US 20170058290A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0058290 A1 Evdokimov et al. (43) Pub. Date: Mar. 2, 2017

(54) METHODS AND COMPOSITIONS FOR AOIN 37/28 (2006.01) TOLERANCE IN PLANTS AOIN 43/54 (2006.01) AOIN 43/90 (2006.01) (71) Applicant: Monsanto Technology LLC, St. Louis, AOIN 43/653 (2006.01) MO (US) AOIN 43/82 (2006.01) AOIN 43/56 (2006.01) (72) Inventors: Artem G. Evdokimov, Orchard Park, CI2N 9/02 (2006.01) NY (US); Clayton T. Larue, AOIN 43/84 (2006.01) Chesterfield, MO (US); Farhad (52) U.S. Cl. Moshiri, Chesterfield, MO (US); Joel CPC ...... CI2N 15/8274 (2013.01); C12N 9/001 E. Ream, St. Louis, MO (US), Xuefeng (2013.01); C12Y 103/03004 (2013.01); C12N Zhou, St. Louis, MO (US) 15/8241 (2013.01); C12O 1/68 (2013.01); A0IN 37/10 (2013.01); A0IN 37/28 (21) Appl. No.: 15/228,993 (2013.01); A0IN 43/84 (2013.01); A0IN 1-1. 43/90 (2013.01); A0IN 43/653 (2013.01); (22) Filed: Aug. 4, 2016 A0IN 43/82 (2013.01): A0IN 43/56 Related U.S. Application Data (2013.01); A0IN 43/54 (2013.01) (60) Provisional application No. 62/212.716, filed on Sep. 57 ABSTRACT 1, 2015, provisional application No. 62/323,852, filed (57) on Apr. 18, 2016. The invention relates to biotechnology and provides novel O O recombinant DNA molecules and engineered proteins for Publication Classification conferring tolerance to protoporphyrinogen oxidase-inhibi- - - - - (51) Int. Cl. tor . The invention also provides herbicide toler CI2N 5/82 (2006.01) ant transgenic plants, seeds, cells, and plant parts containing CI2O I/68 (2006.01) the recombinant DNA molecules, as well as methods of AOIN 37/10 (2006.01) using the same. Patent Application Publication Mar. 2, 2017 Sheet 1 of 2 US 2017/0058290 A1

Acifluorfen 1 mM & Fumioxazin 1mM : 1mM Formesafen 1mM DMSO 1% E LB control 120 --~

100 ---

8 O

6 O

PPO Wariant

Figure 1 Patent Application Publication Mar. 2, 2017 Sheet 2 of 2 US 2017/0058290 A1

Aciforfen 1 nM. & S300 00M 3DMSO % E3 LB control 120 --~

100 or

6 O i 40 . 20 - 0 i. a c c c c c s s s s s E.d Z Z. Z Z Z. n u) wa N We ask 9. e ge e e se PPO Variant

Figure 2 US 2017/0058290 A1 Mar. 2, 2017

METHODS AND COMPOSITIONS FOR Journal of Bacteriology 174(24):8081-8093 (1992)), HemG HERBICIDE TOLERANCE IN PLANTS (A Sasarman, et al., “Mapping of a new hem gene in Escherichia coli K12 Microbiology 113:297-303 (1979)), CROSS-REFERENCE TO RELATED and Heml (TO Boynton, et al., “Discovery of a gene APPLICATIONS involved in a third bacterial protoporphyrinogen oxidase 0001. This application claims the benefit of priority of activity through comparative genomic analysis and func U.S. Provisional Application No. 62/212.716, filed Sep. 1, tional complementation’ Applied and Environmental Micro 2015, and U.S. Provisional Application No. 62/323,852, biology 77(14):4795-4801 (2011)). This invention provides filed Apr. 18, 2016, the disclosures of which are hereby novel recombinant iPPOs that are members of the HemY incorporated by reference in their entirety. family. Despite over twenty years of research and the number of iPPOs identified to date, a transgenic crop plant comprising a recombinant iPPO has yet to be commercial INCORPORATION OF SEQUENCE LISTING ized. A strong weed control platform depends, in part, on 0002 The sequence listing that is contained in the file continued development of herbicide tolerance trait pack named MONS384 US sequence listing..txt, which is 204 ages. Identifying and utilizing iPPOS to create transgenic kilobytes (measured in MS-Windows) and created on Aug. crop traits therefore represents an advance to . 3, 2016, is filed herewith by electronic submission and incorporated herein by reference. SUMMARY OF THE INVENTION BACKGROUND 0009. In one aspect, the invention provides a recombinant DNA molecule comprising a heterologous promoter oper 0003 Field of the Invention ably linked to a nucleic acid sequence encoding a polypep 0004. The present invention relates to the field of bio tide that has at least 85% sequence identity to an amino acid technology. More specifically, the invention relates to sequence chosen from SEQ ID NOs: 1-2 and SEQ ID NOs: recombinant DNA molecules encoding enzymes that pro 6-12, wherein the polypeptide has herbicide-insensitive pro vide tolerance to herbicides that inhibit protoporphyrinogen toporphyrinogen oxidase activity. In certain embodiments, oxidase. the polypeptide has at least about 85% sequence identity, at 0005 Related Art least about 90% sequence identity, at least 95% sequence 0006 Agricultural crop production often utilizes trans identity, at least 96% sequence identity, at least 97% genic traits created using the methods of biotechnology. A sequence identity, at least 98% sequence identity, or at least heterologous gene, also known as a transgene, can be 99% sequence identity to an amino acid sequence chosen introduced into a plant to produce a transgenic trait. Expres from among SEQ ID NOS:1-2 and SEQ ID NOS:6-12 and sion of the transgene in the plant confers a trait, such as has herbicide-insensitive protoporphyrinogen oxidase activ herbicide tolerance, on the plant. Examples of transgenic ity. In some embodiments there is provided a recombinant herbicide tolerance traits include tolerance, glu DNA molecule, wherein the nucleic acid sequence is fosinate tolerance, and tolerance. With the increase selected from the group consisting of SEQ ID NOS:26-27. of weed species resistant to the commonly used herbicides, 31-32, 36-46, and 47-48. In particular embodiments the new herbicide tolerance traits are needed in the field. Her recombinant DNA molecule encodes a polypeptide compris bicides of particular interest include herbicides that inhibit ing an amino acid sequence selected from the group con protoporphyrinogen oxidase (PPO, EC 1.3.3.4), referred to sisting of SEQ ID NOs: 1-2 and SEQ ID NOS:6-12. A as PPO herbicides. PPO herbicides provide control of a recombinant polypeptide that comprises at least 85% spectrum of herbicide-resistant weeds, thus making a trait sequence identity to the full length of an amino acid conferring tolerance to these herbicides particularly useful in sequence chosen from among SEQID NOs: 1-2 and SEQID a cropping system combined with one or more other herbi NOs:6-12, wherein the polypeptide has herbicide-insensi cide-tolerance trait(s). tive protoporphyrinogen oxidase activity is therefore pro 0007 Protoporphyrinogen oxidase functions in both vided by the invention. and biosynthesis pathways where it con 0010. In certain embodiments a heterologous promoter, verts protoporphyrinogen IX to protoporphyrin IX. Follow for instance, a promoter functional in a plant cell, is operably ing production of protoporphyrin IX, the chlorophyll and linked to the nucleic acid sequence encoding a polypeptide heme biosynthetic pathways diverge with different metal that has at least 85% sequence identity to an amino acid ions being incorporated (iron for heme and magnesium for sequence of the invention, for instance an amino acid chlorophyll). Segments of this pathway are conserved across sequence chosen from SEQ ID NOs: 1-2 and SEQ ID NOs: prokaryotes and eukaryotes, and many of the PPO enzymes 6-12, wherein the polypeptide has herbicide-insensitive pro found across prokaryotes and eukaryotes are relatively simi toporphyrinogen oxidase activity. Such a resulting DNA lar. Some prokaryotes (e.g., cyanobacteria) use this pathway molecule may further comprise a targeting sequence that for chlorophyll and heme production while other prokary functions to localize the polypeptide within a cell. otes (e.g., Escherichia coli) use this pathway for heme 0011. In one aspect, the invention provides a DNA con production. struct comprising a recombinant DNA molecule of the 0008 Herbicide-insensitive protoporphyrinogen oxi invention. In one embodiment, such a DNA construct com dases (iPPOs) have been isolated from a number of pro prises, in operable linkage to a nucleic acid sequence of the karyotes and eukaryotes. On a structural basis, it is believed invention, a targeting sequence that functions to localize the that there are at least three distinct subclasses of PPO polypeptide within a cell. The DNA construct may be enzymes: HemY (M Hansson and L. Hederstedt, “Cloning present in the genome of a transgenic plant, seed, or cell. In and characterization of the Bacillus subtilis hemEHY gene certain embodiments, the polypeptide confers herbicide tol cluster, which encodes protoheme IX biosynthetic enzymes’ erance to the cell, plant, seed, or plant part. US 2017/0058290 A1 Mar. 2, 2017

0012 Another aspect of the invention provides a trans 0019. Further, the invention provides methods of screen genic plant, seed, cell, or plant part comprising a recombi ing for a herbicide tolerance gene comprising: a) expressing nant DNA molecule of the invention or a recombinant a recombinant DNA molecule of the invention in a bacterial polypeptide of the invention. The transgenic plant, seed, cell lacking HemG, wherein the bacterial cell is grown in a cell, or plant part may thus comprise, i.e. display, tolerance heme-free medium in the presence of a PPO herbicide; and to at least one PPO herbicide. In some embodiments, the b) identifying a bacterial cell that displays tolerance to a transgenic plant, seed, cell, or plant part comprises an PPO herbicide. additional transgenic herbicide tolerance trait. 0020. In another aspect, the invention provides a method 0013 Another aspect of the invention provides a method of producing a plant tolerant to a PPO herbicide and at least for conferring herbicide tolerance to a plant, seed, cell, or one other herbicide comprising: a) obtaining a plant com plant part comprising: heterologously expressing a recom prising a recombinant DNA molecule of the invention; b) binant polypeptide of the invention in the plant, seed, cell, crossing the transgenic plant with a second plant comprising or plant part. In some embodiments of the method, the plant, tolerance to the at least one other herbicide, and c) selecting seed, cell, or plant part comprises protoporphyrinogen oxi a progeny plant resulting from said crossing that comprises dase activity conferred by the recombinant polypeptide. In tolerance to a PPO herbicide and the at least one other Some embodiments, the herbicide tolerance is to at least one herbicide is another aspect of the invention. PPO herbicide selected from the group consisting of acif 0021. The invention also provides, in another aspect, a luorfen, , lactofen, fluoroglycofen-ethyl, oxyfluo method for reducing the development of herbicide tolerant rfen, flumioxazin, azafenidin, carfentraZone-ethyl, Sulfentra weeds comprising: a) cultivating in a crop growing envi Zone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen ronment a plant of the present invention that comprises ethyl, and S-3100. tolerance to a PPO herbicide, for instance by comprising a 0014) Another aspect of the invention relates to a method DNA molecule of the present invention, and comprises of plant transformation, comprising the steps of: a) intro tolerance to at least one other herbicide; and b) applying a ducing a recombinant DNA molecule of the invention into a PPO herbicide and at least one other herbicide to the crop plant cell; and b) regenerating a transgenic plant therefrom growing environment, wherein the crop plant is tolerant to that comprises the recombinant DNA molecule. The method the PPO herbicide and the at least one other herbicide. In may further comprise the step of selecting a plant that is certain embodiments of the method, the PPO herbicide may tolerant to at least one PPO herbicide. The method may also be selected from the group consisting of , fome further comprise a step of crossing the regenerated plant safen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumiox with itself or with a second plant and collecting seed from azin, azafenidin, carfentraZone-ethyl, , fluthi the cross. acet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, saflufenacil and S-3100. In some embodiments of the 00.15 Yet another aspect of the invention provides a method, the at least one other herbicide is selected from the method for controlling weeds in a plant growth area, com group consisting of an ACCase inhibitor, an ALS inhibitor, prising contacting a plant growth area comprising the trans an EPSPS inhibitor, a synthetic , a photosynthesis genic plant or seed with at least one PPO herbicide, wherein inhibitor, a glutamine synthesis inhibitor, a HPPD inhibitor, the transgenic plant or seed is tolerant to the PPO herbicide a PPO inhibitor, and a long-chain fatty acid inhibitor. In and wherein weeds are controlled in the plant growth area. particular embodiments, the ACCase inhibitor is an aryloxy 0016. Also provided is a method of identifying a nucleo phenoxy propionate or a cyclohexanedione; the ALS inhibi tide sequence encoding a protein having protoporphyrino tor is a Sulfonylurea, imidazolinone, triazoloyrimidine, or a gen oxidase activity, the method comprising: a) transform triazolinone; the EPSPS inhibitor is glyphosate; the syn ing an E. coli strain having a gene knockout for the native thetic auxin is a , a , a E. coli PPO enzyme with a bacterial expression vector carboxylic acid, or a semicarbazone; the photosynthesis comprising a recombinant DNA molecule encoding a can inhibitor is a triazine, a triazinone, a nitrile, a benzothiadi didate herbicide tolerance protein; and b) growing said azole, or a ; the glutamine synthesis inhibitor is glufo transformed E. coli using a heme-free bacterial medium, sinate; the HPPD inhibitor is an isoxazole, a pyrazolone, or wherein growth using said bacterial medium identifies a a triketone; the PPO inhibitor is a diphenylether, a N-phe protein having protoporphyrinogen oxidase activity. nylphthalimide, an aryl triazinone, or a pyrimidinedione; or 0017. Further provided by the invention is a method of the long-chain fatty acid inhibitor is a chloroacetamide, an identifying a nucleotide sequence encoding a protein having oxyacetamide, or a pyrazole. herbicide-insensitive protoporphyrinogen oxidase activity, the method comprising: a) transforming an E. coli Strain BRIEF DESCRIPTION OF THE DRAWINGS having a gene knockout for the native E. coli PPO enzyme 0022 FIG. 1. Assay results from herbicide bacterial with a bacterial expression vector comprising a recombinant screening system with PPO herbicides. Assay of E. coli DNA molecule encoding a recombinant protein; and b) Hemg (H N10) (SEQ ID NO:76), HemY PPO R1N473 growing said transformed E. coli using a bacterial medium (SEQ ID NO:13), HemYPPOR1N533 (SEQID NO:14); or containing at least one PPO herbicide, wherein growth of HemY PPO R1N171 (SEQ ID NO:15) in the presence of bacteria identifies a protein having herbicide-insensitive acifluorfen, flumioxazin, lactofen, or fomesafen PPO herbi protoporphyrinogen oxidase activity. cides. 0018. Another aspect of the invention relates to a method 0023 FIG. 2. Assay results from herbicide bacterial of Screening for a herbicide tolerance gene comprising: a) screening system with PPO herbicides. Assay of HemY expressing a recombinant DNA molecule of the invention in R2N30 (SEQ ID NO:1), Hem Y R2N40 (SEQ ID NO:2), a plant cell; and b) identifying a plant cell that displays HemY R2N70 (SEQ ID NO:3), Hemy R2N90 (SEQ ID tolerance to a PPO herbicide. NO:4), HemY R2N100 (SEQ ID NO:5), and negative con US 2017/0058290 A1 Mar. 2, 2017

trol (WH) PPO (SEQ ID NO:80) 0051 SEQ ID NOs: 63-67 are the nucleotide sequences in the presence of acifluorfen and S-3100 PPO herbicides encoding the amino acid sequences represented by SEQID NOs: 13-17, codon optimized for monocot expression. BRIEF DESCRIPTION OF THE SEQUENCES 0052 SEQ ID NOS:68-75 are the nucleotide sequences 0024 SEQ ID NO:1 is the amino acid sequence of encoding the amino acid sequences represented by SEQID R2N3O. NOs:18-25, respectively, codon optimized for dicot expres 0025 SEQ ID NO:2 is the amino acid sequence of S1O. R2N40. 0053 SEQ ID NO:76 is the amino acid sequence of E. 0026 SEQ ID NO:3 is the amino acid sequence of coli PPO enzyme HemG (protoporphyrinogen IX dehydro R2N70. genase; GenBank Accession No. WP 021498.199). 0027 SEQ ID NO:4 is the amino acid sequence of 0054 SEQ ID NOS:77-79 are the nucleotide sequences R2N90. encoding the amino acid sequence represented by SEQ ID 0028 SEQ ID NO:5 is the amino acid sequence of NO:76. R2N1OO. 0055 SEQ ID NO:80 is the amino acid sequence of the 0029 SEQ ID NO:6 is the amino acid sequence of a wild-type protoporphyrinogen oxidase from Amaranthus variant of SEQ ID NO: 1 (R2N30). tuberculatus (WH). 0030 SEQID NOS:7-12 are the amino acid sequences of 0056 SEQ ID NO:81 is the nucleotide sequence encod variants of SEQ ID NO:2 (R2N40). ing the amino acid sequence represented by SEQID NO:80, 0031 SEQ ID NO:13 is the amino acid sequence of codon optimized for bacterial E. coli expression. R1N473. 0032 SEQ ID NO:14 is the amino acid sequence of DETAILED DESCRIPTION R1N533. 0057 The following descriptions and definitions are pro 0033 SEQ ID NO:15 is the amino acid sequence of vided to better define the invention and to guide those of R1N171. ordinary skill in the art in the practice of the invention. 0034 SEQ ID NO:16 is the amino acid sequence of Unless otherwise noted, terms are to be understood accord R1N311. ing to conventional usage by those of ordinary skill in the 0035) SEQ ID NO:17 is the amino acid sequence of relevant art. R1N333. 0058. The invention provides novel, recombinant DNA 0036 SEQID NO:18 is a truncated amino acid sequence molecules and proteins that encode herbicide-insensitive of R1 N473. protoporphyrinogen oxidases (iPPOs). For instance, the 0037 SEQID NO:19 is a truncated amino acid sequence invention provides in one embodiment vectors and expres of R1 N533. sion cassettes encoding microbially derived iPPOs for 0038 SEQID NO:20 is a truncated amino acid sequence expression in cells and plants. Methods for producing cells of R1N171. and plants tolerant to PPO herbicides are also provided. The 0039 SEQID NO:21 is a truncated amino acid sequence invention further provides methods and compositions for of R1N333. using protein engineering and bioinformatic tools to obtain 0040 SEQID NO:22 is a truncated amino acid sequence and improve iPPOs. of R1 N473. 0059. In specific aspects, the invention provides recom 0041 SEQID NO:23 is a truncated amino acid sequence binant DNA molecules and proteins. As used herein, the of R1 N533. term “recombinant refers to a non-naturally occurring 0042 SEQID NO:24 is a truncated amino acid sequence DNA, protein, cell, seed, or organism that is the result of of R1N171. genetic engineering and as Such would not normally be 0043 SEQID NO:25 is a truncated amino acid sequence found in nature. A “recombinant DNA molecule' is a DNA of R1N333. molecule comprising a DNA sequence that does not natu 0044 SEQID NOS:26-30 are the native bacterial nucleo rally occur in nature and as such is the result of human tide sequences encoding SEQ ID NOs: 1 through SEQ ID intervention, such as a DNA molecule comprised of at least NO:5, respectively. two DNA molecules heterologous to each other. An example 0045 SEQ ID NOS:31-35 are nucleotide sequences of a recombinant DNA molecule is a DNA molecule pro encoding SEQ ID NOs: 1-5, respectively, codon optimized vided herein encoding herbicide-insensitive protoporphy for dicot expression. rinogen oxidase operably linked to a heterologous regulatory 0046 SEQ ID NOS:36-42 are nucleotide sequences or other element, Such as a heterologous promoter. A encoding SEQ ID NOS:6-12, respectively, codon optimized “recombinant protein’ is a protein comprising an amino acid for dicot expression. sequence that does not naturally occur and as such is the 0047 SEQ ID NOs:43-46 are nucleotide sequences result of human intervention, Such as an engineered protein encoding SEQ ID NO:9, codon optimized for dicot expres or a chimeric protein. A recombinant cell, seed, or organism S1O. is a cell, seed, or organism comprising transgenic DNA, for 0048 SEQ ID NOs:47-51 are nucleotide sequences example a transgenic cell, seed, plant, or plant part com encoding SEQ ID NOs: 1-5, respectively, codon optimized prising a recombinant DNA molecule and therefore pro for monocot expression. duced as a result of plant transformation. 0049 SEQID NOS:52-56 are the native bacterial nucleo 0060. As used herein, the term “genetic engineering tide sequences encoding SEQ ID NOS:13-17. refers to the creation of a non-natural DNA, protein, or 0050 SEQ ID NOS:57-62 are the nucleotide sequences organism that would not normally be found in nature and encoding the amino acid sequences represented by SEQID therefore entails applying human intervention. Genetic engi NOs: 13-17, codon optimized for dicot expression. neering can be used to produce an engineered DNA, protein, US 2017/0058290 A1 Mar. 2, 2017

or organism that was conceived of and created in the Sasarman et al., “Nucleotide sequence of the hemO gene laboratory using one or more of the techniques of biotech involved in the protoporphyrinogen oxidase activity of E. nology Such as molecular biology, protein biochemistry, coli K12 Can J Microbiol 39:1155-1161, 1993). bacterial transformation, and plant transformation. For 0063 As used herein, the term “transgene' refers to a example, genetic engineering can be used to create a chi DNA molecule artificially incorporated into an organisms meric gene comprising at least two DNA molecules heter genome because of human intervention, Such as a plant ologous to each other using one or more of the techniques of transformation method. As used herein, the term “trans molecular biology, Such as gene cloning, DNA ligation, and genic’ means comprising a transgene, for example a “trans DNA synthesis. A chimeric gene may consist of two or more genic plant” refers to a plant comprising a transgene in its heterologous DNA molecules that are operably linked, such genome and a “transgenic trait” refers to a characteristic or as a protein-coding sequence operably linked to a gene phenotype conveyed or conferred by the presence of a expression element such as a transit peptide-coding transgene incorporated into the plant genome. Because of sequence or a heterologous promoter. Genetic engineering Such genomic alteration, the transgenic plant is something can be used to create an engineered protein whose amino distinctly different from the related wild-type plant and the acid sequence was created using one or more of the tech transgenic trait is a trait not naturally found in the wild-type niques of protein engineering, such as protein design using plant. Transgenic plants of the invention comprise the site-directed mutagenesis and directed evolution using ran recombinant DNA molecules and engineered proteins pro dom mutagenesis and DNA shuffling. An engineered protein vided by the invention. may have one or more deletions, insertions, or Substitutions 0064. As used herein, the term "heterologous” refers to relative to the coding sequence of the wild-type protein and the relationship between two or more items derived from each deletion, insertion, or Substitution may consist of one or different sources and thus not normally associated in nature. more amino acids. In another embodiment, an engineered For example, a protein-coding recombinant DNA molecule protein may consist of two heterologous peptides that are is heterologous with respect to an operably linked promoter operably linked, such as an enzyme operably linked to a if such a combination is not normally found in nature. In transit peptide. another example, a protein-coding DNA molecule or a 0061. As used herein, “herbicide-insensitive' or “herbi polypeptide may be heterologously expressed in a plant, cide-insensitive protoporphyrinogen oxidase activity” seed, cell, or plant part if such protein-coding DNA mol means the ability of a protoporphyrinogen oxidase (PPO, EC ecule or polypeptide is not normally expressed in Such a 1.3.3.4) to maintain at least some of its protoporphyrinogen plant, seed, cell, or plant part in nature. A particular recom oxidase activity in the presence of one or more PPO herbi binant DNA molecule may be heterologous with respect to cide(s). The term “protoporphyrinogen oxidase activity” a cell, seed, or organism into which it is inserted when it means the ability to catalyze the six-electron oxidation would not naturally occur in that particular cell, seed, or (removal of electrons) of protoporphyrinogen IX to form organism. A particular polypeptide may be heterologous protoporphyrin IX, that is, to catalyze the dehydrogenation with respect to a cell, seed, or organism in which it is of protoporphyrinogen to form protoporphyrin. Enzymatic expressed when it would not naturally occur in that particu activity of a protoporphyrinogen oxidase can be measured lar cell, seed, or organism. by any means known in the art, for example, by an enzy 0065. As used herein, the term "isolated’ refers to at least matic assay in which the production of the product of partially separating a molecule from other molecules typi protoporphyrinogen oxidase or the consumption of the Sub cally associated with it in its natural state. In one embodi strate of protoporphyrinogen oxidase in the presence of one ment, the term "isolated’ refers to a DNA molecule that is or more PPO herbicide(s) is measured via fluorescence, high separated from the nucleic acids that normally flank the performance liquid chromatography (HPLC), or mass spec DNA molecule in its natural state. For example, a DNA trometry (MS). Another example of an assay for measuring molecule encoding a protein that is naturally present in a enzymatic activity of a protoporphyrinogen oxidase is a bacterium would be an isolated DNA molecule if it was not bacterial assay, such as the assays described herein, whereby within the DNA of the bacterium from which the DNA a recombinant protoporphyrinogen oxidase is expressed in a molecule encoding the protein is naturally found. Thus, a bacterial cell otherwise lacking PPO activity and the ability DNA molecule fused to or operably linked to one or more of the recombinant protoporphyrinogen oxidase to comple other DNA molecule(s) with which it would not be associ ment this knockout phenotype is measured. Herbicide-in ated in nature, for example as the result of recombinant sensitivity may be complete or partial insensitivity to a DNA or plant transformation techniques, is considered iso particular herbicide, and may be expressed as a percent (%) lated herein. Such molecules are considered isolated even tolerance or insensitivity to a particular PPO herbicide. As when integrated into the chromosome of a host cell or used herein, an "herbicide-insensitive protoporphyrinogen present in a nucleic acid solution with other DNA molecules. oxidase” or “iPPO” exhibits herbicide-insensitivity in the 0066. As used herein, the term “protein-coding DNA presence of one or more PPO herbicide(s). molecule' refers to a DNA molecule comprising a nucleo 0062. As used herein, a “hemG knockout strain” means tide sequence that encodes a protein. A "protein-coding an organism or cell of an organism, Such as E. coli, that lacks sequence” means a nucleic acid sequence that encodes a HemG activity to the extent that it is unable to grow on protein. A "sequence” means a sequential arrangement of heme-free growth medium, or Such that its growth is detect nucleotides or amino acids. The boundaries of a protein ably impaired in the absence of heme relative to an other coding sequence may be determined by a translation start wise isogenic strain comprising a functional HemG. AhemO codon at the 5'-terminus and a translation stop codon at the knockout strain of for instance, E. coli may be prepared in 3'-terminus. A protein-coding molecule may comprise a view of knowledge in the art, for instance in view of the E. nucleic acid sequence encoding an amino acid sequence. As coli hemO sequence (Ecogene Accession No. EG11485; used herein, “transgene expression', 'expressing a trans US 2017/0058290 A1 Mar. 2, 2017

