|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 ., “ - Type 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 ) 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

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Coenzyme A thioester hydrolysis ,” FEBS Lett. 516 ( 1 - 3 ) : 161 - 163 Wood et al. , “ A challenge for 21st century molecular biology and (2002 ) . biochemistry : what are the causes of obligate autotrophy and methanotrophy ? , ” FEMS Microbiol. Rev . 28 : 335 - 352 ( 2004 ) . * cited by examiner U . S . Patent Jun . 26 , 2018 Sheet 1 of 24 US 10 ,006 ,055 B2 Isoprene OPP Dimethylally Diphosphate oppoynoportoda MevalonateopMevalonate Diphosphate Y

o

F OPP

HO,C M IsopentenylDiphosphate de OHDE ou

1.FIGFIG.1 ,HO Mevalonate

C

° 3-Hydroxy methylglutaryl ???\, ?? SCOATBHO,CSCOA COA

? Co4-y AcetylCOAAAcetoacetyl-CoA SCOA

2 UU. S. .S . PatentPatent Junmoson . 26 , 2018 somSheet 2kan of 24 US 10 ,006 ,055 B2

F TOH Crotonaldehyde Crotylalcohol F Top topp HT OH >Butadiene

O D

Crotonate 2-butenyl4phosphate LoomSCOA Crotonyl-CoA 2-butenyl4diphosphate

FIG.2

SCOA o

??? Acetoacetyl-CoA3-Hydroxybutyryl-CoA COA COA COA COA Glutacony-COA Glutaryl-CoA WhatIscoaSCOA NH30 3-AminobutyrylCoA 4-HydroxybutyrylCoA ?? HO ??

NovelCrotylAlcoholPathwaytoButadiene A

2SCOAAcetylCoA U . S . Patent Jun . 26 , 2018 Sheet 3 of 24 US 10 , 006 , 055 B2

1-Hydroxy2butenyl4 OPP2Butenyl-4 diphosphate Erythritol-2,4cyclodiphosphate diphosphate Butadiene PP W

E Q O

POZ.pr.02 O LE HO OPP

H

Butenyl4-diphosphateW OP FIG.3 HOVYOPP-CytwwwH0Y0PP-Cyt ErythroseErythrose cytidine'Phospho(5-4Cytidine2diphospho)-erythritol OH OH .Ho HI OHErythritol HO OH HOY OH

- y B

OH OH OH YOP Erythrose-4phosphate HOVYOP phosphateErythritol-4 A OH U . S . Patent Jun . 26 , 2018 Sheet 4 of 24 US 10 , 006 , 055 B2

Butadiene FIG.4

-

A 2-Butenyl4-diphosphate Butenyl4-diphosphate PPOPPO

G

-OH OH 0 5-Hydroxy3~OXO 0 o

M 345PDP 0 3,5-Dioxopentanoate Pentanoate PPO

OHF OH– OH

0

Malonyl-CoA.Acetyl ??? OH 3-OxoglutarylCoA B 3-HydroxyglutarylCoA C E AL 3,5-Dihydroxy 345PP 0 CoALLOHCOAL 3-Hydroxy5oxopentanoate pentanoate OH COACOA COA PO

J atent Jun . 26 , 2018 Sheet 5 of 24 US 10 ,006 ,055 B2

Crotylalcohol ~ Crotonaldehyde ?? Crotonate D SCOA VSCOA Crotonyl-COA

C FIG.5 SCOA ? VSCOA 3-HydroxybutyrylCOA ??

Gii=OmiSCOAB SCOA Acetoacetyl-CoA

A SCOA AcetylCOA

2 U . S . Patent Jun . 26 , 2018 Sheet 6 of 24 US 10 ,006 ,055 B2

Acetyl - CoA synthetase or Acetate kinase, Phosphotransacetylase - ATP Acetyl- CoA r Acetate Citrate lyase ATP - Citrate lyase Citrate Oxaloacetate - ATP Aconitase 2[ H Malate dehydrogenase D - socitrate

Malate socitrate dehydrogenase 2[ H ] , CO2 Fumarase a - ketoglutarate Alpha- ketoglutarate : Fumarate ferredoxin Fumarate oxidoreductase reductase Succinyl- CoA 2CH) , - C02 Succinyl- CoA Succinate transferase or Succinyl- CoA synthetase 2 Ferredoxin (RD ) 2 Ferredoxin (Ox ) - ATP NAD (P )+ V NAD( P ) Ferredoxin :NAD (P ) + oxidoreductase CO + H20 2[ H ] + CO2 CO dehydrogenase H2 + 2[ H ] Hydrogenase FIG . 6 U . S . Patent Jun . 26 , 2018 Sheet 7 of 24 US 10 , 006 , 055 B2

Phosphoenolpyruvate Pyruvate PEP carboxylase Pyruvate Pyruvate :ferredoxin Pep cotonylaseof Malic carboxylase oxidoreductase PEP carboxykinose enzyme Armate or HATP 2011 Pyruvate formate lyase , COM formate dehydogenase Acetyl- CoA synthetase Acetate kinase , Phosphotransacetylase CO2 -ATP Acetyl - CoA tom Acetate Citrate lyase ATP - Citrate lyase Citrate Oxaloacetate ATP Aconitase 2H Malate dehydrogenase Isocitrate D - socitrate Acetyl- CoA lyase Malate pe w Glyoxylate + Malate synthase dehydrogenasesocitrate 2H ), CO2 Fumarase Alpha - ketoglutaratea : - ketoglutarate Fumarate ferredoxin Fumarate Oxidoreductaseoxidoreductase reductosectose Succinyl- CoA 241, - C07 2[ H ] Succinyl- COA Succinate transferase or Succinyl- CoA synthetase 2 Ferredoxin( RD ) 2 Ferredoxin( Ox ) - ATP NAD (P ) + I NAD (P ) H Ferredoxin: NAD (P ) + oxidoreductase CO 002 CO dehydrogenase , + 2 [H ] H2 ? 2H + H2 hydrogenase , + 2[ H ] + NATP FIG . 7 U . S . Patent Jun . 26 , 2018 Sheet 8 of 24 US 10 , 006 , 055 B2 wwwwwwwwww 8FIG. U . S . Patent Jun . 26 , 2018 Sheet 9 of 24 US 10 ,006 ,055 B2

105120

90

Time(s) 456075

COoxidation -PZA335.2(3:31pm)ACS90.2(3:42pm) -PZA335.3(5:38pm) -PZA33S.1(:58pm)-ACS90.1(2:07pm) -AC590.3(5:50pm)-ACS91.3(5:45pm) TT 30

-

- 15 -

- wil 0 0 0.3 0.1 0.35 0.05 ) 578nm( Abs FIG.9 105120

90

456075 Time(s) PZA33S.supe4(6:34pm) COoxidation -PZA335.3(5:38pm) -Moorella.supe4(6:38pm) -Moorella.2(3:38pm) -Moorella.3(5:42pm) .PZA3352(3:31pm) -Moorella.4(6:56pm) 30 15

be 0 anawe ot-0.57 ) 578nm ( Abs U . S . Patent Jun . 26 , 2018 Sheet 10 of 24 US 10 ,006 ,055 B2

Acetyl- CoA synthetase Acetate kinase, Phosphotransacetylase - ATP Acetyl- CoA L Acetate Citrate lyase ATP - Citrate lyase * Citrate Oxaioacetate - ATP Aconitase 2[ 1h Malote dehydrogenase D - Isocitrate Isocitrate Malate dehydrogenase2 [H ) , CO Fumarase a - ketoglutarate Alpha - ketoglutarate: Fumarate ferredoxin fumarate oxidoreductase reductase Succinyl- CoA 2[ H ] ,- com 2 [H ] Succinyl - COA Succinate transferase or Succinyl - CoA synthetase 2 Ferredoxin (RD ) 2 Ferredoxin( OX ) - ATP NAD (P )+ I NAD (P )H Ferredoxin: NAD (P ) + oxidoreductase CO+ H2O + 2[ H] +CO2 CO dehydrogenase H22 [H ] Hydrogenose FIG . 10A U . S . Patent Jun . 26 , 2018 Sheet 11 of 24 US 10 , 006 , 055 B2

CrotonaldehydeET Crotylalcohol Crotonate Crotonyl-COA

106.FIGFIG.10B ? 3-HydroxybutyrylCOA iii??. 2SCOAÀWscoaBVSCOACNSCOADMH Acetoacetyl-COA

AcetylCOA U . S . Patent Jun . 26 , 2018 Sheet 12 of 24 US 10 ,006 ,055 B2

Acetyl- CoA synthetase or Acetate kinase , Phosphotransacetylase - ATP Acetyl - CoA Acetate Citrate lyase ATP - Citrate lyase - Citrate Oxaioacetate - ATP Aconitase 2[ HhMalate Malate dehydrogenase D - socitrate socitrate Molate dehydrogenase2 [H ) , CO2 Fumarase a - ketoglutarate Alpha - ketoglutarate : Fumarate ferredoxin Fumarate oxidoreductase reductase Succinyl- CoA 2[ H ) , CO2 2[ H ] Succinyl - CoA Succinate transferase or Succinyl -CoA synthetase 2 Ferredoxin (RD ) 2 Ferredoxin( OX ) - ATP NAD (P )+ I NAD (P )H Ferredoxin :NAD (P ) + oxidoreductase CO + H20 + 2[ H ]+ CO2 CO dehydrogenase H2 - 2[ 4 ] Hydrogenose FIG . 11A atent Jun . 26 , 2018 Sheet 13 of 24 US 10 ,006 ,055 B2

Pp OH Crotonaldehyde E V Crotylalcohol Butadiene ?? Crotonate D SCOASOA 2-butenyl4phosphate CMSCOADCrotonyl-COA 2-butenyl4diphosphate FIG.11B SCOA O 3-HydroxybutyrylCoA OH ?????roxybay?ycoA B ???SCOAAcetoacety-COA Acetoacetyl-COA

onsii?SCOAA SCOA AcetylCOA

2 atent Jun . 26 , 2018 Sheet 14 of 24 US 10 ,006 ,055 B2

ATGGCAGTGGATTCACCGGATGAGCGGCTACAGCGCCGCATTGCACAGTTGTTTGCAGAAGATG AGCAGGTCAAGGCCGCACGTCCGCTCGAAGCGGTGAGCGCGGCGGTGAGCGCGCCCGGTATGCG GCTGGCGCAGATC6ccccCACTGTHAT G6ccGGTTACC?CGAcccccccccccccGGGCAGCGT GCGTTCGAACTGAACACCGACGACGCGACGGGCCGCACCTCGCTGCGGTTACTTCCCCGATTCG AGACCATCACCTATCGCGAACTGTGGCAGCGAGTCGGCGAGGTTGCCGCGGCCTGGCATCATGA TCCCGAGAACCCCTTGCGCGCAGGTGATTTCGTCGCCCTGCTCGGCTTCACCAGCATCGACTAC GCCACCCTCGACCTGGCCGATATCCACCTCGGCGCGGTTACCGTGCCGTTGCAGGCCAGCGCGG CGGTGTCCCAGCTGATCGCTATCCTCACCGAGACTTCGCCGCGGCTGCTCGCCTCGACCCCGGA GCACC } CGA ??????? } ??AGE ?CC ?????????CAC?????????AC?AC {?? G???? } C GACE ACC???????????????ACCA?????????CT CAA ???????????CGC0????? ACGCGGGCAGCTTGGTGATCGTCGAAACGCTCGATGCCGTGCGTGCCCGGGGCCGCGACTTACC ?????????????? } ?? ??????????????C?????????????? ????? ACAC??c? G?CAGOACC?GAA??CC????????????????????? ??? } ????????????? ??CAGG GGAACTCGATGCTGCAGGGGAACTCGCAACGGGTCGGGATCAATCTCAACTACATGCCGATGAG CCACATCGCCGGTCGCATATCGCTGTTCGGCGTGCTCGCTCGCGGTGGCACCGCATACTTCGCG ?CCAAGA?CGACA ]?????ACT? ???AAGAC? ????? ??? A?? ?????c?AGA ??? TCGTCCCGCGCGTGTGCGACATGGTCTTCCAGCGCTATCAGAGCGAGCTGGACCGGCGCTCGGT GGCGGGCGCCGACCTGGACACGCTCGATCGGGAAGTGAAAGCCGACCTCCGGCAGAACTACCTC GGTGGGCGCTTCCTGGTGGCGGTCGTCGGCAGCGCGCCGCTGGCCGCGGAGATGAAGACGTTCA ????????? } CC CGA ? ???AC } ?CA?GA???? AC??????ACC????????CGCAAG??? GCTGCTCGACAACCAGATCCAGCGGCCGCCGGTGCTCGATTACAAGCTCGTCGACGTGCCCGAA C G & G } ACT ????ACC??????????? { ??????????????? } ??? ??AAGG?????ACCA C?? } { ?????CTAC ] ACAA?????????????????????AT? } } C???????????? } CTA ?AAGACCG???? ? ??????????????GA?????????????0 ? ??CGAccG C?CAA AA } ?? G????????? 0?CA?????AG { ????ACC??????CA } C { £GA???c? } { { C???A GCAGCCCGCTGATCCGGCAGATCTTCATCTACGGCAGCAGCGAACGTTCCTATCTGCTCGCGGT GA ???????????????????????????????ACcG?CACT{ ???? } ?????????? ?AAG } cG? ?CA?CGCA CGCCAA??????????????????? ??????NE ¥c???????? ??? ??? { c?AGAC??????? { ?ACCA } c?CCAA????? ??c? ?????? ??????????? G?? CCCCAATCTGAAGGAACGCTACGGCGCTCAGCTGGAGCAGATGTACACCGATCTCGCGACAGGC C??GCcGA ????????????? } ????????????????????????????? } ??AAAcc??CA ??????CA?CG???????????????????C ???????? ??????c?????????ACT CA CGAC? } ??????GA ! } c?? } T { ?????? } ?¥c? ?? C?????? } ???????? { C }¥000 ??C??????CC??????????? G???????????????????????????????AAT EACA TTGAGGCGGAACGCAACTCGGGCGCGAAGCGTCCCACCTTCACCTCGGTGCACGGCGGCGGTTC C????? ???????????? } ?? GAcc?? ?GACAAGE CATCG????????Acc???????????0 GACAGCATTCCGCACGCGCCGGTGCCAGCGCAGACGGTGCTGCTGACCGGCGCGAACGGCTACC TCGGCCGGTTCCTGTGCCTGGAATGGCTGGAGCGGCTGGACAAGACGGGTGGCACGCTGATCTG FIG . 12A atent Jun . 26 , 2018 Sheet 15 of 24 US 10 ,006 , 055 B2

CGTCGTGCGCGGTAGTGACGCGGCCGCGGCCCGTAAACGGCTGGACTCGGCGTTCGACAGCGGC GATCCCGGCCTGCTCGAGCACTACCAGCAACTGGCCGCACGGACCCTGGAAGTCCTCGCCGGTG AEA¥ ?????ACC?GAATC¥??? % AC ACGC % AC ????????? { { ????????CcGT??? ccTGATCGTCCATcccccccccTT GGTCAACCACCTCCTICCCTACACCCAGCTGTTCGGcccc A? ??? ??????????????AAA C??????? ?????ATC????????????AA???????ACC ACCTGTCGACCGTCGGAGTGGCCGACCAGGTCGACCCGGCGGAGTATCAGGAGGACAGCGACGT C??????? { GA????????????T?? } ?????????? } AC????????? ACCGCAA??????? TGGGCGGGGGAGGTCCTGCTGCGCGAAGCACACGATCTGTGTGGCTTGCCGGTCGCGGTGTTCC GTTCGGACATGATCCTGGCGCACAGCCGGTACGCGGGTCAGCTCAACGTCCAGGACGTGTTCAC CCGGCTGATCCTCAGCCTGGTCGCCACCGGCATCGCGCCGTACTCGTTCTACCGAACCGACGCG GACGGCAACCGGCAGCGGGCCCACTATGACGGCTTGCCGGCGGACTTCACGGCGGCGGCGATCA CCGCGCTCGGCATCCAAGCCACCGAAGGCTICCGGACCTACGACGTGCTCAATCCGTACGACGA TGGCATCTC?CTCGATGAATICGTCGACTGQ??????QAATCCGGCCACCCGATCCAGCGCATC ACCGACTACAGCGACTGGTTCCACCGTTTCGAGACGGCGATCCGCGCGCTGCCGGAAAAGCAAC GCCAGGccTCGGTGCTGCCGTTGCTGGACGCCTACCGCAACCCCTGCCCGGCGGTccGcGGCGC GATACTCCCGGCCAAGGAGTTCCAAGCGGCGGTGCAAACAGCCAAAATCGGTCCGGAACAGGAC ATCCCGCATTTGTCCGCGCCACTGATCGATAAGTACGTCAGCGATCTGGAACTGCTTCAGCTGCwww C T G VAIV T FIG . 12A cont. mavdspder Igrriaglfaedequkaarpleavsaavsapgmrlagiaatvmagyadr poagar afelntddatgrtslrllprfetityrelworvgevaga whhdpenplragdfvallgftsidy atldladihlgavtvplqasoavsqlioiltetsprllastpehldaovecllagttper lvyf dyhpedddgraafesarrrladagslvivetldavrargrdlpaaplfvpdtdddplalliyts gstgtpkgamytnrlaotmwagnsmlagnsarvginlnympmshiagrislfgvlarggtayfa aksdms tlfediglvrpteiffvprvcdmvfgryqseldrrsvagadldtidrevkadlranyl ggr flvavvgsaplaaemkt fmesvidlplhdgygsteagasvlldqiqrppvidyklvdvpe lgyfrtdrphprgelllkaet tipyykrpevtaeifdedgfyktgdivaelendr lvyverrn nviklsqgefvtvahleavfassplirqifiygssersyllavivptddolrgrdtatiksola esiqriakdanlqpyeiprdflietepftiangllsgiakllrpnlkerygaqleqmytdlatg qodellalrredodlpvletysraakamlgvasodmrpdohftdlagdsisalsfsnllheifg vevpvgvvvspanelrdlanyiegernsgakrptftsvhgggseiraodltidkfidartlaaa dsiphopvpaqtvlltgangylgrflclewler ldk tggtlicvvrgsdagdarkrldsafdsg dpgllehyaqlaortlevlogdigdpolglddatworlaetvdlivhpaalvnhvlpytqlfgp nyugtaeivrlaitarrkpvtylstvgvadgvdpaeyqedsdvremsavr vyresyangygnsk wagevllreahdlcglpvavfrsdmilahsr yagolnvqdvftrlilslvatgiapysfyrtda dgnrarahydglpadftaaditalgigategfr tydvinpyddgisldefvdwlvesghpiqri tdysdwfhr fetairalpekor qasvlplldayrnpcpavrgailpakefqaavatakigped iphlsaplidkyvsdlellqll * FIG . 12B U . S . Patent Jun . 26 , 2018 Sheet 16 of 24 US 10 ,006 ,055 B2

ATGATTGAAACCATTCTGCCTGCAGGCGTTGAAAGCGCAGAACTGCTGGAATATCCGGAAGATCTTAA TGAAAGCACATCCGGCAGAAGAACATCTGATTGCCAAAAGCGTTGAAAAACGTCGTCGTGATTT TATTGGTGCACGTCATTGTGCACGTCTGGCACTGGCAGAACTGGGTGAACCTCCGGTTGCAATT GGTAAAGGTGAACGTGGTGCACCGATTTGGCCTCGTGGTGTTGTTGGTAGCCTGACCCATTGTG ATGGTTATCGTGCAGCAGCAGTTGCACATAAAATGCGCTTTCGCAGCATTGGTATTGATGCAGAT A ACCGCATGCAACCCTGCCGGAAGGTGTTCTGGATAGCGTTAGCCTGCCGCCGGAACGTGAATGG CTGAAAACCACCGATAGCGCACTGCATCTGGATCGTCTGCTGTTTTGTGCAAAAGAAGCCACCT ATA AAAAGCCLAA GGfGGCCSCFGACA CACGIGGCYGGGI IIGAAGAAGCCCATALYACCAAVA LA } } } GA TA T AATTGAAGATGGTAGCGCAGATAGCGGTAATGGCACCTTTCATAGCGAACTGCTGGTTCCGGGTASAVVUN

AMAT * CAGACCAATGATGGTGGTACACCGCTGCTGAGCTTTGATGGTCGTTGGCTGATTGCAGATGGTTVUNUA TTATTCTGACCGCAATTGCCTATGCCTAAT T ATTA FIG . 13A

mietilpagvesaelleypedlkohpaeehliaksvekrrrdfigarhcarlaloelgeppvai gkger gapiwprgvvgslthcdgyrogavankmr frsigidaephatipegvldsvslpperew Ikttdsalhldr llfcakeatykawwpltarwlgfeeahitfeiedgsadsgngtfhsellvpg qindggtpllsfdgrp wliadgfiltoiayaf + * FIG . 13B atent Jun . 26 , 2018 Sheet 17 of 24 US 10 ,006 ,055 B2 atgaccagcgatgttcacgacgccacagacggcgtcaccgadaccgcactcgacgacgagcagtcgacccgccgcat cgccgagctgtacgccaccgatcccgagttcgccgccgccgcaccgttgcccgecgtggtcgacgcggcgcacaaac ccgggctgcggctggcagagatcctgcagaccctgttcaccggctacggtgaccgcccggcgctgggataccgcgcc cgtgaactggccaccgacgagggcgggcgcaccgtgacgcgtctgctgccgcggttcgacaccctcacctacgccca ggtgtggtcgcgcgtgcoagcggtcgccgcggccctgcgecacaocttcgcgcagccgatctoccccggcgacgccg togogocgatcggtttcgcgagtcccgattacctgocgctggotctcgtatgcgcctocctgggcctcgtgagtgtt ccgctgcagcocaacgcoccggtcagccggctcgccccgatcctggccgaggtcgoaccgcggatcctcaccgtgag cgccgaotacctcgacctcgcagtcgaatccgtgcgggacgtcoactcggtgtcgcagctcgtggtgttcgaccato accccgaggtcgacgaccaccgcgacgcactggcccgcgcgcgtgaacaactcgccggcoagggcatcgccgtcaco accctggacgcgatcgccgacgagggcgccgggctgccggccgaaccgatctacaccgccgaccatgatcagcgcct cgcgatgatcctgtacacctcgggttccaccggcgcacccaagggtgcgatgtacaccgaggcgatggtggcgcggo tgtggaccatgtcgttcatcacgggtgaccccacgccggtcatcaacgtcaacttcatgccgctcaoccacctgggc gggcgcatccccatttccaccgccgtgcagaacggtggaaccagttacttogtaccggaatccgocatgtccocgct gttcgaggatctcgcgctggtgcgcccgaccgaac tcggcctggttccgcgcgtcgccgacatgctctaccagcaco acctcgccoccgtcgoccgcctggtcacgcogggcgccgacgoactgaccgccgagoagcaggccggtgccgaactg cgtgagcaggtgctcggcggacgcgigotcoccggattcgtcagcaccgcaccgctggccgcggagatgagggcgtt cctcgacatcaccctgggcgcacacatcgtcgacggctacgggctcaccgagaccggcgccgtgacacgcgocggtg tgatcgtgcggccaccggtgatcgactacoagctgatcgacgttcccgaactcggctacttcagcaccgacoagccc taccogcgtggcgaactgctggtcaggtcgcaaacgctgactoccgggtactocaagcgccccgaggtcaccgcgag cgtcttcgaccgggacggctoctoccacaccggcgacgtcatggccgagaccgcacccgaccacctggtgtocgtgg accgtcgcoacoacgtcctcadactcgcgcagggcgagttogtggcggtcgccaacctggaggcggtgttctccggo goggcgctggtgcgccagatcttcgtgtacggcaacagcgagcgcagtttccttctggccgtggtggtcccgacgcc ggaggcgctcgagcagtocgatccggccgcgctcaaggccgcgctggccgactcgctgcagcgcaccgcacgcgacg ccgaactgcoatcctacgaggtgccggccgatttcatcgtcgagaccgagccgttcagcgccgccaacgggctgctg togggtgtcggagoactgctgcggcccaocctcaaagaccgctacgggcagcgcctggagcagatgtacgccgatat cgcggccacgcaggccaoccagtigcgcgaactgeggcgcgcggccgccacacaaccggtgotcgacaccctcocco aggccgctgccacgatcctcggcaccgggogcgaggtggcatccgacgcccacttcaccgacctgggcggggattcc ctgtcggcgctgacactttcgoacctgctgagcgatttcttcggtttcgaogttcccgtcggcaccatcgtgaocco ggccaccaacctcgcccaoctcgcccagcocatcgaggcgcagcgcaccgcgggtgaccgcaggccgagtttcacca cogtgcacggcgcggacgccaccgagatccgggcgagtgagctgaccctggacaagttcatcgacgccgaoacgcto cgggccgcaccgggtctgcccaaggtcaccaccgagecacggacggtgttgctctcgggcgccaacggctggctggg ccggttcctcacgtigcagtggctggaacgcctggcacctgtcggcggcoccctcatcacgatcgtgcggggccgcg acgacgccgcggcccgcgcacggctgacccaggcctacgacaccgatcccgagttgtcccgccgcttcgccgagctg gocgaccgccacctgcgggtggtcgccggtgacatcggcgacccgaotctgggcctcocacccgogatctggcaccg gctcgccgccgaggtcgacctggtggtgcatccggcagcgctggtcoaccacgtgctcccctaccogcogctgttcg gccccaacgtcgtgggcacggccgaggtgatcaagctggccctcaccgaacggotcaagcccgtcocgtacctgtcc accgtgtcggtggccatggggatccccgacttcgaggaggacggcgacatccggaccgtgagcccggtgcgcccgct cgacggcggatacgccaacggctacggcaacagcaagtgggccggcgaggtgctgctgcgggaggcccacgotctgt gcgggctgcccgtggcgacgttccgctcggacatgatcctggcgcatccgcgctaccgcggtcaggtcaacgtgcca FIG . 14A U . S . Patent Jun . 26 , 2018 Sheet 18 of 24 US 10 ,006 ,055 B2

gacotgttcacgcgactcctgttgagcctcttgatcaccggcgtcgcgccgcggtcgttctacatcggagacggtga gcgcccgcgggcgcactaccccggcctgocggtcgatttcgtggccgaggcggtcacgocgctcggcgcgcagcago gcgagggatacgtgtcctacgacgtgatgaacccgcacgacgacgggatctccctggatgtgttogtggactggctg atccgggcgggccatccgatcgoccgggtcgacgactacgacgactgggtgcgtcggttcgagaccgcgttgaccgo gcttcccgagaagcgccgcgcacagaccgtactgccgetgctgcacgcgttccgcgctccgcaggcaccgttgcgcg gcgcacccgaacccacggaggtgttccacgccgoggtgcgcaccgcgaaggtgggcccgggagacatcccgcacctog g gacgaggcgctgatcgacaagtacatacgcgotctgcgtgagtteggtctgatctaa FIG . 14A Cont.

MTSDVHDATDGVTETALDDEQSTRRIAELYATDPEFAAAAPLPAVDAAHKPGLRLAEILQTLFTGYGDRPALGYRA RELATDEGGRTVTRLLPRFDTLTYAQVWSRVQAVAAALRHNFAQPIYPGDAVATIGFASPDYLTLDLVCAYLGLVSV PLQHNAPVSRLAPILAEVEPRILTVSAEYLDLAVESVRDVNSVSQLVVFDHHPEVDDHRDALARAREQLAGKGIAVT TLDAIADEGAGLPAEPIYTADHDQRLAMILYTSGSTGAPKGAMYTEAMVARLWTMSFITGOPTPVINVNFMPLNHLG GRIPISTAVQNGGTSYFVPESOMSTLFEDLALVRPTELGLVPRVADMLYQHHLATVDRLVTQGADELTAEKQAGAEL REQVLGGRVITGFVSTAPLAAEMRAFLDITLGAHIVDGYGLTETGAVTRDGVI VRPPVIDYKLIDVPELGYFSTOKP YPRGELLVRSQTLTPGYYKRPEVTASVFORDGYYHTGDVMAETAPDHL VYVORRNNVLKLAQGEFVAVANLEAVFSG AALVRQIFVYGNSERSFLLAVVVPTPEALEQYDPAALKAALADSLQRTARDAELQSYEVPADFIVETEPFSAANGLL SGVGKLLRPNLKDRYGQRLEQMYADIAATQANQLRELRRAAATOPVIDTLTQAAATILGTGSEVASDAHFTDLGGOS LSALTLSNLLSOFFGFEVPVGTIVNPATNLAQLAQHIEAQRTAGORRPSFTTVHGADATEIRASELTLDKFIDAETLT RAAPGLPKVTTEPRTVLLSGANGWLGRFLTLQWLERLAPVGGTLITI VRGRDDAAARARLTQAYDTDPELSRRFAEL ADRHLRVVAGDIGDPNLGLTPE I WHRLAAEVDLVVHPAALVNHVLPYRQLFGPNVVGTAEVIKLALTER IKPVTYLS TVSVAMGIPDFEEDGDIRTVSPVRPLDGGYANGYGNSKWAGEVLLREAHDLCGLPVATERSOMILAHPRYRGQVNVPF F DMFTRLLLSLLITGVAPRSFYIGOGERPRAHYPGL TVDFVAEAVTTLGAQQREGYVSYDVMNPHDDGISLOVFVDWLUntu DIET HAFRA PRC HUA VIP CIALIALT L 11 IVOWAT LUO E P CYT AANVRANVR 20 DEALIDKYIRDLREFGLI FIG . 14B U . S . Patent Jun . 26 , 2018 Sheet 19 of 24 US 10 ,006 , 055 B2

atgtcgactgccacccatgacgoocgoctcgaccgtcgcgtccacgaactcatcgccaccgacccgcaattcgccgc cgcccaacccgacccggcgatcoccgccgccctcgaacagcccgggctgcggctgccgcagatcatccgcaccgtgc togacggctacgccgaccggccggcgctgggacagcgcgtggtggagttcgtcacggacgccaagaccgggcgcacg toggcgcagctgctcccccgcttcgagaccatcacgtacagcgaagtagcgcagcgtgtttcggcgctgggccgcgc cctgtccgacgacgcggtgcoccccggcgaccgggtgtgcgtgctgggcticaacagcgtcgactacgccaccotcg acatggcgctgggcgccatcggcgccgtctcggtgccgctgcagoccogcgcggcoatcogctcgctgcagccgoto gtggccgagoccgogcccaccctcatcgcgtccagcgtgaaccogctgtccgacgcggtgcagctgotcaccggcgc cgagcaggcgcccacccggctggtggtgttcgactaccacccgcaggtcgocgaccagcgcgaggccgtccaggocg ccgoggcgcggctgtccagcaccggcgtggccgtccagacgctggccgagctgctggagcgcggcaaggacctgccc gccgtcgcggagccgcccgccgocgaggactcgctggccctgctgatctacacctccgggtccaccggcgcccccaO gggcgcgatgtacccocagagcoacgtcggcoagotgtggcgccgcggcagcaagaactggtteggcgagagcgccg cgtcgatcaccctgoacttcatgccgotgagccocgtgatgggccgaagcatcctctacggcacgctgggcaacggo ggcaccgcctacttcgccgcccgcagcgacctgtccaccctcttgaggacctcgagctggtgeggcccaccgagct coacttcgtcccgcggatctgggagacgctglacggcgaattccagcgtcaggtcgagoggcggctctccgaggccgO t o gggacgccggcgaacgtcgcgccgtcgaggccgaggtgctggccgagcagcgccagtacctgctgggcgggcggtto accttcgcgotgacgggctcggcgcccatctcgccggagctgcgcoactgggtcgogtcgctgctcgaaatgcacct gotggacggctacggctccaccgaggccggaotggtgttgttcgocggggagattcagcgcccgccggtgotcgact acaagctggtcgacgtgccggacctgggctacttcogcaccgaccggccgcatccgcgcggcgagctgctgctgcgc accgagaacatgttcccgggctactacaagcgggccgadaccaccgcgggcgtcttcgacgaggacggctactacco caccggcgacgtgttcgccgagotcgccccggaccggctggtctacgtcgaccgccgcaacaacgtgctcaagctgg cgcogggcgaattogtcacgctggccoagctggaggcggtgtteggcaocagcccgctgatccgccagotctacgtc tacggcoacagcgcccagccctacctgctggcggtcgtggtgcccaccgaggaggcgctggcctcgggtgaccccga gacgctcaagcccaagatcgccgactcgctgcagcaggtcgccaaggaggccggcctgcagtcctacgaggtgccgc gcgacttcatcatcgagaccaccccgttcagcctggaaaacggtctgctgaccgggatccggaagctggcgtggccg daactgaagcagcactacggggaacggctggagcagotgtacgccgacctggccgccggacaggccaacgagctggc cgagctgcgccgcoacggtgcccaggcgccggtgttgcagaccgtgagccgcgccgcgggcgccatgctgggttcgg ccgcctccgocctgtcccccgacgcccacttcoccgatctgggcggagactcgttgtcggcgttgacattcggcaac ctgctgcgcgagatcttcgacgtcgacgtgccggtaggcgtgatcgtcagccoggccaacgacctggcggccatcgc gagctacatcgaggccgagoggcagggcagcaagcgcccgacgttcgcctcggtgcacggccgggacgcgoccgtgg tgcgcgccgccgacctgacgctggacaagttcctcgacgccgagocgctggccgccgcgccgaacctgcccaagccg gccaccgoggtgcgcoccgtgctgctgaccggcgccaccggcttcctgggccgctacctggccctggaatggctggo gcggotggacatggtggacggcoaggtcatcgccctggtccgggcccgctccgocgaggaggcacgcgcccggctgg acaagaccttcgacagcggcgacccgogactgctcgcgcactaccagcagctggccgccgotcacctggaggtcato gccggcgacaagggcgaggccaatctgggcctgggccaagacgtttggcaocgactggccgacacggtcgacgtgat cgtcgaccccgccgcgctggtcaoccacgtgttgccgtocagcgagctgttcgggcccaacgccctgggcaccgcgg agctgatecggctggcgctgacgtccaagcagoagccgtocacctacgtgtccaccatcggcgtgggcgaccagato gogccgggcaagttcgtcgagaacgccgocatccggcagatgagcgccacccgggcgatcaacgacagctacgccaa cggctatggcaocagcaagtgggccggcgaggtgctgctgcgcgaggcgcacgacctgtgcgggctgcccgtcgcgg tgttccgctgcgacatgatcctggccgacaccocgtatgccgggcagctcaacctgccggacatgttcacccggctg FIG . 15A U . S . Patent Jun . 26 , 2018 Sheet 20 of 24 US 10 ,006 ,055 B2

atgctgagcctggtggccaccgggatcgcgcccggetcgttctacgagctcgacgccgacggcaoccggcagcgggc gcactacgacggcctgccggtcgagt tcatcgccgcggcgatctcgocgctgggttcgcagatcaccgacagcgoca ccggcttccagacctaccacgtgatgaacccctacgotgacggcgtcggtctggacgogtacgtcgattggctggtg gocgccggctattegatcgagcggatcgccgactactccgaatggctgcggcggttcgagacctcgctgcgggccct gccggaccggcagcgccagtactcgctgctgccgctgctgcacaactaccgcacgccggagaagccgatcaacgggt cgotagctcccaccgacgtgttccgggcagoggtgcaggoggcgaaaotcggccccgacodagocattccgcacgtg tcgccgccggtcatcgtcaagtocatcaccgacctgcagctgctcgggctgctctaa FIG . 15A Cont.

MSTATHDERLORRVHELIATDPQFAAAQPOPAITAALEQPGLRLPQIIRTVLDGYADRPALGQRVVEFVTDAKTGRT SAQLLPRFETITYSEVAQRVSALGRALSDDAVHPGDRVCVLGFNSVDYATIDMALGAIGAVSVPLQTSAAISSLQPIPRE 1 V T S 1 VAETEPTLIASSVNQLSDAVQLITGAEQAPTRLVVFDYHPQVDDQREAVQDAAARLSSTGVAVOTLAELLERGKDLP AVAEPPADEDSLALLIYTSGSTGAPKGAMYPQSNVGKMWRRGSKNWFGESAASITLNFMPMSHVMGRSILYGTLGNG GTAYFAARSOLSTLLEDLELVRPTELNFVPRIWETLYGEFQRQVERRLSEAGDAGERRAVEAEVLAEQRQYLLGORF TFAMTGSAPISPELRNWVESLLEMHLMDGYGSTEAGMVLFDGE IQRPPVIDYKLVDVPDLGYFSTORPHPRGELLLR TENMFPGYYKRAETTAGVFDEDGYYRTGDVFAELAPDRLVYVDRRNNVLKLAQGEFVTLAKLEAVFGNSPLIRQIYVR Y YGNSAQPYLLAVVPTEEALASGDPETLKPKIADSLQQVAKEAGLQSYEVPRDFIIETTPFSLENGLLTGIRKLAWP KLKQHYGERLEQMYADLAAGQANELAELRRNGAQAPVLQTVSRAAGAMLGSAASDLSPDAHFTDLGGDSLSALTFGN LLREIFDVDVPVGVIVSPANDLAAIASYIEAERQGSKRPTFASVHGRDATVRAADLTLDKFLDAETLAAAPNLPKP ATEVRTVLLTGATGFLGRYLALEWLERMOMVDGKVIALVRARSDEEARARLOKTFDSGDPKLLAHYQQLAADHLEVI AGDKGEANLGLGQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNALGTAELIRLALTSKQKPYTYVSTIGVGDQIBU INI Antil itu EPGKFVENADIRQMSATRAINDSYANGYGNSKWAGEVLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMFTRL MLSLVATGIAPGSFYELDADGNRQRAHYDGLPVEFIAAAISTLGSQITDSDTGFQTYHVMNPYODGVGLDEYVDWLV DAGYSIERIADYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIAPTDVFRAAVQEAKIGPDKDIPHVDVRAAVDVCD SPPVIVKYITOLQLLGLL FIG . 15B U . S . Patent Jun . 26 , 2018 Sheet 21 of 24 6 , 055 B2

atgtcgccoatcacgcgtgaagagcggctcgagcgccgcotccoggacctctacgccaacgacccgcagttcgccgc cgccaaacccgccacggcgotcaccgcogcootcgagcggccgggtctaccgctococcagatcatcgagaccgtca tgaccggatacgccgatcggccggctctcgctcagcgctcggtcgoottogtgaccgocgccggcoccggccocaco acgctgcgactgctcccccacttcgaaaccatcagctacggcgogetttgggaccgcatcagcgcactggccgacgt gctcagcoccgaacagacggtgaaaccgggcgaccgggtctgcttgttgggcttcaacagcgtcgactacgccacga tcgacatgactttggcgcggctgggcgcggtggccgtaccactgcagaccagcgcggcgotaacccagctgcagccg atcgtcgccgogacccagcccaccatgatcgcggccagcgtcgacgcactcgctgacgccaccgoottggctctgtc cggtcagaccgctococgagtcctggtgttcgoccaccaccogcaggttgacgcacoccgcgcagcggtcgaatccg cccgggagcgcctggccggctcggcggtcgtcgogaccctggccgaggccatcgcgcgcggcgocgtgccccgcggt gcgtccgccggctcggcgcccggcaccgatgtgtccgocgactcgctcgcgctactgotctocacctcgggcagcac gggtgcgcccaagggcgcgatgtacccccgacgcoacgttgcgaccttctggcgcaagcgcocctggttcgaoggcg gctacgagccgtcgatcacgctgaacttcotgccaatgagccacgtcatgggccgccaaatcctgtacggcacgctg tocaatggcggcaccgcctacttcgtggcgoagagcgatctctccaccttgttcgaagacctggcgctggtgcggcc caccgagctgaccttcgtgccgcgcgtgtgggacatggtgttcgacgagtttcagagtgaggtcgaccgccgcctgg tcgocggcgccgaccgggtcgcgctcgaagcccoggtcoaggccgagatacgcaocgacgtgctcggtggacggtat accagcgcoctgoccggctccgcccctatctccgocgagotgaaggcgtgggtcgaggagctgctcgacatgcatct ggtcgagggctacggctccaccgaggccgggotgatectgatcgacggagccatteggcgcccggcggtactcgoct acaagctggtcgatgttcccgacctgggétacttcctgaccgoccggccacatccgcggggcgagttgctggtcaag accgatagtttgttcccgggctactaccagcgagccgaagtcaccgccgacgtgttcgatgctgacggcttctaccg gaccggcgacatcatggccgaggtcggccccgaacogticgtgtocctcgaccgccgcaacaacgtgttgaagctgt cgcagggcgagttegtcaccgtctccaaaclcgaagcggtgtttggcgacagcccactggtacggcagotctacatc tacggcaacagcgcccgtgcctacctgttggcggtgotcgtccccacccaggaggcgctggacgccgtgcctgtcga ggagetcoaggcgcggctgggcgactcgctgcaogaggtcgcagoggccgccggcctgcagtcctacgogatcccgc gogacttcatcatcgadacaacaccatggocgctggagaacggcctgctcaccggcatccgcaagttggccaggccg cagctgaaaoogcottacggcgagctlctcgagcagotctocacggacctggcacacggccaggccgacgaactgeg ctcgctgcgccooogcggtgccgatgcgccggtgctggtgocggtgtgccgtgcggcggccgcgctgttgggcggca gcgcctctgacgtccagcecgatgcgcacttcaccgatttgggcggcgactcgctgtcggcgctgtcgttcaccaac ctgctgcacgagatcttcgacatcgaagtgccggtgggcgtcotcgtcagccccgccaacgacttgcaggccctggo cgactacgtcgaggeggctcgcaaacccggctcgtcacggccgaccttcgcctcggtccacggcgcctcgaatgggc oggtcaccgaggtgcotgccggtgacctgtccctggacooattcatcgatgccgcaaccctggccgoagctccccgg ctgcccgccgcocacocccaagtgcgcoccgtgctgctgoccogcgccaccggcttcctcgggcgctacctggccct ggaatggctggagoggatggocctggtcgacggcaoactgatctgcctggtccgggccoogtccgacaccgaagcac gggcgcggctggacaagacgttcgacagcggcgaccccgoactgctggcccactaccgcgcactggccggcgaccac ctcgaggtgctcgccggtgacaagggcgaagccgacctcggactggaccggcagacctggcoacgcctggccgacac ggtcgacctgatcgtcgaccccgcggccctggtcaaccacgtactgccatacagccagctgttcgggcccaacgcgc tgggcaccgccgagctgctgcggctggcgctcocctccaagatcaagccctacagctacocctcgacaatcggtgtc gccgaccagatcccgccgtcggcgttcaccgaggacgccgacotccgggtcatcagcgccacccgcgcggtcgacgo cagctacgecoatggctactogaacogcoogtgggccggcgaggtgctgttgcgcgoggcgcatgacctgtgtggcc tgccggttgcggtgticcgctgcgocatgatcctggccgacaccacatgggcgggacagctcaotgtgccggacotgO 0 ttcaccoggatgatectgagcctggcggccaccggtotcgcgccgggttcgttctatgagcttgeggccgacggcgcOf FIG . 16A U . S . Patent Jun . 26 , 2018 Sheet 22 of 24 US 10 ,006 ,055 B2

ccggcaacgcgcccactatgacggtctgcccgtcgagttcotcgccgaggcgotttcgactttgggtgcgcogagcc9 g aggatggtttccacacglatcacgtgotgaacccctacgacgacggcatcggoctcgacgagttcgtcgactggctc aacgagtccggttgccccatccagcgcatcgctgactatggcgactggctgcagcgettcgaaaccgcactgcgcgc actgcccgatcggcagcggcacagctcactgctgccgctgttgcacaactatcggcagccggagoggcccgtccgcg ggtcgatcgcccctaccgatcgcttccgggcagcggtgcaagaggccaagatcggccccgacaaagacattccgcac gtcggcgcgccgatcatcgtgaagtacgtcagcgacctgcgcctacteggcctgctctaa FIG . 16A cont.

MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTATTAA ITAV TLRLLPHFETISYGEL11 WDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPPAVAVDI IVAETQPTMIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGOVPRG ASAGSAPGTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKRTWFEGGYEPSITLNFMPMSHVMGRQILYGTL CNGGTAYFVAKSOLSTLFEDLALVRPTELTFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRY TSALTGSAP ISDEMKAWVEELLOMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTORPHPRGELLVK TOSLFPGYYQRAEVTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLCVTAVE ATIVI SQGEFVTVSKLEAVFGDSPLVRQIYI YGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSYE IPRDFIIETTPWTLENGLLTGIRKLARP QLKKHYGELLEQIYTDLAHGQADELRSLRQSGADAPVLVTVCRAAAALLGGSASDVQPDAHFTDLGGDSLUN SALSETN LLHEIFDIEVPVGVIVSPANDLQALADYVEAARKPGSSRPTFASVHGASNGQVTEVHAGDLSLDKFIDAATLAEAPR LPAANTQVRTVLLTGATGFLGRYLALEWLERMDLVDGKLICLVRAKSDTEARARLDKTFDSGDPELLAHYRALAGDH LEVLAGOKGEADLGLDRQTWQRLADTVDLIVDPAALVNHVLPYSQLFGPNALGTAELLRLALTSKIKPYSYTSTIGV ADQIPPSAFTEDADIRVISATRAVDDSYANGYSNSKWAGEVLLRE AHDLCGLPVAVFRCDMILADTTWAGQLNVPDM FTRMILSLAATGIAPGSFYELAADGARQRAHYDGLPVEFIAEAISTLGAQSQDGFHTYHVMNPYDDGIGLDEFVDWL NESGCPIQRIADYGDWLQRFETALRALPDRQRHSSLLPLLHNYRQPERPVRGSTAPTORFRAAVQEAKIGPDKDIPHUN AVALAK VGAPI IVKYVSDLRLLGLL FIG . 16B atent Jun . 26 , 2018 Sheet 23 of 24 US 10 ,006 ,055 B2 atgagcaccgcaacccatgatgaacgtctggatcgtcgtgttcatgaactgattgcaaccgato cgcagtttgcagcagcacagccggatcctgcaottaccgcagcactggaacagcctggtctgcg tctgccgcagattattogtaccgttctggatggttatgcagatcgtccggcactgggtcagogt gttgttgootttgttaccgatgcaaooaccggtcgtaccogcgcocagctgctgcctcgttttg+ +

O aaaccattacctatagcgaagttgcacagcgtgttagcgcactgggtcgtgcactgagtgatgaw tgcagttcatccgggtgatcgtgtttgtgttctgggttttogtagcgttgattatgccaccattc so gatotggcactgggtgcoattggtgcagttagcgttccgctgcagaccagcgcogcaattagcat O C gcctgcagccgattgttgcagagaccgaaccgacectgattgcaagcagogttaatcagctgtcO agatgcagttcagctgattaccggtgcagaacaggcaccgacccgtctggttgtttttgattat+ catccgcaggttgatgatcagcgtgaagcagttcoggatgcagcagcacgtctgagcogcaccg gtgttgcagttcagaccctggcagaac tgctggaacgtggtaaagatctgcctgcagttgcaga accgcctgcagatgoagatagcctggcactgctgatttataccagcggtagcacaggtgcaccg agoggtgcaotgtatccgcagagcaatgttggtagaatgtggcgtcgtggtagcogadottggt ttggtgaaagcgcagcaagcattaccctgaatttcatgccgatgagccatgttatgggtcgtag cattctgtatggcaccctgggtootggtggcaccgcotattttgcagcacgtagcgatctgago accctgctggaagatctggaactggttcgtccgaccgaactgaattttgttccgcgtatttggg aaaccctgtatggtgaatttcagcgtcaggttgaacgtcgtctgagcgaagctggcgatgccgg tgaacgtcgtgcagttgaagcagoagttctggcagaacagcgtcagtatctgctgggtggtigt tttacctttgcoatgaccggtagcgcoccgottagtccggoactgcgtaattgggtO tgoaagcc tactagooatgcatctgatggatggctatggtagcaccgaagcoggtatggticigttgtttgatgg cgadottcagcgtccgcctgtgottgat tataoactggttgatgttccogatctgggttatttt agcaccgatogtccgcatccgcgtggtgaactgctgctgcgtaccgaaaotatgtttccgggtt

+ attatoaacgtgcagaaaccaccgcaggcgtttttgatgaagatggttattatcgtaccggtgaO O tgtgtttgcogaaat tgcaccggatcgtctggtttatgttgotcgtcgtaataatgttctgaga+ ctggcacagggtgaatttgtgoccctggccogactggaagcagtttttogtaatagtccgctga+ ttcgtcagatttotgtgtatggtaatagcgcacagccgtatctgctggcagttgttgttccgac cgaagaggcactggcaagcggtgatecggaaaccctgaaaccgaaaattgcagatagcctgcag caggttgcaaaagaagcaggtctgcagagctatgaagttccgcgtgattttattattgaaacca ccccgtttagcctggaaaatggtctgctgaccogtattogtagactggcatggccgaoactgoa acagcattatggtgaacgcctggaacagatgtatgcagatctggcagcaggtcaggcaaatgaa ctggccgaactgcgtcgtaatggtgcacaggcoccggttctgcagoccgttagccgtgcagccg gtgcaatgctgggtagcgcagccagcgatctgagtccggotgcacottttaccgotctgggt99 tgatagcctgagcgcactgacctttogtaatctgctgcgtgaaatttttgatgttgatgtocco+ gttggtgttattgttagtccggctaatgatctggcagccattgcaagctatottgaagcagaac gtcogggtagcocacgtccgacctttgcaagcgttcatggtcgtgatgcoaccgttgttogtgcO agcagatctgaccctggataaatttctggatgcagaaaccctggcagcagcaccgaatctgccg> aaaccogcoaccgaagttogtaccgtgctgctgacaggtgcaaccggttttctgggtogttatcO tggcactggaatggctggaacgtatggatatggttgatggtaaagttattgcactggttogtgcC ccgtogtgatgaagaagcocgcgcacgtctggotagaocctttgatogtggtgatccgaaacto ctggcacattatcagcagctggctgcagotcatctggaagttattgccggtgatagaggtgaag cagatctgggtctgggtcaggatgtttggcagcgtctggcagataccgttgatgttattgtgga FIG . 17A atent Jun . 26 , 2018 Sheet 24 of 24 US 10 ,006 ,055 B2

tccggcagcactggttaatcatgttctgccgtatagcgaactgtttggtccgoatgcactgggcC C g accgcagaactgattcgtctggcactgaccagcaaacagaaaccgtatacctatgttagcocca ttggtgttggcgatcagattgaaccgggtaaatttgttgaaaatgccgatottcgtcagatgag cgcoocccgtgcaattaatgatagctatgcaaotggctacggcaatagcogatgggcaggcgoo0 U o gttctgctgcgcgaagcacatgatctgtgtggtctgccggttgcagtttttcgttgtgatatga ttctggccgataccacctatgcaggtcagctgaatctgccggatatgtttacccgtctgatgct+ gagcctggttgcaaccogtattgcoccgggtogcttttotgaactggatgcagatggtaatcgt cagcgtgcacattatgatggcctgccggttgaatttattgcagcagccattagcaccctgggtt cacagattaccgatagcgataccggttttcagacctatcatgttatgaacccgtatgatgatgg tgttggtctggat gaatatgttgattggctggttgatgecggttatagcottgaacgtattgcat gattatagcgaatggctgcgtcgctttgagacctcactgcgtgcactgccggatcgtcagcgccw agtatagcctgctgccgctgctgcacaattatcgtacaccggaaaaaccgat taotggtagcat tgcaccgaccgatgtttttogtgcagccgttcaagaagccaaaattggtccggatadagotatt cogcatgttagccctccggtgattgttagatatattaccgatctgcagctgctgggtctgctgt 00 FIG 17A cont.

