiamenensis , Sinorhizobium medicae and Sinorhizobium fredii, as the Streptomyces xinghaiensis, Streptomyces xylophagus, Strep - microbes that are classified as the genus Azorhizobium , tomyces yanglinensis , Streptomyces yanii, Streptomyces yat- Azorhizobium caulinodans , as the microbes that are classi ensis , Streptomyces yeochonensis , Streptomyces yerevanen - fied as the genus Brucella , Brucella melitensis, Brucella sis , Streptomyces yogyakartensis , Streptomyces 25 abortus, Brucella suis , Brucella ovis , Brucella canis , Bru yokosukanensis , Streptomyces youssoufiensis , Streptomyces cella microti, Brucella pinnipedialis and Brucella ceti , as the yunnanensis , Streptomyces zaomyceticus, Streptomyces zel microbes that are classified as the genus Aurantimonas, ensis , and Streptomyces zinciresistens. Aurantimonas manganoxydans , as the microbes that are [Genus Geobacillus ] classified as the genus Bradyrhizobium , Bradyrhizobium Examples of the microbes that are classified as the genus 30 japonicum , as the microbes that are classified as the genus Geobacillus include Geobacillus caldoproteolyticus, Geo - Agromonas, Agromonas oligotrophica , as the microbes that bacillus caldoxylosilyticus , Geobacillus debilis, Geobacillus are classified as the genus Rhodopseudomonas , Rho galactosidasius, Geobacillus gargensis , Geobacillus juras - dopseudomonas palustris , as the microbes that are classified sicus, Geobacillus kaustophilus, Geobacillus lituanicus, as the genus Nitrobacter, Nitrobacter winogradskyi and Geobacillus pallidus, Geobacillus stearothermophilus, Geo - 35 Nitrobacter hamburgensis , as the microbes that are classi bacillus stromboliensis, Geobacillus subterraneus, Geoba fied as the genus Methylobacterium , Methylobacterium cillus tepidamans, Geobacillus thermocatenulatus, Geoba extorquens, Methylobacterium radiotolerans and Methyl cillus thermodenitrificans, Geobacillus thermodenitrificans obacterium nodulans, as the microbes that are classified as subsp . calidus , Geobacillus thermoglucosidasius, Geobacil - the genus Rhodomicrobium , Rhodomicrobium vannielii , as lus thermoleovorans, Geobacillus toebii , Geobacillus uzen - 40 the microbes that are classified as the genus Pelagibacte sis , Geobacillus vulcani , and Geobacillus zalihae. rium , Pelagibacterium halotolerans , as the microbes that are [Genus Rhodococcus] classified as the genus Parvibaculum , Parvibaculum lava Examples of the microbes that are classified as the genus mentivorans, as the microbes that are classified as the genus Rhodococcus include Rhodococcus rhodochrous, Rhodo - Parvularcula , Parvularcula bermudensis , as the microbes coccus erythropolis, Rhodococcus equi, Rhodococcus rho - 45 that are classified as the genus Burkholderia , Burkholderia dnii , Rhodococcus corallinus, Rhodococcus rubropertinc mallei, Burkholderia pseudomallei , Burkholderia thailand tus, Rhodococcus coprophilus, Rhodococcus globerulus, ensis , Burkholderia vietnamiensis , Burkholderia cenocepa Rhodococcus chlorophenolicus, Rhodococcus luteus, Rho cia , Burkholderia ambifaria , Burkholderia multivorans , dococcus aichiensis , Rhodococcus chubuensis , Rhodococ - Burkholderia cepacia , Burkholderia xenovorans, Burkhold cus maris , and Rhodococcus fascines . 50 eria phymatum , Burkholderia phytofirmans, Burkholderia Other Preferred Species of Prokaryotes ] glumae, Burkholderia rhizoxinica , Burkholderia gladioli , Further , as the microbes that are classified as the genus Burkholderia phenoliruptrix and Burkholderia oklahomen Magnetospirillum , Magnetospirillum magneticum , as the s is, as the microbes that are classified as the genus Ralstonia , microbes that are classified as the genus Rhodospirillum , Ralstonia solanacearum , Ralstonia pickettii and Ralstonia Rhodospirillum rubrum , Rhodospirillum centenum and Rho - 55 eutropha , as the microbes that are classified as the genus dospirillum photometricum , as the microbes that are classi Cupriavidus, Cupriavidus metallidurans , Cupriavidus tai fied as the genus Azospirillum , Azospirillum lipoferum and wanensis and Cupriavidus necator, as the microbes that are Azospirillum brasilense , as the microbes that are classified classified as the genus Polynucleobacter, Polynucleobacter as the genus Tistrella , Tistrella mobilis , as the microbes that necessarius, as the microbes that are classified as the genus are classified as the genus Acidiphilium , Acidiphilium cryp - 60 Bordetella , Bordetella pertussis , Bordetella parapertussis , tum and Acidiphilium multivorum , as the microbes that are Bordetella petrii and Bordetella avium , as the microbes that classified as the genus Rhodobacter, Rhodobacter spha - are classified as the genus Taylorella , Taylorella equigeni eroides and Rhodobacter capsulatus, as the microbes that talis , as the microbes that are classified as the genus Alicy are classified as the genus Ruegeria , Ruegeria pomeroyi , as cliphilus, Alicycliphilus denitrificans , as the microbes that the microbes that are classified as the genus Roseobacter, 65 are classified as the genus Delftia , Delftia acidovorans , as Roseobacter denitrificans and Roseobacter litoralis , as the the microbes that are classified as the genus Ramlibacter , microbes that are classified as the genus Dinoroseobacter, Ramlibacter tataouinensis , as the microbes that are classi US 10 , 294 ,500 B2 33 34 fied as the genus Rhodoferax , Rhodoferax ferrireducens, as alteromonas haloplanktis, Pseudoalteromonas atlantica and the microbes that are classified as the genus Variovorax, Pseudoalteromonas tunicata , as the microbes that are clas Variovorax paradoxus, as the microbes that are classified as sified as the genus Vibrio , Vibrio parahaemolyticus, Vibrio the genus Polaromonas, Polaromonas naphthalenivorans, vulnificus, Vibrio harveyi, Vibrio furnissii, Vibrio tubiashii, as the microbes that are classified as the genus Acidovorax , 5 Vibrio sinaloensis , Vibrio rotiferianus , Vibrio orientalis , Acidovorax citrulli, Acidovorax ebreus and Acidovorax ave Vibrio harveyi, Vibrio coralliilyticus , Vibrio caribbenthicus , nae , as the microbes that are classified as the genus Vermin Vibrio brasiliensis and Vibrio alginolyticus, as the microbes ephrobacter, Verminephrobacter eiseniae , as the microbes that are classified as the genus Photobacterium , Photobac that are classified as the genus Herminiimonas, Herminii - terium profundum , as the microbes that are classified as the monas arsenicoxydans , as the microbes that are classified as 10 genus Aeromonas , Aeromonas hydrophila , Aeromonas sal the genus Herbaspirillum , Herbaspirillum seropedicae , as monicida and Aeromonas veronii , as the microbes that are the microbes that are classified as the genus Collimonas, classified as the genus Salinisphaera , Salinisphaera sha Collimonas fungivorans , as the microbes that are classified banensis , as the microbes that are classified as the genus as the genus Chromobacterium , Chromobacterium viola Legionella , Legionella pneumophila and Legionella long ceum , as the microbes that are classified as the genus 15 beachae , as the microbes that are classified as the genus Laribacter, Laribacter hongkongensis, as the microbes that Coxiella , Coxiella burnetii , as the microbes that are classi are classified as the genus Pseudogulbenkiania , Pseudogul- fied as the genus Desulfococcus, Desulfococcus oleovorans, benkiania ferrooxidans, as the microbes that are classified as as the microbes that are classified as the genus Desulfobac the genus Nitrosomonas, Nitrosomonas europaea , as the terium , Desulfobacterium autotrophicum , as the microbes microbes that are classified as the genus Nitrosospira , 20 that are classified as the genus Desulfatibacillum , Desulfati Nitrosospira multiformis , as the microbes that are classified bacillum alkenivorans , as the microbes that are classified as as the genus Aromatoleum , Aromatoleum aromaticum , as the the genus Desulfobulbus, Desulfobulbus propionicus , as the microbes that are classified as the genus Dechloromonas , microbes that are classified as the genus Desulfarculus, Dechloromonas aromatica , as the microbes that are classi- Desulfarculus baarsii , as the microbes that are classified as fied as the genus Azospira ( Dechlorosoma ), Azospira oryzae 25 the genus Geobacter, Geobacter metallireducens , Geobacter (Dechlorosoma suillum ) , as the microbes that are classified uraniireducens and Geobacter bemidjiensis , as the microbes as the genus Rheinheimera , Rheinheimera nanhaiensis , that are classified as the genus Syntrophobacter, Syntropho Nitrosococcus , Nitrosococcus oceani, Halorhodospira , bacter fumaroxidans, as the microbes that are classified as Halorhodospira halophila , as the microbes that are classi- the genus Syntrophus, Syntrophus aciditrophicus , as the fied as the genus Xanthomonas, Xanthomonas campestris , 30 microbes that are classified as the genus Desulfomonile , Xanthomonas axonopodis , Xanthomonas oryzae, Desulfomonile tiedjei, as the microbes that are classified as Xanthomonas albilineans and Xanthomonas citri , as the the genus Bdellovibrio , Bdellovibrio bacteriovorus and microbes that are classified as the genus Stenotrophomonas, Bdellovibrio exovorus, as the microbes that are classified as Stenotrophomonas maltophilia , as the microbes that are the genus Bacteriovorax, Bacteriovorax marinus, as the classified as the genus Pseudoxanthomonas, Pseudox - 35 microbes that are classified as the genus Stigmatella , Stig anthomonas suwonensis and Pseudoxanthomonas spadix , as matella aurantiaca , as the microbes that are classified as the the microbes that are classified as the genus Francisella , genus Myxococcus, Myxococcus xanthus and Myxococcus Francisella tularensis and Francisella novicida , as the fulvus , as the microbes that are classified as the genus microbes that are classified as the genus Cycloclasticus, Anaeromyxobacter, Anaeromyxobacter dehalogenans, as the Cycloclasticus zancles , as themicrobes that are classified as 40 microbes that are classified as the genus Sorangium , Soran the genus Hahella , Hahella chejuensis , as the microbes that gium cellulosum , as the microbes that are classified as the are classified as the genus Halomonas, Halomonas elongata , genus Haliangium , Haliangium ochraceum , as the microbes as the microbes that are classified as the genus Alcanivorax , that are classified as the genus Acidobacterium , Acidobac Alcanivorax borkumensis and Alcanivorax dieselolei, as the terium capsulatum , as the microbes that are classified as the microbes that are classified as the genus Kangiella , Kangi- 45 genus Granulicella , Granulicella tundricola , as the ella koreensis , as the microbes that are classified as the microbes that are classified as the genus Ilumatobacter, genus Azotobacter, Azotobacter vinelandii , as the microbes Ilumatobacter coccineum , as themicrobes that are classified that are classified as the genus Psychrobacter, Psychrobacter as the genus Streptosporangium , Streptosporangium arcticus and Psychrobacter cryohalolentis , as the microbes roseum , as the microbes that are classified as the genus that are classified as the genus Alishewanella , Alishewanella 50 Nocardiopsis, Nocardiopsis dassonvillei , as the microbes jeotgali, as the microbes that are classified as the genus that are classified as the genus Thermobifida , Thermobifida Alteromonas , Alteromonas macleodii , as the microbes that fusca , as the microbes that are classified as the genus are classified as the genus Glaciecola , Glaciecola nitratire Thermomonospora , Thermomonospora curvata , as the ducens, Glaciecola psychrophila and Glaciecola punicea , as microbes that are classified as the genus Pseudonocardia , the microbes that are classified as the genus Marinobacter, 55 Pseudonocardia dioxanivorans, as the microbes that are Marinobacter aquaeolei , Marinobacter hydrocarbonoclas - classified as the genus Amycolatopsis , Amycolatopsis medi ticus , Marinobacter adhaerens, Marinobacter algicola and terranei, as the microbes that are classified as the genus Marinobacter manganoxydans, as the microbes that are Saccharomonospora , Saccharomonospora viridis and Sac classified as the genus Marinobacterium , Marinobacterium charomonospora xinjiangensis , as the microbes that are stanieri , as the microbes that are classified as the genus 60 classified as the genus Saccharopolyspora , Saccharopoly Saccharophagus, Saccharophagus degradans, as the spora erythraea and Saccharopolyspora spinosa , as the microbes that are classified as the genus Ferrimonas, Fer - microbes that are classified as the genus Thermobispora , rimonas balearica , as the microbes that are classified as the Thermobispora bispora , as the microbes that are classified genus Idiomarina , Idiomarina loihiensis and Idiomarina as the genus Actinosynnema , Actinosynnema mirum , as the baltica , as the microbes that are classified as the genus 65 microbes that are classified as the genus Micromonospora , Colwellia , Colwellia psychrerythraea , as the microbes that Micromonospora aurantiaca , as the microbes that are clas cassified as the genus Pseudoalteromonas, Pseudo - sited as the genus alipora , alipora tropica and US 10 ,294 ,500 B2 35 36 Salinispora arenicola , as the microbes that are classified as landii , as the microbes that are classified as the genus the genus Verrucosispora , Verrucosispora maris , as the Alkaliphilus, Alkaliphilus metalliredigens and Alkaliphilus microbes that are classified as the genus Kribbella , Krib oremlandii , as the microbes that are classified as the genus bella flavida , as the microbes that are classified as the genus Syntrophomonas , Syntrophomonas wolfei, as the microbes Corynebacterium , Corynebacterium jeikeium , Corynebacte - 5 that are classified as the genus Syntrophothermus, Syntro rium urealyticum , Corynebacterium glutamicum , Coryne - phothermus lipocalidus , as the microbes that are classified bacterium ammoniagenes and Corynebacterium kroppenst- as the genus Eubacterium , Eubacterium rectale and Eubac edtii , as the microbes that are classified as the genus t erium limosum , as the microbes that are classified as the Nocardia , Nocardia farcinica , Nocardia brasiliensis and genus Desulfitobacterium , Desulfitobacterium hafniense , as Nocardia cyriacigeorgica , as the microbes that are classified 10 the microbes that are classified as the genus Desulfotomacu as the genus Gordonia , Gordonia bronchialis , Gordonia lum , Desulfotomaculum reducens , as the microbes that are neofelifaecis and Gordonia terrae , as the microbes that are classified as the genus Pelotomaculum , Pelotomaculum classified as the genus Dietzia , Dietzia cinnamea , as the thermopropionicum , as themicrobes that are classified as the microbes that are classified as the genus Mycobacterium , genus Butyrivibrio , Butyrivibrio proteoclasticus , as the Mycobacterium tuberculosis , Mycobacterium bovis, Myco - 15 microbes that are classified as the genus Roseburia , Rose bacterium leprae , Mycobacterium avium , Mycobacterium buria hominis, Oscillibacter, Oscillibacter valericigenes , smegmatis , Mycobacterium ulcerans, Mycobacterium van Thermoanaerobacter, Thermoanaerobacter tengcongensis , baalenii, Mycobacterium gilvum , Mycobacterium absces - Carboxydothermus, Carboxydothermus hydrogenoformans , sus , Mycobacterium marinu , Mycobacterium massiliense , as the microbes that are classified as the genus Natranaero Mycobacterium phlei, Mycobacterium thermoresistibile , 20 bius, Natranaerobius thermophilus , as the microbes that are Mycobacterium tusciae , Mycobacterium xenopi and Myco - classified as the genus Haliscomenobacter, Haliscomeno bacterium rhodesiae , as the microbes that are classified as bacter hydrossis , as the microbes that are classified as the the genus Amycolicicoccus, Amycolicicoccus subflavus, as genus Porphyromonas, Porphyromonas gingivalis and Por the microbes that are classified as the genus Tsukamurella , phyromonas asaccharolytica , as the microbes that are clas Tsukamurella paurometabola , as the microbes that are clas - 25 sified as the genus Odoribacter, Odoribacter splanchnicus , sified as the genus Segniliparus , Segniliparus rotundus , as as the microbes that are classified as the genus Spirosoma , the microbes that are classified as the genus Microbacte - Spirosoma linguale , as the microbes that are classified as the rium , Microbacterium testaceum , as the microbes that are genus Runella , Runella slithyformis , as the microbes that are classified as the genus Micrococcus, Micrococcus luteus , as classified as the genus Deinococcus, Deinococcus radio the microbes that are classified as the genus Arthrobacter, 30 durans , Deinococcus geothermalis, Deinococcus deserti , Arthrobacter arilaitensis, Arthrobacter chlorophenolicus, Deinococcus maricopensis , Deinococcus proteolyticus and Arthrobacter globiformis and Arthrobacter phenanthreniv - Deinococcus gobiensis , as the microbes that are classified as orans , as the microbes that are classified as the genus the genus Thermus, Thermus thermophilus , Thermus agnati Renibacterium , Renibacterium salmoninarum , as the cus, Thermus flavus, Thermus caldophilus, Thermus ruder microbes that are classified as the genus Kocuria , Kocuria 35 and Thermus scotoductus, as the microbes that are classified rhizophila , as the microbes that are classified as the genus as the genus Meiothermus, Meiothermus ruber and Meio Kytococcus, Kytococcus sedentarius , as the microbes that thermus silvanus , as the microbes that are classified as the are classified as the genus Cellulomonas , Cellulomonas fimi, genus Oceanithermus, Oceanithermus profundus , as the as the microbes that are classified as the genus Intrasporan - microbes that are classified as the genus Marinithermus , gium , Intrasporangium calvum , as the microbes that are 40 Marinithermus hydrothermalis , as the microbes that are classified as the genus Serinicoccus, Serinicoccus profundi, classified as the genus Gemmatimonas, Gemmatimonas as the microbes that are classified as the genus Frankia , aurantiaca , as the microbes that are classified as the genus Frankia alni, as the microbes that are classified as the genus Fusobacterium , Fusobacterium nucleatum , as the microbes Acidothermus, Acidothermus cellulolyticus, as the microbes that are classified as the genus Ilyobacter, Ilyobacter poly that are classified as the genus Nakamurella , Nakamurella 45 tropus, as the microbes that are classified as the genus multipartita , as the microbes that are classified as the genus Roseiflexus, Roseiflexus castenholzii, as the microbes that Geodermatophilus , Geodermatophilus obscurus , as the are classified as the genus Herpetosiphon , Herpetosiphon microbes that are classified as the genus Stackebrandtia , aurantiacus , as the microbes that are classified as the genus Stackebrandtia nassauensis , as the microbes that are clas - Thermomicrobium , Thermomicrobium roseum , as the sified as the genus Catenulispora , Catenulispora acidiphila , 50 microbes that are classified as the genus Thermotoga , Ther as the microbes that are classified as the genus Rubrobacter, motoga lettingae , as the microbes that are classified as the Rubrobacter xylanophilus , as the microbes that are classified genus Thermosipho , Thermosipho melanesiensis and Ther as the genus Conexibacter, Conexibacter woesei , as the mosipho africanus , as the microbes that are classified as the microbes that are classified as the genus Oceanobacillus, genus Fervidobacterium , Fervidobacterium nodosum , as the Oceanobacillus iheyensis , as the microbes that are classified 55 microbes that are classified as the genus Deferribacter , as the genus Lysinibacillus, Lysinibacillus sphaericus, as the Deferribacter desulfuricans , as the microbes that are clas microbes that are classified as the genus Halobacillus, sified as the genus Calditerrivibrio , Calditerrivibrio nitrore Halobacillus halophilus, as the microbes that are classified ducens, as the microbes that are classified as the genus as the genus Alicyclobacillus, Alicyclobacillus acidocal - Flexistipes, Flexistipes sinusarabici , as the microbes that are darius , as the microbes that are classified as the genus 60 classified as the genus Metallosphaera , Metallosphaera Kyrpidia , Kyrpidia tusci, as the microbes that are classified sedula , as the microbes that are classified as the genus as the genus Brevibacillus, Brevibacillus choshinensis , as Aeropyrum , Aeropyrum pernix , as the microbes that are the microbes that are classified as the genus Lactobacillus, classified as the genus Pyrobaculum , Pyrobaculum aerophi Lactobacillus buchneri , as the microbes that are classified as lum , Pyrobaculum islandicum , Pyrobaculum calidifontis the genus Clostridium , Clostridium acetobutylicum , 65 and Pyrobaculum neutrophilum , as the microbes that are Clostridium perfringens, Clostridium kluyveri , Clostridium classified as the genus Caldivirga , Caldivirga maquilingen cellulovorans, Clostridium difficile and Clostridium stick - sis , as the microbes that are classified as the genus Vulca US 10 ,294 ,500 B2 37 38 nisaeta , Vulcanisaeta distributa , as the microbes that are the genus Monilinia , and the genus Sclerotinia , as the classified as the genus Acidilobus, Acidilobus saccharo - microbes that are classified as the genus Pezizales, the genus vorans , as the microbes that are classified as the genus Peziza , the genus Morchella , the genus Helvella , the genus Haloarcula , Haloarcula marismortui, as the microbes that Ascodesmis , the genus Ascobolus, and the genus Tuber , as are classified as the genus Haloquadratum , Haloquadratum 5 the microbes that are classified as the genus Polyporales , the walsbyi, as the microbes that are classified as the genus genusMycoleptodonoides , the genus Mycoaciella , the genus Natronomonas, Natronomonas pharaonis , as the microbes Parmastomyces and the genus Postia , as the microbes that that are classified as the genus Halorubrum , Halorubrum are classified as the genus Agaricales , the genus Arthromy lacusprofundi, as the microbes that are classified as the ces, the genus Mycenoporella , the genus Laccaria , the genus genus Haloterrigena , Haloterrigena turkmenica , as the 10 Moniliophthora , the genus Coprinopsis and the genusS microbes that are classified as the genus Natrialba, Natri- chizophyllum , as the microbes that are classified as the alba magadii, as the microbes that are classified as the genus genus Pucciniales, the genus Puccinia , the genus Melamp Halalkalicoccus , Halalkalicoccus jeotgali, as the microbes sora , the genus Cronartium , the genus Cyttaria , the genus that are classified as the genus Halogeometricum , Halogeo Coleosporium , the genus Blastospora and the genus Rav metricum borinquense , as the microbes that are classified as 15 enelia , as the microbes that are classified as the genus the genus Thermoplasma, Thermoplasma acidophilum and Magnaporthales , the genus Magnaporthe , as the microbes Thermoplasma volcanium , as the microbes that are classified that are classified as the genus Sordariales, the genus as the genus Picrophilus, Picrophilus torridus, as the Podospora , the genus Neurospora , the genus Sordaria , the microbes that are classified as the genus Ferroplasma, genus Chaetomium , the genus Thielavia , the genus Myce Ferroplasma acidarmanus, as the microbes that are classi - 20 liophthora and the genus Monotosporella , as the microbes fied as the genus Archaeoglobus , Archaeoglobus fulgidus that are classified as the genus Microascales , the genus and Archaeoglobus veneficus, as the microbes that are Microascus , the genus Nodulosphaeria , the genus Okeano classified as the genus Ferroglobus, Ferroglobus placidus, myces, and the genus Ceratocystis , as the microbes that are as the microbes that are classified as the genus Polymor - classified as the genus Hypocreales, the genus Fusarium , the phum , Polymorphum gilvum , as the microbes that are clas - 25 genus Nectria , the genus Hypocrea , the genus Hypomyces, sified as the genus Micavibrio , Micavibrio aeruginosavorus, the genus Trichoderma , the genus Cordyceps , the genus as the microbes that are classified as the genus Simiduia , Claviceps , and the genus Acremonium , as the microbes that Simiduia agarivorans, as the microbes that are classified as are classified as the genus Sporidiobolales , the genus Rho the genuptthrepthrixchodnia the microbe s pridium , the genu dotra, the genuridib that are classified as the genus Thiomonas , Thiomonas 30 lus , and the genus Sporobolomyces , as the microbes that are intermedia , as the microbes that are classified as the genus classified as the genus Exobasidiales , the genus Graphiola , Rubrivivax, Rubrivivax gelatinosus, as the microbes that are the genus Exobasidium , and genus Dicellomyces , as the classified as the genus Methylibium , Methylibium petro - microbes that are classified as the genus Malasseziales , the leiphilum , and as the microbes that are classified as the genus Malassezia , as the microbes that are classified as the genus Anaerococcus, Anaerococcus prevotii are particularly 35 genus Onygenales , the genus Gymnoascus , the genus preferable . Eremascus, the genus Arthroderma , the genus Coccidioides , [ Preferred Order of Eukaryotes ] the genus Paracoccidioides, the genus Uncinocarpus, the Examples of the microbes which belong to the eukaryotes genus Trichophyton , and the genus Ajellomyces , as the include microbes belonging to Eurotiales , Saccharomyc - microbes that are classified as the genus Laboulbeniales, the etales, Capnodiales, Pleosporales , Ustilaginales, Tremella - 40 genus Rhachomyces , the genus Rickia , the genus Stigmato les , Cystofilobasidiales , Helotiales, Pezizales , Polyporales, myces, and the genus Ceratomyces , as the microbes that are Agaricales, Pucciniales , Magnaporthales , Sordariales , classified as the genusMucorales , gensus Absidia , the genus Microascales , Hypocreales, Sporidiobolales , Exobasidiales , Mucor, the genus Rhizomucor, and the genus Rhizopus can Malasseziales, Onygenales, Laboulbeniales, or Mucorales . be mentioned . Further , examples of the particularly preferred microbes 45 As themicrobes that are classified as Eurotiales, the genus which allow experimental confirmation of the production of Neopetromyces, the genus Aspergillus, the genus Neosarto methacrylic acid derivatives include the microbes belonging rya , the genus Byssochlamys , the genus Emericella , the to Eurotiales and Saccharomycetaleslog . genus Eupenicillium , the genus Eurotium , the genus Hemi [ Preferred Family and the Genus of Eukaryotes ] carpenteles, the genus Penicillium , the genus Talaromyces , As the microbes that are classified as Capnodiales , the 50 and the genus Monascus can be mentioned . genus Mycosphaerella , the genus Zygophiala , the genus As the microbes that are classified as Saccharomycetales , Zymoseptoria , the genus Cladosporium and the genus Cap - the genus Arthroascus , the genus Guilliermondella , the nodium , as the microbes that are classified as Pleosporales , genus Clavispora , the genus Metschnikowia , the genus the genus Preussia , the genus Brachycladium , the genus Lipomyces, the genus Endomyces , the genus Ascoidea , the Cochliobolus, the genus Pleospora , the genus Pleospora , 55 genus Cephaloascus, the genus Wickerhamiella , the genus the genus Leptosphaeria , the genus Leptosphaerulina , the Zygoascus , the genus Trichomonascus , the genus Aciculo genus Phaeosphaeria and the genus Pyrenophora , as the conidium , the genus Ambrosiozyma, the genus Blastobotrys, microbes that are classified as the genus Ustilaginales , the the genus Botryozyma , the genus Brettanomyces, the genus genus Pseudozyma, the genus Tolyposporium and the genus Myxozyma , the genus Ogataea , the genus Komagataella , the Ustilago , as the microbes that are classified as the genus 60 genus Trigonopsis, the genus Schizoblastosporion , and the Tremellales, the genus Bulleromyces, the genus Filobasi- genus Sympodiomyces can be mentioned diella (Cryptococcus ) , the genus Bullera , the genus Fello . Further , as the microbes belonging to Saccharomycetales , myces , the genus Kockovaella and the genus Trichosporon , preferred examples include the microbes that are classififed as the microbes that are classified as the genus Cystofiloba - as Saccharomycodaceae , Pichiaceae, Dipodascaceae or sidiales, microbe Mrakia , the genus Xanthophyllomyces and 65 mitosporic Saccharomycetales. Examples of the microbes genus Udeniomyces , as the microbes that are classified as the belonging to that family include the genus Hanseniaspora , genus Helotiales , the genus Botryotinia , the genus Monilia , the genus Kloeckera , the genus Saccharomycodes , the genus US 10 , 294 ,500 B2 39 40 Nadsonia , the genus Ashbya , the genus Citeromyces , the ces kluyveri, Saccharomyces kudriavzevii, Saccharomyces genus Dekkera , the genus Issatchenkia , the genus Kluyvero - kunashirensis, Saccharomyces martiniae , Saccharomyces myces , the genus Pachysolen , the genus Saccharomyces, the mikatae, Saccharomyces naganishii , Saccharomyces noval genus Saturnispora , the genus Tetrapisispora , the genus varietatis, Saccharomyces paradoxus, Saccharomyces pas Torulaspora , the genus Zygosaccharomyces , the genus 5 torianus , Saccharomyces rosinii , Saccharomyces rouxii, Zygotorulaspora , the genus Eremothecium , the genus Saccharomyces stellatus, Saccharomyces servazzii, Saccha Lachancea , the genus Vanderwaltozyma, the genus Naumo romyces spencerorum , Saccharomyces transvaalensis , Sac vozyma, the genus Kazachstania , the genus Eremothecium , charomyces turicensis , Saccharomyces yakushimaensis var. the genus Williopsis , the genus Pichia , the genus Yarrowia , yakushimaensis, Saccharomyces unisporus, and Saccharo the genus Dipodascus, the genus Arxula , the genus Galac - 10 myces uvarum . tomyces, the genus Geotrichum , the genus Babjevia , the [Genus Candida ] genus Debaryomyces, the genus Schwanniomyces, the genus Examples of the microbes that are classified as the genus Priceomyces, the genus Yamadazyma, the genus Scheffer - Candida include Candida aaseri, Candida africana , Can somyces, the genus Meyerozyma, the genus Lodderomyces , dida aglyptinia , Candida agrestis , Candida akabanensis , the genus Sporopachydermia , and the genus Candida . 15 Candida alai, Candida albicans, Candida alishanica , Can Among those belonging to the eukarytoes, examples of dida allociferrii , Candida amapae, Candida ambrosiae, other genus include the genus Botryosphaeria , the genus Candida amphixiae, Candida anatomiae, Candida ancuden Helicomyces, the genus Aureobasidium , the genus Seleno sis, Candida andamanensis , Candida anglica , Candida phoma , the genus Melanotaenium , the genus Otospora , the anneliseae, Candida antillancae, Candida anutae, Candida genus Glomus, the genus Allomyces, the genus Rhizidiomy- 20 apicola , Candida apis, Candida apis var. apis , Candida ces , the genus Hyphochytrium , the genus Schizosaccharo - aquaetextoris , Candida arabinofermentans, Candida myces, the genus Filobasidium , the genus Lophodermium , arcana, Candida ascalaphidarum , Candida asiatica , Can the genus Dothidea , the genus Oidium , the genus Amor dida atbi, Candida atakaporum , Candida athensensis , Can photheca , the genus Scytalidium , the genus Taphrina , the dida atlantica , Candida atmosphaerica , Candida auringi genus Strangospora , the genus Chytridium , the genus Pneu - 25 ensis , Candida auris , Candida aurita , Candida mocystis , the genus Sclerotium , the genus Neolecta , the austromarina , Candida azyma, Candida barrocoloradensis , genus Septobasidium , the genus Xylaria , the genus Pesta Candida batistae , Candida beechii , Candida bertae , Can lotiopsis , the genus Phomopsis , the genus Sydowiella , the dida berthetii, Candida bituminiphila , Candida blackwellae , genus Coniochaetidium , the genus Sporothrix , the genus Candida blankii , Candida blattae, Candida blattariae , Can Moleospora , the genus Verticillium , the genus Microascales , 30 dida bohiensis , Candida boidinii , Candida bokatorum , Can the genus Leucosporidium , the genus Microstroma, and the dida boleticola , Candida bolitotheri, Candida bombi, Can genus Thraustochytrium . dida bombicola , Candida bondarzewiae, Candida bovina , Among them , the preferred microbes are the microbes Candida bribrorum , Candida brumptii , Candida buenavis which have been demonstrated to have an enzyme for the taensis, Candida buinensis , Candida bullrunensis, Candida metabolism pathway of branched amino acids, that is , 35 butyri, Candida cacaoi, Candida canberraensis , Candida microbes classified as the genus Zymoseptoria , the genus cantarellii , Candida cariosilignicola , Candida carpophila , Phaeosphaeria , the genus Pyrenophora , the genus Ustilagoago , Candida caryicola , Candida caseinolytica , Candida castel the genus Ashbya , the genus Kluyveromyces , the genus lii, Candida castrensis , Candida catenulata , Candida cel Saccharomyces, the genus Yarrowia , the genus Debaryomy lulolytica , Candida cerambycidarum , Candida chauliodes, ces , the genus Scheffersomyces , the genus Meyerozyma, the 40 Candida chickasaworum , Candida chilensis , Candida chi genus Lodderomyces , the genus Candida , the genus Filoba ropterorum , Candida choctaworum , Candida chrysomeli sidiella (Cryptococcus ), the genus Botryotinia , the genus darum , Candida cidri , Candida ciferrii, Candida cleri Sclerotinia , the genus Tuber , the genus Postia , the genus darum , Candida coipomensis, Candida colliculosa , Laccaria , the genus Moniliophthora , the genus Coprinopsis , Candida conglobata , Candida corydali , Candida cylindra the genus Schizophyllum , the genus Puccinia , the genus 45 cea , Candida dajiaensis , Candida danieliae , Candida dav Magnaporthe , the genus Podospora , the genus Neurospora , enportii, Candida davisiana , Candida deformans , Candida the genus Sordaria , the genus Thielavia , the genus Myce dendrica , Candida dendronema, Candida derodonti , Can liophthora , the genus Fusarium , the genus Nectria , the dida deserticola , Candida diddensiae, Candida digboiensis , genus Malassezia , the genus Aspergillus, the genus Neosar Candida diversa , Candida dosseyi, Candida drimydis, Can torya , the genus Arthroderma, the genus Coccidioides, the 50 dida drosophilae, Candida dubliniensis, Candida easanen genus Paracoccidioides , the genus Uncinocarpus, the genus sis , Candida edax, Candida elateridarum , Candida elegans , Trichophyton , or the genus Ajellomyces . Candida emberorum , Candida endomychidarum , Candida Further , examples of the particularly preferred microbes entomophila , Candida eppingiae , Candida eremophlia , which allow experimental confirmation of the production of Candida ergatensis , Candida ernobii, Candida etchellsii , methacrylic acid derivatives include the microbes that are 55 Candida ethanolica , Candida famata , Candida famata var. classified as the genus Saccharomyces , the genus Candida, famata , Candida fennica , Candida fermentati , Candida fer or the genus Aspergillus. menticarens, Candida floricola , Candida fluviatilis , Can [Preferred Species of Eukaryotes ] dida fragi, Candida fragicola , Candida freyschussii , Can [Genus Saccharomyces ] dida friedrichii , Candida frijolensensis , Candida fructus, Examples of the microbes that are classified as the genus 60 Candida fukazawae, Candida fukuyamanensis , Candida Saccharomyces include Saccharomyces barnettii , Saccharo - fungicola , Candidafusiformata , Candida galacta , Candida myces bayanus, Saccharomyces bayanus var. uvarum , Sac - galis , Candida gatunensis, Candida geochares, Candida charomyces boulardii, Saccharomyces bulderi, Saccharo germanica , Candida ghanaensis , Candida gigantensis , Can myces cariocanus , Saccharomyces cerevisiae , dida glabrata , Candida glaebosa , Candida globosa , Can Saccharomyces castellii, Saccharomyces dairenensis , Sac - 65 dida glucosophila , Candida golubevii, Candida gorgasii , charomyces diastaticus, Saccharomyces douglasii, Saccha - Candida gosingica , Candida gotoi, Candida gropengiesseri , romyces exiguus , Saccharomyces humaticus, Saccharomy- Candida guaymorum , Candida guilliermondii , Candida US 10 , 294 ,500 B2 42 haemulonii, Candida halonitratophila , Candida halophila , santamariae, Candida santamariae var . membranifaciens, Candida hasegawae , Candida hellenica , Candida holmii, Candida santamariae var . santamariae , Candida santja Candida homilentoma , Candida hordei, Candida hsintzi cobensis , Candida sanyiensis, Candida saraburiensis , Can buensis , Candida humilis , Candida hungarica , Candida dida savonica , Candida schatavii, Candida sekii, Candida hungchunana , Candida incommunis , Candida inconspicua , 5 sequanensis , Candida sergipensis , Candida shehatae , Can Candida ingens, Candida insectalens, Candida insecta - dida shehatae var . insectosa , Candida shehatae var. lignosa , mans , Candida insectorum , Candida insectosa , Candida Candida shehatae var. shehatae , Candida siamensis , Can intermedia , Candida intermedia var. intermedia , Candida dida silvae, Candida silvanorum , Candida silvatica , Can ipomoeae , Candida ishiwadae , Candida japonica , Candida dida silvicultrix, Candida sinolaborantium , Candida sithep jaron , Candida frescandidaanshihenis , Candidansandidat eCandidamagucandida jiufengensis , Candida kanchanaburiensis , Candida kaohsi- smithsonii , Candida sojae , Candida solani, Candida soli ungensis , Candida karawaiewii , Candida kashinagacola , cola, Candida sonckii , Candida songkhlaensis , Candida Candida kazuoi, Candida kefyr, Candida khaoyaiensis , Can sonorensis , Candida sophiaereginae, Candida sorbophila , dida khmerensis, Candida kofuensis , Candida krabiensis , Candida sorbosa , Candida sorbosivorans, Candida sor Candida krissii , Candida kruisii , Candida krusei, Candida 15 boxylosa , Candida spandovensis , Candida sphaerica , Can krusoides, Candida kunorum , Candida kunwiensis, Candida dida stauntonica , Candida stellata , Candida stellimalicola , labiduridarum , Candida lactis -condensi , Candida laem - Candida stri , Candida succiphila , Candida suecica , Can sonensis, Candida lambica , Candida lassenensis, Candida dida suthepensis , Candida suwanaritii, Candida suzukii, laureliae , Candida leandrae , Candida lessepsii, Candida Candida takamatsuzukensis , Candida takata , Candida levantina , Candida lidongshanica , Candida lignicola , Can - 20 taliae , Candida tammaniensis , Candida tannotolerans, Can dida lignohabitans, Candida lignophila , Candida lignosa , dida tanticharoeniae , Candida tanzawaensis , Candida Candida lipolytica , Candida litsaeae , Candida llanquihuen - taoyuanica , Candida tartarivorans, Candida temnochilae , sis , Candida lodderae , Candida lundiana , Candida lusita Candida tenuis , Candida tepae , Candida terebra , Candida niae, Candida lycoperdinae, Candida lyxosophila , Candida terraborum , Candida tetrigidarum , Candida thailandica , maesa , Candida magnoliae, Candida maltosa , Candida 25 Candida thaimueangensis , Candida theae , Candida thermo mannitofaciens, Candida maris, Candida maritima, Can - phila , Candida tilneyi , Candida tolerans, Candida torresii , dida maxii , Candida melibiosica , Candida melinii, Candida Candida tritomae , Candida tropicalis , Candida tropicalis membranifaciens, Candida mesenterica , Candida metapsi var. tropicalis , Candida trypodendroni , Candida tsuchiyae , losis , Candida methanolovescens, Candida methanosor Candida tsukubaensis , Candida tumulicola , Candida ulmi, bosa , Candida methylica , Candida michaelii, Candida mill - 30 Candida utilis, Candida vaccinii , Candida valdiviana , Can eri, Candida mogii , Candida mokoenaii , Candida dida valida , Candida vanderwaltii, Candida variabilis, molischiana , Candida montana , Candida morakotiae, Can Candida vartiovaarae, Candida veronae, Candida versati dida mucifera , Candida multigemmis , Candida musae, Can - lis , Candida vinaria , Candida vini, Candida viswanathii, dida mycetangii , Candida naeodendra , Candida nakhon - Candida wanchemiae , Candida wickerhamii, Candida wou ratchasimensis , Candida nanaspora , Candida natalensis, 35 nanorum , Candida wyomingensis , Candida xestobii , Can Candida neerlandica , Candida nemodendra , Candida neu - dida xyloterini, Candida yuanshanicus, Candida yuchorum , stonensis , Candida nitrativorans, Candida nitratophila , Candida zemplinina, Candida zeylanoides , and Candida Candida nodaensis , Candida nonsorbophila , Candida nor zeylanoides var. zeylanoides. vegensis , Candida norvegica , Candida novakii , Candida (Genus Aspergillus obtusa var . obtusa , Candida odintsovae, Candida oleophila , 40 Examples of the microbes that are classified as the genus Candida olivae, Candida ontarioensis , Candida ooitensis , Aspergillus include Aspergillus acanthosporus, Aspergillus Candida orba , Candida oregonensis, Candida orthopsilosis , acolumnaris , Aspergillus aculeatus, Aspergillus aeneus, Candida ortonii , Candida ovalis, Candida oxycentoniae , Aspergillus affinis , Aspergillus allahabadii, Aspergillus Candida pallodes, Candida palmioleophila , Candida alliaceus, Aspergillus alutaceus, Aspergillus ambiguus, palmyrensis , Candida paludigena , Candida panamensis , 45 Aspergillus amstelodami, Aspergillus amylovorus, Aspergil Candida panamericana , Candida parapolymorpha , Can lus anomalus, Aspergillus anthodesmis, Aspergillus apica , dida parapsilosis, Candida parapsilosis var. parapsilosis, Aspergillus appendiculatus, Aspergillus arenarius , Aspergil Candida pararugosa , Candida pattaniensis , Candida pel - lus asperescens , Aspergillus athecius , Aspergillus aurantio tata , Candida peoriaensis , Candida petrohuensis , Candida brunneus, Aspergillus auratus, Aspergillus aureus var. aci phangngensis , Candida picachoensis , Candida piceae, Can - 50 dus, Aspergillus aureus var. minor, Aspergillus aureofulgens, dida pignaliae , Candida pimensis, Candida pini, Candida Aspergillus aureolatus, Aspergillus aureolus, Aspergillus pintolopesii , Candida pintolopesii var . pintolopesii, Can - auricomus, Aspergillus avenaceus , Aspergillus awamori, dida pintolopesii var. sloofiae, Candida pinus, Candida Aspergillus awamori var . fumeus, Aspergillus awamori var . plutei, Candida polymorpha , Candida pomiphila , Candida fuscus, Aspergillus awamori var . minimus, Aspergillus ponderosae , Candida populi, Candida powellii , Candida 55 awamori var. piceus, Aspergillus batatas, Aspergillus prachuapensis, Candida prunicola , Candida pseudofloscu bicolor, Aspergillus biplanus, Aspergillus bisporus , Asper lorum , Candida pseudoglaebosa, Candida pseudohaemulo gillus bombycis , Aspergillus brasiliensis , Aspergillus brevi nii, Candida pseudointermedia , Candida pseudojiufengen - pes, Aspergillus bridgeri , Aspergillus brunneo -uniseriatus , sis , Candida pseudolambica , Candida pseudovanderkliftii, Aspergillus brunneo -uniseriatus var. nanus, Aspergillus Candida psychrophila , Candida pulcherrima , Candida 60 caelatus, Aspergillus caesiellus, Aspergillus caespitosus , pyralidae, Candida quercitrusa , Candida quercuum , Can Aspergillus campestris, Aspergillus candidus, Aspergillus dida railenensis, Candida ralunensis , Candida ranongensis , carbonarius , Aspergillus carneus, Aspergillus carnoyi, Candida ratchasimensis , Candida restingae, Candida Aspergillus cellulosae , Aspergillus cervinus, Aspergillus reversa , Candida rhagii, Candida robnettiae, Candida chevalieri, Aspergillus chevalieri var. intermedius, Asper rugopelliculosa , Candida rugosa , Candida rugosa var. 65 gillus chrysellus, Aspergillus cinnamomeus, Aspergillus rugosa , Candida sagamina , Candida saitoana , Candida citrisporus, Aspergillus clavatoflavus, Aspergillus clavato sake, Candida salmanticensis , Candida sanitii , Candida nanica , Aspergillus clavatonanicus, Aspergillus clavatus, US 10 , 294 ,500 B2 43 44 Aspergillus cleistominutus , Aspergillus conicus, Aspergillus Aspergillus pseudodeflectus, Aspergillus pseudoglaucus, conjunctus, Aspergillus coremiiformis , Aspergillus corruga - Aspergillus pseudotamarii , Aspergillus pulverulentus, tus, Aspergillus crassihyphae , Aspergillus cremeus, Asper - Aspergillus pulvinus, Aspergillus puniceus, Aspergillus gillus cristatus, Aspergillus crustosus, Aspergillus crystalli quadricinctus, Aspergillus quadrilineatus , Aspergillus nus, Aspergillus deflectus, Aspergillus dimorphicus , 5 quercinus, Aspergillus quercinus var . petrakii, Aspergillus Aspergillus diversus, Aspergillus dorothicus , Aspergillus raperi , Aspergillus recurvatus, Aspergillus repens, Aspergil duricaulis , Aspergillus eburneocremeus, Aspergillus echinu - lus restrictus , Aspergillus rhizopodus, Aspergillus robustus, latus , Aspergillus egyptiacus, Aspergillus elegans , Aspergil - Aspergillus ruber , Aspergillus rugulosus, Aspergillus saitoi lus ellipticus, Aspergillus elongatus, Aspergillus fennelliae, var . kagoshimaensis , Aspergillus salviicola , Aspergillus Aspergillus ficuum , Aspergillus fischeri , Aspergillus fischeri 10 sclerotiorum , Aspergillus sepultus, Aspergillus silvaticus, var . brasiliensis, Aspergillus fischeri var . glaber, Aspergillus Aspergillus sojae , Aspergillus sparsus, Aspergillus spathu fischeri var. spinosus, Aspergillus flaschentraegeri, Asper - latus, Aspergillus spectabilis, Aspergillus spelunceus, Asper gillus flavipes, Aspergillus flavofurcatus , Aspergillus flavus , gillus speluneus, Aspergillus spinulosus , Aspergillus stella Aspergillus flavus var. columnaris, Aspergillus flavus var. tus, Aspergillus stellatus var. astellatus, Aspergillus flavus , Aspergillus flavus var. sojae, Aspergillus flocculosus, 15 stramenius, Aspergillus striatus, Aspergillus stromatoides , Aspergillus floriformis , Aspergillus foetidus, Aspergillus foe Aspergillus subolivaceus, Aspergillus subsessilis , Aspergil tidus var . acidus, Aspergillus foetidus var. pallidus, Asper lus sulphureus, Aspergillus sulphureus var. minimus, Asper gillus foveolatus, Aspergillus fresenii, Aspergillus fruticulo - gillus sunderbanii , Aspergillus sydowii, Aspergillus tamarii , sus, Aspergillus fumaricus, Aspergillus fumigatus, Aspergillus tardus, Aspergillus terreus, Aspergillus terreus Aspergillus fumigatus var . acolumnaris, Aspergillus fumiga - 20 var. africanus, Aspergillus terreus var. aureus, Aspergillus tus var. ellipticus, Aspergillus fumigatus var. fumigatus, terreus var . baedijnii , Aspergillus terreus var . globosus, Aspergillus fumigatus var. helvolus, Aspergillus funiculosus, Aspergillus terreus var. terreus, Aspergillus terricola , Asper Aspergillus giganteus, Aspergillus glaber , Aspergillus glau gillus terricola var . americana , Aspergillus terricola var. cus, Aspergillus gorakhpurensis , Aspergillus gracilis , Asper indicus, Aspergillus testaceocolorans, Aspergillus thermo gillus granulatus, Aspergillus granulosus, Aspergillus gym - 25 mutatus, Aspergillus thomii , Aspergillus togoensis , Asper nosardae , Aspergillus halophilicus, Aspergillus helicothrix , gillus tonophilus, Aspergillus toxicarius, Aspergillus tubin Aspergillus hennebergii , Aspergillus heterocaryoticus, gensis, Aspergillus umbrosus, Aspergillus unguis , Aspergillus heteromorphus , Aspergillus heterothallicus, Aspergillus unilateralis , Aspergillus usamii mut. shi Aspergillus iizukae , Aspergillus implicatus, Aspergillus rousamii , Aspergillus usamii var. shiro -usamii , Aspergillus insuetus, Aspergillus insulicola , Aspergillus inuii , Aspergil - 30 ustus, Aspergillus uvarum , Aspergillus vadensis , Aspergillus lus itaconicus, Aspergillus ivoriensis , Aspergillus janus, varians, Aspergillus versicolor, Aspergillus violaceofuscus, Aspergillus janus var . brevis , Aspergillus janus var. janus, Aspergillus violaceus, Aspergillus viridinutans, Aspergillus Aspergillus japonicus , Aspergillus japonicus var. atrofuscus, vitis , Aspergillus vitis var. montevidensis , Aspergillus wentii , Aspergillus japonicus var . viridiflavus, Aspergillus kam Aspergillus wentii var . minimus, Aspergillus zhaoqingensis , barensis , Aspergillus kanagawaensis , Aspergillus kawachii, 35 and Aspergillus zonatus . Aspergillus lanosus, Aspergillus lentulus, Aspergillus lepo [Other Preferred Species of Eukaryotes ] ris , Aspergillus leucocarpus , Aspergillus longivesica , Asper . Further , as the microbes that are classified as the genus gillus luchuensis , Aspergillus lucknowensis , Aspergillus Zymoseptoria , Zymoseptoria tritici, as the microbes that are luteo -niger , Aspergillus malignus, Aspergillus malodoratus, classified as the genus Phaeosphaeria , Phaeosphaeria nodo Aspergillus manginii, Aspergillus medius , Aspergillus mel - 40 rum , as the microbes that are classified as the genus Pyreno leus, Aspergillus microcephalus, Aspergillus microviridici - phora , Pyrenophora teres , as the microbes that are classified trinus, Aspergillus microcysticus, Aspergillus minimus, as the genus Ustilago, Ustilago maydis, as the microbes that Aspergillus miyokoensis , Aspergillus montevidensis, Asper - are classified as the genus Ashbya , Ashbya gossypii ( Er gillus multicolor, Aspergillus mutabilis , Aspergillus nidu emothecium gossypii) , as the microbes that are classified as lans , Aspergillus nidulans var . acristatus , Aspergillus nidu - 45 the genus Kluyveromyces, Kluyveromyces lactis , as the lans var . dentatus, Aspergillus nidulans var. echinulatus, microbes that are classified as the genus Yarrowia , Yarrowia Aspergillus nidulans var . latus, Aspergillus nidulans var . lipolytica , as the microbes that are classified as the genus roseus , Aspergillus niger, Aspergillus niger mut. cinna - Debaryomyces, Debaryomyces hansenii , as the microbes momeus, Aspergillus niger var . intermedius, Aspergillus that are classified as the genus Scheffersomyces , Scheffer niger var. macrosporus, Aspergillus niger var. nanus, Asper - 50 somyces stipitis , as the microbes that are classified as the gillus niger var. niger, Aspergillus niger var . niger f. hen - genus Meyerozyma, Meyerozyma guilliermondii, as the nebergii , Aspergillus niger var. phoenicis , Aspergillus niger microbes that are classified as the genus Lodderomyces, mut. schiemanni, Aspergillus niveoglaucus , Aspergillus Lodderomyces elongisporus, as the microbes that are clas niveus, Aspergillus niveus ver. indica , Aspergillus nomius, sified as the genus Filobasidiella (Cryptococcus ), Crypto Aspergillus nutans, Aspergillus ochraceoroseus , Aspergillus 55 coccus neoformans , Cryptococcus gattii and Cryptococcus ochraceus , Aspergillus ornatus, Aspergillus oryzae , Asper - amylolentus , as the microbes that are classified as the genus gillus oryzae var. brunneus , Aspergillus oryzae var. effusus, Botryotinia , Botryotinia fuckeliana , as the microbes that are Aspergillus oryzae var. magnasporus , Aspergillus oryzae classified as the genus Sclerotinia , Sclerotinia sclerotiorum , var. oryzae, Aspergillus oryzae var. sporoflavus, Aspergillus as the microbes that are classified as the genus Tuber, Tuber oryzae var. variabilis , Aspergillus oryzae var. viridis , Asper - 60 melanosporum , as the microbes that are classified as the gillus ostianus, Aspergillus pallidus , Aspergillus panamen - genus Postia , Postia placenta , as the microbes that are sis , Aspergillus paradoxus, Aspergillus parasiticus, Asper - classified as the genus Laccaria , Laccaria bicolor, as the gillus parasiticus var. globosus, Aspergillus parvathecius, microbes that are classified as the genus Moniliophthora , Aspergillus parvulus , Aspergillus penicillioformis , Aspergil - Moniliophthora perniciosa , as the microbes that are classi lus penicillioides, Aspergillus persii, Aspergillus petrakii , 65 fied as the genus Coprinopsis , Coprinopsis cinerea , as the Aspergillus peyronelii, Aspergillus phialiseptus, Aspergillus microbes that are classified as the genus Schizophyllum , phoenicis, Aspergillus proliferans , Aspergillus protuberus, Schizophyllum commune , as the microbes that are classified US 10 ,294 ,500 B2 45 46 as the genus Puccinia , Puccinia graminis , as the microbes [Correction of Nov. 27 , 2013 Based on Rule 91 ] that are classified as the genus Magnaporthe, Magnaporthe ( i ) B25 - 2 strain ( The National Institute of Technology and oryzae , as the microbes that are classified as the genus Evaluation (NITE ), Patent Microorganisms Depository ( zip Podospora , Podospora anserina , as the microbes that are code 292 -0818 , 2 - 5 - 8 Kazusakamatari, Kisarazu - shi, Chiba classified as the genus Neurospora , Neurospora crassa , as 5 ken , 292 - 0818 , Japan ), Accession number : NITE BP - 1451) , the microbes that are classified as the genus Sordaria , D22 - 1 strain (Accession number: NITE BP - 1452 ) , D43 - 1 Sordaria macrospora , as the microbes that are classified as strain (Accession number: NITE BP - 1453 ) , D25 strain (Ac the genus Thielavia , Thielavia terrestris , as the microbes cession number: NITE BP - 1454 ), D26 strain ( Accession that are classified as the genus Myceliophthora , Mycelio - number : NITE BP - 1455 ) , D29 strain (Accession number : phthora thermophila , as the microbes that are classified as 10 NITE BP - 1456 ), D41 - 2 strain (Accession number : NITE the genus Fusarium , Fusarium graminearum , as the BP - 1457 ) , and Pseudomonas putida NBRC12996 which microbes that are classified as the genus Nectria , Nectria belong to the genus Pseudomonas , or haematococca , as the microbes that are classified as the ( ii ) G1 strain (Accession number : NITE BP - 1458 ), G2 strain genus Malassezia , Malassezia globosa , as the microbes that ( Accession number : NITE BP - 1459 ) strain , R1 strain ( Ac are classified as the genus Neosartorya , Neosartorya fisch - 15 cession number : NITE BP - 1460 ), Bacillus subtilis eri , as the microbes that are classified as the genus Arthro - NBRC12210 , Bacillus badius ATCC14574 , Bacillus mega derma, Arthroderma benhamiae , as the microbes that are terium NBRC15308 , and Bacillus simplex ATCC49097 classified as the genus Coccidioides, Coccidioides immitis which belong to the genus Bacillus and Coccidioides posadasii, as the microbes that are clas (iii ) B13 strain ( Accession number: NITE BP - 1461) which sified as the genus Paracoccidioides , Paracoccidioides 20 belongs to the genus Sphingobacterium . brasiliensis , as the microbes that are classified as the genus ( iv ) Comamonas terrigena NBRC13299 which belongs to Uncinocarpus, Uncinocarpus reesii , as the microbes that are the genus Comamonase . classified as the genus Trichophyton , Trichophyton verru ( v ) Brevundimonas diminuta ATCC11568 , Brevundimonas cosum , as the microbes that are classified as the genus vesicularis ATCC11426 or Brevundimonas subvibrioides Ajellomyces , Ajellomyces capsulatus , as the microbes that 25 NBRC16000 which belongs to the genus Brevundimonas. are classified as the genus Hypocrea , Hypocrea rufa , as the (vi ) Sphingomonas paucimobilis NBRC 13935 which microbes that are classified as the genus Acremonium , belongs to the genus Sphingomonas. Acremonium chrysogenum , as the microbes that are classi- ( vii ) Ochrobactrum sp . NBRC12951, Ochrobactrum inter fied as the genus Penicillium , Penicillium camembertii , as m edium NBRC15820 , Ochrobactrum intermedium the microbes that are classified as the genus Ogataea , 30 NBRC13694 , Ochrobactrum anthropic ATCC49237 , Ogataea minuta , as the microbes that are classified as the Ochrobactrum grignonense NBRC 102586 or Ochrobac genus Komagataella , Komagataella pastoris , as the trum lupini NBRC 102587 which belongs to the genus microbes that are classified as the genus Trigonopsis , Trigo - Ochrobactrum . nopsis variabilis , as the microbes that are classified as the ( viii) Pedobacter heparinus NBRC12017 which belongs to genus Schizosaccharomyces, Schizosaccharomyces pombe , 35 the genus Pedobacter. and as the microbes that are classified as the genus Scyta - (ix ) Paenibacillus sp . NBRC 13157 which belongs to the lidium , Scytalidium thermophirum can be mentioned . genus Paenibacillus. Among the aforementioned prokaryotes and eukaryotes, ( x ) Achromobacter denitrificans NBRC 12669 which examples of the particularly preferred microbes which allow belongs to the genus Achromobacter experimental confirmation of the production of methacrylic 40 ( xi) Acinetobacter haemolyticus ATCC17906 or Acineto acid derivatives include the microbes which belong to the bacter junii ATCC17908 which belongs to the genus Acine genus Pseudomonas, the genus Bacillus, the genus Sphin - tobacter . gobacterium , the genus Comamonas, the genus Brevundi - (xii ) Shewanella fodinae NBRC105216 which belongs to monas, the genus Sphingomonas , the genus Ochrobactrum , the genus Shewanella. the genus Pedobacter , the genus Paenibacillus, the genus 45 (xiii ) Listonella anguillarum ATCC19264 which belongs to Achromobacter , the genus Acinetobacter , the genus the genus Listonella . Shewanella , the genus Listonella , the genus Agrobacterium , ( xiv ) Agrobacterium luteum NBRC15768 which belongs to the genus Mesorhizobium , the genus Rhizobium , the genus the genus Agrobacterium . Paracoccus, the genus Xanthobacter, the genus Streptomy (xv )Mesorhizobium loti ATCC700743 which belongs to the ces , the genus Geobacillus, the genus Rhodococcus, the 50 genus Mesorhizobium . genus Saccharomyces, the genus Candida , or the genus (xvi ) Rhizobium leguminosarum ATCC10004 which Aspergillus. belongs to the genus Rhizobium . Among them , more preferred are the microbes which (xvii ) Paracoccus aminophilus NBRC16710 which belongs belong to the genus Sphingobacterium , the genus Comamo- to the genus Paracoccus. nas, the genus Brevundimonas, the genus Sphingomonas , the 55 (xviii ) Xanthobacter autotrophicus ATCC35674 which genus Ochrobactrum , the genus Pedobacter , the genus Pae - belongs to the genus Xanthobacter nibacillus, the genus Achromobacter, the genus Acineto - ( xix ) Streptomyces griseus NBRC13350 which belongs to bacter , the genus Shewanella , the genus Listonella , the the genus Streptomyces. genus Agrobacterium , the genus Mesorhizobium , the genus (xx ) Geobacillus stearothermophilus NBRC 12983 which Paracoccus , the genus Xanthobacter , the genus Geobacil - 60 belongs to the genus Geobacillus . lus, the genus Rhodococcus, or the genus Candida . (xxi ) Rhodococcus erythropolis can be mentioned . More [ Preferred Microbial Strains preferred examples of the strain include Rhodococcus eryth Particularly preferred examples of the microbes having ropolis PR -4 strain , Rhodococcus erythropolis KA2 - 5 -1 the ability to produce methacrylic acid , which are used for strain , Rhodococcus erythropolis IGTS8 strain , Rhodococ the method for producing methacrylic acid of the invention , 65 cus erythropolis D - 1 strain , Rhodococcus erythropolis H - 2 include the following microbial strains having high ability to strain , Rhodococcus erythropolis N1- 36 strain , Rhodococ produce methacrylic acid (see , Examples described below ) . cus erythropolis 1- 19 strain , Rhodococcus erythropolis US 10 ,294 ,500 B2 47 48 ECRD - 1 strain , Rhodococcus erythropolis B1 strain , Rho - or isobutyric acid , and property for producing methacrylic dococcus erythropolis SY - 1 strain , Rhodococcus erythropo acid . It is also possible to select them from existing type lis UM3 strain , Rhodococcus erythropolis UM9 strain , and culture according to the same procedure . Rhodococcus erythropolis TO9 strain . Particularly preferred [Derivative Strain ] examples include Rhodococcus erythropolis PR -4 strain . 5 A derivative strain which is derived either naturally or by ( xxii ) Saccharomyces cerevisiae NBRC1136 , Saccharomy- a chemical or physical treatment from the microbes having ces cerevisiae NBRC2347 or Saccharomyces paradoxus the ability to produce methacrylic acid ( for example , the NBRC 10609 which belongs to the genus Saccharomyces. aforementioned microbial strains of (i ) to (xxiv )) and main ( xxiii ) Candida utilis NBRC1086 or Candida parapsilosis tains the ability to produce methacrylic acid by using ATCC22019 which belongs to the genus Candida . 10 renewable raw materials and / or biomass as a carbon source ( xxiv ) Aspergillus niger ATCC6275 , Aspergillus flavus and /or energy source can be also used as microbes . The NBRC8558 or Aspergillus oryzae NBRC4255 which derivative strain encompasses the “ variant strain ” and “ gene belongs to the genus Aspergillus. modified strain ” that are described below . [Method for Selecting Microbes by Screening or the Like ] [ Variant Strain ] The microbes having the ability to produce methacrylic 15 The variant strain can be obtained by causing a genetic acid can be identified from soil , river water, lake water , sea variation in the microbes having the ability to produce water , sludge , animal, plant, insect , or the like based on methacrylic acid according to a chemical or physical treat screening such as a flat plate separation or direct culture ment ( for example , y ray irradiation ) . method by using the assimilating property of amino acids or [Gene Modified Strain ] fatty acids. 20 The genemodified strain is a strain of the microbes having Specifically , according to evaluation of the assimilating the ability to produce methacrylic acid with enhanced activ property of valine or isobutyric acid and / or the property for ity or deleted or lowered activity as described below . producingmethacrylic acid , the microbes appropriate for the Enhanced activity means that the expression amount of an invention can be selected . The assimilating property of enzyme gene (regardless of its origin ) is increased in valine or isobutyric acid can be evaluated by observing the 25 microbes based on the gene incorporated from outside of the growth on a synthetic medium which uses 0 . 1 to 1 % valine microbial cells to the microbes. In addition to the incorpo or isobutyric acid as a carbon source . The property for ration of a gene encoding an enzyme from outside of the producing methacrylic acid can be evaluated by analyzing microbial cells to inside of the microbial cells , enhancing the the production of methacrylic acid in the aforementioned promoter activity of an enzyme gene which is included on broth . Alternatively , the evaluation can be made by contact- 30 the microbes on genome, expressing strongly an enzyme ing the cells obtained by the culture with a solution con - gene by substituting with other promoter , and enhancing the taining valine or isobutyric acid at 0 . 1 to 1 % and analyzing activity of an enzyme gene as a result of lowering or the production of methacrylic acid in the same reaction inactivating the activity of the repressor of the enzyme gene solution . The reaction can be performed at 30° C . with are included . Meanwhile , the gene introduction or substitu shaking for 5 to 120 hours . Preferably, it is the microbes 35 tion of a gene on genome can be performed according to a having the ability to produce methacrylic acid derivatives at general method . 0 .04 ppm or more as determined by the aforementioned The gene modified strain can be a modified strain having evaluation . gene modification for deleting or lowering the activity of an Alternatively , the microbes appropriate for the invention enzyme which suppresses the reaction for synthesizing can be selected according to mycological properties of 40 methacrylic acid . “ Deleting ” or “ lowering ” the activity microbes (that is, cultural property ,morphological property , means that completely removed or lowered expression of the physiological property , chemosystematic property , and enzyme gene , and in addition to an occurrence of substitu genetic property ) . For example , the microbes of the inven t ion , deletion , or insertion in the enzyme gene , suppressing tion can be selected in view of the identity of the rRNA gene the activity of the promoter of the enzyme gene which is of the microbes. Specifically , the selection can be made from 45 contained on a gene of the microbes , suppressing the expres the microbes having rRNA gene which includes a nucleotide sion of the enzyme gene by substituting with other promoter , sequence having the identity of 81 % or more , preferably and lowering the activity of an enzyme gene as a result of 88 % or more , more preferably 95 % or more , more prefer - enhancing or activating the activity of the repressor of the ably 97 % or more , more preferably 98 % or more , and even enzyme are included . Meanwhile , those gene modifications more preferably 99 . 5 % or more to any one of the nucleotide 50 can be performed to a general method . sequences described in SEQ ID NOS . 1 to 49. Examples of the preferred modified gene include a modi Further , the microbes appropriate for the invention can be fied gene having at least one of the following ( a ) and ( b ) . efficiently narrowed by selecting from microbes belonging (a ) According to introduction of the BCKAD gene and /or to the taxonomic order including the order , family , or the ACD gene , the activity of producing methacrylic acid genus described above and combining the information of 55 is enhanced . rRNA gene . ( b ) According to deletion of inactivation of enoyl COA Meanwhile , with regard to the method for analyzing and hydratase gene , 3 -hydroxyisobutyryl CoA hydrolase identifying the rRNA gene, reference can be made to “ Deter gene , and / or 3 -hydroxyisobutyric acid dehydrogenase mination method of 16S rRNA gene sequence ( In The gene , the activity of producing methacrylic acid is Isolation and Characterization of Actinomycetes , pp . 60 enhanced . The deletion or inactivation is performed by 88 - 117 , Edited by The Society Actinomycetes, Japan , 2001 ) substitution , deletion , or insertion in the entire or partial ” , “ Bulletin of Japanese Society of Microbial Ecology Vol. nucleotide sequence of a gene . 10 , 31 -42 , 1995 ” , and “ Japanese Pharmacopoeia , 16 revised [Gene Recombinant Strain ] edition ; Method for quick identification of microbes based Further, a strain newly given with an ability to produce on gene analysis ” ), or the like. Preferably , the microbes 65 methacrylic acid by using renewable raw materials and /or appropriate for the invention are selected by combining biomass as a carbon source and /or energy source according those mycological properties , property of assimilating valine to introduction of an enzyme involved with methacrylic acid US 10 , 294 ,500 B2 49 50 synthesis , which is identified from microbes having the (7 ) Microbes which belong to the genus Ochrobactrum and ability to produce methacrylic acid , to microbes not having have 16S rDNA containing the nucleotide sequence with the the ability to produce methacrylic acid can be also used as identity of 81 % or more , preferably 88 % or more , more microbes . With regard to the production of the gene recom preferably 95 % or more , more preferably 97 % or more , binant strain , it can be performed with the samemethod as 5 more preferably 98 % or more , and even more preferably the production of a gene modified strain described above . 99 . 5 % or more to any one of the partial nucleotide sequence [ Homologous Strain ] of 16S rDNA of the strain of ( vii ) above (SEQ ID NOS . 22 to 26 ). Further , a strain which is a homologous strain of the ( 8 ) Microbes which belong to the genus Pedobacter and microbes having the ability to produce methacrylic acid ( for 10 have 16S rDNA containing the nucleotide sequence with the example , the aforementioned microbial strains of (i ) to " identity of 81 % or more , preferably 88 % or more , more ( xxiv ) ) and maintains the ability to produce methacrylic acid preferably 95 % or more, more preferably 97 % or more , by using renewable raw materials and /or biomass as a more preferably 98 % or more , and even more preferably carbon source and/ or energy source can be also used as 99 . 5 % or more to any one of the partial nucleotide sequence microbes . The homologous strain can be obtained by per - 15 of 16S rDNA of the strain of ( viii ) above (SEO ID NO . 27 ) . forming molecular phylogenetic analysis based on the base ( 9 ) Microbes which belong to the genus Paenibacillus and sequence information of nucleic acid , for example . Specifi have 16S rDNA containing the nucleotide sequence with the cally , the homologous strain can be obtained by a homology identity of 81 % or more , preferably 88 % or more , more analysis of the nucleotide sequence of ribosomal RNA gene preferably 95 % or more , more preferably 97 % or more , ( rRNA gene ; hereinbelow , rDNA ) . 20 more preferably 98 % or more , and even more preferably Examples of the preferred homologous strain include the 99 . 5 % or more to the partial nucleotide sequence of 16S followings. rDNA of the strain of ( ix ) above (SEQ ID NO . 28 ). ( 1 ) Microbes which belong to the genus Pseudomonas and ( 10 ) Microbes which belong to the genus Achromobacter have 16S rDNA containing the nucleotide sequence with the and have 16S rDNA containing the nucleotide sequence with identity of 81 % or more , preferably 88 % or more , more 25 the identity of 81 % or more , preferably 88 % or more, more preferably 95 % or more , more preferably 97 % or more , preferably 95 % or more , more preferably 97 % or more, more preferably 98 % or more , and even more preferably more preferably 98 % or more, and even more preferably 99 . 5 % ormore to any one of the partial nucleotide sequence 99. 5 % or more to the partial nucleotide sequence of 16S of 16S rDNA of the strain of ( i ) above (SEQ ID NOS . 1 to rDNA of the strain of ( x ) above (SER ID NO . 29 ) . 8 ). 30 ( 11 ) Microbes which belong to the genus Acinetobacter and ( 2 ) Microbes which belong to the genus Bacillus and have have 16S rDNA containing the nucleotide sequence with the 16S rDNA containing the nucleotide sequence with the identity of 81 % or more, preferably 88 % or more , more identity of 81 % or more , preferably 88 % or more , more preferably 95 % or more , more preferably 97 % or more, preferably 95 % or more , more preferably 97 % or more , more preferably 98 % or more , and even more preferably more preferably 98 % or more , and even more preferably 35 99 . 5 % or more to any one of the partial nucleotide sequence 99 . 5 % ormore to any one of the partial nucleotide sequence of 16S rDNA of the strain of (xi ) above (SEQ ID NOS . 30 of 16S rDNA of the strain of ( ii ) above ( SEQ ID NOS . 9 to and 31 ) . 15 ) . ( 12 ) Microbes which belong to the genus Shewanella and ( 3 ) Microbes which belong to the genus Sphingobacterium have 16S rDNA containing the nucleotide sequence with the and have 16S rDNA containing the nucleotide sequence with 40 identity of 81 % or more , preferably 88 % or more , more the identity of 81 % or more , preferably 88 % or more , more preferably 95 % or more, more preferably 97 % or more , preferably 95 % or more , more preferably 97 % or more, more preferably 98 % or more , and even more preferably more preferably 98 % or more , and even more preferably 99 . 5 % or more to the partial nucleotide sequence of 16S 99 . 5 % or more to the partial nucleotide sequence of 16S rDNA of the strain of ( xii) above (SEQ ID NO . 32 ) . rDNA of the strain of ( iii ) above (SEQ ID NO . 16 ) . 45 (13 ) Microbes which belong to the genus Listonella and ( 4 ) Microbes which belong to the genus Comamonas and have 16S rDNA containing the nucleotide sequence with the have 16S rDNA containing the nucleotide sequence with the identity of 81 % or more , preferably 88 % or more , more identity of 81 % or more , preferably 88 % or more, more preferably 95 % or more, more preferably 97 % or more , preferably 95 % or more , more preferably 97 % or more , more preferably 98 % or more, and even more preferably more preferably 98 % or more , and even more preferably 50 99 . 5 % or more to the partial nucleotide sequence of 16S 99 . 5 % or more to the partial nucleotide sequence of 16S rDNA of the strain of (xiii ) above (SEO ID NO . 33 ) . rDNA of the strain of (iv ) above (SEQ ID NO . 17 ) . ( 14 ) Microbes which belong to the genus Agrobacterium and ( 5 ) Microbes which belong to the genus Brevundimonas and have 16S rDNA containing the nucleotide sequence with the have 16S rDNA containing the nucleotide sequence with the identity of 81 % or more , preferably 88 % or more , more identity of 81 % or more , preferably 88 % or more , more 55 preferably 95 % or more , more preferably 97 % or more , preferably 95 % or more , more preferably 97 % or more , more preferably 98 % or more , and even more preferably more preferably 98 % or more , and even more preferably 99 . 5 % or more to the partial nucleotide sequence of 16S 99. 5 % or more to any one of the partial nucleotide sequence rDNA of the strain of ( xiv ) above ( SEQ ID NO . 34 ) . of 16S rDNA of the strain of ( v ) above (SEQ ID NOS . 18 ( 15 ) Microbes which belong to the genus Mesorhizobium to 20 ) . 60 and have 16S rDNA containing the nucleotide sequence with ( 6 ) Microbes which belong to the genus Sphingomonas and the identity of 81 % or more , preferably 88 % or more , more have 16S rDNA containing the nucleotide sequence with the preferably 95 % or more , more preferably 97 % or more , identity of 81 % or more , preferably 88 % or more, more more preferably 98 % or more , and even more preferably preferably 95 % or more , more preferably 97 % or more , 99 . 5 % or more the partial nucleotide sequence of 16S rDNA more preferably 98 % or more, and even more preferably 65 of the strain of (xv ) above (SEQ ID NO . 35 ). 99 . 5 % or more to the partial nucleotide sequence of 16S ( 16 ) Microbes which belong to the genus Rhizobium and rDNA of the strain of (vi ) above (SEQ ID NO . 21) . have 16S rDNA containing the nucleotide sequence with the US 10 , 294 ,500 B2 51 52 identity of 81 % or more, preferably 88 % or more , more As described herein , the “ identity ” of a sequence indicates preferably 95 % or more , more preferably 97 % or more , the percentage that is obtained by , in case of a nucleotide more preferably 98 % or more , and even more preferably sequence , aligning two nucleotide sequences for comparison 99 . 5 % or more to the partial nucleotide sequence of 16S such that they are in match with each other as much as rDNA of the strain of (xvi ) above (SEQ ID NO . 36 ). 5 possible and dividing the number of nucleotides in match by ( 17 ) Microbes which belong to the genus Paracoccus and the number of total nucleotides . For the aligning , a gap is have 16S rDNA containing the nucleotide sequence with the suitably inserted to one or both sequences for comparison , if identity of 81 % or more, preferably 88 % or more , more necessary . The aligning of a sequence can be performed by preferably 95 % or more , more preferably 97 % or more , using a known program such as BLAST, FASTA , or more preferably 98 % or more , and even more preferably 10 CLUSTALW . In case of inserting a gap , the number of total 99 . 5 % or more to any one of the partial nucleotide sequence nucleotides corresponds to the number of nucleotides after of 16S rDNA of the strain of (xvii ) above (SEQ ID NO . 37 ) . counting one gap as one nucleotide . When the number of ( 18 ) Microbes which belong to the genus Xanthobacter and total nucleotides counted as above is different between two have 16S rDNA containing the nucleotide sequence with the sequences for comparison , the identity ( % ) is calculated by identity of 81 % or more , preferably 88 % or more , more 15 dividing the number of nucleotides in match by the number preferably 95 % or more , more preferably 97 % or more , of total nucleotides with longer length . The same applies to more preferably 98 % or more , and even more preferably the identity of an amino acid sequence . 99. 5 % or more to the partial nucleotide sequence of 16S Further, according to the method for producing meth rDNA of the strain of (xviii ) above (SEQ ID NO . 38 ). acrylic acid and / or a methacrylic acid ester of the invention , ( 19 ) Microbes which belong to the genus Streptomyces and 20 it is also possible that, with a step of using recombinant have 16S rDNA containing the nucleotide sequence with the microbes in which AAT gene and /or ACD gene is introduced identity of 81 % or more , preferably 88 % or more, more to the microbes having the ability to produce methacrylic preferably 95 % or more , more preferably 97 % or more, acid and contacting them with renewable raw materials more preferably 98 % or more , and even more preferably and / or biomass , methacrylic acid and /or a methacrylic acid 99 . 5 % or more to any one of the partial nucleotide sequence 25 ester is produced . of 16S rDNA of the strain of (xix ) above (SEQ ID NO . 39 ). [Production of Recombinant Microbes in which AAT Gene ( 20 ) Microbes which belong to the genus Geobacillus and is Introduced to the Microbes Having Ability to Produce have 16S rDNA containing the nucleotide sequence with the Methacrylic Acid ] identity of 81 % or more , preferably 88 % or more, more [AAT ] preferably 95 % or more , more preferably 97 % or more , 30 AAT is an enzyme having a catalytic function of trans more preferably 98 % or more , and even more preferably ferring an acyl group of acyl- CoA to alcohols or phenols to 99 .5 % or more to the partial nucleotide sequence of 16S synthesize ester. AAT is believed to be involved with pro rDNA of the strain of (xx ) above (SEQ ID NO . 40 ) . duction of esters in various fruits . AAT is known to be (21 ) Microbes which have 16S rDNA containing the nucleo present in a plant such as Zingiberales (banana ) , Rosales tide sequence with the identity of 81 % or more , preferably 35 (strawberry , apple , pear, and peach ) , Cucurbitales (melon ) , 88 % or more, more preferably 95 % or more , more prefer - Ericales (kiwi ) , Lamiales ( olive ) , Solanales ( tomato ), and ably 97 % or more, more preferably 98 % or more , and even Sapindales (lemon and mango ) . more preferably 99. 5 % or more to the partial nucleotide Regardless of the type and origin , the AAT used in the sequence of 16S rDNA of the Rhodococcus erythropolis invention is not particularly limited if it is a catalyst derived PR - 4 strain ( SEQ ID NO . 41) . 40 from a living microorganism having the ability to produce ( 22 ) Microbes which belong to the genus Saccharomyces methacrylic acid by using methacrylyl- CoA and alcohols or and have LSUrDNA containing the nucleotide sequence phenols as a raw material. As an enzyme source , those with the identity of 81 % or more , preferably 88 % or more derived from a plant are preferable. Among them , those more preferably 95 % or more , more preferably 97 % or classified as an angiosperm plant are preferable. more , more preferably 98 % or more , and even more pref- 45 The AAT suitable for the invention can be selected from erably 99 . 5 % or more to any one of the partial nucleotide the aforementioned plants according to the following sequence of LSUrDNA of the strain of (xxii ) above ( SEQ ID method . A suitable area of a tissue is obtained by cutting , if NOS . 42 to 44 ) . necessary . To the cut area , a solution containing methacry ( 23 ) Microbes which belong to the genus Candida and have lyl- CoA and alcohols or phenols is added , shaken , and LSUrDNA containing the nucleotide sequence with the 50 allowed to proceed with the reaction for a certain time. By identity of 81 % or more , preferably 88 % or more , more confirming the presence or absence of a methacrylic acid preferably 95 % or more , more preferably 97 % or more, ester in the reaction solution using GC (gas chromatogra more preferably 98 % or more , and even more preferably phy ) , the synthetic activity can be confirmed . Specifically , 99 .5 % or more to any one of the partial nucleotide sequence fruit flesh or fruit skin is cut and added with a solution of LSUrDNA of the strain of ( xxiii ) above (SEQ ID NOS. 55 containing 1 to 10 mM methacrylyl- CoA , 0 . 35 M KCl, and 45 and 46 ). 5 to 50x molar amount of n - butanol, and shaken for 1 to 10 ( 24 ) Microbes which belong to the genus Aspergillus and hours at 30° C . Once the reaction is completed , by confirm have LSUrDNA containing the nucleotide sequence with the ing the presence or absence of a methacrylic acid ester by identity of 81 % or more , preferably 88 % or more , more GC , the AAT applicable to the invention can be selected . preferably 95 % or more , more preferably 97 % or more , 60 The alcohols or phenols are a compound represented by more preferably 98 % or more, and even more preferably the following formula “ R — OH ” . Because the structure of 99 .5 % or more to any one of the partial nucleotide sequence the alcohols or phenols corresponds to methacrylic acid ofLSUrDNA of the strain of (xxiv ) above (SEQ ID NOS. 47 ester, the structure has the same definition as R of Formula to 49 ) . 1 above , and it represents a linear or branched hydrocarbon Further, the homologous strain can be selected from 65 group having 1 to 20 carbon atoms. The hydrocarbon group existing type culture based on the information in which can be a saturated or unsaturated noncyclic type , or a mycological properties are suitably combined . saturated or unsaturated cyclic type . Preferably, it is a linear US 10 ,294 ,500 B2 53 54 or branched unsubstituted alcohol, aralkyl alcohols or phe raceae, the genus Ananas as those belonging to Bromeli nols having 1 to 10 carbon atoms. Particularly preferred aceae, the genus Oryza , the genus Triticum , the genus examples include alkyl alcohol having 1 to 8 carbon atoms Hordeum , the genus Zea , the genus Sorghumm and the genus such as a methyl group , an ethyl group , a n - propyl group , an Brachypodium as those belonging to Poaceae , the genus isopropyl group , a n - butyl group , an isobutyl group , a 5 Cocos as those belonging to Arecaceae, the genus Vanda as sec -butyl group , a tert - butyl group , a n -pentyl alcohol, those belonging to Orchidaceae , the genus Iris as those isopentyl alcohol, tert- pentyl alcohol, n -hexyl alcohol, iso - belonging to Iridaceae , the genus Ribes as those belonging hexyl alcohol, 2 -hexyl alcohol, dimethylbutyl alcohol, or to Grossulariaceae, the genus Gypsophila as those belonging ethylbutyl alcohol, heptyl alcohol, octyl alcohol, or 2 - eth to Caryophyllaceae , the genus Vitis as those belonging to ylhexyl alcohol, benzyl alcohol, and phenol. 10 Vitaceae , the genus Malpighia as those belonging to Specific examples of the AAT enzyme source which is Malpighiaceae , the genus Passiflora as those belonging to suitable for the invention include those belonging to any Passifloraceae , the genus Ricinus as those belonging to order selected from a group consisting of Zingiberales, Euphroniaceae , the genus Populus as those belonging to Rosales, Ericales , Cucurbitales , Brassicales , Laurales , Salicaceae , the genus Averrhoa as those belonging to Oxali Poales , Arecales, Asparagales , Saxifragales, Caryophyllales , 15 daceae, the genus Medicago , the genus Lupinus, the genus Vitales, Malpighiales , Oxalidales, Fabales , Sapindales , Mal - Glycine, and the genus Clitoria as those belonging to vales , Myrtales , Ranunculales , Solanales , Lamiales, Genti - Fabaceae, the genus Citrus and the genus Aegle as those anales, and Asterales. Among them , preferred are those belonging to Rutaceae , the genus Litchi as those belonging belonging to any order selected from a group consisting of to Sapindaceae , the genus Mangifera as those belonging to Zingiberales, Rosales, Ericales , Cucurbitales , Brassicales, 20 Anacardiaceae , the genus Durio and the genus Theobroma and Laurales. as those belonging to Malvaceae, the genus Punica as those Preferred are as follows; Musaceae and Zingiberaceae as belonging to Lythraceae , the genus Clarkia as those belong those belonging to the order Zingiberales, Rosaceae and ing to Onagraceae, the genus Psidium as those belonging to Moraceae as those belonging to the order Rosales, Erica - Myrtaceae , the genus Actaea as those belonging to Ranun ceae , Actinidiaceae , Ebenaceae and Theaceae as those 25 culaceae , the genus Papaver as those belonging to Papav belonging to the order Ericales , Cucurbitaceae as those eraceae , the genus the genus Solanum , the genus Capsicum , belonging to the order Cucurbitales , Caricaceae and Bras - the genus Nicotiana and the genus Petunia as those belong sicaceae as those belonging to the order Brassicales , Lau - ing to Solanaceae, the genus Olea as those belonging to raceae as those belonging to the order Laurales , Bromeli - Oleaceae , the genus Glandularia as those belonging to aceae and Poaceae as those belonging to the order Poales, 30 Verbenaceae, the genus Salvia as those belonging to Lami Arecaceae as those belonging to the order Arecales , Orchi- aceae , the genus Rauvolfia and the genus Catharanthus as daceae and Iridaceae as those belonging to the order Aspara - those belonging to Apocynaceae , and the genus Chamae gales , Grossulariaceae as those belonging to the order Saxi- melum as those belonging to Asteraceae . Among them , a fragales, Caryophyllaceae as those belonging to the order plant belonging to the genus Musaceae, the genus fragaria , Caryophyllales, Vitaceae as those belonging to the order 35 the genus Malus , the genus Purunus, the genus Pyrus , the Vitales , Malpighiaceae , Passifloraceae, Euphorbiaceae and genus Vaccinium hirtum , the genus Bower actinidia , the Salicaceae as those belonging to the order Malpighiales, genus Cucumis , the genus Carica or the genus Persea is Oxalidaceae as those belonging to the order Oxalidales, more preferable . A plant belonging to the genus Musaceae , Fabaceae as those belonging to the order Fabales , Rutaceae , the genus Malus, the genus Purunus, the genus Pyrus , the Sapindaceae and Anacardiaceae as those belonging to the 40 genus Vaccinium hirtum , the genus Bower actinidia , the order Sapindales , Malvaceae as those belonging to the order genus Cucumis , the genus Carica or the genus Persea is Malvales , Lythraceae , Onagraceae and Myrtaceae as those particularly preferable . A plant belonging to the genus belonging to the order Myrtales , Ranunculaceae and Papav - Musaceae , the genus Malus , the genus Pyrus , the genus eraceae as those belonging to the order Ranunculales, Solan - Bower actinidia , the genus Cucumis , the genus Carica , or aceae as those belonging to the order Solanales , Oleaceae , 45 the genus Persea is particularly preferable . Verbenaceae and Lamiaceae as those belonging to the order Further, specific examples of the particularly preferred are Lamiales , Apocynaceae as those belonging to the order as follows: Musaxparadisiaca , Musa basjoo , Musa coc Gentianales , and Asteraceae as those belonging to the order cinea and Musa acuminate as those belonging to the genus Asterales . Homologous species of the aforementioned plant Musa , Zingiber officinale as those belonging to the genus can be also used . Among them , more preferred is a plant 50 Zingiber , Fragariaxananassa , Fragaria virginiana , Fra belonging to Musaceae , Rosaceae, Ericaceae , Actinidiaceae , garia chiloensis and Fragaria vesca as those belonging to Cucurbitaceae, Caricaceae or Lauraceae . the genus Fragaria , Malus pumila , Malus domestica , Malus Specifically , preferred are as follows : the genus Musa as baccata , Malus halliana , Malus floribunda and Malus those belonging to Musaceae , the genus Zingiber as those prunifolia as those belonging to the genus Malus, Prunus belonging to Zingiberales , the genus Fragaria , the genus 55 mume, Prunus avium , Prunus persica , Prunus armeniaca , Malus , the genus Prunus , the genus Pyrus , the genus Erio - Prunus dulcis , Prunus salicina and Prunus domestica as botrya , the genus Chaenomeles , the genus Rubus and the those belonging to the genus Prunus, Pyrus communis , genus Rosa as those belonging to Rosaceae , the genus Ficus Pyrus pyrifolia , Pyrus calleryana and Pyrus pyraster as as those belonging to Moraceae , the genus Vaccinium as those belonging to the genus Pyrus, Eriobotrya japonica as those belonging to Ericaceae , the genus Actinidia as those 60 those belonging to the genus Eriobotrya , Chaenomeles belonging to Bower actinidia , the genus Diospyros as those sinensis as those belonging to the genus Chaenomeles , belonging to Ebenaceae , the genus Camellia as those Rubus idaeus and Rubus fruticosus as those belonging to the belonging to Theaceae , the genus Cucumis and the genus genus Rubus, Rosa rugosa as those belonging to the genus Citrullus as those belonging to Cucurbitaceae , the genus Rosa , Ficus carica as those belonging to the genus Ficus , Carica and the genus Vasconcellea as those belonging to 65 Vaccinium corymbosum ( Vaccinium angustifolium ) , Vac Caricaceae , the genus Arabidopsis as those belonging to cinium myrtillus, Vaccinium vitis - idaea and Vaccinium oxy Brassicaceae , the genus Persea as those belonging to Lau coccos as those belonging to the genus Vaccinium hirtum , US 10 , 294 ,500 B2 55 56 Actinidia chinensis (Actinidia deliciosa ) , Actinidia arguta , kiwi, melon , papaya , and avocado are more preferable . Actinidia rufa and Actinidia polygama as those belonging to Banana , apple , pear, kiwi, melon , papaya , and avocado are the genus Bower actinidia , Diospyros kaki as those belong particularly preferable. ing to the genus Diospyros, Camellia sinensis as those In the invention , plant classification is based on the APG belonging to the genus Camellia , Cucumis sativus, Cucumis 5 plant classification system III (Botanical Journal of the melo , Cucumis anguria and Cucumis metulifer as those Linnean Society , 2009 , 161, 105121) . belonging to the genus Cucumis , Citrullus lanatus as those At the time of selecting AAT, it is also possible that the belonging to the genus Citrullus, Carica papaya as those AAT gene is isolated , introduced to a common host vector belonging to the genus Carica , Vasconcellea cundinamar system , for example , and then evaluated and selected by censis as those belonging to the genus Vasconcellea , Ara - 1 using microbes that are transformed with the vector system . bidopsis thaliana and Arabidopsis lyrata as those belonging For example, several AAT genes have been known ( for to the genus Arabidopsis , Persea americana as those example , see Patent Document 7 ) . Based on the information , belonging to the genus Persea , Ananas comosu as those the gene can be isolated by producing a DNA probe , belonging to the genus Ananas, Oryza sativa as those 15 producing a primer used for PCR , for example , and per belonging to the genus Oryza , Triticum aestivum as those forming PCR . It is also possible that the entire nucleotide belonging to the genus Triticum , Hordeum vulgare as those sequence of AAT gene is synthesized by a general method . belonging to the genus Cerealia , Zea mays as those belong . According to the same method as described above , it is ing to the genus Zea , Sorghum bicolor as those belonging to possible to determine whether or not the genetic information the genus Sorghum , Brachypodium distachyon as those 20 has the activity of synthesizing a methacrylic acid ester of belonging to the genus Brachypodium , Cocos nucifera a known AAT. Meanwhile, with regard to the AAT with those belonging to the genus palm tree , Vanda hybrid unclear genetic information , it is possible that the genetic cultivar as those belonging to the genus Vanda, Irisxhol- information can be obtained based on genetic engineering of landica as those belonging to the genus Iris , Ribes nigrum a protein resulting from purification of the AAT . as those belonging to the genus Ribes, Gypsophila panicu - 25 As for the AAT gene preferred in the invention , it is not lata (Gypsophila elegans ) as those belonging to the genus particularly limited if the translated product has an ability to Gypsophila , Vitis vinifera ( Vitis labrusca ) as those belonging produce a methacrylic acid ester , and it is suitably selected to the genus Vits , Malpighia glabra as those belonging to the from the AAT enzyme sources described above . Examples of the particularly preferred include the AAT gene derived from genus Malpighia coccigera , Passiflora edulis as thoseas 30 an apple (SEQ ID NO . 77 ) , the AAT gene derived from belonging to the genus Passiflora , Ricinus communis as 30 strawberry (SEQ ID NO . 79 ) , and the AAT gene derived those belonging to the genus Ricinus, Populus trichocarpa from strawberry (SEQ ID NO . 81) . as those belonging to the genus Populus, Averrhoa caram Meanwhile , the AAT gene of the invention also encom bola as those belonging to the genus Carambola , Medicago passes a gene which contains an amino acid sequence of truncatula as those belonging to the genus Medicago , LupLupi ! - 35 having one or several substitutions, deletions, or additions of nus albus as those belonging to the genus Lupinus, Glycine the amino acids in the amino acid sequence of the wild type max as those belonging to the genus Glycine mas, Clitoria and encodes a protein having an activity of producing a ternatea as those belonging to the genus Clitoria , Citrus methacrylic acid ester from methacrylyl- CoA and alcohol . limon , Citrus sudachi, Citrus sphaerocarpa , Citrusxpara - As described herein , the term “ several” indicates 1 to 40 , disi, Citrus junos, Citrus aurantifolia , Citrus unshiu and 40 preferably 1 to 20 , and more preferably 10 or less . In order Citrus sinensis as those belonging to the genus Citrus, Aegle to introduce a mutation to a gene , a kit for introducing marmelos as those belonging to the genus Aegle, Litchi mutation which uses a site specific mutagenesis , such as chinensis as those belonging to the genus Litchi, Mangifera QuikChangeTM Site -Directed Mutagenesis Kit (manufac indica as those belonging to the genus Mangifera , Durio tured by Stratagene ) , GeneTailorTM Site -Directed Mutagen zibethinus as those belonging to the genus Durio , Theo - 45 esis System (manufactured by Invitrogen ), TaKaRa Site broma cacao as those belonging to the genus Theobroma, Directed Mutagenesis System (manufactured by Takara Bio Punica granatum as those belonging to the genus Punica , Inc ., Mutan - K , Mutan - Super Express Km or the like ), can be fairy fans (Clarkia breweri) and Red ribbons ( Clarkia con used based on a known method like Kunkel method or cinna ) as those belonging to the genus Clarkia , Psidium Gapped duplex method . Alternatively , the entire gene having guajava as those belonging to the genus Psidium , Actaea 50 a sequence in which a mutation is contained can be artifi racemosa as those belonging to the genus Actaea , Papaver cially synthesized . somniferum , Papaver orientale and Papaver bracteatum as In the invention , identification of the nucleotide sequence those belonging to the genus Papaver , Solanum lycopersi of a DNA can be performed by determining the sequence by cum as those belonging to the genus Solanum , Capsicum a commonly used method . For example, the sequence can be annuum and Capsicum chinense as those belonging to the 55 identified based on Sanger method by using a suitable DNA genus Capsicum , Nicotiana tabacum (Nicotiana attenuata ) sequencer. as those belonging to the genus Nicotiana , Petuniaxhybrida Further, the AAT gene of the invention encompasses a as those belonging to the genus Petunia , Olea europaea as gene which exhibits the identity of 90 % or more , preferably those belonging to the genus Olea , Glandulariaxhybrida as 95 % or more , more preferably 99 . 5 % or more , and even those belonging to the genus Glandularia , Salvia splendens 60 more preferably 99 . 9 % or more to the protein consisting of as those belonging to the genus Salvia , Rauvolfia serpentina an amino acid sequence of the wild type and encodes a as those belonging to the genus Rauwolfia , Catharanthus protein having an activity of producing a methacrylic acid roseus as those belonging to the genus Catharanthus, and ester from methacrylyl- CoA and alcohol. Chamaemelum nobile as those belonging to Chamaemelum . Further, the AAT gene of the invention encompasses a Among them , banana, strawberry, apple , Japanese apricot, 65 gene which hybridizes under stringent conditions to a poly pear, blueberry , kiwi, melon , papaya , and avocado are pref- nucleotide having a nucleotide sequence complementary to erable . Banana , apple , Japanese apricot, pear , blueberry, the nucleotide sequence of the wild type and encodes a US 10 ,294 , 500 B2 57 58 protein having an activity of producing a methacrylic acid sequence for expression regulation . Although many host and ester from methacrylyl- CoA and alcohol. vector systems are known, they can be developed by the In the specification , reference can be made to “Molecular samemethod , if necessary . Cloning , A Laboratory Manual 2nd ed .” (Cold Spring Har As for the vector for introduction into microbes which bor Laboratory Press (1989 ) ) , “ Current Protocols in Molecu - 5 belong to the genus Rhodococcus, a known vector such as lar Biology ” ( John Wiley & Sons ( 1987 - 1997 ) ) or the like pK1, pK2, PK3, pK4 , or pSJ034 ( see JP - A No . 10 -337185 ) , with regard to the method for hybridization . PSJO23 and PSJ002 ( see JP - A No . 10 - 24867 ) can be used In the specification , the stringent conditions may be a (but not limited thereto ) . Further , a known vector modified condition in which the hybridization is performed by incu - to have a desired constitution can be also used . PSJO23 has bating a nylon membrane having immobilized DNA with a 10 been deposited with National Institute of Advanced Indus probe in a solution containing 6xSSC ( 1xSSC indicates 8 .76 trial Science and Technology as the transformant Rhodococ g of sodium chloride and 4 .41 g of sodium citrate are cus rhodochrous ATCC12674 /PSJO23 (FERM BP -6232 ) as dissolved in 1 liter water) , 1 % SDS , 100 ug /ml salmon Rhodococcus rhodochrous ATCC12674 /pSJO23 (FERM sperm DNA , 0 . 1 % bovine serum albumin , 0 . 1 % polyvi- BP -6232 ) . nylpyrrolidone , and 0 . 