gene”, “protein expression', and “expressing a protein’ 0069. In one embodiment of the invention, a DNA con mean the production of a protein through the process of struct provided herein includes a nucleic acid sequence transcribing a DNA molecule into messenger RNA (mRNA) encoding a targeting sequence that is operably linked to a and translating the mRNA into polypeptide chains, which heterologous nucleic acid sequence encoding a polypeptide are ultimately folded into proteins. A protein-coding DNA molecule that has herbicide-insensitive protoporphyrinogen molecule may be operably linked to a heterologous promoter oxidase activity, whereby the targeting sequence facilitates in a DNA construct for use in expressing the protein in a cell localizing the polypeptide molecule within the cell. Target transformed with the recombinant DNA molecule. As used ing sequences are known in the art as signal sequences, herein, “operably linked' means two or more DNA mol targeting peptides, localization sequences, and transit pep ecules or two or more polypeptides linked in manner so that tides. An example of a targeting sequence is a chloroplast one may affect the function of the other. Operably-linked transit peptide (CTP), a mitochondrial targeting sequence DNA molecules or operably-linked polypeptides may be (MTS), or a dual chloroplast and mitochondrial targeting part of a single contiguous molecule and may or may not be peptide. By facilitating protein localization within the cell, adjacent. For example, a promoter is operably linked with a the targeting sequence may increase the accumulation of protein-coding DNA molecule in a DNA construct where the recombinant protein, protect the protein from proteolytic two DNA molecules are so arranged that the promoter may degradation, and/or enhance the level of herbicide tolerance, affect the expression of the transgene. and thereby reduce levels of injury in the transgenic cell, 0067. As used herein, a “DNA construct” is a recombi seed, or organism after herbicide application. nant DNA molecule comprising two or more heterologous 0070 CTPs and other targeting molecules that may be DNA sequences. DNA constructs are useful for transgene used in connection with the present invention are known in expression and may be comprised in vectors and plasmids. the art and include, but are not limited to, the Arabidopsis DNA constructs may be used in vectors for the purpose of thaliana EPSPS CTP (Klee et al., Mol Gen Genet. 210:437 transformation, that is the introduction of heterologous DNA 442, 1987), the Petunia hybrida EPSPS CTP (della-Cioppa into a host cell, to produce transgenic plants and cells, and et al., PNAS 83:6873-6877, 1986), the cab-m7 signal as such may also be contained in the plastid DNA or sequence (Becker et al., Plant Mol Biol. 20:49-60, 1992: genomic DNA of a transgenic plant, seed, cell, or plant part. PCT WO 97/41228), a mitochondrial pre-sequence (e.g. As used herein, a “vector” means any recombinant DNA Silva Filho et al., Plant Mol Biol 30:769-780, 1996), and the molecule that may be used for the purpose of bacterial or pea glutathione reductase signal sequence (Creissen et al., plant transformation. Recombinant DNA molecules as set Plant J. 8:167-175, 1995; PCT WO 97/41228). forth in the sequence listing, can, for example, be inserted 0071 Recombinant DNA molecules of the present inven into a vector as part of a construct having the recombinant tion may be synthesized and modified by methods known in DNA molecule operably linked to a gene expression element the art, either completely or in part, where it is desirable to that functions in a plant to affect expression of the engi provide sequences useful for DNA manipulation (Such as neered protein encoded by the recombinant DNA molecule. restriction enzyme recognition sites or recombination-based General methods useful for manipulating DNA molecules cloning sites), plant-preferred sequences (such as plant for making and using recombinant DNA constructs and plant codon usage or Kozak consensus sequences), or sequences transformation vectors are well known in the art and useful for DNA construct design (such as spacer or linker described in detail in, for example, handbooks and labora sequences). The present invention includes recombinant tory manuals including Michael R. Green and Joseph Sam DNA molecules and engineered proteins having at least 70% brook, “Molecular Cloning: A Laboratory Manual” (Fourth sequence identity, at least 80% sequence identity, at least Edition) ISBN: 978-1-93.6113-42-2, Cold Spring Harbor 85% sequence identity, at least 90% sequence identity, at Laboratory Press, NY (2012). The components for a DNA least 95% sequence identity, at least 96% sequence identity, construct, or a vector comprising a DNA construct, include at least 97% sequence identity, at least 98% sequence one or more gene expression elements operably linked to a identity, and at least 99% sequence identity to any of the transcribable nucleic acid sequence, Such as the following: recombinant DNA molecule or amino acid sequences pro a promoter for the expression of an operably linked DNA, an vided herein, and having herbicide-insensitive protoporphy operably linked protein-coding DNA molecule, and an oper rinogen oxidase activity. As used herein, the term "percent ably linked 3' untranslated region (UTR). Gene expression sequence identity” or “96 sequence identity” refers to the elements useful in practicing the present invention include, percentage of identical nucleotides or amino acids in a linear but are not limited to, one or more of the following type of polynucleotide or amino acid sequence of a reference elements: promoter, 5' UTR, enhancer, leader, cis-acting ("query) sequence (or its complementary strand) as com element, intron, targeting sequence, 3' UTR, and one or pared to a test (“Subject”) sequence (or its complementary more selectable marker transgenes. Strand) when the two sequences are optimally aligned (with 0068. The DNA constructs of the invention may include appropriate nucleotide or amino acid insertions, deletions, or a promoter operably linked to a protein-coding DNA mol gaps totaling less than 20 percent of the reference sequence ecule provided by the invention, whereby the promoter over the window of comparison). Optimal alignment of drives expression of the recombinant protein molecule. sequences for aligning a comparison window are well Promoters useful in practicing the present invention include known to those skilled in the art and may be conducted by those that function in a cell for expression of an operably tools such as the local homology algorithm of Smith and linked polynucleotide. Such as a bacterial or plant promoter. Waterman, the homology alignment algorithm of Needle Plant promoters are varied and well known in the art and man and Wunsch, the search for similarity method of include, for instance, those that are inducible, viral, Syn Pearson and Lipman, and by computerized implementations thetic, constitutive, temporally regulated, spatially regu of these algorithms such as GAP BESTFIT. FASTA, and lated, and/or spatio-temporally regulated. TFASTA available as part of the Sequence Analysis software US 2017/0058290 A1 Mar. 2, 2017

package of the GCG(R) Wisconsin Package(R) (Accelrys Inc., TABLE 1-continued San Diego, Calif.), MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, Wis. 53715), and MUSCLE (version 3.6) (RC Amino Acid Substitutions. Edgar, “MUSCLE: multiple sequence alignment with high Conservative Conservative accuracy and high throughput Nucleic Acids Research Residue Substitutions Residue Substitutions 32(5):1792-7 (2004)) for instance with default parameters. Gln ASn Ser Thr; Gly An “identity fraction’ for aligned segments of a test Cys Ser Thr Ser; Val sequence and a reference sequence is the number of identical Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe components that are shared by the two aligned sequences His ASn; Glin Wal Ile: Leu divided by the total number of components in the portion of Ile Leu; Val the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented 0072. As used herein, “wild-type' means a naturally as the identity fraction multiplied by 100. The comparison of occurring similar, but not identical, version. A "wild-type one or more sequences may be to a full-length sequence or DNA molecule' or “wild-type protein’ is a naturally occur a portion thereof, or to a longer sequence. Engineered ring version of the DNA molecule or protein, that is, a proteins may be produced by changing (that is, modifying) version of the DNA molecule or protein pre-existing in a wild-type protein to produce a new protein with modified nature. An example of a wild-type protein useful for com characteristic(s) e.g. a particular cellular localization pattern, parison with the engineered proteins provided by the inven Such as targeted to the chloroplast or mitochondria, or a tion is the protoporphyrinogen oxidase from Arabidopsis novel combination of useful protein characteristics, such as thaliana. A “wild-type plant' is a non-transgenic plant of the altered V. K. K. ICso, Substrate specificity, inhibitor/ same type as the transgenic plant, and as Such is genetically herbicide specificity, substrate selectivity, the ability to distinct from the transgenic plant comprising the herbicide interact with other components in the cell Such as partner tolerance trait. Examples of a wild-type plant useful for proteins or membranes, and protein stability, among others. comparison with transgenic maize plants are non-transgenic Modifications may be made at specific amino acid positions LH244 maize (ATCC deposit number PTA-1173) and in a protein and may be a Substitution of the amino acid 01DKD2 inbred maize (I294213) (ATCC deposit number found at that position in nature (that is, in the wild-type PTA-7859). For transgenic plants an exemplary protein) with a different amino acid. Engineered proteins comparative line would be non-transgenic A3555 soy provided by the invention thus provide a new protein with (ATCC deposit number PTA-10207), and for transgenic one or more altered protein characteristics relative to a cotton plants an exemplary comparative line would be similar protein found in nature. In one embodiment of the non-transgenic Coker 130 (Plant Variety Protection Number invention, an engineered protein has altered protein charac 8900252). teristics, such as those that result in decreased sensitivity to one or more herbicides as compared to a similar wild-type Transgenic Plants & Herbicides protein or improved ability to confer herbicide tolerance on 0073. One aspect of the invention includes transgenic a transgenic plant expressing the engineered protein to one plant cells, transgenic plant tissues, transgenic plants, and or more herbicides. In one embodiment, the invention pro transgenic seeds that comprise the recombinant DNA mol vides an engineered protein, and the recombinant DNA ecules and engineered proteins provided by the invention. molecule encoding it, comprising at least one amino acid These cells, tissues, plants, and seeds comprising the recom substitution selected from Table 1 and having at least about binant DNA molecules and engineered proteins exhibit 70% sequence identity, about 80% sequence identity, about herbicide tolerance to one or more PPO herbicide(s), and, 85% sequence identity, about 90% sequence identity, about optionally, tolerance to one or more additional herbicide(s). 95% sequence identity, about 96% sequence identity, about 0074 Suitable methods for transformation of host plant 97% sequence identity, about 98% sequence identity, and cells for use with the current invention include virtually any about 99% sequence identity to any of the engineered amino method by which DNA can be introduced into a cell (for acid sequences provided herein, including but not limited to example, where a recombinant DNA construct is stably SEQ ID NOs: 1-2 and 6-12. Amino acid mutations may be integrated into a plant chromosome) and are well known in made as a single amino acid Substitution in the protein or in the art. An exemplary and widely utilized method for combination with one or more other mutation(s). Such as one introducing a recombinant DNA construct into plants is the or more other amino acid Substitution(s), deletions, or addi Agrobacterium transformation system, which is well known tions. Mutations may be made by any method known to to those of skill in the art. Another exemplary method for those of skill in the art. introducing a recombinant DNA construct into plants is insertion of a recombinant DNA construct into a plant TABLE 1. genome at a pre-determined site by methods of site-directed integration. Site-directed integration may be accomplished Amino Acid Substitutions. by any method known in the art, for example, by use of Conservative Conservative Zinc-finger nucleases, engineered or native meganucleases, Residue Substitutions Residue Substitutions TALE-endonucleases, or an RNA-guided endonuclease (for example a CRISPR/Cas9 system). Transgenic plants can be Ala Ser Leu Ile: Val Arg Lys Lys Arg: Gln regenerated from a transformed plant cell by the methods of ASn Gln: His Met Leu: Ile plant cell culture. A transgenic plant homozygous with Asp Glu Phe Met; Leu: Tyr respect to a transgene (that is, two allelic copies of the transgene) can be obtained by self-pollinating (selfing) a US 2017/0058290 A1 Mar. 2, 2017 transgenic plant that contains a single transgene allele with is equivalent to 453.592 grams. Herbicide rates can be itself, for example an R0 plant, to produce R1 seed. One converted between English and metric as: (1b ai/ac) multi fourth of the R1 seed produced will be homozygous with plied by 1.12=(kg ai/ha) and (kg ai/ha) multiplied by 0.89= respect to the transgene. Plants grown from germinating R1 (1b ai/ac). seed can be tested for Zygosity, using a SNP assay, DNA sequencing, or a thermal amplification assay that allows for TABLE 2 the distinction between heterozygotes and homozygotes, referred to as a Zygosity assay. Exemplary PPO Herbicides 0075. As used herein, a “PPO inhibitor herbicide' or PPO Herbicide Chemical Family 1X Rate “PPO herbicide' is a chemical that targets and inhibits the acifluorfen Diphenylethers 420 gai?ha enzymatic activity of a protoporphyrinogen oxidase (PPO). fomesafen Diphenylethers 420 gai?ha which catalyzes the dehydrogenation of protoporphyrinogen lactofen Diphenylethers 7-220 gai?ha IX to form protoporphyrin IX, which is the precursor to fluoroglycofen-ethyl Diphenylethers 15-40 gai?ha oxyfluorfen Diphenylethers 0.28-2.24 kg ai?ha heme and chlorophyll. Inhibition of protoporphyrinogen flumioxazin N-phenylphthalimide 70 gai?ha oxidase causes formation of reactive oxygen species, result aZafenidin Triazolinone 240 gai?ha ing in cell membrane disruption and ultimately the death of carfentraZone-ethyl Triazolinone 4-36 gai?ha susceptible cells. PPO herbicides are well-known in the art SulfentraZone Triazolinone 0.1-0.42 kg ai?ha fluthiacet-methyl Thiadiazole 3-15 gai?ha and commercially available. Examples of PPO herbicides oxadiargyl Oxadiazole 50-150 gai?ha include, but are not limited to, diphenylethers (such as oxadiazon Oxadiazole 2.24-4.48 kg ai?ha acifluorfen, its salts and esters, , , its salts pyraflufen-ethyl Phenylpyrazole 6-12 gai?ha and esters, ethoxyfen, its salts and esters, fluoronitrofen, saflufenacil Pyrimidine dione 25-50 g/ha furyloxyfen, halosafen, chlomethoxyfen, fluoroglycofen, its S-3100 Pyrimidine dione 5-80 g?ha salts and esters, lactofen, its salts and esters, oxyfluorfen, and fomesafen, its salts and esters); thiadiazoles (such as 0077. Herbicide applications may be sequentially or tank fluthiacet-methyl and thidiazimin); pyrimidinediones or mixed with one, two, or a combination of several PPO phenyluracils (such as benzfendizone, , ethyl 3 herbicides or any other compatible herbicide. Multiple -2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-di applications of one herbicide or of two or more herbicides, oXo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy-2-pyridy in combination or alone, may be used over a growing season loxyacetate (CAS Registry Number 353292-31-6 and to areas comprising transgenic plants of the invention for the referred to herein as S-3100), flupropacil, saflufenacil, and control of a broad spectrum of dicot weeds, monocot weeds, tiafenacil); phenylpyrazoles (such as fluaZolate, pyraflufen or both, for example, two applications (such as a pre and pyraflufen-ethyl); oxadiazoles (such as oxadiargyl and planting application and a post-emergence application or a oxadiazon); triazolinones (such as azafenidin, bencarba pre-emergence application and a post-emergence applica Zone, carfentraZone, its salts and esters, and SulfentraZone); tion) or three applications (such as a pre-planting applica oxazolidinediones (such as pentoxazone); N-phenylphthal tion, a pre-emergence application, and a post-emergence imides (such as cinidon-ethyl, flumiclorac, flumiclorac-pen application or a pre-emergence application and two post tyl, and flumioxazin); benzoxazinone derivatives (such as emergence applications). 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3- 0078. As used herein, “tolerance' or “herbicide toler oXo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazi ance' means a plant, seed, or cells ability to resist the toxic nane-2,4-dione); flufenpyr and flufenpyr-ethyl; pyraclonil; effects of an herbicide when applied. Herbicide tolerant and profluaZol. Protoporphyrinogen oxidases and cells, crops can continue to grow and are unaffected or minimally seeds, plants, and plant parts provided by the invention affected by the presence of the applied chemical. As used exhibit herbicide tolerance to one or more PPO herbicide(s). herein, an “herbicide tolerance trait” is a transgenic trait 0076. Herbicides may be applied to a plant growth area imparting improved herbicide tolerance to a plant as com comprising the plants and seeds provided by the invention as pared to the wild-type plant. Contemplated plants which a method for controlling weeds. Plants and seeds provided might be produced with an herbicide tolerance trait of the by the invention comprise an herbicide tolerance trait and as present invention could include, for instance, any plant such are tolerant to the application of one or more PPO including crop plants such as Soybean (e.g. Glycine max), herbicides. The herbicide application may be the recom corn (maize), cotton (Gossypium sp.), and canola, among mended commercial rate (1X) or any fraction or multiple others. thereof. Such as twice the recommended commercial rate 007.9 The transgenic plants, progeny, seeds, plant cells, (2x). Herbicide rates may be expressed as acid equivalent and plant parts of the invention may also contain one or per pound per acre (Tb ae?acre) or acid equivalent per gram more additional transgenic traits. Additional transgenic traits per hectare (gae/ha) or as pounds active ingredient per acre may be introduced by crossing a plant containing a trans (Tb ai/acre) or grams active ingredient per hectare (g ai/ha), gene comprising the recombinant DNA molecules provided depending on the herbicide and the formulation. The herbi by the invention with another plant containing one or more cide application comprises at least one PPO herbicide. The additional transgenic trait(s). As used herein, "crossing plant growth area may or may not comprise weed plants at means breeding two individual plants to produce a progeny the time of herbicide application. A herbicidally effective plant. Two transgenic plants may thus be crossed to produce dose of PPO herbicide(s) for use in an area for controlling progeny that contain the transgenic traits from each parent. weeds may consist of a range from about 0.1x to about 30x As used herein “progeny’ means the offspring of any label rate(s) over a growing season. The 1x label rate for generation of a parent plant, and transgenic progeny com some exemplary PPO herbicides is provided in Table 2. One prise a DNA construct provided by the invention and inher (1) acre is equivalent to 2.47 105 hectares and one (1) pound ited from at least one parent plant. Alternatively, additional US 2017/0058290 A1 Mar. 2, 2017 transgenic trait(s) may be introduced by co-transforming a northern blotting, PCR, and DNA sequencing; biochemical DNA construct for that additional transgenic trait(s) with a assays, such as detecting the presence of a protein product, DNA construct comprising the recombinant DNA molecules for example, by immunological means (ELISAS and western provided by the invention (for example, with all the DNA blots) or by enzymatic function; plant part assays, such as constructs present as part of the same vector used for plant leaf or root assays; and also, by analyzing the phenotype of transformation) or by inserting the additional trait(s) into a the whole plant. transgenic plant comprising a DNA construct provided by I0082 Introgression of a transgenic trait into a plant the invention or vice versa (for example, by using any of the genotype is achieved as the result of the process of backcross methods of plant transformation or gene editing on a trans conversion. A plant genotype into which a transgenic trait genic plant or plant cell). Such additional transgenic traits has been introgressed may be referred to as a backcross include, but are not limited to, increased insect resistance, converted genotype, line, inbred, or hybrid. Similarly a plant increased water use efficiency, increased yield performance, genotype lacking the desired transgenic trait may be referred increased drought resistance, increased seed quality, to as an unconverted genotype, line, inbred, or hybrid. improved nutritional quality, hybrid seed production, and I0083. Having described the invention in detail, it will be herbicide tolerance, in which the trait is measured with apparent that modifications, variations, and equivalent respect to a wild-type plant. Exemplary additional herbicide embodiments are possible without departing the scope of the tolerance traits may include transgenic or non-transgenic invention defined in the appended claims. Furthermore, it tolerance to one or more herbicides such as ACCase inhibi should be appreciated that the examples in the present tors (for example aryloxyphenoxy propionates and cyclo disclosure are provided as non-limiting examples. hexanediones), ALS inhibitors (for example sulfonylureas, imidazolinones, triazoloyrimidines, and triazolinones) EXAMPLES EPSPS inhibitors (for example glyphosate), synthetic (for example phenoxys, benzoic acids, carboxylic acids, I0084. The following examples are included to demon semicarbazones), photosynthesis inhibitors (for example tri strate embodiments of the invention. It should be appreci azines, triazinones, nitriles, benzothiadiazoles, and ), ated by those of skill in the art that the techniques disclosed glutamine synthesis inhibitors (for example ), in the examples that follow represent techniques discovered HPPD inhibitors (for example isoxazoles, pyrazolones, and by the inventors to function well in the practice of the triketones), PPO inhibitors (for example diphenylethers, invention, and thus can be considered to constitute preferred N-phenylphthalimide, aryl triazinones, and pyrimidin modes for its practice. However, those of skill in the art ediones), and long-chain fatty acid inhibitors (for example should, in light of the present disclosure, appreciate that chloroacetamindes, oxyacetamides, and pyrazoles), among many changes can be made in the specific embodiments others. Exemplary insect resistance traits may include resis which are disclosed and still obtain a like or similar result tance to one or more insect members within one or more of without departing from the concept, spirit, and scope of the the orders of Lepidoptera, Coleoptera, Hemiptera, and invention. More specifically, it will be apparent that certain Homoptera, among others. Such additional transgenic traits agents which are both chemically and physiologically are known to one of skill in the art; for example, a list of such related may be substituted for the agents described herein traits is provided by the United States Department of Agri with the same or similar result achieved. All such similar culture's (USDA) Animal and Plant Health Inspection Ser Substitutes and modifications apparent to those skilled in the vice (APHIS). art are deemed to be within the spirit, scope, and concept of 0080 A cell transformed with a polynucleotide of the the invention as defined by the appended claims. present invention, such as an expression construct, may be selected for the presence of the polynucleotide or its Example 1 encoded enzymatic activity before or after regenerating Such a cell into a transgenic plant. Transgenic plants comprising Microbial Protoporphyrinogen Oxidase Discovery Such a polynucleotide may thus be selected for instance by I0085 Novel protoporphyrinogen oxidases were identi identifying a transgenic plant that comprises the polynucle fied from microbial sequence databases using bioinformatic otide or the encoded enzymatic activity, and/or displays an methods and a novel protoporphyrinogen oxidase bacterial altered trait relative to an otherwise isogenic control plant. screening system. Three sequences representing a diverse Such a trait may be, for example, tolerance to a PPO range of HemY microbial protoporphyrinogen oxidases and herbicide. the sequence for the PPO herbicide sensitive HemY proto 0081 Transgenic plants and progeny that contain a trans oxidase from Arabidopsis thaliana were used genic trait provided by the invention may be used with any to identify new putative protoporphyrinogen oxidase breeding methods that are known in the art. In plant lines sequences from microbial sequence databases. The use of comprising two or more transgenic traits, the transgenic these four diverse sequences with bioinformatic tools per traits may be independently segregating, linked, or a com mitted skewing of search results toward sequences that are bination of both in plant lines comprising three or more more similar to microbial protoporphyrinogen oxidases than transgenic traits. Back-crossing to a parental plant and plant protoporphyrinogen oxidases in order to increase the out-crossing with a non-transgenic plant are also contem likelihood of identifying PPO herbicide tolerant protopor plated, as is vegetative propagation. Descriptions of breed phyrinogen oxidases. ing methods that are used for different traits and crops are I0086 Ninety-nine putative protoporphyrinogen oxidases well known to those of skill in the art. To confirm the of the HemY PPO family were identified using this method. presence of the transgene(s) in a particular plant or seed, a The sequences encoding these putative HemYPPO enzymes variety of assays may be performed. Such assays include, for were compared using phylogenetic tree mapping. Forty-four example, molecular biology assays, such as Southern and putative HemY PPO enzymes were selected for further US 2017/0058290 A1 Mar. 2, 2017 analysis due to their representation of individual unique liquid media supplemented with a PPO herbicide to identify clustered members on the phylogenetic tree. The coding protoporphyrinogen oxidases that were not sensitive to the sequences for the forty-four selected putative HemY PPO PPO herbicide. enzymes were cloned into bacterial expression vectors for 0090. The hemC knockout E. coli strain was transformed analysis in a E. coli hemC knockout screen described below. with the bacterial expression vectors containing the con 0087. A protoporphyrinogen oxidase bacterial screening firmed protoporphyrinogen oxidases and cultured in LB system was created to test recombinant proteins for proto liquid media. A saturating amount of the purified crystalline porphyrinogen oxidase activity. This screening system used form of one of five different PPO herbicides (acifluorfen, a functional rescue assay in an E. coli strain that contained flumioxazin, lactofen, fomesafen, and S-3100), representing a gene knockout for the E. coli PPO enzyme (HemG, SEQ three different PPO chemistry subclasses, was added to the ID NO: 76). The hemC knockout E. coli strain showed media. Recombinant proteins were expressed and the E. coli minimal growth on classical bacterial media (e.g., LB growth rates were measured. Growth curves (OD600) were media), but growth rates recovered when the bacterial media measured for the different variants in the presence and was supplemented with free heme or when a recombinant absence of the PPO herbicides at selected time-points (e.g., protoporphyrinogen oxidase was expressed in the E. coli. eight hours). The growth of a transformed hemC knockout The hemO knockout E. coli strain could thus be used with E. coli strain on LB media in the presence of a PPO recombinant protein expression to quickly and easily assay herbicide indicates a protoporphyrinogen oxidase that is an proteins for protoporphyrinogen oxidase activity. herbicide-insensitive protoporphyrinogen oxidase (iPPO). 0091. The novel putative protoporphyrinogen oxidases 0088. The hemC knockout E. coli strain was transformed were used with this assay to test for insensitivity to PPO with the bacterial expression vectors containing the putative herbicides. Expression of E. coli HemG (SEQ ID NO: 76), protoporphyrinogen oxidases and plated on LB media. HemY PPO R1N473 (SEQ ID NO: 13), Hemy PPO Recombinant proteins were expressed in E. coli and growth R1N533 (SEQID NO: 14); or Hemy PPOR1N171 (SEQID rates were measured. Growth of the transformed hemO NO: 15) conferred normal growth rates on the hemC knock knockout E. coli strain on LB media indicated an amino acid out E. coli Strain in minimal media in the presence of sequence that confirmed as a functioning protoporphyrino acifluorfen, flumioxazin, lactofen, or fomesafen PPO herbi gen oxidase. Using this assay, a large number of novel or cides, therefore indicating that these proteins were highly engineered proteins can be screened to confirm and measure herbicide-insensitive protoporphyrinogen oxidases (iPPO). protoporphyrinogen oxidase activity. Ten of the forty-four Data are provided in FIG. 1. Bacteria transformed with putative novel PPO enzymes rescued the hemC knockout E. constructs encoding HemY PPO R1 N311 (SEQ ID NO:16) coli strain, confirming their activity as protoporphyrinogen or HemY PPO R1 N333 (SEQ ID NO:17) did not grow in oxidases, and were selected for further characterization. the presence of any of acifluorfen, flumioxazin, lactofen, or Table 3 provides the SEQ ID NOS corresponding to the ten fomesafen, indicating that these proteins did not confer selected HemY PPO variants, the E. coli HemG, and the A. tolerance to any of these four PPO herbicides. Expression of tuberculatus PPO. HemY R2N30 (SEQ ID NO: 1) or Hem Y R2N40 (SEQ ID NO:2) conferred normal growth rates on the hemC knockout TABLE 3 E. coli strain in minimal media in the presence of acifluorfen and S-3100, but at higher levels these two enzymes showed SEQ ID NOS corresponding to HenY PPO variants sensitivity to both herbicides. The growth rate was slower Monocot for the hemC knockout E. coli strain containing HemY Bacterial Dicot codon codon R2N70 (SEQ ID NO:3), HemY R2N90 (SEQID NO:4), or PPO Protein DNA optimized optimized HemY R2N100 (SEQID NO:5), but tolerance to acifluorfen R2N3O 1, 6 26 31, 36 47 and S-3100 was much better than for the hemO knockout E. R2N40 2, 7, 8, 9, 10, 27 32, 37, 38, 48 coli strain containing either the R2N30 (SEQ ID NO:1) and 11, 12 39, 40, 41, 42, 43, 44, 45, 46 R2N40 (SEQ ID NO:2). Data are provided in FIG. 2. The R2N70 3 28 33 49 hemC knockout E. coli strain expressing the A. tuberculatus R2N90 4 29 34 50 PPO (SEQ ID NO:80) was used as a negative control and R2N100 5 30 35 51 was sensitive to all of the PPO herbicides. Using this assay, R1N473 13, 18, 22 52 57, 68, 72 63 R1NS33 14, 19, 23 53 58, 69, 73 64 a large number of novel or engineered proteins can be R1N171 15, 20, 24 S4 59, 70, 74 65 screened to confirm protoporphyrinogen oxidase activity in R1N311 16 55 60 66 the presence of PPO herbicide(s). R1N333 17, 21, 25 56 61, 62, 71, 75 67 E. Coi HemG 76 77 78 79 Example 3 A. tuberculatus 8O 81 na na Protoporphyrinogen Oxidase (PPO). Enzyme Assay 0092 Novel protoporphyrinogen oxidases were charac Example 2 terized using an enzyme assay. This assay used recombinant HemY PPO proteins with plant plastid extract and PPO Protoporphyrinogen Oxidase Inhibitor Insensitivity Substrate to identify protoporphyrinogen oxidases that had activity in the presence of a PPO herbicide. 0089 Novel protoporphyrinogen oxidases that are toler 0093 Plant plastid extract was produced used etioplasts ant to PPO herbicides were identified using an herbicide and chloroplasts that were prepared from etiolated cotyle bacterial screening system. This screening system used a dons (soybean, Glycine max), etiolated leaves/coleoptiles growth assay of the hemC knockout E. coli strain in LB (corn, Zea mays) and unfolded apical leaves (A. tubercula US 2017/0058290 A1 Mar. 2, 2017

