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C12N 90008, C12N 9.0006, C12N 9001: E US0093.21701 B2 (12) United States Patent (10) Patent No.: US 9,321,701 B2 Burk et al. (45) Date of Patent: Apr. 26, 2016 (54) MICROORGANISMS AND METHODS FOR 2003/0233218 A1 12/2003 Schilling THE BIOSYNTHESIS OF BUTADENE 38885 A. 58: ManaSSO ca.al 2004/OO72723 A1 4/2004 Palsson et al. (75) Inventors: Mark J. Burk, San Diego, CA (US); 2009/0047719 A1 2/2009 Burgard et al. Anthony P. Burgard, Bellefonte, PA 2010.0003716 A1 1/2010 Cervin et al. (US); Jun Sun, San Diego, CA (US); 2010/0330635 Al 12/2010 Burgard et al. Robin E. Osterhout, San Diego, CA 392 A: i: Ewar 3. S. PritiD4- Pharkya, San Diego, CA 2014/O155567 A1 6, 2014 BurklKC et al. (73) Assignee: Genomatica, Inc., San Diego, CA (US) FOREIGN PATENT DOCUMENTS - GB 5251.98 8, 1940 (*) Notice: Subject to any disclaimer, the term of this WO WO O2/O55995 T 2002 patent is extended or adjusted under 35 WO WOO3/106998 12/2003 U.S.C. 154(b) by 0 days. WO WO 2007 141208 12/2007 WO WO 2008/115840 9, 2008 WO WO 2010.006O76 1, 2010 (21) Appl. No.: 13/365,132 WO WO 2010/031079 3, 2010 WO WO 2011/O52718 5, 2011 (22) Filed: Feb. 2, 2012 WO 2011 14.0171 11, 2011 (65) Prior Publication Data OTHER PUBLICATIONS US 2012/O225466A1 Sep. 6, 2012 Egloff et al. Conversion of oxygen derivatives of hydrocarbons into O O butadiene, Chem. Rev., (1945), 36(1), 63-141.* Related U.S. Application Data Aberhart and Hsu, "Stereospecific hydrogen loss in the conversion of (60) y inal application No. 61/438,947, filed on Feb. The2H7 stereochemistry isobutyrate to B-hydroxyisobutyrateof B-hydroxyisobutyrate in Pseudomonas dehydrogenase” putida. J. s Chem. Soc. Perkin II 6:1404-1406 (1979). (51) Int. Cl Adams, “Exploratory Catalytic Oxidations with Bismuth we Molybdate.” J. Catal. 10:355-361 (1968). CI2P 5/02 (2006.01) Ajawi et al., “Thiamin pyrophosphokinase is required for thiamin N 7. 3.08: cofactor activation in Arabidopsis.” Plant Mol. Biol. 65(1-2): 151-162 (2007). (Epub Jul. 5, 2007). C7H 2L/04 (2006.01) Alber et al., “Malonyl-Coenzyme A reductase in the modified CD7C I/24 (2006.01) 3-hydroxypropionate cycle for autotrophic carbon fixation in CI2P 7/04 (2006.01) archaeal Metallosphaera and Sulfolobus spp.” J. Bacteriol. (52) U.S. Cl. 188(24):8551-8559 (2006). CPC. C07CC07C 1/24 2521/04 (2013,01); (2013.01); C12P 704 C07C (2013.01); 2521/12 EooE. Coo""E" opacter spinaerodes. E. Mot. E.Microbiot. (2013.01); COC 252 1/16 (2013.01); COC Alincicek et al., LytB protein catalyzes the terminal step of the 2523/10 (2013.01); C07C2523/28 (2013.01); 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid C07C2523/31 (2013.01); C07C 2523/745 biosynthesis.” FEBS Lett. 532(3):437.440 (2002). (2013.01); C07C 2527/167 (2013.01); C07C Anderson et al., “Isopentenyl diphosphate:dimethylallyl diphosphate 2527/173 (2013.01) isomerase. An improved purification of the enzyme and isolation of (58) Field of Classification Search the gene from Saccharomyces cerevisiae,” J. Biol. Chem. CPC .............. C12P 7/04, C12P 7/16; C12P 5/026, 26432) 19169-1975 (1989). C12P 7/00; C12P 2203/00; C12N 15/52; Atsumi et al., Metabolic engineer.g. of Escherichia coli for C12N 90008, C12N 9.0006, C12N 9001: E""SE"E"Suml et al., on-Termentative painways Ior synthesis o di2. IN E. N 2. branched-chain higher alcohols as biofuels.” Nature 451 (7174):86 CO7C 1767. CO7C 252/04 Co7C 89 (2008). 2527,167, CO7C 2527,173 (Continued) See application file for complete search history. (56) References Cited Primary Examiner — Iqbal H Chowdhury (74) Attorney, Agent, or Firm — Jones Day U.S. PATENT DOCUMENTS 5,413,922 A 5/1995 Matsuyama et al. (57) ABSTRACT 5,849,970 A 12/1998 Fall et al. The invention provides non-naturally occurring microbial 7,127.379 B2 10/2006 Palsson et al. 7,947483 B2 5/2011 Burgard et al. organisms having a butadiene pathway. The invention addi 8,580,543 B2 11/2013 Burk et al. tionally provides methods of using Such organisms to produce 2002/0012939 A1 1/2002 Palsson butadiene. 2002/0168654 A1 11/2002 Maranas et al. 2003/0059792 A1 3/2003 Palsson et al. 2003,0224363 A1 12, 2003 Park et al. 20 Claims, 4 Drawing Sheets US 9,321,701 B2 Page 2 (56) References Cited Burgard et al., “Optiknock: a bilevel programming framework for identifying gene knockout strategies for microbial strain optimiza OTHER PUBLICATIONS tion.” Biotechnol. Bioeng. 84(6):647-657 (2003). Buu et al., “Functional characterization and localization of acetyl Basson et al., "Saccharomyces cerevisiae contains two functional CoA hydrolase, Achlp, in Saccharomyces cerevisiae, "J. Biol. Chem. genes encoding 3-hydroxy-3-methylglutaryl-coenzyme A 278: 17203-17209 (2003). Byres et al., “Crystal structures of Trypanosoma bruceii and reductase.” Proc. Natl. Acad. Sci. U.S. A. 83(15):5563-5567 (1986). 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Botella-Pavia et al., “Regulation of carotenoid biosynthesis in plants: Colonna et al., “Synthesis and radiocarbon evidence of terephthalate evidence for a key role of hydroxymethylbutenyl diphosphate polyesters completely prepared from renewable resources.” Green reductase in controlling the Supply of plastidial isoprenoid precur Chemistry 13:2543-2548 (2011). sors.” Plant J. 40(2): 188-199 (2004). Corthesy-Theulaz et al., "Cloning and Characterization of Bower et al., “Cloning, sequencing, and characterization of the Bacil Helicobacter pylori Succinyl CoA: Acetoacetate CoA-transferase, a lus subtilis biotin biosynthetic operon.” J. Bacteriol. 178(14):4122 Novel Prokaryotic Member of the CoA-transferase Family.” J. Biol. 4130 (1996). Chem. 272(41): 25659-25667 (1997). Boynton et al., “Cloning, sequencing, and expression of clustered Crans et al., “Glycerol Kinase: Substrate Specificity.” J. Am. 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