USOO9580731B2

(12) United States Patent (10) Patent No.: US 9,580,731 B2 B0tes et al. (45) Date of Patent: Feb. 28, 2017

(54) METHODS OF PRODUCING 7-CARBON 8,361,769 B1 1/2013 Koch et al. CHEMICALS VA C1 CARBON CHAIN 200-83 R 58. et al ELONGATIONASSOCATED WITH 2010.0035309 A1 2, 2010 Rh et al. COENZYME B SYNTHESIS 2010/0151536 A1 6/2010 Baynes et al. 2010/0203600 A1 8, 2010 Dubois (71) Applicant: INVISTA North America S.ár.l., 2010/02986 12 A1 11/2010 Behrouzian et al. Wilmington, DE (US) 2010. 0317069 A1 12/2010 Burk et al. 2011 0171699 A1 7/2011 Raemakers-Franken et al. (72) Inventors: Adriana Leonora Botes, Rosedale East 58.263 A. 1939 El (GB); Alex Van Eck Conradie, 2012/0101009 A1 4/2012 Beatty Eaglescliffe (GB); Changlin Chen, 2013,0065279 A1 3, 2013 Burk et al. Ingleby Barwick (GB); Paul S. 2013,0183728 A1 7/2013 Botes 2013,0210.090 A1 8, 2013 Pearlman et al. Pearlman, Thornton, PA (US) 2013/0217081 A1 8/2013 Pearlman et al. 2013,0224807 A1 8, 2013 Pearlman et al. (73) Assignee: INVISTA NORTH AMERICA 2013/0267012 A1 10, 2013 Steen et al. S.A.R.L., Wilmington, DE (US) 2014/0186902 A1 7, 2014 Botes et al. 2014/O186904 A1 7/2014 Botes et al. (*) Notice: Subject to any disclaimer, the term of this 338E. A. 23: s al patent 1s lists adjusted under 35 2014/0193864 A1 7, 2014 BotesOCS et al.a U.S.C. 154(b) by 0 days. 2014/0193865 A1 7, 2014 Botes et al. 2014,01969.04 A1 7/2014 Fontenelle et al. (21) Appl. No.: 14/138,971 2014,0199.737 A1 7, 2014 Botes et al.

(22) Filed: Dec.e Afa23, 9 2013 (Continued) FOREIGN PATENT DOCUMENTS (65) Prior Publication Data US 2014/0193863 A1 Jul. 10, 2014 Wo woooooo; "38. Related U.S. Application Data (Continued) (60) Provisional application No. 61/747,406, filed on Dec. OTHER PUBLICATIONS 31, 2012, provisional application No. 61/829,088, filed on May 30, 2013. Kizer L. et al. Application of Functional Genomics to Pathway Optimization for Increased Isoprenoid Production. 2008. Applied (51) Int. Cl. and Environmental Microbiology, vol. 74, No. 10. p. 3229-3241.* Prather KLJ et al. De novo biosynthetic pathways: rational design CI2P I3/00 (2006.01) of microbial chemical factories. 2008. Current Opinion in Biotech CI2P 7/18 (2006.01) nology. 19:468-474.* CI2P 7/42 (2006.01) “Enterococcus faecalis V583 bifuntional acetaldehyde-CoA/Alco CI2P 7/44 (2006.01) hol Dehydrogenase.” biocyc.org, retrieved on Jun. 19, 2014. http:// CI2N 15/52 (2006.01) biocyc.org/EFAE226185/N EW-IMAGE?type=ENZYM (52) U.S. Cl. E&object-GH11-877-MONOMER, 9 pages. “Information on EC 1.2.1.57—butanal dehydrogenase,” CPC ...... CI2P 13/001 (2013.01); C12N 15/52 .org, retrieved on Jun. 19, 2014. http:// brenda-en (2013.01); CI2P 7/18 (2013.01): CI2P 7/42 Zymes.org/php/result flat.php4?ecno=1.2.1.57, 6 pages. (2013.01); C12P 7/44 (2013.01); C12P 13/005 “BRENDA The comprehensive Information System.” (2013.01) Jul. 2011, retrieved on Sep. 19, 2014. http://web.archive.org/web/ (58) Field of Classification Search 2011 1009205602/http://www.brenda-enzymes.org/, 1 page. None (Continued) See application file for complete search history. Primary Examiner — Paul Holland (56) References Cited (74) Attorney, Agent, or Firm — Finnegan, Henderson, U.S. PATENT DOCUMENTS Farabow, Garrett & Dunner, LLP, Carla A. Mouta-Bellum

2.439,513. A 4, 1948 Hambleta et alla (57) ABSTRACT 2,557,282 A 6, 1951 Hamblet et al. This document describes biochemical pathways for produc 3. Sg A 3. 8. s Jr. et al ing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyhep 2970 10 A 2/1961 Gilby, Jr. et al. tanoic acid, heptamethylenediamine or 1,7-heptanediol by 3,023,238 A 2, 1962 Chapman et al. forming One Or tWO terminal functional groups, each CO 3,338,959 A 8, 1967 Sciance et al. prised of carboxyl, amine or hydroxyl group, in a C7 3,365.490 A 1, 1968 Arthur et al. aliphatic backbone . These pathways, metabolic 3,515,751 A g 1970 re 1 engineering and cultivation Strategies described herein rely 3.653. A 1923 Aka et al. on the C1 elongation enzymes or homolog associated with 6.255.451 B1 7/2001 Kochet al. coenzyme B biosynthesis. 6,372,939 B1 4/2002 Bunnel et al. 8,088,607 B2 1/2012 Burgard et al. 16 Claims, 26 Drawing Sheets US 9,580,731 B2 Page 2

(56) References Cited Binieda et al., “Purification, characterization, DNA Sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas medocin U.S. PATENT DOCUMENTS 35,” Biochem J., 1999, 340:793-801. Bond-Watts et al., "Biochemical and Structural Characterization of 2014/0248673 A1 9, 2014 Botes et al. 2015,0111262 A1 4/2015 Botes et al. the trans-Enoly-CoA Reductase from Treponema denticola,” Bio 2015,0267211 A1 9, 2015 Botes et al. chemistry, 2012, 51:6827-6837. 2015/0307854 A1 10/2015 Botes et al. Bordes et al., “ Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight FOREIGN PATENT DOCUMENTS into the denaturation mechanisms,” Journal of Biotechnology, 2011, 156: 117-124. WO WO 2008,145.737 12/2008 Batting, "Substrate Specificity of the 3-Methylaspartate Ammonia WO WO 2009/121066 1, 2009 Reaction: Observation of Differential Relative Reaction Rates WO WO 2009.113853 9, 2009 for Substrate- Pairs.” Biochemistry, 1988, 27:2953-2955. WO WO 2009.113855 9, 2009 WO WO 2009,140159 11/2009 Boylan et al., “Functional identification of the fatty acid reductase WO WO 2009,140695 11/2009 components encoded in the luminescence operon of Vibrio fischeri.” WO WO 2009,140696 11/2009 Journal of Bacteriology, 1985, 163(3): 1186-1190. WO WO 2009,151728 12/2009 Boylan et al., “Lux C, D and E genes of the Vibrio fischeri WO WO 2010/068944 6, 2010 luminescence operon code for the reductase, , and WO WO 2010/068953 6, 2010 synthetase enzymes involved in aldehyde biosynthsis.” Photochem WO WO 2010/071759 6, 2010 istry and photobiology, 1989, 49:681-688. WO WO 2010/104390 9, 2010 Bramer et al., “The methylcitric acid pathway in Ralstonia eutropha: WO WO 2010/104391 9, 2010 WO WO 2010, 129936 11 2010 new genes identified involved in propionate metabolism,” Micro WO WO 2010, 132845 11 2010 biology 2001, 147:2203-2214. WO WO 2011/OO3O34 1, 2011 Breithaupt et al., “Crystal structure of 12-oxophytodienoate WO WO 2011/031146 3, 2011 reductase 3 from tomato: self-inhibition by dimerization.” Proc WO WO 2011/031147 3, 2011 Natl. Acad Sci. USA, 2006, 103: 14337-14342. WO WO 2012/031910 3, 2012 Brigham et al., “Engineering Ralstonia eutropha for Production of WO WO 2012/071439 5, 2012 Isobutanol from CO2, H2, and O2,” Advanced Biofuels and WO WO 2012,094.425 T 2012 Bioproducts 2013, Chapter 39, pp. 1065-1090. WO WO 2012, 174430 12/2012 Brzostowicz et al., “ mRNA differential display in a microbial WO WO 2012/177721 12/2012 enrichment culture: simultaneous identification of three WO WO 2013/OO3744 1, 2013 cyclohexanonemonooxygenases from three species.' Applied and WO WO 2013/0285.19 2, 2013 WO WO 2013/082542 6, 2013 Environmental Microbiology, 2003, 69: 334-342. WO WO 2013/090837 6, 2013 Brzostowicz et al., “Identification of two gene clusters involved in WO WO 2013,0968.98 6, 2013 cyclohexanone oxidation in Brevibacterium epidermidis strain WO WO 2014/031724 2, 2014 HCU,” Applied and Microbiological Biotechnology, 2002, 58:781 WO WO 2014/093865 6, 2014 789. WO WO 2014f105,788 T 2014 Buckel et al., “Glutaconate CoA-transferase from Acidaminococcus WO WO 2014f105790 T 2014 fermentans.' Eur J. Biochem, 1981, 118:315-321. WO WO 2014f105793 T 2014 Budde et al., “Roles of Multiple Acetoacetyl WO 2O15036.050 3, 2015 Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonis eutropha H16.” J Bacteriol. 2010, 192(20):5319-5328. OTHER PUBLICATIONS Bugg et al., “The emerging role for bacteria in lignin degradation Alber et al., “Malonyl-coenzyme A reductase in the modified and bio-product formation.” Curr Opin Biotechnol 2011, 22(3):394 3-hydroxypropionate cycle for autotrophic carbon fixation in 400. archaeal Metallosphaera and Sulfolobus spp. J. Bacteriology, Buhler et al., “Occurrence and the possible physiological role of 2006, 188:8551-8559. 2-enoate reductases.” FEBS Letters, 1980, 109:244-246. Anton et al., Polyamides, Fibers, Encyclopedia of Polymer Science Bult et al., “Complete genome sequence of the and Engineering, 2001, 11:409-445. methanogenicarchaeon, Methanococcus jannaschii.' Science, 1996, AZuma et al., “Naphthalene—a constituent of Magnolia flowers.” 273: 1058-1073. Phytochemistry, 1996, 42:999-1004. Bunik et al., “Kinetic properties of the 2-oxoglutarate Barker et al., “Enzymatic reactions in the degradation of dehydrogenase complex from AZotobacter vinelandii evidence for 5-aminovalerate by Clostridium aminovalercum.” J Biol Chem. the formation of a precatalytic complex with 2-oxoglutarate.” Eur J 1987, 262(19): 8994-9003. Biochem., 267(12):3583-3591, Jun. 2000. Becker et al., “Metabolic flux engineering of L-lysine production in Cantu et al., “Thioesterases: A new perspective based on their Corynebacterium glutamicum—over expression and modification primary and tertiary structures.” Protein Science 2010, 19:1281 of G6P dehydrogenase,” J Biotechnol. 2007, 132(2):99-109. 1295. Bellmann et al., “Expression control and specificity of the basic Chayabutra and Ju, “Degradation of n-hexadecane and its metabo amino acid exporter LysE of Corynebacterium glutamicum, Micro lites by Pseudomonas aeruginosa under microaerobic and anaerobic biology 2001, 147: 1765-1774. denitrifying conditions.” Appl Environ Microbiol. 66(2):493-498, Bennett et al., “Purification and properties of 6-caprolactone Feb. 2000. from Acinetobacter NCIB 98.71 and Nocardia globevula Cheesbrough and Kolattukudy, “Alkane biosynthesis by CL1.” Journal of General Microbiology, 1988 134: 161-168. decarbonylation of aldehydes catalyzed by a particulate preparation Bernstein et al., “Transfer of the high-GC cyclohexane caboxylate from Pisum sativum.” PNAS USA, 1984, 81 (21):6613-7. degradation pathway from Rhodopseudomonas palustris to Chen et al., “Termites fumigate their nests with naphthalene.” Escherichia coli for production of biotin.” Metabolic Engineering, Nature, 1998, 392:558-559. May 2008, 10(3–4): 131-140. Cheng et al., “Genetic Analysis of a Gene Cluster for Cyclohexanol Berthold et al., “Structure of the branched-chain keto acid Oxidation in Acinetobacter sp. Strain SE19 by In Vitro Transposi decarboxylase (KdcA) from Lactococcus lactis provides insights tion.” Journal of Bacteriology, 2000, 182(17):4744-4751. into the structural basis for the chemoselective and enantioselective Coon, “Omega Oxygenases: nonheme-iron enzymes and P450 carboligation reaction.” Acta Crystallographica Sec. D, 2007. cytochromes. Biochemical & Biophysical Research Communica D63:1217-1224. tions, 2005,338:378-385. US 9,580,731 B2 Page 3

(56) References Cited aciditrophicus Strain SB in Syntrophic Association with H2-Using Microorganisms,” Applied and Environ. Microbiol. Apr. 2001, OTHER PUBLICATIONS 67(4): 1728-1738. Eurich et al., "Cloning and characterization of three fatty alcohol Cronan and Lin, “Synthesis of the O.co-dicarboxylic acid precursor oxidase genes from Candida tropicalis strain ATCC 20336.” of biotin by the canonical fatty acid biosynthetic pathway,” Current Applied & Environmental Microbiology, 2004, 70(8): 4872-4879. Opinion in Chem Biol., 2011, 15:407-413. Ferreira et al. “A member of the Sugar transporter family, Stillp is Cryle and Schlichting, "Structural insights from a P450 Carrier the glycerol/H=Symporter in Saccharomyces cerevisiae.” Molecular Protein complex reveal how specificity is achieved in the P450Biol Biology of the Cell, American Society for Cell Biology, Apr. 1, ACP complex,” Proceedings of the National Academy of Sciences, 2005, 16(4):2068-2076. Oct. 2008, 105(41): 15696-15701. Fickers et al., “Carbon and nitrogen sources modulate lipase pro Cryle et al., “Carbon-carbon bond cleavage by cytochrome duction in the yeast Yarrowia lipolytica,” Journal of Applied Micro P450BioI (CYP107H1) E1.” Chemical Communications, Jan. 2004, biology, 2004, 96:742-9. 86-87. Fickers et al., “The lipases from Yarrowia lipolytica: Genetics, production, regulation, biochemical characterization and Cryle, "Selectivity in a barren landscape: the P450BioI-ACP com biotechnological applications.” Biotechnology Advances, 2011, 29: plex,” Biochemical Society Transactions, Aug. 2010, 38(4):934 632-644. 939. Fuchs et al., “Microbial degradation of aromatic compounds—from Da Silva et al., “Glycerol: A promising and abundant carbon Source one strategy to four,” Nat Rev Microbiol., Oct. 3, 2011:9(11):803 for industrial microbiology,” Biotechnology Advances, 2009, 816, Oct. 2011. 27:30-39. Fukui et al., “Expression and Characterization of (R-Specific Enoly Daisy et al., “Naphthalene, an insect repellent, is produced by Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosyn Muscodor vitigenus, a novel endophytic fungus,” Microbiology, thesis by Aeromonas caviae,” J Bacteriol. 1998, 180(3):667-673. 2002, 148:3737-3741. Funhoff et al., “CYP153A6, a Soluble P450 Oxygenase Catalyzing Dalby, “Optimizing enzyme function by directed evolution.” Cur Terminal-Alkane Hydroxylation.” J Bacteriol. 2006, 188(14):5220 rent Opinion in Structural Biology, 2003, 13, 500-505. 5227. Davis et al., “Overproduction of acetyl-CoA carboxylase activity Funhoff et al., “Expression and Characterization of (R)-Specific increases the rate of fatty acid biosynthesis in Escherichia coli,” J. Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biol. Chem..., 2000, 275(37): 28593-28598. Biosynthesis by Aeromonas caviae,” J. Bacteriol., 2006, Day et al., “Partial purification and properties of acyl-CoA reductase 188(14):5220-5227. from Clostridum butyricum.” Archives of Biochemistry and Bio Gallus and Schink, “Anaerobic degradation of pimelate by newly physics, 1978, 190(1):322-331. isolated denitrifying bacteria.” Microbiology, 1994, 140:409-416. Deana et al., “Substrate specificity of a dicarboxyl-CoA: Gasmi et al., “A molecular approach to optimize hIFN C2b expres Dicarboxylic acid coenzyme . A transferase from rat liver mito sion and secretion in Yarrowia lipolytica.” Appl Microbiol Biotechnol, 2011, 89:109-119. chondria, Biochem Int., 1992, 26:767-773. GenBank Accession No. AAA24664.1, Mar. 25, 1993, 1 page. Dekishima et al., “Extending Carbon Chain Length of 1-Butanol GenBank Accession No. AAA24665.1, Apr. 26, 1993, 1 page. Pathway for 1-Hexanol Synthesis from Glucose by Engineered GenBank Accession No. AAA57874.1, Nov. 21, 2011, 2 pages. Escherichia coli,” J. Am. Chem. Soc., Aug. 2011, 133(30): 11399 GenBank Accession No. AAA69178.1, Jul. 1, 1995, 1 page. 11401. GenBank Accession No. AAB35106, Nov. 1995, 1 page. Dellomonaco et al., “Engineered reversal of the beta-oxidation GenBank Accession No. AAB60068.1, dated Jul. 1995, 1 page. cycle for the synthesis of fuels and chemicals.” Nature, Jan. 2011, GenBank Accession No. AAB98494.1, Oct. 23, 2009, 2 pages. 476(7360):355-359. GenBank Accession No. AAB99007.1, Oct 23, 2009, 2 pages. Deshmukh and Mungre, “Purification and properties of 2-amino GenBank Accession No. AAB99277.1, Oct. 23, 2009. adipate: 2-oxoglutarate aminotransferase from bovine kidney.” GenBank Accession No. AAC23921, Apr. 23, 2003, 2 pages. Biochem J, 1989, 26.1(3):761-768. GenBank Accession No. AAC76437.1, dated Oct. 2010, 2 pages. Doan et al., “Functional expression of five Arabidopsis fatty acyl GenBank Accession No. AAF02538.1, Oct. 20, 1999, 2 pages. CoA reductase genes in Escherichia coli,” J. Plant Physiology, GenBank Accession No. AAG081911, Jan. 31, 2014, 2 pages. 2009, 166:787-796. GenBank Accession No. AAK73167.2, retrieved May 19, 2014, 1 Dobritzsch et al., “High resolution crystal structure of pyruvate page. decarboxylase from Zymomonas mobilis. Implications for Substrate GenBank Accession No. AAN37290.1, retrieved May 19, 2014, 1 activation in pyruvate decarboxylases,” J. Biol. Chem., 1998, page. 273:20196-20204. GenBank Accession No. AAO77182, Mar. 28, 2003, 1 page. Donoghue and Trudgill, “The Metabolism of Cyclohexanol by GenBank Accession No. AAQ59697.1, Jan. 31, 2014, 2 pages. Acinetobacter NCIB9871, Eur J. Bochem., 1975, 60:1-7. GenBank Accession No. AAS11092.1, Mar. 5, 2010, 1 page. Drevland et al., “Enzymology and Evolution of the Pyruvate GenBank Accession No. AAS43086.1, dated Nov. 2011, 1 page. Pathway to 2-Oxobutyrate in Methanocaldococcus jannaschii.” J. GenBank Accession No. AAT43726, retrieved May 19, 2014, 1 Bacteriol. Apr. 2007, 189(12):4391-4400. page. Drevland et al., “Methanogen homoaconitase catalyzes both GenBank Accession No. AAW66853.1, Feb. 12, 2005, 1 page. hydrolyase reactions in coenzyme B biosynthesis,” J Biol Chem. GenBank Accession No. AAY39893.1, Jan. 31, 2014, 2 pages. Oct. 2008, 283: 28888-28896. GenBank Accession No. AB005294, Feb. 2000, 2 pages. Egmond et al., “Fusarium Solani pisi cutinase.” Biochimie, Nov. GenBank Accession No. ABA81135.1, Jan. 28, 2014, 2 pages. 2000, 82(11): 1015-1021. GenBank Accession No. ABC76100.1, Mar. 11, 2010, 1 page. Elkins et al., “Substrate Specificity of the RND-Type Multidrug GenBank Accession No. ABC76101.1, Mar. 11, 2010, 1 page. Efflux Pumps AcrB and AcrD of Esherichia coli is Determined GenBank Accession No. ABC76114.1, Mar. 11, 2010, 1 page. Predominately by Two Large Periplasmic Looops.” J Bacteriol. GenBank Accession No. ABC76260.1, Mar. 11, 2010, 1 page. 2002, 184(23):6490-6499. GenBank Accession No. ABC76948.1, Mar. 11, 2010, 1 page. Elshahed et al., “Benzoate Fermentation by the Anaerobic bacte GenBank Accession No. ABC76949.1, Mar. 11, 2010, 1 page. rium Syntrophus aciditrophicus in the Absence of Hydrogen-Using GenBank Accession No. ABC77793.1, Mar. 11, 2010, 1 page. Microorganisms,” Applied and Environ Microbiology, 2001, GenBank Accession No. ABC77794.1, Mar. 11, 2010, 1 page. 67(12):5520-5525. GenBank Accession No. ABC77898.1, Mar. 11, 2010, 1 page. Elshahed et al., “Metabolism of Benzoate, Cyclohex-1-ene GenBank Accession No. ABC77899.1, Mar. 11, 2010, 1 page. Carboxylate, and Cyclohexane Carboxylate by Syntrophus GenBank Accession No. ABC77900. 1, Mar. 11, 2010, 1 page. US 9,580,731 B2 Page 4

(56) References Cited Gonzalez-Lopez. "Genetic control of extracellular protease synthe sis in the yeast Yarrowia lipolytica,” Genetics, 2002, 160: 417-427. OTHER PUBLICATIONS Graupner et al., “Identification of the gene encoding Sulfopyruvate decarboxylase, an enzyme involved in biosynthesis of coenzyme GenBank Accession ABC78517.1, Mar. 11, 2010, 1 page. M. J. Bacterial., 2000, 182: 4862-4867. GenBank Accession ABC78756.1, Mar. 11, 2010, 1 page. Guerrillot et al., “Purification and Characterization of Two Alde GenBank Accession ABC78863. 1, Mar. 11, 2010, 1 page. hyde Dehydrogenases from Pseudomonas aeruginosa, Eur, J. GenBank Accession ABC7888.1.1, Mar. 11, 2010, 1 page. Biochem. 1977, 81:185-192. GenBank Accession . ABC7895.0.1, Mar. 11, 2010, 1 page. Hall, “The Contribution of Horizontal Gene Transfer to the Evolu GenBank Accession . ABE47158.1, Jan. 26, 2014, 1 page. tion of Fugi.” Duke University Libraries, May 10, 2007, 163 pages. GenBank Accession . ABE47159.1, Jan. 28, 2014, 2 pages. Hall, “Asymmetric bioreduction of activated alkenes using cloned GenBank Accession . ABE47160.1, Jan. 28, 2014, 1 page. 12-oxophytodienoate reductase isoenzymes OPR-1 and OPR-3 GenBank Accession . ABI83656.1, Jan. 3, 2007, 1 page. from Lycopersicon esculentum (tomato): a striking change of GenBank Accession . ABJ63754.1, dated Mar. 2010, 1 page. stereoselectivity,” Agnew Chem Int. Ed., 2007, 46:3934-3937. GenBank Accession . ABK71854.1, Jan. 31, 2014, 2 pages. Han et al., “Oxaloacetate , the C-C bond lyase of oxalate GenBank Accession . ABK75684.1, Jan. 31, 2014, 2 pages. secreting fungi.” J. Biol. Chem. 2007, 282:9581-9590. GenBank Accession . ACC40567. 1, Jan. 31, 2014, 2 pages. Harrison and Harwood, “The pimFABCDE operon from GenBank Accession . ACJO6772.1, Dec. 4, 2009, 1 page. Phodopseudomonas palustris mediates dicarboxylic acid degrada GenBank Accession . ADG98140.1, Jan. 28, 2014, 2 pages. tion and participates in anaerobic benzoate degradation,” Microbi GenBank Accession . ADK1958.1.1, Sep. 20, 2010, 2 pages. ology, 2005, 151:727-736. GenBank Accession . AEA39183.1, Apr. 4, 2011, 1 page. Harwood and Parales, “The beta-ketoadipate pathway and the GenBank Accession . AJO 12480.1, Apr. 2005, 2 pages. biology of self-identity.” Ann. Rev. Microbiol., 1996, 50:553-590. GenBank Accession . AY143338, Apr. 2003, 5 pages. Harwood et al., “Anaerobic metabolism of aromatic compounds via GenBank Accession . AY495697, Mar. 2004, 3 pages. the benzoyl-CoA pathway.” FEMS Microbiology Reviews, 1999, GenBank Accession . BAB91331.1, retrieved May 19, 2014, 1 22:439-458. page. Hasson et al., “The crystal structure of benzoylformate GenBank Accession . BAC06606, Aug. 1, 2002, 1 page. decarboxylase at 1.6A resolution-Diversity of catalytic residues in GenBank Accession . BAD69624, Sep. 2005, 1 page. ThDP-dependent enzymes.” Biochemistry, 1998, 37:9918-9930. GenBank Accession . BAF92773, Nov. 27, 2007, 1 page. Hayaishi et al., “Enzymatic Studies on the Metabolism of GenBank Accession No. BAF943.04.1, retrieved May 19, 2014, 1 page. fi-Alanine,” J. Biol. Chem., 1961, 236, p. 781-790. GenBank Accession . CAA44858.1, Apr. 28, 1992, 1 page. Haywood et al., "Characterization of two 3-ketothiolases possessing GenBank Accession . CAA81612.1, Apr. 18, 2005, 2 pages. differing Substrate specificities in the polyhydroxyalkanoate syn GenBank Accession . CAA90836.1, Apr. 18, 2005, 2 pages. thesizing organism Alcaligenes eutrophus.” FEMS Microbiology GenBank Accession . CAB13029.2, Nov. 20, 1997, 2 pages. Letters 1988, 52(1-2):91-96. GenBank Accession . CAC48239.1, Apr. 15, 2005, 2 page. He et al., “Nocardia sp. carboxylic acid reductase: cloning, expres GenBank Accession . CAE26094.1, Apr. 17, 2005, 2 pages. sion, and characterization of a new aldehyde fam GenBank Accession . CAE26097.1, Apr. 17, 2005, 2 pages. ily.” Applied and Environmental Microbiology, 2004, 70: 1874 GenBank Accession . CAHO4396.1, Apr. 7, 2005, 1 page. 1881. GenBank Accession . CAH04397.1, Apr. 7, 2005, 2 pages. Heath et al., “The enoyl-acyl-carrier-protein reductases FabI and GenBank Accession . CAHO4398.1, Apr. 7, 2005, 1 page. FabL from Bacillus subtilis,” J Biol Chem., 275(51):40 128-401.33, GenBank Accession . CCC78182.1, dated Jul. 2011, 1 page. Dec. 22, 2000. GenBank Accession . D84432, replaced by Q9SKC9.1, Feb. Hermann et al., “Industrial production of amino acids by coryneform 2005, 2 pages. bacteria.” J Biotechnol. 2003, 104(1-3): 155-172. GenBank Accession . EFV11917.1, Sep. 9, 2013, 2 pages. Hess et al., “Extremely thermostable esterases from the GenBank Accession . EIV11143.1, Jun. 19, 2012, 2 pages. thermoacidophilic euryarchaeon Picrophilus torridus.” GenBank Accession . JA114148, Apr. 2011, 1 page. Extremophiles, 2008, 12:351-364. GenBank Accession . JA114151, Apr. 2011, 1 page. Ho and Weiner, “Isolation and characterization of an aldehyde GenBank Accession . JA114154, Apr. 2011, 1 page. dehydrogenase encoded by the aldB gene of Escherichia coli,” J. GenBank Accession . JA114157, Apr. 2011, 1 page. Bacteriol., 2005, 187(3):1067-1073. GenBank Accession . L42023, Oct. 2009, 285 pages. Hoffmeister et al., “Mitochondrial trans-2-enoyl-CoA reductase of GenBank Accession . NM 001246944, Dec. 2011, 2 pages. wax ester fermentation from Euglena gracilis defines a new family GenBank Accession . NM 001247852, Dec. 2011, 2 pages. of enzymes involved in lipid synthesis,” J Biol Chem. 280(6):4329 GenBank Accession . NM 133240, Feb. 25, 2002, 2 pages. 4338. Epub Nov. 29, 2004. GenBank Accession . P22822, Mar. 1, 1992, 1 page. Hofvander et al., “A prokaryotic acyl-CoA reductase performing GenBank Accession . P94129 (replaced by Q6F7B8), Mar. 1, reduction of fatty acyl-CoA to fatty alcohol.” FEBS Letters, 2001, 2004. 1 page. 585:3538-3543. GenBank Accession No. S48141, May 1993, 2 pages. Holden et al., “Chorismate lyase: kinetics and engineering for GenBank Accession No. XM 001827609, Mar. 2011, 2 pages. stability.” Biochim Biophys Acta., Jan. 31, 2002, 1594(1):160-167. GenBank Accession No. YP 001394144.1, Jul. 26, 2007, 1 page. Hooks et al., “Long-chain acyl-CoA oxidases of Arabidopsis.” Plant GenBank Accession No. YP 400611, Nov. 10, 2005, 2 pages. J., 1999, 20:1-13. GenBank Accession No. YP 959486, Jan. 3, 2007, 2 pages. Hotta et al., “Extremely Stable and Versatile Carboxylesterase from GenBank Accession No YP 959769, Jan. 3, 2007, 2 pages. a Hyperthermophilic Archaeon.' Applied and Environmental Gerbling et al., “A new acyl-CoA synthetase, located in higher plant Microbiology, 2002, 68(8):3925-3931. cytosol.” J Plant Physiol, 1994, 143:561-564. Howell et al., “Alpha-keto acid chain elongation reactions involved Gloeckler et al., “Cloning and characterization of the Bacillus in the biosynthesis of coenzyme B (7-mercaptoheptanoyl threonine Sphaericus genes controlling the bioconversion of pimlate into phosphate) in methanogenic Archaea,” Biochemistry, 1989, 37: dethiobiotin, Gene, 1990, 87:63-70. 101.08-101.17. Gloerich et al., “Peroxisomal trans-2-enoyl-CoA reductase is Howell et al., “Identification of enzymes homologous to isocitrate involed in phytol degradation.” FEBS Letters 2006, 580:2092-2096. dehydrogenase that are involved in coenzyme Band leucine bio Gocke et al., “Comparative characterization of ThPP-dependent synthesis in methanoarchaea.” J Bacteriol. Sep. 2000, 182:5013 decarboxylases,” J. Mol. Cat. B: Enzymatic, 2009, 61:30-35. SO16. US 9,580,731 B2 Page 5