tus) generally by the procedure described by Grossmann (about five minutes). The reaction vial was flushed with (2010). Soybean (A3555) and corn (LH244) seeds were argon and centrifuged briefly to pellet the remaining sodium placed between two sheets of moist germination paper amalgam. The Supernatant solution was diluted 1:1 (VfV) (Anchor Paper Company, Saint Paul, Minn.) in a beaker of with a solution of 0.1M DTT and 0.5M Tris-HCl, pH 7.5 and water in continuous darkness for eight to ten days. A. the vial flushed with argon. The protogen solution was split tuberculatus plants were grown for 30 days in the green into Smaller aliquots into 0.5 ml polypropylene capped tubes house. Tissue was collected, placed between moist sheets of which were flushed with argon immediately after the aliquot paper towels and ground to fine powder with a mortar and was added. Capped tubes were covered with aluminum foil pestle in liquid nitrogen. Homogenization buffer (50 mM and stored at -80° C. For the enzyme assay, the thawed Tris-HCl, pH 7.4, 500 mM sucrose, 1 mM EDTA, 1 mM protogen aliquots were stored covered on ice, and used on magnesium chloride, and 2 g/liter bovine serum albumin) the same day. The concentration of protogen in the prepa was added to the frozen powder at 4:1 (ml homogenization ration was calculated by Subtracting the proto concentration, buffer to g fresh weight tissue), mixed vigorously and as measured by fluorescence HPLC (method described by filtered through four layers of pre-moistened Miracloth. The Matsumoto, 1994), in the final protogen solution (typically filtrate was centrifuged at 9299 g for five minutes. The pellet about 1% of starting material) from the proto concentration was resuspended in homogenization buffer and centrifuged in the starting material and assuming no significant impu at 150 g for two minutes. The supernatant solution was rities in either sample. Protogen prepared and stored under centrifuged at 4000 g for fifteen minutes. All centrifugation these conditions was stable at least six months. steps were carried out a 4° C. The pellet (intact plastid 0096. A PPO enzyme assay was conducted to measure fraction) was resuspended in 50 mM Tris-HCl (pH 7.4), 2 PPO activity using the plant plastid extract and bacterial mM EDTA and 20% (v/v) glycerin and stored in aliquots at extract preparations with the PPO substrate. PPO activity -80° C. Total protein in plastid preparations was measured was measured generally as described by Grossmann (2010). by the method of Bradford (1976) with bovine serum Ten microliters of either plastid extract (40 ug total protein) albumin as the standard. or bacterial extract (1.1 ug total protein for E. coli HemG 0094) Recombinant PPO enzymes were expressed in E. (SEQ ID NO: 76); or 45 to 70 ug total protein for R1 N473 coli HemG knockout cell line and extracted for the enzyme (SEQ ID NO:13), R1N171 (SEQID NO:15), R1N533 (SEQ assay. Bacterial cells from an overnight culture were used to ID NO:14), R2N30 (SEQ ID NO:1), R2N40 (SEQ ID inoculate 20 ml of fresh media. These cultures were allowed NO:2), R2N90 (SEQID NO:4), or R2N100 (SEQID NO:5)) to grow for approximately 48 hrs at 20°C. to a dense culture. was added to assay buffer (100 mM Tris-HCl, pH 7.4, 5 mM Bacterial cells were collected by centrifugation and the cell DTT, 1 mM EDTA and 0.085% (v/v) Tween 80) with buffer pellets stored at -80° C. until enzyme assays were per or with S-3100 (added as a two-microliter volume from a formed. Frozen bacterial pellets were resuspended in extrac 100x stock solution prepared in acetone). Analytical-grade tion buffer (50 mM Tris-HCl, pH 7.6, 1 mM EDTA & 1 mM S-3100 was provided by Sumitomo Chemical Company. All MgCl) and sonicated (Sonics VibraCellTM) for 30 seconds assays were run in a final concentration of 1% (v/v) acetone. in an ice bath with a one-minute rest period between cycles. The extracts (plastid or bacterial), buffer, and S-3100 were For E. coli transformed with a construct encoding E. coli incubated at 30° C. (plant extracts) or 37° C. (bacterial HemG (SEQ ID NO: 76) the broken cells were centrifuged extracts) for five minutes before addition of two microliters at 200 g for 2 minutes at 4° C. and the supernatant solution of protogen to initiate the assay. All assays were done in a was used for PPO enzyme assays after dilution with extrac 96-well black polystyrene microtiter plate (Costar R. 3925, tion buffer. Frozen cell pellets prepared from bacteria trans Corning, Inc., Corning, N.Y.) at a final volume of 200 formed with a construct encoding R1N473 (SEQ ID microliters. After protogen addition (3 uM for ICso mea NO:13), R1N171 (SEQ ID NO:15), R1N533 (SEQ ID Surements; variable for K, measurements) to all wells, the NO:14), R2N30 (SEQ ID NO:1), R2N40 (SEQ ID NO:2), plate was incubated at 30° C. (plant extracts) or 37° C. R2N90 (SEQ ID NO:4), or R2N100 (SEQ ID NO:5) were (bacterial extracts) before initiating data collection. Fluores centrifuged at 9400 g for 10 minutes at 4°C. The pellet cence over time was measured at 30° C. (plant extracts) or fraction from this Supernatant solution was prepared by 37° C. (bacterial extracts) with excitation and emission ultracentrifugation at 100,000 g for one hour at 4°C. to 7° wavelengths of 405 mm and 630 mm, respectively, in a C. and resuspended in extraction buffer for PPO assays. SpectraMax(R) M5 Multi-Mode Microplate Reader (Molecu Total protein was measured by the method of Bradford lar Devices, Sunnyvale, Calif.). An assay blank was run by (1976) with bovine serum albumin as the standard. adding heat-inactivated (five minutes at 100° C.) extract to 0095. The PPO substrate protoporphyrinogen IX (proto the assay mixture. PPO apparent K values were calculated gen) was prepared by reduction of commercially available using rectangular hyperbola curve-fitting using the Soft protoporphyrin with sodium mercury amalgam as described ProR kinetics software package (Molecular Devices, Sunny by Jacobs and Jacobs (1999). Protoporphyrin (proto) was vale, Calif.). The S-3100 ICso values were determined added to 0.01N potassium hydroxide in 20% ethanol and graphically from the semi logarithmic plot of S-3100 con stirred in the dark until dissolved (about 40 minutes). A centration versus PPO activity. volume of 0.8 ml of proto was placed in a 2-ml polypro 0097 Substrate (protoporphyrinogen) binding affinity pylene vial with a screw-top cap containing an O-ring, and was measured as the K. Enzyme activity sensitivity to the about 1 g (a spatula tipful, oil drained off) of sodium PPO herbicide S-3100 was measured as the concentration mercury amalgam (Product Number 45.1908, Sigma-Al giving 50% inhibition of control activity (ICs). The K for drich, St. Louis, Mo., stored under oil) was added. The tube the plant PPO enzymes (A. tuberculatus, soybean, or corn) was capped and mixed vigorously with a Vortex mixer and and the bacterial PPO enzymes (E. coli HemG (SEQID NO: vented about every 30 seconds by loosening the cap until the 76), R1N473 (SEQ ID NO:13), R1N171 (SEQ ID NO:15), Solution was no longer fluorescing red under a UV light R1N533 (SEQID NO:14), R2N30 (SEQ ID NO:1), R2N40 US 2017/0058290 A1 Mar. 2, 2017

(SEQ ID NO:2), R2N90 (SEQ ID NO:4), or R2N100 (SEQ Mutagenesis Kit (Agilent Technologies, Santa Clara, Calif.). ID NO:5)) were similar, ranging from 0.7 LM to 2.0 uM. The resulting library of mutagenized bacterial expression Each of the three plant PPO enzymes were sensitive to vectors was used to transform the hemO knockout E. coli S-3100 with an ICs of 0.003 to 0.009 uM. The bacterial strain and this was plated onto LB media plates containing PPO enzymes R2N30 (SEQID NO:1) and R2N40 (SEQ ID acifluorifen. Bacterial colonies that grew on the herbicide NO:2) had an ICs of 0.02 uM and 0.04 uM, respectively, medium were selected, the transformation plasmids were and were 10-fold less sensitive to the herbicide than the plant purified, and the mutant PPO genes were sequenced. Engi PPO enzymes. The bacterial PPO enzymes E. coli HemG neered HemYPPOR2N40 enzymes are provided as SEQID (SEQID NO: 76), R1N473 (SEQID NO:13), R1N171 (SEQ NO:7-12. ID NO:15), R1N533 (SEQ ID NO:14), R2N90 (SEQ ID NO:4), and R2N100 (SEQID NO:5) had an ICs of greater than 100 uM and were measured as insensitive to the Example 5 herbicide. Data are provided in Table 4. Expression and Testing of PPO Enzymes in TABLE 4 Soybean Plants PPO Enzymatic Activity 0100. The microbial HemY PPO enzymes were Source K. LM S-3100 ICso, M expressed in transgenic soybean plants, and the transgenic A. tuberculatus 0.7 O.OO9 plants were analyzed for PPO herbicide tolerance. A set of Soybean 1.8 O.OO)4 constructs for high-throughput screening were produced Corn 2.0 O.OO3 E. Coi HemG 1.6 >100 with the same promoter element and 3' UTR operably linked R1N473 1.2 >100 to one of ten different cassettes encoding HemY PPO R1N171 O.2 >100 enzymes R1N171 (SEQ ID NO: 20); R1N473 (SEQ ID R1NS33 0.4 >100 NO:18); R1N533 (SEQID NO:19); R2N30 (SEQID NO:1, R2N3O O.8 O.O2 R2N40 O.8 O.04 6); R2N40 (SEQ ID NO:2, 7); R2N40opt (SEQ ID NO:9, R2N90 2.8 >100 10-12); R2N70 (SEQ ID NO:3); R2N90 (SEQ ID NO:4); R2N100 0.4 >100 R2N100 (SEQ ID NO:5); and R1N333 (SEQ ID NO:21) operably linked to one of 39 different transit peptides. For plant transformation, the nucleotide sequences encoding the HemY PPO enzymes were codon optimized for dicot Example 4 expression. This permitted the side-by-side comparison of Enzymatic Optimization of Protoporphyrinogen the seven different HemY PPO enzymes with thirty-nine different targeting peptides using the same promoter and Oxidases 3'UTR elements for gene expression 0098 Protein optimization may used to improve or alter the enzymatic properties of protoporphyrinogen oxidases. 0101 The plant transformation constructs were used to One or more methods of protein engineering may be used to transform soybean excised embryos (germplasm A3555) optimize the enzymes. Non-limiting examples of protein using A. tumefaciens and standard methods known in the art. engineering approaches include Alanine-Scanning Muta Four hundred explants were inoculated for each construct tions; Homology-Scanning Mutations; Prof Gly Scanning resulting in twelve containers per construct. A sterile PPO Mutations; Region Swaps or Mutations; and combinations herbicide solution was used for herbicide tolerance testing. of these various techniques (see, M Lehmann and M Wyss, The herbicide solution consisted of 0.3 g of S-3100 in crop Current Opinion in Biotechnology 12(4):371-375 (2001): B oil concentrate (5.0 mL) and 495 mL of deionized water. Van den Burg and VGH Eijsink, Current Opinion in Bio This was filtered through a 0.45 micron NalgeneR Rapid technology 13(4):333-337 (2002); and Weiss et al., Proceed FlowTM Tissue Culture Filter Unit and Surfactant-Free Cel ings of the National Academy of Sciences USA 97(16): lulose Acetate membrane filter unit (VWR. Radnor, Pa., 8950-8954 (2000)). Engineered protoporphyrinogen oxidase USA). The resulting sterile solution was shaken before nucleic acid sequences may be synthesized and cloned into application. a bacterial expression vector and used to transform the 0102 At five weeks post-transformation, four of the hemC knockout E. coli strain for the initial high-throughput twelve plant containers per construct were sprayed with two bacterial rescue screen as described in Example 1. The passes of the sterile PPO herbicide solution. The treated engineered proteins that rescue the hemC knockout E. coli plantlets were then enclosed in the container and received at strain may be screened for sensitivity to one or more PPO least 15 hours of light exposure post spray each day for four herbicide(s) using the bacterial growth assay as described in days. At the end of day four post application of S-3100, the Example 2. The engineered proteins that exhibit tolerance to treated plantlets were photographed and scored on a visual PPO herbicides in the second screen may then be expressed scale of green coloration (green coloration was representa as recombinant protein in a bacterial expression system, and tive of healthy photosynthetic plant tissue as compared to enzyme characterization may be done using the purified photo-bleached tissue) versus damage. The scoring values protein as described in Example 3. Engineered proteins that were 0 for poor tolerance, high damage, low green color are insensitive to PPO herbicides may be selected for ation; 1 for some tolerance, average damage, moderate green cloning into plant transformation vectors and this may be coloration; and 2 for good tolerance, low damage, high used to produce transgenic plants for in planta testing. green coloration. The results of herbicide application of 0099. A library of randomly mutagenized R2N40 coding S-3100 at five weeks is presented in Table 5, where n.d. sequences were produced using a GeneMorph R. II Random indicates the analysis was not conducted. The results indi US 2017/0058290 A1 Mar. 2, 2017 12 cated that in this high-throughput Screening a number of eleven weeks to the RO plants are presented in Table 6, constructs comprising HemY PPO enzymes R1N473 (SEQ where n.d. indicates the analysis was not conducted. The ID NO:18); R1N533 (SEQ ID NO:19); R2N30 (SEQ ID results indicated that plants expressing a number of con NO:1, 6); R2N40 (SEQ ID NO:2, 7); R2N4Oopt (SEQ ID structs comprising HemY PPO enzymes R2N30 (SEQ ID NO:9, 10-12); and R2N70 (SEQ ID NO:3) provided toler NO:1, 6); R2N40 (SEQ ID NO:2, 7); and R2N40opt (SEQ ance to the PPO herbicide. The results indicated that in this ID NO:9, 10-12) provided tolerance to the PPO herbicide high-throughput screening the HemY PPO enzymes with an injury rating below the non-transgenic control. R1N171 (SEQID NO:20); R2N90 (SEQID NO:4); R2N100 Plants expressing the HemY PPO enzyme R2N30 (SEQ ID (SEQ ID NO:5); and R1N333 (SEQ ID NO:21) did not NO:1, 6) provided herbicide tolerance in 16 of the 19 provide tolerance to the PPO herbicide. constructs tested, with injury ratings for these constructs of TABLE 5

Tolerance score to S-3100 at 5 weeks for HemYPPO Targeting Peptide R1N171 R1N473 R1N533 R2N30 R2N40 R2N40 opt

TP1 O 2 O 2 l il.C. il.C. O n.d. TP2 O O 2 n.d. l n.d. O O O TP3 O O 1 l n.d. 1 l. O O TP4 n.d. O 2 l O O l. O O TP5 1 n.d. n.d. n.d. 1 l O O n.d. TP6 n.d. n.d. l l. O O TP7 O O 1 il.C. 2 l. il.C. n.d. O TP8 1 n.d. 2 O 1 1 O TP9 1 n.d. 2 O O 1 n.d. 1 TP10 1 2 l n.d. O 1 n.d. TP11 O O O 2 l l n.d. O TP12 O O l 2 1 O 1 TP13 O O O n.d l O O n.d. O TP14 n.d. l n.d O 1 O TP15 2 Il. l O O TP16 2 2 l O n.d. O TP17 O n.d. l il.C. O O TP18 O O O il.C. O 1 1 O TP19 O n.d. 2 1 1 O TP2O O 2 l O n.d. n.d. TP21 O O n.d. l n.d. O 1 O O TP22 n.d. 2 l n.d. O O O TP23 l n.d. 1 O 1 TP24 n.d. O 2 2 l n.d. O O O TP25 O O 2 l 1 n.d. O O TP26 O l 1 n.d. 1 O TP27 O O n.d. l 2 O O 1 TP28 O 2 O 2 l n.d. O 1 O TP29 O O l O O O 1 TP30 n.d. il.C. O O O O TP31 O O O 1 1 TP32 O il.C. O l. O O O TP33 O 2 O n.d. 2 1 O O O TP34 n.d. n.d n.d. O 1 n.d. n.d. n.d. 1 TP35 O O 1 1 n.d. n.d. n.d. TP36 n.d. n.d. O 1 n.d. n.d. O 1 TP37 O O O 1 2 n.d. O n.d. TP38 n.d. n.d. 2 n.d. 2 n.d. n.d. n.d. n.d. TP39 1 1 2 2 1 n.d. n.d. n.d.

0103 Plants in the non-sprayed containers corresponding 7% to 25%. Plants expressing the HemY PPO enzymes to constructs having a high passing score of 2 and a few fails R2N40 (SEQ ID NO:2, 7) and R2N40opt (SEQ ID NO:9, as negative controls were transplanted at approximately 7 10-12) provided herbicide tolerance in 8 of the 11 constructs weeks post transformation. The RO plants were grown in a tested, with injury ratings for these constructs of 20% to greenhouse under long-day nursery conditions (18 hr light at 80° F. then 6 hr dark at 74°F.) for approximately four weeks. 25%. The results indicated that in this high-throughput At eleven weeks, the RO plants were sprayed with two screening plants expressing the HemY PPO enzymes passes of the same herbicide solution (0.3 g of S-3100) R1N171 (SEQ ID NO:20); R1N473 (SEQ ID NO:18): described above. Herbicide injury ratings were collected R1N533 (SEQ ID NO:19); R2N70 (SEQ ID NO:3); and seven days after treatment. Any injury rating of 30% or R1N333 (SEQ ID NO:21) had an injury rating of 30% or above was equivalent to non-transgenic soybean injury above equivalent to non-transgenic control injury ratings ratings. The results of the herbicide tolerance application at and thus did not provide tolerance to the PPO herbicide.