MSTATHDERLORRVHELIATDPOFAAAQPDPAITAALEQPGLRLPQIIRTVLDGYADRPALGQR VCVEFVTDAKTGRTSAQLLPRFETITYSEVAQRVSALGRALSDDAVHPGDRVCVLGENSVDYATI T DMALGAIGAVSVPLOTSAAISSLOPIVAETEPTLIASSVNQLSDAVQLITGAEQAPTRLVVFDYT HPQVODQREAVQDAAARLSSTGVAVQTLAELLERGKDLPAVAEPPADEDSLALLIYTSGSTGAPVA KGAMYPQSNVGKMWRRGSKNWFGESAASITLNFMPMSHVMGRSILYGTLGNGGTAYFAARSDLS TLLEDLELVRPTELNFVPRIWETLYGEFQRQVERRLSEAGDAGERRAVEAEVLAEQRQYLLGGR FIFAMTGSAPISPELRNWVESLLEMHLMDGYGSTEAGMVLFDGEIQRPPVIDYKLVDVPDLGYF STDRPHPRGELLLRTENMFPGYYKRAETTAGVFDEDGYYRTGDVFAEIAPDRLVYVDRRNNVLK LAQGEFVTLAKLEAVFGNSPLIRQIYVYGNSAQPYLLAVVVPTEEALASGDPETLKPKIADSLQ QVAKEAGLQSYEVPRDFIIETTPFSLENGLLTGIRKLAWPKLKQHYGERLEQMYADLAAGQANE1 E LAELRRNGAOAPVLOTVSRAAGAM GSAASOL SPDAHFTDLGGDSLSALTEGNLLRE IFDVDYP VGVIVSPANDLAAIASYIEAEROGSKRPTFASVHGRDATVVRAADLTLDKFLDAETLAAAPNLP KPATEVRTVLLTGATGFLGRYLALEWLERMDMVDGKVIALVRARSDEEARARLDKTFDSGDPKL LAHYQQLAADHLEVIAGDKGEANLGLGQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNALG L MVTV/ TNIAIT TAELIRLALTSKQKPYTYVSTIGVGDQIEPGKFVENADIRQMSATRAINDSYANGYGNSKWAGETT T I VLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMF TRLMLSLVATGIAPGSFYELDADGNR QRAHYDGLPVEFIAAAISTLGSQITDSDTGFQTYHVMNPYDDGVGLDEYVDWLVDAGYSIERIA YDTDCVO DOVE DYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIAPTDVFRAAVQEAKIGPDKDIINITUN VIVKYY 1 FIG . 17B US 10 ,006 ,055 B2