(56) References Cited Invitation to Pay Fees in International Application No. PCT/ US2013/077420, mailed May 13, 2014, 9 pages. OTHER PUBLICATIONS Invitation to Pay Fees in International Application No. PCT/ US2013/077423, mailed May 13, 2014, 10 pages. Hugler et al., “Malonyl-coenzyme A reductase from Chloroflexus Invitation to Pay Fees in International Application No. PCT/ aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for US2013/077430, mailed Aug. 25, 2014, 9 pages. autotrophic CO(2) fixation.” J. Bacteriology, 2002, 184:2404-2410. Ishige et al., “Wax Ester Production from n-Alkanes by Huhn et al., “Identification of the membrane protein Such and its Acinetobacter sp. Strain M-1: Ultrastructure of Cellular Inclusions role in Succinate transport in Corynebacterium glutamicum,’ Appl and Role of Acyl Coenzyme A Reductase.” Appl. Envtl. Microbil Microbiol Biotechnol. 2011, 89(2):327-335. Hunt et al., “Characterization of an acyl-CoA thioesterase that ogy, 2002, 68: 1192-1195. functions as a major regulator of peroxisomal lipid metabolism,” J. Ishikawa et al., “The pathway via D-galacturonate/L-galactonate is Biol Chem, 2002, 277:1128-1138. significant for ascorbate biosynthesis in Euglena gracilis: identifi International Preliminary Report on Patentability for International cation and functional characterization of aldonolactonase,” Journal Application No. PCT/US2012/069934, mailed Jun. 17, 2014, 15 of Biologiocal Chemistry, 2008, 283:31133-31141. pageS. Iwaki et al., “Cloning and Characterization of a Gene Cluster International Preliminary Report on Patentability in International Involved in Cyclopentanol Metabolism in Comamonas sp. Strain Application No. PCT/US2012/042777, mailed Jan. 10, 2013, 22 NCIMB 98.72 and Biotransformations Effected by Escherichia pageS. coli-Expressed Cyclopentanone 1,2-Monooxygenase.' Appl International Preliminary Report on Patentability in International Environ Microbiol., 2002, 68(11):5671-5684, 14 pages. Application No. PCT/US2012/044984, mailed Jan. 28, 2014, 11 Iwaki et al., “Identification of a Transcriptional Activator (ChnR) pageS. and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol International Search Report and Written Opinion for International Catabolic Pathway in Acinetobacter sp. Strain NCIMB 98.71 and Application No. PCT/US2012/069934, mailed Jan. 17, 2014, 21 Localization of the Genes That Encode Them.” Appl. Environ. pageS. Microbiol., 1999, 65(11):5158-5162. International Search Report and Written Opinion in International Izumi et al., “Structure and Mechanism of HpcG, a Hydratase in the Application No. PCT/US2012/042747, mailed Jan. 14, 2013, 19 Homoprotocatechuate Degradation Pathway of Escherichia coli,” J. pageS. Mol. Biol., 2007, 370:899-911. International Search Report and Written Opinion in International Izumi et al., “The pimeloyl-CoA synthetase responsible for the first Application No. PCT/US2012/042777, mailed Sep. 11, 2012, 9 step in biotin biosythesis by microorganisms,” Agr. Biol. Chem. pageS. 1974, 38:2257-2262. International Search Report and Written Opinion in International Jacob et al., “Glutaconate CoA-transferase from Acidamiococcus Application No. PCT/US2012/044984, mailed Dec. 17, 2013, 17 fermenians: the crystal structure reveals homology with other pages. CoA-,” Structure, 1997, 5:415-426. International Search Report and Written Opinion in International Jang et al., “Bio-based production of C2-C6 platform chemicals.” Application No. PCT/US2012/071472, mailed Dec. 17, 2013, 17 Biotechnol. & Bioengineering, 2012, 109(10):2437-2459. pageS. Jarboe, “YohD: a broad-substrate range aldehyde reductase with International Search Report and Written Opinion in International various applications in production of biorenewable fuels and chemi Application No. PCT/US2013/075058, mailed Sep. 15, 2014, 17 cals.” Appl Microbiol Biotechnol., 2011, 89(2):249-257. pageS. Jaremko et al., “The initial metabolic conversion of levulinic acid in International Search Report and Written Opinion in International Cupriavidus necator.” J. Biotechnol., 2011, 155(3):293-298. Application No. PCT/US2013/075087, mailed Aug. 4, 2014, 18 Jeyakanthan et al., “Substrate specificity determinants of the pageS. methanogen homoaconitase enzyme: structure and function of the International Search Report and Written Opinion in International small subunit,” Biochemistry, 2010, 49:2687-2696. Application No. PCT/US2013/077411, mailed Sep. 24, 2014, 18 Jing et al., “Phylogenetic and experimental characterization of an pageS. acyl-ACP thioesterase family reveals significant diversity in enzy International Search Report and Written Opinion in International matic specificity and activity.” BMC Biochemistry, 2011, 12:44, 16 Application No. PCT/US2013/077413, mailed Jul 22, 2014, 20 pageS. pageS. Joon-Young et al., “Production of 1.2-Propanediol from Glycerol in International Search Report and Written Opinion in International Saccharomyces cerevisiae,” J. Microbiology and Biotechnology, Application No. PCT/US2013/077419, mailed Jun. 16, 2014, 19 May 19, 2011, 21 (8):846-853. pageS. Kakugawa et al., “Purification and Characterization of a Lipase International Search Report and Written Opinion in International from the Glycolipid-Producing Yeast Kurtzmanomyces sp I-11.” Application No. PCT/US2013/077420, mailed Jul. 21, 2014, 21 Bioscience Biotechnology Biochemistry, 2002, 66(5): 978-985. pageS. Kato and Asano, "Cloning, nucleotide sequencing, and expression International Search Report and Written Opinion in International of the 2-methylasparatate ammonia-lyase gene from Citrobacter Application No. PCT/US2013/077423, mailed Jul. 21, 2014, 22 amalonaticus strain YG-1002.” Appl. Microbiol Biotechnol, 1998, pageS. 50:468-474. International Search Report and Written Opinion in International Kaulmann et al., “Substrate spectrum of omega-transaminase from Application No. PCT/US2013/077445, mailed Sep. 15, 2014, 17 Chromobacterium violaceum DSM30191 and its potential for pageS. biocatalysis,” Enzyme Microb Technol. 2007, 41:628-637. Invitation to Pay Additional Fees in International Application No. Kikuchi et al., “Characterization of a second lysine decarboxylase PCT/US2013/075058, mailed Jul. 7, 2014, 7 pages. isolated from Escherichia coli,” J Bacteriol, 1997, 179(14): 4486 Invitation to Pay Additional Fees in International Application No. 4489. PCT/US2013/07745, mailed Jul. 7, 2014, 9 pages. Kim et al., “Cloning and characterization of a cyclohexanone Invitation to Pay Fees in International Application No. PCT/ monooxygenase gene from Arthrobacter sp. L661. Biotechnology US2013/075087, mailed May 16, 2014, 9 pages. Bioprocess Engineering, 2008, 13:40-47. Invitation to Pay Fees in International Application No. PCT/ Kim, “Purification and properties of a diamine alpha-ketoglutarate US2013/0774 11, mailed Jul. 16, 2014, 9 pages. transaminase from Escherichia coli,” J Biol Chem 1964, Invitation to Pay Fees in International Application No. PCT/ 239(3):783-786. US2013/077413, mailed May 12, 2014, 9 pages. Kitzing et al., “The 1.3 Acrystal structure of the flavoprotein YoM Invitation to Pay Fees in International Application No. PCT/ reveals a novel class of Old Yellow Enzymes,” J. Biol. Chem., 2005, US2013/077419, mailed Apr. 16, 2014, 9 pages. 280:27904-27913. US 9,580,731 B2 Page 6

(56) References Cited Lopez-Sanchez et al., “Tetralin-Induced and ThnR-Regulated Alde hyde Dehydrogenase and 3-Oxidation Genes in Sphingomonas OTHER PUBLICATIONS macrogolitabida Strain TFA.” Appl. Environ. Microbiol., 2010, 76(1): 110-118. Koch et al., “Products of Enzymatic Reduction of Benzoyl-CoA. A Luo et al., “Production of 3-hydroxypropionic acid through Key Reaction in Anaerobic Aromatic Metabolism.” Eur, J. Bio propionaldehyde dehydrogenase PduP mediated biosynthetic path chemistry, Jan. 1993, 211(3):649-661. way in Klebsiella pneumoniae.” Bioresource Technoogy, 2012, Koch et al., “In Vivo Evolution of Butane Oxidation by Terminal 103: 1-6. Alkane Hydroxylases AlkB and CYP153A6.” Appl. Environ. Litke-Eversloh & Steinbichel, "Biochemical and molecular char Microbiol., 2009, 75(2):337-344. acterization of a Succinate semialdehyde dehydrogenase involved in Kockelkorn and Fuchs, "Malonic semialdehyde reductase, Succinic the catabolism of 4-hydroxybutyric acid in Ralstonia eutropha.” semialdehyde reductase, and Succinyl-coenzyme A reductase from FEMS Microbiology Letters, 1999, 181(1):63-71. Metallosphaera Sedula: enzymes of the autotrophic Mack and Buckel, "Conversion of glutaconate CoA-transferase 3-hydroxypropionate/4-hydroxybutyrate cycle in Sulfolobales,” J. from Acidaminococcus fermentans into an acyl-CoA hydrolase by Bacteriology, 2009, 191:6352-6362. site-directed mutagenesis.” FEBS Letters, 1997, 405:209-212. Maeda et al., “Purification and characterisation of a biodegradable Kolattukudy, “Enzymatic synthesis of fatty alcohols in Brassica plastic-degrading enzyme from Aspergillus oryzae, Applied and oleracea.” Archives of Biochemistry and Biophysics, 1971, Environmental Biotechnology, 2005, 67: 778-788. 142(2):701-709. Mahadik et al., “Production of acidic lipase by Aspergillus niger in Köpke et al., “2,3-Butanediol Production by Acetogenic Bacteria, solid state fermentation.” Process Biochemistry, 2002, 38: 715-721. an Alternative Route to Chemical Synthesis, Using Industrial Waste Martin and Prather, “High-titer production of monomeric Gas.” Appl Environ Microbiol., 2011, 77(15):5467-5475. hydroxyvalerates from levulinic acide Pseudomonas putida,” J. Kulkarni and Kanekar, 'Bioremediation of epsilon-caprolactam Biotechnol., 2009, 139: 61-67. from nylon-6 waste water by use of Pseudomonas aeruginosa MCM Martinez et al., “Fusarium Solani cutinase is a lipolytic enzyme with B-407. Curr. Microbiol., 1998, 37:191-194. a catalytic serine accessible to solvent.” Nature, 1992, 356:615-618. Kung et al., “Cyclohexanecarboxyl-coenzyme A (CoA) and Matsumoto et al., “A new pathway for poly(3-hydroxybutyrate) cyclohex-1-ene-1-carboxyl-CoA dehydrogenases, two enzymes production in Escherichia coli and Corynebacterium glutamicum by involved in the fermentation of benzoate and crotonate in functional expression of a new acetoacetyl-coenzyme a synthase.” Syntrophus aciditrophicus,” J Bacteriol. 195(14):3193-3200, Epub Biosci. Biotechnol. Biochem., 2011, 75(2):364-366. May 10, 2013. Mawal and Deshmukh, "Alpha-aminoadipate and kynurenine Lan et al., "Oxygen-tolerant coenzyme A-acylating aldehyde aminotransferase activities from rat kidney. Evidence for separate dehydrogenase facilitates efficient photosynthetic n-butanol biosyn identity,” J. Biol Chem, 1991, 266(4):2573-2575. thesis in cyanobacteria.” Energy Environ Sci., 2013, 6:2672-2681. McAndrew et al., “Structural basis for substrate fatty acyl chain Larroy et al., "Characterization of the Saccharomyces cerevisiae specificity: crystal structure of human very-long-chain acyl-CoA YMR318C (ADH6) gene product as a broad specificity NADPH dehydrogenase,” J. Biol. Chem., 2008, 283:9435-9443. dependent alcohol dehydrogenase: relevance in aldehyde reduc Meijnen et al., “Improved p-hydroxybenzoate production by engi tion.” Biochem J., 2002, 361 (Pt 1):163-172. neered Pseudomonasputida S12 by using a mixed-substrate feeding Le Dall et al., “Multiple-copy integration in the yeast Yarrowia strategy.” Appl. Microbiol. Biotechnol., 2011, 90:885-893. lipolytica,” Current Genetics, 1994 26:38-44. Mhetras et al., “Purification and characterization of acidic lipase Lee and Meighen, “Cysteine-286 as the site of acylation of the from Aspergillus niger NCIM 1207,” Bioresource Technology, LUX-specific fatty acyl-CoA reductase.” Biochim Biophys Acta, 2009, 100: 1486-1490. 1997, 1338:215-222. Millar et al., “CUT1, an Arabidopsis Gene Required for Cuticular Lee et al., “Metabolic Engineering of Pentose Phosphate Pathway in Wax Biosynthesis and Pollen Fertility, Encodes a Very-Long-Chain Ralstonia eutropha for Enhanced Biosynthesis of Poly-8- Fatty Acid Condensing Enzyme.” The Plant Cell, May 1999, hydroxybutyrate.” Biotechnology Progress, 2003, 19(5): 1444-1449. 11(5):825-838, retrieved on Sep. 30, 2014. http://www.planticell. Lee et al., “Synthesis of pure meso-2,3-butanediol from crude org/content/11/5/825.full. glycerol using an engineered metabolic pathway in Escherichia Miyazaki et al., “Alpha-Aminoadipate aminotransferase from an coli,” Appl Biochem Biotechnol., 2012, 166(7): 1801-1813. extremely thermophilic bacterium. Thermus thermophilus,” Micro Li et al., “Cupriavidus necator JMP 134 rapidly reduces furfural biology, 2004, 150(7): 2327-2334. through a Zn-dependent alcohol dehydrogenase,” Biodegradation, Mo et al., “Connecting extracellular metabolomic measurements to 2011, 22:1215-1225. intracellular flux states in yeast.” BMC Systems Biology, 2009, Lim et al., “Amplification of the NADPH-related genes Zwfand gnd 3(37): 1-17. for the oddball biosynthesis of PHB in an E. coli transformant Mouttaki et al., “Cyclohexane Carboxylate and Benzoate Formation harboring a cloned phbCAB operon.” J Bioscience and Bioengi from Crotonate in Sytrophus aciditrophicus.” Applied and Environ neering, 2002, 93(6):543-549. Microbiology, Feb. 2007, 73(3):930-938. Lin and Cronan, "Closing in on complete pathways of biotin Murphy et al., “Fusarium polycaprolactone depolymerase is biosynthesis.” Molecular Biosystems, 2011, 7: 1811-1821. cutinase.” Appl. Environm. Microbiol., 1996, 62:456-460. Lin et al., “Biotin Sythesis Begins by Hijacking the Fatty Acid Naggert et al., “Cloning, sequencing, and characterization of Synthetic Pathway,” Nature Chem Biol. Sep. 2010, 6:682-688. Escherichia coli thioesterase II,” J. Biol. Chem., 1991, Lin et al., “The BioC O-Methyltransferase Catalyzed Methyl 266(17): 11044-11050. Esterification of Malonyl-Acyl Carrier Protein, an Essential Step in Ney?akh, “The Multidrug Efflux Transporter of Bacillus subtilis is Biotin Synthesis,” Journal of Biological Chemistry, Sep. 2012, a Structural and Functional Homolog of the Staphylococcus NorA 287(44):370 10-37020. Protein.” Antimicrob Agents Chemother, 1992, 36(2):484-485. Lin, “Biotin Synthesis in Escherichia coli.” PhD Dissertation, Ng et al., “Quinolone Resistance Mediated by norA: Physiologic University of Illinois at Urbana-Champaign, 2012, 140 pages. Characterization and Relationship to flaB, a Quinolone Resistance Liu and Chen, “Production and characterization of medium-chain Locus on the Staphylococcus aureus Chromosome.' Antimicrob length polyhydroxyalkanoate with high 3-hydroxytetradecanoate Agents Chemother, 1994, 38(6): 1345-1355. monomer content by fadB and fadA knockout mutant of Nicol et al., “Bioconversion of crude glycerol by fungi.” Applied Pseudomonas putida KT2442,” Appl. Microbiol. Biotechnol., 2007. Microbiology and Biotechnology, Feb. 10, 2012, 93(5): 1865-1875. 76(5): 1153-1159. Nieder and Shapiro, “Physiological function of the Pseudomonas Liu et al., “Two novel metal-independent long-chain alkyl alcohol putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: dehydrogenases from Geobacillus thermodenitrificans NG80-2.” monoterminal Oxidation of alkanes and fatty acids,” J. Bacteriol. Microbiology, 2009, 155:2078-2085. 1975, 122(1):93-98. US 9,580,731 B2 Page 7

(56) References Cited Ramsay et al., “Use of a Nylon Manufacturing Waste as an Industrial Fermentation Substrate.” Applied and Environmental OTHER PUBLICATIONS Microbiology, 1986, 52(1): 152-156. Ray et al., "Cocrystal structures of diaminopimelate decarboxylase: Nishimaki et al., “Studies on the Metabolism of Unsaturated Fatty mechanism, evolution, and inhibition of an antibiotic resistance Acids. XIV. 1 Purification and Properties of NADPH-Dependent accessory factor.” Structure, 2002, 10(11): 1499-1508. trans-2-Enoyl-CoA Reductase of Escherichia coli K-12.” J. Rea et al., “Structure and Mechanism of HpcH: A Metal Ion Biochem., 1984, 95:1315-1321. Dependent Class II Aldolase from the Homoprotocatechuate Deg Nomura et al., “Expression of 3-Ketoacyl-Acyl Carrier Protein radation Pathway of Escherichia coli,” J. Mol. Biol., 2007, 373:866 Reductase (fabG) Genes Enhances Production of 876. Polyhydroxyalkanoate Copolymer from Glucose in Recombinant Rohdich et al., “Enoate reductases of Clostridia. Cloning, sequenc Escherichia coli JM109.” Appl. Environ. Microbiol., 2005, ing, and expression.” J. Biol. Chem., 2001, 276:5779-5787. 71(8):4297-4306. Roje, “Vitamin B biosynthesis in plants.” Phytochemistry, 2007. Ohashi et al., “Continuous production of lactic acid from molasses 68:1904-1921. by perfusion culture of Lactococcus lactis using a stirred ceramic Roujeinikova et al., “Structural studies of fatty acyl-(acyl carrier membrane reactor.” J. Bioscience and Bioengineering, 1999, protein) thioesters reveal a hydrophobic binding cavity that can 87(5):647-654. expand to fit longer substrates,” J Mol Biol. 365(1):135-145, Epub Okuhara et al., “Formation of Glutaric and Adipic Acids from Sep. 23, 2006. n-Alkanes with Odd and Even Nos. of Carbons by Candida Ryu et al., “A novel synthesis of beta-trichlorostannyl ketones tropicalis OH23,” Agr. Biol. Chem., 1971, 35(9): 1376-1380. from siloxycyclopropanes and their facile dehydrostannation Onakunle et al., “The formation and substrate specificity of bacterial affording 2-methylene ketones,” JOC, 1986, 51:2389-2391. lactonases capable of enantioselective resolution of racemic Salcher and Lingens, "Regulation of phospho-2-keto-3-deoxy lactones,” Enzyme and Microbial Technology, 1997, 21: 245-251. heptonate aldolase (DAHP synthase) and anthranilate synthase of Oppenheim and Dickerson, "Adipic Acid.” Kirk-Othmer Encyclo Pseudomonas aureofaciens,” J Gen Microbiol. 121(2):473-476, pedia of Chemical Technology, 2003. Dec. 1980. Ouchi et al., “Dual roles of a conserved pair, Arg23 and Ser20, in Sambrook et al., Molecular Cloning a Laboratory Manual, Cold recognition of multiple Substrates in alpha-aminoadipate Spring Harbor Laboratory Press, 2001. aminotransferase from Thermus thermophilus.” Biochem Biophys Samsonova et al., “Molecular cloning and characterization of Res Commun, 2009, 388(1):21-27. Escherichia coli K12 ygG gene.” BMC Microbiology, 2003, 3:2. Palosaari and Rogers, “Purification and properties of the inducible Sanders et al., “Characterization of the human (D-Oxidation pathway coenzyme A-linked butyraldehyde dehydrogenase from Clostridium for co-hydroxy-very-long-chain fatty acids.” FASEB Journal, 2008, acetobutylicum.” J. Bacteriol., 1988, 170(7):2971-2976. 22(6):2064-2071. Papanikolaou et al., "Citric acid production by Yarrowia lipolytica Sanders et al., “Evidence for two enzymatic pathways for co-oxi dation of docosanoic acid in rat liver microsomes,” J. Lipid cultivated on olive-mill wastewater-based media,” Bioresource Research, 2005, 46(5):1001-1008. Technol., 2008, 99(7):2419-2428. Satoh et al., “Enzyme-catalyzed poly(3-hydroxybutyrate) synthesis Parthasarthy et al., “Substrate specificity of 2-hydroxyglutaryl-CoA from acetate with CoA recycling and NADPH regeneration in dehydratase from Clostiridium symbiosum: Toward a bio-based vitro.” J Bioscience and Bioengineering, 2003, 95(4):335-341. production of adipic acid.” Biochemistry, 2011, 50:3540-3550. Scheller et al., “Generation of the Soluble and Functional Cytosolic Pelletier and Harwood et al., “2-Hydroxycyclohexanecarboxyl Domain of Microsomal Cytochrome P450 52A3.” J Biol Chem. coenzyme A dehydrogenase, an enzyme characteristic of the anaero 1994, 269(17): 12779-12783. bic benzoate degradation pathway used by Rhodopseudomonas Schirmer et al., “Microbial Biosythesis of Alkanes,” Science, 2010, palustris,” J Bacteriol., 182(10):2753-2760, May 2000. 329:559-562. Pérez-Pantoja et al., “Metabolic reconstruction of aromatic com Schwartz et al., “A proteomic view of the facultatively pounds degradation from the genome of the amazing pollutant chemolithoautotrophic lifestyle of Ralstonia eutropha H16.” degrading bacterium Cupriavidus necator JMP134.” FEMS Proteomics, 2009, 9:5132-5142. Microbiol. Rev., 2008, 32:736-794. Seedorf et al., “The genome of Clostridium kluyveri, a strict Peterson et al., “The Thermal Stability of the Fusarium solani pisi anaerobe with unique metabolic features.” Proc. Natl. Acad. Sci. Cutinase as a Function of pH.” BioMed Research International, USA, 2008, 105(6): 2128-2133. 2001, 1.2:62-69. Shapiro et al., “Remarkable Diversity in the Enzymes Catalyzing Pignede et al., “Autocloning and Amplification of LIP2 in Yarrowia the Last Step in Synthesis of the Pimelate Moiety of Biotin.” lipolytica.” Appl. Environ. Microbiol, 2000 66:3283-3289. PLoSOne, Nov. 2012, 7(11):e49440, 11 pages. Pignede et al., “Characterization of an extracellular lipase encoded Shen et al., “Driving Forces Enable High-Titer Anaerobic 1-Butanol by LIP2 in Yarrowia lipolytica,” Journal of Bacteriology, 2000, 182: Synthesis in Escherichia coli.” Appl. Environ. Microbiol., 2011, 2802-2810. 77(9):2905-2915. Ploux et al., “Investigation of the first step of biotin biosynthsis in Shikata et al., “A novel ADP-forming succinyl-CoA synthetase in Bacillus sphaericus: Purification and characterization of the Thermococcus kodakaraensis structurally related to the archaeal pimloyl-CoA sythase, and uptake of pimelate.” Biochem J., 1992, nucleoside diphosphate-forming acetyl-CoA synthetases,” J. Biol. 287:685-690. Chem, 2007, 282(37):26963-26970. Prybylski et al., “Third-generation feed stocks for the clean and Siegert et al., “Exchanging the Substrate specificities of pyruvate Sustainable biotechnological production of bulk chemicals: synthe decarboxylase from Zymomonas mobilis and benzoylformate sis of 2-hydroxyisobutyric acid.” Energy, Sustainability and Society, decarboxylase from Pseudomonas putida,” Port. Eng. Des. Sel. 2012, 2:11. 2005, 18:345-357. Qian et al., “Metabolic engineering of Escherichia coli for the Simon et al., “Chiral Compounds Synthesized by Biocatalytic production of cadaverine: a five carbon diamine.” Biotechnol Reductions New Synthetic Methods (51).” Angew Chem Ed Bioeng, 2011, 108(1):93-103. Engl., 1985, 24:539-553. Qiu et al., “Crystal structure and substrate specificity of the Simon, “Properties and mechanistic aspects of newly found redox B-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylo enzymes from anaerobes Suitable for bioconversions on preparatory coccus aureus.” Protein Sci., 2005, 14(8):2087-2094. scale.” Pure and Appl. Chem, 1992, 64: 1181-1186. Rajashekhara et al., “Propionyl-coenzyme A synthetases of Slater et al., “Multiple B-Ketothiolases Mediate Poly(B- Ralstonia Solanacearum and Salmonella choleraesuis display atypi Hydroxyalkanoate) Copolymer Synthesis in Ralstonia eutropha.” J cal kenetics.” FEBS Letters, 2004, 556:143-147. Bacteriol., 1998, 180(8): 1979-1987. US 9,580,731 B2 Page 8

(56) References Cited White, “A novel biosynthesis of medium chain length alpha ketodicarboxylic acids in methanogenic archaebacteria.” Archivers OTHER PUBLICATIONS of Biochemistry and Biophysics, 1989, 270: 691-697. White, “Biosynthesis of the 7-mercaptoheptanoic acid subunit of Smith et al., "Complete genome sequence of Methanobacterium component B (7-mercaptoheptanoyl)threonine phosphate of thermoautotrophicum deltaH: functional analysis and comparative methanogenic bacteria.” Biochemistry, 1989, 28: 860-865. genomics,” J Bacteriol., 1997, 179: 7135-7155. White, “Steps in the conversion of a-ketosuberate to Smith et al., “Structural analysis of ligand binding and in 7-mercaptoheptanoic acid in methanogenic bacteria, Biochemistry, chorismate lyase.” Archives of Biochemistry and Biophysics, Jan. 1989, 28:9417-9423. 2006, 445(1):72-80. Widmann et al., “Structural classification by the Lipase Engineering Stok et al., “Expression, Purification, and Characterization of BioI: Database: a case study of Candida antarctica lipase A.” BMC A Carbon-Carbon Bond Cleaving Cytochrome P450 Involved in Genomics, 2010, 11:123-130. Biotin Biosynthesis in Bacillus Subtilis.” Archives of Biochemistry Willis et al., “Characterization of a fatty acyl-CoA reductase from and Biophysics, Dec. 2000, 384(2):351-360. Marinobacter aquaeolei VT8: a bacterial enzyme catalyzing the Strassner et al., “A homolog of old yellow enzyme in tomato. reduction of fatty acyl-CoA to fatty alcohol.” Biochemistry, 2011, Spectral properties and Substrate specificity of the recombinant 50:10550-10558. protein.” J. Biol. Chem. 1999, 274:35067-35073. Wilson and Bouwer, "Biodegradation of aromatic compounds under Stueckler, “Stereocomplementary bioreduction of alphabeta-un mixed oxygen? denitrifying conditions: a review.” J Ind Microbiol Saturated dicarboxylic acids and dimethyl esters using enoate Biotechnol., 18(2-3): 116-130, Feb.-Mar. 1997. reductases: enzyme- and Substrate-based stereocontrol.” Org. Lett. Wischgoll et al., “Structural basis for promoting and preventing 2007, 9:5409-5411. decarboxylation in glutaryl-coenzyme. A dehydrogenases.” Bio Suzuki et al., “Acetylputrescine deacetylase from Micrococcus chemistry, 2010, 49:5350-5357. luteus K-11.” BBA General Subjects, 1986, 882(1): 140-142. Woolridge et al., “Efflux of the natural polyamine spermidine Suzuki et al., “Antimicrobial Activity of Meropenem Against Main facilitated by the Bacillus subtilis multidrug transporter Blt.” J Biol Bacterial Species Isolated from Patient Blood in 2006,” J. Antibiot. Chem., 1997, 272(14):8864-8866. 2007, 60(6):380-387. Xiong et al., “A bio-catalytic approach to aliphatic ketones,” Sci Suzuki et al., “GriC and GrilD Constitute a Carboxylic Acid Rep. 2:311, Epub Mar. 13, 2012. Reductase Involved in Grixazone Biosynthesis in Streptomyces Yang et al., “Value-added uses for crude glycerol—a byproduct of griseus,” J. Antibiot., 2007, 60(6):380-387. biodiesel production.” Biotechnology for Biofuels, 2012. 5:13. Tomita et al., “Mechanism for multiple-substrates recognition of Yonaha et al., “4-Aminobutyrate : 2-oxoglutarate aminotransferase alpha-aminoadipate aminotransferase from Thermus thermophilus.” of Streptomyces griseus: Purification and properties.' Eur, J. Proteins, 2009, 75(2):348-359. Biochem., 1985, 146:101-106. Tseng et al., “Biosynthesis of chiral 3-hydroxyvalerate from single Zhang et al., “Expanding metabolism for biosynthesis of nonnatural propionate-unrelated carbon sources in metabolically engineered E. alcohols.” Proc Natl Acad Sci U S A., 105(52):20653-20658 Epub coli, Microbial Cell Factories, 2010, 9:96. Dec. 8, 2008. US Non-Final Office Action in U.S. Appl. No. 13/524,883, mailed Zhuang et al., “Divergence of function in the hot dog fold enzyme Nov. 29, 2013, 13 pages. Superfamily: the bacterial thioesterase Yei A.” Biochemistry, 2008, US Non-Final Office Action in U.S. Appl. No. 13/715,981, mailed 47(9):2789-2796. Jun. 27, 2014, 23 pages. Zomorrodi et al., “Improving the iMM904 S. Cerevisiae metabolic US Notice of Allowance in U.S. Appl. No. 13/524,883, mailed May model using essentiality and synthetic lethality data.” BMC Sys 29, 2014, 7 pages. tems Biology, Dec. 2010, 4(1): 1-15. Vamecq et al., “The microsomal dicarboxylyl-CoA synthetase.” Aimin et al., “Nocardia sp. carboxylic acid reductase: cloning, Biochem J., 1985, 230:683-693. expression, and characterization of a new aldehyde oxidoreductase Van Beilen and Funhoff, “Expanding the alkane oxygenase toolbox: family.” Appl. Environ. Microbiol., 2004, 70: 1874-1881. new enzymes and Applications.” Curr. Opin. Biotechnol., 2005, Aloulou et al., “Purification and biochemical characterization of the 16:308-314. LIP2 lipase from Yarrowia lipolytica,” Biochim. Biophys. Acta, Venkitasubramanian et al., “Aldehyde oxidoreductase as a 2007, 1771:228-237. biocatalyst: Reductions of vanillic acid.” Enzyme and Microbial Atsumi et al., “Acetolactate synthase from Bacillus Subtilisserves as Technology, 2008, 42:130-137. a 2-ketoisovalerate decarboxylase from isobutanol synthesis in Vioque et al., Resolution and purification of an aldehyde-generating Escherichi coli.” Applied and Environ. Microbiol., 2009, and an alcohol-generating fatty-acyl-CoA reductase from Pea leaves 75(19):6306-6311. (Pisum sativum L). Archives of Biochemistry and Biophysics, 1997. Bergler et al., “Protein EnvM is the NADH-dependent enoyl-ACP 340(1):64-72. reductase (FabI) of Escherichia coli,” J. Bio Chem, 1993, Wahlen et al., “Purification, characterization and potential bacterial 269(8):5493-5496. wax production role of an NADPH-dependent fatty aldehyde Chinese Office Action in Chinese Application No. 2012800401576, reductase from Marinobacter aquaeolei VT8.” Appl. Environ mailed Oct. 17, 2014, 7 pages. (with English Translation). Microbiol, 2009, 75:2758-2764. Eikmanns and Buckel, “Properties of 5-hydroxyvalerate CoA Wang and Kolattukudy, “Solubilization and purification of alde transferase from Clostridium aminovalericum,’ Biol. Chem, 1990, hyde-generation fatty acyl-CoA reductase from green alga 371:1077-1082. Botryococcus braunii.” FEBS Letters, 1995, 370:15-18. Fonknechten et al., "Clostridium sticklandii, a specialist in amino Wee et al., “Biotechnological Production of Lactic Acid and Its acid degradation: revisiting its metabolism through its genome Recent Applications.” Food Technol. Biotechnol., 2006, 44(2):163 sequence.” BMC Genomics, 2010, 11:1-12. 172. GenBank Accession No. AAA23536, Apr. 26, 1993, 1 page. Westin et al., “Molecular cloning and characterization of two mouse GenBank Accession No. AAA92347.1, Mar. 15, 1996, 1 page. peroxisome proliferator-activated receptor alpha (PPARalpha)- GenBank Accession No. AAB991.00, Aug. 27, 1996, 2 pages. regulated peroxisomal acyl-CoA thioesterases,” J. Biol Chem, 2004, GenBank Accession No. AE000666.1, Jan. 5, 2006, 309 pages. 279:21841-21848. GenBank Accession No. D87518, Jul. 31, 1997, 2 pages. Westin et al., “The identification of a Succinyl-CoA thioesterase GenBank Accession No. HQ418483.1, Apr. 4, 2011, 2 pages. Suggests a novel pathway for Succinate production in peroxisomes.” GenBank Accession No. JA114119.1, Apr. 19, 2011, 1 page. J. Biol Chem, 2005, 280:38125-38132. GenBank Accession No. MJ0663, Oct. 1, 2014, 4 pages. White and Kelly, "Purification and Properties of Diaminopimelate GenBank Accession No. NC 013156.1, Jun. 10, 2013, 2 pages. Decarboxylase From Escherichia Coli,” Biochem J., 1965, 96:75 GenBank Accession No. NC 014122.1, Jun. 10, 2013, 2 pages. 84. GenBank Accession No. NC 015562.1, Jun. 10, 2013, 2 Pages. US 9,580,731 B2 Page 9

