|HAI LUI AUN ACUTULUS010006055B2 LUNIIT MOONHITI (12 ) United States Patent (10 ) Patent No. : US 10 , 006 , 055 B2 Burk et al. (45 ) Date of Patent: Jun . 26 , 2018 ( 54 ) MICROORGANISMS FOR PRODUCING 2002/ 0168654 A1 11/ 2002 Maranas et al. 2003 / 0059792 Al 3 /2003 Palsson et al . BUTADIENE AND METHODS RELATED 2003 /0087381 A1 5 / 2003 Gokarn THERETO 2003 / 0224363 Al 12 /2003 Park et al . 2003 / 0233218 Al 12 /2003 Schilling (71 ) Applicant: Genomatica , Inc. , San Diego , CA (US ) 2004 / 0009466 AL 1 /2004 Maranas et al. 2004 / 0029149 Al 2 /2004 Palsson et al. ( 72 ) Inventors : Mark J . Burk , San Diego , CA (US ) ; 2004 / 0072723 A1 4 /2004 Palsson et al. Anthony P . Burgard , Bellefonte , PA 2004 / 0152159 Al 8 / 2004 Causey et al . 2005 /0042736 A1 2 / 2005 San et al . (US ) ; Robin E . Osterhout , San Diego , 2005 / 0079482 A1 4 / 2005 Maranas et al . CA (US ) ; Jun Sun , San Diego , CA 2006 / 0046288 Al 3 / 2006 Ka - Yiu et al. ( US ) ; Priti Pharkya , San Diego , CA 2006 / 0073577 A1 4 / 2006 Ka - Yiu et al . (US ) 2007 /0184539 Al 8 / 2007 San et al . 2009 / 0047718 Al 2 / 2009 Blaschek et al . 2009 / 0047719 Al 2 / 2009 Burgard et al . (73 ) Assignee : Genomatica , Inc ., San Diego , CA (US ) 2009 /0191593 A1 7 / 2009 Burk et al . 2010 / 0003716 A1 1 / 2010 Cervin et al. ( * ) Notice : Subject to any disclaimer , the term of this 2010 /0184171 Al 7 /2010 Jantama et al. patent is extended or adjusted under 35 2010 /0304453 Al 12 / 2010 Trawick et al . 2010 / 0330635 Al 12 / 2010 Burgard et al . U . S . C . 154 (b ) by 0 days . days . 2011 /0008858 AL 1 / 2011 Osterhout et al. 2011/ 0008861 A1 1 / 2011 Berry et al. ( 21) Appl. No. : 14 /869 , 872 2011 / 0045563 AL 2 / 2011 Melis 2011/ 0201089 Al * 8 / 2011 Burgard C12N 9 / 0006 ( 22 ) Filed : Sep . 29 , 2015 435 / 243 2011/ 0300597 A112 / 2011 Burk et al . (65 ) ) Prior Publication Data 2012 /0225466 AL 9 / 2012 Burk et al . US 2016 /0244786 A1 Aug . 25, 2016 2013 /0011891 A11 / 2013 Burk et al . FOREIGN PATENT DOCUMENTS Related U . S . Application Data WO WO 2002 /055995 7 / 2002 (62 ) Division of application No . 13/ 527 ,440 , filed on Jun . WO WO 2002 /061115 8 / 2002 WO WO 2003 / 106998 12 / 2003 19 , 2012 , now Pat . No . 9 , 169 ,486 . WO WO 2004 /018621 3 / 2004 (60 ) Provisional application No . 61/ 502 , 264, filed on Jun . WO WO 2005 /047498 5 /2005 28 , 2011 . (Continued ) ( 51 ) Int. Cl. OTHER PUBLICATIONS C12P 7 /04 ( 2006 .01 ) C12N 15 / 52 ( 2006 .01 ) Aberhart and Hsu , “ Stereospecific hydrogen loss in the conversion C12P 5 /02 ( 2006 . 01 ) of [2H7 ] isobutyrate to B -hydroxyisobutyrate in Pseudomonas C12N 15 / 70 ( 2006 .01 ) putida . The stereochemistry of B -hydroxyisobutyrate C12P 7 / 16 ( 2006 .01 ) dehydrogenase, ” J . Chem . Soc . [ Perkinl ] 6 ; 1404 - 1406 ( 1979 ) . (52 ) U . S . CI. Adams et al ., “ Oxidoreductase - Type Enzymes and Redox Proteins Involved in Fermentative Metabolisms of Hyperthermophilic CPC .. C12P 7704 ( 2013 .01 ) ; C12N 15 /52 Archaea ,” Archaea . Adv. Protein Chem . 48 : 101 - 180 ( 1996 ) . (2013 . 01 ) ; C12N 15 / 70 (2013 . 01 ) ; C12P 5 / 026 Ajjawi et al . , “ Thiamin pyrophosphokinase is required for thiamin ( 2013 .01 ) ; C12P 7 / 16 ( 2013 . 