1 % Ficoll for 20 hours at 65° C . , but 15 Insertion of the AAT gene to a vector can be performed by it is not limited thereto . A skilled person in the pertinent art using a genetic recombination technique which is known to can set the conditions for hybridization by considering , in a skilled person in the pertinent art. Examples of the method addition to the conditions like salt concentration of a buffer , which can be used include a method of using restriction with temperature , or the like , various conditions such as probe a restriction enzyme and a ligation kit , a method of using concentration , probe length , or reaction time . For example , 20 topoisomerase , and a method of using In Fusion kit ( Takara with regard to cleaning condition after the hybridization , a Bio ) . The gene to be inserted to a vector is inserted by condition like “ 1xSSC , 0 . 1 % SDS , 65° C .” , “ 0 . 5xSSC , 0 . 1 % ligating it to the downstream of a promoter , which can SDS , 50° C .” can be mentioned as a more stringent condition regulate transcription and translation of a protein encoded by than “ 2xSSC , 0 . 1 % SDS , 42° C . ” , “ 1xSSC , 0 . 1 % SDS , 37° each gene in a host organism . Further , if required for C ." . 25 insertion , a suitable linker may be added . Further , if neces Further , in the invention , the AAT gene also encompasses sary, a terminator sequence , an enhancer sequence , a splic a gene consisting of a nucleotide sequence having the ing signal sequence , a signal sequence for adding polyA , a identity of 80 % or more , preferably 90 % or more , and most ribosome- binding sequence such as SD sequence or Kozak preferably 95 % or more to the nucleotide sequence of the sequence , and a selection marker gene , which can be used in wild type when calculation is made by using BLAST ( for 30 a host microbe to which a gene is introduced , can be added . example , default , that is , parameters of initial setting ) and Examples of the selection marker gene include , in addition encoding the protein having an activity of producing a to a drug - resistant gene such as ampicillin resistant gene , methacrylic acid ester from methacrylyl- CoA and alcohol. tetracycline resistant gene , neomycin resistant gene , Further, the codon of the AAT gene may be modified in kanamycin resistant gene, or chloramphenicol resistant response to the codon usage frequency of the microbes that 35 gene , a gene involved with cellular biosynthesis of nutrients are used for transformation . such as amino acid or nucleic acid , and a gene encoding a [ Production of Recombinant Microbes Introduced with AAT fluorescent protein such as luciferase . Part of the amino acid Genel sequence encoded by DNA can be also substituted in con By introducing the DNA encoding the AAT gene to the junction with the insertion . microbes having the ability to produce methacrylic acid as 40 In view of the above, it is particularly preferable to use , a host and performing transcription and translation into a in an example of the invention , PLK005 obtained by per protein in the microbes , methacrylic acid ester can be forming a mutagenic treatment of pK4 as a vector for the produced . It is also possible that a gene of the enzyme which genus Rhodococcus . By ligating and inserting the AAT gene enhances the methacrylic acid producing activity ( for such that it is located 3 ' downstream of the promoter of example , gene like ACD and BCKAD ) can be simultane - 45 PLK005, an expression plasmid vector for expressing the ously introduced . AAT gene as caused by the promoter can be constructed . The gene introduction method is not particularly limited . In the vector, the AAT gene alone or any one gene selected However , the DNA to be introduced is preferably in a state from a group consisting of enzyme genes which enhance the in which it is bound to a vector . Specifically, the AAT gene activity of producing methacrylic acid can be inserted . It is is bound to the vector in a state in which it can be expressed 50 also possible that two or more genes are inserted . When used in a host cell , and introduced to the host cell . Examples of in relation to the gene to be introduced to a vector , the term the vector include those capable of self -replication in host “ two or more ” means that 2 to 5 , 2 to 4 , and preferably 2 to microbes and those capable of binding to the genome of 3 genes can be inserted . Further , when two or more genes are microbes . However , as long as the AAT gene is maintained , inserted to a single vector, it is preferable that those genes it is not limited and a vector that is suitable for each microbe 55 form an operon . As described herein , the term “ operon ” can be used . In case of bacteria of the genus Rhodococcus means a unit of nucleic acids consisting of one or more and analog bacteria , it is also possible to insert a DNA genes that are transcribed under control of the same pro fragment containing the AAT gene that can be expressed to moter. a genome by taking advantage of the property that a DNA The aforementioned gene , and preferably the gene present introduced by electroporation or the like can be easily bound 60 in the form of a vector , is introduced to a host microbe to a genome . When it is desired to introduce plural genes according to a method known to a person skilled in the containing the AAT gene , it can be bound to one or plural pertinent art . Examples of the method for introducing a expression vectors, each in expressible state . recombinant vector to a host microbe is not particularly The vector which may be used in the invention encom - limited if it is a method suitable for the host microbe . passes a plasmid vector, a phage (virus ) vector, a cosmid 65 Examples thereof include electroporation , spheroplasting, vector, and an artificial chromosome vector, and it may also lithium acetate method , calcium phosphate method , lipofec contain one or several selectable marker genes and suitable tion , and transconjugation . US 10 , 294 ,500 B2 59 60 ( 2 ) Step for Producing Methacrylic Acid produced . The concentration is , for example , 0 .001 to 1 . 6 ( 2 - 1 ) Production of Methacrylic Acid by Culture ( w / v ) % , preferably 0 . 005 to 1 . 3 ( w / v ) % , and more pref With the method for producing methacrylic acid accord erably 0 .01 to 1 (w /v ) % . The reason for using them at 0 .01 ing to the invention , methacrylic acid can be obtained in ( w / v ) % or higher is to have enhanced property of the culture by culturing the aforementioned microbes in an 5 microbes to produce methacrylic acid , and the reason for aqueous medium containing renewable raw materials and / or using them at 1 ( w / v ) % or lower is that no significant biomass . increase in effect is observed even when they are added at [Medium ] higher concentration . The aqueous medium containing renewable raw materials In addition to them , a trace amount of a metal, a vitamin , and / or biomass ( that is , medium ) which is used for the 10 or the like can be added to the medium , if necessary . Further, culture of microbes is a solid medium or a liquid medium if necessary, various organic substances, inorganic sub containing sufficient nutrients which include at least one stances , surfactants , that are required for growth of kind of carbon source , on which the microbes can prolifer - microbes, or anti - foaming agents that are generally used can ate . According to one embodiment, the medium is prepared be added to the medium . advantageously to have pH and salt concentration that are 15 Culture Conditions optimum for survival and proliferation of cells . Seeding of the microbes on the medium can be performed The renewable raw materials and biomass are not par- by a known method of the related art . The culture method is ticularly limited if it can produce methacrylic acid . The not particularly limited either , and a known method such as renewable raw materials and biomass can be a plant mate shaking culture , aeration and agitation culture , or static rial, an animalmaterial , or a biodegradable waste . Examples 20 culture can be used . of the preferred renewable raw materials and biomass Conditions for culturing microbes are not particularly include polypeptone , meat extract, yeast extract , corn steep limited as long as the microbes can grow and produce liquor, or an extract of bean or the like . The extract is used methacrylic acid . Culture can be performed under aerobic as it is or after partial purification . The renewable raw conditions or anaerobic conditions. materials can be polysaccharides , oligosaccharides and 25 A pH , temperature , and culture time are not particularly monosaccharides such as glucose , galactose , mannose, fruc - limited as long as they are the conditions allowing growth of tose , xylose , or arabinose , or derivatives thereof. Further , as the microbes and production of methacrylic acid . pH is the renewable raw materials and biomass , lipids, amino preferably 3 to 10 , more preferably 4 to 9 , and even more acids , organic acids, and alcohols that are generated by preferably 5 to 8 . The temperature is preferably 10 to 45° C . , derivatization or metabolism of those sugars can be used . 30 more preferably 15 to 40° C ., and even more preferably 20 Examples of the preferred renewable raw materials include to 35º C . The culture time is preferably 10 to 1000 hours , sugars such as glucose , fructose, or xylose ; amino acids such more preferably 15 to 480 hours, and even more preferably as valine , alanine, leucine , lysine , or glutamic acid ; acids 20 to 240 hours . such as acetic acid , pyruvic acid , lactic acid , acetoacetic Those culture conditions are selected or optimized for acid , acetolactic acid , 2 -oxoisovaleric acid , butyric acid , 35 each strain so as to maximize the ratio of the methacrylic isobutyric acid , propionic acid , malic acid , fumaric acid , acid production amount compared to the use amount of the citric acid , or succinic acid ; and alcohols such as ethanol, renewable raw materials and / or biomass . Meanwhile , by n - propanol, isopropanol, n - butanol , or isobutanol. The suitably controlling the amount of carbon sources and cul renewable raw materials can be used either singly or in ture conditions, the methacrylic acid production amount can combination of two or more types . 40 be also controlled . A concentration of the renewable raw materials and / or As the conditions preferred for accumulating methacrylic biomass in the medium is not particularly limited as long as acid at 0 .04 ppm or higher , the reaction is allowed to occur methacrylic acid can be produced . The concentration is , for for three hours or longer at conditions of pH 5 . 5 to 7 . 5 , while example, 0 .05 to 20 ( w / v ) % , preferably 0 . 1 to 15 ( w / v ) % , maintaining directly or indirectly the concentration of the and more preferably 0 . 2 to 10 ( w / v ) % . The reason for using 45 renewable raw materials and /or biomass at 0 . 1 % or higher them at 0 . 2 ( w / v ) % or higher is to have enhanced property and adjusting the temperature in the range of 20 to 40° C . of the microbes to produce methacrylic acid , and the reason Further , within the range that death ratio does not increase for using them at 10 ( w / v ) % or lower is that no significant as the environment of broth becomes inappropriate for increase in effect is observed even when they are added at growth of microbes or cultured cells , the concentration of higher concentration . 50 the microbes in broth is preferably maintained at high level An inorganic nitrogen source or an inorganic metal salts in terms of obtaining efficient productivity . For example , by may be added to the medium . As the inorganic nitrogen maintaining it at 2 g / l or more in dry weight, good produc source , ammonium salts of inorganic acid or organic acid tion efficiency is obtained and the accumulated concentra such as ammonium chloride , ammonium sulfate , ammonium tion of the product can be increased . acetate , or ammonium phosphate are used . 55 ( 2 - 2 ) Production of Methacrylic Acid Based on Reaction of A concentration of the nitrogen source in the medium is Resting Cells not particularly limited as long as methacrylic acid can be For the method for producing methacrylic acid according produced . The concentration is , for example , 0 . 01 to 10 to the invention , the following method can be also adopted ( w / v ) % , preferably 0 .05 to 8 ( w / v ) % , and more preferably in addition to the method which involves performing , in an 0 . 1 to 4 ( w / v ) % . 60 aqueous medium containing renewable raw materials and / or Examples of the inorganic metal salt which can be used biomass , culture of microbes accompanied with prolifera include monobasic potassium phosphate, dibasic potassium tion . The microbes may or may not have a proliferation phosphate , magnesium phosphate , magnesium sulfate , property, and methacrylic acid can be also produced based sodium chloride , ferrous sulfate , manganese sulfate , copper on a resting cell reaction which is substantially not accom sulfate , and calcium carbonate . 65 panied with proliferation , by contacting the microbes cul A concentration of the inorganic salts in the medium is not tured in advance with an aqueous medium containing renew particularly limited as long as methacrylic acid can be able raw materials and /or biomass. US 10 , 294 ,500 B2 61 62 [Reaction Solution ] pyl ether, dibutyl ether , tert - butyl methyl ether, and dime The renewable raw materials that are contained in an thoxyethane ), and an ester solvent ( for example , methyl aqueous medium containing renewable raw materials and / or formate , methyl acetate , ethyl acetate , butyl acetate , and biomass used for the resting cell reaction (that is , reaction methyl propionate ) . By adding the organic solvent in solution ) and the concentration can be the same as those for 5 advance , methacrylic acid produced may migrate to an the production of methacrylic acid by culture . organic phase to have efficient progress of the reaction . To the reaction solution , inorganic metal salt or the like The reaction conditions are suitably selected or optimized may be added . Examples of the inorganic metal salt which for each strain so as to maximize the ratio of the methacrylic can be used include monobasic potassium phosphate , diba - acid production amount compared to the use amount of the sic potassium phosphate, magnesium phosphate , magnesium 10 renewable raw materials and / or biomass . Meanwhile , by sulfate , sodium chloride , ferrous sulfate , manganese sulfate , suitably controlling the amount of carbon sources and reac copper sulfate , and calcium carbonate . tion conditions, the methacrylic acid production amount can A concentration of the inorganic salts in the reaction be also controlled . solution is not particularly limited as long as methacrylic With regard to the method for producing methacrylic acid acid can be produced . The concentration is , for example , 15 according to the invention , the aforementioned production 0 .0001 to 2 ( w / v ) % , preferably 0 .0003 to 1 . 3 ( w / v ) % , and of methacrylic acid based on culture and production of more preferably 0 .001 to 1 ( w / v ) % . methacrylic acid based on the resting cell reaction can be In addition to them , a trace amount of a metal, a vitamin , suitably combined . According to the combination of the two or the like is added to the reaction solution . Further, if methods, more efficient production of methacrylic acid can necessary , various organic substances , inorganic substances, 20 be achieved . Further, for the method for producing meth surfactants , that are required for the reaction or anti - foaming acrylic acid according to the invention , a method of directly agents that are generally used can be added to the reaction contacting the microbes with renewable raw materials and / solution . or biomass can be also adopted , in addition to the method of [Reaction Conditions ] contacting the microbes with an aqueous medium containing For the resting cell reaction , the broth of the microbes 25 renewable raw materials and / or biomass , which is prepared which have been cultured in advance may be used as it is , as a medium or solution for the resting cell reaction . or the cells collected by filtration or centrifugal separation As the conditions preferred for accumulating methacrylic are used . The collected microbes can be used at any micro - acid at 0 .04 ppm or higher, the reaction is allowed to occur bial concentration after re -suspending them in a suitable for three hours or longer at conditions of pH 5 . 5 to 7 . 5 , while buffer solution or the like. Examples of the buffer solution 30 maintaining directly or indirectly the concentration of the which may be used include physiological saline , potassium renewable raw materials and / or biomass at 0 . 1 % or higher phosphate buffer solution , tris - hydrochloric acid buffer solu and adjusting the temperature in the range of 20 to 40° C . tion , glycine - sodium hydroxide buffer solution , and borate - Further, the concentration of the microbes in reaction solu sodium hydroxide buffer solution . tion is preferably maintained at high level in terms of Further, a processed product of the collected microbes 35 obtaining efficient productivity . For example , by maintain ( for example , disrupted product, co -enzymes , or purified ing it at 2 g /l or more in dry weight, good production enzymes ) can be used for the resting cell reaction . Further , efficiency is obtained and the accumulated concentration of it is also possible that the microbes or their processed the product can be increased . products are immobilized on a suitable carrier by a known ( 2 - 3 ) Recovery of Methacrylic Acid method and the immobilized product is used for the reaction . 40 The methacrylic acid produced in a medium or reaction The conditions for culturing microbes are notparticularly solution and its production amount can be detected and limited as long as it allows production of methacrylic acid measured by using a common method like high speed liquid A culture can be performed under aerobic conditions or chromatography and LC -MS . anaerobic conditions . The reaction method is not particu - Methacrylic acid can be isolated and purified from the larly limited either , and a known method such as shaking 45 medium or reaction solution by suitably using, if necessary reaction , aeration and agitation reaction , or static reaction in combination , a known process like filtration , centrifugal can be used separation , vacuum concentration , ion exchange or adsorp ApH , temperature , and reaction time are not particularly tive chromatography , solvent extraction , distillation , and limited as long as they are the conditions allowing the crystallization . production of methacrylic acid . The pH is preferably 3 to 10 , 50 Methacrylic acid ester can be produced from the obtained more preferably 4 to 9 , and even more preferably 5 to 8 . The methacrylic acid based on an esterification . Further, accord temperature is preferably 10 to 45° C . , more preferably 15 ing to genetic introduction of an enzyme involved with to 40° C . , and even more preferably 20 to 35° C . The culture esterification of methacrylic acid to microbes having the time is preferably 10 to 180 hours, more preferably 15 to 150 ability to produce a methacrylic acid ester ( for example , hours , and even more preferably 20 to 120 hours . 55 microbial strains of ( i ) to (xxiv ) described above ), it is also Further , for purpose of isolating methacrylic acid pro - possible to produce a methacrylic acid ester. duced , it is also possible to add an organic solvent in ( 3 ) Step for Producing Methacrylic Acid Ester advance and performing the reaction in a biphasic system . ( 3 - 1 ) Production of Methacrylic Acid Ester by Culture Examples of the organic acid include a linear, branched , or I n the invention , production of methacrylic acid ester is cyclic and saturated or unsaturated aliphatic hydrocarbon , a 60 performed by producing and accumulating methacrylic acid saturated or unsaturated aromatic hydrocarbon or the like , ester in cultured cells or culture by culturing the microbes and they can be used either singly or as a mixture of two or having the ability of producing methacrylic acid and the more types . Specific examples include a hydrocarbon sol genetic recombinant microbes, obtained by introducing the vent ( for example , pentane , hexane , cyclohexane , benzene , AAT gene to the derivative strain , in an aqueous medium toluene , and xylene) , a halogenated hydrocarbon solvent (for 65 containing the renewable raw materials and /or biomass , and example , methylene chloride and chloroform ) , an ether collecting methacrylic acid ester from the cultured cells , ovenfor example , diethylether , dipropylether , dispro culture , or vapor phase of the culture container . US 10 , 294 ,500 B2 63 64 [Medium ] Examples of the inorganic metal salt which can be used The aqueous medium containing renewable raw materials include monobasic potassium phosphate , dibasic potassium and /or biomass ( that is , medium ) which is used for culturing phosphate , magnesium phosphate , magnesium sulfate , the microbes is a solid medium or a liquid medium contain - sodium chloride , ferrous sulfate , manganese sulfate , copper ing sufficient nutrients which include at least one kind of 5 sulfate , and calcium carbonate . carbon source, on which the microbes can proliferate . A concentration of the inorganic salts in themedium is not According to one embodiment, the medium is prepared particularly limited as long as methacrylic acid ester can be produced . The concentration is , for example , 0 . 001 to 1 . 6 advantageously to have pH and salt concentration that are ( w / v ) % , preferably 0 .005 to 1 . 3 ( w / v ) % , and more pref optimum for survival and proliferation of cells . 10 erably 0 .01 to 1 (w / v ) % . The reason for using them at 0. 01 The renewable raw materials and biomass are not par ( w / v ) % or higher is to have enhanced property of the ticularly limited if it can produce methacrylic acid ester . The microbes to produce methacrylic acid , and the reason for renewable raw materials and biomass can be a plant mate using them at 1 ( w / v ) % or lower is that no significant rial, an animalmaterial , or a biodegradable waste . Examples increase in effect is observed even when they are added at of the preferred renewable raw materials and biomass 15 higher concentration include polypeptone, meat extract, yeast extract, corn steep In addition to them , a trace amount of a metal, a vitamin , liquor, or an extract of bean or the like . The extract is used or the like can be added to the medium , if necessary . Further , as it is or after partial purification . The renewable raw if necessary , various organic substances, inorganic sub materials can be polysaccharides , oligosaccharides and stances , surfactants , that are required for growth of monosaccharides such as glucose , galactose , mannose , fruc - 20 microbes, or anti - foaming agents that are generally used can tose , xylose , or arabinose, or derivatives thereof. Further, as be added to the medium . the renewable raw materials and biomass , lipids, amino Culture Conditions ] acids , organic acids, and alcohols that are generated by seeding of the microbes on the medium can be per derivatization or metabolism of those sugars can be used formed by a known method of the related art. The culture Examples of the preferred renewable raw materials include 25 method is not particularly limited either , and a known sugars such as glucose , fructose , or xylose ; amino acids such method such as shaking culture , aeration and agitation as valine , alanine, leucine , lysine, or glutamic acid ; acids culture , or static culture can be used . such as acetic acid , pyruvic acid , lactic acid , acetoacetic The conditions for culturing microbes are not particularly acid , acetolactic acid , 2 -oxoisovaleric acid , butyric acid , limited as long as the microbes can grow and produce isobutyric acid , propionic acid , malic acid , fumaric acid , 30 methacrylic acid ester . Culture can be performed under citric acid , or succinic acid ; and alcohols such as ethanol, aerobic conditions or anaerobic conditions. n - propanol , isopropanol, n - butanol , or isobutanol. The A pH , temperature, and culture time are not particularly renewable raw materials can be used either singly or in limited as long as they are the conditions allowing growth of combination of two or more types . the microbes and production of methacrylic acid ester. The A concentration of the renewable raw materials and / or 35 pH is preferably 3 to 10 , more preferably 4 to 9 , and even biomass in the medium is not particularly limited as long as more preferably 5 to 8 . The temperature is preferably 10 to methacrylic acid ester can be produced . The concentration 45° C . , more preferably 15 to 40° C ., and even more is , for example , 0 .05 to 20 (w /v ) % , preferably 0 . 1 to 15 preferably 20 to 35º C . The culture time is preferably 10 to ( w / v ) % , and more preferably 0 . 2 to 10 ( w / v ) % . The reason 1000 hours, more preferably 15 to 480 hours, and even more for using them at 0 . 2 ( w / v ) % or higher is to have enhanced 40 preferably 20 to 240 hours . property of the microbes to produce methacrylic acid , and Those culture conditions are selected or optimized for the reason for using them at 10 ( w / v ) % or lower is that no each strain so as to maximize the ratio of the methacrylic significant increase in effect is observed even when they are acid ester production amount compared to the use amount of added at higher concentration . the renewable raw materials and / or biomass. Meanwhile, by To the medium , alcohols or phenols are added depending 45 suitably controlling the amount of carbon sources and cul on desired methacrylic acid ester . As for the alcohols or ture conditions, the methacrylic acid ester production phenols , those described before are preferably used . The amount can be also controlled . alcohols or phenols can be used either singly or in combi As the conditions preferred for accumulating methacrylic nation of two or more types . acid ester at 0 .04 ppm or higher, the reaction is allowed to A concentration of the alcohols or phenols in the medium 50 occur for three hours or longer at conditions of pH 5 . 5 to 7 . 5 , is not particularly limited as long as methacrylic acid ester while maintaining directly or indirectly the concentration of can be produced . The concentration is , for example , 0 .01 to the renewable raw materials and / or biomass at 0 . 1 % or 20 ( w / v ) % , preferably 0 .05 to 10 ( w / v ) % , and more higher and the concentration of the alcohols or phenols at preferably 0 . 1 to 5 ( w / v ) % . Further , the alcohols or phenols 0 . 1 % or higher and adjusting the temperature in the range of can be added in advance to the medium , or continuously or 55 20 to 40° C . Further , within the range that death ratio does intermittently added two or more divided times while per - not increase as the environment of broth becomes inappro forming the culture . priate for growth of microbes or cultured cells , the concen An inorganic nitrogen source or an inorganic metal salts tration of the microbes in broth is preferably maintained at may be added to the medium . As the inorganic nitrogen high level in terms of obtaining efficient productivity . For source , ammonium salts of inorganic acid or organic acid 60 example , by maintaining it at 2 g / l or more in dry weight , such as ammonium chloride , ammonium sulfate , ammonium good production efficiency is obtained and the accumulated acetate , or ammonium phosphate are used . concentration of the product can be increased . A concentration of the nitrogen source in the medium is (3 - 2 ) Production of Methacrylic Acid Ester Based on Reac not particularly limited as long as methacrylic acid ester can tion of Resting Cells be produced . The concentration is , for example , 0 .01 to 10 65 For the method for producing a methacrylic acid ester ( w / v ) % , preferably 0 .05 to 8 ( w / v ) % , and more preferably according to the invention , the following method can be also 0 . 1 to 4 ( w / v ) % . adopted in addition to the method which involves perform US 10 , 294 ,500 B2 65 66 ing , in an aqueous medium containing renewable raw mate Further , for purpose of isolating methacrylic acid ester rials and / or biomass , culture of microbes accompanied with produced , it is also possible to add an organic solvent in proliferation . The microbes may or may not have a prolif advance and performing the reaction in a biphasic system . eration property , and methacrylic acid ester can be also Examples of the organic acid include a linear , branched , or produced based on a resting cell reaction which is substan - 5 cyclic and saturated or unsaturated aliphatic hydrocarbon , a tially not accompanied with proliferation , by contacting the saturated or unsaturated aromatic hydrocarbon or the like , and they can be used either singly or as a mixture of two or microbes cultured in advance with an aqueous medium more types . Specific examples include a hydrocarbon sol containing renewable raw materials and/ or biomass . vent ( for example , pentane , hexane, cyclohexane , benzene , [Reaction Solution ] an 10 toluene, and xylene ), a halogenated hydrocarbon solvent ( for The renewable raw materials that are contained in an example , methylene chloride and chloroform ) , an ether aqueous medium containing renewable raw materials and / or solvent ( for example , diethyl ether, dipropyl ether, diisopro biomass used for the resting cell reaction ( that is , reaction pyl ether , dibutyl ether , tert -butyl methyl ether , and dime solution ) and the concentration can be the same as those for thoxyethane ), and an ester solvent ( for example , methyl the production of methacrylic acid ester by culture . 15 formate , methyl acetate , ethyl acetate , butyl acetate, and The alcohols or phenols that are used for the resting cell methyl propionate ) . By adding the organic solvent in reaction and the concentration can be the same as those for advance . methacrylic acid ester produced may migrate to an the production of methacrylic acid ester by culture . organic phase to have efficient progress of the reaction . To the reaction solution , inorganic metal salt or the like The reaction conditions are suitably selected or optimized may be added . Examples of the inorganic metal salt which 20 for each strain so as to maximize the ratio of the methacrylic can be used include monobasic potassium phosphate , diba - acid ester production amount compared to the use amount of sic potassium phosphate , magnesium phosphate , magnesium the renewable raw materials and / or biomass . Meanwhile , by sulfate , sodium chloride , ferrous sulfate , manganese sulfate , suitably controlling the amount of carbon sources and reac copper sulfate , and calcium carbonate . tion conditions , the methacrylic acid ester production A concentration of the inorganic salts in the reaction 25 amount can be also controlled solution is not particularly limited as long as methacrylic As the conditions preferred for accumulating methacrylic acid ester can be produced . The concentration is , for acid ester at 0 .04 ppm or higher , the reaction is allowed to example , 0 .0001 to 2 (w /v ) % , preferably 0 .0003 to 1. 3 ( w / v ) occur for three hours or longer at conditions of pH 5 .5 to 7. 5 , % , and more preferably 0 .001 to 1 ( w / v ) % . while maintaining directly or indirectly the concentration of In addition to them , a trace amount of a metal, a vitamin , 30 the renewable raw materials and /or biomass at 0 . 1 % or or the like is added to the reaction solution . Further , if higher and the concentration of the alcohols or phenols at necessary , various organic substances , inorganic substances , 0 .1 % or higher and adjusting the temperature in the range of surfactants , that are required for the reaction or anti- foaming 20 to 40° C . Further , the concentration of the microbes in agents that are generally used can be added to the reaction reaction liquid is preferably maintained at high level in solution . 35 terms of obtaining efficient productivity . For example , by [Reaction Conditions ] maintaining it at 2 g /l or more in dry weight, good produc For the resting cell reaction , the broth of the microbes tion efficiency is obtained and the accumulated concentra which have been cultured in advance may be used as it is , tion of the product can be increased . or the cells collected by filtration or centrifugal separation For performing the method for producing a methacrylic are used . The collected microbes can be used at any micro - 40 acid ester according to the invention , the aforementioned bial concentration after re - suspending them in a suitable production of methacrylic acid ester based on culture and buffer solution or the like . Examples of the buffer solution production of methacrylic acid ester based on the resting cell which may be used include physiological saline , potassium reaction can be suitably combined . According to the com phosphate buffer solution , tris -hydrochloric acid buffer solu - bination of the two methods, more efficient production of tion , glycine -sodium hydroxide buffer solution , and borate - 45 methacrylic acid ester can be achieved . Further, for the sodium hydroxide buffer solution . method for producing a methacrylic acid ester according to Further , a processed product of the collected microbes the invention , a method of directly contacting the microbes ( for example , disrupted product, co - enzymes , or purified with renewable raw materials and / or biomass can be also enzymes ) can be used for the resting cell reaction . Further , adopted , in addition to the method of contacting the it is also possible that the microbes or their processed 50 microbes with an aqueous medium containing renewable products are immobilized on a suitable carrier by a known raw materials and / or biomass, which is prepared as a method and the immobilized product is used for the reaction . medium or solution for the resting cell reaction . The conditions for the reaction are not particularly limited (3 - 3 ) Recovery of Methacrylic Acid Ester as long as it allows production of methacrylic acid ester. The T he methacrylic acid ester produced in a medium or reaction can be performed under aerobic conditions or 55 reaction solution and its production amount can be detected anaerobic conditions. The reaction method is not particu - and measured by using a common method like high speed larly limited either , and a known method such as shaking liquid chromatography and LC -MS . Further , the methacrylic reaction , aeration and agitation reaction , or static reaction acid ester vaporized in the reaction container or the vapor can be used . phase of the reaction container ( that is, head space part) and ApH , temperature , and reaction time are not particularly 60 the production amount can be defected and measured by limited as long as they are the conditions allowing produc - using a common method like gas chromatography . tion of methacrylic acid ester. The pH is preferably 3 to 10 , Methacrylic acid ester can be isolated and purified from more preferably 4 to 9 , and even more preferably 5 to 8 . The the medium or reaction solution by suitably using , if nec temperature is preferably 10 to 45° C . , more preferably 15 essary in combination , a known process like filtration , to 40° C . , and even more preferably 20 to 35° C . The culture 65 centrifugal separation , vacuum concentration , ion exchange time is preferably 5 to 180 hours, more preferably 10 to 150 or adsorptive chromatography , solvent extraction , distilla hours , and even more preferably 15 to 120 hours . tin , andcrystallization . US 10 , 294 ,500 B2 67 68 2 . Enzymes Involved with Synthesis of Methacrylic Acid Open Reading Frame (ORF ) , which is annotated to acyl and their Genes COA dehydrogenase , has been known . Hereinbelow , the enzymes that are involved with synthe - Against the genome sequence information of the two sis of methacrylic acid and their genes as one aspect of the bacteria , homology search was performed for the amino acid invention will be described in detail . 5 sequence of human ACAD8. Genes of the total six kinds of The inventors of the invention found that , as a result of ORF found to have the homology were amplified and conducting extensive searches for microbes having the abil obtained by PCR and ligated to the expression vector for E . ity to produce methacrylic acid derivatives, methacrylic acid coli. By using them , a recombinant was produced , and can be produced by using microbes . Based on history of the expression of the homolog protein in the recombinant and searches and techniques of the related art , the synthetic route 10 the ACD activity of the recombinant cell extract were is presumably the decomposition pathway of valine . determined In view of the relationship with a genetic disease , studies [ ACD ] on the decomposition pathway of valine are made for a As a result , a very high activity was obtained from the human or a rat (Methods in Enzymology, 324 : 241- 258 recombinant introduced with the gene named PA _ acd1 from ( 2000 ) ) . However , little is known about the enzymes of the 15 Pseudomonas aeruginosa PAO1 and the gene named decomposition pathway of valine in microbes , and the RE _ acdl from Rhodococcus erythropolis PR4 (SEQ ID details have remained unclear. With regard to the mold NOS . 51 and 53 ) . PA acdl and RE acdl exhibited 57 % Aspergillus nidulans, there are results demonstrating that identity to each other in terms of the amino acid sequence scdA gene product is involved with metabolism of isoleu - encoded by them (SEQ ID NOS. 50 and 52 ). Meanwhile , the cine , valine , and short chain fatty acid and has an activity of 20 amino acid - level identity of PA _ acd2 , RE _ acd2 not acyl COA dehydrogenase acting on several metabolic path - observed with any activity or acdH ( Streptomyces ) observed ways , based on the analysis of the variant strain deficient of only with weak activity to PA _ acdl and RE _ acdl observed scdA gene (Fungal Genet. Biol. 45 : 180 - 189 ( 2008 ) ) . Those with high activity is as described below . PA _ acd2 exhibited results are an indirect evaluation , and it remained uncertain the identity of 40 % and 41 % to PA _ acdl and RE acdi , whether or not the enzyme is actually effective for producing 25 respectively . RE _ acd2 exhibited the identity of 36 % to methacrylic acid . Other than that, there is the information PA _ acdl and RE _ acdl , respectively . acdH exhibited the only about the genus Streptomyces , which are actinomycetes identity of 38 % and 36 % to PA _ acd1 and RE _ acd1 , respec as one type of bacteria , with regard to the enzyme derived tively . microbes (Microbiology 145 : 2323 - 2334 ( 1999 ) ) . In the In other words , as one of ACD useful for synthesis of literature, ACD gene ( acdH ) is cloned from Streptomyces 30 methacrylic acid , there are ( A ) the enzyme consisting of the coelicolor , and by using partially purified enzyme which has amino acid sequence (SEQ ID NO . 50 or 52 ) encoded by been produced from recombinant E . coli , production of PA _ acdl or RE _ acd1 and ( B ) the enzyme consisting of an methacrylyl- CoA from isobutyryl- CoA was confirmed . The amino acid which exhibits the amino acid - level identity of inventors of the invention also followed the test to recognize 45 % or more , more preferably 60 % or more, and even more the significant expression of the enzyme protein in a recom - 35 preferably 80 % or more to the amino acid sequence (SEQ ID binant which uses E . coli as a host. However, regarding the NO . 50 or 52 ) encoded by PA _ acdl or RE _ acdl and ACD activity , only an extremely weak activity was recog - exhibiting the ACD activity . Alternatively , it is ( C ) the nized and it was determined that the enzyme is not suitable enzyme having the ACD activity and consisting of an amino for synthesis of methacrylic acid . acid sequence with deletion , substitution , or addition of one Accordingly , for the purpose of using effectively the 40 or several amino acids in the amino acid sequence ( SEQ ID enzyme involved with synthesis of methacrylic acid in NO . 50 or 52 ) encoded by PA _ acd1 or RE _ acdl . microbes , the inventors made investigations about the Further , PA _ acdl and RE _ acdl exhibited 69 % identity enzyme gene of other microbes . As a result , the following for the 70 amino acids from the C terminus of the amino acid useful enzymes were found . sequence encoded by them ( SEQ ID NOS . 50 and 52 ). Among the microbes having the ability to produce meth - 45 Meanwhile , with regard to PA _ acd2 , RE _ acd2 not observed acrylic acid as described in the invention , the genus with any activity or acdH ( Streptomyces ) observed only with Pseudomonas and the genus Rhodococcus were selected . weak activity , the amino acid - level homology of 70 amino With regard to the microbes of the genus Pseudomonas, acids from the C terminus of PA _ acdl and RE _ acdl that Pseudomonas putida and Pseudomonas aeruginosa are have been observed with high activity are as described known to have a valine - assimilating property (Bacteriol . 50 below . PA _ acd2 exhibited the identity of 43 % and 43 % to Review 40 : 42 -54 ( 1976 ) ) . Although the valine -assimilating PA _ acd1 and RE _ acd1 , respectively . RE _ acd2 exhibited the property is not known for the microbes of the genus Rho - identity of 38 % to PA _ acdl and RE _ acd1 , respectively . dococcus, the inventors of the invention experimentally acdH exhibited the identity of 43 % and 38 % to PA _ acdl and confirmed that they have an ability to produce methacrylic RE _ acd1, respectively . acid . Among them , for Pseudomonas aeruginosa PAO1 and 55 In the invention , another embodiment of the ACD which Rhodococcus erythropolis PR4 which have a known genome is useful for the synthesis of methacrylic acid is ( D ) the sequence, list -up of candidates was performed by homology enzyme having the ACD activity and consisting of an amino search under the assumption that they have ACD believed to acid sequence which exhibits the identity of 60 % or more , be involved with synthesis of methacrylic acid . more preferably 65 % or more, and even more preferably With regard to the presence of a homolog gene of bacteria 60 80 % or more to the amino acid sequence of the 70 amino which exhibits high identity to ACAD8 which becomes acid residues from C terminus of the amino acid sequence human ACD , there have been descriptions in the literature encoded by PA acdl or RE acdl (SEO ID NO . 50 or 52 ) . ( J .Mol . Evol. 69 : 176 - 193 (2009 ) ) (however , the activity has One of the ACD gene particularly useful for the method not been confirmed experimentally ) . Further , for the two for producing methacrylic acid of the invention is a gene bacteria of Pseudomonas aeruginosa PAO1 and Rhodococ- 65 selected from the group consisting of the followings . cus erythropolis PR4 which have a known genome ( a ) a gene encoding the protein consisting of an amino acid sequence, only the information regarding the presence of sequence represented by SEQ ID NO . 50 or 52 , US 10 , 294 ,500 B2 69 70 ( b ) a gene consisting of a nucleotide sequence represented polypeptides having an amino acid sequence represented by by SEQ ID NO . 51 or 52 , SEQ ID NOS . 54 , 56 , 58 , and 60 and encoding the protein ( c ) a gene encoding the protein consisting of an amino acid having the BCKAD activity . sequence, which exhibits the identity of 45 % or more to the (h ) a gene consisting of an amino acid sequence with protein consisting of an amino acid sequence represented by 5 deletion , substitution , and /or addition of one or several SEQ ID NO . 50 or 52, and having the ACD activity , and amino acids in the amino acid sequence represented by SEQ ( d ) a gene encoding the protein consisting of an amino acid ID NOS . 54 , 56 , 58 , and 60 , and encoding the protein having sequence with deletion , substitution , or addition of one or the BCKAD activity . several amino acids in the amino acid sequence represented ( i) a gene encoding the four polypeptides having an amino by SEQ ID NO . 50 or 52 and having the ACD activity. * * 10 acid sequence represented by SEQ ID NOS . 62 , 64 , 66 , and 68 . In the invention , ACD is not particularly limited if it is ( ) a gene consisting of a nucleotide sequence represented by useful as an enzyme for synthesizing methacrylic acid and SEQ ID NOS . 63, 65 , 67, and 69 . also derived from microbes having the ability to produce ( k ) a gene capable of hybridizing under stringent conditions methacrylic acid as described in the invention . 15 to a complementary sequence of the gene encoding the four More preferably , it is those derived from Pseudomonas or polypeptides having an amino acid sequence represented by Rhodococcus erythropolis , and examples of the preferred SEQ ID NOS . 62, 64, 66 , and 68 and encoding the protein hodococcus erythropolis PR - 4 strain , Rho - having the BCKAD activity . dococcus erythropolis KA2- 5 - 1 strain , Rhodococcus eryth ( 1) a gene consisting of an amino acid sequence with ropolis IGTS8 strain , Rhodococcus erythropolis D - 1 strain , 20 deletion , substitution , and /or addition of one or several Rhodococcus erythropolis H - 2 strain , Rhodococcus eryth amino acids in the amino acid sequence represented by SEO ropolis N1- 36 strain , Rhodococcus erythropolis 1 - 19 strain , ID NOS . 62 , 64 , 66 , and 68 , and encoding the protein having Rhodococcus erythropolis ECRD - 1 strain , Rhodococcus the BCKAD activity. erythropolis B1 strain , Rhodococcus erythropolis SY - 1 ( m ) a gene encoding the three polypeptides having an amino strain , Rhodococcus erythropolis UM3 strain , Rhodococcus 25 acid sequence represented by SEQ ID NOS. 70 , 72 , and 74 . erythropolis UM9 strain and Rhodococcus erythropolis T09 ( n ) a gene consisting of a nucleotide sequence represented by SEQ ID NOS . 71 , 73 , and 75 . strain . Particularly preferred is Rhodococcus erythropolis ( o ) a gene capable of hybridizing under stringent conditions PR - 4 strain . to a complementary sequence of the gene encoding the three Hereinabove, the ACD as one of the enzymes involvedthe 30 polypeptides having an amino acid sequence represented by with synthesis of methacrylic acid and obtainment of the 30 SEQ ID NOS. 70, 72 , and 74 and encoding the protein gene thereof will be described . having the BCKAD activity . TBCKADI (p ) a gene consisting of an amino acid sequence with Next, descriptions are made with regard to BCKAD deletion , substitution , and / or addition of one or several which is involved with synthesis of methacrylic acid .| 35 amino acids in the amino acid sequence represented by SEQ BCKAD is an enzyme capable of producing isobutyryl- CoA ID NOS . 70 , 72 , and 74, and encoding the protein having the from 2 - oxoisovaleric acid . From the microbes of the inven BCKAD activity . tion having the ability to produce methacrylic acid , BCKAD In the invention , the gene encoding BCKAD is not limited and the gene thereof can be obtained . to the aforementioned sequences, and a gene having a BCKAD derived from Pseudomonas putida or 40 nucleotide sequence which has the identity of about 50 % or Pseudomonas aeruginosa is a complex consisting of four more , preferably about 60 % or more , more preferably about polypeptides (SEQ ID NOS. 54, 56 , 58 and 60 ) , and their 70 % or more , even more preferably about 80 % or more , genes (SEQ ID NOS. 55 , 57 , 59 and 61) are known to form particularly preferably about 90 % or more , particularly even an operon (Methods in Enzymology (2000 ) 324 : 129 - 138 ) . more preferably about 95 % or more , and most preferably As a method for obtainment, a vector for expressing 45 about 98 % or more to the nucleotide sequence described BCKAD can be constructed , for example , by designing a with the aforementioned SEQ ID NOs is also included in the primer for amplifying the entire BCKAD operon from the gene encoding BCKAD as long as a protein having the genome sequence of Pseudomonas aeruginosa PAO1, BCKAD activity is encoded by it . amplifying the entire BCKAD operon by PCR reaction In the invention , examples of the BCKAD include those using the genomic DNA as a template , and ligating it to an 50 having an amino acid sequence represented by SEQ ID expression vector for E . coli. By using the vector, a recom - NOS . 54 , 56 , 58 , and 60 , an amino acid sequence repre binant can be produced and the BCKAD gene and BCKAD sented by SEQ ID NOS. 62 , 64 , 66 , and 68, or an amino acid can be obtained . sequence represented by SEQ ID NOS . 