US 2017/0058290 A1 Mar. 2, 2017 14

- Continued

85 90 95 His Ala Val Ser Pro His Pro Phe Lys Ile Leu Gln Ser Ala Tyr Ile 1OO 105 11 O Ser Gly Gly Ala Lys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Lys Ala 115 12 O 125 Ala Ala Pro Glu Gly Glu Glu Thr Val Ser Ser Phe Val Thr Arg Arg 13 O 135 14 O Phe Gly Lys Glu Ile Asn Asp Tyr Lieu. Phe Glu Pro Val Lieu. Ser Gly 145 150 155 160 Ile Tyr Ala Gly Asn. Pro Asp Lieu Met Ser Val Gly Glu Val Lieu Pro 1.65 17O 17s Met Leu Pro Gln Trp Glu Gln Lys Tyr Gly Ser Val Thr Glin Gly Lieu. 18O 185 19 O Lieu Lys Asn Lys Gly Ala Met Gly Gly Arg Lys Ile Ile Ala Phe Lys 195 2OO 2O5 Gly Gly Asn Ala Thr Lieu. Thir Asn Arg Lieu. Glin Ser Lieu. Lieu. Ser Gly 21 O 215 22O Lys Ile Arg Phe Asn. Cys Ala Val Thr Gly Val Thr Arg Gly Ala Asp 225 23 O 235 24 O Asp Tyr Ile Val Glin Tyr Thr Glu Asn Gly Asn Thr Ala Met Lieu. Asn 245 250 255 Ala Ser Arg Val Ile Phe Thir Thr Pro Ala Tyr Ser Thr Ala Val Ala 26 O 265 27 O Ile Glin Ala Lieu. Asp Ala Ser Lieu Ala Thr His Lieu. Ser Asp Val Pro 27s 28O 285 Tyr Pro Arg Met Gly Val Lieu. His Lieu. Gly Phe Gly Ala Glu Ala Arg 29 O 295 3 OO Glin Lys Ala Pro Ala Gly Phe Gly Phe Lieu Val Pro His Ala Ala Gly 3. OS 310 315 32O Llys His Phe Lieu. Gly Ala Ile Cys Asn. Ser Ala Ile Phe Pro Ser Arg 3.25 330 335 Val Pro Thr Gly Lys Val Lieu Phe Thr Val Phe Leu Gly Gly Ala Arg 34 O 345 35. O Glin Glu Gln Lieu. Phe Asp Gln Lieu. Gly Pro Glu Lys Lieu. Glin Glin Thr 355 360 365 Val Val Lys Glu Lieu Met Glu Lieu. Leu Gly Lieu. Thir Thr Pro Pro Glu 37 O 375 38O Met Glin Arg Phe Ser Glu Trp Asn Arg Ala Ile Pro Gln Lieu. Asn. Wall 385 390 395 4 OO Gly Tyr Ala Glin Thr Arg Glin Glin Ile Gly Val Phe Glu Glin Arg Tyr 4 OS 41O 415

Pro Gly Ile Arg Lieu Ala Gly Asn Tyr Val Thr Gly Val Ala Val Pro 42O 425 43 O

Ala Ile Ile Glin Ala Ala Lys Gly Tyr Cys 435 44 O

<210s, SEQ ID NO 2 &211s LENGTH: 448 212. TYPE: PRT <213> ORGANISM: Chitinophaga pinensis

<4 OOs, SEQUENCE: 2 US 2017/0058290 A1 Mar. 2, 2017 15

- Continued Met Ser Asp Glin Pro Val Lieu. Ile Val Gly Ala Gly Lieu. Ser Gly Lieu. 1. 5 1O 15 Ser Ile Ala Tyr Glu Lieu Gln Lys Lieu. Glin Val Pro Tyr Glin Val Lieu. 2O 25 3O Glu Val Ser Gly His Ser Gly Gly Wal Met Lys Ser Lieu. Arg Lys Asp 35 4 O 45 Gly Phe Glu Lieu. Asp Ala Gly Ala Asn. Thir Ile Ala Ala Ser Pro Glu SO 55 6 O Ile Lieu Ala Tyr Phe Thir Ser Lieu. Gly Lieu. Glu Asn. Glu Ile Lieu. Glin 65 70 7s 8O Ala Thr Ala Ala Ser Llys His Arg Phe Lieu Val Arg Arg Arg Glin Lieu 85 90 95 His Ala Val Ser Pro His Pro Phe Lys Ile Met Ser Ser Pro Tyr Lieu. 1OO 105 11 O Ser Arg Gly Ser Llys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Llys Pro 115 12 O 125 Val Val Ala Ser Gly Glu Glu Thr Val Thr Asp Phe Ile Thr Arg Arg 13 O 135 14 O Phe Asn Arg Glu Ile Ala Glu Tyr Val Phe Asp Pro Val Lieu. Ser Gly 145 150 155 160 Ile Tyr Ala Gly Asn. Pro Asp Gln Met Ser Ile Ala Glu Val Lieu Pro 1.65 17O 17s Ala Lieu Pro Arg Trp Glu Arg Glu Tyr Gly Ser Val Thr Lys Gly Lieu 18O 185 19 O Met Lys Asp Llys Gly Ala Met Gly Gly Arg Lys Ile Ile Ser Phe Lys 195 2OO 2O5 Gly Gly Asn Glin Lieu. Lieu. Thir Asn Arg Lieu. Glin Glin Lieu. Lieu. Thir Thr 21 O 215 22O Pro Val Arg Phe Asn Cys Llys Val Thr Gly Ile Thr Ala Ser Asn Gly 225 23 O 235 24 O Gly Tyr Ile Val Ser Ala Val Glu Asp Gly Val Ser Glu Ser Tyr Thr 245 250 255 Ala Ser Arg Val Ile Lieu. Thir Thr Pro Ala Tyr Ser Ala Ala Ala Thr 26 O 265 27 O Ile Thr Asn Lieu. Asp Ala Ala Thir Ala Ala Lieu. Lieu. Asn. Glu Ile His 27s 28O 285 Tyr Pro Arg Met Gly Val Lieu. His Lieu. Gly Phe Asp Ala Thir Ala Lieu. 29 O 295 3 OO Pro Glin Pro Lieu. Asp Gly Phe Gly Phe Lieu Val Pro Asn Ala Glu Asn 3. OS 310 315 32O Met His Phe Lieu. Gly Ala Ile Cys Asn Ala Ala Ile Phe Pro Asp Llys 3.25 330 335

Ala Pro Pro Gly Lys Ile Lieu. Phe Thr Val Phe Lieu. Gly Gly Ala Arg 34 O 345 35. O

Gln Glu Ser Leu Phe Asp Gln Met Thr Pro Glu Ala Leu Glin Glin Glin 355 360 365

Wal Wal Ser Glu Wal Met Ser Lieu. Lieu. His Lieu. Ser Ala Pro Pro Wall 37 O 375 38O

Met Gln His Phe Ser Ser Trp Asn Lys Ala Ile Pro Gln Lieu. Asn Val 385 390 395 4 OO Gly His Val Lys Lieu. Arg Arg Ala Val Glu Ala Phe Glu Lys Llys Tyr US 2017/0058290 A1 Mar. 2, 2017 16

- Continued

4 OS 41O 415 Pro Gly Ile His Leu Ser Gly Asn Tyr Lieu Gln Gly Val Ala Ile Pro 42O 425 43 O Ala Lieu. Lieu. Glin His Ala Ala Ala Lieu Ala Ala Ser Lieu Lys Lys Asn 435 44 O 445

<210s, SEQ ID NO 3 &211s LENGTH: 470 212. TYPE: PRT <213> ORGANISM; Bacillus subtilis

<4 OOs, SEQUENCE: 3 Met Ser Asp Gly Lys Lys His Val Val Ile Ile Gly Gly Gly Ile Thr 1. 5 1O 15 Gly Lieu Ala Ala Ala Phe Tyr Met Glu Lys Glu Ile Lys Glu Lys Asn 2O 25 3O Lieu Pro Lieu. Glu Lieu. Thir Lieu Val Glu Ala Ser Pro Arg Val Gly Gly 35 4 O 45 Lys Ile Glin Thr Val Llys Lys Asp Gly Tyr Ile Ile Glu Arg Gly Pro SO 55 6 O Asp Ser Phe Lieu. Glu Arg Llys Llys Ser Ala Pro Gln Lieu Val Lys Asp 65 70 7s 8O Lieu. Gly Lieu. Glu. His Lieu. Lieu Val Asn. Asn Ala Thr Gly Glin Ser Tyr 85 90 95 Val Lieu Val Asn Arg Thr Lieu. His Pro Met Pro Lys Gly Ala Val Met 1OO 105 11 O Gly Ile Pro Thr Lys Ile Ala Pro Phe Val Ser Thr Gly Lieu. Phe Ser 115 12 O 125 Lieu. Ser Gly Lys Ala Arg Ala Ala Met Asp Phe Ile Lieu Pro Ala Ser 13 O 135 14 O Llys Thir Lys Asp Asp Glin Ser Lieu. Gly Glu Phe Phe Arg Arg Arg Val 145 150 155 160 Gly Asp Glu Val Val Glu Asn Lieu. Ile Glu Pro Lieu Lleu Ser Gly Ile 1.65 17O 17s Tyr Ala Gly Asp Ile Asp Llys Lieu Ser Leu Met Ser Thr Phe Pro Glin 18O 185 19 O Phe Tyr Glin Thr Glu Gln Lys His Arg Ser Lieu. Ile Leu Gly Met Lys 195 2OO 2O5 Llys Thr Arg Pro Glin Gly Ser Gly Glin Glin Lieu. Thir Ala Lys Lys Glin 21 O 215 22O Gly Glin Phe Glin Thr Lieu Ser Thr Gly Lieu Gln Thr Lieu Val Glu Glu 225 23 O 235 24 O Ile Glu Lys Glin Lieu Lys Lieu. Thir Lys Val Tyr Lys Gly. Thir Lys Val 245 250 255 Thir Lys Lieu. Ser His Ser Gly Ser Gly Tyr Ser Lieu. Glu Lieu. Asp Asn 26 O 265 27 O Gly Val Thir Lieu. Asp Ala Asp Ser Val Ile Val Thr Ala Pro His Lys 27s 28O 285 Ala Ala Ala Gly Met Lieu. Ser Glu Lieu Pro Ala Ile Ser His Lieu Lys 29 O 295 3 OO

Asn Met His Ser Thr Ser Val Ala Asn Val Ala Lieu. Gly Phe Pro Glu 3. OS 310 315 32O US 2017/0058290 A1 Mar. 2, 2017 17

- Continued Gly Ser Val Glin Met Glu. His Glu Gly Thr Gly Phe Val Ile Ser Arg 3.25 330 335 Asn Ser Asp Phe Ala Ile Thr Ala Cys Thir Trp Thr Asn Llys Llys Trip 34 O 345 35. O Pro His Ala Ala Pro Glu Gly Lys Thr Lieu. Lieu. Arg Ala Tyr Val Gly 355 360 365 Lys Ala Gly Asp Glu Ser Ile Val Asp Lieu. Ser Asp Asn Asp Ile Ile 37 O 375 38O Asn. Ile Val Lieu. Glu Asp Lieu Lys Llys Val Met Asn. Ile Asin Gly Glu 385 390 395 4 OO Pro Glu Met Thr Cys Val Thr Arg Trp His Glu Ser Met Pro Glin Tyr 4 OS 41O 415 His Val Gly His Lys Glin Arg Ile Lys Glu Lieu. Arg Glu Ala Lieu Ala 42O 425 43 O Ser Ala Tyr Pro Gly Val Tyr Met Thr Gly Ala Ser Phe Glu Gly Val 435 44 O 445 Gly Ile Pro Asp Cys Ile Asp Glin Gly Lys Ala Ala Val Ser Asp Ala 450 45.5 460 Lieu. Thir Tyr Lieu Phe Ser 465 470

<210s, SEQ ID NO 4 &211s LENGTH: 470 212. TYPE PRT <213> ORGANISM: Bacillus pumilus <4 OOs, SEQUENCE: 4 Met His Asp Asin Glin Llys His Lieu Val Ile Ile Gly Gly Gly Ile Thr 1. 5 1O 15 Gly Lieu Ala Ala Ala Phe Tyr Lieu. Glu Lys Glu Val Glu Glu Lys Gly 2O 25 3O Lieu Pro Ile Glin Ile Ser Lieu. Ile Glu Ala Ser Pro Arg Lieu. Gly Gly 35 4 O 45 Lys Ile Glin Thir Lieu. Tyr Lys Asp Gly Tyr Ile Ile Glu Arg Gly Pro SO 55 6 O Asp Ser Phe Lieu. Glu Arg Llys Val Ser Gly Pro Gln Lieu Ala Lys Asp 65 70 7s 8O Val Gly Lieu. Ser Asp Gln Lieu Val Asn. Asn. Glu Thr Gly Glin Ala Tyr 85 90 95 Val Lieu Val Asn. Glu Lys Lieu. His Pro Met Pro Llys Gly Ala Wal Met 1OO 105 11 O Gly Ile Pro Thr Glin Ile Ser Pro Phe Ile Thir Thr Gly Lieu. Phe Ser 115 12 O 125

Val Ala Gly Lys Ala Arg Ala Ala Met Asp Phe Val Lieu Pro Llys Ser 13 O 135 14 O

Lys Glin Thr Glu Asp Glin Ser Lieu. Gly Glu Phe Phe Arg Arg Arg Val 145 150 155 160

Gly Asp Glu Val Val Glu Asn Lieu. Ile Glu Pro Lieu Lleu Ser Gly Ile 1.65 17O 17s

Tyr Ala Gly Asp Ile Asp Arg Lieu. Ser Lieu Met Ser Thr Phe Pro Glin 18O 185 19 O

Phe Tyr Glin Thr Glu Gln Gln His Arg Ser Lieu. Ile Leu Gly Met Lys 195 2OO 2O5 US 2017/0058290 A1 Mar. 2, 2017 18

- Continued

Llys Ser Glin Gln His Ala Lys Ala Glin Glin Val Thr Ala Lys Lys Glin 21 O 215 22O Gly Glin Phe Glin Thir Ile Asin Glin Gly Lieu. Glin Ser Lieu Val Glu Ala 225 23 O 235 24 O Val Glu Gly Lys Lieu Lys Lieu. Thir Thr Val Tyr Lys Gly Thr Llys Val 245 250 255 Lys Glin Ile Glu Lys Thr Asp Gly Gly Tyr Gly Lieu Gln Lieu. Asp Ser 26 O 265 27 O Gly Glin Thr Lieu Phe Ala Asp Ser Ala Ile Val Thr Thr Pro His Glin 27s 28O 285 Ser Ile Tyr Ser Met Phe Pro Lys Glu Ala Gly Lieu. Glu Tyr Lieu. His 29 O 295 3 OO Asp Met Thir Ser Thr Ser Val Ala Thr Val Ala Lieu. Gly Phe Lys Asp 3. OS 310 315 32O Glu Asp Wal His Asn. Glu Tyr Asp Gly. Thr Gly Phe Val Ile Ser Arg 3.25 330 335 Asn Ser Asp Phe Ser Ile Thr Ala Cys Thr Trp Thr Asn Llys Llys Trp 34 O 345 35. O Pro His Thr Ala Pro Llys Gly Lys Thr Lieu. Lieu. Arg Ala Tyr Val Gly 355 360 365 Lys Ala Gly Asp Glu Ser Ile Val Glu Glin Ser Asp Ser Glin Ile Val 37 O 375 38O Ser Ile Val Lieu. Glu Asp Lieu Lys Lys Ile Met Asp Ile Lys Ala Asp 385 390 395 4 OO Pro Glu Lieu. Thir Thr Val Thr Arg Trp Llys Thr Ser Met Pro Glin Tyr 4 OS 41O 415 His Val Gly His Gln Lys Ala Ile Ser Asn Met Arg Glu Thr Phe Lys 42O 425 43 O Gln Ser Tyr Pro Gly Val Tyr Ile Thr Gly Ala Ala Phe Glu Gly Val 435 44 O 445 Gly Ile Pro Asp Cys Ile Asp Glin Gly Lys Ala Ala Ile Ser Glu Ala 450 45.5 460 Val Ser Tyr Lieu Phe Ser 465 470

<210s, SEQ ID NO 5 &211s LENGTH: 470 212. TYPE: PRT <213> ORGANISM: Bacillus pumilus <4 OOs, SEQUENCE: 5 Met His Asp Asin Glin Llys His Lieu Val Ile Ile Gly Gly Gly Ile Thr 1. 5 1O 15

Gly Lieu Ala Ala Ala Phe Tyr Lieu. Glu Lys Glu Val Glu Glu Lys Gly 2O 25 3O

Lieu Pro Ile Glin Ile Ser Lieu. Ile Glu Ala Ser Pro Arg Lieu. Gly Gly 35 4 O 45

Lys Ile Glin Thir Lieu. Tyr Lys Asp Gly Tyr Ile Ile Glu Arg Gly Pro SO 55 6 O

Asp Ser Phe Lieu. Glu Arg Llys Val Ser Gly Pro Gln Lieu Ala Lys Asp 65 70 7s 8O

Val Gly Lieu. Ser Asp Gln Lieu Val Asn. Asn. Glu Thr Gly Glin Ala Tyr US 2017/0058290 A1 Mar. 2, 2017 19

- Continued

85 90 95 Val Lieu Val Asn Glu Thir Lieu. His Pro Met Pro Lys Gly Ala Val Met 1OO 105 11 O Gly Ile Pro Thr Glin Ile Ser Pro Phe Ile Thir Thr Gly Lieu. Phe Ser 115 12 O 125 Val Ala Gly Lys Ala Arg Ala Ala Met Asp Phe Val Lieu Pro Llys Ser 13 O 135 14 O Lys Glin Thr Glu Asp Glin Ser Lieu. Gly Glu Phe Phe Arg Arg Arg Val 145 150 155 160 Gly Asp Glu Val Val Glu Asn Lieu. Ile Glu Pro Lieu Lleu Ser Gly Ile 1.65 17O 17s Tyr Ala Gly Asp Ile Asp Arg Lieu. Ser Lieu Met Ser Thr Phe Pro Glin 18O 185 19 O Phe Tyr Glin Thr Glu Gln Lys His Arg Ser Lieu. Ile Leu Gly Met Lys 195 2OO 2O5 Llys Ser Glin Gln His Ala Lys Ala Glin Glin Val Thr Ala Lys Lys Glin 21 O 215 22O Gly Glin Phe Glin Thir Ile Asin Glin Gly Lieu. Glin Ala Lieu Val Glu Ala 225 23 O 235 24 O Val Glu Ser Lys Lieu Lys Lieu. Thir Thir Ile Tyr Lys Gly Thr Llys Val 245 250 255 Lys Glin Ile Glu Lys Thr Asp Gly Gly Tyr Gly Val Glin Lieu. Asp Ser 26 O 265 27 O Gly Glin Thr Lieu. Leu Ala Asp Ser Ala Ile Val Thr Thr Pro His Glin 27s 28O 285 Ser Ile Tyr Ser Met Phe Pro Lys Glu Ala Gly Lieu. Glu Tyr Lieu. His 29 O 295 3 OO Asp Met Thir Ser Thr Ser Val Ala Thr Val Ala Lieu. Gly Phe Lys Glu 3. OS 310 315 32O Glu Asp Wal His Asn. Glu Tyr Asp Gly. Thr Gly Phe Val Ile Ser Arg 3.25 330 335 Asn Ser Asp Phe Ser Ile Thr Ala Cys Thr Trp Thr Asn Llys Llys Trp 34 O 345 35. O Pro His Thr Ala Pro Llys Gly Lys Thr Lieu. Lieu. Arg Ala Tyr Val Gly 355 360 365 Lys Ala Gly Asp Glu Ser Ile Val Glu Glin Ser Asp His Glin Ile Val 37 O 375 38O Ser Ile Val Lieu. Glu Asp Lieu Lys Lys Ile Met Asp Ile Lys Ala Asp 385 390 395 4 OO Pro Glu Lieu. Thir Thr Val Thr Arg Trp Llys Thr Ser Met Pro Glin Tyr 4 OS 41O 415 His Val Gly His Gln Lys Ala Ile Ser Asn Met Arg Glu Thr Phe Lys 42O 425 43 O

Gln Ser Tyr Pro Gly Val Tyr Ile Thr Gly Ala Ala Phe Glu Gly Val 435 44 O 445

Gly Ile Pro Asp Cys Ile Asp Glin Gly Lys Ala Ala Ile Ser Glu Ala 450 45.5 460

Val Ser Tyr Lieu Phe Ser 465 470

<210s, SEQ ID NO 6 US 2017/0058290 A1 Mar. 2, 2017 20

- Continued

&211s LENGTH: 439 212. TYPE: PRT <213> ORGANISM: Xanthomonas campestris

<4 OOs, SEQUENCE: 6 Glin Pro Val Ile Ile Ala Gly Ala Gly Ile Ala Gly Lieu. Ser Ile Ala 1. 5 1O 15 Tyr Glu Lieu. Glin Gln Lys Gly Ile Pro Tyr Glu Ile Met Glu Ala Ser 2O 25 3O Ser Tyr Ala Gly Gly Val Val Lys Ser Lieu. His Ile Asp Gly Tyr Glu 35 4 O 45 Lieu. Asp Ala Gly Pro Asn. Ser Lieu Ala Ala Ser Ala Ala Phe Met Ala SO 55 6 O Tyr Ile Asp Gln Lieu. Gly Lieu. Glin Asp Glin Val Lieu. Glu Ala Ala Ala 65 70 7s 8O Ala Ser Lys Asn Arg Phe Lieu Val Arg Asn Asp Llys Lieu. His Ala Val 85 90 95 Ser Pro His Pro Phe Lys Ile Leu Glin Ser Ala Tyr Ile Ser Gly Gly 1OO 105 11 O Ala Lys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Lys Ala Ala Ala Pro 115 12 O 125 Glu Gly Glu Glu Thr Val Ser Ser Phe Val Thr Arg Arg Phe Gly Lys 13 O 135 14 O Glu Ile ASn Asp Tyr Lieu Phe Glu Pro Val Lieu Ser Gly Ile Tyr Ala 145 150 155 160 Gly Asn Pro Asp Leu Met Ser Val Gly Glu Val Lieu Pro Met Leu Pro 1.65 17O 17s Glin Trp Glu Glin Llys Tyr Gly Ser Val Thr Glin Gly Lieu. Lieu Lys Asn 18O 185 19 O Lys Gly Ala Met Gly Gly Arg Lys Ile Ile Ala Phe Lys Gly Gly Asn 195 2OO 2O5 Ala Thr Lieu. Thir Asn Arg Lieu. Glin Ser Lieu. Lieu. Ser Gly Lys Ile Arg 21 O 215 22O Phe Asn. Cys Ala Val Thr Gly Val Thr Arg Gly Ala Asp Asp Tyr Ile 225 23 O 235 24 O Val Glin Tyr Thr Glu Asn Gly Asn. Thir Ala Met Lieu. Asn Ala Ser Arg 245 250 255 Val Ile Phe Thr Thr Pro Ala Tyr Ser Thr Ala Val Ala Ile Glin Ala 26 O 265 27 O Lieu. Asp Ala Ser Lieu Ala Thr His Lieu. Ser Asp Val Pro Tyr Pro Arg 27s 28O 285 Met Gly Val Lieu. His Lieu. Gly Phe Gly Ala Glu Ala Arg Glin Lys Ala 29 O 295 3 OO

Pro Ala Gly Phe Gly Phe Leu Val Pro His Ala Ala Gly Llys His Phe 3. OS 310 315 32O

Lieu. Gly Ala Ile Cys Asn Ser Ala Ile Phe Pro Ser Arg Val Pro Thr 3.25 330 335

Gly Llys Val Lieu. Phe Thr Val Phe Lieu. Gly Gly Ala Arg Glin Glu Glin 34 O 345 35. O

Lieu. Phe Asp Gln Lieu. Gly Pro Glu Lys Lieu. Glin Glin Thr Val Val Lys 355 360 365