MICROORGANISMS FOR PRODUCING described herein under conditions and for a sufficient period BUTADIENE AND METHODS RELATED of time to produce butadiene or crotyl alcohol. THERETO BRIEF DESCRIPTION OF THE DRAWINGS This application is a divisional application of U . S . patent 5 application Ser. No. 13 /527 ,440 , filed Jun . 19 , 2012 , now FIG . 1 shows a natural pathway to isoprenoids and issued U . S . Pat. No. 9 , 169 , 486 , and claims the benefit of terpenes . Enzymes for transformation of the identified sub priority of U . S . Provisional application Ser . No . 61/ 500 , 130 , strates to products include: A . acetyl -CoA :acetyl -CoA acyl filed Jun . 22 , 2011 , and U . S . Provisional application Ser. No . transferase , B . hydroxymethylglutaryl -CoA synthase , C . 61/ 502 , 264 , filed Jun . 28 , 2011 , the entire contents of each 10 3 - hydroxy - 3 -methylglutaryl - CoA reductase (alcohol form of which are incorporated herein by reference . ing ), D . , E . phosphomevalonate kinase , The instant application contains a Sequence Listing which F . diphosphomevalonate decarboxylase, G . isopentenyl has been submitted via EFS - Web and is hereby incorporated diphosphate isomerase , H . isoprene synthase . by reference in its entirety . Said ASCII copy, created on wayMay 1516 FIG . 2 shows exemplary pathways for production of 10 , 2016 , is named 12956 - 272 - 999SequenceListing . txt and butadiene from acetyl- CoA , glutaconyl- CoA , glutaryl- CoA , is 77 ,835 bytes in size . 3 -aminobutyryl - CoA or 4 -hydroxybutyryl - CoA via crotyl alcohol . Enzymes for transformation of the identified sub BACKGROUND OF THE INVENTION strates to products include: A . acetyl- CoA : acetyl- CoA acyl 20 transferase , B . acetoacetyl- CoA reductase, C . 3 -hydroxybu The present invention relates generally to biosynthetic tyryl- CoA dehydratase , D . crotonyl- CoA reductase processes, and more specifically to organisms having buta - (aldehyde forming ), E . crotonaldehyde reductase (alcohol diene or crotyl alcohol biosynthetic capability . formforming ), F . crotyl alcoholkinase , G . 2 -butenyl - 4 -phosphate Over 25 billion pounds of butadiene ( 1 , 3 -butadiene , BD ) kinase , H . butadiene synthase , I . crotonyl- CoA hydrolase , are produced annually and is applied in the manufacture of 25 synthetase, transferase , J. crotonate reductase , K . crotonyl polymers such as synthetic rubbers and ABS resins, and CoA reductase (alcohol forming ), L . glutaconyl- CoA decar chemicals such as hexamethylenediamine and 1 , 4 -butane - boxylase , M . , glutaryl- CoA dehydrogenase, N . 3 -aminobu diol. Butadiene is typically produced as a by -product of the tyryl -CoA deaminase , O . 4 -hydroxybutyryl - CoA steam cracking process for conversion of petroleum feed - dehydratase , P . crotyl alcohol diphosphokinase . stocks such as naphtha , liquefied petroleum gas, ethane or 30 FIG . 3 shows exemplary pathways for production of natural gas to ethylene and other olefins. The ability to butadiene from erythrose - 4 -phosphate . Enzymes for trans formation of the identified substrates to products include : A . manufacture butadiene from alternative and /or renewable Erythrose - 4 -phosphate reductase , B . Erythritol- 4 -phosphate feedstocks would represent a major advance in the quest for cytidylyltransferase , C . 4 - (cytidine 5 ' -diphospho ) - erythritol more sustainable chemical production processes 1 , 35 kinase , D . Erythritol 2 ,4 -cyclodiphosphate synthase , E . One possible way to produce butadiene renewably 1 -Hydroxy - 2 -butenyl 4 -diphosphate synthase , F. 1 -Hy involves fermentation of sugars or other feedstocks to pro droxy - 2 -butenyl 4 - diphosphate reductase , G . Butenyl duce diols , such as 1 , 4 - butanediol or 1 , 3 - butanediol, which 4 - diphosphate isomerase, H . Butadiene synthase I. Eryth are separated , purified , and then dehydrated to butadiene in rose - 4 - phosphate kinase , J . Erythrose reductase , K . Eryth a second step involving metal- based catalysis. Direct fer - 40 ritol kinase . mentative production of butadiene from renewable feed FIG . 4 shows an exemplary pathway for production of stocks would obviate the need for dehydration steps and butadiene from malonyl- CoA plus acetyl- CoA . Enzymes for butadiene gas (bp - 4 .4° C . ) would be continuously emitted transformation of the identified substrates to products from the fermenter and readily condensed and collected . include : A . malonyl - CoA : acetyl - CoA acyltransferase , B . Developing a fermentative production process would elimi- 45 3 -oxoglutaryl -CoA reductase (ketone -reducing ), C . 3 -hy nate the need for fossil- based butadiene and would allow droxyglutaryl- CoA reductase (aldehyde forming ) , D . 3 - hy substantial savings in cost , energy , and harmful waste and droxy - 5 - oxopentanoate reductase , E . 3 ,5 - dihydroxypen emissions relative to petrochemically -derived butadiene . tanoate kinase , F . 3H5PP kinase , G . 3H5PDP decarboxylase , Microbial organisms and methods for effectively produc - H . butenyl 4 -diphosphate isomerase , I. butadiene synthase , ing butadiene or crotyl alcohol from cheap renewable feed - 50 J. 3 -hydroxyglutaryl - CoA reductase (alcohol forming) , K . stocks such as molasses, sugar cane juice , and sugars 3 -oxoglutaryl - CoA reductase ( aldehyde forming ) , L . 3 , 5 derived from biomass sources, including agricultural and dioxopentanoate reductase (ketone reducing ), M . 3 ,5 - dioxo wood waste , as well as C1 feedstocks such as syngas and pentanoate reductase (aldehyde reducing ), N . 5 -hydroxy - 3 carbon dioxide , are described herein and include related oxopentanoate reductase , O . 3 - oxo - glutaryl- CoA reductase advantages. 55 (CoA reducing and alcohol forming ). Compound abbrevia tions include : 3H5PP = 3 -Hydroxy - 5 -phosphonatooxypen SUMMARY OF THE INVENTION tanoate and 3H5PDP = 3 -Hydroxy -5 - [hydroxy (phospho nooxy phosphoryl] oxy pentanoate . The invention provides non - naturally occurring microbial FIG . 5 shows an exemplary pathway for production of organisms containing butadiene or crotyl alcohol pathways 60 crotyl alcohol from acetyl- CoA . Enzymes for transformation comprising at least one exogenous nucleic acid encoding a of the identified substrates to products include: A . acetyl butadiene or crotyl alcohol pathway enzyme expressed in a CoA : acetyl- CoA acyltransferase, B . acetoacetyl - CoA reduc sufficient amount to produce butadiene or crotyl alcohol. tase , C . 3 - hydroxybutyryl- CoA dehydratase, D . crotonyl The invention additionally provides methods of using such CoA reductase ( aldehyde forming ) , E . crotonaldehyde microbial organisms to produce butadiene or crotyl alcohol, 65 reductase (alcohol forming ), F . crotonyl- CoA reductase ( al by culturing a non - naturally occurring microbial organism cohol forming ), G . crotonyl -CoA hydrolase , synthetase , containing butadiene or crotyl alcohol pathways as transferase , and H . crotonate reductase . US 10 , 006 , 055 B2 FIG . 6 shows the reverse TCA cycle for fixation of CO2 FIG . 17A shows the nucleotide sequence (SEQ ID NO : on carbohydrates as substrates . The enzymatic transforma- 11 ) of carboxylic acid reductase designated 891GA , and tions are carried out by the enzymes as shown . FIG . 17B shows the encoded amino acid sequence (SEQ ID FIG . 7 shows the pathway for the reverse TCA cycle NO : 12 ) . coupled with carbon monoxide dehydrogenase and hydro - 5 genase for the conversion of syngas to acetyl- CoA . DETAILED DESCRIPTION OF THE FIG . 8 shows Western blots of 10 micrograms ACS90 INVENTION ( lane 1) , ACS91 ( lane2 ) , Mta98 /99 ( lanes 3 and 4 ) cell extracts with size standards ( lane 5 ) and controls of M . The present invention is directed to the design and pro thermoacetica CODH (Moth _ 1202/ 1203 ) or Mtr 10 duction of cells and organisms having biosynthetic produc (Moth _ 1197 ) proteins ( 50 , 150 , 250 , 350 , 450 , 500 , 260750 , tion capabilities for butadiene or crotyl alcohol. The inven 900, and 1000 ng ) . tion , in particular, relates to the design of microbial FIG . 9 shows CO oxidation assay results . Cells ( M . organism capable of producing butadiene or crotyl alcohol thermoacetica or E . coli with the CODH / ACS operon ; 1 by introducing one or more nucleic acids encoding a buta ACS90 or ACS91 or empty vector: PZA33S ) were grown diene or a crotyl alcohol pathway enzyme. and extracts prepared . Assays were performed at 55° C . at In one embodiment , the invention utilizes in silico stoi various times on the day the extracts were prepared . Reduc - chiometric models of Escherichia coli metabolism that tion of methylviologen was followed at 578 nm over a 120 identify metabolic designs for biosynthetic production of sec time course . 20 butadiene or crotyl alcohol . The results described herein FIGS . 10A and B show exemplary pathways to crotyl indicate that metabolic pathways can be designed and alcohol. FIG . 10A shows the pathways for fixation of CO2 recombinantly engineered to achieve the biosynthesis of to acetyl- CoA using the reductive TCA cycle . FIG . 10B butadiene or crotyl alcohol in Escherichia coli and other shows exemplary pathways for the biosynthesis of crotyl cells or organisms. Biosynthetic production of butadiene or alcohol from acetyl - CoA ; the enzymatic transformations 255 crotyl alcohol, for example , for the in silico designs can be shown are carried out by the following enzymes: A . acetyl confirmed by construction of strains having the designed COA :acetyl - CoA acyltransferase , B . acetoacetyl- CoA reduc metabolic genotype . These metabolically engineered cells or tase, C . 3 -hydroxybutyryl - CoA dehydratase , D . crotonyl organisms also can be subjected to adaptive evolution to CoA reductase ( aldehyde forming ) , E . crotonaldehyde further augment butadiene or crotyl alcohol biosynthesis , reductase (alcohol forming ) , F. crotonyl- CoA reductase (al - 30 including under conditions approaching theoretical maxi cohol forming ), G . crotonyl -CoA hydrolase , synthetase , mum growth . transferase , and H . crotonate reductase . In certain embodiments , the butadiene biosynthesis char FIGS . 11A and 11 B show exemplary pathways to buta acteristics of the designed strains make them genetically diene. FIG . 11A shows the pathways for fixation of CO2 to stable and particularly useful in continuous bioprocesses . acetyl- CoA using the reductive TCA cycle . FIG . 11B shows 35 . Separate strain design strategies were identified with incor exemplary pathways for the biosynthesis of butadiene from poration of different non -native or heterologous reaction acetyl - CoA ; the enzymatic transformations shown are car capabilities into E . coli or other host organisms leading to ried out by the following enzymes: A . acetyl- CoA :acetyl butadiene producing metabolic pathways from acetyl- CoA , COA acyltransferase , B . acetoacetyl- CoA reductase, C . 3 -hy glutaconyl- CoA , glutaryl - CoA , 3 - aminobutyryl- CoA , 4 -hy droxybutyryl- CoA dehydratase , D . crotonyl- CoA reductase 40 droxybutyrylafoxybu - CoA , erythrose -4 - phosphate or malonyl- CoA ( aldehyde forming ), E . crotonaldehyde reductase (alcohol plus acetyl -CoA . In silico metabolic designs were identified forming ) , F . crotonyl- CoA reductase ( alcohol forming ), G . that resulted in the biosynthesis of butadiene in microorgan crotonyl- CoA hydrolase , synthetase , transferase , H . croto isms from each of these substrates or metabolic intermedi nate reductase , I . crotyl alcohol kinase , J. 2 -butenyl - 4 ates . phosphate kinase , K . butadiene synthase , L . crotyl alcohol 45 Strains identified via the computational component of the diphosphokinase . platform can be put into actual production by genetically FIG . 12A shows the nucleotide sequence (SEQ ID NO : 1 ) engineering any of the predicted metabolic alterations , of carboxylic acid reductase from Nocardia iowensis which lead to the biosynthetic production of butadiene or (GNM _ 720 ) , and FIG . 12B shows the encoded amino acid other intermediate and /or downstream products . In yet a sequence (SEQ ID NO : 2 ) . further embodiment, strains exhibiting biosynthetic produc FIG . 13A shows the nucleotide sequence ( SEQ ID NO : 3 ) tion of these compounds can be further subjected to adaptive of phosphpantetheine transferase, which was codon opti evolution to further augment product biosynthesis . The mized , and FIG . 13B shows the encoded amino acid levels of product biosynthesis yield following adaptive sequence (SEQ ID NO : 4 ) . evolution also can be predicted by the computational com FIG . 14A shows the nucleotide sequence (SEQ EOID ID NO .: 5 ) 55ss pouponent of the system . of carboxylic acid reductase from Mycobacterium smegma The maximum theoretical butadiene yield from glucose is tis mc( 2 ) 155 ( designated 890 ), and FIG . 14B shows the 1 . 09 mol/mol ( 0 . 33 g / g ) . encoded amino acid sequence (SEQ ID NO : 6 ) . FIG . 15A shows the nucleotide sequence ( SEO ID NO : 7 ) 11C6H1206 = 12C4H6+ 18CO2 + 30H20 of carboxylic acid reductase from Mycobacterium avium 60 The pathways presented in FIGS . 2 and 4 achieve a yield subspecies paratuberculosis K - 10 (designated 891) , and of 1 . 0 moles butadiene per mole of glucose utilized . Increas FIG . 15B shows the encoded amino acid sequence (SEQ ID ing product yields to theoretical maximum value is possible NO : 8 ). if cells are capable of fixing CO2 through pathways such as FIG . 16A shows the nucleotide sequence (SEQ ID NO : 9 ) the reductive (or reverse ) TCA cycle or the Wood -Ljungdahl of carboxylic acid reductase from Mycobacterium marinum 65 pathway. Organisms engineered to possess the pathway M ( designated 892 ) , and FIG . 16B shows the encoded amino depicted in FIG . 3 are also capable of reaching near theo acid sequence (SEQ ID NO : 10 ) . retical maximum yields of butadiene . US 10 ,006 , 055 B2 As used herein , the term “ non - naturally occurring " when As used herein , the term “ substantially anaerobic ” when used in reference to a microbial organism or microorganism used in reference to a culture or growth condition is intended of the invention is intended to mean that the microbial to mean that the amount of oxygen is less than about 10 % organism has at least one genetic alteration not normally of saturation for dissolved oxygen in liquid media . The term found in a naturally occurring strain of the referenced 5 also is intended to include sealed chambers of liquid or solid species , including wild - type strains of the referenced spe - medium maintained with an atmosphere of less than about cies . Genetic alterations include , for example , modifications 1 % oxygen . introducing expressible nucleic acids encoding metabolic “ Exogenous” as it is used herein is intended to mean that polypeptides , other nucleic acid additions, nucleic acid the referenced molecule or the referenced activity is intro deletions and / or other functional disruption of the microbial 10 duced into the host microbial organism . The molecule can be organism ' s genetic material. Such modifications include , for introduced , for example , by introduction of an encoding example , coding regions and functional fragments thereof , nucleic acid into the host genetic material such as by for heterologous , homologous or both heterologous and integration into a host chromosome or as non -chromosomal homologous polypeptides for the referenced species . Addi genetic material such as a plasmid . Therefore , the term as it tionalmodifications include , for example , non - coding regu - 15 is used in reference to expression of an encoding nucleic latory regions in which the modifications alter expression of acid refers to introduction of the encoding nucleic acid in an a gene or operon . Exemplary metabolic polypeptides include expressible form into the microbial organism . When used in enzymes or proteins within a butadiene or crotyl alcohol reference to a biosynthetic activity , the term refers to an biosynthetic pathway . activity that is introduced into the host reference organism . A metabolic modification refers to a biochemical reaction 20 The source can be , for example , a homologous or heterolo that is altered from its naturally occurring state . Therefore , gous encoding nucleic acid that expresses the referenced non - naturally occurring microorganisms can have genetic activity following introduction into the host microbial modifications to nucleic acids encoding metabolic polypep organism . Therefore , the term " endogenous” refers to a tides , or functional fragments thereof . Exemplary metabolic referenced molecule or activity that is present in the host. modifications are disclosed herein . 25 Similarly , the term when used in reference to expression of As used herein , the term “ butadiene , ” having the molecu - an encoding nucleic acid refers to expression of an encoding lar formula C H , and a molecular mass of 54. 09 g /mol ( see nucleic acid contained within the microbial organism . The FIGS. 2 -4 ) ( IUPAC name Buta - 1, 3 -diene ) is used inter term “ heterologous” refers to a molecule or activity derived changeably throughout with 1 , 3 -butadiene , biethylene , from a source other than the referenced species whereas erythrene , divinyl, vinylethylene . Butadiene is a colorless , 30 “ homologous” refers to a molecule or activity derived from non corrosive liquefied gas with a mild aromatic or gasoline the host microbial organism . Accordingly , exogenous like odor. Butadiene is both explosive and flammable expression of an encoding nucleic acid of the invention can because of its low flash point. utilize either or both a heterologous or homologous encod As used herein , the term “ isolated ” when used in refer - ing nucleic acid . ence to a microbial organism is intended to mean an organ - 35 It is understood that when more than one exogenous ism that is substantially free of at least one component as the nucleic acid is included in a microbial organism that the referenced microbial organism is found in nature . The term more than one exogenous nucleic acids refers to the refer includes a microbial organism that is removed from some or e nced encoding nucleic acid or biosynthetic activity , as all components as it is found in its natural environment. The discussed above . It is further understood , as disclosed term also includes a microbial organism that is removed 40 herein , that such more than one exogenous nucleic acids can from some or all components as the microbial organism is be introduced into the host microbial organism on separate found in non - naturally occurring environments . Therefore , nucleic acid molecules , on polycistronic nucleic acid mol an isolated microbial organism is partly or completely ecules , or a combination thereof , and still be considered as separated from other substances as it is found in nature or as more than one exogenous nucleic acid . For example , as it is grown , stored or subsisted in non - naturally occurring 45 disclosed herein a microbial organism can be engineered to environments . Specific examples of isolated microbial express two or more exogenous nucleic acids encoding a organisms include partially pure microbes , substantially desired pathway enzyme or protein . In the case where two pure microbes and microbes cultured in a medium that is exogenous nucleic acids encoding a desired activity are non - naturally occurring. introduced into a host microbial organism , it is understood As used herein , the terms " microbial, ” “ microbial organ - 50 that the two exogenous nucleic acids can be introduced as a ism ” or “ microorganism ” are intended to mean any organism single nucleic acid , for example , on a single plasmid , on that exists as a microscopic cell that is included within the separate plasmids, can be integrated into the host chromo domains of archaea , bacteria or eukarya . Therefore , the term some at a single site or multiple sites , and still be considered is intended to encompass prokaryotic or eukaryotic cells or as two exogenous nucleic acids . Similarly , it is understood organisms having a microscopic size and includes bacteria , 55 that more than two exogenous nucleic acids can be intro archaea and eubacteria of all species as well as eukaryotic duced into a host organism in any desired combination , for microorganisms such as yeast and fungi . The term also example , on a single plasmid , on separate plasmids , can be includes cell cultures of any species that can be cultured for integrated into the host chromosome at a single site or the production of a biochemical. multiple sites, and still be considered as two or more As used herein , the term “ COA ” or “ coenzyme A ” is 60 exogenous nucleic acids, for example three exogenous intended to mean an organic cofactor or prosthetic group nucleic acids . Thus , the number of referenced exogenous (nonprotein portion of an enzyme) whose presence is nucleic acids or biosynthetic activities refers to the number required for the activity of many enzymes (the apoenzyme ) of encoding nucleic acids or the number of biosynthetic to form an active enzyme system . Coenzyme A functions in activities, not the number of separate nucleic acids intro certain condensing enzymes , acts in acetyl or other acyl 65 duced into the host organism . group transfer and in fatty acid synthesis and oxidation , The non - naturally occurring microbial organisms of the pyruvate oxidation and in other acetylation . invention can contain stable genetic alterations, which refers US 10 ,006 , 055 B2 to microorganisms that can be cultured for greater than five first species can be considered an ortholog to either or both generations without loss of the alteration . Generally, stable of the exonuclease or the from the second genetic alterations include modifications that persist greater species and vice versa . than 10 generations, particularly stable modifications will In contrast, paralogs are homologs related by , for persist more than about 25 generations, and more particu - 5 example , duplication followed by evolutionary divergence larly, stable genetic modifications will be greater than 50 and have similar or common , but not identical functions . generations, including indefinitely. Paralogs can originate or derive from , for example , the same species or from a different species. For example, microsomal Those skilled in the art will understand that the genetic epoxide hydrolase ( epoxide hydrolase I ) and soluble epoxide alterations, including metabolic modifications exemplified+ 10 hydrolase ( epoxide hydrolase II ) can be considered paralogs herein , are described with reference to a suitable host because they represent two distinct enzymes , co - evolved organism such as E . coli and their corresponding metabolic from a common ancestor , that catalyze distinct reactions and reactions or a suitable source organism for desired genetic have distinct functions in the same species . Paralogs are material such as genes for a desired metabolic pathway. proteins from the same species with significant sequence However , given the complete genome sequencing of a wide 15 similarity to each other suggesting that they are homolo variety of organisms and the high level of skill in the area of gous , or related through co - evolution from a common ances genomics , those skilled in the art will readily be able to tor. Groups of paralogous protein families include HipA apply the teachings and guidance provided herein to essen - homologs , luciferase genes, peptidases, and others . tially all other organisms. For example , the E . colimetabolic A nonorthologous gene displacement is a nonorthologous alterations exemplified herein can readily be applied to other 20 gene from one species that can substitute for a referenced species by incorporating the same or analogous encoding gene function in a different species . Substitution includes , nucleic acid from species other than the referenced species . for example , being able to perform substantially the same or Such genetic alterations include , for example , genetic altera - a similar function in the species of origin compared to the tions of species homologs , in general, and in particular, referenced function in the different species . Although gen orthologs , paralogs or nonorthologous gene displacements . 25 erally , a nonorthologous gene displacement will be identi An ortholog is a gene or genes that are related by vertical fiable as structurally related to a known gene encoding the descent and are responsible for substantially the same or referenced function , less structurally related but functionally identical functions in different organisms. For example , similar genes and their corresponding gene products never mouse epoxide hydrolase and human epoxide hydrolase can theless will still fall within the meaning of the term as it is be considered orthologs for the biological function of hydro - 30 used herein . Functional similarity requires , for example, at lysis of epoxides . Genes are related by vertical descent least some structural similarity in the active site or binding when , for example , they share sequence similarity of suffi region of a nonorthologous gene product compared to a gene cient amount to indicate they are homologous , or related by encoding the function sought to be substituted . Therefore , a evolution from a common ancestor. Genes can also be nonorthologous gene includes, for example , a paralog or an considered orthologs if they share three - dimensional struc - 35 unrelated gene . ture but not necessarily sequence similarity , of a sufficient Therefore , in identifying and constructing the non - natu amount to indicate that they have evolved from a common rally occurring microbial organisms of the invention having ancestor to the extent that the primary sequence similarity is butadiene or crotyl alcohol biosynthetic capability, those not identifiable. Genes that are orthologous can encode skilled in the art will understand with applying the teaching proteins with sequence similarity of about 25 % to 100 % 40 and guidance provided herein to a particular species that the amino acid sequence identity . Genes encoding proteins identification of metabolic modifications can include iden sharing an amino acid similarity less that 25 % can also be tification and inclusion or inactivation of orthologs . To the considered to have arisen by vertical descent if their three extent that paralogs and / or nonorthologous gene displace dimensional structure also shows similarities . Members of ments are present in the referenced microorganism that the serine protease family of enzymes , including tissue 45 encode an enzyme catalyzing a similar or substantially plasminogen activator and elastase , are considered to have similar metabolic reaction , those skilled in the art also can arisen by vertical descent from a common ancestor. utilize these evolutionally related genes . Orthologs include genes or their encoded gene products Orthologs , paralogs and nonorthologous gene displace that through , for example , evolution , have diverged in ments can be determined by methods well known to those structure or overall activity . For example , where one species 50 skilled in the art . For example, inspection of nucleic acid or encodes a gene product exhibiting two functions and where amino acid sequences for two polypeptides will reveal such functions have been separated into distinct genes in a sequence identity and similarities between the compared second species , the three genes and their corresponding sequences . Based on such similarities , one skilled in the art products are considered to be orthologs . For the production can determine if the similarity is sufficiently high to indicate of a biochemical product , those skilled in the art will 55 the proteins are related through evolution from a common understand that the orthologous gene harboring the meta - ancestor. Algorithms well known to those skilled in the art, bolic activity to be introduced or disrupted is to be chosen such as Align , BLAST , Clustal W and others compare and for construction of the non - naturally occurring microorgan - determine a raw sequence similarity or identity , and also ism . An example of orthologs exhibiting separable activities determine the presence or significance of gaps in the is where distinct activities have been separated into distinct 60 sequence which can be assigned a weight or score . Such gene products between two or more species or within a algorithms also are known in the art and are similarly single species. A specific example is the separation of applicable for determining nucleotide sequence similarity or elastase proteolysis and plasminogen proteolysis , two types identity . Parameters for sufficient similarity to determine of serine protease activity, into distinct molecules as plas - relatedness are computed based on well known methods for minogen activator and elastase . A second example is the 65 calculating statistical similarity, or the chance of finding a separation of mycoplasma 5 '- 3 ' exonuclease and Drosophila similar match in a random polypeptide , and the significance DNA polymerase III activity . The DNA polymerase from the of the match determined . A computer comparison of two or US 10 , 006 , 055 B2 10 more sequences can , if desired , also be optimized visually produce butadiene, the butadiene pathway including an by those skilled in the art. Related gene products or proteins acetyl- CoA : acetyl- CoA acyltransferase , an acetoacetyl - CoA can be expected to have a high similarity , for example , 25 % reductase, a 3 - hydroxybutyryl - CoA dehydratase , a butadi to 100 % sequence identity . Proteins that are unrelated can ene synthase , a crotonyl -CoA reductase (alcohol forming ) have an identity which is essentially the same as would be 5 and a crotyl alcohol diphosphokinase (FIG . 2 , steps A - C , K , expected to occur by chance , if a database of sufficient size P , H ) . In one aspect, the non -naturally occurring microbial is scanned ( about 5 % ) . Sequences between 5 % and 24 % organism includes a microbial organism having a butadiene may or may not represent sufficient homology to conclude pathway having at least one exogenous nucleic acid encod that the compared sequences are related . Additional statis - ing butadiene pathway enzymes expressed in a sufficient tical analysis to determine the significance of such matches 10 amount to produce butadiene , the butadiene pathway includ given the size of the data set can be carried out to determine ing an acetyl- CoA :acetyl - CoA acyltransferase , an the relevance of these sequences . acetoacetyl- CoA reductase, a 3 -hydroxybutyryl - CoA dehy Exemplary parameters for determining relatedness of two dratase , a crotonaldehyde reductase (alcohol forming ) , a or more sequences using the BLAST algorithm , for crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase , a example , can be as set forth below . Briefly , amino acid 15 butadiene synthase , a crotonyl- CoA hydrolase , synthetase , sequence alignments can be performed using BLASTP or transferase and a crotonate reductase , ( FIG . 2 , steps A - C , version 2 . 0 . 8 (Jan . 5 , 1999 ) and the following parameters : I, J , E , F , G , H ) . In one aspect , the non - naturally occurring Matrix : 0 BLOSUM62 ; gap open : 11; gap extension : 1; microbial organism includes a microbial organism having a X _ dropoff : 50 ; expect : 10 . 0 ; wordsize : 3 ; filter: on . Nucleic butadiene pathway having at least one exogenous nucleic acid sequence alignments can be performed using BLASTN 20 acid encoding butadiene pathway enzymes expressed in a version 2 . 0 .6 ( Sep . 16 , 1998 ) and the following parameters : sufficient amount to produce butadiene , the butadiene path Match : 1 ; mismatch : - 2 ; gap open : 5 ; gap extension : 2 ; way including an acetyl- CoA : acetyl- CoA acyltransferase , X _ dropoff : 50 ; expect: 10 . 0 ; wordsize : 11 ; filter : off. Those an acetoacetyl- CoA reductase, a 3 -hydroxybutyryl - CoA skilled in the art will know what modifications can be made dehydratase , a crotonaldehyde reductase (alcohol forming ) , to the above parameters to either increase or decrease the 25 a butadiene synthase , a crotonyl -CoA hydrolase , synthetase stringency of the comparison , for example , and determine or transferase , a crotonate reductase and a crotyl alcohol the relatedness of two or more sequences . diphosphokinase (FIG . 2 , steps A - C , I , J , E , P , H ) . In one In some embodiments , the invention provides a non aspect, the non - naturally occurring microbial organism naturally occurring microbial organism , including a micro - includes a microbial organism having a butadiene pathway bial organism having a butadiene pathway having at least 30 having at least one exogenous nucleic acid encoding buta one exogenous nucleic acid encoding a butadiene pathway diene pathway enzymes expressed in a sufficient amount to enzyme expressed in a sufficient amount to produce buta - produce butadiene , the butadiene pathway including an diene , the butadiene pathway including an acetyl -CoA : acetyl- CoA : acetyl -COA acyltransferase , an acetoacetyl- COA acetyl- CoA acyltransferase , an acetoacetyl- CoA reductase , a reductase, a 3 -hydroxybutyryl - CoA dehydratase , a crotonyl 3 - hydroxybutyryl- CoA dehydratase , a crotonyl- CoA reduc - 35 CoA reductase (aldehyde forming) , a crotonaldehyde reduc tase (aldehyde forming ) , a crotonaldehyde reductase ( alco - tase ( alcohol forming ) , a butadiene synthase and a crotyl hol forming ), a crotyl alcohol kinase , a 2 -butenyl - 4 -phos alcohol diphosphokinase (FIG . 2 , steps A - E , P , H ) . In one phate kinase , a butadiene synthase , a crotonyl- CoA aspect, the non - naturally occurring microbial organism hydrolase , synthetase, or transferase, a crotonate reductase , includes a microbial organism having a butadiene pathway a crotonyl- CoA reductase ( alcohol forming ) , a glutaconyl - 40 having at least one exogenous nucleic acid encoding buta COA decarboxylase , a glutaryl -CoA dehydrogenase , an diene pathway enzymes expressed in a sufficient amount to 3 - aminobutyryl- CoA deaminase , a 4 -hydroxybutyryl - CoA produce butadiene, the butadiene pathway including a dehydratase or a crotyl alcohol diphosphokinase (FIG . 2 ) . In glutaconyl- CoA decarboxylase , a crotonyl- CoA reductase one aspect, the non - naturally occurring microbial organism (aldehyde forming ) , a crotonaldehyde reductase (alcohol includes a microbial organism having a butadiene pathway 45 forming ) , a crotyl alcohol kinase , a 2 - butenyl - 4 - phosphate having at least one exogenous nucleic acid encoding buta kinase and a butadiene synthase (FIG . 2 , steps L , D - H ) . In diene pathway enzymes expressed in a sufficient amount to one aspect, the non -naturally occurring microbial organism produce butadiene , the butadiene pathway including an includes a microbial organism having a butadiene pathway acetyl - CoA : acetyl - CoA acyltransferase , an acetoacetyl- CoA having at least one exogenous nucleic acid encoding buta reductase , a 3 -hydroxybutyryl -CoA dehydratase , a crotonyl - 50 diene pathway enzymes expressed in a sufficient amount to CoA reductase ( aldehyde forming ), a crotonaldehyde reduc - produce butadiene , the butadiene pathway including a tase (alcohol forming) , a crotyl alcohol kinase , a 2 - butenyl- glutaconyl -CoA decarboxylase , a crotyl alcohol kinase , a 4 -phosphate kinase and a butadiene synthase ( FIG . 2 , steps 2 -butenyl - 4 -phosphate kinase , a butadiene synthase and A - H ) . In one aspect, the non -naturally occurring microbial crotonyl -CoA reductase ( alcohol forming ) (FIG . 2 , steps L , organism includes a microbial organism having a butadiene 55 K , F , G , H ) . In one aspect, the non - naturally occurring pathway having at least one exogenous nucleic acid encod microbial organism includes a microbial organism having a ing butadiene pathway enzymes expressed in a sufficient butadiene pathway having at least one exogenous nucleic amount to produce butadiene , the butadiene pathway includ - acid encoding butadiene pathway enzymes expressed in a ing an acetyl- CoA :acetyl - CoA acyltransferase , an sufficient amount to produce butadiene , the butadiene path acetoacetyl- CoA reductase , a 3 -hydroxybutyryl - CoA dehy - 60 way including a glutaconyl -CoA decarboxylase , a butadiene dratase , a crotyl alcohol kinase , a 2 - butenyl- 4 - phosphate synthase , a crotonyl -CoA reductase ( alcohol forming ) and a kinase , a butadiene synthase and crotonyl- CoA reductase crotyl alcohol diphosphokinase ( FIG . 2 , steps L , K , P , H ) . In ( alcohol forming ) ( FIG . 2 , steps A - C , K , F , G , H ) . In one one aspect , the non - naturally occurring microbial organism aspect, the non - naturally occurring microbial organism includes a microbial organism having a butadiene pathway includes a microbial organism having a butadiene pathway 65 having at least one exogenous nucleic acid encoding buta having at least one exogenous nucleic acid encoding buta diene pathway enzymes expressed in a sufficient amount to diene pathway enzymes expressed in a sufficient amount to produce butadiene, the butadiene pathway including a US 10 ,006 , 055 B2 glutaconyl- CoA decarboxylase, a crotonaldehyde reductase one aspect, the non - naturally occurring microbial organism ( alcohol forming ) , a crotyl alcohol kinase , a 2 - butenyl- 4 includes a microbial organism having a butadiene pathway phosphate kinase , a butadiene synthase , a crotonyl- CoA having at least one exogenous nucleic acid encoding buta hydrolase , synthetase , or transferase and a crotonate reduc - diene pathway enzymes expressed in a sufficient amount to tase (FIG . 2 , steps L , I , J , E , F , G , H ) . In one aspect, the 5 produce butadiene, the butadiene pathway including a 3 -hy non - naturally occurring microbial organism includes a droxybutyryl- CoA dehydratase , a crotonyl- CoA reductase microbial organism having a butadiene pathway having at ( aldehyde forming ), a crotonaldehyde reductase ( alcohol least one exogenous nucleic acid encoding butadiene path - forming ) , a butadiene synthase , a glutaryl -CoA dehydroge way enzymes expressed in a sufficient amount to produce nase and a crotyl alcohol diphosphokinase ( FIG . 2 , steps M , butadiene , the butadiene pathway including a glutaconyl- 10 C , D , E , P , H ) . In one aspect , the non - naturally occurring COA decarboxylase , a crotonaldehyde reductase ( alcohol microbial organism includes a microbial organism having a forming ) , a butadiene synthase , a crotonyl - CoA hydrolase , butadiene pathway having at least one exogenous nucleic synthetase or transferase , a crotonate reductase and a crotyl acid encoding butadiene pathway enzymes expressed in a alcohol diphosphokinase (FIG . 2 , steps L , I, J , E , P , H ) . In sufficient amount to produce butadiene , the butadiene path one aspect, the non - naturally occurring microbial organism 15 way including an 3 - aminobutyryl - CoA deaminase , a croto includes a microbial organism having a butadiene pathway nyl -CoA reductase ( aldehyde forming ) , a crotonaldehyde having at least one exogenous nucleic acid encoding buta - reductase (alcohol forming ), a crotyl alcohol kinase , a diene pathway enzymes expressed in a sufficient amount to 2 -butenyl - 4 -phosphate kinase and a butadiene synthase produce butadiene , the butadiene pathway including a 3 -hy - (FIG . 2 , steps N , D - H ) . In one aspect, the non - naturally droxybutyryl- CoA dehydratase , a crotonyl- CoA reductase 20 occurring microbial organism includes a microbial organism (aldehyde forming ), a crotonaldehyde reductase (alcohol having a butadiene pathway having at least one exogenous forming ) , a butadiene a glutaconyl- CoA decarboxylase and nucleic acid encoding butadiene pathway enzymes a crotyl alcohol diphosphokinase ( FIG . 2 , steps L , C , D , E , expressed in a sufficient amount to produce butadiene, the P , H ) . In one aspect , the non - naturally occurring microbial butadiene pathway including an 3 - aminobutyryl- CoA organism includes a microbial organism having a butadiene 25 deaminase , a crotyl alcohol kinase , a 2 - butenyl - 4 - phosphate pathway having at least one exogenous nucleic acid encod - kinase , a butadiene synthase and crotonyl -CoA reductase ing butadiene pathway enzymes expressed in a sufficient (alcohol forming ) (FIG . 2 , steps N , K , F , G , H ) . In one amount to produce butadiene , the butadiene pathway includ aspect, the non - naturally occurring microbial organism ing a glutaryl- CoA dehydrogenase , a crotonyl -CoA reduc - includes a microbial organism having a butadiene pathway tase ( aldehyde forming ) , a crotonaldehyde reductase ( alco - 30 having at least one exogenous nucleic acid encoding buta hol forming ) , a crotyl alcohol kinase , a 2 -butenyl - 4 - diene pathway enzymes expressed in a sufficient amount to phosphate kinase and a butadiene synthase ( FIG . 2 , steps M , produce butadiene , the butadiene pathway including an D - H ) . In one aspect, the non - naturally occurring microbial 3 -aminobutyryl - CoA deaminase , a butadiene synthase , a organism includes a microbial organism having a butadiene crotonyl- CoA reductase ( alcohol forming ) and a crotyl alco pathway having at least one exogenous nucleic acid encod - 35 hol diphosphokinase ( FIG . 2 , steps N , K , P , H ) . In one ing butadiene pathway enzymes expressed in a sufficient aspect, the non - naturally occurring microbial organism amount to produce butadiene , the butadiene pathway includ - includes a microbial organism having a butadiene pathway ing a glutaryl- CoA dehydrogenase, a crotyl alcohol kinase , having at least one exogenous nucleic acid encoding buta a 2 -butenyl - 4 -phosphate kinase, a butadiene synthase and diene pathway enzymes expressed in a sufficient amount to crotonyl -CoA reductase ( alcohol forming ) (FIG . 2 , steps M , 40 produce butadiene , the butadiene pathway including an K , F , G , H ). In one aspect , the non -naturally occurring 3 -aminobutyryl - CoA deaminase , a crotonaldehyde reduc microbial organism includes a microbial organism having a tase (alcohol forming ), a crotyl alcohol kinase , a 2 -butenyl butadiene pathway having at least one exogenous nucleic 4 -phosphate kinase , a butadiene synthase , a crotonyl -CoA acid encoding butadiene pathway enzymes expressed in a hydrolase , synthetase , or transferase and a crotonate reduc sufficient amount to produce butadiene, the butadiene path - 45 tase (FIG . 2 , steps N , I, J , E , F , G , H ) . In one aspect, the way including a glutaryl- CoA dehydrogenase , a butadiene non - naturally occurring microbial organism includes a synthase , a crotonyl- CoA reductase (alcohol forming ) and a microbial organism having a butadiene pathway having at crotyl alcohol diphosphokinase ( FIG . 2 , steps M , K , P , H ) . least one exogenous nucleic acid encoding butadiene path In one aspect, the non -naturally occurring microbial organ - way enzymes expressed in a sufficient amount to produce ism includes a microbial organism having a butadiene path - 50 butadiene , the butadiene pathway including an 3 -aminobu way having at least one exogenous nucleic acid encoding tyryl - CoA deaminase , a crotonaldehyde reductase ( alcohol butadiene pathway enzymes expressed in a sufficient amount forming ), a butadiene synthase , a crotonyl- CoA hydrolase , to produce butadiene , the butadiene pathway including a synthetase or transferase , a crotonate reductase and a crotyl glutaryl- CoA dehydrogenase , a crotonaldehyde reductase alcohol diphosphokinase (FIG . 2 , steps N , I , J , E , P , H ) . In ( alcohol forming ) , a crotyl alcohol kinase , a 2 -butenyl - 4 - 55 one aspect , the non -naturally occurring microbial organism phosphate kinase , a butadiene synthase , a crotonyl- CoA includes a microbial organism having a butadiene pathway hydrolase , synthetase, or transferase and a crotonate reduc - having at least one exogenous nucleic acid encoding buta tase ( FIG . 2 , steps M , I , J , E , F , G , H ) . In one aspect, the diene pathway enzymes expressed in a sufficient amount to non -naturally occurring microbial organism includes a produce butadiene , the butadiene pathway including a 3 -hy microbial organism having a butadiene pathway having at 60 droxybutyryl -CoA dehydratase , a crotonyl -CoA reductase least one exogenous nucleic acid encoding butadiene path - ( aldehyde forming ) , a crotonaldehyde reductase (alcohol way enzymes expressed in a sufficient amount to produce forming ), a butadiene synthase , a 3 - aminobutyryl- CoA butadiene, the butadiene pathway including a glutaryl- CoA deaminase and a crotyl alcohol diphosphokinase ( FIG . 2 , dehydrogenase , a crotonaldehyde reductase (alcohol form - steps N , C , D , E , P , H ) . In one aspect , the non - naturally ing ) , a butadiene synthase , a crotonyl -CoA hydrolase , syn - 65 occurring microbial organism includes a microbial organism thetase or transferase, a crotonate reductase and a crotyl having a butadiene pathway having at least one exogenous alcohol diphosphokinase (FIG . 2 , steps M , I , J , E , P , H ) . In nucleic acid encoding butadiene pathway enzymes US 10 ,006 ,055 B2 13 14 expressed in a sufficient amount to produce butadiene, the ing butadiene pathway enzymes expressed in a sufficient butadiene pathway including a 4 - hydroxybutyryl- CoA dehy amount to produce butadiene, the butadiene pathway includ dratase , a crotonyl- CoA reductase ( aldehyde forming ) , a ing an erythrose - 4 - phosphate reductase , an erythritol- 4 crotonaldehyde reductase (alcohol forming) , a crotyl alcohol phosphate cytidylyltransferase , a 4 - ( cytidine 5 ' - diphospho ) kinase , a 2 - butenyl- 4 -phosphate kinase and a butadiene 5 erythritol kinase , an erythritol 2 , 4 -cyclodiphosphate synthase ( FIG . 2 , steps O , D - H ) . In one aspect, the non - synthase , a 1 - hydroxy - 2 -butenyl 4 - diphosphate synthase , a naturally occurring microbial organism includes a microbial 1 -hydroxy - 2 -butenyl 4 -diphosphate reductase and a butadi organism having a butadiene pathway having at least one ene synthase (FIG . 3 , steps A - F , and H ) . In one aspect , the exogenous nucleic acid encoding butadiene pathway non -naturally occurring microbial organism includes a enzymes expressed in a sufficient amount to produce buta - 10 microbial organism having a butadiene pathway having at diene , the butadiene pathway including a 4 -hydroxybutyryl - least one exogenous nucleic acid encoding butadiene path COA dehydratase , a crotyl alcohol kinase, a 2 -butenyl - 4 - way enzymes expressed in a sufficient amount to produce phosphate kinase , a butadiene synthase and crotonyl- CoA butadiene, the butadiene pathway including an erythrose - 4 reductase (alcohol forming ) ( FIG . 2 , steps O , K , F , G , H ) . In phosphate reductase , an erythritol - 4 - phosphate cytidylyl one aspect, the non -naturally occurring microbial organism 15 transferase , a 4 - ( cytidine 5 ' - diphospho ) - erythritol kinase , an includes a microbial organism having a butadiene pathway erythritol 2 , 4 - cyclodiphosphate synthase , a 1 - hydroxy - 2 having at least one exogenous nucleic acid encoding buta - butenyl 4 - diphosphate synthase , a 1 - hydroxy - 2 - butenyl diene pathway enzymes expressed in a sufficient amount to 4 - diphosphate reductase , a butenyl 4 - diphosphate isomerase produce butadiene , the butadiene pathway including a 4 -hy - and butadiene synthase ( FIG . 3 , steps A - H ) . In one aspect , droxybutyryl- CoA dehydratase , a butadiene synthase , a 20 the non -naturally occurring microbial organism includes a crotonyl - CoA reductase ( alcohol forming ) and a crotyl alco - microbial organism having a butadiene pathway having at hol diphosphokinase (FIG . 2 , steps O , K , P , H ) . In one least one exogenous nucleic acid encoding butadiene path aspect , the non - naturally occurring microbial organism way enzymes expressed in a sufficient amount to produce includes a microbial organism having a butadiene pathway butadiene, the butadiene pathway including an erythritol- 4 having at least one exogenous nucleic acid encoding buta - 25 phosphate cytidylyltransferase , a 4 - cytidine 5 ' - diphospho ) diene pathway enzymes expressed in a sufficient amount to erythritol kinase , an erythritol 2 , 4 -cyclodiphosphate syn produce butadiene , the butadiene pathway including a 4 -hy - thase , a 1 -hydroxy - 2 - butenyl 4 - diphosphate synthase , a droxybutyryl- CoA dehydratase , a crotonaldehyde reductase 1 -hydroxy - 2 -butenyl 4 -diphosphate reductase , a butadiene ( alcohol forming ) , a crotyl alcohol kinase , a 2 - butenyl- 4 - synthase , an erythrose - 4 - phosphate kinase , an erythrose phosphate kinase , a butadiene synthase , a crotonyl -CoA 30 reductase and a erythritol kinase ( FIG . 3 , steps I , J , K , B - F , hydrolase , synthetase , or transferase and a crotonate reduc - H ) . In one aspect, the non - naturally occurring microbial tase (FIG . 2 , steps O , I, J , E , F , G , H ) . In one aspect , the organism includes a microbial organism having a butadiene non - naturally occurring microbial organism includes a pathway having at least one exogenous nucleic acid encod microbial organism having a butadiene pathway having at ing butadiene pathway enzymes expressed in a sufficient least one exogenous nucleic acid encoding butadiene path - 35 amount to produce butadiene , the butadiene pathway includ way enzymes expressed in a sufficient amount to produce ing an erythritol- 4 -phosphate cytidylyltransferase , a 4 - cy butadiene, the butadiene pathway including a 4 - hydroxybu - tidine 5 '- diphospho ) -erythritol kinase, an erythritol 2 ,4 -cy tyryl- CoA dehydratase , a crotonaldehyde reductase (alcohol clodiphosphate synthase , a 1 -hydroxy - 2 -butenyl forming ), a butadiene synthase , a crotonyl- CoA hydrolase , 4 -diphosphate synthase , a 1 -hydroxy - 2 -butenyl 4 - diphos synthetase or transferase , a crotonate reductase and a crotyl 40 phate reductase , a butenyl 4 -diphosphate isomerase , a buta alcohol diphosphokinase (FIG . 2 , steps O , I , J , E , P , H ) . In diene synthase , an erythrose - 4 -phosphate kinase , an eryth one aspect , the non -naturally occurring microbial organism rose reductase and an erythritol kinase (FIG . 3 , steps I , J , K , includes a microbial organism having a butadiene pathway B - H ) . having at least one exogenous nucleic acid encoding buta In some embodiments , the invention provides a non diene pathway enzymes expressed in a sufficient amount to 45 naturally occurring microbial organism , including a micro produce butadiene , the butadiene pathway including a 3 -hy - bial organism having a butadiene pathway having at least droxybutyryl- CoA dehydratase , a crotonyl- CoA reductase one exogenous nucleic acid encoding a butadiene pathway ( aldehyde forming) , a crotonaldehyde reductase (alcohol enzyme expressed in a sufficient amount to produce buta forming ), a butadiene synthase , a 4 -hydroxybutyryl - CoA diene , the butadiene pathway including a malonyl- CoA : dehydratase and a crotyl alcohol diphosphokinase ( FIG . 2 , 50 acetyl- CoA acyltransferase , an 3 -oxoglutaryl - CoA reductase steps L , C , D , E , P , H ) . (ketone - reducing ) , a 3 - hydroxyglutaryl- CoA reductase (al In some embodiments , the invention provides a non dehyde forming ), a 3 -hydroxy -5 -oxopentanoate reductase , a naturally occurring microbial organism , including a micro - 3 , 5 - dihydroxypentanoate kinase , a 3 -hydroxy - 5 -phosphona bial organism having a butadiene pathway having at least tooxypentanoate kinase , a 3 - hydroxy - 5 - [hydroxy ( phospho one exogenous nucleic acid encoding a butadiene pathway 55 nooxy ) phosphoryl] oxy pentanoate decarboxylase , a butenyl enzyme expressed in a sufficient amount to produce buta - 4 -diphosphate isomerase , a butadiene synthase , a 3 - hy diene, the butadiene pathway including an erythrose - 4 - droxyglutaryl- CoA reductase ( alcohol forming ) , an 3 -oxo phosphate reductase , an erythritol - 4 - phosphate cytidylyl glutaryl- CoA reductase ( aldehyde forming ) , a 3 , 5 - dioxopen transferase, a 4 -( cytidine 5' - diphospho ) - erythritol kinase , an tanoate reductase (ketone reducing) , a 3 ,5 - dioxopentanoate erythritol 2 , 4 - cyclodiphosphate synthase , a 1 -hydroxy - 2 - 60 reductase (aldehyde reducing ) , a 5 - hydroxy - 3 -oxopentano butenyl 4 - diphosphate synthase , a 1 -hydroxy - 2 - butenyl a te reductase or an 3 - oxo - glutaryl- CoA reductase (COA 4 - diphosphate reductase , a butenyl 4 -diphosphate reducing and alcohol forming ) (FIG . 4 ) . In one aspect, the isomerase , a butadiene synthase , an erythrose - 4 - phosphate non - naturally occurring microbial organism includes a kinase , an erythrose reductase or an erythritol kinase ( FIG . microbial organism having a butadiene pathway having at 3 ). In one aspect , the non -naturally occurring microbial 65 least one exogenous nucleic acid encoding butadiene path organism includes a microbial organism having a butadiene way enzymes expressed in a sufficient amount to produce pathway having at least one exogenous nucleic acid encod - butadiene , the butadiene pathway including a malonyl- CoA : US 10 ,006 , 055 B2 15 16 acetyl- CoA acyltransferase , an 3 - oxoglutaryl- CoA reductase hyde to crotyl alcohol , crotyl alcohol to 2 - betenyl- phos (ketone - reducing ) , a 3 - hydroxyglutaryl - CoA reductase ( al- phate , 2 -betenyl - phosphate to 2 -butenyl - 4 - diphosphate , dehyde forming ) , a 3 -hydroxy - 5 - oxopentanoate reductase , a 2 -butenyl - 4 -diphosphate to butadiene , erythrose - 4 -phos 3 , 5 - dihydroxypentanoate kinase , a 3 -hydroxy - 5 - phosphona phate to erythritol - 4 -phosphate , erythritol- 4 - phosphate to tooxypentanoate kinase , a 3 -hydroxy - 5 - [hydroxy (phospho - 5 4 - ( cytidine 5 '- diphospho ) - erythritol, 4 - cytidine 5 '- diphos nooxy )phosphoryl ] oxy pentanoate decarboxylase , a butenyl pho ) - erythritol to 2 -phospho - 4 - ( cytidine 5 ' -diphospho ) 4 - diphosphate isomerase and a butadiene synthase (FIG . 4 , erythritol, 2 - phospho - 4 - ( cytidine 5 ' - diphospho ) - erythritol to steps A - I ). In one aspect, the non -naturally occurring micro - erythritol- 2 , 4 -cyclodiphosphate , erythritol- 2 , 4 - cyclodiphos bial organism includes a microbial organism having a buta - phate to 1- hydroxy - 2 -butenyl 4 - diphosphate, 1 - hydroxy -2 diene pathway having at least one exogenous nucleic acid 10 butenyl 4 - diphosphate to butenyl 4 -diphosphate , butenyl encoding butadiene pathway enzymes expressed in a suffi - 4 -diphosphate to 2 - butenyl 4 -diphosphate , 1 -hydroxy - 2 cient amount to produce butadiene , the butadiene pathway butenyl 4 - diphosphate to 2 -butenyl 4 - diphosphate , 2 - bute including a malonyl- CoA :acetyl - CoA acyltransferase , a 3 , 5 - nyl 4 -diphosphate to butadiene , malonyl- CoA and acetyl dihydroxypentanoate kinase, a 3 -hydroxy - 5 -phosphona CoA to 3 -oxoglutaryl - CoA , 3 -oxoglutaryl - CoA to tooxypentanoate kinase , a 3 -hydroxy -5 - [hydroxy (phospho - 15 3 -hydroxyglutaryl - CoA to 3 -hydroxy -5 - oxopentanoate , nooxy )phosphoryl ] oxy pentanoate decarboxylase , a butenyl 3 -hydroxy - 5 -oxopentanoate to 3 ,5 - dihydroxy pentanoate , 4 - diphosphate isomerase , a butadiene synthase , an 3 - oxo 3 , 5 - dihydroxy pentanoate to 3 -hydroxy - 5 -phosphonatooxy glutaryl- CoA reductase ( aldehyde forming ) , a 3 , 5 -dioxopen pentanoate , 3 -hydroxy - 5 - phosphonatooxypentanoate to tanoate reductase ( aldehyde reducing ) and a 5 -hydroxy - 3 3 - hydroxy - 5 - [hydroxy (phosphonooxy )phosphoryl ] oxy pen oxopentanoate reductase . (FIG . 4 , steps A , K , M , N , E , F , G , 20 tanoate , 3 -hydroxy - 5 -[ hydroxy (phosphonooxy ) phosphoryl] H , I ) . In one aspect, the non -naturally occurring microbial oxy pentanoate to butenyl 4 - biphosphate , glutaconyl- CoA to organism includes a microbial organism having a butadiene crotonyl- CoA , glutaryl - CoA to crotonyl- CoA , 3 -aminobu pathway having at least one exogenous nucleic acid encod tyryl- CoA to crotonyl- CoA , 4 -hydroxybutyryl - CoA to ing butadiene pathway enzymes expressed in a sufficient crotonyl - CoA , crotonyl- CoA to crotonate , crotonate to cro amount to produce butadiene , the butadiene pathway includ - 25 tonaldehyde , crotonyl- CoA to crotyl alcohol, crotyl alcohol ing a malonyl- CoA : acetyl- CoA acyltransferase , a 3 -hy - to 2 - butenyl - 4 - diphosphate , erythrose - 4 - phosphate to eryth droxy - 5 - oxopentanoate reductase, a 3 , 5 -dihydroxypentano - rose , erythrose to erythritol, erythritol to erythritol - 4 - phos ate kinase , a 3 -Hydroxy - 5 - phosphonatooxypentanoate phate , 3 - oxoglutaryl- CoA to 3 , 5 -dioxopentanoate , 3 , 5 -di kinase , a 3 -Hydroxy - 5 - [hydroxy (phosphonooxy ) phospho - oxopentanoate to 5 -hydroxy - 3 -oxopentanoate , 5 -hydroxy - 3 ryl] oxy pentanoate decarboxylase , a butenyl 4 -diphosphate 30 oxopentanoate to 3 , 5 - dihydroxypentanoate , 3 -oxoglutaryl isomerase , a butadiene synthase , an 3 -oxoglutaryl - CoA COA to 5 -hydroxy - 3 -oxopentanoate , 3 , 5 - dioxopentanoate to reductase ( aldehyde forming) and a 3 , 5 -dioxopentanoate 3 -hydroxy - 5 - oxopentanoate, 3 -hydroxyglutaryl - CoA to 3 , 5 reductase ( ketone reducing ) . ( FIG . 4 , steps A , K , L , D , E , F , dihydroxypentanoate and oxaloacetate to malate , malate to G , H , I ) . In one aspect, the non -naturally occurring microbial fumarate , fumarate to succinate, succinate to succinyl- CoA , organism includes a microbial organism having a butadiene 35 succinyl -CoA to a -ketoglutarate , a -ketoglutarate to D - iso pathway having at least one exogenous nucleic acid encod citrate , D - isocitrate to succinate , D - isocitrate to glyoxylate , ing butadiene pathway enzymes expressed in a sufficient glyoxylate and acetyl- CoA to malate , D -isocitrate to citrate , amount to produce butadiene , the butadiene pathway includ - citrate to acetate , citrate to oxaloacetate , citrate to acetyl ing a malonyl- CoA :acetyl - CoA acyltransferase , a 3 , 5 -dihy - COA , acetyl -CoA to pyruvate, pyruvate to phosphoenolpyru droxypentanoate kinase , a 3 - hydroxy - 5 - phosphonatooxy - 40 vate , pyruvate to oxaloacetate , pyruvate to malate , phospho pentanoate kinase , a 3 -hydroxy - 5 - [hydroxy ( phosphonooxy ) enolpyruvate to oxaloacetate . One skilled in the art will phosphoryl] oxy pentanoate decarboxylase , a butenyl understand that these are merely exemplary and that any of 4 - diphosphate isomerase , a butadiene synthase , a 5 -hy - the substrate - product pairs disclosed herein suitable to pro droxy - 3 -oxopentanoate reductase and a 3 -oxo - glutaryl- CoA duce a desired product and for which an appropriate activity reductase (CoA reducing and alcohol forming ) . (FIG . 4 , 45 is available for the conversion of the substrate to the product steps A , O , N , E , F , G , H , I) . In one aspect, the non -naturally can be readily determined by one skilled in the art based on occurring microbial organism includes a microbial organism the teachings herein . Thus, the invention provides a non having a butadiene pathway having at least one exogenous naturally occurring microbial organism containing at least nucleic acid encoding butadiene pathway enzymes one exogenous nucleic acid encoding an enzyme or protein , expressed in a sufficient amount to produce butadiene , the 50 where the enzyme or protein converts the substrates and butadiene pathway including a malonyl- CoA : acetyl- CoA products of a butadiene or a crotyl alcohol pathway , such as acyltransferase , an 3 - oxoglutaryl - CoA reductase ( ketone - that shown in FIGS. 2 - 7 and 10 - 11 . reducing ) , a 3 , 5 - dihydroxypentanoate kinase, a 3 -hydroxy - While generally described herein as a microbial organism 5 -phosphonatooxypentanoate kinase , a 3 -hydroxy -5 - [hy - that contains a butadiene or a crotyl alcohol pathway, it is droxy (phosphonooxy )phosphoryl ] oxy pentanoate 55 understood that the invention additionally provides a non decarboxylase , a butenyl 4 - diphosphate isomerase , a buta naturally occurring microbial organism comprising at least diene synthase and a 3 -hydroxyglutaryl - CoA reductase (al one exogenous nucleic acid encoding a butadiene or a crotyl cohol forming) . (FIG . 4 , steps A , B , J , E , F , G , H , I) . alcohol pathway enzyme expressed in a sufficient amount to In an additional embodiment, the invention provides a produce an intermediate of a butadiene or a crotyl alcohol non - naturally occurring microbial organism having a buta - 60 pathway. For example , as disclosed herein , a butadiene diene or a crotyl alcohol pathway , wherein the non -naturally pathway is exemplified in FIGS . 2 - 4 . Therefore , in addition occurring microbial organism comprises at least one exog - to a microbial organism containing a butadiene pathway that enous nucleic acid encoding an enzyme or protein that produces butadiene, the invention additionally provides a converts a substrate to a product selected from the group non -naturally occurring microbial organism comprising at consisting of acetyl- CoA to acetoacetyl - CoA , acetoacetyl- 65 least one exogenous nucleic acid encoding a butadiene COA to 3 -hydroxybutyryl - CoA , 3 -hydroxybutyryl - CoA to pathway enzyme, where the microbial organism produces a crotonyl -CoA , crotonyl- CoA to crotonaldehyde, crotonalde - butadiene pathway intermediate , for example , acetoacetyl US 10 ,006 , 055 B2 17 18 COA , 3 - hydroxybutyryl -CoA , crotonyl -CoA , crotonalde - been developed , and most designs are based on partial hyde , crotyl alcohol, 2 -betenyl - phosphate , 2 -butenyl - 4 - oxidation , where limiting oxygen avoids full combustion , of diphosphate , erythritol- 4 -phosphate , 4 - cytidine organic materials at high temperatures (500 -1500° C . ) to 5' -diphospho )- erythritol, 2 - phospho -4 -( cytidine 5 ' -diphos provide syngas as a 0 . 5 : 1 - 3 : 1 H / CO mixture . In addition to pho ) - erythritol, erythritol- 2 , 4 - cyclodiphosphate , 1 -hydroxy - 5 coal, biomass of many types has been used for syngas 2 -butenyl 4 -diphosphate , butenyl 4 -diphosphate , 2 -butenyl production and represents an inexpensive and flexible feed 4 -diphosphate , 3 - oxoglutaryl- CoA , 3 -hydroxyglutaryl - CoA , stock for the biological production of renewable chemicals 3 - hydroxy - 5 -oxopentanoate , 3 , 5 - dihydroxy pentanoate , and fuels . Carbon dioxide can be provided from the atmo 3 -hydroxy - 5 -phosphonatooxypentanoate , 3 -hydroxy - 5 - [hy - sphere or in condensed from , for example, from a tank droxy (phosphonooxy )phosphoryl ]oxy pentanoate , croto - 10 cylinder, or via sublimation of solid CO2. Similarly , CO and nate , erythrose , erythritol, 3, 5 -dioxopentanoate or 5 -hy - hydrogen gas can be provided in reagent form and /or mixed droxy - 3 -oxopentanoate . in any desired ratio . Other gaseous carbon forms can It is understood that any of the pathways disclosed herein , include , for example , methanol or similar volatile organic as described in the Examples and exemplified in the Figures , solvents . including the pathways of FIGS . 2 - 7 and 10 - 11 , can be 15 The components of synthesis gas and / or other carbon utilized to generate a non - naturally occurring microbial sources can provide sufficient CO , , reducing equivalents , organism that produces any pathway intermediate or prod - and ATP for the reductive TCA cycle to operate . One turn of uct, as desired . As disclosed herein , such a microbial organ the RTCA cycle assimilates two moles of CO2 into one mole ism that produces an intermediate can be used in combina - of acetyl - CoA and requires 2 ATP and 4 reducing equiva tion with another microbial organism expressing 20 lents . CO and /or H2 can provide reducing equivalents by downstream pathway enzymes to produce a desired product. means of carbon monoxide dehydrogenase and hydrogenase However , it is understood that a non -naturally occurring enzymes, respectively . Reducing equivalents can come in microbial organism that produces a butadiene or crotyl the form of NADH , NADPH , FADH , reduced quinones, alcohol pathway intermediate can be utilized to produce the reduced ferredoxins, thioredoxins and reduced flavodoxins . intermediate as a desired product . 25 The reducing equivalents , particularly NADH , NADPH , and This invention is also directed , in part to engineered reduced ferredoxin , can serve as cofactors for the RTCA biosynthetic pathways to improve carbon flux through a cycle enzymes, for example , malate dehydrogenase , fumar central metabolism intermediate en route to butadiene or a te reductase , alpha - ketoglutarate : ferredoxin oxidoreductase crotyl alcohol. The present invention provides non -naturally (alternatively known as 2 - oxoglutarate :ferredoxin oxi occurring microbial organisms having one or more exog - 30 doreductase , alpha - ketoglutarate synthase , or 2 - oxoglutarate enous genes encoding enzymes that can catalyze various synthase ), pyruvate : ferredoxin oxidoreductase and isocitrate enzymatic transformations en route to butadiene or crotyl dehydrogenase . The electrons from these reducing equiva alcohol. In some embodiments , these enzymatic transforma - lents can alternatively pass through an ion - gradient produc tions are part of the reductive tricarboxylic acid (RTCA ) ing electron transport chain where they are passed to an cycle and are used to improve product yields , including but 35 acceptor such as oxygen , nitrate , oxidized metal ions , pro not limited to , from carbohydrate -based carbon feedstock . tons, or an electrode . The ion - gradient can then be used for In numerous engineered pathways , realization of maxi - ATP generation via an ATP synthase or similar enzyme. mum product yields based on carbohydrate feedstock is The reductive TCA cycle was first reported in the green hampered by insufficient reducing equivalents or by loss of sulfur photosynthetic bacterium Chlorobium limicola ( Ev reducing equivalents and /or carbon to byproducts . In accor- 40 ans et al. , Proc . Natl. Acad. Sci. U . S. A . 55 : 928 - 934 ( 1966 ) ). dance with some embodiments , the present invention Similar pathways have been characterized in some prokary increases the yields of butadiene or crotyl alcohol by ( a ) otes (proteobacteria , green sulfur bacteria and thermophillic enhancing carbon fixation via the reductive TCA cycle , Knallgas bacteria ) and sulfur- dependent archaea (Hugler et and / or (b ) accessing additional reducing equivalents from al. , J. Bacteriol. 187: 3020 - 3027 (2005 ; Hugler et al. , Envi gaseous carbon sources and / or syngas components such as 45 ron . Microbiol. 9 :81 - 92 ( 2007 ) . In some cases , reductive and CO , CO , , and / or H , . In addition to syngas , other sources of oxidative (Krebs ) TCA cycles are present in the same such gases include, but are not limited to , the atmosphere , organism (Hugler et al ., supra (2007 ) ; Siebers et al ., J . either as found in nature or generated . Bacteriol. 186 : 2179 - 2194 ( 2004 ) ) . Some methanogens and The CO , - fixing reductive tricarboxylic acid (RTCA ) obligate anaerobes possess incomplete oxidative or reduc cycle is an endergenic anabolic pathway of CO , assimilation 50 tive TCA cycles that may function to synthesize biosynthetic which uses reducing equivalents and ATP (FIG . 6 ). One turn intermediates ( Ekiel et al. , J . Bacteriol. 162 : 905 - 908 (1985 ) ; of the RTCA cycle assimilates two moles of CO , into one Wood et al. , FEMS Microbiol. Rev. 28 : 335 - 352 ( 2004 ) ) . mole of acetyl -CoA , or fourmoles of CO2 into one mole of The key carbon - fixing enzymes of the reductive TCA oxaloacetate . This additional availability of acetyl- CoA cycle are alpha -ketoglutarate : ferredoxin oxidoreductase , improves the maximum theoretical yield of product mol- 55 pyruvate : ferredoxin oxidoreductase and isocitrate dehydro ecules derived from carbohydrate -based carbon feedstock . genase . Additional carbon may be fixed during the conver Exemplary carbohydrates include but are not limited to sion of phosphoenolpyruvate to oxaloacetate by phospho glucose , sucrose , xylose , arabinose and glycerol. enolpyruvate carboxylase or phosphoenolpyruvate In some embodiments , the reductive TCA cycle , coupled carboxykinase or by conversion of pyruvate to malate by with carbon monoxide dehydrogenase and / or hydrogenase 60 malic enzyme . enzymes , can be employed to allow syngas , CO2, CO , H , , Many of the enzymes in the TCA cycle are reversible and and / or other gaseous carbon source utilization by microor - can catalyze reactions in the reductive and oxidative direc ganisms. Synthesis gas ( syngas ) , in particular is a mixture of tions. However , some TCA cycle reactions are irreversible in primarily H , and CO , sometimes including some amounts of vivo and thus different enzymes are used to catalyze these CO2, that can be obtained via gasification of any organic 65 reactions in the directions required for the reverse TCA feedstock , such as coal, coal oil , natural gas, biomass , or cycle . These reactions are : ( 1 ) conversion of citrate to waste organic matter. Numerous gasification processes have oxaloacetate and acetyl- CoA , ( 2 ) conversion of fumarate to US 10 ,006 , 055 B2 19 20 succinate, and ( 3 ) conversion of succinyl- CoA to alpha - ketoglutarate: ferredoxin oxidoreductase . Additionally, such ketoglutarate . In the TCA cycle, citrate is formed from the an organism can also include at least one exogenous enzyme condensation of oxaloacetate and acetyl- CoA . The reverse selected from a carbon monoxide dehydrogenase , a hydro reaction , cleavage of citrate to oxaloacetate and acetyl - CoA , genase , a NAD ( P ) H :ferredoxin oxidoreductase , and a ferre is ATP -dependent and catalyzed by ATP -citrate lyase , or 5 doxin , expressed in a sufficient amount to allow the utiliza citryl- CoA synthetase and citryl- CoA lyase . Alternatively, tion of ( 1 ) CO , ( 2 ) CO , , ( 3 ) H , , ( 4 ) CO , and H ,, ( 5 ) CO and citrate lyase can be coupled to acetyl- CoA synthetase , an CO2, ( 6 ) CO and H2, or ( 7 ) CO , CO2, and H , to produce a acetyl - CoA transferase , or phosphotransacetylase and product . acetate kinase to form acetyl- CoA and oxaloacetate from In some embodiments a non -naturally occurring micro citrate . The conversion of succinate to fumarate is catalyzed 10 bial organism having a butadiene or crotyl alcohol pathway by succinate dehydrogenase while the reverse reaction is further includes at least one exogenous nucleic acid encod catalyzed by fumarate reductase . In the TCA cycle succinyl- ing a reductive TCA pathway enzyme expressed in a suffi COA is formed from the NAD ( P ) + dependent decarboxy - cient amount to enhance carbon flux through acetyl- CoA . lation of alpha -ketoglutarate by the alpha -ketoglutarate The at least one exogenous nucleic acid is selected from an dehydrogenase complex . The reverse reaction is catalyzed 15 ATP - citrate lyase , citrate lyase , a fumarate reductase , a by alpha -ketoglutarate : ferredoxin oxidoreductase . pyruvate : ferredoxin oxidoreductase , isocitrate dehydroge An organism capable of utilizing the reverse tricarboxylic nase , aconitase, and an alpha - ketoglutarate : ferredoxin oxi acid cycle to enable production of acetyl- CoA -derived prod - doreductase . ucts on 1 ) CO , 2 ) CO , and H , 3 ) CO and CO2, 4 ) synthesis In some embodiments a non -naturally occurring micro gas comprising CO and H2, and 5 ) synthesis gas or other 20 bial organism having a butadiene or crotyl alcohol pathway gaseous carbon sources comprising CO , CO2, and H , can includes at least one exogenous nucleic acid encoding an include any of the following enzyme activities : ATP - citrate enzyme expressed in a sufficient amount to enhance the lyase , citrate lyase , aconitase , isocitrate dehydrogenase , availability of reducing equivalents in the presence of car alpha -ketoglutarate :ferredoxin oxidoreductase , succinyl bon monoxide and /or hydrogen , thereby increasing the yield COA synthetase , succinyl -CoA transferase , fumarate reduc - 25 of redox - limited products via carbohydrate - based carbon tase , fumarase , malate dehydrogenase , acetate kinase , phos - feedstock . The at least one exogenous nucleic acid is photransacetylase , acetyl- CoA synthetase , acetyl- CoA selected from a carbon monoxide dehydrogenase , a hydro transferase , pyruvate : ferredoxin oxidoreductase , NAD ( P ) H : genase , an NAD ( P ) H : ferredoxin oxidoreductase , and a ferredoxin oxidoreductase , carbon monoxide dehydroge ferredoxin . In some embodiments, the present invention nase , hydrogenase , and ferredoxin ( see FIG . 7 ) . Enzymes 30 provides a method for enhancing the availability of reducing and the corresponding genes required for these activities are equivalents in the presence of carbon monoxide or hydrogen described herein . thereby increasing the yield of redox - limited products via Carbon from syngas or other gaseous carbon sources can carbohydrate - based carbon feedstock , such as sugars or be fixed via the reverse TCA cycle and components thereof. gaseous carbon sources, the method includes culturing this Specifically , the combination of certain carbon gas - utiliza - 35 non - naturally occurring microbial organism under condi tion pathway components with the pathways for formation tions and for a sufficient period of time to produce butadiene of butadiene or crotyl alcohol from acetyl- CoA results in or crotyl alcohol. high yields of these products by providing an efficient In some embodiments , the non - naturally occurringmicro mechanism for fixing the carbon present in carbon dioxide, bial organism having a butadiene or crotyl alcohol pathway fed exogenously or produced endogenously from CO , into 40 includes two exogenous nucleic acids, each encoding a acetyl - CoA . reductive TCA pathway enzyme. In some embodiments , the In some embodiments , a butadiene or crotyl alcohol non - naturally occurring microbial organism having a buta pathway in a non -naturally occurring microbial organism of diene or crotyl alcohol pathway includes three exogenous the invention can utilize any combination of ( 1 ) CO , ( 2 ) nucleic acids each encoding a reductive TCA pathway CO ) , ( 3 ) Hy, or mixtures thereof to enhance the yields of 45 enzyme . In some embodiments , the non - naturally occurring biosynthetic steps involving reduction , including addition to microbial organism includes three exogenous nucleic acids driving the reductive TCA cycle . encoding an ATP - citrate lyase , a fumarate reductase , and an In some embodiments a non - naturally occurring micro - alpha -ketoglutarate : ferredoxin oxidoreductase . In some bial organism having a butadiene or crotyl alcohol pathway embodiments , the non -naturally occurring microbial organ includes at least one exogenous nucleic acid encoding a 50 ism includes three exogenous nucleic acids encoding a reductive TCA pathway enzyme. The at least one exogenous citrate lyase , a fumarate reductase , and an alpha -ketoglut nucleic acid is selected from an ATP - citrate lyase , citrate arate : ferredoxin oxidoreductase . In some embodiments , the lyase , a fumarate reductase , isocitrate dehydrogenase , aco - non - naturally occurring microbial organism includes four nitase , and an alpha -ketoglutarate : ferredoxin oxidoreduc exogenous nucleic acids encoding a pyruvate : ferredoxin tase ; and at least one exogenous enzyme selected from a 55 oxidoreductase ; a phosphoenolpyruvate carboxylase or a carbon monoxide dehydrogenase , a hydrogenase , a NAD ( P ) phosphoenolpyruvate carboxykinase , a CO dehydrogenase ; H : ferredoxin oxidoreductase , and a ferredoxin , expressed in and an H2 hydrogenase . In some embodiments , the non a sufficient amount to allow the utilization of ( 1 ) CO , ( 2 ) naturally occurring microbial organism includes two exog CO2, ( 3 ) H2, ( 4 ) CO2 and H2, (5 ) CO and CO2, ( 6 ) CO and enous nucleic acids encoding a CO dehydrogenase and an H2, or ( 7 ) CO , CO2, and H2. 60 H2 hydrogenase . In some embodiments a method includes culturing a In some embodiments , the non - naturally occurring micro non - naturally occurring microbial organism having a buta - bial organisms having a butadiene or crotyl alcohol pathway diene or crotyl alcohol pathway also comprising at least one further include an exogenous nucleic acid encoding an exogenous nucleic acid encoding a reductive TCA pathway enzyme selected from a pyruvate: ferredoxin oxidoreductase , enzyme. The at least one exogenous nucleic acid is selected 65 an aconitase , an isocitrate dehydrogenase, a succinyl- CoA from an ATP - citrate lyase , citrate lyase , a fumarate reduc - synthetase , a succinyl -CoA transferase , a fumarase , a malate tase, isocitrate dehydrogenase , aconitase , and an alpha - dehydrogenase, an acetate kinase , a phosphotransacetylase , US 10 ,006 ,055 B2 21 22 an acetyl- CoA synthetase, an NAD (P ) H : ferredoxin oxi wherein the at least one exogenous nucleic acid is selected doreductase , and combinations thereof. from a pyruvate : ferredoxin oxidoreductase , a phospho In some embodiments , the non -naturally occurring micro - enolpyruvate carboxylase , a phosphoenolpyruvate car bial organism having a butadiene or crotyl alcohol pathway boxykinase , a CO dehydrogenase , and an H , hydrogenase ; further includes an exogenous nucleic acid encoding an 5 or ( c ) at least one exogenous nucleic acid encodes an enzyme selected from carbon monoxide dehydrogenase , enzyme selected from a CO dehydrogenase , an H , hydro acetyl- CoA synthase , ferredoxin , NAD ( P ) H : ferredoxin oxi- genase , and combinations thereof. In such a microbial doreductase and combinations thereof. organism , a butadiene pathway can comprise a butadiene In some embodiments , the non -naturally occurring micro - pathway disclosed herein . For example , the butadien path bial organism having a butadiene or crotyl alcohol pathway 10 way can be selected from : (i ) an acetyl - CoA :acetyl - CoA utilizes a carbon feedstock selected from ( 1 ) CO , ( 2 ) CO , , acyltransferase, an acetoacetyl- CoA reductase , a 3 - hydroxy ( 3 ) CO , and H2, ( 4 ) CO and H2, or ( 5 ) CO , CO2, and Hz. In butyryl- CoA dehydratase , a crotonyl- CoA reductase (alde some embodiments , the non - naturally occurring microbial hyde forming ), a crotonaldehyde reductase (alcohol form organism having a butadiene or crotyl alcohol pathway ing ) , a crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase utilizes hydrogen for reducing equivalents . In some embodi - 15 and a butadiene synthase ; ( ii ) an acetyl- CoA : acetyl- CoA ments , the non -naturally occurring microbial organism hav - acyltransferase , an acetoacetyl -CoA reductase , a 3 -hydroxy ing a butadiene or crotyl alcohol pathway utilizes CO for butyryl- CoA dehydratase , a crotyl alcohol kinase , a 2 -bute reducing equivalents . In some embodiments , the non - natu - nyl- 4 -phosphate kinase , a butadiene synthase and crotonyl rally occurring microbial organism having a butadiene or CoA reductase ( alcohol forming ) ; ( iii ) an acetyl - CoA : acetyl crotyl alcohol pathway utilizes combinations of CO and 20 COA acyltransferase , an acetoacetyl- CoA reductase , a hydrogen for reducing equivalents . 3 -hydroxybutyryl - CoA dehydratase , a butadiene synthase , a In some embodiments , the non -naturally occurring micro crotonyl- CoA reductase (alcohol forming ) and a crotyl alco bial organism having a butadiene or crotyl alcohol pathway hol diphosphokinase ; ( iv ) an acetyl- CoA : acetyl- CoA acyl further includes one or more nucleic acids encoding an transferase , an acetoacetyl- CoA reductase , a 3 -hydroxybu enzyme selected from a phosphoenolpyruvate carboxylase , 25 tyryl- CoA dehydratase , a crotonaldehyde reductase (alcohol a phosphoenolpyruvate carboxykinase , a pyruvate carboxy - forming ), a crotyl alcohol kinase , a 2 -butenyl - 4 - phosphate lase , and a malic enzyme . kinase , a butadiene synthase , a crotonyl- CoA hydrolase , In some embodiments , the non -naturally occurring micro - synthetase or transferase and a crotonate reductase ; ( v ) an bial organism having a butadiene or crotyl alcohol pathway acetyl -CoA : acetyl - COA acyltransferase , an acetoacetyl -COA further includes one or more nucleic acids encoding an 30 reductase , a 3 -hydroxybutyryl - CoA dehydratase , a croton enzyme selected from a malate dehydrogenase , a fumarase , aldehyde reductase ( alcohol forming ) , a butadiene synthase , a fumarate reductase , a succinyl- CoA synthetase , and a a crotonyl- CoA hydrolase, synthetase or transferase , a cro succinyl -CoA transferase . tonate reductase and a crotyl alcohol diphosphokinase ; (vi ) In some embodiments , the non -naturally occurring micro an acetyl- CoA :acetyl - CoA acyltransferase , an acetoacetyl bial organism having a butadiene or crotyl alcohol pathway 35 CoA reductase , a 3 -hydroxybutyryl - CoA dehydratase , a further includes at least one exogenous nucleic acid encod - crotonyl- CoA reductase (aldehyde forming ) , a crotonalde ing a citrate lyase , an ATP - citrate lyase , a citryl- CoA syn hyde reductase ( alcohol forming ) , a butadiene synthase and thetase , a citryl- CoA lyase , an aconitase , an isocitrate dehy - a crotyl alcohol diphosphokinase . ( vii ) a glutaconyl -COA drogenase , a succinyl- COA synthetase , a succinyl - CoA decarboxylase , a crotonyl- CoA reductase (aldehyde form transferase , a fumarase , a malate dehydrogenase , an acetate 40 ing ) , a crotonaldehyde reductase (alcohol forming ) , a crotyl kinase , a phosphotransacetylase , an acetyl - CoA synthetase, alcohol kinase , a 2 -butenyl - 4 -phosphate kinase and a buta and a ferredoxin . diene synthase . ( viii ) a glutaconyl- CoA decarboxylase , a It is understood by those skilled in the art that the crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase , a above -described pathways for increasing product yield can butadiene synthase and crotonyl- CoA reductase (alcohol be combined with any of the pathways disclosed herein , 45 forming ) ; ( ix ) a glutaconyl -CoA decarboxylase , a butadiene including those pathways depicted in the figures . One skilled synthase , a crotonyl- CoA reductase ( alcohol forming and a in the art will understand that, depending on the pathway to crotyl alcohol diphosphokinase ; ( x ) a glutaconyl -CoA decar a desired product and the precursors and intermediates of boxylase, a crotonaldehyde reductase ( alcohol forming ), a that pathway , a particular pathway for improving product crotyl alcohol kinase , a 2 - butenyl- 4 -phosphate kinase , a yield , as discussed herein above and in the examples , or 50 butadiene synthase , a crotonyl- CoA hydrolase, synthetase , combination of such pathways, can be used in combination or transferase and a crotonate reductase ; (xi ) a glutaconyl with a pathway to a desired product to increase the yield of COA decarboxylase , a crotonaldehyde reductase (alcohol that product or a pathway intermediate. forming ) , a butadiene synthase , a crotonyl -CoA hydrolase , In one embodiment, the invention provides a non -natu synthetase or transferase , a crotonate reductase and a crotyl rally occurring microbial organism , comprising a microbial 55 alcohol diphosphokinase ; (xii ) a 3 -hydroxybutyryl - CoA organism having a butadiene pathway comprising at least dehydratase , a crotonyl- CoA reductase (aldehyde forming) , one exogenous nucleic acid encoding a butadiene pathway a crotonaldehyde reductase ( alcohol forming) , a butadiene a enzyme expressed in a sufficient amount to produce buta - glutaconyl - CoA decarboxylase and a crotyl alcohol diphos diene. Such a microbial organism can further comprise ( a ) a phokinase ; ( xiii ) a glutaryl- CoA dehydrogenase, a crotonyl reductive TCA pathway comprising at least one exogenous 60 CoA reductase ( aldehyde forming ) , a crotonaldehyde reduc nucleic acid encoding a reductive TCA pathway enzyme , tase (alcohol forming ) , a crotyl alcohol kinase, a 2 - butenyl wherein the at least one exogenous nucleic acid is selected 4 - phosphate kinase and a butadiene synthase ; (xiv ) a from an ATP - citrate lyase , a citrate lyase, a citryl- CoA glutaryl - CoA dehydrogenase , a crotyl alcohol kinase , a synthetase , a citryl- CoA lyase , a fumarate reductase , and an 2 - butenyl- 4 -phosphate kinase , a butadiene synthase and alpha- ketoglutarate : ferredoxin oxidoreductase; ( b ) a reduc - 65 crotonyl -CoA reductase ( alcohol forming ) ; (xv ) a glutaryl tive TCA pathway comprising at least one exogenous COA dehydrogenase , a butadiene synthase , a crotonyl- COA nucleic acid encoding a reductive TCA pathway enzyme, reductase ( alcohol forming ) and a crotyl alcohol diphospho US 10 ,006 , 055 B2 23 24 kinase ; ( xvi) a glutaryl- CoA dehydrogenase , a crotonalde - 4 - phosphate cytidylyltransferase , a 4 - cytidine 5 '- diphos hyde reductase (alcohol forming) , a crotyl alcohol kinase, a pho ) - erythritol kinase , an erythritol 2 ,4 - cyclodiphosphate 2 -butenyl - 4 -phosphate kinase , a butadiene synthase , a croto synthase , a 1 -hydroxy - 2 -butenyl 4 - diphosphate synthase , a nyl- CoA hydrolase , synthetase , or transferase and a croto 1 -hydroxy - 2 -butenyl 4 -diphosphate reductase, a butadiene nate reductase ; ( xvii ) a glutaryl -CoA dehydrogenase , a cro - 5 synthase , an erythrose - 4 - phosphate kinase , an erythrose tonaldehyde reductase ( alcohol forming ) , a butadiene reductase and a erythritol kinase ; (xxxiv ) an erythritol- 4 synthase , a crotonyl- CoA hydrolase , synthetase or trans - phosphate cytidylyltransferase , a 4 -( cytidine 5 ' - diphospho ) ferase, a crotonate reductase and a crotyl alcohol diphos - erythritol kinase , an erythritol 2 , 4 -cyclodiphosphate syn phokinase ; ( xviii ) a 3 - hydroxybutyryl - CoA dehydratase , a thase , a 1 -hydroxy - 2 -butenyl 4 - diphosphate synthase , a crotonyl- CoA reductase (aldehyde forming ) , a crotonalde - 10 1 -hydroxy -2 -butenyl 4 -diphosphate reductase , a butenyl hyde reductase (alcohol forming ) , a butadiene synthase , a 4 - diphosphate isomerase , a butadiene synthase , an eryth glutaryl- CoA dehydrogenase and a crotyl alcohol diphos - rose -4 -phosphate kinase , an erythrose reductase and an phokinase ; (xix ) an 3 -aminobutyryl - CoA deaminase, a erythritol kinase; (xxxv ) a malonyl- CoA :acetyl - CoA acyl crotonyl- CoA reductase (aldehyde forming ) , a crotonalde- transferase , an 3 - oxoglutaryl- CoA reductase (ketone - reduc hyde reductase (alcohol forming ) , a crotyl alcohol kinase , a 15 ing ) , a 3 -hydroxyglutaryl - CoA reductase (aldehyde form 2 -butenyl - 4 -phosphate kinase and a butadiene synthase ; (xx ) ing ) , a 3 -hydroxy - 5 - oxopentanoate reductase , a 3 , 5 an 3 - aminobutyryl- CoA deaminase , a crotyl alcohol kinase , dihydroxypentanoate kinase , a 3 - hydroxy - 5 a 2 -butenyl - 4 -phosphate kinase , a butadiene synthase and phosphonatooxypentanoate kinase , a 3 -hydroxy -5 - [hydroxy crotonyl -CoA reductase ( alcohol forming ) ; ( xxi) an 3 - amin (phosphonooxy ) phosphoryl] oxy pentanoate decarboxylase , obutyryl- CoA deaminase , a butadiene synthase , a crotonyl- 20 a butenyl 4 - diphosphate isomerase and a butadiene synthase ; CoA reductase (alcohol forming ) and a crotyl alcohol ( xxxvi) a malonyl- CoA :acetyl - COA acyltransferase , a 3 , 5 diphosphokinase ; ( xxii ) an 3 -aminobutyryl - CoA deaminase , dihydroxypentanoate kinase , a 3 -hydroxy - 5 - phosphona a crotonaldehyde reductase (alcohol forming ), a crotyl alco tooxypentanoate kinase, a 3 -hydroxy - 5 - [hydroxy (phospho hol kinase , a 2 -butenyl - 4 - phosphate kinase , a butadiene nooxy ) phosphoryl] oxy pentanoate decarboxylase, a butenyl synthase , a crotonyl- CoA hydrolase , synthetase or trans- 25 4 - diphosphate isomerase , a butadiene synthase , an 3 - Oxo ferase and a crotonate reductase ; ( xxiii ) an 3 - aminobutyryl - glutaryl - CoA reductase (aldehyde forming ) , a 3 , 5 -dioxopen COA deaminase , a crotonaldehyde reductase (alcohol form - tanoate reductase (aldehyde reducing) and a 5 - hydroxy - 3 ing) , a butadiene synthase , a crotonyl- CoA hydrolase , oxopentanoate reductase ; (xxxvii ) a malonyl- CoA : acetyl synthetase or transferase , a crotonate reductase and a crotyl COA acyltransferase , a 3 -hydroxy - 5 -oxopentanoate alcohol diphosphokinase ; ( xxiv ) a 3 -hydroxybutyryl - CoA 30 reductase , a 3 , 5 - dihydroxypentanoate kinase , a 3 -Hydroxy dehydratase , a crotonyl - CoA reductase (aldehyde forming ) , 5 - phosphonatooxypentanoate kinase , a 3 -Hydroxy - 5 - [hy a crotonaldehyde reductase (alcohol forming ), a butadiene droxy (phosphonooxy )phosphoryl ] oxy pentanoate decar synthase , a 3 - aminobutyryl- CoA deaminase and a crotyl boxylase , a butenyl 4 - diphosphate isomerase , a butadiene alcohol diphosphokinase ; (XXV ) a 4 -hydroxybutyryl - CoA synthase , an 3 -oxoglutaryl - CoA reductase ( aldehyde form dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , 35 ing ) and a 3 , 5 - dioxopentanoate reductase (ketone reducing ) ; a crotonaldehyde reductase (alcohol forming ) , a crotyl alco - ( xxxviii ) a malonyl -CoA :acetyl -CoA acyltransferase , a 3 , 5 hol kinase , a 2 -butenyl - 4 - phosphate kinase and a butadiene dihydroxypentanoate kinase , a 3 -hydroxy - 5 -phosphona synthase ; ( xxvi ) a 4 -hydroxybutyryl - CoA dehydratase , a tooxypentanoate kinase , a 3 -hydroxy - 5 - [hydroxy (phospho crotyl alcohol kinase , a 2 -butenyl - 4 - phosphate kinase , a nooxy ) phosphoryl] oxy pentanoate decarboxylase, a butenyl butadiene synthase and crotonyl- CoA reductase (alcohol 40 4 -diphosphate isomerase , a butadiene synthase , a 5 -hy forming ) ; (xxvii ) a 4 -hydroxybutyryl - CoA dehydratase , a droxy - 3 - oxopentanoate reductase and a 3 -oxo - glutaryl- CoA butadiene synthase , a crotonyl- CoA reductase (alcohol reductase (CoA reducing and alcohol forming ) ; and ( xxxix ) forming ) and a crotyl alcohol diphosphokinase ; (xxviii ) a a butadiene pathway comprising a malonyl -CoA :acetyl 4 -hydroxybutyryl - CoA dehydratase , a crotonaldehyde COA acyltransferase , an 3 -oxoglutaryl - CoA reductase (ke reductase (alcohol forming ) , a crotyl alcohol kinase , a 45 tone - reducing ) , a 3 , 5 - dihydroxypentanoate kinase , a 3 - hy 2 -butenyl - 4 - phosphate kinase , a butadiene synthase , a croto - droxy - 5 - phosphonatooxypentanoate kinase , a 3 - hydroxy - 5 nyl- CoA hydrolase, synthetase or transferase and a crotonate [hydroxy (phosphonooxy )phosphoryl ]oxy pentanoate reductase ; ( xxix ) a 4 -hydroxybutyryl - CoA dehydratase , a decarboxylase , a butenyl 4 -diphosphate isomerase , a buta crotonaldehyde reductase ( alcohol forming ) , a butadiene diene synthase and a 3 -hydroxyglutaryl - CoA reductase (al synthase , a crotonyl- CoA hydrolase , synthetase or trans - 50 cohol forming ) . ferase , a crotonate reductase and a crotyl alcohol diphos - In such microbial organisms of the invention , a microbial phokinase; (xxx ) a 3 -hydroxybutyryl -CoA dehydratase , a organism comprising (a ) can further comprise an exogenous crotonyl - CoA reductase (aldehyde forming ) , a crotonalde - nucleic acid encoding an enzyme selected from a pyruvate : hyde reductase ( alcohol forming ) , a butadiene synthase , a ferredoxin oxidoreductase, an aconitase , an isocitrate dehy 4 -hydroxybutyryl - CoA dehydratase and a crotyl alcohol 55 drogenase , a succinyl- CoA synthetase , a succinyl- CoA diphosphokinase ; ( xxxi) an erythrose - 4 -phosphate reduc - transferase , a fumarase , a malate dehydrogenase , an acetate tase , an erythritol - 4 - phosphate cytidylyltransferase , a 4 - ( cy - kinase , a phosphotransacetylase , an acetyl- CoA synthetase , tidine 5 '- diphospho ) -erythritol kinase , an erythritol 2 ,4 - cy an NAD ( P ) H : ferredoxin oxidoreductase, ferredoxin , and clodiphosphate synthase , a 1 -hydroxy - 2 -butenyl combinations thereof. In addition , a microbial organism 4 -diphosphate synthase , a 1 - hydroxy - 2 -butenyl 4 - diphos - 60 comprising ( b ) can further comprise an exogenous nucleic phate reductase and a butadiene synthase ; (xxxii ) an eryth - acid encoding an enzyme selected from an aconitase , an rose - 4 -phosphate reductase , an erythritol - 4 -phosphate cyti - isocitrate dehydrogenase , a succinyl- CoA synthetase , a suc dylyltransferase , a 4 -( cytidine 5' - diphospho ) -erythritol cinyl- CoA transferase , a fumarase , a malate dehydrogenase , kinase , an erythritol 2 , 4 - cyclodiphosphate synthase , a 1 -hy - and combinations thereof. droxy - 2 -butenyl 4 - diphosphate synthase , a 1 - hydroxy - 2 - 65 In a particular embodiment, such a microbial organism butenyl 4 - diphosphate reductase , a butenyl 4 -diphosphate can comprise two , three , four, five , six or seven exogenous isomerase and a butadiene synthase ; ( xxxiii ) an erythritol nucleic acids each encoding a butadiene pathway enzyme. US 10 ,006 , 055 B2 25 26 For example , such a microbial organism can comprise ferase , a crotonate reductase and a crotyl alcohol diphos exogenous nucleic acids encoding each of the enzymes phokinase ; (xviii ) a 3 -hydroxybutyryl - CoA dehydratase , a selected from : ( i) an acetyl- CoA :acetyl - CoA acyltrans crotonyl- CoA reductase (aldehyde forming ) , a crotonalde ferase , an acetoacetyl- CoA reductase, a 3 - hydroxybutyryl hyde reductase ( alcohol forming ) , a butadiene synthase , a COA dehydratase , a crotonyl -CoA reductase ( aldehyde form - 5 glutaryl -CoA dehydrogenase and a crotyl alcohol diphos ing ) , a crotonaldehyde reductase ( alcohol forming ) , a crotyl phokinase ; (xix ) an 3 - aminobutyryl- CoA deaminase , a alcohol kinase , a 2 -butenyl - 4 -phosphate kinase and a buta crotonyl -CoA reductase (aldehyde forming ), a crotonalde diene synthase ; ( ii ) an acetyl -CoA : acetyl - CoA acyltrans hyde reductase ( alcohol forming ), a crotyl alcohol kinase , a ferase , an acetoacetyl- CoA reductase , a 3 -hydroxybutyryl - 2 -butenyl - 4 -phosphate kinase and a butadiene synthase ; (xx ) CoA dehydratase , a crotyl alcohol kinase, a 2 -butenyl - 4 - 10 an 3 -aminobutyryl - CoA deaminase , a crotyl alcohol kinase , phosphate kinase , a butadiene synthase and crotonyl - CoA a 2 -butenyl - 4 -phosphate kinase , a butadiene synthase and reductase ( alcohol forming) ; ( iii ) an acetyl- CoA :acetyl - CoA crotonyl - CoA reductase ( alcohol forming ); (xxi ) an 3 - amin acyltransferase , an acetoacetyl- CoA reductase , a 3 -hydroxy obutyryl -CoA deaminase, a butadiene synthase , a crotonyl butyryl- CoA dehydratase , a butadiene synthase , a crotonyl- CoA reductase ( alcohol forming ) and a crotyl alcohol CoA reductase (alcohol forming ) and a crotyl alcohol 15 diphosphokinase ; (xxii ) an 3 -aminobutyryl - CoA deaminase , diphosphokinase ; (iv ) an acetyl- CoA :acetyl - CoA acyltrans a crotonaldehyde reductase (alcohol forming ) , a crotyl alco ferase, an acetoacetyl- CoA reductase, a 3 -hydroxybutyryl - hol kinase, a 2 -butenyl - 4 - phosphate kinase , a butadiene COA dehydratase , a crotonaldehyde reductase ( alcohol form - synthase , a crotonyl- CoA hydrolase , synthetase or trans ing) , a crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate ferase and a crotonate reductase ; (xxiii ) an 3 - aminobutyryl kinase , a butadiene synthase , a crotonyl- CoA hydrolase , 20 COA deaminase, a crotonaldehyde reductase ( alcohol form synthetase or transferase and a crotonate reductase ; ( v ) an ing ), a butadiene synthase , a crotonyl -COA hydrolase , acetyl- CoA : acetyl - CoA acyltransferase , an acetoacetyl- CoA synthetase or transferase , a crotonate reductase and a crotyl reductase , a 3 -hydroxybutyryl - CoA dehydratase , a croton alcohol diphosphokinase ; (xxiv ) a 3 -hydroxybutyryl - CoA aldehyde reductase (alcohol forming ) , a butadiene synthase , dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , a crotonyl - CoA hydrolase , synthetase or transferase , a cro - 25 a crotonaldehyde reductase (alcohol forming) , a butadiene tonate reductase and a crotyl alcohol diphosphokinase ; ( vi ) synthase , a 3 - aminobutyryl- CoA deaminase and a crotyl an acetyl- CoA : acetyl -CoA acyltransferase , an acetoacetyl alcohol diphosphokinase ; ( XXV ) a 4 -hydroxybutyryl - CoA CoA reductase , a 3 - hydroxybutyryl- CoA dehydratase , a dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , crotonyl -CoA reductase ( aldehyde forming ) , a crotonalde - a crotonaldehyde reductase ( alcohol forming ) , a crotyl alco hyde reductase ( alcohol forming ) , a butadiene synthase and 30 hol kinase , a 2 -butenyl - 4 - phosphate kinase and a butadiene a crotyl alcohol diphosphokinase ; ( vii ) a glutaconyl- CoA synthase ; (xxvi ) a 4 -hydroxybutyryl - CoA dehydratase , a decarboxylase , a crotonyl- CoA reductase (aldehyde form - crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase, a ing ), a crotonaldehyde reductase ( alcohol forming ) , a crotyl butadiene synthase and crotonyl- CoA reductase (alcohol alcohol kinase , a 2 -butenyl - 4 -phosphate kinase and a buta forming) ; (xxvii ) a 4 -hydroxybutyryl - CoA dehydratase , a diene synthase ; (viii ) a glutaconyl- CoA decarboxylase , a 35 butadiene synthase , a crotonyl - CoA reductase ( alcohol crotyl alcohol kinase , a 2 - butenyl- 4 -phosphate kinase , a forming ) and a crotyl alcohol diphosphokinase ; ( xxviii ) a butadiene synthase and crotonyl -CoA reductase ( alcohol 4 -hydroxybutyryl -CoA dehydratase , a crotonaldehyde forming ) ; ( ix ) a glutaconyl- CoA decarboxylase , a butadiene reductase ( alcohol forming ) , a crotyl alcohol kinase , a synthase, a crotonyl- CoA reductase (alcohol forming ) and a 2 -butenyl - 4 -phosphate kinase, a butadiene synthase , a croto crotyl alcohol diphosphokinase ; (x ) a glutaconyl- CoA decar - 40 nyl -CoA hydrolase, synthetase or transferase and a crotonate boxylase , a crotonaldehyde reductase ( alcohol forming ), a reductase ; (xxix ) a 4 -hydroxybutyryl - CoA dehydratase , a crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase , a crotonaldehyde reductase (alcohol forming ) , a butadiene butadiene synthase , a crotonyl- CoA hydrolase, synthetase , synthase , a crotonyl- CoA hydrolase , synthetase or trans or transferase and a crotonate reductase; (xi ) a glutaconyl- ferase , a crotonate reductase and a crotyl alcohol diphos COA decarboxylase , a crotonaldehyde reductase (alcohol 45 phokinase ; ( XXX ) a 3 -hydroxybutyryl -CoA dehydratase , a forming ), a butadiene synthase , a crotonyl- CoA hydrolase , crotonyl- CoA reductase (aldehyde forming ) , a crotonalde synthetase or transferase , a crotonate reductase and a crotyl hyde reductase (alcohol forming ) , a butadiene synthase , a alcohol diphosphokinase ; ( xii ) a 3 -hydroxybutyryl - CoA 4 -hydroxybutyryl - CoA dehydratase and a crotyl alcohol dehydratase , a crotonyl- CoA reductase (aldehyde forming ), diphosphokinase ; (xxxi ) an erythrose - 4 - phosphate reduc a crotonaldehyde reductase (alcohol forming ), a butadiene a 50 tase , an erythritol- 4 -phosphate cytidylyltransferase , a 4 - (cy glutaconyl- CoA decarboxylase and a crotyl alcohol diphos - tidine 5 - diphospho ) - erythritol kinase , an erythritol 2 ,4 - cy phokinase; (xiii ) a glutaryl- CoA dehydrogenase , a crotonyl- clodiphosphate synthase , a 1 -hydroxy - 2 -butenyl CoA reductase (aldehyde forming ), a crotonaldehyde reduc 4 -diphosphate synthase , a 1 -hydroxy - 2 -butenyl 4 -diphos tase (alcohol forming ), a crotyl alcohol kinase, a 2 -butenyl phate reductase and a butadiene synthase ; (xxxii ) an eryth 4 -phosphate kinase and a butadiene synthase ; (xiv ) a 55 rose- 4 -phosphate reductase, an erythritol- 4 -phosphate cyti glutaryl- CoA dehydrogenase , a crotyl alcohol kinase , a dylyltransferase , a 4 - ( cytidine 5 - diphospho )- erythritol 2 -butenyl - 4 -phosphate kinase, a butadiene synthase and kinase, an erythritol 2 ,4 - cyclodiphosphate synthase , a 1 -hy crotonyl- CoA reductase ( alcohol forming ) ; ( xv ) a glutaryl droxy - 2 -butenyl 4 - diphosphate synthase , a 1 -hydroxy - 2 COA dehydrogenase , a butadiene synthase , a crotonyl- CoA butenyl 4 -diphosphate reductase, a butenyl 4 -diphosphate reductase (alcohol forming ) and a crotyl alcohol diphospho - 60 isomerase and a butadiene synthase ; ( xxxiii ) an erythritol kinase ; ( xvi) a glutaryl- CoA dehydrogenase , a crotonalde - 4 -phosphate cytidylyltransferase , a 4 - cytidine 5 '- diphos hyde reductase (alcohol forming ), a crotyl alcohol kinase , a pho ) -erythritol kinase , an erythritol 2 ,4 -cyclodiphosphate 2 -butenyl - 4 -phosphate kinase, a butadiene synthase , a croto synthase , a 1 -hydroxy - 2 - butenyl 4 -diphosphate synthase , a nyl- CoA hydrolase , synthetase, or transferase and a croto - 1 -hydroxy - 2 - butenyl 4 -diphosphate reductase , a butadiene nate reductase ; (xvii ) a glutaryl- CoA dehydrogenase, a cro - 65 synthase , an erythrose -4 - phosphate kinase , an erythrose tonaldehyde reductase ( alcohol forming ) , a butadiene reductase and a erythritol kinase ; (xxxiv ) an erythritol - 4 synthase , a crotonyl- CoA hydrolase, synthetase or trans phosphate cytidylyltransferase , a 4 -( cytidine 5 -diphospho ) US 10 ,006 , 055 B2 27 28 erythritol kinase , an erythritol 2 ,4 - cyclodiphosphate syn exogenous nucleic acid encoding a reductive TCA pathway thase , a 1 -hydroxy - 2 -butenyl 4 -diphosphate synthase , a enzyme, wherein the at least one exogenous nucleic acid is 1 -hydroxy - 2 -butenyl 4 - diphosphate reductase , a butenyl selected from an ATP - citrate lyase, a citrate lyase , a citryl 4 -diphosphate isomerase , a butadiene synthase , an eryth COA synthetase , a citryl -CoA lyase , a fumarate reductase , rose - 4 - phosphate kinase , an erythrose reductase and an 5 and an alpha - ketoglutarate : ferredoxin oxidoreductase ; ( b ) a erythritol kinase ; ( xxxv ) a malonyl- CoA :acetyl - CoA acyl- reductive TCA pathway comprising at least one exogenous transferase, an 3 -oxoglutaryl - CoA reductase (ketone - reduc - nucleic acid encoding a reductive TCA pathway enzyme, ing ) , a 3 -hydroxyglutaryl - CoA reductase (aldehyde form wherein the at least one exogenous nucleic acid is selected ing ) , a 3 - hydroxy - 5 - oxopentanoate reductase , a 3 , 5 - from a pyruvate: ferredoxin oxidoreductase , a phospho dihydroxypentanoate kinase , a 3 -hydroxy - 5 - 10 enolpyruvate carboxylase , a phosphoenolpyruvate car phosphonatooxypentanoate kinase, a 3 -hydroxy - 5 - [ hydroxy boxykinase , a CO dehydrogenase, and an H , hydrogenase ; ( phosphonooxy )phosphoryl ] oxy pentanoate decarboxylase , or ( c ) at least one exogenous nucleic acid encodes an a butenyl 4 - diphosphate isomerase and a butadiene synthase ; enzyme selected from a CO dehydrogenase , an H , hydro (xxxvi ) a malonyl -CoA :acetyl - CoA acyltransferase , a 3 , 5 - genase , and combinations thereof. dihydroxypentanoate kinase, a 3 -hydroxy - 5 - phosphona - 15 In such a microbial organism , the crotyl alcohol pathway tooxypentanoate kinase , a 3 -hydroxy - 5 - [hydroxy (phospho - can be selected from any of those disclosed herein and in the nooxy ) phosphoryl] oxy pentanoate decarboxylase , a butenyl figures. For example , the crotyl alcohol pathway can be 4 - diphosphate isomerase, a butadiene synthase , an 3 - oxo selected from ( i) an acetyl- CoA :acetyl - CoA acyltransferase ; glutaryl- CoA reductase ( aldehyde forming) , a 3 , 5 - dioxopen - an acetoacetyl- CoA reductase ; a 3 -hydroxybutyryl - CoA tanoate reductase (aldehyde reducing ) and a 5 -hydroxy - 3 - 20 dehydratase ; a crotonyl- CoA hydrolase , synthase , or trans oxopentanoate reductase ; (xxxvii ) a malonyl- CoA :acetyl - ferase ; a crotonate reductase; and a crotonaldehyde reduc CoA acyltransferase , a 3 -hydroxy - 5 - oxopentanoate tase ( alcohol forming ); (ii ) an acetyl - CoA :acetyl - CoA acyl reductase , a 3 ,5 - dihydroxypentanoate kinase, a 3 - Hydroxy - transferase; an acetoacetyl- CoA reductase ; a 5 -phosphonatooxypentanoate kinase, a 3 -Hydroxy -5 - [hy - 3 -hydroxybutyryl - CoA dehydratase; a crotonyl- CoA reduc droxy (phosphonooxy ) phosphoryl] oxy pentanoate decar - 25 tase (aldehyde forming ) ; and a crotonaldehyde reductase boxylase , a butenyl 4 -diphosphate isomerase , a butadiene ( alcohol forming ) ; ( iii ) an acetyl - CoA :acetyl - CoA acyltrans synthase , an 3 -oxoglutaryl - CoA reductase (aldehyde form - ferase ; an acetoacetyl- CoA reductase; a 3 -hydroxybutyryl ing ) and a 3 , 5 -dioxopentanoate reductase (ketone reducing) ; COA dehydratase ; and a crotonyl- CoA reductase (alcohol ( xxxviii ) a malonyl- CoA :acetyl - CoA acyltransferase , a 3 , 5 - forming ) ; ( iv ) a glutaconyl- CoA decarboxylase ; a crotonyl dihydroxypentanoate kinase , a 3 -hydroxy - 5 - phosphona - 30 COA hydrolase , synthase , or transferase ; a crotonate reduc tooxypentanoate kinase , a 3 -hydroxy - 5 - [hydroxy ( phospho - tase ; and a crotonaldehyde reductase (alcohol forming ) ; ( v ) nooxy )phosphoryl ] oxy pentanoate decarboxylase , a butenyl a glutaconyl- CoA decarboxylase ; a crotonyl- CoA reductase 4 - diphosphate isomerase , a butadiene synthase , a 5 -hy - (aldehyde forming ) ; and a crotonaldehyde reductase ( alco droxy - 3 -oxopentanoate reductase and a 3 -oxo - glutaryl- CoA hol forming ) ; and ( vi) a glutaconyl- CoA decarboxylase ; and reductase (CoA reducing and alcohol forming ) ; and ( xxxix ) 35 a crotonyl -CoA reductase (alcohol forming ) . ( vii) a glutaryl a butadiene pathway comprising a malonyl- CoA :acetyl - COA dehydrogenase; a crotonyl -CoA hydrolase, synthase , or COA acyltransferase , an 3 -oxoglutaryl - CoA reductase (ke transferase ; a crotonate reductase ; and a crotonaldehyde tone - reducing ) , a 3 , 5 -dihydroxypentanoate kinase , a 3 - hy - reductase (alcohol forming ) ; ( viii ) a glutaryl- CoA dehydro droxy - 5 -phosphonatooxypentanoate kinase , a 3 -hydroxy - 5 genase; a crotonyl- CoA reductase (aldehyde forming ) ; and a [hydroxy (phosphonooxy )phosphoryl ] oxy pentanoate 40 crotonaldehyde reductase (alcohol forming ); ( ix ) a glutaryl decarboxylase, a butenyl 4 - diphosphate isomerase , a buta - COA dehydrogenase ; and a crotonyl - CoA reductase (alcohol diene synthase and a 3 -hydroxyglutaryl - CoA reductase ( al- forming) ; ( x ) a 3 - aminobutyryl -CoA deaminase ; a crotonyl cohol forming ) . COA hydrolase , synthase , or transferase ; a crotonate reduc Such microbial organisms of the invention can comprise tase ; and a crotonaldehyde reductase ( alcohol forming) ; ( xi) two, three , four or five exogenous nucleic acids each encod - 45 a 3 - aminobutyryl -CoA deaminase ; a crotonyl- CoA reduc ing enzymes of ( a ), ( b ) or ( c ). For example , a microbial tase (aldehyde forming ) ; and a crotonaldehyde reductase organism comprising (a ) can comprise three exogenous ( alcohol forming ); ( xii ) a 3 - aminobutyryl- CoA deaminase; nucleic acids encoding ATP - citrate lyase or citrate lyase , a and a crotonyl- CoA reductase ( alcohol forming) ; ( xiii ) a fumarate reductase , and an alpha - ketoglutarate : ferredoxin 4 - hydroxybutyryl- CoA dehydratase ; a crotonyl - CoA hydro oxidoreductase ; a microbial organism comprising ( b ) can 50 lase , synthase , or transferase ; a crotonate reductase ; and a comprise four exogenous nucleic acids encoding pyruvate : crotonaldehyde reductase (alcohol forming ) ; ( xiv ) a 4 -hy ferredoxin oxidoreductase, a phosphoenolpyruvate carboxy - droxybutyryl- CoA dehydratase ; a crotonyl -CoA reductase lase or a phosphoenolpyruvate carboxykinase , a CO dehy - (aldehyde forming ) ; and a crotonaldehyde reductase ( alco drogenase , and an H2 hydrogenase ; or a microbial organism hol forming) ; and (xv ) a 4 -hydroxybutyryl - CoA dehy comprising ( c ) can comprise two exogenous nucleic acids 55 dratase ; and a crotonyl- CoA reductase ( alcohol forming ) . encoding CO dehydrogenase and H2 hydrogenase . The Such a microbial organism of the invention comprising invention further provides methods for producing butadiene ( a ) can further comprise an exogenous nucleic acid encoding by culturing such non -naturally occurring microbial organ an enzyme selected from a pyruvate: ferredoxin oxidoreduc isms under conditions and for a sufficient period of time to tase , an aconitase , an isocitrate dehydrogenase , a succinyl produce butadiene . 60 COA synthetase , a succinyl- CoA transferase , a fumarase , a The invention additionally provides a non -naturally malate dehydrogenase , an acetate kinase , a phosphotrans occurring microbial organism , comprising a microbial acetylase , an acetyl- CoA synthetase , an NAD ( P ) H : ferre organism having a crotyl alcohol pathway comprising at doxin oxidoreductase , ferredoxin , and combinations thereof. least one exogenous nucleic acid encoding a crotyl alcohol Such a microbial organism comprising ( b ) can further com pathway enzyme expressed in a sufficient amount to produce 65 prise an exogenous nucleic acid encoding an enzyme crotyl alcohol. Such a microbial organism can further com - selected from an aconitase , an isocitrate dehydrogenase , a prise (a ) a reductive TCA pathway comprising at least one succinyl- CoA synthetase , a succinyl- CoA transferase, a US 10 , 006 ,055 B2 29 30 fumarase , a malate dehydrogenase , and combinations sources enumerated above will be referred to herein , col thereof. Such a microbial organism can comprise two , three , lectively , as “ uptake sources. ” Uptake sources can provide four , five , six or seven exogenous nucleic acids each encod - isotopic enrichment for any atom present in the product ing a crotyl alcohol pathway enzyme. butadiene or crotyl alcohol or butadiene or crotyl alcohol For example , the microbial organism can comprise exog - 5 pathway intermediate , or for side products generated in enous nucleic acids encoding each of the enzymes selected reactions diverging away from a butadiene or crotyl alcohol from ( 1 ) an acetyl -CoA : acetyl -CoA acyltransferase ; an pathway . Isotopic enrichment can be achieved for any target acetoacetyl- CoA reductase ; a 3 -hydroxybutyryl - CoA dehy - atom including , for example , carbon , hydrogen , oxygen , dratase ; a crotonyl- CoA hydrolase , synthase , or transferase ; , sulfur , phosphorus , chloride or other halogens . a crotonate reductase ; and a crotonaldehyde reductase ( alco - 10 In some embodiments , the uptake sources can be selected hol forming ) ; ( ii ) an acetyl- CoA :acetyl - CoA acyltransferase ; to alter the carbon - 12 , carbon - 13 , and carbon - 14 ratios . In an acetoacetyl- CoA reductase ; a 3 -hydroxybutyryl - CoA some embodiments , the uptake sources can be selected to dehydratase ; a crotonyl- CoA reductase (aldehyde forming ) ; alter the oxygen - 16 , oxygen - 17 , and oxygen - 18 ratios . In and a crotonaldehyde reductase ( alcohol forming ) ; ( iii ) an some embodiments , the uptake sources can be selected to acetyl- CoA : acetyl - CoA acyltransferase ; an acetoacetyl- CoA 15 alter the hydrogen , deuterium , and tritium ratios. In some reductase ; a 3 -hydroxybutyryl - CoA dehydratase ; and a embodiments , the uptake sources can be selected to alter the crotonyl -CoA reductase (alcohol forming ) ; ( iv ) a glutaco - nitrogen - 14 and nitrogen - 15 ratios. In some embodiments , nyl- CoA decarboxylase ; a crotonyl -CoA hydrolase, syn the uptake sources can be selected to alter the sulfur -32 , thase , or transferase ; a crotonate reductase ; and a crotonal- sulfur- 33 , sulfur - 34 , and sulfur- 35 ratios . In some embodi dehyde reductase (alcohol forming ) ; ( v ) a glutaconyl- CoA 20 ments , the uptake sources can be selected to alter the decarboxylase ; a crotonyl- CoA reductase ( aldehyde form - phosphorus - 31, phosphorus - 32 , and phosphorus - 33 ratios . ing) ; and a crotonaldehyde reductase ( alcohol forming ) ; (vi ) In some embodiments , the uptake sources can be selected to a glutaconyl- CoA decarboxylase; and a crotonyl- CoA reduc alter the chlorine - 35 , chlorine- 36 , and chlorine - 37 ratios. tase ( alcohol forming ) ; (vii ) a glutaryl- CoA dehydrogenase ; In some embodiments , a target isotopic ratio of an uptake a crotonyl -CoA hydrolase , synthase , or transferase ; a croto - 25 source can be obtained via synthetic chemical enrichment of nate reductase ; and a crotonaldehyde reductase ( alcohol the uptake source . Such isotopically enriched uptake sources forming ) ; (viii ) a glutaryl- CoA dehydrogenase ; a crotonyl- can be purchased commercially or prepared in the labora CoA reductase ( aldehyde forming ) ; and a crotonaldehyde tory . In some embodiments , a target isotopic ratio of an reductase (alcohol forming ) ; ( ix ) a glutaryl -CoA dehydro - uptake source can be obtained by choice of origin of the genase ; and a crotonyl- CoA reductase ( alcohol forming ) ; ( x ) 30 uptake source in nature . In some such embodiments , a a 3 - aminobutyryl- CoA deaminase ; a crotonyl- CoA hydro - source of carbon , for example , can be selected from a fossil lase , synthase , or transferase ; a crotonate reductase ; and a fuel -derived carbon source, which can be relatively depleted crotonaldehyde reductase ( alcohol forming ) ; (xi ) a 3 - amin - of carbon - 14 , or an environmental carbon source , such as obutyryl- CoA deaminase ; a crotonyl- CoA reductase (alde - CO2, which can possess a larger amount of carbon - 14 than hyde forming ); and a crotonaldehyde reductase (alcohol 35 its petroleum -derived counterpart. forming ) ; (xii ) a 3 -aminobutyryl - CoA deaminase ; and a Isotopic enrichment is readily assessed by mass spectrom crotonyl - CoA reductase ( alcohol forming ) . (xiii ) a 4 -hy etry using techniques known in the art such as Stable Isotope droxybutyryl- CoA dehydratase ; a crotonyl- CoA hydrolase , Ratio Mass Spectrometry (SIRMS ) and Site - Specific Natu synthase , or transferase ; a crotonate reductase ; and a cro ral Isotopic Fractionation by Nuclear Magnetic Resonance tonaldehyde reductase ( alcohol forming) ; (xiv ) a 4 -hydroxy - 40 (SNIF - NMR ) . Such mass spectral techniques can be inte butyryl- CoA dehydratase ; a crotonyl- CoA reductase ( alde grated with separation techniques such as liquid chromatog hyde forming) ; and a crotonaldehyde reductase ( alcohol raphy (LC ) and / or high performance liquid chromatography forming ) ; and (xv ) a 4 -hydroxybutyryl - CoA dehydratase ; (HPLC ) . and a crotonyl- CoA reductase (alcohol forming ) . In some embodiments , the present invention provides Such microbial organisms of the invention can comprise 45 butadiene or crotyl alcohol or a butadiene or crotyl alcohol two , three , four or five exogenous nucleic acids each encod - intermediate that has a carbon - 12 , carbon - 13 , and carbon - 14 ing enzymes of ( a ), ( b ) or (c ) . For example , a microbial ratio that reflects an atmospheric carbon uptake source . In organism comprising ( a ) can comprise three exogenous some such embodiments , the uptake source is CO , . In some nucleic acids encoding ATP - citrate lyase or citrate lyase , a embodiments , the present invention provides butadiene or fumarate reductase , and an alpha -ketoglutarate : ferredoxin 50 crotyl alcohol or a butadiene or crotyl alcohol intermediate oxidoreductase ; a microbial organism comprising ( b ) can that has a carbon - 12 , carbon - 13 , and carbon - 14 ratio that comprise four exogenous nucleic acids encoding pyruvate : reflects petroleum - based carbon uptake source . In some ferredoxin oxidoreductase , a phosphoenolpyruvate carboxy - embodiments , the present invention provides butadiene or lase or a phosphoenolpyruvate carboxykinase , a CO dehy - crotyl alcohol or a butadiene or crotyl alcohol intermediate drogenase , and an H , hydrogenase ; or a microbial organism 55 that has a carbon - 12 , carbon - 13, and carbon - 14 ratio that is comprising ( c ) can comprise two exogenous nucleic acids obtained by a combination of an atmospheric carbon uptake encoding CO dehydrogenase and H2 hydrogenase . The source with a petroleum - based uptake source . Such combi invention additionally provides methods for producing cro - nation of uptake sources is one means by which the carbon tyl alcohol, comprising culturing the non - naturally occurring 12 , carbon - 13 , and carbon - 14 ratio can be varied . microbial organism under conditions and for a sufficient 60 The invention is described herein with general reference period of time to produce crotyl alcohol. to the metabolic reaction , reactant or product thereof, or with In some embodiments , the carbon feedstock and other specific reference to one or more nucleic acids or genes cellular uptake sources such as phosphate , ammonia , sulfate , encoding an enzyme associated with or catalyzing , or a chloride and other halogens can be chosen to alter the protein associated with , the referenced metabolic reaction , isotopic distribution of the atoms present in butadiene or 65 reactant or product . Unless otherwise expressly stated crotyl alcohol or any butadiene or crotyl alcohol pathway herein , those skilled in the art will understand that reference intermediate. The various carbon feedstock and other uptake to a reaction also constitutes reference to the reactants and US 10 , 006 , 055 B2 31 32 products of the reaction . Similarly , unless otherwise for a desired biosynthetic pathway, then expressible nucleic expressly stated herein , reference to a reactant or product acids for the deficient enzyme ( s ) or protein ( s ) are introduced also references the reaction , and reference to any of these into the host for subsequent exogenous expression . Alterna metabolic constituents also references the gene or genes tively , if the chosen host exhibits endogenous expression of encoding the enzymes that catalyze or proteins involved in 5 some pathway genes , but is deficient in others , then an the referenced reaction , reactant or product . Likewise , given encoding nucleic acid is needed for the deficient enzyme( s ) the well known fields ofmetabolic biochemistry , enzymol- or protein ( s ) to achieve butadiene biosynthesis . Thus, a ogy and genomics , reference herein to a gene or encoding non - naturally occurring microbial organism of the invention nucleic acid also constitutes a reference to the corresponding can be produced by introducing exogenous enzyme or encoded enzyme and the reaction it catalyzes or a protein 10 protein activities to obtain a desired biosynthetic pathway or associated with the reaction as well as the reactants and a desired biosynthetic pathway can be obtained by introduc products of the reaction . ing one or more exogenous enzyme or protein activities that, As disclosed herein , the intermediates crotonate , 3 , 5 - together with one or more endogenous enzymes or proteins , dioxopentanoate , 5 - hydroxy - 3 - oxopentanoate , 3 -hydroxy - 5 . produces a desired product such as butadiene . oxopentanoate, 3 -oxoglutaryl - CoA and 3 -hydroxyglutaryl - 15 Host microbial organisms can be selected from , and the COA , as well as other intermediates , are carboxylic acids, non -naturally occurring microbial organisms generated in , which can occur in various ionized forms, including fully for example , bacteria , yeast, fungus or any of a variety of protonated , partially protonated , and fully deprotonated other microorganisms applicable to fermentation processes . forms. Accordingly, the suffix " -ate , " or the acid form , can Exemplary bacteria include species selected from Escheri be used interchangeably to describe both the free acid form 20 chia coli , Klebsiella oxytoca , Anaerobiospirillum succin as well as any deprotonated form , in particular since the iciproducens, Actinobacillus succinogenes, Mannheimia ionized form is known to depend on the pH in which the succiniciproducens, Rhizobium etli, Bacillus subtilis, compound is found . It is understood that carboxylate prod - Corynebacterium glutamicum , Gluconobacter oxydans, ucts or intermediates includes ester forms of carboxylate Zymomonas mobilis , Lactococcus lactis , Lactobacillus plan products or pathway intermediates , such as O - carboxylate 25 tarum , Streptomyces coelicolor, Clostridium acetobutyli and S - carboxylate esters. O - and S - carboxylates can include cum , Pseudomonas fluorescens, and Pseudomonas putida . lower alkyl, that is C1 to 06 , branched or straight chain Exemplary yeasts or fungi include species selected from carboxylates . Some such O - or S - carboxylates include , Saccharomyces cerevisiae , Schizosaccharomyces pombe, without limitation , methyl, ethyl, n - propyl, n -butyl , i -propyl , Kluyveromyces lactis , Kluyveromyces marxianus , Aspergil sec -butyl , and tert -butyl , pentyl, hexyl O - or S -carboxylates , 30 lus terreus , Aspergillus niger , Pichia pastoris , Rhizopus any of which can further possess an unsaturation , providing arrhizus, Rhizopus oryzae , Yarrowia lipolytica , and the like . for example , propenyl, butenyl, pentyl, and hexenyl O - or E . coli is a particularly useful host organism since it is a well S - carboxylates . O - carboxylates can be the product of a characterized microbial organism suitable for genetic engi biosynthetic pathway. Exemplary O - carboxylates accessed neering . Other particularly useful host organisms include via biosynthetic pathways can include , without limitation : 35 yeast such as Saccharomyces cerevisiae . It is understood that methyl crotonate ; methy - 3 , 5 - dioxopentanoate ; methyl- 5 -hy - any suitable microbial host organism can be used to intro droxy - 3 -oxopentanoate ; methyl- 3 -hydroxy - 5 -oxopentano duce metabolic and / or genetic modifications to produce a ate ; 3 -oxoglutaryl - CoA , methyl ester ; 3 -hydroxyglutaryl - desired product. COA , methyl ester ; ethyl crotonate ; ethyl- 3 , 5 - Depending on the butadiene or crotyl alcohol biosynthetic dioxopentanoate ; ethyl- 5 - hydroxy - 3 - oxopentanoate ; ethyl- 40 pathway constituents of a selected host microbial organism , 3 - hydroxy - 5 -oxopentanoate ; 3 -oxoglutaryl - CoA , ethyl the non - naturally occurring microbial organisms of the ester ; 3 -hydroxyglutaryl - CoA , ethyl ester ; n -propyl croto - invention will include at least one exogenously expressed nate ; n -propyl - 3 , 5 - dioxopentanoate ; n -propyl - 5 -hydroxy - 3 butadiene or crotyl alcohol pathway -encoding nucleic acid oxopentanoate ; n -propyl - 3 -hydroxy - 5 - oxopentanoate ; and up to all encoding nucleic acids for one or more 3 -oxoglutaryl - CoA , n -propyl ester ; and 3 -hydroxyglutaryl - 45 butadiene or crotyl alcohol biosynthetic pathways . For COA , n -propyl ester. Other biosynthetically accessible example , butadiene biosynthesis can be established in a host O - carboxylates can include medium to long chain groups, deficient in a pathway enzyme or protein through exogenous that is C7 -C22 , O -carboxylate esters derived from fatty expression of the corresponding encoding nucleic acid . In a alcohols , such heptyl, octyl, nonyl, decyl, undecyl, lauryl , host deficient in all enzymes or proteins of a butadiene tridecyl, myristyl, pentadecyl, cetyl, palmitoyl, heptadecyl, 50 pathway, exogenous expression of all enzyme or proteins in stearyl , nonadecyl, arachidyl, heneicosyl, and behenyl alco - the pathway can be included , although it is understood that hols , any one of which can be optionally branched and / or all enzymes or proteins of a pathway can be expressed even contain unsaturations. O - carboxylate esters can also be if the host contains at least one of the pathway enzymes or accessed via a biochemical or chemical process , such as proteins . For example , exogenous expression of all enzymes esterification of a free carboxylic acid product or transes - 55 or proteins in a pathway for production of butadiene can be terification of an o - or S - carboxylate . S -carboxylates are included , such as an acetyl- CoA :acetyl - CoA acyltransferase , exemplified by COA S - esters, cysteinyl S -esters , alkylthio an acetoacetyl- CoA reductase, a 3 -hydroxybutyryl - CoA esters , and various aryl and heteroaryl thioesters . dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , The non -naturally occurring microbial organisms of the a crotonaldehyde reductase ( alcohol forming ) , a crotyl alco invention can be produced by introducing expressible 60 hol kinase , a 2 - butenyl- 4 - phosphate kinase and a butadiene nucleic acids encoding one or more of the enzymes or synthase ( FIG . 2 , steps A - H ) . proteins participating in one or more butadiene or crotyl Given the teachings and guidance provided herein , those alcohol biosynthetic pathways . Depending on the host skilled in the art will understand that the number of encoding microbial organism chosen for biosynthesis , nucleic acids nucleic acids to introduce in an expressible form will , at for some or all of a particular butadiene or crotyl alcohol 65 least , parallel the butadiene or crotyl alcohol pathway defi biosynthetic pathway can be expressed . For example, if a ciencies of the selected host microbial organism . Therefore , chosen host is deficient in one or more enzymes or proteins a non -naturally occurring microbial organism of the inven US 10 , 006 , 055 B2 33 34 tion can have one , two , three , four, five , six , seven , eight, endogenous gene can be engineered to incorporate an induc nine or ten , up to all nucleic acids encoding the enzymes or ible regulatory element, thereby allowing the regulation of proteins constituting a butadiene or crotyl alcohol biosyn - increased expression of an endogenous gene at a desired thetic pathway disclosed herein . In some embodiments , the time. Similarly , an inducible promoter can be included as a non - naturally occurring microbial organisms also can 5 regulatory element for an exogenous gene introduced into a include other genetic modifications that facilitate or opti - non - naturally occurring microbial organism . mize butadiene or crotyl alcohol biosynthesis or that confer It is understood that, in methods of the invention , any of other useful functions onto the host microbial organism . One the one or more exogenous nucleic acids can be introduced such other functionality can include, for example , augmen - into a microbial organism to produce a non - naturally occur tation of the synthesis of one or more of the butadiene or 10 ring microbial organism of the invention . The nucleic acids crotyl alcohol pathway precursors such as acetyl - CoA , can be introduced so as to confer , for example, a butadiene glutaconyl- CoA , glutaryl- CoA , 3 - aminobutyryl- CoA , 4 - hy - or crotyl alcohol biosynthetic pathway onto the microbial droxybutyryl- CoA , erythrose - 4 - phosphate or malonyl- CoA . organism . Alternatively, encoding nucleic acids can be intro Generally , a host microbial organism is selected such that duced to produce an intermediate microbialorganism having it produces the precursor of a butadiene or crotyl alcohol 15 the biosynthetic capability to catalyze some of the required pathway , either as a naturally produced molecule or as an reactions to confer butadiene or crotyl alcohol biosynthetic engineered product that either provides de novo production capability . For example , a non -naturally occurring microbial of a desired precursor or increased production of a precursor organism having a butadiene biosynthetic pathway can naturally produced by the host microbial organism . For comprise at least two exogenous nucleic acids encoding example , acetyl- CoA , glutaconyl- CoA , glutaryl- CoA , 20 desired enzymes or proteins, such as the combination of a 3 - aminobutyryl- CoA , 4 -hydroxybutyryl -CoA , erythrose - 4 - crotyl alcohol kinase and a butadiene synthase , or alterna phosphate or malonyl- CoA are produced naturally in a host tively a 4 - ( cytidine 5 '- diphospho ) - erythritol kinase and a organism such as E . coli. A host organism can be engineered butadiene synthase , or alternatively a 1- hydroxy - 2 -butenyl to increase production of a precursor, as disclosed herein . In 4 -diphosphate synthase and a butadiene synthase , or alter addition , a microbial organism that has been engineered to 25 natively a 3 - hydroxy - 5 - phosphonatooxypentanoate kinase produce a desired precursor can be used as a host organism and a butadiene synthase , or alternatively a crotonyl- CoA and further engineered to express enzymes or proteins of a hydrolase and a crotyl alcohol diphosphokinase , or alterna butadiene or crotyl alcohol pathway . tively an erythrose reductase and butadiene synthase , or In some embodiments , a non - naturally occurring micro - alternatively an 3 - oxo - glutaryl - CoA reductase (CoA reduc bial organism of the invention is generated from a host that 30 ing and alcohol forming ) and 3 -Hydroxy -5 - [hydroxy contains the enzymatic capability to synthesize butadiene or (phosphonooxy )phosphoryl ]oxy pentanoate decarboxylase , crotyl alcohol. In this specific embodiment it can be useful or alternative an ATP - citrate lyase and butadiene synthase , to increase the synthesis or accumulation of a butadiene or or alternatively a pyruvate : ferredoxin oxidoreductase and a a crotyl alcohol pathway product to , for example, drive crotyl alcohol diphosphokinase , or alternatively a CO dehy butadiene or crotyl alcohol pathway reactions toward buta - 35 drogenase and a butadiene synthase , and the like . Thus , it is diene or crotyl alcohol production . Increased synthesis or understood that any combination of two or more enzymes or accumulation can be accomplished by , for example , over- proteins of a biosynthetic pathway can be included in a expression of nucleic acids encoding one or more of the non - naturally occurring microbial organism of the inven above -described butadiene or crotyl alcohol pathway tion . Similarly , it is understood that any combination of three enzymes or proteins . Overexpression the enzyme or 40 or more enzymes or proteins of a biosynthetic pathway can enzymes and / or protein or proteins of the butadiene or crotyl be included in a non -naturally occurring microbial organism alcohol pathway can occur, for example , through exogenous of the invention , for example , a crotyl alcohol kinase , a expression of the endogenous gene or genes , or through 2 -butenyl - 4 -phosphate kinase and a butadiene synthase , or exogenous expression of the heterologous gene or genes . alternatively a 1 - hydroxy - 2 -butenyl 4 - diphosphate synthase , Therefore , naturally occurring organisms can be readily 45 a 1 -hydroxy - 2 -butenyl 4 - diphosphate reductase , and a buta generated to be non - naturally occurring microbial organisms diene synthase , or alternatively an 3 - oxoglutaryl- CoA reduc of the invention , for example , producing butadiene or crotyl tase , a 3 -hydroxy - 5 - oxopentanoate reductase , and a butadi alcohol, through overexpression of one , two , three , four, ene synthase , or alternatively an acetyl- CoA :acetyl - COA five , six , seven , eight, nine , or ten , that is , up to all nucleic acyltransferase , a crotyl alcohol kinase and a butadiene acids encoding butadiene or crotyl alcohol biosynthetic 50 synthase , or alternatively a glutaconyl- CoA decarboxylase , a pathway enzymes or proteins . In addition , a non -naturally crotonyl- CoA reductase (alcohol forming ), and a crotyl occurring organism can be generated by mutagenesis of an alcohol diphosphokinase , or alternatively a an erythrose - 4 endogenous gene that results in an increase in activity of an phosphate kinase , a 4 - ( cytidine 5 ' -diphospho ) -erythritol enzyme in the butadiene or crotyl alcohol biosynthetic kinase and a 1- hydroxy - 2 -butenyl 4 -diphosphate synthase, pathway . 55 or alternatively a 3 , 5 - dioxopentanoate reductase (aldehyde In particularly useful embodiments , exogenous expres - reducing ) , a butenyl 4 - diphosphate isomerase , and a buta sion of the encoding nucleic acids is employed . Exogenous diene synthase, or alternatively a citrate lyase , a fumarate expression confers the ability to custom tailor the expression reductase , and a butadiene synthase , or alternatively a phos and / or regulatory elements to the host and application to phoenolpyruvate carboxylase , a CO dehydrogenase , and a achieve a desired expression level that is controlled by the 60 butadiene synthase , or alternatively an alpha - ketoglutarate : user. However, endogenous expression also can be utilized ferredoxin oxidoreductase , an H2 hydrogenase , and a crotyl in other embodiments such as by removing a negative alcohol diphosphokinase , and so forth , as desired , so long as regulatory effector or induction of the gene ’ s promoter when the combination of enzymes and /or proteins of the desired linked to an inducible promoter or other regulatory element. biosynthetic pathway results in production of the corre Thus , an endogenous gene having a naturally occurring 65 sponding desired product. Similarly , any combination of inducible promoter can be up - regulated by providing the four, such as a crotonaldehyde reductase ( alcohol forming ) , appropriate inducing agent, or the regulatory region of an a crotyl alcoholkinase , a 2 -butenyl - 4 - phosphate kinase and US 10 ,006 , 055 B2 35 36 a butadiene synthase , or alternatively a 1 -hydroxy - 2 -butenyl version of one pathway intermediate to another pathway 4 - diphosphate synthase , a 1 -hydroxy - 2 -butenyl 4 - diphos intermediate or the product. Alternatively , butadiene also phate reductase , a butenyl 4 - diphosphate isomerase and can be biosynthetically produced from microbial organisms butadiene synthase , or alternatively a 3 -hydroxy - 5 -phospho through co - culture or co - fermentation using two organisms natooxypentanoate kinase , a 3 -hydroxy - 5 - hydroxy 5 in the same vessel, where the first microbial organism (phosphonooxy ) phosphoryl] oxy pentanoate kinase , a bute - produces a butadiene intermediate and the second microbial nyl 4 -diphosphate isomerase and a butadiene synthase , or organism converts the intermediate to butadiene . alternatively an erythrose - 4 - phosphate reductase , an eryth - Given the teachings and guidance provided herein , those ritol- 4 -phosphate cytidylyltransferase , a 4 - ( cytidine skilled in the art will understand that a wide variety of 5 - diphospho ) - erythritol kinase and a butadiene synthase , or 10 combinations and permutations exist for the non - naturally alternatively an 3 - aminobutyryl -CoA deaminase , a crotonyl occurring microbial organisms and methods of the invention CoA reductase (alcohol forming ) , a crotyl alcohol diphos together with other microbial organisms, with the co -culture phokinase and a butadiene synthase , or alternatively an of other non -naturally occurring microbial organisms having erythrose reductase , a 4 - ( cytidine 5 ' - diphospho ) - erythritol subpathways and with combinations of other chemical and / kinase , an erythritol 2 , 4 - cyclodiphosphate synthase and a 15 or biochemical procedures well known in the art to produce 1 -hydroxy - 2 -butenyl 4 - diphosphate reductase , or alterna - butadiene or crotyl alcohol. tively a malonyl- CoA :acetyl - CoA acyltransferase , a 3 -hy - Sources of encoding nucleic acids for a butadiene or droxyglutaryl- CoA reductase ( alcohol forming) , a butenyl crotyl alcohol pathway enzyme or protein can include , for 4 -diphosphate isomerase and a butadiene synthase , or alter example , any species where the encoded gene product is natively citrate lyase, a fumarate reductase , an alpha -keto - 20 capable of catalyzing the referenced reaction . Such species glutarate : ferredoxin oxidoreductase , and a butadiene syn - include both prokaryotic and eukaryotic organisms includ thase , or alternatively a phosphoenolpyruvate ing , but not limited to , bacteria , including archaea and carboxykinase , a CO dehydrogenase , an H2 hydrogenase eubacteria , and eukaryotes, including yeast , plant, insect, and a crotyl alcohol diphosphokinase , or alternatively a animal, and mammal, including human . Exemplary species pyruvate : ferredoxin oxidoreductase , a phosphoenolpyruvate 25 for such sources include, for example , Escherichia coli , carboxylase , a phosphoenolpyruvate carboxykinase, and a Acetobacter aceti, Acidaminococcus fermentans , Acineto glutaconyl- CoA decarboxylase , or more enzymes or proteins bacter baylyi, Acinetobacter calcoaceticus , Acinetobacter of a biosynthetic pathway as disclosed herein can be sp . ADP1, Acinetobacter sp . Strain M - 1, Actinobacillus included in a non -naturally occurring microbial organism of succinogenes, Aeropyrum pernix , Allochromatium vinosum the invention , as desired , so long as the combination of 30 DSM 180 , Anaerobiospirillum succiniciproducens, Aquifex enzymes and / or proteins of the desired biosynthetic pathway aeolicus, Aquifex aeolicus, Arabidopsis thaliana , Arabidop results in production of the corresponding desired product. sis thaliana col, Archaeoglobus fulgidus, Archaeoglobus In addition to the biosynthesis of butadiene or croty1 fulgidus DSM 4304, Aromatoleum aromaticum EbN1, alcohol as described herein , the non - naturally occurring Ascaris suum , Aspergillus nidulans, Azoarcus sp . CIB , Azo microbial organisms and methods of the invention also can 35 arcus sp . T , Azotobacter vinelandii DJ, Bacillus cereus, be utilized in various combinations with each other and with Bacillus megaterium , Bacillus subtilis , Balnearium lithotro other microbial organisms and methods well known in the phicum , Bos Taurus, BRC 13350 , Brucella melitensis, Bur art to achieve product biosynthesis by other routes . For kholderia ambifaria AMMD , Burkholderia phymatum , example , one alternative to produce butadiene other than use butyrate - producing bacterium L2 - 50 , Campylobacter curvus of the butadiene producers is through addition of another 40 525 .92 , Campylobacter jejuni, Candida albicans, Candida microbial organism capable of converting a butadiene path - magnolia , Carboxydothermus hydrogenoformans, Chloro way intermediate to butadiene . One such procedure bium phaeobacteroides DSM 266 , Chlorobium limicola , includes, for example , the fermentation of a microbial Chlorobium tepidum , Chloroflexus aurantiacus, Citrobacter organism that produces a butadiene pathway intermediate . youngae ATCC 29220 , Clostridium acetobutylicum , The butadiene pathway intermediate can then be used as a 45 Clostridium aminobutyricum , Clostridium beijerinckii , substrate for a second microbial organism that converts the Clostridium beijerinckii NCIMB 8052 , Clostridium beijer butadiene pathway intermediate to butadiene . The butadiene inckii NRRL B593 , Clostridium botulinum C str . Eklund , pathway intermediate can be added directly to another Clostridium carboxidivorans P7 , Clostridium cellulolyticum culture of the second organism or the original culture of the H10 , Clostridium kluyveri, Clostridium kluyveri DSM 555 , butadiene pathway intermediate producers can be depleted 50 Clostridium novyiNT, Clostridium pasteurianum , of these microbial organisms by, for example , cell separa - Clostridium saccharoperbutylacetonicum , Corynebacterium tion , and then subsequent addition of the second organism to glutamicum , Corynebacterium glutamicum ATCC 13032 , the fermentation broth can be utilized to produce the final Cupriavidus taiwanensis , Cyanobium PCC7001, Desulfovi product without intermediate purification steps . brio africanus , Desulfo Vibrio desulfuricans G20 , Desulfo In other embodiments, the non -naturally occurring micro - 55 vibrio desulfuricans subsp . desulfuricans str . ATCC 27774 , bial organisms and methods of the invention can be Desulfovibrio fructosovorans JJ , Desulfovibrio vulgaris str . assembled in a wide variety of subpathways to achieve Hildenborough , Dictyostelium discoideum AX4 DSM 266 , biosynthesis of, for example, butadiene or crotyl alcohol. In Enterococcus faecalis , Erythrobacter sp . NAP1 , Escheri these embodiments, biosynthetic pathways for a desired chia coli K12 , Escherichia coli str . K - 12 substr. MG1655 , product of the invention can be segregated into different 60 Eubacterium rectale ATCC 33656 , Fusobacterium nuclea microbial organisms, and the different microbial organisms tum , Fusobacterium nucleatum subsp . nucleatum ATCC can be co - cultured to produce the final product. In such a 25586 , Geobacillus thermoglucosidasius, Geobacter metal biosynthetic scheme, the product of one microbial organism lireducens GS -15 , Geobacter sulfurreducens, Haematococ is the substrate for a second microbial organism until the cus pluvialis , Haemophilus influenza , Haloarcula marismor final product is synthesized . For example , the biosynthesis 65 tui, Haloarcula marismortui ATCC 43049 , Helicobacter of butadiene can be accomplished by constructing a micro - pylori, Helicobacter pylori 26695 , Homo sapiens, Hydrog bial organism that contains biosynthetic pathways for con enobacter thermophilus, Klebsiella pneumonia , Klebsiella US 10 ,006 ,055 B2 37 38 pneumonia , Lactobacillus plantarum , Leuconostoc mes alcohol described herein with reference to a particular enteroides, Leuconostoc mesenteroides, Mannheimia suc - organism such as E . coli can be readily applied to other ciniciproducens , marine gamma proteobacterium microorganisms, including prokaryotic and eukaryotic HTCC2080 , Metallosphaera sedula , Methanocaldococcus organisms alike . Given the teachings and guidance provided jannaschii , Methanosarcina thermophila , Methanothermo - 5 herein , those skilled in the art will know that a metabolic bacter thermautotrophicus, Methylobacterium extorquens, alteration exemplified in one organism can be applied Moorella thermoacetica , Mus musculus , Mycobacterium equally to other organisms. avium subsp . paratuberculosis K - 10 , Mycobacterium bovis In some instances, such as when an alternative butadiene BCG , Mycobacterium marinum M , Mycobacterium smeg - or crotyl alcohol biosynthetic pathway exists in an unrelated matis, Mycobacterium smegmatis MC2 155 ,Mycobacterium 10 species , butadiene or crotyl alcohol biosynthesis can be tuberculosis , Mycoplasma pneumoniae M129, Nocardia conferred onto the host species by, for example , exogenous farcinica IFM 10152 , Nocardia iowensis ( sp . NRRL 5646 ) , expression of a paralog or paralogs from the unrelated Nostoc sp . PCC 7120 , Oryctolagus cuniculus, Paracoccus species that catalyzes a similar, yet non - identical metabolic denitrificans, Pelobacter carbinolicus DSM 2380 , Peloto - reaction to replace the referenced reaction . Because certain maculum thermopropionicum , Penicillium chrysogenum , 15 differences among metabolic networks exist between differ Populus alba , Populus tremulaxPopulus alba , Porphyromo ent organisms , those skilled in the art will understand that nas ingivalis, Porphyromonas gingivalis W83 , Pseudomo the actual gene usage between different organisms may nas aeruginosa , Pseudomonas aeruginosa PAO1, differ . However, given the teachings and guidance provided Pseudomonas fluorescens, Pseudomonas fluorescens Pf - 5 , herein , those skilled in the art also will understand that the Pseudomonas knackmussii (B13 ), Pseudomonas putida , 20 teachings andmethods of the invention can be applied to all Pseudomonas putida E23 , Pseudomonas putida KT2440 , microbial organisms using the cognate metabolic alterations Pseudomonas sp , Pueraria Montana , Pyrobaculum aerophi- to those exemplified herein to construct a microbial organ lum str. IM2, Pyrococcus furiosus, Ralstonia eutropha , ism in a species of interest that will synthesize butadiene or Ralstonia eutropha H16 , Ralstonia metallidurans, Rattus crotyl alcohol. norvegicus, Rhodobacter capsulatus , Rhodobacter 25 Methods for constructing and testing the expression levels spaeroides, Rhodococcus rubber, Rhodopseudomonas of a non - naturally occurring butadiene or crotyl alcohol palustris , Rhodopseudomonas palustris , Rhodopseudomo - producing host can be performed , for example , by recom nas palustris CGA009 , Rhodospirillum rubrum , Roseburia binant and detection methods well known in the art. Such intestinalis L1 - 82 , Roseburia inulinivorans DSM 16841 , methods can be found described in , for example , Sambrook Roseburia sp . A2 - 183 , Roseiflexus castenholzii , Saccharo - 30 et al ., Molecular Cloning : A Laboratory Manual , Third Ed. , myces cerevisiae , Saccharomyces cerevisiae , Saccharopoly - Cold Spring Harbor Laboratory, New York (2001 ) ; and spora rythraea NRRL 2338 , Salmonella enteric , Salmonella Ausubel et al ., Current Protocols in Molecular Biology, John enterica subsp . , rizonae serovar, Salmonella typhimurium , Wiley and Sons , Baltimore , Md . ( 1999 ) . Schizosaccharomyces pombe , Simmondsia chinensis , Exogenous nucleic acid sequences involved in a pathway Sinorhizobium meliloti, Sordaria macrospora , Staphylococ - 35 for production of butadiene or crotyl alcohol can be intro cus ureus, Streptococcus pneumonia , Streptomyces coeli - duced stably or transiently into a host cell using techniques color, Streptomyces griseus subsp . griseus, Streptomyces well known in the art including, but not limited to , conju griseus subsp . griseus NBRC 13350 , Streptomyces sp . ACT- gation , electroporation , chemical transformation , transduc 1 , Sulfolobus acidocalarius, Sulfolobus shibatae , Sulfolobus tion , transfection , and ultrasound transformation . For exog solfataricus, Sulfolobus sp . strain 7 , Sulfolobus tokodaii, 40 enous expression in E . coli or other prokaryotic cells , some Sulfurihydrogenibium subterraneum , Sulfurimonas denitri- nucleic acid sequences in the genes or cDNAs of eukaryotic ficans , Synechocystis sp . strain PCC6803 , Syntrophus, cidi- nucleic acids can encode targeting signals such as an N - ter trophicus, Thauera aromatica , Thermoanaerobacter brockii minal mitochondrial or other targeting signal, which can be HTD4, Thermoanaerobacter tengcongensis MB4, Thermo - removed before transformation into prokaryotic host cells , if crinis albus, Thermosynechococcus elongates, Thermotoga 45 desired . For example , removal of a mitochondrial leader maritime, Thermotoga maritime MSB8, Thermus hermophi- sequence led to increased expression in E . coli (Hoffmeister lus HB8 , Thermus thermophilus, Thermus thermophilus, et al. , J . Biol. Chem . 280 :4329 - 4338 ( 2005 ) ). For exogenous Thiobacillus denitrificans, Thiocapsa roseopersicina , expression in yeast or other eukaryotic cells , genes can be Trichomonas vaginalis G3, Trichosporonoides megachilien - expressed in the cytosol without the addition of leader sis , Trypanosoma brucei , Tsukamurella paurometabola 50 sequence , or can be targeted to mitochondrion or other DSM 20162 , Yarrowia lipolytica , Yersinia intermedia ATCC organelles, or targeted for secretion , by the addition of a 29909 , Zea mays , Zoogloea ramigera , Zygosaccharomyces suitable targeting sequence such as a mitochondrial targeting rouxii , Zymomonas mobilis, as well as other exemplary or secretion signal suitable for the host cells . Thus , it is species disclosed herein are available as source organisms understood that appropriate modifications to a nucleic acid for corresponding genes . However, with the complete 55 sequence to remove or include a targeting sequence can be genome sequence available for now more than 550 species incorporated into an exogenous nucleic acid sequence to (with more than half of these available on public databases impart desirable properties. Furthermore , genes can be sub such as the NCBI) , including 395 microorganism genomes jected to codon optimization with techniques well known in and a variety of yeast , fungi , plant, and mammalian t he art to achieve optimized expression of the proteins. genomes, the identification of genes encoding the requisite 60 An expression vector or vectors can be constructed to butadiene or crotyl alcohol biosynthetic activity for one or include one or more butadiene or crotyl alcohol biosynthetic more genes in related or distant species , including for pathway encoding nucleic acids as exemplified herein oper example , homologues , orthologs , paralogs and nonortholo - ably linked to expression control sequences functional in the gous gene displacements of known genes, and the inter - host organism . Expression vectors applicable for use in the change of genetic alterations between organisms is routine 65 microbial host organisms of the invention include , for and well known in the art. Accordingly , the metabolic example , plasmids , phage vectors , viral vectors , episomes alterations allowing biosynthesis of butadiene or crotyl and artificial chromosomes , including vectors and selection US 10 ,006 , 055 B2 39 40 sequences or markers operable for stable integration into a kinase (FIG . 2 , steps A - C , K , P , H ). In one aspect , the host chromosome. Additionally , the expression vectors can method includes a microbial organism having a butadiene include one or more selectable marker genes and appropriate pathway including an acetyl- CoA :acetyl - CoA acyltrans expression control sequences. Selectable marker genes also ferase , an acetoacetyl- CoA reductase, a 3 -hydroxybutyryl can be included that, for example , provide resistance to 5 COA dehydratase , a crotonaldehyde reductase (alcohol form antibiotics or toxins, complement auxotrophic deficiencies , ing ) , a crotyl alcohol kinase , a 2 -butenyl - 4 - phosphate or supply critical nutrients not in the culture media . Expres - kinase , a butadiene synthase , a crotonyl- CoA hydrolase , sion control sequences can include constitutive and induc - synthetase , or transferase and a crotonate reductase , ( FIG . 2 , ible promoters , transcription enhancers , transcription termi- steps A - C , I , J , E , F , G , H ) . In one aspect, the method nators , and the like which are well known in the art. When 10 includes a microbial organism having a butadiene pathway two or more exogenous encoding nucleic acids are to be including an acetyl- CoA :acetyl - CoA acyltransferase , an co -expressed , both nucleic acids can be inserted , for acetoacetyl- CoA reductase, a 3 -hydroxybutyryl - CoA dehy example , into a single expression vector or in separate dratase , a crotonaldehyde reductase ( alcohol forming ) , a expression vectors . For single vector expression , the encod butadiene synthase , a crotonyl- CoA hydrolase , synthetase or ing nucleic acids can be operationally linked to one common 15 transferase , a crotonate reductase and a crotyl alcohol expression control sequence or linked to different expression diphosphokinase (FIG . 2 , steps A - C , I, J , E , P , H ) . In one control sequences, such as one inducible promoter and one aspect , the method includes a microbial organism having a constitutive promoter. The transformation of exogenous butadiene pathway including an acetyl- CoA : acetyl- CoA nucleic acid sequences involved in a metabolic or synthetic acyltransferase , an acetoacetyl -CoA reductase , a 3 -hydroxy pathway can be confirmed using methods well known in the 20 butyryl -CoA dehydratase , a crotonyl- CoA reductase ( alde art . Such methods include , for example , nucleic acid analy - hyde forming ) , a crotonaldehyde reductase (alcohol form sis such as Northern blots or polymerase chain reaction ing ) , a butadiene synthase and a crotyl alcohol ( PCR ) amplification of mRNA , or immunoblotting for diphosphokinase (FIG . 2 , steps A - E , P, H ). In one aspect , the expression of gene products , or other suitable analytical method includes a microbial organism having a butadiene methods to test the expression of an introduced nucleic acid 25 pathway including a glutaconyl- CoA decarboxylase , a croto sequence or its corresponding gene product . It is understood nyl- CoA reductase (aldehyde forming ), a crotonaldehyde by those skilled in the art that the exogenous nucleic acid is reductase (alcohol forming ), a crotyl alcohol kinase , a expressed in a sufficient amount to produce the desired 2 -butenyl - 4 - phosphate kinase and a butadiene synthase product, and it is further understood that expression levels (FIG . 2 , steps L , D - H ) . In one aspect, the method includes can be optimized to obtain sufficient expression using meth - 30 a microbial organism having a butadiene pathway including ods well known in the art and as disclosed herein . a glutaconyl- CoA decarboxylase , a crotyl alcohol kinase , a In some embodiments , the invention provides a method 2 - butenyl- 4 -phosphate kinase, a butadiene synthase and for producing butadiene that includes culturing a non - crotonyl- CoA reductase ( alcohol forming ) ( FIG . 2 , steps L , naturally occurring microbial organism , including a micro K , F , G , H ) . In one aspect, the method includes a microbial bial organism having a butadiene pathway , the butadiene 35 organism having a butadiene pathway including a glutaco pathway including at least one exogenous nucleic acid nyl- CoA decarboxylase , a butadiene synthase , a crotonyl encoding a butadiene pathway enzyme expressed in a suf- CoA reductase (alcohol forming) and a crotyl alcohol ficient amount to produce butadiene , the butadiene pathway diphosphokinase ( FIG . 2 , steps L , K , P , H ) . In one aspect, the including an acetyl- CoA :acetyl - CoA acyltransferase, an method includes a microbial organism having a butadiene acetoacetyl- CoA reductase, a 3 -hydroxybutyryl - CoA dehy - 40 pathway including a glutaconyl- CoA decarboxylase, a cro dratase , a crotonyl - CoA reductase ( aldehyde forming ) , a tonaldehyde reductase ( alcohol forming ) , a crotyl alcohol crotonaldehyde reductase (alcohol forming ) , a crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase , a butadiene syn kinase , a 2 -butenyl - 4 -phosphate kinase , a butadiene syn - thase , a crotonyl- CoA hydrolase , synthetase , or transferase thase , a crotonyl- CoA hydrolase , synthetase , or transferase , and a crotonate reductase (FIG . 2 , steps L , I , J , E , F , G , H ) . a crotonate reductase , a crotonyl -CoA reductase (alcohol 45 In one aspect, the method includes a microbial organism forming ), a glutaconyl - CoA decarboxylase , a glutaryl- CoA having a butadiene pathway including a glutaconyl- CoA dehydrogenase , an 3 - aminobutyryl- CoA deaminase, a 4 -hy - decarboxylase , a crotonaldehyde reductase (alcohol form droxybutyryl- CoA dehydratase or a crotyl alcohol diphos - ing ) , a butadiene synthase , a crotonyl- CoA hydrolase , syn phokinase (FIG . 2 ) . In one aspect, the method includes a thetase or transferase , a crotonate reductase and a crotyl microbial organism having a butadiene pathway including 50 alcohol diphosphokinase (FIG . 2 , steps L , I, J, E , P , H ). In an acetyl- CoA : acetyl -CoA acyltransferase , an acetoacetyl- one aspect , the method includes a microbial organism hav CoA reductase , a 3 - hydroxybutyryl- CoA dehydratase , a ing a butadiene pathway including a 3 - hydroxybutyryl- CoA crotonyl- CoA reductase ( aldehyde forming ), a crotonalde dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , hyde reductase ( alcohol forming ), a crotyl alcohol kinase , a a crotonaldehyde reductase ( alcohol forming ), a butadiene a 2 -butenyl - 4 -phosphate kinase and a butadiene synthase 55 glutaconyl- CoA decarboxylase and a crotyl alcohol diphos ( FIG . 2 , steps A - H ) . In one aspect , the method includes a phokinase (FIG . 2 , steps L , C , D , E , P , H ) . In one aspect, the microbial organism having a butadiene pathway including method includes a microbial organism having a butadiene an acetyl -CoA :acetyl -CoA acyltransferase , an acetoacetyl- pathway including a glutaryl -CoA dehydrogenase , a croto CoA reductase , a 3 -hydroxybutyryl - CoA dehydratase , a cro - nyl- CoA reductase (aldehyde forming ), a crotonaldehyde tyl alcohol kinase , a 2 - butenyl- 4 - phosphate kinase , a buta - 60 reductase alcohol forming ) , a crotyl alcohol kinase , a diene synthase and crotonyl- CoA reductase (alcohol 2 -butenyl - 4 -phosphate kinase and a butadiene synthase forming ) ( FIG . 2 , steps A - C , K , F , G , H ) . In one aspect, the (FIG . 2 , steps M , D - H ) . In one aspect, the method includes method includes a microbial organism having a butadiene a microbial organism having a butadiene pathway including pathway including an acetyl- CoA :acetyl - CoA acyltrans a glutaryl- CoA dehydrogenase, a crotyl alcohol kinase , a ferase , an acetoacetyl- CoA reductase , a 3 -hydroxybutyryl - 65 2 -butenyl - 4 -phosphate kinase, a butadiene synthase and COA dehydratase , a butadiene synthase , a crotonyl- CoA crotonyl -CoA reductase ( alcohol forming ) ( FIG . 2 , steps M , reductase (alcohol forming ) and a crotyl alcohol diphospho - K , F , G , H ). In one aspect, the method includes a microbial US 10 , 006 , 055 B2 41 42 organism having a butadiene pathway including a glutaryl pathway including a 4 - hydroxybutyryl- CoA dehydratase, a COA dehydrogenase , a butadiene synthase , a crotonyl - CoA butadiene synthase , a crotonyl- CoA reductase (alcohol reductase (alcohol forming ) and a crotyl alcohol diphospho forming ) and a crotyl alcohol diphosphokinase (FIG . 2 , steps kinase (FIG . 2 , steps M , K , P , H ) . In one aspect, the method O , K , P, H ) . In one aspect , the method includes a microbial includes a microbial organism having a butadiene pathway 5 organism having a butadiene pathway including a 4 -hy including a glutaryl- CoA dehydrogenase , a crotonaldehyde droxybutyryl - CoA dehydratase , a crotonaldehyde reductase reductase ( alcohol forming) , a crotyl alcohol kinase , a (alcohol forming ) , a crotyl alcohol kinase , a 2 -butenyl - 4 2 -butenyl - 4 - phosphate kinase , a butadiene synthase , a croto nyl- CoA hydrolase, synthetase , or transferase and a croto phosphate kinase , a butadiene synthase , a crotonyl -CoA nate reductase ( FIG . 2 , steps M , I, J, E , F , G , H ). In one 10 hydrolase , synthetase , or transferase and a crotonate reduc aspect , the method includes a microbial organism having a tase (FIG . 2, steps O , I, J, E , F, G , H ). In one aspect, the butadiene pathway including a glutaryl- CoA dehydroge method includes a microbial organism having a butadiene nase , a crotonaldehyde reductase (alcohol forming ), a buta pathway including a 4 -hydroxybutyryl - CoA dehydratase, a diene synthase , a crotonyl- CoA hydrolase , synthetase or crotonaldehyde reductase (alcohol forming ) , a butadiene transferase . a crotonate reductase and a crotyl alcohol 15 synthase , a crotonyl- CoA hydrolase , synthetase or trans diphosphokinase ( FIG . 