US009045789B2

(12) United States Patent (10) Patent No.: US 9,045,789 B2 Nishi0 et al. (45) Date of Patent: Jun. 2, 2015

(54) METHOD FOR PRODUCING ATARGET CI2Y 102/01026 (2013.01); C12Y-301/01068 SUBSTANCE BY FERMENTATION (2013.01); C12Y402/01082 (2013.01); CI2P 7/58 (2013.01); C12P 13/001 (2013.01); (71) Applicant: AJINOMOTO CO., INC., Tokyo (JP) (Continued) (72) Inventors: Yousuke Nishio, Kanagawa (JP); Youko (58) Field of Classification Search Yamamoto, Kanagawa (JP); Kazuteru None Yamada, Kanagawa (JP); Kosuke See application file for complete search history. Yokota, Kanagawa (JP) (56) References Cited (73) Assignee: AJINOMOTO CO., INC., Tokyo (JP) U.S. PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this 5,977,331 A 11/1999 Asakura et al. patent is extended or adjusted under 35 6,197.559 B1 3/2001 Moriya et al. U.S.C. 154(b) by 0 days. (Continued) (21) Appl. No.: 13/914,872 FOREIGN PATENT DOCUMENTS

(22) Filed: Jun. 11, 2013 EP 1577396 9, 2005 JP 2005-261433 9, 2005 (65) Prior Publication Data OTHER PUBLICATIONS US 2013/O295621 A1 Nov. 7, 2013 Weimberg, “Pentose oxidation by Pseudomonas fragi'. Journal of Related U.S. Application Data Biological Chemistry, vol. 236, No. 3, pp. 629-635, 1961.* (63) Continuation of application No. PCT/JP2012/078725, (Continued) filed on Nov. 6, 2012. (60) Provisional application No. 61/558,685, filed on Nov. Primary Examiner — Rebecca Prouty 11, 2011. Assistant Examiner — Richard Ekstrom (74) Attorney, Agent, or Firm — Shelly Guest Cermak; (30) Foreign Application Priority Data Cermak Nakajima & McGowan LLP Nov. 11, 2011 (JP) ...... 2011-247031 (57) ABSTRACT A target Substance can be produced by culturing a bacterium (51) Int. C. having an ability to produce 2-ketoglutaric acidora derivative CI2PI3/4 (2006.01) thereof, and an ability to produce Xylonic acid from Xylose, CI2P 7/50 (2006.01) which is imparted with xylonate dehydratase activity, 2-keto (Continued) 3-deoxyXylonate dehydratase activity and 2-ketoglutaric semialdehyde dehydrogenase activity, or in which these (52) U.S. C. activities are enhanced, in a medium containing Xylose as a CPC, C12P 13/14 (2013.01); C12P 7/50 (2013.01); carbon Source to produce and accumulate the target Substance CI2N 15/52 (2013.01); C12N 9/0006 in the medium, and collecting the target Substance from the (2013.01); C12N 9/0008 (2013.01); C12N 9/16 medium. (2013.01); C12Y 103/05001 (2013.01); CI2N 9/88 (2013.01); CI2Y 101/01 113 (2013.01); 6 Claims, 4 Drawing Sheets

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(51) Int. Cl. Hartman, A. L., et al., “The Complete Genome Sequence of Haloferax volcanii DS2, a Model Archaeon. PLoS ONE CI2N 15/52 (2006.01) 2010:5(3),e9605:1-20. CI2N 9/04 (2006.01) Hosoya, S., et al., “Identification and characterization of the Bacillus CI2N 9/02 (2006.01) subtilis D-glucarate?galactarate utilization operon yebCDEFGHJ.” CI2N 9/16 (2006.01) FEMS Microbiol. Lett. 2002:210:193-199. Johnsen, U., et al., “Novel Xylose Dehydrogenase in the Halophilic CI2N 9/88 (2006.01) Archaeon Haloarcula marismortui.’ J. Bacteriol. CI2P 7/58 (2006.01) 2004; 186(18):6198-6207. CI2PI3/00 (2006.01) Johnsen, U., et al., “D-Xylose Degradation Pathway in the Halophilic CI2PI3/04 (2006.01) Archaeon Haloferax volcanii.” J. Biol. Chem. 2009:284(40):27290 CI2PI3/10 (2006.01) 273O3. Kawaguchi, H., et al., “Engineering of a Xylose Metabolic Pathway CI2PI3/24 (2006.01) in Corynebacterium glutamicum.” Appl. Environmen. Microbiol. (52) U.S. Cl. 2006:72(5):3418-3428. CPC ...... CI2PI3/04 (2013.01): CI2P 13/10 Meijnen, J.-P. et al., “Establishment of Oxidative D-Xylose Metabo (2013.01); CI2P 13/24 (2013.01) lism in Pseudomonas putida S12.” Appl. Environmen. Microbiol. 2009;75(9):2784-2791. (56) References Cited Meisenzahl. A. C., et al., “Isolation and Characterization of a Xylose Dependent Promoter from Caulobacter crescentus.” J. Bacteriol. U.S. PATENT DOCUMENTS 1997; 179(3):592-600. Nichols, N. N., et al., “Use of catabolite repression mutants for 6,267,309 B1 7/2001 Chieffalo et al. fermentation of Sugar mixtures to ethanol. Appl. Microbiol. 6,331419 B1 12/2001 Moriya et al. Biotechnol. 2001:56:120-125. 6,682,912 B2 1/2004 Moriya et al. Nierman, W. C., et al., “Complete genome sequence of Caulobacter 6,962,805 B2 11/2005 Asakura et al. crescentus.” PNAS 2001:98(7):4136-4141. 7,037,690 B2 5, 2006 Hara et al. Nygård, Y, et al., “Bioconversion of D-xylose to D-xylonate with 7,090,998 B2 8/2006 Ishikawa et al. Kluyveromyces lactis.” Metabolic Engineering 2011:13:383-391. 7,205,132 B2 4/2007 Hirano et al. Sasaki, M., et al., “Engineering of pentose transport in 7,244,581 B2 * 7/2007 Sode ...... 435/14 Corynebacterium glutamicum to improve simultaneous utilization of 7,344,874 B2 3/2008 Hara et al. mixed sugars.” Appl. Microbiol. Biotechnol. 2009:85:105-115. 7,695,946 B2 4/2010 USuda et al. Song, S., et al., “Organization and Regulation of the D-Xylose 7,696,315 B2 4/2010 USuda et al. Operons in Escherichia coli K-12: XylR Acts as a Transcriptional 7,785,845 B2 * 8/2010 Hara et al...... 435/110 Activator.” J. Bacteriol. 1997; 179(22):7025-7032. 7,785,858 B2 8, 2010 Kozlov et al. Stephens, C., et al., “Regulation of D-Xylose Metabolism in 7,794,989 B2 9/2010 Nakamura et al. Caulobacter crescentus by a Lacl-Type Repressor.” J. Bacteriol. 7,915,018 B2 3/2011 Rybak et al. 2007; 189(24):8828-8834. 7.923,226 B2 * 4/2011 Frost ...... 435,158 Tao, H., et al., “Engineering a Homo-Ethanol Pathway in Escherichia 7,927,844 B2 4/2011 Nakamura et al. coli: Increased Glycolytic Flux and Levels of Expression of RE42,350 E 5, 2011 IZuiet al. Glycolytic Genes during Xylose Fermentation.” J. Bacteriol. 8,003,367 B2 8/2011 Marchenko et al. 2001; 183(10):2979-2988. 8,012,722 B2 9, 2011 Chinen et al. Toivari, M. H., et al., “Microbial D-xylonate production.” Appl. 8,058,035 B2 11/2011 Hara et al. Microbiol. Biotechnol. 2012.96:1-8. 8,129,151 B2 3/2012 Moriya et al. Toivari, M. H., et al., "Saccharomyces cerevisiae engineered to pro 8, 192,963 B2 6, 2012 Nishio et al. duce D-xylonate.” Appl. Microbiol. Biotechnol. 2010;88:751-760. 8,206,954 B2 6/2012 Takikawa et al. Watanabe, S., et al., “Enzyme Catalysis and Regulation: 8,222,007 B2 7, 2012 Hara et al. O-Ketoglutaric Semialdehyde Dehydrogenase Isozymes Involved in 8,278,074 B2 10/2012 Nakamura et al. Metabolic Pathways of D-Glucarate, D-Galactarate, and Hydroxy 2005/0214913 A1 9, 2005 Marchenko et al. L-proline: Molecular and Metabolic Convergent Evolution.” J. Biol. 2005/0233308 A1 10, 2005 Nishio et al. Chem. 2007:282:6685-6695. 2007,0004014 A1 1/2007 Tsuji et al. Thanbichler, M., et al., “A comprehensive set of plasmids for vanil 2009/0286290 A1* 11/2009 Hara et al...... 435/107 late- and xylose-inducible gene expression in Caulobacter 2010, O190217 A1 7, 2010 Doi et al. crescentus.” Nucl. Acids Res. 2007:35(20),e 137: 1-16. 2011/0076730 A1 3f2011 Frost et al...... 435/106 Aghaie, A., et al., “Metabolism and Bioenergetics: New Insights into 2012/0129233 A1 5/2012 Tajima et al. the Alternative d-Glucarate Degradation Pathway,” J. Biol. Chem. 2013/0217078 A1* 8/2013 Tang et al...... 435/99 2008:283(23): 15638-15646. 2013/0260423 A1* 10, 2013 Knudsen et al...... 435/99 Watanabe, S., et al., “Enzyme Catalysis and Regulation: Identifica tion and Characterization of I-Arabonate Dehydratase, I-2-Keto-3- OTHER PUBLICATIONS deoxyarabonate Dehydratase, and I-Arabinolactonase Involved in an Alternative Pathway of I-Arabinose Metabolism: Novel Evolution Berghall, S., et al., “Identification in the mould Hypocreajecorina of ary Insight Into Sugar Metabolism.” J. Biol. Chem. a gene encoding an NADP+:D-xylose dehydrogenase.” FEMS 2006:281 (44):33521-33536. Microbiol. Lett., 2007:277:249-253. Gopinath, V., et al., “Amino acid production from rice Straw and Brouns, S. J. J., et al., “Identification of the Missing Links in wheat bran hydrolysates by recombinant pentose-utilizing Prokaryotic Pentose Oxidation Pathways: Evidence for Enzyme Corynebacterium glutamicum.” Appl. Microbiol. Biotechnol. Recruitment. J. Bio. Chem. 2006:281:27378-27388. 2011;92:985-996. Dahms, A. S., et al., “D-xylose Dehydrogenase.” Methods in Enzy Liu, H., et al., “High yield production of D-xylonic acid from mology 1982;89:226-228. D-xylose using engineered Escherichia coli,” Bioresource Technol. Ely, B., “Genetics of Caulobacter crescentus.” Methods in Enzymol 2012; 115:244-248. ogy 1991:204:372-384. Stephens, C., et al., “Genetic Analysis of a Novel Pathway for Fernandes, S., et al., “Xylose reductase from the thermophilic fungus D-Xylose Metabolism in Caulobacter crescentus.” J. Bacteriol. Talaromyces emersonii: cloning and heterologous expression of the 2007; 189(5): 218 1-2185. native gene (Texr) and a double mutant (TexrK271 R+N273DI) with International Search Report for PCT Patent App. No. PCT/JP2012/ altered coenzyme specificity.” J. Biosci. 2009:34(6):881-890. 078725 (Jan. 22, 2013). Gonzalez, R., et al., “Global Gene Expression Differences Associ International Preliminary Report on Patentability for PCT Patent ated with Changes in Glycolytic Flux and Growth Rate in App. No. PCT/JP2012/078725 (May 22, 2014). Escherichia coli during the Fermentation of Glucose and Xylose.” Biotechnol. Prog. 2002:18:6-20. * cited by examiner U.S. Patent Jun. 2, 2015 Sheet 1 of 4 US 9,045,789 B2

Fig. 1

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100 O 20 40 60 80 100 U.S. Patent Jun. 2, 2015 Sheet 2 of 4 US 9,045,789 B2

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I/5 U.S. Patent Jun. 2, 2015 Sheet 3 of 4 US 9,045,789 B2

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U.S. Patent Jun. 2, 2015 Sheet 4 of 4 US 9,045,789 B2

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st 20 a ctss 15 10 5 O control corXylA YCbD XylA(Hbo) XylA(Abr) US 9,045,789 B2 1. 2 METHOD FOR PRODUCING ATARGET tose, Sucrose, blackstrap molasses, starch hydrolysate, and so SUBSTANCE BY FERMENTATION forth as a carbon Source, but they are relatively expensive, and use of biomass raw materials derived from plants and the like This application is a Continuation of, and claims priority has also advanced in recent years. under 35 U.S.C. S 120 to, International Application No. PCT/ Although raw materials including edible portions such as JP2012/078725, filed Nov. 6, 2012, and claims priority there starch and fats and oils are mainly used as Such biomass raw through under 35 U.S.C. S 119 to Japanese Patent Application materials at present, it is necessary to shift Such biomass raw No. 2011-247031, filed Nov. 11, 2011, and U.S. Provisional materials to those which include non-edible portions, specifi Patent Application No. 61/558,685, filed Nov. 11, 2011, the cally, cellulose, hemicellulose, lignin, and so forth in the entireties of which are incorporated by reference herein. Also, 10 the Sequence Listing filed electronically herewith is hereby future. Non-edible biomass such as cellulose and hemicellu incorporated by reference (File name: 2013-06-11T US lose are converted into pentoses or hexoses via a pretreatment 471 Seq List: File size: 263 KB: Date recorded: Jun. 11, using heat or acid, and a saccharification treatment using a 2013). cellulase enzyme, and then they can be used as raw materials 15 in fermentation (Japanese Patent Laid-open based on PCT BACKGROUND OF THE INVENTION application (Kohyo) No. 9-507386 and Japanese Patent Laid open based on PCT application No. 11-506934). If mixed 1. Field of the Invention saccharides of pentoses or hexoses are used as the raw mate The present invention relates to a method for producing a rials for amino acid fermentation etc., Escherichia coli pref target Substance such as L-amino acids by fermentation using erentially assimilates glucose, and as a result, the phenomena a microorganism. More precisely, the method for producing a of two-step proliferation (diauxy), delayed growth etc. have target Substance by fermentation uses Xylose as a raw mate been observed (Nichols N. N. et al., Appl. Microbiol. Bio rial. technol., 2001 July, 56(1-2): 120-125 and Gonzalez, R., Bio 2. Brief Description of the Related Art technol. Prog., 2002 January-February, 18(1):6-20) Methods for producing target Substances such as L-amino 25 In Escherichia coli, a xylose assimilation pathway utilizing acids by fermentation using a bacterium include methods of Xylose isomerase encoded by the XylA gene and Xylulokinase using a wild-type bacterium (wild-type strain), methods of encoded by the XylB gene is known, and it is also known that using an auxotrophic strain derived from a wild-type strain, L-amino acids can be produced from Xylose by introducing methods of using a metabolic regulation mutant strain derived that pathway into Escherichia coli or Corynebacterium from a wild-type strain which is resistant to various drugs, 30 glutamicum (Tao H. et al., J. Bacteriol., 2001 May, 183 (10): methods of using a strain having properties of both aux 2979-2988, European Patent No. 1577396, Gopinath, V. et otrophic strain and metabolic regulation mutant, and so forth. al., Appl. Microbiol. Biotechnol., 2011 Jul., 28). For example, L-glutamic acid is mainly produced by fer It has also been reported that Caulobacter Crescentus and mentation using an L-glutamic acid-producing bacterium of Haloferax volcanii utilize a pathway of converting Xylose into the so-called coryneform bacteria belonging to the genus 35 2-ketoglutaric acid via Xylonic acid in five steps, not using the Brevibacterium, Corynebacterium or Microbacterium or a conventionally known pathway as described above mutant strain thereof (refer to, for example, Akashi K. et al., (Stephens, C. et al., J. Bacteriol., 2007 March, 189 (5):2181 Amino Acid Fermentation, Japan Scientific Societies Press, 2185). Moreover, examples of expression of that pathway in pp. 195-215, 1986). As methods for producing L-glutamic Escherichia coli are also known (Huaiwei, Let al., Bioresour acid by using other strains, methods utilizing a microorgan 40 Technol., 2011 Aug. 22, U.S. Pat. No. 7.923.226). ism belonging to the genus Bacillus, Streptomyces, Penicil lium, or the like (refer to, for example, U.S. Pat. No. 3,220. SUMMARY OF THE INVENTION 929), methods utilizing a microorganism belonging to the genus Pseudomonas, Arthrobacter, Serratia, Candida, or the Aspects of the Invention like (refer to, for example, U.S. Pat. No. 3,563,857), methods 45 utilizing a microorganism belonging to the genus Bacillus, or An aspect of the present invention is to provide a microor Aerobacter aerogenes (currently Enterobacter aerogenes), or ganism that can efficiently produce a target Substance such as the like (refer to, for example, Japanese Patent Publication L-glutamic acid in a medium containing Xylose, and a method (Kokoku) No. 32-9393), methods utilizing a mutant strain of for producing a target Substance using such a microorganism. Escherichia coli (refer to, for example, Japanese Patent Laid 50 open (Kokai) No. 5-244970), and so forth are known. Fur Means for Achieving the Aspects thermore, methods of producing L-glutamic acid using a microorganism belonging to the genus Klebsiella, Erwinia, The development of a microorganism by utilizing the path Pantoea, or Enterobacter (refer to, e.g., Japanese Patent Laid way of converting Xylose into 2-ketoglutaric acid via Xylonic openNo. 2000-106869, Japanese Patent Laid-openNo. 2000 55 acid for the purpose of developing an amino acid-producing 1891.69 and Japanese Patent Laid-open No. 2000-189175) bacterium having a pentose- or hexose-assimilating ability by have also been disclosed. breeding is described. As a result, a microorganism express In recent years, recombinant DNA techniques have been ing such a pathway as described above can efficiently assimi used in the production of target Substances by fermentation. late xylose. For example, L-amino acid productivity of a bacterium is 60 It is an aspect of the present invention to provide a method improved by enhancing expression of a gene encoding an for producing a target Substance comprising culturing a bac L-amino acid biosynthetic enzyme (U.S. Pat. No. 5,168,056 terium having an ability to produce the target Substance in a and U.S. Pat. No. 5,776.736), or by enhancing uptake of a medium containing Xylose so that the target Substance accu carbon Source into the L-amino acid biosynthesis system mulates in the medium, and collecting the target Substance (U.S. Pat. No. 5,906,925). 65 from the medium, wherein: Conventional industrial production of substances by fer the target Substance is 2-ketoglutaric acid or a derivative mentation typically employ saccharides, i.e., glucose, fruc thereof, US 9,045,789 B2 3 4 the bacterium has an ability to produce xylonic acid from It is a further aspect of the present invention to provide the Xylose, and activities of the enzymes Xylonate dehydratase, method as described above, wherein the bacterium is Coryne 2-keto-3-deoxyXylonate dehydratase and 2-ketoglutaric bacterium glutamicum. semialdehyde dehydrogenase have been imparted to or It is a further aspect of the present invention to provide the enhanced in the bacterium. 5 method as described above, wherein the 2-ketoglutaric acid It is a further aspect of the present invention to provide the derivative is a Substance selected from the group consisting of method as described above, wherein said activities are L-glutamic acid, L-glutamine, L-arginine, L-citrulline, L-or imparted to or enhanced in the bacterium by introducing nithine, L-proline, putrescine, and Y-aminobutyric acid. expressible forms of genes coding for the enzymes into the bacterium. 10 BRIEF DESCRIPTION OF THE DRAWINGS It is a further aspect of the present invention to provide the method as described above, FIG. 1 depicts graphs showing results of a growth comple wherein the genes are derived from, or native to, a micro mentation test for C. crescentus-derived NXA operon-ex organism belonging to a genus selected from the group con 15 pressing strain using an iccd gene-deficient strain. El-O.KG, sisting of Caulobacter; Escherichia, Agrobacterium, M9-O.KG, M9-Xyl, and E1-Xyl represent M9 minimal Herbaspirillum, Actinoplanes, Cupriavidus, Pseudomonas, medium or El Synthetic medium containing 2-ketoglutaric Zobellia, Thermobacillus, Arthrobacter, Azospirillum, acid or xylose as the Sole carbon Source, respectively. Halomonas, Bacillus, and Aspergillus. FIG. 2 depicts graphs showing results of L-glutamic acid It is a further aspect of the present invention to provide the production culture of E. coli L-glutamic acid-producing method as described above, wherein the bacterium can pro strain expressing the E. coli ccrNXA operon. duce Xylonic acid from Xylose because of any one of the FIG. 3 depicts graphs showing results of L-glutamic acid following characteristics: production culture of a ccrNXA operon-expressing strain (A) Xylose dehydrogenase activity, or xylose dehydroge using a strain deficient in the Xylose assimilation pathway nase activity and Xylonolactonase activity have been 25 characteristic to E. coli as a host. imparted to or enhanced in the bacterium, or FIG. 4 depicts graphs showing results of L-glutamic acid (B) the bacterium has glucose dehydrogenase activity that production culture of a ccrNXA operon-expressing strain can catalyze a reaction producing Xylonic acid from Xylose. utilizing a medium copy number type plasmid. It is a further aspect of the present invention to provide the FIG. 5 depicts graphs showing results of L-glutamic acid method as described above, wherein the glucose dehydroge 30 production culture of XylD homologue gene-expressing nase uses pyrroloquinoline quinone as a coenzyme, and the strains derived from various kinds of microorganisms. Atu, bacterium has glucose dehydrogenase activity because it has Hse, Amis, and Aor represent Agrobacterium tumefaciens, pyrroloquinoline quinone-producing ability, or it is cultured Herbaspirillum seropedicae, Actinoplanes missouriensis, in a medium containing pyrroloquinoline quinone. and Aspergillus Oryzae, respectively. 35 FIG. 6 depicts graphs showing results of L-glutamic acid It is a further aspect of the present invention to provide the production culture of XylX homologue-expressing strains method as described above, wherein the bacterium can pro derived from various kinds of microorganisms. Art, Atu, Cne, duce Xylonic acid from Xylose because a gene coding for Zga, Tco, and Selo represent Arthrobacter globiformis, Agro Xylose dehydrogenase, or expressible forms of genes coding bacterium tumefaciens, Cupriavidus necator; Zobellia galac for Xylose dehydrogenase and Xylonolactonase have been 40 tanivorans, Thermobacillus composti, and Pseudomonas elo introduced in to said bacterium. dea, respectively. It is a further aspect of the present invention to provide the FIG. 7 depicts graphs showing results of L-glutamic acid method as described above, wherein the bacterium has been production culture of XylA homologue-expressing strains modified so that activity of 2-ketoglutarate dehydrogenase is derived from various kinds of microorganisms. Hbo and Abr reduced. 45 represent Halomonas boliviensis and Azospirillum It is a further aspect of the present invention to provide the brasilense, respectively. method as described above, wherein the bacterium has been further modified so that activity of Succinate dehydrogenase DESCRIPTION OF THE PREFERRED is reduced. EMBODIMENTS It is a further aspect of the present invention to provide the 50 method as described above, wherein the bacterium is an The method in accordance with the presently described enterobacterium or a coryneform bacterium. Subject matter can be a method for producing a target Sub It is a further aspect of the present invention to provide the stance by culturing a bacterium having an ability to produce method as described above, wherein the bacterium is a bac the target Substance in a medium containing Xylose as a terium belonging to the genus Pantoea. 55 carbon Source to produce and accumulate the target Substance It is a further aspect of the present invention to provide the in the medium, and collecting the target Substance from the method as described above, wherein the bacterium is Pantoea medium, wherein: ananatis. the target Substance is 2-ketoglutaric acid or a derivative It is a further aspect of the present invention to provide the thereof, and method as described above, wherein the bacterium is a bac 60 the bacterium has an ability to produce xylonic acid from terium belonging to the genus Escherichia. Xylose, and activities of the enzymes Xylonate dehydratase, It is a further aspect of the present invention to provide the 2-keto-3-deoxyXylonate dehydratase and 2-ketoglutaric method as described above, wherein the bacterium is Escheri semialdehyde dehydrogenase have been imparted to or chia coli. enhanced in the bacterium. It is a further aspect of the present invention to provide the 65 The target Substance can be 2-ketoglutaric acid (O-keto method as described above, wherein the bacterium is a bac glutaric acid, C.KG) or a derivative thereof. Examples of the terium belonging to the genus Corynebacterium. derivative of 2-ketoglutaric acid can include L-glutamic acid, US 9,045,789 B2 5 6 L-glutamine, L-arginine, L-citrulline, L-ornithine, L-proline, aforementioned enzymes. Furthermore, these enzymatic putrescine, and Y-aminobutyric acid. activities can also be determined by measuring Xylonic acid The “ability to produce a target Substance' can mean an produced from Xylose. ability of the bacterium to produce a target Substance to Such Xylonate dehydratase is an enzyme that reversibly cata an extent that the target Substance can be collected from cells 5 lyzes the following reaction (EC4.2.1.82), and can also be or medium, when it is cultured in the medium, and/or an called D-xylo-aldonate dehydratase, D-xylonate dehy ability to produce the target Substance in a larger amount as dratase, or D-Xylonate hydro-lyase. compared to that obtainable with a wild-type strain or a non D-Xylonic acid->2-dehydro-3-deoxy-D-xylonate-- modified strain cultured under the same conditions. The bac HO 10 terium may have an ability to produce two or more kinds of The xylonate dehydratase activity can be measured by, for target Substances. example, mixing a D-Xylonic acid solution and a test sample The target Substance can include a compound in a free form to allow the reaction, then terminating the reaction with addi and/or a salt thereof, for example, sulfate, hydrochloride, tion of a stop solution which includes 1% aqueous solution of carbonate, ammonium salt, sodium salt, potassium salt, and 15 semicarbazidehydrochloride and a 1.5% aqueous solution of so forth. Sodium acetate, and measuring the absorbance of the diluted <1> Bacterium reaction solution at 250 nm (Dahms, A. S., et al., Methods The bacterium in accordance with the presently described Enzymol., 1982, 90 Pt E:302-5). Subject matter can be a bacterium having an ability to produce 2-keto-3-deoxyXylonate dehydratase (2-keto-3-deoxy-Xy xylonic acid from xylose, and in which the activities of lonate dehydratase) is an enzyme that can reversibly catalyze Xylonate dehydratase, 2-keto-3-deoxyXylonate dehydratase, the following reaction (EC4.2.1-). and 2-ketoglutaric semialdehyde dehydrogenase have been 2-Dehydro-3-deoxy-D-xylonate->2-oxoglutaric semi imparted, or in which these activities have been enhanced. aldehyde--H2O The bacterium can be a bacterium that does not inherently The 2-keto-3-deoxyxylonate dehydratase activity can be have the activities of xylonate dehydratase, 2-keto-3-deox 25 measured by, for example, mixing a solution of 2-keto-3- yXylonate dehydratase, and 2-ketoglutaric semialdehyde deoxyxylonic acid as the Substrate and a test sample to allow dehydrogenase, but in which these enzymatic activities can be the reaction, and then measuring the decrease of 2-keto-3- imparted, or it can be a bacterium which inherently has these deoxyxylonic acid. enzymatic activities and in which these enzymatic activities 2-ketoglutaric semialdehyde dehydrogenase is an oxi can be enhanced. 30 The ability to produce xylonic acid from Xylose can be doreductase that can reversibly catalyze the following reac attained by, for example, one or both of tion (EC1.2.1.26). 1) impartation or enhancement of Xylose dehydrogenase 2-Oxoglutaric semialdehyde--NAD(P)->2-oxoglutaric activity, and acid--NAD(P)H 2) possession of glucose dehydrogenase activity that can cata 35 The 2-ketoglutaric semialdehyde dehydrogenase activity lyze the reaction producing Xylonic acid from Xylose. can be measured by, for example, measuring reduction of Examples of a microorganism to which Xylose dehydroge NAD(P). For example, the activity of this enzyme can be nase activity can be imparted or enhanced can include measured by adding 2-ketoglutaric semialdehyde to a mix Escherichia bacteria and coryneform bacteria, and examples ture of pyrophosphoric acid (pH 8.5), NAD(P), and a test of a microorganism that has glucose dehydrogenase include 40 sample, and measuring the absorbance of the reaction mixture Pantoea bacteria, and so forth. at 340 nm (Adams, E., et al., J. Biol. Chem., 1967, 242, In addition to Xylose dehydrogenase activity, Xylonolacto 1802-1814). nase activity may also be enhanced. Xylose dehydrogenase (D-Xylose-1-dehydrogenase) is a The bacteria belonging to these genera will be explained dismutase for a pentose and glucuronic acid, and is an oxi later. 45 doreductase that can reversibly catalyze the following reac Xylonic acid produced from Xylose is converted into 2-ke toglutaric acid by Xylonate dehydratase, 2-keto-3-deoxyxy tion (EC1.1.1.175). lonate dehydratase, and 2-ketoglutaric semialdehyde dehy D-Xylose--NAD(P)->D-xylonolactone--NAD(P)H- drogenase. The pathway in which Xylose is converted into H 2-ketoglutaric acid by Xylonate dehydratase, 2-keto-3-deox 50 The D-xylose-1-dehydrogenase activity can be measured yXylonate dehydratase, and 2-ketoglutaric semialdehyde by, for example, mixing Xylose, a test sample, and NAD(P) to dehydrogenase is also called the Weimberg pathway (J. Biol. allow the reaction, and measuring absorbance of the reaction Chem. 236:629-636). The Weimberg pathway and the path mixture at 340 nm (Stephens, C. et al., J. Bacteriol., 2007, way in which Xylose is converted into Xylonic acid by Xylose 189(5):181-2185). dehydrogenase and/or Xylonolactonase may be collectively 55 The Xylose dehydrogenase of Caulobacter Crescentus can referred to as the NXA (Novel Xylose Assimilation) pathway. catalyze the reaction which converts D-xylose into Xylonic Whether a bacterium has the Weimberg pathway, or this acid. pathway has been introduced into a bacterium can be deter Xylonolactonase is an enzyme that reversibly catalyzes the mined by measuring enzymatic activities of Xylonate dehy following reaction (EC3.1.1.68). dratase, 2-keto-3-deoxyXylonate dehydratase, and 2-ketoglu 60 taric semialdehyde dehydrogenase in an extract of the D-Xylonolactone->D-xylonic acid bacterium, or confirming assimilation of Xylonic acid, which The Xylonolactonase activity can be measured by, for accumulates in a strain not having the Weimberg pathway. example, mixing Xylonolactone and a test sample to allow the Furthermore, whether a bacterium has the NXA pathway or reaction, and quantifying the remaining Xylonolactone this pathway has been introduced into a bacterium can be 65 according to the hydroxamate method (Appl. Microbiol. Bio determined by measuring enzymatic activities of xylose technol., 29:375-379, 1988: Appl. Microbiol. Biotechnol., dehydrogenase and/or Xylonolactonase in addition to the 27:333-336, 1988). US 9,045,789 B2 7 8 The genes coding for the enzymes Xylonate dehydratase, Furthermore, as for 2-keto-deoxyxylonate dehydratase, 2-keto-3-deoxyXylonate dehydratase, and 2-ketoglutaric XylX gene homologues of bacteria belonging to the genus semialdehyde dehydrogenase can be derived from, or native Agrobacterium, Pseudomonas, Zobellia, Thermobacillus, or to, any microorganism having the Weimberg pathway, and Arthrobacter, such as Agrobacterium tumefaciens, Cupriavi examples include, for example, genes derived from, or native dus necator, Pseudomonas elodea, Zobellia galactanivorans, to, a microorganim Such as a bacterium belonging to the Thermobacillus composti, and Arthrobacter globiformis, genus Caulobacter; Escherichia, Agrobacterium, Herbaspir may also be used. illum, Actinoplanes, Cupriavidus, Pseudomonas, Zobellia, Furthermore, as for 2-ketoglutaric semialdehyde dehydro Thermobacillus, Arthrobacter, Azospirillum, Halomonas, genase, genes of bacteria belonging to the genus Azospiril Bacillus, or a filamentous fungus belonging to the genus 10 lum, Halomonas, or Bacillus, such as XylA gene homologues Aspergillus. of Azospirillum brasilense and Halomonas boliviensis, and An example of the Caulobacter bacteria can include Cau ycbD of Bacillus subtilis, may also be used. lobacter crescentus. The nucleotide sequences of the aforementioned genes and As Caulobacter Crescentus, the CB-15 strain and the amino acid sequences encoded by them are shown in Table CB-13 strain are known, and are stored at the AmericanType 15 11. Culture Collection (Address: P.O. Box 1549, Manassas, Va. In Caulobacter crescentus, the genes of the five enzymes of 20108, United States of America) as ATCC 19089 and ATCC the NXA pathway constitute an operon structure as described 33532, respectively. Furthermore, the NA-1000 strain (J. later. The nucleotide sequence of this operon is registered at Bacteriol., 192:3678-88, 2010) and the K31 strain can also be GenBank as Accession No. AAK22808 Caulobacter Cres used. centus. The nucleotide sequence of this operon is shown in The genome sequences of the Caulobacter Crescentus SEQ ID NO. 23. The amino acid sequences of 2-keto-3- CB15, NA1000, and K31 strains are registered as GenBank deoxyxylonate dehydratase, 2-ketoglutaric semialdehyde Accession Nos. AE005673, CP001340, and CP000927, dehydrogenase, Xylose dehydrogenase, and Xylonolactonase respectively. encoded by this operon are shown in SEQID NOS: 24 to 27, The genes of the enzymes of the Caulobacter Crescentus 25 respectively. Furthermore, the nucleotide sequence of the CB15, NA1000, and K31 strains are registered at GenBank XylD gene in this operon, and the amino acid sequences of with the following gene symbols. xylonate dehydratase encoded by it are shown in SEQ ID TABLE 1 Gene symbol (GenBank Gene Enzyme EC number CB15 NA1OOO K31 xylD Xylonate dehydratase EC4.2.1.82 CC 0823 CCNA 00866 Caul 4000 xy|X 2-keto-3- EC: 4.2.1 CC 0822. CCNA OO865 deoxyxylonate dehydratase XylA 2-Ketoglutaric EC: 1.2.1.26 CC 0821 CCNA 00864 Caul 4001 semialdehyde dehydrogenase xylB Xylose EC: 1.1.1.175 CC 0820 CCNA OO863 Caul 4002 dehydrogenase Xylc Xylonolactonase EC: 3.11.68 CC 0819 CCNA 00862 Caul 4003