Glu Lieu Met Glu Lieu. Leu Gly Lieu. Thir Thr Pro Pro Glu Met Glin Arg US 2017/0058290 A1 Mar. 2, 2017 21

- Continued

37 O 375 38O Phe Ser Glu Trp Asn Arg Ala Ile Pro Glin Lieu. Asn Val Gly Tyr Ala 385 390 395 4 OO Gln Thr Arg Glin Glin Ile Gly Val Phe Glu Glin Arg Tyr Pro Gly Ile 4 OS 41O 415 Arg Lieu Ala Gly Asn Tyr Val Thr Gly Val Ala Val Pro Ala Ile Ile 42O 425 43 O Glin Ala Ala Lys Gly Tyr Cys 435

<210s, SEQ ID NO 7 &211s LENGTH: 445 212. TYPE: PRT <213> ORGANISM: Chitinophaga pinensis

<4 OO > SEQUENCE: 7 Glin Pro Val Lieu. Ile Val Gly Ala Gly Lieu. Ser Gly Lieu. Ser Ile Ala 1. 5 1O 15 Tyr Glu Lieu Gln Lys Lieu. Glin Val Pro Tyr Glin Val Lieu. Glu Val Ser 2O 25 3O Gly His Ser Gly Gly Wal Met Lys Ser Lieu. Arg Lys Asp Gly Phe Glu 35 4 O 45 Lieu. Asp Ala Gly Ala Asn. Thir Ile Ala Ala Ser Pro Glu Ile Lieu Ala SO 55 6 O Tyr Phe Thir Ser Lieu. Gly Lieu. Glu Asn. Glu Ile Lieu. Glin Ala Thr Ala 65 70 7s 8O Ala Ser Lys His Arg Phe Lieu Val Arg Arg Arg Glin Lieu. His Ala Val 85 90 95 Ser Pro His Pro Phe Lys Ile Met Ser Ser Pro Tyr Lieu Ser Arg Gly 1OO 105 11 O Ser Lys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Llys Pro Val Val Ala 115 12 O 125 Ser Gly Glu Glu Thr Val Thr Asp Phe Ile Thr Arg Arg Phe Asn Arg 13 O 135 14 O Glu Ile Ala Glu Tyr Val Phe Asp Pro Val Leu Ser Gly Ile Tyr Ala 145 150 155 160 Gly Asn Pro Asp Gln Met Ser Ile Ala Glu Val Lieu Pro Ala Lieu Pro 1.65 17O 17s Arg Trp Glu Arg Glu Tyr Gly Ser Val Thir Lys Gly Lieu Met Lys Asp 18O 185 19 O Lys Gly Ala Met Gly Gly Arg Lys Ile Ile Ser Phe Lys Gly Gly Asn 195 2OO 2O5 Glin Lieu. Lieu. Thir Asn Arg Lieu. Glin Glin Lieu. Lieu. Thir Thr Pro Val Arg 21 O 215 22O

Phe Asn Cys Llys Val Thr Gly Ile Thr Ala Ser Asn Gly Gly Tyr Ile 225 23 O 235 24 O

Val Ser Ala Val Glu Asp Gly Val Ser Glu Ser Tyr Thr Ala Ser Arg 245 250 255

Val Ile Lieu. Thir Thr Pro Ala Tyr Ser Ala Ala Ala Thr Ile Thr Asn 26 O 265 27 O Lieu. Asp Ala Ala Thr Ala Ala Lieu. Lieu. Asn. Glu Ile His Tyr Pro Arg 27s 28O 285 US 2017/0058290 A1 Mar. 2, 2017 22

- Continued Met Gly Val Lieu. His Leu Gly Phe Asp Ala Thr Ala Leu Pro Glin Pro 29 O 295 3 OO Lieu. Asp Gly Phe Gly Phe Leu Val Pro Asn Ala Glu Asn Met His Phe 3. OS 310 315 32O Lieu. Gly Ala Ile Cys Asn Ala Ala Ile Phe Pro Asp Lys Ala Pro Pro 3.25 330 335 Gly Lys Ile Lieu. Phe Thr Val Phe Lieu. Gly Gly Ala Arg Glin Glu Ser 34 O 345 35. O Lieu. Phe Asp Gln Met Thr Pro Glu Ala Leu Gln Glin Glin Val Val Ser 355 360 365

Glu Wal Met Ser Lieu Lleu. His Lieu. Ser Ala Pro Pro Wal Met Glin His 37 O 375 38O Phe Ser Ser Trp Asn Lys Ala Ile Pro Glin Lieu. Asn Val Gly His Val 385 390 395 4 OO Llys Lieu. Arg Arg Ala Val Glu Ala Phe Glu Lys Llys Tyr Pro Gly Ile 4 OS 41O 415 His Lieu. Ser Gly Asn Tyr Lieu. Glin Gly Val Ala Ile Pro Ala Lieu. Lieu. 42O 425 43 O Glin His Ala Ala Ala Lieu Ala Ala Ser Lieu Lys Lys Asn 435 44 O 445

<210s, SEQ ID NO 8 &211s LENGTH: 448 212. TYPE PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 8 Met Ser Asp Glin Pro Val Lieu. Ile Val Gly Ala Gly Lieu. Ser Gly Lieu. 1. 5 1O 15 Ser Ile Ala Tyr Glu Lieu Gln Lys Lieu. Glin Val Pro Tyr Glin Val Lieu. 2O 25 3O Glu Val Ser Gly His Ser Gly Gly Wal Met Lys Ser Lieu. Arg Lys Asp 35 4 O 45 Gly Phe Glu Lieu. Asp Ala Gly Ala Asn. Thir Ile Ala Thir Ser Pro Glu SO 55 6 O Ile Lieu Ala Tyr Phe Thir Ser Lieu. Gly Lieu. Glu Asn. Glu Ile Lieu. Glin 65 70 7s 8O Ala Thr Ala Thir Ser Llys His Arg Phe Lieu Val Arg Arg Arg Glin Lieu 85 90 95 His Ala Val Ser Pro His Pro Phe Lys Ile Met Ser Ser Pro Tyr Lieu. 1OO 105 11 O Cys Arg Gly Ser Llys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Llys Pro 115 12 O 125

Val Val Ala Ser Gly Glu Glu Thr Val Thr Asp Phe Ile Thr Arg Arg 13 O 135 14 O

Phe Asn Arg Glu Ile Ala Glu Tyr Val Phe Asp Pro Val Lieu. Ser Gly 145 150 155 160

Ile Tyr Ala Gly Asn. Pro Asp Gln Met Ser Ile Ala Glu Val Lieu Pro 1.65 17O 17s

Ala Lieu Pro Arg Trp Glu Arg Glu Tyr Gly Ser Val Thir Lys Gly Lieu 18O 185 19 O US 2017/0058290 A1 Mar. 2, 2017 23

- Continued Met Lys Asp Llys Gly Ala Met Gly Gly Arg Lys Ile Ile Ser Phe Lys 195 2OO 2O5 Gly Gly Asn Glin Lieu. Lieu. Thir Asn Arg Lieu. Glin Glin Lieu. Lieu. Thir Thr 21 O 215 22O Pro Val Arg Phe Asn Cys Llys Val Thr Gly Ile Thr Ala Ser Asn Gly 225 23 O 235 24 O Gly Tyr Ile Val Ser Ala Val Glu Asp Gly Val Ser Glu Ser Tyr Thr 245 250 255 Ala Ser Arg Val Ile Lieu. Thir Thr Pro Ala Tyr Ser Ala Ala Ala Thr 26 O 265 27 O Ile Thr Asn Lieu. Asp Ala Ala Thir Ala Ala Lieu. Lieu. Asn. Glu Ile His 27s 28O 285 Tyr Pro Arg Met Gly Val Lieu. His Lieu. Gly Phe Asp Ala Thir Ala Lieu. 29 O 295 3 OO Pro Glin Pro Lieu. Asp Gly Phe Gly Phe Lieu Val Pro Asn Ala Glu Asn 3. OS 310 315 32O Met His Phe Lieu. Gly Ala Ile Cys Asn Ala Ala Ile Phe Pro Asp Llys 3.25 330 335 Ala Pro Pro Gly Lys Ile Lieu. Phe Thr Val Phe Lieu. Gly Gly Ala Arg 34 O 345 35. O Gln Glu Ser Leu Phe Asp Gln Met Thr Pro Glu Ala Leu Glin Glin Glin 355 360 365

Wal Wal Ser Glu Wal Met Ser Lieu Lleu. His Lieu Ser Ala Pro Pro Wall 37 O 375 38O Met Gln His Phe Ser Ser Trp Asn Lys Ala Ile Pro Gln Lieu. Asn Val 385 390 395 4 OO Gly His Val Lys Lieu. Arg Arg Ala Val Glu Ala Phe Glu Lys Llys Tyr 4 OS 41O 415 Pro Gly Ile His Leu Ser Gly Asn Tyr Lieu Gln Gly Val Ala Ile Pro 42O 425 43 O Ala Lieu. Lieu. Glin His Ala Ala Ala Lieu Ala Ala Ser Lieu Lys Lys Asn 435 44 O 445

<210s, SEQ ID NO 9 &211s LENGTH: 445 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 9 Glin Pro Val Lieu. Ile Val Gly Ala Gly Lieu. Ser Gly Lieu. Ser Ile Ala 1. 5 1O 15 Tyr Glu Lieu Gln Lys Lieu. Glin Val Pro Tyr Glin Val Lieu. Glu Val Ser 2O 25 3O Gly His Ser Gly Gly Wal Met Lys Ser Lieu. Arg Lys Asp Gly Phe Glu 35 4 O 45

Lieu. Asp Ala Gly Ala Asn. Thir Ile Ala Thir Ser Pro Glu Ile Lieu Ala SO 55 6 O

Tyr Phe Thir Ser Lieu. Gly Lieu. Glu Asn. Glu Ile Lieu. Glin Ala Thr Ala 65 70 7s 8O Thir Ser Llys His Arg Phe Lieu Val Arg Arg Arg Glin Lieu. His Ala Val 85 90 95 US 2017/0058290 A1 Mar. 2, 2017 24

- Continued Ser Pro His Pro Phe Lys Ile Met Ser Ser Pro Tyr Lieu. Cys Arg Gly 1OO 105 11 O Ser Lys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Llys Pro Val Val Ala 115 12 O 125 Ser Gly Glu Glu Thr Val Thr Asp Phe Ile Thr Arg Arg Phe Asn Arg 13 O 135 14 O Glu Ile Ala Glu Tyr Val Phe Asp Pro Val Leu Ser Gly Ile Tyr Ala 145 150 155 160 Gly Asn Pro Asp Gln Met Ser Ile Ala Glu Val Lieu Pro Ala Lieu Pro 1.65 17O 17s Arg Trp Glu Arg Glu Tyr Gly Ser Val Thir Lys Gly Lieu Met Lys Asp 18O 185 19 O Lys Gly Ala Met Gly Gly Arg Lys Ile Ile Ser Phe Lys Gly Gly Asn 195 2OO 2O5 Glin Lieu. Lieu. Thir Asn Arg Lieu. Glin Glin Lieu. Lieu. Thir Thr Pro Val Arg 21 O 215 22O Phe Asn Cys Llys Val Thr Gly Ile Thr Ala Ser Asn Gly Gly Tyr Ile 225 23 O 235 24 O Val Ser Ala Val Glu Asp Gly Val Ser Glu Ser Tyr Thr Ala Ser Arg 245 250 255 Val Ile Lieu. Thir Thr Pro Ala Tyr Ser Ala Ala Ala Thr Ile Thr Asn 26 O 265 27 O Lieu. Asp Ala Ala Thr Ala Ala Lieu Lieu. ASn Glu Ile His Tyr Pro Arg 27s 28O 285 Met Gly Val Lieu. His Leu Gly Phe Asp Ala Thr Ala Leu Pro Glin Pro 29 O 295 3 OO Lieu. Asp Gly Phe Gly Phe Leu Val Pro Asn Ala Glu Asn Met His Phe 3. OS 310 315 32O Lieu. Gly Ala Ile Cys Asn Ala Ala Ile Phe Pro Asp Lys Ala Pro Pro 3.25 330 335 Gly Lys Ile Lieu. Phe Thr Val Phe Lieu. Gly Gly Ala Arg Glin Glu Ser 34 O 345 35. O Lieu. Phe Asp Gln Met Thr Pro Glu Ala Leu Gln Glin Glin Val Val Ser 355 360 365

Glu Wal Met Ser Lieu Lleu. His Lieu. Ser Ala Pro Pro Wal Met Glin His 37 O 375 38O Phe Ser Ser Trp Asn Lys Ala Ile Pro Glin Lieu. Asn Val Gly His Val 385 390 395 4 OO Llys Lieu. Arg Arg Ala Val Glu Ala Phe Glu Lys Llys Tyr Pro Gly Ile 4 OS 41O 415 His Lieu. Ser Gly Asn Tyr Lieu. Glin Gly Val Ala Ile Pro Ala Lieu. Lieu. 42O 425 43 O

Glin His Ala Ala Ala Lieu Ala Ala Ser Lieu Lys Lys Asn 435 44 O 445

<210s, SEQ ID NO 10 &211s LENGTH: 445 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 10 US 2017/0058290 A1 Mar. 2, 2017 25

- Continued

Glin Pro Val Lieu. Ile Val Gly Ala Gly Lieu. Ser Gly Lieu. Ser Ile Ala 1. 5 1O 15 Tyr Glu Lieu Gln Lys Lieu. Glin Val Pro Tyr Glin Val Lieu. Glu Val Ser 2O 25 3O Gly His Ser Gly Gly Wal Met Lys Ser Lieu. Arg Lys Asp Gly Phe Glu 35 4 O 45 Lieu. Asp Ala Gly Ala Asn. Thir Ile Ala Thir Ser Pro Glu Ile Lieu Ala SO 55 6 O Tyr Phe Thir Ser Lieu. Gly Lieu. Glu Asn. Glu Ile Lieu. Glin Ala Thr Ala 65 70 7s 8O Ala Ser Lys His Arg Phe Lieu Val Arg Arg Arg Glin Lieu. His Ala Val 85 90 95 Ser Pro His Pro Phe Lys Ile Met Ser Ser Pro Tyr Lieu Ser Arg Gly 1OO 105 11 O Ser Lys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Llys Pro Val Val Ala 115 12 O 125 Ser Gly Glu Glu Thr Val Thr Asp Phe Ile Thr Arg Arg Phe Asn Arg 13 O 135 14 O Glu Ile Ala Glu Tyr Val Phe Asp Pro Val Leu Ser Gly Ile Tyr Ala 145 150 155 160 Gly Asn Pro Asp Gln Met Ser Ile Ala Glu Val Lieu Pro Ala Lieu Pro 1.65 17O 17s Arg Trp Glu Arg Glu Tyr Gly Ser Val Thr Lys Gly Lieu Met Lys Asp 18O 185 19 O Lys Gly Ala Met Gly Gly Arg Lys Ile Ile Ser Phe Lys Gly Gly Asn 195 2OO 2O5 Glin Lieu. Lieu. Thir Asn Arg Lieu. Glin Glin Lieu. Lieu. Thir Thr Pro Val Arg 21 O 215 22O Phe Asn Cys Llys Val Thr Gly Ile Thr Ala Ser Asn Gly Gly Tyr Ile 225 23 O 235 24 O Val Ser Ala Val Glu Asp Gly Val Ser Glu Ser Tyr Thr Ala Ser Arg 245 250 255 Val Ile Lieu. Thir Thr Pro Ala Tyr Ser Ala Ala Ala Thr Ile Thr Asn 26 O 265 27 O Lieu. Asp Ala Ala Thr Ala Ala Lieu. Lieu. Asn. Glu Ile His Tyr Pro Arg 27s 28O 285 Met Gly Val Lieu. His Leu Gly Phe Asp Ala Thr Ala Leu Pro Glin Pro 29 O 295 3 OO Lieu. Asp Gly Phe Gly Phe Leu Val Pro Asn Ala Glu Asn Met His Phe 3. OS 310 315 32O Lieu. Gly Ala Ile Cys Asn Ala Ala Ile Phe Pro Asp Lys Ala Pro Pro 3.25 330 335

Gly Lys Ile Lieu. Phe Thr Val Phe Lieu. Gly Gly Ala Arg Glin Glu Ser 34 O 345 35. O

Lieu. Phe Asp Gln Met Thr Pro Glu Ala Leu Gln Glin Glin Val Val Ser 355 360 365

Glu Wal Met Ser Lieu Lleu. His Lieu. Ser Ala Pro Pro Wal Met Glin His 37 O 375 38O

Phe Ser Ser Trp Asn Lys Ala Ile Pro Glin Lieu. Asn Val Gly His Val 385 390 395 4 OO

Llys Lieu. Arg Arg Ala Val Glu Ala Phe Glu Lys Llys Tyr Pro Gly Ile US 2017/0058290 A1 Mar. 2, 2017 26

- Continued

4 OS 41O 415 His Lieu. Ser Gly Asn Tyr Lieu. Glin Gly Val Ala Ile Pro Ala Lieu. Lieu. 42O 425 43 O Glin His Ala Ala Ala Lieu Ala Ala Ser Lieu Lys Lys Asn 435 44 O 445

<210s, SEQ ID NO 11 &211s LENGTH: 445 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 11 Glin Pro Val Lieu. Ile Val Gly Ala Gly Lieu. Ser Gly Lieu. Ser Ile Ala 1. 5 1O 15 Tyr Glu Lieu Gln Lys Lieu. Glin Val Pro Tyr Glin Val Lieu. Glu Val Ser 2O 25 3O Gly His Ser Gly Gly Wal Met Lys Ser Lieu. Arg Lys Asp Gly Phe Glu 35 4 O 45 Lieu. Asp Ala Gly Ala Asn. Thir Ile Ala Thir Ser Pro Glu Ile Lieu Ala SO 55 6 O Tyr Phe Thir Ser Lieu. Gly Lieu. Glu Asn. Glu Ile Lieu. Glin Ala Thr Ala 65 70 7s 8O Thir Ser Llys His Arg Phe Lieu Val Arg Arg Arg Gln Lieu. His Ala Val 85 90 95 Ser Pro His Pro Phe Lys Ile Met Ser Ser Pro Tyr Lieu. Cys Arg Gly 1OO 105 11 O Ser Lys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Llys Pro Val Val Ala 115 12 O 125 Ser Gly Glu Glu Thr Val Thr Asp Phe Ile Thr Arg Arg Phe Asn Arg 13 O 135 14 O Glu Ile Ala Glu Tyr Val Phe Asp Pro Val Leu Ser Gly Ile Tyr Ala 145 150 155 160 Gly Asn Pro Asp Gln Met Ser Ile Ala Glu Val Lieu Pro Ala Lieu Pro 1.65 17O 17s Arg Trp Glu Arg Glu Tyr Gly Ser Val Thir Lys Gly Lieu Met Lys Asp 18O 185 19 O Lys Gly Ala Met Gly Gly Arg Lys Ile Ile Ser Phe Lys Gly Gly Asn 195 2OO 2O5 Glin Lieu. Lieu. Thir Asn Arg Lieu. Glin Glin Lieu. Lieu. Thir Thr Pro Val Arg 21 O 215 22O Phe Asn Cys Llys Val Thr Gly Ile Thr Ala Ser Asn Gly Gly Tyr Ile 225 23 O 235 24 O

Val Ser Ala Val Glu Asp Gly Val Ser Glu Ser Tyr Thr Ala Ser Arg 245 250 255

Val Ile Lieu. Thir Thr Pro Ala Tyr Ser Ala Ala Ala Thr Ile Thr Asn 26 O 265 27 O

Lieu. Asp Ala Ala Thr Ala Ala Lieu. Lieu. Asn. Glu Ile His Tyr Pro Arg 27s 28O 285

Met Gly Val Lieu. His Leu Gly Phe Asp Ala Thr Ala Leu Pro Glin Pro 29 O 295 3 OO

Lieu. Asp Gly Phe Gly Phe Leu Val Pro Asn Ala Glu Asn Met His Phe US 2017/0058290 A1 Mar. 2, 2017 27

- Continued

3. OS 310 315 32O

Lell Gly Ala Ile Cys Asn Ala Ala Ile Phe Pro Asp Ala Pro Pro 3.25 330 335

Gly Lys Ile Leu Phe Thr Val Phe Leu Gly Gly Ala Arg Glin Glu Ser 34 O 345 35. O

Lell Phe Asp Gln Met Thr Pro Glu Ala Leu Glin Glin Glin Wall Wall Ser 355 360 365

Glu Wal Met Ser Lieu. Lieu. His Lieu Ser Ala Pro Pro Wall Met Glin His 37 O 375

Phe Ser Ser Trp Asn Lys Ala Ile Pro Glin Luell Asn Wall Gly His Wall 385 390 395 4 OO

Lieu. Arg Arg Ala Val Glu Ala Phe Glu Pro Gly Ile 4 OS 41O 415

His Lieu. Ser Gly Asn Tyr Lieu. Glin Gly Val Ala Ile Thir Ala Luell Luell 42O 425 43 O

Glin His Ala Ala Ala Lieu Ala Ala Ser Lieu. Asn 435 44 O 445

<210s, SEQ ID NO 12 &211s LENGTH: 445 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 12

Pro Val Lieu. Ile Val Gly Ala Gly Lieu. Ser Gly Lell Ser Ile Ala 5 1O 15

Glu Lieu. Glin Llys Lieu. Glin Val Pro Tyr Glin Wall Lell Glu Wall Ser 2O 25

Gly His Ser Gly Gly Val Met Lys Ser Leu Arg Asp Gly Phe Glu 35 4 O 45

Lell Asp Ala Gly Ala Asn. Thir Ile Ala Thr Ser Pro Glu Ile Luell Ala SO 55 6 O

Tyr Phe Thir Ser Lieu. Gly Lieu. Glu Asn Glu Ile Lell Glin Ala Thir Ala 65 70

Thir Ser Lys His Arg Phe Lieu Val Arg Arg Arg Glin Lell His Ala Wall 85 90 95

Ser Pro His Pro Phe Lys Ile Met Ser Ser Pro Lell Cys Arg Gly 1OO 105 11 O

Ser Llys Trp Arg Lieu. Phe Thr Glu Arg Phe Arg Pro Wall Wall Ala 115 12 O 125

Ser Gly Glu Glu Thr Val Thr Asp Phe Ile Thir Arg Arg Phe Asn Arg 13 O 135 14 O

Glu Ile Ala Glu Tyr Val Phe Asp Pro Val Luell Ser Gly Ile Ala 145 150 155 160

Gly Asn Pro Asp Glin Met Ser Ile Ala Glu Wall Lell Pro Ala Luell Pro 1.65 17O 17s

Arg Trp. Glu Arg Glu Tyr Gly Ser Val Thr Gly Lell Met Asp 18O 185 19 O

Gly Ala Met Gly Gly Arg Lys Ile Ile Ser Phe Lys Gly Gly Asn 195 2OO 2O5

Glin Lieu. Lieu. Thir Asn Arg Lieu. Glin Glin Lieu. Luell Thir Thir Pro Wall Arg US 2017/0058290 A1 Mar. 2, 2017 28

- Continued

21 O 215 22O Phe Asn Tyr Llys Val Thr Gly Ile Thr Ala Ser Asn Gly Gly Tyr Ile 225 23 O 235 24 O Val Ser Ala Val Glu Asp Gly Val Ser Glu Ser Tyr Thr Ala Ser Arg 245 250 255 Val Ile Lieu. Thir Thr Pro Ala Tyr Ser Ala Ala Ala Thr Ile Thr Asn 26 O 265 27 O Lieu. Asp Ala Ala Thr Ala Ala Lieu. Lieu. Asn. Glu Ile His Tyr Pro Arg 27s 28O 285 Met Gly Val Lieu. His Leu Gly Phe Asp Ala Thr Ala Leu Pro Glin Pro 29 O 295 3 OO Lieu. Asp Gly Phe Gly Phe Leu Val Pro Asn Ala Glu Asn Met His Phe 3. OS 310 315 32O Lieu. Gly Ala Ile Cys Asn Ala Ala Ile Phe Pro Asp Lys Ala Pro Pro 3.25 330 335 Gly Lys Ile Lieu. Phe Thr Val Phe Lieu. Gly Gly Ala Arg Glin Glu Ser 34 O 345 35. O Lieu. Phe Asp Gln Met Thr Pro Glu Ala Leu Gln Glin Glin Val Val Ser 355 360 365