2 , steps M , I, J, E , P , H ). In one ferase, a crotonate reductase and a crotyl alcohol diphos aspect , the method includes a microbial organism having a phokinase (FIG . 2 , steps O , I , J , E , P , H ) . In one aspect, the butadiene pathway including a 3 -hydroxybutyryl - CoA dehy - method includes a microbial organism having a butadiene dratase , a crotonyl- CoA reductase (aldehyde forming ) , a pathway including a 3 -hydroxybutyryl -CoA dehydratase , a crotonaldehyde reductase ( alcohol forming ), a butadiene 20 crotonyl- CoA reductase (aldehyde forming ) , a crotonalde synthase , a glutaryl- CoA dehydrogenase and a crotyl alcohol hyde reductase ( alcohol forming ) , a butadiene synthase , a diphosphokinase (FIG . 2 , steps M , C , D , E , P , H ) . In one 4 -hydroxybutyryl - CoA dehydratase and a crotyl alcohol aspect , the method includes a microbial organism having a diphosphokinase (FIG . 2 , steps O , C , D , E , P , H ) . butadiene pathway including an 3 -aminobutyryl - COA In some embodiments, the invention provides a method deaminase , a crotonyl- CoA reductase (aldehyde forming ) , a 25 for producing butadiene that includes culturing a non crotonaldehyde reductase (alcohol forming ) , a crotyl alcohol naturally occurring microbial organism , including a micro kinase , a 2 - butenyl- 4 -phosphate kinase and a butadiene bial organism having a butadiene pathway, the butadiene synthase ( FIG . 2 , steps N , D - H ) . In one aspect, the method pathway including at least one exogenous nucleic acid includes a microbial organism having a butadiene pathway encoding a butadiene pathway enzyme expressed in a suf including an 3 -aminobutyryl - CoA deaminase , a crotyl alco - 30 ficient amount to produce butadiene, the butadiene pathway hol kinase , a 2 -butenyl - 4 - phosphate kinase , a butadiene including an erythrose - 4 - phosphate reductase , an erythritol synthase and crotonyl- CoA reductase (alcohol forming) 4 -phosphate cytidylyltransferase, a 4 - (cytidine 5 '- diphos (FIG . 2 , steps N , K , F , G , H ) . In one aspect, the method pho ) -erythritol kinase , an erythritol 2 , 4 -cyclodiphosphate includes a microbial organism having a butadiene pathway synthase , a 1 - hydroxy - 2 -butenyl 4 -diphosphate synthase, a including an 3 - aminobutyryl- CoA deaminase , a butadiene 35 1 -hydroxy - 2 -butenyl 4 - diphosphate reductase , a butenyl synthase , a crotonyl- CoA reductase ( alcohol forming ) and a 4 -diphosphate isomerase , a butadiene synthase , an eryth crotyl alcohol diphosphokinase (FIG . 2 , steps N , K , P , H ). In rose -4 - phosphate kinase , an erythrose reductase or an eryth one aspect, the method includes a microbial organism hav ritol kinase (FIG . 3 ) . In one aspect, the method includes a ing a butadiene pathway including an 3 - aminobutyryl- CoA microbial organism having a butadiene pathway including deaminase , a crotonaldehyde reductase (alcohol forming ) , a 40 an erythrose - 4 - phosphate reductase , an erythritol - 4 -phos crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase , a phate cytidylyltransferase , a 4 - ( cytidine 5 '- diphospho ) butadiene synthase , a crotonyl- CoA hydrolase, synthetase , erythritol kinase , an erythritol 2 , 4 -cyclodiphosphate syn or transferase and a crotonate reductase ( FIG . 2 , steps N , I , thase , a 1 - hydroxy - 2 -butenyl 4 - diphosphate synthase , a J , E , F , G , H ) . In one aspect, the method includes a microbial 1 -hydroxy - 2 -butenyl 4 -diphosphate reductase and a butadi organism having a butadiene pathway including an 3 - amin - 45 ene synthase ( FIG . 3 , steps A - F , and H ) . In one aspect, the obutyryl- CoA deaminase , a crotonaldehyde reductase ( alco - method includes a microbial organism having a butadiene hol forming ), a butadiene synthase , a crotonyl- CoA hydro - pathway including an erythrose - 4 - phosphate reductase , an lase , synthetase or transferase , a crotonate reductase and a erythritol- 4 - phosphate cytidylyltransferase , a 4 - ( cytidine crotyl alcohol diphosphokinase ( FIG . 2 , steps N , I , J , E , P , 5 ' - diphospho ) - erythritol kinase , an erythritol 2 , 4 - cyclo H ) . In one aspect , the method includes a microbial organism 50 diphosphate synthase , a 1 -hydroxy - 2 -butenyl 4 - diphosphate having a butadiene pathway including a 3 -hydroxybutyryl - synthase , a 1 -hydroxy - 2 - butenyl 4 - diphosphate reductase , a COA dehydratase , a crotonyl- CoA reductase (aldehyde form - butenyl 4 - diphosphate isomerase and butadiene synthase ing ) , a crotonaldehyde reductase (alcohol forming ), a buta (FIG . 3 , steps A - H ) . In one aspect , the method includes a diene synthase , a 3 - aminobutyryl -CoA deaminase and a microbial organism having a butadiene pathway including crotyl alcohol diphosphokinase (FIG . 2 , steps N , C , D , E , P , 55 an erythritol- 4 - phosphate cytidylyltransferase, a 4 - ( cytidine H ) . In one aspect, themethod includes a microbial organism 5 '- diphospho ) -erythritol kinase , an erythritol 2 , 4 - cyclo having a butadiene pathway including a 4 -hydroxybutyryl - diphosphate synthase , a 1 -hydroxy - 2 -butenyl 4 - diphosphate CoA dehydratase , a crotonyl- CoA reductase (aldehyde form synthase, a 1 - hydroxy - 2 -butenyl 4 - diphosphate reductase , a ing ) , a crotonaldehyde reductase (alcohol forming ), a crotyl butadiene synthase , an erythrose -4 -phosphate kinase , an alcohol kinase , a 2 -butenyl - 4 -phosphate kinase and a buta - 60 erythrose reductase and a erythritol kinase ( FIG . 3 , steps I , diene synthase (FIG . 2, steps O , D - H ). In one aspect , the J, K , B - F , H ). In one aspect, the method includes a microbial method includes a microbial organism having a butadiene organism having a butadiene pathway including an erythri pathway including a 4 -hydroxybutyryl - CoA dehydratase , a tol- 4 - phosphate cytidylyltransferase , a 4 -( cytidine crotyl alcohol kinase , a 2 - butenyl- 4 -phosphate kinase , a 5 ' - diphospho ) - erythritol kinase , an erythritol 2 , 4 - cyclo butadiene synthase and crotonyl- CoA reductase (alcohol 65 diphosphate synthase, a 1 -hydroxy -2 -butenyl 4 -diphosphate forming ) (FIG . 2 , steps O , K , F , G , H ) . In one aspect, the synthase , a 1 - hydroxy - 2 -butenyl 4 - diphosphate reductase , a method includes a microbial organism having a butadiene butenyl 4 - diphosphate isomerase , a butadiene synthase , an US 10 ,006 , 055 B2 43 44 erythrose - 4 - phosphate kinase , an erythrose reductase and an a 3 - hydroxyglutaryl -CoA reductase ( alcohol forming ). (FIG . erythritol kinase (FIG . 3 , steps I , J, K , B - H ) . 4 , steps A , B , J , E , F , G , H , I ). In some embodiments , the invention provides a method In some embodiments , the invention provides a method for producing butadiene that includes culturing a non - for producing butadiene that includes culturing a non naturally occurring microbial organism , including a micro - 5 naturally occurring microbial organism as described herein , bial organism having a butadiene pathway, the butadiene including a microbial organism having a butadiene pathway pathway including at least one exogenous nucleic acid comprising at least one exogenous nucleic acid encoding a encoding a butadiene pathway enzyme expressed in a suf- butadiene pathway enzyme expressed in a sufficient amount ficient amount to produce butadiene , the butadiene pathway to produce butadiene. Such a microbial organism can further including a malonyl- CoA :acetyl -CoA acyltransferase , an 10 comprise ( a ) a reductive TCA pathway comprising at least 3 -oxoglutaryl - CoA reductase (ketone -reducing ) , a 3 -hy one exogenous nucleic acid encoding a reductive TCA droxyglutaryl - CoA reductase (aldehyde forming ) , a 3 -hy - pathway enzyme, wherein the at least one exogenous nucleic droxy - 5 - oxopentanoate reductase , a 3 , 5 - dihydroxypentano - acid is selected from an ATP - citrate lyase , a citrate lyase , a ate kinase , a 3 -hydroxy - 5 -phosphonatooxypentanoate citryl- CoA synthetase, a citryl- CoA lyase , a fumarate reduc kinase , a 3 - hydroxy - 5 - [hydroxy (phosphonooxy ) phospho - 15 tase , and an alpha -ketoglutarate : ferredoxin oxidoreductase ; ryl ]oxy pentanoate decarboxylase , a butenyl 4 -diphosphate ( b ) a reductive TCA pathway comprising at least one exog isomerase , a butadiene synthase , a 3 -hydroxyglutaryl - CoA enous nucleic acid encoding a reductive TCA pathway reductase (alcohol forming ) , an 3 - oxoglutaryl- CoA reduc- enzyme, wherein the at least one exogenous nucleic acid is tase (aldehyde forming ), a 3 ,5 - dioxopentanoate reductase selected from a pyruvate :ferredoxin oxidoreductase , a phos (ketone reducing ), a 3 , 5 -dioxopentanoate reductase ( alde - 20 phoenolpyruvate carboxylase , a phosphoenolpyruvate car hyde reducing ), a 5 - hydroxy - 3 - oxopentanoate reductase or boxykinase, a CO dehydrogenase, and an H , hydrogenase ; an 3 - oxo - glutaryl- CoA reductase (CoA reducing and alcohol or ( c ) at least one exogenous nucleic acid encodes an forming ) ( FIG . 4 ). In one aspect, the method includes a enzyme selected from a CO dehydrogenase, an H , hydro microbial organism having a butadiene pathway including a genase , and combinations thereof. In such a microbial malonyl -CoA : acetyl -CoA acyltransferase , an 3 -oxoglutaryl - 25 organism , a butadiene pathway can comprise a butadiene CoA reductase (ketone -reducing ) , a 3 -hydroxyglutaryl - CoA pathway disclosed herein . For example , the butadien path reductase (aldehyde forming ) , a 3 - hydroxy - 5 - oxopentanoate way can be selected from : ( i ) an acetyl- CoA :acetyl - CoA reductase , a 3 , 5 -dihydroxypentanoate kinase , a 3 -hydroxy acyltransferase , an acetoacetyl- CoA reductase, a 3 -hydroxy 5 -phosphonatooxypentanoate kinase , a 3 -hydroxy - 5 - [hy - butyryl- CoA dehydratase , a crotonyl- CoA reductase (alde droxy ( phosphonooxy phosphoryl] oxy pentanoate decar - 30 hyde forming ) , a crotonaldehyde reductase (alcohol form boxylase , a butenyl 4 - diphosphate isomerase and a ing ) , a crotyl alcohol kinase , a 2 -butenyl - 4 - phosphate kinase butadiene synthase ( FIG . 4 , steps A - I) . In one aspect , the and a butadiene synthase ; ( ii ) an acetyl - CoA :acetyl - CoA method includes a microbial organism having a butadiene acyltransferase , an acetoacetyl - CoA reductase , a 3 -hydroxy pathway including a malonyl- CoA : acetyl -CoA acyltrans butyryl- CoA dehydratase , a crotyl alcohol kinase , a 2 -bute ferase, a 3 , 5 - dihydroxypentanoate kinase , a 3 -hydroxy - 5 - 35 nyl- 4 -phosphate kinase , a butadiene synthase and crotonyl phosphonatooxypentanoate kinase, a 3- hydroxy - 5 -[ hydroxy CoA reductase (alcohol forming ); ( iii ) an acetyl- CoA :acetyl (phosphonooxy )phosphoryl ] oxy pentanoate decarboxylase , COA acyltransferase, an acetoacetyl- CoA reductase, a a butenyl 4 -diphosphate isomerase, a butadiene synthase, an 3 -hydroxybutyryl - CoA dehydratase , a butadiene synthase , a 3 -oxoglutaryl - CoA reductase (aldehyde forming ) , a 3 , 5 crotonyl- CoA reductase ( alcohol forming ) and a crotyl alco dioxopentanoate reductase (aldehyde reducing ) and a 5 -hy - 40 hol diphosphokinase ; ( iv ) an acetyl - CoA : acetyl- CoA acyl droxy - 3 -oxopentanoate reductase . ( FIG . 4 , steps A , K , M , N , transferase , an acetoacetyl -CoA reductase , a 3 -hydroxybu E , F , G , H , I) . In one aspect , the method includes a microbial tyryl- CoA dehydratase , a crotonaldehyde reductase (alcohol organism having a butadiene pathway including a malonyl- forming ), a crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate CoA :acetyl - CoA acyltransferase , a 3 -hydroxy - 5 -oxopen - kinase , a butadiene synthase , a crotonyl- CoA hydrolase , tanoate reductase , a 3 , 5 - dihydroxypentanoate kinase , a 45 synthetase or transferase and a crotonate reductase ; ( v ) an 3 -Hydroxy - 5 - phosphonatooxypentanoate kinase , a 3 -Hy - acetyl- CoA :acetyl - CoA acyltransferase , an acetoacetyl- CoA droxy - 5 - [hydroxy ( phosphonooxy )phosphoryl ] oxy pentano - reductase , a 3 -hydroxybutyryl - CoA dehydratase , a croton ate decarboxylase , a butenyl 4 -diphosphate isomerase, a aldehyde reductase (alcohol forming ) , a butadiene synthase , butadiene synthase , an 3 -oxoglutaryl - CoA reductase ( alde - a crotonyl- CoA hydrolase , synthetase or transferase , a cro hyde forming ) and a 3 , 5 - dioxopentanoate reductase (ketone 50 tonate reductase and a crotyl alcohol diphosphokinase ; ( vi ) reducing ) . ( FIG . 4 , steps A , K , L , D , E , F , G , H , I ) . In one an acetyl- CoA :acetyl - CoA acyltransferase , an acetoacetyl aspect , the method includes a microbial organism having a COA reductase, a 3 -hydroxybutyryl - CoA dehydratase , a butadiene pathway including a malonyl- CoA :acetyl - CoA crotonyl- CoA reductase ( aldehyde forming) , a crotonalde acyltransferase , a 3 , 5 - dihydroxypentanoate kinase , a 3 -hy - hyde reductase (alcohol forming ) , a butadiene synthase and droxy - 5 -phosphonatooxypentanoate kinase , a 3 -hydroxy - 5 - 55 a crotyl alcohol diphosphokinase . ( vii ) a glutaconyl- CoA [hydroxy (phosphonooxy ) phosphoryl] oxy pentanoate decar - decarboxylase , a crotonyl- CoA reductase (aldehyde form boxylase , a butenyl 4 -diphosphate isomerase , a butadiene ing ) , a crotonaldehyde reductase (alcohol forming ), a crotyl synthase, a 5 -hydroxy - 3 -oxopentanoate reductase and a alcohol kinase , a 2 -butenyl - 4 - phosphate kinase and a buta 3 - oxo - glutaryl- CoA reductase (CoA reducing and alcohol diene synthase . (viii ) a glutaconyl- CoA decarboxylase , a forming ) . (FIG . 4 , steps A , O , N , E , F , G , H , I ) . In one aspect, 60 crotyl alcohol kinase , a 2 -butenyl - 4 -phosphate kinase , a the method includes a microbial organism having a butadi- butadiene synthase and crotonyl- CoA reductase ( alcohol ene pathway including a malonyl - CoA : acetyl- CoA acyl- forming ) ; ( ix ) a glutaconyl- CoA decarboxylase , a butadiene transferase , an 3 -oxoglutaryl - CoA reductase (ketone - reduc synthase, a crotonyl- CoA reductase (alcohol forming ) and a ing ) , a 3 , 5 - dihydroxypentanoate kinase , a 3 - hydroxy - 5 - crotyl alcohol diphosphokinase ; ( x ) a glutaconyl- CoA decar phosphonatooxypentanoate kinase, a 3- hydroxy - 5 -[ hydroxy 65 boxylase , a crotonaldehyde reductase (alcohol forming ), a ( phosphonooxy )phosphoryl ] oxy pentanoate decarboxylase , crotyl alcohol kinase , a 2 - butenyl- 4 -phosphate kinase , a a butenyl 4 - diphosphate isomerase, a butadiene synthase and butadiene synthase , a crotonyl- CoA hydrolase, synthetase , US 10 ,006 ,055 B2 45 46 or transferase and a crotonate reductase ; (xi ) a glutaconyl- ferase , a crotonate reductase and a crotyl alcohol diphos COA decarboxylase , a crotonaldehyde reductase ( alcohol phokinase ; ( xxx ) a 3 -hydroxybutyryl - CoA dehydratase, a forming ), a butadiene synthase , a crotonyl- CoA hydrolase, crotonyl - CoA reductase ( aldehyde forming ), a crotonalde synthetase or transferase, a crotonate reductase and a crotyl hyde reductase ( alcohol forming ) , a butadiene synthase , a alcohol diphosphokinase ; ( xii) a 3 - hydroxybutyryl - CoA 5 4 -hydroxybutyryl - CoA dehydratase and a crotyl alcohol dehydratase , a crotonyl- CoA reductase (aldehyde forming ), diphosphokinase ; (xxxi ) an erythrose - 4 - phosphate reduc a crotonaldehyde reductase ( alcohol forming) , a butadiene a tase , an erythritol- 4 - phosphate cytidylyltransferase , a 4 - cy glutaconyl- CoA decarboxylase and a crotyl alcohol diphos tidine 5 ' - diphospho ) - erythritol kinase , an erythritol 2 , 4 - cy phokinase ; ( xiii ) a glutaryl - CoA dehydrogenase , a crotonyl- clodiphosphate synthase , a 1 - hydroxy - 2 - butenyl CoA reductase (aldehyde forming ), a crotonaldehyde reduc - 10 4 -diphosphate synthase , a 1- hydroxy - 2 -butenyl 4 -diphos tase (alcohol forming ) , a crotyl alcohol kinase , a 2 - butenyl- phate reductase and a butadiene synthase ; ( xxxii ) an eryth 4 - phosphate kinase and a butadiene synthase ; ( xiv ) a rose - 4 - phosphate reductase , an erythritol - 4 - phosphate cyti glutaryl- CoA dehydrogenase , a crotyl alcohol kinase , a dylyltransferase , a 4 -( cytidine 5 '- diphospho ) - erythritol 2 -butenyl - 4 -phosphate kinase, a butadiene synthase and kinase , an erythritol 2 ,4 - cyclodiphosphate synthase , a 1 -hy crotonyl- CoA reductase (alcohol forming ); (xv ) a glutaryl- 15 droxy - 2- butenyl 4 -diphosphate synthase , a 1 - hydroxy - 2 COA dehydrogenase , a butadiene synthase , a crotonyl- CoA butenyl 4 -diphosphate reductase , a butenyl 4 - diphosphate reductase (alcohol forming ) and a crotyl alcohol diphospho - isomerase and a butadiene synthase ; ( xxxiii ) an erythritol kinase ; (xvi ) a glutaryl- CoA dehydrogenase , a crotonalde - 4 -phosphate cytidylyltransferase, a 4 - ( cytidine 5' - diphos hyde reductase (alcohol forming ), a crotyl alcohol kinase, a pho )- erythritol kinase , an erythritol 2 , 4 -cyclodiphosphate 2 - butenyl- 4 - phosphate kinase , a butadiene synthase , a croto - 20 synthase , a 1 -hydroxy - 2 -butenyl 4 - diphosphate synthase , a nyl- CoA hydrolase, synthetase , or transferase and a croto - 1 -hydroxy - 2 - butenyl 4 -diphosphate reductase , a butadiene nate reductase ; (xvii ) a glutaryl - CoA dehydrogenase , a cro - synthase , an erythrose -4 - phosphate kinase , an erythrose tonaldehyde reductase ( alcohol forming ) , a butadiene reductase and a erythritol kinase; (xxxiv ) an erythritol- 4 synthase, a crotonyl- CoA hydrolase, synthetase or trans - phosphate cytidylyltransferase, a 4 - (cytidine 5 '- diphospho ) ferase , a crotonate reductase and a crotyl alcohol diphos - 25 erythritol kinase , an erythritol 2 , 4 -cyclodiphosphate syn phokinase ; (xviii ) a 3 -hydroxybutyryl - CoA dehydratase , a thase , a 1 -hydroxy - 2 - butenyl 4 - diphosphate synthase , a crotonyl -CoA reductase (aldehyde forming ) , a crotonalde - 1 - hydroxy - 2 -butenyl 4 - diphosphate reductase , a butenyl hyde reductase ( alcohol forming ), a butadiene synthase , a 4 -diphosphate isomerase , a butadiene synthase, an eryth glutaryl - CoA dehydrogenase and a crotyl alcohol diphos - rose - 4 - phosphate kinase , an erythrose reductase and an phokinase ; ( xix ) an 3 -aminobutyryl - CoA deaminase, a 30 erythritol kinase ; (xxxv ) a malonyl - CoA :acetyl - CoA acyl crotonyl- CoA reductase ( aldehyde forming ), a crotonalde - transferase , an 3 - oxoglutaryl- CoA reductase (ketone - reduc hyde reductase (alcohol forming ), a crotyl alcohol kinase, a ing ), a 3 -hydroxyglutaryl - CoA reductase ( aldehyde form 2 -butenyl - 4 - phosphate kinase and a butadiene synthase ; (xx ) ing ), a 3 -hydroxy - 5 - oxopentanoate reductase , a 3 , 5 an 3 - aminobutyryl- CoA deaminase , a crotyl alcohol kinase , dihydroxypentanoate kinase , a 3 -hydroxy - 5 a 2 -butenyl - 4 -phosphate kinase, a butadiene synthase and 35 phosphonatooxypentanoate kinase , a 3 -hydroxy - 5 - [hydroxy crotonyl -CoA reductase ( alcohol forming ) ; ( xxi) an 3 - amin (phosphonooxy )phosphoryl ] oxy pentanoate decarboxylase , obutyryl- CoA deaminase , a butadiene synthase , a crotonyl a butenyl 4 -diphosphate isomerase and a butadiene synthase ; CoA reductase (alcohol forming ) and a crotyl alcohol (xxxvi ) a malonyl- CoA :acetyl -CoA acyltransferase, a 3, 5 diphosphokinase ; ( xxii ) an 3 -aminobutyryl - CoA deaminase , dihydroxypentanoate kinase , a 3 - hydroxy - 5 - phosphona a crotonaldehyde reductase (alcohol forming ), a crotyl alco - 40 tooxypentanoate kinase, a 3 -hydroxy -5 -[ hydroxy (phospho hol kinase , a 2 - butenyl- 4 -phosphate kinase , a butadiene nooxy ) phosphoryl] oxy pentanoate decarboxylase, a butenyl synthase , a crotonyl- CoA hydrolase , synthetase or trans - 4 -diphosphate isomerase , a butadiene synthase , an 3 - oxo ferase and a crotonate reductase ; ( xxiii ) an 3 - aminobutyryl glutaryl- CoA reductase ( aldehyde forming ) , a 3 , 5 - dioxopen COA deaminase, a crotonaldehyde reductase ( alcohol form - tanoate reductase ( aldehyde reducing ) and a 5 - hydroxy - 3 ing ) , a butadiene synthase , a crotonyl -CoA hydrolase , 45 oxopentanoate reductase ; ( xxxvii ) a malonyl -CoA :acetyl synthetase or transferase , a crotonate reductase and a crotyl COA acyltransferase , a 3 -hydroxy - 5 -oxopentanoate alcohol diphosphokinase ; ( xxiv ) a 3 -hydroxybutyryl - CoA reductase , a 3 , 5 - dihydroxypentanoate kinase , a 3 -Hydroxy dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , 5 -phosphonatooxypentanoate kinase, a 3 - Hydroxy - 5 - [hy a crotonaldehyde reductase (alcohol forming ), a butadiene droxy (phosphonooxy ) phosphoryl] oxy pentanoate decar synthase , a 3 -aminobutyryl - CoA deaminase and a crotyl 50 boxylase , a butenyl 4 -diphosphate isomerase, a butadiene alcohol diphosphokinase ; (XXV ) a 4 -hydroxybutyryl - CoA synthase , an 3 - oxoglutaryl - CoA reductase (aldehyde form dehydratase , a crotonyl- CoA reductase ( aldehyde forming ), ing ) and a 3 , 5 -dioxopentanoate reductase (ketone reducing) ; a crotonaldehyde reductase (alcohol forming) , a crotyl alco (xxxviii ) a malonyl -CoA :acetyl - CoA acyltransferase, a 3 , 5 hol kinase , a 2 -butenyl - 4 - phosphate kinase and a butadiene dihydroxypentanoate kinase , a 3 -hydroxy - 5 -phosphona synthase; (xxvi ) a 4 -hydroxybutyryl - CoA dehydratase , a 55 tooxypentanoate kinase, a 3 -hydroxy -5 -[ hydroxy (phospho crotyl alcohol kinase , a 2 -butenyl -4 -phosphate kinase , a nooxy )phosphoryl ]oxy pentanoate decarboxylase, a butenyl butadiene synthase and crotonyl- CoA reductase (alcohol 4 - diphosphate isomerase , a butadiene synthase , a 5 -hy forming ); (xxvii ) a 4 -hydroxybutyryl - CoA dehydratase, a droxy - 3 - oxopentanoate reductase and a 3 -oxo - glutaryl- CoA butadiene synthase , a crotonyl- CoA reductase (alcohol reductase (CoA reducing and alcohol forming ) ; and ( xxxix ) forming ) and a crotyl alcohol diphosphokinase ; ( xxviii ) a 60 a butadiene pathway comprising a malonyl - CoA : acetyl 4 -hydroxybutyryl - CoA dehydratase, a crotonaldehyde COA acyltransferase , an 3 -oxoglutaryl - CoA reductase (ke reductase (alcohol forming) , a crotyl alcohol kinase , a tone- reducing) , a 3 , 5 - dihydroxypentanoate kinase, a 3 - hy 2 -butenyl - 4 -phosphate kinase , a butadiene synthase , a croto - droxy - 5 - phosphonatooxypentanoate kinase, a 3 - hydroxy - 5 nyl- CoA hydrolase , synthetase or transferase and a crotonate [ hydroxy ( phosphonooxy )phosphoryl ]oxy pentanoate reductase; (xxix ) a 4 -hydroxybutyryl - CoA dehydratase, a 65 decarboxylase , a butenyl 4 -diphosphate isomerase , a buta crotonaldehyde reductase ( alcohol forming ) , a butadiene diene synthase and a 3 -hydroxyglutaryl - CoA reductase ( al synthase , a crotonyl- CoA hydrolase, synthetase or trans cohol forming ) . US 10 ,006 , 055 B2 47 48 In some embodiments , the invention provides a method reductase and a crotyl alcohol diphosphokinase ; ( xii) a for producing butadiene that includes culturing a non - 3 -hydroxybutyryl - CoA dehydratase , a crotonyl -CoA reduc naturally occurring microbial organism as described herein , tase (aldehyde forming ), a crotonaldehyde reductase (alco including a microbial organism comprising ( a ) as described hol forming ) , a butadiene a glutaconyl- CoA decarboxylase above , which can further comprise an exogenous nucleic 5 and a crotyl alcohol diphosphokinase ; ( xiii) a glutaryl -CoA acid encoding an enzyme selected from a pyruvate : ferre - dehydrogenase , a crotonyl- CoA reductase (aldehyde form doxin oxidoreductase , an aconitase , an isocitrate dehydro ing ), a crotonaldehyde reductase ( alcohol forming ) , a crotyl genase , a succinyl - CoA synthetase , a succinyl- CoA trans alcohol kinase , a 2 -butenyl - 4 - phosphate kinase and a buta ferase , a fumarase , a malate dehydrogenase , an acetate diene synthase; (xiv ) a glutaryl- CoA dehydrogenase , a crotyl kinase , a phosphotransacetylase , an acetyl- CoA synthetase , 10 alcohol kinase , a 2 - butenyl- 4 - phosphate kinase , a butadiene an NAD ( P ) H : ferredoxin oxidoreductase , ferredoxin , and synthase and crotonyl- CoA reductase (alcohol forming ) ; combinations thereof. In addition , a microbial organism (xv ) a glutaryl -CoA dehydrogenase, a butadiene synthase , a comprising ( b ) as described above can further comprise an crotonyl -CoA reductase (alcohol forming ) and a crotyl alco exogenous nucleic acid encoding an enzyme selected from hol diphosphokinase ; (xvi ) a glutaryl -CoA dehydrogenase, a an aconitase , an isocitrate dehydrogenase , a succinyl- CoA 15 crotonaldehyde reductase ( alcohol forming ) , a crotyl alcohol synthetase , a succinyl - CoA transferase , a fumarase , a malate kinase , a 2 -butenyl - 4 - phosphate kinase , a butadiene syn dehydrogenase, and combinations thereof. thase , a crotonyl- CoA hydrolase, synthetase , or transferase In a particular embodiment, such a microbial organism and a crotonate reductase ; (xvii ) a glutaryl- CoA dehydro used in a method for producing butadiene can comprise two, genase , a crotonaldehyde reductase (alcohol forming ) , a three , four, five , six or seven exogenous nucleic acids each 20 butadiene synthase , a crotonyl- CoA hydrolase , synthetase or encoding a butadiene pathway enzyme. For example, such a transferase , a crotonate reductase and a crotyl alcohol microbial organism can comprise exogenous nucleic acids diphosphokinase ; (xviii ) a 3 -hydroxybutyryl - CoA dehy encoding each of the enzymes selected from : ( i ) an acetyl dratase , a crotonyl- CoA reductase ( aldehyde forming ), a COA : acetyl -COA acyltransferase , an acetoacetyl -CoA reduc - crotonaldehyde reductase (alcohol forming ) , a butadiene tase , a 3 - hydroxybutyryl- CoA dehydratase , a crotonyl- CoA 25 synthase , a glutaryl- CoA dehydrogenase and a crotyl alcohol reductase ( aldehyde forming) , a crotonaldehyde reductase diphosphokinase ; ( xix ) an 3 - aminobutyryl- CoA deaminase , (alcohol forming ), a crotyl alcohol kinase , a 2 - butenyl- 4 - a crotonyl- CoA reductase ( aldehyde forming ) , a crotonalde phosphate kinase and a butadiene synthase ; ( ii ) an acetyl- hyde reductase (alcohol forming) , a crotyl alcohol kinase , a COA : acetyl -COA acyltransferase , an acetoacetyl -CoA reduc - 2 -butenyl - 4 - phosphate kinase and a butadiene synthase ; ( xx ) tase, a 3 - hydroxybutyryl- CoA dehydratase, a crotyl alcohol 30 an 3 - aminobutyryl- CoA deaminase , a crotyl alcohol kinase , kinase , a 2 - butenyl - 4 - phosphate kinase , a butadiene syn - a 2 -butenyl - 4 -phosphate kinase , a butadiene synthase and thase and crotonyl- CoA reductase ( alcohol forming ) ; (iii ) an crotonyl -CoA reductase ( alcohol forming ); (xxi ) an 3 -amin acetyl- CoA :acetyl - CoA acyltransferase , an acetoacetyl- CoA obutyryl- CoA deaminase , a butadiene synthase , a crotonyl reductase , a 3 -hydroxybutyryl - CoA dehydratase, a butadi CoA reductase ( alcohol forming ) and a crotyl alcohol ene synthase , a crotonyl- CoA reductase ( alcohol forming ) 35 diphosphokinase ; ( xxii ) an 3 - aminobutyryl - CoA deaminase , and a crotyl alcohol diphosphokinase ; ( iv ) an acetyl - CoA : a crotonaldehyde reductase ( alcohol forming ), a crotyl alco acetyl- CoA acyltransferase , an acetoacetyl- CoA reductase , a hol kinase , a 2 -butenyl - 4 -phosphate kinase , a butadiene 3 -hydroxybutyryl - CoA dehydratase , a crotonaldehyde synthase, a crotonyl- CoA hydrolase , synthetase or trans reductase ( alcohol forming ) , a crotyl alcohol kinase , a ferase and a crotonate reductase ; (xxiii ) an 3 - aminobutyryl 2 -butenyl - 4 -phosphate kinase, a butadiene synthase, a croto - 40 COA deaminase , a crotonaldehyde reductase (alcohol form nyl- CoA hydrolase , synthetase or transferase and a crotonate ing ) , a butadiene synthase , a crotonyl- CoA hydrolase , reductase ; (v ) an acetyl- CoA :acetyl - CoA acyltransferase , an synthetase or transferase , a crotonate reductase and a crotyl acetoacetyl- CoA reductase, a 3 -hydroxybutyryl - CoA dehy - alcohol diphosphokinase ; ( xxiv ) a 3 -hydroxybutyryl - CoA dratase , a crotonaldehyde reductase ( alcohol forming ) , a dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , butadiene synthase , a crotonyl -CoA hydrolase , synthetase or 45 a crotonaldehyde reductase ( alcohol forming ) , a butadiene transferase , a crotonate reductase and a crotyl alcohol synthase , a 3 - aminobutyryl -CoA deaminase and a crotyl diphosphokinase ; ( vi) an acetyl- CoA : acetyl- CoA acyltrans - alcohol diphosphokinase ; ( XXV ) a 4 -hydroxybutyryl - CoA ferase, an acetoacetyl- CoA reductase , a 3 -hydroxybutyryl dehydratase , a crotonyl- CoA reductase ( aldehyde forming ) , CoA dehydratase , a crotonyl - CoA reductase ( aldehyde form - a crotonaldehyde reductase ( alcohol forming ) , a crotyl alco ing ) , a crotonaldehyde reductase (alcohol forming ), a 50 hol kinase , a 2 - butenyl- 4 - phosphate kinase and a butadiene butadiene synthase and a crotyl alcohol diphosphokinase ; synthase ; (xxvi ) a 4 -hydroxybutyryl - CoA dehydratase , a ( vii ) a glutaconyl- CoA decarboxylase , a crotonyl- CoA crotyl alcohol kinase , a 2 - butenyl - 4 - phosphate kinase , a reductase ( aldehyde forming) , a crotonaldehyde reductase butadiene synthase and crotonyl- CoA reductase (alcohol (alcohol forming ) , a crotyl alcohol kinase , a 2 -butenyl - 4 - forming ) ; (xxvii ) a 4 -hydroxybutyryl - CoA dehydratase, a phosphate kinase and a butadiene synthase ; (viii ) a glutaco - 55 butadiene synthase , a crotonyl- CoA reductase (alcohol nyl- CoA decarboxylase , a crotyl alcoholkinase , a 2 - butenyl forming ) and a crotyl alcohol diphosphokinase ; ( xxviii ) a 4 - phosphate kinase, a butadiene synthase and crotonyl- CoA 4 -hydroxybutyryl - CoA dehydratase , a crotonaldehyde reductase ( alcohol forming ) ; ( ix ) a glutaconyl- CoA decar reductase (alcohol forming ) , a crotyl alcohol kinase , a boxylase , a butadiene synthase, a crotonyl -CoA reductase 2 -butenyl - 4 -phosphate kinase , a butadiene synthase , a croto ( alcohol forming ) and a crotyl alcohol diphosphokinase ; ( x ) 60 nyl- CoA hydrolase , synthetase or transferase and a crotonate a glutaconyl- CoA decarboxylase , a crotonaldehyde reduc - reductase ; ( xxix ) a 4 -hydroxybutyryl -CoA dehydratase , a tase (alcohol forming) , a crotyl alcohol kinase , a 2 -butenyl - crotonaldehyde reductase (alcohol forming ) , a butadiene 4 -phosphate kinase , a butadiene synthase , a crotonyl- CoA synthase , a crotonyl- CoA hydrolase, synthetase or trans hydrolase , synthetase , or transferase and a crotonate reduc ferase , a crotonate reductase and a crotyl alcohol diphos tase ; (xi ) a glutaconyl- CoA decarboxylase , a crotonaldehyde 65 phokinase ; (xxx ) a 3 -hydroxybutyryl - CoA dehydratase, a reductase (alcohol forming ) , a butadiene synthase , a croto - crotonyl- CoA reductase ( aldehyde forming ) , a crotonalde nyl- CoA hydrolase , synthetase or transferase , a crotonate hyde reductase ( alcohol forming ) , a butadiene synthase , a US 10 , 006 , 055 B2 49 50 4 -hydroxybutyryl - CoA dehydratase and a crotyl alcohol described above . For example , a microbial organism com diphosphokinase ; ( xxxi) an erythrose - 4 - phosphate reduc - prising ( a ) can comprise thee exogenous nucleic acids tase , an erythritol- 4 -phosphate cytidylyltransferase , a 4 - ( cy encoding ATP - citrate lyase or citrate lyase , a fumarate tidine 5 '- diphospho ) -erythritol kinase , an erythritol 2 ,4 - cy reductase , and an alpha -ketoglutarate : ferredoxin oxi clodiphosphate synthase , a 1 -hydroxy - 2 - butenyl 5 doreductase ; a microbial organism comprising ( b ) can com 4 - diphosphate synthase , a 1 -hydroxy - 2 - butenyl 4 -diphos - prise four exogenous nucleic acids encoding pyruvate : ferre phate reductase and a butadiene synthase ; (xxxii ) an eryth doxin oxidoreductase , a phosphoenolpyruvate carboxylase rose - 4 -phosphate reductase , an erythritol- 4 -phosphate cyti or a phosphoenolpyruvate carboxykinase , a CO dehydroge dylyltransferase , a 4 - ( cytidine 5 ' -diphospho ) - erythritol nase, and an H2 hydrogenase ; or a microbial organism kinase , an erythritol 2 , 4 - cyclodiphosphate synthase , a 1 -hy - 10 comprising ( c ) can comprise two exogenous nucleic acids droxy - 2 -butenyl 4 - diphosphate synthase , a 1 -hydroxy - 2 - encoding CO dehydrogenase and H2 hydrogenase . The butenyl 4 -diphosphate reductase, a butenyl 4 -diphosphate invention further providesmethods for producing butadiene isomerase and a butadiene synthase ; (xxxiii ) an erythritol - by culturing such non - naturally occurring microbial organ 4 - phosphate cytidylyltransferase , a 4 - ( cytidine 5 - diphos - isms under conditions and for a sufficient period of time to pho ) - erythritol kinase , an erythritol 2 , 4 - cyclodiphosphate 15 produce butadiene. synthase , a 1 - hydroxy - 2 -butenyl 4 - diphosphate synthase , a In some embodiments, the invention provides a method 1 - hydroxy - 2 -butenyl 4 -diphosphate reductase , a butadiene for producing crotyl alcohol that includes culturing a non synthase , an erythrose - 4 - phosphate kinase , an erythrose naturally occurring microbial organism as described herein , reductase and a erythritol kinase ; ( xxxiv ) an erythritol- 4 - including a microbial organism having a crotyl alcohol phosphate cytidylyltransferase, a 4 -( cytidine 5 '- diphospho )- 20 pathway comprising at least one exogenous nucleic acid erythritol kinase , an erythritol 2 , 4 - cyclodiphosphate syn - encoding a crotyl alcohol pathway enzyme expressed in a thase , a 1 - hydroxy - 2 - butenyl 4 - diphosphate synthase , a sufficient amount to produce crotyl alcohol. Such a micro 1 -hydroxy - 2 -butenyl 4 -diphosphate reductase , a butenyl bial organism can further comprise (a ) a reductive TCA 4 - diphosphate isomerase , a butadiene synthase , an eryth pathway comprising at least one exogenous nucleic acid rose - 4 - phosphate kinase , an erythrose reductase and an 25 encoding a reductive TCA pathway enzyme, wherein the at erythritol kinase ; ( xXxv ) a malonyl -CoA :acetyl - CoA acyl- least one exogenous nucleic acid is selected from an ATP transferase, an 3 -oxoglutaryl - CoA reductase (ketone - reduc - citrate lyase , a citrate lyase, a citryl- CoA synthetase , a ing ), a 3 -hydroxyglutaryl - CoA reductase (aldehyde form citryl- CoA lyase , a fumarate reductase , and an alpha -keto ing ) , a 3 - hydroxy - 5 - oxopentanoate reductase , a 3 , 5 - glutarate : ferredoxin oxidoreductase ; ( b ) a reductive TCA dihydroxypentanoate kinase , a 3 -hydroxy - 5 - 30 pathway comprising at least one exogenous nucleic acid phosphonatooxypentanoate kinase, a 3 -hydroxy - 5 - [ hydroxy encoding a reductive TCA pathway enzyme, wherein the at ( phosphonooxy )phosphoryl ] oxy pentanoate decarboxylase , least one exogenous nucleic acid is selected from a pyruvate : a butenyl 4 - diphosphate isomerase and a butadiene synthase ; ferredoxin oxidoreductase , a phosphoenolpyruvate carboxy ( xxxvi ) a malonyl- CoA :acetyl - CoA acyltransferase , a 3 , 5 lase , a phosphoenolpyruvate carboxykinase , a CO dehydro dihydroxypentanoate kinase , a 3 -hydroxy - 5 - phosphona - 35 genase , and an H , hydrogenase ; or ( c ) at least one exogenous tooxypentanoate kinase , a 3 -hydroxy - 5 -[ hydroxy (phospho - nucleic acid encodes an enzyme selected from a CO dehy nooxy )phosphoryl ] oxy pentanoate decarboxylase , a butenyl drogenase , an H , hydrogenase , and combinations thereof. 4 - diphosphate isomerase , a butadiene synthase , an 3 - oxo - In such a microbial organism used in a method for glutaryl- CoA reductase ( aldehyde forming ) , a 3 , 5 - dioxopen producing crotyl alcohol, the crotyl alcohol pathway can be tanoate reductase (aldehyde reducing ) and a 5 -hydroxy - 3 - 40 selected from any of those disclosed herein and in the oxopentanoate reductase ; (xxxvii ) a malonyl- CoA : acetyl- figures. For example , the crotyl alcohol pathway can be CoA acyltransferase, a 3 -hydroxy - 5 -oxopentanoate selected from (i ) an acetyl- CoA :acetyl - CoA acyltransferase ; reductase , a 3 , 5 - dihydroxypentanoate kinase, a 3 -Hydroxy an acetoacetyl- CoA reductase ; a 3 -hydroxybutyryl - CoA 5 - phosphonatooxypentanoate kinase, a 3 -Hydroxy - 5 - [hy dehydratase ; a crotonyl- CoA hydrolase , synthase , or trans droxy ( phosphonooxy phosphorylloxy pentanoate decar - 45 ferase ; a crotonate reductase ; and a crotonaldehyde reduc boxylase , a butenyl 4 -diphosphate isomerase , a butadiene tase (alcohol forming ); ( ii ) an acetyl- CoA :acetyl - CoA acyl synthase , an 3 -oxoglutaryl - CoA reductase ( aldehyde form - transferase ; an acetoacetyl - CoA reductase ; a ing ) and a 3 , 5 -dioxopentanoate reductase (ketone reducing ) ; 3 -hydroxybutyryl - CoA dehydratase ; a crotonyl- CoA reduc ( xxxviii ) a malonyl- CoA :acetyl - CoA acyltransferase , a 3 , 5 - tase (aldehyde forming ) ; and a crotonaldehyde reductase dihydroxypentanoate kinase, a 3 -hydroxy - 5 - phosphona - 50 (alcohol forming ) ; (iii ) an acetyl- CoA :acetyl - CoA acyltrans tooxypentanoate kinase , a 3 -hydroxy - 5 - [ hydroxy ( phospho - ferase ; an acetoacetyl- CoA reductase ; a 3 - hydroxybutyryl nooxy )phosphoryl ] oxy pentanoate decarboxylase , a butenyl COA dehydratase ; and a crotonyl- CoA reductase (alcohol 4 - diphosphate isomerase , a butadiene synthase , a 5 -hy - forming ) ; ( iv ) a glutaconyl- CoA decarboxylase ; a crotonyl droxy - 3 - oxopentanoate reductase and a 3 -oxo - glutaryl- CoA COA hydrolase , synthase , or transferase ; a crotonate reduc reductase (CoA reducing and alcohol forming) ; and ( xxxix ) 55 tase ; and a crotonaldehyde reductase (alcohol forming ) ; ( v ) a butadiene pathway comprising a malonyl- CoA :acetyl a glutaconyl - CoA decarboxylase ; a crotonyl- CoA reductase COA acyltransferase , an 3 -oxoglutaryl - CoA reductase (ke (aldehyde forming ) ; and a crotonaldehyde reductase (alco tone - reducing) , a 3 , 5 - dihydroxypentanoate kinase , a 3 -hy hol forming ); and (vi ) a glutaconyl - CoA decarboxylase ; and droxy - 5 -phosphonatooxypentanoate kinase , a 3 - hydroxy - 5 - a crotonyl- CoA reductase ( alcohol forming ) . ( vii ) a glutaryl [hydroxy (phosphonooxy ) phosphoryl] oxy pentanoate 60 COA dehydrogenase ; a crotonyl -CoA hydrolase , synthase , or decarboxylase , a butenyl 4 -diphosphate isomerase , a buta transferase ; a crotonate reductase ; and a crotonaldehyde diene synthase and a 3 -hydroxyglutaryl - CoA reductase (al - reductase (alcohol forming ) ; ( viii ) a glutaryl- CoA dehydro cohol forming ). genase ; a crotonyl- CoA reductase ( aldehyde forming) ; and a In some aspects, the invention provides a method for crotonaldehyde reductase (alcohol forming ) ; ( ix ) a glutaryl producing butatiene , wherein the microbial organisms of the 65 COA dehydrogenase ; and a crotonyl -CoA reductase ( alcohol invention comprise two , three , four or five exogenous forming ) ; ( x ) a 3 - aminobutyryl- CoA deaminase ; a crotonyl nucleic acids each encoding enzymes of ( a ) , ( b ) or ( c ) as COA hydrolase, synthase , or transferase ; a crotonate reduc US 10 ,006 , 055 B2 51 tase; and a crotonaldehyde reductase ( alcohol forming ); ( xi) crotonaldehyde reductase ( alcohol forming ) ; ( xiv ) a 4 - hy a 3 -aminobutyryl - CoA deaminase ; a crotonyl- CoA reduc droxybutyryl- CoA dehydratase ; a crotonyl- CoA reductase tase (aldehyde forming ); and a crotonaldehyde reductase (aldehyde forming ) ; and a crotonaldehyde reductase (alco ( alcohol forming ) ; ( xii ) a 3 - aminobutyryl- CoA deaminase ; hol forming ) ; and ( xv ) a 4 -hydroxybutyryl - CoA dehy and a crotonyl -CoA reductase ( alcohol forming ) ; ( xiii ) a 5 dratase ; and a crotonyl -CoA reductase ( alcohol forming ) . 4 -hydroxybutyryl - CoA dehydratase ; a crotonyl -CoA hydro - Such microbial organisms used in a method for producing lase, synthase , or transferase ; a crotonate reductase ; and a crotyl alcohol as disclosed herein can comprise two , three , crotonaldehyde reductase (alcohol forming ) ; (xiv ) a 4 -hy - four or five exogenous nucleic acids each encoding enzymes droxybutyryl- CoA dehydratase ; a crotonyl- CoA reductase of ( a ), ( b ) or ( c ) . For example , a microbial organism com (aldehyde forming ) ; and a crotonaldehyde reductase ( alco - 10 prising ( a ) can comprise three exogenous nucleic acids hol forming ) ; and ( xv ) a 4 - hydroxybutyryl- CoA dehy - encoding ATP - citrate lyase or citrate lyase , a fumarate dratase ; and a crotonyl- CoA reductase ( alcohol forming ) . reductase, and an alpha -ketoglutarate :ferredoxin oxi In some aspects , the invention provides a method for doreductase ; a microbial organism comprising (b ) can com producing crotyl alcohol, where a microbial organism com prise four exogenous nucleic acids encoding a pyruvate : prising ( a ) can further comprise an exogenous nucleic acid 15 ferredoxin oxidoreductase , a phosphoenolpyruvate encoding an enzyme selected from a pyruvate : ferredoxin carboxylase or a phosphoenolpyruvate carboxykinase , a CO oxidoreductase , an aconitase , an isocitrate dehydrogenase , a dehydrogenase, and an H , hydrogenase ; or a microbial succinyl -CoA synthetase , a succinyl -CoA transferase , a organism comprising ( c ) can comprise two exogenous fumarase , a malate dehydrogenase , an acetate kinase, a nucleic acids encoding a CO dehydrogenase and an H2 phosphotransacetylase , an acetyl- CoA synthetase , an NAD 20 hydrogenase . ( P ) H :ferredoxin oxidoreductase , ferredoxin , and combina Suitable purification and /or assays to test for the produc tions thereof. In some aspects , such a microbial organism tion of butadiene can be performed using well known used in a method for producing crotyl alcohol include a methods. Suitable replicates such as triplicate cultures can microbial organism comprising ( b ) , which can further com - be grown for each engineered strain to be tested . For prise an exogenous nucleic acid encoding an enzyme 25 example , product and byproduct formation in the engineered selected from an aconitase , an isocitrate dehydrogenase , a production host can be monitored . The final product and succinyl -CoA synthetase, a succinyl - CoA transferase , a intermediates , and other organic compounds, can be ana fumarase , a malate dehydrogenase , and combinations lyzed by methods such as HPLC (High Performance Liquid thereof. Such a microbial organism can comprise two, three , Chromatography ), GC -MS (Gas Chromatography -Mass four, five , six or seven exogenous nucleic acids each encod - 30 Spectroscopy ) and LC -MS ( Liquid Chromatography -Mass ing a crotyl alcohol pathway enzyme. Spectroscopy ) or other suitable analytical methods using For example , the microbial organism used in the methods routine procedures well known in the art. The release of for producing crotyl alcohol as disclosed herein can com - product in the fermentation broth can also be tested with the prise exogenous nucleic acids encoding each of the enzymes culture supernatant. Byproducts and residual glucose can be selected from ( i ) an acetyl- CoA :acetyl - CoA acyltransferase ; 35 quantified by HPLC using , for example , a refractive index an acetoacetyl -CoA reductase; a 3 -hydroxybutyryl -CoA detector for glucose and alcohols , and a UV detector for dehydratase ; a crotonyl- CoA hydrolase , synthase, or trans organic acids (Lin et al. , Biotechnol. Bioeng . 90 :775 -779 ferase ; a crotonate reductase ; and a crotonaldehyde reduc- ( 2005 ) ) , or other suitable assay and detection methods well tase (alcohol forming ) ; ( ii ) an acetyl- CoA :acetyl -CoA acyl- known in the art . The individual enzyme or protein activities transferase; an acetoacetyl- CoA reductase ; a 40 from the exogenous DNA sequences can also be assayed 3 -hydroxybutyryl - CoA dehydratase ; a crotonyl- CoA reduc using methods well known in the art . For typical Assay tase (aldehyde forming ) ; and a crotonaldehyde reductase Methods, see Manual on Hydrocarbon Analysis (ASTM ( alcohol forming ); (iii ) an acetyl- CoA :acetyl - CoA acyltrans- Manula Series , A . W . Drews, ed ., 6th edition , 1998 , Ameri ferase ; an acetoacetyl- CoA reductase ; a 3 - hydroxybutyryl - can Society for Testing and Materials , Baltimore , Md. COA dehydratase ; and a crotonyl- CoA reductase ( alcohol 45 The butadiene can be separated from other components in forming ) ; (iv ) a glutaconyl - CoA decarboxylase ; a crotonyl - the culture using a variety ofmethods well known in the art . COA hydrolase , synthase , or transferase ; a crotonate reduc - Such separation methods include , for example , extraction tase ; and a crotonaldehyde reductase ( alcohol forming ) ; ( v ) procedures as well as methods that include continuous a glutaconyl -CoA decarboxylase ; a crotonyl - CoA reductase liquid - liquid extraction , pervaporation , membrane filtration , ( aldehyde forming ) ; and a crotonaldehyde reductase ( alco - 50 membrane separation , reverse osmosis , electrodialysis , dis hol forming ) ; (vi ) a glutaconyl- CoA decarboxylase ; and a tillation , crystallization , centrifugation , extractive filtration , crotonyl -CoA reductase ( alcohol forming) ; ( vii ) a glutaryl ion exchange chromatography , size exclusion chromatogra COA dehydrogenase ; a crotonyl- CoA hydrolase , synthase , or phy, adsorption chromatography , and ultrafiltration . All of transferase ; a crotonate reductase ; and a crotonaldehyde the above methods are well known in the art . reductase ( alcohol forming ) ; (viii ) a glutaryl- CoA dehydro - 55 Any of the non - naturally occurring microbial organisms genase ; a crotonyl- CoA reductase ( aldehyde forming) ; and a described herein can be cultured to produce and / or secrete crotonaldehyde reductase (alcohol forming ) ; ( ix ) a glutaryl- the biosynthetic products of the invention . For example, the COA dehydrogenase ; and a crotonyl - CoA reductase ( alcohol butadiene producers can be cultured for the biosynthetic forming ) ; ( x ) a 3 - aminobutyryl- CoA deaminase ; a crotonyl- production of butadiene. COA hydrolase , synthase , or transferase ; a crotonate reduc - 60 For the production of butadiene or crotyl alcohol, the tase ; and a crotonaldehyde reductase (alcohol forming ) ; ( xi) recombinant strains are cultured in a medium with carbon a 3 - aminobutyryl - CoA deaminase ; a crotonyl- CoA reduc - source and other essential nutrients . It is sometimes desir tase (aldehyde forming) ; and a crotonaldehyde reductase able and can be highly desirable to maintain anaerobic ( alcohol forming ) ; ( xii ) a 3 - aminobutyryl - CoA deaminase ; conditions in the fermenter to reduce the cost of the overall and a crotonyl- CoA reductase ( alcohol forming ) . ( xiii ) a 65 process . Such conditions can be obtained , for example , by 4 -hydroxybutyryl - CoA dehydratase ; a crotonyl -CoA hydro - first sparging themedium with nitrogen and then sealing the lase , synthase , or transferase ; a crotonate reductase ; and a flasks with a septum and crimp- cap . For strains where US 10 ,006 , 055 B2 53 54 growth is not observed anaerobically, microaerobic or sub necessary reducing equivalents ( see for example , Drake , stantially anaerobic conditions can be applied by perforating Acetogenesis , pp . 3 - 60 Chapman and Hall , New York , the septum with a small hole for limited aeration . Exemplary (1994 )) . This can be summarized by the following equation : anaerobic conditions have been described previously and are well -known in the art. Exemplary aerobic and anaerobic 5 2002 + 4H2nADP + nPi - CH3COOH + 2H2O +NATP conditions are described , for example , in United State pub Hence , non -naturally occurring microorganisms possess lication 2009 /0047719 , filed Aug . 10 , 2007 . Fermentations ing the Wood -Ljungdahl pathway can utilize CO , and H , can be performed in a batch , fed -batch or continuous man mixtures as well for the production of acetyl - CoA and other ner , as disclosed herein . desired products. If desired , the pH of the medium can be maintained at a 10 The Wood -Ljungdahl pathway is well known in the art desired pH , in particular neutral pH , such as a pH of around and consists of 12 reactions which can be separated into two 7 by addition of a base , such as NaOH or other bases , or acid , branches : ( 1 ) methyl branch and ( 2 ) carbonyl branch . The as needed to maintain the culture medium at a desirable pH methyl branch converts syngas to methyl - tetrahydrofolate The growth rate can be determined by measuring optical (methyl - THF ) whereas the carbonyl branch converts density using a spectrophotometer (600 nm ), and the glucose 15 methyl - THF to acetyl - CoA . The reactions in the methyl uptake rate by monitoring carbon source depletion over branch are catalyzed in order by the following enzymes or time . proteins : ferredoxin oxidoreductase , formate dehydroge The growth medium can include , for example, any car nase , formyltetrahydrofolate synthetase , methenyltetrahy bohydrate source which can supply a source of carbon to the drofolate cyclodehydratase , methylenetetrahydrofolate non - naturally occurring microorganism . Such sources 20 dehydrogenase and methylenetetrahydrofolate reductase . include , for example , sugars such as glucose , xylose , arab - The reactions in the carbonyl branch are catalyzed in order inose , galactose, mannose , fructose , sucrose and starch . by the following enzymes or proteins: methyltetrahydrofo Other sources of carbohydrate include , for example , renew - late : corrinoid protein methyltransferase ( for example , able feedstocks and biomass. Exemplary types of biomasses AcsE ) , corrinoid iron - sulfur protein , nickel- protein assem that can be used as feedstocks in the methods of the 25 bly protein ( for example , AcsF ) , ferredoxin , acetyl- COA invention include cellulosic biomass , hemicellulosic bio - synthase , carbon monoxide dehydrogenase and nickel - pro mass and lignin feedstocks or portions of feedstocks . Such tein assembly protein ( for example, CooC ) . Following the biomass feedstocks contain , for example , carbohydrate sub - teachings and guidance provided herein for introducing a strates useful as carbon sources such as glucose , xylose , sufficient number of encoding nucleic acids to generate a arabinose , galactose , mannose , fructose and starch . Given 30 butadiene or crotyl alcohol pathway , those skilled in the art the teachings and guidance provided herein , those skilled in will understand that the same engineering design also can be the art will understand that renewable feedstocks and bio performed with respect to introducing at least the nucleic mass other than those exemplified above also can be used for acids encoding the Wood -Ljungdahl enzymes or proteins culturing the microbial organisms of the invention for the absent in the host organism . Therefore , introduction of one production of butadiene or crotyl alcohol. 35 or more encoding nucleic acids into themicrobial organisms In addition to renewable feedstocks such as those exem - of the invention such that the modified organism contains the plified above, the butadiene or crotyl alcohol microbial complete Wood - Ljungdahl pathway will confer syngas uti organisms of the invention also can be modified for growth lization ability . on syngas as its source of carbon . In this specific embodi Additionally, the reductive ( reverse ) tricarboxylic acid ment, one or more proteins or enzymes are expressed in the 40 cycle coupled with carbon monoxide dehydrogenase and /or butadiene or crotyl alcohol producing organisms to provide hydrogenase activities can also be used for the conversion of a metabolic pathway for utilization of syngas or other CO , CO2 and / or H , to acetyl - CoA and other products such gaseous carbon source . as acetate . Organisms capable of fixing carbon via the Synthesis gas, also known as syngas or producer gas, is reductive TCA pathway can utilize one or more of the the major product of gasification of coal and of carbona - 45 following enzymes : ATP citrate lyase , citrate lyase , aconi ceous materials such as biomass materials , including agri- tase , isocitrate dehydrogenase , alpha -ketoglutarate : ferre cultural crops and residues. Syngas is a mixture primarily of doxin oxidoreductase , succinyl- CoA synthetase , succinyl H , and CO and can be obtained from the gasification of any COA transferase , fumarate reductase , fumarase , malate organic feedstock , including but not limited to coal, coal oil, dehydrogenase , NAD ( P ) H : ferredoxin oxidoreductase , car natural gas, biomass , and waste organic matter. Gasification 50 bon monoxide dehydrogenase , and hydrogenase . Specifi is generally carried out under a high fuel to oxygen ratio . cally, the reducing equivalents extracted from CO and /or H2 Although largely H , and CO , syngas can also include CO , by carbon monoxide dehydrogenase and hydrogenase are and other gases in smaller quantities . Thus , synthesis gas utilized to fix CO , via the reductive TCA cycle into acetyl provides a cost effective source of gaseous carbon such as COA or acetate . Acetate can be converted to acetyl- CoA by CO and , additionally , CO2. 55 enzymes such as acetyl- CoA transferase, acetate kinase ! The Wood - Ljungdahl pathway catalyzes the conversion phosphotransacetylase , and acetyl- CoA synthetase . Acetyl of CO and H , to acetyl - CoA and other products such as COA can be converted to the butadiene or crotyl alcohol acetate . Organisms capable of utilizing CO and syngas also precursors , glyceraldehyde- 3 - phosphate , phosphoenolpyru generally have the capability of utilizing CO2 and CO2/ H vate , and pyruvate , by pyruvate : ferredoxin oxidoreductase mixtures through the same basic set of enzymes and trans - 60 and the enzymes of gluconeogenesis . Following the teach formations encompassed by the Wood - Ljungdahl pathway . ings and guidance provided herein for introducing a suffi H2- dependent conversion of CO2 to acetate by microorgan - cient number of encoding nucleic acids to generate a buta isms was recognized long before it was revealed that CO diene or a crotyl alcohol pathway , those skilled in the art will also could be used by the same organisms and that the same understand that the same engineering design also can be pathways were involved . Many acetogens have been shown 65 performed with respect to introducing at least the nucleic to grow in the presence of CO , and produce compounds such acids encoding the reductive TCA pathway enzymes or as acetate as long as hydrogen is present to supply the proteins absent in the host organism . Therefore , introduction US 10 , 006 , 055 B2 55 56 of one or more encoding nucleic acids into the microbial at intracellular concentrations of 5 - 10 mM or more as well organisms of the invention such that the modified organism as all other concentrations exemplified herein . It is under contains a reductive TCA pathway can confer syngas utili stood that, even though the above description refers to zation ability. intracellular concentrations, butadiene or crotyl alcohol pro Accordingly , given the teachings and guidance provided 5 ducing microbial organisms can produce butadiene or crotyl herein , those skilled in the art will understand that a non - alcohol intracellularly and/ or secrete the product into the naturally occurring microbial organism can be produced that culture medium . secretes the biosynthesized compounds of the invention In addition to the culturing and fermentation conditions when grown on a carbon source such as a carbohydrate . disclosed herein , growth condition for achieving biosynthe Such compounds include , for example , butadiene and any of 10 sis of butadiene or crotyl alcohol can include the addition of the intermediate metabolites in the butadiene pathway . All an osmoprotectant to the culturing conditions . In certain that is required is to engineer in one or more of the required embodiments , the non - naturally occurring microbial organ enzyme or protein activities to achieve biosynthesis of the isms of the invention can be sustained , cultured or fermented desired compound or intermediate including , for example , as described herein in the presence of an osmoprotectant . inclusion of some or all of the butadiene biosynthetic 15 Briefly , an osmoprotectant refers to a compound that acts as pathways . Accordingly , the invention provides a non - natu - an osmolyte and helps a microbial organism as described rally occurring microbial organism that produces and /or herein survive osmotic stress . Osmoprotectants include , but secretes butadiene when grown on a carbohydrate or other are not limited to , betaines , amino acids , and the sugar carbon source and produces and / or secretes any of the trehalose . Non - limiting examples of such are glycine intermediate metabolites shown in the butadiene pathway 20 betaine, praline betaine, dimethylthetin , dimethyl slfonio when grown on a carbohydrate or other carbon source . The proprionate , 3 - dimethylsulfonio - 2 -methylproprionate , pipe butadiene producing microbial organisms of the invention colic acid , dimethylsulfonioacetate , choline , L -carnitine and can initiate synthesis from an intermediate , for example , ectoine . In one aspect , the osmoprotectant is glycine betaine . acetoacetyl- CoA , 3 - hydroxybutyryl - CoA , crotonyl- CoA , It is understood to one of ordinary skill in the art that the crotonaldehyde , crotyl alcohol, 2 - betenyl - phosphate , 25 amount and type of osmoprotectant suitable for protecting a 2 -butenyl -4 -diphosphate , erythritol- 4 -phosphate , 4 - cyti- microbial organism described herein from osmotic stress dine 5 ' -diphospho ) - erythritol, 2 -phospho - 4 - (cytidine will depend on the microbial organism used . The amount of 5 '- diphospho )- erythritol , erythritol- 2 ,4 -cyclodiphosphate , osmoprotectant in the culturing conditions can be, for 1 -hydroxy - 2 -butenyl 4 - diphosphate , butenyl 4 - diphosphate , example , no more than about 0 . 1 mM , no more than about 2 - butenyl 4 - diphosphate , 3 -oxoglutaryl - CoA , 3 -hydroxy - 30 0 . 5 mm , no more than about 1 . 0 mm , no more than about 1 . 5 glutaryl- CoA , 3 -hydroxy - 5 -oxopentanoate , 3 , 5 - dihydroxy m M , no more than about 2 . 0 mM , no more than about 2 . 5 pentanoate , 3 -hydroxy -5 - phosphonatooxypentanoate, 3 -hy - mM , no more than about 3 . 0 mm , no more than about 5 . 0 droxy - 5 - hydroxy ( phosphonooxy phosphoryl] oxy pentano - mm , no more than about 7 . 0 mm , no more than about 10 ate , crotonate , erythrose , erythritol, 3 , 5 - dioxopentanoate or mM , no more than about 50 mM , no more than about 100 5 - hydroxy - 3 -oxopentanoate . 35 mM or no more than about 500 mM . The non -naturally occurring microbial organisms of the In some embodiments , the carbon feedstock and other invention are constructed using methods well known in the cellular uptake sources such as phosphate , ammonia , sulfate , art as exemplified herein to exogenously express at least one chloride and other halogens can be chosen to alter the nucleic acid encoding a butadiene or a crotyl alcohol path isotopic distribution of the atoms present in butadiene or way enzyme or protein in sufficient amounts to produce 40 crotyl alcohol or any butadiene or crotyl alcohol pathway butadiene or crotyl alcohol. It is understood that the micro - intermediate . The various carbon feedstock and other uptake bial organisms of the invention are cultured under conditions sources enumerated above will be referred to herein , col sufficient to produce butadiene or crotyl alcohol. Following lectively , as " uptake sources . " Uptake sources can provide the teachings and guidance provided herein , the non -natu - isotopic enrichment for any atom present in the product rally occurring microbial organisms of the invention can 45 butadiene or crotyl alcohol or butadiene or crotyl alcohol achieve biosynthesis ofbutadiene or crotyl alcohol resulting pathway intermediate including any butadiene or crotyl in intracellular concentrations between about 0 .001 -2000 alcohol impurities generated in diverging away from the mM or more . Generally , the intracellular concentration of pathway at any point. Isotopic enrichment can be achieved butadiene or crotyl alcohol is between about 3 - 1500 mm , for any target atom including , for example , carbon , hydro particularly between about 5 - 1250 mM and more particu - 50 gen , oxygen , nitrogen , sulfur, phosphorus , chloride or other larly between about 8 - 1000 mm , including about 10 mm , halogens . 100 mM , 200 mM , 500 mm , 800 mM , or more. Intracellular In some embodiments , the uptake sources can be selected concentrations between and above each of these exemplary to alter the carbon - 12 , carbon - 13 , and carbon - 14 ratios. In ranges also can be achieved from the non -naturally occur- some embodiments , the uptake sources can be selected to ring microbial organisms of the invention . 55 alter the oxygen - 16 , oxygen - 17 , and oxygen - 18 ratios . In In some embodiments , culture conditions include anaero - some embodiments , the uptake sources can be selected to bic or substantially anaerobic growth or maintenance con alter the hydrogen , deuterium , and tritium ratios . In some ditions . Exemplary anaerobic conditions have been embodiments , the uptake sources can be selected to alter the described previously and are well known in the art . Exem - nitrogen - 14 and nitrogen - 15 ratios . In some embodiments , plary anaerobic conditions for fermentation processes are 60 the uptake sources can be selected to alter the sulfur- 32 , described herein and are described , for example , in U . S . sulfur- 33 , sulfur- 34 , and sulfur- 35 ratios . In some embodi publication 2009/ 0047719, filed Aug . 10 , 2007 . Any of these ments , the uptake sources can be selected to alter the conditions can be employed with the non -naturally occur- phosphorus- 31, phosphorus -32 , and phosphorus -33 ratios . ring microbial organisms as well as other anaerobic condi- In some embodiments , the uptake sources can be selected to tions well known in the art . Under such anaerobic or 65 alter the chlorine -35 , chlorine - 36 , and chlorine -37 ratios. substantially anaerobic conditions, the butadiene or crotyl In some embodiments, the isotopic ratio of a target atom alcohol producers can synthesize butadiene or crotyl alcohol can be varied to a desired ratio by selecting one or more US 10 , 006 , 055 B2 57 58 uptake sources. An uptake source can be derived from a results , for example , measured by ASM , are calculated using natural source , as found in nature , or from a man -made the internationally agreed upon definition of 0 . 95 times the source , and one skilled in the art can select a natural source , specific activity of NBS Oxalic Acid I (SRM 4990b ) nor a man -made source , or a combination thereof, to achieve a malized to 813CVPDB = - 19 per mil . This is equivalent to an desired isotopic ratio of a target atom . An example of a 5 absolute ( AD 1950 ) 140 /12C ratio of 1 . 176 + 0 .010x10 - 12 man -made uptake source includes, for example , an uptake ( Karlen et al. , Arkiv Geofysik , 4 : 465- 471 (1968 ) ) . The source that is at least partially derived from a chemical standard calculations take into account the differential synthetic reaction . Such isotopically enriched uptake uptake of one isotope with respect to another, for example , sources can be purchased commercially or prepared in the the preferential uptake in biological systems of C12 over C13 laboratory and / or optionally mixed with a natural source of 10 over C ' , and these corrections are reflected as a Fm the uptake source to achieve a desired isotopic ratio . In some corrected for $ 13 . embodiments , a target atom isotopic ratio of an uptake An oxalic acid standard ( SRM 4990b or HOx 1 ) was made source can be achieved by selecting a desired origin of the from a crop of 1955 sugar beet. Although there were 1000 uptake source as found in nature . For example , as discussed lbs made , this oxalic acid standard is no longer commer herein , a natural source can be a biobased derived from or 15 cially available . The Oxalic Acid II standard (HOX 2 ; synthesized by a biological organism or a source such as N . I . S . T designation SRM 4990 C ) was made from a crop of petroleum -based products or the atmosphere . In some such 1977 French beet molasses . In the early 1980 ' s , a group of embodiments , a source of carbon , for example , can be 12 laboratories measured the ratios of the two standards. The selected from a fossil fuel- derived carbon source , which can ratio of the activity of Oxalic acid II to 1 is 1 . 2933 + 0 .001 be relatively depleted of carbon - 14 , or an environmental or 20 (the weighted mean ) . The isotopic ratio of HOx II is - 17 . 8 atmospheric carbon source , such as CO , , which can possess per mille . ASTM D6866 - 11 suggests use of the available a larger amount of carbon - 14 than its petroleum -derived Oxalic Acid II standard SRM 4990 C (Hox2 ) for the modern counterpart . standard ( see discussion of original vs. currently available The unstable carbon isotope carbon - 14 or radiocarbon oxalic acid standards in Mann , Radiocarbon , 25 ( 2 ) :519 -527 makes up for roughly 1 in 1012 carbon atoms in the earth ' s 25 ( 1983 )) . A Fm = 0 % represents the entire lack of carbon - 14 atmosphere and has a half- life of about 5700 years . The atoms in a material, thus indicating a fossil (for example , stock of carbon is replenished in the upper atmosphere by a petroleum based ) carbon source . A Fm = 100 % , after correc nuclear reaction involving cosmic rays and ordinary nitro - tion for the post- 1950 injection of carbon - 14 into the atmo gen ( 14N ) . Fossil fuels contain no carbon - 14 , as it decayed sphere from nuclear bomb testing , indicates an entirely long ago . Burning of fossil fuels lowers the atmospheric 30 modern carbon source . As described herein , such a “ mod carbon - 14 fraction , the so - called “ Suess effect” . ern ” source includes biobased sources. Methods of determining the isotopic ratios of atoms in a As described in ASTM D6866 , the percentmodern carbon compound are well known to those skilled in the art. Isotopic (PMC ) can be greater than 100 % because of the continuing enrichment is readily assessed by mass spectrometry using but diminishing effects of the 1950s nuclear testing pro techniques known in the art such as accelerated mass 35 grams, which resulted in a considerable enrichment of spectrometry (AMS ) , Stable Isotope Ratio Mass Spectrom - carbon - 14 in the atmosphere as described in ASTM D6866 etry (SIRMS ) and Site - Specific Natural Isotopic Fraction 11 . Because all sample carbon - 14 activities are referenced to ation by Nuclear Magnetic Resonance (SNIF - NMR ) . Such a “ pre - bomb " standard , and because nearly all new biobased mass spectral techniques can be integrated with separation products are produced in a post- bomb environment, all pMC techniques such as liquid chromatography ( LC ) , high per - 40 values (after correction for isotopic fraction ) must be mul formance liquid chromatography (HPLC ) and / or gas chro - tiplied by 0 .95 ( as of 2010 ) to better reflect the true biobased matography, and the like . content of the sample . A biobased content that is greater than In the case of carbon , ASTM D6866 was developed in the 103 % suggests that either an analytical error has occurred , United States as a standardized analytical method for deter - or that the source of biobased carbon is more than several mining the biobased content of solid , liquid , and gaseous 45 years old . samples using radiocarbon dating by the American Society ASTM D6866 quantifies the biobased content relative to for Testing and Materials ( ASTM ) International. The stan - the material' s total organic content and does not consider the dard is based on the use of radiocarbon dating for the inorganic carbon and other non -carbon containing sub determination of a product' s biobased content. ASTM stances present. For example, a product that is 50 % starch D6866 was first published in 2004 , and the current active 50 based material and 50 % water would be considered to have version of the standard is ASTM D6866 - 11 ( effective Apr. 1 , a Biobased Content = 100 % ( 50 % organic content that is 2011 ) . Radiocarbon dating techniques are well known to 100 % biobased ) based on ASTM D6866 . In another those skilled in the art, including those described herein . example , a product that is 50 % starch -based material, 25 % The biobased content of a compound is estimated by the petroleum - based , and 25 % water would have a Biobased ratio of carbon - 14 (14C ) to carbon - 12 ( 12C ) . Specifically , the 55 Content= 66 . 7 % ( 75 % organic content but only 50 % of the Fraction Modern ( Fm ) is computed from the expression : product is biobased ) . In another example , a product that is Fm = ( S - B )/ ( M - B ) , where B , S and M represent the 140 /12C 50 % organic carbon and is a petroleum -based product would ratios of the blank , the sample and the modern reference , be considered to have a Biobased Content = 0 % (50 % organic respectively . Fraction Modern is a measurement of the carbon but from fossil sources ) . Thus , based on the well deviation of the 140 /12C ratio of a sample from “ Modern .” 60 known methods and known standards for determining the Modern is defined as 95 % of the radiocarbon concentration biobased content of a compound or material, one skilled in ( in AD 1950 ) of National Bureau of Standards (NBS ) Oxalic the art can readily determine the biobased content and /or Acid I i . e . , standard reference materials (SRM ) 4990b ) prepared downstream products that utilize of the invention normalized to 81 %CVPDR = - 19 per mil (Olsson , The use of having a desired biobased content. Oxalic acid as a Standard . in , Radiocarbon Variations and 65 Applications of carbon -14 dating techniques to quantify Absolute Chronology , Nobel Symposium , 12th Proc ., John bio -based content of materials are known in the art (Currie Wiley & Sons, New York (1970 )) . Mass spectrometry et al. , Nuclear Instruments and Methods in Physics Research US 10 ,006 , 055 B2 59 60 B , 172 : 281 - 287 ( 2000 ) ) . For example , carbon - 14 dating has a bioderived product of butadiene or crotyl alcohol , or an been used to quantify bio -based content in terephthalate - intermediate thereof, to generate a desired product are well containing materials (Colonna et al ., Green Chemistry , known to those skilled in the art , as described herein . The 13 : 2543 - 2548 (2011 ) ) . Notably, polypropylene terephthalate invention further provides a polymer, synthetic rubber , resin , ( PPT ) polymers derived from renewable 1 , 3 - propanediol 5 chemical, monomer , fine chemical, agricultural chemical , or and petroleum - derived terephthalic acid resulted in Fm pharmaceutical having a carbon - 12 versus carbon - 13 versus values near 30 % (i . e . , since 3/ 11 of the polymeric carbon carbon - 14 isotope ratio of about the same value as the CO , derives from renewable 1 , 3 - propanediol and 8 / 11 from the that occurs in the environment , wherein the polymer , syn fossil end member terephthalic acid ) (Currie et al. , supra , thetic rubber, resin , chemical, monomer, fine chemical, 2000 ) . In contrast , polybutylene terephthalate polymer 10 agricultural chemical, or pharmaceutical is generated derived from both renewable 1 , 4 -butanediol and renewable directly from or in combination with bioderived butadiene or terephthalic acid resulted in bio -based content exceeding crotyl alcohol or a bioderived butadiene or crotyl alcohol 90 % (Colonna et al. , supra , 2011) . intermediate as disclosed herein . Accordingly , in some embodiments , the present invention Butadiene is a chemical commonly used in many com provides butadiene or crotyl alcohol or a butadiene or crotyl 15 mercial and industrial applications . Non - limiting examples alcohol intermediate that has a carbon - 12 , carbon - 13, and of such applications include production of polymers, such as carbon - 14 ratio that reflects an atmospheric carbon , also synthetic rubbers and ABS resins , and chemicals , such as referred to as environmental carbon , uptake source . For hexamethylenediamine and 1 , 4 -butanediol . Accordingly, in example , in some aspects the butadiene or crotyl alcohol or some embodiments , the invention provides a biobased poly a butadiene or crotyl alcohol intermediate can have an Fm 20 mer, synthetic rubber, resin , or chemical comprising one or value of at least 10 % , at least 15 % , at least 20 % , at least more bioderived butadiene or bioderived butadiene interme 25 % , at least 30 % , at least 35 % , at least 40 % , at least 45 % , diate produced by a non - naturally occurring microorganism at least 50 % , at least 55 % , at least 60 % , at least 65 % , at least of the invention or produced using a method disclosed 70 % , at least 75 % , at least 80 % , at least 85 % , at least 90 % , herein . at least 95 % , at least 98 % or as much as 100 % . In some such 25 Crotyl alcohol is a chemical commonly used in many embodiments , the uptake source is CO2. In some embodi - commercial and industrial applications. Non - limiting ments , the present invention provides butadiene or crotyl examples of such applications include production of crotyl alcohol or a butadiene or crotyl alcohol intermediate that has halides , esters , and ethers , which in turn are chemical are a carbon - 12 , carbon - 13 , and carbon - 14 ratio that reflects chemical intermediates in the production ofmonomers , fine petroleum -based carbon uptake source . In this aspect, the 30 chemicals , such as sorbic acid , trimethylhydroquinone, cro butadiene or crotyl alcohol or a butadiene or crotyl alcohol tonic acid and 3 -methoxybutanol , agricultural chemicals , intermediate can have an Fm value of less than 95 % , less and pharmaceuticals . Crotyl alcohol can also be used as a than 90 % , less than 85 % , less than 80 % , less than 75 % , less precursor in the production of 1 , 3 -butadiene . Accordingly , in than 70 % , less than 65 % , less than 60 % , less than 55 % , less some embodiments , the invention provides a biobased than 50 % , less than 45 % , less than 40 % , less than 35 % , less 35 monomer, fine chemical , agricultural chemical , or pharma than 30 % , less than 25 % , less than 20 % , less than 15 % , less ceutical comprising one or more bioderived crotyl alcohol or than 10 % , less than 5 % , less than 2 % or less than 1 % . In bioderived crotyl alcohol intermediate produced by a non some embodiments , the present invention provides butadi naturally occurring microorganism of the invention or pro ene or crotyl alcohol or a butadiene or crotyl alcohol duced using a method disclosed herein . intermediate that has a carbon - 12 , carbon - 13 , and carbon - 14 40 As used herein , the term “ bioderived ” means derived ratio that is obtained by a combination of an atmospheric from or synthesized by a biological organism and can be carbon uptake source with a petroleum -based uptake source . considered a renewable resource since it can be generated by Using such a combination of uptake sources is one way by a biological organism . Such a biological organism , in par which the carbon - 12 , carbon - 13 , and carbon - 14 ratio can be ticular the microbial organisms of the invention disclosed varied , and the respective ratios would reflect the propor - 45 herein , can utilize feedstock or biomass , such as , sugars or tions of the uptake sources. carbohydrates obtained from an agricultural, plant, bacterial, Further , the present invention relates to the biologically or animal source . Alternatively , the biological organism can produced butadiene or crotyl alcohol or butadiene or crotyl utilize atmospheric carbon . As used herein , the term alcohol intermediate as disclosed herein , and to the products " biobased ” means a product as described above that is derived therefrom , wherein the butadiene or crotyl alcohol 50 composed , in whole or in part, of a bioderived compound of or a butadiene or crotyl alcohol intermediate has a carbon the invention . A biobased or bioderived product is in contrast 12 , carbon - 13 , and carbon - 14 isotope ratio of about the same to a petroleum derived product, wherein such a product is value as the CO , that occurs in the environment. For derived from or synthesized from petroleum or a petro example , in some aspects the invention provides bioderived chemical feedstock . butadiene or crotyl alcohol or a bioderived butadiene or 55 In some embodiments , the invention provides a biobased crotyl alcohol intermediate having a carbon - 12 versus car- polymer, synthetic rubber , resin , or chemical comprising bon - 13 versus carbon - 14 isotope ratio of about the same bioderived butadiene or bioderived butadiene intermediate , value as the CO , that occurs in the environment, or any of wherein the bioderived butadiene or bioderived butadiene the other ratios disclosed herein . It is understood , as dis - intermediate includes all or part of the butadiene or butadi closed herein , that a product can have a carbon - 12 versus 60 ene intermediate used in the production of polymer, syn carbon - 13 versus carbon - 14 isotope ratio of about the same thetic rubber, resin , or chemical. Thus , in some aspects , the value as the CO , that occurs in the environment, or any of invention provides a biobased polymer , synthetic rubber, the ratios disclosed herein , wherein the product is generated resin , or chemical comprising at least 2 % , at least 3 % , at from bioderived butadiene or crotyl alcohol or a bioderived least 5 % , at least 10 % , at least 15 % , at least 20 % , at least butadiene or crotyl alcohol intermediate as disclosed herein , 65 25 % , at least 30 % , at least 35 % , at least 40 % , at least 50 % , wherein the bioderived product is chemically modified to at least 60 % , at least 70 % , at least 80 % , at least 90 % , at least generate a final product. Methods of chemically modifying 95 % , at least 98 % or 100 % bioderived butadiene or bio US 10 ,006 , 055 B2 61 62 derived butadiene intermediate as disclosed herein . Addi centration in the medium remains between 0 and 10 % of tionally , in some aspects , the invention provides a biobased saturation . Substantially anaerobic conditions also includes polymer , synthetic rubber , resin , or chemical wherein the growing or resting cells in liquid medium or on solid agar butadiene or butadiene intermediate used in its production is inside a sealed chamber maintained with an atmosphere of a combination of bioderived and petroleum derived butadi- 5 less than 1 % oxygen . The percent of oxygen can be main ene or butadiene intermediate . For example , a biobased tained by, for example, sparging the culture with an N /CO polymer, synthetic rubber , resin , or chemical can be pro - mixture or other suitable non -oxygen gas or gases . duced using 50 % bioderived butadiene and 50 % petroleum The culture conditions described herein can be scaled up derived butadiene or other desired ratios such as 60 % / 40 % , and grown continuously for manufacturing of butadiene or 70 % /30 % , 80 % /20 % , 90 % / 10 % , 95 % / 5 % , 100 % / 0 % , 40 % / 10 crotyl alcohol. Exemplary growth procedures include, for 60 % , 30 % /70 % , 20 % /80 % , 10 % / 90 % of bioderived /petro example , fed -batch fermentation and batch separation ; fed leum derived precursors , so long as at least a portion of the batch fermentation and continuous separation , or continuous product comprises a bioderived product produced by the fermentation and continuous separation . All of these pro microbial organisms disclosed herein . It is understood that cesses are well known in the art. Fermentation procedures methods for producing polymer , synthetic rubber, resin , or 15 are particularly useful for the biosynthetic production of chemical using the bioderived butadiene or bioderived buta - commercial quantities of butadiene or crotyl alcohol. Gen diene intermediate of the invention are well known in the art. erally , and as with non - continuous culture procedures , the In some embodiments , the invention provides a biobased continuous and / or near - continuous production of butadiene monomer, fine chemical, agricultural chemical, or pharma - or crotyl alcohol will include culturing a non -naturally ceutical comprising bioderived crotyl alcohol or bioderived 20 occurring butadiene or crotyl alcohol producing organism of crotyl alcohol intermediate , wherein the bioderived crotyl the invention in sufficient nutrients and medium to sustain alcohol or bioderived crotyl alcohol intermediate includes and / or nearly sustain growth in an exponential phase . Con all or part of the crotyl alcohol or crotyl alcohol intermediate tinuous culture under such conditions can include , for used in the production of monomer, fine chemical , agricul example , growth for 1 day, 2 , 3 , 4 , 5 , 6 or 7 days or more . tural chemical, or pharmaceutical. Thus, in some aspects , the 25 Additionally , continuous culture can include longer time invention provides a biobased monomer , fine chemical, periods of 1 week , 2 , 3 , 4 or 5 or more weeks and up to agricultural chemical, or pharmaceutical comprising at least several months . Alternatively , organisms of the invention 2 % , at least 3 % , at least 5 % , at least 10 % , at least 15 % , at can be cultured for hours , if suitable for a particular appli least 20 % , at least 25 % , at least 30 % , at least 35 % , at least cation . It is to be understood that the continuous and / or 40 % , at least 50 % , at least 60 % , at least 70 % , at least 80 % , 30 near - continuous culture conditions also can include all time at least 90 % , at least 95 % , at least 98 % or 100 % bioderived intervals in between these exemplary periods . It is further crotyl alcohol or bioderived crotyl alcohol intermediate as understood that the time of culturing the microbial organism disclosed herein . Additionally, in some aspects, the inven - of the invention is for a sufficient period of time to produce tion provides a biobased monomer , fine chemical, agricul - a sufficient amount of product for a desired purpose . tural chemical, or pharmaceutical wherein the crotyl alcohol 35 Fermentation procedures are well known in the art. or crotyl alcohol intermediate used in its production is a Briefly , fermentation for the biosynthetic production of combination of bioderived and petroleum derived crotyl butadiene or crotyl alcohol can be utilized in , for example , alcohol or crotyl alcohol intermediate . For example , a fed - batch fermentation and batch separation ; fed -batch fer biobased monomer, fine chemical, agricultural chemical , or mentation and continuous separation , or continuous fermen pharmaceutical can be produced using 50 % bioderived 40 tation and continuous separation . Examples of batch and crotyl alcohol and 50 % petroleum derived crotyl alcohol or continuous fermentation procedures are well known in the other desired ratios such as 60 % / 40 % , 70 % / 30 % , 80 % / 20 % , art . 90 % / 10 % , 95 % / 5 % , 100 % / 0 % , 40 % /60 % , 30 % / 70 % , 20 % In addition to the above fermentation procedures using the 80 % , 10 % / 90 % of bioderived /petroleum derived precursors , butadiene or crotyl alcohol producers of the invention for so long as at least a portion of the product comprises a 45 continuous production of substantial quantities of butadiene bioderived product produced by the microbial organisms or crotyl alcohol, the butadiene or crotyl alcohol producers disclosed herein . It is understood thatmethods for producing also can be , for example, simultaneously subjected to chemi monomer, fine chemical, agricultural chemical, or pharma- cal synthesis procedures to convert the product to other ceutical using the bioderived crotyl alcohol or bioderived compounds or the product can be separated from the fer crotyl alcohol intermediate of the invention are well known 50 mentation culture and sequentially subjected to chemical or in the art. enzymatic conversion to convert the product to other com The culture conditions can include, for example , liquid pounds , if desired . culture procedures as well as fermentation and other large To generate better producers , metabolic modeling can be scale culture procedures . As described herein , particularly utilized to optimize growth conditions. Modeling can also be useful yields of the biosynthetic products of the invention 55 used to design gene knockouts that additionally optimize can be obtained under anaerobic or substantially anaerobic utilization of the pathway (see , for example, U . S . patent culture conditions . publications US 2002 /0012939 , US 2003 / 0224363 , US As described herein , one exemplary growth condition for 2004 /0029149 , US 2004 /0072723 , US 2003 / 0059792 , US achieving biosynthesis of butadiene or crotyl alcohol 2002 /0168654 and US 2004 /0009466 , and U . S . Pat. No . includes anaerobic culture or fermentation conditions. In 60 7 ,127 ,379 ). Modeling analysis allows reliable predictions of certain embodiments , the non -naturally occurring microbial the effects on cell growth of shifting the metabolism towards organisms of the invention can be sustained , cultured or more efficient production of butadiene or crotyl alcohol. fermented under anaerobic or substantially anaerobic con - One computationalmethod for identifying and designing ditions . Briefly , anaerobic conditions refers to an environ - metabolic alterations favoring biosynthesis of a desired ment devoid of oxygen . Substantially anaerobic conditions 65 product is the OptKnock computational framework (Bur include, for example , a culture , batch fermentation or con - gard et al. , Biotechnol. Bioeng . 84 :647 -657 ( 2003 ) ) . Opt tinuous fermentation such that the dissolved oxygen con Knock is a metabolic modeling and simulation program that US 10 ,006 , 055 B2 64 suggests gene deletion or disruption strategies that result in can reach the same result in many different ways . Biological genetically stable microorganisms which overproduce the systems are designed through evolutionary mechanisms that target product . Specifically , the framework examines the have been restricted by fundamental constraints that all complete metabolic and / or biochemical network of a micro - living systemsmust face . Therefore , constraints -based mod organism in order to suggest genetic manipulations that 5 eling strategy embraces these general realities . Further , the force the desired biochemical to become an obligatory ability to continuously impose further restrictions on a byproduct of cell growth . By coupling biochemical produc - network model via the tightening of constraints results in a tion with cell growth through strategically placed gene reduction in the size of the solution space , thereby enhancing deletions or other functional gene disruption , the growth the precision with which physiological performance or phe selection pressures imposed on the engineered strains after 10 notype can be predicted . long periods of time in a bioreactor lead to improvements in Given the teachings and guidance provided herein , those performance as a result of the compulsory growth - coupled skilled in the art will be able to apply various computational biochemical production . Lastly, when gene deletions are frameworks for metabolic modeling and simulation to constructed there is a negligible possibility of the designed design and implement biosynthesis of a desired compound strains reverting to their wild -type states because the genes 15 in host microbial organisms. Such metabolic modeling and selected by OptKnock are to be completely removed from simulation methods include , for example , the computational the genome. Therefore , this computational methodology can systems exemplified above as SimPheny® and OptKnock . be used to either identify alternative pathways that lead to For illustration of the invention , somemethods are described biosynthesis of a desired product or used in connection with herein with reference to the OptKnock computation frame the non - naturally occurring microbial organisms for further 20 work for modeling and simulation . Those skilled in the art optimization of biosynthesis of a desired product. will know how to apply the identification , design and Briefly, OptKnock is a term used herein to refer to a implementation of the metabolic alterations using OptKnock computational method and system for modeling cellular to any of such other metabolic modeling and simulation metabolism . The OptKnock program relates to a framework computational frameworks and methods well known in the of models and methods that incorporate particular con - 25 art . straints into flux balance analysis ( FBA ) models . These The methods described above will provide one set of constraints include , for example , qualitative kinetic infor - metabolic reactions to disrupt. Elimination of each reaction mation , qualitative regulatory information , and /or DNA within the set or metabolic modification can result in a microarray experimental data . OptKnock also computes desired product as an obligatory product during the growth solutions to various metabolic problems by , for example , 30 phase of the organism . Because the reactions are known , a tightening the flux boundaries derived through flux balance solution to the bilevel OptKnock problem also will provide models and subsequently probing the performance limits of the associated gene or genes encoding one or more enzymes metabolic networks in the presence of gene additions or that catalyze each reaction within the set of reactions . deletions . OptKnock computational framework allows the Identification of a set of reactions and their corresponding construction of model formulations that allow an effective 35 genes encoding the enzymes participating in each reaction is query of the performance limits of metabolic networks and generally an automated process , accomplished through cor provides methods for solving the resulting mixed - integer relation of the reactions with a reaction database having a linear programming problems. The metabolic modeling and relationship between enzymes and encoding genes . simulation methods referred to herein as OptKnock are Once identified , the set of reactions that are to be dis described in , for example , U . S . publication 2002/ 0168654 , 40 rupted in order to achieve production of a desired product filed Jan . 10 , 2002 , in International Patent No . PCT/ USO2 / are implemented in the target cell or organism by functional 00660 , filed Jan . 10 , 2002 , and U . S . publication 2009 / disruption of at least one gene encoding each metabolic 0047719 , filed Aug . 10 , 2007 . reaction within the set. One particularly useful means to Another computational method for identifying and achieve functional disruption of the reaction set is by designing metabolic alterations favoring biosynthetic pro - 45 deletion of each encoding gene . However, in some instances , duction of a product is a metabolic modeling and simulation it can be beneficial to disrupt the reaction by other genetic system termed SimPheny® . This computational method and aberrations including, for example , mutation , deletion of system is described in , for example , U . S . publication 2003 / regulatory regions such as promoters or cis binding sites for 0233218 , filed Jun . 14 , 2002 , and in International Patent regulatory factors, or by truncation of the coding sequence Application No . PCT /US03 / 18838 , filed Jun . 13 , 2003 . 50 at any of a number of locations. These latter aberrations , SimPheny® is a computational system that can be used to resulting in less than total deletion of the gene set can be produce a network model in silico and to simulate the flux useful, for example , when rapid assessments of the coupling of mass , energy or charge through the chemical reactions of of a product are desired or when genetic reversion is less a biological system to define a solution space that contains likely to occur. any and all possible functionalities of the chemical reactions 55 To identify additional productive solutions to the above in the system , thereby determining a range of allowed described bilevel OptKnock problem which lead to further activities for the biological system . This approach is referred sets of reactions to disrupt or metabolic modifications that to as constraints - based modeling because the solution space can result in the biosynthesis , including growth - coupled is defined by constraints such as the known stoichiometry of biosynthesis of a desired product, an optimization method , the included reactions as well as reaction thermodynamic 60 termed integer cuts , can be implemented . This method and capacity constraints associated with maximum fluxes proceeds by iteratively solving the OptKnock problem through reactions . The space defined by these constraints exemplified above with the incorporation of an additional can be interrogated to determine the phenotypic capabilities constraint referred to as an integer cut at each iteration . and behavior of the biological system or of its biochemical Integer cut constraints effectively prevent the solution pro components . 65 cedure from choosing the exact same set of reactions iden These computational approaches are consistent with bio - tified in any previous iteration that obligatorily couples logical realities because biological systems are flexible and product biosynthesis to growth . For example , if a previously US 10 , 006 , 055 B2 65 66 identified growth - coupled metabolic modification specifies tionally , optimization methods can be applied to increase the reactions 1 , 2 , and 3 for disruption , then the following activity of an enzyme or protein and /or decrease an inhibi constraint prevents the same reactions from being simulta - tory activity , for example , decrease the activity of a negative neously considered in subsequent solutions. The integer cut regulator. method is well known in the art and can be found described 5 One such optimization method is directed evolution . in , for example , Burgard et al. , Biotechnol. Prog . 17 : 791 - Directed evolution is a powerful approach that involves the 797 ( 2001) . As with all methods described herein with introduction of mutations targeted to a specific gene in order reference to their use in combination with the OptKnock to improve and / or alter the properties of an enzyme. computational framework for metabolic modeling and simu - Improved and /or altered enzymes can be identified through lation , the integer cut method of reducing redundancy in 10 the development and implementation of sensitive high iterative computational analysis also can be applied with throughput screening assays that allow the automated other computational frameworks well known in the art screening of many enzyme variants ( for example , > 104 ) . including , for example , SimPheny® . Iterative rounds of mutagenesis and screening typically are Themethods exemplified herein allow the construction of performed to afford an enzyme with optimized properties . cells and organisms that biosynthetically produce a desired 15 Computational algorithms that can help to identify areas of product, including the obligatory coupling of production of the gene for mutagenesis also have been developed and can a target biochemical product to growth of the cell or organ - significantly reduce the number of enzyme variants that need ism engineered to harbor the identified genetic alterations . to be generated and screened . Numerous directed evolution Therefore , the computational methods described herein technologies have been developed ( for reviews, see Hibbert allow the identification and implementation of metabolic 20 et al ., Biomol. Eng 22 : 11 - 19 ( 2005 ); Huisman and Lalonde , modifications that are identified by an in silico method In Biocatalysis in the pharmaceutical and biotechnology selected from OptKnock or SimPheny® . The set of meta - industries pgs. 717 -742 ( 2007 ) , Patel (ed . ) , CRC Press ; bolic modifications can include , for example , addition of one Otten and Quax . Biomol. Eng 22 : 1 - 9 (2005 ) ; and Sen et al . , or more biosynthetic pathway enzymes and /or functional Appl Biochem . Biotechnol 143 : 212 - 223 ( 2007) ) to be effec disruption of one or more metabolic reactions including , for 25 tive at creating diverse variant libraries, and these methods example , disruption by gene deletion . have been successfully applied to the improvement of a wide As discussed above , the OptKnock methodology was range of properties across many enzyme classes . Enzyme developed on the premise that mutant microbial networks characteristics that have been improved and / or altered by can be evolved towards their computationally predicted directed evolution technologies include , for example : selec maximum - growth phenotypes when subjected to long peri - 30 tivity /specificity , for conversion of non - natural substrates ; ods of growth selection . In other words, the approach temperature stability , for robust high temperature process leverages an organism ' s ability to self -optimize under selec - ing; pH stability , for bioprocessing under lower or higher pH tive pressures . The OptKnock framework allows for the conditions ; substrate or product tolerance , so that high exhaustive enumeration of gene deletion combinations that product titers can be achieved ; binding (Kn ) , including force a coupling between biochemical production and cell 35 broadening substrate binding to include non -natural sub growth based on network stoichiometry . The identification strates ; inhibition ( K ; ) , to remove inhibition by products , of optimal gene /reaction knockouts requires the solution of substrates, or key intermediates; activity (kcat ) , to increases a bilevel optimization problem that chooses the set of active enzymatic reaction rates to achieve desired flux ; expression reactions such that an optimal growth solution for the levels , to increase protein yields and overall pathway flux ; resulting network overproduces the biochemical of interest 40 oxygen stability , for operation of air sensitive enzymes (Burgard et al. , Biotechnol. Bioeng . 84 :647 - 657 ( 2003 ) ) . under aerobic conditions ; and anaerobic activity , for opera An in silico stoichiometric model of E . coli metabolism tion of an aerobic enzyme in the absence of oxygen . can be employed to identify essential genes for metabolic Described below in more detail are exemplary methods pathways as exemplified previously and described in , for that have been developed for the mutagenesis and diversi example , U .S . patent publications US 2002 /0012939 , US 45 fication of genes to target desired properties of specific 2003 /0224363 , US 2004 /0029149 , US 2004 /0072723 , US enzymes . Such methods are well known to those skilled in 2003 / 0059792 , US 2002 /0168654 and US 2004 /0009466 , the art . Any of these can be used to alter and /or optimize the and in U . S . Pat. No . 7 ,127 , 379. As disclosed herein , the activity of a butadiene or crotyl alcohol pathway enzyme or OptKnock mathematical framework can be applied to pin - protein . point gene deletions leading to the growth -coupled produc - 50 EpPCR (Pritchard et al. , J Theor. Biol. 234 :497 -509 tion of a desired product. Further , the solution of the bilevel (2005 ) ) introduces random point mutations by reducing the OptKnock problem provides only one set of deletions. To fidelity ofDNA polymerase in PCR reactions by the addition enumerate all meaningful solutions, that is , all sets of of Mn2+ ions, by biasing dNTP concentrations, or by other knockouts leading to growth -coupled production formation , conditional variations. The five step cloning process to an optimization technique , termed integer cuts, can be 55 confine the mutagenesis to the target gene of interest implemented . This entails iteratively solving the OptKnock involves : 1 ) error -prone PCR amplification of the gene of problem with the incorporation of an additional constraint interest; 2 ) restriction enzyme digestion ; 3 ) gel purification referred to as an integer cut at each iteration , as discussed of the desired DNA fragment; 4 ) ligation into a vector; 5 ) above . transformation of the gene variants into a suitable host and As disclosed herein , a nucleic acid encoding a desired 60 screening of the library for improved performance . This activity of a butadiene or crotyl alcohol pathway can be method can generate multiple mutations in a single gene introduced into a host organism . In some cases , it can be simultaneously , which can be useful to screen a larger desirable to modify an activity of a butadiene or crotyl number of potential variants having a desired activity . A high alcohol pathway enzyme or protein to increase production of number of mutants can be generated by EpPCR , so a butadiene or crotyl alcohol. For example , known mutations 65 high - throughput screening assay or a selection method , for that increase the activity of a protein or enzyme can be example , using robotics , is useful to identify those with introduced into an encoding nucleic acid molecule . Addi- desirable characteristics . US 10 ,006 , 055 B2 67 68 Error -prone Rolling Circle Amplification (epRCA ) (Fujii full - length diverse strands hybridized to the scaffold , which et al ., Nucleic Acids Res . 32 : e145 ( 2004 ) ; and Fujii et al ., contains U to preclude amplification . The scaffold then is Nat. Protoc . 1 : 2493 -2497 ( 2006 ) ) has many of the same destroyed and is replaced by a new strand complementary to elements as epPCR except a whole circular plasmid is used the diverse strand by PCR amplification . The method as the template and random 6 -mers with exonuclease resis - 5 involves one strand ( scaffold ) that is from only one parent tant thiophosphate linkages on the last 2 nucleotides are used while the priming fragments derive from other genes ; the to amplify the plasmid followed by transformation into cells parent scaffold is selected against . Thus, no reannealing with in which the plasmid is re -circularized at tandem repeats . parental fragments occurs . Overlapping fragments are Adjusting the Mn2+ concentration can vary the mutation rate somewhat. This technique uses a simple error- prone , single - 10 trimmed with an exonuclease. Otherwise , this is conceptu step method to create a full copy of the plasmid with 3 - 4 ally similar to DNA shuffling and StEP. Therefore, there mutations/ kbp . No restriction enzyme digestion or specific should be no siblings, few inactives, and no unshuffled primers are required . Additionally, this method is typically parentals . This technique has advantages in that few or no available as a commercially available kit . parental genes are created and many more crossovers can DNA or Family Shuffling (Stemmer . Proc Natl Acad Sci 15 result relative to standard DNA shuffling . USA 91 : 10747 - 10751 (1994 ) ) ; and Stemmer, Nature 370 : Recombined Extension on Truncated templates (RETT ) 389 -391 (1994 ) ) typically involves digestion of two or more entails template switching of unidirectionally growing variant genes with nucleases such as Dnase I or Endo V to strands from primers in the presence of unidirectional generate a pool of random fragments that are reassembled by ssDNA fragments used as a pool of templates (Lee et al. , J. cycles of annealing and extension in the presence of DNA 20 Molec . Catalysis 26 : 119 - 129 ( 2003 ) ) . No DNA endonu polymerase to create a library of chimeric genes . Fragments cleases are used . Unidirectional ssDNA is made by DNA prime each other and recombination occurs when one copy polymerase with random primers or serial deletion with primes another copy ( template switch ) . This method can be exonuclease. Unidirectional ssDNA are only templates and used with > 1 kbp DNA sequences . In addition to mutational not primers . Random priming and exonucleases do not recombinants created by fragment reassembly, this method 25 introduce sequence bias as true of enzymatic cleavage of introduces point mutations in the extension steps at a rate DNA shuffling /RACHITT . RETT can be easier to optimize similar to error -prone PCR . The method can be used to than StEP because it uses normal PCR conditions instead of remove deleterious , random and neutral mutations . very short extensions . Recombination occurs as a compo Staggered Extension (StEP ) ( Zhao et al. , Nat. Biotechnol. nent of the PCR steps , that is, no direct shuffling . This 16 : 258 - 261 ( 1998 ) ) entails template priming followed by 30 method can also be more random than StEP due to the repeated cycles of 2 step PCR with denaturation and very absence of pauses. short duration of annealing / extension ( as short as 5 sec ). In Degenerate Oligonucleotide Gene Shuffling (DOGS ) Growing fragments anneal to different templates and extend degenerate primers are used to control recombination further , which is repeated until full - length sequences are between molecules ; (Bergquist and Gibbs, Methods Mol. made . Template switching means most resulting fragments 35 Biol 352 : 191 - 204 ( 2007 ) ; Bergquist et al. , Biomol. Eng have multiple parents . Combinations of low - fidelity poly - 22 :63 - 72 (2005 ) ; Gibbs et al. , Gene 271 : 13 - 20 (2001 ) ) this merases ( Taq and Mutazyme ) reduce error -prone biases can be used to control the tendency of other methods such because of opposite mutational spectra . as DNA shuffling to regenerate parental genes. This method In Random Priming Recombination (RPR ) random can be combined with random mutagenesis (epPCR ) of sequence primers are used to generate many short DNA 40 selected gene segments . This can be a good method to block fragments complementary to different segments of the tem - the reformation of parental sequences . No endonucleases are plate (Shao et al. , Nucleic Acids Res 26 : 681 -683 (1998 )) . needed . By adjusting input concentrations of segments Base misincorporation and mispriming via epPCR give made , one can bias towards a desired backbone . This method point mutations. Short DNA fragments prime one another allows DNA shuffling from unrelated parents without based on homology and are recombined and reassembled 45 restriction enzyme digests and allows a choice of random into full - length by repeated thermocycling . Removal of mutagenesis methods. templates prior to this step assures low parental recombi Incremental Truncation for the Creation of Hybrid nants. This method , like most others , can be performed over Enzymes (ITCHY ) creates a combinatorial library with 1 multiple iterations to evolve distinct properties . This tech - base pair deletions of a gene or gene fragment of interest nology avoids sequence bias, is independent of gene length , 50 (Ostermeier et al. , Proc . Natl . Acad . Sci . USA 96 : 3562- 3567 and requires very little parent DNA for the application . ( 1999 ) ; and Ostermeier et al. , Nat . Biotechnol . 17 : 1205 In Heteroduplex Recombination linearized plasmid DNA 1209 ( 1999 ) ) . Truncations are introduced in opposite direc is used to form heteroduplexes that are repaired by mismatch tion on pieces of 2 different genes . These are ligated together repair (Volkov et al , Nucleic Acids Res. 27 : e18 ( 1999 ); and and the fusions are cloned . This technique does not require Volkov et al. , Methods Enzymol. 328 :456 - 463 ( 2000 ) ) . The 55 homology between the 2 parental genes . When ITCHY is mismatch repair step is at least somewhat mutagenic . Het combined with DNA shuffling , the system is called eroduplexes transform more efficiently than linear homodu - SCRATCHY (see below ) . A major advantage of both is no plexes . This method is suitable for large genes and whole need for homology between parental genes; for example , operons. functional fusions between an E . coli and a human gene Random Chimeragenesis on Transient Templates (RA - 60 were created via ITCHY. When ITCHY libraries are made , CHITT ) (Coco et al. , Nat. Biotechnol . 19: 354 -359 ( 2001) ) all possible crossovers are captured . employs Dnase I fragmentation and size fractionation of Thio - Incremental Truncation for the Creation of Hybrid single stranded DNA ( ssDNA ) . Homologous fragments are Enzymes ( THIO - ITCHY ) is similar to ITCHY except that hybridized in the absence of polymerase to a complementary phosphothioate dNTPs are used to generate truncations ssDNA scaffold . Any overlapping unhybridized fragment 65 (Lutz et al. , Nucleic Acids Res 29 : E16 ( 2001 ) ) . Relative to ends are trimmed down by an exonuclease . Gaps between ITCHY , THIO - ITCHY can be easier to optimize , provide fragments are filled in and then ligated to give a pool of more reproducibility , and adjustability . US 10 ,006 , 055 B2 69 70 SCRATCHY combines two methods for recombining the technique works well with very short fragments ( 86 bp ) genes, ITCHY and DNA shuffling (Lutz et al. , Proc . Natl . and has a low error rate . The chemical cleavage of DNA Acad . Sci. USA 98 : 11248 - 11253 (2001 ) ) . SCRATCHY used in this technique results in very few unshuffled clones . combines the best features of ITCHY and DNA shuffling . In Sequence Homology - Independent Protein Recombina First , ITCHY is used to create a comprehensive set of 5 tion (SHIPREC ) , a linker is used to facilitate fusion between fusions between fragments of genes in a DNA homology . two distantly related or unrelated genes . Nuclease treatment independent fashion . This artificial family is then subjected is used to generate a range of chimeras between the two to a DNA - shuffling step to augment the number of cross - genes . These fusions result in libraries of single - crossover overs . Computational predictions can be used in optimiza - hybrids (Sieber et al. , Nat. Biotechnol. 19 : 456 - 460 ( 2001 )) . tion . SCRATCHY is more effective than DNA shuffling 10 This produces a limited type of shuffling and a separate when sequence identity is below 80 % . process is required for mutagenesis . In addition , since no In Random Drift Mutagenesis (RNDM ) mutations are homology is needed , this technique can create a library of made via epPCR followed by screening / selection for those chimeras with varying fractions of each of the two unrelated retaining usable activity (Bergquist et al ., Biomol. Eng . parent genes . SHIPREC was tested with a heme -binding 22 :63 -72 ( 2005 ) ) . Then , these are used in DOGS to generate 15 domain of a bacterial CP450 fused to N - terminal regions of recombinants with fusions between multiple active mutants a mammalian CP450 ; this produced mammalian activity in or between active mutants and some other desirable parent. a more soluble enzyme. Designed to promote isolation of neutral mutations ; its In Gene Site Saturation MutagenesisTM (GSSMTM ) the purpose is to screen for retained catalytic activity whether or starting materials are a supercoiled dsDNA plasmid contain not this activity is higher or lower than in the original gene . 20 ing an insert and two primers which are degenerate at the RNDM is usable in high throughput assays when screening desired site of mutations (Kretz et al . , Methods Enzymol. is capable of detecting activity above background . RNDM 388 : 3 - 11 (2004 )) . Primers carrying the mutation of interest, has been used as a front end to DOGS in generating anneal to the same sequence on opposite strands of DNA . diversity . The technique imposes a requirement for activity The mutation is typically in the middle of the primer and prior to shuffling or other subsequent steps ; neutral drift 25 flanked on each side by approximately 20 nucleotides of libraries are indicated to result in higher /quicker improve - correct sequence . The sequence in the primer is NNN or ments in activity from smaller libraries . Though published NNK ( coding ) and MNN (noncoding ) ( N = all 4 , K = G , T , using epPCR , this could be applied to other large - scale M = A , C ) . After extension , Dpnl is used to digest dam mutagenesis methods. methylated DNA to eliminate the wild - type template . This Sequence Saturation Mutagenesis (SeSaM ) is a random 30 technique explores all possible amino acid substitutions at a mutagenesis method that: 1 ) generates a pool of random given locus ( that is , one codon ). The technique facilitates the length fragments using random incorporation of a phospho - generation of all possible replacements at a single - site with thioate nucleotide and cleavage ; this pool is used as a no nonsense codons and results in equal to near - equal template to 2 ) extend in the presence of “ universal” bases representation of most possible alleles . This technique does such as inosine ; 3 ) replication of an inosine - containing 35 not require prior knowledge of the structure , mechanism , or complement gives random base incorporation and , conse - domains of the target enzyme. If followed by shuffling or quently , mutagenesis (Wong et al ., Biotechnol. J . 3 :74 -82 Gene Reassembly , this technology creates a diverse library ( 2008 ); Wong et al ., Nucleic Acids Res. 32 :e26 (2004 ); and of recombinants containing all possible combinations of Wong et al ., Anal. Biochem . 341 : 187 - 189 ( 2005 ) ) . Using single- site up - mutations. The usefulness of this technology this technique it can be possible to generate a large library 40 combination has been demonstrated for the successful evo of mutants within 2 to 3 days using simple methods . This lution of over 50 different enzymes, and also for more than technique is non - directed in comparison to the mutational one property in a given enzyme. bias of DNA . Differences in this approach Combinatorial Cassette Mutagenesis (CCM ) involves the makes this technique complementary (or an alternative ) to use of short oligonucleotide cassettes to replace limited epPCR . 45 regions with a large number of possible amino acid sequence In Synthetic Shuffling , overlapping oligonucleotides are alterations (Reidhaar - Olson et al. Methods Enzymol. 208 : designed to encode " all genetic diversity in targets" and 564 - 586 ( 1991 ) ; and Reidhaar -Olson et al. Science 241 :53 allow a very high diversity for the shuffled progeny ( Ness et 57 ( 1988 ) ). Simultaneous substitutions at two or three sites al. , Nat . Biotechnol. 20 : 1251 - 1255 (2002 ) ) . In this tech - are possible using this technique . Additionally , the method nique, one can design the fragments to be shuffled . This aids 50 tests a large multiplicity of possible sequence changes at a in increasing the resulting diversity of the progeny. One can limited range of sites. This technique has been used to design sequence / codon biases to make more distantly related explore the information content of the lambda repressor sequences recombine at rates approaching those observed DNA -binding domain . with more closely related sequences . Additionally, the tech - Combinatorial Multiple Cassette Mutagenesis (CMCM ) nique does not require physically possessing the template 55 is essentially similar to CCM except it is employed as part genes . of a larger program : 1 ) use of epPCR at high mutation rate Nucleotide Exchange and Excision Technology NexT to 2 ) identify hot spots and hot regions and then 3 ) extension exploits a combination of dUTP incorporation followed by by CMCM to cover a defined region of protein sequence treatment with uracil DNA glycosylase and then piperidine space (Reetz et al ., Angew . Chem . Int. Ed Engl. 40 : 3589 to perform endpoint DNA fragmentation (Muller et al. , 60 3591 ( 2001 ) ) . As with CCM , this method can test virtually Nucleic Acids Res . 33 : e117 ( 2005 ) ) . The gene is reas - all possible alterations over a target region . If used along sembled using internal PCR primer extension with proof - with methods to create random mutations and shuffled reading polymerase . The sizes for shuffling are directly genes , it provides an excellentmeans of generating diverse , controllable using varying dUPT: :dTTP ratios . This is an shuffled proteins . This approach was successful in increas end point reaction using simple methods for uracil incorpo - 65 ing, by 51- fold , the enantioselectivity of an enzyme. ration and cleavage . Other nucleotide analogs , such as In the Mutator Strains technique, conditional ts mutator 8 - oxo - guanine , can be used with this method . Additionally, plasmids allow increases of 20 to 4000 - x in random and US 10 ,006 , 055 B2 71 natural mutation frequency during selection and block accu calculate coupling interactions at each position . Structural mulation of deleterious mutations when selection is not tolerance toward amino acid substitution is a measure of required (Selifonova et al. , Appl. Environ . Microbiol. coupling. Ultimately , this technology is designed to yield 67 :3645 -3649 (2001 )) . This technology is based on a plas desired modifications of protein properties while maintain mid - derived mutD5 gene , which encodes a mutant subunit 5 ing the integrity of structural characteristics . The method of DNA polymerase III. This subunit binds to endogenous computationally assesses and allows filtering of a very large DNA polymerase III and compromises the proofreading number of possible sequence variants ( 1050 ). The choice of ability of polymerase III in any strain that harbors the sequence variants to test is related to predictions based on plasmid . A broad - spectrum of base substitutions and frame the most favorable thermodynamics. Ostensibly only stabil shift mutations occur. In order for effective use , the mutator " ity or properties that are linked to stability can be effectively plasmid should be removed once the desired phenotype is addressed with this technology . The method has been suc achieved ; this is accomplished through a temperature sen cessfully used in some therapeutic proteins, especially in sitive ( ts ) origin of replication , which allows for plasmid engineering immunoglobulins . In silico predictions avoid curing at 41° C . It should be noted that mutator strains have 16 testing extraordinarily large numbers of potential variants . been explored for quite some time ( see Low et al. , J . Mol. Predictions based on existing three - dimensional structures Biol. 260 :359 - 3680 ( 1996 ) ). In this technique , very high are more likely to succeed than predictions based on hypo spontaneous mutation rates are observed . The conditional thetical structures . This technology can readily predict and property minimizes non -desired background mutations. This allow targeted screening of multiple simultaneous muta technology could be combined with adaptive evolution to 20 tions , something not possible with purely experimental enhance mutagenesis rates and more rapidly achieve desired technologies due to exponential increases in numbers . phenotypes . Iterative Saturation Mutagenesis ( ISM ) involves : 1 ) using Look - Through Mutagenesis (LTM ) is a multidimensional knowledge of structure / function to choose a likely site for mutagenesis method that assesses and optimizes combina - enzyme improvement; 2 ) performing saturation mutagenesis torial mutations of selected amino acids (Rajpal et al . , Proc . 25 at chosen site using a mutagenesis method such as Strata Natl. Acad . Sci. USA 102 :8466 -8471 (2005 ) ) . Rather than gene QuikChange (Stratagene ; San Diego Calif .) ; 3 ) screen saturating each site with all possible amino acid changes, a ing /selecting for desired properties ; and 4 ) using improved set of nine is chosen to cover the range of amino acid clone ( s ), start over at another site and continue repeating R - group chemistry . Fewer changes per site allows multiple until a desired activity is achieved (Reetz et al ., Nat . Protoc . sites to be subjected to this type ofmutagenesis . A > 800 - fold 30 2 : 891- 903 ( 2007 ) ; and Reetz et al. , Angew . Chem . Int. Ed increase in binding affinity for an antibody from low nano - Engl . 45 :7745 -7751 ( 2006 ) ) . This is a proven methodology , molar to picomolar has been achieved through this method . which assures all possible replacements at a given position This is a rational approach to minimize the number of are made for screening / selection . random combinations and can increase the ability to find Any of the aforementioned methods for mutagenesis can improved traits by greatly decreasing the numbers of clones be used alone or in any combination . Additionally , any one to be screened . This has been applied to antibody engineer - or combination of the directed evolution methods can be ing , specifically to increase the binding affinity and / or used in conjunction with adaptive evolution techniques , as reduce dissociation . The technique can be combined with described herein . either screens or selections . 40 It is understood that modifications which do not substan Gene Reassembly is a DNA shuffling method that can be tially affect the activity of the various embodiments of this applied to multiple genes at one time or to create a large invention are also provided within the definition of the library of chimeras (multiple mutations ) of a single gene invention provided herein . Accordingly, the following ( Tunable GeneReassemblyTM ( TGRTM ) Technology sup - examples are intended to illustrate but not limit the present plied by Verenium Corporation ). Typically this technology is 45 invention . used in combination with ultra - high - throughput screening to query the represented sequence space for desired improve ments . This technique allows multiple gene recombination Example 1 independent of homology . The exact number and position of cross - over events can be pre- determined using fragments 50 designed via bioinformatic analysis . This technology leads Pathways for Producing Butadiene to a very high level of diversity with virtually no parental gene reformation and a low level of inactive genes. Com bined with GSSMTM , a large range of mutations can be Disclosed herein are novel processes for the direct pro tested for improved activity . The method allows “ blending " » duction of butadiene using engineered non - natural microor and “ fine tuning” of DNA shuffling, for example , codon ganisms that possess the enzymes necessary for conversion usage can be optimized . of common metabolites into the four carbon diene , 1 , 3 In Silico Protein Design Automation (PDA ) is an optimi- butadiene . One novel route to direct production of butadiene zation algorithm that anchors the structurally defined protein entails reduction of the known butanol pathway metabolite backbone possessing a particular fold , and searches crotonyl - CoA to crotyl alcohol via reduction with aldehyde sequence space for amino acid substitutions that can stabi and alcohol dehydrogenases, followed by phosphorylation lize the fold and overall protein energetics (Hayes et al. , with to afford crotyl pyrophosphate and subsequent Proc . Natl. Acad . Sci. USA 99 : 15926 - 15931 ( 2002 ) ) . This conversion to butadiene using isoprene synthases or variants technology uses in silico structure - based entropy predictions 65 thereof (see FIG . 2 ) . Another route (FIG . 3 ) is a variant of in order to search for structural tolerance toward protein the well- characterized DXP pathway for isoprenoid biosyn amino acid variations . Statistical mechanics is applied to thesis . In this route , the substrate lacks a 2 -methyl group and US 10 ,006 ,055 B2 73 74 provides butadiene rather than isoprene via a butadiene - continued synthase . Such a butadiene synthase can be derived from an isoprene synthase usingmethods , such as directed evolution , Protein Genbank ID GI number Organism as described herein . Finally , FIG . 4 shows a pathway to hbd P52041. 2 18266893 Clostridium acetobutylicum butadiene involving the substrate 3 - hydroxyglutaryl- CoA , HSD17B10 002691 . 3 3183024 Bos Taurus which serves as a surrogate for the natural mevalonate phbB P23238 . 1 130017 Zoogloea ramigera pathway substrate 3 -hydroxy - 3 -methyl - glutaryl- CoA phaB YP _ 353825 . 1 77464321R hodobacter sphaeroides ( shown in FIG . 1 ) . Enzyme candidates for steps A - P of FIG . 2 , steps A - K of FIG . 3 and steps A - 0 of FIG . 4 are provided 10 A number of similar enzymes have been found in other below . species of Clostridia and in Metallosphaera sedula (Berg et Acetyl- CoA : Acetyl - CoA Acyltransferase (FIG . 2 , Step A ) al. , Science . 318 : 1782 - 1786 ( 2007 ) ) . Acetoacetyl- CoA thiolase converts two molecules of acetyl- CoA into one molecule each of acetoacetyl- CoA and Protein GenBank ID GI number Organism CoA . Exemplary acetoacetyl- CoA thiolase enzymes include 15 hbd NP _ 349314 . 1 NP _ 349314 . 1 Clostridium the gene products of atoB from E . coli (Martin et al. , Nat. acetobutylicum Biotechnol 21 :796 - 802 ( 2003 ) ), th1A and th1B from C . hbd AAM14586 . 1 AAM14586 . 1 Clostridium acetobutylicum (Hanai et al ., Appl Environ Microbiol beijerinckii Msed 1423 YP _ 001191505 YP _ 001191505 Metallosphaera 73 :7814 -7818 (2007 ) ; Winzer et al. , J. Mol. Microbiol sedula Biotechnol 2 : 531 - 541 ( 2000 ) ) , and ERG10 from S. cerevi- - Msed _ 0399 YP _ 001190500 YP _ 001190500 Metallosphaera siae (Hiser et al. , J . Biol. Chem . 269 :31383 -31389 ( 1994 ) ). sedula Msed _ 0389 YP _ 001190490 YP _ 001190490 Metallosphaera sedula Msed _ 1993 YP _ 001192057 YP _ 001192057 Metallosphaera Protein GenBank ID GI number Organism 25 sedula AtoB NP 416728 16130161 Escherichia coli Thl? NP 349476 . 1 15896127 Clostridium acetobutylicum ThlB N P _ 149242. 1 15004782 Clostridium acetobutylicum 3 -Hydroxybutyryl - CoA Dehydratase (FIG . 2 , Step C ) ERG10 NP _ 015297 6325229 Saccharomyces cerevisiae 3 - Hydroxybutyryl- CoA dehydratase (EC 4 . 2 . 1 . 55 ), also called crotonase , is an enoyl -CoA hydratase that reversibly Acetoacetyl- CoA Reductase (FIG . 2 , Step B ) 30 dehydrates 3 -hydroxybutyryl - CoA to form crotonyl- CoA . Acetoacetyl- CoA reductase catalyzing the reduction of Crotonase enzymes are required for n -butanol formation in acetoacetyl - CoA to 3 -hydroxybutyryl -CoA participates in some organisms, particularly Clostridial species , and also the acetyl - CoA fermentation pathway to butyrate in several comprise one step of the 3 -hydroxypropionate / 4 -hydroxy species of Clostridia and has been studied in detail ( Jones et 35 butyrate cycle in thermoacidophilic Archaea of the genera al. , Microbiol Rev. 50 : 484 -524 (1986 ) ) . The enzyme from Sulfolobus, Acidianus, and Metallosphaera . Exemplary Clostridium acetobutylicum , encoded by hbd , has been genes encoding crotonase enzymes can be found in C . cloned and functionally expressed in E . coli ( Youngleson et acetobutylicum (Atsumi et al ., Metab Eng . 10 : 305 -311 al. , J Bacteriol. 171: 6800 -6807 ( 1989 ) ) . Additionally , sub (2008 ) ; Boynton et al. , J Bacteriol. 178 : 3015 - 3024 ( 1996 ) ) , units of two fatty acid oxidation complexes in E . coli , 40 C . kluyveri (Hillmer et al. , FEBS Lett . 21 : 351 - 354 ( 1972 ) ), encoded by fadB and fad ) , function as 3 -hydroxyacyl - COA and Metallosphaera sedula (Berg et al. , Science 318 : 1782 dehydrogenases (Binstock et al ., Methods Enzymol. 71 Pt 1786 (2007a )) though the sequence of the latter gene is not C :403 - 411 (1981 ) ) . Yet other gene candidates demonstrated known . The enoyl- CoA hydratase of Pseudomonas putida, to reduce acetoacetyl - CoA to 3 - hydroxybutyryl- CoA are encoded by ech , catalyzes the conversion of crotonyl- CoA to phbB from Zoogloea ramigera (Ploux et al. , Eur. J Biochem . 45 3 -hydroxybutyryl - CoA (Roberts et al. , Arch Microbiol. 117 : 174 : 177 - 182 ( 1988 ) ) and phaB from Rhodobacter spha 99- 108 ( 1978 ) ) . Additional enoyl- CoA hydratase candidates eroides ( Alber et al ., Mol. Microbiol 61 :297 - 309 (2006 ) ). are phaA and phaB , of P . putida , and paaA and paaB from The former gene candidate is NADPH -dependent , its P . fluorescens ( Olivera et al ., Proc . Natl . Acad . Sci U . S . A nucleotide sequence has been determined (Peoples et al ., 50 95 :6419 -6424 ( 1998 )) . Lastly , a number of Escherichia coli Mol . Microbiol 3 : 349 - 357 ( 1989 ) ) and the gene has been 50 genes have been shown to demonstrate enoyl- CoA hydratase expressed in E . coli. Substrate specificity studies on the gene functionality including maoC (Park et al ., J Bacteriol. 185 : led to the conclusion that it could accept 3 -oxopropionyl 5391- 5397 (2003 ) ) , paaF ( Ismail et al. , Eur. J Biochem . COA as a substrate besides acetoacetyl- CoA (Ploux et al. , 270 :3047 -3054 (2003 ) ; Park et al. , Appl. Biochem . Biotech supra , ( 1988 ) ). Additional gene candidates include Hbd1 55 nol 113 - 116 : 335 - 346 (2004 ) ; Park et al. , Biotechnol Bioeng ( C - terminal domain ) and Hbd2 ( N - terminal domain ) in 86 :681 -686 ( 2004 ) ) and paaG (Ismail et al. , supra , ( 2003 ) ; Clostridium kluyveri (Hillmer and Gottschalk , Biochim . Park and Lee , supra , (2004 ) ; Park and Yup , supra, (2004 ) ). Biophys . Acta 3334 : 12 - 23 ( 1974 ) ) and HSD17B10 in Bos These proteins are identified below . taurus (WAKIL et al . , J Biol. Chem . 207: 631 -638 ( 1954 ) ) . Protein GenBank ID GI Number Organism Protein Genbank ID GI number Organism crt NP _ 349318 . 1 15895969 Clostridium acetobutylicum crt1 YP _ 001393856 . 1 153953091 Clostridium kluyveri fadB P21177 . 2 119811 Escherichia coli ech NP 745498 . 1 26990073 Pseudomonas puti da fadJ P77399 . 1 3334437 Escherichia coli paaA NP _ 745427 . 1 26990002 Pseudomonas putida Hbd2 EDK34807. 1 146348271 Clostridium kluyveri ???? NP 745426 . 1 26990001 Pseudomonas puti da Hbd1 EDK32512 . 1 146345976 Clostridium kluyveri phaA ABF82233 . 1 106636093 Pseudomonas fluorescens US 10 ,006 ,055 B2 75 76 -continued aldehyde dehydrogenase, an enzyme catalyzing the reduc tion and concurrent dephosphorylation of aspartyl- 4 -phos Protein GenBank ID GI Number Organism phate to aspartate semialdehyde. Additional gene candidates phaB ABF82234 . 1 106636094 Pseudomonas fluorescens can be found by sequence homology to proteins in other maoC NP _ 415905 . 1 16129348 Escherichia coli organisms including Sulfolobus solfataricus and Sulfolobus paar NP 415911 . 1 16129354 Escherichia coli acidocaldarius. Yet another candidate for COA - acylating paag NP _ 415912 . 1 16129355 Escherichia coli aldehyde dehydrogenase is the ald gene from Clostridium beijerinckii ( Toth , Appl. Environ . Microbiol . 65 : 4973 -4980 Crotonyl- CoA Reductase ( Aldehyde Forming) (FIG . 2 , Step 10 (1999 ) . This enzyme has been reported to reduce acetyl- CoA D ) and butyryl- CoA to their corresponding aldehydes . This Several acyl -CoA dehydrogenases are capable of reduc gene is very similar to eutE that encodes acetaldehyde ing an acyl- CoA to its corresponding aldehyde . Thus they dehydrogenase of Salmonella typhimurium and E . coli can naturally reduce crotonyl- CoA to crotonaldehyde or can ( Toth , Appl. Environ . Microbiol . 65: 4973 - 4980 ( 1999 ) . be engineered to do so . Exemplary genes that encode such 15 These proteins are identified below . enzymes include the Acinetobacter calcoaceticus acr1 encoding a fatty acyl- CoA reductase (Reiser et al . , J . Bac teriol. 179 :2969 - 2975 ( 1997 ) ) , the Acinetobacter sp . M - 1 Protein GenBank ID GI Number Organism fatty acyl- CoA reductase ( Ishige et al ., Appl. Environ . Msed _ 0709 YP _ 001190808 . 1 146303492 Metallosphaera sedula Microbiol. 68 :1192 -1195 ( 2002 )) , and a CoA - and NADP- 20 Mer NP _ 378167 . 1 15922498 Sulfolobus tokodaii NP _ 343563 . 1 15898958 Sulfolobus solfataricus dependent succinate semialdehyde dehydrogenase encoded asdSaci - 2 .2370 YP 256941. 1 70608071 Sulfolobus by the sucD gene in Clostridium kluyveri ( Sohling et al. , J acidocaldarius Bacteriol. 178 :871 - 880 ( 1996 ); Sohling et al. , J. Bacteriol. Ald AAT66436 49473535 Clostridium beijerinckii 178 : 871- 80 ( 1996 )) ) . SucD of P . gingivalis is another suc - 25 eutE AAA80209 687645 Salmonella typhimurium cinate semialdehyde dehydrogenase ( Takahashi et al. , ? eutE P77445 2498347 Escherichia coli Bacteriol. 182: 4704 - 4710 ( 2000 ) ). These succinate semial dehyde dehydrogenases were specifically shown in ref . Crotonaldehyde Reductase (Alcohol Forming ) (FIG . 2 , Step (Burk et al. , WO / 2008 / 115840 : ( 2008 ) ) to convert 4 -hy - E ) droxybutyryl- CoA to 4 -hydroxybutanal as part of a pathway 30 Enzymes exhibiting crotonaldehyde reductase (alcohol to produce 1 , 4 -butanediol . The enzyme acylating acetalde forming ) activity are capable of forming crotyl alcohol from hyde dehydrogenase in Pseudomonas sp , encoded by bphG crotonaldehyde . The following enzymes can naturally pos is yet another capable enzyme as it has been demonstrated sess this activity or can be engineered to exhibit this activity . to oxidize and acylate acetaldehyde , propionaldehyde , Exemplary genes encoding enzymes that catalyze the con butyraldehyde, isobutyraldehyde and formaldehyde (Pow - 35 version of an aldehyde to alcohol ( i. e ., alcohol dehydroge lowski et al. , J . Bacteriol. 175 : 377 - 385 ( 1993 ) ) . nase or equivalently aldehyde reductase ) include alrA encod ing a medium -chain alcohol dehydrogenase for C2- C14 Protein GenBank ID GI Number Organism ( Tani et al. , Appl. Environ . Microbiol. 66 : 5231- 5235 40 (2000 ) ) , ADH2 from Saccharomyces cerevisiae (Atsumi et acr1 YP _ 047869 . 1 50086359 Acinetobacter calcoaceticus acr1 AAC45217 1684886 Acinetobacter baylyi al. , Nature 451: 86 -89 ( 2008 ) ) , yqhD from E . coli which has acr1 BAB85476 . 1 18857901 Acinetobacter sp . Strain M - 1 preference for molecules longer than C (3 ) (Sulzenbacher et sucD P38947 . 1 172046062 Clostridium kluyveri sucD NP _ 904963 . 1 34540484 Porphyromonas gingivalis al ., J. Mol. Biol. 342 :489 -502 ( 2004 )) , and bdh I and bdh II bphG BAA03892 . 1 425213 Pseudomonas sp 45 from C . acetobutylicum which converts butyraldehyde into butanol (Walter et al. , J. Bacteriol. 174 : 7149- 7158 ( 1992 )) . An additional enzyme type that converts an acyl- CoA to ADH1 from Zymomonas mobilis has been demonstrated to its corresponding aldehyde is malonyl -CoA reductase which have activity on a number of aldehydes including formal transforms malonyl- CoA to malonic semialdehyde . Malo - dehyde , acetaldehyde, propionaldehyde, butyraldehyde , and nyl- CoA reductase is a key enzyme in autotrophic carbon 50 acrolein (Kinoshita , Appl. Microbiol. Biotechnol. 22 : 249 fixation via the 3 -hydroxypropionate cycle in thermoaci dophilic archael bacteria (Berg et al. , Science 318 : 1782 254 (1985 ) ). Cbei _ 2181 from Clostridium beijerinckii 1786 ( 2007b ) ; Thauer, 318 : 1732 - 1733 ( 2007 )) . The enzyme NCIMB 8052 encodes yet another useful alcohol dehydro utilizes NADPH as a cofactor and has been characterized in genase capable of converting crotonaldehyde to crotyl alco Metallosphaera and Sulfolobus spp ( Alber et al ., J. Bacte - » riol. 188 :8551 - 8559 (2006 ) ; Hugler et al . , J . Bacteriol. 184 : 2404 - 2410 ( 2002 ) . The enzyme is encoded by Msed _ 0709 in Metallosphaera sedula ( Alber et al. , supra, Protein GenBank ID GI Number Organism (2006 ) ; Berg et al. , supra , (2007b )) . A gene encoding a alrA BAB12273 . 1 9967138 Acinetobacter sp . Strain M - 1 malonyl- CoA reductase from Sulfolobus tokodaii was ADH2 NP _ 014032 . 1 6323961 Saccharomyces cloned and heterologously expressed in E . coli ( Alber et al. , cerevisiae supra , (2006 ) ) . Although the aldehyde dehydrogenase func yqhD NP _ 417484 . 1 16130909 Escherichia coli bdh I NP _ 349892. 1 15896543 Clostridium tionality of these enzymes is similar to the bifunctional acetobutylicum dehydrogenase from Chloroflexus aurantiacus, there is little 65 bah NP 349891 . 1 15896542 Clostridium sequence similarity . Both malonyl- CoA reductase enzyme acetobutylicum candidates have high sequence similarity to aspartate - semi US 10 ,006 ,055 B2 77 78 -continued - continued Protein GenBank ID GI Number Organism Enzyme Commission adha YP _ 162971. 1 56552132 Zymomonas mobilis Number Enzyme Name Cbei_ 2181 YP _ 001309304 . 1 150017050 Clostridium beijerinckii 5 NCIMB 8052 2 . 7 . 1 . 40 2 . 7 . 1 . 41 glucose - 1 - phosphate phosphodismutase 2 . 7 . 1 .42 riboflavin phosphootransferase Enzymes exhibiting 4 - hydroxybutyrate dehydrogenase 2 . 7 . 1 .43 2 . 7 . 1 .44 galacturonokinase activity (EC 1 . 1 . 1 .61 ) also fall into this category . Such 2 . 7 . 1 . 45 2 -dehydro - 3 -deoxygluconokinase enzymes have been characterized in Ralstonia eutropha 2 . 7 . 1 .46 L - arabinokinase (Bravo et al. , J. Forensic Sci. 49: 379 - 387 (2004 )) , 2 . 7 . 1 .47 D - 2 . 7 . 1. 48 Clostridium kluyveri (Wolff et al. , Protein Expr. Purif . 2 . 7 . 1 . 49 hydroxymethylpyrimidine kinase 6 : 206 - 212 ( 1995 ) ) and Arabidopsis thaliana ( Breitkreuz et 2 . 7 . 1 .50 hydroxyethylthiazole kinase 15 2 . 7 . 1 . 51 L - fuculokinase al. , J. Biol. Chem . 278 :41552 -41556 (2003 )) . 2 . 7 . 1 . 52 2 . 7 . 1 . 53 L - 2 . 7 . 1 . 54 D - arabinokinase Protein GenBank ID GI Number Organism 2 . 7 . 1 .55 allose kinase 2 . 7 . 1 . 56 1 - 4hbd YP _ 726053 . 1 113867564 Ralstonia eutropha H16 2 . 7 . 1 . 58 2 -dehydro - 3 -deoxygalactonokinase 4hbd L219021 . 1 46348486 Clostridium kluyveri DSM 555 - 2 . 7 . 1 . 59 N -acetylglucosamine kinase 4hbd Q94B07 75249805 Arabidopsis thaliana 2 . 7 . 1 .60 N - acylmannosamine kinase 2 . 7 . 1. 61 acyl- phosphate - hexose phosphotransferase 2 . 7 . 1 .62 phosphoramidate - hexose phosphotransferase Crotyl Alcohol Kinase ( FIG . 2 , Step F ) 2 . 7 . 1 .63 polyphosphate - glucose phosphotransferase 2 . 7 . 1 .64 inositol 3 -kinase Crotyl alcohol kinase enzymes catalyze the transfer of a 25 2 . 7 . 1 . 65 scyllo - inosamine 4 - kinase phosphate group to thene nydroxylhydroxyl group of01 crotylcroyl dicon01alcohol. 22 . 7 . 11 . 66 The enzymes described below naturally possess such activ 2 . 7 . 1 .67 1 - phosphatidylinositol 4 - kinase 2 .7 . 1 .68 1 -phosphatidylinositol - 4 -phosphate 5 -kinase ity or can be engineered to exhibit this activity . Kinases that 2 . 7 . 1 .69 protein - Np -phosphohistidine - sugar phosphotransferase catalyze transfer of a phosphate group to an alcohol group 2 . 7 . 1 . 70 identical to EC 2 . 7 . 1 . 37 . are members of the EC 2 . 7 . 1 enzyme class. The table below 30 2. 7 . 1. 71 lists several useful kinase enzymes in the EC 2 . 7 . 1 enzyme 2 . 7 . 1 . 72 streptomycin 6 -kinase 2 . 7 . 1 . 73 class . 2 . 7 . 1. 74 2 . 7 . 1 . 76 2 . 7 . 1 . 77 nucleoside phosphotransferase Enzyme 35 2 . 7 . 1. 78 polynucleotide 5 ' - hydroxyl- kinase Commission 2 . 7 . 1. 79 diphosphate - glycerol phosphotransferase Number Enzyme Name 2 . 7 . 1 . 80 diphosphate - serine phosphotransferase 2 . 7 . 1 . 81 2 . 7 . 1 . 1 2 . 7 . 1 . 82 2 . 7 . 1 . 2 2 . 7 . 1 . 83 2 . 7 . 1 . 3 ketohexokinase 40 2 . 7 . 1 . 84 alkylglycerone kinase 2 . 7 . 1 . 4 2 . 7 . 1 .85 B - glucoside kinase 2 . 7 . 1 . 5 2 . 7 . 1 . 86 NADH kinase 2 . 7 . 1 . 6 2 . 7 . 1 .87 streptomycin 3 " -kinase 2 . 7 . 1 . 7 2 . 7 . 1 . 88 dihydrostreptomycin - 6 -phosphate 3 ' a -kinase 2 . 7 . 1 . 8 2 . 7 . 1 .89 2 . 7 . 1 . 10 2 . 7 . 1 . 90 diphosphate - fructose- 6 -phosphate 1 -phosphotransferase 2 . 7 . 1 . 11 6 -phosphofructokinase 45 2 . 7 . 1 . 91 sphinganine kinase 2 . 7 . 1 . 12 2 . 7 . 1 . 92 5 - dehydro - 2 - deoxygluconokinase 2 . 7 . 1 . 13 dehydrogluconokinase 2 . 7 . 1 .93 alkylglycerol kinase 2 . 7 . 1 . 14 2 . 7 . 1 .94 2 . 7 . 1 . 15 2 . 7 . 1 . 95 2 . 7 . 1 . 16 ribulokinase 2 . 7 . 1 . 100 S -methyl - 5 - thioribose kinase 2 . 7 . 1 . 17 xylulokinase 50 2 . 7 . 1. 101 tagatose kinase 2 . 7 . 1 . 18 phosphoribokinase 2 . 7 . 1 . 102 hamamelose kinase 2 . 7 . 1 . 19 2 . 7 . 1 . 103 viomycin kinase 2 . 7 . 1 . 20 2 . 7 . 1 . 105 6 -phosphofructo - 2 -kinase 2 . 7 . 1 . 21 2 . 7 . 1 . 106 glucose - 1, 6 -bisphosphate synthase 2 . 7 . 1 . 22 ribosylnicotinamide kinase 2 . 7 . 1 . 107 2 . 7 . 1 . 23 NAD + kinase 55 2 . 7 . 1 . 108 2 . 7 . 1 . 24 dephospho - CoA kinase 2 . 7 . 1 . 113 2 . 7 . 1 . 25 adenylyl- sulfate kinase 2 . 7 . 1 . 114 AMP -thymidine kinase 2 . 7 . 1 . 26 2 . 7 . 1 . 118 ADP - thymidine kinase 2 . 7 . 1 . 27 erythritol kinase 2 . 7 . 1 . 119 2 . 7 . 1 . 28 hygromycin - B 7 " - O - kinase 2 . 7 . 1 . 29 glycerone kinase 2 . 7 . 1 . 121 phosphoenolpyruvate - glycerone phosphotransferase 2 .7 . 1. 30 glycerol kinase 60 2 . 7 . 1 . 122 xylitol kinase 2 . 7 . 1 . 31 2 . 7 . 1 . 127 inositol- trisphosphate 3 - kinase 2 . 7 . 1 . 32 2 . 7 . 1 . 130 tetraacyldisaccharide 4 '- kinase 2 . 7 . 1 . 33 2 . 7 . 1 . 134 inositol- tetrakisphosphate 1 -kinase 2 . 7 . 1 . 34 pantetheine kinase 2 . 7 . 1 . 136 macrolide 2 '- kinase 2 . 7 . 1 . 35 2 . 7 . 1 . 137 phosphatidylinositol 3 -kinase 2 . 7 . 1 . 36 mevalonate kinase 65 2 . 7 . 1. 138 2 . 7 . 1 . 39 homoserine kinase 2 . 7 . 1 . 140 inositol -tetrakisphosphate 5 - kinase US 10 ,006 ,055 B2 79 80 -continued -continued Enzyme Protein GenBank ID GI Number Organism Commission Enzyme Name glpK2 NP _ 229230 . 1 15642775 Thermotoga maritime MSB8 Number Gut1 NP _ 011831. 1 82795252 Saccharomyces cerevisiae 2 . 7 . 1 . 142 glycerol- 3 -phosphate - glucose phosphotransferase 2 . 7 . 1 . 143 diphosphate - purine nucleoside kinase 2 . 7 . 1 . 144 tagatose - 6 -phosphate kinase Homoserine kinase is another possible candidate that can 2 . 7 . 1 . 145 2 . 7 . 1 . 146 ADP - dependent phosphofructokinase lead to the phosphorylation of 3 , 5 - dihydroxypentanoate . 2 . 7 . 1 . 147 ADP - dependent glucokinase 10 This enzyme is also present in a number of organisms 2 . 7 . 1 . 148 4 - ( cytidine 5 '- diphospho ) - 2 - C -methyl - D - erythritol kinase including E . coli , Streptomyces sp , and S. cerevisiae . Homo 2 . 7 . 1 . 149 1 -phosphatidylinositol - 5 -phosphate 4 - kinase 2 . 7 . 1 . 150 1 - phosphatidylinositol- 3 -phosphate 5 - kinase serine kinase from E . coli has been shown to have activity 2 . 7 . 1 . 151 inositol - polyphosphate multikinase on numerous substrates, including , L - 2 - amino , 1, 4 -butane 2 . 7 . 1 . 153 phosphatidylinositol -4 , 5 -bisphosphate 3 -kinase diol, aspartate semialdehyde, and 2 -amino - 5 -hydroxyvaler 2 . 7 . 1 . 154 phosphatidylinositol - 4 - phosphate 3 -kinase 15 2 . 7 . 1 . 156 adenosylcobinamide kinase ate (Huo et al ., Biochemistry 35 :16180 - 16185 ( 1996 ); Huo 2 . 7 . 1 . 157 N -acetylgalactosamine kinase et al ., Arch . Biochem . Biophys. 330 : 373 -379 ( 1996 )) . This 2 . 7 . 1 . 158 inositol- pentakisphosphate 2 -kinase 2 .7 . 1 . 159 inositol- 1 ,3 ,4 - trisphosphate 5 /6 -kinase enzyme can act on substrates where the carboxyl group at 2 . 7 . 1 . 160 2 ' -phosphotransferase the alpha position has been replaced by an ester or by a 2 . 7 . 1 . 161 CTP - dependent riboflavin kinase 2 . 7 . 1 . 162 N - acetylhexosamine 1 - kinase 20 hydroxymethyl group . The gene candidates are: 2 . 7 . 1 . 163 hygromycin B 4 - O - kinase 2 . 7 . 1 . 164 O -phosphoseryl - tRNASec kinase Protein GenBank ID GI Number Organism thrB BAB96580. 2 85674277 Escherichia A good candidate for this step is mevalonate kinase (EC 25- coli K12 2 .7 . 1. 36 ) that phosphorylates the terminal hydroxyl group of SACTIDRAFT _ 4809 ZP _ 06280784. 1 282871792 Streptomyces the methyl analog , mevalonate , of 3 , 5 -dihydroxypentanote . sp . ACT- 1 Some gene candidates for this step are erg12 from S. Thr1 AAA35154 . 1 172978 Saccharomyces cerevisiae , mvk from Methanocaldococcus jannaschi, MVK serevisiae from Homo sapeins, and mvk from Arabidopsis thaliana 30 col. 2 - Butenyl -4 -Phosphate Kinase (FIG . 2 , Step G ) 2 - Butenyl- 4 - phosphate kinase enzymes catalyze the transfer of a phosphate group to the phosphate group of Protein GenBank ID GI Number Organism 2 -butenyl - 4 - phosphate . The enzymes described below natu erg12 CAA39359 . 1 3684 Sachharomyces cerevisiae 35 rally possess such activity or can be engineered to exhibit mvk Q58487 . 1 2497517 Methanocaldococcus jannaschii this activity. Kinases that catalyze transfer of a phosphate mvk AAH16140 . 1 16359371 Homo sapiens group to another phosphate group are members of the EC M \mvk NP _ 851084 . 1 30690651 Arabidopsis thaliana 2 .7 .4 enzyme class . The table below lists several useful kinase enzymes in the EC 2 . 7 . 4 enzyme class . Glycerol kinase also phosphorylates the terminal 40 hydroxyl group in glycerol to form glycerol- 3 - phosphate . Enzyme This reaction occurs in several species, including Escheri Commission chia coli, Saccharomyces cerevisiae , and Thermotoga mar Number Enzyme Name itima . The E . coli glycerol kinase has been shown to accept 2 . 7 . 4 . 1 alternate substrates such as dihydroxyacetone and glyceral 2 . 7 . 4 . 2 phosphomevalonate kinase dehyde (Hayashi et al ., J Biol. Chem . 242 : 1030 - 1035 2 . 7 . 4 . 3 2 . 7 . 4 . 4 nucleoside -phosphate kinase ( 1967 )) . T, maritime has two glycerolkinases (Nelson et al ., 2 . 7 . 4 . 6 nucleoside - diphosphate kinase Nature 399 :323 - 329 ( 1999 )) . Glycerol kinases have been 2 . 7 . 4 . 7 phosphomethylpyrimidine kinase shown to have a wide range of substrate specificity . Crans 50 2. 7. 4 .8 2 . 7 . 4 . 9 dTMP kinase and Whiteside studied glycerol kinases from four different 2 . 7 . 4 . 10 nucleoside -triphosphate - adenylate kinase organisms (Escherichia coli, S . cerevisiae, Bacillus stearo 2 . 7 . 4 . 11 (deoxy )adenylate kinase thermophilus, and Candida mycoderma ) ( Crans et al. , J . Am . 2 . 7 . 4 . 12 T2 - induced deoxynucleotide kinase 2 . 7 . 4 . 13 (deoxy )nucleoside - phosphate kinase Chem . Soc. 107: 7008 - 7018 ( 2010 ) ; Nelson et al. , supra , 2 . 7 . 4 . 14 ( 1999) ) . They studied 66 different analogs of glycerol and 2 . 7 . 4 . 15 thiamine -diphosphate kinase concluded that the enzyme could accept a range of substitu 2 . 7 . 4 . 16 thiamine -phosphate kinase 2 . 7 . 4 . 17 3 - phosphoglyceroyl- phosphate - polyphosphate ents in place of one terminal hydroxyl group and that the phosphotransferase hydrogen atom at C2 could be replaced by a methyl group . 2 . 7 . 4 . 18 farnesyl - diphosphate kinase Interestingly , the kinetic constants of the enzyme from all 602. 7 .4 . 19 5 -methyldeoxycytidine - 5 '- phosphate kinase 2 . 7 . 4 . 20 dolichyl- diphosphate - polyphosphate phosphotransferase four organisms were very similar. The gene candidates are : 2 . 7 . 4 .21 inositol- hexakisphosphate kinase 2 . 7 . 4 .22 UMP kinase 2 . 7 . 4 .23 ribose 1 ,5 - bisphosphate phosphokinase Protein GenBank ID GI Number Organism 2 . 7 . 4 . 24 diphosphoinositol -pentakisphosphate kinase glpK AP _ 003883 . 1 89110103 Escherichia coli K12 65 glpK1 NP 228760 . 1 15642775 Thermotoga maritime MSB8 Phosphomevalonate kinase enzymes are of particular interest. Phosphomevalonate kinase (EC 2 . 7 . 4 . 2 ) catalyzes US 10 ,006 ,055 B2 87 82 the analogous transformation to 2 - butenyl- 4 -phosphate kinase . This enzyme is encoded by erg8 in Saccharomyces Protein GenBank ID GI Number Organism cerevisiae ( Tsay et al. , Mol . Cell Biol. 11 :620 -631 ( 1991 )) hibch Q5XIE6 . 2 146324906 Rattus norvegicus and mvaK2 in Streptococcus pneumoniae, Staphylococcus hibch QONVY1. 2 146324905 Homo sapiens aureus and Enterococcus faecalis (Doun et al. , Protein Sci. > hibch P28817 . 2 2506374 Saccharomyces cerevisiae 14 : 1134 - 1139 ( 2005 ) ; Wilding et al. , J Bacteriol. 182 :4319 BC _ 2292 AP09256 29895975 Bacillus cereus 4327 (2000 ) ). The Streptococcus pneumoniae and Entero coccus faecalis enzymes were cloned and characterized in E . Several eukaryotic acetyl- CoA (EC 3 . 1 . 2 . 1 ) coli (Pilloff et al. , J Biol. Chem . 278 :4510 -4515 ( 2003903 ) ;. 10 have broad substrate specificity and thus represent suitable Doun et al. , Protein Sci. 14 : 1134 - 1139 ( 2005 ) ) . candidate enzymes. For example , the enzyme from Rattus norvegicus brain (Robinson et al. , Res. Commun . 71 :959 965 ( 1976 ) ) can react with butyryl- CoA , hexanoyl- CoA and Protein GenBank ID GI Number Organism malonyl- CoA . Though its sequence has not been reported , Erg8 AAA34596 . 1 171479 Saccharomyaromyces cerevisiae 15 the enzyme from the mitochondrion of the pea leaf also has mvaK2 AAGO2426 . 1 9937366 Staphylococcus aureus a broad substrate specificity , with demonstrated activity on mvaK2 AAG02457 . 1 9937409 Streptococcus pneumoniae acetyl - CoA , propionyl- CoA , butyryl- CoA , palmitoyl -CoA , mvaK2 AAG02442 . 1 9937388 Enterococcus faecalis oleoyl- CoA , succinyl- CoA , and crotonyl- CoA (Zeiher et al. , Plant . Physiol. 94 :20 -27 ( 1990 ) ). The acetyl- CoA hydrolase , Butadiene Synthase (FIG . 2 , Step H ) ACH1, from S . cerevisiae represents another candidate Butadiene synthase catalyzes the conversion of 2 -butenyl hydrolase (Buu et al ., J. Biol. Chem . 278 : 17203 - 17209 4 - diphosphate to 1 , 3 - butadiene. The enzymes described (2003 ) ) . These proteins are identified below . below naturally possess such activity or can be engineered to exhibit this activity . Isoprene synthase naturally catalyzes 25 Protein GenBank ID GI Number Organism the conversion of dimethylallyl diphosphate to isoprene, but can also catalyze the synthesis of 1 , 3 -butadiene from 2 - bute acot12 NP _ 570103 . 1 18543355 Rattus norvegicus nyl- 4 - diphosphate . Isoprene synthases can be found in sev ACH1NP _ 009538 6319456 Saccharomyces cerevisiae eral organisms including Populus alba (Sasaki et al. , FEBS Another candidate hydrolase is the human dicarboxylic Letters , 2005 , 579 ( 11) , 2514 - 2518 ) , Pueraria montana acid thioesterase , acot8 , which exhibits activity on glutaryl ( Lindberg et al. , Metabolic Eng, 2010 , 12 ( 1 ) , 70 -79 ; Shar COA , adipyl - CoA , suberyl- CoA , sebacyl -CoA , and dode key et al. , Plant Physiol. , 2005 , 137 ( 2 ) , 700 -712 ) , and canedioyl- CoA (Westin et al ., J Biol. Chem . 280 : 38125 Populus tremulaxPopulus alba (Miller et al. , Planta , 2001, 38132 (2005 ) ) and the closest E . coli homolog , tesB , which 213 (3 ), 483 -487 ) . Additional isoprene synthase enzymes are 35 can also hydrolyze a broad range of CoA thioesters (Na described in (Chotani et al. , WO /2010 /031079 , Systems et al. , J Biol. Chem . 266 : 11044 - 11050 (1991 ) ). A similar Using Cell Culture for Production of Isoprene; Cervin et al ., enzyme has also been characterized in the rat liver ( Deana US Patent Application 20100003716 , Isoprene Synthase et al. , Biochem . Int. 26 : 767 - 773 ( 1992 )) . Other potential E . Variants for Improved Microbial Production of Isoprene ) . 10 coli thioester hydrolases include the gene products of tesA (Bonner et al. , Chem . 247 :3123 -3133 ( 1972 )) , ybgC (Kuz netsova et al. , FEMSMicrobiol Rev 29 : 263 - 279 (2005 ) ; and Protein GenBank ID GI Number Organism ( Zhuang et al. , FEBS Lett . 516 : 161- 163 (2002 ) ) , paal (Song ispS BAD98243 . 1 63108310 Populus alba et al . , J Biol. Chem . 281: 11028 - 11038 (2006 ) ) , and ybdB ispS AAQ84170 . 1 35187004 Pueraria montana 45 (Leduc et al. , J Bacteriol. 189 :7112 -7126 ( 2007 )) . These ispS CAC35696 . 1 13539551 Populus tremula x Populus alba proteins are identified below . Crotonyl- CoA Hydrolase , Synthetase , Transferase (FIG . 2 , Step I) Protein GenBank ID GI Number Organism 50 tesB NP _ 414986 16128437 Escherichia coli Crotonyl- CoA hydrolase catalyzes the conversion of acot8 CAA15502 3191970 Homo sapiens crotonyl - CoA to crotonate . The enzymes described below acot8 NP _ 570112 51036669 Rattus norvegicus naturally possess such activity or can be engineered to tes A NP 415027 16128478 Escherichia coli ybgC NP _ 415264 16128711 Escherichia coli exhibit this activity . 3 - Hydroxyisobutyryl- CoA hydrolase paal NP _ 415914 16129357 Escherichia coli efficiently catalyzes the conversion of 3 -hydroxyisobutyryl - 55 ybdB NP _ 41512916128580 Escherichia coli COA to 3 -hydroxyisobutyrate during valine degradation ( Shimomura et al. , J Biol Chem . 269 : 14248 -14253 ( 1994 ) ) . Yet another candidate hydrolase is the glutaconate COA Genes encoding this enzyme include hibch of Rattus nor - transferase from Acidaminococcus fermentans . This enzyme vegicus (Shimomura et al. , supra ; Shimomura et al ., Meth - so was transformed by site -directed mutagenesis into an acyl ods Enzymol. 324 :229 - 240 ( 2000 ) ) and Homo sapiens (Shi COA hydrolase with activity on glutaryl- CoA , acetyl- CoA momura et al. , supra ). The H . sapiens enzyme also accepts and 3 - butenoyl- CoA (Mack et al ., FEBS . Lett. 405 : 209 -212 3 -hydroxybutyryl - CoA and 3 -hydroxypropionyl - CoA as (1997 ) ) . This suggests that the enzymes encoding succinyl substrates (Shimomura et al. , supra ) . Candidate genes by COA : 3 -ketoacid - CoA and acetoacetyl- COA : sequence homology include hibch of Saccharomyces cer - 65 acetyl- CoA transferases can also serve as candidates for this evisiae and BC _ 2292 of Bacillus cereus. These proteins are reaction step but would require certain mutations to change identified below . their function . These proteins are identified below . US 10 ,006 ,055 B2 83 84 Additional exemplary CoA - include the rat dicar Protein GenBank ID GI Number Organism boxylate -CoA ligase for which the sequence is yet unchar gctA CAA57199 559392 Acidaminococcus acterized ( Vamecq et al. , Biochemical Journal 230 :683 -693 fermentans ( 1985 ) ) , either of the two characterized phenylacetate - COA gotB CAA57200 559393 Acidaminococcus 5 ligases from P . chrysogenum (Lamas -Maceiras et al. , Bio fermentans chem . J . 395 : 147 - 155 (2005 ) ; Wang et al. , Biochem Biophy Res Commun 360 ( 2 ): 453 -458 (2007 ) ) , the phenylacetate Crotonyl- CoA synthetase catalyzes the conversion of COA ligase from Pseudomonas putida (Martinez - Blanco et crotonyl- CoA to crotonate . The enzymes described below al. , J . Biol. Chem . 265 : 7084 - 7090 ( 1990 ) ) , and the 6 - car naturally possess such activity or can be engineered to 10 boxyhexanoate - CoA ligase from Bacillus subtilis (Bower et exhibit this activity . One candidate enzyme, ADP - forming al. , J . Bacteriol . 178 ( 14 ): 4122 - 4130 ( 1996 ) ) . Additional acetyl- CoA synthetase ( ACD , EC 6 .2 . 1. 13 ), couples the candidate enzymes are acetoacetyl -CoA synthetases from conversion of acyl- CoA esters to their corresponding acids Mus musculus ( Hasegawa et al. , Biochim Biophys Acta with the concurrent synthesis of ATP. Several enzymes with 16 1779 : 414 -419 (2008 ) ) and Homo sapiens (Ohgami et al. , broad substrate specificities have been described in the Biochem Pharmacol 65: 989 - 994 ( 2003 )) which naturally literature . ACD I from Archaeoglobus fulgidus, encoded by catalyze the ATP -dependant conversion of acetoacetate into AF1211 , was shown to operate on a variety of linear and acetoacetyl - CoA . These proteins are identified below . branched - chain substrates including acetyl- CoA , propionyl COA , butyryl- CoA , acetate , propionate , butyrate , isobutyry - 20 ate , isovalerate , succinate , fumarate , phenylacetate , Protein GenBank ID GI Number Organism indoleacetate (Musfeldt et al. , J Bacteriol 184 :636 -644 ( 2002) ) . The enzyme from Haloarcula marismortui (anno phl CAJ15517 . 1 77019264 Penicillium chrysogenum tated as a succinyl- CoA synthetase ) accepts propionate , phlB ABS19624 . 1 152002983 Penicillium chrysogenum butyrate , and branched -chain acids (isovalerate and isobu - 25 paaF AAC24333 . 2 22711873 Pseudomonas putida tyrate ) as substrates, and was shown to operate in the bioW NP _ 390902 . 2 50812281 Bacillus subtilis forward and reverse directions (Brasen et al. , Arch Microbiol AACS NP __ 084486 . 1 2131352021313520 Mus musculus 182 :277 - 287 (2004 ) ) . The ACD encoded by PAE3250 from AACS NP 076417 . 2 31982927 Homo sapiens hyperthermophilic crenarchaeon Pyrobaculum aerophilum showed the broadest substrate range of all characterized 30 ACDs, reacting with acetyl- CoA , isobutyryl -CoA (preferred Crotonyl- CoA transferase catalyzes the conversion of substrate ) and phenylacetyl -CoA (Brasen et al. , supra ). The crotonyl - CoA to crotonate . The enzymes described below enzymes from A . fulgidus, H . marismortui and P . aerophi naturally possess such activity or can be engineered to lum have all been cloned , functionally expressed , and char - 35 exhibit this activity . Many transferases have broad specific acterized in E . coli (Musfeldt et al. , supra ; Brasen et al ., " ity and thus can utilize CoA acceptors as diverse as acetate , supra ) . These proteins are identified below . succinate , propionate , butyrate , 2 -methylacetoacetate , 3 -ke tohexanoate , 3 -ketopentanoate , valerate , crotonate , 3 -mer captopropionate , propionate , vinylacetate , butyrate, among Protein GenBank ID GI Number Organism 40 others . For example , an enzyme from Roseburia sp . A2 - 183 AF1211 NP _ 070039 .1 11498810 Archaeoglobus fulgidus was shown to have butyryl- CoA :acetate :CoA transferase DSM 4304 and propionyl - CoA : acetate : COA transferase activity (Char SCS YP _ 135572 . 1 55377722 Haloarcula marismortui ATCC 43049 rier et al. , Microbiology 152 , 179 - 185 ( 2006 )) . Close PAE3250 NP _ 560604 .1 18313937 Pyrobaculum aerophilum homologs can be found in , for example , Roseburia intesti str . IM2 45 nalis L1- 82 , Roseburia inulinivorans DSM 16841, Eubac terium rectale ATCC 33656 . Another enzyme with propio Another candidate CoA synthetase is succinyl- CoA syn nyl- CoA transferase activity can be found in Clostridium thetase . The sucCD genes of E . coli form a succinyl- CoA propionicum (Selmer et al. , Eur J Biochem 269 , 372 -380 synthetase complex which naturally catalyzes the formations (2002 ) ) . This enzyme can use acetate , ( R ) - lactate , ( S ) of succinyl- CoA from succinate with the concaminant con 30 lactate , acrylate , and butyrate as the COA acceptor (Selmer sumption of one ATP, a reaction which is reversible in vivo et al. , Eur J Biochem 269, 372 - 380 ( 2002 ); Schweiger and (Buck et al. , Biochem . 24 :6245 -6252 ( 1985 ) ) . These proteins Buckel, FEBS Letters , 171 ( 1 ) 79 - 84 (1984 ) ) . Close homologs can be found in , for example , Clostridium novyi are identified below . NT, Clostridium beijerinckii NCIMB 8052 , and Clostridium 55 botulinum C str. Eklund . YgfH encodes a propionyl COA : Protein GenBank ID GI Number Organism succinate CoA transferase in E . coli (Haller et al. , Biochem istry , 39 ( 16 ) 4622 - 4629 ) . Close homologs can be found in , succ NP _ 415256 . 1 16128703 Escherichia coli sucD AAC73823 . 1 1786949 Escherichia coli for example, Citrobacter youngae ATCC 29220 , Salmonella enterica subsp . arizonae serovar, and Yersinia intermedia ATCC 29909 . These proteins are identified below .