In addition, the Xylose dehydrogenase gene and the 2-ke NOS: 28 and 29, respectively. The nucleotide sequence of toglutaric semialdehyde dehydrogenase gene of Caulobacter 45 SEQID NO: 28 corresponds to the positions 5509 to 7296 of crescentus can be referred to as ccrXylB and ccrXylA, respec the sequence of SEQID NO. 23. tively. Although two sites are Suggested as the start codon of Furthermore, examples of the enzymes of the NXA (Novel xylX, the positions 1175 to 1177 are described as the start Xylose Assimilation) pathway can include, besides those of codon in SEQID NO. 23. Two start codons are suggested also Caulobacter bacteria, for example, Xylose dehydrogenase of 50 for xylD, and the positions 1 to 3 or the positions 13 to 15 of Hypocrea jecorina (Trichoderma ressei) (FEMS Microbiol. Lett., 277, 249-254, 2007); yebD of Bacillus subtilis (2-ke SEQ ID NO: 28 may be used as the start codon. When the toglutaric semialdehyde dehydrogenase, type.III), 2-ketoglu positions 13 to 15 are considered as the start codon, the amino taric semialdehyde dehydrogenase (type I) of Pseudomonas acid sequence of xylonate dehydratase of SEQ ID NO: 29 putida, 2-ketoglutaric semialdehyde dehydrogenase, type, begins from the Leu at the position 5. typeII, typeIII of Azospirillum brasilense (J. Bac. Chem. 55 Although glucose dehydrogenase can reversibly catalyze 282,6685-6695, 2007 for these), and their homologues. the following reaction (EC1.1.1.119), the phrase “glucose In particular, as the Xylonate dehydratase gene, the yhC dehydrogenase that can catalyze the reaction which produces gene and yagF gene of a bacterium belonging to the genus Xylonic acid from Xylose can mean an enzyme that can Escherichia such as Escherichia coli can be used. TheyhC convert D-xylose into D-Xylonolactone by using pyrrolo gene of Escherichia coli is shown in SEQID NO:34, and the 60 quinoline quinone as a coenzyme. yagF gene of Escherichia coli is shown in SEQID NO: 36. Furthermore, as for Xylonate dehydratase, XylD gene homo B-D-Glucose--NADP->D-glucono-1,5-lactone-- logues of microorganisms belonging to the genus Agrobac NADPH-H terium, Herbaspirillum, Actinoplanes, or Aspergillus, such as Pyrroloquinoline quinone can be produced by a native Agrobacterium tumefaciens, Herbaspirillum seropedicae, 65 ability possessed by the microorganism, or can be added to Actinoplanes missouriensis, and Aspergillus Oryzae, may the medium (Appl. Environ. Microbiol., 2009 May, 75(9) also be used. 2784-2791). US 9,045,789 B2 10 In the bacterium that produces 2-ketoglutaric acid or a Accession No. NC 003450 and the amino acid sequence of derivative thereof, the decomposition pathway of 2-ketoglu the Elo subunit encoded thereby are disclosed in European taric acid can be attenuated or deleted. To attenuate or delete Patent Application Laid-open No. 2100957 A1. the decomposition pathway of 2-ketoglutaric acid, the activi Genes coding for each of the C-KGDH subunits, and the ties or activity of C-ketoglutarate dehydrogenase and/or suc- 5 gene cluster containing them may be generically called the cinate dehydrogenase are(is) attenuated or deleted. The C-ke “genes coding for C.-KGDH'. toglutarate dehydrogenase, which can henceforth also be The Succinate dehydrogenase, which can also be referred referred to as “C.-KGDH', activity can mean an activity of to as “SDH, is the enzyme EC: 1.3.99.1, which can reversibly catalyzing the reaction in which C-ketoglutaric acid (2-oxo catalyze the following reaction. SDH activity can mean the glutaric acid) is oxidatively decarboxylated to generate suc- 10 activity for catalyzing this reaction: cinyl-CoA. The aforementioned reaction is catalyzed by three kinds of enzyme subunits, C-KGDH (Elo, C.-ketoglutarate Succinic acid--FAD->fumaric acid--FADH dehydrogenase, EC: 1.2.4.2), dihydrolipoamide S-Succinyl SDH is made up of three or four subunit structures, depend transferase (E2O, EC: 2.3.1.61), and dihydrolipoamide dehy ing on type of microorganism, and the activity thereof can be drogenase (E3, EC: 1.8.1.4). That is, these three subunits cata- 15 decreased or deleted by modifying at least one of these pro lyze the following reactions, respectively, and the collective teins so that it does not normally function. Specifically, SDH activity of catalyzing a reaction by a combination of these is made up of the following Subunits (names of genes coding three reactions can be called the C-KGDH activity. The for the Subunits are described in parentheses), and the mem C.-KGDH activity can be confirmed by measurement accord brane anchor protein is encoded solely by schC or by schC ing to the method of Shiio et al. (Isamu Shiio and Kyoko and SdhD depending on species. Ujigawa-Takeda, Agric. Biol. Chem., 44 (8), 1897-1904, SDHA: flavoprotein subunit (sdhA) 1980). SDHB: Fe S protein subunit (sdhB) Elo: 2-oxoglutarate+dihydrolipoyllysine-residue Succinyl SDHC: membrane anchor protein (sdho) transferaselipoyllysine-dihydrolipoyllysine-residue succi SDHD: membrane anchor protein (sdhD) nyltransferaseS-succinyldihydrolipoyllysine--CO 25 Furthermore, the SDH subunit complex may have the E2O: CoA--enzyme N6-(S-succinyldihydrolipoyl) activities of both SDH and fumarate reductase. For example, lysine=succinyl-CoA--enzyme N6-(dihydrolipoyl)lysine the SDH subunit complex of coryneform bacteria has the E3: protein N6-(dihydrolipoyl)lysine--NAD protein N6-(li activities of both SDH and fumarate reductase (WO2005/ poyl)lysine+NADH+H" 021770). C-KGDH can also be called oxoglutarate dehydrogenase 30 The SDH activity can be confirmed by measuring reduc or 2-oxoglutarate dehydrogenase. tion of 2,6-dichloroindophenol (DCIP) as an indicative index. In Enterobacteriaceae bacteria such as Pantoea ananatis, A specific method is described in Tatsuki Kurokawa and the protein subunits having these three enzymatic activities, Junshi Sakamoto, Arch. Microbiol. (2005) 183:317-324. respectively, form a complex. The subunits are encoded by The genes coding for the SDH subunits, and the operon SucA, such3 and lpd, respectively, and the SucA and Such 35 containing them may be generically called the 'genes coding genes are present downstream from the Succinate dehydroge for SDH. nase iron-sulfur protein gene (sdhB) (U.S. Pat. No. 6,331, As genes coding for SDH of enterobacteria, the nucleotide 419). Although these genes are described as genes of Entero sequences of Such genes of Pantoea anamatis and the amino bacter agglomerans AJ13355 in the aforementioned patent, acid sequences of the subunits are disclosed in WO2008/ this strain was later reclassified into Pantoea anamatis. 40 O75483. As genes coding for C.-KGDH of enterobacteria, the nucle As the genes coding for SDH of coryneform bacteria, for otide sequences of the SucA gene, the Such gene and the SucC example, there are disclosed the sequences of the Sdh operon and the amino acid sequences of the Subunits of Pantoea of Corynebacterium glutamicum (GenBank accession No. anamatis are disclosed in European Patent Application Laid NCg 10359 (sdh(C) NCg10360 (sdhA) NCg10361 (sdhB)), open No. 2100957 A1. Furthermore, the sucA, such3 and sucC 45 and the Sdh operon of Brevibacterium flavum (Japanese genes coding for C.-KGDH of Escherichia coli have been Patent Laid-open No. 2005-095169, European Patent Appli opened to public as Genbank NP 415254 and NP 415255, cation Laid-open No. 1672077 A1, WO2008/075483). respectively. For reducing or deleting the activities of C-KGDH and In coryneform bacteria, the Elo subunit is encoded by the SDH, the methods described later for reduction of activity of odha gene (registered as NCg 11084 of GenBank Accession 50 an enzyme that catalyzes a reaction branching from the bio No. NC 003450, which is also called the sucA gene), and the synthesis pathway of L-glutamic acid, and produces other E3 subunit is encoded by the lpd gene (GenBank Accession compounds, can be used. No. Y 16642). On the other hand, it is estimated that the E2o Furthermore, an activity of an enzyme that incorporates subunit is encoded by the odh A gene together with the Elo Xylose into cells may further be enhanced in the microorgan subunit as a bifunctional protein (Usuda et al., Microbiology, 55 1S. 142,3347-3354, 1996), or encoded by the gene registered as An example of an enzyme that can catalyze incorporation NCg 12126 of GenBank Accession No. NC 003450, which of Xylose into cells can include D-xylose permease, and an is different from the odh A gene. Therefore, although the example of a gene which encodes for D-Xylose permease can odha gene can code for the Elo Subunit, it can also code for include the xylE gene. The nucleotide sequence of the xylE E2O. 60 gene of Escherichia coli coding for D-Xylose permease, and The nucleotide sequence of the odh A gene of Brevibacte the amino acid sequence encoded by this gene are shown in rium lactofermentum and the amino acid sequence of the Elo SEQ ID NOS:30 and 31, respectively. subunit encoded thereby (WO2006/028298), the nucleotide Furthermore, Xylose isomerase (XylA) and Xylulose kinase sequence of the aforementioned NCg 12126 of GenBank (XylB) can be attenuated in the microorganism. The Xylose Accession No. NC 003450 and the amino acid sequence of 65 isomerase (XylA) gene and Xylulose kinase (XylB) gene of the E2O subunit encoded thereby, as well as the nucleotide Escherichia coli are disclosed as NC000913.1 gi:16131436 sequence of the aforementioned NCg 11084 of GenBank and 16131435, respectively. US 9,045,789 B2 11 12 Xylonic acid may accumulate in the medium, and espe addition, transformation of microorganisms can also be per cially in Escherichia coli, the activity of xylonate dehydratase formed by the electric pulse method (Japanese Patent Laid can be further enhanced. For example, the activity can be 10 open No. 2-207791). umol/min/mg protein or higher, 15 Limol/min/mg protein or The target enzyme gene can also be introduced into the higher, or 17 umol/min/mg protein or higher. 5 chromosome of the host microorganism. The target enzyme Methods for imparting activity of a target enzyme to a gene can be introduced into a chromosome of a microorgan microorganism or for increasing activity of a target enzyme of ism randomly using a transposon or Mini-Mu (Japanese a microorganism will be explained below. Patent Laid-open No. 2-109985, U.S. Pat. No. 5,882,888, When the activity of a target enzyme is not native to the European Patent Publication No. 805867 B1), or by homolo 10 gous recombination using a sequence present on the chromo chosen microorganism, the activity of the target enzyme can Somal DNA in multiple copies as a target, Such as repetitive be imparted to the microorganism by introducing the gene DNA, and an inverted repeat located at the end of a transpos encoding the target enzyme into the microorganism. Further able element. Alternatively, a target gene can be introduced more, when the microorganism has the activity of the target into a chromosome by using the Red driven integration enzyme, the activity can be increased by introducing a non 15 method (WO2005/010175). Moreover, a target gene can also native target enzyme gene, increasing the copy number of the be introduced into a chromosome by transduction using a endogenous target enzyme gene, or modifying an expression phage such as P1 phage, or by using a conjugative transfer control sequence Such as a promoter of the target enzyme vector. Furthermore, it is also possible to introduce a target gene to increase expression of the gene. The expression enzyme gene using a gene unnecessary for production of “introduce a target enzyme gene' can mean not only to intro target substance as a target, as described in WO03/040373. duce a target enzyme gene into a microorganism in which One or plural copies of the target enzyme gene can be intro activity of the target enzyme is not native, but also to intro duced into a target sequence by Such methods as described duce a foreign target enzyme gene into a microorganism above. having activity of the target enzyme, and also to introduce an Transfer of a target gene on a chromosome can be con endogenous target enzyme gene into a microorganism having 25 firmed by Southern hybridization using a probe having a activity of the target enzyme to increase expression of the sequence complementary to the target gene or a part thereof. endogenous target enzyme gene. Although it is sufficient that a copy number of the intro In order to introduce a target enzyme gene, for example, the duced target gene is not less than 1, the copy number can be 2 target enzyme gene is cloned into an appropriate vector, and or more, 3 or more, or 5 or more. As for the xylonate dehy a host microorganism is transformed with the obtained vector. 30 dratase gene as the target gene, in particular, 2 or more copies Examples of the vector which can be used for transforma of the gene can be introduced. tion can include a plasmid which can autonomously replicate Furthermore, the activity of a target enzyme gene can be in the chosen microorganism. Examples of a plasmid autono optimized by Substituting or mutating an expression control mously replicable in a microorganism belonging to the family sequence such as a promoter of the target enzyme gene in Enterobacteriaceae include puC19, puC18, pBR322, 35 combination as described later. In particular, the Xylonate RSF1010, pHSG299, pHSG298, pHSG399, pHSG398, dehydratase gene can be overexpressed by Substituting or pSTV28, pSTV29, pTWV228, p.TWV229 (pHSG, pSTV and mutating an expression control sequence instead of or pTWV series vectors are available from Takara Bio), together with the aforementioned increase of the copy num pMW 119, pMW 118, pMW219, pMW218 (pMW series vec ber. tors are available from Nippon Gene), and so forth. Further 40 Examples of the method for increasing expression of a more, plasmids for coryneform bacteria include p AM330 target enzyme gene include replacing an expression control (Japanese Patent Laid-open No. 58-67699), pHM1519 (Japa sequence such as a promoter of the target enzyme gene with nese Patent Laid-open No. 58-77895), pSFK6 (Japanese one having an appropriate strength on a chromosomal DNA Patent Laid-openNo. 2000-262288), pVK7 (U.S. Patent Pub or a plasmid to enhance expression of the gene. For example, lished Application No. 2003/0175912), p.AJ655, p.AJ611, 45 the thr promoter, lac promoter, trp promoter, trc promoter, pI, pAJ1844 (Japanese Patent Laid-open No. 58-192900), pCG1 promoter, tac promoter, and so forth are known as frequently (Japanese Patent Laid-open No. 57-134500), pCG2 (Japa used promoters. Furthermore, variants of the tac promoter nese Patent Laid-open No. 58-35197), pCG4, pCG11 (Japa used in the examples described below (PtacA promoter, nese Patent Laid-open No. 57-183799), pHK4 (Japanese PtacB promoter) can also be used. Methods for evaluating the Patent Laid-open No. 5-7491), and so forth. 50 strength of promoters and strong promoters are described in Examples of transformation methods include treating the paper of Goldstein and Doi (Goldstein, M.A. and Doi R. recipient cells with calcium chloride to increase permeability H., 1995, Prokaryotic promoters in biotechnology, Biotech for DNA, which has been reported for Escherichia coli K-12 nol. Annu. Rev. 1, 105-128), and so forth. (Mandel, M. and Higa, A., J. Mol. Biol., 1970, 53:159-162), Furthermore, it is also possible to substitute several nucle preparing competent cells from cells which are at the growth 55 otides into the promoter region of a gene to strengthen it, as phase, followed by transformation with DNA, which has been disclosed in International Publication WOOO/18935. Substi reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. tution of an expression control sequence can be performed in and Young, F. E., 1977, Gene, 1:153-167), and so forth. Alter the same manner as, for example, that of the gene Substitution natively, a method of making DNA-recipient cells into pro using a temperature-sensitive plasmid. Examples of vectors toplasts or spheroplasts, which can easily take up recombi 60 having a temperature-sensitive replication origin and effec nant DNA, followed by introducing recombinant DNA into tive in Escherichia coli and Pantoea anamatis include, for the DNA-recipient cells, which is known to be applicable to example, the temperature-sensitive plasmid pMAN997 Bacillus subtilis, actinomycetes, and yeast (Chang, S, and described in International Publication WO99/03988, deriva Choen, S.N., 1979, Mol. Gen. Genet., 168:111-115; Bibb, M. tives thereof, and so forth. Furthermore, substitution of an J. Ward, J. M. and Hopwood, O.A., 1978, Nature, 274:398 65 expression control sequence can also be performed by a 400; Hinnen, A., Hicks, J. B. and Fink, G. R., 1978, Proc. method utilizing a linear DNA such as the method called Natl. Sci., USA, 75:1929-1933) can also be employed. In "Red-driven integration using Red recombinase of W phage US 9,045,789 B2 13 14 (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad. Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Sci. USA. 97:6640-6645), and the method using a combina Asp for ASn, substitution of Asn. Glu or Gln for Asp, substi tion of the Red-driven integration method and the W phage tution of Seror Ala for Cys, substitution of Asn., Glu, Lys, His, excision system (Cho, E. H., Gumport, R.I., Gardner, J. F. J. Asp or Arg for Gln, Substitution of Gly, ASn, Gln, Lys or Asp Bacteriol. 184: 5200-5203 (2002)) (refer to WO2005/ for Glu, substitution of Pro for Gly, substitution of Asn. Lys, 010175). Modification of an expression control sequence can Gln, Arg or Tyr for His, substitution of Leu, Met, Val or Phe be combined with increasing the copy number of a gene. for Ile, substitution of Ile, Met, Val or Phe for Leu, substitu Furthermore, it is known that substitution of several nucle tion of Asn. Glu, Gln, His or Arg for Lys, substitution of Be, otides in a spacer between the ribosome-binding site (RBS) Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Be or and translation initiation codon, especially a sequence imme 10 diately upstream from the initiation codon, greatly affects the Leu for Phe, substitution of Thror Ala for Ser, substitution of mRNA translation efficiency, and therefore this sequence can Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substi be modified to improve the translation amount. tution of His, Phe or Trp for Tyr, and substitution of Met, Ile When a target gene is introduced into the aforementioned or Leu for Val. The aforementioned amino acid substitutions, amplification plasmid or chromosome, any promoter can be 15 deletions, insertions, additions, inversions or the like may be used to express the gene so long as the chosen promoter a result of a naturally-occurring mutation or a variation due to functions in the chosen microorganism. The promoter can be an individual difference or a difference of species of a micro the native promoter for the chosen gene, or a modified pro organism from which the genes are derived (mutant or vari moter. Expression of a gene can also be controlled by Suitably ant). Such proteins can be obtained by, for example, modify choosing a promoter that strongly functions in the chosen ing a nucleotide sequence of a wild-type target enzyme gene microorganism, or by making the -35 and -10 regions of the by site-specific mutagenesis so that the amino acid residues at promoter closer to the consensus sequence. the specific sites of the encoded protein include substitutions, Whether a target enzyme activity is enhanced or not can be deletions, insertions, or additions of amino acid residues. confirmed by comparing the target enzyme activities of a Furthermore, such a protein having a conservative muta modified strain to a parent or non-modified strain. If the target 25 tion as described above may have a homology of for enzyme activity of the modified Strain is increased as com example, 80% or more, 90% or more, 95% or more, 97% or pared to the parent or non-modified Strain, the target enzyme more, 98% or more, or 99% or more, to the entire amino acid activity is enhanced. Furthermore, when the parent strain sequence, and having a function equivalent to that of the does not have the target enzyme activity, if the target enzyme wild-type protein. In this specification, “homology” can activity can be detected in the modified strain, the target 30 mean “identity”. enzyme activity is enhanced. So long as the wild-type target enzyme gene codes for Such The target enzyme gene can be obtained by PCR using an amino acid sequence as described above, it is not limited to oligonucleotides prepared on the basis of the aforementioned genes of Caulobacter crescentus, Haloferax volcanii, and the sequence information or sequence information of gene or like, but it may be any that have an equivalent codon for an protein known for the microorganism as primers, or hybrid 35 arbitrary codon. ization using an oligonucleotide prepared on the basis of the The wild-type gene can also be a DNA that is able to aforementioned sequence information as a probe from a chro hybridize with a nucleotide sequence complementary to the mosomal DNA or chromosomal DNA library of a microor nucleotide sequence of each enzyme gene, or a probe that can ganism having the target enzyme. be prepared from the complementary sequence, under strin Moreover, the target enzyme and the gene coding for it may 40 gent conditions, and codes for a protein having functions be a homologue or artificial modification thereof, or a protein equivalent to those of the wild-type target enzyme. The “strin having a conservative mutation, or a gene coding for it, so gent conditions' can refer to conditions under which a so long as the enzymatic activity is maintained. called specific hybrid is formed, and a non-specific hybrid is Such a homologue, artificial modification thereof, or a not formed. Examples of the stringent conditions include protein having a conservative mutation or genes coding for 45 those under which highly homologous DNAs hybridize to these can be referred to as a conservative variant. each other, for example, DNAs not less than 80% homolo The conservative variant of a target enzyme may be, for gous, not less than 90% homologous, not less than 95% example, a protein having the aforementioned amino acid homologous, not less than 97% homologous, not less than sequence of the enzyme, but can include Substitution, dele 98% homologous, or not less than 99% homologous, hybrid tion, insertion, addition or the like of one or several amino 50 ize to each other, and DNAs less homologous than the above acid residues at one or several positions. do not hybridize to each other, or conditions corresponding to Although the number of the “one or several amino acid washing of typical Southern hybridization, i.e., conditions of residues may differ depending on the position in the three washing once, or 2 or 3 times, at a salt concentration and dimensional structure or the types of amino acid residues of temperature of 1xSSC, 0.1% SDS at 60° C., 0.1xSSC, 0.1% the protein, specifically, it can be 1 to 20, 1 to 10, or 1 to 5. The 55 SDS at 60° C., or 0.1XSSC, 0.1% SDS at 68° C. conservative mutation is typically a conservative substitution. As the probe, a part of a sequence that is complementary to The conservative substitution is a mutation wherein substitu the target enzyme gene may also be used. Such a probe can be tion takes place mutually among Phe, Trp, and Tyr, if the prepared by PCR using oligonucleotides prepared on the Substitution site is an aromatic amino acid; among Leu, Ile basis of a known gene sequence as primers and a DNA frag and Val, if the substitution site is a hydrophobic amino acid; 60 ment containing the nucleotide sequence as a template. For between Gln and Asn., if the substitution site is a polar amino example, when a DNA fragment having a length of about 300 acid; among Lys, Arg and His, if the Substitution site is a basic bp is used as the probe, the washing conditions of the hybrid amino acid; between Asp and Glu, if the Substitution site is an ization may be, for example, 50° C., 2xSSC and 0.1% SDS. acidic amino acid; and between Ser and Thr, if the substitu The aforementioned descriptions concerning conservative tion site is an amino acid having a hydroxyl group. Substitu 65 variants of the above-mentioned proteins and genes coding tions considered conservative Substitutions can include, spe for them can be similarly applied to the other genes described cifically, substitution of Ser or Thr for Ala, substitution of below for the bacteria that produce target substances. US 9,045,789 B2 15 16 The microorganism can inherently have an ability to pro using the assigned registration numbers listed in the cata duce a target Substance, or the ability may be imparted by logue of the ATCC (www.atcc.org/). The AJ12340 strain was breeding using a mutation method, a recombinant DNA tech deposited on Oct. 27, 1987 at National Institute of Bioscience nique, or the like. and Human Technology of Agency of Industrial Science and Microorganisms can include, but are not limited to, bacte Technology (currently independent administrative agency, ria belonging to the family Enterobacteriaceae such as those National Institute of Technology and Evaluation, Interna of genera Escherichia, Pantoea, and Enterobacter, coryne tional Patent Organism Depositary, Tsukuba Central 6, 1-1, form bacteria Such as Corynebacterium glutamicum and Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Brevibacterium lactofermentum, and Bacillus bacteria such Japan), as the accession number of FERM BP-1539 based on as Bacillus subtilis. 10 Budapest Treaty. Coryneform bacteria include those bacteria having been Microorganisms belonging to the family Enterobacteri originally classified into the genus Brevibacterium, but are aceae can include, but are not limited to, bacteria belonging to now classified into the genus Corynebacterium (Int. J. Syst. the genera Escherichia, Enterobacter, Pantoea, Klebsiella, Bacteriol. 41,255 (1981)), and include bacteria belonging to Serratia, Erwinia, Salmonella, Morganella or the like and the genus Brevibacterium, which is closely related to the 15 which are able to produce a target Substance. Specifically, genus Corynebacterium. Examples of Such coryneform bac bacteria belonging to the family Enterobacteriaceae accord teria are listed below. ing to the classification shown in NCBI (National Center for Corynebacterium acetoacidophilum Biotechnology Information) database (www.ncbi.nlm.nih. Corynebacterium acetoglutamicum gov/htbin-post/Taxonomy/ Corynebacterium alkanolyticum wgetorg?mode=Tree&id=1236&lvl=3&keep–1&srch Corynebacterium callunae mode=1&unlock) can be used. Among the bacteria of the Corynebacterium glutamicum family Enterobacteriaceae, bacteria belonging to the genus Corynebacterium lilium Escherichia, Enterobacter, or Pantoea can be used as a parent Corynebacterium melassecola strain. Corynebacterium thermoaminogenes (Corynebacterium 25 Escherichia bacteria which can be used as the parent strain efficiens) include, but are not limited to, Escherichia bacteria reported Corynebacterium herculis by Neidhardt et al. (Neidhardt, F. C. et al., Escherichia coli Brevibacterium divaricatum and Salmonella Tiphimurium, American Society for Micro Brevibacterium flavum biology, Washington D.C., 1029 table 1), such as Escherichia Brevibacterium immariophilum 30 coli. Specific examples of Escherichia coli include Escheri Brevibacterium lactofermentum (Corynebacterium chia coli W3110 (ATCC 27325), and MG1655 (ATCC glutamicum) 47076) strains, which are derived from the wild-type (proto Brevibacterium roseum type) Escherichia coli K12 strain, and so forth. Brevibacterium saccharolyticum In particular, Pantoea bacteria, Erwinia bacteria, and Brevibacterium thiogenitalis 35 Enterobacter bacteria are classified as Y-proteobacteria, and Corynebacterium ammoniagenes are taxonomically very close to one another (J. Gen. Appl. Brevibacterium album Microbiol. December 1997, 43(6), 355-361; International Brevibacterium cerinum Journal of Systematic Bacteriology, October 1997, pp. 1061 Microbacterium ammoniaphilum 1067). In recent years, some bacteria belonging to the genus Specific examples of these bacteria include the following: 40 Enterobacter were reclassified as Pantoea agglomerans, Pan Corynebacterium acetoacidophilum ATCC 13870 toea dispersa, or the like, on the basis of DNA-DNA hybrid Corynebacterium acetoglutamicum ATCC 15806 ization experiments etc. (International Journal of Systematic Corynebacterium alkanolyticum ATCC 21511 Bacteriology, July 1989, 39(3). p. 337-345). Furthermore, Corynebacterium callunae ATCC 15991 Some bacteria belonging to the genus Erwinia were re-clas Corynebacterium glutamicum ATCC 13020, ATCC 45 sified as Pantoea ananas or Pantoea stewarti (refer to Inter 13032, ATCC 13060 national Journal of Systematic Bacteriology, January 1993, Corynebacterium lilium ATCC 15990 43(1), pp. 162-173). In addition, Pantoea ananas was then Corynebacterium melassecola ATCC 17965 further re-classified as Pantoea anamatis. Corynebacterium thermoaminogenes AJ 12340 (FERM Examples of the Enterobacter bacteria include Entero BP-1539) 50 bacter agglomerans (currently re-classified as Pantoea Corynebacterium herculis ATCC 13868 ananatis etc.), Enterobacter aerogenes, and so forth. Specifi Brevibacterium divaricatum ATCC 14020 cally, the Strains exemplified in European Patent Application Brevibacterium flavum ATCC 13826, ATCC 14067 Laid-open No. 952221 can be used. A typical strain of the Brevibacterium immariophilum ATCC 14068 genus Enterobacter is Enterobacter agglomeranses ATCC Brevibacterium lactofermentum ATCC 13869 (Coryne 55 12287 (currently re-classified as Pantoea ananatis). bacterium glutamicum ATCC 13869) Typical strains of the Pantoea bacteria include Pantoea Brevibacterium roseum ATCC 13825 ananatis, Pantoea stewartii, Pantoea agglomerans, and Pan Brevibacterium saccharolyticum ATCC 14066 toea citrea. Specific examples include the following strains: Brevibacterium thiogenitalis ATCC 19240 Pantoea ananatis AJ13355 (FERM BP-6614, European Brevibacterium ammoniagenes ATCC 6871, ATCC 6872 60 Patent Application Laid-open No. 0952221) Brevibacterium album ATCC 15111 Pantoea ananatis AJ13356 (FERM BP-6615, European Brevibacterium cerinum ATCC 15112 Patent Application Laid-open No. 0952221) Microbacterium ammoniaphilum ATCC 15354 Although these strains are described as Enterobacter These strains are available from the AmericanType Culture agglomerans in European Patent Application Laid-open No. Collection (ATCC) (Address: P.O. Box 1549, Manassas, Va. 65 0952221, they have been reclassified as Pantoea anamatis on 20108, United States of America). That is, a registration num the basis of nucleotide sequence analysis of 16S rRNA etc., as ber is assigned to each of the strains. Strains can be ordered described above. US 9,045,789 B2 17 18 The Pantoea anamatis AJ13355 strain was isolated from Hereinafter, methods for imparting an ability to produce a soil in Iwata-shi, Shizuoka, Japan as a strain that can prolif target Substance to Such microorganisms as described above, erate in a medium containing L-glutamic acid and a carbon or methods for enhancing an ability to produce a target Sub source at low pH. The SC17 strain was selected as a low stance of Such microorganisms are described. viscous substance-producing mutant strain from the AJ13355 To impart an ability to produce a target Substance, methods strain (U.S. Pat. No. 6,596.517). The Pantoea ananatis conventionally employed in the breeding of coryneform bac AJ13355 strain was deposited at the National Institute of teria or bacteria of the genus Escherichia (see Amino Acid Bioscience and Human-Technology, Agency of Industrial Fermentation”, Gakkai Shuppan Center (Ltd.), 1st Edition, Science and Technology, Ministry of International Trade and published May 30, 1986, pp. 77-100) can be used. Such 10 methods include acquisition of an auxotrophic mutant, a tar Industry (currently, National Institute of Technology and get Substance analogue-resistant Strain, or a metabolic regu Evaluation, International Patent Organism Depositary, lation mutant, construction of a recombinant strain in which Address: Tsukuba Central 6, 1-1, Higashi 1-Chome, expression of a target Substance biosynthesis enzyme is Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Feb. 19, 1998 enhanced, and so forth. In the breeding of target Substance and assigned an accession number of FERM P-16644. The 15 producing bacteria, imparted properties such as an aux deposit was then converted to an international deposit under otrophic mutation, analogue resistance, or metabolic regula the provisions of Budapest Treaty on Jan. 11, 1999 and tion mutation may be one or more. The expression of target assigned an accession number of FERM BP-6614. The Pan Substance biosynthesis enzyme(s) can be enhanced alone or toea anamatis SC 17 strain was given the private number in combinations of two or more. Furthermore, imparting AJ416, and deposited on Feb. 4, 2009 at National Institute of properties such as an auxotrophic mutation, analogue resis Technology and Evaluation, International Patent Organism tance, or metabolic regulation mutation may be combined Depository (Address: Tsukuba Central 6, 1-1, Higashi with enhancing the biosynthesis enzymes. 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan), and An auxotrophic mutant strain, analogue-resistant strain, or assigned an accession number of FERM BP-11091. metabolic regulation mutant strain with an ability to produce Examples of L-glutamic acid-producing Pantoea anamatis 25 a target Substance can be obtained by Subjecting a parent bacteria further include SC17 sucA/RSFCPG+pSTVCB, strain or wild-type strain to conventional mutatagenesis. Such AJ13601, NP106, and NA1 strains. The SC17sucA/RS as exposure to X-rays or UV irradiation, or treatment with a FCPG+pSTVCB strain was obtained by introducing the plas mutagen Such as N-methyl-N'-nitro-N-nitrosoguanidine, mid RSFCPG containing the citrate synthase gene (gltA), etc., and then selecting those which exhibit autotrophy, ana phosphoenolpyruvate carboxylase gene (prpC), and 30 logue resistance, or a metabolic regulation mutation and glutamate dehydrogenase gene (gdh A) derived from Escheri which also have an ability to produce a target Substance. chia coli, and the plasmid pSTVCB containing the citrate Moreover, a target substance-producing bacterium can also synthase gene (gltA) derived from Brevibacterium lactofer be obtained by enhancing activity of a biosynthesis enzyme of mentum, into the SC17sucA strain, which is a SucA gene the target Substance by gene recombination. deficient strain derived form the SC17 strain (U.S. Pat. No. 35 Hereinafter, examples of a method for imparting an ability 6,596.517). The AJ13601 strain was selected from the to produce a target Substance and microorganisms to which an SC17sucA/RSFCPG+pSTVCB strain for its resistance to ability to produce a target Substance is imparted will be L-glutamic acid of high concentration at a low pH. Further explained. more, the NP106 strain was derived from the AJ13601 strain Examples of a method for imparting or enhancing an abil by eliminating the RSFCPG+pSTVCB plasmid (WO2010/ 40 ity to produce a target Substance by breeding can include, for 027045). The AJ13601 strain was deposited at National Insti example, a method of modifying a microorganism so that tute of Technology and Evaluation, International Patent expression of a gene coding for an enzyme involved in bio Organism Depositary (Tsukuba Central 6, 1-1, Higashi synthesis of a target Substance is enhanced. For example, 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postal code: 305 examples of enzymes involved in L-glutamic acid biosynthe 8566) on Aug. 18, 1999, and assigned an accession number 45 sis include glutamate dehydrogenase (gdh A), glutamine Syn FERM P-17516. Then, the deposit was converted into an thetase (glnA), glutamate synthetase (gltBD), aconitate international deposit under the provisions of the Budapest hydratase (acna, acnB), citrate synthase (gltA), phospho Treaty on Jul. 6, 2000, and assigned an accession number enolpyruvate carboxylase (ppc), pyruvate carboxylase, pyru FERM BP-7207. This strain was originally identified as vate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, Enterobacter agglomerans when it was isolated, and depos 50 pykF), phosphoenolpyruvate synthase (ppSA), enolase (eno), ited as Enterobacter agglomerans. However, it was recently phosphoglyceromutase (pgmA, pgmI), phosphoglycerate re-classified as Pantoea anamatis on the basis of nucleotide kinase (pgk), glyceraldehyde-3-phophate dehydrogenase sequencing of 16S rRNA and so forth. (gapA), triose phosphate isomerase (tpiA), fructose bisphos The NA1 strain is a strain corresponding to the NP106 phate aldolase (fbp), phosphofructokinase (pfkA, pfkB), glu strain having RSFPPG (WO2008/020654) in which the gltA 55 cose phosphate isomerase (pgi), methyl citrate synthase gene of RSFCPG described above is replaced with the methyl (prpC), and so forth. The gene names are in the parentheses citrate synthase gene (prpC) (WO2010/027045). following the enzyme names (the same shall apply to the Examples of the Erwinia bacteria include Erwinia amylo following descriptions). vora and Erwinia carotovora, and examples of the Klebsiella Expression of the aforementioned genes can be enhanced bacteria include Klebsiella planticola. Specific examples 60 by the method described for the enhancement of the activities include the following strains: of the enzymes of the aforementioned NXA pathway. Erwinia amylovora ATCC 15580 Examples of microorganisms which can be modified so Erwinia Carotovora ATCC 15713 that expression of the citrate synthase gene, pyruvate dehy Klebsiella planticola AJ13399 (FERM BP-6600, Euro drogenase gene, and/or glutamate dehydrogenase gene is/are pean Patent Laid-open No. 955368) 65 enhanced can include the microorganisms described in Klebsiella planticola AJ13410 (FERM BP-6617, Euro WO00/18935, European Patent Application Laid-open No. pean Patent Laid-open No. 955368). 