01 ) cofactor activation in Arabidopsis , ” Plant Mol. Biol. 65 ( 1 - 2 ) : 151 ( 58 ) Field of Classification Search 162 (2007 ) . (Epub Jul. 5 , 2007 ) . None Alber et al . , “Malonyl -Coenzyme A reductase in the modified See application file for complete search history. 3 -hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp ., " J . Bacteriol. References Cited 188 ( 24 ) : 8551 - 8559 ( 2006 ) . ( 56 ) Alber et al. , “ Study of an alternate glyoxylate cycle for acetate U . S . PATENT DOCUMENTS assimilation by Rhodobacter sphaeroides ,” Mol. Microbiol. 61 ( 2 ): 297 - 309 ( 2006 ) . 5 ,413 , 922 A 5 / 1995 Matsuyama et al . 5 , 830 ,716 A 11/ 1998 Kojima et al. (Continued ) 5 , 849 , 970 A 12 / 1998 Fall et al. 5 , 958 , 745 A 9 / 1999 Gruys et al. Primary Examiner — Iqbal H Chowdhury 6 , 117 ,658 A 9 / 2000 Dennis et al . ( 74 ) Attorney , Agent, or Firm — Jones Day 6 , 455 ,284 B1 9 / 2002 Gokarn et al . 7 , 127, 379 B2 10 / 2006 Palsson et al . ABSTRACT 7 , 223 , 567 B2 5 / 2007 Ka - Yiu et al. (57 ) 7 , 244 , 610 B2 7 / 2007 San et al. The invention provides non -naturally occurring microbial 7 , 256, 016 B2 8 / 2007 San et al. organisms having a butadiene or crotyl alcohol pathway . The 7 , 262 , 046 B2 8 / 2007 Ka - Yiu et al . 7 ,309 ,597 B2 12 / 2007 Liao et al. invention additionally provides methods of using such 7 , 718 , 417 B2 5 / 2010 Millis et al. organisms to produce butadiene or crotyl alcohol. 7 , 947, 483 B2 5 / 2011 Burgard et al . 2002 /0012939 AL 1 /2002 Palsson 14 Claims, 24 Drawing Sheets US 10 ,006 , 055 B2 Page 2 References Cited nucleatum ) . Comparison with the glutaconyl- CoA decarboxylases ( 56 ) from gram - positive bacteria , " Arch . Microbiol. 154 ( 4 ) : 362 - 369 ( 1990 ) . FOREIGN PATENT DOCUMENTS Bekal et al . , “ Purification of Leuconostoc mesenteroides Citrate WO WO 2006 /031424 3 / 2006 Lyase and Cloning and Characterization of the citCDEFG Gene WO WO 2006 / 034156 3 / 2006 Cluster, " J . Bacteriol. 180 :647 -654 ( 1998 ) . WO WO 2007 / 141208 12 / 2007 Berg et al. , “ A 3 -Hydroxypropionate / 4 -Hydroxybutyrate WO WO 2008 / 080124 7 / 2008 Autotrophic Carbon Dioxide Assimilation Pathway in Archaea, " WO WO 2008 / 115840 9 / 2008 Science 318 ( 5857 ) 1782 - 1786 (2007 ) . wo WO 2008 / 131286 10 / 2008 Bergquist and Gibbs, “ Degenerate oligonucleotide gene shuffling, " WO WO 2009 /076676 6 / 2009 Meth . Mol. Biol. 352 : 191 - 204 ( 2007 ) . WO WO 2009 /094485 7 / 2009 Bergquist et al ., “ Degenerate oligonucleotide gene shuffling WO WO 2009 / 111513 9 / 2009 (DOGS ) and random drift mutagenesis (RNDM ) : Two complemen WO WO 2010 / 031077 3 / 2010 tary techniques for enzyme evolution ,” Biomol. Eng . 22 :63 - 72 WO WO 2010 /031079 3 / 2010 ( 2005 ) . WO WO 2011 /052718 5 / 2011 Bernhard et al. , “ Functional and structural role of the cytochrome b wo WO 2011 / 140171 11 / 2011 subunit of the membrane -bound hydrogenase complex of Alcaligenes eutrophus H16 , ” Eur. J . Biochem . 248 , 179 - 186 ( 1997 ) . OTHER PUBLICATIONS Binstock and Schulz , “ Fatty acid oxidation complex from Escherichia coli ," Methods Enzymol. 