(56) References Cited International Preliminary Report on Patentability for International Application No. PCT/US2013/075058, mailed Jun. 25, 2015, 11 OTHER PUBLICATIONS pageS. International Preliminary Report on Patentability for International GenBank Accession No. NP 247129, Jun. 10, 2013, 2 pages. Application No. PCT/US2013/075087, mailed Jun. 25, 2015, 11 GenBank Accession No. NP 247250. Jun. 10, 2013, 2 pages. pageS. GenBank Accession No. NP 247647. Jun. 10, 2013, 2 pages. International Preliminary Report on Patentability for International GenBank Accession No. YP 0.03127480, Jun. 10, 2013, 2 pages. Application No. PCT/US2013/077445, mailed Jul. 9, 2015, 11 GenBank Accession No. YP 0.03128272, Jun. 10, 2013, 2 pages. pageS. GenBank Accession No. YP 003615747, Jun. 10, 2013, 1 page. International Preliminary Report on Patentability for International GenBank Accession No. YP 003615922, Jun. 10, 2013, 2 pages. Application No. PCT/US2013/077420, mailed Jul. 9, 2015, 14 GenBank Accession No. YP 0.04483786, Jul. 6, 2013, 2 pages. pageS. Horning et al., “O-Ketoglutaric Acid.” Organic Syntheses, 1955, 3: International Preliminary Report on Patentability for International 510-512. Application No. PCT/US2013/077419, mailed Jul. 9, 2015, 13 International Search Report and Written Opinion in International pageS. Application No. PCT/US2014/052950, mailed Dec. 3, 2014, 15 International Preliminary Report on Patentability for International pageS. Application No. PCT/US2013/077430, mailed Jul. 9, 2015, 18 International Search Report and Written Opinion in International pageS. Application No. PCT/US2013/077430, mailed Nov. 10, 2014, 23 International Preliminary Report on Patentability for International pageS. Application No. PCT/US2013/077413, mailed Jul. 9, 2015, 13 Invitation to Pay Additional Fees in International Application No. pageS. PCT/US2014/053222, mailed Dec. 15, 2014, 8 pages. International Preliminary Report on Patentability for International Klatte et al., “Redox self-sufficient whole cell biotransformation for Application No. PCT/US2013/0774 11, mailed Jul. 9, 2015, 12 amination of alcohols.” Bioorg & Medicinal Chem, May 2014, 22: pageS. 5578-5585. International Preliminary Report on Patentability for International Lea et al., “Long-chain acyl-CoA dehydrogenase is a key enzyme in Application No. PCT/US2013/077423, mailed Jul. 9, 2015, 14 the mitochondrial B-Oxidation of unsaturated fatty acids.” pageS. Biochmica et Biophysica Acta, 2000, 1485: 121-128. International Search Report and Written Opinion in International Mutti et al., “Amination of ketones by employing two new (S)- Application No. PCT/US2014/053222, mailed Mar. 4, 2015, 18 Selective w-transaminases and the His-tagged W-TA from Vibrio pageS. fluvialis,” Eur, J. Org. Chem, 2012, 1003-1007 (Abstract). International Search Report and Written Opinion in International Prabhu et al., “Lactate and Acrylate Metabolism by Megasphaera Application No. PCT/US2015/031227, mailed Jul. 31, 2015, 40 elsdenii under Batch and Steady-State Conditions.” Applied and pageS. Environ. Microbiology, Sep. 2012, 78(24): 8564-8570. International Search Report and Written Opinion in International Reiser and Somerville, "Isolation of mutants of Acinetobacter Application No. PCT/US2015/036050, mailed Aug. 14, 2015, 38 cal coaceticus deficient in wax ester synthesis and complementation pageS. of on mutation with gene encoding a fatty acyl coenzyme a International Search Report and Written Opinion in International reductase,” J. Bacteriol., 1997, 179:2969-2975. Application No. PCT/US2015/036057, mailed Aug. 14, 2015, 74 Rizzarelli et al., “Evidence for Selective Hydrolysis of Aliphatic pageS. Copolyesters Induced by Lipase Catalysis,” Biomacromolecules, KEGG Enzyme 1.2.99.6 (last viewed on Aug. 17, 2015). 2004, 5:433-444. KEGG Enzyme 3.1.2.14 (last viewed on Aug. 17, 2015). Uniprot Accession No. I5YEB8, Sep. 5, 2012, 1 page. Scheps et al., “Synthesis of omega-hydroxy dodecanoic acid based US Notice of Allowance in U.S. Appl. No. 13/715,981, mailed Dec. on an engineered CYP153A fusion construct.” Microbial Biotech 16, 2014, 23 pages. nology, 2013, 6:694-707. US Non-Final Office Action in U.S. Appl. No. 13/715,826, mailed US Notice of Allowance in U.S. Appl. No. 13/715,981, mailed Apr. Jan. 30, 2015, 24 pages. 6, 2015, 10 pages. Vyazmensky et al., “Isolation and Characterization of Subunits of US Non-Final Office Action in U.S. Appl. No. 14/106,033, mailed Acetohydroxy Acid Synthase Isozyme III and Reconstruction of the Apr. 6, 2015, 37 pages. Holoenzyme.” Biochemistry, 1996, 35:10339-10346. US Non-Final Office Action in U.S. Appl. No. 14/138,827, mailed Zhao et al., “Prediction and characterization of enzymatic activities Apr. 24, 2015, 35 pages. guided by sequence similarity and genome neighborhood net US Non-Final Office Action in U.S. Appl. No. 14/138,971, mailed works,” E-Life, Jun. 2014, 3: 1-32. Jun. 9, 2015, 44 pages. Akita et al., “Highly stable meso-diaminopimelate dehydrogenase US Non-Final Office Action in U.S. Appl. No. 14/490,270, mailed from an Ureibacillus thermosphaericus strain A1 isolated from a Jul. 17, 2015, 49 pages. Japanese compost: purification, characterization and sequencing.” US Non-Final Office Action in U.S. Appl. No. 14/130,117, mailed AMB Express, 2011, 1:43, 8 pages. Aug. 21, 2015, 49 pages. Aursnes et al., “Total Synthesis of the Lipid Mediator PD1(n-3 US Notice of Allowance in U.S. Appl. No. 14/106,124, mailed Dec. DPA): Configurational Assignments and Anti-Inflammatory and 24, 2014, 31 pages. Pro-resolving Actions,” Journal of Natural Products, Feb. 2014, White et al., “Carboxylic acid reductase: a new tungsten enzyme T7:910-916. catalyses the reduction of non-activated carboxylic acids to alde Bordeaux et al., "Catalytic, Mild, and Selective Oxyfunctionaliza hydes,” Eur, J. Biochem., 1989, 184(1):89-96. tion of Linear Alkanes: Current challenges.” Angew. Chem. Int. Ed., "Metabolic engineering.” Wikipedia, Jun. 8, 2014 (Jun. 8, 2014), 2012, 51: 10712-10723. XP002744570, Retrieved from the Internet: URL:https://en. Clomburg et al., “Integrated engineering of Beta-Oxidation reversal wikipedia.org/w/index.php?title=Metabolicengineering and omega-Oxidation pathways for the synthesis of medium chain &oldid=612026466 retrieved on Sep. 15, 2015 last paragraph. omega-functionalized carboxylic acids.” Metabolic Engineering, Akatsuka et al., “The Serratia marcescens bioH gene encodes an Jan. 2015, 28:202-212. esterase, GENE, Jan. 2003, 302:185-192. Gao et al: “A novel meso-diaminopimelate dehydrogenase from Brady et al., “A serine protease triad forms the catalytic centre of a Symbiobacterium thermophilum: overexpression, characterization, triacylglycerol lipase,” Nature, Feb. 1990, 343:767-70. and potential for D-amino acid synthesis.' Applied and Environ Eriksen et al., “Protein Design for Pathway Engineering.” Journal of mental Microbiology, 2012, 78:8595-8600. Structural Biology, Apr. 2013, 185(2):234-242. US 9,580,731 B2 Page 10

(56) References Cited the hinge domains at both sides of the lip domain to interfacial action.” Biotechnol. Prog., 2009, 25:409-16. OTHER PUBLICATIONS niprot Accession No. 032472, Jun. 11, 2014, 2 pages. niprot Accession No. P69909, Jan. 4, 2005, 1 page. International Search Report and Written Opinion in International niprot Accession No. POA6RO, May 14, 2014, 5 pages. Application No. PCT/US2015/036086, mailed Nov. 30, 2015, 18 niprot Accession No. P0A8Z0, Jun. 11, 2014, 3 pages. pageS. niprot Accession No. POAGG2, Jun. 11, 2014, 3 pages. niprot Accession No. P0AEK4. Jun. 11, 2014, 6 pages. International Search Report and Written Opinion in International niprot Accession No. P0A953, Jun. 11, 2014, 4 pages. Application No. PCT/US2015/036092, mailed Nov. 26, 2015, 20 niprot Accession No. POA6O6, Jun. 11, 2014, 3 pages. pageS. niprot Accession No. P0AEK2, May 14, 2014, 4 pages. International Search Report and Written Opinion in International niprot Accession No. P13001, Jun. 11, 2014, 4 pages. Application No. PCT/US2015/036067, mailed Nov. 23, 2015, 30 niprot Accession No. Q5EU90, Feb. 19, 2014, 2 pages. pageS. niprot Accession No. Q73Q47. May 14, 2014, 2 pages. Invitation to Pay Fees in International Application No. PCT/ niprot Accession No. Q818X2, Jun. 11, 2014, 2 pages. US2015/036015, mailed Oct. 2, 2015, 9 pages. S Non-Final Office Action in U.S. Appl. No. 14/138,992, mailed Invitation to Pay Fees in International Application No. PCT/ Nov. 17, 2015, 19 pages. US2015/036092, mailed Sep. 21, 2015, 8 pages. US Non-Final Office Action in U.S. Appl. No. 14/139,225, mailed Invitation to Pay Fees in International Application No. PCT/ Dec. 8, 2015, 15 pages. US2015/036067, mailed Sep. 18, 2015, 12 pages. US Final Office Action in U.S. Appl. No. 13/715,826, dated Nov. 5, Karam et al., “Potential applications of enzymes in waste treat 2015, 27 pages. ment, J. Chem. Tech. Biotechnol., 1997, 69:141-53. US Final Office Action in U.S. Appl. No. 14/106,033, dated Nov. 13, Klapa and Stephanopoulos, "Bioreaction Engineering: Modeling 2015, 7 pages. and Control.” 2000, Springer Verlag, Heidelberg, pp. 106-124. Van Hamme et al., “Recent advances in petroleum microbiology,” Moreno-Sanchez et al., “Experimental validation of metabolic path Microbiol. Mol. Biol. Rev., 2003, 67:503-49. way modeling—An illustration with glycolytic segments from Yadav et al., “The future of metabolic engineering and synthetic Entamoeba histolytica.” FEBS Journal, Jul. 2008, 275(13):3454 biology: Towards a systematic practice.” Metabolic Engineering, 3469. Feb. 2012, 14(3):233-241. Palsson, “The challenges of in silico biology,” Nature Biotechnol International Search Report and Written Opinion in International ogy, Nature Publishing Group, US, Nov. 2000, 18(1): 1147-1150. Application No. PCT/US2015/036074, mailed Sep. 9, 2015, 14 Price et al., “Genome-scale models of microbial cells: evaluating pageS. the consequences of constraints.” Nature Reviews. Microbiology, Invitation to Pay Fees in International Application No. PCT/ Nature Publishing Group, GB, Nov. 2004, 2(11):886-897. US2015/036086, mailed Sep. 16, 2015, 7 pages. Shu et al., "Aspergillus niger lipase, heterologous expression in Pichia pastoris, molecular modeling prediction and importance of * cited by examiner