1010755, and so forth. US 9,045,789 B2 19 20 Moreover, a modification for imparting the L-glutamic lase). Examples of microorganisms which have enhanced acid-producing ability may also be performed by reducing or activity of phosphoketolase include the following microor deleting activity of an enzyme that catalyzes a reaction which ganisms (WO2006/016705): branches off from the L-glutamic acid biosynthetic pathway Brevibacterium lactofermentum ATCC 13869AsucA (pVK9 and produces a compound other than L-glutamic acid. Xfp) Examples of Such enzymes can include 2-oxoketoglutarate Brevibacterium lactofermentum ATCC 13869AsucA (pVK9 dehydrogenase, Succinate dehydrogenase, isocitrate lyase, PS2 xpkA) acetohydroxy acid synthase, acetolactate synthase, formate L-Glutamic acid-producing ability can also be imparted by acetyltransferase, lactate dehydrogenase, glutamate decar enhancing the 6-phosphogluconate dehydratase activity, the boxylase, 1-pyrroline dehydrogenase, acetyl-CoA hydrase 10 2-keto-3-deoxy-6-phosphogluconate aldolase activity, or (International Patent Publication WO2006/057450), and so forth. both. An example of a microorganism in which 6-phospho In order to reduce or eliminate the activity of a target gluconate dehydratase activity and the 2-keto-3-deoxy-6- enzyme, a mutation may be introduced into the gene of the phosphogluconate aldolase activity are increased include the enzyme on a genome by a usual mutagenesis method or gene 15 microorganism disclosed in Japanese Patent Laid-open No. recombination technique so that intracellular activity of the 2003-274988. Furthermore, L-glutamic acid-producing abil enzyme is reduced or eliminated. Such a mutation can be ity can also be imparted by amplifying the yhfK and ybjL introduced by, for example, using genetic recombination to genes, which are L-glutamic acid secretion genes (WO2005/ eliminate the gene coding for the enzyme on the genome or to 085419, WO2008/133161). modify an expression control sequence Such as a promoter or As an L-glutamic acid-producing microorganism, a micro the Shine-Dalgarno (SD) sequence. It can also be achieved by organism having an ability to produce L-glutamic acid in a introducing an amino acid substitution (missense mutation), a liquid medium in an amount exceeding the Saturation con stop codon (nonsense mutation), or a frame shift mutation for centration of L-glutamic acid when it is cultured under acidic adding or deleting one or two nucleotides into the regions conditions (henceforth also referred to as an L-glutamic acid coding for the enzyme on the genome, or partially or totally 25 accumulation ability under acidic condition) can be used. For deleting the gene (J. Biol. Chem., 272:8611-8617, 1997). The example, by obtaining a strain in which resistance to enzymatic activity can also be decreased or eliminated by L-glutamic acid in a low pH environmentis improved accord constructing a gene coding for a mutant enzyme, in which the ing to the method described in European Patent Application coding region is totally or partially deleted, and Substituting it Laid-open No. 1078989, the ability to produce L-glutamic for a normal gene on a genome by homologous recombina 30 acid in an amount exceeding the Saturation concentration can tion or the like, or by introducing a transposon or IS factor into be imparted. the gene. Other methods for imparting or enhancing L-glutamic For example, in order to introduce a mutation that acid-producing ability can include methods of imparting decreases or eliminates the activities of the above-mentioned resistance to an organic acid analogue, respiratory inhibitor, enzymes by genetic recombination, the following methods 35 or the like, and methods of imparting sensitivity to a cell wall can be used. A mutant gene can be prepared by modifying a synthesis inhibitor. Examples include a method of imparting partial sequence of a target gene so that it does not encode an monofluoroacetic acid resistance (Japanese Patent Laid-open enzyme that can function normally, and then a bacterium No. 50-113209), a method of imparting adenine resistance or belonging to the family Enterobacteriaceae can be trans thymine resistance (Japanese Patent Laid-open No. formed with a DNA containing the mutant gene to cause 40 57-065198), a method of attenuating urease (Japanese Patent recombination of a corresponding gene on the genome with Laid-open No. 52-038088), a method of imparting malonic the mutant gene to Substitute the mutant gene for the target acid resistance (Japanese Patent Laid-open No. 52-038088), a gene on the genome. Examples of Such gene Substitution method of imparting resistance to benzopyrons or naphtho using homologous recombination include methods of using a quinones (Japanese Patent Laid-openNo. 56-1889), a method linear DNA such as the method called Red-driven integration 45 of imparting HOQNO resistance (Japanese Patent Laid-open (Datsenko, K. A. and Wanner, B. L., 2000, Proc. Natl. Acad. No. 56-140895), a method of imparting C.-ketomalonic acid Sci. USA, 97:6640-6645), and the method utilizing the Red resistance (Japanese Patent Laid-open No. 57-2689), a driven integration in combination with an excisive system method of imparting guanidine resistance (Japanese Patent derived from phage (Cho, E. H., Gumport, R.I., Gardner, J. Laid-open No. 56-35981), a method of imparting sensitivity F., 2002, J. Bacteriol., 184:5200-5203, refer to WO2005/ 50 to penicillin (Japanese Patent Laid-open No. 4-88994), and so 01.0175, Russian Patent Application No. 2006134574), a forth. method of using a plasmid containing a temperature sensitive Specific examples of such resistant bacteria include the replication origin (U.S. Pat. No. 6,303.383, Japanese Patent following strains. Laid-open No. 05-007491), and so forth. Furthermore, such Brevibacterium flavum AJ3949 (FERM BP-2632, refer to site-specific mutagenesis based on gene Substitution using 55 Japanese Patent Laid-open No. 50-113209) homologous recombination can also be performed by using a Corynebacterium glutamicum AJ11628 (FERM P-5736, plasmid which is not able to replicate in a host. refer to Japanese Patent Laid-open No. 57-065198) Furthermore, the ability to produce L-glutamic acid in Brevibacterium flavum AJ11355 (FERM P-5007, refer to coryneform bacteria can also be achieved by a method of Japanese Patent Laid-open No. 56-1889) amplifying theyggB gene (NCgl 1221; NP 600492. Reports 60 Corynebacterium glutamicum AJ1 1368 (FERM P-5020, small-conductance. Igi:19552490, WO2006/070944), and a refer to Japanese Patent Laid-open No. 56-1889) method of introducing a mutantyggB gene in which a muta Brevibacterium flavum AJ11217 (FERM P-4318, refer to tion is introduced into the coding region. Japanese Patent Laid-open No. 57-2869) Examples of methods to enhance L-glutamic acid-produc Corynebacterium glutamicum AJ1 1218 (FERM P-4319, ing ability include introducing genes encoding D-xylulose 65 refer to Japanese Patent Laid-open No. 57-2869) 5-phosphate phosphoketolase and/or fructose-6-phosphate Brevibacterium flavum AJ11564 (FERMBP-5472, refer to phosphoketolase (these are collectively called phosphoketo Japanese Patent Laid-open No. 56-140895) US 9,045,789 B2 21 22 Brevibacterium flavum AJ11439 (FERMBP-5136, refer to the b2682, b2683, b 1242 or b3434 gene is enhanced (Japa Japanese Patent Laid-open No. 56-35981) nese Patent Laid-open No. 2002-300874), and so forth. Corynebacterium glutamicum H7684 (FERM BP-3004, Examples of L-proline-producing strains of coryneform refer to Japanese Patent Laid-open No. 04-88994) bacteria can include the DL-3,4-dehydroproline resistant Brevibacterium lactofermentum AJ11426 (FERMP-5123, strain (FERM BP-1219, U.S. Pat. No. 4,224.409), the strains refer to Japanese Patent Laid-open No. 56-048890) in which citrate synthetase activity increases 1.4 times or Corynebacterium glutamicum AJ11440 (FERM P-5137, more as compared to the parent strains thereof (FERM refer to Japanese Patent Laid-open No. 56-048890) P-5332, FERMP-5333, FERMP-5342, FERMP-5343, Japa Brevibacterium lactofermentum AJ11796 (FERMP-6402, nese Patent No. 1426823), and the strain to which acetic acid refer to Japanese Patent Laid-open No. 58-158192) 10 auxotrophy is imparted (FERM P-5931). Examples of microorganisms having L-glutamine-produc Examples of microorganisms having an L-arginine-pro ducing ability include Escherichia Colimutants strains having ing ability can include bacteria in which glutamate dehydro resistance to O-methylmethionine, p-fluorophenylalanine, genase activity is enhanced, bacteria in which glutamine Syn D-arginine, arginine hydroxamate, AEC (S-(2-aminoethyl)- thetase (glnA) activity is enhanced, and bacteria in which 15 cysteine), C.-methylserine, B-2-thienylalanine, or Sulfaguani glutaminase gene is disrupted (European Patent Application dine (refer to Japanese Patent Laid-openNo. 56-106598). The Laid-open Nos. 1229121 and 1424398). Enhancement of the Escherichia coli strain 237, which contains highly active glutamine synthetase activity can also be attained by disrup N-acetylglutamate synthase having a mutation for resistance tion of the glutamine adenylyltransferase (glnE) or disruption to feedback inhibition by L-arginine (Russian Patent Appli of the PII control protein (glnB). Furthermore, a strain cation No. 2000117677), is also an L-arginine-producing belonging to the genus Escherichia and having a mutant bacterium. The strain 237 was deposited at the Russian glutamine synthetase in which the tyrosine residue in the 397 National Collection of Industrial Microorganisms (VKPM) position is replaced with another amino acid residue is an (GNIIGenetika) on Apr. 10, 2000 under an accession number example of a L-glutamine-producing bacterium (U.S. Patent of VKPMB-7925, and the original deposit was converted to Published Application No. 2003/0148474). 25 an international deposit based on Budapest Treaty on May 18, Other methods for imparting or enhancing the L-glutamic 2001. The Escherichia coli 382 strain, which is a derivative of acid-producing ability can include a method of imparting the 237 strain and is an L-arginine-producing strain having 6-diazo-5-oxo-norleucine resistance (Japanese Patent Laid improved ability to assimilate acetic acid (Japanese Patent open No. 3-232497), a method of imparting purine analogue Laid-open No. 2002-017342), may also be used. The Escheri resistance and methionine Sulfoxide resistance (Japanese 30 chia coli 382 strain was deposited at the Russian National Patent Laid-open No. 61-202694), a method of imparting Collection of Industrial Microorganisms (VKPM) on Apr. 10, C.-ketomalonic acid resistance (Japanese Patent Laid-open 2000 under accession number of VKPMB-7926. No. 56-151495), and so forth. Specific examples of coryne As a microorganism having an L-arginine-producing abil form bacteria having L-glutamic acid-producing ability ity, microorganisms in which the expression amount of one or include the following strains. 35 more genes coding for an L-arginine biosynthetic enzyme is Brevibacterium flavum AJ11573 (FERMP-5492, Japanese increased can also be used. Examples of the L-arginine bio Patent Laid-open No. 56-161495) synthetic enzyme can include one or more enzymes selected Brevibacterium flavum AJ11576 (FERM BP-10381, Japa from N-acetylglutaminate synthetase (argA), N-acetyl nese Patent Laid-open No. 56-151495) glutamyl phosphate reductase (argC), ornithine acetyl trans Brevibacterium flavum AJ12212 (FERMP-8123, Japanese 40 ferase (arg.J), N-acetylglutamate kinase (argB), acetylorni Patent Laid-open No. 61-202694) thine transaminase (arg)), acetylornithine deacetylase Examples of microorganisms having L-proline-producing (argE), ornithine carbamoyl transferase (argF), argininosuc ability can include, for example, bacteria having Y-glutamyl cinic acid synthetase (argG), argininosuccinic acid lyase kinase which is desensitized to feedback inhibition by L-pro (argH), and carbamoyl phosphate synthase (carAB). A line, and bacteria in which L-proline decomposition system is 45 mutant N-acetylglutamate synthase gene (argA) coding for attenuated. The method of modifying bacteria by using a the enzyme in which the amino acid sequence corresponding DNA coding for Y-glutamyl kinase desensitized to feedback to the 15 to 19 positions of the wild-type enzyme is replaced inhibition by L-proline is disclosed in Dandekar, A. M., and the feedback inhibition by L-arginine is thereby canceled Uratsu S. L., J. Bacteriol. 170, 12:5943-5 (1988). Further can be used (European Patent Application Laid-open No. more, examples of the method for obtaining a bacterium in 50 1170361). which the L-proline decomposition system is attenuated can Although the L-arginine-producing coryneform bacteria include, for example, a method of introducing a mutation into are not particularly limited so long as a coryneform bacterium a proline dehydrogenase gene for reducing the enzymatic having an L-arginine-producing ability is chosen, examples activity. Examples of bacteria having L-proline-producing can include wild-type strains of coryneform bacteria; coryne ability include the Escherichia coli NRRL B-12403 strain and 55 form bacteria resistant to certain agents including Sulfa drugs, NRRL B-12404 strain (British Patent No. 2075056), Escheri 2-thiazolealanine, -amino-hydroxyvaleric acid, and so forth; chia coli VKPMB-8012 strain (U.S. Patent Published Appli coryneform bacteria exhibiting auxotrophy for L-histidine, cation No. 2002/0058315), and strains having the mutant L-proline, L-threonine, L-isoleucine, L-methionine or plasmid disclosed in German Patent No. 3127361 or the L-tryptophan in addition to the resistance to 2-thiazolealanine mutant plasmid disclosed in the reference of Bloom F. R. etal. 60 (Japanese Patent Laid-open No. 54-44096); coryneform bac (The 15th Miami Winter Symposium, 1983, p. 34). teria resistant to ketomalonic acid, fluoromalonic acid or Furthermore, microorganisms having L-proline-producing monofluoroacetic acid (Japanese Patent Laid-open No. ability can also include the Escherichia coli 702 strain 57-18989); coryneform bacteria resistant to argininol (Japa (VKPMB-8011), which is a 34-dehydroxyproline and azeti nese Patent Laid-open No. 62-24075); coryneform bacteria dine-2-carboxylate resistant strain, 702ilvA strain (VKPMB 65 resistant to X-guanidine (X represents a derivative of ali 8012 strain), which is an ilvA-deficient strain of the 702 phatic acid oraliphatic chain, Japanese Patent Laid-open No. strain, E. coli strains of which activity of protein encoded by 2-186995), and so forth. US 9,045,789 B2 23 24 A coryneform bacterium having L-arginine-producing erol, fructose, Sucrose, maltose, mannose, galactose, arabi ability can be bred to be resistant to 5-azauracil, 6-azauracil, nose, starch hydrolysates and molasses can be used. In addi 2-thiouracil, 5-fluorouracil, 5-bromouracil, 5-azacytosine, tion, organic acids such as acetic acid and citric acid, and 6-azacytosine and so forth; resistant to arginine hydroxamate alcohols such as ethanol can also be used each alone or in and 2-thiouracil; resistant to arginine hydroxamate and combination with other carbon Sources. 6-azauracil (Japanese Patent Laid-open No. 49-126819); Although the ratio of xylose to other carbon sources is not resistant to a histidine analogue or tryptophan analogue particularly limited, the ratio of xylose:other carbon source (Japanese Patent Laid-open No. 52-114092); auxotrophic for (weight ratio) can be 1:0.1 to 100, 1:0.1 to 10, 1:0.1 to 5, 1:1 at least one of methionine, histidine, threonine, proline, iso to 5, or 1:1 to 3. leucine, lysine, adenine, guanine and uracil (or uracil precur 10 The concentration of the carbon source in the medium is sor) (Japanese Patent Laid-open No. 52-99289); resistant to not particularly limited so long as the concentration is Suit arginine hydroxamate (Japanese Patent Publication No. able for producing the chosen target Substance. However, the 51-6754); auxotrophic for succinic acid or resistant to a concentration of the carbon Source in the medium can be nucleic acid base analogue (Japanese Patent Laid-open No. about 0.1 to 50 w/v '%, about 0.5 to 40 w/v '%, or about 1 to 58-9692); deficient in arginine decomposition ability, resis 15 30%. tant to an arginine antagonist and canavanine and auxotrophic Xylose, or a mixture of Xylose and a hexose Such as glu for lysine (Japanese Patent Laid-open No. 52-8729); resistant cose, can be obtained from a Supply source of biomass that is to arginine, arginine hydroxamate, homoarginine, D-arginine not fully used. Such pentoses and hexoses can be released and canavanine, or resistant to arginine hydroxamate and from biomass by hydrolysis with Steam and/or a acid, 6-azauracil (Japanese Patent Laid-open No. 53-143288); hydrolysis with diluted acid, hydrolysis with an enzyme such resistant to canavanine (Japanese Patent Laid-open No. as cellulase, oran alkaline treatment. When the substrate is a 53-3586), or the like. cellulose-type material, cellulose is hydrolyzed into saccha Specific examples of coryneform bacteria having L-argin rides simultaneously or Successively, and the saccharides can ine-producing ability include the following strains. be used for the production of the target substance. Since Brevibacterium flavum AJ11169 (FERM P-4161) 25 hemicellulose is generally more easily hydrolyzed into sac Brevibacterium lactofermentum AJ12092 (FERMP-7273) charides as compared to cellulose, a cellulose-type material Brevibacterium flavum AJ11336 (FERM P-4939) can be hydrolyzed beforehand, the pentoses separated, and Brevibacterium flavum AJ11345 (FERM P-4948) then the cellulose hydrolyzed by a treatment with steam, acid, Brevibacterium lactofermentum AJ 12430 (FERM alkali, cellulase, or a combination of these, to produce hex BP-2228) 30 OSS. Furthermore, a strain deficient in ArgR, which is an argin Xylose in the medium may also be supplied by converting ine repressor (U.S. Published Patent Application No. 2002/ each of the hexoses to xylose (D-xylose) using a microorgan 0045223), and a strain in which glutamine synthetase activity ism mutated to have a pathway for converting glucose, galac is increased (U.S. Published Patent Application No. 2005/ tose or arabinose into Xylose. 0014236) can also be used. 35 As the nitrogen source, ammonia, urea, ammonium salts L-Citrulline and L-ornithine share common biosynthetic Such as ammonium sulfate, ammonium carbonate, ammo pathways with L-arginine, and the ability to produce L-cit nium chloride, ammonium phosphate and ammonium rulline and L-ornithine can be imparted by increasing the acetate, nitric acid salts and so forth can be used. As the enzymatic activities of N-acetylglutamate syntase (argA), organic trace nutrients, amino acids, vitamins, fatty acids, N-acetylglutamylphosphate reductase (argC), ornithine 40 nucleic acids, nutrients containing the foregoing Substances acetyltransferase (arg.J), N-acetylglutamate kinase (argB), Such as peptone, casamino acid, yeast extract, soybean pro acetylornithine transaminase (arg)), and acetylornithine tein decomposition product and so forth can be used. When an deacetylase (argE) (WO2006/35831). auxotrophic mutant strain that requires an amino acid or the As an Y-aminobutyric acid (GABA)-producing bacterium, like for its growth is used, the required nutrient can be supple a strain in which activity of glutamate decarboxylase is 45 mented. As the mineral salts, phosphoric acid salts, magne enhanced (Microb. Cell Fact., 2010, Nov. 12; 9:85: Amino sium salts, calcium salts, iron salts, manganese salts and so Acids, 2010 November, 39(5):1107-16; U.S. Patent Pub forth can be used. lished Application No. 2010/0324258) can be used. The culture can be performed under aerobic conditions, As a putrescine-producing bacterium, a strain in which while the fermentation temperature can be controlled to be 20 4-hydroxybutyrate reductase, Succinyl-CoA reductase (alde 50 to 45° C., and pH to be 3 to 9. To adjust the pH, an inorganic hyde forming), and 4-hydroxybutyrate dehydrogenase are or organic acidic or alkaline Substance, ammonia gas, and so enhanced (WO2011/047101), and a strain of which y-ami forth can be used. A Substantial amount of the target Substance nobutyraldehyde dehydrogenase is enhanced (FEBS Lett. can be accumulated in the culture medium or cells after 10 to 2005 Aug. 1, 579 (19):4107-12), can be used. 120 hours of culture under such conditions as described <2> Method for Producing Target Substance 55 above. By culturing Such a bacterium as described above in a Moreover, when the target Substance is L-glutamic acid, medium containing Xylose as a carbon Source to produce and the culture can be performed to produce and accumulate accumulate a target Substance in the medium, and collecting L-glutamic acid by precipitating L-glutamic acid in a liquid the target Substance from the medium, the target Substance medium adjusted to satisfy a condition under which can be produced. 60 L-glutamic acid is precipitated. Examples of the condition As the medium used for the culture, a typical media con under which L-glutamic acid is precipitated include, for taining a carbon Source, nitrogen Source and mineral salts as example, pH of 5.0 to 4.0, 4.5 to 4.0, more 4.3 to 4.0, or 4.0. well as organic trace nutrients such as amino acids and Vita In order to simultaneously obtain both improvement of mins as required can be used. Either a synthetic medium or a growth under acidic conditions and efficient precipitation of natural medium may be used. 65 L-glutamic acid, the pH can be 5.0 to 4.0, 4.5 to 4.0, or 4.3 to As the carbon Source, so long as Xylose is present, other 4.0. The culture may be performed at the aforementioned pH carbon sources, for example, Sugars Such as glucose, glyc for the whole culture period or for only a portion of it. US 9,045,789 B2 25 The target Substance collected may contain microbial cells, medium components, moisture, and by-product metabolites *Trace elements of the microorganism in addition to the target Substance. CaCl2.H2O 0.66 g/L Purity of the collected target substance is 50% or higher, 85% ZnSO7H2O 0.18 g/L or higher, or 95% or higher (Japanese Patent No. 1214636, 5 CuSO4.5H2O 0.16 g/L MnSO4HO 0.15 g/L U.S. Pat. Nos. 5,431,933, 4,956,471, 4,777,051, 4,946,654, CoCl26H2O 0.18 g/L 5,840,358, 6.238,714, U.S. Patent Published Application No. HBO 0.10 g/L 2005/0025878). Na-MoO, 0.30 g/L The target substance can be collected from the culture 10 medium after completion of the culture by a combination of MSII-Xylose Medium: conventionally known methods such as ion-exchange resin The same components as those of the MSII-Glucose method (Nagai, H. et al., Separation Science and Technology, medium except that glucose (40 g/L) is replaced with Xylose 39(16), 3691-3710), membrane separation (Japanese Patent (40 g/L). Laid-open Nos. 9-164323 and 9-173.792), crystallization 15 MSII-GX Medium: (WO2008/078448, WO2008/078646), and other methods. The same components as those of the MSII-Glucose Furthermore, when the target substance deposits in the medium except that glucose (40 g/L) is replaced with a mix medium, it can be collected by centrifugation, filtration or the ture of glucose (20 g/L) and Xylose (20 g/L) like. A target Substance deposited in the medium and a target MSII-SX Medium: Substance dissolved in the medium may be isolated together The same components as those of the MSII-Glucose after the target Substance dissolved in the medium is crystal medium except that glucose (40 g/L) is replaced with a mix ture of Sucrose (20 g/L) and Xylose (20 g/L). lized. E1 Synthetic Medium: EXAMPLES 25 Group A Hereafter, the present invention will be still more specifi NHCI 20 nM cally explained with reference to the following non-limiting MgSO4·7H2O 2 mM NaHPO. 40 mM examples. 30 KHPO. 30 mM The medium compositions used in the following examples CaCl2 O.O1 mM are shown below. FeSO7HO O.O1 mM MnSO4 to 5H2O O.O1 mM LB Medium: Citrate 5 mM pH Free 35 Filter-sterilized Bacto tryptone 10 g/L Group B-1 Yeast extract 5 g/L Carbon source 50 (or 100) mM NaCl 5 g/L Filter-sterilized pH 7.0 Group B-2 40 Thiamine HCI 1 mM LBGM9: The same components as those of the LB medium, plus minimal medium components (5 g/L of glucose, 2 mM of b. This component was added to the component of the group magnesium Sulfate, 3 g/L of monopotassium phosphate, 0.5 B-1 after filter sterilization (0.22 Lm). g/L of sodium chloride, 1 g/L of ammonium chloride, 6 g/L of 45 disodium phosphate) Group C MSII-Glucose Medium: MES-NaOH (pH 6.8) 50 mM Filter sterilized (0.22 m) Group A 50 c. Solutions containing the components of the groups A to C Glucose 40 g/L MgSO 7H2O 0.5 g/L at 5-fold higher concentrations were prepared as stock solu Group B tions. CM-Dex Medium: (NH4)2SO 20 g/L 55 NaCl 0.5 g/L Polypeptone 10 g/L Yeast extract 2 g/L Yeast extract 10 g/L CaCl,7HO 0.25 g/L Glucose 5 g/L FeSO4·7H2O 20 mg/L. KH2PO 1 g/L MnSOnHO 20 mg/L. 60 Urea 3 g/L Trace elements 4 mill MgSO4·7H2O 0.4 g/L L-Lys 200 mg/L FeSO4·7HO 0.01 g/L DL-Met 200 mg/L MnSOC5H2O 0.01 g/L DAP 200 mg/L Bean filtrate 1.2 g/L (T-N) Soybean hydrolysate 65 pH 7.5 adjusted with KOH a. The components of Groups A and B were separately auto claved at 120° C. for 20 minutes, and then mixed. US 9,045,789 B2 27 28 Glc Medium: iv) pMW 119/SmaI (219f: SEQID NO: 7, 219r: SEQID NO: 8) Then, by PCR using the purified PCR products of i) and ii) Glucose 80 g/L (NH4)2SO 30 g/L as the template, as well as PtwVPtacf and 0819r as the prim KH2PO 1 g/L ers, a fragment Ptac XylXccrAccrBC consisting of the fore MgSO4·7H2O 0.4 g/L going PCR products ligated together was amplified. The in FeSO4·7H2O 0.01 g/L MnSOSHO 0.01 g/L fusion reaction was performed with these three of the Vitamin B1 200 g/L obtained Ptac xylXccrAccrBC and the PCR products of iii) Biotin 60 g/L and iv) using Clontech In-Fusion Cloning Kit, E. coli JM109 Bean filtrate 0.48 g/L (T-N) 10 Soybean hydrolysate strain was transformed with the reaction product, and the pH 8.0 adjusted with KOH target plasmid pMW 119 Ptac ccrNXA was obtained from a transformant. Then, by using pMW 119 Ptac ccrNXA as the template, as Xyl Medium (Biotin Restricted) 15 well as PtwVPtacf and 219CC0819r as the primers, ccrNXA The same components as those of the Glc medium except operon containing Ptac was amplified. The in-fusion reaction that glucose (80 g/L) is replaced with xylose (80 g/L), and was performed with the obtained amplified product and minus biotin. pTWV228 which had been digested with SmaI; the E. coli MS Medium: JM109 strain was transformed with the reaction product, and the target plasmid pTWV228Ptac ccrNXA was obtained Group A from a transformant. (2) Construction of Plasmid puT-MuKim Containing Glucose or xylose 40 g/L pUT399 Carrying Kanamycin Resistant Mini-Mu Glucose and xylose (1:1) 40 g/L MgSO 7H2O 1 g/L pUT399 is a plasmid having the replication origin of R6K Group B 25 and the mob region required for conjugative transfer, and is not replicable in a strain which lacks the pir gene (available (NH4)2SO 20 g/L KH2PO 1 g/L from Biomedal, refer to R. Simon., et al., BIO/TECHNOL Yeast Extract 2 g/L OGY NOVEMBER 1983,784-791; U.S. Pat. No. 7,090,998). FeSO4·7H2O 10 mg/L. pCE 1134 (Japanese Patent Laid-open No. 2-109985) is a MnSOnHO 10 mg/L. 30 plasmid containing Mud II 1734, and carries a Km resistance gene and the lacXYZ gene in the Mini-Mu unit. By the The components of Groups A and B were separately auto method described below, a DNA fragment not having the claved at 120° C. for 20 minutes, and then mixed, and 50 g/L lacXYZ region was prepared from the Mini-Mu unit of of calcium carbonate according to the Japan Pharmacopoeia 35 pCE1134, and cloned into puT399. was added. By PCR using pCE 1134 as the template, as well as primers attL-F (SEQ ID NO: 9) and mptII-R (SEQ ID NO: 10), a Example 1 fragment containing the repressor MuCts of the MuAB gene coding for the left end and transposase, and the Kim resistance Introduction of NXA Pathway into Pantoea ananatis 40 gene, was obtained. Furthermore, by using pCE 1134 as the template, as well as primers attR-F (SEQ ID NO: 11) and Construction of plasmid pTWV228Ptac ccrNXA for attR-R (SEQID NO: 12), a fragment containing the right end introduction of NXA Pathway was similarly obtained. Crossover PCR was performed by The NXA pathway has been reported in C. Crescentus using these 2 fragments as the template, as well as the primers (Stephens, C. et al., J. Bacteriol., 189(5):181-2185, 2007). To 45 attl-F and attR-R, and the obtained fragment of about 2.3 kb obtain the genes coding for the enzymes of the NXA pathway was introduced into puT399 at the SmaI site. In this way, the of C. crescentu, the following methods were employed. plasmid puT-Mukim was obtained. The genome of C. Crescentus has a length of about 4 Mb, in Since the Mini-Muunit constructed as described above has which five genes forman operon structure (Journal of Bacte the Kim resistance gene and the 8-base recognizing Not site riology, 189:2181-2185, 2007). The genome was extracted 50 as a cloning site in the transposition unit, various genes can be from the published strain of C. crescentus (CB-15 (ATCC cloned into it. 19089, available from ATCC), and the genes were cloned and (3) Substitution of Drug Resistance Gene of an expression vector for the genes constructed. pTWV228Ptac ccrNXA The expression vectors were constructed by using Clon The ampicillin resistance gene of pTWV228Ptac ccrNXA 55 was replaced with the kanamycin resistance gene by the Red tech In-Fusion Cloning Kit. method. The following four kinds of DNA fragments were ampli By using puT MuKm as the template, as well as primers fied by PCR using chromosomal DNA of C. Crescentus Ap-Km-fw (SEQ ID NO: 13) and Ap-Km-ry (SEQ ID NO: CB-15 (ATCC 19089) for the following i), ii) and iii), and 14), a sequence containing the kanamycin resistance gene pMW 119 for the following iv) as the templates. The primers 60 (ntpII fragment) was amplified. used for PCR are indicated in the parentheses. The PCR was performed by using PrimeSTAR HS Poly i) tac promoter sequence (henceforth referred to as “Ptac'. merase (Takara Bio) according the protocol attached to this PtwvPtacf: SEQID NO: 1,0823Ptacr: SEQID NO: 2) enzyme. ii) Fragment containing XylX, ccrxylA, ccrXylB and Xylc A helper plasmid RSF Red TER (U.S. Patent Published (Ptac0823f: SEQID NO:3,0819r: SEQID NO:4) 65 Application No. 2009/0286290A1, WO2008/075483) was iii) xylD and downstream region thereof of about 120 bp introduced into E. coli JM109 having (0819f: SEQ ID NO:5, 219 cc0819r: SEQID NO: 6) pTWV228Ptac ccrNXA, and the cells were cultured in 50 ml US 9,045,789 B2 29 30 of the LB medium (containing 1 mM IPTG, 100 mg/L of 6(5), 343-345, 2009) to obtain pUC18-xylD in which the Sfil ampicillin, and 25 mg/L of chloramphenicol) at 37° C. until site was removed, and the XylD gene was inserted. OD660 value became 0.4. Separately, pSTV28-Ptac-Ttrp was digested with SmaI in a The aforementioned RSF Red TER is a helper plasmid conventional manner. The in-fusion reaction was performed for inducing w-dependent integration (Red-driven integra 5 with the DNA fragment of the xylD gene and the vector DNA tion, WRed method), and it can induce expression of gam, bet fragment, the E. coli JM109 strain was transformed with the and eXo genes of w with the lad gene. This plasmid also reaction product, and the target plasmid pSTVPtac xylD T contains the levanSucrase gene (sacB), and can eliminate a plasmid from a cell with this gene in a medium containing trp was obtained from a transformant. Sucrose. Furthermore, this plasmid also contains the chloram 10 pSTV28-Ptac-Ttrp was constructed as follows. phenicol resistance gene. A DNA fragment (PtacTtrp) having the tac promoter (hav The cells cultured as described above were collected, ing the sequence of SEQID NO:32) and the sequence of the washed twice with a 10% glycerol solution by centrifugation, trp terminator was synthesized, and ligated between the and suspended 1 mL of a 10% glycerol solution. Then, the KpnI-BamHI sites of the pMW219 vector to obtain cells were transformed with the ntpII fragment obtained 15 pMW219-Ptac-Ttrp. The same amounts of pSTV28 and above by electroporation, and the transformants were sub pMW219-Ptac-Ttrp both digested with KpnI and BamHI jected to selection on the LB agar medium containing 40 were mixed, and ligated, JM109 was transformed with the mg/L of kanamycin. The obtained transformants were inocu ligation product, and a plasmid was extracted from a colony lated on the LB agar medium (containing 1 mM IPTG, 10% that showed Cm resistance. It was confirmed that the obtained Sucrose, and 40 mg/L of kanamycin), and cultured overnight plasmid showed bands of about 400 bp and 3 kbp (correctly at 37° C. to obtain a single clone. It was confirmed that the 389 bp and 2994 bp), which were expected as a result of the obtained transformant could not grow on the LBagar medium double digestion with KpnI and BamHI, and thus pSTV28 containing 100 mg/L of amplicillin, and thereby it was con Ptac-Ttrp was obtained. firmed that the amplicillin resistance gene of (4) L-Glutamic Acid Production with Pantoea ananatis pTWV228Ptac ccrNXA was replaced with the kanamycin 25 Having the NXA Pathway resistance gene. The obtained plasmid was designated pTWVPtac ccrNXA Km. The Panamatis NA1 strain was transformed with pTWVP (4) Construction of Plasmid Containing xylD tac ccrNXA Km by electroporation (refer to U.S. Pat. No. The construction was performed by using Clontech In 6,682.912). For the strain containing pTWVPtac ccrNX Fusion Cloning Kit. 30 A Km, a plate medium with LBGM9 supplemented with First, by PCR using a plasmid containing puC18 in which kanamycin at a final concentration of 40 mg/L was used. each gene was cloned as the template, as well as xylD IFS Cells of the Panamatis NA1 strain and the transformant 5742-10-5 (SEQ ID NO: 15) and xylD IFS 5742-10-6 strain cultured overnight at 34°C. on the LBGM9 plate were (SEQID NO: 16) as the primers, a DNA fragment containing each scraped off in an amount corresponding to "/6 of the xylD was amplified. Specifically, it was cloned into a puC18 35 plate, inoculated into 5 ml of the MSII-Xylose or MSII-GX plasmid in which the Sfil site had been removed by the medium contained in a large test tube, and cultured at 34°C. method described below. and 120rpm for 48 hours, and residual saccharide, amounts of By PCR using the genomic DNA of the C. Crescentus accumulated L-glutamic acid (Glu), and Xylonic acid were CB-15 strain as the template, CC0819-01F 4691-88-7 (SEQ measured. The results are shown in Tables 2 and 3. TABLE 2 Glu production in MSII-GX medium OD660 Consumed Consumed Glu Yield Xylonic Strain (x51) Glc (gL) Xyl (gL) (g/L) (%) acid (gL) NA1 O.129 O.OO1 21.8 22.9 11.5 + 0.1 25.7 O.3 18.4 O2 NA1/pTWV228Ptac ccrNXA Km O.134 O.OO6 21.8 22.9 31.3 0.1 69.8 O1 O.O