71 ( Pt C ) :403 - 411 ( 1981 ) . Alber et al. , “ Malonyl- Coenzyme A Reductase in the Modified Bisswanger, “ Substrate Specificity of the Pyruvate Dehydrogenase 3 -Hydroxypropionate Cycle for Autotrophic Carbon Fixation in Complex from Escherichia coli ,” H . , J . Biol. Chem . 256 : 815 - 82 Archaeal Metallosphaera and Sulfolobus spp ., " J. Bacteriol. ( 1981 ) . Blaschkowski et al. , “ Routes of Flavodoxin and Ferredoxin Reduc 188 : 8551 - 8559 ( 2006 ) . tion in Escherichia coli, ” Eur. J . Biochem . 123 : 563 - 569 ( 1982 ) . Altincicek et al. , " LytB protein catalyzes the terminal step of the Blazquez et al. , “ Identification and analysis of a glutaryl - CoA 2 - C -methyl - D - erythritol- 4 -phosphate pathway of isoprenoid bio dehydrogenase- encoding gene and its cognate transcriptional regu synthesis , ” FEBS Lett . 532 ( 3 )437 -440 ( 2002 ) . lator from Azoarcus sp . CIB , " Environ . Microbiol. 10 ( 2 ): 474 -482 Anderson et al ., “ Isopentenyl diphosphate : dimethylallyl (2008 ). diphosphate isomerase . An improved purification of the enzyme and Bochar et al. , “ 3 -hydroxy - 3 -methylglutaryl coenzyme A reductase isolation of the gene from Saccharomyces cerevisiae, " J . Biol. of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Chem . 264 (32 ) : 19169 - 19175 ( 1989 ) . Escherichia coli of the hmgA gene , and purification and kinetic Andersson et al. , “ Effect of different carbon sources on the produc characterization of the gene product ,” J . Bacteriol. 179 ( 11 ) :3632 tion of succinic acid using metabolically engineered Escherichia 3638 ( 1997 ). coli, ” Biotechnol. Prog . 23 ( 2 ) : 381 - 388 ( 2007 ) . Bock et al. , “ Purification and Characterization of Two Extremely Aoshima and Igarashi, “ A novel oxalosuccinate - forming enzyme Thermostable Enzymes, Phosphate Acetyltransferase and Acetate involved in the reductive carboxylation of 2 - oxoglutarate in Kinase , from the Hyperthermophilic Eubacterium Thermotoga Hydrogenobacter thermophilus TK - 6 ," Mol. Microbiol. 62 :748 -759 maritime, " J . Bacteriol. 181: 1861- 1867 ( 1999 ) . (2006 ) . Boiangiu et al. , “ Sodium Ion Pumps and Hydrogen Production in Aoshima and Igarashi, “ Nondecarboxylating and Decarboxylating Glutamate Fermenting Anaerobic Bacteria , ” J . Mol. Microbiol. Isocitrate Dehydrogenases : Oxalosuccinate Reductase as an Ances Biotechnol . 10 : 105 - 119 ( 2005 ) . tral Form of Isocitrate Dehydrogenase, ” J . Bacteriol. 190 :2050 Bonner and Bloch , “ Purification and properties of fatty acyl 2055 ( 2008 ) . thioesterase I from Escherichia coli, ” J. Biol. Chem . 247 ( 10 ) :3123 Aoshima et al ., “ A novel biotin protein required for reductive 3133 ( 1972 ) . carboxylation of 2 -oxoglutarate by isocitrate dehydrogenase in Botella -Pavia et al. , “ Regulation of carotenoid biosynthesis in Hydrogenobacter thermophilus TK - 6 ," Mol. Microbiol. 51: 791 - 798 plants : evidence for a key role of hydroxymethylbutenyl ( 2004 ) . diphosphate reductase in controlling the supply of plastidial Aoshima et al ., “ A novel enzyme, citryl- CoA lyase , catalysing the isoprenoid precursors , ” Plant J . 40 ( 2 ) : 188 - 199 (2004 ) . second step of the citrate cleavage reaction