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US 9,580,731 B2 1. 2 METHODS OF PRODUCING 7-CARBON CoA and glutaryl-CoA by several bacteria has been charac CHEMICALS VA C1 CARBON CHAIN terized comprehensively (Harwood and Parales, Annual ELONGATION ASSOCATED WITH Review of Microbiology, 1996, 50, 553-590). COENZYME B SYNTHESIS The optimality principle states that microorganisms regu late their biochemical networks to Support maximum bio CROSS-REFERENCE TO RELATED mass growth. Beyond the need to express heterologous APPLICATIONS pathways in a host organism, directing carbon flux towards C7 building blocks that serve as carbon sources rather than This application claims priority of U.S. Provisional Appli to biomass growth constituents, contradicts the optimality cation Ser. No. 61/747,406, filed Dec. 31, 2012, and U.S. 10 principle. For example, transferring the 1-butanol pathway Provisional Application Ser. No. 61/829,088, filed May 30, from Clostridium species into other production strains has 2013. The contents of the prior applications are incorporated often fallen short by an order of magnitude compared to the herein by reference in their entirety. production performance of native producers (Shen et al., 15 Appl. Environ. Microbiol., 2011, 77(9), 2905-2915). TECHNICAL FIELD The efficient synthesis of the seven carbon aliphatic This invention relates to methods for biosynthesizing one backbone precursor is a key consideration in synthesizing or more of pimelic acid, 7-aminoheptanoic acid, heptameth C7 building blocks prior to forming terminal functional ylenediamine and 1,7-heptanediol (hereafter “C7 building groups, such as carboxyl, amine or hydroxyl groups, on the blocks”) from oxoglutarate or oxiadipate using one or more C7 aliphatic backbone. isolated enzymes Such as synthases, dehydratases, hydratases, dehydrogenases, reductases, thioesterases, SUMMARY reversible CoA , CoA transferases, deacetylases, and transaminases or using recombinant host cells expressing This document is based at least in part on the discovery one or more Such enzymes. 25 that it is possible to construct biochemical pathways for producing a seven carbon chain aliphatic backbone precur BACKGROUND Sor, in which one or two functional groups, i.e., carboxyl, amine, or hydroxyl, can be formed, leading to the synthesis Nylons are polyamides which are sometimes synthesized of one or more of pimelic acid, 7-aminoheptanoate, 7-hy by the condensation polymerisation of a diamine with a 30 droxyheptanoate, heptamethylenediamine, and 1.7-heptane dicarboxylic acid. Similarly, nylons may be produced by the diol (hereafter “C7 building blocks). Pimelic acid and pime condensation polymerisation of lactams. Aubiquitous nylon late, 7-hydroxyheptanoic acid and 7-hydroxyheptanoate, is Nylon 6.6, which is produced by reaction of hexameth and 7-aminoheptanoic and 7-aminoheptanoate are used ylenediamine (HMD) and adipic acid. Nylon 6 is produced interchangeably herein to refer to the compound in any of its by a ring opening polymerisation of caprolactam (Anton & 35 neutral or ionized forms, including any salt forms thereof. It Baird, Polyamides Fibers, Encyclopedia of Polymer Science is understood by those skilled in the art that the specific form and Technology, 2001). will depend on pH. These pathways, metabolic engineering Nylon 7 and Nylon 7.7 represent novel polyamides with and cultivation strategies described herein rely on the C1 value-added characteristics compared to Nylon 6 and Nylon elongation enzymes or homologs associated with the Coen 6.6. Nylon 7 is produced by polymerisation of 7-aminohep 40 Zyme B biosynthesis pathway of methanogens. tanoic acid, whereas Nylon 7.7 is produced by condensation In the face of the optimality principle, it Surprisingly has polymerisation of pimelic acid and heptamethylenediamine. been discovered that appropriate non-natural pathways, No economically viable petrochemical routes exist to pro feedstocks, host microorganisms, attenuation strategies to ducing the monomers for Nylon 7 and Nylon 7.7. the host’s biochemical network and cultivation strategies Given no economically viable petrochemical monomer 45 may be combined to efficiently produce one or more C7 feedstocks, biotechnology offers an alternative approach via building blocks. biocatalysis. Biocatalysis is the use of biological catalysts, In some embodiments, the C7 aliphatic backbone for Such as enzymes, to perform biochemical transformations of conversion to one or more C7 building blocks is 7-oxohep organic compounds. tanoate (also known as pimelate semialdehyde) or pimeloyl Both bioderived feedstocks and petrochemical feedstocks 50 CoA (also known as 6-carboxyhexanoyl-CoA), which can are viable starting materials for the biocatalysis processes. be formed from 2-oxoadipate via two cycles of C1 carbon Accordingly, against this background, it is clear that there chain elongation. Pimelate semialdehyde and pimeloyl-CoA is a need for methods for producing pimelic acid, 7-amino also can be formed from 2-oxoglutarate via three cycles of heptanoic acid, heptamethylenediamine, 7-hydroxyhep C1 carbon chain elongation (2-oxoacid elongation cycles) tanoic acid and 1,7-heptanediol (hereafter “C7 building 55 (i.e., from 2-oxoglutarate to 2-oxoSuberate, followed by blocks”) wherein the methods are biocatalyst-based. decarboxylation to either pimelate semialdehyde or However, no wild-type prokaryote or eukaryote naturally pimeloyl-CoA). See FIG. 1. overproduces or excretes C7 building blocks to the extra In some embodiments, a terminal carboxyl group can be cellular environment. Nevertheless, the metabolism of enzymatically formed using a decarboxylase, a thioesterase, pimelic acid has been reported. 60 an , a 6-oxohexanoate dehydroge The dicarboxylic acid, pimelic acid, is converted effi nase, a 7-oxoheptanoate dehydrogenase, a reversible CoA ciently as a carbon Source by a number of bacteria and yeasts (e.g., a reversible Succinyl-CoA-ligase), or a CoA via B-oxidation into central metabolites. 3-oxidation of transferase (e.g., a glutaconate CoA-transferase). See FIG. 1 CoEnzyme A (CoA) activated pimelate to CoA-activated and FIG. 2. 3-oxopimelate facilitates further catabolism via, for 65 In some embodiments, a terminal amine group can be example, pathways associated with aromatic Substrate deg enzymatically formed using a co-transaminase or a deacety radation. The catabolism of 3-oxopimeloyl-CoA to acetyl lase. See FIG. 3 and FIG. 4. US 9,580,731 B2 3 4 In some embodiments, a terminal hydroxyl group can be In some embodiments, the non-biological feedstock is or enzymatically formed using a 4-hydroxybutyrate dehydro derives from natural gas, syngas, CO/H, methanol, etha genase, 5-hydroxypentanoate dehydrogenase or a 6-hy nol, benzoate, non-volatile residue (NVR) or a caustic wash droxyhexanoate dehydrogenase or an alcohol dehydroge waste stream from cyclohexane oxidation processes, or a nase. See FIG. 5 and FIG. 6. terephthalic acid/isophthalic acid mixture waste stream. In one aspect, this document features a method for bio This document also features a recombinant host that synthesizing a product selected from the group consisting of includes at least one exogenous nucleic acid encoding (i) a pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, (homo), citrate synthase, (ii) a (homo), citrate dehydratase heptamethylenediamine and 1,7-heptanediol. The method and a (homo),aconitate hydratase, (iii) an iso(homo), citrate includes enzymatically synthesizing a seven carbon chain 10 dehydrogenase, and (iv) an indolepyruvate decarboxylase or aliphatic backbone from 2-oxoglutarate via three cycles of a 2-oxoglutarate dehydrogenase complex, the host produc 2-oxoacid carbon chain elongation and enzymatically form ing pimeloyl-CoA or pimelate semialdehyde. ing one or two terminal functional groups selected from the A recombinant host producing pimeloyl-CoA further can group consisting of carboxyl, amine, and hydroxyl groups in include at least one exogenous nucleic acid encoding one or the backbone, thereby forming the product. The seven 15 more of a thioesterase, an aldehyde dehydrogenase, a 7-OXo carbon chain aliphatic backbone can be pimeloyl-CoA or heptanoate dehydrogenase, a 6-oxohexanoate dehydroge pimelate semialdehyde. Each of the three cycles of 2-ox nase, a CoA-transferase, a reversible CoA-ligase (e.g., a oacid chain elongation can include using a (homo), citrate reversible Succinyl-CoA-ligase), an acetylating aldehyde synthase, a (homo), citrate dehydratase, a (homo),aconitate dehydrogenase, or a carboxylate reductase, the host produc hydratase and an iso(homo), citrate dehydrogenase to form ing pimelic acid or pimelate semialdehyde. In any of the 2-oxoSuberate from 2-oxoglutarate. The 2-oxoSuberate can recombinant hosts expressing a carboxylate reductase, a be converted to pimelate semialdehyde by an indolepyruvate phosphopantetheinyl transferase also can be expressed to decarboxylase or converted to pimeloyl-CoA by a 2-oxo enhance the activity of the carboxylate reductase. glutarate dehydrogenase complex. A recombinant host producing pimelate semialdehyde The two terminal functional groups can be the same (e.g., 25 further can include at least one exogenous nucleic acid amine or hydroxyl) or can be different (e.g., a terminal amine encoding a co-transaminase, and producing 7-aminoheptano and a terminal carboxyl group; or a terminal hydroxyl group ate. and a terminal carboxyl group). A recombinant host producing pimelate semialdehyde A ()-transaminase or a deacetylase can enzymatically further can include at least one exogenous nucleic acid form an amine group. The co-transaminase can have at least 30 encoding a 4-hydroxybutyrate dehydrogenase, a 5-hydroxy 70% sequence identity to any one of the amino acid pentanoate dehydrogenase or a 6-hydroxyhexanoate dehy sequences set forth in SEQID NO. 8-13. drogenase, the host producing 7-hydroxyheptanoic acid. A 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypen A recombinant host producing pimelate semialdehyde, tanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or 7-aminoheptanoate, or 7-hydroxyheptanoic acid further can an alcohol dehydrogenase can enzymatically form a 35 include a carboxylate reductase, a co-transaminase, a hydroxyl group. deacetylase, an N-acetyl transferase, or an alcohol dehydro A thioesterase, an aldehyde dehydrogenase, a 7-oxohep genase, the host producing heptamethylenediamine. tanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a A recombinant host producing 7-hydroxyheptanoic acid CoA-transferase (e.g. a glutaconate CoA transferase), or a further can include at least one exogenous nucleic acid reversible CoA-ligase (e.g., a reversible Succinate-CoA 40 encoding a carboxylate reductase or an alcohol dehydroge ligase) can enzymatically forms a terminal carboxyl group. nase, the host producing 1.7-heptanediol. The thioesterase can have at least 70% sequence identity to The recombinant host can be a prokaryote, e.g., from the the amino acid sequence set forth in SEQ ID NO: 1. genus Escherichia Such as Escherichia coli; from the genus A carboxylate reductase and a phosphopantetheinyl trans Clostridia such as Clostridium liungdahli, Clostridium ferase can form a terminal aldehyde group as an intermedi 45 autoethanogenium or Clostridium kluyveri; from the genus ate in forming the product. The carboxylate reductase can Corynebacteria Such as Corynebacterium glutamicum; from have at least 70% sequence identity to any one of the amino the genus Cupriavidus Such as Cupriavidus necator or acid sequences set forth in SEQ ID NO. 2-7. Cupriavidus metallidurans; from the genus Pseudomonas Any of the methods can be performed in a recombinant Such as Pseudomonas fluorescens, Pseudomonas putida or host by fermentation. The host can be subjected to a culti 50 Pseudomonas Oleavorans; from the genus Delftia acido Vation strategy under anaerobic, micro-aerobic or mixed vorans, from the genus Bacillus such as Bacillus subtilis; oxygen/denitrification cultivation conditions. The host can from the genes Lactobacillus such as Lactobacillus del be cultured under conditions of nutrient limitation. The host brueckii; from the genus Lactococcus Such as Lactococcus can be retained using a ceramic hollow fiber membrane to lactis or from the genus Rhodococcus such as Rhodococcus maintain a high cell density during fermentation. The final 55 equi. electron acceptor can be an alternative to oxygen Such as The recombinant host can be a eukaryote, e.g., a eukary nitrates. ote from the genus Aspergillus such as Aspergillus niger; In any of the methods, the hosts tolerance to high from the genus Saccharomyces such as Saccharomyces concentrations of a C7 building block can be improved cerevisiae; from the genus Pichia Such as Pichia pastoris; through continuous cultivation in a selective environment. 60 from the genus Yarrowia Such as Yarrowia lipolytica, from The principal carbon source fed to the fermentation can the genus Issatchenkia Such as Issathenkia Orientalis, from derive from biological or non-biological feedstocks. In some the genus Debaryomyces Such as Debaryomyces hansenii, embodiments, the biological feedstock is, includes, or from the genus Arxula Such as Arxula adenoinivorans, or derives from, monosaccharides, disaccharides, lignocellu from the genus Kluyveromyces Such as Kluyveromyces lac lose, hemicellulose, cellulose, lignin, levulinic acid and 65 tis. formic acid, triglycerides, glycerol, fatty acids, agricultural Any of the recombinant hosts described herein further can waste, condensed distillers’ solubles, or municipal waste. include one or more of the following attenuated enzymes: a US 9,580,731 B2 5 6 polymer synthase, a NADPH-specific L-glutamate dehydro FIG. 2 is a schematic of exemplary biochemical pathways genase, a NADPH-consuming transhydrogenase, a leading to pimelate using pimeloyl-CoA or pimelate semi pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase aldehyde as a central precursor. that degrades C7 building blocks and their precursors; a FIG.3 is a schematic of exemplary biochemical pathways glutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase. leading to 7-aminoheptanoate using pimeloyl-CoA, pime Any of the recombinant hosts described herein further can late or pimelate semialdehyde as a central precursor. overexpress one or more genes encoding: a 6-phosphoglu FIG. 4 is a schematic of an exemplary biochemical conate dehydrogenase; a transketolase; a feedback resistant pathways leading to heptamethylenediamine using 7-amino shikimate kinase; a feedback resistant 2-dehydro-3-deoxy heptanoate, 7-hydroxyheptanoate or pimelate semialdehyde 10 as a central precursor. phosphoheptanate aldose; a puridine nucleotide transhydro FIG. 5 is a schematic of exemplary biochemical pathways genase; a glyceraldehyde-3P-dehydrogenase; a malic leading to 7-hydroxyheptanoate using pimelate, pimeloyl enzyme; a glucose-6-phosphate dehydrogenase; a fructose CoA or pimelate semialdehyde as a central precursor. 1.6 diphosphatase; a ferredoxin-NADP reductase, a L-ala FIG. 6 is a schematic of an exemplary biochemical nine dehydrogenase; a NADH-specific L-glutamate dehy 15 pathway leading to 1.7-heptanediol using 7-hydroxyhep drogenase; a diamine transporter, a dicarboxylate trans tanoate as a central precursor. porter, and/or a multidrug transporter. FIG. 7 contains the amino acid sequences of an Escheri The reactions of the pathways described herein can be chia colithioesterase encoded by tesB (See GenBank Acces performed in one or more cell (e.g., host cell) strains (a) sion No. AAA24665.1, SEQ ID NO: 1), a Mycobacterium naturally expressing one or more relevant enzymes, (b) marinum carboxylate reductase (See Genbank Accession genetically engineered to express one or more relevant No. ACC40567.1, SEQID NO: 2), a Mycobacterium smeg enzymes, or (c) naturally expressing one or more relevant matis carboxylate reductase (See Genbank Accession No. enzymes and genetically engineered to express one or more ABK71854.1, SEQ ID NO. 3), a Segniliparus rugosus relevant enzymes. Alternatively, relevant enzymes can be carboxylate reductase (See Genbank Accession No. extracted from any of the above types of host cells and used 25 EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis in a purified or semi-purified form. Extracted enzymes can carboxylate reductase (See Genbank Accession No. optionally be immobilized to a solid substrate such as the ABK75684.1, SEQ ID NO: 5), a Mycobacterium massil floors and/or walls of appropriate reaction vessels. More iense carboxylate reductase (See Genbank Accession No. over, Such extracts include lysates (e.g., cell lysates) that can EIV11143.1, SEQ ID NO: 6), a Segniliparus rotundus be used as Sources of relevant enzymes. In the methods 30 carboxylate reductase (See Genbank Accession No. provided by the document, all the steps can be performed in ADG98140.1, SEQ ID NO: 7), a Chromobacterium viola cells (e.g., host cells), all the steps can be performed using ceum ()-transaminase (See Genbank Accession No. extracted enzymes, or Some of the steps can be performed in AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa cells and others can be performed using extracted enzymes. co-transaminase (See Genbank Accession No. AAG08191.1, Many of the enzymes described herein catalyze reversible 35 SEQ ID NO: 9), a Pseudomonas syringae ()-transaminase reactions, and the reaction of interest may be the reverse of (See Genbank Accession No. AAY39893.1, SEQ ID NO: the described reaction. The schematic pathways shown in 10), a Rhodobacter sphaeroides ()-transaminase (see Gen FIGS. 1-6 illustrate the reaction of interest for each of the bank Accession No. ABA81135.1, SEQ ID NO: 11), an intermediates. Escherichia coli ()-transaminase (see Genbank Accession Unless otherwise defined, all technical and scientific 40 No. AAA57874.1, SEQ ID NO: 12), a Vibrio fluvialis terms used herein have the same meaning as commonly co-transaminase (see Genbank Accession No. AEA39183.1, understood by one of ordinary skill in the art to which this SEQ ID NO: 13), a Bacillus subtilis phosphopantetheinyl invention pertains. Although methods and materials similar transferase (see Genbank Accession No. CAA44858.1, SEQ or equivalent to those described herein can be used to ID NO:14), or a Nocardia sp. NRRL 5646 phosphopanteth practice the invention, Suitable methods and materials are 45 einyl transferase (see Genbank Accession No. ABI83656.1, described below. All publications, patent applications, pat SEQ ID NO:15). ents, and other references mentioned herein are incorporated FIG. 8 is a bar graph of the relative absorbance at 412 nm by reference in their entirety. In case of conflict, the present after 20 minutes of released CoA as a measure of the activity specification, including definitions, will control. In addition, of a thioesterase for converting pimeloyl-CoA to pimelate the materials, methods, and examples are illustrative only 50 relative to the empty vector control. and not intended to be limiting. FIG. 9 is a bar graph Summarizing the change in absor The details of one or more embodiments of the invention bance at 340 nm after 20 minutes, which is a measure of the are set forth in the accompanying drawings and the descrip consumption of NADPH and activity of carboxylate reduc tion below. Other features, objects, and advantages of the tases relative to the enzyme only controls (no Substrate). invention will be apparent from the description and the 55 FIG. 10 is a bar graph of the change in absorbance at 340 drawings, and from the claims. The word “comprising in nm after 20 minutes, which is a measure of the consumption the claims may be replaced by “consisting essentially of or of NADPH and the activity of carboxylate reductases for with “consisting of according to standard practice in patent converting pimelate to pimelate semialdehyde relative to the law. empty vector control. 60 FIG. 11 is a bar graph of the change in absorbance at 340 DESCRIPTION OF DRAWINGS nm after 20 minutes, which is a measure of the consumption of NADPH and the activity of carboxylate reductases for FIG. 1 is a schematic of an exemplary biochemical converting 7-hydroxyheptanoate to 7-hydroxyheptanal rela pathway leading to pimelate semialdehyde or pimeloyl-CoA tive to the empty vector control. using the 2-oxoacid chain elongation pathway associated 65 FIG. 12 is a bar graph of the change in absorbance at 340 with coenzyme B synthesis and 2-oxoglutarate or 2-oxoa nm after 20 minutes, which is a measure of the consumption dipate as a central metabolite. of NADPH and the activity of carboxylate reductases for US 9,580,731 B2 7 8 converting N7-acetyl-7-aminoheptanoate to N7-acetyl-7- host once in the host. It is important to note that non aminoheptanal relative to the empty vector control. naturally-occurring nucleic acids can contain nucleic acid FIG. 13 is a bar graph of the change in absorbance at 340 Subsequences or fragments of nucleic acid sequences that nm after 20 minutes, which is a measure of the consumption are found in nature provided the nucleic acid as a whole does of NADPH and activity of carboxylate reductases for con not exist in nature. For example, a nucleic acid molecule verting pimelate semialdehyde to heptanedial relative to the containing a genomic DNA sequence within an expression empty vector control. vector is non-naturally-occurring nucleic acid, and thus is FIG. 14 is a bar graph Summarizing the percent conver exogenous to a host cell once introduced into the host, since sion after 4 hours of pyruvate to L-alanine (mol/mol) as a that nucleic acid molecule as a whole (genomic DNA plus measure of the co-transaminase activity of the enzyme only 10 vector DNA) does not exist in nature. Thus, any vector, controls (no Substrate). autonomously replicating plasmid, or virus (e.g., retrovirus, FIG. 15 is a bar graph of the percent conversion after 4 adenovirus, or herpes virus) that as a whole does not exist in hours of pyruvate to L-alanine (mol/mol) as a measure of the nature is considered to be non-naturally-occurring nucleic ()-transaminase activity for converting 7-aminoheptanoate acid. It follows that genomic DNA fragments produced by to pimelate semialdehyde relative to the empty vector con 15 PCR or restriction endonuclease treatment as well as cDNAs trol. are considered to be non-naturally-occurring nucleic acid FIG. 16 is a bar graph of the percent conversion after 4 since they exist as separate molecules not found in nature. It hours of L-alanine to pyruvate (mol/mol) as a measure of the also follows that any nucleic acid containing a promoter ()-transaminase activity for converting pimelate semialde sequence and polypeptide-encoding sequence (e.g., cDNA hyde to 7-aminoheptanoate relative to the empty vector or genomic DNA) in an arrangement not found in nature is control. non-naturally-occurring nucleic acid. A nucleic acid that is FIG. 17 is a bar graph of the percent conversion after 4 naturally-occurring can be exogenous to a particular host hours of pyruvate to L-alanine (mol/mol) as a measure of the microorganism. For example, an entire chromosome iso ()-transaminase activity for converting heptamethylenedi lated from a cell of yeast X is an exogenous nucleic acid with amine to 7-aminoheptanal relative to the empty vector 25 respect to a cell of yeast y once that chromosome is control. introduced into a cell of yeast y. FIG. 18 is a bar graph of the percent conversion after 4 In contrast, the term "endogenous” as used herein with hours of pyruvate to L-alanine (mol/mol) as a measure of the reference to a nucleic acid (e.g., a gene) (or a protein) and ()-transaminase activity for converting N7-acetyl-1,7-di a host refers to a nucleic acid (or protein) that does occur in aminoheptane to N7-acetyl-7-aminoheptanal relative to the 30 (and can be obtained from) that particular host as it is found empty vector control. in nature. Moreover, a cell "endogenously expressing a FIG. 19 is a bar graph of the percent conversion after 4 nucleic acid (or protein) expresses that nucleic acid (or hours of pyruvate to L-alanine (mol/mol) as a measure of the protein) as does a host of the same particular type as it is ()-transaminase activity for converting 7-aminoheptanol to found in nature. Moreover, a host "endogenously produc 7-oxoheptanol relative to the empty vector control. 35 ing’ or that "endogenously produces a nucleic acid, pro tein, or other compound produces that nucleic acid, protein, DETAILED DESCRIPTION or compound as does a host of the same particular type as it is found in nature. This document provides enzymes, non-natural pathways, For example, depending on the host and the compounds cultivation strategies, feedstocks, host microorganisms and 40 produced by the host, one or more of the following enzymes attenuations to the host's biochemical network, which gen may be expressed in the host in addition to a (homo), citrate erates a seven carbon chain aliphatic backbone from central synthase, a (homo), citrate dehydratase, a (homo)naconitate metabolites in which one or two terminal functional groups hydratase and an iso(homo), citrate dehydrogenase: a may be formed leading to the synthesis of pimelic acid, indolepyruvate decarboxylase or 2-oxoglutarate dehydroge 7-aminoheptanoic acid, heptamethylenediamine or 1.7-hep 45 nase complex, a thioesterase, a reversible CoA-ligase (e.g., tanediol (referred to as “C7 building blocks' herein). As a reversible Succinyl-CoA-ligase), a CoA-transferase (e.g., a used herein, the term “central precursor is used to denote glutaconate CoA-transferase), an acetylating aldehyde dehy any metabolite in any metabolic pathway shown herein drogenase, a 6-oxohexanoate dehydrogenase, a 7-oxohep leading to the synthesis of a C7 building block. The term tanoate dehydrogenase, an aldehyde dehydrogenase, a car “central metabolite' is used herein to denote a metabolite 50 boxylate reductase, a co-transaminase, a N-acetyl that is produced in all microorganisms to Support growth. transferase, an alcohol dehydrogenase, a deacetylase, a Host microorganisms described herein can include endog 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentano enous pathways that can be manipulated Such that one or ate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase. more C7 building blocks can be produced. In an endogenous In recombinant hosts expressing a carboxylate reductase, a pathway, the host microorganism naturally expresses all of 55 phosphopantetheinyl transferase also can be expressed as it the enzymes catalyzing the reactions within the pathway. A enhances activity of the carboxylate reductase. host microorganism containing an engineered pathway does For example, a recombinant host can include at least one not naturally express all of the enzymes catalyzing the exogenous nucleic acid encoding a (homo), citrate synthase, reactions within the pathway but has been engineered Such a (homo), citrate dehydratase, a (homo),aconitate hydratase, that all of the enzymes within the pathway are expressed in 60 and an iso(homo), citrate dehydrogenase, a indolepyruvate the host. decarboxylase and a 2-oxoglutarate dehydrogenase com The term “exogenous” as used herein with reference to a plex, and produce pimelate semialdehyde or pimeloyl-CoA. nucleic acid (or a protein) and a host refers to a nucleic acid Such recombinant hosts further can include at least one that does not occur in (and cannot be obtained from) a cell exogenous nucleic acid encoding one or more of a thio of that particular type as it is found in nature or a protein 65 esterase, an aldehyde dehydrogenase, a 7-oxoheptanoate encoded by Such a nucleic acid. Thus, a non-naturally dehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA occurring nucleic acid is considered to be exogenous to a transferase, a reversible CoA-ligase, an acetylating aldehyde US 9,580,731 B2 10 dehydrogenase, or a carboxylate reductase and produce For example, a carboxylate reductase described herein pimelic acid or pimelate semialdehyde. For example, a can have at least 70% sequence identity (homology) (e.g., at recombinant host producing pimeloyl-acp or pimeloyl least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) CoA further can include a thioesterase, a reversible Co to the amino acid sequence of a Mycobacterium marinum ligase (e.g., a reversible Succinyl-CoA ligase), or a CoA (see Genbank Accession No. ACC40567.1, SEQID NO: 2), transferase (e.g., a glutaconate CoA-transferase) and pro a Mycobacterium Smegmatis (see Genbank Accession No. duce pimelic acid. For example, a recombinant host produc ABK71854.1, SEQID NO:3), a Segniliparus rugosus (see ing pimeloyl-CoA further can include an acetylating alde Genbank Accession No. EFV11917.1, SEQ ID NO: 4), a hyde dehydrogenase and produce pimelate semilaldehyde. Mycobacterium smegmatis (see Genbank Accession No. For example, a recombinant host producing pimelate further 10 ABK75684.1, SEQ ID NO: 5), a Mycobacterium massil can include a carboxylate reductase and produce pimelate iense (see Genbank Accession No. EIV11143.1, SEQ ID semialdehyde. NO: 6), or a Segniliparus rotundus (see Genbank Accession A recombinant hosts producing pimelic acid or pimelate No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase. semialdehyde further can include at least one exogenous See, FIG. 7. nucleic acid encoding a co-transaminase and produce 15 For example, a co-transaminase described herein can have 7-aminoheptanoate. In some embodiments, a recombinant at least 70% sequence identity (homology) (e.g., at least host producing pimeloyl-CoA includes a carboxylate reduc 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to tase and a co-transaminase to produce 7-aminoheptanoate. the amino acid sequence of a Chronobacterium violaceum A recombinant host producing pimelate or pimelate semi (see Genbank Accession No. AAQ59697.1, SEQID NO: 8), aldehyde further can include at least one exogenous nucleic a Pseudomonas aeruginosa (see Genbank Accession No. acid encoding a 6-hydroxyhexanoate dehydrogenase, a AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae 5-hydroxypentanoate dehydrogenase or a 4-hydroxybu (see Genbank Accession No. AAY39893.1, SEQ ID NO: tyrate dehydrogenase, and produce 7-hydroxyheptanoic 10), a Rhodobacter sphaeroides (see Genbank Accession acid. In some embodiments, a recombinant host producing No. ABA81135.1, SEQID NO: 11), an Escherichia coli (see pimeloyl-CoA includes an acetylating aldehyde dehydroge 25 Genbank Accession No. AAA57874.1, SEQID NO: 12), or nase, and a 6-hydroxyhexanoate dehydrogenase, a 5-hy a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, droxypentanoate dehydrogenase or a 4-hydroxybutyrate SEQ ID NO: 13) ()-transaminase. Some of these dehydrogenase to produce 7-hydroxyheptanoate. In some ()-transaminases are diamine ()-transaminases. embodiments, a recombinant host producing pimelate For example, a phosphopantetheinyl transferase described includes a carboxylate reductase and a 6-hydroxyhexanoate 30 herein can have at least 70% sequence identity (homology) dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a (e.g., at least 75%, 80%, 85%, 90%. 95%, 97%, 98%, 99%, 4-hydroxybutyrate dehydrogenase to produce 7-hydroxy or 100%) to the amino acid sequence of a Bacillus subtilis heptanoate. phosphopantetheinyl transferase (see Genbank Accession A recombinant hosts producing 7-aminoheptanoate, 7-hy No. CAA44858.1, SEQID NO:14) or a Nocardia sp. NRRL droxyheptanoate or pimelate semialdehyde further can 35 5646 phosphopantetheinyl transferase (see Genbank Acces include at least one exogenous nucleic acid encoding a sion No. ABI83656.1, SEQ ID NO:15). See FIG. 7. ()-transaminase, a deacetylase, a N-acetyl transferase, or an The percent identity (homology) between two amino acid alcohol dehydrogenase, and produce heptamethylenedi sequences can be determined as follows. First, the amino amine. For example, a recombinant host producing 7-hy acid sequences are aligned using the BLAST 2 Sequences droxyheptanoate can include a carboxylate reductase with a 40 (B12seq) program from the stand-alone version of BLASTZ phosphopantetheline transferase enhancer, a co-transaminase containing BLASTP version 2.0.14. This stand-alone ver and an alcohol dehydrogenase. sion of BLASTZ can be obtained from Fish & Richardson's A recombinant host producing 7-hydroxyheptanoic acid web site (e.g., www.fr.com/blast?) or the U.S. governments further can include one or more of a carboxylate reductase National Center for Biotechnology Information web site with a phosphopantetheline transferase enhancer and an 45 (www.ncbi.nlm.nih.gov). Instructions explaining how to use alcohol dehydrogenase, and produce 17-heptanediol. the B12seq program can be found in the readme file accom Within an engineered pathway, the enzymes can be from panying BLASTZ. B12seq performs a comparison between a single source, i.e., from one species or genus, or can be two amino acid sequences using the BLASTP algorithm. To from multiple sources, i.e., different species or genera. compare two amino acid sequences, the options of B12seq Nucleic acids encoding the enzymes described herein have 50 are set as follows: -i is set to a file containing the first amino been identified from various organisms and are readily acid sequence to be compared (e.g., C:\Seq 1.txt); - is set to available in publicly available databases such as GenBank or a file containing the second amino acid sequence to be EMBL. compared (e.g., C:\Seq2.txt); -p is set to blastp; -o is set to Any of the enzymes described herein that can be used for any desired file name (e.g., C:\output.txt); and all other production of one or more C7 building blocks can have at 55 options are left at their default setting. For example, the least 70% sequence identity (homology) (e.g., at least 75%, following command can be used to generate an output file 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the containing a comparison between two amino acid amino acid sequence of the corresponding wild-type sequences: C:\B12seq -ic:\Seq1.txt - c.:\Seq2.txt -p blastp -o enzyme. It will be appreciated that the sequence identity can c:\output.txt. If the two compared sequences share homol be determined on the basis of the mature enzyme (e.g., with 60 ogy (identity), then the designated output file will present any signal sequence removed). those regions of homology as aligned sequences. If the two For example, a thioesterase described herein can have at compared sequences do not share homology (identity), then least 70% sequence identity (homology) (e.g., at least 75%, the designated output file will not present aligned sequences. 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the Similar procedures can be following for nucleic acid amino acid sequence of an Escherichia coli thioesterase 65 sequences except that blastn is used. encoded by tesB (see GenBank Accession No. Once aligned, the number of matches is determined by AAA24665.1, SEQ ID NO: 1). See FIG. 7. counting the number of positions where an identical amino US 9,580,731 B2 11 12 acid residue is presented in both sequences. The percent amino acid sequence other than (a). A heterologous identity (homology) is determined by dividing the number of sequence can be, for example a sequence used for purifica matches by the length of the full-length polypeptide amino tion of the recombinant protein (e.g., FLAG, polyhistidine acid sequence followed by multiplying the resulting value (e.g., hexahistidine), hemagglutinin (HA), glutathione-S- by 100. It is noted that the percent identity (homology) value transferase (GST), or maltosebinding protein (MBP)). Het is rounded to the nearest tenth. For example, 78.11, 78.12. erologous sequences also can be proteins useful as detect 78.13, and 78.14 is rounded down to 78.1, while 78.15, able markers, for example, luciferase, green fluorescent 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also protein (GFP), or chloramphenicol acetyltransferase (CAT). is noted that the length value will always be an integer. In some embodiments, the fusion protein contains a signal It will be appreciated that a number of nucleic acids can 10 sequence from another protein. In certain host cells (e.g., encode a polypeptide having a particular amino acid yeast host cells), expression and/or secretion of the target sequence. The degeneracy of the genetic code is well known protein can be increased through use of a heterologous to the art; i.e., for many amino acids, there is more than one signal sequence. In some embodiments, the fusion protein nucleotide triplet that serves as the codon for the amino acid. can contain a carrier (e.g., KLH) useful, e.g., in eliciting an For example, codons in the coding sequence for a given 15 immune response for antibody generation) or ER or Golgi enzyme can be modified Such that optimal expression in a apparatus retention signals. Heterologous sequences can be particular species (e.g., bacteria or fungus) is obtained, using of varying length and in some cases can be a longer appropriate codon bias tables for that species. sequences than the full-length target proteins to which the Functional fragments of any of the enzymes described heterologous sequences are attached. herein can also be used in the methods of the document. The Engineered hosts can naturally express none or some term “functional fragment” as used herein refers to a peptide (e.g., one or more, two or more, three or more, four or more, fragment of a protein that has at least 25% (e.g., at least: five or more, or six or more) of the enzymes of the pathways 30%; 40%; 50%: 60%; 70%; 75%: 80%; 85%: 90%; 95%; described herein. Thus, a pathway within an engineered host 98%: 99%; 100%; or even greater than 100%) of the activity can include all exogenous enzymes, or can include both of the corresponding mature, full-length, wild-type protein. 25 endogenous and exogenous enzymes. Endogenous genes of The functional fragment can generally, but not always, be the engineered hosts also can be disrupted to prevent the comprised of a continuous region of the protein, wherein the formation of undesirable metabolites or prevent the loss of region has functional activity. intermediates in the pathway through other enzymes acting This document also provides (i) functional variants of the on Such intermediates. Engineered hosts can be referred to enzymes used in the methods of the document and (ii) 30 as recombinant hosts or recombinant host cells. As described functional variants of the functional fragments described herein recombinant hosts can include nucleic acids encoding above. Functional variants of the enzymes and functional one or more of a synthase, a dehydratase, a hydratase, a fragments can contain additions, deletions, or Substitutions dehydrogenase, a thioesterase, a reversible CoA-ligase, a relative to the corresponding wild-type sequences. Enzymes CoA-transferase, a reductase, deacetylase, N-acetyl trans with substitutions will generally have not more than 50 (e.g., 35 ferase or a co-transaminase as described in more detail not more than one, two, three, four, five, six, seven, eight, below. nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) amino acid In addition, the production of one or more C7 building Substitutions (e.g., conservative Substitutions). This applies blocks can be performed in vitro using the isolated enzymes to any of the enzymes described herein and functional described herein, using a lysate (e.g., a cell lysate) from a fragments. A conservative Substitution is a Substitution of 40 host microorganism as a source of the enzymes, or using a one amino acid for another with similar characteristics. plurality of lysates from different host microorganisms as Conservative substitutions include substitutions within the the source of the enzymes. following groups: valine, alanine and glycine; leucine, Enzymes Generating the C7 Aliphatic Backbone for Con valine, and isoleucine; aspartic acid and glutamic acid; version to C7 Building Blocks asparagine and glutamine; serine, cysteine, and threonine; 45 As depicted in FIG. 1, a C7 aliphatic backbone for lysine and arginine; and phenylalanine and tyrosine. The conversion to a C7 building block can be formed from nonpolar hydrophobic amino acids include alanine, leucine, 2-oxoglutarate via three cycles of 2-oxoacid carbon chain isoleucine, valine, proline, phenylalanine, tryptophan and elongation associated with Coenzyme B biosynthesis methionine. The polar neutral amino acids include glycine, enzymes. A C7 aliphatic backbone for conversion to a C7 serine, threonine, cysteine, tyrosine, asparagine and gluta 50 building block also can be formed from 2-oxoadipate via mine. The positively charged (basic) amino acids include two cycles of C1 carbon chain elongation associated with arginine, lysine and histidine. The negatively charged Coenzyme B biosynthesis enzymes. (acidic) amino acids include aspartic acid and glutamic acid. In some embodiments, a C1 carbon chain elongation Any substitution of one member of the above-mentioned cycle comprises a synthetase, a dehydratase, a dehydratase, polar, basic or acidic groups by another member of the same 55 and a dehydrogenase. For example, each elongation cycle group can be deemed a conservative substitution. By con can use a (homo), citrate dehydratase, a (homo),aconitate trast, a nonconservative Substitution is a Substitution of one hydratase and an iso(homo), citrate dehydrogenase. amino acid for another with dissimilar characteristics. In some embodiments, a (homo), citrate synthase can be Deletion variants can lack one, two, three, four, five, six, classified, for example, under EC 2.3.3.14 or EC 2.3.3.13. seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 60 Such as the gene product of akSA from Methanocaldococcus 20 amino acid segments (of two or more amino acids) or jannaschii (see Genbank Accession No. AAB98494.1). non-contiguous singleamino acids. Additions (addition vari In some embodiments, the combination of (homo), citrate ants) include fusion proteins containing: (a) any of the dehydratase and (homo),aconitate hydratase may be classi enzymes described herein or a fragment thereof; and (b) fied, for example, under EC 4.2.1.- (e.g., EC 4.2.1.114, EC internal or terminal (C or N) irrelevant or heterologous 65 4.2.1.36 or EC 4.2.1.33), such as the gene product of aksD amino acid sequences. In the context of such fusion proteins, from Methanocaldococcus jannaschii (see, Genbank Acces the term "heterologous amino acid sequences’ refers to an sion No. AAB99007.1) or gene product of aksh from US 9,580,731 B2 13 14 Methanocaldococcus jannaschii (see, Genbank Accession 1281-1295; Zhuang et al., Biochemistry, 2008, 47(9), 2789 No. AAB99277.1). The gene products of aksD and aksh are 2796; or Naggert et al., J. Biol. Chem., 1991, 266(17), subunits of an enzyme classified under EC 4.2.1.114. 11044-11050). Homologs in 2-oxoacid chain elongation from the branch In some embodiments, the second terminal carboxyl chain amino acid synthesis pathway this is also true, e.g. The 5 group leading to the synthesis of pimelic acid is enzymati gene products of LeuC and Leul) are subunits of an enzyme cally formed by an aldehyde dehydrogenase classified, for classified under EC 4.2.1.33. example, under EC 1.2.1.3 (see, for example, Guerrillot & In some embodiments, an iso(homo), citrate dehydroge Vandecasteele, Eur: J. Biochem., 1977, 81, 185-192). nase may be classified, for example, under EC 1.1.1.- Such In some embodiments, the second terminal carboxyl as EC 1.1.1.85, EC 1.1.1.87 or EC 1.1.1.286, such as the 10 group leading to the synthesis of pimelic acid is enzymati gene product of aksh from Methanocaldococcus jannaschii cally formed by a 6-oxohexanoate dehydrogenase or a (see, Genbank Accession No. ACA28837.1). 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, In some embodiments, the product of two or three chain Such as the gene product of ChinE from Acinetobacter sp. or elongation cycles, 2-oxo-Suberate, can be decarboxylated by ThnG from Sphingomonas macrogolitabida (see, for an indolepyruvate decarboxylase classified, for example, 15 example, Iwaki et al., Appl. Environ. Microbiol., 1999, under EC 4.1.1.43 or EC 4.1.1.74 such as the indole-3- 65(11), 5158-5162; or Lopez-Sanchez et al., Appl. Environ. pyruvate decarboxylase from Salmonella typhimurium (see, Microbiol., 2010, 76(1), 110-118). For example, a 6-oxo for example, Genbank Accession No. CAC48239.1 in which hexanoate dehydrogenase can be classified under EC residue 544 can be a leucine or an alanine) 1.2.1.63. For example, a 7-oxoheptanoate dehydrogenase 2-oxo-Suberate also can be decarboxylated by a 2-oxo can be classified under EC 1.2.1.-. glutarate dehydrogenase complex comprised of enzymes In some embodiments, the second terminal carboxyl homologous to enzymes classified, for example, under EC group leading to the synthesis of pimelic acid is enzymati 1.2.4.2, EC 1.8.1.4, and EC 2.3.1.61. The 2-oxoglutarate cally formed by a CoA-transferase Such as a glutaconate dehydrogenase complex contains multiple copies of a 2-oxo CoA-transferase classified, for example, under EC 2.8.3.12 glutarate dehydrogenase classified, for example, under EC 25 Such as from Acidaminococcus fermentans. See, for 1.2.4.2 bound to a core of dihydrolipoyllysine-residue suc example, Buckel et al., 1981, Eur: J. Biochem., 118:315-321. cinyltransferases classified, for example, under EC 2.3.1.61, In some embodiments, the second terminal carboxyl which also binds multiple copies of a dihydrolipoyl dehy group leading to the synthesis of pimelic acid is enzymati drogenase classified, for example, under EC 1.8.1.4. cally formed by a reversible CoA-ligase such as a Succinate Several analogous 2-oxoacid chain elongation pathway 30 CoA ligase classified, for example, under EC 6.2.1.5 such as are utilized by microorganisms in producing branch chain from Thermococcus kodakaraensis. See, for example, amino acids, lysine, and Coenzyme B (see, for example, Shikata et al., 2007, J. Biol. Chem., 282(37):26963-26970. Drevland et al., 2007, J. Bacteriol., 189(12), 4391-4400). Enzymes Generating the Terminal Amine Groups in the Using the chain elongation enzymes for Coenzyme B bio Biosynthesis of C7 Building Blocks synthesis (see, for example, Drevland et al., 2008, J. Biol. 35 As depicted in FIG. 3 and FIG. 4, terminal amine groups Chem., 283 (43), 28888-28896; Howell et al., 2000, J. Bac can be enzymatically formed using a co-transaminase or a teriol., 182(7), 5013-5016), the chain of 2-oxoglutrate can deacetylase. be elongated to the C8 dicarboxylic acid, 2-oxosuberate. In some embodiments, the first or second terminal amine Similarly, using the chain elongation enzymes associated group leading to the synthesis of 7-aminoheptanoic acid is with branch chain amino acid biosynthesis, 2-isopropyl 40 enzymatically formed by a co-transaminase classified, for malate synthase encoded by LeuA, can be engineered to example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC accept longer chain substrates allowing chain elongation to 2.6.1.48, or EC 2.6.1.82 such as that obtained from Chro C7/C8 (Zhang et al., 2008, Proc. Natl. Acad. Sci., 105(52), mobacterium violaceum (Genbank Accession No. 20653-20658). AAQ59697.1, SEQ ID NO: 8), Pseudomonas aeruginosa A mutant variant of the indolepyruvate decarboxylase 45 (Genbank Accession No. AAG081911, SEQ ID NO: 9), from Salmonella typhimurium has been engineered Success Pseudomonas Syringae (Genbank Accession No. fully to selectively accept longer chain length substrates. AAY39893.1, SEQ ID NO: 10), Rhodobacter sphaeroides The L544A mutation of the sequence provided in Genbank (Genbank Accession No. ABA81135.1, SEQ ID NO: 11), Accession No. CAC48239.1 allowed for 567 times higher Escherichia coli (Genbank Accession No. AAA57874.1, selectivity towards the C7 2-oxoacid than towards the C5 50 SEQ ID NO: 12), Vibrio Fluvialis (Genbank Accession No. 2-oxoacid (see, Xiong et al., 2012, Scientific Reports, 2: AAA57874.1, SEQ ID NO: 13), Streptomyces griseus, or 311). The 2-oxoglutarate dehydrogenase complex has dem Clostridium viride. Some of these ()-transaminases are onstrated activity for 2-oxoglutate and 2-oxoadipate (Bunik diamine ()-transaminases (e.g., SEQ ID NO:12). For et al., 2000, Eur: J. Biochem., 267, 3583-3591). example, the co-transaminases classified, for example, under Enzymes Generating the Terminal Carboxyl Groups in the 55 EC 2.6.1.29 or EC 2.6.1.82 may be diamine ()-transami Biosynthesis of C7 Building Blocks aSCS. As depicted in FIG. 2, a terminal carboxyl group can be The reversible ()-transaminase from Chromobacterium enzymatically formed using an thioesterase, an aldehyde violaceum (Genbank Accession No. AAQ59697.1, SEQ ID dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxo NO: 8) has demonstrated analogous activity accepting hexanoate dehydrogenase, a CoA-transferase or a reversible 60 6-aminohexanoic acid as amino donor, thus forming the first CoA-ligase. terminal amine group in adipate semialdehyde (Kaulmann et In some embodiments, the second terminal carboxyl al., Enzyme and Microbial Technology, 2007, 41, 628-637). group leading to the synthesis of a C7 building block is The reversible 4-aminobubyrate:2-oxoglutarate transami enzymatically formed by a thioesterase classified under EC nase from Streptomyces griseus has demonstrated analogous 3.1.2.-, such as the gene product of YciA, tesB (Genbank 65 activity for the conversion of 6-aminohexanoate to adipate Accession No. AAA24665.1, SEQ ID NO: 1) or Acot13 semialdehyde (Yonaha et al., Eur: J. Biochem., 1985, 146: (see, for example, Cantu et al., Protein Science, 2010, 19. 101-106). US 9,580,731 B2 15 16 The reversible 5-aminovalerate transaminase from sion to 2-oxo-pimelate by an iso(homo), citrate dehydroge Clostridium viride has demonstrated analogous activity for nase classified under, for example, EC 1.1.1.85, EC 1.1.1.87, the conversion of 6-aminohexanoate to adipate semialde or EC 1.1.1.286 (e.g., Genbank Accession No. hyde (Barker et al., J. Biol. Chem., 1987, 262(19), 8994 AAB98494.1); followed by conversion to (Homo) citrate by 9003). 