Protein GenBank ID GI Number Organismsin Ach1 AAX19660 . 1 60396828 Roseburia sp . A2 - 183 ROSINTL182 _ 07121 ZP _ 04743841. 2 257413684 Roseburia intestinalis L1 -82 US 10 ,006 ,055 B2 85 86 - continued Protein GenBank ID GI Number Organism ROSEINA2194 _ 03642 ZP _ 03755203. 1 225377982 Roseburia inulinivorans DSM 16841 EUBREC _ 3075 YP _ 002938937 . 1 238925420 Eubacterium rectale ATCC 33656 pct CAB77207 . 1 7242549 Clostridium propionicum NT01CX _ 2372 YP _ 878445 . 1 118444712 Clostridium novyi NT Cbei _ 4543 YP _ 001311608 . 1 150019354 Clostridium beijerinckii NCIMB 8052 CBC _ A0889 ZP _ 02621218 . 1 168186583 Clostridium botulinum C str. Eklund ygfH NP _ 417395 . 1 16130821 Escherichia coli str. K - 12 substr . MG1655 CIT292 _ 04485 ZP _ 03838384 . 1 227334728 Citrobacter youngae ATCC 29220 SARI_ 04582 YP _ 001573497. 1 161506385 Salmonella enterica subsp . arizonae serovar yinte0001 _ 14430 ZP _ 04635364 . 1 238791727 Yersinia intermedia ATCC 29909