70 , 72 , and 74 . With regard to the BCKAD of the invention , there is no In the invention , the BCKAD is not limited to those particular limitation on the gene thereof, if it is useful as one 55 having the aforementioned sequences, and a protein having enzyme useful for synthesis of methacrylic acid and is the BCKAD activity and containing an amino acid sequence derived from microbes having the ability to produce meth - which has the identity of about 50 % or more , preferably acrylic acid as described in the invention . However , about 60 % or more , more preferably about 70 % or more , examples thereof include a gene selected from the group even more preferably about 80 % or more, particularly consisting of the following ( e ) to ( h ) , ( i ) to ( 1 ) , or ( m ) to ( p ) . 60 preferably about 90 % or more , particularly even more ( e ) a gene encoding the four polypeptides having an amino preferably about 95 % or more , and most preferably about acid sequence represented by SEQ ID NOS . 54 , 56 , 58 , and 98 % or more to the amino acid sequence described with the 60 . aforementioned SEQ ID NOs is also included in the gene ( f ) a gene consisting of a nucleotide sequence represented by encoding BCKAD . SEQ ID NOS . 55 , 57 , 59 , and 61. 65 The identity value described above is obtained by running ( g ) a gene capable ofhybridizing under stringent conditions a program for analyzing homology with use ofGENETYX to a complementary sequence of the gene encoding the four (manufactured by GENETYX Corporation ), which is a US 10 ,294 ,500 B2 71 72 software for sequence analysis . Parameters at that time are Further, in response to the aforementioned deletion , sub the same as the default setting initial setting ) . stitution , addition , and / or insertion of the amino acid In the BCKAD of the invention , a protein having the sequence , a nucleotide sequence having a mutation like BCKAD activity and containing an amino acid sequence deletion , substitution , addition , and /or insertion occurred in with deletion , substitution , and /or addition of one or several 5 several nucleotides of the nucleotide sequence represented amino acids in the amino acid sequence represented by SEO by SEQ ID NOS . 55, 57, 59 , and 61, the nucleotide sequence represented by SEQ ID NOS . 63 , 65 , 67 , and 69 , or the ID NOS . 54 , 56 , 58 , and 60 , the amino acid sequence nucleotide sequence represented by SEQ ID NOS . 71, 73 , represented by SEQ ID NOS . 62 , 64 , 66 , and 68 , or the and 75 is also included in the gene which encodes BCKAD , amino acid sequence represented by the amino acid 10 as long as it encodes the protein having the BCKAD activity sequence represented by SEQ ID NOS . 70, 72 , and 74 is also described in the invention . Meanwhile , the number of the included . nucleotides that are subjected to deletion , substitution , addi More specific examples thereof include a protein contain tion , and / or insertion is 30 or less , preferably 15 or less, and ing the following amino acid sequence and having the particularly preferably 6 or less. Further , a DNA capable of BCKAD activity . 15 hybridizing, under a stringent condition , to a DNA consist (i ) An amino acid sequence with deletion of 1 to 20 ( for ing of a nucleotide sequence complementary to the gene example , 1 to 10 , preferably 1 to 5 , and even more preferably consisting of the nucleotide sequence represented by SEO 1 to 2 ) amino acids in the amino acid sequence represented ID NOS. 55 , 57 , 59 , and 61 . the gene consisting of the by SEQ ID NOS. 54 , 56 , 58 , and 60 , the amino acid nucleotide sequence represented by SEQ ID NOS. 63, 65 , sequence represented by SEQ ID NOS . 62 , 64 , 66 , and 68 , 20 67 , and 69 , or the gene consisting of the nucleotide sequence or the amino acid sequence represented by SEQ ID NOS. 70 , represented by SEQ ID NOS. 71, 73 , and 75 is also included 72 , and 74 . in the gene which encodes BCKAD , as long as it encodes the ( ii ) An amino acid sequence with substitution of 1 to 20 ( for protein having the BCKAD activity . example , 1 to 10 , preferably 1 to 5 , and even more preferably Hereinabove , descriptions are made regarding enzyme 1 to 2 ) amino acids in the amino acid sequence represented 25 proteins and obtainment of their genes for ACD and by SEQ ID NOS. 54 , 56 , 58 , and 60 , the amino acid BCKAD , as an example of an enzyme that is involved with sequence represented by SEQ ID NOS . 62, 64 , 66 , and 68 , synthesis of methacrylic acid . In the invention , other or the amino acid sequence represented by SEQ ID NOS . 70 , enzymes and genes thereof that are involved either directly 72 , and 74 with other amino acids . or indirectly with synthesis of methacrylic acid can be also ( iii ) An amino acid sequence with addition of 1 to 20 ( for 30 obtained in a similar manner. example , 1 to 10 , preferably 1 to 5 , and even more preferably For example , enzymes involved with synthesis of meth 1 to 2 ) amino acids in the amino acid sequence represented acrylic acid can be purified by using a common method with by SEQ ID NOS. 54 , 56 , 58 , and 60 , the amino acid a use of the enzyme activity as an indicator. Further, the sequence represented by SEQ ID NOS . 62 , 64 , 66 , and 68, genetic information can be obtained by a genetic engineer or the amino acid sequence represented by SEQ ID NOS . 70 , 35 ing method based on the proteins . Alternatively , the entire 72 , and 74 . genome sequence is determined for the microbes having the ( iv ) An amino acid sequence with insertion of 1 to 20 ( for ability to synthesize methacrylic acid as described in the example , 1 to 10 , preferably 1 to 5 , and even more preferably invention . Selection can be made by searching a gene 1 to 2 ) amino acids to the amino acid sequence represented sequence having high homology by using homology search by SEO ID NOS . 54 , 56 , 58 , and 60 , the amino acid 40 based on known information about the sequence of a gene sequence represented by SEQ ID NOS . 62 , 64 , 66 , and 68, which encodes the target enzyme, isolating or synthesizing or the amino acid sequence represented by SEQ ID NOS. 70 , the entire gene with a known method and introducing it to 72 , and 74 . a general host vector system , expressing the candidate ( v ) An amino acid sequence having a combination of ( i) to protein using microbes transformed with the vector system , ( iv ) . 45 and evaluating the activity of the target enzyme . Meanwhile , When one or more amino acids of the amino acid due to the availability of a next generation sequencer, a sequence are substituted , it is preferable to have conserva - skilled person in the pertinent art can easily analyze the tive substitution between similar amino acid residues . For entire genome sequence of microbes . example, amino acids are classified into hydrophobic amino Further, the microbes having the ability to synthesize acids ( A , I , L , M , F , P , W , Y , V ) , hydrophilic amino acids ( R , 50 methacrylic acid as described in the invention include an D , N , C , E , Q , G , H , K , S , T ) , amino acids having aliphatic acceptor of electrons, which are released in accordance with side chains ( G , A , V , L , I , P ) , amino acids having side chains the dehydrogenation of ACD , proteins relating to the trans that contain a hydroxy group ( S , T , Y ) , amino acids having fer system , and the genes thereof, and they are also included side chains that contain a sulfur atom ( C , M ), amino acids in the invention . having side chains that contain carboxylic acid and amide 55 In addition , the enzymes involved with synthesis of ( D , N , E , Q ) , amino acids having side chains that contain a methacrylic acid , which are obtained from microbes having base ( R , K , H ), amino acids having side chains that contain the ability to synthesize methacrylic acid can produce , either aromatics ( H , F , Y , W ), based on the characteristics of the singly or collectively with others, methacrylic acid deriva side chains . The amino acids classified into each group are tives and intermediates thereof. For example , with BCKAD , known to have a high possibility of maintaining the activity 60 isobutyryl - CoA can be produced from 2 -oxoisovaleric acid , of the polypeptide when substitution is made between them , and with ACD , methacrylyl- CoA can be produced from and it is preferable to have mutual substitution between such isobutyryl- CoA . Thus, by combining the reactions of both amino acids . Examples include substitution between glycine BCKAD and ACD , it becomes possible to produce meth and proline, glycine and alanine or valine , leucine and acrylyl- CoA from 2 -oxoisovaleric acid . Alternatively , by isoleucine , glutaminic acid and glutamine , asparaginic acid 65 adding an enzyme which has an activity of hydrolyzing AAT and asparagine , cysteine and threonine, threonine and serine or methacrylyl- CoA , each of methacrylic acid ester and or alanine , and lysine and arginine . methacrylic acid can be produced . US 10 ,294 , 500 B2 73 74 The enzymes involved with synthesis ofmethacrylic acid and suspended in 0 . 3 mL sterilized water . 0 . 1 mL of the can be obtained by isolating the gene as described above , supernatant was added to the LB liquid medium and cultured producing a recombinant ( transformant) by using a general at 30° C . for 48 hours. A suitable amount of the culture host vector, and culturing the recombinant. Examples of the supernatant was added to 10 ml liquid medium . The com host include E . coli , the genus Rhodococcus, the genus 5 position of the medium is shown below . Pseudomonas, the genus Corynebacterium , the genus Bacil K2HPO4 1 . 0 g /1 lus, the genus Streptococcus, and the genus Streptomyces as MgSO4. 7H2O 0 . 2 g /1 bacteria , Saccharomyces , the genus Candida , the genus FeSO4. 7H20 0 .01 g / 1 Shizosaccharomyces, and the genus Pichia as yeast, and the CaCl , 0 .01 g/ 1 genus Aspergillus as mold . For a case in which obtainment 10 L - Valine 5 . 0 g / 1 of the enzyme (protein ) is the object, using E . coli is pH 7 .0 preferred due to convenience and good efficiency. It is also Shake culture was performed at 30° C . for 3 to 7 days . The possible to express plural enzymes involved with synthesis cultured strain was transferred to the same medium and of methacrylic acid in the same host. With regard to the acclimated culture was performed for several times. After obtainment of the enzymes from culture of a transformant, 15 that, they were inoculated on an agar medium , that is , the collection can be made by disruption , extraction , or cen same medium added with 1 .5 % agar, and cultured at 30° C . trifugal separation of the cells after recovery . for 48 hours to form a colony . Using a platinum loop , the By contacting the enzymes obtained accordingly with a colony was scraped and inoculated on an agar medium ( LB compound as a raw material under conditions allowing the medium , 1 . 5 % agar ) and cultured at 30° C . for 48 hours to enzymes to work , each target product can be obtained . 20 obtain the isolated strains (G1 strain , G2 strain ) . The mycological properties of the isolated strains are EXAMPLES shown in the following table . In the table , “ + ” indicates positive, “ _ ” indicates negative and “ + w ” indicates a weak Hereinbelow , the invention is explained in greater detail response . on the basis of the examples , but the invention is not limited 25 ( 1 Mycological Properties of Pseudomonas sp . B25 - 2 to them . Example 1: Search for Methacrylic Acid -Producing TABLE 1- 1 Microbes 1. Morphological properties 30 Culture conditions Nutrient agar medium 30° C . Using the soils and insects which have been collected Shape of cell Bacillus ( 0 . 6 to 0 . 7 x from different areas in Japan as a source ofmicrobes , search 1 .5 to 2 .0 um ) for methacrylic acid -producing microbes was conducted Presence or absence of cell pleomorphism T based on enrichment culture . Because the metabolitic inter Motility (attachment state of flagella ) + Presence or absence of spore ( spore 1 mediates of valine include methacrylyl - CoA , which is a 35 location ) derivative of methacrylic acid , a medium containing L - va 2 . Cultural properties line was used and the screening was performed using the assimilation property of valine as an indicator. Culture conditions Nutrient agar medium 30° C . [ Isolation from Soils ] Color Pale yellow color An appropriate amount of soils was added to 5 mL of 40 GlossPigment production sterilized water and stirred . After allowing it to stand , a Culture conditions Nutrient broth medium 30° C . suitable amount of the supernatant was added to 10 ml liquid Presence surface growth Presence or absence of turbidity in medium . The composition of the medium is shown below . medium K HPO , 1. 0 ul Culture conditions Gelatin stab culture 30° C . MgSO . .7H , O 0 . 2 g / 1 45 Growth state FeSO4. 7H2O 0 .01 g / l Gelatin liquefaction Culture conditions Litmus milk 30° C . CaC1, 0 .01 g / 1 Solidification L - Valine 5 . 0 g / 1 Liquefaction pH 7 . 0 3 . Physiological properties Shake culture was performed at 30° C . for 3 to 7 days. The 50 Gram stain grown strain was transferred to the same medium and Nitrate salt reduction acclimated culture was performed for several times. After Denitrification that, they were inoculated on an agar medium , that is , the MR test LLL+ VP test same medium added with 1 . 5 % agar, and cultured at 30° C . Indole production for 48 hours to form a colony . Using a platinum loop , the 55 Hydrogen sulfide production colony was scraped and inoculated on an agar medium (LB Hydrolysis of starch medium , 1 . 5 % agar ) and cultured at 30° C . for 48 hours to Use of citric acid (Koser ) + obtain the isolated strains (B25 - 2 strain , D22 - 1 strain , D43 - 1 ( Christensen ) + strain , D25 strain , D26 strain , D29 strain , D41 - 2 strain , R1 Use of inorganic Nitrate salt + strain , B13 strain ). The composition of the LB medium is as 60 nitrogen source Ammonium salt + follows: 1 % bactotrypton , 0 . 5 % bactoyeast extract , and 0 . 5 % NaCl. [ Isolation from Insect ] TABLE 1 - 2
The collected Carabus insulicola was kept for 2 days in Urease activity | starvation state , and the body of the insect was washed with 65 Catalase + 95 % ethanol and sterilized physiological saline , two times Oxidase + for each . The abdomen part of the insect body was separated US 10 ,294 ,500 B2 75 76 TABLE 1- 2 -continued TABLE 2 - 1 - continued Growth range + Presence or absence of cell pleomorphism pH + Motility ( attachment state of flagella ) + ( Ciliate ) + Presence or absence of spore (spore Growth range + 5 location ) Temperature + 2 . Cultural properties (°C .) I Culture conditions Nutrient agar medium 30° C . Anaerobic growing property + Color Pale yellow color O•F Test (oxidation / fermentation ) Gloss 4 . Acid production gas production from carbohydrates 10 Pigment production Culture conditions Nutrient broth medium 30° C . L - Arabinose D - Xylose Presence or absence of surface growth D -Glucose D -Mannose Presence or absence of turbidity in D - Fructose D -Galactose medium Maltose Saccharose Culture conditions Gelatin stab culture 30° C . Lactose - - Trehalose 15 Growth state + D - Sorbitol - - D -Mannitol Gelatin liquefaction Inositol - - Glycerin Fiiii Culture conditions Litmus milk 30° C . 5 . Other physiological properties Solidification Liquefaction B -Galactosidase activity | 3 . Physiological properties Arginine dehydrolase activity +
Lysine decarboxylase activity 1 Gram stain Tryptophan deaminase activity Nitrate salt reduction Gelatinase activity Denitrification MR test TIL+ VP test According to a common method , the partial nucleotide Indole production sequence of 16S rDNA of the B25 - 2 strain (SEO ID NO . 1 ) 25 Hydrogen sulfide production Hydrolysis of starch was determined . Use of citric acid (Koser ) The partial nucleotide sequence of 16S rDNA of the (Christensen ) + B25 - 2 strain exhibited high homology to the nucleotide Use of inorganic Nitrate salt + sequence of 16S rDNA of Pseudomonas, and it exhibited the nitrogen source Ammonium salt + highest identity of 99 .2 % to the Pseudomonas orizihabitans 30 IAM ( Institute of Molecular and Cellular Biology Culture Collection (University of Tokyo )) 1568 strain TABLE 2 - 2 (NBRC102199 ) and Pseudomonas japonica IAM 15071 strain (NBRC 103040 ) (NBRC strain is obtainable from Urease activity | Biotechnology Center of National Institute of Technology 35 Catalase + Oxidase + and Evaluation , Independent Administrative Institution ) . Growth range + As a result of a brief molecular phylogenetic analysis pH + based on the partial nucleotide sequence of 16S rDNA , B25 - 2 strain is included in a cluster formed of the species of Growth range + Temperature + Pseudomonas . Further, unlike any other known species , 40 ( ° C . ) B25 - 2 strain showed an independentmolecular phylogenetic position in the cluster of Pseudomonas. Anaerobic growing property + B25 - 2 strain does not reduce a nitrate salt, exhibits an O•F Test (oxidation / fermentation ) arginine hydrolase activity , does not hydrolyze gelatin , but 4 . Acid production / gas production from carbohydrates assimilates glucose, D -mannose , potassium gluconate , or the 45 L - Arabinose D - Xylose like . Further , it does not assimilate L - arabinose and D -man D -Glucose D -Mannose D - Fructose D -Galactose nitol, produces a fluorescent pigment in Kings’ B agar Maltose Saccharose +Ììì medium , shows motility, shows a positive response for both Lactose Trehalose the catalase reaction and oxidase reaction , and does not D - Sorbitol D -Mannitol hydrolyze starch . Those properties are believed not to con - 50 Inositol - | - Glycerin + w / tradict with the properties of Pseudomonas, but they are not 5. Other physiological properties in match with the properties of any other known species . B -Galactosidase activity | As such , B25 - 2 strain was identified as Pseudomonas sp . Arginine dehydrolase activity + On Nov. 7 , 2012 , B25 - 2 strain was subjected to international Lysine decarboxylase activity + deposition with National Institute of Technology and Evalu - 55 Tryptophan deaminase activity | ation (NITE ) , Independent Administrative Institution ( zip Gelatinase activity | code : 292 - 0818 , Kazusakamatari, Kisarazu -shi , Chiba , Japan , 2 - 5 - 8 ), under accession number of NITE BP - 1451 . According to a common method , the partial nucleotide ( 2 ) Mycological Properties of Pseudomonas sp . D22 - 1 sequence of 16S rDNA of the D22 - 1 strain (SEQ ID NO . 2 ) 60 was determined . TABLE 2 - 1 The partial nucleotide sequence of 16SDNÁf the D22 - 1 strain exhibited high homology to the nucleotide 1 . Morphological properties sequence of 16S rDNA of Pseudomonas, and it exhibited the Culture conditions Nutrient agar medium 30° C . highest identity of 98 . 8 % to the Pseudomonas agarici Shape of cell Bacillus (0 .7 to 0 . 8 x 65 LMG2112 strain . 1 . 2 to 2 . 0 um ) As a result of a brief molecular phylogenetic analysis based on the partial nucleotide sequence of 16S rDNA , US 10 , 294 , 500 B2 77 78 2 - strains included in cluster formed of the species of TABLE 3 - 2 - continued Pseudomonas . Further , although D22 -1 strain formed a Anaerobic growing property cluster with Pseudomonas abietaniphila , a distance is rec O•F Test (oxidation / fermentation ) + ognized between them . 4 . Acid production gas production from carbohydrates D22 - 1 strain does not reduce a nitrate salt , exhibits an 5 arginine hydrolase activity but no urease activity , does not L - Arabinose + D - Xylose D -Glucose + D -Mannose hydrolyze gelatin but assimilates glucose , D -mannose and D - Fructose T D -Galactose N - acetyl - D - glucosamine , or the like. Further , it does not Maltose 1 Saccharose assimilate L -arabinose and D -mannitol , does not produce a Lactose Trehalose D - Sorbitol D -Mannitol fluorescent pigment in Kings ' B agar medium , shows motil - 10 Inositol - / Glycerin ity , and shows a positive response to both the catalase 5 . Other physiological properties reaction and oxidase reaction . Those properties are believed to be in match with the properties of Pseudomonas, but they B -Galactosidase activity are not in match with the properties of any other known Arginine dehydrolase activity 1+ species . 15 Lysine decarboxylase activity As such , D22 - 1 strain was identified as Pseudomonas sp . Tryptophan deaminase activity On Nov . 7 , 2012 , D22 - 1 strain was subjected to international Gelatinase activity deposition under accession number of NITE BP -1452 . ( 3 ) Mycological Properties of Pseudomonas umsongensis According to a common method , the partial nucleotide D43 - 1 20 sequence of 16S rDNA of the D43 - 1 strain (SEQ ID NO . 3 ) was determined . TABLE 3 -1 The partial nucleotide sequence of 16S rDNA of the 1 . Morphological properties D43 - 1 strain exhibited high homology to the nucleotide sequence of 16S rDNA of Pseudomonas, and it exhibited the Culture conditions Nutrient agar medium 30° C . 25 highest identity of 100 % to the Pseudomonas umsongensis Shape of cell Bacillus ( 0 . 8 to 0 . 9 x 1 . 2 to 1. 5 um ) Ps33 - 10 strain . Presence or absence of cell pleomorphism As a result of a brief molecular phylogenetic analysis Motility ( attachment state of flagella ) + (Ciliate ) based on the partial nucleotide sequence of 16S rDNA , Presence or absence of spore ( spore location ) D43 - 1 strain is included in a cluster formed of the species of 2. Cultural properties 30 Pseudomonas . Further , D43- 1 strain formed a cluster with Pseudomonas umsongensis and Pseudomonas mohnii , and it Culture conditions Nutrient agar medium 30° C . exhibited the samemolecular phylogenetic position as those Color Pale yellow color Gloss two species . Pigment production D43 - 1 strain reduces a nitrate salt , exhibits an arginine Culture conditions Nutrient broth medium 30° C . hydrolase activity but no urease activity , does not hydrolyze Presence or absence of surface growth T Presence or absence of turbidity in + gelatin but assimilates glucose, L - arabinose and D -mannose , medium or the like . Further , it does not assimilate D -mannitol and Culture conditions Gelatin stab culture 30° C . N -acetyl - D - glucosamine , produces a fluorescent pigment in Growth state Gelatin liquefaction 40 Kings ' B agar medium , showsmotility , and shows a positive Culture conditions Litmus milk 30° C . response to both the catalase reaction and oxidase reaction . Solidification Liquefaction Those properties are almost in match with the properties of 3 . Physiological properties Pseudomonas umsongensis . As such , D43 - 1 strain was identified as Pseudomonas Gram stain 45 umsongensis . On Nov . 7 , 2012 , D43 - 1 strain was subjected Nitrate salt reduction +I to international deposition under accession number of NITE Denitrification MR test BP - 1453 . VP test ( 4 ) Mycological Properties of Pseudomonas sp . D25 Indole production Hydrogen sulfide production Hydrolysis of starch 50 TABLE 4 - 1 Use of citric acid (Koser ) +III 1 . Morphological properties ( Christensen ) Use of inorganic Nitrate salt Culture conditions Nutrient agar medium 30° C . nitrogen source Ammonium salt Shape of cell Bacillus ( 0 . 8 to 0 . 9 x 55 1 . 2 to 2 . 0 um ) Presence or absence of cell pleomorphism Motility ( attachment state of flagella ) + ( Ciliate ) TABLE 3 - 2 Presence or absence of spore ( spore location ) Urease activity 2 . Cultural properties Catalase Oxidase +| Culture conditions Nutrient agar medium 30° C . Growth range Color Pale yellow color PH Gloss + Pigment production Growth range Culture conditions Nutrient broth medium 30° C . Temperature + Presence or absence of surface growth (°C . ) 65 Presence or absence of turbidity in T medium US 10 , 294 ,500 B2 79 80 TABLE 4 - 1- continued to both the catalase reaction and oxidase reaction . Those properties are believed to be in match with the properties of Culture conditions Gelatin stab culture 30° C . Growth state + Pseudomonas, but a difference is recognized with Gelatin liquefaction Pseudomonas putida , Pseudomonas fuscovaginae , Culture conditions Litmus milk 30° C . 5 Pseudomonas asplenii and Pseudomonas agarici for which Solidification high homology has been demonstrated in terms of the Liquefaction assimilation property or producing a fluorescent pigment . 3 . Physiological properties As such , D25 strain was identified as Pseudomonas sp . Gram stain On Nov . 7 , 2012 , D25 strain was subjected to international Nitrate salt reduction Denitrification 10 deposition under accession number of NITE BP - 1454 . MR test LLLL+ ( 5 ) Mycological Properties of Pseudomonas sp . D26 VP test Indole production TABLE 5 - 1 Hydrogen sulfide production Hydrolysis of starch 15 1 . Morphological properties Use of citric acid (Koser ) + ( Christensen ) + Culture conditions Nutrient agar medium 30° C . Use of inorganic Nitrate salt + Shape of cell Bacillus ( 0 . 7 to 0 . 8 x nitrogen source Ammonium salt + 1 . 2 to 1 . 5 um ) Presence or absence of cell pleomorphism - Motility (attachment state of flagella ) + (Ciliate ) 20 Presence or absence of spore ( spore location ) TABLE 4 - 2 2 . Cultural properties 1 Urease activity Culture conditions Catalase + Nutrient agar medium 30° C . Color Pale yellow color Oxidase +
+ 25 Gloss Growth range Pigment production pH + Culture conditions Nutrient broth medium 30° C . Growth range + Presence or absence of surface growth Temperature + Presence or absence of turbidity in medium ( ° C . ) 1 30 Culture conditions Gelatin stab culture 30° C . Anaerobic growing property + Growth state O•F Test (oxidation / fermentation ) + / Gelatin liquefaction 4 . Acid production gas production from carbohydrates Culture conditions Litmus milk 30° C . Solidification Liquefaction L - Arabinose D -Xylose 3 . Physiological properties D - Glucose D -Mannose + 35 D -Galactose + D - Fructose +III Gram stain Maltose Saccharose I Nitrate salt reduction + Lactose Trehalose i Denitrification I D - Sorbitol D -Mannitol I MR test Inositol Glycerin + VP test + 5 . Other physiological properties 40 Indole production B -Galactosidase activity Hydrogen sulfide production Arginine dehydrolase activity Hydrolysis of starch Lysine decarboxylase activity Use of citric acid (Koser ) + Tryptophan deaminase activity (Christensen ) + Gelatinase activity Use of inorganic Nitrate salt + 45 nitrogen source Ammonium salt + According to a common method , the partial nucleotide sequence of 16S rDNA of the D25 strain (SEQ ID NO . 4 ) was determined . TABLE 5 - 2
The partial nucleotide sequence of 16S rDNA of the D25 50 Urease activity T strain exhibited high homology to the nucleotide sequence Catalase + of 16S rDNA of Pseudomonas , and it exhibited the highest Oxidase + identity of 99 . 0 % to the Pseudomonas putida ATCC12633 Growth range + strain , Pseudomonas fuscovaginae MAFF 301177 strain , pH +
Pseudomonas asplenii ATCC 23835 strain and Pseudomo - 55 Growth range + nas agarici LMG 2112 strain . Temperature + As a result of a brief molecular phylogenetic analysis ( C . ) based on the partial nucleotide sequence of 16S rDNA , D25 AWN Anaerobic growing property + strain is included in a cluster formed of the species of O•F Test (oxidation / fermentation ) +X Pseudomonas. Further, D25 strain showed , in the cluster of 4 . Acid production gas production from carbohydrates Pseudomonas , an independent molecular phylogenetic posi- 60 tion unlike any other known species . L - Arabinose + D - Xylose D25 strain reduces a nitrate salt , exhibits an arginine D - Glucose + D -Mannose hydrolase activity but no urease activity , does not hydrolyze D - Fructose ¡ D - Galactose Maltose ¡ Saccharose +TL gelatin but assimilates glucose , L - arabinose and D -mannitol , Lactose Ï Trehalose or the like . Further , it does not assimilate D -mannose or 65 D -Sorbitol Ï D -Mannitol maltose, does not produce a fluorescent pigment in Kings ' B Inositol Ï Glycerin agar medium , showsmotility , and shows a positive response US 10 ,294 ,500 B2 81 82 TABLE 5 - 2 - continued TABLE 6 - 1 - continued 5 . Other physiological properties Culture conditions Litmus milk 30° C . Solidification B -Galactosidase activity Liquefaction Arginine dehydrolase activity 5 3 . Physiological properties T Lysine decarboxylase activity Tryptophan deaminase activity Gram stain L Gelatinase activity Nitrate salt reduction + Denitrification L MR test According to a common method , the partial nucleotide 10 VP test + Indole production sequence of 16S rDNA of the D26 strain (SEQ ID NO . 5 ) Hydrogen sulfide production L was determined . Hydrolysis of starch The partial nucleotide sequence of 16S rDNA of the D26 Use of citric acid (Koser ) + strain exhibited high homology to the nucleotide sequence ( Christensen ) + 16 . Use of inorganic Nitrate salt + of 16S rDNA of Pseudomonas , and it exhibited the highest 15 nitrogen source Ammonium salt + identitf8. 6 ttheudomonas putid 12633 strain , Pseudomonas fuscovaginae MAFF 301177 strain and Pseudomonas asplenii ATCC 23835 strain . As a result of a brief molecular phylogenetic analysis TABLE 6 - 2 based on the partial nucleotide sequence of 16S rDNA , D2620 Urease activity strain is included in a cluster formed of the species of Catalase + Oxidase + Pseudomonas. Further, D25 strain forms a cluster with Growth range + Pseudomonas putida , Pseudomonas fuscovaginae , pH + Pseudomonas asplenii and Pseudomonas agarici, and Growth range + among them , it showed the same molecular phylogenetic Temperature position as the three species , that is , Pseudomonas putida , (°C . ) + W Pseudomonas fuscovaginae and Pseudomonas asplenii .
D26 strain reduces a nitrate salt , does not exhibit an Anaerobic growing property *+ arginine hydrolase activity and urease activity , does not O•F Test (oxidation / fermentation ) hydrolyze gelatin but assimilates glucose, L - arabinose and 4 . Acid production / gas production from carbohydrates D -mannose , or the like . Further, it does not assimilate L - Arabinose + D - Xylose maltose and adipic acid , produces a fluorescent pigment in D -Glucose + D -Mannose Kings ' B agar medium , shows motility, shows a positive D - Fructose - / D -Galactose response to both the catalase reaction and oxidase reaction , as Maltose - - Saccharose p?i?i Lactose - - Trehalose and does not hydrolyze starch . Although those properties D - Sorbitol - / D -Mannitol have a similarity to those of Pseudomonas putida , Inositol - / Glycerin + w / Pseudomonas fuscovaginae and Pseudomonas asplenii for 5 . Other physiological properties which the homologous property has been demonstrated , B - Galactosidase activity differences are also confirmed . In particular , reducing a 10 Arginine dehydrolase activity nitrate salt but not exhibiting the arginine hydrolase activity Lysine decarboxylase activity TL+ is different from those species . Tryptophan deaminase activity As such , D26 strain was identified as Pseudomonas sp . Gelatinase activity On Nov . 7 , 2012 , D26 strain was subjected to international deposition under accession number of NITE BP - 1455 . 45 According to a common method , the partial nucleotide (6 ) Mycological Properties of Pseudomonas sp . D29 sequence of 16S rDNA of the D29 strain (SEQ ID NO . 6 ) was determined . TABLE 6 - 1 The partial nucleotide sequence of 16S rDNA of the D29 1. Morphological properties strain exhibited high homology to the nucleotide sequence - 50 of 16S rDNA of Pseudomonas, and it exhibited the highest Culture conditions Nutrient agar medium 30° C . identity of 99 .6 % to the Pseudomonas vancouverensis DhA Shape of cell Bacillus ( 0 . 7 to 0 . 8 x 1 . 5 to 2 . 0 um ) 51 strain . Presence or absence of cell pleomorphism As a result of a brief molecular phylogenetic analysis Motility ( attachment state of flagella ) + (Ciliate ) based on the partial nucleotide sequence of 16S rDNA , D29 Presence or absence of spore (spore 55 strain forms a cluster with Pseudomonas umsongensis , location ) Pseudomonas mohnii, Pseudomonas graminis , and among 2 . Cultural properties them , it showed the samemolecular phylogenetic position as Culture conditions Nutrient agar medium 30° C . Pseudomonas umsongensis and Pseudomonas mohnii. Color Pale yellow color D29 strain reduces a nitrate salt, exhibits an arginine Gloss + Pigment production 60 hydrolase activity but no urease activity , does not hydrolyze Culture conditions Nutrient broth medium 30° C . gelatin but assimilates glucose, L - arabinose and D -mannose , Presence or absence of surface growth or the like. Further , it does not assimilate N - acetyl- D Presence or absence of turbidity in + glucosamine , maltose and adipic acid , does not produce a medium Culture conditions Gelatin stab culture 30° C . fluorescent pigment in Kings ' B agar medium , shows motil Growth state + 65 ity , and shows a positive response to both the catalase Gelatin liquefaction | reaction and oxidase reaction . Although those properties have a similarity to those of Pseudomonas umsongensis and US 10 , 294 ,500 B2 83 84 Pseudomonas mohnii for which the homologous property TABLE 7 - 2 - continued has been demonstrated , differences are also confirmed . In particular, not exhibiting the arginine hydrolase activity is 5 . Other physiological properties different from those two species while reducing a nitrate salt B -Galactosidase activity L is different from the properties of Pseudomonas mohnii 5 Arginine dehydrolase activity + Lysine decarboxylase activity + As such , D29 strain was identified as Pseudomonas sp . Tryptophan deaminase activity On Nov . 7, 2012 , D29 strain was subjected to international Gelatinase activity | deposition under accession number of NITE BP - 1456 . ( 7 ) Mycological Properties of Pseudomonas sp . D41- 2 10 According to a common method , the partial nucleotide sequence of 16S rDNA of the D41- 2 strain (SEQ ID NO . 7 ) TABLE 7 - 1 was determined . 1. Morphological properties The partial nucleotide sequence of 16S rDNA of the Culture conditions Nutrient agar medium 30° C . 15 D41- 2 strain exhibited high homology to the nucleotide Shape of cell Bacillus ( 0 . 7 to 0 . 8 x sequence of 16S rDNA of Pseudomonas , and it exhibited the 1. 2 to 1. 5 um ) highest identity of 99 . 8 % to the Pseudomonas vancouveren Presence or absence of cell pleomorphism sis DhA -51 strain . Motility (attachment state of flagella ) + ( Ciliate ) Presence or absence of spore (spore As a result of a brief molecular phylogenetic analysis location ) 20 based on the partial nucleotide sequence of 16S rDNA, 2 . Cultural properties D41 - 2 is included in a cluster formed of the species Culture conditions Nutrient agar medium 30° C . Pseudomonas . Further , D41 - 2 strain forms a cluster with Color Pale yellow color Pseudomonas vancouverensis and Pseudomonas moorei, Gloss + Pigment production and it showed the samemolecular phylogenetic position as Culture conditions Nutrient broth medium 30° C . 25 those two species . Presence or absence of surface growth D41 - 2 strain reduces a nitrate salt , exhibits an arginine Presence or absence of turbidity in hydrolase activity , does nothydrolyze gelatin but assimilates medium Culture conditions Gelatin stab culture 30° C . glucose , L - arabinose and D -mannose , or the like. Further, it Growth state + does not assimilate maltose and adipic acid , produces a Gelatin liquefaction 30 fluorescent pigment in Kings ' B agar medium , shows motil Culture conditions Litmus milk 30° C . ity, and shows a positive response to both the catalase Solidification Liquefaction reaction and oxidase reaction . Although those properties 3 . Physiological properties have a similarity to those of Pseudomonas vancouverensis 35 and Pseudomonas moorei for which the homology has been Gram stain L demonstrated , differences are also confirmed . In particular, Nitrate salt reduction + producing a fluorescent pigment and reducing a nitrate salt Denitrification are different from the properties of Pseudomonas moorei MR test L while exhibiting the arginine hydrolase activity is different VP test + Indole production from the properties of Pseudomonas vancouverensis .
Hydrogen sulfide production L As such , D41- 2 strain was identified as Pseudomonas sp . Hydrolysis of starch T On Nov . 7 , 2012 , D41 - 2 strain was subjected to international Use of citric acid (Koser ) deposition under accession number of NITE BP - 1457 . (Christensen ) + Use of inorganic Nitrate salt + ( 8 ) Mycological Properties of Bacillus thuringiensis G1 nitrogen source Ammonium salt + TABLE 8 - 1 1. Morphological properties TABLE 7 - 2 Culture conditions Nutrient agar medium 30° C . Urease activity 50 Shape of cell Bacillus ( 1 . 0 x 1 . 5 to 2 . 5 um ) Catalase Presence or absence of cell pleomorphism Oxidase |+ Motility attachment state of fagella ) + ( Peritrichous flagella ) Growth range Presence or absence of spore ( spore + ( Center to closed to end ) pH location ) 2 . Cultural properties Growth range 55 Temperature + Culture conditions Nutrient agar medium 30° C . ( ° C . ) Color Cream color Gloss Anaerobic growing property + Pigment production O•F Test (oxidation / fermentation ) + / Culture conditions Nutrient broth medium 30° C . 4 . Acid production / gas production from carbohydrates Presence or absence of surface growth 60 Presence or absence of turbidity in L - Arabinose + - D - Xylose medium D -Glucose D -Mannose Culture conditions Gelatin stab culture 30° C . D - Fructose D - Galactose Growth state + Maltose Saccharose Gelatin liquefaction Lactose Trehalose Culture conditions Litmus milk 30° C . D - Sorbitol D -Mannitol F?ítt 65 Solidification Inositol Glycerin + w / Liquefaction US 10 , 294 ,500 B2 85 86 TABLE 8 - 1 - continued As such , G1 strain was identified as Bacillus thuringien sis. On Nov. 7 , 2012, G1 strain was subjected to interna 3 . Physiological properties tional deposition under accession number of NITE BP - 1458 . Gram stain + Nitrate salt reduction (9 ) Mycological Properties of Bacillus thuringiensis G2 Denitrification + MR test + TABLE 9 - 1 VP test + Indole production 1 . Morphological properties Hydrogen sulfide production L Hydrolysis of starch + 10 Culture conditions Nutrient agar medium 30° C . Use of citric acid (Koser ) L Shape of cell Bacillus ( 1 . 0 x 1 . 5 to 2 . 5 um ) (Christensen ) + Presence or absence of cell pleomorphism Use of inorganic Nitrate salt Motility ( attachment state of flagella ) + (Peritrichous flagella ) nitrogen source Ammonium salt TL Presence or absence of spre spor + ( Center to close to end ) location ) 2 . Cultural properties 15 Culture conditions Nutrient agar medium 30° C . TABLE 8 - 2 Color Cream color Gloss Urease activity Pigment production Catalase + Culture conditions Nutrient broth medium 30° C . Oxidase + 20 Presence or absence of surface growth Growth range + Presence or absence of turbidity in pH + medium + Culture conditions Gelatin stab culture 30° C . Growth range + Growth state + Temperature + Gelatin liquefaction + ( ° C . ) + 25 Culture conditions Litmus milk 30° C .
Anaerobic growing property + Solidification O•F Test (oxidation / fermentation )?Tour Liquefaction 4 . Acid production gas production from carbohydrates 3 . Physiological properties
L - Arabinose - - D -Xylose Gram stain + D -Glucose +/ D -Mannose 30 Nitrate salt reduction D - Fructose +/ D -Galactose Denitrification + Maltose + / Saccharose MR test + Lactose - - Trehalose IIII+ VP test + D - Sorbitol D -Mannitol Indole production L Inositol Glycerin Hydrogen sulfide production 5. Other physiological properties 35 Hydrolysis of starch + Use of citric acid (Koser ) L B - Galactosidase activity | ( Christensen ) + Arginine dehydrolase activity + Lysine decarboxylase activity Use of inorganic Nitrate salt L Tryptophan deaminase activity nitrogen source Ammonium salt | Gelatinase activity + 40 According to a common method , the partial nucleotide TABLE 9 - 2 sequence of 16S rDNA of the G1 strain (SEQ ID NO . 9 ) was Urease activity | determined Catalase + The partial nucleotide sequence of 16S rDNA of the G1 45 Oxidase + strain exhibited high homology to the nucleotide sequence Growth range + of 16S rDNA of Bacillus, and it exhibited the highest pH + + identity of 99. 8 % to the Bacillus thuringiensis ATCC 10792 Growth range + strain . Temperature + As a result of a brief molecular phylogenetic analysis 50 (°C . ) + based on the partial nucleotide sequence of 16S rDNA, G1 Anaerobic growing property + strain is included in a cluster formed of the species of O•F Test (oxidation / fermentation ) Bacillus . Further , G1 strain forms a cluster with Bacillus 4 . Acid production gas production from carbohydrates thuringiensis and both exhibits the same molecular phylo genetic position . 55 L - Arabinose - / D -Xylose G1 strain ferments glycerol, ribose, and glucose , does not D -Glucose + D -Mannose D - Fructose T+III D - Galactose ferment D - xylose , L -Xylose , and galactose , does not exhibit Maltose Saccharose III+ the B - galactosidase activity or urease activity, uses citric Lactose Trehalose acid , does not produce indole , produces acetoin , does not D - Sorbitol D -Mannitol reduce a nitrate salt , does not grow at 45° C . , grows under 60 Inositol Glycerin + anaerobic conditions, and hydrolyzes starch . Although those 5 . Other physiological properties properties are almost in match with those of Bacillus B -Galactosidase activity Arginine dehydrolase activity + thuringiensis for which the homology has been demon Lysine decarboxylase activity strated , not reducing a nitrate salt is different from the Tryptophan deaminase activity L typical properties of Bacillus thuringiensis . With regard to 65 Gelatinase activity + the physiological biochemical differences, the possibility of having a difference at strain level cannot be ruled out. US 10 , 294 ,500 B2 87 88 According to a common method , the partial nucleotide TABLE 10 - 2 sequence of 16S rDNA of the G2 strain (SEQ ID NO . 10 ) Urease activity was determined . Catalase + The partial nucleotide sequence of 16S rDNA of the G2 Oxidase strain exhibited high homology to the nucleotide sequence 5 Growth range of 16S rDNA of Bacillus, and it exhibited the highest ?? + + identity of 99 . 2 % to the Bacillus thuringiensis ATCC 17092 Growth range + Tocour + strain . Temperature As a result of a brief molecular phylogenetic analysis ( ° C . ) + L based on the partial nucleotide sequence of 16S rDNA , G2 Anaerobic growing property T strain is included in a cluster formed of the species of O•F Test (oxidation / fermentation ) Bacillus. Further, G2 strain forms a cluster with Bacillus 4 . Acid production gas production from carbohydrates thuringiensis and both exhibits the same molecular phylo L - Arabinose - / D - Xylose + / genetic position . 15 D - Glucose ++ / D -Mannose - / G2 strain ferments glycerol, ribose and glucose , does not D - Fructose + / D - Galactose - - Maltose + Saccharose +
+ ferment D - xylose , L -xylose , and galactose , does not exhibit Lactose L Trehalose the B - galactosidase activity or urease activity , uses citric D - Sorbitol L D -Mannitol + acid , does not produce indole , produces acetoin , does not Inositol | Glycerin ++ reduce a nitrate salt , does not grow at 45° C ., grows under 20 5 . Other physiological properties anaerobic conditions, and hydrolyzes starch . Although those B -Galactosidase activity properties are almost in match with those of Bacillus LysineArginine decarboxylase dehydrolase activity thuringiensis for which the homology has been demon Tryptophan deaminase activity strated , not reducing a nitrate salt is different from the Gelatinase activity typical properties of Bacillus thuringiensis . With regard to 25 the physiological biochemical differences, the possibility of having a difference at strain level cannot be ruled out. According to a common method , the partial nucleotide As such , G2 strain was identified as Bacillus thuringien sequence of 16S rDNA of the R1 strain (SEQ ID NO . 11 ) sis . On Nov . 7 , 2012 , G2 strain was subjected to interna was determined . tional deposition under accession number of NITE BP - 1459030. 30 The partial nucleotide sequence of 16S rDNA of the R1 ( 10 ) Mycological Properties of Bacillus sp . R1 strain exhibited high homology to the nucleotide sequence of 16S rDNA of Bacillus, and it exhibited the highest identity of 98 . 9 % to the Bacillus oceanisediminis H2 strain . TABLE 10 - 1 As a result of a brief molecular phylogenetic analysis 1. Morphological properties 35 based on the partial nucleotide sequence of 16S rDNA , R1 strain is included in a cluster formed of the species of Culture conditions Nutrient agar medium 30° C . Shape of cell Bacillus ( 0 . 7 to 0 . 8 x Bacillus. Further, R1 strain forms a cluster with Bacillus 1 . 5 to 2 . 5 um ) oceanisediminis and they are found to be homologous to Presence or absence of cell pleomorphism each other. Motility (attachment state of flagella ) + ( Peritrichous flagella ) 100 R1 strain oxidizes glycerol, ribose and D -xylose , does not Presence or absence of spore ( spore + (Center to close to end ) 40 location ) oxidize D -arabinose and L -Xylose , does not exhibit the 2 . Cultural properties arginine hydrolase activity , hydrolyzes gelatin , reduces a nitrate salt, forms a spore , does not grow at 45° C . , and does Culture conditions Nutrient agar medium 30° C . not hydrolyze starch . Although those properties are similar Color Pale yellow color Gloss 45 to those of Bacillus oceanisediminis for which the homology Pigment production has been demonstrated , a difference is also recognized . In Culture conditions Nutrient broth medium 30° C . particular, not exhibiting the arginine hydrolase activity , not Presence or absence of surface growth growing at 45° C . , and not hydrolyzing starch are different Presence or absence of turbidity in medium from the properties of Bacillus oceanisediminis . Culture conditions Gelatin stab culture 30° C . 50 As such , R1 strain was identified as Bacillus sp . On Nov . Growth state + 7 , 2012 , R1 strain was subjected to international deposition Gelatin liquefaction Culture conditions Litmus milk 30° C . under accession number of NITE BP - 1460 . idification (11 ) Mycological Properties of Sphingobacterium sp . B13 Liquefaction 3 . Physiological properties 55 TABLE 11 - 1 Gram stain 1 . Morphological properties Nitrate salt reduction Denitrification Culture conditions Nutrient agar medium 30° C . MR test Shape of cell Bacillus (0 . 6 to 0 .77 x VP test +IIIIIIIIII 1 . 2 to 1 . 5 um ) Indole production 60 Presence or absence of cell pleomorphism Hydrogen sulfide production Motility ( attachment state of flagella ) Hydrolysis of starch Presence or absence of spore (spore Use of citric acid (Koser ) location ) ( Christensen ) 2 . Cultural properties Use of inorganic Nitrate salt nitrogen source Ammonium salt 65 Culture conditions Nutrient agar medium 30° C . Color Yellow color US 10 , 294 ,500 B2 89 90 TABLE 11 - 1 - continued As such , B13 strain was identified as Sphingobacterium sp . On Nov . 7 , 2012 , R1 strain was subjected to international Gloss + deposition under accession number of NITE BP - 1461. Pigment production + Culture conditions Nutrient broth medium 30° C . Presence or absence of surface growth T 5 Example 2 : Production of Methacrylic Acid Using Presence or absence of turbidity in + Synthetic Medium medium Culture conditions Gelatin stab culture 30° C . Growth state + Using the isolated strains obtained from Example 1 , Gelatin liquefaction production ofmethacrylic acid in a synthetic medium was Culture conditions Litmus milk 30° C . 10 performed . Solidification Liquefaction +1 D22 - 1 strain , D41 - 2 strain or D43 - 1 strain was grown on 3 . Physiological properties an agar medium (LB medium , 1 . 5 % agar ) and one platinum loop of the cells was inoculated on 10 ml liquid medium . Gram stain The composition of the medium is shown below . Under Nitrate salt reduction 15 aerobic conditions at 30° C . with a rotary shaker (230 rpm ) , Denitrification MR test culture was performed for 19 to 22 hours (pre -culture ) . VP test K HPO , 1 . 0 g / l Indole production MgSO4. 7H2O 0 . 2 g / l Hydrogen sulfide production FeSO4. 7H2O 0 .01 g /1 Hydrolysis of starch CaCl2 0 .01 g / 1 Use of citric acid (Koser ) IIII+IIIII 20 ( Christensen ) L - Valine 5 . 0 g /1 Use of inorganic Nitrate salt pH 7 . 0 nitrogen source Ammonium salt 2 ml of the pre - culture was inoculated to 100 mL liquid medium ( 100 mlmedium / 500 ml volume conical flask ) . The composition of the medium is the same as that of the pre - culture medium . Under aerobic conditions at 30° C . with TABLE 11 - 2 a rotary shaker ( 230 rpm ) , culture was performed for 44 to 72 hours (main culture ) . Urease activity + After the main culture , the main culture was subjected to Catalase + Oxidase + centrifugal separation (12000 rpm , 10 min ) and the super Growth range 30 natant fraction was fractionated . Compounds contained the pH + culture supernatant were analyzed by HPLC . Conditions for + HPLC analysis are as follows. Growth range + Apparatus : e2695 (manufactured by Waters ) Temperature Tocour + Column : Ion exclusion type polymer column T - 132 - E for (°C .) + 35 analysis of organic acids (manufactured by WAKO ) Anaerobic growing property ? + Eluent : 0 . 1 % ( v / v ) phosphoric acid solution O•F Test (oxidation / fermentation ) Flow rate : 0 .5 ml/min 4 . Acid production gas production from carbohydrates Column temperature : 40° C .