TABLE 3 Glu production in MSII-Xylose medium OD660 Consumed Glu Yield Xylonic Strain (x51) Xyl (gL) (gL) (%) acid (gL) NA1 O.O19 OOO6 O.O O.O O.O O.O NA1/pTWV228Ptac ccrNXA Km O.O69 O.OO8 410 32.70.9 80.5 - 2.2 10.1 - 1.9

ID NO: 17) and CC0819-01R 5659-9-1 (SEQID NO: 18), 60 When the Panamatis NA1 strain was cultured with the as well as CC0819-02F 5659-9-2 (SEQ ID NO: 19) and mixed carbon source of glucose and xylose (MSII-GX medium), the glutamic acid yield was 25.7% (Table 2). In this CC0819-02R 4691-88-10 (SEQID NO: 20) as the primers, case, accumulation of Xylonic acid was observed, and thus it fragments of 1130 bp and 653 bp were amplified, respec was suggested that most of xylose was converted into Xylonic tively. Then, puC18 which had been digested with SmaI, and 65 acid. It is estimated that the accumulation of xylonic acid with the two amplified fragments described above, Were the Panamatis NA1 strain was provided by the activity of assembled by the in vitro assembly method (Nature Methods, glucose dehydrogenase of Panamatis. US 9,045,789 B2 31 32 On the other hand, when the Panamatis NA1 strain con TABLE 4-continued taining pTWVPtac ccrNXA Km was cultured with the mixed carbon source of glucose and xylose (MSII-GX Growth in the M9 medium), the glutamic acid yield was significantly higher as Vector Promoter Gene xylose medium compared to the parent strain (yield: 69.9%). If it is taken into ccrxylB xylic consideration that the parent strain hardly produces glutamic xylD acid from Xylose, and it is assumed that the glutamic acid pMW119 Ptac xylx yield from glucose of the strain containing pTWVPtac ccrxylA ccrNXA Km is equivalent to that of the parent strain, the ccrxylB 10 xylic yield of glutamic acid produced from Xylose via the NXA xylD pathway is about 86%. In fact, when the culture was per pTWV228 Ptac xylx formed with xylose as the sole carbon source (MSII-Xylose), ccrxylA the strain containing pTWVPtac ccrNXA Km produced ccrxylB Glu at a yield of 80% (Table 3). xylic 15 xylD Example 2 (2) Expression of NXA Pathway in E. Coli L-Glutamic Introduction of NXA Pathway into Escherichia coli Acid-Producing Strain AS E. coli L-glutamic acid-producing strain, Expression of NXA Pathway in E. Coli MG1655AsucA (U.S. Patent Published Application No. By using a strain deficient in isocitrate dehydrogenase 2005/0106688), which is an OKGDH-deficient strain, was (Aicd), which is an enzyme of the TCA cycle and produces used. pMW119 Ptac ccrNXA or pMW119 as a control was C.KG from isocitric acid, expression of the NXA pathway was introduced into the above strain to obtain MG 1655ASucA/ attempted by growth complementation in a minimal medium pMW1 19Ptac ccrNXA and MG1655AsucA/pMW 119. containing Xylose as the Sole carbon Source. Since the iccd 25 These strains were each cultured as a flask culture in the MS gene-deficient strain cannot produce C.KG, it cannot grow in culture medium containing glucose (40 g/L), Xylose (40 g/L). a minimal medium containing Xylose as the Sole carbon source. However, if the ability to produce C.KG from xylose or glucose and Xylose (20g/L each) as the carbon Source. The can be imparted by introducing the NXA pathway, then this culture was performed for 24 hours in the medium containing strain acquires the ability to grow in Such a medium. 30 only glucose as the carbon source, and for 48 hours in the Specifically, the JW1122 strain, which is an iccd gene other media. The results are shown in FIG. 2. The numerals deficient strain of Keio Collection (cgsc.biology.yale.edu/ 325, 425 and 513 attached to the strain names shown in the Person.php?ID99553, available from E. coli Genetic figure are clone numbers. Resource Center at Yale CGSC, The Coli Genetic Stock Cen When a mixture of glucose and Xylose was used as the ter), was used as a host bacterial strain, and by introducing 35 carbon source, while the control strain (MG1655ASucA/ and expressing the NXA pathway in that strain using a plas pMW 119) accumulated 15 to 16 g/L of L-glutamic acid, the mid, it was examined whether the NXA pathway could func ccrNXA operon-expressing strain (MG1655ASucA/ tion also in E. coli. pMW1 19Ptac ccrNXA) produced about 12 g/L L-glutamic In Table 4, the constructed plasmids and the results of acid of, and thus tended to show reduced accumulation and growth complementation in the iccd gene-deficient strain are 40 yield of L-glutamic acid. Also when xylose was used as the shown. It was confirmed that the strain introduced with a sole carbon source, the same result was obtained. As for plasmid pMW 119 Ptac ccrNXA (prepared in Example 1) by-products, the resulting culture was analyzed for organic containing the NXA pathway operon (XylX, ccrXylA, acids and Xylonic acid. As a result, it was found that acetic ccrxylB, Xylc, xylD) and the tac promoter in combination acid and Xylonic acid were mainly produced. could grow on the M9 minimal medium (plate) (Sambrook, J. 45 (3) Analysis of Rate-Limiting Point of NXA Pathway in E. et al., Molecular Cloning, Cold Spring Harbor Laboratory Coli Press (1989)) containing xylose as the sole carbon source. Since xylonic acid, which is an intermediate of the NXA A similar study was also performed using liquid culture. pathway, was detected in the culture Supernatant of the Growth (O.D.) in the M9 minimal medium and the E1 syn ccrNXA operon-expressing E. coli L-glutamic acid-produc thetic medium containing Xylose or C.KG as the sole carbon 50 ing bacterium as described above, it was considered that it Source was measured over time by using a culture apparatus was highly possible that a part of incorporated Xylose was for 36 samples. The results are shown in FIG.1. Like the plate assimilated via the NXA pathway. Furthermore, the follow culture, the NXA pathway-introduced strain favorably grew ing problems were estimated. on the M9 or E1 medium containing xylose as the sole carbon i) While xylose incorporated into the cells may be assimi Source, whereas the vector control strain did not grow in Such 55 lated by both the Xylose-assimilating system characteristic to a medium. It was considered that these results were obtained E. coli and the NXA pathway, a certain amount of xylose may because the strain grew by assimilating xylose via the NXA be utilized by the E. coli system due to the difference in pathway, i.e., the NXA pathway derived from C. Crescentus activity or substrate specificity of the first enzyme of the E. functioned also in E. coli. coli system, Xylose isomerase (XylA), and the first enzyme of 60 the NXA pathway, xylose dehydrogenase (XDH), and thus TABLE 4 the flow rate of metabolic flux using the NXA pathway may become Smaller. Growth in the M9 ii) There may be a rate-limiting point in the NXA pathway, Vector Promoter Gene xylose medium or an unknown bypass pathway, and therefore OKG may not pTWV229 Native xy|X X 65 be produced. ccrxylA It was considered that the problem of i) might be amelio rated by increasing the amounts of the enzymes of the NXA US 9,045,789 B2 33 34 pathway by replacing the low-copy NXA operon expression duced L-glutamic acid in an amount Substantially equivalent vector (pMW 119) to a medium copy number type vector to that observed with the control strain. However, when the (pTWV228), and thereby increasing the uptake amount of the mixed culture system of glucose and Xylose was used, the substrate into the ccrNXA pathway. accumulation of L-glutamic acid tended to decrease. Further It was also considered that the problem of ii) might be 5 more, results of sibling strains also fluctuated. One of the overcome by improving the strain by breeding based on conceivable reasons is the elimination of the medium copy analysis of rate-limiting point and results thereof. number expression vector. Furthermore, like the strains On the basis of the above considerations, the following was described above, accumulation of xylonic acid was observed. performed: In order to confirm whether the activities of the enzymes of a) construction of a strain from a strain deficient in the E. 10 the ccrNXA pathway were increased by the increase in copy coli-specific Xylose-assimilating pathway (ASucAAXylA) as number of the NXA operon, the activity of XDH, which is the a host, in which the ccrNXA operon is expressed and thus first enzyme of the NXA pathway, was measured. The results carbon flux is forced through the NXA pathway, and evalua are shown in Table 5. “7513 and “1110 in the Strain names tion thereof by culture, mentioned in Table 5 are clone numbers. b) construction of accrNXA operon expression vector using 15 a medium copy number vector, construction of a strain using TABLE 5 Such a expression vector, and evaluation thereof by culture, and Results of XDH (Xylose dehydrogenase) activity measurement c) analysis of rate-limiting point. Specific activity By deleting the E. coli-specific Xylose-assimilating gene (mol/min.img xylA from MG 1655ASucA according to the w-Red method Strain protein Relative activity using primers xylA-H1P1-5742-5-1 (SEQ ID NO: 21) and MG1655AsucApMW 119 ND xylA-H2P2-5742-5-2 (SEQ ID NO: 22), MG1655AsucApMW ccrNXA7513 21.5 1.O MG1655AsucAAxylA strain WaS obtained. MG1655AsucApMW228 ND pMW1 19Ptac ccrNXA was introduced into this strain to 25 MG1655AsucApMW ccrNXA1110 140.4 6.5 obtain accrNXA operon-expressing Strain deficient in XylA. ND: Not Detected The results of the L-glutamic acid production culture per Note: formed by culturing the ccrNXA operon-expressing strain The relative activity was indicated as relative activity based on the specific activity of deficient in XylA in the same manner as that described in the ccrNXA7513 taken as 1. above section (3) are shown in FIG. 3. In FIG. 3, “ccrNXA' 30 The medium copy number expression vector-introduced represents pMW 119Ptac ccrNXA, and the following numer strain showed about 7 times higher XDH activity as compared als represent the clone numbers. to the low copy number expression vector-introduced strain. Whereas the vector control strain of the AsucAAxylA It had not been confirmed how xylose was actually distributed strain could not assimilate xylose, and could form cells and within the cells at the branching point of the xylose isomerase produce L-glutamic acid from only glucose, the ccrNXA 35 (XylA) characteristic to E. coli and XDH, and thus it was also operon-expressing strain showed consumption of xylose and still considered that the activity of XDH, which is the first production of L-glutamic acid, which was considered to be enzyme of the NXA pathway, might be insufficient. However, derived form xylose. However, it was found that the since the increase of the XDH activity did not improve L-glutamic acid accumulation amount thereof was Smaller L-glutamic acid accumulation, and on the basis of accumu than that obtained with the model strain (ASucA strain), and it 40 lation of Xylonic acid, and so forth, it was considered that accumulated Xylonic acid, which is a metabolic intermediate either one or more of enzymes of the NXA pathway might be of the ccrNXA pathway. From these results, it was suggested rate-limiting. that the metabolic flux of the whole ccrNXA pathway might Therefore, the rate-limiting point of the NXA pathway was be insufficient. Furthermore, since by-production of CKG analyzed. Since Xylonic acid accumulates as a metabolic was not observed, it was considered that supply of NADPH 45 intermediate, it was considered at least that the rate-limiting required for expression of the activity of GDH did not pose point might exist in the pathway from Xylonic acid to CKG any problem at this stage. rather than the metabolic pathway from Xylose to xylonic Then, accrNXA operon expression vector was constructed acid. Furthermore, in the structure of the ccrNXA operon, using a medium copy number vector. The ccrNXA operon whereas the enzymes of the pathway from Xylose to Xylonic containing the tac promoter region was amplified by using 50 acid are encoded by the genes located at the third and fourth pMW119 Ptac ccrNXA as the template, as well as PtwVPtacf positions, the enzymes of the pathway from Xylonic acid to (SEQ ID NO: 1) and 0819r (SEQ ID NO: 4) as the primers. CKG are encoded by the genes located at the first, second and pTWV228 was digested with SmaI, and used together with fifth positions. The activity of XDH, of which gene is located the PCR fragment of the ccrNXA operon containing the tac at the third position in the operon, had been detected in vitro, promoter region to perform the in-fusion reaction, the E. coli 55 and thus it was considered that if activity of the enzyme JM109 strain was transformed with the reaction product, and encoded by the gene located at the fifth position in the operon the target plasmid pTWVPtac ccrNXA was obtained from a (XylD) could be further detected, it might serve as circum transformant. This plasmid was introduced into the stantial evidence of transcription and translation of the whole MG 1655ASucAstrain, and the obtained strain was cultured in NXA operon. Therefore, it was estimated that one of the three the same manner as that described in the above section (3). 60 reactions from Xylonic acid to C.KG constituted a rate-limit The results are shown in FIG. 4. In the figure, "AsucA' ing point, and the following experiments were conducted. represents the MG 1655AsucA strain, and “?pTWV and “/v” Plasmids pSTVPtac xylD Ttrp, pSTVPtac xylx Ttrp, mean that the strain harbored pTWV228. Furthermore, and pSTVPtac ccrxylA Ttrp that express the xylD, xylX. pTWV110 to pTWV 119 indicate clone numbers of and ccrxylA genes, respectively, were prepared as follows. pTWV228Ptac ccrNXA. 65 pSTV28-Ptac-xylx-Ttrp was prepared by constructing an When only glucose was used as the carbon Source, the xylX fragment by PCR using puC18-xylX, which is a plas medium copy number ccrNXA operon-expressing strain pro mid prepared by cloning xylX lacking the Sfil site into US 9,045,789 B2 35 36 pUC18, as the template, as well as xylx-IFS-5742-10-1 (SEQ as well as CC0819-02F 5659-9-2 (SEQ ID NO: 19) and ID NO:38) and xylx-IFA-5742-10-2 (SEQID NO:39) as the CC0819-02R 4691-88-10 (SEQID NO: 20) as the primers, primers, and cloning the obtained plasmid into pSTV28-Ptac fragments of 1130 bp and 653 bp were amplified, respec Ttrp which had been digested with SmaI by the in-fusion tively. Then, puC18 digested with SmaI, and two of the cloning method. aforementioned amplified fragments were assembled by the pSTV28-Ptac-ccrxylA-Ttrp was prepared by constructing in vitro assembly method (Nature Methods, 6(5), 343-345, accrxylA fragment by PCR using puC18-ccrxylA, which is 2009) to obtain puC18-xylD in which the Sfil site was a plasmid prepared by cloning ccrXylA lacking the Sfil site removed, and the XylD gene was inserted. into puC18, as the template, as well as xylA IFS 5742-10-3 Crude enzyme extracts were prepared from the ccrNXA (SEQ ID NO: 40) and xylA IFA 5742-10-4 (SEQ ID NO: 10 41) as the primers, and cloning the obtained plasmid into operon-expressing strain (MG1655AsucA/ pSTV28-Ptac-Ttrp digested with SmaI by the in-fusion clon pTWV228Ptac ccrNXA), and the strains harboring each of ing method. the plasmids expressing one of the aforementioned XylD. pSTV28-Ptac-xylD-Ttrp was prepared by constructing an xylX, and ccrxylA genes, respectively (MG1655ASucA/ xylD fragment by PCR using puC18-xylD, which is a plas 15 pSTVPtac xylD Ttrp, MG1655AsucA/pSTVPtac xylX T mid prepared by cloning xylD lacking the Sfil site into trp, and MG 1655ASucA/pSTVPtac ccrxylA Ttrp), and then pUC18, as the template, as well as xylD IFS 5742-10-5 each of the crude enzyme extracts of the strain harboring only (SEQ ID NO: 42) and xylD IFA 5742-10-6 (SEQ ID NO: the vector, or the strains expressing one of the XylD, Xyl X, and 43) as the primers, and cloning the obtained plasmid into ccrXylA genes was added to the crude enzyme extract of the pSTV28-Ptac-Ttrp digested with SmaI by the in-fusion clon ccrNXA operon-expressing strain, and the activity for pro ing method. ducing CKG from Xylonic acid of each mixture was mea The aforementioned plasmids puC18-xylX. pUC18 sured. The results are shown in Table 6. In Table 6, “1110 ccrxylA, and pUC18-xylD were prepared as described below, next to the strain name is the clone number. respectively. By PCR using the genomic DNA of the C. Crescentus 25 TABLE 6 CB-15 strain as the template, CC0823-01F 4691-87-1 (SEQ ID NO:44) and CC0823-01R 4691-87-2 (SEQID NO: 45), Measurement results of activity for producing CKG from Xylonic acid as well as CC0823-02F 4691-87-3 (SEQ ID NO: 46) and O4 sucAlpTWVPtac ccrNXA 1110 CC0823-02R 4691-87-4 (SEQ ID NO: 47) as the primers, (2) JM109 DSTVPiac Ttrip fragments of 900 bp and 280 bp were amplified, respectively. 30 Then, puC18 digested with SmaI, and two of the aforemen CFE (O -- O)) None xylD xy|X ccrxylA tioned amplified fragments were assembled by the in vitro Specific activity 1.35 3.48 1.34 1.91 assembly method (Nature Methods, 6(5), 343-345, 2009) to (mol/min.img-protein) obtain puC18-xylX in which the Sfil site was removed, and 1.O 2.6 1.O 1.4 the XylX gene was inserted. 35 Note: By PCR using the genomic DNA of the C. Crescentus The relative activity is indicated as relative activity based on the specific activity of the CB-15 strain as the template, CC0822-01F 4691-87-5 (SEQ ccrNXA1110 and pSTV28-Ptac-Ttrp mixed system taken as 1. ID NO: 48) and CC0822-01 R 5659-8-7 (SEQID NO: 49), When the crude enzyme extract of the strain expressing CC0822-02F 5659-8-8 (SEQ ID NO: 50) and CC0822 only the XylD gene was added to the system, an increase in the 02R 5659-8-9 (SEQID NO:51), CC0822-03F 5659-8-10 40 CKG-producing activity was observed. From this result, it (SEQ ID NO. 52) and CC0822-03R 5659-8-11 (SEQ ID was suggested that the Xylonate dehydratase (XylD) encoded NO: 53), CC0822-04F 5659-8-12 (SEQ ID NO. 54) and by the xylD gene constituted a rate-limiting point of the NXA CC0822-04R 5659-8-13 (SEQ ID NO: 55), as well as pathway constructed in E. coli by heterogenous expression. CC0822-05F 5659-8-14 (SEQ ID NO. 56) and CC0822 Since the metabolic flux of the whole pathway might be 05R 4691-87-14 (SEQID NO:57) as the primers, five frag 45 improved by further enhancing the XylD gene expression in ments, 11 v02 (175 bp), 12v02 (325 bp), 13 v02 (260 bp), the ccrNXA operon-expressing strain as suggested by the 14v02 (193 bp), and 15v02 (544 bp), were amplified, respec measurement of the enzymatic activity, a strain was con tively. Then, two of the fragments, 11 v02 and 12v02, were structed with increased expression of the xylD gene by intro ligated by crossover PCR using these two fragments as the ducing a XylD gene expression vector into the ccrNXA template, as well as CC0822-01 F 4691-87-5 and CC0822 50 operon-expressing strain, and evaluating L-glutamic acid 02R 5659-8-9 as the primers. Similarly, two of the frag production using glucose and Xylose as the carbon Source. As ments, 13 v02 and 14v02, were ligated by crossover PCR the ccrNXA operon-expressing strain, MG 1655ASucA/ using these two fragments as the template, as well as pMW1 19Ptac ccrNXA and MG1655ASucA/ CC0822-03F 5659-8-12 and CCO822-04R 5659-8-13 as pTWV228Ptac ccrNXA were used. the primers. These two of fragments and the aforementioned 55 The culture was performed in the same manner as that 15v02 fragment were ligated by the in vitro assembly method described in the aforementioned section (3). (Nature Methods, 6(5), 343-345, 2009). The obtained ligated The results are shown in Table 7. The strain with increased fragment was amplified by PCR using it as the template, as expression of the XylD gene showed markedly improved well as CCO822-01 F 4691-87-5 and CCO822-05R 4691 L-glutamic acid accumulation and yield. L-Glutamic acid 87-14 as the primers. Then, puC18 digested with SmaI, and 60 accumulation was 23 to 25 g/L in contrast to 15 to 16 g/L in the aforementioned ligated fragment was assembled by the in the control strain, and the yield based on the consumed sac vitro assembly method (Nature Methods, 6(5), 343-345, charide reached 57 to 60% in contrast to 37 to 40% for the 2009) to obtain puC18-ccrxy1A in which the Sfil site was control Strain. Xylonic acid, which is a metabolic intermedi removed, and the ccrXylA gene was inserted. ate, was not seen in the XylD gene expression-enhanced By PCR using the genomic DNA of the C. Crescentus 65 strain. On the other hand, the effect of enhancing xylD gene CB-15 strain as the template, CC0819-01F 4691-88-7 (SEQ expression was seen only in the expression strain carrying a ID NO: 17) and CC0819-01R 5659-9-1 (SEQID NO: 18), medium copy number type ccrNXA operon expression vec US 9,045,789 B2 37 38 tor, and the effect was not seen in the expression strain car Peftu XylxABCD fw (SEQID NO: 60) and Peftu xylxAB rying the low copy number type Vector. From these results, it CD ry (SEQID NO: 61) to obtain a fragment containing the was considered that removal of the rate-limiting point of this pathway by increasing the activity of the whole NXA path xylXABCD sequence of C. Crescentus. This PCR was per way by increasing the copy number of the vectors and further formed by using PrimeSTAR GXL Polymerase according to enhancement of xylD gene expression provided the improve the protocol attached to this enzyme. ment in the amount of L-glutamic acid produced. In addition, Then, the Peftu fragment and the fragment containing the activity for producing CKG from Xylonic acid of thexylD gene expression-enhanced strain was increased about 10 xylxABCD obtained above were mixed with pVK9 treated times as compared to that observed before the enhancement 10 with PstI and BamHI, and used to perform the in-fusion (Table 8). The numerals “1110”, “17” and “19' next to the reaction according to the protocol of Clontech In-fusion HD strain names mentioned in Tables 7 and 8 are the clone num Cloning Kit. pVK9 is a shuttle vector obtained by blunt bers. ending pHSG299 (Takara Bio) at the AvalI site, and inserting