An additional candidate enzyme is the two -unit enzyme encoded by pcal and pca ) in Pseudomonas, which has been Protein GenBank ID GI Number Organism shown to have 3 - oxoadipyl- CoA / succinate transferase activ ato A P76459 . 1 2492994 Escherichia coli K12 atoD P76458. 1 2492990 Escherichia coli K12 ity (Kaschabek et al. , supra ). Similar enzymes based on 25 actA YP _ 226809. 1 62391407 Corynebacterium glutamicum homology exist in Acinetobacter sp . ADP1 (Kowalchuk et ATCC 13032 al. , Gene 146 : 23 - 30 ( 1994 )) and Streptomyces coelicolor. cg0592 YP _ 224801. 1 62389399 Corynebacterium glutamicum ATCC 13032 Additional exemplary succinyl - CoA : 3 : oxoacid - CoA trans - ctfA NP _ 149326 . 1 15004866 Clostridium acetobutylicum ferases are present in Helicobacter pylori (Corthesy - Theulaz ctfB NP _ 149327 . 1 15004867 Clostridium acetobutylicum ctfA AAP42564 . 1 31075384 Clostridium et al ., J. Biol. Chem . 272 :25659 -25667 ( 1997 )) and Bacillus saccharoperbutylacetonicum subtilis (Stols et al ., Protein . Expr. Purif . 53 :396 -403 ctfB AAP42565 . 1 31075385 Clostridium (2007 ) ). These proteins are identified below . saccharoperbutylacetonicum 35 The above enzymes can also exhibit the desired activities Protein GenBank ID GI Number Organism on crotonyl- CoA . Additional exemplary transferase candi pcal AAN69545 . 1 24985644 Pseudomonas putida dates are catalyzed by the gene products of cat1, cat2, and pca ) NP _ 746082 . 1 26990657 Pseudomonas putida cat3 of Clostridium kluyveri which have been shown to pcal YP _ 046368 . 1 50084858 Acinetobacter sp . ADP1 an exhibit succinyl- CoA , 4 -hydroxybutyryl - CoA , and butyryl pca ) AAC37147 . 1 141776 Acinetobacter sp . ADP1 COA transferase activity , respectively ( Seedorf et al. , supra ; pcal NP _ 630776 . 1 21224997 Streptomyces coelicolor Sohling et al. , Eur. J Biochem . 212 : 121- 127 ( 1993 ); Sohling pca ) NP _ 630775 . 1 21224996 Streptomyces coelicolor HPAG1_ 0676 YP _ 627417 108563101 Helicobacter pylori et al. , J Bacteriol. 178 :871 - 880 ( 1996 )) . Similar CoA trans HPAG1_ 0677 YP _ 627418 108563102 Helicobacter pylori ferase activities are also present in Trichomonas vaginalis SCOA NP _ 391778 16080950 Bacillus subtilis 45 (van Grinsven et al. , J . Biol. Chem . 283 : 1411 - 1418 ( 2008 ) ) ScoB NP _ 391777 16080949 Bacillus subtilis and Trypanosoma brucei (Riviere et al. , J. Biol . Chem . 279 : 45337 -45346 (2004 )) . These proteins are identified A COA transferase that can utilize acetate as the COA below . acceptor is acetoacetyl- CoA transferase , encoded by the E . 50 coli atoa ( alpha subunit ) and atoD (beta subunit ) genes Protein GenBank ID GI Number Organism ( Vanderwinkel et al. , Biochem . Biophys. Res Commun . cat1 P38946 . 1 729048 Clostridium kluyveri 33: 902 - 908 (1968 ); Korolev et al. , Acta Crystallogr. D Biol cat2 P38942 . 2 172046066 Clostridium kluyveri Crystallo . 58 :2116 -2121 ( 2002 )) . This enzyme has also been cat3 EDK35586 . 1 146349050 Clostridium kluyveri shown to transfer the CoA moiety to acetate from a variety » TVAG _ 395550 XP _ 001330176 123975034 Trichomonas ofbranched and linear acyl - CoA substrates , including isobu vaginalis G3 tyrate (Matthies et al ., Appl Environ Microbiol 58 : 1435 Tb11 .02 . 0290 XP _ 828352 71754875 Trypanosoma brucei 1439 ( 1992 ) ) , valerate ( Vanderwinkel et al. , supra ) and butanoate (Vanderwinkel et al. , supra ). Similar enzymes 6 The glutaconate - CoA - transferase ( EC 2 . 8 . 3 . 12 ) enzyme exist in Corynebacterium glutamicum ATCC 13032 ( Dun from anaerobic bacterium Acidaminococcus fermentans can et al. , Appl Environ Microbiol 68 : 5186 - 5190 ( 2002 ) ) , reacts with diacid glutaconyl- CoA and 3 -butenoyl - CoA Clostridium acetobutylicum ( Cary et al. , Appl Environ (Mack et al. , FEBS Lett . 405 : 209 -212 ( 1997 ) ) . The genes Microbiol 56 : 1576 - 1583 (1990 ) ) , and Clostridium saccha encoding this enzyme are gctA and getB . This enzyme has roperbutylacetonicum (Kosaka et al. , Biosci. Biotechnol 65 reduced but detectable activity with other CoA derivatives Biochem . 71: 58 -68 ( 2007 ) ) . These proteins are identified including glutaryl- CoA , 2 -hydroxyglutaryl - CoA , adipyl below . CoA and acrylyl -CoA (Buckel et al. , Eur. J. Biochem . US 10 , 006 , 055 B2 87 88 118 :315 - 321 (1981 )) . The enzyme has been cloned and An additional enzyme candidate found in Streptomyces expressed in E . coli (Mack et al ., Eur. J. Biochem . 226 :41 - 51 griseus is encoded by the griC and grid genes . This enzyme ( 1994 )) . These proteins are identified below . is believed to convert 3 - amino - 4 -hydroxybenzoic acid to 3 -amino - 4 -hydroxybenzaldehyde as deletion of either griC or griD led to accumulation of extracellular 3 - acetylamino Protein GenBank ID GI Number Organism 4 -hydroxybenzoic acid , a shunt product of 3 - amino - 4 - hy gctA CAA57199 . 1 559392 Acidaminococcus fermentans droxybenzoic acid metabolism ( Suzuki, et al. , J . Antibiot. gctB CAA57200 . 1 559393 Acidaminococcus fermentans 60 ( 6 ): 380 - 387 (2007 ) ). Coexpression of griC and griD with SGR _665 , an enzyme similar in sequence to the Nocardia 10 Crotonate Reductase ( FIG . 2 , Step J ) iowensis npt, can be beneficial. Crotonate reductase enzymes are capable of catalyzing the conversion of crotonate to crotonaldehyde . The enzymes described below naturally possess such activity or can be Protein GenBank ID GI Number Organism engineered to exhibit this activity . Carboxylic acid reductase 15 griC YP _ 001825755 . 1 182438036 Streptomyces griseus subsp . catalyzes the magnesium , ATP and NADPH -dependent griseus NBRC 13350 reduction of carboxylic acids to their corresponding alde Grid YP _ 001825756 . 1 182438037 Streptomyces griseus subsp . hydes ( Venkitasubramanian et al. , J . Biol. Chem . 282 : 478 griseus NBRC 13350 485 ( 2007 ) ). This enzyme, encoded by car, was cloned and functionally expressed in E . coli (Venkitasubramanian et al. , 20 J . Biol. Chem . 282 : 478 - 485 ( 2007 ) ). Expression of the npt An enzyme with similar characteristics , alpha -aminoadi gene product improved activity of the enzyme via post pate reductase ( AAR , EC 1 . 2 . 1 .31 ) , participates in lysine transcriptionalmodification . The npt gene encodes a specific biosynthesis pathways in some fungal species . This enzyme phosphopantetheine transferase (PPTase ) that converts the naturally reduces alpha - aminoadipate to alpha -aminoadipate inactive apo - enzyme to the active holo -enzyme . The natural semialdehyde. The carboxyl group is first activated through substrate of this enzyme is vanillic acid , and the enzyme the ATP - dependent formation of an adenylate that is then exhibits broad acceptance of aromatic and aliphatic sub reduced by NAD ( P ) H to yield the aldehyde and AMP. Like strates ( Venkitasubramanian et al. , in Biocatalysis in the CAR , this enzyme utilizes magnesium and requires activa Pharmaceutical and Biotechnology Industries, ed . R . N . 30 tion by a PPTase . Enzyme candidates for AAR and its Patel , Chapter 15 , pp . 425 - 440 , CRC Press LLC , Boca corresponding PPTase are found in Saccharomyces cerevi Raton , Fla . (2006 )) . siae (Morris et al. , Gene 98 : 141 - 145 ( 1991) ) , Candida albicans (Guo et al. , Mol. Genet. Genomics 269 :271 -279 ( 2003 )) , and Schizosaccharomyces pombe (Ford et al. , Curr. Protein GenBank ID GI Number Organism 35 Genet. 28 : 131- 137 (1995 ) ). The AAR from S . pombe exhib Car AAR91681. 1 40796035 Nocardia iowensis ited significant activity when expressed in E . coli (Guo et al ., ( sp . NRRL 5646 ) Yeast 21 : 1279 - 1288 ( 2004 ) ) . The AAR from Penicillium Npt ABI83656 . 1 114848891 Nocardia iowensis chrysogenum accepts S - carboxymethyl- L -cysteine as an ( sp . NRRL 5646 ) alternate substrate , but did not react with adipate , L - gluta 40 mate or diaminopimelate (Hijarrubia et al. , J. Biol . Chem . Additional car and npt genes can be identified based on 278 :8250 - 8256 ( 2003 ) ) . The gene encoding the P . chryso sequence homology genum PPTase has not been identified to date .