L - Arabinose - - D - Xylose | Sample injection amount: 10 ul D -Glucose + D -Mannose + / - 40 Time for analysis : 40 min D - Fructose I D -Galactose ++ / Detector: UV Maltose + Saccharose L The sample for analysis was suitably diluted with the Lactose + Trehalose L eluent, filtered with a DISMIC - 13CP Cellulose Acetate 0 . 2 D - Sorbitol I D -Mannitol L um filter (manufactured by ADVANTEC ) , and used . As a I Glycerin + Inositol 5. Other physiological properties result of the analysis , a peak was observed at the same 45 elution time (31 . 15 min ) as methacrylic acid . In “ Table 12” , B -Galactosidase activity the concentration of methacrylic acid which is in the super Arginine dehydrolase activity natant of each culture at the time of terminating the main Lysine decarboxylase activity +III Tryptophan deaminase activity culture is shown . Gelatinase activity 50 TABLE 12 According to a common method , the partial nucleotide Microbial Time for pre- Time for main Concentration of sequence of 16S rDNA of the B13 strain (SEQ ID NO . 16 ) strain culture ( hr ) culture ( hr ) methacrylic acid (ppm ) was determined . D22 - 1 22 12 0 . 05 The partial nucleotide sequence of 16S rDNA of the B132 55 D41- 2 44 0 . 06 strain exhibited high homology to the nucleotide sequence D43 - 1 0 .06 of 16S rDNA of Sphingobacterium , and it exhibited the highest identity of 97 . 4 % to the Sphingobacterium siyan gense SY1 strain . GC /MS analysis was performed for the above peak . B13 strain does not reduce a nitrate salt , does not ferment Conditions for the GC /MS analysis are as follows. glucose . exhibits the urease and B - galactosidase activity . 60 Apparatus: 6890 / 5875A (manufactured by Agilent Tech hydrolyzes exculin and gelatin , assimilates glucose , D -man an - nologies )) Column: DB - FFAP column (manufactured by nose and maltose , does not assimilate L -arabinose and J & W ) D -mannitol , does not exhibit motility , exhibits the catalase Carrier : He activity , and does not produce indole . Although those prop - Oven temperature : after 50° C . ( 1 min ) , temperature was erties are believed to be in match with the properties of 65 raised to 150° C . at 5° C ./ min , and again raised to 250° C . Sphingobacterium , no known species having the properties at 20° C . /min . that are in match with those of B13 strain was found . Inlet temperature : 200° C . US 10 , 294 ,500 B2 91 92 Column flow rate : 1 ml/ min ( constant flow mode ) (230 rpm ), culture was performed for 69 to 76 hours (main Split ratio : 1 /50 culture ) . Sample injection amount: 1 ul After the main culture, 40 mL of the main broth was Detector : MSD (EI ) transferred to a conical tube with volume of 50 mL and The obtained total ion chromatograph is shown in FIG .25 separated by centrifuge (12000 rpm , 10 min ) to obtain the and the mass spectrum of the observed peak is shown in FIG . cells . To the cells , 10 mL solution for resting cell reaction 3 . As a result of the analysis , it was able to confirm that the was added to perform the resting cell reaction . The compo peak observed at the same retention time ( 17 . 15 min ) as sition of the solution for resting cell reaction includes 5 . 0 g /l methacrylic acid corresponds to methacrylic acid . L -valine , 50 mM phosphate buffer, and pH 7 . 0 . The reaction 10 was performed for 24 hours under aerobic conditions at 30° Example 3 : Production of Methacrylic Acid Using C . with a rotary shaker (230 rpm ) . Natural Medium According to the method shown in Example 2 , the com Using the isolated strains obtained from Example 1 , pounds contained the solution for resting cell reaction were production of methacrylic acid in a natural medium was 1615 analyzedand . As a result , a peak was observed at the same performed elution time (31 . 15 min ) as methacrylic acid . In “ Table 13 ” , G2 strain was grown on an agar medium (LB medium , the concentration of methacrylic acid which is in each 1 .5 % agar ) and one platinum loop of the cells was inoculated reaction solution at the time of terminating the resting cell on 10 ml liquid medium . The composition of the medium is reaction is shown . shown below . Under aerobic conditions at 37° C . with a 20 rotary shaker TABLE 13 (230 rpm ), culture was performed for 24 hours (pre - culture ) . Microbial Resting cell Concentration of Tryptone (manufactured by Becton , Dickinson and Com strain reaction OD methacrylic acid (ppm ) pany ) 10 g/ 1 B25 - 2 0 . 2 Yeast extract (manufactured by Becton , Dickinson and 25 D22 - 1 0 .??= 3 Company ) 5 g / 1 D43- 1 NaCl 10 g /l D25 1 . 0 pH 7 .0 D26 0 . 8 D29 0 . 2 1 ml of the pre - culture was inoculated to 100 mL liquid D41 - 2 0 . 7 medium ( 100 mlmedium /500 ml volume conical flask ). The 30 G1 10 composition of the medium is the same as that of the G2 10 pre -culture medium . Under aerobic conditions at 37° C . with R1 12 a rotary shaker (230 rpm ) , culture was performed for 24 B13 10 hours (main culture ). After the main culture, the main culture was subjected to 35 centrifugal separation ( 12000 rpm , 10 min ) and the super Example 5 : Production of Methacrylic Acid in natant fraction was fractionated . Compounds contained the Synthetic Medium Using Genus Pseudomonas culture supernatant were analyzed by the method described in Example 2 . As a result , a peak was observed at the same Pseudomonas putida NBRC12996 was grown on an agar elution time (31 . 15 min ) as methacrylic acid . The concen - 40 medium (LB medium , 1 . 5 % agar ) and one platinum loop of tration of methacrylic acid in the culture was 1 . 0 ppm . the cells was inoculated on 10 ml liquid medium . Under aerobic conditions at 30° C . with a rotary shaker (230 rpm ) , Example 4 : Production of Methacrylic Acid Based culture was performed for 1 to 4 days ( pre - culture ). The on Resting Cell Reaction composition of the medium for the pre -culture is described 45 below . Using the isolated strains obtained from Example 1 , Na2HPO4 (anhydrous ) 6 .0 g /l production of methacrylic acid was performed based on KH2PO4 3 . 0 g /1 resting cell reaction . NaCl 0 . 5 g /1 B25 -2 strain , D22 - 1 strain , D43 - 1 strain , D25 strain , D26 NH _ C1 1 .0 g /1 strain , D29 strain , D41 - 2 strain , G1 strain , G2 strain , R1 50 1M MgSO4.7H , O 1 ml strain , and B13 strain were grown on an agar medium (LB 1M CaCl, 0 . 1 ml medium , 1 . 5 % agar ) and one platinum loop of the cells was 1 % Thiamine 1 ml inoculated on 10 ml liquid medium . The composition of the Glucose 2 . 0 g / 1 medium is shown below . Under aerobic conditions at 30° C . 1 ml of the pre - culture was inoculated to 100 mL liquid with a rotary shaker ( 230 rpm ) , culture was performed for 19 55 medium ( 100 ml medium / 500 ml volume conical flask ) . to 22 hours (pre -culture ). Under aerobic conditions at 30° C . with a rotary shaker (230 K HPO . 1 . 0 g / l rpm ), culture was performed for 2 days (main culture ). The MgSO4. 7H2O 0 . 2 g /l composition of the medium for themain culture is described FeSO4. 7H2O 0 .01 g / l below . CaC1, 0 .01 g / 1 60 Na2HPO4 ( anhydrous ) 6 . 0 g /1 L - Valine 5 . 0 g /l KH2PO4 3 . 0 g /1 pH 7 . 0 NaCl 0 .5 g /1 2 ml of the pre -culture was inoculated to 100 mL liquid NH4Cl 1 . 0 g /l medium ( 100 mlmedium / 500 ml volume conical flask ) . The 1M MgSO4. 7H20 1 ml composition of the medium is the same as that of the 65 1M CaCl, 0 . 1 ml pre -culture medium . Under aerobic conditions at 30° C . with 1 % Thiamine 1 ml a rotary shaker L - Valine 2 . 0 g /1 US 10 , 294 , 500 B2 93 94 After the main culture , the main broth was separated by Under aerobic conditions at 30° C . with a rotary shaker ( 230 centrifuge ( 7500 rpm , 15 min ) and the supernatant fraction rpm ) , culture was performed for 2 days (main culture ) . The was subjected to a filtration treatment which uses a DIS composition of the medium for the main culture is described MIC - 13CP Cellulose Acetate 0 . 2 um filter (manufactured by below . ADVANTEC ). The compounds contained in the culture 5 Na HPO4 (anhydrous ) 6 . 0 g /1 supernatant were analyzed according to two kinds of analy KH ,POA 3 . 0 g /l sis conditions. Conditions for HPLC analysis are as follows. NaCl 0 . 5 g / 1 Analysis condition A NH C1 1 . 0 g / 1 Apparatus: e2695 (manufactured by Waters ) Column: Ion exclusion type polymer column T - 132 - E for 10 1M MgSO4. 7H20 1 ml analysis of organic acids (manufactured by WAKO ) 1M CaCl, 0 . 1 ml Eluent: 0 . 1 % ( v / v ) phosphoric acid solution 1 % Thiamine 1 ml Flow rate : 0 .5 ml/min L - Valine 2 . 0 g /1 Column temperature : 40° C . Glucose 2 .0 g / 1 Sample injection amount : 10 ul 15 After the main culture , the main broth was separated by Time for analysis : 40 min centrifuge ( 7500 rpm , 15 min ) and the supernatant fraction Detector: UV was subjected to a filtration treatment which uses a DIS Analysis Condition B MIC - 13CP Cellulose Acetate 0 . 2 um filter (manufactured by Apparatus : JASCO UV - 970 , CO - 960, PU - 980 , DG - 1580 ADVANTEC ) . The compounds contained in the culture 54 (manufactured by JASCO , Japan ) 20 supernatant were analyzed according to the analysis condi Column : Inertsil RODS- 3V (manufactured by GL Sci- tion A described in Example 5 . As a result , a peak was ences ) observed at the same elution time as methacrylic acid . In Eluent: 20 % (v / v ) methanol, 0 .2 % ( v/ v ) phosphoric acid “ Table 14 ” , the concentration ofmethacrylic acid which is in solution each culture supernatant at the time of terminating the main Flow rate : 1 . 0 ml/min 25 culture is shown . Column temperature : 40° C . Sample injection amount : 5 ul TABLE 14 Time for analysis : 15 min Number of Number of Number of Detector: UV days for days for days for Concentration As a result of the analysis , a peak was observed at the 30 plate pre - main of same elution time as methacrylic acid for both the analysis culture culture culture methacrylic condition A and the analysis condition B . The concentration Microbial strain (days ) (days ) (days ) acid (ppm ) of methacrylic acid in the supernatant of the culture was 0 . 2 Brevundimonas 3 3 2 0 . 1 ppm at the time of terminating the main culture . subvibrioides 33 NBRC16000 Example 6 : Production of Methacrylic Acid in Ochrobactrum 1 . 0 grignonense 3 3 2 1. 0 Synthetic Medium Using Genus Brevundimonas , NBRC102586 Genus Ochrobactrum , and Genus Paracoccus Ochrobactrum ??? 0 . 2 lupini Brevundimonas subvibrioides NBRC 16000 , Ochrobac - 40 NBRC102587 trum grignonense NBRC102586 , Ochrobactrum lupini Paracoccus ? 0 . 3 NBRC102587 , and Paracoccus aminophilus NBRC 16710 aminophilus were grown on an agar medium (plate culture ) . The com NBRC16710 position of the medium for plate culture is shown below . Polypeptone (manufactured by Becton , Dickinson and 45 Company ) 10 g Example 7 : Production of Methacrylic Acid in Yeast extract (manufactured by Becton , Dickinson and Synthetic Medium Using Genus Sphingomonas, Company ) 2 g Genus Ochrobactrum , Genus Paenibacillus, and MgSO4 .7H201 g Genus Mesorhizobium Distilled water 11 50 Ager 15 g Sphingomonas paucimobilis NBRC13935 , Ochrobactrum pH 7 . 0 intermedium NBRC15820 , Paenibacillus sp . NBRC13157 , Cells grown by plate culture were inoculated in 10 ml Mesorhizobium loti ATCC700743 were grown on a nutrient liquid medium ( one platinum loop ) and cultured under agar medium (manufactured by Becton , Dickinson and aerobic conditions at 30° C . with a rotary shaker ( 230 rpm ) 55 Company , agar 1 . 5 % ). The grown cells were inoculated ( one for 3 to 4 days (pre -culture ). The composition of the medium platinum loop ) to 10 ml liquid medium and cultured under for the pre - culture is described below . aerobic conditions at 30° C . with a rotary shaker (230 rpm ) Na2HPO4 (anhydrous ) 6 .0 g /1 for 1 to 4 days ( pre - culture ) . The composition of themedium KH ,POA 3 . 0 g / l for the pre - culture is described below . NaCl 0 .5 g /l 60 Na HPO4 (anhydrous ) 6 . 0 g /1 NH C1 1 .0 g / l KH2PO4 3 . 0 g /1 IM MgSO4. 7H2O 1 ml NaCl 0 . 5 g /1 1M CaCl2 0 . 1 ml NH4Cl 1 . 0 g /l 1 % Thiamine 1 ml IM MgSO4. 7H2O 1 ml Glucose 2 . 0 g / l 65 M CaCl, 0 . 1 ml 1 ml of the pre - culture was inoculated to 100 mL liquid 1 % Thiamine 1 ml medium ( 100 ml medium / 500 ml volume conical flask ). Glucose 2 .0 g /1 US 10 ,294 ,500 B2 95 96 1 ml of the pre -culture was inoculated to 100 mL liquid L - Valine 2 . 0 g /1 medium ( 100 ml medium / 500 ml volume conical flask ). Glucose 2 .0 g/ 1 Under aerobic conditions at 30° C . with a rotary shaker (230 After the main culture , the main broth was separated by rpm ), culture was performed for 3 to 6 days (main culture ). centrifuge (7500 rpm , 15 min ) and the supernatant fraction The composition of the medium for the main culture is 5 was subjected to a filtration treatment which uses a DIS described below . MIC - 13CP Cellulose Acetate 0 . 2 um filter (manufactured by Na2HPO4 (anhydrous ) 6 . 0 g /1 ADVANTEC ) . The compounds contained in the culture KH PO4 3 . 0 g / 1 supernatant were analyzed according to the two kinds of NaCl 0 . 5 g / 1 analysis condition described in Example 5 . As a result , a NH C1 1 . 0 g / 1 peak was observed at the same elution time as methacrylic IM MgSO4 . 7H2O 1 ml acid for both the analysis condition A and the analysis 1M CaCl, 0 . 1 ml condition B . Concentration ofmethacrylic acid in the culture 1 % Thiamine 1 ml supernatant at the time of terminating the main reaction was L - Valine 2 . 0 g / l 2 .0 ppm and 2 .4 ppm for the analysis condition A and the Glucose 2 . 0 g / 1 15 analysis condition B , respectively . After the main culture , the main broth was separated by centrifuge (7500 rpm , 15 min ) and the supernatant fraction Example 9 : Production of Methacrylic Acid in was subjected to a filtration treatment which uses a DIS Natural Medium Using Genus Ochrobactrum MIC - 13CP Cellulose Acetate 0 . 2 um filter (manufactured by ADVANTEC ) . The compounds contained in the culture 20 Ochrobactrum sp . NBRC 12951 was grown on a nutrient supernatant were analyzed according to the analysis condi agar medium (manufactured by Becton , Dickinson and tion B described in Example 5 . As a result , a peak was Company , agar 1 . 5 % ) . The grown cells were inoculated (one observed at the same elution time as methacrylic acid . In platinum loop ) to 100 mL liquid medium ( 100 mlmedium / “ Table 15 ” , the concentration ofmethacrylic acid which is in 500 ml volume conical flask ) and cultured under aerobic each culture supernatant at the time of terminating the main 25 conditions at 30° C . with a rotary shaker ( 230 rpm ) for 8 culture is shown . days (main culture ) . Composition of the medium for the main culture is described below . TABLE 15 Nutrientmedium 8 g / 1 L - Valine 2 . 0 g / 1 Number of Number of Number of 30 After the main culture, the main broth was separated by days for days for days for Concentration plate pre - main of centrifuge ( 7500 rpm , 15 min ) and the supernatant fraction culture culture culture methacrylic was subjected to a filtration treatment which uses a DIS Microbial strain (days ) (days ) (days ) acid (ppm ) MC- 13CPlusAcetate02umfilter manufacturedby ADVANTEC ) . The compounds contained in the culture Sphingomonas ??? ? 0 . 2 paucimobilis supernatant were analyzed according to the analysis condi NBRC13935 tion B described in Example 5 . As a result, a peak was Ochrobactrum 3 3 3 0 . 6 observed at the same elution time as methacrylic acid . intermedium Concentration of methacrylic acid in the culture supernatant NBRC15820 Paenibacillus at the time of terminating the main reaction was 3 . 1 ppm . sp . NBRC13157 3 2 3 0. 1 40 Mesorhizobium 6430. 2 Example 10 : Production of Methacrylic Acid in loti ATCC700743 Natural Medium Using Genus Brevundimonas Brevundimonas diminuta ATCC11568 was grown on a 45 nutrient agar medium ( manufactured by Becton , Dickinson Example 8 : Production of methacrylic acid in and Company, agar 1 . 5 % ) . The grown cells were inoculated synthetic medium using genus Bacillus ( one platinum loop ) to 10 mL nutrient medium and cultured under aerobic conditions at 30° C . with a rotary shaker ( 230 Bacillus badius ATCC 14574 was grown on a nutrient rpm ) for 1 day ( pre - culture ). The pre - culture and 40 % agar medium (manufactured by Becton , Dickinson and 50 glycerol were mixed with each other at 1 : 1 and subjected to Company , agar 1 . 5 % ) . The grown cells were inoculated ( one cryopreservation at - 80° C . platinum loop ) to 10 ml liquid medium and cultured under The pre - culture which has been remained under cryo aerobic conditions at 30° C . with a rotary shaker ( 230 rpm ) preservation was thawed at room temperature and 0 . 5 mL for 1 day (pre - culture ) . was inoculated to 100 ml liquid medium ( 100 ml medium / 1 ml of the pre -culture was inoculated to 100 mL liquid 55 500 ml volume conical flask ) . It was then cultured under medium (100 ml medium / 500 ml volume conical flask ) . aerobic conditions at 30° C . with a rotary shaker (230 rpm ) Under aerobic conditions at 30° C . with a rotary shaker (230 for 2 days (main culture ) . Composition of the medium for rpm ), culture was performed for 8 days (main culture ). the main culture is described below . Composition of the medium for the main culture is described Nutrient medium 8 g / 1 below . 60 L - Valine 2 . 0 g /1 Na HPO4 (anhydrous ) 6 . 0 g/ l After the main culture , the main broth was separated by KH2PO4 3 . 0 g / 1 centrifuge ( 7500 rpm , 15 min ) and the supernatant fraction NaCl 0 .5 g / 1 was subjected to a filtration treatment which uses a DIS NH _ C1 1 . 0 g / 1 MIC - 13CP Cellulose Acetate 0 . 2 um filter (manufactured by IM MgSO4. 7H2O 1 ml 65 ADVANTEC ) . The compounds contained in the culture IM CaCl2 0 . 1 ml supernatant were analyzed according to the analysis condi 1 % Thiamine 1 ml tion B described in Example 5 . As a result, a peak was US 10 , 294 ,500 B2 97 98 observed at the same elution time as methacrylic acid and water , and 1 ml of the resultant was inoculated to 100 mL the concentration of methacrylic acid in the culture super liquid medium ( 100 ml medium /500 ml volume conical natant at the time of terminating the main culture was 37 .0 flask ) and cultured under aerobic conditions at 24° C . with ppm . a rotary shaker (230 rpm ) for 2 days (main culture ). Com 5 position of the medium for the main culture is described Example 11 : Production of Methacrylic Acid Using below . Yeast L - Valine 2 . 0 g /1 NaNO3 2. 5 g/ 1 Candida utilis NBRC 1086 was grown on a nutrient agar K2HPO4 1 .25 g / 1 medium (manufactured by Becton , Dickinson and Com - 10 MgSO4 .7H2O 0 .63 g / 1 pany , agar 1 . 5 % ) . The grown cells were inoculated ( one KC1 0 .63 g / 1 platinum loop ) to 10 mL liquid medium and cultured under FeSO4. 7H2O 0 .013 g / 1 Wheat bran 5 g /1 aerobic conditions at 24° C . with a rotary shaker ( 230 rpm ) pH 7 .0 for 1 day ( pre - culture ). Composition of the medium for the After the main culture , the main broth was separated by pre -culture is described below . 15 centrifuge ( 750rpm , 15minand the supernatant fraction Yeast extract (manufactured by Becton , Dickinson and was subjected to a filtration treatment which uses a DIS Company ) 20 g/ 1 MIC - 13CP Cellulose Acetate 0 . 2 um filter (manufactured by Polypeptone (manufactured by Becton , Dickinson and ADVANTEC ) . The compounds contained in the culture Company ) 20 g /1 supernatant were analyzed according to the analysis condi Glucose 20 g / 1 20 tion A and the condition B described in Example 5 . As a 1 ml of the pre -culture was inoculated to 100 mL liquid result , a peak was observed at the same elution time as medium ( 100 ml medium /500 ml volume conical flask ) and methacrylic acid for both the condition A and the condition cultured under aerobic conditions at 24° C . with a rotary B . In “ Table 16 ” , concentration ofmethacrylic acid which is shaker ( 230 rpm ) for 2 days (main culture ) . Composition of in the supernatant of each culture at the time of terminating the medium for the main culture is described below . 25 the main culture is shown . Glucose 2 . 0 g / l L - Valine 2 . 0 g /l TABLE 16 (NH4 ) 2SO4 6 . 0 g /l KCI 2 . 4 g /1 Concentration of methacrylic acid (ppm ) NaCl 0 . 12 g/ 1 30 Analysis Analysis H3PO4 3 . 0 g /1 Microbial strain condition A condition B MgSO4. 7H2O 2 . 4 g / l Aspergillus flavus 0 . 3 0 . 5 FeSO2. 7H , 0 0 .01 g /1 NBRC8558 ZnSO4.7H20 0 . 12 g / 1 Aspergillus oryzae 0. 1 0 . 04 MnSO20 .4 -6H2O 0 .024 g /1 35 NBRC4255 CuSO4. 5H2O 0 . 006 g / l CaCl, 0 .12 g/ 1 Vitamin Mix 30 ml Example 13 : Production of Methacrylic Acid Based Composition of Vitamin Mix is shown below . on Resting Cell Reaction Using Genus Biotin 0 . 2 g /1 40 Sphingomonas, Genus Pedobacter , Genus Bacillus, Calcium pantothenate 2 g/ 1 and Genus Listonella Folic acid 0 . 002 g /l Thiamine hydrochloride 0 . 4 g / 1 Sphingomonas paucimobilis NBRC13935 , Pedobacter Rivoflavin 0 .2 g / 1 heparinus NBRC12017 , Bacillus subtilis NBRC12210 , and Nicotinic acid 0 . 4 g / l 45 Listonella anguillarum ATCC19264 were grown on a nutri Pyridoxine hydrochloride 0 . 4 g / 1 ent agar medium (manufactured by Becton , Dickinson and Inositol 1 g / l Company, agar 1 . 5 % ) . The cells were inoculated ( one plati p - Aminobenzoic acid 0 . 2 g / l num loop ) to 10 mL liquid medium ( one platinum loop ) and After the main culture , the main broth was separated by cultured under aerobic conditions at 30° C . with a rotary centrifuge ( 7500 rpm . 15 min ) and the supernatant fraction 50 shaker ( 230 rpm ) for 1 to 3 days ( pre - culture ). Composition of the medium for the pre - culture is described below . was subjected to a filtration treatment which uses a DIS Na2HPO4 ( anhydrous ) 6 . 0 g / l MIC - 13CP Cellulose Acetate 0 . 2 um filter (manufactured by KH PO4 3 . 0 g / 1 ADVANTEC ) . The compounds contained in the culture NaCl 0 . 5 g /1 supernatant were analyzed according to the analysis condi NH _ C1 1 .0 g /1 tion A described in Example 5 . As a result , a peak was 55 1M MgSO4 .7H2O 1 ml observed at the same elution time as methacrylic acid and IM CaCl, 0 . 1 ml the concentration of methacrylic acid in the culture super 1 % Thiamine 1 ml natant at the time of terminating the main culture was 0 . 1 Glucose 2 . 0 g / l ppm . 1 ml of the pre - culture was inoculated to 100 mL liquid 60 medium ( 100 mlmedium / 500 ml volume conical flask ) and Example 12 : Production of Methacrylic Acid Using cultured under aerobic conditions at 30° C . with a rotary Mildew shaker (230 rpm ) for 3 to 6 days (main culture ) . Composi tion of the medium for the main culture is described below . Aspergillus flavus NBRC8558, Aspergillus oryzae Na2HPO4 ( anhydrous) 6 .0 g / l NBRC4255 was grown on a potato dextrose agar medium 65 KH2PO4 3. 0 g /1 (manufactured by Nissui Pharmaceutical Co . , Ltd . ) . The NaCl 0 . 5 g / 1 grown mycelia and spores were suspended in 5 ml sterilized NH4Cl 1 . 0 g / 1 US 10 , 294 ,500 B2 99 100 1M MgSO4. 7H2O 1 ml MgSO4 7 . H ,01 g 1M Caci, 0 . 1 ml Distilled water 1 1 1 % Thiamine 1 ml L - Valine 2 .0 g / 1 Ager 15 g Glucose 2 . 0 g /l 5 pH 7 . 0 After the main culture , the entire volume of the main Cells grown by plate culture were inoculated ( one plati culture was transferred to a centrifuge tube with volume of num loop ) to 10 ml liquid medium and cultured under 225 ml and then centrifuged (5000 rpm , 15 min ). By aerobic conditions at 30° C . with a rotary shaker (230 rpm ) removing the supernatant, the cells were obtained . To the for 3 to 4 days (pre - culture ). Composition of the medium for cells , 40 ml of 50 mM phosphate buffer (pH 7 .0 ) were added 10 the pre - culture is described below . and the cells were suspended and centrifuged again under Na2HPO4 ( anhydrous) 6 .0 g / l the same conditions . By removing the supernatant, the KH2PO4 3 . 0 g /1 washed cells were obtained . To the washed cells , 3 ml of 50 NaCl 0 . 5 g /1 mM phosphate buffer ( pH 7 . 0 ) were added to prepare a cell 15 suspension . To a conical tube with volume of 50 mL , 3 ml NH4Cl 1. 0 g /l of the cell suspension and 3 ml of the solution for resting cell 1M MgSO4. 7H2O 1 ml reaction were added to perform the resting cell reaction . The 1M CaCl , 0 . 1 ml reaction was allowed to occur under aerobic conditions at 30° C . with a rotary shaker (230 rpm ) for 24 hours . Com - 20 1 % Thiamine 1 ml position of the solution for resting cell reaction includes 5 . 0 20 Glucose 2 .0 g/ l g / 1 L -valine and 50 mM phosphate buffer (pH 7 . 0 ) . 1 ml of the pre - culture was inoculated to 100 mL liquid Five hours and twenty - four hours after starting the resting medium ( 100 ml medium /500 ml volume conical flask ) . cell reaction , 1 .6 ml of the reaction solution were collected . Under aerobic conditions at 30° C . with a rotary shaker ( 230 The reaction solution was separated by centrifuge ( 15000 25 rpm ) , culture was performed for 2 days (main culture ) . rpm , 5 min ) and the supernatant fraction was subjected to a Composition of themedium for the main culture is described filtration treatment which uses a DISMIC - 13CP Cellulose below . Acetate 0 . 2 um filter (manufactured by ADVANTEC ). The compounds contained in the culture supernatant were ana Na2HPO4 ( anhydrous ) 6 . 0 g /l lyzed according to the analysis condition A described in 30 KH2PO4 3 .0 g /1 Example 5 . As a result , a peak was observed at the same NaCl 0 .5 g /1 elution time as methacrylic acid . In “ Table 17” , concentra NH4Cl 1. 0 g / 1 tion of methacrylic acid which is in the supernatant of the reaction solution of the resting cell reaction is shown . 1M MgSO4. 7H2O 1 ml 35 1M CaCl , 0 . 1 ml TABLE 17 1 % Thiamine 1 ml Number of Number of Concentration of L - Valine 2 . 0 g /l days for days for methacrylic acid (ppm ) Glucose 2 .0 g/ l pre -culture main culture After After 40 After the main culture , the entire volume of the main Microbial strain (days ) ( days) 5 hours 24 hours culture was transferred to a centrifuge tube with volume of 225 ml and then centrifuged (5000 rpm , 15 min ) By remov Sphingomonas 0 .2 0 . 1 paucimobilis ing the supernatant, the cells were obtained . To the cells , 40 NBRC 13935 ml of 50 mM phosphate buffer (pH 7 . 0 ) were added and the Pedobacter 1 . 2 2 . 8 heparinus 45 cells were suspended and centrifuged again under the same NBRC12017 conditions. By removing the supernatant, the washed cells Bacillus 1 0. 0 04. 4 were obtained . To the washed cells , 3 ml of 50 mm subtilis phosphate buffer (pH 7 . 0 ) were added to prepare a cell NBRC12210 Listonella ND 0 . 4 suspension . To a conical tube with volume of 50 mL , 3 ml anguillarum 50 of the cell suspension and 3 ml of the solution for resting cell ATCC19264 reaction were added to perform the resting cell reaction . The reaction was allowed to occur under aerobic conditions at (ND : Not Detected ) 30° C . with a rotary shaker ( 230 rpm ) for 24 hours . Com position of the solution for resting cell reaction includes 5 . 0 Example 14 : Production of Methacrylic Acid Based 55 g /l L -valine and 50 mM phosphate buffer (pH 7 .0 ). on Resting Cell Reaction Using Genus Five hours and twenty - four hours after starting the resting Ochrobactrum and Genus Paracoccus cell reaction , 1 .6 ml of the reaction solution were collected . The reaction solution was separated by centrifuge ( 15000 Ochrobactrum grignonense NBRC102586 , Ochrobac - rpm , 5 min ) and the supernatant fraction was subjected to a trum lupini NBRC102587, and Paracoccus aminophilus 60 filtration treatment which uses a DISMIC - 13CP Cellulose NBRC16710 were grown on an agar medium (plate culture ). Acetate 0 .2 um filter (manufactured by ADVANTEC ). The Composition of the medium for plate culture is shown compounds contained in the culture supernatant were ana below . lyzed according to the analysis condition A described in Polypeptone (manufactured by Becton , Dickinson and Example 5 . As a result , a peak was observed at the same Company ) 10 g 65 elution time as methacrylic acid . In “ Table 18 ” , concentra Yeast extract (manufactured by Becton , Dickinson and tion of methacrylic acid which is in the supernatant of the Company ) 2 g reaction solution of the resting cell reaction is shown . US 10 , 294 ,500 B2 101 102 TABLE 18 solution were analyzed according to the analysis condition A described in Example 5 . As a result, a peak was observed at Number of Number of Concentration of the same elution time as methacrylic acid . In “ Table 19” , days for days for methacrylic acid (ppm ) concentration of methacrylic acid which is in the supernatant pre -culture main culture After After 5 of the reaction solution of the resting cell reaction is shown . Microbial strain (days ) ( days ) 5 hours 24 hours Ochrobactrum 2 1 . 4 18 . 1 TABLE 19 grignonense NBRC 102586 Concentration of Ochrobactrum 1 . 0 49 methacrylic acid (ppm ) lupini 3 2 1 .0 4910. Microbial strain NBRC 102587 Bacillus megaterium NBRC15308 0 . 1 Paracoccus 0 . 3 2 . 5 Bacillus simplex ATCC49097 0 . 1 aminophilus 4 2 0. 3 2. 5 Streptomyces griseus NBRC13350 0 . 1 NBRC16710 15 Example 15 : Production of Methacrylic Acid Based Example 16 : Production of Methacrylic Acid Based on Resting Cell Reaction Using Genus Bacillus and on Resting Cell Reaction Using Genus Genus Streptomyces Comamonas, Genus Acinetobacter, and Genus 20 Xanthobacter Bacillus megaterium NBRC15308 , Bacillus simplex ATCC49097 , and Streptomyces griseus NBRC 13350 were Comamonas terrigena NBRC13299 , Acinetobacter junii grown on a nutrient agar medium (manufactured by Becton , ATCC17908 , and Xanthobacter autotrophicus ATCC35674 Dickinson and Company, agar 1 . 5 % ) . The cells were inocu - os were grown on a nutrient agar medium (manufactured by lated (one platinum loop ) to 10 mL liquid medium (one Becton , Dickinson and Company, agar 1. 5 % ). The cells platinum loop ) and cultured under aerobic conditions at 30° were inoculated (one platinum loop ) to 10 mL nutrient C . with a rotary shaker ( 230 rpm ) for 1 day ( pre - culture) . medium (one platinum loop ) and cultured under aerobic 1 ml of the pre -culture was inoculatedculated to 100 mlmL liquid conditions at 30°30°C C . with a rotary shaker ( 230 rpm ) for 1 day medium ( 100 mlmedium /500 ml volume conical flask ) and 30 pre -culture ). cultured under aerobic conditions at 30° C . with a rotary 1 ml of the pre -culture was inoculated to 100 mL liquid shaker ( 230 rpm ) for 2 days (main culture ) . Composition of medium ( 100 mlmedium /500 ml volume conical flask ) and the medium for the main culture is described below . cultured under aerobic conditions at 30° C . with a rotary Na ,HPO , (anhydrous ) 6 . 0 g / l shaker (230 rpm ) for 2 days (main culture ). Composition of KH , PO , 3 . 0 g /1 35 the medium for the main culture is described below . NaCl 0 . 5 g /1 Nutrientmedium 8 g/ 1 NH _C1 1 .0 g /1 IM MgSO4. 7H2O 1 ml L - Valine 2 . 0 g / 1 1M CaCl2 0 . 1 ml After the main culture , the entire volume of the main 1 % Thiamine 1 ml an culture was transferred to a centrifuge tube with volume of L - Valine 2 .0 g /1 225 ml and then centrifuged (5000 rpm , 15 min ). By Glucose 2 . 0 g / l removing the supernatant, the cells were obtained . To the After the main culture , the entire volume of the main cells , 40 ml of 50 mM phosphate buffer (pH 7 . 0 ) were added culture was transferred to a centrifuge tube with volume of and the cells were suspended and centrifuged again under 225 and the centrifuged (5000rpm , 15min15 min ) . By 4545 the same conditions. By removing the supernatant, the removing the supernatant , the cells were obtained . To the washed cells were obtained . To the washed cells , 3 mlof 50 cells . 40 mlof 50 mM phosphate buffer (pH 7 . 0 ) were added mM phosphate buffer (pH 7 .0 ) were added to prepare a cell and the cells were suspended and centrifuged again under suspension . To a conical tube with volume of 50 mL , 3 ml the same conditions . By removing the supernatant. the of the cell suspension and 3 mlof the solution for resting cell washed cells were obtained . To the washed cells, 3 ml of 50 50 reaction were added to perform the resting cell reaction . The mM phosphate buffer ( pH 7 . 0 ) were added to prepare a cell reaction was allowed to occur under aerobic conditions at suspension . To a conical tube with volume of 50 mL , 3 ml 30° C . with a rotary shaker (230 rpm ) for 24 hours . Com of the cell suspension and 3 ml of the solution for resting cell position of the solution for resting cell reaction includes 5 .0 reaction were added to perform the resting cell reaction . The g / l L - valine or isobutyric acid and 50 mM phosphate buffer reaction was allowed to occur under aerobic conditions at 55 (pH 7 . 0 ) . 30° C . with a rotary shaker (230 rpm ) for 24 hours . Com Five hours and twenty - four hours after starting the resting position of the solution for resting cell reaction includes 5 . 0 cell reaction , 1 . 6 ml of the reaction solution were collected . g / 1 L -valine and 50 mM phosphate buffer (pH 7 . 0 ) . The reaction solution was separated by centrifuge ( 15000 Five hours (Streptomyces griseus NBRC13350 ) or rpm , 5 min ) and the supernatant fraction was subjected to a twenty - four hours ( Bacillus megaterium NBRC 15308 and 60 filtration treatment which uses a DISMIC - 13CP Cellulose Bacillus simplex ATCC49097 ) after starting the resting cell Acetate 0 . 2 um filter (manufactured by ADVANTEC ) . The reaction , 1. 6 mlof the reaction solution were collected . The compounds contained in the culture supernatant were ana reaction solution was separated by centrifuge (15000 rpm , 5 lyzed according to the analysis condition A described in min ) and the supernatant fraction was subjected to a filtra - Example 5 . As a result, a peak was observed at the same tion treatment which uses a DISMIC - 13CP Cellulose 65 elution time as methacrylic acid . In “ Table 20 ” , concentra Acetate 0 . 2 um filter (manufactured by ADVANTEC ) . The tion of methacrylic acid which is in the supernatant of the compounds contained in the supernatant of the reaction reaction solution of the resting cell reaction is shown . US 10 ,294 ,500 B2 103 104 TABLE 20 TABLE 21 Concentration of Concentration of methacrylic acid ( ppm ) Number of days methacrylic acid (ppm ) Substrate for After After for main After After Microbial strain resting reaction 5 hours 24 hours Microbial strain culture ( days ) 5 hours 24 hours Comamonas terrigena Isobutyric acid 1 . 7 5 .2 Ochrobactrum sp . 2. 9 5 . 7 NBRC13299 NBRC12951 Acinetobacter junii Valine ND 0. 3 Ochrobactrum intermedium 4 3 3. . 44 . 1414. . 44 ATCC17908 A ND 10 NBRC13694 Xanthobacter autotrophicus Valine 0 . 1 0 . 1 Ochrobactrum anthropi 4 070 . 7 11 . . 6 ATCC35674 ATCC49237 (ND : Not Detected ) 15 TABLE 22 Example 17 : Production of Methacrylic Acid Based on Resting Cell Reaction Using Genus Number of days Concentration of Ochrobactrum , Genus Achromobacter , and Genus for main methacrylic Acinetobacter Microbial strain culture (days ) acid (ppm ) Achromobacter denitrificans 3 0 . 3 20 NBRC12669 Ochrobactrum sp . NBRC12951, Ochrobactrum interme Acinetobacter haemolyticus ?? 0 . 1 dium NBRC13694 , Ochrobactrum anthropi ATCC49237 , ATCC17906 Achromobacter denitrificans NBRC12669 , and Acineto bacter haemolyticus ATCC 17906 were grown on a nutrient agar medium (manufactured by Becton , Dickinson and 25 Company , agar 1 . 5 % ) . The grown cells were inoculated ( one Example 18 : Production of Methacrylic Acid Based platinum loop ) to 100 mL liquid medium ( 100 ml medium / on Resting Cell Reaction Using Genus Shewanella 500 ml volume conical flask ) and cultured under aerobic Shewanella fodinae NBRC 105216 was grown on a conditions at 30° C . with a rotary shaker ( 230 rpm ) for 3 to 30 nutrient agar medium (manufactured by Becton , Dickinson 4 days (main culture ) . Composition of the medium for the 30 and Company, agar 1 . 5 % ) . The grown cells were inoculated main culture is described below . ( one platinum loop ) to 100 mL liquid medium ( 100 ml Nutrient medium 8 g / l medium / 500 ml volume conical flask ) and cultured under L -Valine 2 .0 g /l aerobic conditions at 30° C . with a rotary shaker (230 rpm ) After the main culture , the entire volume of the mainin 35 for 3 days (main culture) . Composition of the medium for culture was transferred to a centrifuge tube with volume of the main culture is described below . 225 ml and then centrifuged ( 5000 rpm , 15 min ). By Polypeptone (manufactured by Becton , Dickinson and removing the supernatant, the cellss were obtained . To the CorCompany ) 10 g cells , 40 ml of 50 mM phosphate buffer (pH 7. 0 ) were added Yeast extract (manufactured by Becton , Dickinson and and the cells were suspended and centrifuged again under 40 Company ) 2 g the same conditions. By removing the supernatant, the MgSO4. 7H20 1 g washed cells were obtained . To the washed cells , 3 ml of 50 Sea water (Daigo ' s artificial sea water SP for marine mM phosphate buffer ( pH 7 .0 ) were added to prepare a cell microalgae ,manufactured by Nihon Seiyaku Co . Japan ) 750 spennanical tube with volume of50L, 3mm of the cell suspension and 3 mlof the solution for resting cell 45 Distilled water 250 ml reaction were added to perform the resting cell reaction . The L - Valine 2 . 0 g /1 reaction was allowed to occur under aerobic conditions at pH 727 . 2 - 7 . 4 30° C . with a rotary shaker ( 230 rpm ) for 24 hours. Com After the main culture , the entire volume of the main position of the solution for resting cell reaction includes 5 .0 culture was transferred to a centrifuge tube with volume of g /l L - valine or isobutyric acid and 50 mM phosphate buffer 50 225 ml and then centrifuged ( 5000 rpm , 15 min ). By ( pH 7 . 0 ) . removing the supernatant, the cells were obtained . To the cells , 40 ml of 50 mM phosphate buffer ( pH 7 . 0 ) were added Five hours and twenty - four hours ( genus Ochrobactrum ) and the cells were suspended and centrifuged again under or five hours ( Achromobacter denitrificans NBRC 12669 the same conditions. By removing the supernatant, the and Acinetobacter haemolyticus ATCC17906 ) after starting washed cells were obtained . To the washed cells , 3 ml of 50 the resting cell reaction , 1 . 6 ml of the reaction solution were mM phosphate buffer (pH 7 . 0 ) were added to prepare a cell collected . The reaction solution was separated by centrifuge suspension . To a conical tube with volume of 50 mL, 3 ml ( 15000 rpm , 5 min ) and the supernatant fraction was sub of the cell suspension and 3 ml of the solution for resting cell jected to a filtration treatment which uses a DISMIC - 13CP reaction were added to perform the resting cell reaction . The Cellulose Acetate 0 .2 um filter (manufactured by ADVAN - 60 reaction was allowed to occur under aerobic conditions at TEC ). The compounds contained in the culture supernatant 30° C . with a rotary shaker (230 rpm ) for 24 hours . Com were analyzed according to the analysis condition A position of the solution for resting cell reaction includes 5 . 0 described in Example 5 . As a result , a peak was observed at g /1 L -valine and 50 mM phosphate buffer (pH 7. 0 ). the same elution time as methacrylic acid . In “ Table 21 ” and Twenty - four hours after starting the resting cell reaction , " Table 22 ” concentration of methacrylic acid which is in the 65 1 .6 ml of the reaction solution were collected . The reaction supernatant of the reaction solution of the resting cell solution was separated by centrifuge ( 15000 rpm , 5 min ) and reaction is shown . the supernatant fraction was subjected to a filtration treat US 10 , 294 ,500 B2 105 106 ment which uses a DISMIC - 13CP Cellulose Acetate 0 . 2 um was observed at the same elution time as methacrylic acid filter (manufactured by ADVANTEC ). The compounds con and the concentration ofmethacrylic acid was 0 . 1 ppm after tained in the supernatant of the reaction solution were 5 hours and 0 . 1 ppm after 24 hours . analyzed according to the analysis condition A described in Example 5 . As a result, a peak was observed at the same 5 Example 20 : Production of methacrylic acid based elution time as methacrylic acid and the concentration of on resting cell reaction using genus Brevundimonas methacrylic acid was 0 . 1 ppm . and genus Rhizobium Example 19: Production of Methacrylic Acid Based Brevundimonas vesicularis ATCC11426 and Rhizobium on Resting Cell Reaction Using Genus 10 leguminosarum ATCC10004 were grown on a nutrient agar Agrobacterium medium (manufactured by Becton , Dickinson and Com Agrobacterium luteum NBRC15768 was grown at 25° C . pany, agar 1. 5 % ). The grown cells were inoculated (one on an agar medium (plate culture ) . Composition of the platinum loop ) to 10 mL liquid medium and cultured under medium for plate culture is shown below . 15 aerobic conditions at 30° C . with a rotary shaker ( 230 rpm ) Polypeptone (manufactured by Becton , Dickinson and for 4 days (pre -culture ) . Composition of the medium for the Company ) 10 g main culture is described below . The pre - culture and 40 % Yeast extract (manufactured by Becton , Dickinson and glycerol were mixed with each other at 1 : 1 and subjected to Company ) 2 g cryopreservation at - 80° C . MgSO4. 7H2O1g 20 Na2HPO4 ( anhydrous ) 6 . 0 g /1 Sea water (Daigo 's artificial sea water SP for marine KH2PO4 3 . 0 g /l microalgae , manufactured by Nihon Seiyaku Co . Japan ) 750 NaCl 0 . 5 g / 1 ml NH C1 1 . 0 g /l Distilled water 250 ml 1M MgSO4. 7H2O 1 ml Ager 15 g 25 1M CaCl , 0 .1 ml pH 7 . 2 -7 .4 1 % Thiamine 1 ml The grown cells were inoculated to 100 mL liquid Glucose 2 . 0 g /1 medium ( 100 mlmedium / 500 ml volume conical flask ) and The pre - culture which has been remained under cryo cultured under aerobic conditions at 30° C . with a rotary preservation was thawed at room temperature and 0 . 5 ml shaker ( 230 rpm ) for 3 days (main culture ) . Composition of 30 was inoculated to 100 ml liquid medium ( 100 mlmedium / the medium for the main culture is described below . Polypeptone (manufactured by Becton , Dickinson and 500 ml volume conical flask ) . It was then cultured under Company ) 10 g aerobic conditions at 30° C . with a rotary shaker ( 230 rpm ) Yeast extract (manufactured by Becton , Dickinson and for 2 days (main culture ) . Composition of the medium for Company ) 2 g the main culture is described below . MgSO4. 7H20 1g Nutrient medium 8 g /1 Sea water (Daigo ' s artificial sea water SP for marine L - Valine 2 .0 g /1 microalgae, manufactured by Nihon Seiyaku Co . Japan ) 750 After the main culture , the entire volume of the main ml culture was transferred to a centrifuge tube with volume of Distilled water 250 ml 225 ml and then centrifuged (5000 rpm , 15 min ). By L - Valine 2 .0 g /1 removing the supernatant, the cells were obtained . To the pH 7 . 2 - 7 . 4 cells , 40 ml of 50 mM phosphate buffer ( pH 7 . 0 ) were added After the main culture , the entire volume of the main and the cells were suspended and centrifuged again under culture was transferred to a centrifuge tube with volume of the same conditions. By removing the supernatant, the 225 ml and then centrifuged (5000 rpm , 15 min ). By 45 washed cells were obtained . To the washed cells , 3 mlof 50 removing the supernatant, the cells were obtained . To the mM phosphate buffer ( pH 7 .0 ) were added to prepare a cell cells , 40 ml of 50 mM phosphate buffer (pH 7 . 0 ) were added suspension . To a conical tube with volume of 50 mL , 3 ml and the cells were suspended and centrifuged again under of the cell suspension and 3 mlof the solution for resting cell the same conditions . By removing the supernatant, the reaction were added to perform the resting cell reaction . The washed cells were obtained . To the washed cells , 3 ml of 50 50 reaction was allowed to occur under aerobic conditions at mM phosphate buffer ( pH 7 . 0 ) were added to prepare a cell 30° C . with a rotary shaker ( 230 rpm ) for 24 hours . Com suspension . To a conical tube with volume of 50 mL, 3 ml position of the solution for resting cell reaction includes 5 . 0 of the cell suspension and 3 mlof the solution for resting cell g / 1 L - valine and 50 mM phosphate buffer (pH 7 . 0 ) . reaction were added to perform the resting cell reaction . The Five hours ( Brevundimonas vesicularis ATCC11426 ) or reaction was allowed to occur under aerobic conditions at 55 twenty -four hours ( Rhizobium leguminosarum ATCC 30° C . with a rotary shaker (230 rpm ) for 24 hours . Com 10004 ) after starting the resting cell reaction , 1 .6 ml of the position of the solution for resting cell reaction includes 5 . 0 reaction solution were collected . The reaction solution was g / l L - valine and 50 mM phosphate buffer (pH 7 . 