Glu Wal Met Ser Lieu Lleu. His Lieu. Ser Ala Pro Pro Wal Met Glin His 37 O 375 38O Phe Ser Ser Trp Asn Lys Ala Ile Pro Glin Lieu. Asn Val Gly His Val 385 390 395 4 OO Llys Lieu. Arg Arg Ala Val Glu Ala Phe Glu Lys Llys Tyr Pro Gly Ile 4 OS 41O 415 His Lieu. Ser Gly Asn Tyr Lieu. Glin Gly Val Ala Ile Pro Ala Lieu. Lieu. 42O 425 43 O Glin His Ala Ala Ala Lieu Ala Ala Ser Lieu Lys Lys Asn 435 44 O 445

<210s, SEQ ID NO 13 &211s LENGTH: 477 212. TYPE: PRT <213> ORGANISM: Paenibacillus macerans

<4 OOs, SEQUENCE: 13 Met Ser Lys Lys Ile Ala Val Ile Gly Gly Gly Ile Thr Gly Lieu. Ser 1. 5 1O 15 Val Ala Tyr Tyr Val Arg Llys Lieu. Lieu. Arg Glu Glin Gly Val Asn Ala 2O 25 3O Gly Val Thir Lieu Val Glu Glin Ser Asp Arg Lieu. Gly Gly Lys Ile Arg 35 4 O 45 Ser Lieu. Arg Arg Asp Gly Phe Thir Ile Glu Glin Gly Pro Asp Ser Met SO 55 6 O

Ile Ala Arg Llys Pro Ala Ala Lieu. Glu Lieu. Ile Arg Glu Lieu. Gly Lieu. 65 70 7s 8O

Glu Asp Llys Lieu Ala Gly. Thir Asn Pro Glin Ala Lys Arg Ser Tyr Ile 85 90 95 Lieu. His Arg Gly Lys Phe His Pro Met Pro Pro Gly Lieu Met Leu Gly 1OO 105 11 O

Ile Pro Thr Gln Met Trp Pro Met Val Lys Thr Gly Lieu. Leu Ser Pro 115 12 O 125 US 2017/0058290 A1 Mar. 2, 2017 29

- Continued Ala Gly Lys Lieu. Arg Ala Ala Met Asp Lieu. Lieu Lleu Pro Ala Arg Arg 13 O 135 14 O Gly Gly Gly Asp Glu Ser Lieu. Gly Gly Phe Ile Arg Arg Arg Lieu. Gly 145 150 155 160 Arg Glu Val Lieu. Glu Gln Met Thr Glu Pro Lieu. Lieu Ala Gly Ile Tyr 1.65 17O 17s Ala Gly Asp Thr Glu Gln Leu Ser Leu Lys Ala Thr Phe Pro Glin Phe 18O 185 19 O Met Glu Met Glu Arg Llys His Arg Ser Lieu. Ile Lieu. Gly Lieu. Lieu Ala 195 2OO 2O5 Gly Lys Lys Gln Pro Pro Arg Pro Gly Gly Ser Glin Val Pro Leu Pro 21 O 215 22O Lys Ala Ala Glin Thr Ser Met Phe Lieu. Thir Lieu. Thr Gly Gly Lieu. Glu 225 23 O 235 24 O Gly Lieu. Thr Glu Ala Lieu. Glu Glu Ser Lieu. Ser Glu Glu Lys Ile Ile 245 250 255 Thr Gly Glin Ala Val Thr Gly Lieu Ser Glin Glin Glu Ala Gly Tyr Glu 26 O 265 27 O Lieu. Asn Lieu. Ser Gly Gly Glu Arg Lieu. Asn Ala Asp Gly Val Ile Lieu. 27s 28O 285 Ala Val Pro Ala Phe Ala Ala Ala Arg Lieu. Lieu. Asp Gly Val Pro Glu 29 O 295 3 OO Ala Ala Tyr Lieu. Glu Arg Ile Arg Tyr Val Ser Val Ala ASn Lieu Ala 3. OS 310 315 32O Phe Ala Tyr Arg Arg Glu Asp Val Pro His Asp Lieu. Asn Gly Ser Gly 3.25 330 335 Val Lieu. Ile Pro Arg Gly Glu Gly Arg Met Ile Thr Ala Ile Thr Trp 34 O 345 35. O Val Ser Ser Lys Trp Lieu. His Ser Ala Pro Gly Asp Lys Ala Lieu. Lieu 355 360 365 Arg Ala Tyr Ile Gly Arg Lieu. Gly Asp Glu Ala Trp Thr Ala Met Cys 37 O 375 38O Arg Ala Asp Ile Glu Arg Arg Val Ala Ala Glu Lieu. Arg Asp Lieu. Lieu 385 390 395 4 OO Gly Ile Ala Ala Ser Pro Lieu. Phe Cys Glu Lieu Ala Ala Lieu Pro Glu 4 OS 41O 415 Ser Met Pro Glin Tyr Pro Val Gly His Val Glu Arg Lieu. Glu Ala Leu 42O 425 43 O Arg Gly Ala Lieu. Cys Arg Ala Lys Pro Gly Lieu Lleu Lieu. Cys Gly Ala 435 44 O 445 Gly Tyr Ala Gly Val Gly Ile Pro Asp Cys Ile Arg Glin Gly Lys Glu 450 45.5 460

Ala Ala Glu Ser Met Ala Ala Tyr Lieu. Arg Asp Gly Arg 465 470 47s

<210s, SEQ ID NO 14 &211s LENGTH: 493 212. TYPE: PRT <213> ORGANISM: Paenibacillus thiaminolyticus

<4 OOs, SEQUENCE: 14 Met Lys Ala Lieu. Arg Llys Lieu Val Val Ile Gly Gly Gly Ile Thr Gly 1. 5 1O 15 US 2017/0058290 A1 Mar. 2, 2017 30

- Continued

Lieu. Ser Ala Ala Phe Tyr Ala Lieu Lys Glin Ala Asp Glu Glu Gly Glin 2O 25 3O Pro Ile Ser Val Thir Ile Ile Glu Glin Ser Asp Arg Lieu. Gly Gly Lys 35 4 O 45 Ile Glin Thr Lieu. Arg Lys Glu Gly CyS Val Ile Glu Lys Gly Pro Asp SO 55 6 O Ser Phe Lieu Ala Arg Llys Lieu Pro Met Ile Asp Lieu Ala Arg Asp Lieu. 65 70 7s 8O Gly Met Asp Ser Glu Lieu Val Ala Thr Asn. Pro His Ala Lys Llys Thr 85 90 95 Tyr Ile Lieu. Arg Arg Gly Llys Lieu. Tyr Arg Met Pro Pro Gly Lieu Val 1OO 105 11 O Lieu. Gly Ile Pro Thr Glu Lieu. Gly Pro Phe Ala Lys Thr Gly Lieu. Ile 115 12 O 125 Ser Pro Trp Gly Llys Lieu. Arg Ala Ala Met Asp Lieu. Phe Ile Llys Pro 13 O 135 14 O His Pro Ala Asp Glu Asp Glu Ser Val Gly Ala Phe Lieu. Asp Arg Arg 145 150 155 160 Lieu. Gly Arg Glu Val Thr Glu. His Ile Ala Glu Pro Lieu. Lieu Ala Gly 1.65 17O 17s Ile Tyr Ala Gly Asp Lieu. Glin Ala Lieu. Ser Lieu. Glin Ala Thr Phe Pro 18O 185 19 O Glin Phe Ala Glin Val Glu Arg Llys His Gly Gly Lieu. Ile Arg Gly Met 195 2OO 2O5 Lys Ala Ser Arg Glin Ala Gly Glin Ser Val Pro Gly Lieu Pro Asp Wall 21 O 215 22O Ala Lys Gly Thr Met Phe Lieu. Thr Phe Arg Asn Gly Lieu. Thir Ser Leu 225 23 O 235 24 O Val Glu Arg Lieu. Glu Glu Thir Lieu. Arg Asp Arg Ala Glu Lieu. Cys Lieu 245 250 255 Gly Ile Gly Ala Glu Gly Phe Glu Lys Arg Glu Asp Gly Thr Tyr Lieu. 26 O 265 27 O Val Arg Lieu. Ser Asp Gly Ser Arg Lieu. Glin Ala Asp Ala Val Ile Val 27s 28O 285 Thir Thr Pro Ser Tyr His Ala Ala Ser Leu Lieu. Glu Glu. His Val Asp 29 O 295 3 OO Ala Ser Ala Lieu. Glin Ala Ile Arg His Val Ser Val Ala Asn Val Val 3. OS 310 315 32O Ser Val Phe Asp Arg Lys Glin Val Asn. Asn Glin Phe Asp Gly Thr Gly 3.25 330 335 Phe Val Ile Ser Arg Arg Glu Gly Arg Ala Ile Thr Ala Cys Thir Trp 34 O 345 35. O Thir Ser Val Lys Trp Pro His Thr Ser Arg Gly Asp Llys Lieu. Ile Ile 355 360 365 Arg Cys Tyr Ile Gly Arg Ala Gly Asp Glu Glu Arg Val Asp Trp Pro 37 O 375 38O

Asp Glu Ala Lieu Lys Arg Thr Val Arg Ser Glu Lieu. Arg Glu Lieu. Lieu 385 390 395 4 OO Asp Ile Asp Ile Asp Pro Glu Phe Val Glu Ile Thr Arg Lieu. Arg His 4 OS 41O 415 US 2017/0058290 A1 Mar. 2, 2017 31

- Continued Ser Met Pro Glin Tyr Pro Val Gly His Val Glin Ala Ile Arg Ser Lieu. 42O 425 43 O Arg Asp Glu Val Gly Arg Thr Lieu Pro Gly Val Phe Lieu Ala Gly Glin 435 44 O 445 Pro Tyr Glu Gly Val Gly Met Pro Asp Cys Val Arg Ser Gly Arg Asp 450 45.5 460

Ala Ala Glu Ala Ala Wal Ser Ala Met Glin Ala Met Ser Thr Glu Pro 465 470 47s 48O Glu Ala Pro Ala Glu Asp Ala Ala Thr Gly Thr Ala Gly 485 490

<210s, SEQ ID NO 15 &211s LENGTH: 474 212. TYPE: PRT <213> ORGANISM: Paenibacillus polymyxa <4 OOs, SEQUENCE: 15 Met Gly Asp Llys Lys Arg Arg Val Val Val Val Gly Gly Gly Lieu. Thir 1. 5 1O 15 Gly Lieu. Ser Ala Ala Phe Tyr Ile Arg Llys His Tyr Arg Glu Ala Gly 2O 25 3O Val Glu Pro Val Ile Thr Lieu Val Glu Lys Ser Ser Ser Met Gly Gly 35 4 O 45 Met Ile Glu Thir Lieu. His Arg Asp Gly Phe Val Ile Glu Lys Gly Pro 50 55 60 Asp Ser Phe Lieu Ala Arg Llys Thr Ala Met Ile Asp Lieu Ala Lys Glu 65 70 7s 8O Lieu. Glu Ile Asp His Glu Lieu Val Ser Glin Asn. Pro Glu Ser Lys Llys 85 90 95 Thr Tyr Ile Met Glin Arg Gly Lys Lieu. His Pro Met Pro Ala Gly Lieu. 1OO 105 11 O Val Lieu. Gly Ile Pro Thr Glu Lieu. Arg Pro Phe Lieu. Arg Ser Gly Lieu 115 12 O 125 Val Ser Pro Ala Gly Llys Lieu. Arg Ala Lieu Met Asp Phe Val Ile Pro 13 O 135 14 O Pro Arg Arg Thir Thr Glu Asp Glu Ser Lieu. Gly Tyr Met Ile Glu Arg 145 150 155 160 Arg Lieu. Gly Ala Glu Val Lieu. Glu Asn Lieu. Thr Glu Pro Lieu. Lieu Ala 1.65 17O 17s Gly Ile Tyr Ala Gly Asp Met Arg Arg Lieu. Ser Lieu. Glin Ala Thr Phe 18O 185 19 O Pro Glin Phe Gly Glu Val Glu Arg Asp Tyr Gly Ser Lieu. Ile Arg Gly 195 2OO 2O5 Met Met Thr Gly Arg Llys Pro Ala Glu Thr His Thr Gly Thr Lys Arg 21 O 215 22O

Ser Ala Phe Lieu. Asn. Phe Arg Glin Gly Lieu. Glin Ser Lieu Val His Ala 225 23 O 235 24 O

Lieu Val His Glu Lieu. Glin Asp Wall Asp Glin Arg Lieu. Asn. Thir Ala Val 245 250 255

Llys Ser Lieu. Glin Arg Lieu. Asp Gly Ala Glin Thr Arg Tyr Arg Val Glu 26 O 265 27 O

Lieu. Gly Asn Gly Glu Met Lieu. Glu Ala Asp Asp Val Val Val Thr Val 27s 28O 285 US 2017/0058290 A1 Mar. 2, 2017 32

- Continued

Pro Thr Tyr Val Ala Ser Glu Lieu. Leu Lys Pro His Val Asp Thr Ala 29 O 295 3 OO Ala Lieu. Asp Ala Ile Asn Tyr Val Ser Val Ala Asn Val Val Lieu Ala 3. OS 310 315 32O Phe Glu Lys Lys Glu Val Glu. His Val Phe Asp Gly Ser Gly Phe Lieu. 3.25 330 335 Val Pro Arg Lys Glu Gly Arg Asn Ile Thr Ala Cys Thr Trp Thr Ser 34 O 345 35. O Thir Lys Trp Lieu. His Thir Ser Pro Asp Asp Llys Val Lieu. Lieu. Arg Cys 355 360 365 Tyr Val Gly Arg Ser Gly Asp Glu Glin Asn Val Glu Lieu Pro Asp Glu 37 O 375 38O Ala Lieu. Thir Asn Lieu Val Lieu Lys Asp Lieu. Arg Glu Thir Met Gly Ile 385 390 395 4 OO Glu Ala Val Pro Ile Phe Ser Glu Ile Thr Arg Lieu. Arg Llys Ser Met 4 OS 41O 415 Pro Glin Tyr Pro Val Gly His Lieu. Glin His Ile Ala Ala Lieu. Arg Glu 42O 425 43 O Glu Lieu. Gly Ser Llys Lieu Pro Gly Val Tyr Ile Ala Gly Ala Gly Tyr 435 44 O 445 Glu Gly Val Gly Lieu Pro Asp Cys Ile Arg Glin Ala Lys Glu Met Ser 450 45.5 460 Val Glin Ala Thr Glin Glu Lieu Ala Ala Asp 465 470

<210s, SEQ ID NO 16 &211s LENGTH: 470 212. TYPE: PRT <213> ORGANISM: Bacillus atrophaeus

<4 OOs, SEQUENCE: 16 Met Ser Asp Gly Llys Llys His Lieu Val Ile Ile Gly Gly Gly Ile Thr 1. 5 1O 15 Gly Lieu Ala Ser Ala Phe Tyr Met Glu Lys Glu Ile Arg Glu Lys Asn 2O 25 3O Lieu Pro Lieu. Ser Val Thir Lieu Val Glu Ala Ser Pro Arg Val Gly Gly 35 4 O 45 Lys Ile Glin Thir Ala Arg Lys Asp Gly Tyr Ile Ile Glu Arg Gly Pro SO 55 6 O Asp Ser Phe Lieu. Glu Arg Llys Llys Ser Ala Pro Glu Lieu Val Glu Asp 65 70 7s 8O Lieu. Gly Lieu. Glu. His Lieu. Lieu Val Asn. Asn Ala Thr Gly Glin Ser Tyr 85 90 95

Val Lieu Val Asn Glu Thir Lieu. His Pro Met Pro Lys Gly Ala Val Met 1OO 105 11 O

Gly Ile Pro Thr Lys Ile Ala Pro Phe Met Ser Thr Gly Lieu. Phe Ser 115 12 O 125

Phe Ser Gly Lys Ala Arg Ala Ala Met Asp Phe Val Lieu Pro Ala Ser 13 O 135 14 O

Llys Pro Lys Glu Asp Glin Ser Lieu. Gly Glu Phe Phe Arg Arg Arg Val 145 150 155 160

Gly Asp Glu Val Val Glu Asn Lieu. Ile Glu Pro Lieu Lleu Ser Gly Ile US 2017/0058290 A1 Mar. 2, 2017 33

- Continued

1.65 17O 17s Tyr Ala Gly Asp Ile Asp Arg Lieu. Ser Lieu Met Ser Thr Phe Pro Glin 18O 185 19 O Phe Tyr Glin Thr Glu Gln Lys His Arg Ser Lieu. Ile Leu Gly Met Lys 195 2OO 2O5 Llys Thr Arg Pro Glin Gly Ser Gly Glin Arg Lieu. Thir Ala Lys Lys Glin 21 O 215 22O Gly Glin Phe Glin Thr Lieu Lys Thr Gly Lieu Gln Thr Lieu Val Glu Glu 225 23 O 235 24 O Lieu. Glu Asn Glin Lieu Lys Lieu. Thir Lys Val Tyr Lys Gly. Thir Lys Val 245 250 255 Thir Asn. Ile Ser Arg Gly Glu Lys Gly Cys Ser Ile Ala Lieu. Asp Asn 26 O 265 27 O Gly Met Thir Lieu. Asp Ala Asp Ala Ala Ile Val Thir Ser Pro His Lys 27s 28O 285 Ser Ala Ala Gly Met Phe Pro Asp Lieu Pro Ala Val Ser Glin Lieu Lys 29 O 295 3 OO Asp Met His Ser Thr Ser Val Ala Asn Val Ala Lieu. Gly Phe Pro Glin 3. OS 310 315 32O Glu Ala Val Glin Met Glu. His Glu Gly Thr Gly Phe Val Ile Ser Arg 3.25 330 335 Asn Ser Asp Phe Ser Ile Thr Ala Cys Thr Trp Thr Asn Llys Llys Trp 34 O 345 35 O Pro His Ser Ala Pro Glu Gly Lys Thr Lieu. Lieu. Arg Ala Tyr Val Gly 355 360 365 Lys Ala Gly Asp Glu Ser Ile Val Glu Lieu. Ser Asp Asn. Glu Ile Ile 37 O 375 38O Lys Ile Val Lieu. Glu Asp Lieu Lys Llys Wal Met Lys Ile Lys Gly Glu 385 390 395 4 OO Pro Glu Met Thr Cys Val Thr Arg Trp Asin Glu Ser Met Pro Glin Tyr 4 OS 41O 415 His Val Gly His Lys Glin Arg Ile Llys Llys Val Arg Glu Ala Lieu Ala 42O 425 43 O Ala Ser Tyr Pro Gly Val Tyr Met Thr Gly Ala Ser Phe Glu Gly Val 435 44 O 445 Gly Ile Pro Asp Cys Ile Asp Glin Gly Lys Ser Ala Val Ser Asp Wall 450 45.5 460 Lieu Ala Tyr Lieu. Phe Gly 465 470

<210s, SEQ ID NO 17 &211s LENGTH: 470 212. TYPE: PRT <213> ORGANISM: Bacillus atrophaeus

<4 OOs, SEQUENCE: 17 Met Ser Asp Gly Llys Llys His Lieu Val Ile Ile Gly Gly Gly Ile Thr 1. 5 1O 15

Gly Lieu Ala Ser Ala Phe Tyr Met Glu Lys Glu Ile Arg Glu Lys Asn 2O 25 3O

Lieu Pro Lieu. Ser Val Thir Lieu Val Glu Ala Ser Pro Arg Val Gly Gly 35 4 O 45 US 2017/0058290 A1 Mar. 2, 2017 34

- Continued Lys Ile Glin Thir Ala Arg Lys Asp Gly Tyr Ile Ile Glu Arg Gly Pro SO 55 6 O Asp Ser Phe Lieu. Glu Arg Llys Llys Ser Ala Pro Glu Lieu Val Glu Asp 65 70 7s 8O Lieu. Gly Lieu. Glu. His Lieu. Lieu Val Asn. Asn Ala Thr Gly Glin Ser Tyr 85 90 95 Val Lieu Val Asn Glu Thir Lieu. His Pro Met Pro Lys Gly Ala Val Met 1OO 105 11 O Gly Ile Pro Thr Lys Ile Ala Pro Phe Met Ser Thr Arg Lieu. Phe Ser 115 12 O 125 Phe Ser Gly Lys Ala Arg Ala Ala Met Asp Phe Val Lieu Pro Ala Ser 13 O 135 14 O Llys Pro Lys Glu Asp Glin Ser Lieu. Gly Glu Phe Phe Arg Arg Arg Val 145 150 155 160 Gly Asp Glu Val Val Glu Asn Lieu. Ile Glu Pro Lieu Lleu Ser Gly Ile 1.65 17O 17s Tyr Ala Gly Asp Ile Asp Arg Lieu. Ser Lieu Met Ser Thr Phe Pro Glin 18O 185 19 O Phe Tyr Glin Thr Glu Gln Lys His Arg Ser Lieu. Ile Leu Gly Met Lys 195 2OO 2O5 Llys Thr Arg Pro Glin Gly Ser Gly Glin Glin Lieu. Thir Ala Lys Lys Glin 21 O 215 22O Gly Glin Phe Glin Thr Lieu Lys Thr Gly Lieu Gln Thr Lieu Val Glu Glu 225 23 O 235 24 O Lieu. Glu Asn Glin Lieu Lys Lieu. Thir Lys Val Tyr Lys Gly. Thir Lys Val 245 250 255 Thir Asn. Ile Ser Arg Gly Glu Lys Gly Cys Ser Ile Ala Lieu. Asp Asn 26 O 265 27 O Gly Met Thir Lieu. Asp Ala Asp Ala Ala Ile Val Thir Ser Pro His Lys 27s 28O 285 Ser Ala Ala Gly Met Phe Pro Asp Lieu Pro Ala Val Ser Glin Lieu Lys 29 O 295 3 OO Asp Met His Ser Thr Ser Val Ala Asn Val Ala Lieu. Gly Phe Pro Glin 3. OS 310 315 32O Glu Ala Val Glin Met Glu. His Glu Gly Thr Gly Phe Val Ile Ser Arg 3.25 330 335 Asn Ser Asp Phe Ser Ile Thr Ala Cys Thr Trp Thr Asn Llys Llys Trp 34 O 345 35. O Pro His Ser Ala Pro Glu Gly Lys Thr Lieu. Lieu. Arg Ala Tyr Val Gly 355 360 365 Lys Ala Gly Asp Glu Ser Ile Val Glu Lieu. Ser Asp Asn. Glu Ile Ile 37 O 375 38O

Lys Ile Val Lieu. Glu Asp Lieu Lys Llys Wal Met Lys Ile Lys Gly Glu 385 390 395 4 OO

Pro Glu Met Thr Cys Val Thr Arg Trp Asin Glu Ser Met Pro Glin Tyr 4 OS 41O 415 His Val Gly His Lys Glin Arg Ile Llys Llys Val Arg Glu Ala Lieu Ala 42O 425 43 O

Ala Ser Tyr Pro Gly Val Tyr Met Thr Gly Ala Ser Phe Glu Gly Val 435 44 O 445

Gly Ile Pro Asp Cys Ile Asp Glin Gly Lys Ser Ala Val Ser Asp Wall US 2017/0058290 A1 Mar. 2, 2017 35

- Continued

450 45.5 460 Lieu Ala Tyr Lieu. Phe Glu 465 470

SEQ ID NO 18 LENGTH: TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Recombinant

SEQUENCE: 18

Lys Ile Ala Ile Gly Gly Gly Ile Thir Gly Lell Ser Wall Ala 15

Tyr Wall Arg Lell Lell Arg Glu Glin Gly Wall Asn Ala Gly Wall 25

Thir Luell Wall Glu Glin Ser Asp Arg Luell Gly Gly Ile Arg Ser Luell 35 4 O 45

Arg Arg Asp Gly Phe Thir Ile Glu Glin Gly Pro Asp Ser Met Ile Ala SO 55 6 O

Arg Pro Ala Ala Lell Glu Luell Ile Arg Glu Lell Gly Luell Glu Asp 65 70

Luell Ala Gly Thir Asn Pro Glin Ala Lys Arg Ser Ile Luell His 85 90 95

Arg Gly Lys Phe His Pro Met Pro Pro Gly Lieu. Met Lieu Gly Ile Pro 105 11 O

Thir Glin Met Trp Pro Met Wall Lys Thir Gly Luell Lell Ser Pro Ala Gly 115 12 O 125