TABLE 7 15 a region autonomously replicable in coryneform bacteria contained in pHK4 (Japanese Patent Laid-open No. Result of evaluation of ccrNXA operon + xylD expressing strain by L Glu production culture 05-007491), which was excised with BamHI and KpnI, and blunt-ended (Japanese Patent Laid-open No. 2007-97573, L-Glu Strain (g/L) Yield (%) O.D.600 U.S. Patent Published Application No. 2005/0196846). E. MG1655AsucApTWV228 15.7 37.6 23.9 coli JM109 was transformed with the in-fusion reaction mix MG1655AsucApTWVccrNXA1110 16.3 39.2 16.6 ture. The transformants were subjected to selection on anagar MG1655AsucApTWVccrNXA/ 16.3 39.2 22.9 pSTVPtacTtrp medium containing the LB medium Supplemented with kana MG1655AsucApTWVccrNXA/xylD17 25.4 61.1 14.3 mycin at a final concentration of 50 mg/L. The target plasmid MG1655AsucApTWVccrNXA/xylD19 24.2 S8.1 16.0 25 pVK9Peftu ccrNXA was obtained from an obtained trans formant. TABLE 8 (2) L-Glutamic Acid Production by Corynebacterium glutamicum Introduced with NXA Pathway Results of measurement of activity for producing CKG from Xylonic acid 30 The C. glutamicum ATCC 13869 strain was transformed Specific activity (mol/min Relative with the aforementioned pVK9Peftu ccrNXA by the electric Strain mg-protein) activity pulse method (Japanese Patent Laid-open No. 2-2077.91). A MG1655AsucApTWV228/pSTVPtacTtrip ND 35 strain introduced with pVK9Peftu ccrNXA was selected on MG1655AsucApTWVccrNXA1110 1.9 1.O an agar medium comprising the CM-Dex medium Supple MG1655AsucApTWVccrNXApSTVxylD17 2O2 10.8 MG1655AsucApTWVccrNXApSTVxylD19 17.1 9.2 mented with kanamycin at a final concentration of 25 mg/L. Furthermore, the C. glutamicum ATCC13869 strain trans Note: The results for MG 1655AsucAlpTWVccrNXA1110 are values obtained in experiments formed with pVK9 was also selected in a similar manner as a using different batches. 40 control Strain. ND: Not Detected Note: Xylose-assimilating ability and L-glutamic acid-produc The relative activity is indicated as relative activity based on the specific activity of ccrNXA1110 taken as 1. ing ability of the obtained transformants were verified by performing culture using a Sakaguchi flask. Cells of each Example 3 45 transformant strain cultured at 31.5° C. for 24 hours on the CM-DeX agar medium Supplemented with kanamycin at a Introduction of NXA Pathway into Corynebacterium final concentration of 25 mg/L were scraped offin an amount glutamicum corresponding to /6 of the plate, and inoculated into 20 mL of 50 the Glc medium contained in a Sakaguchi flask, 1 g of cal Construction of Plasmid pVK9Peftu ccrNXA for Intro cium carbonate sterilized beforehand with hot air was added, duction of NXA Pathway and shaking culture was performed at 31.5° C. and 120 rpm A plasmid having a sequence containing the promoter for 24 hours. The obtained culture medium in a volume of 1 sequence of the elongation factor Tu (EF-Tu) gene, tuf mL was inoculated into 20 mL the Xyl medium (biotin (WO2008/114721, SEQID NO:33, henceforth referred to as 55 restricted) contained in a Sakaguchi flask, 1 g of calcium “Peftu') and xylD ligated downstream from the promoter carbonate sterilized beforehand with hot air was added, and sequence was constructed by using Clontech In-Fusion Clon shaking culture was performed at 31.5°C. and 120 rpm for 73 ing HD Kit (Clontech). First, PCR was performed by using hours. The results are shown in Table 9. the chromosomal DNA of the C. glutamicum ATCC 13869 60 The C. glutamicum ATCC 13869 strain introduced with strain as the template, as well as primers Peftu(Pst) (SEQID pVK9 hardly grew in the Xyl medium, and did not produce NO: 58) and Peftu Rv (SEQID NO. 59) to obtain a fragment L-glutamic acid, either. On the other hand, the C. glutamicum containing the Peftu sequence. This PCR was performed by ATCC 13869 strain containing pVK9Peftu ccrNXA grew in using PrimeSTAR HS Polymerase according to the protocol the Xyl medium, and produced L-glutamic acid. From these attached to this enzyme. 65 results, it was found that introduction of the NXA pathway Furthermore, PCR was performed by using into coryneform bacteria improved Xylose-assimilating abil pTWV228Ptac ccrNXA as the template, as well as primers ity, and Such a strain produced L-glutamic acid from Xylose. US 9,045,789 B2 40 TABLE 9

Consumed OD62O Xyl Glu Yield Xylonic acid Strain (x101) (gL) (g/L) (%) (gL) ATCC13869 pVK9 O.048 O.O13 O O O O ATCC13869, O404 - O.O1O 57.1 O.22 2.5 + 0.3 4.3 O. 445 1.5 pVK9Peftu ccrNXA

10 (3) Construction of Strain in which xylD Gene Expression nomycin resistance gene was obtained from an obtained is Further Enhanced transformant. Furthermore, PCR was performed by using Since the C. glutamicum ATCC 13869 strain harboring pVC7-spc as the template, as well as primers spc(GTG pVK9Peftu ccrNXA was able to produce xylonic acid, it was start)-F (SEQID NO: 67) and spc(stop)-R (SEQID NO: 68) assumed that the activity of the XylD gene product was insuf 15 to amplify the spectinomycin resistance gene. ficient. Therefore, a plasmid expressing of the NXA pathway Separately, PCR was performed by using pVC7 as the was constructed in which thexylD gene was further enhanced template, as well as primers Spc-pVC7-Cm-F (SEQID NO: by introducing one more copy of xylD gene into the pVK9 69) and Spc-pVC7-Cm-R (SEQID NO: 70) to obtain a DNA Peftu ccrNXA plasmid. fragment containing pVC7 in which the chloramphenicol (4) Construction of Plasmid pVS7PmsrA xylD for xylD resistance gene was removed. This DNA fragment and the Gene Expression DNA fragment of the spectinomycin resistance gene obtained A plasmid having a sequence containing the promoter above from pVC7-spc were mixed, and used to perform the sequence of the misrA gene (peptide methionine Sulfoxide in-fusion reaction according to the protocol of Clontech In reductase A) (henceforth referred to as “PrmsrA) and the fusion HD Cloning Kit, and E. coli JM109 was transformed XylD gene of C. Crescentus ligated downstream from the 25 promoter sequence was constructed by using Clontech In with the resulting reaction mixture. The transformants were Fusion Cloning HD Kit. Subjected to selection on an agar medium containing the LB First, PCR was performed by using the chromosomal DNA medium Supplemented with spectinomycin at a final concen of the C. glutamicum ATCC 13869 strain as the template, as tration of 25 mg/L. pVS7 was obtained from an obtained transformant. well as primers PmsrA(Pst) (SEQ ID NO: 62) and PmsrAR 30 (SEQID NO: 63) to obtain a fragment containing the PmsrA (5) Construction of the PlasmidpVK9Peftu ccrNXA+D in sequence. Furthermore, PCR was performed by using which the xylD Gene is Further Enhanced pTWV228Ptac ccrNXA as the template, as well as primers The DNA fragment containing the spectinomycin resis PmsrA xylD fw (SEQ ID NO: 64) and Peftu xylxAB tance gene (Spc) and PmsrA XylD was amplified by using CD ry to obtain a fragment containing the XylD gene 35 pVS7PmsrA XylD as the template, as well as primers sequence of C. crescentus. These PCRs were performed by ME Spc fow (SEQ ID NO: 71) and ME Peftu xylxAB using PrimeSTAR HS Polymerase according to the protocol CD ry (SEQ ID NO: 72). The obtained DNA fragment was attached to this enzyme. inserted into pVK9Peftu ccrNXA in vitro according to the Then, the fragments containing the PmsrA fragment and protocol of EZ-Tn5TM Custom Transposome Construction the xylD gene obtained above were mixed with the shuttle 40 Kit (Epicentre), and the resultant was used to transform E. vector pVS7 (construction method is shown below) treated coli DH5a. The transformants were subjected to selection on with PstI and BamHI, and used to perform the in-fusion an agar medium containing the LB agar medium Supple reaction according to the protocol of Clontech In-fusion HD mented with kanamycin and spectinomycinata final concen Cloning Kit, and then E. coli JM109 was transformed with tration of 50 mg/L and 25 mg/L, respectively. Plasmids were this reaction mixture. The transformants were subjected to 45 extracted from the obtained transformants, and a plasmid for selection on an agar medium containing the LB medium which it was confirmed that the insertion site of the Spc Supplemented with spectinomycin at a final concentration of PmsrA xylD sequence was not on the Peftu ccrNXA 25 mg/L. The target plasmidpVS7PmsrA xylD was obtained sequence by analysis of nucleotide sequence around the Spc from an obtained transformant. PmsrA xylD Sequence WaS designated pVS7 is a plasmid obtained by replacing the chlorampheni 50 pVK9Peftu ccrNXA+D. col resistance gene of pVC7 (Japanese Patent Laid-open No. (6) L-Glutamic Acid Production by a Strain Containing the 2000-201692, European Patent No. 1004671) with a specti NXA Pathway and in which the xylD Gene is Further nomycin resistance gene. The spectinomycin resistance gene Enhanced can be obtained by preparing a plasmid p)G1726 from the pVK9Peftu ccrNXA+D was introduced into the C. Escherichia coli ECE 101E strain sold by Bacillus Genetic 55 glutamicum ATCC 13869 strain by the electric pulse method, Stock Center (BGSC), and taking out the resistance gene and the cells were applied to the CMDex agar medium con from the plasmid as a cassette. By PCR using ploG1726 as the taining 25 mg/L of kanamycin. L-Glutamic acid-producing template, as well as primers SpcR-F (SEQ ID NO: 65) and ability of a strain grown after culture at 31.5°C. was verified SpcR-R (SEQID NO: 66), the spectinomycin resistance gene in the same manner as that of the aforementioned section (2). was amplified. The obtained gene fragment was mixed with 60 The results are shown in Table 10. pVC7 treated with SmaI, a ligation reaction was performed The strain harboring pVK9Peftu ccrNXA+D accumu according to the protocol of Ligation Mix of lated D-Xylonic acid in a smaller amount, but accumulated Takara Bio, and E. coli JM109 was transformed with this L-glutamic acid in a larger amount as compared to the strain reaction mixture. The transformants were subjected to selec harboring pVK9Peftu ccrNXA. By this result, it was dem tion on an agar medium containing the LB medium Supple 65 onstrated that L-glutamic acid could be more efficiently pro mented with spectinomycin at a final concentration of 25 duced from D-xylose via the NXA pathway by further mg/L. pVC7-spc containing pVC7 inserted with the specti enhancing the XylD gene. US 9,045,789 B2 41 42 TABLE 10 L-Glutamic acid production of xylD gene-further enhanced NXA pathway-introduced strain Consumed Xylonic OD62O Xyl L-Glu Yield acid Strain (x101) (g/L) (g/L) (%) (g/L) ATCC13869, O.297 O.OO4 58.3 - 0.11 4.2. O.1 7.10.1 SO2 + 0.1 pVK9Peftu ccrNXA ATCC13869, O.351 O.OO2 71.9 0.66 27.5 + 0.1 38.2O4. 22.7 O.4 pVK9Peftu ccrNXA'P

Example 4 TABLE 12-continued 15 Substitution of NXA Pathway Genes SEQID NO SEQ ID NO (Nucleotide (Amino acid In the aforementioned examples, by using the genes XylX, Abbreviation Sequence) sequence) ccrxy1A, ccrxylB, Xylc, and xylD from the known bacterium XylD(Amis) origina 79 8O C. Crescentus, for which the NXA pathway has been reported, 20 optimize 81 it is demonstrated that glutamic acid can be produced from xylD(Aor) One 83 Xylose via the NXA pathway. In this example, homologue xy|X(Atu) origina 85 86 genes of XylD, XylX, and ccrxylA are obtained from biologi- optimize 87 cal species other than C. Crescentus, and it is investigated xy|X(Cne) origina s 89 whether these genes can replace the genes of C. crescentus. 25 xy|X(Selo) Af 91 92 As the gene sources, the biological species described in Table optimize 93 11 were chosen. The gene symbols of the genes (GenBank) XylX(Zga) origina 94 95 are also shown. The sequence identification numbers of the optimize 96 nucleotide sequences of the genes and the amino acid xy|X(Tco) Some 3. 98 sequences encoded by them used in Sequence Listing are 30 xy|X(Art) origina 100 101 shown in Table 12. In Table 12, "original means nucleotide optimize 102 sequence of naturally occurring gene, and "optimized” means XylA(Abr) origina 103 104 nucleotide sequence of which codons are optimized accord- xylA(Hbo) E.Ef E. 107 ing to the codon usage in E. coli. optimize 108 In the following descriptions, the enzymes encoded by 35 ycbD 109 110 homologues of the XylD, XylX, and XylA genes may be referred to as XylD, XylX, and XylA, respectively. TABLE 11 Organism Classification Abbreviation Gene symbol Agrobacterium tumefaciens 5A C-proteobacteria xylD(Atu) EHJ968.30 Herbaspirillum seropedicae B-proteobacteria xylD(Hse) Hsero 4498 Escherichia coi Y-proteobacteria yjhC ECK4286 Escherichia coi Y-proteobacteria yagF ECKO270 Actinoplanes missouriensis Actinobacteria xylD(Amis) AMIS 27920 Aspergilius Oryzae Fungi xylD(Aor) AOR 1412134 Agrobacterium tumefaciens 5A C-proteobacteria xylx(Atu) EHJ96825 Cipriavidits necator B-proteobacteria xylx(Cne) CNE 2CO3420 Pseudomonas elodea Y-proteobacteria xylx(Selo) ZP 09955741 Zobeliia galactanivorans Bacteroidetes XylX(Zga) Zobellia 2318 Thermobacilius composti Firmicutes xylx(Tco) ZP O8919992.1 Arthrobacter globiformis Actinobacteria xylx(Art) ARGLB 037 02150 Azospirilium brasilense C-proteobacteria xylA(Abr) BAE94276.1 Haiomonas boiviensis Y-proteobacteria xylA(Hbo) ZP 09 188044.1 Bacilius subtiis Firmicutes ycbD BSUO2470

55 TABLE 12 (2) Construction of Plasmids for Detecting XylD, XylX, and XylA Activities, pTWVPtac ccrNXA AxylD Km, SEQID NO SEQID NO pTWVPtac ccrNXA Axyl X Km, and pTWVPtac (Nucleotide (Amino acid ccrNXA AccrxylA Km Abbreviation Sequence) Sequence) 60 The construction was performed by using Clontech In Fusion Cloning Kit. XviDyID(Atu) (Atu Sincedoriginal 7573 74 First, by PCR using pTWVPtac ccrNXA Km as the tem xylD(Hse) original 76 77 plate, as well as Ptac xylxABC F (SEQ ID NO; 111) and optimized 78 Ptac xylxABC R (SEQ ID NO: 112) as the primers, the yhC 34 35 65 DNA fragment except for xylD was amplified. The PCR yagF 36 37 product was used to perform the in-fusion reaction according to the protocol of Clontech In-fusion HD Cloning Kit, the E. US 9,045,789 B2 43 44 coli JM109 strain was transformed with the reaction product, sized, codons were optimized so that the fragment is Suitable and the target plasmidpTWVPtac ccrNXA AxylD Km was for expression in E. coli. Equal amounts of pSTV28 and obtained from a transformant. pET 1.2-Ptac-xylD(Atu)-Ttrp, both of which were digested In the same manner as described above, pTWVPtac with EcoRI and Kipni, were mixed, and aligation reaction was ccrNXA Axylx Km was constructed by using Ptac xylAB performed. Then, JM109 was transformed with the ligation CD F (SEQ ID NO: 113) and Ptac xylABCD R (SEQ ID product, and a plasmid was extracted from a colony showing NO: 114) as the primers, and pTWVPtac ccrNXA Accrxy Cm resistance to obtain pSTV28-Ptac-xylD(Atu)-Ttrp. 1A. Kim was constructed by using Ptac xylXBCD F (SEQID Plasmids for expression of xylX(Atu) or xylA(Hbo), NO: 115) and Ptac xylxBCD R (SEQ ID NO: 116) as the pSTV28-Ptac-xylx(Atu)-Ttrp and pSTV28-Ptac-xylA primers. 10 (Hbo)-Ttrip, were also prepared in the same manner. (3) Construction of Pantoea ananatis for Detecting XylD, A plasmid for expression of xylx(Art), pSTV28-Ptac XylX, and XylA Activities xylX(Art)-Ttrp, was prepared as follows. A DNA fragment The Panamatis NA1 strain was transformed with pTWVP having the sequences of tac promoter, XylX(Art), and trp tac ccrNXA AxylD Km described above by the electropo terminator (Ptac-xylX(Art)-Ttrp) was synthesized, and ration method. The constructed strain was referred to as P. 15 ligated with the pCC1 vector (purchased from Epicentre) ananatis NA2 AxylD. For the culture of the Panamatis NA2 digested with EcoRV to obtain pCC1-Ptac-xylX(Art)-Ttrp. AxylD, a plate medium comprising LBGM9 to which kana When the DNA fragment was synthesized, codons were opti mycin and tetracycline were added at final concentrations of mized so that the fragment is suitable for expression in E. coli. 40 mg/L and 12.5 mg/L, respectively, was used. Equal amounts of pSTV28 and pCC1-Ptac-xylX(Art)-Ttrp, In the same manner, the Panamatis NA1 Strain was trans both of which were digested with EcoRI and Kipni, were formed with pTWVPtac ccrNXA Axylx Km or pTWVP mixed, and ligation reaction was performed. Then, JM109 tac ccrNXA AccrxylA Km to construct Panamatis NA2 was transformed with the ligation product, and a plasmid was AxylX strain and Panamatis NA2 AccrxylA strain. extracted from a colony showing Cm resistance to obtain (4) Construction of xylD, xylX, and xylA Homologue pSTV28-Ptac-xylx(Art)-Ttrp. Expression Plasmids 25 (5) Detection of Activities of XylD Homologues Plasmids for expression of yhC or yagF, pSTV28-Ptac The Panamatis NA2 AxylD strain was transformed with yhC-Ttrp and pSTV28-Ptac-yagF-Ttrp, were prepared as pSTV28-Ptac-Ttrp, pSTV28-Ptac-xylD-Ttrp, pSTV28-Ptac follows. xylD(Atu)-Ttrp, pSTV28-Ptac-xylD(Hse)-Ttrp, pSTV28 pSTV28-Ptac-yhC-Ttrp was prepared by amplifying a Ptac-yhC-Ttrp, pSTV28-Ptac-yagF-Ttrp, pSTV28-Ptac yhC fragment by PCR using the genomic DNA of the E. coli 30 xylD(Amis)-Ttrp, or pSTV28-Ptac-xylD(Aor)-Ttrp by the MG 1655 strain as the template, as well as yhC F (SEQ ID electroporation method (refer to U.S. Pat. No. 6,682,912). For NO: 117) and yhC R (SEQID NO: 118) as the primers, and the culture of the transformants, a plate medium comprising cloning the amplified fragment into pSTV28-Ptac-Ttrp LBGM9 to which kanamycin, tetracycline and chloram digested with SmaI according to the in-fusion cloning phenicol were added at final concentrations of 40 mg/L, 12.5 method. 35 mg/L and 25 mg/L, respectively, was used. pSTV28-Ptac-yagF-Ttrp was prepared in the same manner Cells of each transformant cultured overnight at 34°C. on as that described above by using yagF F (SEQID NO: 119) the LBGM9 plate to which the drugs were added were and yagF R (SEQID NO: 120) as the primers. scraped offin an amount corresponding to "/6 of the cells on A plasmid for expression of xylD(Hse), pSTV28-Ptac the plate, inoculated into 5 ml of the MSII-SX medium con xylD(Hse)-Ttrp, was prepared as follows. A DNA fragment 40 tained in a large test tube, and cultured at 34° C. and 120 rpm having the sequences of tac promoter, XylD(Hse), and trp for 48 hours, and amount of accumulated L-glutamic acid terminator (Ptac-XylD(Hse)-Ttrp) was synthesized, and (Glu) was measured. The results are shown in FIG. 5. ligated with the puC57 vector (purchased from Thermo Fis Whereas the Panamatis NA2 AxylD strain introduced with cher Scientific) digested with EcoRV to obtain pUC57-Ptac pSTV28-Ptac-Ttrp accumulated 6.9 g/L of L-glutamic acid, xylD(Hse)-Ttrp. When the DNA fragment was synthesized, 45 the other transformants accumulated 11.1 to 23.1 g/L of codons were optimized so that the fragment is Suitable for L-glutamic acid. In the strain introduced with pSTV28-Ptac expression in E. coli. Equal amounts of pSTV28 and puC57 Ttrp, L-glutamic acid was hardly produced from Xylose, and Ptac-xylD(Hse)-Ttrp, both of which were digested with thus it was considered that L-glutamic acid was produced EcoRI and Kipni, were mixed, and ligation reaction was per from Xylose via the NXA pathway in the other transformants. formed. Then, JM109 was transformed with the ligation prod 50 That is, it was demonstrated that a xylD homologue derived uct, and a plasmid was extracted from a colony showing Cm from any of the biological species other than C. Crescentus resistance to obtain pSTV28-Ptac-xylD(Hse)-Ttrp. could substitute for xylD. Plasmids for expression of xylD(Amis), xylD(Aor), xylX (6) Detection of Activities of XylX Homologues (Cne), xylX(Zga), xylX(Tco), xylA(Abr), and ycbD, The Panamatis NA2 AxylX strain was transformed with pSTV28-Ptac-xylD(Amis)-Ttrp, pSTV28-Ptac-xylD(Aor)- 55 pSTV28-Ptac-Ttrp, pSTV28-Ptac-xylx-Ttrp, pSTV28-Ptac Ttrp, pSTV28-Ptac-xylx(Cne)-Ttrp, pSTV28-Ptac-xylx xylx(Art)-Ttrp, pSTV28-Ptac-xylx(Atu)-Ttrp, pSTV28 (Zga)-Ttrp, pSTV28-Ptac-xylx(Tco)-Ttrp, pSTV28-Ptac Ptac-xylX(Cne)-Ttrp, pSTV28-Ptac-xylx(Zga)-Ttrp, xylA(Abr)-Ttrp, and pSTV28-Ptac-ycbD-Ttrp, respectively, pSTV28-Ptac-xylx(Tco)-Ttrp, orpSTV28-Ptac-xylx(Selo)- were also prepared in the same manner. Codon optimization Ttrp by the electroporation method. For the culture of the was not carried out for YcbD. 60 transformants, a plate medium comprising LBGM9 to which A plasmid for expression of xylD(Atu), pSTV28-Ptac kanamycin, tetracycline and chloramphenicol were added at xylD(Atu)-Ttrp, was prepared as follows. A DNA fragment final concentrations of 40 mg/L, 12.5 mg/L and 25 mg/L. having the sequences of tac promoter, XylD(Atu), and trp respectively, was used. terminator (Ptac-xylD(Atu)-Ttrp) was synthesized, and Cells of each transformant cultured overnight at 34°C. on ligated with the pET 1.2 vector (purchased from Thermo 65 the LBGM9 plate to which the drugs were added were Fischer Scientific) digested with EcoRV to obtain pET 1.2- scraped offin an amount corresponding to "/6 of the cells on Ptac-xylD(Atu)-Ttrp. When the DNA fragment was synthe the plate, inoculated into 5 ml of the MSII-SX medium con US 9,045,789 B2 45 46 tained in a large test tube, and cultured at 34°C. and 120 rpm the plate, inoculated into 5 ml of the MSII-SX medium con for 48 hours, and the amount of accumulated L-glutamic acid tained in a large test tube, and cultured at 34° C. and 120 rpm (Glu) was measured. The results are shown in FIG. 6. for 48 hours, and amount of accumulated L-glutamic acid Whereas the Panamatis NA2AXylX strain introduced with (Glu) was measured. The results are shown in FIG. 7. 5 Whereas the Panamatis NA2 AccrxylA strain introduced pSTV28-Ptac-Ttrp accumulated 8.7 g/L of L-glutamic acid, with pSTV28-Ptac-Ttrp accumulated 1.0 g/L of L-glutamic the other transformants accumulated 20.5 to 27.8 g/L of acid, the other transformants accumulated 18.1 to 30.7 g/L of L-glutamic acid. In the strain introduced with pSTV28-Ptac L-glutamic acid. In the strain introduced with pSTV28-Ptac Ttrp, L-glutamic acid was hardly produced from Xylose, and Ttrp, L-glutamic acid was hardly produced from Xylose, and thus it was considered that L-glutamic acid was produced 10 thus it was considered that L-glutamic acid was produced from Xylose via the NXA pathway in the other transformants. from Xylose via the NXA pathway in the other transformants. That is, it was demonstrated that a xylX homologue derived That is, it was demonstrated that a ccrXylA homologue from any of the biological species other than C. Crescentus derived from any of the biological species other than C. could substitute for xylx. crescentus could substitute for xylA. (7) Detection of Activities of XylA Homologues 15 The Panamatis NA2 AccrxylA strain was transformed INDUSTRIAL APPLICABILITY with pSTV28-Ptac-Ttrp, pSTV28-Ptac-ccrxylA-Ttrp, pSTV28-Ptac-ycbD-Ttrp, pSTV28-Ptac-xylA(Hbo)-Ttrp, or According to the present invention, a target Substance can pSTV28-Ptac-xylA(Abr)-Ttrp by the electroporation be efficiently produced by fermentation using a Xylose raw method. For the culture of the transformants, a plate medium material. containing LBGM9 to which kanamycin, tetracycline and While the invention has been described in detail with ref chloramphenicol were added at final concentrations of 40 erence to preferred embodiments thereof, it will be apparent mg/L, 12.5 mg/L and 25 mg/L, respectively, was used. to one skilled in the art that various changes can be made, and Cells of each transformant cultured overnight at 34°C. on equivalents employed, without departing from the scope of the LBGM9 plate to which the drugs were added were the invention. Each of the aforementioned documents is scraped offin an amount corresponding to "/6 of the cells on incorporated by reference herein in its entirety.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 12O