0 ) . separated by centrifuge ( 15000 rpm , 5 min ) and the super Five hours and twenty -four hours after starting the resting natant fraction was subjected to a filtration treatment which cell reaction , 1 .6 ml of the reaction solution were collected . 60 uses a DISMIC - 13CP Cellulose Acetate 0 . 2 um filter (manu The reaction solution was separated by centrifuge ( 15000 factured by ADVANTEC ) . The compounds contained in the rpm , 5 min ) and the supernatant fraction was subjected to a supernatant of the reaction solution were analyzed according filtration treatment which uses a DISMIC - 13CP Cellulose to the analysis condition B described in Example 5 . As a Acetate 0 . 2 um filter (manufactured by ADVANTEC ) . The result , a peak was observed at the same elution time as compounds contained in the supernatant of the reaction 65 methacrylic acid and the concentration of methacrylic acid solution were analyzed according to the analysis condition was 0 . 7 ppm for Brevundimonas vesicularis ATCC 11426 and condition B described in Example 5 . As a result, a peak and 1. 6 ppm for Rhizobium leguminosarum ATCC 10004 . US 10 , 294 , 500 B2 107 108 Example 21 : Production of Methacrylic Acid Based rpm ) for 5 days (main culture ). Composition of the medium on Resting Cell Reaction Using Genus for the main culture is described below . Brevundimonas Nutrient medium 8 g / l L - Valine 2 .0 g /1 Brevundimonas diminuta ATCC 11568 was grown on a 5 After the main culture , the entire volume of the main nutrient agar medium (manufactured by Becton , Dickinson culture was transferred to a centrifuge tube with volume of and Company, agar 1 . 5 % ) . The grown cells were inoculated 225 ml and then centrifuged (5000 rpm , 15 min ) . By ( one platinum loop ) to 10 mL nutrient medium and cultured removing the supernatant , the cells were obtained . To the under aerobic conditions at 30° C . with a rotary shaker (230 cells , 40 ml of 50 mM phosphate buffer (pH 7 .0 ) were added rpm ) for 1 day (pre - culture ). The pre -culture and 1040 % 10 and the cells were suspended and centrifuged again under glycerol were mixed with each other at 1 : 1 and subjected to the same conditions. By removing the supernatant, the washed cells were obtained . To the washed cells , 3 ml of 50 cryopreservation at - 80° C . mM phosphate buffer ( pH 7 . 0 ) were added to prepare a cell The pre- culture which has been remained under cryo suspension . To a conical tube with volume of 50 mL , 3 ml preservation was thawed at room temperature and 0 . 5 ml 1615 of the cell suspension and 3 ml of the solution for resting cell was inoculated to 100 ml liquid medium ( 100 mlmedium / reaction were added to perform the resting cell reaction . The 500 ml volume conical flask ) . It was then cultured under reaction was allowed to occur under aerobic conditions at aerobic conditions at 30° C . with a rotary shaker ( 230 rpm ) 30° C . with a rotary shaker ( 230 rpm ) for 24 hours . Com for 2 days (main culture ). Composition of the medium for position of the solution for resting cell reaction includes 5 . 0 the main culture is described below . 20 g / l L -valine and 50 mM phosphate buffer (pH 7 . 0 ) . Nutrient medium 8 g /l Five hours and twenty - four hours after starting the resting L - Valine 2 . 0 g / l cell reaction , 1. 6 mlof the reaction solution were collected . After the main culture , the entire volume of the main The reaction solution was separated by centrifuge ( 15000 culture was transferred to a centrifuge tube with volume of rpm , 5 min ) and the supernatant fraction was subjected to a 225 ml and then centrifuged (5000 rpm , 15 min ) . By 25 filtration treatment which uses a DISMIC - 13CP Cellulose removing the supernatant, the cells were obtained . To the Acetate 0 . 2 um filter (manufactured by ADVANTEC ) . The cells , 40 ml of 50 mM phosphate buffer (pH 7 . 0 ) were added compounds contained in the supernatant of the reaction and the ce r pended and centrifuged again under solution were analyzed according to the analysis condition A the same conditions . By removing the supernatant , the described in Example 5 . As a result, a peak was observed at washed cells were obtained . To the washed cells , 3 mlof 50 30 the same elution time as methacrylic acid and the concen mM phosphate buffer (pH 7 . 0 ) were added to prepare a cell tration of methacrylic acid was 0 . 4 ppm after 5 hours and 0 . 3 suspension . To a conical tube with volume of 50 mL , 3 ml ppm after 24 hours . of the cell suspension and 3 mlof the solution for resting cell reaction were added to perform the resting cell reaction . The Example 23 : Production of Methacrylic Acid Based reaction was allowed to occur under aerobic conditions at 35 on Resting Cell Reaction Using Yeast 30° C . with a rotary shaker ( 230 rpm ) for 24 hours . Com position of the solution for resting cell reaction includes 5 .0 Saccharomyces cerevisiae NBRC1136 , Saccharomyces g / 1 L - valine and 50 mM phosphate buffer ( pH 7 . 0 ) . cerevisiae NBRC2347 , Saccharomyces paradoxus Five hours and twenty - four hours after starting the resting NBRC10609 , and Candida parapsilosis ATCC22019 were cell reaction , 1 . 6 ml of the reaction solution were collected . 40 grown on an agar medium ( plate culture ) . Composition of The reaction solution was separated by centrifuge ( 15000 the medium for plate culture is shown below . The culture rpm , 5 min ) and the supernatant fraction was subjected to a was performed at 30° C . (genus Saccharomyces ) or at 24° C . filtration treatment which uses a DISMIC - 13CP Cellulose (genus Candida ) . Acetate 0 . 2 um filter (manufactured by ADVANTEC ) . The Yeast extract (manufactured by Becton , Dickinson and compounds contained in the supernatant of the reaction 45 Company ) 20 g / l solution were analyzed according to the analysis condition B Polypeptone (manufactured by Becton , Dickinson and described in Example 5 . As a result , a peak was observed at Company ) 20 g/ l the same elution time as methacrylic acid and the concen Glucose 20 g / 1 tration ofmethacrylic acid was 33 .0 ppm after 5 hours and Ager 15 g /1 37 . 0 ppm after 24 hours . 50 The grown cells were inoculated (one platinum loop ) to 10 ml liquid medium and cultured under aerobic conditions Example 22 : Production of Methacrylic Acid Based at 30° C . with a rotary shaker (230 rpm ) for 2 to 7 days on Resting Cell Reaction Using Thermophillic (pre - culture ) . The culture was performed at 30° C . (genus Bacteria Saccharomyces ) or at 24° C . ( genus Candida ), and as a 55 medium for the pre - culture , the medium 1 or 2 was used . Geobacillus stearothermophilus NBRC 12983 was grown Composition of the medium is shown below . on a nutrient agar medium (manufactured by Becton , Dick Medium 1 inson and Company, agar 1 . 5 % ) . The cells were inoculated Yeast extract (manufactured by Becton , Dickinson and ( one platinum loop ) to 10 mL liquid medium and cultured Company ) 20 g / l under aerobic conditions at 37° C . with a rotary shaker (230 60 Polypeptone (manufactured by Becton , Dickinson and rpm ) for 2 days (pre -culture ). Composition of the medium Company ) 20 g / 1 for the pre - culture is described below . Glucose 20 g /1 Nutrient medium 8 g / l Medium 2 Glucose 2 .0 g /l Glucose 2 . 0 g / 1 The grown cells were inoculated to 100 ml liquid medium 65 L - Valine 2 . 0 g / l ( 100 mlmedium /500 ml volume conical flask ) and cultured (NH4 ) 2SO4 6 .0 g / 1 under aerobic conditions at 25° C . with a rotary shaker ( 230 KC1 2 . 4 g / l US 10 ,294 ,500 B2 109 110 NaCl 0 . 12 g /1 TABLE 23 H2PO2 3 .0 g /1 MgSO4. 7H2O 2 . 4 g /l Resting FeSO4. 7H2O 0 .01 g / l Pre - culture Main culture cell ZnSO4. 7H2O 0 . 12 g / 1 Number Number reaction MnSO40 . 4 -6H 0 0 .024 g /1 Microbial strain Medium of days Substrate of days Substrate CuSO4.5H20 0 .006 g / 1 Saccharomyces Medium 1 1 Isobutyric 3 Isobutyric CaCl , 0 .12 g/ 1 cerevisiae acid acid Vitamin Mix 30 ml NBRC1136 Composition of Vitamin Mix is shown below . 10 Saccharomyces Medium 1 Isobutyric 3 Isobutyric cerevisiae acid acid Biotin 0 . 2 g/ 1 NBRC2347 Calcium pantothenate 2 g / 1 Saccharomyces Medium 2 Isobutyric Isobutyric Folic acid 0 .002 g/ 1 paradoxus acid Thiamine hydrochloride 0 . 4 g /l NBRC10609 Riboflavin 0 . 2 g / l 15 Candida Medium2 3 Valine 2 Valine Nicotinic acid 0 . 4 g /l parapsilosis Pyridoxine hydrochloride 0. 4 g /1 ATCC22019 Inositol 1 g / 1 p - Aminobenzoic acid 0 .2 g / l Five hours (Saccharomyces cerevisiae NBRC2347 and 1 ml of the pre -culture was inoculated to 100 ml liquid 20 Candida parapsilosis ATCC22019 ) or twenty -four hours medium ( 100 ml medium / 500 ml volume conical flask ). It (Saccharomyces cerevisiae NBRC1136 and Saccharomyces was then cultured under aerobic conditions at 30° C . with a rotary shaker ( 230 rpm ) for 2 to 3 days (main culture ) . The paradoxus NBRC10609 ) after starting the resting cell reac culture was performed at 30° C . (genus Saccharomyces ) or tion , 1. 6 ml of the reaction solution were collected . The at 24° C . ( genus Candida ) . Composition of the medium for 25 reaction solution was separated by centrifuge ( 15000 rpm , 5 the main culture is described below . min ) and the supernatant fraction was subjected to a filtra Glucose 2 . 0 g /1 L - Valine or Isobutyric acid 2 . 0 g / l tion treatment which uses a DISMIC - 13CP Cellulose (NH2 ) , SO , 6 . 0 g / l Acetate 0 . 2 um filter (manufactured by ADVANTEC ) . The KCI 2 . 4 g / 1 30 compounds contained in the supernatant of the reaction NaCl 0 . 12 g / 1 solution were analyzed according to the analysis condition A H2PO2 3 .0 g /1 MgSO4. 7H2O 2 . 4 g / l described in Example 5 . As a result , a peak was observed at FeSO4. 7H2O 0 .01 g / 1 the same elution time as methacrylic acid . In “ Table 24 ” , ZnSO4.7H2O 0 . 12 g / 1 35 concentration ofmethacrylic acid which is in the supernatant MnSO4. 4 -6H 0 0 .024 g / 1 of the reaction solution of the resting cell reaction is shown . CuSO4. 5H20 0 . 006 g /l CaCl , 0 .12 g/ 1 TABLE 24 Vitamin Mix 30 ml Composition of Vitamin Mix is shown below . 40 Concentration of Biotin 0 . 2 g/ 1 Microbial strain methacrylic acid (ppm ) Calcium pantothenate 2 g / l Saccharomyces cerevisiae NBRC1136 0 . 1 Folic acid 0 .002 g/ 1 Saccharomyces cerevisiae NBRC2347 0 . 2 Thiamine hydrochloride 0 . 4 g / l Saccharomyces paradoxus NBRC10609 0 . 2 Riboflavin 0 .2 g /1 45 Candida parapsilosis ATCC22019 0. 2 Nicotinic acid 0 . 4 g /l Pyridoxine hydrochloride 0 .4 g / l Inositol 1 g / 1 Example 24 : Production of Methacrylic Acid Based p - Aminobenzoic acid 0 .2 g /l on Resting Cell Reaction Using Mildew After the main culture , the entire volume of the main 50 culture was transferred to a centrifuge tube with volume of 225 ml and then centrifuged (5000 rpm , 15 min ) By remov Aspergillus niger ATCC6275 , Aspergillus flavus ing the supernatant, the cells were obtained . To the cells , 40 NBRC8558 , and Aspergillus oryzae NBRC4255 was grown mlof 50 mM phosphate buffer (pH 7 .0 ) were added and the on a potato dextrose agar medium (manufactured by Nissui cells were suspended and centrifuged again under the same 55 Pharmaceutical Co ., Ltd . ). The grown mycelia and spores conditions . By removing the supernatant, the washed cells were suspended in 5 ml sterilized water, and 1 ml of the werebtained . thwashed , 3m 5 MM phosphate buffer (pH 7 . 0 ) were added to prepare a cell resultant was inoculated to 100 mL liquid medium ( 100 ml suspension . To a conical tube with volume of 50 mL , 3 ml medium /500 ml volume conical flask ) and cultured under of the cell suspension and 3 ml of the solution for resting cell 60 aerobic conditions at 24° C . with a rotary shaker ( 230 rpm ) reaction were added to perform the resting cell reaction . The for 2 days (main culture ) . Composition of the medium for reaction was allowed to occur under aerobic conditions at the main culture is described below . 30° C . with a rotary shaker (230 rpm ) for 24 hours . Com position of the solution for resting cell reaction includes 5 . 0 L - Valine 2 .0 g/ 1 g / 1 L - valine or isobutyric acid and 50 mM phosphate buffer 65 NaNO3 2 . 5 g / 1 (pH 7 . 0 ) . In “ Table 23 ” , conditions for the culture and K2HPO4 1. 25 g/ 1 resting cell reaction are shown . MgSO4.7H20 0 .63 g/ 1 US 10 , 294 ,500 B2 111 112 KCI 0 .63 g / 1 “ Table 25 ” , concentration ofmethacrylic acid which is in the FeSO4. 7H2O 0 .013 g / 1 supernatant of the reaction solution of the resting cell Wheat bran 5 g / l reaction is shown . pH 7 . 0 After the main culture , the entire volume of the main TABLE 25 culture was transferred to a centrifuge tube with volume of Concentration of methacrylic acid (ppm ) 225 ml and then centrifuged ( 5000 rpm , 15 min ) . By removing the supernatant , the cells were obtained . To the After 5 hours After 24 hours 10 Analysis Analysis Analysis Analysis cells , 40 ml of 50 mM phosphate buffer ( pH 7 . 0 ) were added condition condition condition condition and the cells were suspended and centrifuged again under Microbial strain ? B ? B the same conditions. By removing the supernatant, the Aspergillus niger ND ND 1 . 7 washed cells were obtained . To the washed cells , 10 ml of ATCC6275 ate buffer in 70 ) were added to prepare a 15 Aspergillus flavus 0. 7 1 . 4 NBRC8558 cell suspension . To a conical tube with volume of 50 mL , the Aspergillus oryzae 0 . 4 0. 8 cell suspension was transferred and 10 ml of the solution for NBRC4255 resting cell reaction were added to perform the resting cell (ND : Not Detected ) reaction . The reaction was allowed to occur under aerobic 20 conditions at 30° C . with a rotary shaker (230 rpm ) for 24 hours . Composition of the solution for resting cell reaction Example 25: Identity Comparison Using Genus includes 5 .0 g /l L -valine and 50 mM phosphate buffer (pH Pseudomonas, Genus Bacillus, Genus 7 . 0 ) . 25 Brevundimonas, Genus Ochrobactrum , and Genus Five hours and twenty - four hours after starting the resting Acinetobacter cell reaction , about 2 ml of the reaction solution were collected . The reaction solution was separated by centrifuge By using the homology analysis program GENETYX ver . ( 15000 rpm . 5 min ) and the supernatant fraction was sub - 2010 , which is a software for processing genetic information jected to a filtration treatment which uses a DISMIC - 13CP by GENETYX CORPORATION , identity between nucleo Cellulose Acetate 0 . 2 uM filter (manufactured by ADVAN tide sequences of 16S rDNA or LSU rDNA in each microbe , TEC ) . The compounds contained in the culture supernatant which are shown in SEQ ID NOS. 1 to 8 , 9 to 15 , 18 to 20 , were analyzed according to the analysis condition A and 35 22 to 26 , 30 to 31 , 42 to 44 , 45 to 46 , and 47 to 49, was condition B described in Example 5 . As a result , a peak was compared . The results are shown in “ Table 26 to 33 .” observed at the same elution time as methacrylic acid . In SEQ ID NO . 1 to 8 TABLE 26 Pseudomonas Pseudomonas Pseudomonas Pseudomonas putida Pseudomonas Pseudomonas Pseudomonas Pseudomonas sp . D29 sp . D41 - 2 sp . 143 - 1 NBRC12996 sp . B25 - 2 sp . B22 - 1 sp . D25 sp . 126 Pseudomonas sp . D29 99 % 98 % 97 % 97 % 96 % 95 % 95 % Pseudomonas sp . D41 - 2 99 % 98 % 97 % 97 % 95 % 96 % 96 % Pseudomonas sp . 143 - 1 98 % 98 % 98 % 98 % 97 % 96 % 96 % Pseudomonas putida NBRC12996 97 % 97 % 98 % 98 % 97 % 98 % 97 % Pseudomonas sp . B25 - 2 97 % 97 % 98 % 98 % 96 % 97 % 97 % Pseudomonas sp . B22 - 1 96 % 95 % 97 % 97 % 96 % 99 % 98 % Pseudomonas sp . D25 95 % 96 % 96 % 98 % 97 % 99 % 98 % Pseudomonas sp . D26 95 % 96 % 96 % 97 % 97 % 98 % 98 %
SEQ ID NO . 9 to 15 TABLE 27 Bacillus Bacillus Bacillus Bacillus subtilis Bacillus badius megaterium simplex Bacillus Bacillus NBRC12210 sp . R1 ATCC14574 NBRC15308 ATCC49097 sp . G1 sp . G2 Bacillus subtilis NBRC12210 93 % 90 % 89 % 89 % 92 % 1 Bacillus sp . R1 93 % 92 % 92 % 91 % 89 % 89 % Bacillus badius ATCC14574 90 % 92 % 89 % 90 % 88 % 88 % Bacillus megaterium NBRC1530889 % 92 % 89 % 92 % 90 % 90 % Bacillus simplex ATCC49097 89 % 91 % 90 % 92 % 89 % 89 % Bacillus sp . G1 92 % 89 % 88 % 90 % 89 % Bacillus sp . G2 91 % 89 % 88 % 90 % 89 % 99 % 99 % US 10 , 294 ,500 B2 113 114 SEQ ID NOS. 18 to 20 SEQ ID NOS . 47 to 49 TABLE 28 TABLE 33 Brevundimonas Brevundimonas Brevundimonas 5 diminuta subvibrioides vesicularis Aspergillus Aspergillus Aspergillus ATCC11568 NBRC16000 ATCC11426 niger flavus oryzae ATCC6275 NBRC8558 NBRC4255 Brevundimonas 96 % 96 % diminuta ATCC11568 10 Aspergillus niger 96 % 96 % Brevundimonas 96 % 95 % subvibrioides ATCC6275 NBRC16000 Aspergillus flavus 96 % 100 % Brevundimonas 96 % 95 % NBRC8558 vesicularis Aspergillus oryzae 96 % ATCC11426 15 96 % 100 % NBRC4255 SEQ ID NOS. 22 to 26 TABLE 29 Ochrobactrum Ochrobactrum Ochrobactrum Ochrobactrum Ochrobactrum intermedium lupini sp . intermedium grignonense NBRC13694 NBRC 102587 NBRC12951 NBRC15820 NBRC102586 Ochrobactrum intermedium NBRC13694 100 % 100 % 98 % 97 % Ochrobactrum lupini NBRC 102587 100 % 100 % 98 % 97 % Ochrobactrum sp . NBRC12951 100 % 100 % 98 % 97 % Ochrobactrum intermedium NBRC15820 98 % 98 % 98 % 97 % Ochrobactrum grignonense NBRC102586 97 % 97 % 97 % 97 %
30 SEQ ID NOS. 30 to 31 Reference Example 1 : Preparation of Recipient PR4KS for Conjugal Transfer TABLE 30 Acinetobacter Acinetobacter Rhodococcus erythropolis PR4 (Biological and genetic haemolyticus junii resource division of The National Institute of Technology ATCC17906 ATCC17908 and Evaluation ; Accession number : NBRC 100887 ) was Acinetobacter haemolyticus 95 % modified by a method described in JP - A No . 2011 -200133 , ATCC17906 and a derivative strain which exhibits resistance to 120 mg / l Acinetobacter junii ATCC17908 95 % chloramphenicol and is deficient of the kanamycin resistant 40 gene was produced and named PR4KS strain . Specifically , to enhance the chloramphenicol resistance, a SEQ ID NOS. 42 to 44 natural mutation was caused by subculturing the strain PR4 while the concentration of chloramphenicol in MYK TABLE 31 45 medium ( 0 . 5 % polypeptone , 0 . 3 % bact yeast extract , 0 . 3 % Saccharomyces Saccharomyces Saccharomyces malt extract , 0 . 2 % KH2PO4, 0 . 2 % K2HPO4) is gradually cerevisiae cerevisiae paradoxus increased from 10 mg/ mL to 120 mg/ mL . Accordingly , the NBRC1136 NBRC2347 NBRC10609 derivative strain RhCmSR - 09 strain having resistance to 120 Saccharomyces 100 % 98 % mg/mL chloramphenicol was obtained . cerevisiae NBRC1136 50 Saccharomyces 100 % 98 % Subsequently , the RhCmSR -09 strain was admixed with cerevisiae NBRC2347 the E . coli strain described in JP - A No . 2011 -200133 , which Saccharomyces 98 % 98 % contains plasmid PKM043 for introducing kanamycin resis paradoxus tant gene deletion mutation , at ratio of 1 : 1. After introducing NBRC10609 55 the PKM043 to the RhCmSR - 09 strain by conjugal transfer, with culture in the MYK agar medium ( 0 . 5 % polypeptone , 0 . 3 % bact yeast extract, 0 . 3 % malt extract , 0 .2 % KH ,PO . . SEQ ID NOS. 45 to 46 0 . 2 % K2HPO4, 1 .5 % agar) containing kanamycin sulfate at 200 mg/ L and chloramphenicol at 50 mg/ L , the homologues TABLE 32 60 recombinant strain having the pKM043 inserted to the Candida genome of the RhCmSR -09 strain was obtained . The Candida utilis parapsilosis homologous recombinant strain was cultured in the MYK NBR186ACC2219 agar medium containing 10 % sucrose to obtain from the Candida utilis NBRC1086 81 % resulting colonies a derivative strain which becomes a Candida parapsilosis ATCC22019 81 % 65 kanamycin - sensitive strain , that is , the mutant derivative strain PR4KS strain deleted with the kanamycin resistant gene . US 10 , 294 , 500 B2 115 116 Reference Example 2 : Cloning of LigD Homolog was observed . With regard to the aforementioned steps for Gene and Production of Plasmid for Gene Deletion producing the plasmid PTJ002 , Wizard Genomic DNA Puri fication Kit (manufactured by Promega ) was used for the LigD homolog gene ( Accession No .: YP _ 002767969 ) of genome extraction form the PR4 strain , Gel/ PCR Purifica the PR4KS strain was the target gene . After amplification by 5 tion Kit (manufactured by FAVORGEN ) was used for puri PCR of about 5 . 4 kb DNA containing peripheral sequences fication of the DNA fragment digested with restrictions of the LigD gene , it was cloned into the plasmid vector enzymes and the PCR product, DNA Ligation Kit < Mighty pK19mobsacB1 described in JP - A No . 2011 - 200133 , in Mix > (manufactured by Takara Bio Inc . ) was used for which the sacB gene is introduced in the downstream or in conjugation between DNAs , and OIAprep miniprep kit the same direction of the kanamycin resistant gene , to obtain 10 (manufactured by OIAGEN ) was used for the plasmid the plasmid PTJ001. Conditions for PCR are as follows . extraction . Reference Example 3 : Production of the PR4KS Primers GB - 138 : Derivative Strain in which LigD Homolog Gene is ( SEO ID NO . 94 ) Deleted 5 ' - GGCCTGCAGGTACCGATCATCACCATCGGTGTC - 3 ' By having the E . coli (Escherichia coli) S17 - 1àpir which GB - 139 : has been transformed with PTJ002 as a donor and PR4KS ( SEO ID NO . 95 ) obtained by the method of Reference example 1 as a 5 ' - GGTCTAGACTGAGCAGTGTTCCAATGCG - 3 ' 20 recipient, conjugal transfer was performed according to the Composition of Reaction Solution method described in JP - A No. 2011 - 200133 to obtain by Sterilized water 22 ul homologous recombination a derivative strain of thirteen 2xPrimeSTAR (manufactured by Takara Bio Inc .) 25 ul strains generated in which LigD homolog gene is deleted . GB - 138 1 ul One strain was selected from the above derivative strain GB - 139 1 ul 25 with deletion and named PR4KSAligD derivative strain . PR4KS genome ( 50 ng/ ul) 1 ul Total volume 50 Example 26 : Production of Plasmid pLK005 for Temperature Cycle Bacteria of Genus Rhodococcus and Plasmid 35 Cycles of the Reaction Including 98° C . for 10 Seconds, pSJ201 for Expression of Nitrile Hydratase Using it 55° C . for 10 Seconds, and 72° C . for 120 Seconds 30 Plasmid PTJ002 for deletion of LigD homolog gene in (1 ) Obtainment and Analysis of PLK005 which the full - length sequence of the LigD homolog gene By using pK4 ( see , JP - A No. 5 -64589 ) , Rhodococcus sp . (about 2 . 3 kb ) inside PTJ001 is deleted to maintain only the N775 (Accession number of FERM BP - 961, Patent Micro upstream and downstream sequences of the LigD homolog organisms Depository Center of National Institute of gene was produced (see , FIG . 4 ) . PTJ002 was produced by 35 Advanced Industrial Science and Technology , Independent transforming E . coli JM109 strain with a PCR product not Administrative Institution ) was transformed by electropora containing the LigD homolog gene , which has been obtained tion . The obtained transformant was inoculated to 10 ml by amplifying internal sequence of PTJ001 using primers MYK medium and cultured at 30° C . for 1 day . According GB - 140 and GB - 141 designed so as to contain both the to UV irradiation on it in a clean bench , a mutagenic sequences near the start codon and the sequence near the 40 treatment was performed . The culture after the mutagenic stop codon of the Ligd homolog gene as a target and to treatment was applied to an agar medium containing 50 to elongate respectively in the upstream direction from the start 400 ug /ml kanamycin and cultured at 30° C . for 3 days . codon or in the downstream direction from the stop codon , Each of the plural colonies shown on the agar medium to yield a cyclic DNA ( see , FIG . 4 ) . PCR conditions are as was cultured on the MYK medium and the plasmid was follows. 45 recovered from the transformant . By using the recovered plasmid , Rhodococcus sp . N775 was transformed again and an examination was made to see if there is any increase in Primers the resistance of the transformant to kanamycin . As a result , GB - 140 : ( SEO ID NO . 96 ) several strains of the transformant were observed with GAGGAAATGGTCACAGGGCGAGAATAGGTTG 50 clearly increased kanamycin resistance . As a result of examining the nucleotide sequence of the GB - 141 : plasmid for which increased kanamycin resistance is ( SEO ID NO . 97 ) observed , a change in the sequence in the upstream region GCCCTGTGACCATTTCCTCATTGTGCTGG of the kanamycin resistant gene of pK4 was observed Composition of Reaction Solution 55 ( eight- nucleotide sequence of GTTGTAGG is repeated ) . Sterilized water 22 ul The plasmid observed with increased kanamycin resistance 2xPrimeSTAR (manufactured by Takara Bio Inc . ) 25 ul was named pLK005. GB - 140 1 ul ( 2 ) Production of PSJ040 GB - 141 1 ul Plasmid pSJ034 was produced from plasmid pSJ023 PTJ001 1 ul 60 according to the method described in JP - A No. 10 -337185 . Total volume 50 ul There are three sites for the restriction enzyme EcoRI in Temperature Cycle PSJ034 , and plasmid PSJ040 was produced in which one of 30 Cycles of the Reaction Including 98° C . for 10 Seconds, those sites is modified to Spel. With regard to the production 50° C . for 10 Seconds, and 72° C . for 180 Seconds method , pSJ034 was partially degraded by the restriction Upon the completion of PCR , determination of the frag - 65 enzyme EcoRI and blunting of the restriction sites were ment was performed by using 1 ul sample and 0 . 7 % agarose performed by using Takara Blunting Kit . In the presence of gel electrophoresis . As a result, the fragment amplification Spel linker , the ligation reaction was performed and the E . US 10 ,294 ,500 B2 117 118 coli JM109 strain was transformed by using the reaction Purification Kit (manufactured by FAVORGEN ) and used solution . After culturing the transformant, the plasmid was for the reaction using In - Fusion HD Cloning Kit which is extracted and the plasmid inserted with Spel linker was described below . selected . Among the three EcoRI sites in PSJ034 , the one (2 ) Ligation Between Vector and Target Fragment Using having Spel linker inserted to EcoRI site, which is located 5 in - Fusion HD Cloning Kit and Transformation downstream of the kanamycin resistant gene, was named By using In -Fusion HD Cloning Kit, ligation between the PSJ040 . above fragment and vector was performed . The reactions ( 3 ) Construction of PSJ201 conditions are described below . By digesting PLK005 with HindIII, a fragment of about Composition of Reaction Solution 2 . 1 kb was produced . Meanwhile , by digesting pSJ040 with 10 5x In - Fusion HD Enzyme Premix 2 ul HindIII , a fragment of about 9 . 8 kb was produced . By using Vector fragment 1 . 5 ul those two fragments , the ligation reaction was performed DNA fragment 11 ul and the E . coli JM109 strain was transformed by using the DNA fragment 22 ul reaction solution . After culturing the transformant, the plas - 15 D . W . 3 .5 ul mid was extracted and , as a result of determining the Total 10 ul nucleotide sequence , the plasmid having the mutated After incubating for 15 minutes at 50° C ., the above sequence derived from PLK005 ( repetition ofGTTGTAGG ) reaction solution was cooled on ice and used for transfor but basically the same sequence as pSJ040 other than that mation of E . coli JM109 strain . Selection of the E . coli was named pSJ201. 20 transformant was performed on an LB agar medium con taining kanamycin sulfateat5mghereinbew, LB Example 27: Production of Derivative Strain in Km50 agar medium ) . From the obtained transformant , a which RE acd1 /RE echA /RE _ hchA /RE _ mmsB plasmid was prepared by using Mini prep Kit (manufactured gene of PR4KSAligD Derivative Strain is Deleted by QIAGEN ) to obtain the target plasmid . Confirmation of 25 the plasmid was performed by examining the fragment size ( 1 ) Production of Plasmid for Gene Deletion Using in after treatment with the restriction enzyme Xbal and the Fusion Method sequence of the linking region between the insertion frag Production of plasmid for gene deletion using In - Fusion ment and the vector. The target plasmid was named HD Cloning kit (manufactured by Takara Bio Inc . ) which MMA302 . uses RE _ acd1 /RE _ echA / RE _ hchA /RE _ mmsB of PR4KS 30 ( 3 ) Production of Homologous Recombinant Derivative strain as a target gene was performed (see , FIG . 5 ). Strain of PRAKSAligD Derivative Strain and Derivative DNA of the upstream and downstream of the target gene S train with Deleted Gene was amplified by PCR . PCR conditions are as described To 20 ul of competent cells of PRAKSAligD strain , 1 ul of below . PMMA302 was added and incubated on ice for 10 minutes. Entire volume of the solution after the incubation was Primer for fragment 1 transferred to an ice - cooled electroporation cuvette ( 0 . 1 cm ) MMA - 061 : and applied with high voltage of 1 . 5 kV (2002 ) . Immedi ( SEQ ID NO . 98 ) ately thereafter, 600 ul of the LB liquid medium was added CGACTCTAGAGGATCGCTCAGTACATCTACGAGAC 40 and kept for 6 hours at 30° C . 200 ul was added to a LB Km10 agar medium and cultured at 30° C . for 4 days . The MMA - 062 : ( SEQ ID NO . 99 ) grown colonies were streaked on a LB Km10 agar medium AGTGTGAGGAAAGTGTTCCGATCAGTTCAT and , after growing for 4 days, colony PCR was performed Primer for fragment 2 according to the following conditions to confirm the MMA - 063 : 45 homologous recombinant derivative strain . ( SEO ID NO . 100 ) CACTTTCCTCACACTCGTCGAGAGTATGAG Primer MMA - 064 : MMA - 069 : ( SEQ ID NO . 101 ) ( SEO ID NO . 102 ) CGGTACCCGGGGATCAGCGCGACGAACAACGAGAC 50 GCGCATCTACAAGGAAGAGATC Composition of Reaction Solution MMA - 070 : Template (PR4 wild type genomic DNA ) 1 ul ( SEQ ID NO . 103 ) 2x PrimeSTAR Max Premix (manufactured by Takara GCGACGCTCATCGAGATCTC Inc . ) 25 ul 55 Composition of Reaction Solution Fw Primer (20 uM ) 1 ul Template 4 . 0 ul Rv Primer (20 uM ) 1 ul 2x Mighty Amp Buffer (manufactured by Takara Inc .) 5 . 0 D . W . 22 ul Total 50 ul Fw Primer (20 uM ) 0 .25 ul Temperature Cycle 60 Rv Primer ( 20 UM ) 0 .25 ul 30 Cycles of the Reaction Including 98° C . for 10 Seconds, D .W . 0 .3 ul 60° C . for 10 Seconds , and 72° C . for 120 Seconds Mighty Amp DNAPolymerase (manufactured by Takara Upon the completion of PCR , determination of the frag - Inc .) 0 .2 ul ment was performed by using 1 ul sample and 0 . 7 % agarose Total 10 . 0 ul gel electrophoresis . As a result, the fragment amplification 65 Temperature Cycle was observed . The PCR product ( fragment 1 and fragment 30 Cycles of the Reaction Including 98° C . for 10 Seconds 2 ) was subjected to buffer exchange by using Gel/ PCR and 68° C . for 180 Seconds US 10 , 294 , 500 B2 119 120 Colonies recognized to be a homologous recombinant Composition for Restriction Enzyme Treatment Reaction derivative strain was suspended in 200 ulof LB medium and PCR amplified fragment 20 ul 100 ul was applied to LB + 10 % Sucrose agar medium 10xM 5 ul followed by culture for 3 days. From the grown colonies , 0 . 1 % BSA 5 ul those having kanamycin sensitivity were selected and dele - 5 Xbal (manufactured by TAKARA Inc. ) 2 ul tion of the target gene was confirmed from them by colony Sse83871 (manufactured by TAKARA Inc . ) 2 ul PCR . As a result , a strain in which 4 genes , that is , RE _ acdi , D . W . 16 ul RE _ echA , RE _ hchA , RE _mmsB , are deleted from the Total 50 ul PRAKSAligD derivative strain was obtained , and it was Composition for SAP Treatment Reaction 10 Vector fragment (pSJ201 ) 44 ul named DMA008 strain . 10xSAP Buffer 5 ul Example 28: Production of Plasmid for Expression SAP (manufactured by Promega ) 1 ul Total 50 ul of ACD or AAT in Bacteria of genus Rhodococcus Composition for Ligation Reaction 15 Vector fragment 4 ul A plasmid for expression of ACD or AAT in microbes that Target fragment 1 ul belong to the genus Rhodococcus was produced . Ligation Mix (manufactured by TAKARA Inc. ) 3 ul In order to produce a plasmid for expressing each of ACD Total 8 ul or AAT, DNA of each gene was amplified by PCR . PCR By using the reaction solution for ligation as a mixture of conditions are as described below . 20 the above composition , transformation of E . coli JM109 strain was performed . From the transformant obtained , the Primer for RE acdi plasmid was extracted , and by performing agarose electro MMA - 114 phoresis after treatment with the restriction enzyme Xbal (SEQ ID NO . 126 ) and Sse83871, it was confirmed that the fragment of a GGTCTAGAATGTTTACTCTGACCGATGACGAGCG 25 desired size is inserted . Further , according to nucleotide sequence analysis of a linking region between the insertion MMA - 022 ( SEQ ID NO . 115 ) fragment and vector, it is confirmed to be the target plasmid . GGCCTGCAGGCCGTCACGCTTTTCGATCAATAC The plasmid incorporated with RE _ acd1 gene for MPAAT1 gene was named PMMA401 and PAAT301, respectively . Primer for MPAAT1 MMA - 109 30 ( SEO ID NO . 127 ) Example 29: Production of Plasmid for Expression GGTCTAGAATGAAATCATTCTCAGTACTTCAG of Both ACD and AAT for Transformation of Genus Rhodococcus MMA - 045 ( SEO ID NONO . 8383 )) 35 Plasmid production was performed for producing a meth CGGTACGCGCGGATCTTCCAGAG acrylic acid ester using microbes of the genus Rhodococcus, Template for RE _ acd1 which has been obtained in Example 27 . Rhodococcus erythropolis PR4 wild type strain genomic At the downstream of RE _ acdl gene of plasmid DNA PMMA401 for expressing RE _ acdl , the “ nitrilase pro Template for MPAAT1 40 moter + MPAAT1 gene ” fragment, which has been obtained plasmid PAAT001 ( see Example 45 ) by PCR using as a template the plasmid PAAT301 for Composition of Reaction Solution expression of MPAAT1 gene , was inserted . Template 1 ul Amplification of the “ nitrilase promoter +MPAAT1 gene” 2x PrimeSTAR Max Premix (manufactured by TAKARA fragment was performed as described below . Inc . ) 25 ul 45 Fw Primer ( 20 uM ) 1 ul Primer Rv Primer ( 20 UM ) 1 ul MMA - 133 ( Sse - ProFw ) : D . W . 22 ul ( SEO ID NO . 104 ) Total 50 ul TGACCTGCAGGTGCACTCCGCTGCGACATGTATCGA Temperature Cycle : 50 30 Cycles of the Reaction Including 98° C . for 10 Seconds, MMA - 131 ( Sse - 001Rv ) : 60° C . for 10 Seconds, and 72° C . for 60 Seconds ( SEO ID NO . 105 ) Upon the completion of PCR , the PCR product was ACTCTAGCCTGCAGGTCATTGACTAGTTGATCTAAGGTTGTTACA subjected to buffer exchange by using Gel/ PCR Purification Composition for PCR Reaction Kit (manufactured by FAVORGEN ) and the obtained frag - 55 Template (PAAT301 ) 1 ul ment was treated with restriction enzymes Xbal and 2x PrimeSTAR Max Premix (manufactured by TAKARA Sse83871 . Further , PSJ201 produced in Example 26 was also Inc . ) 10 ul treated with the restriction enzymes Xbal and Sse83871. Fw Primer ( 10 uM ) 0 .6 ul After performing ethanol precipitation , it was dissolved Rv Primer ( 10 ?M ) 0 . 6 ul again with sterilized water and subjected to the Shrimp 60 D . W . 7 . 8 ul Alkaline Phosphetase (SAP ) treatment. The PCR fragment Total 20 ul and a large fragment derived from pSJ201 were purified by, Temperature Cycle: after 0 . 7 % agarose gel electrophoresis , using Gel/ PCR Puri - 30 Cycles of the Reaction Including 98° C . for 5 Seconds, fication Kit (manufactured by FAVORGEN ). The reaction 60° C . for 5 Seconds, and 72° C . for 45 Seconds conditions for the restriction enzyme treatment, conditions 65 The “ nitrilase promoter + MPAAT1 gene ” fragment as for SAP treatment, and the ligation reaction conditions are as obtained above was treated with the restriction enzyme follows. Sse83871 . Meanwhile , PMMA401 was also subjected to the US 10 ,294 ,500 B2 121 122 SAP treatment after being treated with Sse83871. Those cal flask ) , and under aerobic conditions at 30° C . with a DNA fragments were purified , after performing 0 .7 % aga - rotary shaker (230 rpm ) , culture was performed for 3 days rose gel electrophoresis , by using Gel/ PCR Purification Kit (main culture ) . (manufactured by FAVORGEN ) . The conditions for restric After the main culture , 40 ul of the main - broth was tion enzyme treatment reaction and the conditions for liga - 5 transferred to a conical tube with volume of 50 mL and tion reaction are as described below . subjected to centrifugal separation ( 12000 rpm , 10 min ) to Composition for the Restriction Enzyme Treatment Reac obtain cells . The following reaction was performed by using tion (AAT Fragment) the cells . To a conical tube with volume of 50 mL , 1 mL of PCR amplification fragment 40 ul the reaction solution was added and the reaction was per 10 formed for 24 hours under aerobic conditions at 30° C . with 10xM buffer 5 ul a rotary shaker ( 180 rpm ) . 0 . 1 % BSA 4 ul Composition of Reaction Solution Sse83871 (manufactured by TAKARA Inc .) 1 ul OD630 = 10 bacterial cells ( final concentration ) Total 50 ul 5 . 0 g /1 2 -oxoisovaleric acid ( final concentration ) Composition for the restriction enzyme treatment reaction" 1515 50 mM phosphate buffer / pH 7 . 0 ( final concentration ) ( Vector fragment) After the reaction , an appropriate amount of the superna PMMA401 ( vector) 3 ul tant of the reaction solution was collected and subjected to 10xM buffer 4 ul the analysis according to the following HPLC conditions. As 0 . 1 % BSA 4 ul a result, methacrylic acid of 12 ppm was detected . AP 1 ul 20 Conditions for HPLC Analysis Sse83871 (manufactured by Promega ) 1 ul Apparatus: Waters 2695 D . W . 27 ul Column : ICsep USP L - 17 , 04 .0 mmx250 mm Total 40 ul Mobile phase : 0 .01 N H2SO4 Composition for Ligation Reaction Flow amount: 0 .35 ul/ min pMMA401 1 ul 25 Run time: 20 min Insertion fragment 2 ul Column temperature : 40° C . Ligation Mix (manufactured by TAKARA Inc. ) 3 ul Sample temperature : 20° C . Total 6 ul Detection : UV 210 nm By using the ligation reaction solution which has been mixed according to the above composition , transformation 30 Example 31: Production of Butyl Methacrylate of E . coli JM109 strain was performed . From the obtained Using Genus Rhodococcus transformant, the plasmid was extracted , and by performing DMA008 strain obtained from Example 27 ( 3 ) was trans agarose electrophoresis after treatment with the restriction formed with each of the plasmid PACDAAT1, PACDAAT2 , enzyme Sse83871, it was confirmed that the fragment ofOsa a 3536 PACDAAT3 , PACDAAT4 , PACDAAT6 , and PACDAAT8 . desired size is inserted . Further, according to nucleotide The obtained recombinant (DMA008 / PACDAAT1 , sequence analysis of a linking region of the insertion frag - DMA008 /PACDAAT2 . DMA008 /PACDAAT3 , DMA008 / ment of the obtained plasmid , it was confirmed to be the PACDAAT4 , DMA008 /PACDAAT6 and DMA008 / target plasmid , and the plasmid was named PACDAAT1 . By PACDAAT8 ) was used for producing a methacrylic acid using the same method as described above , total six plas - 40 ester based on the resting cell reaction . One platinum loop mids with a different sequence for co - expressing ACD and inoculation was made to 2 mL of LB Km10 liquid medium AAT (PACDAAT2 , PACDAATU , PACDAAT4 , (wassermann test tube ) , and cultured for 2 days under PACDAAT6 , and PACDAAT8 ) were prepared ( see , FIG . 6 ) . aerobic conditions at 30° C . using a rotary shaker ( 180 rpm ) (pre - culture ) . 1 ul of the pre - broth was inoculated to 100 mL Example 30 : Production ofMethacrylic Acid Using 45 LB Km10 (100 ulmedium /500 ul volume conical flask ), and Genus Rhodococcus under aerobic conditions at 30° C . with a rotary shaker ( 230 rpm ) , culture was performed for 3 days (main culture ) . DMA008 strain obtained from Example 27 ( 3 ) was trans - Meanwhile, as a control, plasmid DMA008 /pLK005 was formed with the plasmid PMMA401. The obtained recom used . binant (DMA008 /pMMA401 ) was used for producing meth - 50 After the main culture , 40 ul of the main - broth was acrylic acid based on the resting cell reaction . One platinum transferred to a conical tube with volume of 50 mL and loop inoculation was made to 2 mL of M9/ Frc /Km10 liquid subjected to centrifugal separation ( 12000 rpm , 10 min ) to medium shown below (wassermann test tube) , and cultured obtain cells . The following reaction was performed by using for 2 days under aerobic conditions at 30° C . using a rotary the cells. To a glass sample bottle with volume of 10 mL , 1 shaker ( 180 rpm ) (pre -culture ). 55 mL of the reaction solution was added and the reaction was M9/ Frc /Km10 Liquid Medium performed for 18 hours under aerobic conditions at 30° C . 6 .0 g/ l Na2HPO4 with a rotary shaker ( 180 rpm ) . 3 .0 g /1 KH2PO4 Composition of Reaction Solution 0 . 5 g /1 NaCl OD630 = 10 bacterial cells ( final concentration ) 0NC1 . 60 5 . 0 g / 1 2 -oxoisovaleric acid ( final concentration ) 1 mM MgSO4.7H20 40 mM alcohol ( final concentration ) 1 mM CaCl2 50 mM phosphate buffer / pH 7 . 5 ( final concentration ) 0 .01 g /1 Thiamine As an alcohol, n -butanol was used . 2 . 0 g / l Fructose After the reaction , 1 mL acetonitrile was added to the 10 ug /1 Kanamycin 65 reaction solution and mixed well . After filtration using a 1 ul of the pre -broth was inoculated to 100 mLM9/ Frc / syringe filter DISMIC /hole diameter 0 . 45 um (manufactured Km10 liquid medium ( 100 ul medium /500 ul volume coni - by ADVANTEC ), analysis was made by the HPLC analysis US 10 , 294 , 500 B2 123 124 described in Example 49 . In Table 34 , the results of ana - 50 uL of genomic DNA was obtained by using Wizard lyzing the product after 18 hours are shown . Genomic DNA Purification Kit (manufactured by Promega ). Production of Butyl Methacrylate by Rhodococcus Recom < Cloning into Expression Vector > binant which Co -Expresses ACD and AAT By using the genomic DNA as a template and designing 5 an oligonucleotide such that it is in the form in which a TABLE 34 restriction enzyme recognition site , which is easily intro ducible to an expression vector , is added , a DNA fragment Recombinant Production amount of butyl methacrylate (UM ) containing a gene presumed to encode ACD was produced DMA008 /pLK005 by PCR method . DMA008 /PACDAAT1 7 .51 DMA008 / PACDAAT2 2 . 06 DMA008 / PACDAAT3 4 . 34 TABLE 36 DMA008 / PACDAAT4 0 . 46 DMA008 /PACDAAT6 2 . 18 Isobutyryl COA Obtained plasmid DMA008 / PACDAATS 0 .52 - 15 dehydrogenase _ PCR Primer pTrc99A homolog Forward Reverse Vector PET Vector Example 32 : Production of Methacrylic Acid Ester PA acd1 MMA -003 MMA -004 PMMA002 PMMA102 Using Genus Rhodococcus PA _ acd2 MMA -020 MMA- 006 PMMA003 PMMA103 PA acd3 MMA -018 MMA- 008 PMMA004 PMMA104 DMA008 strain obtained from Example 27 ( 3 ) was trans - 20 PA acd4 MMA -019 MMA - 010 PMMA005 PMMA105 formed with plasmid PACDAAT1. The obtained recombi nant (DMA008 / PACDAAT1) was used for producing a methacrylic acid ester based on the resting cell reaction . Further, as a control, plasmid DMA008 /pLK005 was used . Oligonucleotide primer By using the method described in Example 31, the recom - 25 MMA - 003 : binant was cultured to obtain the cells . ( SEO ID NO . 106 ) Composition of Reaction Solution 5 ' - GACCCATGGATTTCGACCTCACCGAAGAAC - 3 OD630 = 10 microbial cells ( final concentration ) MMA - 004 : 5 . 0 g /1 2 - oxoisovaleric acid ( final concentration ) ( SEQ ID NO . 107 ) 40 mM alcohol ( final concentration ) 30 5 ' - GCCCTGCAGGATGCGATGGTTCGCGGCGTTC - 3 ! 50 mM phosphate buffer /pH 7 . 5 ( final concentration ) MMA - 020 : As an alcohol, n -butanol , isobutanol, and 2 - ethylhexylal ( SEQ ID NO . 108 ) cohol were used . 5 ' - GGACATGTTTCGTGATCCGGAAACCCTGAAC - 3 ' After the reaction , 1 mL acetonitrile was added to the 25 MMA - 006 : reaction solution and mixed well . After filtration using a 35 ( SEO ID NO . 109 ) syringe filter DISMIC /hole diameter 0 . 45 um (manufactured 5 ' - GGCCTGCAGGCGAAGGATCGACGCTAGCCCTG - 3 ' by ADVANTEC ) , analysis was made by the HPLC analysis described in Example 9B . In Table 35 , the results of ana MMA - 018 : lyzing the product after 18 hours are shown. ( SEO ID NO . 110 ) Production of Methacrylic Acid Ester by Rhodococcus 40 51 - GGACATGTTTCCCTGCGAAGAAGAGATCCAG - 3 ! Recombinant which Co - Expresses ACD and AAT MMA - 008 : ( SEO ID NO . 111 ) TABLE 35 5 ' - GGCCTGCAGGCGCCGTTGCGGAAACGACGG - 3 ! MMA - 019 : Production amount (MM ) 45 ( SEQ ID NO . 112 ) Butyl Isobutyl 2 - Ehtylhexyl 5 ' - GGCCATGGTACCGAGAACCCTGTTCAGCTC - 3 ' Recombinant methacrylate methacrylate methacrylate MMA - 010 : ( SEQ ID NO . 113 ) DMA008 /pLK005 5 ' - GGCCTGCAGGCTGGACGAGGAGGTGCTCGC - 3 DMA008/ PACDAAT1 0. 01 0 .006 0 .02 50 Composition of Reaction Solution Sterilized water 22 uL Example 33 : Cloning of ACD Homolog (ACD ) 2x PrimeSTAR (manufactured by Takara Bio Inc .) 25 uL Gene from Pseudomonas aeruginosa PAO1 and Forward primer 1 uL Production of Recombinant with High Expression 55 Reverse primer 1 uL Genomic DNA 1 L Isolation of a gene which is involved with synthesis of Total volume 50 UL methacrylic acid in microbes of the genus Pseudomonas Temperature Cycle : having an ability to produce methacrylic acid was per - 30 Cycles of the Reaction Including 98° C . for 10 Seconds, formed . 