Luell Arg Ala Ala Met Asp Lel Luell Luell Pro Ala Arg Arg Gly Gly 13 O 135 14 O

Gly Asp Glu Ser Lell Gly Gly Phe Ile Arg Arg Arg Lell Gly Arg Glu 145 150 155 160

Wall Luell Glu Glin Met Thir Glu Luell Luell Ala Gly Ile Ala Gly 1.65 17O 17s

Asp Thir Glu Glin Lell Ser Lell Ala Thir Phe Pro Glin Phe Met Glu 18O 185 19 O

Met Glu Arg His Arg Ser Lel Ile Luell Gly Lell Lell Ala Gly 195

Glin Pro Pro Arg Pro Gly Gly Ser Glin Wall Pro Lell Pro Ala 21 O 215 22O

Ala Glin Thir Ser Met Phe Lell Thir Luell Thir Gly Lell Glu Gly Luell 225 23 O 24 O

Thir Glu Ala Luell Glu Glu Ser Luell Ser Glu Ile Ile Thir Gly 245 250 255

Glin Ala Wall Thir Gly Lell Ser Glin Glin Glu Gly Glu Luell Asn 26 O 265 27 O

Lell Ser Gly Gly Glu Arg Lell Asn Ala Asp Wall Ile Luell Ala Wall 28O 285

Pro Ala Phe Ala Ala Ala Arg Luell Luell Asp Wall Pro Glu Ala Ala 29 O 295 3 OO

Tyr Luell Glu Arg Ile Arg Wall Ser Wall Asn Lell Ala Phe Ala 3. OS 310 32O

Arg Arg Glu Asp Wall Pro His Asp Luell ASn Gly Ser Gly Wall Luell US 2017/0058290 A1 Mar. 2, 2017 36

- Continued

3.25 330 335

Ile Pro Arg Gly Glu Gly Arg Met Ile Thr Ala Ile Thir Trp Wall Ser 34 O 345 35. O

Ser Llys Trp Lieu. His Ser Ala Pro Gly Asp Ala Lell Luell Arg Ala 355 360 365

Ile Gly Arg Lieu. Gly Asp Glu Ala Trip Thir Ala Met Arg Ala 37 O 375

Asp Ile Glu Arg Arg Val Ala Ala Glu Lieu. Arg Asp Lell Luell Gly Ile 385 390 395 4 OO

Ala Ala Ser Pro Lieu. Phe Cys Glu Lieu Ala Ala Lell Pro Glu Ser Met 4 OS 41O 415

Pro Gln Tyr Pro Val Gly His Val Glu Arg Luell Glu Ala Luell Arg Gly 42O 425 43 O

Ala Lieu. Cys Arg Ala Lys Pro Gly Lieu. Lieu. Luell Gly Ala Gly Tyr 435 44 O 445

Ala Gly Val Gly Ile Pro Asp Cys Ile Arg Glin Gly Lys Glu Ala Ala 450 45.5 460

Glu Ser Met Ala Ala Tyr Lieu. Arg Asp Gly Arg 465 470 47s

<210s, SEQ ID NO 19 &211s LENGTH: 489 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 19

Arg Llys Lieu Val Val Ile Gly Gly Gly Ile Thir Gly Lell Ser Ala Ala 1. 5 1O 15

Phe Tyr Ala Lieu Lys Glin Ala Asp Glu Glu Gly Glin Pro Ile Ser Wall 2O 25

Thir Ile Ile Glu Glin Ser Asp Arg Lieu. Gly Gly Ile Glin Thir Luell 35 4 O 45

Arg Lys Glu Gly Cys Val Ile Glu Lys Gly Pro Asp Ser Phe Luell Ala SO 55 6 O

Arg Llys Lieu Pro Met Ile Asp Lieu Ala Arg Asp Lell Gly Met Asp Ser 65 70

Glu Lieu Val Ala Thr Asn Pro His Ala Lys Thir Ile Luell Arg 85 90 95

Arg Gly Lys Lieu. Tyr Arg Met Pro Pro Gly Luell Wall Lell Gly Ile Pro 1OO 105 11 O

Thir Glu Lieu. Gly Pro Phe Ala Lys Thr Gly Luell Ile Ser Pro Trp Gly 115 12 O 125

Lieu. Arg Ala Ala Met Asp Lieu. Phe Ile Pro His Pro Ala Asp 13 O 135 14 O

Glu Asp Glu Ser Val Gly Ala Phe Lieu. Asp Arg Arg Lell Gly Arg Glu 145 150 155 160

Wall Thr Glu. His Ile Ala Glu Pro Lieu. Lieu. Ala Gly Ile Ala Gly 1.65 17O 17s

Asp Lieu. Glin Ala Lieu. Ser Lieu. Glin Ala Thr Phe Pro Glin Phe Ala Glin 18O 185 19 O

Wall Glu Arg Llys His Gly Gly Lieu. Ile Arg Gly Met Ala Ser Arg US 2017/0058290 A1 Mar. 2, 2017 37

- Continued

195 2OO 2O5 Glin Ala Gly Glin Ser Val Pro Gly Lieu Pro Asp Val Ala Lys Gly. Thir 21 O 215 22O Met Phe Lieu. Thir Phe Arg Asn Gly Lieu. Thir Ser Lieu Val Glu Arg Lieu. 225 23 O 235 24 O Glu Glu Thir Lieu. Arg Asp Arg Ala Glu Lieu. Cys Lieu. Gly Ile Gly Ala 245 250 255 Glu Gly Phe Glu Lys Arg Glu Asp Gly. Thir Tyr Lieu Val Arg Lieu. Ser 26 O 265 27 O Asp Gly Ser Arg Lieu. Glin Ala Asp Ala Val Ile Val Thir Thr Pro Ser 27s 28O 285 Tyr His Ala Ala Ser Lieu. Lieu. Glu Glu. His Val Asp Ala Ser Ala Lieu. 29 O 295 3 OO Glin Ala Ile Arg His Val Ser Val Ala Asn Val Val Ser Val Phe Asp 3. OS 310 315 32O Arg Lys Glin Val Asn. Asn Glin Phe Asp Gly Thr Gly Phe Val Ile Ser 3.25 330 335 Arg Arg Glu Gly Arg Ala Ile Thr Ala Cys Thir Trp Thir Ser Val Lys 34 O 345 35. O Trp Pro His Thir Ser Arg Gly Asp Llys Lieu. Ile Ile Arg Cys Tyr Ile 355 360 365 Gly Arg Ala Gly Asp Glu Glu Arg Val Asp Trp Pro Asp Glu Ala Lieu. 370 375 380 Lys Arg Thr Val Arg Ser Glu Lieu. Arg Glu Lieu. Lieu. Asp Ile Asp Ile 385 390 395 4 OO Asp Pro Glu Phe Val Glu Ile Thr Arg Lieu. Arg His Ser Met Pro Glin 4 OS 41O 415 Tyr Pro Val Gly His Val Glin Ala Ile Arg Ser Lieu. Arg Asp Glu Val 42O 425 43 O Gly Arg Thr Lieu Pro Gly Val Phe Leu Ala Gly Glin Pro Tyr Glu Gly 435 44 O 445 Val Gly Met Pro Asp Cys Val Arg Ser Gly Arg Asp Ala Ala Glu Ala 450 45.5 460

Ala Wal Ser Ala Met Glin Ala Met Ser Thr Glu Pro Glu Ala Pro Ala 465 470 47s 48O Glu Asp Ala Ala Thr Gly. Thir Ala Gly 485

<210s, SEQ ID NO 2 O &211s LENGTH: 469 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 2O Arg Arg Val Val Val Val Gly Gly Gly Lieu. Thr Gly Lieu. Ser Ala Ala 1. 5 1O 15 Phe Tyr Ile Arg Llys His Tyr Arg Glu Ala Gly Val Glu Pro Val Ile 2O 25 3O

Thr Lieu Val Glu Lys Ser Ser Ser Met Gly Gly Met Ile Glu. Thir Lieu. 35 4 O 45 His Arg Asp Gly Phe Val Ile Glu Lys Gly Pro Asp Ser Phe Lieu Ala US 2017/0058290 A1 Mar. 2, 2017 38

- Continued

SO 55 6 O Arg Llys Thir Ala Met Ile Asp Lieu Ala Lys Glu Lieu. Glu Ile Asp His 65 70 7s 8O Glu Lieu Val Ser Glin Asn Pro Glu Ser Lys Llys Thr Tyr Ile Met Glin 85 90 95 Arg Gly Lys Lieu. His Pro Met Pro Ala Gly Lieu Val Lieu. Gly Ile Pro 1OO 105 11 O Thr Glu Lieu. Arg Pro Phe Lieu. Arg Ser Gly Lieu Val Ser Pro Ala Gly 115 12 O 125 Llys Lieu. Arg Ala Lieu Met Asp Phe Val Ile Pro Pro Arg Arg Thir Thr 13 O 135 14 O Glu Asp Glu Ser Lieu. Gly Tyr Met Ile Glu Arg Arg Lieu. Gly Ala Glu 145 150 155 160 Val Lieu. Glu Asn Lieu. Thr Glu Pro Lieu. Lieu Ala Gly Ile Tyr Ala Gly 1.65 17O 17s Asp Met Arg Arg Lieu. Ser Lieu. Glin Ala Thr Phe Pro Glin Phe Gly Glu 18O 185 19 O Val Glu Arg Asp Tyr Gly Ser Lieu. Ile Arg Gly Met Met Thr Gly Arg 195 2OO 2O5 Llys Pro Ala Glu Thr His Thr Gly Thr Lys Arg Ser Ala Phe Lieu. Asn 21 O 215 22O Phe Arg Glin Gly Lieu. Glin Ser Lieu Val His Ala Lieu Val His Glu Lieu. 225 23 O 235 24 O Glin Asp Val Asp Glin Arg Lieu. Asn. Thir Ala Val Llys Ser Lieu. Glin Arg 245 250 255 Lieu. Asp Gly Ala Glin Thr Arg Tyr Arg Val Glu Lieu. Gly Asn Gly Glu 26 O 265 27 O Met Leu Glu Ala Asp Asp Val Val Val Thr Val Pro Thr Tyr Val Ala 27s 28O 285 Ser Glu Lieu. Lieu Lys Pro His Val Asp Thir Ala Ala Lieu. Asp Ala Ile 29 O 295 3 OO Asn Tyr Val Ser Val Ala Asn Val Val Lieu Ala Phe Glu Lys Lys Glu 3. OS 310 315 32O Val Glu. His Val Phe Asp Gly Ser Gly Phe Lieu Val Pro Arg Lys Glu 3.25 330 335 Gly Arg Asn Ile Thr Ala Cys Thr Trp Thr Ser Thr Lys Trp Lieu. His 34 O 345 35. O Thir Ser Pro Asp Asp Llys Val Lieu. Lieu. Arg Cys Tyr Val Gly Arg Ser 355 360 365 Gly Asp Glu Glin Asn Val Glu Lieu Pro Asp Glu Ala Lieu. Thir Asn Lieu. 37 O 375 38O

Val Lieu Lys Asp Lieu. Arg Glu Thir Met Gly Ile Glu Ala Val Pro Ile 385 390 395 4 OO

Phe Ser Glu Ile Thr Arg Lieu. Arg Llys Ser Met Pro Glin Tyr Pro Val 4 OS 41O 415

Gly His Lieu Gln His Ile Ala Ala Lieu. Arg Glu Glu Lieu. Gly Ser Lys 42O 425 43 O

Lieu Pro Gly Val Tyr Ile Ala Gly Ala Gly Tyr Glu Gly Val Gly Lieu. 435 44 O 445

Pro Asp Cys Ile Arg Glin Ala Lys Glu Met Ser Val Glin Ala Thr Glin 450 45.5 460 US 2017/0058290 A1 Mar. 2, 2017 39

- Continued

Glu Lieu Ala Ala Asp 465

<210s, SEQ ID NO 21 &211s LENGTH: 465 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 21 Llys His Lieu Val Ile Ile Gly Gly Gly Ile Thr Gly Lieu Ala Ser Ala 1. 5 1O 15 Phe Tyr Met Glu Lys Glu Ile Arg Glu Lys Asn Lieu Pro Lieu. Ser Val 2O 25 3O Thir Lieu Val Glu Ala Ser Pro Arg Val Gly Gly Lys Ile Glin Thr Ala 35 4 O 45 Arg Lys Asp Gly Tyr Ile Ile Glu Arg Gly Pro Asp Ser Phe Lieu. Glu SO 55 6 O Arg Llys Llys Ser Ala Pro Glu Lieu Val Glu Asp Lieu. Gly Lieu. Glu. His 65 70 7s 8O Lieu. Lieu Val Asn. Asn Ala Thr Gly Glin Ser Tyr Val Lieu Val Asn. Glu 85 90 95 Thr Lieu. His Pro Met Pro Lys Gly Ala Val Met Gly Ile Pro Thr Lys 1OO 105 11 O Ile Ala Pro Phe Met Ser Thr Arg Lieu. Phe Ser Phe Ser Gly Lys Ala 115 12 O 125 Arg Ala Ala Met Asp Phe Val Lieu Pro Ala Ser Llys Pro Lys Glu Asp 13 O 135 14 O Glin Ser Lieu. Gly Glu Phe Phe Arg Arg Arg Val Gly Asp Glu Val Val 145 150 155 160 Glu Asn Lieu. Ile Glu Pro Lieu. Lieu. Ser Gly Ile Tyr Ala Gly Asp Ile 1.65 17O 17s Asp Arg Lieu Ser Leu Met Ser Thr Phe Pro Glin Phe Tyr Glin Thr Glu 18O 185 19 O Glin Llys His Arg Ser Lieu. Ile Lieu. Gly Met Lys Llys Thr Arg Pro Glin 195 2OO 2O5 Gly Ser Gly Glin Gln Lieu. Thir Ala Lys Lys Glin Gly Glin Phe Glin Thr 21 O 215 22O Lieu Lys Thr Gly Lieu. Glin Thr Lieu Val Glu Glu Lieu. Glu Asn Glin Lieu. 225 23 O 235 24 O Llys Lieu. Thir Lys Val Tyr Lys Gly Thr Llys Val Thr Asn. Ile Ser Arg 245 250 255 Gly Glu Lys Gly Cys Ser Ile Ala Lieu. Asp Asn Gly Met Thr Lieu. Asp 26 O 265 27 O

Ala Asp Ala Ala Ile Val Thir Ser Pro His Llys Ser Ala Ala Gly Met 27s 28O 285

Phe Pro Asp Leu Pro Ala Val Ser Gln Leu Lys Asp Met His Ser Thr 29 O 295 3 OO

Ser Val Ala Asn. Wall Ala Lieu. Gly Phe Pro Glin Glu Ala Val Glin Met 3. OS 310 315 32O

Glu. His Glu Gly Thr Gly Phe Val Ile Ser Arg Asn Ser Asp Phe Ser 3.25 330 335 US 2017/0058290 A1 Mar. 2, 2017 40

- Continued

Ile Thr Ala Cys Thr Trp Thr Asn Llys Llys Trp Pro His Ser Ala Pro 34 O 345 35. O

Glu Gly Lys Thir Lieu. Lieu. Arg Ala Tyr Val Gly Ala Gly Asp Glu 355 360 365

Ser Ile Val Glu Lieu. Ser Asp Asn. Glu Ile Ile Lys Ile Wall Luell Glu 37 O 375

Asp Lieu Lys Llys Wal Met Lys Ile Lys Gly Glu Pro Glu Met Thir Cys 385 390 395 4 OO

Wall Thr Arg Trp Asn Glu Ser Met Pro Glin His Wall Gly His 4 OS 41O 415

Glin Arg Ile Llys Llys Val Arg Glu Ala Lieu. Ala Ala Ser Tyr Pro 42O 425 43 O

Wall Tyr Met Thr Gly Ala Ser Phe Glu Gly Wall Gly Ile Pro Asp 435 44 O 445

Ile Asp Glin Gly Llys Ser Ala Val Ser Asp Wall Lell Ala Luell Phe 450 45.5 460

Glu 465

<210s, SEQ ID NO 22 &211s LENGTH: 473 212. TYPE: PRT <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 22

Ile Ala Val Ile Gly Gly Gly Ile Thr Gly Luell Ser Wall Ala Tyr 1. 5 1O 15

Wall Arg Llys Lieu. Lieu. Arg Glu Glin Gly Val ASn Ala Gly Wall Thir Luell 2O 25

Wall Glu Glin Ser Asp Arg Lieu. Gly Gly Lys Ile Arg Ser Luell Arg Arg 35 4 O 45

Asp Gly Phe Thir Ile Glu Glin Gly Pro Asp Ser Met Ile Ala Arg SO 55 6 O

Pro Ala Ala Lieu. Glu Lieu. Ile Arg Glu Lieu. Gly Lell Glu Asp Luell 65 70

Ala Gly. Thir Asn Pro Glin Ala Lys Arg Ser Ile Lell His Arg Gly 85 90 95

Phe His Pro Met Pro Pro Gly Lieu Met Luell Gly Ile Thir Glin 1OO 105

Met Trp Pro Met Val Lys Thr Gly Lieu. Leu Ser Pro Ala Luell 115 12 O 125

Arg Ala Ala Met Asp Lieu. Lieu. Lieu Pro Ala Arg Arg Gly Gly Asp 13 O 135 14 O

Glu Ser Lieu. Gly Gly Phe Ile Arg Arg Arg Luell Gly Arg Wall Luell 145 150 155 160

Glu Gln Met Thr Glu Pro Leu Lleu Ala Gly Ile Tyr Ala Asp Thir 1.65 17O 17s

Glu Gln Leu Ser Lieu Lys Ala Thr Phe Pro Glin Phe Met Met Glu 18O 185

Arg Llys His Arg Ser Lieu. Ile Lieu. Gly Lieu. Luell Ala Gly Glin 195 2OO 2O5 US 2017/0058290 A1 Mar. 2, 2017 41

- Continued

Pro Pro Arg Pro Gly Gly Ser Glin Val Pro Leu Pro Lys Ala Ala Glin 21 O 215 22O Thir Ser Met Phe Lieu. Thir Lieu. Thr Gly Gly Lieu. Glu Gly Lieu. Thr Glu 225 23 O 235 24 O Ala Lieu. Glu Glu Ser Lieu. Ser Glu Glu Lys Ile Ile Thr Gly Glin Ala 245 250 255 Val Thr Gly Lieu. Ser Glin Glin Glu Ala Gly Tyr Glu Lieu. Asn Lieu. Ser 26 O 265 27 O Gly Gly Glu Arg Lieu. Asn Ala Asp Gly Val Ile Lieu Ala Val Pro Ala 27s 28O 285 Phe Ala Ala Ala Arg Lieu. Lieu. Asp Gly Val Pro Glu Ala Ala Tyr Lieu. 29 O 295 3 OO Glu Arg Ile Arg Tyr Val Ser Val Ala Asn Lieu Ala Phe Ala Tyr Arg 3. OS 310 315 32O Arg Glu Asp Val Pro His Asp Lieu. Asn Gly Ser Gly Val Lieu. Ile Pro 3.25 330 335 Arg Gly Glu Gly Arg Met Ile Thr Ala Ile Thir Trp Val Ser Ser Lys 34 O 345 35. O Trp Lieu. His Ser Ala Pro Gly Asp Lys Ala Lieu. Lieu. Arg Ala Tyr Ile 355 360 365 Gly Arg Lieu. Gly Asp Glu Ala Trp Thir Ala Met Cys Arg Ala Asp Ile 37 O 375 38O Glu Arg Arg Val Ala Ala Glu Lieu. Arg Asp Lieu. Lieu. Gly Ile Ala Ala 385 390 395 4 OO Ser Pro Leu Phe Cys Glu Lieu Ala Ala Leu Pro Glu Ser Met Pro Glin 4 OS 41O 415 Tyr Pro Val Gly His Val Glu Arg Lieu. Glu Ala Lieu. Arg Gly Ala Lieu. 42O 425 43 O Cys Arg Ala Lys Pro Gly Lieu. Lieu. Lieu. Cys Gly Ala Gly Tyr Ala Gly 435 44 O 445 Val Gly Ile Pro Asp Cys Ile Arg Glin Gly Lys Glu Ala Ala Glu Ser 450 45.5 460 Met Ala Ala Tyr Lieu. Arg Asp Gly Arg 465 470

<210s, SEQ ID NO 23 &211s LENGTH: 487 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 23 Lieu Val Val Ile Gly Gly Gly Ile Thr Gly Lieu Ser Ala Ala Phe Tyr 1. 5 1O 15

Ala Lieu Lys Glin Ala Asp Glu Glu Gly Glin Pro Ile Ser Val Thir Ile 2O 25 3O

Ile Glu Glin Ser Asp Arg Lieu. Gly Gly Lys Ile Glin Thr Lieu. Arg Llys 35 4 O 45

Glu Gly Cys Val Ile Glu Lys Gly Pro Asp Ser Phe Lieu Ala Arg Llys SO 55 6 O

Lieu Pro Met Ile Asp Lieu Ala Arg Asp Lieu. Gly Met Asp Ser Glu Lieu. 65 70 7s 8O US 2017/0058290 A1 Mar. 2, 2017 42

- Continued

Val Ala Thir Asn Pro His Ala Lys Llys Thr Tyr Ile Lieu. Arg Arg Gly 85 90 95 Lys Lieu. Tyr Arg Met Pro Pro Gly Lieu Val Lieu. Gly Ile Pro Thr Glu 1OO 105 11 O Lieu. Gly Pro Phe Ala Lys Thr Gly Lieu. Ile Ser Pro Trp Gly Lys Lieu. 115 12 O 125 Arg Ala Ala Met Asp Lieu. Phe Ile Llys Pro His Pro Ala Asp Glu Asp 13 O 135 14 O Glu Ser Val Gly Ala Phe Lieu. Asp Arg Arg Lieu. Gly Arg Glu Val Thr 145 150 155 160 Glu. His Ile Ala Glu Pro Lieu. Lieu Ala Gly Ile Tyr Ala Gly Asp Lieu. 1.65 17O 17s

Glin Ala Lieu. Ser Leul Glin Ala Thir Phe Pro Glin Phe Ala Glin Wall Glu 18O 185 19 O Arg Llys His Gly Gly Lieu. Ile Arg Gly Met Lys Ala Ser Arg Glin Ala 195 2OO 2O5 Gly Glin Ser Val Pro Gly Lieu Pro Asp Val Ala Lys Gly Thr Met Phe 21 O 215 22O Lieu. Thir Phe Arg Asn Gly Lieu. Thir Ser Lieu Val Glu Arg Lieu. Glu Glu 225 23 O 235 24 O Thir Lieu. Arg Asp Arg Ala Glu Lieu. Cys Lieu. Gly Ile Gly Ala Glu Gly 245 250 255 Phe Glu Lys Arg Glu Asp Gly Thr Tyr Lieu Val Arg Lieu. Ser Asp Gly 26 O 265 27 O Ser Arg Lieu. Glin Ala Asp Ala Val Ile Val Thr Thr Pro Ser Tyr His 27s 28O 285 Ala Ala Ser Lieu. Lieu. Glu Glu. His Val Asp Ala Ser Ala Lieu. Glin Ala 29 O 295 3 OO Ile Arg His Val Ser Val Ala Asn Val Val Ser Val Phe Asp Arg Llys 3. OS 310 315 32O Glin Val Asn. Asn Glin Phe Asp Gly Thr Gly Phe Val Ile Ser Arg Arg 3.25 330 335 Glu Gly Arg Ala Ile Thr Ala Cys Thr Trp Thir Ser Val Lys Trp Pro 34 O 345 35. O His Thir Ser Arg Gly Asp Llys Lieu. Ile Ile Arg Cys Tyr Ile Gly Arg 355 360 365 Ala Gly Asp Glu Glu Arg Val Asp Trp Pro Asp Glu Ala Lieu Lys Arg 37 O 375 38O Thr Val Arg Ser Glu Lieu. Arg Glu Lieu. Lieu. Asp Ile Asp Ile Asp Pro 385 390 395 4 OO Glu Phe Val Glu Ile Thr Arg Lieu. Arg His Ser Met Pro Glin Tyr Pro 4 OS 41O 415 Val Gly His Val Glin Ala Ile Arg Ser Lieu. Arg Asp Glu Val Gly Arg 42O 425 43 O

Thr Lieu Pro Gly Val Phe Leu Ala Gly Glin Pro Tyr Glu Gly Val Gly 435 44 O 445

Met Pro Asp Cys Val Arg Ser Gly Arg Asp Ala Ala Glu Ala Ala Val 450 45.5 460

Ser Ala Met Glin Ala Met Ser Thr Glu Pro Glu Ala Pro Ala Glu Asp 465 470 47s 48O US 2017/0058290 A1 Mar. 2, 2017 43