5 a (Homo), citrate synthase classified, for example, under EC In some embodiments, a terminal amine group leading to 2.3.3.14 or EC 2.3.3.13 (see, e.g., Genbank Accession No. the synthesis of 7-aminoheptanoate or heptamethylenedi AAB98494.1); followed by conversion to iso(homo) citrate amine is enzymatically formed by a diamine ()-transami by a (homo), citrate dehydratase and a (homo),aconitate nase. For example, the second terminal amino group can be hydratase classified, for example, under EC 4.2.1.114, EC enzymatically formed by a diamine ()-transaminase classi 10 fied, for example, under EC 2.6.1.29 or classified, for 4.2.1.36 or EC 4.2.1.33 (e.g., Genbank Accession Nos. example, under EC 2.6.1.82. Such as the gene product of AAB99007.1 and AAB99277.1); followed by conversion to YgG from E. coli (Genbank Accession No. AAA57874.1, 2-oxo-Suberate by an iso(homo), citrate dehydrogenase clas SEQ ID NO: 12). sified under, for example, EC 1.1.1.85, EC 1.1.1.87, or EC The gene product of ygG accepts a broad range of 15 1.1.1.286 (e.g., Genbank Accession No. AAB98494.1). diamine carbon chain length Substrates, such as putrescine, Pimeloyl-CoA can be produced by conversion of 2-oxo cadaverine and spermidine (see, for example, Samsonova et Suberate to pimeloyl-CoA by a 2-oxoglutarate dehydroge al., BMC Microbiology, 2003, 3:2). nase complex containing enzymes classified, for example, The diamine ()-transaminase from E. coli Strain B has under EC 1.2.4.2, EC 1.8.1.4 and EC 2.3.1.61. Pimelate demonstrated activity for 1.7 diaminoheptane (Kim, The semialdehyde can be produced by conversion of 2-oxo Journal of Chemistry, 1964, 239(3), 783-786). Suberate to pimelate semialdehyde by a decarboxylase clas In some embodiments, the second terminal amine group sified, for example, EC 4.1.1.- (e.g., EC 4.1.1.43 and EC leading to the synthesis of heptamethylenediamine is enzy 4.1.1.74) (see e.g., Genbank Accession No. CAC48239.1). matically formed by a deacetylase Such as acetylputrescine See, FIG. 1. deacetylase classified, for example, under EC 3.5.1.62. The 25 In some embodiments, pimelate semialdehyde or acetylputrescine deacetylase from Micrococcus luteus K-11 pimeloyl-CoA can be synthesized from the central metabo accepts abroad range of carbon chain length substrates. Such lite, 2-oxoadipate, as described in FIG. 1. as acetylputrescine, acetylcadaverine and N-acetylspermi Pathways Using Pimeloyl-CoA or Pimelate Semialdehyde dine (see, for example, Suzuki et al., 1986, BBA—General as Central Precursors to Pimelate Subjects, 882(1): 140-142). 30 In some embodiments, pimelic acid is synthesized from Enzymes Generating the Terminal Hydroxyl Groups in the the central precursor, pimeloyl-CoA, by conversion of Biosynthesis of C7 Building Blocks pimeloyl-CoA to pimelate semialdehyde by an acetylating As depicted in FIG. 5 and FIG. 6, a terminal hydroxyl aldehyde dehydrogenase classified, for example, under EC group can be enzymatically formed using an alcohol dehy 1.2.1.10 such as the gene product of PduB or PduP (see, for drogenase. 35 example, Lan et al., 2013, Energy Environ. Sci., 6:2672 In some embodiments, the second terminal hydroxyl 2681); followed by conversion to pimelic acid by a 7-oxo group leading to the synthesis of 1.7 heptanediol is enzy heptanoate dehydrogenase classified, for example, under EC matically formed by an alcohol dehydrogenase classified, 1.2.1.- Such as the gene product of ThnG, a 6-oxohexanoate for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2, dehydrogenase classified, for example, under EC 1.2.1.- EC 1.1.1.21, or EC 1.1.1.184). 40 Such as the gene product of ChinE, or an aldehyde dehydro Biochemical Pathways genase (classified, for example, under C 1.2.1.3). See FIG. Pathways Using 2-Oxoadipate as Central Metabolite to C7 2. Building Blocks, Pimelate semialdehyde and pimeloyl-CoA In some embodiments, pimelic acid is synthesized from In some embodiments, pimeloyl-CoA or pimelate semi the central precursor, pimeloyl-CoA, by conversion of aldehyde can be synthesized from the central metabolite, 45 pimeloyl-CoA to pimelate by a thioesterase classified, for 2-oxoglutarate, by three cycles of 2-oxoacid chain elonga example, under EC 3.1.2.- Such as the gene products of tion by conversion of 2-oxoglutrate to (Homo), citrate by a YciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID (Homo), citrate synthase classified, for example, under EC NO: 1) or Acot13. See FIG. 2. 2.3.3.14 or EC 2.3.3.13 (see, e.g., AkSA, Genbank Accession In some embodiments, pimelate is synthesized from the No. AAB98494.1); followed by conversion to iso(homo) 50 central precursor, pimeloyl-CoA, by conversion of citrate by a (homo), citrate dehydratase and a (homo), aco pimeloyl-CoA to pimelate by a CoA-transferase such as a nitate hydratase classified, for example, under EC 4.2.1.114, glutaconate CoA-transferase classified, for example, under EC 4.2.1.36 or EC 4.2.1.33 (see, e.g., AksD and Aksh, EC 2.83.12. See FIG. 2. Genbank Accession Nos. AAB99007.1 and AAB99277.1): In some embodiments, pimelate is synthesized from the followed by conversion to 2-oxoadipate by an iso(homo) 55 central precursor, pimeloyl-CoA, by conversion of citrate dehydrogenase classified, for example, under EC pimeloyl-CoA to pimelate by a reversible CoA-ligase such 1.1.1.85, EC 1.1.1.87 or EC 1.1.1.286 (see, e.g., Akse, as a reversible Succinate CoA-ligase classified, for example, Genbank Accession No. AAB98494.1); followed by con under EC 6.2.1.5. See FIG. 2. version to (Homo) citrate by a (Homo), citrate synthase In some embodiments, pimelate is synthesized from the classified, for example, under EC 2.3.3.14 or EC 2.3.3.13 60 central precursor, pimelate semialdehyde, by conversion of (see, e.g., Genbank Accession No. AAB98494.1); followed pimelate semialdehyde to pimelate by a 6-oxohexanoate by conversion to iso(homo) citrate (also known as 1-hy dehydrogenase or a 7-oxoheptanoate dehydrogenase (clas droxypentane-1,2,5-tricarboxylate or threo-iso(homo) cit sified, for example, under EC 1.2.1.-) such as the gene rate) by a (homo), citrate dehydratase and a (homo),aconi product of ThnG or ChinE, or an aldehyde dehydrogenase tate hydratase classified, for example, under EC 4.2.1.114, 65 (classified, for example, under EC 1.2.1.3). See FIG. 2. EC 4.2.1.36 or EC 4.2.1.33 (see, e.g., Genbank Accession Pathways Using Pimeloyl-CoA or Pimelate Semialdehyde Nos. AAB99007.1 and AAB99277.1); followed by conver as Central Precursor to 7-Aminoheptanoate US 9,580,731 B2 17 18 In some embodiments, 7-aminoheptanoate is synthesized carboxylate reductase classified, for example, under EC from the central precursor, pimeloyl-CoA, by conversion of 1.2.99.6 such as the gene product of car (see above) in pimeloyl-CoA to pimelate semialdehyde by an acetylating combination with a phosphopantetheline transferase aldehyde dehydrogenase classified, for example, EC enhancer (e.g., encoded by a Sfp gene from Bacillus subtilis 1.2.1.10, such as the gene product of PduB or PduP; fol or mpt gene from Nocardia) or the gene product of GriC & lowed by conversion of pimelate semialdehyde to 7-amino GrilD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); heptanoate by a co-transaminase classified, for example, followed by conversion of 7-aminoheptanal to 7-aminohep under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48 or tanol by a ()-transaminase classified, for example, under EC EC 2.6.182. See FIG. 3. 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC In some embodiments, 7-aminoheptanoate is synthesized 10 2.6.1.82 such as SEQID NOs:8-13, see above; followed by from the central precursor, pimelate semialdehyde, by con conversion to 7-aminoheptanal by an alcohol dehydrogenase version of pimelate semialdehyde to 7-aminoheptanoate by classified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, a co-transaminase (e.g., EC 2.6.1.18, EC 2.6.1.19, or EC EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene 2.6.1.48). See FIG. 3. product of YMR318C (classified, for example, under EC In some embodiments, 7-aminoheptanoate is synthesized 15 1.1.1.2, see Genbank Accession No. CAA90836.1) or YahD from the central precursor, pimelate, by conversion of pime (from E. coli, GenBank Accession No. AAA69178.1) (Liu et late to pimelate semialdehyde by a carboxylate reductase al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, classified, for example, under EC 1.2.99.6 such as the gene Biochem J., 361 (Pt 1), 163-172: Jarboe, 2011, Appl. Micro product of car in combination with a phosphopantetheline biol. Biotechnol., 89(2), 249-257) or the protein having transferase enhancer (e.g., encoded by a Sfp (Genbank GenBank Accession No. CAA81612.1 (from Geobacillus Accession No. CAA44858.1, SEQ ID NO:14) gene from Stearothermophilus); followed by conversion to heptameth Bacillus subtilis or npt (Genbank Accession No. ylenediamine by a co-transaminase classified, for example, ABI83656.1, SEQ ID NO:15) gene from Nocardia) or the under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, gene products of GriC and Gril) from Streptomyces griseus; or EC 2.6.1.82 such as SEQ ID NOs:8-13, see above. See followed by conversion of pimelate semialdehyde to 25 FIG. 4. 7-aminoheptanoate by a co-transaminase (e.g., EC 2.6.1.18, In some embodiments, heptamethylenediamine is synthe EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.29, EC 2.6.1.82 such as sized from the central precursor, 7-aminoheptanoate, by SEQ ID NOs:8-13). The carboxylate reductase can be conversion of 7-aminoheptanoate to N7-acetyl-7-aminohep obtained, for example, from Mycobacterium marinum (Gen tanoate by a N-acetyltransferase such as a lysine N-acetyl bank Accession No. ACC40567.1, SEQ ID NO: 2), Myco 30 transferase classified, for example, under EC 2.3.1.32; fol bacterium smegmatis (Genbank Accession No. lowed by conversion to N7-acetyl-7-aminoheptanal by a ABK71854.1, SEQID NO:3), Segniliparus rugosus (Gen carboxylate reductase classified, for example, under EC bank Accession No. EFV11917.1, SEQ ID NO: 4), Myco 1.2.99.6 such as the gene product of car (see above) in bacterium smegmatis (Genbank Accession No. combination with a phosphopantetheline transferase ABK75684.1, SEQ ID NO. 5), Mycobacterium massiliense 35 enhancer (e.g., encoded by a Sfp gene from Bacillus subtilis (Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or or mpt gene from Nocardia) or the gene product of GriC & Segniliparus rotundus (Genbank Accession No. Gril); followed by conversion to N7-acetyl-1,7-diaminohep ADG98140.1, SEQ ID NO: 7). See FIG. 3. tane by a co-transaminase classified, for example, under EC Pathway Using 7-Aminoheptanoate, 7-Hydroxyheptanoate 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC or Pimelate Semialdehyde as Central Precursor to Heptam 40 2.6.1.46, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see ethylenediamine above; followed by conversion to heptamethylenediamine In some embodiments, heptamethylenediamine is synthe by an acetylputrescine deacylase classified, for example, sized from the central precursor, 7-aminoheptanoate, by under EC 3.5.1.62. See, FIG. 4. conversion of 7-aminoheptanoate to 7-aminoheptanal by a In some embodiments, heptamethylenediamine is synthe carboxylate reductase classified, for example, under EC 45 sized from the central precursor, pimelate semialdehyde, by 1.2.99.6 such as the gene product of car (see above) in conversion of pimelate semialdehyde to heptanedial by a combination with a phosphopantetheline transferase carboxylate reductase classified, for example, under EC enhancer (e.g., encoded by a Sfp (Genbank Accession No. 1.2.99.6 such as the gene product of car (see above) in CAA44858.1, SEQ ID NO:14) gene from Bacillus subtilis combination with a phosphopantetheline transferase or mpt (Genbank Accession No. ABI83656.1, SEQ ID 50 enhancer (e.g., encoded by a Sfp gene from Bacillus subtilis NO:15) gene from Nocardia) or the gene product of GriC & or mpt gene from Nocardia) or the gene product of GriC & GrilD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); Gril); followed by conversion to 7-aminoheptanal by a followed by conversion of 7-aminoheptanal to heptameth ()-transaminase classified, for example, under EC 2.6.1.18, ylenediamine by a co-transaminase (e.g., classified, for EC 2.6.1.19, or EC 2.6.1.48; followed by conversion to example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 55 heptamethylenediamine by a co-transaminase classified, for 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOS:8-13, see example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC above). See FIG. 4. 2.6.1.48, EC 2.6.1.46, or EC 2.6.1.82 such as SEQ ID The carboxylate reductase encoded by the gene product of NOs:8-13, see above. See FIG. 4. car and the phosphopantetheline transferase enhancer npt or Pathways Using Pimelate or Pimelate Semialdehyde as Sfp has broad Substrate specificity, including terminal 60 Central Precursor to 1.7-Heptanediol difunctional C4 and C5 carboxylic acids (Venkitasubrama In some embodiments, 7-hydroxyheptanoate is synthe nian et al., Enzyme and Microbial Technology, 2008, 42, sized from the central precursor, pimelate, by conversion of 130-137). pimelate to pimelate semialdehyde by a carboxylate reduc In some embodiments, heptamethylenediamine is synthe tase classified, for example, under EC 1.2.99.6 such as the sized from the central precursor, 7-hydroxyheptanoate 65 gene product of car (see above) in combination with a (which can be produced as described in FIG. 5), by conver phosphopantetheline transferase enhancer (e.g., encoded by a sion of 7-hydroxyheptanoate to 7-hydroxyheptanal by a Sfp gene from Bacillus subtilis or npt gene from Nocardia) US 9,580,731 B2 19 20 or the gene product of GriC & Gril); followed by conversion 2012, 166, 1801-1813; Yang et al., Biotechnology for Bio to 7-hydroxyheptanoate by a dehydrogenase classified, for fiels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotech example, under EC 1.1.1.- Such as a 6-hydroxyhexanoate mol., 2011, 90, 885-893). dehydrogenase classified, for example, under EC 1.1.1.258 The efficient catabolism of lignocellulosic-derived levu Such as the gene from of ChnD or a 5-hydroxypentanoate linic acid has been demonstrated in several organisms such dehydrogenase classified, for example, under EC 1.1.1.- as Cupriavidus necator and Pseudomonas putida in the Such as the gene product of CpnD (see, for example, Iwaki synthesis of 3-hydroxyvalerate via the precursor propanoyl et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684) CoA (Jaremko and Yu, Journal of Biotechnology, 2011, 155, or a 4-hydroxybutyrate dehydrogenase such as gab) (see, 2011, 293–298; Martin and Prather, Journal of Biotechnol 10 ogy, 2009, 139, 61-67). for example, Lütke-Eversloh & Steinbichel, 1999, FEMS The efficient catabolism of lignin-derived aromatic com Microbiology Letters, 181(1):63-71). See FIG. 5. Pimelate pounds such as benzoate analogues has been demonstrated semialdehyde also can be produced from pimeloyl-CoA in several microorganisms such as Pseudomonas putida, using an acetylating aldehyde dehydrogenase as described Cupriavidus necator (Bugg et al., Current Opinion in Bio above. See, also FIG. 5. 15 technology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMS In some embodiments, 1.7 heptanediol is synthesized Microbiol. Rev., 2008, 32, 736-794). from the central precursor, 7-hydroxyheptanoate, by con The efficient utilization of agricultural waste, such as version of 7-hydroxyheptanoate to 7-hydroxyheptanal by a olive mill waste water has been demonstrated in several carboxylate reductase classified, for example, under EC microorganisms, including Yarrowia lipolytica (Papaniko 1.2.99.6 such as the gene product of car (see above) in laou et al., Bioresour: Technol., 2008, 99(7), 2419-2428). combination with a phosphopantetheline transferase The efficient utilization of fermentable sugars such as enhancer (e.g., encoded by a Sfp gene from Bacillus subtilis monosaccharides and disaccharides derived from cellulosic, or mpt gene from Nocardia) or the gene product of GriC & hemicellulosic, cane and beet molasses, cassava, corn and Gril); followed by conversion of 7-hydroxyheptanal to 1.7 other agricultural Sources has been demonstrated for several heptanediol by an alcohol dehydrogenase classified, for 25 microorganism such as Escherichia coli, Corynebacterium example, under EC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, glutamicum and Lactobacillus delbrueckii and Lactococcus EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of lactis (see, e.g., Hermann et al., Journal of Biotechnology, YMR318C or Yahl) (see, e.g., Liu et al., Microbiology, 2003, 104, 155-172; Wee et al., Food Technol. Biotechnol., 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 2006, 44(2), 163-172; Ohashi et al., Journal of Bioscience 361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Bio 30 and Bioengineering, 1999, 87(5), 647-654). technol., 89(2), 249-257) or the protein having GenBank The efficient utilization of furfural, derived from a variety Accession No. CAA81612.1 (from Geobacillus Stearother of agricultural lignocellulosic sources, has been demon mophilus). See, FIG. 6. strated for Cupriavidus necator (Li et al., Biodegradation, Cultivation Strategy 2011, 22, 1215-1225). In some embodiments, the cultivation strategy entails 35 In some embodiments, the non-biological feedstock can achieving an aerobic, anaerobic, micro-aerobic, or mixed be or can derive from natural gas, syngas, CO/H, metha oxygen/denitrification cultivation condition. Enzymes char nol, ethanol, benzoic acid, non-volatile residue (NVR), a acterized in vitro as being oxygen sensitive require a micro caustic wash waste stream from cyclohexane oxidation aerobic cultivation strategy maintaining a very low dis processes, or terephthalic acid/isophthalic acid mixture Solved oxygen concentration (See, for example, Chayabatra 40 Waste StreamS. & Lu-Kwang, Appl. Environ. Microbiol., 2000, 66(2), 4930 The efficient catabolism of methanol has been demon 498; Wilson and Bouwer, 1997, Journal of Industrial Micro strated for the methylotrophic yeast Pichia pastoris. biology and Biotechnology, 18(2-3), 116-130). The efficient catabolism of ethanol has been demonstrated In some embodiments, the cultivation strategy entails for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. nutrient limitation Such as nitrogen, phosphate or oxygen 45 Sci. USA, 2008, 105(6) 2128-2133). limitation. The efficient catabolism of CO and H, which may be In some embodiments, a final electron acceptor other than derived from natural gas and other chemical and petro oxygen Such as nitrates can be utilized. chemical sources, has been demonstrated for Cupriavidus In some embodiments, a cell retention strategy using, for necator (Prybylski et al., Energy, Sustainability and Society, example, ceramic hollow fiber membranes can be employed 50 2012, 2:11). to achieve and maintain a high cell density during either The efficient catabolism of syngas has been demonstrated fed-batch or continuous fermentation. for numerous microorganisms, such as Clostridium liung In some embodiments, the principal carbon Source fed to dahlii and Clostridium autoethanogenium (Köpke et al., the fermentation in the synthesis of one or more C7 building Applied and Environmental Microbiology, 2011, 77(15), blocks can derive from biological or non-biological feed 55 5467-5475). stocks. The efficient catabolism of the non-volatile residue waste In some embodiments, the biological feedstock can be, stream from cyclohexane processes has been demonstrated can include, or can derive from, monosaccharides, disac for numerous microorganisms, such as Delfia acidovorans charides, lignocellulose, hemicellulose, cellulose, lignin, and Cupriavidus necator (Ramsay et al., Applied and Envi levulinic acid and formic acid, triglycerides, glycerol, fatty 60 ronmental Microbiology, 1986, 52(1), 152-156). In some acids, agricultural waste, condensed distillers’ solubles, or embodiments, the host microorganism is a prokaryote. For municipal waste. example, the prokaryote can be a bacterium from the genus The efficient catabolism of crude glycerol stemming from Escherichia Such as Escherichia Coli; from the genus the production of biodiesel has been demonstrated in several Clostridia such as Clostridium liungdahli, Clostridium microorganisms such as Escherichia coli, Cupriavidus neca 65 autoethanogenium or Clostridium kluyveri; from the genus tor, Pseudomonas Oleavorans, Pseudomonas putida and Corynebacteria Such as Corynebacterium glutamicum; from Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotechnol., the genus Cupriavidus Such as Cupriavidus necator or US 9,580,731 B2 21 22 Cupriavidus metallidurans; from the genus Pseudomonas of improving activity, improving specificity, reducing feed Such as Pseudomonas fluorescens, Pseudomonas putida or back inhibition, reducing repression, improving enzyme Pseudomonas Oleavorans; from the genus Delfiia Such as solubility, changing Stereo-specificity, or changing co-factor Delftia acidovorans; from the genus Bacillus such as Bacil specificity. lus subtilis; from the genus Lactobacillus Such as Lactoba In some embodiments, the enzymes in the pathways cillus delbrueckii; or from the genus Lactococcus such as outlined herein can be gene dosed (i.e., overexpressed by Lactococcus lactis. Such prokaryotes also can be a source of having a plurality of copies of the gene in the host organ genes to construct recombinant host cells described herein ism), into the resulting genetically modified organism via that are capable of producing one or more C7 building episomal or chromosomal integration approaches. blocks. 10 In some embodiments, genome-scale system biology In some embodiments, the host microorganism is a techniques such as Flux Balance Analysis can be utilized to eukaryote. For example, the eukaryote can be a filamentous devise genome scale attenuation or knockout strategies for fungus, e.g., one from the genus Aspergillus such as Asper directing carbon flux to a C7 building block. gillus niger. Alternatively, the eukaryote can be a yeast, e.g., Attenuation strategies include, but are not limited to; the one from the genus Saccharomyces such as Saccharomyces 15 use of transposons, homologous recombination (double cerevisiae; from the genus Pichia Such as Pichia pastoris; or cross-over approach), mutagenesis, enzyme inhibitors and from the genus Yarrowia Such as Yarrowia lipolytica; from RNA interference (RNAi). the genus Issatchenkia Such as Issathenkia Orientalis; from In some embodiments, fluxomic, metabolomic and tran the genus Debaryomyces such as Debaryomyces hansenii; Scriptomal data can be utilized to inform or Support genome from the genus Arxula Such as Arxtula adenoinivorans; or scale system biology techniques, thereby devising genome from the genus Kluyveromyces Such as Kluyveromyces lac scale attenuation or knockout strategies in directing carbon tis. Such eukaryotes also can be a source of genes to flux to a C7 building block. construct recombinant host cells described herein that are In some embodiments, the host microorganisms toler capable of producing one or more C7 building blocks. ance to high concentrations of a C7 building block can be Metabolic Engineering 25 improved through continuous cultivation in a selective envi The present document provides methods involving less rOnment. than all the steps described for all the above pathways. Such In some embodiments, the host microorganism's endog methods can involve, for example, one, two, three, four, five, enous biochemical network can be attenuated or augmented six, seven, eight, nine, ten, eleven, twelve or more of Such to ensure the intracellular availability of 2-oxoglutarate (2) steps. Where less than all the steps are included in such a 30 create an NAD" imbalance that may only be balanced via the method, the first, and in Some embodiments the only, step formation of a C7 building block, (3) prevent degradation of can be any one of the steps listed. central metabolites or central precursors leading to and Furthermore, recombinant hosts described herein can including C7 building blocks and (4) ensure efficient efflux include any combination of the above enzymes Such that one from the cell. or more of the steps, e.g., one, two, three, four, five, six, 35 In some embodiments requiring the intracellular avail seven, eight, nine, ten, or more of Such steps, can be ability of 2-oxoglutarate, a PEP carboxykinase or PEP performed within a recombinant host. This document pro carboxylase can be overexpressed in the host to generate vides host cells of any of the genera and species listed and anaplerotic carbon flux into the Krebs cycle towards 2-oxo genetically engineered to express one or more (e.g., two, glutarate (Schwartz et al., 2009, Proteomics, 9,5132-5142). three, four, five, six, seven, eight, nine, 10, 11, 12 or more) 40 In some embodiments requiring the intracellular avail recombinant forms of any of the enzymes recited in the ability of 2-oxoglutarate, a pyruvate carboxylase can be document. Thus, for example, the host cells can contain overexpressed in the host to generated anaplerotic carbon exogenous nucleic acids encoding enzymes catalyzing one flux into the Krebs cycle towards 2-oxoglutarate (Schwartz or more of the steps of any of the pathways described herein. et al., 2009, Proteomics, 9, 5132-5142). In addition, this document recognizes that where enzymes 45 In some embodiments requiring the intracellular avail have been described as accepting CoA-activated Substrates, ability of 2-oxoglutarate, a PEP synthase can be overex analogous enzyme activities associated with acp-bound pressed in the host to enhance the flux from pyruvate to PEP. Substrates exist that are not necessarily in the same enzyme thus increasing the carbon flux into the Krebs cycle via PEP class. carboxykinase or PEP carboxylase (Schwartz et al., 2009, Also, this document recognizes that where enzymes have 50 Proteomics, 9, 5132-5142). been described accepting (R)-enantiomers of Substrate, In some embodiments where the host microorganism uses analogous enzyme activities associated with (S)-enantiomer the lysine biosynthesis pathway via meso-2,6-diaminopime Substrates exist that are not necessarily in the same enzyme late, the genes encoding the synthesis of 2-oxoadipate from class. 2-oxoglutarate are gene dosed into the host. This document also recognizes that where an enzyme is 55 In some embodiments where the host microorganism uses shown to accept a particular co-factor, such as NADPH, or the lysine biosynthesis pathway via 2-oxoadipate, the genes a co-substrate. Such as acetyl-CoA, many enzymes are encoding the synthesis of lysine via meso-2,6-diaminopime promiscuous in terms of accepting a number of different late are gene dosed into the host. co-factors or co-substrates in catalyzing a particular enzyme In some embodiments preventing the degradation of activity. Also, this document recognizes that where enzymes 60 NADH formed during the synthesis of C7 building blocks to have high specificity for e.g., a particular co-factor Such as by-products, an endogenous gene encoding an enzyme. Such NADH, an enzyme with similar or identical activity that has as lactate dehydrogenase, that catalyzes the degradation of high specificity for the co-factor NADPH may be in a pyruvate to lactate such as laha is attenuated (Shen et al., different enzyme class. Appl. Environ. Microbiol., 2011, 77(9), 2905-2915). In Some embodiments, the enzymes in the pathways 65 In some embodiments preventing the degradation of outlined herein are the result of enzyme engineering via NADH formed during the synthesis of C7 building blocks to non-direct or rational enzyme design approaches with aims by-products, an endogenous gene. Such as menaquinol US 9,580,731 B2 23 24 fumarate oxidoreductase, encoding an enzyme that catalyzes Solute transporters such as the lysE transporter from Coryne the degradation of phophoenolpyruvate to Succinate Such as bacterium glutamicum (Bellmann et al., 2001, Microbiology, fraBC is attenuated (see, e.g., Shen et al., 2011, Supra). 147, 1765-1774). In some embodiments preventing the degradation of The efflux of pimelic acid can be enhanced or amplified NADH to by-products formed during the synthesis of C7 5 by overexpressing a dicarboxylate transporter Such as the building blocks, an endogenous gene encoding an enzyme Such transporter from Corynebacterium glutamicum (Huhn that catalyzes the degradation of acetyl-CoA to ethanol Such et al., Appl. Microbiol. & Biotech., 89(2), 327-335). as the alcohol dehydrogenase encoded by adhE is attenuated Producing C7 Building Blocks Using a Recombinant Host (Shen et al., 2011, Supra). Typically, one or more C7 building blocks can be pro In some embodiments, an endogenous gene encoding an 10 enzyme that catalyzes the degradation of pyruvate to ethanol duced by providing a host microorganism and culturing the Such as pyruvate decarboxylase is attenuated. provided microorganism with a culture medium containing In some embodiments, an endogenous gene encoding an a suitable carbon Source as described above. In general, the enzyme that catalyzes the generation of isobutanol Such as culture media and/or culture conditions can be such that the a 2-oxoacid decarboxylase can be attenuated. 15 microorganisms grow to an adequate density and produce a In some embodiments, where a pathway requires excess C7 building block efficiently. For large-scale production NAD" co-factor for C7 building block synthesis, a gene processes, any method can be used such as those described encoding a formate dehydrogenase can be attenuated in the elsewhere (Manual of Industrial Microbiology and Biotech host organism (Shen et al., 2011, Supra). nology, 2" Edition, Editors: A. L. Demain and J. E. Davies, In some embodiments, endogenous enzymes facilitating ASM Press; and Principles of Fermentation Technology, P. the conversion of NADPH to NADH are attenuated, such as F. Stanbury and A. Whitaker, Pergamon). Briefly, a large a NADPH-specific glutamate dehydrogenases Such as clas tank (e.g., a 100 gallon, 200 gallon, 500 gallon, or more sified, for example, under EC 1.4.1.4. tank) containing an appropriate culture medium is inocu In some embodiments, transhydrogenases classified, for lated with a particular microorganism. After inoculation, the example, under EC 1.6.1.1, EC 1.6.1.2 or EC 1.6.1.3, may 25 microorganism is incubated to allow biomass to be pro be attenuated. duced. Once a desired biomass is reached, the broth con In some embodiments, an endogenous gene encoding a taining the microorganisms can be transferred to a second glutamate dehydrogenase (EC 1.4.1.3) that utilizes both tank. This second tank can be any size. For example, the NADH and NADPH as co-factors is attenuated. second tank can be larger, Smaller, or the same size as the In some embodiments using hosts that naturally accumu 30 first tank. Typically, the second tank is larger than the first late polyhydroxyalkanoates, an endogenous gene encoding a such that additional culture medium can be added to the polymer synthase enzyme can be attenuated in the host broth from the first tank. In addition, the culture medium strain. within this second tank can be the same as, or different from, In some embodiments, a L-alanine dehydrogenase can be that used in the first tank. overexpressed in the host to regenerate L-alanine from 35 Once transferred, the microorganisms can be incubated to pyruvate as amino donor for co-transaminase reactions. allow for the production of a C7 building block. Once In some embodiments, a NADH-specific L-glutamate produced, any method can be used to isolate C7 building dehydrogenase can be overexpressed in the host to regen blocks. For example, C7 building blocks can be recovered erate L-glutamate from 2-oxoglutarate as amino donor for selectively from the fermentation broth via adsorption pro ()-transaminase reactions. 40 cesses. In the case of pimelic acid and 7-aminoheptanoic In some embodiments, enzymes such as pimeloyl-CoA acid, the resulting eluate can be further concentrated via dehydrogenase classified under, for example, EC 1.3.1.62; evaporation, crystallized via evaporative and/or cooling am acyl-CoA dehydrogenase classified under, for example, crystallization, and the crystals recovered via centrifugation. EC 1.3.8.7 or EC 1.3.8.1; and/or a glutaryl-CoA dehydro In the case of heptamethylenediamine and 1.7-heptanediol. genase classified under, for example, EC 1.3.8.6 that 45 distillation may be employed to achieve the desired product degrade central metabolites and central precursors leading to purity. and including C7 building blocks can be attenuated. The invention is further described in the following In some embodiments, endogenous enzymes activating example, which does not limit the scope of the invention C7 building blocks via Coenzyme A esterification such as described in the claims. CoA-ligases Such as pimeloyl-CoA synthetase classified 50 under, for example, EC 6.2.1.14 can be attenuated. EXAMPLES In some embodiments, the efflux of a C7 building block across the cell membrane to the extracellular media can be Example 1 enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any asso 55 Enzyme Activity of Thioesterases Using ciated transporter activity for a C7 building block. Pimeloyl-CoA as a Substrate and Forming Pimelic The efflux of heptamethylenediamine can be enhanced or Acid amplified by overexpressing broad Substrate range multi drug transporters such as Blt from Bacillus subtilis (Wool A sequence encoding an N-terminal His tag was added to ridge et al., 1997, J. Biol. Chem., 272(14):8864-8866); AcrB 60 the tesB gene from Escherichia coli that encodes a thio and AcrD from Escherichia coli (Elkins & Nikaido, 2002, J. esterase (SEQID NO 1, see FIG. 7), such that an N-terminal Bacteriol., 184(23), 6490-6499) or NorA from Staphylococ HIS tagged thioesterase could be produced. The modified cus aereus (Ng et al., 1994, Antimicrob Agents Chemother, tesB gene was cloned into a pBT15b expression vector under 38(6), 1345-1355) or Bmr from Bacillus subtilis (Neyfakh, control of the T7 promoter. The expression vector was 1992, Antimicrob Agents Chemother, 36(2), 484-485). 65 transformed into a BL21 DE3 E. coli host. The resulting The efflux of 7-aminoheptanoate and heptamethylenedi recombinant E. coli strain was cultivated at 37° C. in a 500 amine can be enhanced or amplified by overexpressing the mL shake flask culture containing 50 mL. Luria Broth (LB) US 9,580,731 B2 25 26 media and antibiotic selection pressure, with shaking at 230 Enzyme activity in the forward direction (i.e., pimelate rpm. The culture was induced overnight at 17°C. using 0.5 semialdehyde to 7-aminoheptanoate) was confirmed for the mM IPTG. transaminases of SEQ ID NO 10, SEQID NO 11 and SEQ The pellet from the induced shake flask culture was ID NO 13. Enzyme activity assays were performed in a harvested via centrifugation. The pellet was resuspended buffer composed of a final concentration of 50 mM HEPES and lysed in Y-PerTM solution (ThermoScientific, Rockford, buffer (pH=7.5), 10 mM pimelate semialdehyde, 10 mM Ill.). The cell debris was separated from the supernatant via L-alanine and 100 uM pyridoxyl 5' phosphate. Each enzyme centrifugation. The thioesterase was purified from the Super activity assay reaction was initiated by adding a cell free natant using Ni-affinity chromatography and the eluate was extract of the co-transaminase gene product or the empty buffer exchanged and concentrated via ultrafiltration. 10 The enzyme activity assay was performed in triplicate in vector control to the assay buffer containing the pimelate a buffer composed of 50 mM phosphate buffer (pH-7.4), 0.1 semialdehyde and incubated at 25°C. for 4 h, with shaking mM Ellman's reagent, and 667 uM of pimeloyl-CoA (as at 250 rpm. The formation of pyruvate was quantified via Substrate). The enzyme activity assay reaction was initiated RP-HPLC. by adding 0.8 uM of the tesB gene product to the assay 15 The gene product of SEQID NO 10, SEQID NO 11 and buffer containing the pimeloyl-CoA and incubating at 37°C. SEQID NO 13 accepted pimelate semialdehyde as substrate for 20 min. The release of Coenzyme A was monitored by as confirmed against the empty vector control. See FIG. 16. absorbance at 412 nm. The absorbance associated with the The reversibility of the co-transaminase activity was con Substrate only control, which contained boiled enzyme, was firmed, demonstrating that the co-transaminases of SEQ ID Subtracted from the active enzyme assay absorbance and NO 8, SEQID NO 10, SEQID NO 11, and SEQID NO 13 compared to the empty vector control. The gene product of accepted pimelate semialdehyde as Substrate and synthe tesB accepted pimeloyl-CoA as Substrate as confirmed via sized 7-aminoheptanoate as a reaction product. relative spectrophotometry (see FIG. 8) and synthesized pimelate as a reaction product. Example 3 25 Example 2 Enzyme Activity of Carboxylate Reductase Using Pimelate as Substrate and Forming Pimelate Enzyme Activity of (O-Transaminase Using Semialdehyde Pimelate Semialdehyde as Substrate and Forming 7-Aminoheptanoate 30 A sequence encoding a HIS-tag was added to the genes from Segniliparus rugosus and Segniliparus rotundus that A sequence encoding an N-terminal His-tag was added to encode the carboxylate reductases of SEQID NOs: 4 and 7. the genes from Chronobacterium violaceum, Pseudomonas respectively (see FIG. 7), such that N-terminal HIS tagged syringae, Rhodobacter sphaeroides, and Vibrio Fluvialis carboxylate reductases could be produced. Each of the encoding the co-transaminases of SEQID NOs: 8, 10, 11 and 35 13, respectively (see FIG. 7) such that N-terminal HIS modified genes was cloned into a pET Duet expression tagged co-transaminases could be produced. Each of the vector along with a Sfp gene encoding a HIS-tagged phos resulting modified genes was cloned into a pET21a expres phopantetheline transferase from Bacillus subtilis, both sion vector under control of the T7 promoter and each under the T7 promoter. Each expression vector was trans expression vector was transformed into a BL21 DE3 E. coli 40 formed into a BL21 DE3 E. coli host and the resulting host. The resulting recombinant E. coli strains were culti recombinant E. coli strains were cultivated at 37°C. in a 250 vated at 37° C. in a 250 mL shake flask culture containing mL shake flask culture containing 50 mL LB media and 50 mL LB media and antibiotic selection pressure, with antibiotic selection pressure, with shaking at 230 rpm. Each shaking at 230 rpm. Each culture was induced overnight at culture was induced overnight at 37° C. using an auto 16° C. using 1 mM IPTG. 45 induction media. The pellet from each induced shake flask culture was The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from and lysed via Sonication, and the cell debris was separated the Supernatant via centrifugation and the cell free extract from the Supernatant via centrifugation. The carboxylate was used immediately in enzyme activity assays. 50 reductases and phosphopantetheline transferases were puri Enzyme activity assays in the reverse direction (i.e., fied from the Supernatant using Ni-affinity chromatography, 7-aminoheptanoate to pimelate semialdehyde) were per diluted 10-fold into 50 mM HEPES buffer (pH=7.5), and formed in a buffer composed of a final concentration of 50 concentrated via ultrafiltration. mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanoate, Enzyme activity assays (i.e., from pimelate to pimelate 10 mM pyruvate and 100 uM pyridoxyl 5' phosphate. Each 55 semialdehyde) were performed in triplicate in a buffer enzyme activity assay reaction was initiated by adding cell composed of a final concentration of 50 mM HEPES buffer free extract of the co-transaminase gene product or the empty (pH=7.5), 2 mM pimelate, 10 mM MgCl, 1 mM ATP and vector control to the assay buffer containing the 7-amino 1 mM NADPH. Each enzyme activity assay reaction was heptanoate and incubated at 25°C. for 4 h, with shaking at initiated by adding purified carboxylate reductase and phos 250 rpm. The formation of L-alanine from pyruvate was 60 phopantetheline transferase gene products or the empty vec quantified via RP-HPLC. tor control to the assay buffer containing the pimelate and Each enzyme only control without 7-aminoheptanoate then incubated at room temperature for 20 min. The con demonstrated low base line conversion of pyruvate to L-ala sumption of NADPH was monitored by absorbance at 340 nine See FIG. 14. The gene product of SEQID NO 8, SEQ nm. Each enzyme only control without pimelate demon ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 accepted 65 strated low base line consumption of NADPH. See FIG. 9. 7-aminoheptanote as Substrate as confirmed against the The gene products of SEQ ID NO 4 and SEQ ID NO 7, empty vector control. See FIG. 15. enhanced by the gene product of Sfp, accepted pimelate as US 9,580,731 B2 27 28 Substrate, as confirmed against the empty vector control (see culture containing 50 mL LB media and antibiotic selection FIG. 10), and synthesized pimelate semialdehyde. pressure, with shaking at 230 rpm. Each culture was induced overnight at 16° C. using 1 mM IPTG. Example 4 The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended Enzyme Activity of Carboxylate Reductase Using and lysed via sonication. The cell debris was separated from 7-Hydroxyheptanoate as Substrate and Forming the Supernatant via centrifugation and the cell free extract 7-Hydroxyheptanal was used immediately in enzyme activity assays. Enzyme activity assays in the reverse direction (i.e., A sequence encoding a His-tag was added to the genes 10 7-aminoheptanol to 7-oxoheptanol) were performed in a from Mycobacterium marinum, Mycobacterium Smegmatis, buffer composed of a final concentration of 50 mM HEPES Segniliparus rugosus, Mycobacterium Smegmatis, Myco buffer (pH=7.5), 10 mM 7-aminoheptanol, 10 mM pyruvate, bacterium massiliense, and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOS: 2-7— and 100 uM pyridoxyl 5' phosphate. Each enzyme activity respectively (see FIG. 7) such that N-terminal HIS tagged 15 assay reaction was initiated by adding cell free extract of the carboxylate reductases could be produced. Each of the ()-transaminase gene product or the empty vector control to modified genes was cloned into a pET Duet expression the assay buffer containing the 7-aminoheptanol and then vector alongside a Sfp gene encoding a His-tagged phos incubated at 25° C. for 4 h, with shaking at 250 rpm. The phopantetheline transferase from Bacillus subtilis, both formation of L-alanine was quantified via RP-HPLC. under control of the T7 promoter. Each enzyme only control without 7-aminoheptanol had Each expression vector was transformed into a BL21 low base line conversion of pyruvate to L-alanine See FIG. DE3 E. coli host and the resulting recombinant E. coli 14. strains were cultivated at 37° C. in a 250 mL shake flask The gene products of SEQ ID NO 8, 10 & 11 accepted culture containing 50 mL LB media and antibiotic selection 7-aminoheptanol as Substrate as confirmed against the empty pressure, with shaking at 230 rpm. Each culture was induced 25 vector control (see FIG. 19) and synthesized 7-oxoheptanol overnight at 37° C. using an auto-induction media. as reaction product. Given the reversibility of the The pellet from each induced shake flask culture was ()-transaminase activity (see Example 2), it can be con harvested via centrifugation. Each pellet was resuspended cluded that the gene products of SEQID 8, 10 & 11 accept and lysed via sonication. The cell debris was separated from 7-oxoheptanol as Substrate and form 7-aminoheptanol. the Supernatant via centrifugation. The carboxylate reduc 30 tases and phosphopantetheline transferase were purified from Example 6 the Supernatant using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH-7.5) and concen Enzyme Activity of (O-Transaminase Using trated via ultrafiltration. Heptamethylenediamine as Substrate and Forming Enzyme activity (i.e., 7-hydroxyheptanoate to 7-hydroxy 35 7-Aminoheptanal heptanal) assays were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH-7.5), 2 mM 7-hydroxyheptanal, 10 mM MgCl, 1 mM A sequence encoding an N-terminal His-tag was added to ATP and 1 mM NADPH. Each enzyme activity assay the Chronobacterium violaceum, Pseudomonas aeruginosa, reaction was initiated by adding purified carboxylate reduc 40 Pseudomonas Syringae, Rhodobacter sphaeroides, Escheri tase and phosphopantetheline transferase or the empty vector chia coli, and Vibrio fluvialis genes encoding the control to the assay buffer containing the 7-hydroxyheptano co-transaminases of SEQ ID NOs: 8-13, respectively (see ate and then incubated at room temperature for 20 min. The FIG. 7) such that N-terminal HIS tagged co-transaminases consumption of NADPH was monitored by absorbance at could be produced. The modified genes were cloned into a 340 nm. Each enzyme only control without 7-hydroxyhep 45 pET21a expression vector under the T7 promoter. Each tanoate demonstrated low base line consumption of expression vector was transformed into a BL21 DE3 E. coli NADPH. See FIG. 9. host. Each resulting recombinant E. coli strain were culti The gene products of SEQ ID NO 2-7, enhanced by the vated at 37° C. in a 250 mL shake flask culture containing gene product of Sfp, accepted 7-hydroxyheptanoate as Sub 50 mL LB media and antibiotic selection pressure, with strate as confirmed against the empty vector control (see 50 shaking at 230 rpm. Each culture was induced overnight at FIG. 11), and synthesized 7-hydroxyheptanal. 16° C. using 1 mM IPTG. The pellet from each induced shake flask culture was Example 5 harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from Enzyme Activity of (O-Transaminase for 55 the Supernatant via centrifugation and the cell free extract 7-Aminoheptanol, Forming 7-Oxoheptanol was used immediately in enzyme activity assays. Enzyme activity assays in the reverse direction (i.e., A nucleotide sequence encoding an N-terminal His-tag heptamethylenediamine to 7-aminoheptanal) were per was added to the Chronobacterium violaceum, Pseudomo formed in a buffer composed of a final concentration of 50 nas Syringae and Rhodobacter sphaeroides genes encoding 60 mM HEPES buffer (pH=7.5), 10 mM heptamethylenedi the co-transaminases of SEQ ID NOs: 8, 10 and 11, respec amine, 10 mM pyruvate, and 100 uM pyridoxyl 5' phos tively (see FIG. 7) such that N-terminal HIS tagged phate. Each enzyme activity assay reaction was initiated by ()-transaminases could be produced. The modified genes adding cell free extract of the co-transaminase gene product were cloned into a pBT21a expression vector under the T7 or the empty vector control to the assay buffer containing the promoter. Each expression vector was transformed into a 65 heptamethylenediamine and then incubated at 25°C. for 4 h. BL21 DE3 E. coli host. Each resulting recombinant E. coli with shaking at 250 rpm. The formation of L-alanine was strain were cultivated at 37° C. in a 250 mL shake flask quantified via RP-HPLC. US 9,580,731 B2 29 30 Each enzyme only control without heptamethylenedi reaction was initiated by adding a cell free extract of the amine had low base line conversion of pyruvate to L-alanine ()-transaminase or the empty vector control to the assay See FIG. 14. buffer containing the N7-acetyl-1,7-diaminoheptane then The gene products of SEQID NO 8-13 accepted heptam incubated at 25° C. for 4 h, with shaking at 250 rpm. The ethylenediamine as Substrate as confirmed against the empty formation of L-alanine was quantified via RP-HPLC. vector control (see FIG. 17) and synthesized 7-aminohep Each enzyme only control without N7-acetyl-1,7-di tanal as reaction product. Given the reversibility of the aminoheptane demonstrated low base line conversion of ()-transaminase activity (see Example 2), it can be con pyruvate to L-alanine See FIG. 14. cluded that the gene products of SEQ ID 8-13 accept The gene product of SEQ ID NO 8-13 accepted 7-aminoheptanal as Substrate and form heptamethylenedi 10 N7-acetyl-1,7-diaminoheptane as substrate as confirmed amine. against the empty vector control (see FIG. 18) and synthe sized N7-acetyl-7-aminoheptanal as reaction product. Example 7 Given the reversibility of the co-transaminase activity (see example 2), the gene products of SEQ ID 8-13 accept Enzyme Activity of Carboxylate Reductase for 15 N7-acetyl-7-aminoheptanal as substrate forming N7-acetyl N7-Acetyl-7-Aminoheptanoate, Forming 1,7-diaminoheptane. N7-Acetyl-7-Aminoheptanal Example 9 The activity of each of the N-terminal His-tagged car boxylate reductases of SEQ ID NOs: 3, 6, and 7 (see Enzyme Activity of Carboxylate Reductase Using Examples 4, and FIG. 7) for converting N7-acetyl-7-amino Pimelate Semialdehyde as Substrate and Forming heptanoate to N7-acetyl-7-aminoheptanal was assayed in Heptanedial triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM N7-acetyl-7-aminohep The N-terminal His-tagged carboxylate reductase of SEQ tanoate, 10 mM MgCl, 1 mM ATP, and 1 mM NADPH. The 25 ID NO 7 (see Example 4 and FIG. 7) was assayed using assays were initiated by adding purified carboxylate reduc pimelate semialdehyde as Substrate. The enzyme activity tase and phosphopantetheline transferase or the empty vector assay was performed in triplicate in a buffer composed of a control to the assay buffer containing the N7-acetyl-7- final concentration of 50 mM HEPES buffer (pH=7.5), 2 aminoheptanoate then incubated at room temperature for 20 mM pimelate semialdehyde, 10 mM MgCl, 1 mM ATP and min. The consumption of NADPH was monitored by absor 30 1 mM NADPH. The enzyme activity assay reaction was bance at 340 nm. Each enzyme only control without initiated by adding purified carboxylate reductase and phos N7-acetyl-7-aminoheptanoate demonstrated low base line phopantetheline transferase or the empty vector control to the consumption of NADPH. See FIG. 9. assay buffer containing the pimelate semialdehyde and then The gene products of SEQ ID NO 3, 6, and 7, enhanced incubated at room temperature for 20 min. The consumption by the gene product of sfp, accepted N7-acetyl-7-aminohep 35 of NADPH was monitored by absorbance at 340 nm. The tanoate as Substrate as confirmed against the empty vector enzyme only control without pimelate semialdehyde dem control (see FIG. 12), and synthesized N7-acetyl-7-amino onstrated low base line consumption of NADPH. See FIG. heptanal. 9. The gene product of SEQID NO 7, enhanced by the gene Example 8 40 product of Sfp, accepted pimelate semialdehyde as Substrate as confirmed against the empty vector control (see FIG. 13) Enzyme Activity of (O-Transaminase Using and synthesized heptanedial. N7-Acetyl-1,7-Diaminoheptane, and Forming N7-Acetyl-7-Aminoheptanal Other Embodiments 45 The activity of the N-terminal His-tagged co-transami It is to be understood that while the invention has been nases of SEQID NOs: 8-13 (see Example 6, and FIG. 7) for described in conjunction with the detailed description converting N7-acetyl-1,7-diaminoheptane to N7-acetyl-7- thereof, the foregoing description is intended to illustrate aminoheptanal was assayed using a buffer composed of a and not limit the scope of the invention, which is defined by final concentration of 50 mM HEPES buffer (pH-7.5), 10 50 the scope of the appended claims. Other aspects, advantages, mM N7-acetyl-1,7-diaminoheptane, 10 mM pyruvate and and modifications are within the scope of the following 100 uM pyridoxyl 5' phosphate. Each enzyme activity assay claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 15