60 55° C . for 15 Seconds, and 72° C . for 150 Seconds < Production of Genomic DNA > The band of the obtained amplified product was purified Pseudomonas aeruginosa PAO1 strain (NBRC 106052 ) with QIAquick Gel Extraction Kit ( QIAGEN ) . For grown in a LB agar medium ( 1 % bactotrypton , 0 . 5 % bac PA _ acd1 , the purified DNA was digested with the restriction toyeast extract , 0 . 5 % NaCl, 1 . 5 % agar ) was inoculated to 10 enzyme Ncol ( the restriction recognition site is included in mL of a LB liquid medium and cultured under shaking for 65 the forward primer ) and Sse83871 (the restriction recogni 15 hours at 37° C . Once the culture is completed , the cells tion site is included in the reverse primer ) . For PA _ acd2 to were collected by centrifuge from 2 mL of the culture , and PA _ acd4 , the restriction was made with the restriction US 10 ,294 ,500 B2 125 126 enzyme BspHI (the restriction recognition site is included in inoculated to 10 mL of a LB liquid medium and cultured the forward primer ) and Sse83871 ( the restriction recogni under shaking for 36 hours at 30° C . Once the culture is tion site is included in the reverse primer) . Separation was completed , the cells from 2 mL of the culture were collected performed by agarose gel electrophoresis , and the target by centrifuge , and 100 uL of genomic DNA was obtained by band was cut out from the gel and purified . For the purifi - 5 using Wizard Genomic DNA Purification Kit (manufactured cation , Gel/ PCR Purification Kit (manufactured by FAVOR - by Promega ) . GEN ) was used , and 30 uL of sterilized water was used for < Cloning into High Expression Vector > elution . By using the genomic DNA as a template and designing By mixing the purified DNA ( 5 uL ), vector pTrc99A (1 an oligonucleotide such that it is in the form in which a uL ) previously digested with Ncol and Sse83871, distilled 10 restriction enzyme recognition site , which is easily intro water ( 4 uL ) and solution I (DNA Ligation Kit ver. 2 ( Takara ducible to an expression vector , is added , a DNA fragment Bio Inc. ) ) ( 10 uL ) and incubating for 12 hours at 16° C . , containing a gene presumed to encode ACD was produced ligation between the PCR amplified product and the vector by PCR methodm . was made . After inoculating the E . coli JM109 strain to 1 mL of the 15 TABLE 37 LB medium and performing aerobic pre - culture at 37° C . for Isobutyryl COA Obtained plasmid 5 hours , 0 . 4 mL of the culture product was added to 40 mL of the SOB medium ( 2 % bactotrypton , 0 . 5 % bactoyeast dehydrogenase PCR Primer pTrc99A extract, 10 mM NaCl, 2 .5 mM KCl, 1 mM MgSO4, 1 mM Mg and cultured at 18C. for 20 hours . The culture homolog Forward Reverse Vector PET Vector product was collected by centrifugation , added with 13 mL RE acd1 MMA 021 MMA 022 PMMA009 pMMA109 of cooled TF solution (20 mM PIPES -KOH (pH 6 . 0 ) , 200 RE _ acd2 MMA 023 MMA 024 PMMA010 PMMA110 mM KC1, 10 mM CaCl2 , 40 mM MnC1_ ), and allowed to stand for 10 minutes at 0° C . After that, it was centrifuged again to remove the supernatant, and the precipitated E . coli 25 was suspended in 3 . 2 ul cooled TF solution . After adding Oligonucleotide primer 0 .22 ul dimethyl sulfoxide , it was allowed to stand for 10 MMA - 021 : minutes at 0° C . ( SEQ ID NO . 114 ) To 200 uL of the competent cells as prepared above , 10 uL 5 ' - GGACATGTTTACTCTGACCGATGACGAGCG - 3 ! of the ligation solution was added , maintained for 30 min - 30 utes at 0° C . , and applied with a heat shock at 42° C . for 30 MMA - 022 : seconds . After cooling at 0° C . for 2 minutes , 1 mL of the ( SEQ ID NO . 115 ) SOC medium ( 20 mM glucose , 2 % bactotrypton , 0 . 5 % 5 ' - GGCCTGCAGGCCGTCACGCTTTTCGATCAATAC - 3 bactoyeast extract , 10 mM NaCl, 2 . 5 mM KCI, 1 mM MMA - 023 : MgSO4, 1 mM MgCl2) was added and followed by culture ( SEO ID NO . 116 ) under shaking at 37° C . for 1 hour. 5 ' - CCACATGTCCGATTACCTTGCCACCGGAGC - 3 ! After the culture , each was applied in an amount of 100 MMA - 024 : ul to the LBAmp agar medium (LB medium containing ( SEO ID NO . 117 ) ampicillin 100 mg/ 1 and 1 . 5 % agar ) and further cultured at 51 - GGCCTGCAGGATCTTCTTGGGGTTCGTCACAAC - 3 ! 37° C . Plural colonies of the transformant which have been grown on the agar medium were cultured overnight at 37°C . 40 Amplification by a PCR reaction was performed in the on 1 . 5 ul LBAmp medium ( LB medium containing ampi- same manner as Example 33 . cillin at 100 mg/ l) . After collecting the cells , the plasmid The band of the obtained amplified product was purified DNA was prepared by using QIAprep Spin Miniprep kit with QIAquick Gel Extraction Kit ( QIAGEN ) and digested (manufactured by QIAGEN ) . with the restriction enzyme Pcil ( the restriction recognition The nucleotide sequence of the obtained recombinant 45 site is included in the forward primer ) and Sse83871 ( the plasmid DNA was determined by using CEQ DTCS Quick restriction recognition site is included in the reverse primer ). Start Kit and Fluorescence sequence CEO 2000XL DNA The following operations were performed like Example 33 Analysis (both manufactured by BECKMAN COULTER , to obtain plasmid PMMA009 and 010 having pTrc99A as a USA ) , and named plasmid PMMA002 to PMMA005 ( Table 36 ) . vector and plasmid PMMA109 and 110 having pET16b as a ACD homolog gene was also inserted to the pET16bh 50 vector ( Table 37 ). vector according to the same procedure as above , and the obtained plasmids were named PMMA102 to pMMA105 Example 35 : Production of Cell Extract from ( Table 36 ) . Meanwhile , since pET16b has no Sse83871 site , Recombinant in which ACD Homolog Gene is a linker containing the Sse83871 restriction site inserted to Expressed and Analysis of Protein Expression BamHI site of PET16b was prepared in advance , and it was » By using the expression plasmid containing ACD used as a vector. homolog gene produced in Example 33 and 34 , E . coli Example 34 : Cloning of ACD Homolog Gene from JM109 ( for PMMA002 to PMMA005 , PMMA009 and 010 ) Rhodococcus erythropolis PR4 Strain and or E . coli BL21 (DE3 ) ( for PMMA102 to PMMA105, Production of Recombinant with High Expression 60 pMMA109 and 110 ) was transformed . Transformed E . coli JM109 strains were cultured as described below . Isolation of a gene which is involved with synthesis of The recombinant E . coli was inoculated to LB medium methacrylic acid in microbes of the genus Pseudomonas was containing 1 mL of 100 ug /mL ampicillin and subjected to performed . pre - culture at 37° C . for 7 hours . 0 . 1 ul of the culture was < Production of Genomic DNA > 65 harvested and added to 100 ul of the same medium ( con Rhodococcus erythropolis PR4 strain (NBRC100887 ) taining 100 ug /mL ampicillin , 1 mM isopropyl- B - D - thioga grown in a LB agar medium (LB medium , 1 .5 % agar) was lactopyranoside (hereinbelow , IPTG )) and cultured under US 10 , 294 , 500 B2 127 128 shaking at 37° C . for 15 hours . From the obtained culture , TABLE 38 the cells were collected by centrifuge ( 3 , 700xg, 10 minutes , 4° C . ), washed with 10 mM sodium phosphate buffer ( pH Isobutyryl COA 7 .0 ), and suspended in the samebuffer solution . As a control dehydrogenase Activity ( U /mg strain , JM109 /pTrc99A was used . Vector homolog Recombinant protein ) pTrc99A PA _ acd1 JM109 /pMMA002 0 .63 The transformed E . coli BL21 (DE3 ) strain was cultured pire PA _ acd2 JM109/ pMMA003 0 as follows. The recombinant E . coli was added to LB PA acd3 JM109 /pMMA004 < 0 . 01 medium containing 1 mL of 100 ug /mL ampicillin and PA acd4 JM109/ pMMA005 0 .024 subjected to pre -culture at 37° C . for 14 hours . 0 .1 mL of the 10 RE _ acd1 JM109 /pMMA009 0 . 063 culture was collected , added to 100 mL of the samemedium RE _ acd2 JM109 /pMMA010 acdH JM109/ PMMA001 < 0 . 01 ( 100 ug /mL ampicillin ) , and cultured under shaking until pET19b PA _ acd1 BL21 (DE3 )/ pMMA102 0 . 44 OD at 37° C . becomes 0 . 3 . Then , IPTG was added to have PA acd2 BL21( DE3 )/ PMMA103 final concentration of 1 mM and it was cultured again under PA acd3 BL21 (DE3 ) /PMMA104 0 .010 shaking for several hours . From the obtained culture , the 15 PA _ acd4 BL21 (DE3 )/ pMMA105 < 0 . 01 RE _ acd1 BL21 (DE3 )/ pMMA109 0 . 009 cells were collected by centrifuge (3 ,700xg , 10 minutes, 4° RE acd2 BL21 (DE3 )/ pMMA110 C . ), washed with 10 mM sodium phosphate buffer (pH 7 . 0 ) , acdH BL21( DE3 ) / pMMA102 < 0 .01 and suspended in the same buffer solution . As a control strain , BL21 (DE3 ) /PET16b was used . 1 mL was collected from the obtained cell suspension , and the cell extract was prepared as follows. By using ultrasonic Example 37 : Cloning of BCKAD Gene, Production homogenizer VP - 300 (manufactured by DYTEC , Japan ), of Recombinant with High Expression , Preparation disruption was performed for 3 minutes with pulse output at of Cell Extract, and Analysis of Protein Expression interval of 1 second under ice cooling. Next, by performing 25 centrifuge ( 10 ,000xg , 5 minutes , 4° C . ), the obtained super- Gene cloning, production of an expression plasmid , and natant was collected as cell extract. production of a recombinant were performed in the same The protein analysis by SDS gel electrophoresis was manner as Example 33 . The DNA fragment containing the performed as follows. After appropriately diluting the cell entire gene operon which encodes the BCKAD complex extract, it was mixed with a sample solution for polyacry - 30 gene was produced , by using the genomic DNA of lamide gel electrophoresis ( 0 . 1M Tris -HCl (pH 6 . 8 ) , 4 % w / v Pseudomonas aeruginosa PAO1 strain as a template, by SDS, 0 .2M dithiothreitol, 20 % v / v glycerol, trace amount of PCR method with the primer shown in Table 39 . The bromophenol blue ) and heated at 98° C . for 5 minutes for obtained fragment was digested with the restriction enzyme denaturation . 10 % polyacrylamide gel was prepared , and by BspHI and Sse83871, and by inserting to the vector p Trc99A using 5 uL of the denatured sample per lane, the electro com phoretic analysis was performed ( FIG . 7 ) . binant plasmid (PWA108 and pWA008 ) was obtained . Example 36 : Measurement of ACD Activity of Cell TABLE 39 Extract 40 Gene of Obtained plasmid pTrc99A The ACD activity was measured according to production 2 - oxoisovaleric acid PCR Primer PET of methacrylyl- CoA having isobutyryl - CoA as a substrate . dehydrogenase complex Forward Reverse Vector Vector To a solution ( 1 . 84 mL ) containing 1 -methoxy - 5 -methyl - 45 BCKAD MMA 15 MMA 16 PWA108 PWA008 phenazinum methyl sulfate , flavine adenine dinucleotide , and isobutyryl- CoA at final concentration of 6 mM , 0 .4 mM , and 1 mM , respectively in 100 mM sodium phosphate buffer (pH 8 . 0 ) , 0 . 16 ul of the cell extract which has been obtained oligonucleotide primer like Example 35 was added to have 2 ul. After the reaction 50 MAA - 15 : at 37° C . for 30 minutes, protein removal was performed by ( SEO ID NO . 118 ) ultrafiltration using Centricut Ultramini W - 10 (manufac 5 ' - GGCCTGTCATGAGTGATTACGAGCCG - 3 ' . tured by Kurashiki Boseki) , the reaction was terminated and MAA - 16 : analysis by HPLC was performed under following condi ( SEO ID NO . 119 ) tions . The results are shown in Table 38 . It was able to 55 5 ' - CGGCCCTGCAGGTTCGCGGGAATCAGATGTGC - 3 ! confirm that high ACD activity is observed from PA acd1 The recombinant E . coli JM109 /pWA108 as obtained and RE _ acdl and methacrylyl- CoA can be produced from above was cultured in the same manner as Example 35 ( in isobutyryl - CoA by those enzymes. case of this recombinant, high protein expression was Conditions for HPLC Analysis observed even without adding IPTG , and thus, although the reason remains unclear , the culture was performed without Column: Inertsil ODS - 3V , 4 .6 mmx250 mm adding IPTG ) . In case of the recombinant E . coli BL21 Mobile phase : 30 % MeOH , 50 mM H2PO4, pH 5 . 7 ( DE3 ) /pWA008 , the culture was performed in the same Flow amount : 1 .0 ul/ min column temperature : 35º C . manner as Example 35 . Preparation of cell extract and Detection : UV 254 nm (210 nm ) 65 analysis of protein expression by SDS polyacrylamide gel Injection amount 10 ul reaction solution was diluted by 10 electrophoresis were performed in the same manner as times with a mobile phase and measured Example 35 . US 10 , 294 , 500 B2 129 130 Example 38 : Cloning of BCKAD Gene from Example 33 . From the obtained transformant, the recombi Bacteria of Genus Rhodococcus ( Cluster 1 ) and nant plasmid ( PMMA020 ) was obtained . Production of Recombinant with High Expression Example 40 : Measurement of Activity of Cell Because there was no knowledge about BCKAD gene 5 Extract of Recombinant which Expresses BCKAD from the bacteria of the genus Rhodococcus, by performing Gene at High Level homology search for the genome sequence of the Rhodo coccus erythropolis PR4 strain based on the amino acid The BCKAD activity was measured according to produc sequence of the BCKAD complex gene of Pseudomonas tion of methacrylyl- CoA having 2 - oxoisovaleric acid as a aeruginosa PAO1 strain , two candidate gene clusters were substrate . found . They are referred to as Cluster 1 and Cluster 2 . To a solution ( 0 . 7 mL ) containing MgCl2 , thiamine pyro With regard to Cluster 1 , the four genes consist of a acid , COA -SH , and DTT at final concentration of 1 mM , 0 . 2 nucleotide sequence represented by SEO ID NOS . 63 , 65 , mM , 1 mM and 2 mM , respectively in 100 mM sodium 67 , 69 like the Pseudomonas aeruginosa PAO1 strain , and 16 phosphate buffer (pH 7 . 0 ) , 0 . 2 ul of the cell extract obtained the amino acid sequence encoded by them are SEQ ID NOS . like Example 37 was added to have 0 . 9 ul. After the reaction 62 , 64 , 66 , 68 . With regard to Cluster 2 , three genes consist at 37° C . for 30 minutes by adding 0 . 1 mL of calcium of the nucleotide sequence represented by SEQ ID NOS. 71, 2 - oxoisovalerate ( final concentration 4 mM ), protein 73 , and 75 , respectively , and the amino acid encoded by removal was performed by ultrafiltration using Centricut them are SEO ID NOS . 70 , 72 , and 74 , respectively . 20 Ultramini W - 10 (manufactured by Kurashiki Boseki ) , the Cloning of BCKAD complex gene of Cluster 1 , produc - reaction was terminated and analysis by HPLC was per tion of an expression plasmid , and production of a recom - formed under following conditions. As a result , production binant were performed in the samemanner as Example 33 . of isobutyryl -CoA was observed at 0 . 83 mM or 0 . 88 mM for The DNA fragment containing the entire gene operon which JM109/ pWA108 and BL21 (DE3 )/ pWA008 , respectively. encodes the BCKAD complex gene was produced , by using 25 Conditions for HPLC Analysis the genomic DNA of Rhodococcus erythropolis PR4 strain Column : Inertsil ODS - 3V , 4 .6 mmx250 mm as a template , by PCR method with use of the primer shown Mobile phase: 35 % MeOH , 50 mM H3PO4, pH 5 .7 below . The obtained fragment was digested with the restric Flow amount: 1 . 0 ul/ min column temperature : 35° C . tion enzyme BspHI and Sse83871 , and by inserting to the Detection : UV 254 nm (210 nm ) vector pTrc99A in the same manner as Example 33, the 30 Injection amount 10 ul reaction solution was diluted by 10 recombinant plasmid (PMMA019 ) was obtained . times with a mobile phase and measured Example 41: Synthesis of Methacrylyl- CoA from oligonucleotide primer 2 -Oxoisovaleric Acid Using Mixture of Cell MMA - 187 : 35 ( SEO ID NO . 120 ) Extracts of Recombinant with High Expression of 5 ' - GGTCATGACTCTTGTCGAGCCCTTG - 3 BCKAD Gene and Recombinant in which ACD Homolog Gene is Expressed MMA - 140 : ( SEQ ID NO . 121 ) To a solution ( 0 .6 mL ) containing 1 mM MgCl2, 0 . 2 mM 5 ' - GACCTGCAGGTCCTCTTCTGGTCATGGTTC - 3 ! 40 thiamine pyro acid , 1 mM COA -SH , 2 mM DTT , 2 mM nicotine amide adenine nucleotide ( NAD ) , 0 . 04 mM flavine Example 39 : Cloning of BCKAD Gene from adenine dinucleotide ( FAD ), 2 mM valine , each in final Bacteria of Genus Rhodococcus (Cluster 2 ) and concentration in 100 mM sodium phosphate buffer (pH 7 . 0 ) , Production of Recombinant with High Expression 0 . 1 ul of the cell extract ( JM109/ pMMA002 and JM109 / 45 p WA108 ) obtained like Example 35 and 40 was added to Cloning of BCKAD complex gene of Cluster 2 , produc - have 0 . 8 ml. After the reaction at 37° C . for 30 minutes with tion of an expression plasmid , and production of a recom - addition of 0 . 1 mL calcium 2 - oxoisovalerate ( final concen binant were performed as follows. It was produced , by using tration of 4 mM ) , production of isobutyryl - CoA was con the genomic DNA of Rhodococcus erythropolis PR4 strain firmed by HPLC . By adding 0 . 1 mL of 1- methoxy - 5 as a template , by PCR method with use of the primer shown 50 methylphenazinum methyl sulfate ( final concentration 6 below . mM ) , the reaction was allowed to occur again for 3 hours . After the reaction , ultrafiltration was performed by using Oligonucleotide primer Centricut Ultramini W - 10 (manufactured by Kurashiki MMA - 188 : Boseki) . The reaction was terminated by removing proteins ( SEO ID NONO . 122122 )) 55 and analysis by HPLC was performed . As a result , produc 5 ' - AGGAAACAGACCATGATCGACAACCTCGATTA - 3 ! tion of methacrylyl- CoA of 2 mM was observed . MMA - 189 : ( SEQ ID NO . 123 ) Example 42 : Synthesis of Butyl Methacrylate 5 ' - CTTGCATGCCTGCAGGCTCACTCGTTCCTTTTTACAG - 3 ' 60 To a solution ( 1 . 84 mL ) containing 6 mM 1 -methoxy - 5 The above PCR product and vector pTrc99A which has methylphenazinum methyl sulfate , 0 . 4 mM FAD and 1 mM been digested in advance with the Ncol and Sse83871 were isobutyryl- CoA , each in final concentration in 100 mM linked by performing recombination of terminal sequence sodium phosphate buffer ( pH 8 . 0 ) , 0 . 16 ml of the cell extract using In - Fusion HD Cloning Kit (manufactured by Takara having an ACD activity , which has been obtained like Bio ) and following the manufacturer ' s instruction . 2 uL of 65 Example 35 , was added and adjusted to 2 ul. After the the infusion reaction solution was used and transformation reaction at 37° C . for 30 minutes , the analysis was performed of the E . coli JM109 was performed in the same manner as under HPLC conditions shown in Example 41 . As a result , US 10 , 294 ,500 B2 131 132 production ofmethacrylyl - CoA was observed while the peak Temperature Cycle : of isobutyryl- CoA has disappeared . 30 Cycles of the Reaction Including 98° C . for 10 Seconds, By using the reaction solution obtained as above in which 60° C . for 10 Seconds, and 72° C . for 120 Seconds methacrylyl- CoA is produced and reacting it with banana When the PCR is completed , the fragment was deter flesh having the AAT activity and n - butyl alcohol, produc - 5 mined based on 0 . 7 % agarose gel electrophoresis by using tion of butyl methacrylate was observed . Specifically , after 1 ul sample . As a result , amplification of a fragment of about removing the skin , banana flesh was sliced to a thickness of 1 . 1 kb was observed . The PCR product was briefly purified about 1 mm , and cut again to four pieces . 1 g of the sliced by Gel /PCR Purification Kit (manufactured by FAVOR banana , 0 . 9 mL of the reaction solution for synthesizing G EN ) to obtain a PCR fragment. methacrylyl -CoA , 0 . 1 mL of 3 . 5 M KCl solution , and 5 uL 10 Meanwhile , the plasmid p WA108 with the restriction of n -butyl alcohol were added to a 50 ml flask and sealed , enzyme Sse83871 was digested to have a vector -side frag and the reaction was allowed to occur at 30° C . for 2 hours. ment. Both fragments described above were used for the After the reaction , 150 UL was collected from of head space reaction shown below which uses In - Fusion HD Cloning ofthe 100 mL flask and analyzed according to the following 15 Kit . GC conditions. As a result, it was found that butyl meth Composition of Reaction Solution acrylate of 0 .015 mM was produced . 5x In - Fusion HD Enzyme Premix 2 ul Conditions for GC Analysis Vector- side fragment 1 .5 ul Column : DB -WAX , 30 mx0 . 32 mm PCR fragment 1 ul Column temperature : 50° C . 5 min - > 5º C . /min - > 100° C . 20 D . W . 3 . 5 ul ( total 15 min ) Total 10 ul Carrier gas : He After incubating for 15 minutes at 50° C ., the above Inject : 200° C . splitless ( sampling time of 1 min ) reaction solution was cooled on ice and used for transfor Detect: 250° C . FID Injection amount : 150 °L mation of E . coli JM109 strain . Selection of the E . coli Meanwhile , concentration of butyl methacrylate was cal- 25 transformant was performed on an LBAmp medium . From culated as follows: an aqueous solution with known con the obtained transformant, a plasmid was prepared by the centration is prepared , 2 mL of the aqueous solution is added method described in Example 33 to obtain the target plas to a 100 mL flask , and after incubation at 30° C . for 30 min mid . Confirmation of the plasmid was performed by exam collection is made from the head space with the same ining the fragment size after treatment with the restriction method as above, GC analysis is made, and then a calibra - 30 enzyme Xbal and Sse83871 and the sequence of the linking tion curve is constructed . region between the insertion fragment and the vector. The target plasmid was named PMMA201. Example 43 : Production of Plasmid for E . coli for ( 2 ) Production of Plasmid PMMA202 Co - Expressing BCKAD and ACD By using PMMA102 produced in Example 33 as a tem 35 platepla , PA _ acd1 gene was amplified by PCR method . PCR The DNA fragment which contains the ACD gene primers are as described below . ( PA _ acdl gene ) is inserted to the downstream of BCKAD of pWA108 produced in Example 37 by using In - Fusion HD Cloning kit (manufactured by Takara Bio Inc. ) to prepare the Fw Primer plasmid pMMA201 and PMMA202 for co -expressing 40 MMA - 042 BCKAD and ACD . Further , in the same manner , the DNA ( SEQ ID NO . 130 ) fragment which contains the BCKAD gene is inserted to the TAGAGTCGACCTGCACCTCTAGAAATAATTTTGTTTA downstream of PA _ acdl of pMMA002 produced in Rv Primer Example 33 to produce the plasmid PMMA203 and MMA - 040 PMMA204 for co - expression . Details are described below . 45 ( SEQ ID NO . 129 ) ( 1 ) Production of Plasmid PMMA201 GCTTGCATGCCTGCACAGCAGCCAACTCAGCTTCCTTT By using PMMA 102 produced in Example 33 as a tem In the same manner as above , the PCR fragment and the plate , PA _ acd1 gene was amplified by PCR method . PCR plasmid pWA108 digested with the restriction enzyme conditions are as described below . Sse83871 were ligated to each other by using In - Fusion HD 50 Cloning Kit and the target plasmid was named PMMA202. ( 3 ) Production of Plasmid PMMA203 Fw Primer By using p pWA008 produced in Example 37 as a MMA - 039 ( SEO ID NO . 128 ) template , BCKAD gene was amplified by PCR method . TAGAGTCGACCTGCACGAGATCTCGATCCCGCGAAAT PCR primers are as described below . 55 Rv Primer MMA - 040 Fw Primer ( SEQ ID NO . 129 ) MMA - 041 GCTTGCATGCCTGCACAGCAGCCAACTCAGCTTCCTTT EN131 ) CCATCGCATCCTGCACGAGATCTCGATCCCGCGAAAT Composition of Reaction Solution 60 Template (PMMA102 ) 1 ul Rv Primer 2x PrimeSTAR Max Premix (manufactured by TAKARA MMA - 040 Inc .) 25 ul ( SEO ID NO . 129 ) FW Primer ( 20 UM ) 1 ul GCTTGCATGCCTGCACAGCAGCCAACTCAGCTTCCTTT Rv Primer (20 °M ) 1 ul 65 The reaction method and obtaining the PCR fragment are D . W . 22 ul performed in the same manner as those described in ( 1 ) Total 50 ul above . US 10 ,294 , 500 B2 133 134 Meanwhile, the plasmid PMMA002 containing PA _ acd1 Reference Example 4 : ( In Vivo ) Synthesis of gene was digested with the restriction enzyme Sse83871 to Methacrylyl- CoA from 2 -Oxoisovaleric Acid Using give a vector- side fragment . In the same manner as above Recombinant E . coli in which BCKAD and ACD ( 1 ) , both fragments were subjected to the reaction using ar Expressed In - Fusion HD Cloning Kit , and transformation of E . coli 5 JM109 strain was performed by using the reaction solution . By using the recombinant E . coli in which BCKAD and A plasmid was obtained from the obtained transformant, and ACD are expressed , synthesis of methacrylic acid or meth in the same manner as ( 1 ) above , plasmid confirmation was acrylyl- CoA from 2 - oxoisovaleric acid was performed . carried out. The target plasmid was named PMMA203 . JM109/ PMMA201 strain , JM109 /pMMA202 strain , ( 4 ) Production of Plasmid PMMA204 10 JM109 /pMMA203 strain and JM109 /pMMA204 strain were By using pWA008 produced in Example 37 as a template , inoculated to LBAmp medium ( ampicillin 100 mg/ l ) con taining 1 ml of 100 ug/ mL ampicillin and pre -cultured at 37° BCKAD gene was amplified by PCR method . PCR primers C . for 6 hours . 0 . 1 mL of the culture was collected and added are as described below . to the same medium ( containing 1 mM IPTG ) and cultured 15 under shaking at 37° C . for 18 hours . The obtained culture Fw Primer was collected in an amount of 10 mL and added to 90 mL MMA - 043 of the LBAmp medium containing 0 . 5 % calcium 2 -oxois ( SEO ID NO . 132 ) ovaleric acid , followed by culture under shaking at 37° C . CCATCGCATCCTGCACCTCTAGAAATAATTTTGTTTA for 25 hours . Once the culture is completed , the culture was Rv Primer 20 filtered and analysis was made by HPLC according to the MMA - 040 following conditions . ( SEQ ID NO . 129 ) Conditions for HPLC Analysis GCTTGCATGC??GCACACCAGCCAACTCAGC??????? Column : Wakopak Wakobeads- T- 132 -E , 07 .8 mmx300 mm The reaction method and obtaining the PCR fragment are 25 Mobile phase : 0 . 1 % phosphoric acid performed in the same manner as those described in ( 1) Flow amount: 0 . 5 ul/ min above . In the same manner as above, the PCR fragment and column temperature : 40° C . the plasmid PMMA002 which has been digested with the Detection : UV 210 nm restriction enzyme Sse83871 were ligated to each other by Injection amount: 5 ul using In - Fusion HD Cloning Kit and the target plasmidmid was 30 As a result , production ofmethacrylic acid and methacry named PMMA204 . lyl- CoA was not observed but the production of isobutyric acid of 14 . 2 mM was observed . Thus , from the in vivo Example 44 : (In Vitro ) Synthesis of reaction in E . coli, it was shown to be impossible to have Methacrylyl- CoA from 2 -Oxoisovaleric Acid Using synthesis of methacrylic acid or methacrylyl- CoA . Cell Extract of E . coli Recombinant in which BCKAD and ACD are Expressed 35 Concentration of Isobutyric Acid Produced TABLE 40 Cell extract of the JM109/ pMMA204 strain was obtained in the samemanner as the method described in Example 35 . Name of strain Concentration of isobutyric acid (MM ) As a control strain , JM109 / pTrc99A was used . With the 40 JM109 /pMMA201 13 . 5 samemethod as the method described in Example 40 , 0 . 1 ml JM109 /pMMA202 13 . 4 of calcium 2 - oxoisovalerate ( final concentration of 4 mM ) JM109 /pMMA203 13 . 4 was added to the cell extract of JM109/ pMMA204 . After the JM109 /PMMA204 14 . 2 reaction at 37° C . for 30 minutes , production of isobutyryl COA was confirmed by HPLC , and 0 . 1 mL of 1 -methoxy - 45 5 -methylphenazinum methyl sulfate ( final concentration 6 Reference Example 5 : Synthesis of Methacrylic mM ) was added . The reaction was allowed to occur again for Acid from 2 -Oxoisovaleric Acid Using 3 hours . After the reaction , removal of the protein ( termi Recombinant E . coli resting Cells in which nating the reaction ) was performed by ultrafiltration using BCKAD and ACD are Expressed Centricut Ultramini W - 10 (manufactured by Kurashiki According to the samemethod as the method described in Boseki) . Then , analysis was made by HPLC according to the Example 35 , JM109/ pMMA201 strain was cultured . From following conditions . the obtained culture , the cells were collected by centrifuge Conditions for HPLC Analysis ( 3 , 700xg , 10 minutes, 4° C . ) , washed with 10 mM sodium Column : Capcell Pak ODS -UG120 (Shiseido ), particle 55 phosphate buffer (pH 7 . 0 ), and suspended in the same buffer diameter um , 2 . 0 mm I. D . x250 mm solution to obtain cell suspension . By using the cell suspen Mobile phase : 25 % MeOH , 50 mM H3PO4, pH 5 . 7 sion , about 3 ml of the resting cell reaction solution was Flow amount: 1 . 0 ul/ min prepared and the reaction was performed at 30° C ., 180 rpm Column temperature : 35° C . for 24 hours . Detection : UV 254 nm (210 nm ) 60 Composition of Resting Cell Reaction Solution Injection amount 10 ul reaction solution was diluted by 10 50 mM Sodium phosphate buffer solution (pH7 . 0 ) times with a mobile phase and measured 0 . 5 % Calcium 2 - oxoisovalerate As a result , production of 0 . 1 mM methacrylyl- CoA was OD10 Cell suspension . observed . It was possible to confirm that BCKAD and ACD Once the culture is completed , the culture was filtered and expressed by E . coli recombinant can produce methacrylyl- 65 analysis was made by HPLC according to the conditions COA from 2 - oxoisovaleric acid in the presence of described in Reference example 4 . As a result , production of 1 - methoxy - 5 -methylphenazinum methyl. methacrylic acid was not observed but production of isobu US 10 , 294 , 500 B2 135 136 tyric acid of 15 . 2 mM was observed . Similar to Reference TABLE 41 example 4 , it was shown to be impossible to have synthesis of methacrylic acid or methacrylyl- CoA using E . coli in the Production amount of isobutyl methacrylate resting cell reaction . Time Production amount of isobutyl methacrylate (mm )
? 0 . 19 Reference Example 6 : Cloning of ACD Gene 0 . 38 (acdH ) from Streptomyces coelicolor A3 (2 ), ??? 0 .45 Production of Expression Recombinant, Preparation of Cell Extract , Analysis of Protein Expression , and 10 Conditions for GLC Analysis Measurement of ACD Activity Column : DB -WAX , 30 mx0 . 32 mm Column temperature : 50° C .- 5 min - > 5º C ./ min -> 100° C . (total 15 min ) According to the method described in Example 33 to 32 , Carrier gas: He an expression recombinant in which acdH gene derived from Inject: 200° C . splitless ( sampling time 1 min ) Streptomyces coelicolor A3 ( 2 ) is added was prepared , and Detect: 250° C . FID injection amount : 150 ul preparation of cell extract, analysis of protein expression , Meanwhile , concentration of methacrylic acid ester was and measurement of ACD activity were performed . The calculated as follows: an aqueous solution with known results of analyzing the protein expression and the results of concentration is prepared first, 2 mL of the aqueous solution measuring the enzyme activity are shown in FIG . 7 ( C ) and is added to a 100 mL flask , and after incubation at 30° C . for 30 min , collection is made from the head space with the Table 38 , respectively . Meanwhile , oligonucloetides used same method as above , GC analysis is made , and then a for PCR to produce the plasmid are as follows. calibration curve is constructed . Reference Example 8 : Synthesis of Butyl Oligonucleotide primer Methacrylate by AAT MMA - 001 : 25 ( SEO ID NO . 124 ) The same process as Reference example 7 was performed 5 ' - CACCATGGACCACAAGCTCTCCCCCGAAC - 3 ' except that n -butyl alcohol is used instead of isobutyl MMA - 002 : alcohol. The results are shown in Table 42 . ( SEQ ID NO . 125 ) 5 ' - GCCCTGCAGGCTCAGCCCACCAGCCCCAAC - 3 30 TABLE 42 Production amount of butyl methacrylate Reference Example 7 : Synthesis of Isobutyl Methacrylate by AAT Time Production amount of butyl methacrylate (mm ) 35 2 20 0 . 30 After removing the skin , banana flesh was sliced using a cutter to a thickness of about 1 mm , and cut again to four pieces . 2 g of the sliced banana , 2 mL of the solution Reference Example 9 : Synthesis 2 of Butyl containing 2 3 mM methacrylyl- CoA and 0 .35 M KC1, and 40 Methacrylate by AAT 5 uL of isobutyl alcohol were added in order to a 100 ml 2 g of the plant specimen shown in Table 43 , 2 ml of the flask . After sealing , the reaction was allowed to occur at 30° solution containing 2 . 3 mM methacrylyl- CoA and 0 .35 M C . The reaction mixture containing isobutyl methacrylate KC1, and 10 ul of n -butyl alcohol were added in order to a which has been produced after 1, 2 or 3 hours was collected 100 ml flask . After sealing , the reaction was allowed to occur in an amount of 150 ul from the head space of the 100 ml 45 at 30° C . Analysis of methacrylic acid ester was performed flask and then analyzed by GC under following conditions . in the same manner as Reference example 7 . The results are The results are shown in Table 41. shown in Table 43 . TABLE 43 Production amount of butyl methacrylate Reaction Production amount of Plant Part for use time butyl methacrylate (mm ) Strawberry Fruit flesh • sliced to thickness of about 1 mm 0 .010 Kiwi Fruit flesh . sliced to thickness of about 1 mm 0 .012 Apple Fruit skin • sliced to thickness of about 1 mm 0 . 016 Melon Fruit flesh • sliced to thickness of about 1 mm aauw 0 . 015 Pear Fruit skin sliced to thickness of about 1 mm 4 0 . 013 Papaya Fruit skin sliced to thickness of about 1 mm 0 .027 Avocado Fruit skin sliced to thickness of about 1 mm 0 . 035 Blueberry Fruit skin sliced to thickness of about 1 mm aa 0 . 009 Plum Fruit skin sliced to thickness of about 1 mm 4 0 .002 US 10 , 294 , 500 B2 137 138 Reference Example 10 : Synthesis of Ethyl Methacrylate by AAT oligonucleotide primer MMA - 044 : 2 g of the plant specimen shown in Table 44 , 2 ml of the ( SEQ ID NO . 82 ) solution containing 2 3 mM methacrylyl- CoA and 0 .35 M 5 5 ' -GTTTGCACGCCTGCCGTTCGACG - 3 ' KC1, and 6 . 4 mlof ethyl alcoholwere added in order to a 100 ml flask . After sealing , the reaction was allowed to occur at MMA - 045 : 30° C . Analysis of methacrylic acid ester was performed in ( SEQ ID NO . 83 ) the samemanner as Reference example 7 . The results are 5 ' - CGGTACGCGCGGATCTTCCAGAG - 3 shown in Table 44 . TABLE 44 Production amount of ethyl methacrylate Reaction Production amount of Plant Part for use time ethyl methacrylate (mm ) Apple Fruit skin sliced to thickness of about 1 mm 0 . 110 Papaya Fruit skin • sliced to thickness of about 1 mm 0 . 003 Avocado Fruit skin • sliced to thickness of about 1 mm 0 .006
Reference Example 11 : Synthesis of Methyl Composition of Reaction Solution Methacrylate by AAT sterilized water 22 uL 2x PrimeSTAR (manufactured by Takara Bio Inc .) 25 uL 2 g of the plant specimen shown in Table 45 , 2 ml of the 25 Forward primer 1 uL solution containing 2 . 3 mM methacrylyl- CoA and 0 . 35 M Reverse primer 1 uL KC1, and 4 . 4 ul of methyl alcohol were added in order to a Genomic DNA 1 UL 100 ml flask . After sealing , the reaction was allowed to occur TotaTotal volume 50 UL at 30° C . Analysis ofmethacrylic acid ester was performed 30 Temperature Cycle in the samemanner as Reference example 7 . The results are 30 Cycles of the Reaction Including 98° C . for 10 Seconds , shown in Table 45 . 55° C . for 15 Seconds , and 72° C . for 150 Seconds TABLE 45 Production amount of methyl methacrylate Reaction Production amount ofmethyl Plant Part for use time methacrylate (mm ) Apple Fruit skin • sliced to thickness of about 1 mm 0 . 043 Papaya Fruit skin sliced to thickness of about 1 mm 0 . 004 Avocado Fruit skin sliced to thickness of about 1 mm 0 . 007
Example 45 : Production of Recombinant E . coli Band of the amplified product obtained was purified by Having Plant -Derived AAT Gene Incorporated 45 QIAquick Gel Extraction Kit (QIAGEN ) . Each purified Therein DNA was digested with the restriction enzyme Pagl (restric tion recognition site is included in the forward primer ) and Sse83871 ( restriction recognition site is included in the Synthesis of the plant- derived AAT gene represented by reverse primer ). After performing the separation by agarose SEQ ID NOS . 77, 79 and 81 was performed by Takara Bio 50 gel electrophoresis, the target band was cut out from the gel Inc . and purified . For the purification , Gel/ PCR Purification Kit Apple AAT (MPAAT1 ) : amino acid sequence (SEQ ID NO . (manufactured by FAVORGEN ) was used and elution was 76 ), nucleotide sequence (SEQ ID NO . 77 ) made using 30 uL of sterilized water . ID 55 By mixing the purified DNA ( 5 uL ) , the vector pTrc99A Strawberry AAT (SAAT ) : amino acid sequence (SEQ ID 555( 1 uL ) which has been digested in advance with Ncol and NO . 78 ) , nucleotide sequence (SEQ ID NO . 79) Sse83871, distilled water ( 4 uL ) and solution I (DNA Liga Strawberry AAT (VAAT ) : amino acid sequence (SEQ ID tion Kit ver. 2 ( Takara Bio Inc . ) ) ( 10 uL ) and incubating NO . 80 ), nucleotide sequence (SEQ ID NO . 81) them for 12 hours at 16° C ., the PCR amplified product and Those synthetic gene fragments were inserted to the 60the vector were ligated to each other. vector pMD19 , and each of them was named PAAT001 to To 200 uL of the competent cell which has been prepared 003 ( Table 46 ) . By having those AAT001 to 003 as a in the same manner as Example 33 , 10 uL of the above template and designing an oligonucleotide such that it is in ligation solution was added . After keeping it at 0° C . for 30 the form in which a restriction enzyme recognition site , minutes , a heat shock of 42° C . was applied for 30 seconds, which is easily introducible to an expression vector, is 65 and after cooling at 0° C . for 2 minutes , 1 mL of the SOC added , a DNA fragment containing the AAT gene was medium was added followed by culture under shaking at 37° produced by PCR method . C . for 1 hour. US 10 , 294 ,500 B2 139 140 After the culture, each was applied in an amount of 100 sodium phosphate buffer (pH 7 . 0 ), and suspended in the uL to the LBAmp agar medium (LB medium containing same buffer solution so as to have OD6 (630 nm ). As a ampicillin 100 mg/ 1 and 1 . 5 % agar ) and further cultured at control strain , BL21 (DE3 ) /PET16b was used . 37° C . Plural colonies of the transformant which have been ( 3 ) Preparation of Cell Extract 3700 5 cell extract was prepared from the cell suspension grown on the agar medium were cultured overnight at 37° C . 5 obtained above . By using ultrasonic homogenizer VP - 15S on 1 . 5 ml LBAmp medium (LB medium containing ampi (manufactured by DYTEC , Japan ), the cell suspension was cillin at 100 mg/ l) . After collecting the cells , the plasmid disrupted for 1 minute under conditions including output DNA was prepared by using QIAprep Spin Miniprep kit control 4 , DUTY CYCLE 40 % , PULS , TIMER = B mode 10 (manufactured by QIAGEN ) . 10 s . Next, by performing centrifuge ( 10 , 000xg , 5 minutes , 4° The nucleotide sequence of the each obtained recombi C . ), 1 mL of the obtained supernatant was collected ( cell nant plasmid DNA was determined by using CEQ DTCS extract ) . Quick Start Kit and Fluorescence sequence CEQ 2000XL DNA Analysis (both manufactured by BECKMAN Example 47 : Synthesis of Butyl Methacrylate by COULTER , USA ) , and named plasmid PAAT101 to 15 Using Cell Extract of AAT Gene Recombinant PAAT103 ( Table 46 ) . 15 The AAT gene was also inserted to the pET16b vector By using the cell extract which has been prepared by the according to the same operation , and obtained plasmids method described in Example 46 , the following reaction was were named PAAT201 to PAAT203 ( Table 46 ) . Meanwhile , performed . By adding 0 . 2 ml of the cell extract to a 10 because the pET16b does not contain a Sse83871 site , 20 ul- volume sample bottle ( for GC ) added with a septum to pET16b added at BamHI site with a linker containing which 0 . 8 ul of a solution of methacrylyl- CoA and alcohol Sse83871 restriction sequence is produced in advance , and has been added such that the final concentration of the used as a vector . reaction solution includes 7 mM methacrylyl - CoA and 40 . 5 By introducing the plasmid PAAT101 to PAAT103 to mM n -butanol , the reaction was started . The sample bottle JM109 strain , the recombinants JM109/ PAAT101 to 25 added with a septum was incubated at 30° C . for 1 to 5 hours PAAT103 were obtained . By introducing the plasmid for having the reaction . PAAT201 to PAAT203 to BL21 (DE3 ) strain , the recombi- Gas in the head space of sample bottle added with a nant BL21 (DE3 ) /PAAT201 to PAAT203 were obtained . septum was analyzed in the same manner as Reference example 7 . The results are shown in Table 47 . TABLE 46 30 TABLE 47 Plasmid for expressing plant- derived AAT gene Production of butyl methacrylate by using AAT gene SEQ Template Plasmid for expression Production amount (MM ) ID NO . Plant ( gene name) plasmid pTrc99A PET16b 35 Apple (MPAAT1 ) PAAT001 PAAT101 PAAT201 Recombinant 1 Hour 3 Hours 5 Hours 4 Strawberry (SAAT ) PAAT002 PAAT102 PAAT202 JM109 /PAAT102 0 . 001 0 .003 0 . 004 6 Strawberry (VAAT ) PAAT003 PAAT103 PAAT203 JM109 /PAAT103 0 001 0 . 002 BL21 (DE3 )/ PAAT201 0 . 003 0 .014 0 .026 BL21 (DE3 ) /PET16b 40 Example 46 : Preparation of Cell Extract from Recombinant E . coli which AAT is Expressed Example 48 : Synthesis of Methacrylic Acid Ester ( 1 ) Culturing Recombinant E . coli Using pTrc99A as Vector by Using Cell Extract of AAT Gene Recombinant The recombinant E . coli JM109 /PAAT101 to PAAT103 45 obtained from Example 45 was inoculated to LB medium By using methanol, ethanol, or n -butanol as the alcohol containing 1 mL of 100 ug /mL ampicillin and subjected to and the extract derived from BL21 (DE3 ) /PAAT201 ( Apple ) pre - culture at 37° C . for 7 hours . 0 . 1 ml of the culture was as the cell extract, the reaction was performed in the same harvested and added to 100 ml of the same medium ( con manner as Example 47. The results of analyzing the product taining 100 ug /mL ampicillin . 1 mM IPTG ) and cultured 50 after 5 hours are shown in Table 48 . under shaking at 37° C . for 15 hours . From the obtained culture , the cells were collected by centrifuge ( 3 ,700xg , 10 TABLE 48 minutes , 4° C . ), washed with 10 mM sodium phosphate Production of methacrylic acid ester by using buffer ( pH 7 .0 ) , and suspended in the same buffer solution . AAT gene recombinant As a control strain , JM109 /pTrc99A was used . 55 ( 2 ) Culturing Recombinant E . coli Using PET16b as Vector Production amount after 5 hours (mm ) The recombinant E . coli BL21 (DE3 )/ PAAT201 to Methyl Ethyl Butyl PAAT203 obtained from Example 45 was inoculated to LB Recombinant methacrylate methacrylate methacrylate medium containing 1 mL of 100 ug /mL ampicillin and subiected to pre -culture at 37° C . for 14 hours . 0 . 1 ml of the 60 BL21 (DE3 ) /PAAT 201 0 .021 0 .045 0 .091 culture was harvested and added to 100 ml of the same medium ( containing 100 ug /mL ampicillin ) and cultured under shaking at 37° C . until OD at 37° C . becomes 0 .3 . Example 49 : Synthesis 2 of Methacrylic Acid Ester Then , IPTG was added to have the final concentration of 1 by Using Cell Extract of AAT Gene Recombinant mM followed by further culture under shaking for several 65 hours. From the obtained culture , the cells were collected by By using isobutanol, phenol , benzyl alcohol or 2 -ethyl centrifuge ( 3 ,700xg , 10 minutes, 4° C . ) , washed with 10 mM hexyl alcohol as the alcohol and the cell extract of BL21 US 10 ,294 , 500 B2 141 142 (DE3 )/ PAAT201 ( Apple ) obtained from Example 46 , the observed . In other words , the AAT derived from yeast was following reaction was performed . not observed with the ability to produce a methacrylic acid By adding 0 . 2 ml of the cell extract to a 10 ul- volume ester . sample bottle ( for GC ) added with a septum to which 0 . 8 ml Production of Ester by Using Yeast AAT Gene Recombinant of a solution of methacrylyl- CoA and alcohol has been 5 added such that the final concentration of the reaction TABLE 51 solution includes 1 mM methacrylyl - CoA and 40 mM alco hol, the reaction was started . The sample bottle added with Production amount (mm ) a septum was incubated at 30° C . for 1 to 5 hours for having Butyl methacrylate Butyl acetate the reaction . When the reaction is completed , 1 mL of 10 acetonitrile was added to the reaction solution in the sample 1353013 bottled added with a septum followed by mixing well. After Recombinant Hour Hours Hours Minutes Hour Hours JM109 /PAAT105 0 0 0 0 .089 0 . 145 0 . 170 filtration using a syringe filter DISMIC / hole diameter 0 . 45 JM109 /PAAT106 0 0 0 0 . 104 0 . 189 0 .290 um (manufactured by ADVANTEC ) , it was subjected to10 15 JM109 /pTrc99A 0 0 0 0 0 HPLC analysis . In Table 49, the results of analyzing the OOO product after 5 hours are shown . Synthesis of Methacrylic Acid Ester ( IsobutylMethacrylate , Phenyl Methacrylate , Benzyl Methacrylate , and 2 - Ethyl INDUSTRIAL APPLICABILITY hexyl Methacrylate ) by Using Cell Extract of AAT Gene 20 According to the method for producing methacrylic acid Recombinant of the invention , methacrylic acid can be produced from recyclable biogenous resources ( renewable raw materials ) TABLE 49 by using microbes without depending on a chemicalmethod . Production amount after 5 hours (mm ) According to the ACH method of the related art , a treatment of oxidizing acetone cyanhydrin by acid is required so that Isobutyl Phenyl Benzyl 2 - Ethylhexyl a large amount of waste acid is generated . Further , as Recombinant methacrylate methacrylate methacrylate methacrylate separation or purification is needed for each step , it causes BL21( DE3 ) 0 .009 0 . 001 0 . 17 0 . 31 high energy consumption . Meanwhile , according to the PAAT201 30 method for producing methacrylic acid of the invention , methacrylic acid can be produced efficiently and simply in Conditions for HPLC Analysis a single step directly from a biomass . In addition , a less load Apparatus: Waters 2695 is applied in terms of safety , environment, facility , works , Column: Shiseido CAPCELL PAK C18 UG120 5 um and cost . Further , by converting the raw materials of meth Mobile phase: 65 % MeOH , 0 . 2 % phosphoric acid 35 acrylic acid to those derived from a biomass , it becomes also Flow amount: 0 .25 ul /min possible to reduce the carbon dioxide discharge amount to an column temperature : 35° C . environment. Detection : UV 210 nm SEQ ID NO . 82 : MMA -044 Injection amount: 10 uL SEO ID NO . 83 : MMA - 045 40 SEQ ID NO . 84 : MMA - 003 Comparative Example 1 SEQ ID NO . 85 : MMA - 004 SEQ ID NO . 88 : MMA -031 Reaction for Synthesis of Methacrylic Acid Ester SEQ ID NO . 89 : MMA -032 by Using Cell Extract of Yeast -Derived AAT Gene SEQ ID NO . 92 : MAA - 15 Recombinant 45 SEQ ID NO . 93 : MAA - 16 SEQ ID NO . 94 : GB - 138 Plasmids for expressing yeast -derived AAT gene were SEQ ID NO . 95 : GB - 139 prepared in the same maneras Example45ab50 ), SEN . 96GB - 14 after transforming E . coli using them , a recombinant SEQ ID NO . 97 : GB - 141 expressing AAT was obtained . 50 SEQ ID NO . 98 : MMA - 061 Plasmid for Expressing Yeast- Derived AAT Gene SEQ ID NO . 99 : MMA -062 SEO ID NO . 100 : MMA - 063 TABLE 50 SEO ID NO . 101 : MMA - 064 SEQ ID NO . 102 : MMA -069 Template Plasmid for expression 55 SEO ID NO . 103 : MMA - 070 SEQ ID NO . Gene name plasmid pTrc99A PET16b SEQ ID NO . 104 : MMA - 133 SEO ID NO . 105 : MMA - 131 133 ATF1 PAATO05 PAAT105 PAAT205 SEQ ID NO . 106 : MMA - 003 135 ATF2 PAAT006 PAAT106 PAAT206 SEQ ID NO . 107 : MMA - 004 60 SEQ ID NO . 108 : MMA - 020 The cell extract was prepared in the same manner as SEQ ID NO . 109 : MMA -006 Example 46 and the reaction for synthesizing butyl meth - SEQ ID NO . 110 : MMA -018 acrylate was performed by having methacrylyl -CoA and SEQ ID NO . 111 : MMA - 008 n -butanol as a substrate in the same manner as Example 47 . SEQ ID NO . 112 : MMA -019 As a result , no production of butyl methacrylate was 65 SEQ ID NO . 113 : MMA -010 observed . Meanwhile , when acetyl- CoA and n - butanol are SEQ ID NO . 114 : MMA -021 used as a substrate , production of butyl acetate was SEQ ID NO . 115 : MMA -022 US 10 , 294 ,500 B2 143 144 SEN16 :MA - 023 SEQ ID NO . 125 : MMA - 002 SEQ ID NO . 117 : MMA - 024 SEQ ID NO . 126 : MMA - 114 SEQ ID NO . 118 : MAA - 15 SEO ID NO . 127 : MMA - 109 SEO ID NO . 119 : MAA - 16 SEQ ID NO . 120 : MMA - 187 SEQ ID NO . 128 : MMA -039 SEQ ID NO . 121 : MMA - 140 SEQ ID NO . 129 : MMA -040 SEO ID NO . 122 : MMA - 188 SEQ ID NO . 130 : MMA -042 SEQ ID NO . 123 : MMA - 189 SEQ ID NO . 131: MMA -041 SEQ ID NO . 124 : MMA -001 SEQ ID NO . 132: MMA- 043
