US007799545B2 (12) United States Patent (10) Patent No.: US 7,799,545 B2 Burgard et al. (45) Date of Patent: Sep. 21, 2010 (54) MICROORGANISMS FOR THE 4,082,788 A 4/1978 Mims PRODUCTION OF ADPCACID AND OTHER 4,190.495 A 2/1980 Curtiss COMPOUNDS 4,301,077 A 1 1/1981 Pesa et al. 4,624,920 A 11/1986 Inoue et al. (75)75 Inventors: Anthony P. Burgard, Bellefonte, PA 4,871,6674,652,685 A 10/19893, 1987 ImadaCawse etet al.al. (US); Priti Pharkya, San Diego, CA (US); Robin E. Osterhout, San Diego, (Continued) CA (US) FOREIGN PATENT DOCUMENTS (73) Assignee: Genomatica, Inc., San Diego, CA (US) CN 1358 841 T 2002 *) NotOt1Ce: Subjubject to anyy d1Sclaimer,disclai theh term off thithis (Continued)ontinue patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 47 days. Alhapel et al., “Molecular and functional analysis of nicotinate (21) Appl. No.: 12/413,355 catabolism in Eubacterium barkeri,’ Proc. Natl. Acad. Sci. USA 103(33):12341-12346 (2006). (22) Filed: Mar. 27, 2009 (Continued) (65) Prior Publication Data Primary Examiner Nashaat T Nashed US 2009/0305364 A1 Dec. 10, 2009 (74) Attorney, Agent, or Firm McDermott Will & Emery • ws LLP Related U.S. Application Data (57) ABSTRACT (60) Provisional application No. 61/040,059, filed on Mar. 27, 2008. The invention provides a non-naturally occurring microbial organism having an adipate, 6-aminocaproic acid or capro (51) Int. Cl. lactam pathway. The microbial organism contains at least one CI2P 7/40 (2006.01) exogenous nucleic acid encoding an enzyme in the respective CI2N L/20 (2006.01) adipate, 6-aminocaproic acid or caprolactam pathway. The (52) U.S. Cl. ................ 435/136: 435/252.3:435/252.33 invention additionally provides a method for producing adi (58) Field of Classification Search ....................... None pate, 6-aminocaproic acid or caprolactam. The method can See application file for complete search history. include culturing an adipate, 6-aminocaproic acid or capro lactam producing microbial organism, where the microbial (56) References Cited organism expresses at least one exogenous nucleic acid encoding an adipate, 6-aminocaproic acid or caprolactam U.S. PATENT DOCUMENTS pathway enzyme in a sufficient amount to produce the respec 3,513,209 A 5, 1970 Clement tive product, under conditions and for a sufficient period of 3,912,586 A 10/1975 Kaneyuki et al. time to produce adipate, 6-aminocaproic acid or caprolactam. 3,965, 182 A 6, 1976 Worrel 4,048,196 A 9, 1977 Broecker et al. 14 Claims, 9 Drawing Sheets } Adiate HO-----o- {} Atipate-caliase k a ..., avir. PP Adipyl-CoA --- scoA &city:-&c., a defydrogenasem A. 39 s w S-(arboxy-2-pentenoyl-CoA HO---- S-3c)a Enoyl-CoA hydratase / 32. 3-ydroxyadipyl-CoA "yr-Yss-CoAHg O 3-isdiriyyacyl-CoA deihydrogenase N. : s: K NA 3-(xeadiyi-CoAw or 'scsC. Y - 3 a. US 7,799,545 B2 Page 2 U.S. PATENT DOCUMENTS 2006/0099578 A1 5, 2006 Wallace et al. 2006, O11081.0 A1 5/2006 Rajgarhia et al. 5,079,143 1, 1992 Klein et al. 2006/0172399 A1 8, 2006 Nomoto et al. 5,143,833 9, 1992 Datta 2006/0281 156 Al 12/2006 Aoyama et al. 5,143,834 9, 1992 Glassner et al. 2007/0042476 A1 2/2007 Lee et al. 5,168,055 12, 1992 Datta et al. 2007/00929.57 A1 4/2007 Donaldson et al. 5,173.429 12, 1992 Gaddy et al. 2007/0111294 A1 5/2007 Burgard et al. 5, 182,199 1, 1993 Hartley 2007/0117191 A1 5/2007 Kamachi et al. 5, 192,673 3, 1993 Jain et al. 2007/O184539 A1 8, 2007 San et al. 5,403,721 4, 1995 Ward, Jr. et al. 2007/025941.0 A1 11/2007 Donaldson et al. 5,413,922 5, 1995 Matsuyama et al. 2008/0274522 A1 11/2008 Bramucci et al. 5,416,020 5, 1995 Severson et al. 2008/0293125 A1 11/2008 Subbian et al. 5,457,040 10, 1995 Jarry et al. 2009/0047719 A1 2/2009 Burgard et al. 5,478,952 12, 1995 Schwartz 2009, OO75351 A1 3/2009 Burk et al. 5,487.987 1, 1996 Frost et al. 2009/0305364 Al 12/2009 Burgard et al. 5,504,004 4, 1996 Guettler et al. 5,521,075 5, 1996 Guettler et al. FOREIGN PATENT DOCUMENTS 5,573.931 11, 1996 Guettler et al. 5,616,496 4, 1997 Frost et al. EP O 494 O78 A1 7, 1992 5,686,276 11, 1997 Laffend et al. EP 1473368 A1 11, 2004 5,700.934 12, 1997 