<21 Os SEQ ID NO 1 &211s LENGTH: 286 212s. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs SEQUENCE: 1 Met Ser Glin Ala Lieu Lys Asn Lieu. Lieu. Thir Lieu. Lieu. Asn Lieu. Glu Lys 1. 5 1O 15 US 9,580,731 B2 31 32 - Continued

Ile Glu Glu Gly Lell Phe Arg Gly Glin Ser Glu Asp Lell Gly Luell Arg 25

Glin Wall Phe Gly Gly Glin Wall Wall Gly Glin Ala Lell Tyr Ala Ala 35 4 O 45

Glu Thir Wall Pro Glu Glu Arg Luell Wall His Ser Phe His Ser Phe SO 55 6 O

Lell Arg Pro Gly Asp Ser Pro Ile Ile Asp Wall Glu Thir 65 70

Lell Arg Asp Gly Asn Ser Phe Ser Ala Arg Arg Wall Ala Ala Ile Glin 85 90 95

Asn Gly Pro Ile Phe Met Thir Ala Ser Phe Glin Ala Pro Glu 105 11 O

Ala Gly Phe Glu His Glin Thir Met Pro Ser Ala Pro Ala Pro Asp 115 12 O 125

Gly Luell Pro Ser Glu Thir Glin Ile Ala Glin Ser Lell Ala His Luell Luell 13 O 135 14 O

Pro Pro Wall Luell Asp Phe Ile Asp Arg Pro Luell Glu Wall 145 150 155 160

Arg Pro Wall Glu Phe His Asn Pro Luell Lys Gly His Wall Ala Glu Pro 1.65

His Arg Glin Wall Trp Ile Arg Ala Asn Gly Ser Wall Pro Asp Asp Luell 18O 185 19 O

Arg Wall His Glin Lell Lell Gly Ala Ser Asp Lell Asn Phe Luell 195

Pro Wall Ala Lieu Gln Pro His Gly Ile Gly Phe Lieu Glu Pro Gly Ile 21 O 215

Glin Ile Ala Thir Ile Asp His Ser Met Trp Phe His Arg Pro Phe Asn 225 23 O 235 24 O

Lell Asn Glu Trp Lell Lell Ser Wall Glu Ser Thir Ser Ala Ser Ser 245 250 255

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

Wall Ala Ser Thir Wall Glin Glu Gly Wall Met Arg Asn His Asn 27s 28O 285

<210s, SEQ ID NO 2 &211s LENGTH: 1174 212. TYPE : PRT <213> ORGANISM: Mycobacterium marinum

<4 OOs, SEQUENCE: 2

Met Ser Pro Ile Thir Glu Glu Arg Luell Glu Arg Arg Ile Glin Asp 1. 5 1O 15

Lell Tyr Ala Asn Asp Pro Glin Phe Ala Ala Ala Pro Ala Thir Ala 2O 25 3O

Ile Thir Ala Ala Ile Glu Arg Pro Gly Luell Pro Lell Pro Glin Ile Ile 35 4 O 45

Glu Thir Wall Met Thir Gly Tyr Ala Asp Arg Pro Ala Lell Ala Glin Arg SO 55 6 O

Ser Wall Glu Phe Wall Thir Asp Ala Gly Thir Gly His Thir Thir Luell Arg 65 70

Lell Luell Pro His Phe Glu Thir Ile Ser Tyr Gly Glu Lell Trp Asp Arg 85 90 95

Ile Ser Ala Luell Ala Asp Wall Luell Ser Thir Glu Glin Thir Wall Pro 1OO 105 11 O US 9,580,731 B2 33 34 - Continued

Gly Asp Arg Wall Lell Lell Gly Phe Asn Ser Wall Asp Tyr Ala Thir 115 12 O 125

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

Thir Ser Ala Ala Ile Thir Glin Luell Glin Pro Ile Wall Ala Glu Thir Glin 145 150 155 160

Pro Thir Met Ile Ala Ala Ser Wall Asp Ala Luell Ala Asp Ala Thir Glu 1.65 17O 17s

Lell Ala Luell Ser Gly Glin Thir Ala Thir Arg Wall Lell Wall Phe Asp His 18O 185 19 O

His Arg Glin Wall Asp Ala His Arg Ala Ala Wall Glu Ser Ala Arg Glu 195

Arg Luell Ala Gly Ser Ala Wall Wall Glu Thir Luell Ala Glu Ala Ile Ala 21 O 215

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

Asp Wall Ser Asp Asp Ser Lell Ala Luell Luell Ile Thir Ser Gly Ser 245 250 255

Thir Gly Ala Pro Gly Ala Met Tyr Pro Arg Arg Asn Wall Ala Thir 26 O 265 27 O

Phe Trp Arg Arg Thir Trp Phe Glu Gly Gly Tyr Glu Pro Ser Ile 28O 285

Thir Luell Asn Phe Met Pro Met Ser His Wall Met Gly Arg Glin Ile Luell 29 O 295 3 OO

Tyr Gly Thir Luell Asn Gly Gly Thir Ala Tyr Phe Wall Ala Ser 3. OS 310 315

Asp Luell Ser Thir Lell Phe Glu Asp Luell Ala Luell Wall Arg Pro Thir Glu 3.25 330 335

Lell Thir Phe Wall Pro Arg Wall Trp Asp Met Wall Phe Asp Glu Phe Glin 34 O 345 35. O

Ser Glu Wall Asp Arg Arg Lell Wall Asp Gly Ala Asp Arg Wall Ala Luell 355 360 365

Glu Ala Glin Wall Ala Glu Ile Arg Asn Asp Wall Lell Gly Gly Arg 37 O 375

Tyr Thir Ser Ala Lell Thir Gly Ser Ala Pro Ile Ser Asp Glu Met Lys 385 390 395 4 OO

Ala Trp Wall Glu Glu Lell Lell Asp Met His Luell Wall Glu Gly Tyr Gly 4 OS 415

Ser Thir Glu Ala Gly Met Ile Luell Ile Asp Gly Ala Ile Arg Arg Pro 425 43 O

Ala Wall Luell Asp Lell Wall Asp Wall Pro Asp Lell Gly Tyr Phe 435 44 O 445

Lell Thir Asp Arg Pro His Pro Arg Gly Glu Luell Lell Wall Thir Asp 450 45.5 460

Ser Luell Phe Pro Gly Tyr Glin Arg Ala Glu Wall Thir Ala Asp Wall 465 470 47s 48O

Phe Asp Ala Asp Gly Phe Arg Thir Gly Asp Ile Met Ala Glu Wall 485 490 495

Gly Pro Glu Glin Phe Wall Luell Asp Arg Arg Asn Asn Wall Luell SOO 505

Lell Ser Glin Gly Glu Phe Wall Thir Wall Ser Lell Glu Ala Wall Phe 515 52O 525 US 9,580,731 B2 35 36 - Continued

Gly Asp Ser Pro Lell Wall Arg Glin Ile Ile Tyr Gly Asn Ser Ala 53 O 535 54 O

Arg Ala Luell Lell Ala Wall Ile Wall Pro Thir Glin Glu Ala Luell Asp 5.45 550 555 560

Ala Wall Pro Wall Glu Glu Lell Ala Arg Luell Gly Asp Ser Luell Glin 565 st O sts

Glu Wall Ala Lys Ala Ala Gly Luell Glin Ser Glu Ile Pro Arg Asp 585 59 O

Phe Ile Ile Glu Thir Thir Pro Trp Thir Luell Glu Asn Gly Luell Luell Thir 595 605

Gly Ile Arg Lell Ala Arg Pro Glin Luell Lys Lys His Gly Glu 610 615

Lell Luell Glu Glin Ile Tyr Thir Asp Luell Ala His Gly Glin Ala Asp Glu 625 630 635 64 O

Lell Arg Ser Luell Arg Glin Ser Gly Ala Asp Ala Pro Wall Luell Wall Thir 645 650 655

Wall Arg Ala Ala Ala Ala Luell Luell Gly Gly Ser Ala Ser Asp Wall 660 665 67 O

Glin Pro Asp Ala His Phe Thir Asp Luell Gly Gly Asp Ser Luell Ser Ala 675 685

Lell Ser Phe Thir Asn Lell Lell His Glu Ile Phe Asp Ile Glu Wall Pro 69 O. 695 7 OO

Wall Gly Wall Ile Wall Ser Pro Ala Asn Asp Luell Glin Ala Luell Ala Asp 7 Os

Wall Glu Ala Ala Arg Pro Gly Ser Ser Arg Pro Thr Phe Ala 72 73 O 73

Ser Wall His Gly Ala Ser Asn Gly Glin Wall Thir Glu Wall His Ala Gly 740 74. 7 O

Asp Luell Ser Luell Asp Phe Ile Asp Ala Ala Thir Lell Ala Glu Ala 760 765

Pro Arg Luell Pro Ala Ala Asn Thir Glin Wall Arg Thir Wall Luell Luell Thir 770 775

Gly Ala Thir Gly Phe Lell Gly Arg Luell Ala Lell Glu Trp Luell Glu 79 O 79.

Arg Met Asp Luell Wall Asp Gly Luell Ile Lell Wall Arg Ala 805 810 815

Ser Asp Thir Glu Ala Arg Ala Arg Luell Asp Thir Phe Asp Ser Gly 825 83 O

Asp Pro Glu Luell Lell Ala His Tyr Arg Ala Luell Ala Gly Asp His Luell 835 84 O 845

Glu Wall Luell Ala Gly Asp Lys Gly Glu Ala Asp Lell Gly Luell Asp Arg 850 855 860

Glin Thir Trp Glin Arg Lell Ala Asp Thir Wall Asp Lell Ile Wall Asp Pro 865 87O 88O

Ala Ala Luell Wall Asn His Wall Luell Pro Tyr Ser Glin Lell Phe Gly Pro 885 890 895

Asn Ala Luell Gly Thir Ala Glu Luell Luell Arg Luell Ala Lell Thir Ser 9 OO 905 91 O

Ile Pro Ser Thir Ser Thir Ile Gly Wall Ala Asp Glin Ile 915 92 O 925

Pro Pro Ser Ala Phe Thir Glu Asp Ala Asp Ile Arg Wall Ile Ser Ala 93 O 935 94 O

Thir Arg Ala Wall Asp Asp Ser Ala Asn Gly Ser Asn Ser US 9,580,731 B2 37 38 - Continued

945 950 955 96.O Trp Ala Gly Glu Val Lieu. Lieu. Arg Glu Ala His Asp Lieu. Cys Gly Lieu. 965 97O 97. Pro Val Ala Val Phe Arg Cys Asp Met Ile Leu Ala Asp Thir Thir Trp 98O 985 99 O Ala Gly Glin Lieu. Asn Val Pro Asp Met Phe Thr Arg Met Ile Leu Ser 995 1OOO 1 OOS Lieu Ala Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glu Lieu Ala Ala 1010 1 O15 1 O2O Asp Gly Ala Arg Glin Arg Ala His Tyr Asp Gly Lieu Pro Val Glu Phe 1025 103 O 1035 104 O Ile Ala Glu Ala Ile Ser Thr Lieu. Gly Ala Glin Ser Glin Asp Gly Phe 1045 1OSO 105.5 His Thr Tyr His Val Met Asn Pro Tyr Asp Asp Gly Ile Gly Lieu. Asp 106 O 1065 1OO Glu Phe Val Asp Trp Lieu. Asn. Glu Ser Gly Cys Pro Ile Glin Arg Ile 1075 108O 1085 Ala Asp Tyr Gly Asp Trp Lieu. Glin Arg Phe Glu Thir Ala Lieu. Arg Ala 1090 1095 11OO Lieu Pro Asp Arg Glin Arg His Ser Ser Lieu. Lieu Pro Lieu. Lieu. His Asn 1105 111 O 1115 112O Tyr Arg Gln Pro Glu Arg Pro Val Arg Gly Ser Ile Ala Pro Thr Asp 1125 113 O 1135 Arg Phe Arg Ala Ala Val Glin Glu Ala Lys Ile Gly Pro Asp Lys Asp 114 O 1145 1150 Ile Pro His Val Gly Ala Pro Ile Ile Val Lys Tyr Val Ser Asp Leu 1155 1160 1165 Arg Lieu. Lieu. Gly Lieu. Lieu. 1170

<210s, SEQ ID NO 3 &211s LENGTH: 1173 212. TYPE: PRT <213> ORGANISM: Mycobacterium smegmatis

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

Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Lieu Lleu Pro Arg Phe 85 90 95

Asp Thir Lieu. Thir Tyr Ala Glin Val Trp Ser Arg Val Glin Ala Val Ala 1OO 105 11 O Ala Ala Lieu. Arg His Asn. Phe Ala Glin Pro Ile Tyr Pro Gly Asp Ala 115 12 O 125

Val Ala Thir Ile Gly Phe Ala Ser Pro Asp Tyr Lieu. Thir Lieu. Asp Lieu 13 O 135 14 O US 9,580,731 B2 39 40 - Continued

Wall Ala Lell Gly Lell Wall Ser Wall Pro Lell Glin His Asn Ala 145 150 155 160

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

Lell Thir Wall Ser Ala Glu Luell Asp Luell Ala Wall Glu Ser Wall Arg 18O 185 19 O

Asp Wall Asn Ser Wall Ser Glin Luell Wall Wall Phe Asp His His Pro Glu 195

Wall Asp Asp His Arg Asp Ala Luell Ala Arg Ala Arg Glu Glin Luell Ala 21 O 215

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

Ala Gly Luell Pro Ala Glu Pro Ile Thir Ala Asp His Asp Glin Arg 245 250 255

Lell Ala Met Ile Lell Thir Ser Gly Ser Thir Gly Ala Pro Gly 26 O 265 27 O

Ala Met Tyr Thir Glu Ala Met Wall Ala Arg Luell Trp Thir Met Ser Phe 27s 285

Ile Thir Gly Asp Pro Thir Pro Wall Ile Asn Wall Asn Phe Met Pro Luell 29 O 295 3 OO

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

Gly Thir Ser Phe Wall Pro Glu Ser Asp Met Ser Thir Luell Phe Glu 3.25 330 335

Asp Lieu Ala Lieu Wall Arg Pro Thr Glu Lieu Gly Lieu Wall Pro Arg Wall 34 O 345 35. O

Ala Asp Met Luell Glin His His Luell Ala Thir Wall Asp Arg Luell Wall 355 360 365

Thir Glin Gly Ala Asp Glu Lell Thir Ala Glu Glin Ala Gly Ala Glu 37 O 375

Lell Arg Glu Glin Wall Lell Gly Gly Arg Wall Ile Thir Gly Phe Wall Ser 385 390 395 4 OO

Thir Ala Pro Luell Ala Ala Glu Met Arg Ala Phe Lell Asp Ile Thir Luell 4 OS 415

Gly Ala His Ile Wall Asp Gly Gly Luell Thir Glu Thir Gly Ala Wall 425 43 O

Thir Arg Asp Gly Wall Ile Wall Arg Pro Pro Wall Ile Asp Luell 435 44 O 445

Ile Asp Wall Pro Glu Lell Gly Phe Ser Thir Asp Pro Pro 450 45.5 460

Arg Gly Glu Luell Lell Wall Arg Ser Glin Thir Luell Thir Pro Gly Tyr 465 470 48O

Arg Pro Glu Wall Thir Ala Ser Wall Phe Asp Arg Asp Gly Tyr 485 490 495

His Thir Gly Asp Wall Met Ala Glu Thir Ala Pro Asp His Luell Wall SOO 505

Wall Asp Arg Arg Asn Asn Wall Luell Luell Ala Glin Gly Glu Phe Wall 515 525

Ala Wall Ala Asn Lell Glu Ala Wall Phe Ser Gly Ala Ala Luell Wall Arg 53 O 535 54 O

Glin Ile Phe Wall Gly Asn Ser Glu Arg Ser Phe Lell Luell Ala Wall 5.45 550 555 560

Wall Wall Pro Thir Pro Glu Ala Luell Glu Glin Tyr Asp Pro Ala Ala Luell US 9,580,731 B2 41 42 - Continued

565 st O sts

Ala Ala Luell Ala Asp Ser Luell Glin Arg Thir Ala Arg Asp Ala Glu 58O 585 59 O

Lell Glin Ser Glu Wall Pro Ala Asp Phe Ile Wall Glu Thir Glu Pro 595 605

Phe Ser Ala Ala Asn Gly Lell Luell Ser Gly Wall Gly Lys Luell Luell Arg 610 615

Pro Asn Luell Asp Arg Gly Glin Arg Luell Glu Glin Met Ala 625 630 635 64 O

Asp Ile Ala Ala Thir Glin Ala Asn Glin Luell Arg Glu Lell Arg Arg Ala 645 650 655

Ala Ala Thir Glin Pro Wall Ile Asp Thir Luell Thir Glin Ala Ala Ala Thir 660 665 67 O

Ile Luell Gly Thir Gly Ser Glu Wall Ala Ser Asp Ala His Phe Thir Asp 675 685

Lell Gly Gly Asp Ser Lell Ser Ala Luell Thir Luell Ser Asn Luell Luell Ser 69 O. 695 7 OO

Asp Phe Phe Gly Phe Glu Wall Pro Wall Gly Thir Ile Wall Asn Pro Ala 7 Os

Thir Asn Luell Ala Glin Lell Ala Glin His Ile Glu Ala Glin Arg Thir Ala 72 73 O 73

Gly Asp Arg Arg Pro Ser Phe Thir Thir Wall His Gly Ala Asp Ala Thir 740 74. 7 O

Glu Ile Arg Ala Ser Glu Lell Thir Luell Asp Phe Ile Asp Ala Glu 755 76 O 76.5

Thir Luell Arg Ala Ala Pro Gly Luell Pro Wall Thir Thir Glu Pro Arg 770 775

Thir Wall Luell Luell Ser Gly Ala Asn Gly Trp Luell Gly Arg Phe Luell Thir 79 O 79.

Lell Glin Trp Luell Glu Arg Lell Ala Pro Wall Gly Gly Thir Luell Ile Thir 805 810 815

Ile Wall Arg Gly Arg Asp Asp Ala Ala Ala Arg Ala Arg Luell Thir Glin 825 83 O

Ala Asp Thir Asp Pro Glu Luell Ser Arg Arg Phe Ala Glu Luell Ala 835 84 O 845

Asp Arg His Luell Arg Wall Wall Ala Gly Asp Ile Gly Asp Pro Asn Luell 850 855 860

Gly Luell Thir Pro Glu Ile Trp His Arg Luell Ala Ala Glu Wall Asp Luell 865

Wall Wall His Pro Ala Ala Lell Wall Asn His Wall Lell Pro Arg Glin 885 890 895

Lell Phe Gly Pro Asn Wall Wall Gly Thir Ala Glu Wall Ile Lys Luell Ala 9 OO 905 91 O

Lell Thir Glu Arg Ile Pro Wall Thir Luell Ser Thir Wall Ser Wall 915 92 O 925

Ala Met Gly Ile Pro Asp Phe Glu Glu Asp Gly Asp Ile Arg Thir Wall 93 O 935 94 O

Ser Pro Wall Arg Pro Lell Asp Gly Gly Tyr Ala Asn Gly Gly Asn 945 950 955 96.O

Ser Trp Ala Gly Glu Wall Luell Luell Arg Glu Ala His Asp Luell 965 97.