- Continued Ala Ala Thr Gly Thr Ala Gly 485

<210s, SEQ ID NO 24 &211s LENGTH: 467 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 24 Val Val Val Val Gly Gly Gly Lieu. Thr Gly Leu Ser Ala Ala Phe Tyr 1. 5 1O 15 Ile Arg Llys His Tyr Arg Glu Ala Gly Val Glu Pro Val Ile Thr Lieu. 2O 25 3O Val Glu Lys Ser Ser Ser Met Gly Gly Met Ile Glu Thir Lieu. His Arg 35 4 O 45 Asp Gly Phe Val Ile Glu Lys Gly Pro Asp Ser Phe Lieu Ala Arg Llys SO 55 6 O Thir Ala Met Ile Asp Lieu Ala Lys Glu Lieu. Glu Ile Asp His Glu Lieu. 65 70 7s 8O Val Ser Glin Asn Pro Glu Ser Lys Llys Thr Tyr Ile Met Glin Arg Gly 85 90 95 Lys Lieu. His Pro Met Pro Ala Gly Lieu Val Lieu. Gly Ile Pro Thr Glu 1OO 105 11 O Lieu. Arg Pro Phe Lieu. Arg Ser Gly Lieu Val Ser Pro Ala Gly Lys Lieu. 115 12 O 125 Arg Ala Lieu Met Asp Phe Val Ile Pro Pro Arg Arg Thir Thr Glu Asp 13 O 135 14 O Glu Ser Lieu. Gly Tyr Met Ile Glu Arg Arg Lieu. Gly Ala Glu Val Lieu. 145 150 155 160 Glu Asn Lieu. Thr Glu Pro Lieu. Lieu Ala Gly Ile Tyr Ala Gly Asp Met 1.65 17O 17s Arg Arg Lieu. Ser Lieu. Glin Ala Thr Phe Pro Glin Phe Gly Glu Val Glu 18O 185 19 O Arg Asp Tyr Gly Ser Lieu. Ile Arg Gly Met Met Thr Gly Arg Llys Pro 195 2OO 2O5 Ala Glu Thir His Thr Gly Thr Lys Arg Ser Ala Phe Lieu. Asn Phe Arg 21 O 215 22O Glin Gly Lieu. Glin Ser Lieu Val His Ala Lieu Val His Glu Lieu. Glin Asp 225 23 O 235 24 O Val Asp Glin Arg Lieu. Asn. Thir Ala Wall Lys Ser Lieu. Glin Arg Lieu. Asp 245 250 255 Gly Ala Glin Thr Arg Tyr Arg Val Glu Lieu. Gly Asn Gly Glu Met Lieu. 26 O 265 27 O

Glu Ala Asp Asp Val Val Val Thr Val Pro Thr Tyr Val Ala Ser Glu 27s 28O 285 Lieu. Lieu Lys Pro His Val Asp Thir Ala Ala Lieu. Asp Ala Ile Asn Tyr 29 O 295 3 OO

Val Ser Val Ala Asn Val Val Lieu Ala Phe Glu Lys Lys Glu Val Glu 3. OS 310 315 32O His Val Phe Asp Gly Ser Gly Phe Lieu Val Pro Arg Lys Glu Gly Arg 3.25 330 335 US 2017/0058290 A1 Mar. 2, 2017 44

- Continued Asn Ile Thr Ala Cys Thr Trp Thr Ser Thr Lys Trp Lieu. His Thr Ser 34 O 345 35. O Pro Asp Asp Llys Val Lieu. Lieu. Arg Cys Tyr Val Gly Arg Ser Gly Asp 355 360 365 Glu Glin Asn Val Glu Lieu Pro Asp Glu Ala Lieu. Thir Asn Lieu Val Lieu. 37 O 375 38O Lys Asp Lieu. Arg Glu Thir Met Gly Ile Glu Ala Val Pro Ile Phe Ser 385 390 395 4 OO Glu Ile Thr Arg Lieu. Arg Lys Ser Met Pro Glin Tyr Pro Val Gly His 4 OS 41O 415 Lieu. Glin His Ile Ala Ala Lieu. Arg Glu Glu Lieu. Gly Ser Lys Lieu Pro 42O 425 43 O Gly Val Tyr Ile Ala Gly Ala Gly Tyr Glu Gly Val Gly Lieu Pro Asp 435 44 O 445 Cys Ile Arg Glin Ala Lys Glu Met Ser Val Glin Ala Thr Glin Glu Lieu. 450 45.5 460 Ala Ala Asp 465

<210s, SEQ ID NO 25 &211s LENGTH: 463 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223 OTHER INFORMATION: Recombinant

<4 OOs, SEQUENCE: 25 Lieu Val Ile Ile Gly Gly Gly Ile Thr Gly Lieu Ala Ser Ala Phe Tyr 1. 5 1O 15 Met Glu Lys Glu Ile Arg Glu Lys Asn Lieu Pro Lieu. Ser Val Thir Lieu. 2O 25 3O Val Glu Ala Ser Pro Arg Val Gly Gly Lys Ile Glin Thr Ala Arg Llys 35 4 O 45 Asp Gly Tyr Ile Ile Glu Arg Gly Pro Asp Ser Phe Lieu. Glu Arg Llys SO 55 6 O Llys Ser Ala Pro Glu Lieu Val Glu Asp Lieu. Gly Lieu. Glu. His Lieu. Lieu. 65 70 7s 8O Val Asn. Asn Ala Thr Gly Glin Ser Tyr Val Lieu Val Asn. Glu Thir Lieu 85 90 95 His Pro Met Pro Lys Gly Ala Val Met Gly Ile Pro Thr Lys Ile Ala 1OO 105 11 O Pro Phe Met Ser Thr Arg Lieu Phe Ser Phe Ser Gly Lys Ala Arg Ala 115 12 O 125 Ala Met Asp Phe Val Lieu Pro Ala Ser Llys Pro Llys Glu Asp Glin Ser 13 O 135 14 O

Lieu. Gly Glu Phe Phe Arg Arg Arg Val Gly Asp Glu Val Val Glu Asn 145 150 155 160

Lieu. Ile Glu Pro Lieu Lleu Ser Gly Ile Tyr Ala Gly Asp Ile Asp Arg 1.65 17O 17s

Lieu. Ser Leu Met Ser Thr Phe Pro Glin Phe Tyr Glin Thr Glu Gln Lys 18O 185 19 O His Arg Ser Lieu. Ile Lieu. Gly Met Lys Llys Thr Arg Pro Glin Gly Ser 195 2OO 2O5 US 2017/0058290 A1 Mar. 2, 2017 45

- Continued Gly Glin Glin Lieu. Thir Ala Lys Lys Glin Gly Glin Phe Glin Thr Lieu Lys 21 O 215 22O Thr Gly Lieu. Glin Thr Lieu Val Glu Glu Lieu. Glu Asn Gln Lieu Lys Lieu. 225 23 O 235 24 O Thr Llys Val Tyr Lys Gly Thr Llys Val Thr Asn Ile Ser Arg Gly Glu 245 250 255 Lys Gly Cys Ser Ile Ala Lieu. Asp Asn Gly Met Thr Lieu. Asp Ala Asp 26 O 265 27 O Ala Ala Ile Val Thr Ser Pro His Llys Ser Ala Ala Gly Met Phe Pro 27s 28O 285 Asp Lieu Pro Ala Val Ser Glin Lieu Lys Asp Met His Ser Thir Ser Val 29 O 295 3 OO Ala Asn. Wall Ala Lieu. Gly Phe Pro Glin Glu Ala Val Glin Met Glu. His 3. OS 310 315 32O Glu Gly Thr Gly Phe Val Ile Ser Arg Asn Ser Asp Phe Ser Ile Thr 3.25 330 335 Ala Cys Thir Trp Thr Asn Lys Llys Trp Pro His Ser Ala Pro Glu Gly 34 O 345 35. O Llys Thr Lieu. Lieu. Arg Ala Tyr Val Gly Lys Ala Gly Asp Glu Ser Ile 355 360 365 Val Glu Lieu. Ser Asp Asn. Glu Ile Ile Lys Ile Val Lieu. Glu Asp Lieu 37 O 375 38O Llys Llys Wal Met Lys Ile Lys Gly Glu Pro Glu Met Thr Cys Val Thr 385 390 395 4 OO Arg Trp Asn Glu Ser Met Pro Glin Tyr His Val Gly His Lys Glin Arg 4 OS 41O 415 Ile Llys Llys Val Arg Glu Ala Lieu Ala Ala Ser Tyr Pro Gly Val Tyr 42O 425 43 O Met Thr Gly Ala Ser Phe Glu Gly Val Gly Ile Pro Asp Cys Ile Asp 435 44 O 445 Glin Gly Lys Ser Ala Val Ser Asp Val Lieu Ala Tyr Lieu. Phe Glu 450 45.5 460

<210s, SEQ ID NO 26 &211s LENGTH: 1329 &212s. TYPE: DNA <213> ORGANISM: Xanthomonas campestris

<4 OOs, SEQUENCE: 26 atgcaaacac agc.ccgittat cattgc.cggc gcc.gg tattg ccggact aag tatagcttac 6 O gaattacagc agaaaggc at tcc ct atgaa at catggagg cct Cttic ct a tigcaggaggc 12 O gttgttgaaat cattacat at tatggittat gaactggatg Ctggc cctaa titcgctggCC 18O gCatctgcag catt catggc titat atcgat Caactgggitt to aggacca ggt attggaa 24 O gctg.cggctg C cagtaagaa cc.gctttctg gtcagaaatgataaattgca to agitatcg 3OO

C Cacat coct ttalagatact gcagt cagca tatat cagtg gtggcgc.cala gtggcgtctg 360 ttcacaga aa gattitcgaaa agcggcc.gct C cqgagggag aggaaac agt at Ctt CCttt 42O gtgaccc.gcc gttittggaaa ggagat caat gactaccttt ttgaaccc.gt gctttctggit 48O atatatgcag gtaatcCtga t ctgatgtca gttggtgaag tactgcctat gctgccacala 54 O tgggagcaaa aatacggtag tittacgcag ggacticctga agaataaagg agctatgggt 6OO

US 2017/0058290 A1 Mar. 2, 2017 83

- Continued acatggacca acaagaagtg gcc.gcacagc gct Coggagg ggaaaac act tct Cogagca 108 O tacgtaggca aggc.cgggga cagt caatt gttgagct Ct c cacaatga aat cattaaa 114 O at agttctgg aggat.cttaa galaggtaatg aagataaagg gggaacctga aatgacgtgt 12 OO gttaccc.gct ggaatgagtic aatgc.cccag taccatgtgg gacacaa.gca gaggataaag 126 O alaggtgaggg aggcgctcgc tigcgt.cctac C cagggg.tct acatgacagg agcgagttitt 132O gagggggtgg gtatt cc.cga citgitat.cgac Caggg taagt cqgcagtgtc. tacgtgctic 1380 gcttacct at tccagtag 1398

<210s, SEQ ID NO 76 &211s LENGTH: 181 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 76 Met Lys Thr Lieu. Ile Leu Phe Ser Thr Arg Asp Gly Glin Thr Arg Glu 1. 5 1O 15 Ile Ala Ser Tyr Lieu Ala Ser Glu Lieu Lys Glu Lieu. Gly Ile Glin Ala 2O 25 3O Asp Wall Ala Asn. Wal His Arg Ile Glu Glu Pro Gln Trp Glu Asn Tyr 35 4 O 45 Asp Arg Val Val Ile Gly Ala Ser Ile Arg Tyr Gly. His Tyr His Ser SO 55 6 O Ala Phe Glin Glu Phe Wall Lys Llys His Ala Thr Arg Lieu. Asn. Ser Met 65 70 7s 8O Pro Ser Ala Phe Tyr Ser Val Asn Lieu Val Ala Arg Llys Pro Glu Lys 85 90 95 Arg Thr Pro Gln Thr Asn Ser Tyr Ala Arg Llys Phe Leu Met Asn Ser 1OO 105 11 O Glin Trp Arg Pro Asp Arg Cys Ala Val Ile Ala Gly Ala Lieu. Arg Tyr 115 12 O 125 Pro Arg Tyr Arg Trp Tyr Asp Arg Phe Met Ile Llys Lieu. Ile Met Lys 13 O 135 14 O Met Ser Gly Gly Glu Thr Asp Thr Arg Lys Glu Val Val Tyr Thr Asp 145 150 155 160 Trp Glu Glin Val Ala Asn. Phe Ala Arg Glu Ile Ala His Lieu. Thir Asp 1.65 17O 17s Llys Pro Thir Lieu Lys 18O

<210s, SEQ ID NO 77 &211s LENGTH: 546 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 77 gtgaaaac at taattcttitt citcaacaagg gacggacaaa cdc.gc.gagat togcct cotac 6 O

Ctggct tcgg aactgaaaga actggggat C Caggcggatgtc.gc.caatgt gcaccgcatt 12 O gaagaaccac agtgggaaaa citatgaccgt gtggt cattg gtgcttctat it cqctatggit 18O

Cactaccatt Cagcgttcca gga atttgtc. aaaaaacatg cacgcggct gaatticgatg 24 O cc.gagcgc.ct titt act cogt gaatctggtg gcgc.gcaaac C9gagaa.gcg tactic cacag 3OO

US 2017/0058290 A1 Mar. 2, 2017 85

- Continued

<4 OOs, SEQUENCE: 80 Met Gly Asn. Ile Ser Glu Arg Glu Glu Pro Thir Ser Ala Lys Arg Val 1. 5 1O 15 Ala Val Val Gly Ala Gly Val Ser Gly Lieu Ala Ala Ala Tyr Lys Lieu 2O 25 3O Llys Ser His Gly Lieu. Ser Val Thir Lieu. Phe Glu Ala Asp Ser Arg Ala 35 4 O 45 Gly Gly Lys Lieu Lys Thr Val Lys Lys Asp Gly Phe Ile Trp Asp Glu SO 55 6 O Gly Ala Asn Thr Met Thr Glu Ser Glu Ala Glu Val Ser Ser Lieu. Ile 65 70 7s 8O Asp Asp Lieu. Gly Lieu. Arg Glu Lys Glin Glin Lieu Pro Ile Ser Glin Asn 85 90 95 Lys Arg Tyr Ile Ala Arg Asp Gly Lieu Pro Val Lieu Lleu Pro Ser Asn 1OO 105 11 O Pro Ala Ala Lieu. Lieu. Thir Ser Asn. Ile Lieu. Ser Ala Lys Ser Lys Lieu. 115 12 O 125 Glin Ile Met Lieu. Glu Pro Phe Lieu. Trp Arg Llys His Asn Ala Thr Glu 13 O 135 14 O Lieu. Ser Asp Glu. His Val Glin Glu Ser Val Gly Glu Phe Phe Glu Arg 145 150 155 160 His Phe Gly Lys Glu Phe Val Asp Tyr Val Ile Asp Pro Phe Val Ala 1.65 170 175 Gly Thr Cys Gly Gly Asp Pro Glin Ser Leu Ser Met His His Thr Phe 18O 185 19 O Pro Glu Val Trp Asn Ile Glu Lys Arg Phe Gly Ser Val Phe Ala Gly 195 2OO 2O5 Lieu. Ile Glin Ser Thr Lieu Lleu Ser Lys Lys Glu Lys Gly Gly Glu Asn 21 O 215 22O Ala Ser Ile Llys Llys Pro Arg Val Arg Gly Ser Phe Ser Phe Glin Gly 225 23 O 235 24 O Gly Met Glin Thr Lieu Val Asp Thr Met Cys Lys Glin Lieu. Gly Glu Asp 245 250 255 Glu Lieu Lys Lieu. Glin Cys Glu Val Lieu. Ser Lieu. Ser Tyr Asn Gln Lys 26 O 265 27 O Gly Ile Pro Ser Lieu. Gly Asn Trp Ser Val Ser Ser Met Ser Asn Asn 27s 28O 285 Thir Ser Glu Asp Glin Ser Tyr Asp Ala Val Val Val Thr Ala Pro Ile 29 O 295 3 OO Arg Asn Val Lys Glu Met Lys Ile Met Llys Phe Gly Asn Pro Phe Ser 3. OS 310 315 32O

Lieu. Asp Phe Ile Pro Glu Val Thr Tyr Val Pro Leu Ser Val Met Ile 3.25 330 335

Thir Ala Phe Llys Lys Asp Llys Wall Lys Arg Pro Lieu. Glu Gly Phe Gly 34 O 345 35. O

Val Lieu. Ile Pro Ser Lys Glu Gln His Asn Gly Lieu Lys Thr Lieu. Gly 355 360 365

Thr Lieu Phe Ser Ser Met Met Phe Pro Asp Arg Ala Pro Ser Asp Met 37 O 375 38O

Cys Lieu. Phe Thir Thr Phe Val Gly Gly Ser Arg Asn Arg Llys Lieu Ala 385 390 395 4 OO

US 2017/0058290 A1 Mar. 2, 2017

- Continued ggccataact atgatagogt gctg.cgc.gca attgataaga tiggaaaagga t ctg.ccgggc 1380 ttcttctatg cgggcaiacca taaaggcggc ctgagcgtgg gcaaag.cgat ggcgagcggc 144 O tgcaaag.cgg C9gaactggit gattagct at Ctggatagoc at atttatgt gaaaatggat 15OO gagalagaccg cgtga 1515

What is claimed is: 17. The method of claim 15, wherein the herbicide 1. A recombinant DNA molecule comprising a heterolo tolerance is to at least one PPO herbicide selected from the gous promoter operably linked to a nucleic acid molecule group consisting of acifluorfen, fomesafen, lactofen, fluo encoding a protein that has at least 85% sequence identity to roglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, car amino acid sequence selected from the group consisting of fentraZone-ethyl, SulfentraZone, fluthiacet-methyl, oxadiar SEQ ID NOS:1-2 and SEQ ID NOS:6-12, wherein the gyl, oxadiazon, pyraflufen-ethyl, saflufenacil and S-3100. protein has herbicide-insensitive protoporphyrinogen oxi 18. A method of plant transformation, comprising the dase activity. steps of 2. The recombinant DNA molecule of claim 1, wherein a) introducing the recombinant DNA molecule of claim 1 the nucleic acid molecule comprises a sequence selected into a plant cell; and from the group consisting of SEQ ID NOS:26-27, 31-32, b) regenerating a plant therefrom that comprises the 36-46, and 47-48. recombinant DNA molecule. 3. The recombinant DNA molecule of claim 1, wherein 19. The method of claim 18, further comprising the step the protein comprises a sequence selected from the group of selecting a plant that is tolerant to at least one PPO consisting of SEQ ID NOS:1-2 and SEQ ID NOS:6-12. herbicide. 4. The recombinant DNA molecule of claim 1, wherein 20. The method of claim 18, further comprising the step the heterologous promoter is functional in a plant cell. of crossing the regenerated plant with itself or with a second 5. The recombinant DNA molecule of claim 4, wherein plant and collecting seed from the cross. the nucleic acid molecule is operably linked to a DNA 21. A method for controlling weeds in a plant growth area, molecule encoding a targeting sequence that functions to comprising contacting a plant growth area comprising the localize an operably linked protein within a cell. transgenic plant or seed of claim 11 with at least one PPO 6. A DNA construct comprising the recombinant DNA herbicide, wherein the transgenic plant or seed is tolerant to molecule of claim 1. the PPO herbicide and wherein at least a first weed is 7. The DNA construct of claim 6, wherein the recombi controlled in the plant growth area by the PPO herbicide. nant DNA molecule further comprises an operably linked 22. A method of identifying a nucleotide sequence encod DNA molecule encoding a targeting sequence that functions ing a protein having protoporphyrinogen oxidase activity, to localize the protein within a cell. the method comprising: 8. The DNA construct of claim 7, wherein the protein a) transforming an E. coli strain having a gene knockout confers PPO herbicide tolerance to said cell. for the native E. coli PPO enzyme with a bacterial 9. The DNA construct of claim 6, wherein the DNA expression vector comprising a recombinant DNA mol construct is present in the genome of a transgenic plant, ecule encoding a candidate protein; and seed, or cell. b) growing said transformed E. coli using a heme-free 10. A recombinant polypeptide that comprises at least bacterial medium, wherein growth using said bacterial 85% sequence identity to the full length of a sequence medium identifies a protein having protoporphyrinogen chosen from SEQ ID NOs: 1-2 and SEQ ID NOS:6-12, oxidase activity. wherein the recombinant polypeptide has herbicide-insensi 23. A method of identifying a nucleotide sequence encod tive protoporphyrinogen oxidase activity. ing a protein having herbicide-insensitive protoporphyrino 11. A transgenic plant, seed, cell, or plant part comprising gen oxidase activity, the method comprising: the recombinant DNA molecule of claim 1. a) transforming an E. coli strain having a gene knockout 12. The transgenic plant, seed, cell, or plant part of claim for the native E. coli PPO enzyme with a bacterial 11, wherein the transgenic plant, seed, cell, or plant part expression vector comprising a recombinant DNA mol comprises an additional transgenic herbicide tolerance trait. ecule encoding a recombinant protein; and 13. The transgenic plant, seed, cell, or plant part of claim b) growing said transformed E. coli using a bacterial 11, defined as comprising herbicide tolerance to at least one medium containing at least one PPO herbicide, wherein PPO herbicide. growth of bacteria identifies a protein having herbicide 14. A transgenic plant, seed, cell, or plant part comprising insensitive protoporphyrinogen oxidase activity. the recombinant polypeptide of claim 10. 24. A method of Screening for a herbicide tolerance gene 15. A method for conferring PPO herbicide tolerance to a comprising: plant, seed, cell, or plant part comprising: heterologously a) expressing the recombinant DNA molecule of claim 1 expressing in said plant, seed, cell, or plant part the recom in a plant cell; and binant polypeptide of claim 10. b) identifying a plant cell that displays tolerance to a PPO 16. The method of claim 15, wherein said plant, seed, cell, herbicide. or plant part comprises protoporphyrinogen oxidase activity 25. A method of screening for a herbicide tolerance gene conferred by the recombinant polypeptide. comprising: US 2017/0058290 A1 Mar. 2, 2017

a) expressing a recombinant DNA molecule of claim 1 in safen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioX a bacterial cell lacking HemG, wherein the bacterial azin, azafenidin, carfentraZone-ethyl, SulfentraZone, fluthi cell is grown in a heme-free medium in the presence of acet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, a PPO herbicide; and saflufenacil and S-3100. b) identifying a bacterial cell that displays tolerance to a 29. The method of claim 27, wherein the at least one other PPO herbicide. herbicide is selected from the group consisting of an 26. A method of producing a plant tolerant to a PPO ACCase inhibitor, an ALS inhibitor, an EPSPS inhibitor, a herbicide and at least one other herbicide comprising: synthetic auxin, a photosynthesis inhibitor, a glutamine a) obtaining a plant according to claim 11; and synthesis inhibitor, a HPPD inhibitor, a PPO inhibitor, and b) crossing the transgenic plant with a second plant a long-chain fatty acid inhibitor. comprising tolerance to the at least one other herbicide, and 30. The method of claim 29, wherein the ACCase inhibi c) selecting a progeny plant resulting from said crossing tor is an aryloxyphenoxy propionate or a cyclohexanedione; that comprises tolerance to a PPO herbicide and the at the ALS inhibitor is a sulfonylurea, imidazolinone, triaz least one other herbicide. oloyrimidine, or a triazolinone; the EPSPS inhibitor is 27. A method for reducing the development of herbicide glyphosate; the synthetic auxin is a phenoxy herbicide, a tolerant weeds comprising: benzoic acid, a carboxylic acid, or a semicarbazone; the a) cultivating in a crop growing environment a plant photosynthesis inhibitor is a triazine, a triazinone, a nitrile, according to claim 12; and a benzothiadiazole, or a urea; the glutamine synthesis inhibi b) applying a PPO herbicide and at least one other tor is glufosinate; the HPPD inhibitor is an isoxazole, a herbicide to the crop growing environment, wherein the pyrazolone, or a triketone; the PPO inhibitor is a dipheny crop plant is tolerant to the PPO herbicide and the at lether, a N-phenylphthalimide, an aryl triazinone, or a least one other herbicide. pyrimidinedione; or the long-chain fatty acid inhibitor is a 28. The method of claim 27, wherein the PPO herbicide chloroacetamide, an oxyacetamide, or a pyrazole. is selected from the group consisting of acifluorfen, fome k k k k k