USOO9708632B2 (12) United States Patent (10) Patent No.: US 9,708.632 B2 Burgard et al. (45) Date of Patent: *Jul.18, 2017 (54) ORGANISMS FOR THE PRODUCTION OF (56) References Cited 1,3-BUTANEDIOL U.S. PATENT DOCUMENTS (71) Applicant: Genomatica, Inc., San Diego, CA (US) 5,512,669 A 4/1996 Sinskey et al. 5,661,026 A 8/1997 Peoples et al. (72) Inventors: Anthony P. Burgard, Bellefonte, PA 5,958,745 A 9/1999 Gruys et al. (US); Mark J. Burk, San Diego, CA 6,515,205 B1 2/2003 Liebergesell et al. (US); Robin E. Osterhout, San Diego, 6,764,851 B2 7/2004 Nikolau et al. CA (US); Priti Pharkya, San Diego, 6,835,820 B2 12/2004 Cannon et al. CA (US) (Continued) (73) Assignee: Genomatica, Inc., San Diego, CA (US) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this W. W. 856 1358. patent is extended or adjusted under 35 WO WO 2008/115840 9, 2008 U.S.C. 154(b) by 0 days. WO WO 2009/O14437 1, 2009 WO WO 2009/094485 T 2009 This patent is Subject to a terminal dis- WO WO 2010/030711 3, 2010 claimer. WO WO 2010/071697 6, 2010 (21) Appl. No.: 14/673,600 OTHER PUBLICATIONS (22) Filed: Mar. 30, 2015 Aberhart and Hsu, "Stereospecific hydrogen loss in the conversion of 2H7 isobutyrate to B-hydroxyisobutyrate in Pseudomonas (65) Prior Publication Data putida. The stereochemistry of B-hydroxyisobutyrate US 2016/0053286 A1 Feb. 25, 2016 dehydrogenase,” J. Chem. Soc. Perkin 16:1404-1406 (1979). (Continued) Related U.S. Application Data Primary Examiner — Robert Mondesi (63) Continuation of application No. 12/772,114, filed on Assistant Examiner — Md. Younus Meah Apr. 30, 2010, now Pat. No. 9,017,983. (74) Attorney, Agent, or Firm — Jones Day (60) Provisional application No. 61/174,473, filed on Apr. (57) ABSTRACT 30, 2009. A non-naturally occurring microbial organism includes a microbial organism having a 1,3-butanediol (1,3-BDO) (51) Int. Cl. pathway having at least one exogenous nucleic acid encod CI2P 7/18 (2006.01) ing a 1,3-BDO pathway enzyme expressed in a sufficient CI2P 7/16 (2006.01) amount to produce 1,3-BDO. The pathway includes an CI2N 9/88 (2006.01) enzyme selected from a 2-amino-4-ketopentanoate (AKP) CI2N IS/00 (2006.01) thiolase, an AKP dehydrogenase, a 2-amino-4-hydroxypen CI2N 15/52 (2006.01) tanoate aminotransferase, a 2-amino-4-hydroxypentanoate CI2N 9/04 (2006.01) oxidoreductase (deaminating), a 2-oxo-4-hydroxypentano C07C 29/80 (2006.01) ate decarboxylase, a 3-hydroxybutyraldehyde reductase, an CI2N 9/02 (2006.01) AKP aminotransferase, an AKP oxidoreductase (deaminat CI2N 9/10 (2006.01) ing), a 2,4-dioxopentanoate decarboxylase, a 3-oxobutyral (52) U.S. Cl. CPC ................ CI2P 7/18 (2013.01); C07C 29/80 dehyde reductase (ketone reducing), a 3-oxobutyraldehyde (2013.01); C12N 9/0006 (2013.01); CI2N reductase (aldehyde reducing), a 4-hydroxy-2-butanone 9/0008 (2013.01); C12N 9/1029 (2013.01); reductase, an AKP decarboxylase, a 4-aminobutan-2-one CI2N 9/1096 (2013.01); C12N 9/88 aminotransferase, a 4-aminobutan-2-one oxidoreductase (2013.01); C12N 15/52 (2013.01); C12Y (deaminating), a 4-aminobutan-2-one ammonia-lyase, a 101/01157 (2013.01); C12Y 101/01 (2013.01); butenone hydratase, an AKP ammonia-lyase, an acetylacry CI2Y 102/01 (2013.01); C12Y 203/01 late decarboxylase, an acetoacetyl-CoA reductase (CoA (2013.01); C12Y 206/01 (2013.01); C12Y dependent, aldehyde forming), an acetoacetyl-CoA reduc 401/01 (2013.01); C12Y402/01 (2013.01); tase (CoA-dependent, alcohol forming), an acetoacetyl-CoA CI2Y 403/01 (2013.01) reductase (ketone reducing), a 3-hydroxybutyryl-CoA (58) Field of Classification Search reductase (aldehyde forming), a 3-hydroxybutyryl-CoA CPC. C12P 7/18: C12N 15/52; C12N 9/88; C12N reductase (alcohol forming), a 4-hydroxybutyryl-CoA dehy 9/0006; C12Y 101/01157; C12Y 401/01; dratase, and a crotonase. A method for producing 1,3-BDO, C12Y 203/O1 includes culturing Such microbial organisms under condi USPC ........ 435/158, 252.2, 252.32, 252.33, 320.1, tions and for a sufficient period of time to produce 1,3-BDO. 435/157 See application file for complete search history. 12 Claims, 7 Drawing Sheets US 9,708.632 B2 Page 2 (56) References Cited Alber et al., “Malonyl-Coenzyme a reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in U.S. PATENT DOCUMENTS archaeal Metallosphaera and Sulfolobus spp.” J. Bacteriol. 188(24):8551-8559 (2006). 6,998.471 B2 2/2006 Hallahan et al. Alber et al., “Study of an alternate glyoxylate cycle for acetate 7,127.379 B2 10/2006 Palsson et al. assimilation by Rhodobacter sphaeroides,” Mol. 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