Gly Luell Pro Wall Ala Thir Phe Arg Ser Asp Met Ile Lell Ala His Pro 98O 985 99 O US 9,580,731 B2 43 44 - Continued

Arg Tyr Arg Gly Glin Val Asn Val Pro Asp Met Phe Thr Arg Lieu. Lieu 995 1OOO 1 OOS Lieu. Ser Leu Lieu. Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr Ile Gly 1010 1 O15 1 O2O Asp Gly Glu Arg Pro Arg Ala His Tyr Pro Gly Lieu. Thr Val Asp Phe 1025 103 O 1035 104 O Val Ala Glu Ala Val Thir Thr Lieu. Gly Ala Glin Glin Arg Glu Gly Tyr 1045 1OSO 105.5 Val Ser Tyr Asp Wal Met Asn Pro His Asp Asp Gly Ile Ser Lieu. Asp 106 O 1065 1OO Val Phe Val Asp Trp Lieu. Ile Arg Ala Gly His Pro Ile Asp Arg Val 1075 108O 1085 Asp Asp Tyr Asp Asp Trp Val Arg Arg Phe Glu Thir Ala Lieu. Thir Ala 1090 1095 11OO Lieu Pro Glu Lys Arg Arg Ala Glin Thr Val Lieu Pro Lieu. Lieu. His Ala 1105 111 O 1115 112O Phe Arg Ala Pro Glin Ala Pro Leu Arg Gly Ala Pro Glu Pro Thr Glu 1125 113 O 1135 Val Phe His Ala Ala Val Arg Thr Ala Lys Val Gly Pro Gly Asp Ile 114 O 1145 1150 Pro His Lieu. Asp Glu Ala Lieu. Ile Asp Llys Tyr Ile Arg Asp Lieu. Arg 1155 1160 1165 Glu Phe Gly Lieu. Ile 1170

<210s, SEQ ID NO 4 &211s LENGTH: 1148 212. TYPE: PRT <213> ORGANISM: Segniliparus rugosus

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

Val Asp Lieu Ala Ala Ala Arg Ser Gly Lieu Val Ser Val Pro Lieu. Glin 115 12 O 125

Ala Gly Ala Ser Lieu Ala Glin Lieu Val Gly Ile Lieu. Glu Glu Thr Glu 13 O 135 14 O

Pro Llys Val Lieu Ala Ala Ser Ala Ser Ser Lieu. Glu Gly Ala Val Ala 145 150 155 160

Cys Ala Lieu Ala Ala Pro Ser Val Glin Arg Lieu Val Val Phe Asp Lieu. 1.65 17O 17s

Arg Gly Pro Asp Ala Ser Glu Ser Ala Ala Asp Glu Arg Arg Gly Ala US 9,580,731 B2 45 46 - Continued

18O 185 19 O

Lell Ala Asp Ala Glu Glu Glin Luell Ala Arg Ala Gly Arg Ala Wall Wall 195

Wall Glu Thir Luell Ala Asp Lell Ala Ala Arg Gly Glu Ala Luell Pro Glu 21 O 215

Ala Pro Luell Phe Glu Pro Ala Glu Gly Glu Asp Pro Lell Ala Luell Luell 225 23 O 235 24 O

Ile Thir Ser Gly Ser Thir Gly Ala Pro Gly Ala Met Tyr Ser 245 250 255

Glin Arg Luell Wall Ser Glin Lell Trp Gly Arg Thir Pro Wall Wall Pro Gly 26 O 265 27 O

Met Pro Asn Ile Ser Lell His Tyr Met Pro Luell Ser His Ser Gly 27s 285

Arg Ala Wall Luell Ala Gly Ala Luell Ser Ala Gly Gly Thir Ala His Phe 29 O 295 3 OO

Thir Ala Asn Ser Asp Lell Ser Thir Luell Phe Glu Asp Ile Ala Luell Ala 3. OS 310 315

Arg Pro Thir Phe Lell Ala Lell Wall Pro Arg Wall Glu Met Luell Phe 3.25 330 335

Glin Ser Glin Arg Gly Glin Asp Wall Ala Glu Lell Arg Glu Arg Wall 34 O 345 35. O

Lell Gly Arg Lell Lell Wall Ala Wall Gly Ser Ala Pro Luell Ser 355 360 365

Pro Met Arg Ala Phe Met Glu Glu Wall Luell Gly Phe Pro Luell Luell 375 380

Asp Gly Ser Thir Glu Ala Luell Gly Wall Met Arg Asn Gly Ile 385 390 395 4 OO

Ile Pro Pro Wall Ile Asp Lys Luell Wall Asp Wall Pro Glu 4 OS 41O 415

Lell Arg Thir Thir Asp Pro Pro Arg Gly Glu Luell 425 43 O

Ile Arg Ser Thir Ser Lell Ile Ser Gly Arg Pro Glu Ile 435 44 O 445

Thir Ala Glu Wall Phe Asp Ala Glin Gly Lys Thir Gly Asp Wall 450 45.5 460

Met Ala Glu Ile Ala Pro Asp His Luell Wall Tyr Wall Asp Arg Ser Lys 465 470 47s

Asn Wall Luell Lell Ser Glin Gly Glu Phe Wall Ala Wall Ala Lys Luell 485 490 495

Glu Ala Ala Tyr Gly Thir Ser Pro Tyr Wall Glin Ile Phe Wall SOO 505

Gly Asn Ser Glu Arg Ser Phe Luell Luell Ala Wall Wall Wall Pro Asn Ala 515 525

Glu Wall Luell Gly Ala Arg Asp Glin Glu Glu Ala Lys Pro Luell Ile Ala 53 O 535 54 O

Ala Ser Luell Glin Ile Ala Glu Ala Gly Lell Glin Ser Glu 5.45 550 555 560

Wall Pro Arg Asp Phe Lell Ile Glu Thir Glu Pro Phe Thir Thir Glin Asn 565 st O sts

Gly Luell Luell Ser Glu Wall Gly Luell Luell Arg Pro Luell Ala 585 59 O

Arg Gly Glu Ala Lell Glu Ala Arg Asp Glu Ile Ala His Gly 595 6OO 605 US 9,580,731 B2 47 48 - Continued

Glin Ala Asp Glu Lieu. Arg Ala Lieu. Arg Asp Gly Ala Gly Glin Arg Pro 610 615 62O Val Val Glu Thr Val Val Arg Ala Ala Val Ala Ile Ser Gly Ser Glu 625 630 635 64 O Gly Ala Glu Val Gly Pro Glu Ala Asn. Phe Ala Asp Lieu. Gly Gly Asp 645 650 655 Ser Lieu. Ser Ala Lieu. Ser Lieu Ala Asn Lieu. Lieu. His Asp Val Phe Glu 660 665 67 O Val Glu Val Pro Val Arg Ile Ile Ile Gly Pro Thr Ala Ser Leu Ala 675 68O 685 Gly Ile Ala Lys His Ile Glu Ala Glu Arg Ala Gly Ala Ser Ala Pro 69 O. 695 7 OO Thir Ala Ala Ser Val His Gly Ala Gly Ala Thr Arg Ile Arg Ala Ser 7 Os 71O 71s 72O Glu Lieu. Thir Lieu. Glu, Llys Phe Lieu Pro Glu Asp Lieu. Lieu Ala Ala Ala 72 73 O 73 Lys Gly Lieu Pro Ala Ala Asp Glin Val Arg Thr Val Lieu. Lieu. Thr Gly 740 74. 7 O Ala Asn Gly Trp Lieu. Gly Arg Phe Lieu Ala Lieu. Glu Gln Lieu. Glu Arg 7ss 760 765 Lieu Ala Arg Ser Gly Glin Asp Gly Gly Lys Lieu. Ile Cys Lieu Val Arg 770 775 78O Gly Lys Asp Ala Ala Ala Ala Arg Arg Arg Ile Glu Glu Thir Lieu. Gly 78s 79 O 79. 8OO Thir Asp Pro Ala Lieu Ala Ala Arg Phe Ala Glu Lieu Ala Glu Gly Arg 805 810 815 Lieu. Glu Val Val Pro Gly Asp Val Gly Glu Pro Llys Phe Gly Lieu. Asp 82O 825 83 O Asp Ala Ala Trp Asp Arg Lieu Ala Glu Glu Val Asp Val Ile Val His 835 84 O 845 Pro Ala Ala Leu Val Asn His Val Lieu Pro Tyr His Gln Leu Phe Gly 850 855 860 Pro Asn Val Val Gly Thr Ala Glu Ile Ile Arg Lieu Ala Ile Thr Ala 865 87O 87s 88O Lys Arg Llys Pro Val Thr Tyr Lieu. Ser Thr Val Ala Val Ala Ala Gly 885 890 895 Val Glu Pro Ser Ser Phe Glu Glu Asp Gly Asp Ile Arg Ala Val Val 9 OO 905 91 O Pro Glu Arg Pro Lieu. Gly Asp Gly Tyr Ala Asn Gly Tyr Gly Asn. Ser 915 92 O 925 Llys Trp Ala Gly Glu Val Lieu. Lieu. Arg Glu Ala His Glu Lieu Val Gly 93 O 935 94 O Lieu Pro Val Ala Val Phe Arg Ser Asp Met Ile Lieu Ala His Thr Arg 945 950 955 96.O

Tyr Thr Gly Glin Lieu. Asn Val Pro Asp Glin Phe Thr Arg Lieu Val Lieu. 965 97O 97.

Ser Lieu. Leu Ala Thr Gly Ile Ala Pro Llys Ser Phe Tyr Glin Glin Gly 98O 985 99 O Ala Ala Gly Glu Arg Glin Arg Ala His Tyr Asp Gly Ile Pro Val Asp 995 1OOO 1 OOS

Phe Thr Ala Glu Ala Ile Thr Thr Lieu. Gly Ala Glu Pro Ser Trp Phe 1010 1 O15 1 O2O US 9,580,731 B2 49 - Continued Asp Gly Gly Ala Gly Phe Arg Ser Phe Asp Val Phe Asn Pro His His 1025 103 O 1035 104 O Asp Gly Val Gly Lieu. Asp Glu Phe Val Asp Trp Lieu. Ile Glu Ala Gly 1045 1OSO 105.5 His Pro Ile Ser Arg Ile Asp Asp His Lys Glu Trp Phe Ala Arg Phe 106 O 1065 1OO Glu Thir Ala Val Arg Gly Lieu Pro Glu Ala Glin Arg Gln His Ser Lieu. 1075 108O 1085 Lieu Pro Leu Lieu. Arg Ala Tyr Ser Phe Pro His Pro Pro Val Asp Gly 1090 1095 11OO Ser Val Tyr Pro Thr Gly Llys Phe Glin Gly Ala Val Lys Ala Ala Glin 1105 111 O 1115 112O Val Gly Ser Asp His Asp Val Pro His Lieu. Gly Lys Ala Lieu. Ile Val 1125 113 O 1135 Llys Tyr Ala Asp Asp Lieu Lys Ala Lieu. Gly Lieu. Lieu. 114 O 1145

<210s, SEQ ID NO 5 &211s LENGTH: 11.68 212. TYPE: PRT <213> ORGANISM: Mycobacterium smegmatis

<4 OOs, SEQUENCE: 5 Met Thir Ile Glu Thir Arg Glu Asp Arg Phe Asn Arg Arg Ile Asp His 1. 5 1O 15 Lieu. Phe Glu Thir Asp Pro Glin Phe Ala Ala Ala Arg Pro Asp Glu Ala 2O 25 30 Ile Ser Ala Ala Ala Ala Asp Pro Glu Lieu. Arg Lieu Pro Ala Ala Val 35 4 O 45 Lys Glin Ile Lieu Ala Gly Tyr Ala Asp Arg Pro Ala Lieu. Gly Lys Arg SO 55 6 O Ala Val Glu Phe Val Thr Asp Glu Glu Gly Arg Thir Thr Ala Lys Lieu 65 70 7s 8O Lieu Pro Arg Phe Asp Thir Ile Thr Tyr Arg Glin Lieu Ala Gly Arg Ile 85 90 95 Glin Ala Val Thr Asn Ala Trp His Asn His Pro Val Asn Ala Gly Asp 1OO 105 11 O Arg Val Ala Ile Leu Gly Phe Thr Ser Val Asp Tyr Thr Thr Ile Asp 115 12 O 125 Ile Ala Lieu. Lieu. Glu Lieu. Gly Ala Val Ser Val Pro Lieu. Glin Thir Ser 13 O 135 14 O Ala Pro Val Ala Glin Lieu. Glin Pro Ile Val Ala Glu Thr Glu Pro Llys 145 150 155 160 Val Ile Ala Ser Ser Val Asp Phe Lieu Ala Asp Ala Wall Ala Lieu Val 1.65 17O 17s Glu Ser Gly Pro Ala Pro Ser Arg Lieu Val Val Phe Asp Tyr Ser His 18O 185 19 O

Glu Val Asp Asp Glin Arg Glu Ala Phe Glu Ala Ala Lys Gly Llys Lieu. 195 2OO 2O5

Ala Gly Thr Gly Val Val Val Glu Thir Ile Thr Asp Ala Lieu. Asp Arg 21 O 215 22O

Gly Arg Ser Lieu Ala Asp Ala Pro Lieu. Tyr Val Pro Asp Glu Ala Asp 225 23 O 235 24 O

Pro Leu. Thir Lieu. Lieu. Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Llys 245 250 255 US 9,580,731 B2 51 52 - Continued

Gly Ala Met Tyr Pro Glu Ser Thir Ala Thir Met Trp Glin Ala Gly 26 O 265 27 O

Ser Ala Arg Trp Asp Glu Thir Luell Gly Wall Met Pro Ser Ile Thir 285

Lell Asn Phe Met Pro Met Ser His Wall Met Gly Arg Gly Ile Luell 29 O 295 3 OO

Ser Thir Luell Ala Ser Gly Gly Thir Ala Phe Ala Ala Arg Ser Asp 3. OS 310 315

Lell Ser Thir Phe Lell Glu Asp Luell Ala Luell Wall Arg Pro Thir Glin Luell 3.25 330 335

Asn Phe Wall Pro Arg Ile Trp Asp Met Luell Phe Glin Glu Tyr Glin Ser 34 O 345 35. O

Arg Luell Asp Asn Arg Arg Ala Glu Gly Ser Glu Asp Arg Ala Glu Ala 355 360 365

Ala Wall Luell Glu Glu Wall Arg Thir Glin Luell Luell Gly Gly Arg Phe Wall 37 O 375

Ser Ala Luell Thir Gly Ser Ala Pro Ile Ser Ala Glu Met Ser Trp 385 390 395 4 OO

Wall Glu Asp Luell Lell Asp Met His Luell Luell Glu Gly Tyr Gly Ser Thir 4 OS 415

Glu Ala Gly Ala Wall Phe Ile Asp Gly Glin Ile Glin Arg Pro Pro Wall 42O 425 43 O

Ile Asp Tyr Lell Wall Asp Wall Pro Asp Luell Gly Tyr Phe Ala Thir 435 44 O 445

Asp Arg Pro Pro Arg Gly Glu Luell Luell Wall Lys Ser Glu Glin Met 450 45.5 460

Phe Pro Gly Tyr Lys Arg Pro Glu Ile Thir Ala Glu Met Phe Asp 465 470

Glu Asp Gly Tyr Tyr Arg Thir Gly Asp Ile Wall Ala Glu Luell Gly Pro 485 490 495

Asp His Luell Glu Tyr Lell Asp Arg Arg Asn ASn Wall Lell Lys Luell Ser SOO 505

Glin Gly Glu Phe Wall Thir Wall Ser Luell Glu Ala Wall Phe Gly Asp 515 525

Ser Pro Luell Wall Arg Glin Ile Wall Gly Asn Ser Ala Arg Ser 53 O 535 54 O

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

Asp Gly Asp Glu Lell Ser Arg Ile Ser Asp Ser Lell Glin Asp Ala 565 st O sts

Ala Arg Ala Ala Gly Lell Glin Ser Tyr Glu Ile Pro Arg Asp Phe Luell 585 59 O

Wall Glu Thir Thir Pro Phe Thir Luell Glu Asn Gly Lell Lell Thir Gly Ile 595 605

Arg Lys Luell Ala Arg Pro Lys Luell Ala His Tyr Gly Glu Arg Luell 610 615

Glu Glin Luell Tyr Thir Asp Lell Ala Glu Gly Glin Ala Asn Glu Luell Arg 625 630 635 64 O

Glu Luell Arg Arg Asn Gly Ala Asp Arg Pro Wall Wall Glu Thir Wall Ser 645 650 655

Arg Ala Ala Wall Ala Lell Lell Gly Ala Ser Wall Thir Asp Luell Arg Ser 660 665 67 O US 9,580,731 B2 53 - Continued Asp Ala His Phe Thr Asp Lieu. Gly Gly Asp Ser Lieu. Ser Ala Lieu. Ser 675 68O 685 Phe Ser Asn Lieu. Lieu. His Glu Ile Phe Asp Val Asp Val Pro Val Gly 69 O. 695 7 OO Val Ile Val Ser Pro Ala Thr Asp Lieu Ala Gly Val Ala Ala Tyr Ile 7 Os 71O 71s 72O Glu Gly Glu Lieu. Arg Gly Ser Lys Arg Pro Thr Tyr Ala Ser Val His 72 73 O 73 Gly Arg Asp Ala Thr Glu Val Arg Ala Arg Asp Lieu Ala Lieu. Gly Lys 740 74. 7 O Phe Ile Asp Ala Lys Thr Lieu. Ser Ala Ala Pro Gly Lieu Pro Arg Ser 7ss 760 765 Gly Thr Glu Ile Arg Thr Val Lieu. Lieu. Thr Gly Ala Thr Gly Phe Leu 770 775 78O Gly Arg Tyr Lieu Ala Lieu. Glu Trp Lieu. Glu Arg Met Asp Lieu Val Asp 78s 79 O 79. 8OO Gly Llys Val Ile Cys Lieu Val Arg Ala Arg Ser Asp Asp Glu Ala Arg 805 810 815 Ala Arg Lieu. Asp Ala Thr Phe Asp Thr Gly Asp Ala Thr Lieu. Lieu. Glu 82O 825 83 O His Tyr Arg Ala Lieu Ala Ala Asp His Lieu. Glu Val Ile Ala Gly Asp 835 84 O 845 Lys Gly Glu Ala Asp Lieu. Gly Lieu. Asp His Asp Thir Trp Glin Arg Lieu. 850 855 860 Ala Asp Thr Val Asp Lieu. Ile Val Asp Pro Ala Ala Lieu Val Asn His 865 87O 87s 88O Val Lieu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala Lieu. Gly Thr Ala 885 890 895 Glu Lieu. Ile Arg Ile Ala Lieu. Thir Thr Thr Ile Llys Pro Tyr Val Tyr 9 OO 905 91 O Val Ser Thir Ile Gly Val Gly Glin Gly Ile Ser Pro Glu Ala Phe Val 915 92 O 925 Glu Asp Ala Asp Ile Arg Glu Ile Ser Ala Thr Arg Arg Val Asp Asp 93 O 935 94 O Ser Tyr Ala Asn Gly Tyr Gly Asn. Ser Lys Trp Ala Gly Glu Val Lieu. 945 950 955 96.O Lieu. Arg Glu Ala His Asp Trp Cys Gly Lieu Pro Val Ser Val Phe Arg 965 97O 97. Cys Asp Met Ile Lieu Ala Asp Thir Thr Tyr Ser Gly Glin Lieu. Asn Lieu. 98O 985 99 O Pro Asp Met Phe Thr Arg Lieu Met Leu Ser Leu Val Ala Thr Gly Ile 995 1OOO 1 OOS Ala Pro Gly Ser Phe Tyr Glu Lieu. Asp Ala Asp Gly Asn Arg Glin Arg 1010 1 O15 1 O2O

Ala His Tyr Asp Gly Lieu Pro Val Glu Phe Ile Ala Glu Ala Ile Ser 1025 103 O 1035 104 O

Thir Ile Gly Ser Glin Val Thr Asp Gly Phe Glu Thr Phe His Val Met 1045 1OSO 105.5

Asn Pro Tyr Asp Asp Gly Ile Gly Lieu. Asp Glu Tyr Val Asp Trp Lieu 106 O 1065 1OO Ile Glu Ala Gly Tyr Pro Val His Arg Val Asp Asp Tyr Ala Thir Trp 1075 108O 1085

Lieu. Ser Arg Phe Glu Thir Ala Lieu. Arg Ala Lieu Pro Glu Arg Glin Arg US 9,580,731 B2 55 - Continued

1090 1095 11OO Glin Ala Ser Leu Lleu Pro Leu Lieu. His Asn Tyr Glin Gln Pro Ser Pro 1105 111 O 1115 112O Pro Val Cys Gly Ala Met Ala Pro Thr Asp Arg Phe Arg Ala Ala Val 1125 113 O 1135 Glin Asp Ala Lys Ile Gly Pro Asp Lys Asp Ile Pro His Val Thir Ala 114 O 1145 1150 Asp Val Ile Val Llys Tyr Ile Ser Asn Lieu Gln Met Lieu. Gly Lieu. Lieu 1155 1160 1165

<210s, SEQ ID NO 6 &211s LENGTH: 1185 212. TYPE: PRT <213> ORGANISM: Mycobacterium massiliense

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

Pro Ser Ala Gly Asp Asp Pro Lieu Ala Lieu. Lieu. Ile Tyr Thir Ser Gly 245 250 255 Ser Thr Gly Ala Pro Llys Gly Ala Met His Ser Glu Asn. Ile Val Arg 26 O 265 27 O

Arg Trp Trp Ile Arg Glu Asp Wal Met Ala Gly Thr Glu Asn Lieu Pro 27s 28O 285 Met Ile Gly Lieu. Asn Phe Met Pro Met Ser His Ile Met Gly Arg Gly 29 O 295 3 OO US 9,580,731 B2 57 - Continued Thr Lieu. Thir Ser Thr Lieu Ser Thr Gly Gly Thr Gly Tyr Phe Ala Ala 3. OS 310 315 32O Ser Ser Asp Met Ser Thr Lieu Phe Glu Asp Met Glu Lieu. Ile Arg Pro 3.25 330 335 Thir Ala Lieu Ala Lieu Val Pro Arg Val Cys Asp Met Val Phe Glin Arg 34 O 345 35. O Phe Glin Thr Glu Val Asp Arg Arg Lieu Ala Ser Gly Asp Thir Ala Ser 355 360 365 Ala Glu Ala Val Ala Ala Glu Val Lys Ala Asp Ile Arg Asp Asn Lieu 37 O 375 38O Phe Gly Gly Arg Val Ser Ala Val Met Val Gly Ser Ala Pro Leu Ser 385 390 395 4 OO Glu Glu Lieu. Gly Glu Phe Ile Glu Ser Cys Phe Glu Lieu. Asn Lieu. Thir 4 OS 41O 415 Asp Gly Tyr Gly Ser Thr Glu Ala Gly Met Val Phe Arg Asp Gly Ile 42O 425 43 O Val Glin Arg Pro Pro Val Ile Asp Tyr Lys Lieu Val Asp Val Pro Glu 435 44 O 445 Lieu. Gly Tyr Phe Ser Thr Asp Llys Pro His Pro Arg Gly Glu Lieu. Lieu. 450 45.5 460 Lieu Lys Thr Asp Gly Met Phe Leu Gly Tyr Tyr Lys Arg Pro Glu Val 465 470 47s 48O Thr Ala Ser Val Phe Asp Ala Asp Gly Phe Tyr Met Thr Gly Asp Ile 485 490 495 Val Ala Glu Lieu Ala His Asp ASn Ile Glu Ile Ile Asp Arg Arg ASn SOO 505 51O Asn Val Lieu Lys Lieu. Ser Glin Gly Glu Phe Val Ala Wall Ala Thir Lieu 515 52O 525 Glu Ala Glu Tyr Ala Asn Ser Pro Val Val His Glin Ile Tyr Val Tyr 53 O 535 54 O Gly Ser Ser Glu Arg Ser Tyr Lieu. Leu Ala Val Val Val Pro Thr Pro 5.45 550 555 560 Glu Ala Val Ala Ala Ala Lys Gly Asp Ala Ala Ala Lieu Lys Thir Thr 565 st O sts Ile Ala Asp Ser Lieu. Glin Asp Ile Ala Lys Glu Ile Glin Lieu. Glin Ser 58O 585 59 O Tyr Glu Val Pro Arg Asp Phe Ile Ile Glu Pro Gln Pro Phe Thr Glin 595 6OO 605 Gly Asn Gly Lieu. Lieu. Thr Gly Ile Ala Lys Lieu Ala Arg Pro Asn Lieu. 610 615 62O Lys Ala His Tyr Gly Pro Arg Lieu. Glu Gln Met Tyr Ala Glu Ile Ala 625 630 635 64 O Glu Glin Glin Ala Ala Glu Lieu. Arg Ala Lieu. His Gly Val Asp Pro Asp 645 650 655

Llys Pro Ala Lieu. Glu Thr Val Lieu Lys Ala Ala Glin Ala Lieu. Lieu. Gly 660 665 67 O

Val Ser Ser Ala Glu Lieu Ala Ala Asp Ala His Phe Thr Asp Lieu. Gly 675 68O 685

Gly Asp Ser Lieu. Ser Ala Lieu. Ser Phe Ser Asp Lieu. Lieu. Arg Asp Ile 69 O. 695 7 OO

Phe Ala Val Glu Val Pro Val Gly Val Ile Val Ser Ala Ala Asn Asp 7 Os 71O 71s 72O Lieu. Gly Gly Val Ala Lys Phe Val Asp Glu Glin Arg His Ser Gly Gly US 9,580,731 B2 59 - Continued

72 73 O 73 Thr Arg Pro Thr Ala Glu Thr Val His Gly Ala Gly His Thr Glu Ile 740 74. 7 O Arg Ala Ala Asp Lieu. Thir Lieu. Asp Llys Phe Ile Asp Glu Ala Thir Lieu 7ss 760 765 His Ala Ala Pro Ser Leu Pro Lys Ala Ala Gly Ile Pro His Thr Val 770 775 78O Lieu. Lieu. Thr Gly Ser Asn Gly Tyr Lieu. Gly His Tyr Lieu Ala Lieu. Glu 78s 79 O 79. 8OO Trp Lieu. Glu Arg Lieu. Asp Llys Thr Asp Gly Lys Lieu. Ile Val Ile Val 805 810 815 Arg Gly Lys Asn Ala Glu Ala Ala Tyr Gly Arg Lieu. Glu Glu Ala Phe 82O 825 83 O Asp Thr Gly Asp Thr Glu Lieu. Lieu Ala His Phe Arg Ser Lieu Ala Asp 835 84 O 845 Llys His Lieu. Glu Val Lieu Ala Gly Asp Ile Gly Asp Pro Asn Lieu. Gly 850 855 860 Lieu. Asp Ala Asp Thir Trp Glin Arg Lieu Ala Asp Thr Val Asp Val Ile 865 87O 87s 88O Val His Pro Ala Ala Lieu Val Asn His Val Lieu Pro Tyr Asn Glin Lieu 885 890 895 Phe Gly Pro Asn Val Val Gly Thr Ala Glu Ile Ile Llys Lieu Ala Ile 9 OO 905 91 O Thir Thr Lys Ile Llys Pro Val Thr Tyr Lieu Ser Thr Val Ala Val Ala 915 920 925 Ala Tyr Val Asp Pro Thir Thr Phe Asp Glu Glu Ser Asp Ile Arg Lieu 93 O 935 94 O Ile Ser Ala Val Arg Pro Ile Asp Asp Gly Tyr Ala Asn Gly Tyr Gly 945 950 955 96.O Asn Ala Lys Trp Ala Gly Glu Val Lieu. Lieu. Arg Glu Ala His Asp Lieu 965 97O 97. Cys Gly Lieu Pro Val Ala Val Phe Arg Ser Asp Met Ile Lieu Ala His 98O 985 99 O Ser Arg Tyr Thr Gly Glin Lieu. Asn Val Pro Asp Glin Phe Thr Arg Lieu. 995 1OOO 1 OOS Ile Leu Ser Lieu. Ile Ala Thr Gly Ile Ala Pro Gly Ser Phe Tyr Glin 1010 1 O15 1 O2O Ala Glin Thir Thr Gly Glu Arg Pro Lieu Ala His Tyr Asp Gly Lieu Pro 1025 103 O 1035 104 O Gly Asp Phe Thr Ala Glu Ala Ile Thr Thr Lieu. Gly Thr Glin Val Pro 1045 1OSO 105.5 Glu Gly Ser Glu Gly Phe Val Thr Tyr Asp Cys Val Asn Pro His Ala 106 O 1065 1OO

Asp Gly Ile Ser Lieu. Asp Asn. Phe Val Asp Trp Lieu. Ile Glu Ala Gly 1075 108O 1085

Tyr Pro Ile Ala Arg Ile Asp Asn Tyr Thr Glu Trp Phe Thr Arg Phe 1090 1095 11OO Asp Thir Ala Ile Arg Gly Lieu. Ser Glu Lys Glin Lys Gln His Ser Lieu 1105 111 O 1115 112O

Lieu Pro Lieu. Lieu. His Ala Phe Glu Gln Pro Ser Ala Ala Glu Asn His 1125 113 O 1135

Gly Val Val Pro Ala Lys Arg Phe Gln His Ala Val Glin Ala Ala Gly 114 O 1145 1150 US 9,580,731 B2 61 - Continued

Ile Gly Pro Val Gly Glin Asp Gly Thr Thr Asp Ile Pro His Leu Ser 1155 1160 1165 Arg Arg Lieu. Ile Val Llys Tyr Ala Lys Asp Lieu. Glu Gln Lieu. Gly Lieu 1170 1175 118O

Lell 1185

<210s, SEQ ID NO 7 &211s LENGTH: 1186 212. TYPE: PRT <213> ORGANISM: Segniliparus rotundus

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

Thir Lieu. Thir Ala Ile Thir Ala Glu. Thir Ala Pro Thir Lieu. Phe Ala Ala 1.65 17O 17s Ser Ile Glu. His Lieu Pro Thr Ala Val Asp Ala Val Lieu Ala Thr Pro 18O 185 19 O Ser Val Arg Arg Lieu. Lieu Val Phe Asp Tyr Arg Ala Gly Ser Asp Glu 195 2OO 2O5 Asp Arg Glu Ala Val Glu Ala Ala Lys Arg Lys Ile Ala Asp Ala Gly 21 O 215 22O Ser Ser Val Lieu Val Asp Val Lieu. Asp Glu Val Ile Ala Arg Gly Lys 225 23 O 235 24 O Ser Ala Pro Lys Ala Pro Lieu Pro Pro Ala Thr Asp Ala Gly Asp Asp 245 250 255

Ser Leu Ser Leu Lieu. Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Llys 26 O 265 27 O Gly Ala Met Tyr Pro Glu Arg Asn Val Ala His Phe Trp Gly Gly Val 27s 28O 285

Trp Ala Ala Ala Phe Asp Glu Asp Ala Ala Pro Pro Val Pro Ala Ile 29 O 295 3 OO

Asn. Ile Thir Phe Lieu Pro Lieu. Ser His Val Ala Ser Arg Lieu. Ser Lieu 3. OS 310 315 32O

Met Pro Thr Lieu Ala Arg Gly Gly Lieu Met His Phe Val Ala Lys Ser US 9,580,731 B2 63 64 - Continued

3.25 330 335

Asp Luell Ser Thir Lell Phe Glu Asp Luell Luell Ala Arg Pro Thir Asn 34 O 345 35. O

Lell Phe Luell Wall Pro Arg Wall Wall Glu Met Luell Tyr Glin His Tyr Glin 355 360 365

Ser Glu Luell Asp Arg Arg Gly Wall Glin Asp Gly Thir Arg Glu Ala Glu 37 O 375

Ala Wall Asp Asp Lell Arg Thir Gly Luell Luell Gly Gly Arg Ile Luell 385 390 395 4 OO

Thir Ala Gly Phe Gly Ser Ala Pro Luell Ser Ala Glu Lell Ala Gly Phe 4 OS 415

Ile Glu Ser Luell Lell Glin Ile His Luell Wall Asp Gly Gly Ser Thir 425 43 O

Glu Ala Gly Pro Wall Trp Arg Asp Gly Luell Wall Lys Pro Pro Wall 435 44 O 445

Thir Asp Lell Ile Asp Wall Pro Glu Luell Gly Phe Ser Thir 450 45.5 460

Asp Ser Pro His Pro Arg Gly Glu Luell Ala Ile Thir Glin Thir Ile 465 470

Lell Pro Gly Tyr Arg Pro Glu Thir Thir Ala Glu Wall Phe Asp 485 490 495

Glu Asp Gly Phe Lell Thir Gly Asp Wall Wall Ala Glin Ile Gly Pro SOO 505 51O

Glu Glin Phe Ala Wall Asp Arg Arg ASn Wall Lell Luell Ser 515 525

Glin Gly Glu Phe Wall Thir Lell Ala Luell Glu Ala Ala Ser Ser 53 O 535 54 O

Ser Pro Luell Wall Arg Glin Lell Phe Wall Gly Ser Ser Glu Arg Ser 5.45 550 555 560

Luell Luell Ala Wall Ile Wall Pro Thir Pro Asp Ala Lell Lys Phe 565 st O sts

Gly Wall Gly Glu Ala Ala Ala Ala Luell Gly Glu Ser Luell Glin 585 59 O

Ile Ala Arg Asp Glu Gly Lell Glin Ser Glu Wall Pro Arg Asp Phe 595 605

Ile Ile Glu Thir Asp Pro Phe Thir Wall Glu ASn Gly Lell Luell Ser Asp 610 615

Ala Arg Ser Lell Arg Pro Luell Glu His Gly Glu Arg 625 630 635 64 O

Lell Glu Ala Met Tyr Glu Luell Ala Asp Gly Glin Ala Asn Glu Luell 645 650 655

Arg Asp Ile Arg Arg Gly Wall Glin Glin Arg Pro Thir Lell Glu Thir Wall 660 665 67 O

Arg Arg Ala Ala Ala Ala Met Luell Gly Ala Ser Ala Ala Glu Ile 675 685

Pro Asp Ala His Phe Thir Asp Luell Gly Gly Asp Ser Lell Ser Ala Luell 69 O. 695 7 OO

Thir Phe Ser Asn Phe Lell His Asp Luell Phe Glu Wall Asp Wall Pro Wall 7 Os 72O

Gly Wall Ile Wall Ser Ala Ala Asn Thir Luell Gly Ser Wall Ala Glu His 72 73 O 73

Ile Asp Ala Glin Lell Ala Gly Gly Arg Ala Arg Pro Thir Phe Ala Thir 740 74. 7 O US 9,580,731 B2 65 - Continued

Val His Gly Lys Gly Ser Thir Thir Ile Lys Ala Ser Asp Lieu. Thir Lieu 7ss 760 765 Asp Llys Phe Ile Asp Glu Glin Thr Lieu. Glu Ala Ala Lys His Lieu Pro 770 775 78O Llys Pro Ala Asp Pro Pro Arg Thr Val Lieu. Lieu. Thr Gly Ala Asn Gly 78s 79 O 79. 8OO Trp Lieu. Gly Arg Phe Lieu Ala Lieu. Glu Trp Lieu. Glu Arg Lieu Ala Pro 805 810 815 Ala Gly Gly Lys Lieu. Ile Thir Ile Val Arg Gly Lys Asp Ala Ala Glin 82O 825 83 O Ala Lys Ala Arg Lieu. Asp Ala Ala Tyr Glu Ser Gly Asp Pro Llys Lieu 835 84 O 845 Ala Gly His Tyr Glin Asp Lieu Ala Ala Thir Thr Lieu. Glu Val Lieu Ala 850 855 860 Gly Asp Phe Ser Glu Pro Arg Lieu. Gly Lieu. Asp Glu Ala Thir Trp Asn 865 87O 87s 88O Arg Lieu Ala Asp Glu Val Asp Phe Ile Ser His Pro Gly Ala Lieu Val 885 890 895 Asn His Val Lieu Pro Tyr Asn Gln Leu Phe Gly Pro Asn Val Ala Gly 9 OO 905 91 O Val Ala Glu Ile Ile Llys Lieu Ala Ile Thir Thr Arg Ile Llys Pro Val 915 92 O 925 Thr Tyr Lieu Ser Thr Val Ala Val Ala Ala Gly Val Glu Pro Ser Ala 93 O 935 94 O Lieu. Asp Glu Asp Gly Asp Ile Arg Thr Val Ser Ala Glu Arg Ser Val 945 950 955 96.O Asp Glu Gly Tyr Ala Asn Gly Tyr Gly Asn. Ser Llys Trp Gly Gly Glu 965 97O 97. Val Lieu. Lieu. Arg Glu Ala His Asp Arg Thr Gly Lieu Pro Val Arg Val 98O 985 99 O Phe Arg Ser Asp Met Ile Leu Ala His Glin Llys Tyr Thr Gly Glin Val 995 1OOO 1 OOS Asn Ala Thir Asp Glin Phe Thr Arg Lieu Val Glin Ser Lieu. Lieu Ala Thr 1010 1 O15 1 O2O Gly Lieu Ala Pro Llys Ser Phe Tyr Glu Lieu. Asp Ala Glin Gly Asn Arg 1025 103 O 1035 104 O Glin Arg Ala His Tyr Asp Gly Ile Pro Val Asp Phe Thr Ala Glu Ser 1045 1OSO 105.5 Ile Thir Thr Lieu. Gly Gly Asp Gly Lieu. Glu Gly Tyr Arg Ser Tyr Asn 106 O 1065 1OO Val Phe Asin Pro His Arg Asp Gly Val Gly Lieu. Asp Glu Phe Val Asp 1075 108O 1085 Trp Lieu. Ile Glu Ala Gly His Pro Ile Thr Arg Ile Asp Asp Tyr Asp 1090 1095 11OO