<21 Oc SEO ID NO 1 <211 LENGTH: 4 O <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <22 Os FEATURE; OTHER INFORMATION: primer PtwvPtacf <4 OOs SEQUENCE: 1

gaatt cqagc ticggtaccca gatct coct g ttgacaatta 4 O

SEO ID NO 2 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: primer O323Ptacr <4 OOs SEQUENCE: 2

aggaatt cac to acgcc cac cct cotgtgt galaattgtta 4 O

SEO ID NO 3 LENGTH: 39 TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: primer Ptaco 823f <4 OOs SEQUENCE: 3

taacaattt C acacaggagg gtgggcgtga gtgaatt CC 39

SEO ID NO 4 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: primer O319r US 9,045,789 B2 47 48 - Continued

<4 OOs, SEQUENCE: 4 gcgggg.cgt.g. C9gttaga.ca toggacct catgctgggg 4 O

<210s, SEQ ID NO 5 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer og 19f <4 OOs, SEQUENCE: 5

CCCC agcatg aggtocgc.ca ttct aaccg cacgc.ccc.gc 4 O

<210s, SEQ ID NO 6 &211s LENGTH: 36 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer 219 cc O819r <4 OOs, SEQUENCE: 6

Ctctagagga t c ccct tcag cqtttggcga C9gaga 36

<210s, SEQ ID NO 7 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer 21.9f < 4 OO SEQUENCE: 7 ggggat.cctic tagagt cac C 21

<210s, SEQ ID NO 8 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer 219r <4 OOs, SEQUENCE: 8 gggt accgag Ctcgaattica C 21

<210s, SEQ ID NO 9 &211s LENGTH: 35 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer attL-F <4 OOs, SEQUENCE: 9 tatattgatt cactitgaagt acgaaaaaaa ccggg 35

<210s, SEQ ID NO 10 &211s LENGTH: 45 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer ntpII-R

<4 OOs, SEQUENCE: 10

Cctgcaggcg gcc.gct cata gaaggcggcg gtggaatcga aat Ct 45

<210s, SEQ ID NO 11 &211s LENGTH: 45 US 9,045,789 B2 49 - Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer attR-F <4 OOs, SEQUENCE: 11 gcggcc.gc.ct gcaggcc.cat gtaatgaata aaaag cagta attaa 45

<210s, SEQ ID NO 12 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer attR-R <4 OOs, SEQUENCE: 12 tgaagcggcg cacgaaaaac gcgaaag.cgt. 3 O

<210s, SEQ ID NO 13 &211s LENGTH: 8O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer Ap-Km-fw <4 OOs, SEQUENCE: 13 ttaccalatgc titaatcagtg aggcaccitat ct cagogatc tdtct atttic aagctt cacg 6 O

Ctgcc.gcaag cacticagggc 8O

<210 SEQ ID NO 14 &211s LENGTH: 8O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer Ap-Km-rv

<4 OOs, SEQUENCE: 14 atgagtatt c aacatttic cq tdtcgc.cctt attcc ctittt ttgcggcatt cqct cataga 6 O aggcggcggit gaatcgaala 8O

<210s, SEQ ID NO 15 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylD IFS 5742-10-5 <4 OOs, SEQUENCE: 15 acacaaggag act cocatgt Cta accgcac gcc.ccgc.cgg 4 O

<210s, SEQ ID NO 16 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylD IFS 5742-10-6

<4 OOs, SEQUENCE: 16 ggaactggcg gct Coctoag tigttgttggc ggggcagctt 4 O

<210s, SEQ ID NO 17 &211s LENGTH: 45 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: US 9,045,789 B2 51 52 - Continued <223> OTHER INFORMATION: primer CCO819 - O1F 4691-88-7 <4 OOs, SEQUENCE: 17 gtcgacticta gaggat CCCC atgtc.t.aacc gcacgcc.ccg ccggit 45

<210s, SEQ ID NO 18 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO819 - O1R 5659-9-1 <4 OOs, SEQUENCE: 18 aaccaggaac ccggcc ttct ctdc 24

<210s, SEQ ID NO 19 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCo819 - O2F 5659-9-2 <4 OOs, SEQUENCE: 19 tcc.cgtacca cago.cgctg gcag 24

<210s, SEQ ID NO 2 O &211s LENGTH: 44 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO819 - O2R 4691-88-10 <4 OOs, SEQUENCE: 2O cgaatticgag Ctcggit accc ticagtggttg tdgcggggga gCtt 44

<210s, SEQ ID NO 21 &211s LENGTH: 68 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylA-H1P1-5742-5-1

<4 OOs, SEQUENCE: 21 acga catcat coat cacccg cqgcattacc tdattatgga gttcaatatg tdaagcc togc 6 O ttitt titat 68

<210s, SEQ ID NO 22 &211s LENGTH: 68 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylA-H2P2-5742-5-2

<4 OOs, SEQUENCE: 22 atcgggccaa C9gactgcac agittagcc.gt tatttgtcga acagataatg cgctcaagtt 6 O agtataaa 68

<210s, SEQ ID NO 23 &211s LENGTH: 829 6 &212s. TYPE: DNA <213s ORGANISM: Caulobacter crescentus 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1175) . . (2329 22 Os. FEATURE:

US 9,045,789 B2 67 68 - Continued

Gly Wall Arg Asp Ser His Trp Asn Asn Pro Glu Pro Glu Wall Wall 195

Lell Luell Asp Gly Ser Gly Luell Ile Arg Gly Ala Ala Luell Gly Asn 21 O 215

Asp Wall Asn Luell Arg Asp Phe Glu Gly Arg Ser Ala Lell Luell Luell Ser 225 23 O 235 24 O

Ala Asp Asn Asn Ala Ser Ala Ile Gly Pro Phe Phe Arg 245 250 255

Lell Phe Asp Glu Thir Phe Gly Luell Asp Asp Wall Arg Ser Ala Glu Wall 26 O 265 27 O

Glu Luell Lys Ile Thir Gly Arg Asp Asn Phe Wall Lell Asp Gly Ser 27s 285

Asn Met Ser Luell Ile Ser Arg Asp Pro Ala Wall Lell Ala Gly Glin Ala 29 O 295 3 OO

Tyr Gly Glin His Glin Pro Asp Gly Phe Ala Lell Phe Luell Gly 3. OS 310 315

Thir Met Phe Ala Pro Ile Glin Asp Arg Asp Thir Pro Gly Glin Gly Phe 3.25 330 335

Thir His Wall Gly Asp Arg Wall Arg Wall Ser Thir Pro Lys Luell Gly 34 O 345 35. O

Wall Luell Glu Asn Glu Wall Thir Thir Asp Ala Lys Pro Trp Thir 355 360 365

Phe Gly Ile Ser Ala Lell Ile Arg Asn Luell Ala Gly Arg Gly Luell Luell 37 O 375 38O

SEO ID NO 25 LENGTH: 478 TYPE : PRT ORGANISM: Caulobacter Crescentus

< 4 OOs SEQUENCE: 25

Met Thr Asp Thir Lell Arg His Ile Gly Gly Glu Arg Wall Ala Ala 1. 5 15

Asp Ala Pro Ala Glu Ser Lell Asn Pro Ser ASn Thir Asn Asp Wall Wall 2O 25

Ala Wall Pro Met Gly Gly Glin Ala Glu Wall Asp Ala Ala Wall Asp 35 4 O 45

Ala Ala Arg Ala Phe Pro Ala Trp Ala Asp Ala Ser Pro Glu Wall SO 55 6 O

Arg Ser Asp Luell Lell Asp Wall Gly Ser Thir Ile Ile Ala Arg Ser 65 70

Ala Asp Ile Gly Arg Lell Lell Ala Arg Glu Glu Gly Thir Luell Ala 85 90 95

Glu Gly Ile Gly Glu Thir Wall Arg Ala Gly Arg Ile Phe Lys Phe 105 11 O

Ala Gly Glu Ala Lell Arg Arg His Gly Glin ASn Lell Glu Ser Thir Arg 115 12 O 125

Pro Gly Wall Glu Ile Glin Thir Arg Glin Ala Wall Gly Wall Gly 13 O 135 14 O

Lell Ile Thir Pro Trp Asn Phe Pro Ile Ala Ile Pro Ala Trp Ala 145 150 155 160

Ala Pro Ala Luell Ala Phe Gly Asn Thir Wall Wall Ile Pro Ala Gly 1.65 17O 17s

Pro Thir Pro Ala Thir Ala Asn Wall Luell Ala Asp Ile Met Ala Glu Cys 18O 185 19 O US 9,045,789 B2 69 70 - Continued

Gly Ala Pro Ala Gly Wall Phe Asn Met Luell Phe Gly Arg Gly Ser Met 195

Gly Asp Ala Luell Ile His Asp Wall Asp Gly Wall Ser Phe Thir 21 O 215 22O

Gly Ser Glin Gly Wall Gly Ala Glin Wall Ala Ala Ala Ala Wall Ala Arg 225 23 O 235 24 O

Glin Ala Arg Wall Glin Lell Glu Met Gly Gly Asn Pro Luell Ile Wall 245 250 255

Lell Asp Asp Ala Asp Lell Glu Arg Ala Wall Ala Ile Ala Luell Asp Gly 26 O 265 27 O

Ser Phe Phe Ala Thir Gly Glin Arg Thir Ala Ser Ser Arg Luell Ile 27s 285

Wall Glin Asp Gly Ile His Asp Phe Wall Ala Lell Lell Ala Glu 29 O 295 3 OO

Wall Ala Ala Luell Arg Wall Gly Asp Ala Luell Asp Pro Asn Thir Glin Ile 3. OS 310 315

Gly Pro Ala Wall Ser Glu Asp Glin Met Glu Thir Ser Arg Tyr Ile 3.25 330 335

Asp Ile Ala Ala Ser Glu Gly Gly Arg Wall Wall Thir Gly Gly Asp Arg 34 O 345 35. O

Ile Luell Asp Asn Pro Gly Trp Wall Arg Pro Thir Luell Ile Ala 355 360 365

Asp Thir Glin Ala Gly Met Arg Ile Asn Asn Glu Glu Wall Phe Gly Pro 37 O 375

Wall Ala Ser Thir Ile Arg Wall Ser Glu Glu Ala Luell Glu Ile 385 390 395 4 OO

Ala Asn Gly Wall Glu Phe Gly Luell Ser Ala Gly Ile Ala Thir Thir Ser 4 OS 415

Lell His Ala Arg His Phe Glin Arg Ala Arg Ala Gly Met Thir 425 43 O

Met Wall Asn Luell Ala Thir Ala Gly Wall Asp His Wall Pro Phe Gly 435 44 O 445

Gly Thir Ser Ser Ser Tyr Gly Ala Arg Glu Glin Gly Phe Ala Ala 450 45.5 460

Wall Glu Phe Phe Thir Glin Thir Thir Ser Tyr Ser Trp Ser 465 470 47s

<210s, SEQ ID NO 26 &211s LENGTH: 248 212. TYPE : PRT &213s ORGANISM: Caulobacter Crescentus

<4 OOs, SEQUENCE: 26

Met Ser Ser Ala Ile Pro Ser Luell Lys Gly Arg Wall Wall Ile 1. 5 15

Thir Gly Gly Gly Ser Gly Ile Gly Ala Gly Luell Thir Ala Gly Phe Ala 25

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

Arg Ala Luell Glu Ala Glu Lell Ala Gly Ser Pro Ile Pro Pro Wall SO 55 6 O

Lys Arg Asp Lell Met Asn Luell Glu Ala Ile Ala Wall Phe Ala 65 70 8O

Glu Ile Gly Asp Wall Asp Wall Luell Wall Asn ASn Ala Gly Asn Asp Asp US 9,045,789 B2 71 72 - Continued

85 90 95

Arg His Luell Ala Asp Wall Thir Gly Ala Tyr Trp Asp Glu Arg Ile 1OO 105 11 O

Asn Wall Asn Luell Arg His Met Luell Phe Thir Glin Ala Wall Ala Pro 115 12 O 125

Gly Met Gly Gly Gly Ala Wall Ile Asn Phe Gly Ser Ile 13 O 135 14 O

Ser Trp His Luell Gly Lell Glu Asp Luell Wall Luell Glu Thir Ala Lys 145 150 155 160

Ala Gly Ile Glu Gly Met Thir Arg Ala Luell Ala Arg Glu Luell Gly Pro 1.65 17O 17s

Asp Asp Ile Arg Wall Thir Wall Wall Pro Gly Asn Wall Lys Thir 18O 185 19 O

Arg Glin Glu Trp Thir Pro Glu Gly Glu Ala Glin Ile Wall Ala 195

Ala Glin Luell Gly Arg Ile Wall Pro Glu Asn Wall Ala Ala Luell 21 O 215 22O

Wall Luell Phe Luell Ala Ser Asp Asp Ala Ser Luell Thir Gly His Glu 225 23 O 235 24 O

Trp Ile Asp Ala Gly Trp Arg 245

SEO ID NO 27 LENGTH: 289 TYPE : PRT ORGANISM; Caulobacter crescents

< 4 OOs SEQUENCE: 27

Met Thir Ala Glin Wall Thir Wall Trp Asp Luell Ala Thir Luell Gly 1. 5 15

Glu Gly Pro Ile Trp His Gly Asp Thir Luell Trp Phe Wall Asp Ile 2O 25

Glin Arg Lys Ile His Asn Tyr His Pro Ala Thir Gly Glu Arg Phe Ser 35 4 O 45

Phe Asp Ala Pro Asp Glin Wall Thir Phe Luell Ala Pro Ile Wall Gly Ala SO 55 6 O

Thir Gly Phe Wall Wall Gly Lell Thir Gly Ile His Arg Phe His Pro 65 70

Ala Thir Gly Phe Ser Lell Lell Luell Glu Wall Glu Asp Ala Ala Luell Asn 85 90 95

Asn Arg Pro Asn Asp Ala Thir Wall Asp Ala Glin Gly Arg Luell Trp Phe 105 11 O

Gly Thir Met His Asp Gly Glu Glu Asn Asn Ser Gly Ser Luell Tyr Arg 115 12 O 125

Met Asp Luell Thir Gly Wall Ala Arg Met Asp Arg Asp Ile Ile Thir 13 O 135 14 O

Asn Gly Pro Wall Ser Pro Asp Gly Lys Thir Phe His Thir Asp 145 150 155 160

Thir Luell Glu Thir Ile Ala Phe Asp Luell Ala Glu Asp Gly Luell 1.65 17O 17s

Lell Ser Asn Lys Arg Wall Phe Wall Glin Phe Ala Lell Gly Asp Asp Wall 18O 185 19 O

Pro Asp Gly Ser Wall Wall Asp Ser Glu Gly Lell Trp Thir Ala 195 2OO 2O5

US 9,045,789 B2 77 78 - Continued

515 525 aac acc ggc cgc tgc gac gcc gt C gac gag gcg acg at C gcc gcg 632 Asn Thir Gly Arg Asp Ala Luell Wall Asp Glu Ala Thir Ile Ala Ala 53 O 535 54 O cgc aag cag gac ggc atc. gcg gtt cc c gcc a CC atg acg Ccc tig Arg Lys Glin Asp Gly Ile Pro Ala Wall Pro Ala Thir Met Thir Pro Trp 5.45 550 555 560

Cag gala at C tac cgc gcc CaC gcc agt cag citc. gac a CC ggc ggc gtg 728 Glin Glu Ile Arg Ala His Ala Ser Glin Luell Asp Thir Gly Gly Val 565 st O sts

Ctg gag ttic gcg gtc aag tac cag gac Ctg gC9 gcc aag Ctg cc c cqc 776 Lell Glu Phe Ala Wall Glin Asp Luell Ala Ala Luell Pro Arg 58O 585 59 O

CaC aac CaC tga 788 His Asn His 595

SEQ ID NO 29 LENGTH: 595 TYPE : PRT ORGANISM: Caulobacter Crescentus

< 4 OOs SEQUENCE: 29

Met Arg Ser Ala Lell Ser Asn Arg Thir Pro Arg Arg Phe Arg Ser Arg 1. 5 15

Asp Trp Phe Asp Asn Pro Asp His Ile Asp Met Thir Ala Luell Tyr Lieu. 25

Glu Arg Phe Met Asn Tyr Gly Ile Thr Pro Glu Glu Lieu Arg Ser Gly 35 4 O 45

Pro Ile Ile Gly Ile Ala Glin Thir Gly Ser Asp Ile Ser Pro Cys SO 55 6 O

Asn Arg Ile His Lell Asp Lell Wall Glin Arg Wall Arg Asp Gly Ile Arg 65 70 8O

Asp Ala Gly Gly Ile Pro Met Glu Phe Pro Wall His Pro Ile Phe Glu 85 90 95

Asn Arg Arg Pro Thir Ala Ala Luell Asp Arg Asn Lell Ser Tyr Lieu. 105 11 O

Gly Luell Wall Glu Thir Lell His Gly Tyr Pro Ile Asp Ala Wall Wall Lieu 115 12 O 125

Thir Thir Gly Asp Thir Thir Pro Ala Gly Ile Met Ala Ala Thr 13 O 135 14 O

Thir Wall Asn Ile Pro Ala Ile Wall Luell Ser Gly Gly Pro Met Lieu. Asp 145 150 155 160

Gly Trp His Glu Asn Glu Lell Wall Gly Ser Gly Thir Wall Ile Trp Arg 1.65 17O 17s

Ser Arg Arg Lys Lell Ala Ala Gly Glu Ile Thir Glu Glu Glu Phe Ile 18O 185 19 O

Asp Arg Ala Ala Ser Ser Ala Pro Ser Ala Gly His Cys Asn Thir Met 195

Gly Thir Ala Ser Thir Met Asn Ala Wall Ala Glu Ala Lell Gly Luell Ser 21 O 215 22O

Lell Thir Gly Ala Ala Ile Pro Ala Pro Tyr Arg Glu Arg Gly Glin 225 23 O 235 24 O

Met Ala Thir Gly Glin Arg Ile Wall Asp Lell Ala Asp Asp 245 250 255

Wall Pro Luell Asp Ile Lell Thir Glin Ala Phe Glu Asn Ala Ile US 9,045,789 B2 79 80 - Continued

26 O 265 27 O

Ala Luell Wall Ala Ala Ala Gly Gly Ser Thir ASn Ala Glin Pro His Ile 285

Wall Ala Met Ala Arg His Ala Gly Wall Glu Ile Thir Ala Asp Asp Trp 29 O 295 3 OO

Arg Ala Ala Asp Ile Pro Luell Ile Wall ASn Met Glin Pro Ala Gly 3. OS 310 315

Luell Gly Glu Arg Phe His Arg Ala Gly Gly Ala Pro Ala Wall 3.25 330 335

Lell Trp Glu Luell Lell Glin Glin Gly Arg Luell His Gly Asp Wall Luell Thir 34 O 345 35. O

Wall Thir Gly Lys Thir Met Ser Glu Asn Luell Glin Gly Arg Glu Thir Ser 355 360 365

Asp Arg Glu Wall Ile Phe Pro His Glu Pro Lell Ala Glu Ala 37 O 375

Gly Phe Luell Wall Lell Lys Gly Asn Luell Phe Asp Phe Ala Ile Met Lys 385 390 395 4 OO

Ser Ser Wall Ile Gly Glu Glu Phe Arg Lys Arg Lell Ser Glin Pro 4 OS 415

Gly Glin Glu Gly Wall Phe Glu Ala Arg Ala Ile Wall Phe Asp Gly Ser 425 43 O

Asp Asp Tyr His Arg Ile Asn Asp Pro Ala Lell Glu Ile Asp Glu 435 44 O 445

Arg Cys Ile Luell Wall Ile Arg Gly Ala Gly Pro Ile Gly Trp Pro Gly 450 45.5 460

Ser Ala Glu Wall Wall Asn Met Glin Pro Pro Asp His Lell Luell Lys 465 470

Gly Ile Met Ser Lell Pro Thir Luell Gly Asp Gly Arg Glin Ser Gly Thir 485 490 495

Ala Asp Ser Pro Ser Ile Lell Asn Ala Ser Pro Glu Ser Ala Ile Gly SOO 505

Gly Gly Luell Ser Trp Lell Arg Thir Gly Asp Thir Ile Arg Ile Asp Luell 515 525

Asn Thir Gly Arg Asp Ala Luell Wall Asp Glu Ala Thir Ile Ala Ala 53 O 535 54 O

Arg Glin Asp Gly Ile Pro Ala Wall Pro Ala Thir Met Thir Pro Trp 5.45 550 555 560

Glin Glu Ile Arg Ala His Ala Ser Glin Luell Asp Thir Gly Gly Wall 565 st O sts

Lell Glu Phe Ala Wall Glin Asp Luell Ala Ala Luell Pro Arg 58O 585 59 O

His Asn His 595

SEQ ID NO 3 O LENGTH: 1476 TYPE: DNA ORGANISM: Escherichia coli FEATURE: NAME/KEY: CDS LOCATION: (1) ... (1476)

<4 OOs, SEQUENCE: 3 O atgaat acc cag tat aat tcc agt tat at a titt tog att acc tta gtc 48 Met Asn Thr Glin Tyr Asn Ser Ser Tyr Ile Phe Ser Ile Thr Lieu Val 1. 5 15

US 9,045,789 B2 83 - Continued

3.25 330 335 gat aaa titt ggit cit aag cca citg caa att atc ggc gca ct c gga atg O56 Asp Llys Phe Gly Arg Llys Pro Lieu. Glin Ile Ile Gly Ala Lieu. Gly Met 34 O 345 35. O gca at C ggt atg titt agc ctic ggit acc gcg ttt tac act cag gCa cc.g 104 Ala Ile Gly Met Phe Ser Lieu. Gly Thr Ala Phe Tyr Thr Glin Ala Pro 355 360 365 ggt att gtg gcg Cta Ctg tcg atg Ctg tt C tat gtt gcc gcc titt gcc 152 Gly Ile Val Ala Lieu Lleu Ser Met Lieu. Phe Tyr Val Ala Ala Phe Ala 37 O 375 38O atg tcc tig ggit ccg gta to tdg gta ctg. citg tcg gaa at C tt C cc.g 2OO Met Ser Trp Gly Pro Val Cys Trp Val Lieu. Leu Ser Glu Ile Phe Pro 385 390 395 4 OO aat gct att cqt ggit aaa gcg Ctg gca at C goggtg gcg gCC cag tig 248 Asn Ala Ile Arg Gly Lys Ala Lieu Ala Ile Ala Val Ala Ala Glin Trp 4 OS 41O 415 citg gog aac tac titc gtc. tcc tigg acc titc ccd atg atg gac aaa aac 296 Lieu Ala Asn Tyr Phe Val Ser Trp Thr Phe Pro Met Met Asp Lys Asn 42O 425 43 O tcc togg ct g g to goc cat titc. cac aac ggit titc. tcc tac togg att tac 344 Ser Trp Leu Val Ala His Phe His Asn Gly Phe Ser Tyr Trp Ile Tyr 435 44 O 445 ggt tdt atg ggc gtt Ctg gca gca citg titt atg tog aaa titt gtC cc.g 392 Gly Cys Met Gly Val Lieu Ala Ala Leu Phe Met Trp Llys Phe Val Pro 450 45.5 460 gala acc aaa ggit aaa acc Ctt gag gag ctg gaa gog Ct c to gaa cc.g 44 O Glu Thir Lys Gly Llys Thr Lieu. Glu Glu Lieu. Glu Ala Lieu. Trp Glu Pro 465 470 475 48O gala acg aag aaa aca Caa caa act gct acg ctg. taa 476 Glu Thir Lys Llys Thr Glin Gln Thr Ala Thr Lieu. 485 490

<210s, SEQ ID NO 31 &211s LENGTH: 491 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 31 Met Asn Thr Glin Tyr Asn Ser Ser Tyr Ile Phe Ser Ile Thr Lieu Val 1. 5 1O 15 Ala Thr Lieu. Gly Gly Lieu Lleu Phe Gly Tyr Asp Thr Ala Val Ile Ser 2O 25 3O Gly Thr Val Glu Ser Lieu. Asn Thr Val Phe Val Ala Pro Glin Asn Lieu. 35 4 O 45 Ser Glu Ser Ala Ala Asn. Ser Lieu. Lieu. Gly Phe Cys Val Ala Ser Ala SO 55 6 O Lieu. Ile Gly Cys Ile Ile Gly Gly Ala Lieu. Gly Gly Tyr Cys Ser Asn 65 70 7s 8O

Arg Phe Gly Arg Arg Asp Ser Lieu Lys Ile Ala Ala Val Lieu. Phe Phe 85 90 95

Ile Ser Gly Val Gly Ser Ala Trp Pro Glu Lieu. Gly Phe Thir Ser Ile 1OO 105 11 O

Asn Pro Asp Asn Thr Val Pro Val Tyr Lieu Ala Gly Tyr Val Pro Glu 115 12 O 125

Phe Val Ile Tyr Arg Ile Ile Gly Gly Ile Gly Val Gly Lieu Ala Ser 13 O 135 14 O

Met Leu Ser Pro Met Tyr Ile Ala Glu Lieu Ala Pro Ala His Ile Arg 145 150 155 160 US 9,045,789 B2 85 - Continued

Gly Lys Lieu Val Ser Phe Asin Glin Phe Ala Ile Ile Phe Gly Glin Lieu. 1.65 17O 17s Lieu Val Tyr Cys Val Asn Tyr Phe Ile Ala Arg Ser Gly Asp Ala Ser 18O 185 19 O Trp Lieu. Asn Thr Asp Gly Trp Arg Tyr Met Phe Ala Ser Glu. Cys Ile 195 2OO 2O5 Pro Ala Lieu. Leu Phe Leu Met Leu Lleu Tyr Thr Val Pro Glu Ser Pro 21 O 215 22O Arg Trp Lieu Met Ser Arg Gly Lys Glin Glu Glin Ala Glu Gly Ile Lieu 225 23 O 235 24 O Arg Lys Ile Met Gly Asn. Thir Lieu Ala Thr Glin Ala Val Glin Glu Ile 245 250 255 Llys His Ser Lieu. Asp His Gly Arg Llys Thr Gly Gly Arg Lieu. Lieu Met 26 O 265 27 O Phe Gly Val Gly Val Ile Val Ile Gly Val Met Leu Ser Ile Phe Glin 27s 28O 285 Glin Phe Val Gly Ile Asn Val Val Lieu. Tyr Tyr Ala Pro Glu Val Phe 29 O 295 3 OO Llys Thr Lieu. Gly Ala Ser Thr Asp Ile Ala Lieu. Lieu. Glin Thir Ile Ile 3. OS 310 315 32O Val Gly Val Ile Asn Lieu. Thir Phe Thr Val Leu Ala Ile Met Thr Val 3.25 330 335 Asp Llys Phe Gly Arg Llys Pro Lieu. Glin Ile Ile Gly Ala Lieu. Gly Met 34 O 345 35. O Ala Ile Gly Met Phe Ser Lieu. Gly Thr Ala Phe Tyr Thr Glin Ala Pro 355 360 365 Gly Ile Val Ala Lieu Lleu Ser Met Lieu. Phe Tyr Val Ala Ala Phe Ala 37 O 375 38O Met Ser Trp Gly Pro Val Cys Trp Val Lieu. Leu Ser Glu Ile Phe Pro 385 390 395 4 OO Asn Ala Ile Arg Gly Lys Ala Lieu Ala Ile Ala Val Ala Ala Glin Trp 4 OS 41O 415 Lieu Ala Asn Tyr Phe Val Ser Trp Thr Phe Pro Met Met Asp Lys Asn 42O 425 43 O Ser Trp Leu Val Ala His Phe His Asn Gly Phe Ser Tyr Trp Ile Tyr 435 44 O 445 Gly Cys Met Gly Val Lieu Ala Ala Leu Phe Met Trp Llys Phe Val Pro 450 45.5 460 Glu Thir Lys Gly Llys Thr Lieu. Glu Glu Lieu. Glu Ala Lieu. Trp Glu Pro 465 470 47s 48O Glu Thir Lys Llys Thr Glin Gln Thr Ala Thr Lieu. 485 490

<210s, SEQ ID NO 32 &211s LENGTH: 399 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic DNA PtacTtrip

<4 OOs, SEQUENCE: 32 ggtaccagat Ctc cctgttg acaattaatc atcggct cta taatgtgtgg aatcgtgagc 6 O ggataacaat ttcacacaag gagactic.ccg ggagcc.gc.ca gttcc.gctgg C9gcatttta 12 O actittctitta atgaag.ccgg aaaaatccta aatt cattta at atttatct ttttaccgtt 18O