Protein GenBank ID GI Number Organism fadD9 YP _ 978699. 1 121638475 Mycobacterium bovis BCG BCG _ 2812c YP _ 978898 . 1 121638674 Mycobacterium bovis BCG nfa20150 YP _ 118225 . 1 54023983 Nocardia farcinica IFM 10152 nfa40540 YP _ 120266 . 1 54026024 Nocardia farcinica IFM 10152 SGR 6790 YP _ 001828302 . 1 182440583 Streptomyces griseus subsp . griseus NBRC 13350 SGR _ 665 YP 001822177 . 1 182434458 Streptomyces griseus subsp . griseus NBRC 13350 MSMEG _ 2956 YP _ 887275 . 1 118473501 Mycobacterium smegmatis MC2 155 MSMEG _ 5739 YP _ 889972. 1 118469671 Mycobacterium smegmatis MC2 155 MSMEG _ 2648 YP _ 8869851 . 118471293 Mycobacterium smegmatis MC2 155 MAP10400 NP _ 9599741 . 41407138 Mycobacterium avium subsp . paratuberculosis K - 10 MAP2899c NP _ 961833 . 1 41408997 Mycobacterium avium subsp . paratuberculosis K - 10 MMAR _ 2117 YP _ 001850422 . 1 183982131 Mycobacterium marinum M MMAR 2936 YP _ 001851230. 1 183982939 Mycobacterium marinum M MMAR 1916 YP _ 001850220 . 1 183981929 Mycobacterium marinum M TpauDRAFT _ 33060 ZP _ 04027864 . 1 227980601 Tsukamurella paurometabola DSM 20162 TpauDRAFT _ 20920 ZP _ 04026660 . 1 227979396 Tsukamurella paurometabola DSM 20162 CPCC7001 _ 1320 ZP _ 05045132. 1 254431429 Cyanobium PCC7001 DDBDRAFT _ 0187729 XP _ 636931. 1 66806417 Dictyostelium discoideum AX4 US 10 ,006 ,055 B2 89 90 Glutaconyl- CoA Decarboxylase (FIG . 2 , Step L ) Protein GenBank ID GI Number Organism Glutaconyl -CoA decarboxylase enzymes, characterized in glutamate -fermenting anaerobic bacteria , are sodium - ion LYS2 AAA34747 . 1 171867 Saccharomyces cerevisiae LYS5 P50113 . 1 1708896 Saccharomyces cerevisiae translocating decarboxylases that utilize biotin as a cofactor LYS2 AAC02241. 1 2853226 Candida albicans 5 and are composed of four subunits (alpha , beta , gamma, and LYS5 AAO26020 . 1 28136195 Candida albicans delta ) ( Boiangiu et al. , J Mol. Microbiol Biotechnol 10 : 105 Lyslp P40976 . 3 13124791 Schizosaccharomyces pombe 119 (2005 ); Buckel , Biochim Biophys Acta . 1505 : 15 - 27 Lys7p 010474 . 1 1723561 Schizosaccharomyces pombe ( 2001 ) ) . Such enzymes have been characterized in Fuso Lys2 CAA74300 . 1 3282044 Penicillium chrysogenum bacterium nucleatum (Beatrix et al. , Arch Microbiol. 154 : 362 - 369 ( 1990 ) ) and Acidaminococcus fermentans (Braune et al. , Mol . Microbiol 31: 473 -487 ( 1999 )) . Analogs to the F. Crotonyl- CoA Reductase ( Alcohol Forming ) (FIG . 2 , Step nucleatum glutaconyl- CoA decarboxylase alpha, beta and K ) delta subunits are found in S . aciditrophicus . A gene anno Crotonaldehyde reductase (alcohol forming ) enzymes tated as an enoyl- CoA dehydrogenase , syn _ 00480 , another catalyze the 2 reduction steps required to form crotyl alcohol , GCD , is located in a predicted operon between a biotin from crotonyl- CoA . Exemplary 2 - step oxidoreductasesctases that 15 carboxyl carrier ( syn _ 00479 ) and a glutaconyl- CoA decar convert an acyl - CoA to an alcohol are provided below . Such boxylase alpha subunit ( syn _ 00481 ) . The protein sequences enzymes can naturally convert crotonyl- CoA to crotyl alco for exemplary gene products can be found using the follow hol or can be engineered to do so . These enzymes include ing GenBank accession numbers shown below . those that transform substrates such as acetyl- CoA to ethanol 20 ( e . g . , adhE from E . coli (Kessler et al. , FEBS . Lett . 281 : 59 Protein GenBank ID GI Number Organism 63 ( 1991 ) ) ) and butyryl- CoA to butanol ( e . g . adhE2 from C . gedA CAA49210 49182 Acidaminococcus fermentans acetobutylicum (Fontaine et al ., J Bacteriol. 184 : 821 -830 gedC AAC69172 3777506 Acidaminococcus fermentans (2002 )) ) . The adhE2 enzyme from C . acetobutylicum was gcdD AAC69171 3777505 Acidaminococcus fermentans specifically shown in ref (Burk et al. , supra , ( 2008 ) ) to 25 gedB AAC69173 3777507 Acidaminococcus fermentans FNO200 AAL94406 19713641 Fusobacterium nucleatum produce BDO from 4 -hydroxybutyryl - CoA . In addition to FN0201 AAL94407 19713642 Fusobacterium nucleatum reducing acetyl- CoA to ethanol, the enzyme encoded by FNO204 AAL9441019713645 Fusobacterium nucleatum adhE in Leuconostoc mesenteroides has been shown to oxide syn _ 00479 YP _ 462066 85859864 Syntrophus aciditrophicus syn __ 00481 YP _ 462068 85859866 Syntrophus aciditrophicus the branched chain compound isobutyraldehyde to isobu 30 syn _ 01431 YP _ 460282 85858080 Syntrophus aciditrophicus tyryl- CoA (Kazahaya et al. , J. Gen . Appl. Microbiol. 18 :43 - 30 syn _ 00480 ABC7789985722956 Syntrophus aciditrophicus 55 ( 1972 ) ; Koo et al. , Biotechnol . Lett . 27 : 505 -510 ( 2005 ) ) . Glutaryl - CoA Dehydrogenase (FIG . 2 Step M ) Protein GenBank ID GI Number Organism Glutaryl- CoA dehydrogenase (GCD , EC 1 . 3 . 99 . 7 and EC - 35 4 . 1. 1 . 70 ) is a bifunctional enzyme that catalyzes the oxida adhE NP _ 415757 . 1 16129202 Escherichia coli adhE2 AAK09379 . 1 12958626 Clostridium acetobutylicum tive decarboxylation of glutaryl- CoA to crotonyl- CoA (FIG . adhE AAV66076 . 1 55818563 Leuconostoc mesenteroides 3 , step 3 ) . Bifunctional GCD enzymes are homotetramers that utilize electron transfer flavoprotein as an electron acceptor ( Hartel et al. , Arch Microbiol. 159 : 174 - 181 Another exemplary enzyme can convert malonyl- CoA to 40 (1993 )) . Such enzymes were first characterized in cell 3 - HP. An NADPH -dependent enzyme with this activity has extracts of Pseudomonas strains KB740 and K172 during been characterized in Chloroflexus aurantiacus where it growth on aromatic compounds (Hartel et al. , supra, ( 1993 ) ) , participates in the 3 -hydroxypropionate cycle (Hugler et al. , but the associated genes in these organisms is unknown . supra , (2002 ); Strauss et al. , 215: 633 -643 ( 1993) ) . This Genes encoding glutaryl- CoA dehydrogenase (gcdH ) and its enzyme, with a mass of 300 kDa, is highly substrate -specific 45 cognate transcriptional regulator (gcdR ) were identified in and shows little sequence similarity to other known oxi Azoarcus sp . CIB (Blazquez et al. , Environ Microbiol. doreductases (Hugler et al ., supra , ( 2002) ) . No enzymes in 10 : 474 -482 (2008 ) ). An Azoarcus strain deficient in gcdH other organisms have been shown to catalyze this specific activity was used to identify the a heterologous gene gcdH reaction ; however there is bioinformatic evidence that other from Pseudomonas putida (Blazquez et al. , supra , (2008 ) ) . organisms can have similar pathways (Klatt et al. , Environon 50 The cognate transcriptional regulator in Pseudomonas Microbiol. 9 : 2067 - 2078 ( 2007) ) . Enzyme candidates in putida has not been identified but the locus PP 0157 has a other organisms including Roseiflexus castenholzii, Eryth high sequence homology ( > 69 % identity ) to the Azoarcus robacter sp . NAP1 and marine gamma proteobacterium enzyme. Additional GCD enzymes are found in Pseudomo nas fluorescens and Paracoccus denitrificans (Husain et al. , HTCC2080 can be inferred by sequence similarity . 55 J Bacteriol. 163: 709 - 715 ( 1985 )) . The human GCD has been extensively studied , overexpressed in E . coli ( Dwyer et al. , Biochemistry 39: 11488 - 11499 (2000 ) ) , crystallized , and the Protein GenBank ID GI Number Organism catalytic mechanism involving a conserved glutamate resi mcr AAS20429 . 1 42561982 Chloroflexus due in the active site has been described ( Fu et al. , Bio aurantiacus Rcas _ 2929 YP _ 001433009. 1 156742880 Roseiflexus 60 chemistry 43 : 9674 - 9684 ( 2004 ) ) . A GCD in Syntrophus castenholzii aciditrophicus operates in the CO , - assimilating direction NAP1 _ 02720 ZP _ 010391790105 . 1 85708113 Erythrobacter during growth on crotonate (Mouttaki et al . , Appl Environ sp . NAP1 MGP2080 _ 00535 ZP _ 01626393 .1 119504313 marine gamma Microbiol . 73: 930 - 938 ( 2007 ) ) ) . Two GCD genes in S . proteobacterium aciditrophicus were identified by protein sequence homol HTCC2080 65 ogy to the Azoarcus GedH : syn _ 00480 (31 % ) and syn _ 01146 ( 31 % ) . No significant homology was found to the Azoarcus GdR regulatory protein . The protein US 10 , 006 , 055 B2 91 92 sequences for exemplary gene products can be found using -continued the following GenBank accession numbers shown below . Protein GenBank ID GI Number Organism Msed _ 1321 YP _ 001191403 146304087 Metallosphaera sedula Protein GenBank ID GI Number Organism Msed _ 1220 YP _ 001191305 146303989 Metallosphaera sedula gcdH ABM69268 . 1 123187384 Azoarcus sp . CIB gedR ABM69269 . 1 123187385 Azoarcus sp . CIB gcdH AAN65791. 1 24981507 Pseudomonas Crotyl Alcohol Diphosphokinase (FIG . 2 , Step P ) putida KT2440 Crotyl alcohol diphosphokinase enzymes catalyze the PP _ 0157 AAN65790 . 1 24981506 Pseudomonas ( gcdR ) putida KT2440 10 transfer of a diphosphate group to the hydroxyl group of gcdH YP _ 257269. 1 70733629 Pseudomonas crotyl alcohol. The enzymes described below naturally pos fluorescens Pf - 5 sess such activity or can be engineered to exhibit this gcvA YP _ 257268. 1 70733628 Pseudomonas activity . Kinases that catalyze transfer of a diphosphate ( gcdR ) fluorescens Pf- 5 ged YP _ 918172 . 1 119387117 Paracoccus denitrificans group are members of the EC 2 .7 .6 enzyme class . The table gedR YP _ 918173 . 1 119387118 Paracoccus denitrificans 15 below lists several useful kinase enzymes in the EC 2 . 7 . 6 ged AAH02579 . 1 12803505 Homo sapiens enzyme class . syn_ 00480 ABC77899 85722956 Syntrophus aciditrophicus syn _ 01146 ABC76260 85721317 Syntrophus aciditrophicus Enzyme 20 Commission Number Enzyme Name 3 - Aminobutyryl- CoA Deaminase (FIG . 2 , Step N ) 2 . 7 . 6 . 1 ribose -phosphate diphosphokinase 3 - aminobutyryl- CoA is an intermediate in lysine fermen 2 . 7 .6 . 2 tation . It also can be formed from acetoacetyl- CoA via a 2. 7 .6 .3 2 -amino -4 - hydroxy -6 -hydroxymethyldihydropteridine transaminase or an aminating dehydrogenase . 3 -aminobu - 25 diphosphokinase tyryl- CoA deaminase (or 3 -aminobutyryl - CoA ammonia 272 . 7 . 6 .5 4 GTPnucleotide diphosphokinase diphosphokinase lyase ) catalyzes the deamination of 3 - aminobutyryl- CoA to - form crotonyl- CoA . This reversible enzyme is present in Fusobacterium nucleatum , Porphyromonas gingivalis , Of particular interest are ribose - phosphate diphosphoki Thermoanaerobacter tengcongensis , and several other 30 nase enzymes which have been identified in Escherichia coli organismsand is co - localized with several genes involved in (Hove - Jenson et al. , J Biol Chem , 1986 , 261 ( 15 ) ; 6765 -71 ) lysine fermentation (Kreimeyer et al. , J Biol Chem , 2007, and Mycoplasma pneumoniae M129 (McElwain et al, Inter 282 ( 10 ) 7191 -7197 ) . national Journal of Systematic Bacteriology , 1988, 38 :417 423 ) as well as thiamine diphosphokinase enzymes. Exem 35 plary thiamine diphosphokinase enzymes are found in Protein GenBank ID GI Number Organism Arabidopsis thaliana (Ajjawi , Plant Mol Biol, 2007, 65 ( 1 kal NP _ 602669 . 1 19705174 Fusobacterium nucleatum subsp . 2 ); 151 -62 ). nucleatum ATCC 25586 kal NP _ 905282 . 1 34540803 Porphyromonas gingivalis W83 kal NP _ 622376 . 1 20807205 Thermoanaerobacter 40 Protein GenBank ID GI Number Organism tengcongensis MB4 prs NP 415725 . 1 16129170 Escherichia coli prsA NP _ 109761 . 1 13507812 Mycoplasma pneumoniae 4 -Hydroxybutyryl - CoA Dehydratase (FIG . 2 , Step O ) M129 Several enzymes naturally catalyze the dehydration of TPK1 BAH19964 . 1 222424006 Arabidopsis thaliana col 4 -hydroxybutyryl - CoA to crotonoyl -CoA . This transforma- 45 TPK2 BAH57065 . 1 227204427 Arabidopsis thaliana col tion is required for 4 - aminobutyrate fermentation by Clostridium aminobutyricum (Scherf et al. , Eur. J Biochem . Erythrose - 4 - Phosphate Reductase ( FIG . 3 , Step A ) 215 : 421 -429 ( 1993 ) ) and succinate - ethanol fermentation by In Step A of the pathway , erythrose - 4 -phosphate is con Clostridium kluyveri (Scherf et al . , Arch . Microbiol 161: verted to erythritol- 4 -phosphate by the erythrose - 4 -phos 239 - 245 ( 1994 ) ) . The transformation is also a key step in 50 phate reductase or erythritol- 4 -phosphate dehydrogenase . Archaea , for example , Metallosphaera sedula , as part of the The reduction of erythrose - 4 - phosphate was observed in 3 - hydroxypropionate / 4 -hydroxybutyrate autotrophic carbon Leuconostoc oenos during the production of erythritol dioxide assimilation pathway (Berg et al. , supra , (2007 ) ) . (Veiga - da - Cunha et al ., J Bacteriol. 175 : 3941- 3948 ( 1993 )) . The reversibility of 4 - hydroxybutyryl -CoA dehydratase is NADPH was identified as the cofactor (Veiga - da -Cunha et well - documented (Muh et al. , Biochemistry. 35 : 11710 - 55 al. , supra , ( 1993 )) . However, gene for erythrose - 4 - phosphate 11718 ( 1996 ); Friedrich et al ., Angew . Chem . Int. Ed . Engl. was not identified . Thus , it is possible to identify the 47 :3254 -3257 (2008 ); Muh et al ., Eur. J. Biochem . 248 :380 erythrose - 4 -phosphate reductase gene from Leuconostoc 384 ( 1997 ) ) and the equilibrium constant has been reported oenos and apply to this step . Additionally , enzymes catalyz to be about 4 on the side of crotonoyl- CoA (Scherf and ing similar reactions can be utilized for this step . An Buckel, supra, ( 1993 )) . 60 example of these enzymes is 1 -deoxy - D - xylulose - 5 - phos phate reductoisomerase (EC 1 . 1 . 1 . 267) catalyzing the con version of 1 -deoxy - D - xylulose 5 - phosphate to 2 - C -methyl Protein GenBank ID GI Number Organism D -erythritol - 4 -phosphate , which has one additional methyl AbfD CAB60035 70910046 Clostridium group comparing to Step A . The dxr or isp? genes encode aminobutyricum 65 the 1 -deoxy - D -xylulose - 5 -phosphate reductoisomerase have AbfD YP _ 001396399153955634 Clostridium kluyveri been well studied : the Dxr proteins from Escherichia coli and Mycobacterium tuberculosis were purified and their US 10 ,006 , 055 B2 93 94 crystal structures were determined ( Yajima et al. , Acta kinase . The exact enzyme for this step has not been identi Crystallogr. Sect. F . Struct. Biol. Cryst. Commun . 63 : 466 - fied . However, enzymes catalyzing similar reactions can be 470 ( 2007) ; Mac et al. , J Mol. Biol. 345: 115 - 127 (2005 ); applied to this step . An example is the 4 -diphosphocytidyl Henriksson et al ., Acta Crystallogr. D . Biol. Crystallogr. 2 -C -methylerythritol kinase ( EC 2 .7 .1 .148 ). The 4 -diphos 62: 807 -813 ( 2006 ) ; Henriksson et al ., J Biol. Chem . 282 : 5 phocytidyl- 2 - C -methylerythritol kinase is also in the methy 19905 - 19916 ( 2007 ) ); the Dxr protein from Synechocystis sp lerythritol phosphate pathway for the isoprenoid was studied by site - directed mutagenesis with modified biosynthesis and catalyzes the conversion between 4 - cyti activity and altered kinetics (Fernandes et al. , Biochim . dine 5 '- diphospho )- 2 - C -methyl - D - erythritol and 2 -phospho Biophys. Acta 1764 :223 -229 ( 2006 ); Fernandes et al. , Arch. 4 -( cytidine 5' -diphospho )- 2 - C -methyl - D - erythritol, with an Biochem . Biophys. 444 : 159 - 164 (2005 ) ). Furthermore , glyc - 10 extra methyl group comparing to Step C conversion . The eraldehyde 3 - phosphate reductase YghZ from Escherichia 4 - diphosphocytidyl- 2 - C -methylerythritol kinase is encoded coli catalyzes the conversion between glyceraldehyde by ispE gene and the crystal structures of Escherichia coli , 3 -phosphate and glycerol- 3 - phosphate (Desai et al. , Bio Thermus thermophilus HB8 , and Aquifex aeolicus IspE were chemistry 47 :7983 - 7985 (2008 )) can also be applied to this determined (Sgraja et al. , FEBS J 275 :2779 - 2794 (2008 ) ; step . The following genes can be used for Step A conversion : 13 Miallau et al. , Proc. Natl . Acad. Sci . U . S . A 100 : 9173 -9178 (2003 ); Wada et al ., J Biol. Chem . 278 : 30022 - 30027 ( 2003 ) ). The ispE genes from above organism were cloned Protein GenBank ID GI Number Organism and expressed , and the recombinant proteins were purified for crystallization . The following genes can be used for Step dxr P45568 . 2 2506592 Escherichia coli strain K12 20 dxr A5U6M4. 1 166218269 Mycobacterium tuberculosis 20 C conversion : dxr 055663 . 1 2496789 Synechocystis sp . strain PCC6803 yghz NP _ 417474 .1 16130899 Escherichia coli strain K12 Protein GenBank ID GI Number Organism 25 ispESpE P62615 . 1 50402174 Escherichia coli strain K12 Erythritol- 4 -Phospate Cytidylyltransferase (FIG . 3 , Step B ) ispE P83700 . 1 51316201 Thermus thermophilus HB8 In Step B of the pathway , erythritol - 4 - phosphate is con ispE 067060 . 1 6919911 Aquifex aeolicus verted to 4 - ( cytidine 5 ' - diphospho ) - erythritol by the eryth ritol- 4 -phosphate cytidylyltransferase or 4 -( cytidine Erythritol 2 ,4 -Cyclodiphosphate Synthase (FIG . 3 , Step D ) 5 '- diphospho ) - erythritol synthase . The exact enzyme for this 30 In Step D of the pathway, 2 - phospho - 4 - ( cytidine step has not been identified . However, enzymes catalyzing 5 '- diphospho ) -erythritol is converted to erythritol- 2 , 4 - cyclo similar reactions can be applied to this step . An example is diphosphate by the Erythritol 2 . 4 - cyclodiphosphate syn the 2 - C - methyl- D - erythritol 4 -phosphate cytidylyltrans thase . The exact enzyme for this step has not been identified . ferase or 4 - cytidine 5 '- diphospho ) - 2 - C -methyl - D - erythritol However, enzymes catalyzing similar reactions can be synthase ( EC 2 . 7 . 7 .60 ) . The 2 - C -methyl - D -erythritol 35 applied to this step . An example is the 2 - C -methyl - D 4 -phosphate cytidylyltransferase is in the methylerythritol erythritol 2 ., 4 -cyclodinho cyclodiphosphate synthase (EC 4 . 6 . 1 . 12 ) . The phosphate pathway for the isoprenoid biosynthesis and 2 - C -methyl - D -erythritol 2 , 4 - cyclodiphosphate synthase is catalyzes the conversion between 2 - C -methyl - D - erythritol also in the methylerythritol phosphate pathway for the 4 -phosphate and 4 - ( cytidine 5 ' - diphospho ) - 2 - C -methyl - D isoprenoid biosynthesis and catalyzes the conversion erythritol, with an extra methyl group comparing to Step B 40 between 2 - phospho - 4 - (cytidine 5 'diphospho ) - 2 - C -methyl conversion . The 2 - C -methyl - D - erythritol 4 -phosphate cyti - D -erythritol and 2 - C -methyl - D - erythritol- 2 , 4 -cyclodiphos dylyltransferase is encoded by ispD gene and the crystal phate , with an extra methyl group comparing to step D structure of Escherichia coli IspD was determined (Kemp et conversion . The 2 - C -methyl - D - erythritol 2 , 4 - cyclodiphos al. , Acta Crystallogr. D . Biol. Crystallogr. 57 : 1189 - 1191 phate synthase is encoded by ispF gene and the crystal ( 2001 ) ; Kemp et al. , Acta Crystallogr. D . Biol. Crystallogr. 15 structures of Escherichia coli, Thermus thermophilus, Hae 59: 607 -610 ( 2003) ; Richard et al. , Nat . Struct. Biol. 8 :641 - mophilus influenzae , and Campylobacter ieiuni IspF were 648 ( 2001 )) . The ispD gene from Mycobacterium tubercu - determined (Richard et al. , J Biol. Chem . 277 : 8667 - 8672 losis H37Rv was cloned and expressed in Escherichia coli , ( 2002) ; Steinbacher et al. , J Mol. Biol. 316 :79 - 88 ( 2002 ) ; and the recombinant proteins were purified with N -terminal Lehmann et al. , Proteins 49 : 135 - 138 ( 2002 ) ; Kishida et al. , His- tag ( Shi et al. , Biochem . Mol. Biol. 40 : 911 - 920 ( 2007 ) ) . 5 . Acta Crystallogr. D . Biol. Crystallogr. 59 : 23 - 31 (2003 ) ; Additionally , the Streptomyces coelicolor ispd gene was Gabrielsen et al. , J Biol. Chem . 279 : 52753 - 52761 ( 2004) ) . cloned and expressed in E . coli, and the recombinant pro The ispF genes from above organism were cloned and teins were characterized physically and kinetically (Cane et expressed , and the recombinant proteins were purified for al. , Bioorg . Med . Chem . 9: 1467 - 1477 (2001 ) ) . The follow crystallization . The following genes can be used for Step D ing genes can be used for Step B conversion : 55 conversion :