SEQUENCE LISTING
< 160 > NUMBER OF SEQ ID NOS : 136 < 210 > SEQ ID NO 1 < 211 > LENGTH : 485 ? 2 > TYPE : DNA ? 3 > ORGANISM : Pseudomonas sp . B25 - 2 < 400 > SEQUENCE : 1 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgagaag agcttgctct togattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120 gtttcgaaag gaacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360 gocatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggcagtaagt taataccttg ctgttttgac gttaccgaca gaataagcac cggctaacto 480 tgtgc 485
< 210 > SEO ID NO 2 < 211 > LENGTH : 485 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas sp . 022 - 1 < 400 > SEQUENCE : 2
attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgacggg agcttgctcc 9 ygaattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120 gtctcgaaag ggacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggcattaacc taatacgtta gtgttttgac gttaccgaca gaataagcac cggctaacto 480 tgtgc 485
< 210 > SEQ ID NO 3 < 211 > LENGTH : 485 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas sp . D43 - 1 < 400 > SEQUENCE : 3 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgaagag agcttgctct 60 ctgattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120 gtctcgaaag ggacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 US 10 , 294 ,500 B2 145 146 - continued ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgateca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggcagtaaat taatactttg ctgttttgac gttaccgaca gaataagcac cggctaacto 480 tgtgc 485
< 210 > SEQ ID NO 4 < 211 > LENGTH : 485 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas sp . D25 < 400 > SEQUENCE : 4 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgacagg agcttgctcc 60 tgaattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120 gtttcgaaag gaacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 ccttgcgcta tcagatgage ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggcattaacc taatacgtta gtgttttgac gttaccgaca gaataagcac cggctaacto 480 tgtgc 485
< 210 > SEQ ID NO 5 < 211 > LENGTH : 485 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas sp . D26 < 400 > SEQUENCE : 5 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgamrrg agcttgctcy 60 ykrattcagc ggcggacggg tgagtaatge ctaggaatct gcctggtagt gggggacaac 120 gtttcgaaag gaacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 ccttgcgcta tcagatgagcctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgateca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggcattaacc taatacgtta gtgttttgac gttaccgaca gaataagcac cggctaactc 480 tgtgc 485
< 210 > SEQ ID NO 6 < 211 > LENGTH : 485 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas sp . D29 < 400 > SEQUENCE : 6 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgaagag agcttgctct ctgattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120 gtctcgaaag ggacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 US 10 , 294 ,500 B2 147 148 - continued aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggttgtagat taatactctg caattttgac gttaccgaca gaataagcac cggctaactc 480 tgtgc 485
< 210 > SEO ID NO 7 < 211 > LENGTH : 485 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas sp . 041 - 2 < 400 > SEQUENCE : 7 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgargag agcttgctct 60 ctgattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120 gtttcgaaag gaacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggttgtagat taatactctg caattttgac gttaccgaca gaataagcac cggctaactc 480 tgtgc 485
< 210 > SEQ ID NO 8 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas putida NBRC12996 < 400 > SEQUENCE : 8 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggatgacggg agcttgctcc 60 ttgattcagc ggcggacggg tgagtaatgc ctaggaatct gcctggtagt gggggacaac 120 gtttcgaaag gaacgctaat accgcatacg tcctacggga gaaagcaggg gaccttcggg 180 ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt ggtgaggtaa tggctcacca 240 aggcgacgat ccgtaactgg tctgagagga tgatcagtca cactggaact gagacacggt 300 ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360 gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag cactttaagt tgggaggaag 420 ggcagtaagt taataccttg ctgttttgac gttaccgaca gaataagcac cggctaacto 480 tgtgccagca gccgcggtaa 500
< 210 > SEQ ID NO 9 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Bacillus sp . G1 < 400 > SEQUENCE : 9 gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaatggattg agagcttgct 60 ctcaagaagt tagcggcgga cgggtgagta acacgtgggt aacctgccca taagactggg 120 ataactccgg gaaaccgggg ctaataccgg ataacatttt gaacygcatg gttcgaaatt 180 gaaaggcggc ttcggctgtc acttatggat ggacccgcgt cgcattagct agttggtgag 240 US 10 , 294 ,500 B2 149 150 - continued gtaacggctc accaaggcaa cgatgcgtag ccgacctgag agggtgatog gccacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360 gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt 420 tgttagggaa gaacaagtgc tagttgaata agctggcacc ttgacggtac ctaaccagaa 480 agccacggct aactacgtgc 500
< 210 > SEQ ID NO 10 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Bacillus sp . G2 < 400 > SEQUENCE : 10 gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaatggattr agagcttgct 60 ctyawgaagt tagcggcgga cgggtgagta acacgtgggt aacctgccca taagactggg 120 ataactccgg gaaaccgggg ctaataccgg ataayatttt gaactgcatg gttcgaaatt 180 gaaaggcggc ttcggctgtc acttatggat ggacccgcgt cgcattagct agttggtgag 240 gtaacggctc accaaggcaa cgatgcgtag ccgacctgag agggtgatog gocacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360 gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt 420 tgttagggaa gaacaagtgc tagttgaata agctggcacc ttgacggtac ctaaccagaa 480 agccacggct aactacgtgc 500
< 210 > SEO ID NO 11 < 211 > LENGTH : 497 < 212 > TYPE : DNA < 213 > ORGANISM : Bacillus sp . Ri < 400 > SEQUENCE : 11 gacgaacgct ggcggcgtgc ctaatacatg caagtcgage ggacagatgg gagcttgctc 60 cctgaagtca gcggcggacg ggtgagtaac acgtgggcaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat aattctttcc ctcacatgag ggaaagctga 180 aagatggttt cggctatcac ttacagatgg goccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa aactctgttg 420 ttagggaaga acaagtrccg gagtaactgc cggtrccttg acggtaccta accagaaagc 480 cacggctaac tacgtgc 497
< 210 > SEQ ID NO 12 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Bacillus subtilis NBRC12210 < 400 > SEQUENCE : 12 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc 60 cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat 120 aactccggga aaccggggct aataccggat ggttgtttga accgcatggt tcaaacataa 180 aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt 240 aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 US 10 , 294 ,500 B2 151 152 - continued ctgagacacg goccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg 420 ttagggaaga acaagtaccg ttcgaatagg gcggtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca 500
210SBN13 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Bacillus badius ATCC14574 < 220 > FEATURE : < 221NPAA > NAME / KEY : misc _ feature < 222MHOWNw > LOCATION : (203 ) . . ( 203 ) < NNNN223 > OTHER INFORMATION : n is a , c , g , or t < 400 > SEQUENCE : 13 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacttgacg gaagottgct 60 tccgttcaag ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg 120 gataactccg ggaaaccggg gctaataccg gatattcttt ttcttcgcat gaagaagaat 180 ggaaaggcgg cttttagctg tcncttacag atggacccgc ggcgcattag ctagt tggtg 240 aggtaacggc tcaccaaggc aacgatgcgt agccgacctg agagggtgat cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 acgaaagtct gacggagcaa cgccgcgtga gtgaagaagg ttttcggatc gtaaagctct 420 gttgtcaggg aagaacaagt acggaagtaa ctgtccgtac cttgacggta cctgaccaga 480 aagccacggc taactacgtg 500
< 210 > SEQ ID NO 14 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Bacillus megaterium NBRC15308 < 400 > SEQUENCE : 14 gatgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaactgatta gaagcttgct 60 tctatgacgt tagcggcgga cgggtgagta acacgtgggc aacctgcctg taagactggg 120 ataacttcgg gaaaccgaag ctaataccgg ataggatott ctccttcatg ggagatgatt 180 gaaagatggt ttcggctatc acttacagat gggcccgcgg tgcattagct agttggtgag 240 gtaacggctc accaaggcaa cgatgcatag ccgacctgag agggtgatog gccacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360 gaaagtctga cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt 420 tgttagggaa gaacaagtac aagagtaact gottgtacct tgacggt acc taaccagaaa 480 gccacggcta actacgtgcc 500
< 210 > SEQ ID NO 15 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Bacillus simplex ATCC49097 < 400 > SEQUENCE : 15 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc gaatcgatgg gagcttgctc cctgagatta gcggcggacg ggtgagtaac acgtgggcaa cctgcctata agactgggat 120 aacttcggga aaccggagct aataccggat acgttctttt ctcgcatgag agaagatgga 180 US 10 , 294 ,500 B2 153 154 - continued aagacggttt acgctgtcac ttatagatgg gcccgcggcg cattagctag ttggtgaggt 240 aatggctcac caaggcgacg atgcgtagcc gacctgagag ggtgatcggc cacactggga 300 ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga 360 aagtctgacg gagcaacgcc gcgtgaacga agaaggcctt cgggtcgtaa agttctgttg 420 ttagggaaga acaagtacca gagtaactgc tggtaccttg acggtaccta accagaaagc 480 cacggctaac tacgtgccag 500
< 210 > SEQ ID NO 16 < 211 > LENGTH : 491 < 212 > TYPE : DNA < 213 > ORGANISM : Sphingobacterium sp . B13 < 400 > SEQUENCE : 16 gatgaacgct agcggcaggc ctaatacatg caagtcggac gggatccatc ggagagottg 60 ctcgaagatg gtgagagtgg cgcacgggtg cgtaacgcgt gagcaaccta cctctatcag 120 ggggatagcc tctcgaaaga gagattaaca ccgcataata taatctaccg gcatcgttgg 180 attattaaat atttatagga tagagatggg ctcgcgtgac attagct agt tggtagggta 240 acggcytacc aaggcgacga tgtctagggg ctctgagagg agaatccccc acactggtac 300 tgagacacgg accagactcc tacgggaggc agcagtaagg aatattggtc aatgggcgga 360 agcctgaacc agccatgccg cgtgcaggat gactgcccta tgggttgtaa actgcttttg 420 tccaggaata aacctagata cgagtatcta gctgaatgta ctggaagaat aaggatcggc 480 taactccgtgc 491
< 210 > SEQ ID NO 17 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Comamonas terrigena NBRC13299 < 400 > SEQUENCE : 17 attgaacgct ggcggcatgc tttacacatg caagtcgaac ggcagcacgg acttcggtct 60 ggtggcgagt ggcgaacggg tgagtaatac atcggaacgt goccagttgt gggggataac 120 tactcgaaag agtagctaat accgcatgag aactgaggtt gaaagcaggg gatcgcaaga 180 ccttgcgcaa ctggagoggc cgatggcaga ttaggtagtt ggtgggataa aagcttacca 240 agccgacgat ctgtagctgg tctgagagga cgaccagcca cactgggact gagacacggc 300 ccagactcct acgggaggca gcagtgggga attttggaca atgggcgaaa gcctgateca 360 gcaatgccgc gtgcaggatg aaggccttcg ggttgtaaac tgcttttgta cggaacgaaa 420 agcttcgggt taataccctg gagtcatgac ggtaccgtaa gaataagcac cggctaacta 480 cgtgccagca gccgcggtaa 500
< 210 > SEQ ID NO 18 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Brevundimonas diminuta ATCC11568 < 220 > FEATURE : < 221 > NAME / KEY : misc_ feature < 222 > LOCATION : ( 387 ) . . ( 387 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 400 > SEQUENCE : 18 agcgaacgct ggcggcaggc ctaacacatg caagtcgaac ggacccttcg gggttagtgg 60 cggacgggtg agtaacacgt gggaacgtgc ctttaggttc ggaat agctc ctggaaacgg 120 US 10 , 294 ,500 B2 155 156 - continued gtggtaat gaatgtgcct agattacgccttagggggg18 ctgattagct agttggtgag gtaacggctc accaaggcga cgatcagtag ctggtctgag 240 aggatgacca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 300 gggaatcttg cgcaatgggc gaaagcctga cgcagccatg ccgcgtgaat gatgaaggtc 360 ttaggattgt aaaattcttt caccggngac gataatgacg gtacccggag aagaagcccc 420 ggctaacttc gtgccagcag ccgcggtaat acgaaggggg ctagcgttgc tcggaattac 480 tgggcgtaaa gggcgcgtag 500
< 210 > SEQ ID NO 19 < 211 > LENGTH : 500 < 212 > TYPE : DNA 213 > ORGANISM : Brevundimonas vesicularis ATCC11426 < 400 > SEQUENCE : 19 agcgaacgct ggcggcaggc ctaacacatg caagtcgaac gaactcttcg gagttagtgg 60 cggacgggtg agtaacacgt gggaacgtgc ctttaggttc ggaataactc agggaaactt 120 gtgctaatac cgaatgtgcc cttcggggga aagatttatc gcctttagag cggcccgcgt 180 ctgattagct agttggtgag gtaaaggctc accaaggcga cgatcagtag ctggtctgag 240 aggatgatca gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagtg 300 gggaatcttg cgcaatgggc gaaagcctga cgcagccatg ccgcgtgaat gatgaaggtc 360 ttaggattgt aaaattcttt caccggggac gataatgacg gtacccggag aagaagcccc 420 ggctaacttc gtgccagcag ccgcggtaat acgaaggggg ctagcgttgc tcggaattac 480 tgggcgtaaa gggagcgtag 500
< 210 > SEQ ID NO 20 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Brevundimonas subvibrioides NBRC16000 < 400 > SEQUENCE : 20 agcgaacgct ggcggcaggc ctaacacatg caagtcgaac ggacctctcg gggttagtgg 60 cggacgggtg agtaacacgt gggaacgtgc cttttggttc ggaatagctc ctggaaacgg 120 gtggtaatgc cgaatgtgcc ctttggggga aagatttatc gccattagag cggcccgcgt 180 ctgattagct agttggtgag gtaaaggetc accaaggcta cgatcagtag ctggtctgag 240 aggatgacca gccacattgg gactgagaca cggcccaaac tcctacggga ggcagcagtg 300 gggaatcttg cgcaatgggc gaaagcctga cgcagccatg ccgcgtgtat gatgaaggtc 360 ttaggattgt aaaatacttt caccggtgaa gataatgact gtagccggag aagaagcccc 420 ggctaacttc gtgccagcag ccgcggtaat acgaaggggg ctagcgttgc tcggaattac 480 tgggcgtaaa gggagcgtag 500
< 210 > SEO ID NO 21 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Sphingomonas paucimobilis NBRC13935 < 220 > FEATURE : < 221 > NAME / KEY : misc _ feature < 222 > LOCATION : ( 268 ) . . ( 268 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 400 > SEQUENCE : 21 US 10 , 294 ,500 B2 157 158 - continued aacgaacgct ggcggcatgc ctaacacatg caagtcgaac gaaggcttcg gccttagtgg 60 cgcacgggtg cgtaacgcgt gggaatctgc cottaggttc ggaataacag ctggaaacgg 120 ctgctaatac cggatgatat cgcgagatca aagatttatc gcctgaggat gagcccgcgt 180 tggattaggt agttggtggg gtaaaggcct accaagccga cgatccatag ctggtctgag 240 aggatgatca gccacactgg gactgagnca cggcccagac tcctacggga ggcagcagtg 300 gggaatattg gacaatgggc gaaagcctga tccagcaatg ccgcgtgagt gatgaaggcc 360 ctagggttgt aaagctcttt tacccgggaa gataatgact gtaccgggag aataagcccc 420 ggctaactcc gtgccagcag ccgcggtaat acggaggggg ctagcgttgt tcggaattac 480 tgggcgtaaa gcgcacgtag 500
< 210 > SEQ ID NO 22 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Ochrobactrum sp . NBRC12951 < 400 > SEQUENCE : 22 aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gocccgcaag gggagoggca 60 gacgggtgag taacgcgtgg gaacgtacct tttgctacgg aataactcag ggaaacttgt 120 gctaataccg tatgtgccct tcgggggaaa gatttatcgg caaaggatcg gcccgcgttg 180 gattagctag ttggtgaggt aaaggctcac caaggcgacg atccatagct ggtctgagag 240 gatgatcagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360 agggttgtaa agctctttca ccggtgaaga taatgacggt aaccggagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggatttactg 480 ggcgtaaagc gcacgtaggc 500
< 210 > SEQ ID NO 23 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Ochrobactrum intermedium NBRC15820 < 400 > SEQUENCE : 23 aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gccccgcaag gggagcggca 60 gacgggtgag taacgcgtgg gaacgtacca tttgctacgg aataactcag ggaaacttgt 120 getaataccg tatgtgcccg aaaggggaaa gatttatcgg caaatgatcg gcccgcgttg 180 gattagctag ttggtggggt aaaggcctac caaggcgacg atccatagct ggtctgagag 240 gatgatcagc cacactggga ctgagacacg goccagactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360 agggttgtaa agctctttca ccggtgaaga taatgacggt aaccggagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggatttactg 480 ggcgtaaagc gcacgtaggc 500
< 210 > SEQ ID NO 24 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Ochrobactrum intermedium NBRC13694 < 400 > SEQUENCE : 24 aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gccccgcaag gggagcggca 60 US 10 , 294 ,500 B2 159 160 - continued gacgggtgag taacgcgtgg gaacgtacct tttgctacgg aataactcag ggaaacttgt 120 gctaataccg tatgtgccct tcgggggaaa gatttatcgg caaaggatcg gcccgcgttg 180 gattagctag ttggtgaggt aaaggctcac caaggcgacg atccatagct ggtctgagag 240 gatgatcagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360 agggttgtaa agctctttca ccggtgaaga taatgacggt aaccggagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggatttactg 480 ggcgtaaagc gcacgtaggo 500
< 210 > SEQ ID NO 25 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Ochrobactrum grignonense NBRC102586 < 400 > SEQUENCE : 25 aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gcctcgcaag aggagcggca 60 gacgggtgag taacgcgtgg gaatctacct tttgctacgg aataactcag ggaaacttgt 120 gctaataccg tatgtgccct tttggggaaa gatttatcgg caaaggatga gcccgcgttg 180 gattagctag ttggtagggt aatggcctac caaggcgacg atccatagct ggtctgagag 240 gatgatcagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360 agggttgtaa agctctttca ccggtgaaga taatgacggt aaccggagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggatttactg 480 ggcgtaaagc gcacgtaggc 500
< 210 > SEQ ID NO 26 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Ochrobactrum lupini NBRC102587 < 400 > SEQUENCE : 26 aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gccccgcaag gggagoggca 60 gacgggtgag taacgcgtgg gaacgtacct tttgctacgg aataactcag ggaaacttgt 120 gctaataccg tatgtgccct tcgggggaaa gatttatcgg caaaggatcg gcccgcgttg 180 gattagctag ttggtgaggt aaaggctcac caaggcgacg atccatagct ggtctgagag 240 gatgatcage cacactggga ctgagacacg goccagactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360 agggttgtaa agctctttca ccggtgaaga taatgacggt aaccggagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggatttactg 480 ggcgtaaagc gcacgtaggc 500
< 210 > SEQ ID NO 27 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Pedobacter heparinus NBRC12017 < 400 > SEQUENCE : 27 gatgaacgct agcggcaggc ctaatacatg caagtcgaac gagattaagg ggcttgctcc 60 US 10 , 294 ,500 B2 161 162 - continued ttatgaaagt ggcgcacggg tgcgtaacgc gtatgcaacc taccttaatc agggggatag 120 cccgaagaaa ttcggattaa caccgcataa aaacacagga tagcattatc caatgttcaa 180 atatttatag gattaagatg ggcatgcgtg tcattagcta gttggcgggg taacggccca 240 ccaaggcgac gatgactagg ggatctgaga ggatgaccccccacactggt actgagacac 300 ggaccagact cctacgggag gcagcagtaa ggaatattgg tcaatggagg gaactctgaa 360 ccagccatgc cgcgtgcagg aagacagccc tctgggtcgt aaactgettt tattcgggaa 420 taaacctact tacgtgtaag tagctgaatg taccgaagga ataaggatog gctaactccg 480 tgccagcagc cgcggtaata 500
< 210 > SEQ ID NO 28 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Paenibacillus sp . NBRC13157 < 220 > FEATURE : < 221 > NAME /KEY : misc _ feature < 222 > LOCATION : ( 66 ) . . ( 66 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 220 > FEATURE : < 221 > NAME / KEY : misc _ feature < 222 > LOCATION : ( 70 ) . . ( 70 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 220 > FEATURE : ? 1 > NAME / KEY : misc _ feature ? LOCATION : ( 157 ) . . ( 157 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 220 > FEATURE : ? 21 > NAME / KEY : misc _ feature ? 22 > LOCATION : ( 162 ) . . ( 162 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 220 > FEATURE : < 221 > NAME / KEY : misc _ feature < 222 > LOCATION : ( 174 ) . . ( 174 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 400 > SEQUENCE : 28 gcgcgccgggtgctatacatgcagtcggggcttgaagaagctt6 ttctcngatn gttagcggcg gacgggtgag taacacgtag gcaacctgcc tgtaagatcg 120 ggataactac cggaaacggt agctaagacc ggataancgg tntctccgca tggngagato 180 gtgaaacacg gagcaatctg tggcttacgg atgggcctgc ggcgcattag ctagttggtg 240 aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgaa cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 acgcaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420 gttgccaggg aagaacgcca aggagagtaa ctgctctttg ggtgacggta cctgagaaga 480 aagccccggc taactacgtg 500
< 210 > SEO ID NO 29 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Achromobacter denitrificans NBRC12669 < 400 > SEQUENCE : 29 attgaacgct agcgggatgc cttacacatg caagtcgaac ggcagcacgg acttcggtct 60 ggtggcgagt ggcgaacggg tgagtaatgt atcggaacgt gcccagtagc gggggataac 120 tacgcgaaag cgtagctaat accgcatacg ccctacgggg gaaagcaggg gatcgcaaga 180 ccttgcacta ttggagcggc cgatatcgga ttagctagtt ggtggggtaa cggctcacca 240 aggcgacgat ccgtagctgg tttgagagga cgaccagcca cactgggact gagacacggc 300 US 10 , 294 ,500 B2 163 164 - continued ccagactcct acgggaggca gcagtgggga attttggaca atgggggaaa ccctgatcca 360 gccatcccgc gtgtgcgatg aaggccttcgggttgtaaag cacttttggc aggaaagaaa 420 cgtcgcgggt taataccccg cggaactgac ggtacctgca gaataagcac cggctaacta 480 cgtgccagca gccgcggtaa 500
< 210 > SEQ ID NO 30 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Acinetobacter haemolyticus ATCC17906 < 400 > SEQUENCE : 30 cctggctcag attgaacgct ggcggcaggc ttaacacatg caagtcgagc ggggaagggt 60 accttgctac ctaacctagc ggcggacggg tgagtaatgc ttaggaatct gcctattagt 120 gggggacaac attccgaaag gaatgctaat accgcatacg tcctacggga gaaagcaggg 180 gatcttcgga ccttgcgcta atagatgagc ctaagtcgga ttagctagtt ggtggggtaa 240 aggcctacca aggcgacgat ctgtagcggg totgagagga tgatccgcca cactgggact 300 gagacacggc ccagactcct acgggaggca gcagtgggga atattggaca atgggcggaa 360 gcctgatcca gccatgccgc gtgtgtgaag aaggcctttt ggttgtaaag cactttaagc 420 gaggaggagg ctactctagt taatacctag agatagtgga cgttactcgc agaataagca 480 ccggctaact ctgtgccago 500
< 210 > SEO ID NO 31 < 211 > LENGTH : 500 V< 212 > TYPE : DNA V< 213 > ORGANISM : Acinetobacter junii ATCC17908 V< 220 > FEATURE : V 1 > NAME / KEY : misc _ feature V< 222 > LOCATION : ( 371 ) . . ( 371 ) V< 223 > OTHER INFORMATION : n is a , c , g , or t < 400 > SEQUENCE : 31 cctggctcag attgaacgct ggcggcaggc ttaacacatg caagtcgagc ggagatgagg 6 tgottgcacc ttatcttagc ggcggacggg tgagtaatgc ttaggaatct gcctattagt 120 gggggacaac attccgaaag gaatgctaat accgcatacg tcctacggga gaaagcaggg 180 gatcttcgga ccttgcgcta atagatgagc ctaagtcgga ttagctagtt ggtggggtaa 240 aggcctacca aggcgacgat ctgtagcggg tctgagagga tgatccgcca cactgggact 300 gagacacggc ccagactcct acgggaggca gcagtgggga atattggaca atggggggaa 360 ccctgatcca nccatgccgc gtgtgtgaag aaggccttat ggttgtaaag cactttaagc 420 gaggaggagg ctactgagac taatactctt ggatagtgga cgttactcgc agaataagca 480 ccggctaact ctgtgccago 500
< 210 > SEQ ID NO 32 < 211? > LENGTH : 500 < 212? > TYPE : DNA < 213? > ORGANISM : Shewanella fodinae NBRC105216 < 220 > FEATURE : < 221 > NAME / KEY : misc _ feature < 222 > LOCATION : ( 237 ) . . ( 237 ) < 223 > OTHER INFORMATION : n is a , c , g , or t < 400 > SEQUENCE : 32 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggcagcgggg agtagcttgc 60 US 10 , 294 ,500 B2 165 166 - continued tactctgccg gcgagoggcg gacgggtgag taatgcctgg gaatttgccc attcgagggg 120 gataacagtt ggaaacgact gctaataccg catacgccct aagggggaaa gcaggggaac 180 ttaggtcctt gcgcgaatgg ataagcccag gtgggattag ctagttggtg aggtaanggo 240 tcaccaaggc gacgatctct agctggtctg agaggatgat cagccacact ggaactgaga 300 cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gggaaaccct 360 gatgcagcca tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact ttcagtcagg 420 aggaaggtgg tgtagctaat atctgcacca attgacgtta ctgacagaag aagcaccggc 480 taactccgtg ccagcagccg 500
< 210 > SEQ ID NO 33 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Listonella anguillarum ATCC19264 < 400 > SEQUENCE : 33 attgaacgct ggcggcaggc ctaacacatg caagtcgagc ggcagcacag aggaacttgt 60 tccttgggtg gcgagoggcg gacgggtgag taatgcctag gaaattgccc tgatgtgggg 120 gataaccatt ggaaacgatg gctaataccg catgatgcct acgggccaaa gagggggacc 180 ttcgggcctc tcgcgtcagg atatgcctag gtgggattag ctagttggtg aggtaatggc 240 tcaccaaggc gacgatccct agctggtctg agaggatgat cagccacact ggaactgaga 300 cacggtccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gcgcaagcct 360 gatgcagcca tgccgcgtgt atgaagaagg ccttcgggtt gtaaagtact ttcagtcgtg 420 aggaaggtgg tgttgttaat agcagcatca tttgacgtta gcgacagaag aagcaccggc 480 taactccgtg ccagcagccg 500
< 210 > SEQ ID NO 34 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Agrobacterium luteum NBRC15768T < 400 > SEQUENCE : 34 aacgaacgct ggcggcatgc ttaacacatg caagtcgaac gagatcttcg gatctagtgg 60 cgcacgggtg cgtaacgcgt ggggatctac catagggtgc gga ataactc agagaaattt 120 gagctaatac cgcataatgt cttcggacca aagatttatc gccctttgat gaacccgcgt 180 aggattagct tgttggtgag gtaagagctc accaaggcga cgatctttag ctggtctgag 240 aggatgatca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 300 gggaatattg gacaatgggc gaaagcctga tccagcaatg ccgcgtgagt gatgaaggcc 360 ttagggttgt aaagctcttt taccagggat gataatgaca gtacctggag aataagctcc 420 ggctaactcc gtgccagcag ccgcggtaat acggagggag ctagcgttgt toggaattac 480 tgggcgtaaa gcgcgcgtag 500
< 210 > SEQ ID NO 35 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Mesorhizobium loti ATCC700743 < 400 > SEQUENCE : 35 aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gcctcgcaag aggagoggca 60 gacgggtgag taacgcgtgg gaatctaccc atctctacgg aacaactccg ggaaactgga 120 US 10 , 294 ,500 B2 167 168 - continued
gctaataccg tatacgtcct tcgggagaaa gatttatcgg agatggatga gcccgcgttg 180 gattagctag ttggtggggt aatggcctac caaggcgacg atccatagct ggtctgagag 240 gatgatcagc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcga aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360 agggttgtaa agctctttca acggtgaaga taatgacggt aaccgtagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggaattactg 480 ggcgtaaagc gcacgtaggc 500
< 210 > SEQ ID NO 36 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Rhizobium leguminosarum ATCC10004 < 400 > SEQUENCE : 36 aacgaacgct ggcggcaggc ttaacacatg caagtcgagc gccccgcaag gggagcggca 60 gacgggtgag taacgcgtgg gaacgtaccc tttactacgg aataacgcag ggaaacttgt 120 gctaataccg tatgtgccct ttgggggaaa gatttatcgg taaaggatcg gcccgcgttg 180 gattagctag ttggtggggt aaaggcctac caaggcgacg atccatagct ggtctgagag 240 gatgatcagc cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgagtga tgaaggccct 360 agggttgtaa agctctttca ccggagaaga taatgacggt atccggagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggct agcgttgttc ggaattactg 480 ggcgtaaagc gcacgtaggc 500
< 210 > SEQ ID NO 37 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Paracoccus aminophilus NBRC16710 < 400 > SEQUENCE : 37 aacgaacgct ggcggcaggc ctaacacatg caagtcgagc gcgcccttcg gggtgagcgg 60 cggacgggtg agtaacacgt gggaacatac ccttttctac ggaatagcct cgggaaactg 120 agagtaatac cgtatacgcc cttcggggga aagatttatc ggagaaggat tggcccgcgt 180 tggattaggt agttggtggg gtaatggcct accaagccga cgatccatag ctggtttgag 240 aggatgatca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 300 gggaatctta gacaatgggg gcaaccctga tctagccatg ccgcgtgagt gatgaaggcc 360 ttagggttgt aaagctcttt cagctgggaa gataatgacg gtaccagcag aagaagcccc 420 ggctaactcc gtgccagcag ccgcggtaat acggaggggg ctagcgttgt tcggaattac 480 tgggcgtaaa gcgcacgtag 500
< 210 > SEO ID NO 38 < 211 > LENGTH : 500 < 212 > TYPE : DNA < 213 > ORGANISM : Xanthobacter autotrophicus ATCC35674 < 400 > SEQUENCE : 38 agcgaacgct ggcggcaggc ctaacacatg caagtcgagc goccagcaat gggagcggca 60 gggggagacacgggggattaccatggaggataacccagnac9120 US 10 , 294 ,500 B2 169 170 - continued actaataccg tatgtgccct tcgggggaaa gatttatcgc cattggatga acccgcgtcg 180 gattagctag ttggtgaggt aaaggctcac caaggcgacg atccgtagct ggtctgagag 240 gatgatcagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgga caatgggcgc aagcctgatc cagccatgcc gcgtgtgtga tgaaggcctt 360 agggttgtaa agcactttcg ccggtgaaga taatgacggt aaccggagaa gaagccccgg 420 ctaacttcgt gccagcagcc gcggtaatac gaagggggca agcgttgctc ggaatcactg 480 ggcgtaaagc gcacgtaggc 500
< 210 > SEQ ID NO 39 < 211 > LENGTH : 500 < 212 > TYPE : DNA 213 > ORGANISM : Streptomyces griseus NBRC13350 < 400 > SEQUENCE : 39 acgaacgctg goggcgtgct taacacatge aagtcgaacg atgaagcctt tcggggtgga 60 ttagtggcga acgggtgagt aacacgtggg caatctgccc ttcactctgg gacaagccct 120 ggaaacgggg totaataccg gataacactc tgtcccgcat gggacggggt taaaagctcc 180 ggcggtgaag gatgagcccg cggcctatca gottgttggt ggggtaatgg cctaccaagg 240 cgacgacggg tagccggcct gagagggcga ccggccacac tgggactgag acacggccca 300 gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgaaagcc tgatgcagcg 360 acgccgcgtg agggatgacg gccttcgggt tgtaaacctc tttcagcagg gaagaagcga 420 gagtgacggt acctgcagaa gaagcgccgg ctaactacgt gccagcagcc gcggtaatac 480 gtagggcgca agcgttgtcc 500
< 210 > SEQ ID NO 40 < 211 > LENGTH : 500 TYPE : DNA < 213 > ORGANISM : Geobacillus stearothermophilus NBRC12983 FEATURE : 21 > NAME / KEY : misc _ feature 22 > LOCATION : ( 462 ) . . ( 462 ) < 223 > OTHER INFORMATION : n is a , c , g , or t . < 400 > SEQUENCE : 40 gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggaccggatt ggggcttgcc 60 ttgattcggt cagcggcgga cgggtgagta acacgtgggc aacctgcccg caagaccggg 120 ataactccgg gaaaccggag ctaataccgg ataacaccga agaccgcatg gtcttcggtt 180 gaaaggcggc ctttgggctg tcacttgcgg atgggcccgc ggcgcattag ctagttggtg 240 aggtaacggc tcaccaaggc gacgatgcgt agccggcctg agagggtgac cggccacact 300 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360 gcgaaagcct gacggagcga cgccgcgtga gcgaagaagg ccttcgggtc gtaaagctct 420 gttgtgaggg acgaaggagc gccgttcgaa gagggcggcg cngtgacggt acctcacgag 480 aaagccccgg ctaactacgt 500
< 210 > SEQ ID NO 41 < 211 > LENGTH : 1520 < 212 > TYPE : DNA < 213 > ORGANISM : Rhodococcus erythropolis PR4 < 400 > SEQUENCE : 41 tcaacggaga gtttgatcct ggctcaggac gaacgctggc ggcgtgctta acacatgcaa 60 US 10 , 294 ,500 B2 171 172 - continued gtcgagcggt aaggcctttc ggggtacacg agcggcgaac gggtgagtaa cacgtgggtg 120 atctgccctg cacttcggga taagcctggg aaactgggtc taataccgga tatgacctca 180 ggttgcatga cttggggtgg aaagatttat cggtgcagga tgggcccgcg gcctatcago 240 ttgttggtgg ggtaatggcc taccaaggcg acgacgggta gccgacctga gagggtgacc 300 ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt 360 gcacaatggg cgaaagcctg atgcagcgac gccgcgtgag ggatgacggc cttcgggttg 420 taaacctctt tcagcaggga cgaagcgcaa gtgacggtac ctgcagaaga agcaccggct 480 aactacgtgc cagcagccgc ggtaatacgt agggtgcaag cgttgtccgg aattactggg 540 cgtaaagagt tcgtaggegg tttgtcgcgt cgtttgtgaa aaccagcagc tcaactgctg 600 gottgcaggc gatacgggca gacttgagta ctgcagggga gactggaatt cctggtgtag 660 cggtgaaatg cgcagatatc aggaggaaca ccggtggcga aggcgggtct ctgggcagta 720 actgacgctg aggaacgaaa gcgtgggtag cgaacaggat tagataccct ggtagtccac 780 gccgtaaacg gtgggcgcta ggtgtgggtt ccttccacgg aatccgtgcc gtagctaacg 840 cattaagcgc cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg 900 ggcccgcaca agcggcggag catgtggatt aattcgatgc aacgcgaaga accttacctg 960 ggtttgacat ataccggaaa gctgcagaga tgtggccccc cttgtggtcg gtatacaggt 1020 ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080 aacccctatc ttatgttgcc agcacgttat ggtggggact cgtaagagac tgccggggtc 1140 aactcggagg aaggtgggga cgacgtcaag tcatcatgcc cottatgtcc agggcttcac 1200 acatgctaca atggccagta cagagggctg cgagaccgtg aggtggagcg aatcccttaa 1260 agctggtctc agttcggatc ggggtctgca actcgacccc gtgaagtcgg agtcgctagt 1320 aatcgcagat cagcaacgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380 acgtcatgaa agtcggtaac acccgaagcc ggtggcttaa ccccttgtgg gagggagccg 1440 tcgaaggtgg gatcggcgat tgggacgaag togtaacaag gtagccgtac cggaaggtgc 1500 ggctggatca cctcctttct 1520
< 210 > SEQ ID NO 42 < 211 > LENGTH : 572 < 212 > TYPE : DNA < 213 > ORGANISM : Saccharomyces cerevisiae NBRC1136 < 400 > SEQUENCE : 42 aaaccaaccg ggattgcctt agtaacggcg agtgaagcgg caaaagctca aatttgaaat 60 ctggtacctt cggtgcccga gttgtaattt ggagagggca actttggggc cgttccttgt 120 ctatgttcct tggaacagga cgtcatagag ggtgagaatc ccgtgtggcg aggagtgcgg 180 ttctttgtaa agtgccttcg aagagtcgag ttgtttggga atgcagctct aagtgggtgg 240 taaattccat ctaaagctaa atattggcga gagaccgata gcgaacaagt acagtgatgg 300 aaagatgaaa agaactttga aaagagagtg aaaaagtacg tgaaattgtt gaaagggaag 360 ggcatttgat cagacatggt gttttgtgcc ctctgctcct tgtgggtaggggaatctcgc 420 atttcactgg gccagcatca gttttggtgg caggataaat ccataggaat gtagcttgcc 480 tcggtaagtattatagcctg tgggaatact gccagctggg actgaggact gcgacgtaag 540 tcaaggatgc tggcataatg gttatatgcc gc 572 US 10 ,294 ,500 B2 173 174 - continued
< 210 > SEQ ID NO 43 < 211 > LENGTH : 572 < 212 > TYPE : DNA < 213 > ORGANISM : Saccaromyces cerevisiae NBRC2347 < 400 > SEQUENCE : 43 aaaccaaccg ggattgcctt agtaacggcg agtgaagcgg caaaagctca aatttgaaat 60 ctggtacctt cggtgcccga gttgtaattt ggagagggca actttggggc cgttccttgt 120 ctatgttcct tggaacagga cgtcatagag ggtgagaatc ccgtgtggcg aggagtgcgg 180 ttctttgtaa agtgccttcg aagagtcgag ttgtttggga atgcagctct aagtgggtgg 240 taaattccat ctaaagctaa atattggcga gagaccgata gcgaacaagt acagtgatgg 300 aaagatgaaa agaactttga aaagagagtg aaaaagtacg tgaaattgtt gaaagggaag 360 ggcatttgat cagacatggt gttttgtgcc ctctgctcct tgtgggtagg ggaatctcgc 420 atttcactgg gccagcatca gttttggtgg caggataaat ccataggaat gtagcttgcc 480 tcggtaagta ttatagcctg tgggaatact gccagctggg actgaggact gcgacgtaag 540 tcaaggatgc tggcataatg gttatatgcc gc 572
< 210 > SEQ ID NO 44 < 211 > LENGTH : 568 < 212 > TYPE : DNA < 213 > ORGANISM : Saccharomyces paradoxus NBRC10609 < 400 > SEQUENCE : 44 aaaccaaccg ggattgcctt agtaacggcg agtgaagcgg caaaagctca aatttgaaat ctggtacctt cggtgcccga gttgtaattt ggagagggca actttggggc cgttccttgt 120 ctatgttcct tggaacagga cgtcatagag ggtgagaatc ccgtgtggcg aggagtgcgg 180 ttctatgtaa agtgccttcg aagagtcgag ttgtttggga atgcagctct aagtgggtgg 240 taaattccat ctaaagctaa atattggcga gagaccgata gcgaacaagt acagtgatgg 300 aaagatgaaa agaactttga aaagagagtg aaaaagtacg tgaaattgtt gaaagggaag 360 ggcatttgat cagacatggt gttttgtgcc ctctgctcct tgtgggtagg ggaatctcgc 420 atttcactgg gccagcatca gttttggtgg caggataaat ccgtaggaat gtaacttgct 480 tcgggaagta ttatagcctg cgggaatact gccagctggg actgaggact gcgacgtaag 540 tcaaggatgc tggcataatg gttatatg 568
< 210 > SEQ ID NO 45 < 211 > LENGTH : 577 < 212 > TYPE : DNA < 213 > ORGANISM : Candida utilis NBRC1086 < 400 > SEQUENCE : 45 aaaccaacag ggattgcctc agtaacggcg agtgaagcgg caaaagctca aatttgaaat 60 ctgaggctct cagcccccga gttgtaattt gaagatggtg ttctggcgcc ggccccctgt 120 ctacgttcct tggaacagga catcacagag ggtgagaatc ccgtctggcg gggcggcctg 180 gctccgtgta gagcgccatc gacgagtcga gttgtttggg aatgcagctc taagtgggtg 240 gtaaattcca totaaagcta aatattggcg agagaccgat agcgaacaag tacagtgatg 300 gaaagatgaa aagaactttg aaaagagagt gaaaaagtac gtgaaattgt tgaaagggaa 360 gggtattgga tcagacttgg tgctgtgcga atagcggctc ttcttgggcc gcccactcgc 420 actccaccgg gccagcatcg gtttgggcgg caagacaatg gcgggggaac gtggcactgc 480 US 10 , 294 ,500 B2 175 176 - continued tctcgggcag tgtgtttata gcccccgctg atgttgcctg cctagaccga ggactgeggc 540 ttctgcctag gatgctggcg taatgatcca acaccgc 577
< 210 > SEQ ID NO 46 < 211 > LENGTH : 569 < 212 > TYPE : DNA < 213 > ORGANISM : Candida parapsilosis ATCC22019 < 400 > SEQUENCE : 46 aaaccaacag ggattgcctt agtagcggcg agtgaagcgg caaaagctca aatttgaaat 60 ctggcacttt cagtgtccga gttgtaattt gaagaaggta tctttgggtc tggctcttgt 120 ctatgtttct tggaacagaa cgtcacagag ggtgagaatc ccgtgcgatg agatgtccca 180 gacctatgta aagttccttc gaagagtcga gttgtttggg aatgcagctc taagtgggtg 240 gtaaattcca tctaaagcta aatattggcg agagaccgat agcgaacaag tacagtgatg 300 gaaagatgaa aagaactttg aaaagagagt gaaaaagtac gtgaaattgt tgaaagggaa 360 gggcttgaga tcagacttgg tattttgtat gttactctct cgggggtggc ctctacagtt 420 taccgggcca gcatcagttt gagcggtagg ataagtgcaa agaaatgtgg cactgcttcg 480 gtagtgtgtt atagtctttg togatactgc cagcttagac tgaggactgc ggcttcggcc 540 taggatgttg gcataatgat cttaagtcg 569
< 210 > SEQ ID NO 47 < 211 > LENGTH : 571 < 212 > TYPE : DNA < 213 > ORGANISM : Aspergillus niger ATCC6275 < 400 > SEQUENCE : 47 aaaccaaccg ggattgcctc agtaacggcg agtgaagcgg caagagctca aatttgaaag 60 ctggctcctt cggagtccgc attgtaattt gcagaggatg ctttgggtgc ggcccccgtc 120 taagtgccct ggaacgggcc gtcagagagg gtgagaatcc cgtcttgggc ggggtgtccg 180 tgcccgtgta aagctccttc gacgagtcga gttgtttggg aatgcagctc taaatgggtg 240 gtaaatttca tctaaagcta aatactggcc ggagaccgat agcgcacaag tagagtgatc 300 gaaagatgaa aagcactttg aaaagagagt taaacagcac gtgaaattgt tgaaagggaa 360 gcgcttgcga ccagactcgc ccgcggggtt cagccggcat tcgtgccggt gtacttcccc 420 gtgggcgggc cagcgtcggt ttgggcggcc ggtcaaaggc ccctggaatg tagtgccctc 480 cggggcacct tatagccagg ggtgcaatgc ggccagcctg gaccgaggaa cgcgcttcgg 540 cacggacgct ggcataatgg tcgtaaacga c 571
< 210 > SEO ID NO 48 < 211 > LENGTH : 556 < 212 > TYPE : DNA < 213 > ORGANISM : Aspergillus flavus NBRC8558 < 400 > SEQUENCE : 48 gcggaaggat cattaccgag tgtagggttcctagcgagcc caacctccca cccgtgttta 60 ctgtacctta gttgcttcgg cgggcccgcc attcatggcc gccgggggct ctcagccccg 120 ggcccgcgcc cgccggagac accacgaact ctgtctgatc tagtgaagtc tgagttgatt 180 gtatcgcaat cagttaaaac tttcaacaat ggatctcttg gttccggcat cgatgaagaa 240 cgcagcgaaa tgcgataact agtgtgaatt gcagaattcc gtgaatcatc gagtctttga 300 US 10 , 294 ,500 B2 177 178 - continued acgcacattg cgccccctgg tattccgggg ggcatgcctg tccgagcgtc attgctgccc 360 atcaagcacg gottgtgtgt tgggtcgtcg tcccctctcc gggggggacg ggccccaaag 420 gcagcggcgg caccgcgtcc gatcctcgag cgtatggggc tttgtcaccc gctctgtagg 480 cccggccggc gottgccgaa cgcaaatcaa tctttttcca ggttgacctc ggatcaggta 540 gggatacccg ctgaac 556
< 210 > SEQ ID NO 49 211BNGTH : 554 < 212 > TYPE : DNA < 213 > ORGANISM : Aspergillus oryzae NBRC4255 < 400 > SEQUENCE : 49 tgaacctgcg gaaggatcat taccgagtgt agggttccta gcgagcccaa cctcccaccc 60 gtgtttactg taccttagtt gottcggcgg gcccgccatt catggccgcc gggggctctc 120 agccccgggc ccgcgcccgc cggagacacc acgaactctg tctgatctag tgaagtctga 180 gttgattgta tcgcaatcag ttaaaacttt caacaatgga tctcttggtt ccggcatcga 240 tgaagaacgc agcgaaatge gataactagt gtgaattgca gaattccgtg aatcatcgag 300 tctttgaacg cacattgcgc cccctggt at tccggggggc atgcctgtcc gagcgtcatt 360 gctgcccato aagcacggct tgtgtgttgg gtcgtcgtcc cctctccggg ggggacgggc 420 cccaaaggca gcggcggcac cgcgtccgat cctcgagcgt atggggcttt gtcacccgct 480 ctgtaggccc ggccggcgct tgccgaacgc aaatcaatct tttccaggtt gacctcggat 540 caggtaggga tacc 554
< 210 > SEQ ID NO 50 < 211 > LENGTH : 387 < 212 > TYPE : PRT < 213 > ORGANISM : Pseudomonas aeruginosa PA01 Acdi < 400 > SEQUENCE : 50 Met Asp Phe Asp Leu Thr Glu Glu Gln Arq Leu Leu Val Glu Ser Ala 15
Arg Ala Phe Ala Arq His Glu Leu Ala Pro Lys Ala Ala Asp Trp Asp 25 Arg Asp His His Phe Pro Val Glu Val Ile Arg Ala Ala Ala Glu Gin 40 45 Gly Tyr Leu Gly Leu Tyr Ile Ala Glu Glu Asp Gly Gly Leu Gly Leu Ser Arg Leu Ser Thr Ser Leu Ile Phe Glu Gin Leu Ala Ala Gly Cys 65 75 80 Val Ala Thr Thr Ala Tyr Ile Ser Ile His Asn Met Ala Ala Trp Met 95 Leu Ala Ser Phe Gly Asp Ala Ala Leu Lys Glu Ala Trp Leu Pro Gly 105 Leu Ile Gly Gly Glu Ser Leu Ala Ser Tyr Cys Leu Thr Glu Pro Asp 120 125 Ala Gly Ser Asp Ala Ala Arg Leu Arg Thr Arg Ala Arg Arg Glu Gly
Asp Glu Tyr Val Leu Asp Gly Ser Lys Cys Phe Ile Ser Gly Ala Gly 145 155 160 Ser Thr Gin Val Leu Ile Val Met Ala Arg Thr Gly Glu Asp Gly Ala 175 US 10 , 294 ,500 B2 179 180 - continued Arg Gly Ile Ser Cys Phe Leu Val Pro Ala Asp Ala Pro Gly Ile Arg 185 190 Tyr Gly Arg Asn Glu Asp Lys Met Gly Trp Arg Ala Gin Pro Thr Arg 195 200 205 Thr Ile Thr Phe Glu Gly Val Arg Ile Pro Ala Gly Asn Arg Ile Gly 220 Pro Glu Gly Gin Gly Phe Val Tyr Ala Met Lys Gly Leu Asp Gly Gly 225 240 Arg Leu Asn Ile Ala Ser Cys Ser Leu Gly Ala Ala Gln Ala Ala Leu
Glu Gln Ser Met Arq Tyr Val Glu Glu Ara Glu Gln Phe Gly Lys Pro 265 270 Leu Ala Thr Phe Gln Ala Leu Gln Phe Lys Leu Ala Asp Met Leu Thr 275 280 285 Glu Leu Thr Ala Ser Arg Gln Met Val Arq Leu Gly Ala His Arq Leu 300 Asp Arg Gly Asp Ala Glu Ala Thr Leu Tyr Cys Ala Met Ala Lys Arq 305 320 Phe Ala Thr Asp Arg Cys Phe Asp Val Cys Asn Glu Ala Leu Gin Leu His Gly Gly Tyr Gly Tyr Leu Asn Asp Tyr Pro Leu Glu Arg Trp Val 345 350 Arg Asp Thr Arg Val His Gin Ile Leu Glu Gly Thr Asn Glu Ile Met 355 360 365 Arg Val Ile Val Ala Arg Arg Leu Leu Glu Gin Gly Gly Met Leu Asp 380 Arg Leu Leu 385
< 210 > SEQ ID NO 51 < 211 > LENGTH : 1164 < 212 > TYPE : DNA < 213 > ORGANISM : Pseudomonas aeruginosa PA01 Acdi < 400 > SEQUENCE : 51 atggatttcg acctcaccga agaacaacgc ctgctggtgg agagcgcccg cgccttcgcc 60 cgccacgaac tggcgccgaa ggcggccgac tgggaccgcg accatcactt cccggtggaa 120 gtcatccgcg ccgccgccga acagggctac ctcggcctgt acatcgccga ggaagacggc 180 ggcctgggcc tgtcgcggct gtccacttcg ctgatcttcg agcaactggc cgccggctgc 240 gtggccacta ccgcctacat cagcatccac aacatggccg cctggatgct cgcctcgttc 300 ggcgacgcgg cgctgaagga ggcctggctg cccggcctga tcggcggcga gtcgctcgcc 360 tcctattgcc tgaccgagcc cgatgccggc tccgacgccg cgcgcctgcg cacccgcgcc 420 cgccgcgagg gcgacgaata cgtgctggac ggcagcaagt gottcatttc cggcgccggc 480 agcacccagg tgctgatcgt catggcgcgc accggcgagg acggcgccag gggcatctcc 540 tgcttcctgg taccggccga cgcgcccggc atccgctacg gccgcaacga ggacaagatg 600 ggctggcgcg cgcagccgac ccgcaccato accttcgaag gcgtgcgcat ccccgccggc 660 aaccgcatcg gcccggaggg ccaaggcttc gtctatgcca tgaaaggcct cgacggcggc 720 cgcctgaaca tcgccagttg ttccctgggc gccgcccagg cggcgctgga gcagtcgatg 780 cgctacgtcg aggagcgcga gcagtteggc aagccgctgg cgaccttcca ggccttgcag 840 ttcaagctcg cogacatgct caccgaactc accgccagcc gccagatggt Ccgcctcggc 900 US 10 , 294 ,500 B2 181 182 - continued
gcccatcggc tggaccgcgg cgacgccgag gcgaccctgt actgcgcaat ggccaagcgc 960 ttcgccaccg accgctgctt cgatgtctgc aacgaggcct tgcaactgca cggcggctac 1020 ggctatctca acgattatcc gctggagcgc tgggtacgcg acacccgcgt gcaccagato 1080 ctcgaaggca ccaacgaaat catgcgggtg atcgtcgccc gccgcctgct ggagcagggc 1140 ggcatgctcg atcgcctgct gtga 1164
< 210 > SEQ ID NO 52 < 211 > LENGTH : 386 < 212 > TYPE : PRT < 213 > ORGANISM : Rhodococcus erythropolis PR4 RE _ Acdi < 400 > SEQUENCE : 52 Met Phe Thr Leu Thr Asp Asp Glu Arg Ala Ile Arg Asp Thr Ala Arg 15 Asp Phe Ala Ala Glu His Leu Ala Pro Asn Ala Val Glu Trp Asp Gln 20 Thr Lys His Phe Pro Val Asp Val Leu Arq Lys Ala Ala Ser Leu Gly 35 40 45 Met Gly Gly Ile Tyr Ile Arg Glu Asp Val Gly Gly Ser Glu Leu Ser
Arg Val Asp Ala Ala Arg Ile Phe Glu Glu Leu Ala Lys Gly Asp Pro 65 80 Ser Ile Ala Ala Tyr Ile Ser Ile His Asn Met Val Thr Trp Met Ile 95 Asp Gin Phe Gly Asn Asp Glu Gin Arg His Lys Trp Val Pro Gly Leu 100 Cys Ser Met Asp Gin Leu Gly Ser Tyr Cys Leu Thr Glu Pro Gly Ala 115 120 125 Gly Ser Asp Ala Ala Gly Leu Ser Thr Lys Ala Val Arg Asp Gly Asp 135 Asp Tyr Ile Leu Asn Gly Val Lys Gin Phe Ile Ser Gly Ala Gly Thr 145 160 Ser Asp Val Tyr Val Val Met Ala Arg Thr Gly Ser Ala Gly Ala Lys 175 Gly Ile Ser Ala Phe Ile Val Pro Lys Asp Ser Pro Gly Leu Ser Phe 180
Gly Ala Asn Glu Val Lys Met Gly Trp Asn Ala Gln Pro Thr Arg Gin 195 200 205 Val Ile Phe Glu Asp Val Arg Val Pro Ala Ala Asn Met Leu Gly Glu Glu Gly Ser Gly Phe Arg Ile Ala Met Lys Gly Leu Asn Gly Gly Arg 225 240 Leu Asn Ile Ala Ala Cys Ser Val Gly Gly Ala Gin Ala Ala Leu Glu 255 Lys Ala Val Ala Tyr Leu Val Asp Arg Lys Ala Phe Gly Ser Ala Leu 260 Ile Glu Ser Gin Ala Leu Gin Phe Gin Leu Ala Asp Met Arg Thr Glu 275 280 285 Leu Glu Ala Ala AraArg Thr LeuLeu Leu TrpTrp AraArg Ala Ala Ala Ala Leu Glu Asp Gly Ala Ser Asp Val Val Glu Leu Cys Ala Met Ala Lys Arg Phe 305 310 320