US 9,045,789 B2 93 94 - Continued

Met Phe Asp Ser Lell Pro Tyr Arg Asn Asp Ala Ser Met Wall Met Arg 13 O 135 14 O

Arg Luell Ile Arg Ser Lell Pro Asp Ala Ala Wall Ile Gly Wall Ala 145 150 155 160

Ser Asp Gly Lell Pro Ala Thir Met Met Ala Lell Ala Ala Glin 1.65

His Asn Ala Thir Wall Lell Wall Pro Gly Gly Ala Thir Luell Pro Ala 18O 185 19 O

Asp Glu Asp Asn Gly Lys Wall Glin Thir Ile Ala Arg Phe

Ala Asn Glu Lell Ser Lell Glin Asp Ala Arg Arg Gly 21 O 215

Ala Ser Ser Gly Gly Gly Glin Phe Lell Thir Ala Gly 225 23 O 235 24 O

Thir Ser Wall Wall Ala Glu Gly Luell Gly Luell Ala Pro His Ser 245 250 255

Ala Luell Pro Ser Gly Glu Pro Wall Trp Arg Glu Ala Arg Ala 26 O 265 27 O

Ser Ala Arg Ala Ala Lell Asn Luell Ser Glin Gly Thir Thir Arg

Glu Ile Luell Thir Asp Ala Ile Glu Asn Ala Met Wall His Ala 29 O 295 3 OO

Ala Phe Gly Gly Ser Thir Asn Luell Luell Luell His Ile Pro Ala Ile Ala 3. OS 310 315

His Glin Ala Gly Cys His Ile Pro Thir Wall Asp Asp Trp Ile Arg Ile 3.25 330 335

Asn Arg Wall Pro Arg Lell Wall Ser Wall Luell Pro Asn Gly Pro Wall 34 O 345 35. O

His Pro Thir Wall Asn Ala Phe Met Ala Gly Gly Wall Pro Glu Wall 355 360 365

Met Luell His Luell Arg Ser Lell Gly Luell Luell His Glu Asp Wall Met Thir 37 O 375

Wall Thir Gly Ser Thir Lell Glu Asn Luell Asp Trp Trp Glu His Ser 385 390 395 4 OO

Glu Arg Arg Glin Arg Phe Glin Luell Luell Luell Asp Glin Glu Glin Ile 4 OS 415

Asn Ala Asp Glu Wall Ile Met Ser Pro Glin Glin Ala Ala Arg Gly 425 43 O

Lell Thir Ser Thir Ile Thir Phe Pro Wall Gly ASn Ile Ala Pro Glu Gly 435 44 O 445

Ser Wall Ile Ser Thir Ala Ile Asp Pro Ser Met Ile Asp Glu Glin 450 45.5 460

Gly Ile His Lys Gly Wall Ala Wall Lell Ser Glu Lys 465 470

Ser Ala Ile Asp Ile His Asp Lys Ile Ala Gly Asp Ile 485 490 495

Lell Wall Ile Ile Gly Wall Gly Pro Ser Gly Thir Met Glu Glu Thir SOO 505

Glin Wall Thir Ser Ala Lell Lys His Luell Ser Gly His Wall 515 52O 525

Ser Luell Ile Thir Asp Ala Arg Phe Ser Gly Wall Ser Thir Gly Ala Cys 53 O 535 54 O

US 9,045,789 B2 99 100 - Continued

SOO 505 51O tac cag ct c acc ticc gcg cta aag cat tog tgg ggc aag acg gtg 584 Gln Lieu. Thir Ser Ala Lieu Llys His Ser Trp Gly Lys Thir Wall 515 52O 525 tcg Ct c at C acc gat gcg cgc ttic tog gtg tcg acg ggc gcc tgc 632 Ser Lieu. Ile Thr Asp Ala Arg Phe Ser Wall Ser Thir Gly Ala Cys 53 O 535 54 O tto ggC Cac gttgtcg CC9 gag gcg Ctg ggc 999 cc.g att ggc aag Phe Gly His Val Ser Pro Glu Ala Lieu Gly Gly Pro Ile Gly Lys 5.45 550 555 560

cgc gat aac gac atc atc gag att gcc gtg gat cgt Ctg acg tta 728 Lell Arg Asp Asn Asp Ile Ile Glu Ile Wall Asp Arg Luell Thir Lel 565 st O sts act ggc agc gtgaac titc atc ggc acc gac aac cc.g Ctg acg 776 Thir Gly Ser Val Asn Phe Ile Gly Thr Asp Asn Pro Luell Thir 58O 585 59 O gaa gag gC gC9 C9C gag Ctg cgg cag acg CaC cc.g gac Ctg 824 Glu Glu Gly Ala Arg Glu Lieu. Arg Glin Thir His Pro Asp Lel 595 605

CaC gcc cac gac titt ttg cc.g gac gac acc cgg Ctg tgg gcg gca Ctg 872 His Ala His Asp Phe Lieu Pro Asp Asp Thir Arg Lell Trp Ala Ala Lel 610 615 62O

Cag tcg gtg agc ggc ggc acc tgg aaa ggc tgt att tat gac acc gat 92 O Glin Ser Val Ser Gly Gly Thr Trp Llys Gly Cys Ile Asp Thir Asp 625 630 635 64 O a.a.a. att at c gag gta att aac gcc ggit a.a.a. a.a.a. gcg citc. gga att taa 96.8 Ile Ile Glu Wall Ile Asn Ala Gly Lys Ala Lell Gly Ile 645 650 655

<210s, SEQ ID NO 37 &211s LENGTH: 655 212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OO > SEQUENCE: 37

Met Thir Ile Glu Lys Ile Phe Thir Pro Glin Asp Asp Ala Phe Tyr Ala 1. 5 1O 15

Wall Ile Thr His Ala Ala Gly Pro Glin Gly Ala Lell Pro Luell Thir Pro 2O 25 3O

Glin Met Lieu Met Glu Ser Pro Ser Gly Asn Luell Phe Gly Met Thir Glin 35 4 O 45

Asn Ala Gly Met Gly Trp Asp Ala Asn Luell Thir Gly Glu Wall SO 55 6 O

Lell Ile Ile Gly Thr Glin Gly Gly Ile Arg Ala Gly Asp Gly Arg Pro 65 70

Ile Ala Leu Gly Tyr His Thr Gly His Trp Glu Ile Gly Met Glin Met 85 90 95

Glin Ala Ala Ala Lys Glu Ile Thr Arg Asn Gly Gly Ile Pro Phe Ala 1OO 105 11 O

Ala Phe Val Ser Asp Pro Cys Asp Gly Arg Ser Glin Gly Thir His Gly 115 12 O 125

Met Phe Asp Ser Leu Pro Tyr Arg Asn Asp Ala Ala Ile Wall Phe Arg 13 O 135 14 O

Arg Lieu. Ile Arg Ser Lieu Pro Thr Arg Arg Ala Wall Ile Gly Wall Ala 145 150 155 160

Thir Cys Asp Llys Gly Lieu Pro Ala Thr Met Ile Ala Lell Ala Ala Met 1.65 17O 17s US 9,045,789 B2 101 102 - Continued

His Asp Luell Pro Thir Ile Lell Wall Pro Gly Gly Ala Thir Luell Pro Pro 18O 185 19 O

Thir Wall Gly Glu Asp Ala Lys Wall Glin Thir Ile Gly Ala Arg Phe 195

Ala Asn His Glu Lell Ser Glin Glu Ala Ala Glu Lell Gly Arg 21 O

Ala Ala Ser Pro Gly Gly Glin Phe Lell Gly Thir Ala Gly 225 23 O 235 24 O

Thir Ser Glin Wall Wall Ala Ala Luell Gly Luell Ala Lell Pro His Ser 245 250 255

Ala Luell Ala Pro Ser Gly Ala Wall Trp Luell Glu Ile Ala Arg Glin 26 O 265 27 O

Ser Ala Arg Ala Wall Ser Luell Asp Ser Arg Gly Ile Thir Thir Arg 285

Asp Ile Luell Ser Asp Ile Glu Asn Ala Met Wall Ile His Ala 29 O 3 OO

Ala Phe Gly Gly Ser Thir Asn Luell Luell Luell His Ile Pro Ala Ile Ala 3. OS 310 315

His Ala Ala Gly Cys Thir Ile Pro Asp Wall Glu His Trp Thir Arg Ile 3.25 330 335

Asn Arg Wall Pro Arg Lell Wall Ser Wall Luell Pro Asn Gly Pro Asp 34 O 345 35. O

His Pro Thir Wall Arg Ala Phe Luell Ala Gly Gly Wall Pro Glu Wall 355 360 365

Met Lieu His Lieu Arg Asp Lieu Gly Lieu Lieu His Lieu Asp Ala Met Thr 37 O 375

Wall Thir Gly Glin Thir Wall Gly Glu Asn Luell Glu Trp Trp Glin Ala Ser 385 390 395 4 OO

Glu Arg Arg Ala Arg Phe Arg Glin Luell Arg Glu Glin Asp Gly Wall 4 OS 415

Glu Pro Asp Asp Wall Ile Lell Pro Pro Glu Ala Ala Gly 425 43 O

Lell Thir Ser Thir Wall Phe Pro Thir Gly ASn Ile Ala Pro Glu Gly 435 44 O 445

Ser Wall Ile Ala Thir Ala Ile Asp Pro Ser Wall Wall Gly Glu Asp 450 45.5 460

Gly Wall His His Thir Gly Arg Wall Arg Wall Phe Wall Ser Glu Ala 465 470

Glin Ala Ile Ala Ile Arg Glu Ile Wall Glin Gly Asp Ile 485 495

Met Wall Wall Ile Gly Gly Gly Pro Ser Thir Gly Met Glu Glu Thir SOO 505 51O

Glin Luell Thir Ser Ala Lell Lys His Ser Trp Gly Thir Wall 515 525

Ser Luell Ile Thir Asp Ala Arg Phe Ser Wall Ser Thir Gly Ala 53 O 535 54 O

Phe Gly His Wall Ser Pro Glu Ala Luell Gly Gly Pro Ile Gly Lys 5.45 550 555 560

Lell Arg Asp Asn Asp Ile Ile Glu Ile Wall Asp Arg Luell Thir Luell 565 sts

Thir Gly Ser Wall Asn Phe Ile Gly Thir Asp Asn Pro Luell Thir Pro 585 59 O

Glu Glu Gly Ala Arg Glu Lell Ala Arg Arg Glin Thir His Pro Asp Luell US 9,045,789 B2 103 104 - Continued

595 6OO 605 His Ala His Asp Phe Lieu Pro Asp Asp Thir Arg Lieu. Trp Ala Ala Lieu. 610 615 62O Gln Ser Val Ser Gly Gly. Thir Trp Lys Gly Cys Ile Tyr Asp Thr Asp 625 630 635 64 O Lys Ile Ile Glu Val Ile Asn Ala Gly Lys Lys Ala Lieu. Gly Ile 645 650 655

<210s, SEQ ID NO 38 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylX IFS 5742-10-1 <4 OOs, SEQUENCE: 38 acacaaggag act cocatgg gcgtgagtga attcc teccg 4 O

<210s, SEQ ID NO 39 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylX IFA 5742-10-2 <4 OOs, SEQUENCE: 39 ggaactggcg gct Cocttag aggaggcc.gc ggc.cggc.cag 4 O

<210 SEQ ID NO 40 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylA. IFS 5742-10-3 <4 OOs, SEQUENCE: 4 O acacaaggag act cocatga ccgacaccct gcgcc attac 4 O

<210s, SEQ ID NO 41 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylA IFA 5742-10-4 <4 OOs, SEQUENCE: 41 ggaactggcg gct Cocttac gaccacgagt aggaggttitt 4 O

<210s, SEQ ID NO 42 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylD IFS 5742-10-5

<4 OOs, SEQUENCE: 42 acacaaggag act cocatgt Cta accgcac gcc.ccgc.cgg 4 O

<210s, SEQ ID NO 43 &211s LENGTH: 4 O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer xylD-IFA 5742-10-6 US 9,045,789 B2 105 106 - Continued

<4 OOs, SEQUENCE: 43 ggaactggcg gct Coctoag tigttgttggc ggggcagctt 4 O

<210s, SEQ ID NO 44 &211s LENGTH: 44 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO823 - 01F 4691-87-1

<4 OOs, SEQUENCE: 44 gtcgacticta gaggat CCCC gtgggcgtga gtgaatticct gcc.g 44

<210s, SEQ ID NO 45 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO823 - O1R 4691-87-2

<4 OOs, SEQUENCE: 45 tatgcc tigtc. citgc.ca.gcac togcc 24

<210s, SEQ ID NO 46 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO823 - O2F 4691-87-3 < 4 OO SEQUENCE: 46 ggcagtgctg gCagga Cagg cata 24

<210s, SEQ ID NO 47 &211s LENGTH: 44 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO823 - O2R 4691-87 - 4 <4 OOs, SEQUENCE: 47 cgaatticgag Ctcggit accc ttagaggagg cc.gcggc.cgg C cag 44

<210s, SEQ ID NO 48 &211s LENGTH: 44 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - O1F 4691-87-5 <4 OOs, SEQUENCE: 48 gtcgacticta gaggat.cccc atgaccgaca ccctg.cgc.ca ttac 44

<210s, SEQ ID NO 49 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCo822 - O1R 5659-8-7

<4 OOs, SEQUENCE: 49 gcgt.cggCCC aggc.cgggala to c 24

<210s, SEQ ID NO 50 &211s LENGTH: 24 US 9,045,789 B2 107 108 - Continued

&212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - O2F 5659-8-8 <4 OOs, SEQUENCE: 50 gtcgacgc.cg cgc.gcaaggc attic 24

<210s, SEQ ID NO 51 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - O2R 5659-8-9 <4 OOs, SEQUENCE: 51 gagggc.cggg gcggcct tcc atgc 24

<210s, SEQ ID NO 52 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - O3F 5659-8-10 <4 OOs, SEQUENCE: 52 citt.ccc.gatc gccatc.ccgg catg 24

<210s, SEQ ID NO 53 &211s LENGTH: 24 & 212 TYPE DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - O3R 5659-8-11 <4 OOs, SEQUENCE: 53

Cagctgcacg cgggcctgac gtgc 24

<210s, SEQ ID NO 54 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - O4F 5659-8-12 <4 OOs, SEQUENCE: 54

24

<210s, SEQ ID NO 55 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - 04R 5659-8-13

<4 OO > SEQUENCE: 55 ggcgacct tc. tcggc.ca.gca gtgc 24

<210s, SEQ ID NO 56 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - O5F 5659-8-14

<4 OOs, SEQUENCE: 56 US 9,045,789 B2 109 110 - Continued ggattcacga Caagttcgtg gcac 24

<210s, SEQ ID NO 57 &211s LENGTH: 44 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer CCO822 - 05R 4691-87-14 <4 OO > SEQUENCE: 57 cgaatticgag Ctcggit accc ttacgaccac gag taggagg ttitt 44

<210s, SEQ ID NO 58 &211s LENGTH: 35 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer Peftu (Pst) <4 OOs, SEQUENCE: 58 cCaagcttgc atgc.ca.gatc gtttagat.cc galagg 35

<210s, SEQ ID NO 59 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer Peftu Rv <4 OO > SEQUENCE: 59 tgitatgtc.ct CCtggaCttic gt 22

<210s, SEQ ID NO 60 &211s LENGTH: 52 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer Peftu xylXABCD fw

<4 OOs, SEQUENCE: 60 cCaggaggac atacaatggg C9tgagtgaa titcctg.ccgg aagattggala ag 52

<210s, SEQ ID NO 61 &211s LENGTH: 39 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer eftu xylXABCD rv

<4 OOs, SEQUENCE: 61 cggit accc.gg ggat.ct cagt ggttgttggcg gggcagott 39

<210s, SEQ ID NO 62 &211s LENGTH: 38 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer PmsrA (Pst)

<4 OOs, SEQUENCE: 62 cCaagcttgc atgc.catttg cgc.ctgcaac gtaggttg 38

<210s, SEQ ID NO 63 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 9,045,789 B2 111 112 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: primer PmsrAR <4 OOs, SEQUENCE: 63 aacaggaatgttc ctitt.cga aaa 23

<210s, SEQ ID NO 64 &211s LENGTH: 41 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer PmsrA xylD fw <4 OOs, SEQUENCE: 64 aggaac attc ctdttatgtc. taaccocacg cccc.gc.cggit t 41

<210s, SEQ ID NO 65 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer SpcR-F

<4 OOs, SEQUENCE: 65

Ctaataacgt aacgtgactg gcaag 25

<210s, SEQ ID NO 66 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: primer SpcR-R

<4 OOs, SEQUENCE: 66 ataagtaaga ttaaccatta gtc 23

<210s, SEQ ID NO 67 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer spc (GTG start) -F <4 OO > SEQUENCE: 67 gtgaggagga t at atttgaa tacatacgaa 3 O

<210s, SEQ ID NO 68 &211s LENGTH: 35 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer spc (stop) -R <4 OOs, SEQUENCE: 68 taattitttitt aatctgtt at ttaaatagitt tatag 35

<210s, SEQ ID NO 69 &211s LENGTH: 42 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: primer Spc-pVC7-Cm-F

<4 OOs, SEQUENCE: 69 atatat cotc ct cactittag ctitcc ttagc ticctgaaaat ct 42 US 9,045,789 B2 113 114 - Continued

SEO ID NO 7 O LENGTH: 45 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer Spc-pVC7-Cm-R

<4 OOs, SEQUENCE: 70 agattaaaaa aattataatt tttittaaggc agittattggit gcc ct 45

SEO ID NO 71 LENGTH: 54 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer ME Spc fw

<4 OOs, SEQUENCE: 71.

Ctgtct Ctta tacacatcto Caagcttgca toccggcc tigttggttg ggtt 54

SEO ID NO 72 LENGTH: 58 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: primer ME Peftu xylXABCD rv

SEQUENCE: 72

Ctgtct Ctta tacacatcto ggt acccggg gat ct cagtg gttgttggcgg ggcagctt 58

SEO ID NO 73 LENGTH: 1785 TYPE: DNA ORGANISM: Agrobacterium tumefaciens FEATURE: NAME/KEY: CDS LOCATION: (1) . . (1785)

< 4 OOs SEQUENCE: 73 atg agc gac gac aag a Ca c cc gtg cgg cgg Ctg cga t cc cag gac tgg 48 Met Ser Asp Asp Lys Thir Pro Wall Arg Arg Luell Arg Ser Glin Asp Trp 1. 5 1O 15 tto gac aac cott gac Cat citc. gac atg acg gC9 citc. tat Ctg gag cgc 96 Phe Asp Asn Pro Asp His Lell Asp Met Thir Ala Lell Tyr Luell Glu Arg 2O 25 3O tto atg aat tat ggc gtg acg cc.g gala gag Ctg cgt t cc ggc aag cc.g 144 Phe Met Asn Tyr Gly Wall Thir Pro Glu Glu Luell Arg Ser Gly Lys Pro 35 4 O 45 gtc at C ggc at C gcg Cag agc ggc agc gac Ctg acg c cc tgc aac cgc 192 Wall Ile Gly Ile Ala Glin Ser Gly Ser Asp Luell Thir Pro Cys Asn Arg SO 55 6 O gtc Cat gt C gala Ctg gtc aag cgg gtg cgc gat ggit atc. cgt. gat gcg 24 O Wall His Wall Glu Lell Wall Lys Arg Wall Arg Asp Gly Ile Arg Asp Ala 65 70 7s ggc ggc at C cc c atc. gag titt cc.g acc Cat cc.g atg tto gala aac tgc 288 Gly Gly Ile Pro Ile Glu Phe Pro Thir His Pro Met Phe Glu Asn 85 90 95 aag cga cc.g aca gcg gcg citt gac cgc aat citt gcc tat citc. agc Ctg 336 Arg Pro Thir Ala Ala Lell Asp Arg Asn Luell Ala Luell Ser Luell 1OO 105 11 O gtg gala gtg citt tac ggit tat CC a citc. gat ggc gtc gtg ttg acc acg 384 Wall Glu Wall Luell Tyr Gly Pro Luell Asp Gly Wall Wall Luell Thir Thir 115 12 O 125

US 9,045,789 B2 117 118 - Continued a CC att citc. gt C att cgc 999 gcc gga cc.g citc. ggc tgg cc.g ggt tog 392 Thir Ile Luell Wall Ile Arg Gly Ala Gly Pro Luell Gly Trp Pro Gly Ser 450 45.5 460 gct gag gt C gt C aac atg Cag cc.g cc.g gac Ctg ttg a.a.a. a.a.a. ggc 44 O Ala Glu Wall Wall Asn Met Glin Pro Pro Asp Lell Lell Gly 465 470 ata acc agc Ctg cc.g a Ca atc. ggc gac ggc cgg Cala tcg gga acc gcg 488 Ile Thir Ser Luell Pro Thir Ile Gly Asp Gly Arg Glin Ser Gly Thir Ala 485 490 495 gac agc cc.g tog atc. citc. aat gcc tog CC a gaa agt gcc gcc ggt gga 536 Asp Ser Pro Ser Ile Lell Asn Ala Ser Pro Glu Ser Ala Ala Gly Gly SOO 505 51O gga Ctg gca tgg citt cgc acg ggt gat gta atc. cgc atc. gac ttic aat 584 Gly Luell Ala Trp Lell Arg Thir Gly Asp Wall Ile Arg Ile Asp Phe Asn 515 525

Cag ggc aag tgc gac gcg ttg gta cc.g gat gca gag citt gct gcc cga 632 Glin Gly Lys Cys Asp Ala Lell Wall Pro Asp Ala Glu Lell Ala Ala Arg 53 O 535 54 O aag gcc gat ggc att cott gcg gtg cott gcg gat gcc acg cc c tgg cag Lys Ala Asp Gly Ile Pro Ala Wall Pro Ala Asp Ala Thir Pro Trp Glin 5.45 550 555 560 cgc at C tat cgt Cala gtg acc cag citt tog gac ggit gcg gtt 728 Arg Ile Arg Glin Ser Wall Thir Glin Luell Ser Asp Gly Ala Wall Luell 565 st O sts gaa 999 gcc gcg gat tto cgc cgg att gcc gag aag atg cc c cgg CaC 776 Glu Gly Ala Ala Asp Phe Arg Arg Ile Ala Glu Met Pro Arg His 58O 585 59 O aac Cat tga Asn His

<210s, SEQ ID NO 74 &211s LENGTH: 594 212. TYPE : PRT <213> ORGANISM: Agrobacterium tumefaciens

<4 OOs, SEQUENCE: 74

Met Ser Asp Asp Lys Thir Pro Wall Arg Arg Luell Arg Ser Glin Asp Trp 1. 5 15

Phe Asp Asn Pro Asp His Lell Asp Met Thir Ala Lell Luell Glu Arg 25 3O

Phe Met Asn Gly Wall Thir Pro Glu Glu Luell Arg Ser Gly Pro 35 4 O 45

Wall Ile Gly Ile Ala Glin Ser Gly Ser Asp Luell Thir Pro Asn Arg SO 55 6 O

Wall His Wall Glu Lell Wall Arg Wall Arg Asp Gly Ile Arg Asp Ala 65 70

Gly Gly Ile Pro Ile Glu Phe Pro Thir His Pro Met Phe Glu Asn 85 90 95

Arg Pro Thir Ala Ala Lell Asp Arg Asn Luell Ala Luell Ser Luell 1OO 105 11 O

Wall Glu Wall Luell Tyr Gly Pro Luell Asp Gly Wall Wall Luell Thir Thir 115 12 O 125

Gly Cys Asp Thir Thir Pro Ser Ala Luell Met Ala Ala Ser Thir Wall 13 O 135 14 O

Asp Ile Pro Ala Ile Wall Lell Ser Gly Gly Pro Met Lell Asp Gly Tyr 145 150 155 160

His Asp Gly Asp Lell Wall Gly Ser Gly Thir Wall Ile Trp Arg Met Arg US 9,045,789 B2 119 120 - Continued

1.65 17O 17s

Arg Gly Ala Gly Glu Ile Thir Arg Glu Glu Phe Luell Glin Ala 18O 185 19 O

Ala Luell Glu Ser Ala Pro Ser Wall Gly His Asn Thir Met Gly Thir 195

Ala Ser Thir Met Asn Ala Ile Ala Glu Ala Luell Gly Met Ser Luell Thir 21 O 215 22O

Gly Gly Ala Ile Pro Ala Ala Arg Glu Arg Gly Glin Met Ala 225 23 O 235 24 O

Arg Thir Gly Arg Arg Ala Wall Glu Luell Wall Wall Glu Asn Ile 245 250 255

Pro Ser Asp Ile Met Thir Arg Glu Ala Phe Luell Asn Ala Ile Arg Wall 26 O 265 27 O

Asn Ser Ala Ile Gly Gly Ser Thir Asn Ala Pro His Luell Ala Ala 27s 285

Met Ala His Ala Gly Wall Glu Luell Arg Glu Asp Trp Glin Wall 29 O 295 3 OO

His Gly Asp Ile Pro Lell Ile Ala Asn Glin Pro Ala Gly Lys 3. OS 310

Trp Luell Gly Glu Lys His Arg Ala Gly Thir Pro Ala Ile Met 3.25 330 335

Trp Glu Luell Lieu Lys Ala Gly Luell Asp Ser Pro Thir Wall 34 O 345 35. O

Thir Gly Lys Thir Wall Ala Glu Asn Luell Asp Arg Glu Ser Thir Asp 355 360 365

Arg Asp Wall Ile Lieu. Pro Tyr Asp Pro Luell Lys Glu Arg Ala Gly 37 O 375

Phe Luell Wall Lieu Lys Gly Asn Luell Phe Asp Phe Ala Ile Met Thir 385 390 395 4 OO

Ser Wall Ile Ser Ala Glu Phe Arg Glin Arg Lell Ser Glu Pro Gly 4 OS 415

Arg Glu Gly Ile Phe Glu Gly Cys Wall Wall Phe Asp Gly Ser Glu 42O 425 43 O

Asp His Ala Arg Ile Asn Asp Pro Ser Luell Asp Ile Asp Glu Arg 435 44 O 445

Thir Ile Luell Wall Ile Arg Gly Ala Gly Pro Luell Gly Trp Pro Gly Ser 450 45.5 460

Ala Glu Wall Wall Asn Met Glin Pro Pro Asp Ala Lell Lell Gly 465 470

Ile Thir Ser Leul Pro Thir Ile Gly Asp Gly Arg Glin Ser Gly Thir Ala 485 490 495

Asp Ser Pro Ser Ile Lell Asn Ala Ser Pro Glu Ser Ala Ala Gly Gly SOO 505

Gly Luell Ala Trp Lieu Arg Thir Gly Asp Wall Ile Arg Ile Asp Phe Asn 515 525

Glin Gly Ala Lell Wall Pro Asp Ala Glu Lell Ala Ala Arg 53 O 535 54 O

Lys Ala Gly Ile Pro Ala Wall Pro Ala Asp Ala Thir Pro Trp Glin 5.45 550 555 560

Arg Ile Arg Glin Ser Wall Thir Glin Luell Ser Asp Gly Ala Wall Luell 565 st O sts

Glu Gly Ala Ala Asp Phe Arg Arg Ile Ala Glu Met Pro Arg His 58O 585 59 O

US 9,045,789 B2 127 128 - Continued

&211s LENGTH: 594 212. TYPE: PRT <213> ORGANISM: Herbaspirillum seropedicae

<4 OO > SEQUENCE: 77 Met Asn Llys Pro Asn Ala Thr Pro Arg Arg Phe Arg Ser Glin Asp Trp 1. 5 1O 15 Phe Asp Asn Pro Asp His Ile Asp Met Thir Ala Lieu. Tyr Lieu. Glu Arg 2O 25 3O Phe Met Asn Tyr Gly Ile Thr Ala Glu Glu Lieu. Arg Ser Gly Arg Pro 35 4 O 45 Ile Ile Gly Ile Ala Glin Ser Gly Ser Asp Ile Ser Pro Cys Asn Arg SO 55 6 O Ile His Lieu. Glu Lieu Ala Lys Arg Val Arg Asp Gly Ile Arg Asp Ala 65 70 7s 8O Gly Gly Ile Pro Met Glu Phe Pro Leu. His Pro Ile Phe Glu Asn Cys 85 90 95 Arg Arg Pro Thr Ala Ala Ile Asp Arg Asn Lieu Ala Tyr Lieu. Gly Lieu 1OO 105 11 O Val Glu Ile Lieu. His Gly Tyr Pro Ile Asp Ala Val Val Lieu. Thir Thr 115 12 O 125 Gly Cys Asp Llys Thr Thr Pro Ser Glin Ile Met Ala Ala Ala Thr Val 13 O 135 14 O Asp Ile Pro Ala Ile Val Lieu. Ser Gly Gly Pro Met Lieu. Asp Gly Trip 145 150 155 160 Met Asp Gly Glu Lieu Val Gly Ser Gly Ser Ala Ile Trp Llys Gly Arg 1.65 17O 17s Llys Lieu. Lieu. Ser Ala Gly Ser Ile Asp Asn. Glu, Llys Phe Lieu. Glu Ile 18O 185 19 O Ala Ala Ala Ser Ala Pro Ser Ser Gly His Cys Asn Thr Met Gly Thr 195 2OO 2O5 Ala Ser Thr Met Asn Ala Met Ala Glu Ala Leu Gly Met Ser Lieu. Thr 21 O 215 22O Gly Cys Ser Ala Ile Pro Ala Pro Tyr Arg Glu Arg Gly Glin Met Ala 225 23 O 235 24 O Tyr Glu Thr Gly Arg Arg Ile Val Gly Met Ala Tyr Glu Asp Lieu. Arg 245 250 255 Pro Ser Ala Ile Lieu. Thir Arg Asp Ala Phe Lieu. Asp Ala Ile Val Val 26 O 265 27 O Asn Ala Ala Ile Gly Gly Ser Thr Asn Ala Glin Pro His Ile Met Ala 27s 28O 285 Met Ala Arg His Ala Gly Val Glu Lieu. Glin Ser Glu Asp Trp Met Lys 29 O 295 3 OO Tyr Gly Tyr Asp Val Pro Lieu. Lieu. Lieu. Asn Met Glin Pro Ala Gly Lys 3. OS 310 315 32O Tyr Lieu. Gly Glu Arg Phe His Arg Ala Gly Gly Val Pro Ala Ile Met 3.25 330 335

Trp Glu Lieu. Glin Glin Ala Gly Lys Lieu. Arg Ala Glu Arg Ile Thr Ala 34 O 345 35. O