Wolters et al. EP 2017344 1, 2009 5,770.435 6, 1998 Donnelly et al. GB 1230276 4f1971 5,807,722 9, 1998 Gaddy et al. GB 1314126 4f1973 5,869,301 2, 1999 Nghiem et al. GB 1344557 1, 1974 5,958,745 9, 1999 Gruys et al. GB 1512751 6, 1978 6,117,658 9, 2000 Dennis et al. JP 50 OO6776 1, 1975 6,133,014 10, 2000 Mukouyama et al. WO WO 82,03854 11, 1982 6,136,577 10, 2000 Gaddy et al. WO WO91f13997 9, 1991 6,159,738 12, 2000 Donnelly et al. WO WO99.06532 2, 1999 6,194.572 2, 2001 Buijs et al. WO WO99,58686 11, 1999 6,214,592 4, 2001 Crouzet et al. WO WOO1 (16346 3, 2001 6,274,790 8, 2001 Kunst et al. WO WO O2/O55995 1, 2002 6,280,986 8, 2001 Hespell et al. WO WO O2/42418 5, 2002 RE37,393 9, 2001 Donnelly et al. WO WO O2/O55995 T 2002 6,340,581 1, 2002 Gaddy et al. WO WO O2/O90312 11, 2002 6,353,100 3, 2002 Guit et al. WO WOO3,O10322 2, 2003 6,432,686 8, 2002 Bulthuis et al. WO WOO3,106691 12/2003 6,448,061 9, 2002 Pan et al. WO WOO3/106998 12/2003 6,455,284 9, 2002 Gokarn et al. WO WO 2005/047498 5, 2005 6,660,857 12, 2003 Agterberg et al. WO WO 2005/068643 7/2005 6,686, 194 2, 2004 Mutzel et al. WO WO 2006/031424 3, 2006 6,686,310 2, 2004 Kourtakis et al. WO WO 2006/034156 3, 2006 6,743,610 6, 2004 Donnelly et al. WO WO 2007 OO1982 1, 2007 6,852,517 2, 2005 Cameron et al. WO WO 2007/03O830 3, 2007 7,127,379 10, 2006 Palsson et al. WO WO 2007 141208 12/2007 7,186.541 3, 2007 Gokarn et al. WO WO 2008/O18930 2, 2008 7,223,567 5/2007 Ka-Yiu et al. WO WO 2008/024023 2, 2008 7,241,594 7/2007 Lee et al. WO WO 2008/027742 3, 2008 7,244,610 7/2007 San et al. WO WO 2008/115840 3, 2008 7,256,016 8, 2007 San et al. WO WO 2008/08O124 T 2008 7,262,046 8, 2007 Ka-Yiu et al. WO WO 2008,137403 11, 2008 7,285.402 10, 2007 Gaddy et al. WO WO 2008,152016 12/2008 7,309,597 12, 2007 Liao et al. WO WO 2009/O14437 1, 2009 7,371,558 5/2008 Cervin et al. WO WO 2009/023493 2, 2009 7,393,676 T/2008 Gokarn et al. WO WO 2009/031766 3, 2009 7,432,091 10, 2008 Yukawa et al WO WO 2009.113853 9, 2009 7,491.520 2, 2009 Raemakers-Franken et al. WO WO 2009.113855 9, 2009 7,569,380 8, 2009 San et al. 2002/0012939 1, 2002 Palsson et al. OTHER PUBLICATIONS 2002fOO40123 4, 2002 Patil et al. 2002fO168654 11, 2002 Maranas Alper et al., “Engineering yeast transcription machinery for 2003/0059792 3, 2003 Palsson et al. improved ethanol tolerance and production.” Science 2003/0O87381 5/2003 Gokarn 314(5805): 1565-1568 (2006). 2003.0113886 6, 2003 Brzostowicz et al. Atsumi et al., “Metabolic engineering of Escherichia coli for 2003/0182678 9, 2003 Mitsky et al. 1-butanol production.” Metab. Eng. 10(6):305-311 (2008). 2003,0224363 12, 2003 Park et al. Bartsch et al., “Molecular analysis of two genes of the Escherichia 2003/0233218 12, 2003 Schilling coli gab cluster: nucleotide sequence of the glutamate:Succinic 2004/OOO9466 1, 2004 Maranas et al. semialdehyde transaminase gene (gabT) and characterization of the 2004.0029149 2, 2004 Palsson et al. Succinic semialdehyde dehydrogenase gene (gabd). J. Bacteriol. 2004/OO72723 4, 2004 Palsson et al. 172(12):7035-7042 (1990). 2005/OO42736 2, 2005 San et al. Bonner and Bloch, “Purification and properties of fatty acyl 2005, 0079482 4, 2005 Maranas et al. thioesterase I from Escherichia coli,” J. Biol. Chem. 247(10):3123 2006/OO73577 4, 2006 Ka-Yiu et al. 3.133 (1972). US 7,799,545 B2 Page 3 Boronin, et al., “Plasmids specifying e-caprolactam degradation in Kanagawa, et al., “Characterization of the 6-aminohexanoate-dimer Pseudomonas strains.” FEMS Microbiol. Lett. 22(3):167-170 (1984). hydrolase from Pseudomonas sp. NK87.” J. Gen. Microbiol. Bower et al., “Cloning, sequencing, and characterization of the Bacil 139(4):787-795 (1993). lus subtilis biotin biosynthetic operon.” J. Bacteriol. 178(14):4122 Kanehisa and Goto, “KEGG: Kyoto Encyclopedia of Genes and 4130 (1996). Genomes database (KEGG).” Nucleic Acids Res. 28(1):27-30 (2000). Boynton, et al., “Cloning, sequencing, and expression of clustered Kaschabek et al., “Degradation of aromatics and chloroaromatics by genes encoding beta-hydroxybutyryl-coenzyme A (CoA) Pseudomonas sp.