Glin Trp Lieu. Ser Arg Phe Glu Thir Ser Lieu. Arg Gly Lieu Pro Glu Ser 1105 111 O 1115 112O Lys Arg Glin Ala Ser Val Lieu Pro Lieu. Lieu. His Ala Phe Ala Arg Pro 1125 113 O 1135

Gly Pro Ala Val Asp Gly Ser Pro Phe Arg Asn Thr Val Phe Arg Thr 114 O 1145 1150 Asp Val Glin Lys Ala Lys Ile Gly Ala Glu. His Asp Ile Pro His Lieu 1155 1160 1165 US 9,580,731 B2 67 - Continued Gly Lys Ala Lieu Val Lieu Lys Tyr Ala Asp Asp Ile Lys Glin Lieu. Gly 1170 1175 118O

Lieu. Luell 1185

<210s, SEQ ID NO 8 &211s LENGTH: 459 212. TYPE: PRT <213> ORGANISM: Chromobacterium violaceum

<4 OOs, SEQUENCE: 8 Met Gln Lys Glin Arg Thir Thir Ser Glin Trp Arg Glu Lieu. Asp Ala Ala 1. 5 1O 15 His His Lieu. His Pro Phe Thr Asp Thr Ala Ser Lieu. Asn Glin Ala Gly 2O 25 3O Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Lieu. Trp Asp Ser Glu 35 4 O 45 Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Lieu. Trp Cys Val Asn. Wall SO 55 6 O Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Glin Met Glu 65 70 7s 8O Glu Lieu Pro Phe Tyr Asn Thr Phe Phe Llys Thir Thr His Pro Ala Val 85 90 95 Val Glu Lieu. Ser Ser Lieu. Lieu Ala Glu Val Thr Pro Ala Gly Phe Asp 1OO 105 11 O Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile 115 120 125 Arg Met Val Arg Arg Tyr Trp Asp Val Glin Gly Llys Pro Glu Lys Llys 13 O 135 14 O Thr Lieu. Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly 145 150 155 160 Ala Ser Lieu. Gly Gly Met Lys Tyr Met His Glu Glin Gly Asp Lieu Pro 1.65 17O 17s Ile Pro Gly Met Ala His Ile Glu Glin Pro Trp Trp Tyr Lys His Gly 18O 185 19 O Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Lieu. 195 2OO 2O5 Glu Glu Lys Ile Lieu. Glu Ile Gly Ala Asp Llys Val Ala Ala Phe Val 21 O 215 22O Gly Glu Pro Ile Glin Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr 225 23 O 235 24 O Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Llys Tyr Asp Val Lieu. Lieu. 245 250 255 Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe 26 O 265 27 O Gly His Gln His Phe Gly Phe Glin Pro Asp Leu Phe Thr Ala Ala Lys 27s 28O 285

Gly Leu Ser Ser Gly Tyr Lieu Pro Ile Gly Ala Val Phe Val Gly Lys 29 O 295 3 OO Arg Val Ala Glu Gly Lieu. Ile Ala Gly Gly Asp Phe Asn His Gly Phe 3. OS 310 315 32O

Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val 3.25 330 335

Ala Ala Lieu. Arg Asp Glu Gly Ile Val Glin Arg Val Lys Asp Asp Ile 34 O 345 35. O US 9,580,731 B2 69 70 - Continued

Gly Pro Tyr Met Glin Lys Arg Trp Arg Glu Thir Phe Ser Arg Phe Glu 355 360 365

His Wall Asp Asp Wall Arg Gly Wall Gly Met Wall Glin Ala Phe Thir Luell 37 O 375

Wall Asn Ala Lys Arg Glu Luell Phe Pro Asp Phe Gly Glu Ile 385 390 395 4 OO

Gly Thir Luell Arg Asp Ile Phe Phe Arg ASn Asn Lell Ile Met Arg 4 OS 415

Ala Gly Asp His Ile Wall Ser Ala Pro Pro Lell Wall Met Thir Arg 42O 425 43 O

Ala Glu Wall Asp Glu Met Lell Ala Wall Ala Glu Arg Cys Luell Glu Glu 435 44 O 445

Phe Glu Glin Thir Lell Ala Arg Gly Luell Ala 450 45.5

SEO ID NO 9 LENGTH: 468 TYPE : PRT ORGANISM: Pseudomonas aeruginosa

< 4 OOs SEQUENCE: 9

Met Asn Ala Arg Lell His Ala Thir Ser Pro Luell Gly Asp Ala Asp Luell 1. 5 1O 15

Wall Arg Ala Asp Glin Ala His Met His Gly His Wall Phe Asp 25

Asp His Arg Wall Asn Gly Ser Lieu Asn Ile Ala Ala Gly Asp Gly Ala 35 4 O 45

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

Met Trp Thir Asn Ile Gly Luell Gly Arg Glu Glu Met Ala Arg Thir 65 70

Wall Ala Glu Glin Thir Arg Lell Luell Ala Tyr Ser Asn Pro Phe Cys Asp 85 90 95

Met Ala Asn Pro Arg Ala Ile Glu Luell Arg Lell Ala Glu Luell 105 11 O

Ala Pro Gly Asp Lell Asp His Wall Phe Luell Thir Thir Gly Gly Ser Thir 115 12 O 125

Ala Wall Asp Thir Ala Ile Arg Luell Met His Tyr Glin Asn Arg 13 O 135 14 O

Gly Arg Ala Lys His Wall Ile Thir Arg Ile Asn Ala His 145 150 155 160

Gly Ser Thir Phe Lell Gly Met Ser Luell Gly Gly Ser Ala Asp Arg 1.65 17O 17s

Pro Ala Glu Phe Asp Phe Lell Asp Glu Arg Ile His His Luell Ala 18O 185 19 O

Pro Tyr Arg Ala Pro Glu Gly Luell Gly Glu Ala Glu Phe Luell 195 2OO

Asp Gly Luell Wall Asp Glu Phe Glu Arg Ile Lell Glu Luell Gly Ala 21 O 215

Asp Arg Wall Gly Ala Phe Ile Ser Glu Pro Wall Phe Gly Ser Gly Gly 225 23 O 235 24 O

Wall Ile Wall Pro Pro Ala Gly His Arg Arg Met Trp Glu Luell 245 250 255

Glin Arg Asp Wall Lell Ile Ser Asp Glu Wall Wall Thir Ser Phe US 9,580,731 B2 71 72 - Continued

26 O 265 27 O

Gly Arg Luell Gly His Phe Phe Ala Ser Glin Ala Wall Phe Gly Wall Glin 27s 285

Pro Asp Ile Ile Lieu. Thir Ala Gly Luell Thir Ser Gly Tyr Glin Pro 29 O 295 3 OO

Lell Gly Ala Cys Ile Phe Ser Arg Arg Ile Trp Glu Wall Ile Ala Glu 3. OS 310 315

Pro Asp Gly Arg Cys Phe Ser His Gly Phe Thir Ser Gly His 3.25 330 335

Pro Wall Ala Cys Ala Ala Ala Luell Lys Asn Ile Glu Ile Ile Glu Arg 34 O 345 35. O

Glu Gly Luell Lieu Ala His Ala Asp Glu Wall Gly Arg Tyr Phe Glu Glu 355 360 365

Arg Luell Glin Ser Luell Arg Asp Luell Pro Ile Wall Gly Asp Wall Arg Gly 37 O 375

Met Arg Phe Met Ala Cys Wall Glu Phe Wall Ala Asp Ala Ser Lys 385 390 395 4 OO

Ala Luell Phe Pro Glu Ser Lell Asn Ile Gly Glu Trp Wall His Luell Arg 4 OS 415

Ala Glin Arg Gly Lell Lell Wall Arg Pro Ile Wall His Luell Asn Wall 42O 425 43 O

Met Ser Pro Leu. Ile Lell Thir Arg Glu Glin Wall Asp Thir Wall Wall 44 O 445

Arg Wall Lel Arg Glu Ser Ile Glu Glu Thir Wall Glu Asp Luell Wall Arg 450 45.5 460

Ala Gly His Arg 465

<210s, SEQ ID NO 10 &211s LENGTH: 45.4 212. TYPE : PRT <213s ORGANISM: Pseudomonas syringae

<4 OOs, SEQUENCE: 10

Met Ser Ala Asn. Asn Pro Glin Thir Luell Glu Trp Glin Ala Luell Ser Ser 1. 5 15

Glu His His Lieu Ala Pro Phe Ser Asp Glin Lell Lys Glu 2O 25 3O

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

Glu Gly Asn Lys Ile Lell Asp Gly Met Ser Gly Lell Trp Wall Ala SO 55 6 O

Ile Gly Tyr Gly Arg Glu Glu Luell Ala Asp Ala Ala Ser Glin Met 65 70 7s

Arg Glu Luell Pro Tyr Asn Luell Phe Phe Glin Thir Ala His Pro Pro 85 90 95

Wall Luell Glu Lieu Ala Ala Ile Ser Asp Ile Ala Pro Glu Gly Met 1OO 105 11 O

Asn His Wall Phe Phe Thir Gly Ser Gly Ser Glu Gly Asn Asp Thir Met 115 12 O 125

Lell Arg Met Val Arg His Tyr Trp Ala Luell Lys Gly Glin Pro Asn 13 O 135 14 O

Lys Thir Ile Ile Ser Arg Wall Asn Gly His Gly Ser Thir Wall Ala 145 150 155 160 US 9,580,731 B2 73 74 - Continued

Gly Ala Ser Luell Gly Gly Met Thir Met His Glu Glin Gly Asp Luell 1.65 17s

Pro Ile Pro Gly Wall Wall His Ile Pro Glin Pro Tyr Trp Phe Gly Glu 18O 185 19 O

Gly Gly Asp Met Thir Pro Asp Glu Phe Gly Ile Trp Ala Ala Glu Glin 195

Lell Glu Ile Lell Glu Luell Gly Wall Glu Asn Wall Gly Ala Phe 21 O 215 22O

Ile Ala Glu Pro Ile Glin Gly Ala Gly Gly Wall Ile Wall Pro Pro Asp 225 23 O 235 24 O

Ser Trp Pro Lys Ile Glu Ile Luell Ser Arg Asp Ile Luell 245 250 255

Phe Ala Ala Asp Glu Wall Ile Gly Phe Gly Arg Thir Ser Glu Trp 26 O 265 27 O

Phe Gly Ser Asp Phe Gly Luell Arg Pro Asp Met Met Thir Ile Ala 27s 28O 285

Gly Luell Thir Ser Gly Tyr Wall Pro Met Gly Gly Lell Ile Wall Arg 29 O 295 3 OO

Asp Glu Ile Wall Ala Wall Lell Asn Glu Gly Gly Asp Phe Asn His Gly 3. OS 310 315

Phe Thir Ser Gly His Pro Wall Ala Ala Ala Wall Ala Luell Glu Asn 3.25 330 335

Ile Arg Ile Luell Arg Glu Glu Ile Wall Glu Arg Wall Arg Ser Glu 34 O 345 35. O

Thr Ala Pro Lieu Gln Arg Lieu Arg Glu Lieu Ser Asp His Pro 355 360 365

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

Wall Asp Thir Thir Arg Glu Arg Thir Asp Gly Ala Gly 385 390 395 4 OO

Met Ile Arg Thir Phe Phe Asp Asn Gly Lell Ile Met Arg Ala 4 OS 41O 415

Wall Gly Asp Thir Met Ile Ile Ala Pro Pro Luell Wall Ile Ser Phe Ala 42O 425 43 O

Glin Ile Asp Glu Lell Wall Glu Lys Ala Arg Thir Lell Asp Luell Thir 435 44 O 445

Lell Ala Wall Luell Glin Gly 450

SEQ ID NO 11 LENGTH: 467 TYPE : PRT ORGANISM: Rhodobacter sphaeroides

< 4 OOs SEQUENCE: 11

Met Thir Arg Asn Asp Ala Thir Asn Ala Ala Gly Ala Wall Gly Ala Ala 1. 5 15

Met Arg Asp His Ile Lell Lell Pro Ala Glin Glu Met Ala Lys Luell Gly 25

Ser Ala Glin Pro Wall Lell Thir His Ala Glu Gly Ile Wall His 35 4 O 45

Thir Glu Asp Gly Arg Arg Lell Ile Asp Gly Pro Ala Gly Met Trp SO 55 6 O

Ala Glin Wall Gly Tyr Gly Arg Arg Glu Ile Wall Asp Ala Met Ala His 65 70 7s 8O US 9,580,731 B2 75 76 - Continued

Glin Ala Met Wall Lell Pro Ala Ser Pro Trp Tyr Met Ala Thir Ser 85 90 95

Pro Ala Ala Arg Lell Ala Glu Ile Ala Thir Lell Thir Pro Gly Asp 105 11 O

Lell Asn Arg Ile Phe Phe Thir Thir Gly Gly Ser Thir Ala Wall Asp Ser 115 12 O 125

Ala Luell Arg Phe Ser Glu Phe Asn Asn Wall Lell Gly Arg Pro Glin 13 O 135 14 O

Lys Arg Ile Ile Wall Arg Asp Gly Tyr His Gly Ser Thir Ala 145 150 155 160

Lell Thir Ala Ala Cys Thir Gly Arg Thir Gly ASn Trp Pro Asn Phe Asp 1.65 17O 17s

Ile Ala Glin Asp Arg Ile Ser Phe Luell Ser Ser Pro Asn Pro Arg His 18O 185 19 O

Ala Gly Asn Arg Ser Glin Glu Ala Phe Luell Asp Asp Lell Wall Glin Glu 195

Phe Glu Asp Arg Ile Glu Ser Luell Gly Pro Asp Thir Ile Ala Ala Phe 21 O 215 22O

Lell Ala Glu Pro Ile Lell Ala Ser Gly Gly Wall Ile Ile Pro Pro Ala 225 23 O 235 24 O

Gly His Ala Arg Phe Ala Ile Cys Glu His Asp Ile Luell 245 250 255

Ile Ser Asp Glu Wall Wall Thir Gly Phe Gly Arg Gly Glu Trp 26 O 265 27 O

Phe Ala Ser Glu Wall Phe Gly Wall Wall Pro Asp Ile Ile Thir Phe 27s 285

Ala Lys Gly Wall Thir Ser Gly Wall Pro Luell Gly Gly Luell Ala Ile 29 O 295 3 OO

Ser Glu Ala Wall Lell Ala Arg Ile Ser Gly Glu Asn Ala Gly Ser 3. OS 310 315

Trp Phe Thir Asn Gly Thir Ser Asn Glin Pro Wall Ala Cys Ala 3.25 330 335

Ala Ala Luell Ala Asn Ile Glu Luell Met Glu Arg Glu Gly Ile Wall Asp 34 O 345 35. O

Glin Ala Arg Glu Met Ala Asp Tyr Phe Ala Ala Ala Lell Ala Ser Luell 355 360 365

Arg Asp Luell Pro Gly Wall Ala Glu Thir Arg Ser Wall Gly Luell Wall Gly 37 O 375

Cys Wall Glin Lell Lell Asp Pro Thir Arg Ala Asp Gly Thir Ala Glu 385 390 395 4 OO

Asp Ala Phe Thir Lell Ile Asp Glu Arg Phe Glu Luell Gly 4 OS 41O 415

Lell Ile Wall Arg Pro Lell Gly Asp Luell Wall Ile Ser Pro Pro Luell 42O 425 43 O

Ile Ile Ser Arg Ala Glin Ile Asp Glu Met Wall Ala Ile Met Arg Glin 435 44 O 445

Ala Ile Thir Glu Wall Ser Ala Ala His Gly Luell Thir Ala Glu Pro 450 45.5 460

Ala Ala Wall 465

<210s, SEQ ID NO 12 &211s LENGTH: 459 US 9,580,731 B2 77 - Continued

212. TYPE: PRT <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 12 Met Asn Arg Lieu Pro Ser Ser Ala Ser Ala Lieu Ala Cys Ser Ala His

Ala Lieu. Asn Lieu. Ile Glu Lys Arg Thr Lieu. Asp His Glu Glu Met Lys 2O 25 3O Ala Lieu. Asn Arg Glu Val Ile Glu Tyr Phe Lys Glu. His Val Asn Pro 35 4 O 45 Gly Phe Lieu. Glu Tyr Arg Llys Ser Val Thir Ala Gly Gly Asp Tyr Gly SO 55 6 O Ala Val Glu Trp Glin Ala Gly Ser Lieu. Asn. Thir Lieu Val Asp Thr Glin 65 70 7s 8O Gly Glin Glu Phe Ile Asp Cys Lieu. Gly Gly Phe Gly Ile Phe Asin Val 85 90 95 Gly His Arg Asin Pro Val Val Val Ser Ala Val Glin Asn Glin Lieu Ala 1OO 105 11 O Lys Glin Pro Lieu. His Ser Glin Glu Lieu. Lieu. Asp Pro Lieu. Arg Ala Met 115 12 O 125 Lieu Ala Lys Thir Lieu Ala Ala Lieu. Thr Pro Gly Llys Lieu Lys Tyr Ser 13 O 135 14 O Phe Phe Cys Asn. Ser Gly Thr Glu Ser Val Glu Ala Ala Lieu Lys Lieu. 145 150 155 160 Ala Lys Ala Tyr Glin Ser Pro Arg Gly Llys Phe Thr Phe Ile Ala Thr 1.65 170 175 Ser Gly Ala Phe His Gly Llys Ser Lieu. Gly Ala Lieu. Ser Ala Thr Ala 18O 185 19 O Lys Ser Thr Phe Arg Llys Pro Phe Met Pro Leu Lleu Pro Gly Phe Arg 195 2OO 2O5 His Val Pro Phe Gly Asn. Ile Glu Ala Met Arg Thr Ala Lieu. Asn. Glu 21 O 215 22O Cys Llys Llys Thr Gly Asp Asp Wall Ala Ala Val Ile Lieu. Glu Pro Ile 225 23 O 235 24 O Gln Gly Glu Gly Gly Val Ile Leu Pro Pro Pro Gly Tyr Lieu. Thir Ala 245 250 255 Val Arg Llys Lieu. Cys Asp Glu Phe Gly Ala Lieu Met Ile Lieu. Asp Glu 26 O 265 27 O Val Glin Thr Gly Met Gly Arg Thr Gly Lys Met Phe Ala Cys Glu. His 27s 28O 285 Glu Asn Val Glin Pro Asp Ile Lieu. Cys Lieu Ala Lys Ala Lieu. Gly Gly 29 O 295 3 OO Gly Val Met Pro Ile Gly Ala Thr Ile Ala Thr Glu Glu Val Phe Ser 3. OS 310 315 32O Val Lieu. Phe Asp Asn Pro Phe Lieu. His Thr Thr Thr Phe Gly Gly Asn 3.25 330 335

Pro Lieu Ala Cys Ala Ala Ala Lieu Ala Thir Ile Asn Val Lieu. Lieu. Glu 34 O 345 35. O

Glin Asn Lieu Pro Ala Glin Ala Glu Gln Lys Gly Asp Met Lieu. Lieu. Asp 355 360 365

Gly Phe Arg Glin Lieu Ala Arg Glu Tyr Pro Asp Lieu Val Glin Glu Ala 37 O 375 38O

Arg Gly Lys Gly Met Lieu Met Ala Ile Glu Phe Val Asp Asn. Glu Ile 385 390 395 4 OO US 9,580,731 B2 79 - Continued

Gly Tyr Asn. Phe Ala Ser Glu Met Phe Arg Glin Arg Val Lieu Val Ala 4 OS 41O 415 Gly. Thir Lieu. Asn. Asn Ala Lys Thir Ile Arg Ile Glu Pro Pro Lieu. Thir 42O 425 43 O Lieu. Thir Ile Glu Glin Cys Glu Lieu Val Ile Lys Ala Ala Arg Lys Ala 435 44 O 445 Lieu Ala Ala Met Arg Val Ser Val Glu Glu Ala 450 45.5

<210s, SEQ ID NO 13 &211s LENGTH: 453 212. TYPE: PRT <213> ORGANISM: Wibrio fluvialis

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

Ala Ile Lieu Pro Ile Lieu. Arg Llys Tyr Asp Ile Pro Val Ile Ser Asp 245 250 255

Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Asn. Thir Trp Gly Cys Val 26 O 265 27 O

Thr Tyr Asp Phe Thr Pro Asp Ala Ile Ile Ser Ser Lys Asn Lieu. Thr 27s 28O 285

Ala Gly Phe Phe Pro Met Gly Ala Val Ile Leu Gly Pro Glu Lieu. Ser 29 O 295 3 OO

Lys Arg Lieu. Glu Thir Ala Ile Glu Ala Ile Glu Glu Phe Pro His Gly US 9,580,731 B2 81 82 - Continued

3. OS 310 315 32O

Phe Thir Ala Ser Gly His Pro Wall Gly Cys Ala Ile Ala Luell Lys Ala 3.25 330 335

Ile Asp Wall Wall Met Asn Glu Gly Luell Ala Glu Asn Wall Arg Arg Luell 34 O 345 35. O

Ala Pro Arg Phe Glu Glu Arg Luell His Ile Ala Glu Arg Pro Asn 355 360 365

Ile Gly Glu Arg Gly Ile Gly Phe Met Trp Ala Lell Glu Ala Wall 37 O 375

Lys Asp Ala Ser Lys Thir Pro Phe Asp Gly Asn Lell Ser Wall Ser 385 390 395 4 OO

Glu Arg Ile Ala Asn Thir Thir Asp Luell Gly Lell Ile Arg Pro 4 OS 415

Lell Gly Glin Ser Wall Wall Lell Pro Pro Phe Ile Lell Thir Glu Ala 425 43 O

Glin Met Asp Glu Met Phe Asp Lys Luell Glu Lys Ala Lell Asp Wall 435 44 O 445

Phe Ala Glu Wall Ala 450

<210s, SEQ ID NO 14 &211s LENGTH: 224 212. TYPE : PRT &213s ORGANISM: Bacillus subtilis

<4 OOs, SEQUENCE: 14

Met Lys Ile Tyr Gly Ile Met Asp Arg Pro Lell Ser Glin Glu Glu 1. 5 15

Asn Glu Arg Phe Met Ser Phe Ile Ser Pro Glu Arg Glu 25

Arg Arg Phe His Glu Asp Ala His Arg Thir Lell Luell Asp 35 4 O 45

Wall Luell Wall Arg Ser Wall Ile Ser Arg Glin Tyr Glin Lell Asp Ser SO 55 6 O

Asp Ile Arg Phe Ser Thir Glin Glu Tyr Gly Lys Pro Ile Pro Asp 65 70

Lell Pro Asp Ala His Phe Asn Ile Ser His Ser Gly Arg Trp Wall Ile 85 90 95

Ala Phe Asp Ser Glin Pro Ile Gly Ile Asp Ile Glu Lys Thir 105 11 O

Pro Ile Ser Luell Glu Ile Ala Lys Arg Phe Phe Ser Lys Thir Glu 115 12 O 125

Ser Asp Luell Luell Ala Asp Asp Glu Glin Thir Asp Phe 13 O 135 14 O

His Luell Trp Ser Met Lys Glu Ser Phe Ile Lys Glin Glu Gly Gly 145 150 155 160

Lell Ser Luell Pro Lell Asp Ser Phe Ser Wall Arg Lell His Glin Asp Gly 1.65 17O 17s

Glin Wall Ser Ile Glu Lell Pro Asp Ser His Ser Pro Tyr Ile 18O 185 19 O

Thir Glu Wall Asp Pro Gly Tyr Met Ala Wall Cys Ala Ala His 195 2O5

Pro Asp Phe Pro Glu Asp Ile Thir Met Wall Ser Tyr Glu Glu Luell Luell 21 O 215 22O US 9,580,731 B2 83 84 - Continued

SEO ID NO 15 LENGTH: 222 TYPE PRT ORGANISM: Nocardia sp. NRRL 5646 <4 OOs, SEQUENCE: 15

Met Ile Glu. Thir Ile Leul Pro Ala Gly Wall Glu Ser Ala Glu Lieu. Luell 1. 5 15

Glu Tyr Pro Glu Asp Lieu. Ala His Pro Ala Glu Glu His Lieu. Ile 25

Ala Lys Ser Wall Glu Arg Arg Arg Asp Phe Ile Gly Ala Arg His 35 4 O 45

Ala Arg Lieu. Ala Lell Ala Glu Lieu. Gly Glu Pro Pro Wall Ala Ile SO 55 6 O

Gly Gly Glu Arg Gly Ala Pro Ile Trp Pro Arg Gly Wall Val Gly 65 70

Ser Luell Thir His Cys Asp Gly Tyr Arg Ala Ala Ala Wall Ala His 85 90 95

Met Arg Phe Arg Ser Ile Gly Ile Asp Ala Glu Pro His Ala Thir Lieu. 105 11 O

Pro Glu Gly Wall Lell Asp Ser Wall Ser Lieu. Pro Pro Glu Arg Glu Trp 115 12 O 125

Lieu Lys Th Thr Asp Ser Ala Lieu. His Lieu. Asp Arg Lell Luell Phe 13 O 135 14 O

Ala Glu Ala Thr Tyr Ala Trp Trp Pro Leu. Thir Ala Arg Trp 145 150 155 16 O

Lieu. Gly Phe Glu Glu Ala His Ile Thir Phe Glu Ile Glu Asp Gly Ser 1.65 17O 17s

Ala Asp Ser Gly Asn Gly Thir Phe His Ser Glu Lieu Lleu. Wall Pro Gly 18O 185 19 O

Gn. Thir Asn Asp Gly Gly Thr Pro Leu Lleu Ser Phe Asp Gly Arg Trp 195

Lell Ile Ala Asp Gly Phe Ile Luell Thir Ala Ile Ala Ala 21 O 215

What is claimed is: dehydrogenase classified under EC 1.1.1.- to form 1. A method for biosynthesizing pimelic acid, 7-amino 45 pimelic acid or 7-hydroxyheptanoate; and/or heptanoate, 7-hydroxyheptanoate, or heptamethylenedi contacting said pimelate semialdehyde or said pimeloyl amine, said method comprising: CoA with an ()-transaminase classified under EC (i) three cycles of 2-oxoacid chain elongation comprising 2.6.1.18, 2.6.1.19, 2.6.1.29, 2.6.1.48, or 2.6.1.82, or a contacting 2-oxoglutarate with a (homo), citrate Syn 50 deacetylase classified under EC 3.5.1.62 to form thase classified under EC 2.3.3.14 or 2.3.3.13, a (homo) 7-aminoheptanoate or heptamethylenediamine. citrate dehydratase classified under EC 4.2.1.114, 2. The method of claim 1, wherein said thioesterase 4.2.1.36, or 4.2.1.33, a (homo)aconitate hydratase, and comprises an amino acid sequence that is at least 90% an iso(homo), citrate dehydrogenase classified under identical to the amino acid sequence set forth in SEQID NO: EC 1.1.85, 1.1.87, or 1.1.1.286, to form 2-oxoSuberate; 55 1. (ii) contacting said 2-oxoSuberate with an indolepyruvate 3. The method of claim 1, wherein said ()-transaminase decarboxylase to form pimelate semialdehyde or con comprises an amino acid sequence that is at least 90% tacting said 2-oxoSuberate with a 2-oxoglutarate dehy sequence identical to the amino acid sequences set forth in drogenase complex to form pimeloyl-CoA, and SEQ ID NOs: 8-13. (iii) contacting said pimelate semialdehyde or said 60 4. The method of claim 1, further comprising contacting pimeloyl-CoA with a thioesterase classified under EC said pimelic acid with a carboxylate reductase to form a 3.1.2, an aldehyde dehydrogenase classified under EC terminal aldehyde group as an intermediate in forming 1.2.1.3, a 7-oxoheptanoate dehydrogenase classified 7-aminoheptanoate. under EC 1.2.1, a 6-oxohexanoate dehydrogenase clas 5. The method of claim 4, wherein said carboxylate sified under EC 1.2.1, a glutaconoate CoA-transferase 65 reductase comprises an amino acid sequence that is at least classified under EC 2.8.3.12, a reversible succinyl 90% sequence identical to the amino acid sequences set CoA-ligase classified under EC 6.2.1.5, or an alcohol forth in SEQ ID NOs: 2-7. US 9,580,731 B2 85 86 6. The method of claim 1, wherein said method is per nol, a pyruvate decarboxylase, a 2-oxoacid decarboxylase formed in a recombinant host cell by fermentation of said generating isobutanol, a formate dehydrogenase, a polymer recombinant host cell. synthase, a NADPH-specific L-glutamate dehydrogenase, a 7. The method of claim 6, wherein said fermentation NADP H-consuming transhydrogenase, a pimeloyl-CoA comprises a principal carbon Source obtained from biologi dehydrogenase; an acyl-CoA dehydrogenase that degrades cal or non-biological feedstocks. C7 building blocks and their precursors; a glutaryl-CoA 8. The method of claim 7, wherein said biological feed dehydrogenase; or a pimeloyl-CoA synthetase; and/or stock is, or obtained from, monosaccharides, disaccharides, wherein said recombinant host cell overexpresses one or lignocellulose, hemicellulose, cellulose, lignin, levulinic more genes encoding: a PEP carboxykinase, a PEP carboxy acid, formic acid, triglycerides, glycerol, fatty acids, agri 10 lase, a pyruvate carboxylase, a PEP synthase, a L-alanine cultural waste, condensed distillers’ solubles, or municipal dehydrogenase; a NADH-specific L-glutamate dehydroge waste; or wherein said non-biological feedstock is, or obtained from, natural gas, syngas, CO/H, methanol, etha nase; a L-glutamine synthetase; a diamine transporter, a nol, benzoate, or terephthalic acid/isophthalic acid mixture dicarboxylate transporter, and/or a multidrug transporter. Waste StreamS. 15 14. The method of claim 1, further comprising contacting 9. The method of claim 6, wherein said recombinant host 7-hydroxyheptanoate with a carboxylate reductase classified cell is a prokaryote. under EC 1.2.99.6 and a dehydrogenase classified under EC 10. The method of claim 9, wherein said prokaryote is a 1.1.1.- to form 1.7 heptanediol. genus selected from the group consisting of Escherichia, 15. The method of claim 9, wherein said prokaryote is Clostridia, Corynebacteria Cupriavidus, Pseudomonas, selected from the group consisting of Escherichia coli, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodo Clostridium ljungdhalii, Clostridium kluyveri, Corynebac COCCS. terium glutamicum, Cupriavidus necator; Cupriavidus met 11. The method of claim 6, wherein said recombinant host allidurans, Pseudomaonas fluorescens, Pseudomonas cell is a eukaryote. putida, Pseudomonas Oleavorans, Delftia acidovorans, 12. The method of claim 11, wherein said eukaryote is a 25 Bacillus subtilis, Lactobacillus delbrueckii, Lactobacillus genus selected from the group consisting of Aspergillus lactis, and Rhodococcus equi. Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryo 16. The method of claim 11, wherein said eukaryote is myces, Arxula, and Kluyveromyces. selected from the group consisting of Aspergillus niger, 13. The method of claim 6, wherein said recombinant host Saccharomyces cerevisiae, Pichia pastoris, Yarrowia cell comprises one or more of the following attenuated 30 lipolytica, Issathenkia Orientalis, Debaryomyces hansenii, enzymes: a lactate dehydrogenase, a menaquinol-fumarate Arcula adenoinivorans, and Kluyveromyces lactis. oxidoreductase, an alcohol dehydrogenase producing etha : : : : :