Thr Gly Llys Thir Met Ala Glu Asn Lieu. Glin Gly Arg Ala Ser Asn Asp 355 360 365

Arg Glu Met Ile Tyr Pro Phe Ala Ala Pro Lieu. Arg Glu Arg Ala Gly 37 O 375 38O

Phe Lieu Val Lieu Lys Gly Asn Lieu. Phe Asp Phe Ala Ile Met Lys Thr

US 9,045,789 B2 135 136 - Continued aala Ct c ct c gala Cag ggg gtc. c9c gac at g g to cig gtc. tcc gac 999 44 O Llys Lieu. Lieu. Glu Glin Gly Val Arg Asp Met Val Arg Val Cys Asp Gly 465 470 47s 48O cgg atgtcg ggit acg gcg tac ggc acg gtg gtc. Ctg cac git C goc 488 Arg Met Ser Gly Thr Ala Tyr Gly Thr Val Val Lieu. His Val Ala Pro 485 490 495 gala gcc gC9 gC9 gC ggg cc.g. Ct c gCC C9g gtC C9C acc ggc gaC atg 536 Glu Ala Ala Ala Gly Gly Pro Lieu Ala Arg Val Arg Thr Gly Asp Met SOO 505 51O atc at C ct c gaC gtc gcg aac C9g cgc ct c gac gcc gac git C cc.g gcc 584 Ile Ile Lieu. Asp Wall Ala Asn Arg Arg Lieu. Asp Ala Asp Val Pro Ala 515 52O 525 gag gag tig gcc gcc C9C gag cc.g. tca CC9 gag gC9 gC9 aaa gCC tac 632 Glu Glu Trp Ala Ala Arg Glu Pro Ser Pro Glu Ala Ala Lys Ala 53 O 535 54 O gcg gC9 cc.g. tcc cqc ggc tigg gag cqt Ct c tac gtc gac acc gtC ggc Ala Ala Pro Ser Arg Gly Trp Glu Arg Lieu. Tyr Val Asp Thr Val Gly 5.45 550 555 560

Cag gcc gac acc ggc gcc gac to gac ttic ctg. c9c ggc gcg agc ggc 728 Glin Ala Asp Thr Gly Ala Asp Cys Asp Phe Lieu. Arg Gly Ala Ser Gly 565 st O sts gac cqc gtc. tcc cqC gag ticc cac ta Asp Arg Val Ser Arg Glu Ser His 58O

<210s, SEQ ID NO 8O &211s LENGTH: 584 212. TYPE: PRT <213> ORGANISM: Actinoplanes missouriensis

<4 OOs, SEQUENCE: 80 Met Asp Lieu. His Arg Pro Arg Llys Lieu. Arg Arg Arg Lieu Ala Met Glin 1. 5 1O 15

Arg Arg Ser Ala Glin Trp Tyr Ala Gly Asp Asp Arg Asn. Ser Tyr Ile 2O 25 3O His Arg Ala Trp Met Arg Arg Gly Lieu Pro Ala Asp Ala Phe Asp Gly 35 4 O 45 Arg Pro His Ile Ala Ile Ala Asn. Thir Ala Ser Asp Lieu. Thr Pro SO 55 6 O

Asn Ala His Phe Asp Glu Val Ala Arg Ser Val Ala Asp Gly Ile His 65 70 7s

Arg Ala Gly Gly Val Ala Lieu. Asn Lieu Pro Val Val Ser Ile Gly Glu 85 90 95

Thr Glin Val Arg Pro Thr Ala Met Lieu. Trp Arg Asn Met Ala Ala Met 1OO 105 11 O

Ala Ile Glu Glu Met Lieu. Arg Ala Asn Pro Ile Asp Gly Val Val Luell 115 12 O 125

Lieu. Gly Gly Cys Asp Llys Thir Ile Pro Ala Lieu Lleu Met Gly Ala Ala 13 O 135 14 O

Ser Val Asp Leu Pro Ala Val Val Met Pro Gly Gly Pro Met Leu Thir 145 150 155 160

Gly Thr Phe Arg Gly Val Pro Leu Gly Cys Gly Thr Asp Val Trp Lys 1.65 17O 17s

Lieu. Ser Glu Glu Val Arg Ala Gly. Thir Lieu. Ser Ala Ala Glu Phe Thir 18O 185 19 O

Arg Ser Glu Ser Ser Met Ile Arg Ser Lys Gly His Cys Asn Thr Met 195 2OO 2O5 US 9,045,789 B2 137 138 - Continued

Gly Thir Ala Ser Thir Met Gly Luell Luell Ala Glu Wall Lell Gly Met Thir 21 O 215 22O

Lell Pro Gly Wall Ala Gly Thir Pro Ala Pro Asp Ser Arg Luell Luell Glu 225 23 O 235 24 O

Ala Ala His Ala Thir Gly Wall Luell Ala Wall Gly Lell Wall Asp Ala Asp 245 250 255

Arg Arg Pro Ser Glin Wall Met Thir Arg Gly Ser Phe Lell Asn Ala Ile 26 O 265 27 O

Wall Ala Luell Ala Ala Lell Gly Gly Ser Thir ASn Ala Wall Wall His Luell 285

Lell Ala Ile Ala Gly Arg Lell Gly Wall Pro Luell Ser Glin Asp Asp Phe 29 O 295 3 OO

Asp Thir Thir Gly Ala Asp Wall Pro Luell Luell Wall Asp Lell Luell Pro Ala 3. OS 310 315

Gly Arg Phe Luell Met Asp Asp Luell Tyr Arg Ala Gly Gly Luell His Ala 3.25 330 335

Wall Luell Ala Glu Wall Arg Asp Luell Luell Asp Pro Ser Ala Ile Thir Wall 34 O 345 35. O

Thir Gly Arg Pro Lell Thir Glu His Luell Gly Asp Ala Arg Wall His Asp 355 360 365

Arg Glu Wall Ile Arg Pro Arg Ala Glu Pro Luell Lell Pro His Ala Gly 37 O 375

Ile Ala Wall Luell Tyr Gly Asn Luell Ala Pro Asp Gly Ala Wall Wall Lys 385 390 395 4 OO

Pro Ala Ala Ala Ser Glu His Lieu Lieu Arg His Arg Gly Pro Ala Wall 4 OS 415

Wall Phe Asp Ser Wall Glu Asp Luell His Ala Arg Lell Asp Asp Pro Asp 425 43 O

Lell Asp Wall Thir Ala Asp Ser Wall Luell Wall Luell Arg Gly Gly Pro 435 44 O 445

Gly Tyr Pro Gly Met Pro Glu Wall Ser ASn Met Pro Luell Pro Ala 450 45.5 460

Lys Luell Luell Glu Glin Gly Wall Arg Asp Met Wall Arg Wall Asp Gly 465 470

Arg Met Ser Gly Thir Ala Gly Thir Wall Wall Lell His Wall Ala Pro 485 490 495

Glu Ala Ala Ala Gly Gly Pro Luell Ala Arg Wall Arg Thir Gly Asp Met SOO 505 51O

Ile Ile Luell Asp Wall Ala Asn Arg Arg Luell Asp Ala Asp Wall Pro Ala 515 525

Glu Glu Trp Ala Ala Arg Glu Pro Ser Pro Glu Ala Ala Ala 53 O 535 54 O

Ala Ala Pro Ser Arg Gly Trp Glu Arg Luell Tyr Wall Asp Thir Wall Gly 5.45 550 555 560

Glin Ala Asp Thir Gly Ala Asp Asp Phe Luell Arg Gly Ala Ser Gly 565 st O sts

Asp Arg Wall Ser Arg Glu Ser His 58O

SEQ ID NO 81 LENGTH: 1755 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic DNA

US 9,045,789 B2 147 148 - Continued

37 O 375

Lell Pro Ala Ile Met Ala Glu Lieu Lieu. Asp Ala Arg Luell Asn. Pro 385 390 395 4 OO

Asp Ala Luell Thr Cys Asn Gly Tyr Thir Wall Ala Glu Asn Wall Arg Asp 4 OS 41O 415

His Thir Trp Asp Arg Arg Met Ile Llys Pro Asn Glu Pro Leu 425 43 O

Lell Glu Asp Ala Gly Phe Lieu. His Lieu. Glin Gly Ser Lell Phe Arg Ser 435 44 O 445

Ala Ile Met Lys Thr Cys Val Ile Ser Glu Pro Phe Arg Glin Llys Phe 450 45.5 460

Lell Glu Asn Pro Lys Asp Pro Asn Ala Phe Glu Gly Thir Wall Wall Wall 465 470 47s 48O

Phe Asp Gly Pro Glu Asp Tyr His His Arg Lieu. Glu Asp Pro Ser Thr 485 490 495

Pro Ile Asp Asp Arg Ser Ile Lieu. Val Met Arg Gly Ala Gly Pro Leu SOO 505

Gly Tyr Pro Gly Ala Ala Glu Val Wall Asn. Met His Pro Pro Gly Arg 515 525

Lell Luell Arg Glin Gly Wall Lys Ser Leu Pro Cys Ile Gly Asp Gly Arg 53 O 535 54 O

Glin Ser Gly Thir Ser Gly Ser Pro Ser Ile Lieu Asn Ala Ser Pro Glu 5.45 550 555 560

Ala Ala Ala Gly Gly Asn Lieu Ala Lieu. Lieu. Glin Asp Gly Asp Arg Lieu. 565 570 575

Arg Wall Asp Lieu. Asn Lys Arg Arg Val Asp Ile Lell Wall Ser Thr Glu 585 59 O

Glu Luell Glu Lys Arg Arg Llys Thr Lieu. Glu Ala Glin Gly Gly Tyr Asp 595 605

Wall Pro Glu Ser Glin Thr Pro Trp Glin Glu Lieu. Phe Arg Arg Glu Thir 610 615

Thir Glin Luell Ser Asp Gly Met Val Lieu. Arg Asp Ala Wall Tyr Glin 625 630 635 64 O

Arg Luell Ala Glin Arg Tyr Glu Asn Pro Arg His Asn His 645 650

SEQ ID NO 84 LENGTH: 1962 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthet ic DNA

<4 OOs, SEQUENCE: 84 atgtcttgcc agagtc.gcac Cagctgttgaa ggttgcagct gttctgatgg cggttct cqt 6 O cc.gc.cggitta at attgaaga ttgcgaaagt gaactgctgg cc.gtacggtg 12 O gaactggaaa aalaccctggc atc tatgcag gatggcc.gtc cgcatgcaaa tgcct ct cqt 18O gcacgtaaac tgcgtag to aaactggttt aattgttgaaa tatgatggcg 24 O ctgtatattg aacgctat ct gaattacggc atcaccc.gtg aagaactgat gagtggcaaa 3OO ccgattatcq gtattgcaca gagtggcago gatctgagcc c catcacctg 360 gaactggcga tgaagg tatt cgtagcgcag gcggt at CC gtttgaattit ccgacccacc cgatt Cagga aacgagcc.gc cgt.ccgaccg cgtgt atcga tcqtaatctg 48O

US 9,045,789 B2 153 154 - Continued

Lieu. Luell 385

<210s, SEQ ID NO 86 &211s LENGTH: 386 212. TYPE: PRT <213> ORGANISM: Agrobacterium tumefaciens <4 OOs, SEQUENCE: 86 Met Thr Gly Ser Arg Ser Ile Asp Ala Thir Ala Ile Lieu Pro Glu Asp 1. 5 1O 15 Phe Glu Asn Ala Lieu. Lieu Val Gly Arg Val Trp Ser Llys Ser Glu Gly 2O 25 3O Gly Pro Cys Pro Val Lieu Lleu Lys Gly Gly Val Lieu. Tyr Asp Lieu. Thir 35 4 O 45 Ser Ile Ser Pro Thr Met Ser Glu Lieu. Leu Glu Lys Thr Asp Leu Val SO 55 6 O Glu Lieu. Lieu Ala Asp Thr Gly Thr Phe Val Ala Lieu. Gly Ala Lieu. Glu 65 70 7s 8O Ala Phe Lieu. Asp Gly Ser Ala Gly Glu Lieu. Lieu Ala Pro Asn Asp Ile 85 90 95 Glin Ala Wall Lys Ala Ala Gly Val Thr Phe Ala Asp Ser Met Lieu. Glu 1OO 105 11 O Arg Val Ile Glu Glu Glin Ala Lys Gly Asp Pro Lieu. Arg Ala Glin Glu 115 12 O 125 Ile Arg Gly Arg Lieu Ala Pro Val Lieu. Gly Asp Asn Lieu Lys Gly Lieu. 13 O 135 14 O Glu Ala Gly Ser Glu Lys Ala Ala Glu Val Lys Llys Lieu. Lieu. Glin Glu 145 150 155 160 Met Gly Lieu. Trp Ser Glin Tyr Lieu. Glu Val Gly Ile Gly Pro Asp Ala 1.65 17O 17s Glu Ile Phe Ser Lys Ala Glin Ala Met Ala Ser Val Gly Cys Gly Ala 18O 185 19 O Lieu. Ile Gly Val His Pro Llys Ser Gln Trp Asn Asn Pro Glu Pro Glu 195 2OO 2O5 Val Val Lieu Ala Ile Ala Ser Asp Gly Arg Ile Val Gly Ala Thir Lieu 21 O 215 22O Gly Asn Asp Val Asn Lieu. Arg Asp Phe Glu Gly Arg Ser Ala Lieu. Lieu. 225 23 O 235 24 O Lieu. Ser Lys Ala Lys Asp Asn. Asn Ala Ser Cys Ala Ile Gly Pro Phe 245 250 255 Ile Arg Lieu. Phe Asp Gly Arg Phe Thir Ile Glu Asp Wall Lys Lys Ala 26 O 265 27 O Glin Ile Ser Lieu. Leu Val Glu Gly Glu Asp Gly Phe Thr Met Thr Gly 27s 28O 285

Ala Ser Ala Met Glin Ala Ile Ser Arg Thr Pro Glu Asn Lieu Ala Ser 29 O 295 3 OO

Glin Lieu. Lieu. Asn Arg Asn His Glin Tyr Pro Asp Gly Ala Val Phe Phe 3. OS 310 315 32O Lieu. Gly Thr Met Phe Ala Pro Wall Lys Asp Arg His Gly Pro Gly Lieu. 3.25 330 335 Gly Phe Thr His Ser Lys Gly Asp Arg Val Glu Ile Ser Thr Pro Llys 34 O 345 35. O

Lieu. Gly Lys Lieu. Ile Asn Trp Val Thir Thr Thr Asp Glu. Cys Pro Glu 355 360 365

US 9,045,789 B2 159 160 - Continued

355 360 365 aac cgc gtg ggc C9C agc gac cgt at C gct cc.g tgg acg ttic ggc gta 1152 Asn Arg Wall Gly Arg Ser Asp Arg Ile Ala Pro Trp Thir Phe Gly Wall 37 O 375 38O cgc gcc Ctg atg gcc aac Ctg gcc gca cgt ggc CaC a CC act ttic tga 12 OO Arg Ala Luell Met Ala Asn Lell Ala Ala Arg Gly His Thir Thir Phe 385 390 395

<210s, SEQ ID NO 89 &211s LENGTH: 399 212. TYPE : PRT &213s ORGANISM: Cupriavidus recator

<4 OOs, SEQUENCE: 89

Met Ala Lieu. Pro Lieu Thir Lell Ser Ala Thir Glin Thir Lell Pro Ala Asp 1. 5 15

Gly Luell Ala Gly Thr Lell Wall Gly Arg Ala Trp Ile Pro Ala Gly Asp 2O 25

Gly Wall Pro Ala Gly Pro Ala Wall Wall Wall Luell Arg Pro Asp Gly Wall 35 4 O 45

Phe Asp Ile Ser Asp Wall Ala Pro Thir Met Ser Thir Lell Luell Glu Glin SO 55 6 O

Asp Asp Pro Lieu. Thir Wall Wall His Asn Ala Pro Gly Arg Trp Ile Gly 65 70

Luell Asp Asp Lieu. Lell Ala Asn Thir Ala Asp Pro His Gly Ser Asn 85 90 95

Wall Ala Arg Lieu. Lieu Ala Pro Cys Asp Lieu. Gln Wall Ile Ala 1OO 105 11 O

Gly Wall Thir Phe Ala Gly Ser Luell Wall Glu Arg Wall Ile Glu Glu 115 12 O 125

Thir Gly Asp Pro Glin Gly Ala Ala Glu Wall Arg Asn Arg Ile 13 O 135 14 O

Ala Luell Val Gly Glu Arg Luell Ser Arg Ile Arg Pro Gly Ser Arg 150 155 160

Ala Gly Glu Lieu. Ala Luell Luell Ile Glu His Gly Met Trp Ser 1.65 17O 17s

Luell Glu Wall Gly Ile Gly Pro Asp Ala Glu Ile Phe Thir 18O 185 19 O

Pro Luell Luell Ser Ala Lell Gly Thir Gly Thir Glu Ile Gly Luell His 195

Pro Gly Ser Ala Trp Asn Asn Pro Glu Pro Glu Ile Wall Luell Ala Ile 21 O 215

Asn Ser Arg Gly Asp Wall Lell Gly Ala Thir Luell Gly Asn Asp Wall Asn 225 23 O 235 24 O

Lell Arg Asp Phe Glu Gly Arg Ser Ala Luell Luell Lell Gly Ala 245 250 255

Asp Asn Asn Gly Ser Ala Ile Gly Pro Phe Lell Arg Luell Phe Asp 26 O 265 27 O

Glin Ser Phe Ser Luell Asp Asp Wall Arg Arg Ala Thir Wall Asp Luell Arg 27s 28O 285

Wall Asp Gly Lieu. Asp Gly Phe Wall Luell Ser Gly Thir Ser Ser Met Asp 29 O 295 3 OO

Glin Ile Thir Arg Asp Pro Lell Glu Luell Ala Glu Glin Ala Met Gly Ala 3. OS 310 315

Thir His Glin Tyr Pro Asp Gly Ala Met Luell Phe Lell Gly Thir Luell Phe

US 9,045,789 B2 165 166 - Continued

citc. gt C aac cgc gtc a CC acg to c aag gcc gcc gcg c cc tgg acg ttic 1056 Lell Wall Asn Arg Val Thir Thir Ser Lys Ala Ala Ala Pro Trp Thir Phe 34 O 345 35. O ggc at C cgc gat Ctg atg cgc aat citc. gcc gcc cgc ggc citt citc. tog 1104 Gly Ile Arg Asp Lieu. Met Arg Asn Luell Ala Ala Arg Gly Luell Luell Ser 355 360 365

Cat to c taa 1113 His Ser 37 O

<210s, SEQ ID NO 92 &211s LENGTH: 370 212. TYPE : PRT &213s ORGANISM: Pseudomonas elodea

<4 OOs, SEQUENCE: 92

Met Lieu Pro Ala Asp His Ala Glin Ala Ile Luell Wall Gly Arg Wall Glin 1. 5 15

Thir Pro Ala Gly Pro Ser Pro Wall Luell Luell Arg Asp Gly Glin Wall Ile 2O 25

Asp Wall Ser Ala Ile Ala Pro Thir Wall Ala Asp Lell Lell Glu Arg Asp 35 4 O 45

Asp Ile Thir Lieu. Ser Gly Thir Wall Luell Cys Ser Wall Asp Ala Luell SO 55 6 O

Gly Thir Ser Ala Pro Glin Wall Luell Ala Pro Wall Asp Luell Glin Cys 65 70

Wall Ala Gly Wall Thr Phe Ala Wall Ser Ala Lieu Glu Arg Wall 85 90 95

Ile Glu Arg Ala Arg Gly Asp Ser Ala Lys Ala Ala Glu Ile Arg 1OO 105 11 O

Gly Asp Glu Ala Wall Gly Ser Gly Ile Arg Ser Wall Wall Pro 12 O 125

Gly Thir Glu Ala Ala Ala Luell Ala Ala Lell Ile Glu Ala Gly 13 O 135 14 O

Met Trp Ser Gln Tyr Lell Glu Wall Ala Ile Gly Pro Asp Ala Glu Wall 145 150 155 160

Phe Thir Ala Pro Wall Lell Ser Ala Met Gly Trp Gly Ala Glu Ile 1.65

Gly Ile Arg Ser Asp Ser Asp Trp Asn Asn Pro Glu Pro Glu Wall Wall 18O 185 19 O

Lell Wall Wall Asp Arg Asn Gly Ala Ile Gly Ala Thir Luell Gly Asn 195

Asp Wall Asn Lieu. Arg Asp Phe Glu Gly Arg Ser Ala Lell Luell Luell Gly 21 O 215

Lys Ala Asp Asn Asn Ala Ser Thir Ala Ile Gly Pro Phe Ile Arg 225 23 O 235 24 O

Lell Phe Asp Asp Gly Phe Thir Met Asp Asp Wall Arg Ser Ala Wall Wall 245 250 255

Asp Luell Thir Ile Asp Gly Pro Glu Gly Arg Lell Ser Gly Thir Asn 26 O 265 27 O

Met Ser Glu Ile Ser Arg Asp Pro Thir Glu Lell Wall Arg Glin Thir 27s 285

Lell Ser Glu His Glin Pro Asp Gly Phe Ala Lell Phe Luell Gly Thir 29 O 295 3 OO

Lell Phe Ala Pro Wall Glin Asp Arg Asp His Pro Gly Arg Gly Phe Thir

US 9,045,789 B2 171 172 - Continued

Ala Pro Thir Glu Asp Arg Asn Gly Lys Gly Lieu Gly Phe Thir His 34 O 345 35. O aag gga gat Cala gtc. aac atc. tct tct tcg cat ttg ggc aca citc. at C 1104 Lys Gly Asp Glin Wall ASn Ile Ser Ser Ser His Lell Gly Thir Luell Ile 355 360 365 aat tgg gtg aac act togt gac caa at a cca aag tgg gaa ttic ggc att 1152 Asn Trp Wall Asn Thr Cys Asp Glin Ile Pro Llys Trp Glu Phe Gly Ile 37 O 375 38O ggit gct titt acg aac tat att gta aaa cqg aat tta a.a.a. tag 1194 Gly Ala Phe Thir Asn Tyr Ile Val Lys Arg Asn Lell 385 390 395

<210s, SEQ ID NO 95 &211s LENGTH: 397 212. TYPE : PRT &213s ORGANISM: Zobellia galactanivorans

<4 OOs, SEQUENCE: 95

Met Asn Tyr Ile Asp Lieu. Asn Arg Lieu. Ile Pro Glu Lell Ala Glu Thir 1. 5 1O 15

Gly Thir Trp Ile Gly Arg Cys Met Val Pro Ala Glin Ala Asn 25 3O

Gly Ile Ala Gly Pro His Val Val Met Ala Arg Gly Ile Tyr 35 4 O 45

Asp Luell Ser Ala His Phe ASn Ser Thir Ser Glu Lell Phe Asn SO 55 6 O

Asn Pro Wall Ser Arg Lieu Lys Ala Lieu. Asn Asp Lell Pro Luell Gly 65 70 75

Ser Luell Asp Ala Lieu Lys Asn Ala Lieu. Tyr Phe Asn Asn Pro 85 90 95

Lell Luell Pro Tyr Ile Ile Ala Pro Asn Asp Ile Glin Ala Ala 105

Gly Wall Thir Phe Ile Llys Ser Lieu. Lieu. Glu Arg Wall Glu Glu 115 12 O 125

Ala Gly Asp Ala Lieu Val Ala Asn Asp Ile Arg Thir Ile 13 O 135 14 O

Tyr Asp Thir Luell Gly Asn Asp Lieu. Ser Llys Val Thir Pro Ser Pro 145 150 155 160

Glu Thir Glu Lieu Lys Glu Glu Lieu Gln Lys Gly Luell Trp Ser 1.65 17O 17s

Glin Tyr Luell Glu Val Gly Ile Gly Lys Asp Ala Glu Wall Phe Thir 18O 185 19 O

Ala Glin Pro Luell Ser Ala Val Gly Phe Gly Ala Glu Ile Gly Wall Luell 195 2OO

Ser Ser Trp Asn Asn Pro Glu Pro Glu Ile Wall Luell Ala Wall 21 O 215

Ser Ser Ser Lys Ile Val Gly Ala Thr Lieu. Gly Asn Asp Wall Asn 225 23 O 235 24 O

Lell Arg Asp Glu Gly Arg Ser Ala Lieu. Lieu. Lell Gly Glu Ala Lys 245 250 255

Asp Glin Asn Gly Ser Cys Ala Ile Gly Pro Lieu. Phe Arg Luell Phe Asp 26 O 265 27 O

Glu Thir Phe Ser Lieu. Asp Asp Wall Lys Asp Cys Asp Wall Met Phe Ser 27s 28O 285

Met Lys Gly Asp Asn. Phe Ala Thir Ser Gly Ser Asn Met Lys 29 O 295 3 OO

US 9,045,789 B2 177 178 - Continued

&211s LENGTH: 298 212. TYPE: PRT <213> ORGANISM: Thermobacillus composti <4 OOs, SEQUENCE: 98 Met Arg Ile Ile Arg Tyr Ile Gly Asp Asp Gly Ala Ala Arg Lieu Ala 1. 5 1O 15 Ala Val Thir Asp Glu Glu Glin Ala Phe Pro Lieu. Arg Ser Pro Asp Phe 2O 25 3O Met Ala Lieu Val Arg Glu Ala Asp Glu Ala Gly Ile Thr Pro Lieu. Glu 35 4 O 45 Ala Val Arg Arg Glin Ile Ala Gly Ala Glin Pro Lieu Pro Gly Asp Trip SO 55 6 O Arg Glu Lieu. Asn Lieu. Lieu. Thr Pro Val Asp Ala Pro Glu Val Trp Ala 65 70 7s 8O Ala Gly Val Thr Tyr Glu Arg Ser Lys Glu Ala Arg Asn. Glu Glu Ser 85 90 95 Lys Gly Ala Ala Thr Gly Asp Glu Thir Phe Tyr Asp Llys Val Tyr Arg 1OO 105 11 O Ala Glu Arg Pro Glu Ile Phe Phe Llys Ser Thr Ser Ala Arg Thr Ala 115 12 O 125 Arg Pro Gly Thr Pro Val Cys Ile Arg Ser Asp Ser Asp Trp Glin Val 13 O 135 14 O Pro Glu Pro Glu Lieu. Gly Ile Val Lieu. Asp Arg Gly Gly Arg Ile Lieu. 145 150 155 160 Gly Tyr Thr Val Gly ASn Asp Met Ser Cys Arg Asp Ile Glu Gly Glu 1.65 17O 17s Asn Pro Lieu. Tyr Lieu Pro Glin Ala Lys Ile Trp Arg Arg Ser Cys Ser 18O 185 19 O Ile Gly Pro Ala Ile Arg Lieu Ala Glu Thr Val Pro Asn Pro Tyr Asp 195 2OO 2O5 Lieu. Thir Ile Thir Cys Arg Ile Tyr Arg Asp Gly Glin Lieu Ala Val Asn 21 O 215 22O Glu Thir Ala Asn Thr Gly Glin Lieu. Arg Arg Llys Lieu. Asp Glu Lieu Ala 225 23 O 235 24 O Ser Phe Lieu Val Arg Asp Asn Val Val Phe Asp Gly Thr Val Lieu. Lieu. 245 250 255 Thr Gly Thr Cys Ile Val Pro Pro Asp Arg Phe Thr Lieu. Glin Pro Gly 26 O 265 27 O Asp Arg Ile Glu Ile Asp Ile Ser Gly Ile Gly Thr Lieu. Ile Asn Pro 27s 28O 285 Val Ala Ala Ala Asp Ala Ala Ile Glin Asp 29 O 295

<210s, SEQ ID NO 99 &211s LENGTH: 897 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: synthetic DNA

<4 OOs, SEQUENCE: 99 atgcgt atta t c cqct at at ttgatgat ggtgcc.gcac gtctggcagc C9ttaccgat 6 O gaagaac agg catttic cqct gcgttct c cq gattt catgg cqctggttcg cgaag.cggat 12 O gaag.cgggta t cacgc.cgct ggaag.cggtg C9tcgc.caga ttgcgggtgc acago.cgctg 18O

US 9,045,789 B2 183 184 - Continued

Lell Pro Asp Pro Ala Ala Ala Wall Arg Ala Ala Lell Lell Asp Pro Ala 65 70

Lell Wall Glin Arg Trp Ala Thir Ala Asp Wall Wall Ala Ala Ser Luell 85 90 95

Ala Asp Ala Ala Arg Pro His Luell Luell Ala Pro Wall Asp Luell Glin 105 11 O

Wall Lys Ala Cys Gly Wall Thir Phe Wall Asp Ser Met Ile Glu Arg 115 12 O 125

Wall Glu Glu Arg Ala Gly Asp Ala Ala Arg Ala Ala Glu Met 135 14 O

Arg Luell Wall Gly Lys Ala Luell Gly Gly Ser Ile Ala Thir Wall Arg 145 150 155 160

Pro Ser Pro Glu Ala Ala Glu Ala Lys Arg Wall Lell Ile Ala Glu 1.65

Gly Luell Trp Ser Glin Lell Glu Wall Gly Ile Gly Pro Asp Pro Glu 18O 185 19 O

Wall Phe Thir Ala Pro Wall Luell Ser Ser Wall Gly Lell Gly Ala Gly 195

Ile Gly Ile Pro Arg Phe Ser Ser Trp Asn ASn Pro Glu Pro Glu Luell 21 O 215 22O

Wall Luell Ile Wall Thir Ser Arg Gly Glu Wall Wall Gly Ala Thir Luell Gly 225 23 O 235 24 O

Asn Asp Wall Asn Lell Arg Asp Wall Glu Gly Arg Ser Ala Luell Luell Luell 245 250 255

Gly Ala Lys Asp Asn Asn Ala Ser Ser Ala Lell Gly Pro Luell Ile 26 O 265 27 O

Arg Luell Phe Asp Gly Ser Phe Thir Wall Asp Thir Lell Arg Glu Glu Glu 27s 285

Ile Luell Luell Arg Wall Glu Gly Luell Asp Gly Lell Lell Glu Gly Arg 29 O 295 3 OO

Asn Thir Luell Ala Arg Ile Ser Arg Pro Phe Glu Glu Lell Wall Ala Ala 3. OS 310 315

Thir Arg Gly Arg His His Glin Pro Asp Gly Phe Ala Luell Phe Thir 3.25 330 335

Gly Thir Luell Phe Ala Pro Thir Glin Asp Arg Asp Glu Pro Gly Glin Gly 34 O 345 35. O

Phe Thir His His Gly Asp Wall Wall Thir Ile Arg Ser Arg His Luell 355 360 365

Gly Ala Luell Ile Asn Arg Wall Gly Thir Ala Glu Glu Lell Pro Glu Trp 37 O 375

Thir Phe Gly Luell Arg Glin Lell Phe Gly Luell Ala Glu Glin Arg Glin 385 390 395 4 OO

Ala Glu Luell Ala Glin Met Glin Glu 4 OS

SEQ ID NO 102 LENGTH: 1227 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic DNA

SEQUENCE: 1 O2 atgaccgc.cc cqc.cgatc.cc gggttcgt.cc gtgcc.gc.cgg tacctctgt t ctgc.cggat