Protein GenBank ID GI Number Organism Protein GenBank ID GI Number Organism ispD Q46893 .3 2833415 Escherichia coli strain K12 ispF P62617 . 1 51317402 Escherichia coli strain K12 ispD A5U8Q7 . 1 166215456 Mycobacterium tuberculosis 60 ispF Q8RQP5. 1 51701599 Thermus thermophilus HB8 ispispD QOLOQ8 . 1 12230289 Streptomyces coelicolor ispF P44815 . 1 1176081 Haemophilus influenzae ispF Q9PM68 . 1 12230305 Campylobacter jejuni 4 - (Cytidine 5 '- Diphospho ) - Erythritol Kinase (FIG . 3 , Step C ) 1 -Hydroxy - 2 -Butenyl 4 - Diphosphate Synthase (FIG . 3 , Step In Step C of the pathway , 4 - ( cytidine 5 - diphospho ) - 65 E ) erythritol is converted to 2 -phospho - 4 -( cytidine 5' - diphos - Step E of FIG . 3 entails conversion of erythritol- 2 ,4 pho) na - erythritol by the 4 - cytidine 5 ' -diphospho ) - erythritol cyclodiphosphateun pret to 1 -hydroxy auto -en 2 -butenyl 4el -diphosphate mundo by US 10 , 006 , 055 B2 95 96 1 - hydroxy - 2 - butenyl 4 -diphosphate synthase . An enzyme isc region involved in assembly of iron - sulfur clusters with this activity has not been characterized to date . This (Grawert et al. , J Am . Chem . Soc . 126 : 12847 - 12855 ( 2004 ) ) . transformation is analogous to the reduction of 2 - C -methyl The gene cluster is composed of the genes icss , icsU , icsA , D -erythritol - 2 ,4 - cyclodiphosphate to 1 -hydroxy - 2 -methyl - hscB , hscA and fdx . Overexpression of these genes was 2 - ( E ) - butenyl 4 - diphosphate by ( E ) - 4 -hydroxy - 3 -methyl - 5 shown to improve the synthetic capability of the iron - sulfur but- 2 - enyl- diphosphate synthase (EC 1 . 17 . 7 . 1 ). This assembly pipeline, required for functional expression of enzyme is an iron -sulfur protein that participates in the iron -sulfur proteins . A similar approach can be applicable in non -mevalonate pathway for isoprenoid biosynthesis found the current application . in bacteria and plants . Most bacterial enzymes including the E . coli enzyme, encoded by ispG , utilize reduced ferredoxin 10 or flavodoxin as an electron donor ( Zepeck et al. , J Org . Protein GenBank ID GI Number Organism Chem . 70 : 9168 - 9174 ( 2005 ) ) . An analogous enzyme from iscs AAT48142 . 1 48994898 Escherichia coli the thermophilic cyanobacterium Thermosynechococcus iscU AAC75582 . 1 1788878 Escherichia coli elongatus BP - 1 , encoded by gcpE , was heterologously iscA AAC75581 . 1 1788877 Escherichia coli expressed and characterized in E . coli (Okada et al. , J Biol. 15 hscB AAC75580 . 1 1788876 Escherichia coli Chem . 280 :20672 - 20679 (2005 ) ). Additional enzyme can hscA AAC75579 . 1 1788875 Escherichia coli didates from Thermus thermophilus and Arabidopsis thali fdx AAC75578 . 1 1788874 Escherichia coli ana have been characterized and expressed in E . coli ( See mann et al. , J Biol. Inorg . Chem . 10 : 131 - 137 ( 2005 ) : Kollas Butenyl 4 -Diphosphate Isomerase ( FIG . 3 , Step G ) et al . , FEBS Lett. 532 : 432 -436 (2002 ) ) . Butenyl 4 -diphosphate isomerase catalyzes the reversible interconversion of 2 - butenyl- 4 - diphosphate and butenyl - 4 diphosphate . The following enzymes can naturally possess Protein GenBank ID GI Number Organism this activity or can be engineered to exhibit this activity . ispG NP _ 417010 . 1 16130440 Escherichia coli Useful genes include those that encode enzymes that inter gcpE NP _ 681786 . 1 22298539 Thermosynechococcus elongatus 25 convert isopenenyl diphosphate and dimethylallyl diphos gepE AA021364 . 1 27802077 Thermus thermophilus phate . These include isopentenyl diphosphate isomerase gepE AA015446 . 1 27462472 Arabidopsis thaliana enzymes from Escherichia coli (Rodriguez - Concepción et al. , FEBS Lett , 473 ( 3 ) :328 - 332 ) , Saccharomyces cerevisiae 1 -Hydroxy - 2 - Butenyl 4 - Diphosphate Reductase (FIG . 3 , 30 (Anderson et al. , J Biol Chem , 1989 , 264 (32 ); 19169- 75 ), Step F ) and Sulfolobus shibatae ( Yamashita et al , Eur J Biochem , The concurrent dehydration and reduction of 1 - hydroxy - 2004 , 271( 6 ) : 1087 - 93 ). The reaction mechanism of isomer 2 -butenyl 4 - diphosphate is catalyzed by an enzyme with ization , catalyzed by the Idi protein of E . coli , has been 1 - hydroxy - 2 -butenyl 4 - diphosphate reductase activity ( FIG . characterized in mechanistic detail ( de Ruyck et al ., J Biol . 3 , Step F ). Such an enzyme will form a mixture of products , 35 Chem . 281 : 17864 - 17869 (2006 ) ). Isopentenyl diphosphate butenyl 4 -diphosphate or 2 - butenyl 4 - diphosphate . An isomerase enzymes from Saccharomyces cerevisiae, Bacil analogous reaction is catalyzed by 4 -hydroxy - 3 -methylbut lus subtilis and Haematococcus pluvialis have been heter 2 - enyl diphosphate reductase ( EC 1 . 17 . 1 . 2 ) in the non ologously expressed in E . coli (Laupitz et al ., Eur. J Bio mevalonate pathway for isoprenoid biosynthesis . This chem . 271 : 2658 - 2669 ( 2004 ) ; Kajiwara et al. , Biochem . J enzyme is an ironron --sulfur sultur protein that utilizesutilizes reducedreduced ferreterre - 40 324 ( Pt 2 ) : 421 -426 ( 1997 ) ) . doxin or flavodoxin as an electron donor. Maximal activity of 4 - hydroxy - 3 -methylbut - 2 - enyl diphosphate reductase E . coli , encoded by ispH , requires both flavodoxin and flavo Protein GenBank ID GI Number Organism doxin reductase (Wolff et al. , FEBS Lett. 541 : 115 - 120 Idi NP _ 417365 . 1 16130791 Escherichia coli ( 2003 ) ; Grawert et al. , J Am . Chem . Soc. 126 : 12847 - 12855 45 IDI1 NP _ 015208 . 1 6325140 Saccharomyces cerevisiae (2004 )) . In the characterized catalytic system , reduced fla Idi BAC82424 . 1 34327946 Sulfolobus shibatae vodoxin is regenerated by the NAD ( P ) + -dependent flavo Idi AAC32209 . 1 3421423 Haematococcus pluvialis doxin reductase . The enzyme from Aquifex aeolicus, Idi BAB32625 . 1 12862826 Bacillus subtilis encoded by lytB , was expressed as a His -tagged enzyme in E . coli and characterized (Altincicek et al. , FEBS Lett. 50 Butadiene Synthase (FIG . 3 , Step H ) 532: 437 -440 ( 2002 ) ) . An analogous enzyme in plants is Butadiene synthase catalyzes the conversion of 2 -butenyl encoded by hdr of Arabidopsis thaliana (Botella -Pavia et al. , 4 -diphosphate to 1 , 3 - butadiene. The enzymes described Plant J 40 : 188 - 199 ( 2004 ) ) . below naturally possess such activity or can be engineered to exhibit this activity . Isoprene synthase naturally catalyzes 55 the conversion of dimethylallyl diphosphate to isoprene , but Protein GenBank ID GI Number Organism can also catalyze the synthesis of 1 , 3 - butadiene from 2 - bute ispH AAL38655 . 1 18652795 Escherichia coli nyl- 4 -diphosphate . Isoprene synthases can be found in sev lytB 067625 . 1 8928180 Aquifex aeolicus eral organisms including Populus alba (Sasaki et al. , FEBS hdr NP _ 567965 . 1 18418433 Arabidopsis thaliana Letters , 579 ( 11 ) , 2514 - 2518 ( 2005 ) ) , Pueraria montana 60 ( Lindberg et al ., Metabolic Eng, 12 ( 1 ): 70 - 79 (2010 ) ; Shar Altering the expression level of genes involved in iron - key et al. , Plant Physiol. , 137 ( 2 ) :700 -712 ( 2005 )) , and sulfur cluster formation can have an advantageous effect on Populus tremulaxPopulus alba (Miller et al ., Planta , 213 the activities of iron - sulfur proteins in the proposed path ( 3 ) :483 - 487 (2001 ) ). Additional isoprene synthase enzymes ways ( for example , enzymes required in Steps E and F of are described in (Chotani et al ., WO /2010 /031079 , Systems FIG . 3 ) . In E . coli, it was demonstrated that overexpression 65 Using Cell Culture for Production of Isoprene ; Cervin et al. , of the iron - sulfur containing protein IspH ( analogous to Step US Patent Application 20100003716 , Isoprene Synthase F of FIG . 3 ) is enhanced by coexpression of genes from the Variants for Improved Microbial Production of Isoprene ). US 10 ,006 ,055 B2 97 98 -continued Protein GenBank ID GI Number Organism Organism ispS BAD98243 . 1 63108310 Populus alba Protein GenBank ID GI Number ispS AAQ84170 . 1 35187004 Pueraria montana er2 BAD90688. 1 60458783 Trichosporonoides megachiliensis ispS CAC35696 . 1 13539551 Populus tremula x Populus alba 5 er3 BAD90689. 1 60458785 Trichosporonoides megachiliensis Erythrose - 4 -Phosphate Kinase (FIG . 3 , Step I) Erythritol Kinase (FIG . 3 , Step K ) In Step I of the pathway , erythrose - 4 - phosphate is con verted to erythrose by the erythrose -4 -phosphate kinase . In In Step K of the pathway, erythritol is converted to industrial fermentative production of erythritol by yeastsIn , 10 erythritol- 4 - phosphate by the erythritol kinase . Erythritol glucose was converted to erythrose - 4 -phosphate through the kinase (EC 2 . 7 . 1 . 27) catalyzes the phosphorylation of eryth pentose phosphate pathway and erythrose -4 -phosphate was ritol. Erythritol kinase was characterized in erythritol utiliz dephosphorylated and reduced to produce erythritol (Moon ing bacteria such as Brucella abortus (Sperry et al. , J et al. , Appl. Microbiol Biotechnol. 86 : 1017 - 1025 ( 2010 ) ) . Bacteriol. 121: 619 -630 ( 1975 ) ) . The eryA gene of Brucella Thus, erythrose - 4 -phosphate kinase is present in many of 15 abortus has been functionally expressed in Escherichia coli these erythritol -producing yeasts, including Trichos - and the resultant EryA was shown to catalyze the ATP poronoides megachiliensis SN -G42 (Sawada et al ., J Biosci . dependent conversion of erythritol to erythritol -4 -phosphate Bioeng . 108 : 385 - 390 ( 2009 ) ) , Candida magnolia (Kohl et (Lillo et al . , Bioorg . Med . Chem . Lett . 13 :737 - 739 ( 2003 )) . al. , Biotechnol. Lett. 25 : 2103 -2105 ( 2003 ) ) , and Torula sp . 2 . The following genes can be used for Step K conversion : (HAJNY et al. , Appl. Microbiol 12 :240 - 246 ( 1964) ; Oh et al. , J . Ind . Microbiol Biotechnol. 26 : 248 - 252 ( 2001 ) ) . How ever , the erythrose - 4 -phosphate kinase genes were not iden Protein GenBank ID GI Number Organism tified yet. There are many polyol with ery Q8YCUS 81850596 Brucella melitensis wide substrate range that can be applied to this step . An , eriA 092NHO 81774560 Sinorhizobium meliloti example is the triose kinase (EC 2 . 7 . 1 .28 ) catalyzing the eryA YP _ 001108625 . 1 134102964 Saccharopolyspora erythraea reversible conversion between glyceraldehydes and glycer NRRL 2338 aldehydes - 3 - phosphate , which are one carbon shorter com paring to Step I . Other examples include the xylulokinase Malonyl - CoA :Acetyl - COA Acyltransferase ( FIG . 4 , Step A ) ( EC 2 .7 . 1. 17 ) or arabinokinase ( EC 2 . 7 . 1 .54 ) that catalyzeszes 30. In Step A of the pathway described in FIG . 4 , malonyl phosphorylation of 5C polyol aldehyde. The following COA and acetyl - CoA are condensed to form 3 -oxoglutaryl genes can be used for Step I conversion : COA by malonyl - CoA :acetyl - CoA acyl transferase , a beta keothiolase . Although no enzyme with activity on malonyl Protein GenBank ID GI Number Organism COA has been reported to date , a good candidate for this 35 transformation is beta - ketoadipyl- CoA thiolase ( EC xylB P09099 . 1 139849 Escherichia coli strain K12 xks 1 P42826 . 2 1723736 Saccharomyces cerevisiae 2 . 3 . 1 . 174 ) , also called 3 -oxoadipyl - CoA thiolase that con xylB P29444 . 1 267426 Klebsiella pneumoniae verts beta -ketoadipyl -CoA to succinyl- CoA and acetyl- CoA , daki Q9HFC5 74624685 Zygosaccharomyces rouxii and is a key enzyme of the beta -ketoadipate pathway for aromatic compound degradation . The enzyme is widespread Erythrose Reductase ( FIG . 3 , Step J) in soil bacteria and fungi including Pseudomonas putida In Step J of the pathway, erythrose is converted to (Harwood et al. , J Bacteriol. 176 :6479 -6488 (1994 )) and erythritol by the erythrose reductase . In industrial fermen - Acinetobacter calcoaceticus (Doten et al ., J Bacteriol. 169 : tative production of erythritol by yeasts , glucose was con 3168 -3174 ( 1987 ) ) . The gene products encoded by pcaF in verted to erythrose - 4 -phosphate through the pentose phos - 45 Pseudomonas strain B13 (Kaschabek et al. , J Bacteriol. phate pathway and erythrose - 4 -phosphate was 184 :207 - 215 (2002 ) ) , phaD in Pseudomonas putida U (Oli dephosphorylated and reduced to produce erythritol (Moon vera et al. , supra , ( 1998 ) ) , paaE in Pseudomonas fluorescens et al. , supra , ( 2010 ) ) . Thus , erythrose reductase is present in ST (Di Gennaro et al . , Arch Microbiol. 88 : 117 - 125 ( 2007 ) ) , many of these erythritol - producing yeasts , including Tricho and paal from E . coli (Nogales et al. , Microbiology, 153 : sporonoides megachiliensis SN -G42 (Sawada et al. , supra , 50 357 - 365 ( 2007 ) ) also catalyze this transformation . Several ( 2009 ) ) , Candida magnolia (Kohl et al. , supra, (2003 ) ) , and beta- ketothiolases exhibit significant and selective activities Torula sp . ( HAJNY et al. , supra , ( 1964 ) ; Oh et al. , supra , in the oxoadipyl- CoA forming direction including bkt from ( 2001) ) . Erythrose reductase was characterized and purified from Torula corallina (Lee et al ., Biotechnol. Prog . 19 : 495 Pseudomonas putida, pcaF and bkt from Pseudomonas 500 ( 2003 ) ; Lee et al. , Appl. Environ . Microbiol 68 : 4534 - 55 aeruginosa PAO1, bkt from Burkholderia ambifaria 4538 (2002 ) ) , Candida magnolia ( Lee et al. , Appl. Environ .- 55 AMMD , paaJ from E . coli , and phaD from P . putida . These Microbiol 69 : 3710 - 3718 (2003 )) and Trichosporonoides enzymes can also be employed for the synthesis of 3 -oxo megachiliensis SN -G42 (Sawada et al ., supra, (2009 )) . Sev glutaryl- CoA , a compound structurally similar to 3 -oxoad eral erythrose reductase genes were cloned and can be ipyl- CoA . applied to Step J . The following genes can be used for Step 60 J conversion : Protein GenBank ID GI Number Organism paaJ NP _ 415915 . 1 16129358 Escherichia coli Protein GenBank ID GI Number Organism pcar AALO2407 17717736947 Pseudomonas knackmussii (B13 ) phaD AAC24332 . 1 3253200 Pseudomonas putida alr ACT78580 . 1 254679867 Candida magnoliae pcaF AAA85138 . 1 506695 Pseudomonas putida er1 BAD90687. 1 60458781 Trichosporonoides megachiliensis pcaF AAC37148 . 1 141777 Acinetobacter calcoaceticus US 10 ,006 ,055 B2 99 100 - continued acetoacetyl- CoA ( EC 2 . 1 . 3 . 9 ). Exemplary acetoacetyl- COA thiolase enzymes include the gene products of atoB from E . Protein GenBank ID GI Number Organism coli (Martin et al. , supra, (2003 )) , th1A and th1B from C . paaE ABF82237 . 1 106636097 Pseudomonas fluorescens acetobutylicum (Hanai et al. , supra , (2007 ) ; Winzer et al. , bkt YP _ 777652 . 1 115360515 Burkholderia ambifaria AMMD 5 supra , (2000 ) ) , and ERG10 from S . cerevisiae (Hiser et al. , bkt AAG06977. 1 9949744 Pseudomonas aeruginosa PAO1 pcaF AAG03617 . 1 9946065 Pseudomonas aeruginosa PAO1 supra , ( 1994 )) . Another relevant beta -ketothiolase is oxopimeloyl- CoA : Protein GenBank ID GI Number Organism glutaryl- CoA acyltransferase (EC 2 . 3 . 1 . 16 ) that combines toB NP _ 416728 16130161 Escherichia coli glutaryl- CoA and acetyl- CoA to form 3 -oxopimeloyl - CoA . thlA NP _ 349476 . 1 15896127 Clostridium acetobutylicum An enzyme catalyzing this transformation is found in Ral thlB NP _ 149242 . 1 15004782 Clostridium acetobutylicum stonia eutropha ( formerly known as Alcaligenes eutrophus) , ERG10 NP _ 015297 6325229 Saccharomyces cerevisiae encoded by genes bktB and bktC (Slater et al. , J . Bacteriol . 180 : 1979 - 1987 ( 1998 ) ; Haywood et al ., FEMS Microbiol- 3 -Oxoglutaryl - CoA Reductase (Ketone -Reducing ) (FIG . 4 , ogy Letters 52 : 91 - 96 (1988 ) . The sequence of the BktB Step B ) protein is known; however, the sequence of the Bkt? protein This enzyme catalyzes the reduction of the 3 - oxo group in has not been reported . The pim operon of Rhodopseudomo - 3 -oxoglutaryl - CoA to the 3 -hydroxy group in Step B of the nas palustris also encodes a beta -ketothiolase , encoded by 20 pathway shown in FIG . 4 . pimB , predicted to catalyze this transformation in the deg 3 - Oxoacyl- CoA dehydrogenase enzymes convert 3 - oxoa radative direction during benzoyl- CoA degradation (Harri - cyl- CoA molecules into 3 -hydroxyacyl - CoA molecules and son et al ., Microbiology 151: 727 -736 (2005 )) . A beta -keto - are often involved in fatty acid beta - oxidation or phenylac thiolase enzyme candidate in S . aciditrophicus was etate catabolism . For example , subunits of two fatty acid identified by sequence homology to bktB ( 43 % identity , 25 oxidation complexes in E . coli , encoded by fadB and fad ) , evalue = le - 93 ) . function as 3 - hydroxyacyl- CoA dehydrogenases (Binstock et al. , Methods Enzymol. 71 Pt C : 403 -411 ( 1981 ) ) . Further more , the gene products encoded by phaC in Pseudomonas Protein GenBank ID GI Number Organism putida U (Olivera et al. , supra , ( 1998 ) ) and paaC in bktB YP _ 725948 11386745 Ralstonia eutropha Pseudomonas fluorescens ST (Di et al. , supra , ( 2007 ) ) pimB CAE29156 39650633 Rhodopseudomonas catalyze the reversible oxidation of 3 -hydroxyadipyl - CoA to palustris form 3 -oxoadipyl - CoA , during the catabolism of phenylac syn _ 02642 YP _ 462685 . 1 85860483 Syntrophus aciditrophicus etate or styrene. In addition , given the proximity in E . coli of paaH to other genes in the phenylacetate degradation Beta -ketothiolase enzymes catalyzing the formation of 35 operon (Nogales et al. , supra , (2007 ) ) and the fact that paaH beta- ketovaleryl - CoA from acetyl- CoA and propionyl- CoA mutants cannot grow on phenylacetate ( Ismail et al ., supra , can also be able to catalyze the formation of 3 -oxoglutaryl ( 2003 ) ) , it is expected that the E . coli paaH gene encodes a COA . Zoogloea ramigera possesses two ketothiolases that 3 -hydroxyacyl - CoA dehydrogenase . can form ß -ketovaleryl - CoA from propionyl- CoA and acetyl- CoA and R . eutropha has a ß - oxidation ketothiolase 4 that is also capable of catalyzing this transformation (Slater Protein GenBank ID GI Number Organism et al . , J . Bacteriol, 180 : 1979 - 1987 ( 1998 ) ) . The sequences of fadB P21177. 2 119811 Escherichia coli these genes or their translated proteins have not been fadJ P77399 . 1 3334437 Escherichia coli ???? NP 415913 . 1 16129356 Escherichia coli reported , but several candidates in R . eutropha , Z . ramigera , 45 phaC NP 745425 . 1 26990000 Pseudomonas putida or other organisms can be identified based on sequence paac homology to bkt? from R . eutropha . These include : ABF82235 . 1 106636095 Pseudomonas fluorescens 3 -Hydroxybutyryl -CoA dehydrogenase, also called acetoacetyl- CoA reductase , catalyzes the reversible NAD ( P ) Protein GenBank ID GI Number Organism 50 H -dependent conversion of acetoacetyl- CoA to 3 -hydroxy phaA YP _ 725941 . 1 113867452 Ralstonia eutropha butyryl- CoA . This enzyme participates in the acetyl- COA h16 _ A1713 YP _ 726205 . 1 113867716 Ralstonia eutropha pcaF YP _ 728366 . 1 116694155 Ralstonia eutropha fermentation pathway to butyrate in several species of h16 _ B1369 YP _ 840888 . 1 116695312 Ralstonia eutropha Clostridia and has been studied in detail ( Jones and Woods, h16 _ A0170 YP _ 724690 . 1 113866201 Ralstonia eutropha supra , ( 1986 ) ) . Enzyme candidates include hbd from C . h16 _ A0462 YP _ 724980 . 1 113866491 Ralstonia eutropha acetobutylicum (Boynton et al. , J . Bacteriol. 178 : 3015 - 3024 h16 _ A1528 YP _ 726028 . 1 113867539 Ralstonia eutropha h16 _ B0381 YP _ 728545 . 1 116694334 Ralstonia eutropha ( 1996 ) ), hbd from C . beijerinckii (Colby et al. , Appl Envi h16 _ B0662 YP _ 728824 . 1 116694613 Ralstonia eutropha ron . Microbiol 58 :3297 -3302 ( 1992 ) ), and a number of h16 _ B0759 YP _ 728921. 1 116694710 Ralstonia similar enzymes from Metallosphaera sedula (Berg et al. , h16 _ B0668 YP _ 728830 . 1 11 6694619 Ralstonia supra , (2007 ) ) . The enzyme from Clostridium acetobutyli h16 _ A1720 YP _ 726212. 1 113867723 Ralstonia e h16 _ A1887 YP _ 726356 . 1 113867867 Ralstonia eutropha 60 ?um , encoded by hbd , has been cloned and functionally phbA P07097 . 4 135759 Zoogloea ramigera expressed in E . coli (Youngleson et al. , supra , (1989 ) ). Yet bktB YP _ 002005382. 1 194289475 Cupriavidus taiwanensis other genes demonstrated to reduce acetoacetyl -CoA to Rmet _ 1362 YP _ 5835141 . 94310304 Ralstonia metallidurans 3 -hydroxybutyryl - CoA are phbB from Zoogloea ramigera Bphy _ 0975 YP _ 001857210 . 1 186475740 Burkholderia phymatum (Ploux et al. , supra , ( 1988 ) ) and phaB from Rhodobacter 65 sphaeroides ( Alber et al. , supra , (2006 )) . The former gene is Additional candidates include beta -ketothiolases that are NADPH -dependent , its nucleotide sequence has been deter known to convert two molecules of acetyl- CoA into mined (Peoples and Sinskey, supra , ( 1989 ) ) and the gene has US 10 ,006 , 055 B2 101 102 been expressed in E . coli. Additional genes include hbd1 Thauer, supra , ( 2007 ) ) . The enzyme utilizes NADPH as a ( C - terminal domain ) and hbd2 ( N - terminal domain ) in cofactor and has been characterized in Metallosphaera and Clostridium kluyveri (Hillmer and Gottschalk , Biochim . Sulfolobus spp ( Alber et al. , supra, ( 2006 ); Hugler et al. , Biophys. Acta 3334 : 12 - 23 ( 1974 ) ) and HSD17B10 in Bos supra, ( 2002) ) . The enzyme is encoded by Msed _ 0709 in taurus (WAKIL et al . , supra , ( 1954 ) ) . 5 Metallosphaera sedula (Alber et al ., supra , (2006 ); Berg et al ., supra, (2007b )) . A gene encoding a malonyl- CoA reduc tase from Sulfolobus tokodaii was cloned and heterologously Protein GenBank ID GI Number Organism expressed in E . coli ( Alber et al. , supra , (2006 ) ). This hbd NP 349314 . 1 15895965 Clostridium enzyme has also been shown to catalyze the conversion of acetobutylicum 10 methylmalonyl- CoA to its corresponding aldehyde (WO / hbd AAM14586 . 1 20162442 Clostridium beijerinckii 2007 / 141208 ) . Although the aldehyde dehydrogenase func Ised _ 1423 YP _ 001191505 146304189 Metallosphaera sedula Msed _ 0399 YP _ 001190500 146303184 Metallosphaera sedula tionality of these enzymes is similar to the bifunctional Msed 0389 YP 001190490 146303174 Metallosphaera sedula dehydrogenase from Chloroflexus aurantiacus, there is little Msed _ 1993 YP _ 001192057 146304741 Metallosphaera sedula sequence similarity . Both malonyl- CoA reductase enzyme hbd2 EDK34807 . 1 146348271 Clostridium kluyveri hbd1 EDK32512 . 1 146345976 Clostridium kluyveri candidates have high sequence similarity to aspartate -semi HSD17B10 002691. 3 3183024 Bos taurus aldehyde dehydrogenase , an enzyme catalyzing the reduc phaB YP _ 353825 . 1 77464321 Rhodobacter sphaeroides tion and concurrent dephosphorylation of aspartyl- 4 -phos phbB P23238 . 1 130017 Zoogloea ramigera phate to aspartate semialdehyde . Additional gene candidates 20 can be found by sequence homology to proteins in other 3 -Hydroxyglutaryl - CoA Reductase (Aldehyde Forming ) organisms including Sulfolobus solfataricus and Sulfolobus ( FIG . 4 , Step C ) acidocaldarius. Yet another acyl -CoA reductase ( aldehyde 3 -hydroxyglutaryl - CoA reductase reduces 3 -hydroxyglu forming ) candidate is the ald gene from Clostridium beijer taryl- CoA to 3 - hydroxy - 5 - oxopentanoate . Several acyl- CoA inckii ( Toth et al. , Appl Environ . Microbiol 65 :4973 - 4980 dehydrogenases reduce an acyl- CoA to its corresponding 25 ( 1999 ) . This enzyme has been reported to reduce acetyl aldehyde ( EC 1 . 2 . 1 ). Exemplary genes that encode such enzymes include the Acinetobacter calcoaceticus acr1 COA and butyryl -CoA to their corresponding aldehydes . encoding a fatty acyl -CoA reductase (Reiser and Somerville , This gene is very similar to eutE that encodes acetaldehyde supra , ( 1997) ), the Acinetobacter sp . M - 1 fatty acyl- CoA dehydrogenase of Salmonella typhimurium and E . coli (Toth reductase ( Ishige et al. , supra , ( 2002 )) , and a COA - and 30 et al. , supra , (1999 ) ) . NADP - dependent succinate semialdehyde dehydrogenase encoded by the sucD gene in Clostridium kluyveri ( Sohling and Gottschalk , supra , ( 1996 ) ; Sohling and Gottschalk , Protein GenBank ID GI Number Organism supra , (1996 )) . SucD of P. gingivalis is another succinate MSED _ 0709 YP _ 001190808 .1 146303492 Metallosphaera sedula NP _ 378167 . 1 15922498 Sulfolobus tokodaii semialdehyde dehydrogenase ( Takahashi et al. , supra , 35 asdmer - 2 NP_ 343563 . 1 15898958 Sulfolobus ( 2000 ) ) . The enzyme acylating acetaldehyde dehydrogenase solfataricus in Pseudomonas sp , encoded by bphG , is yet another as it Saci 2370 YP _ 256941. 1 70608071 Sulfolobus has been demonstrated to oxidize and acylate acetaldehyde , acidocaldarius AAT66436 9473535 Clostridium propionaldehyde, butyraldehyde , isobutyraldehyde and Ald beijerinckii formaldehyde (Powlowski et al ., supra , ( 1993 )) . In addition 40 euth AAA80209 687645 Salmonella to reducing acetyl -CoA to ethanol, the enzyme encoded by typhimurium adhE in Leuconostoc mesenteroides has been shown to eutE P77445 2498347 Escherichia coli oxidize the branched chain compound isobutyraldehyde to isobutyryl- CoA (Koo et al. , Biotechnol Lett . 27 : 505 - 510 (2005 ) ) . Butyraldehyde dehydrogenase catalyzes a similar 45 3 -Hydroxy - 5 - Oxopentanoate Reductase (FIG . 4 , Step D ) reaction , conversion of butyryl- CoA to butyraldehyde, in This enzyme reduces the terminal aldehyde group in solventogenic organisms such as Clostridium saccharoper 3 -hydroxy - 5 - oxopentanoate to the alcohol group . Exem butylacetonicum (Kosaka et al ., Biosci. Biotechnol Biochem . plary genes encoding enzymes that catalyze the conversion 71 :58 -68 (2007 ) ) . of an aldehyde to alcohol ( i. e . , alcohol dehydrogenase or 50 equivalently aldehyde reductase , 1 . 1 . 1 . a ) include alrA encoding a medium -chain alcohol dehydrogenase for Protein GenBank ID GI Number Organism C2 - C14 ( Tani et al. , supra, ( 2000 ) ), ADH2 from Saccharo acr1 YP _ 047869 . 1 50086359 Acinetobacter calcoaceticus myces cerevisiae ( Atsumi et al. , supra , (2008 ) ), yqhD from acr1 AAC45217 1684886 Acinetobacter baylyi acr1 BAB85476 . 1 18857901 Acinetobacter sp . Strain M - 1 55 E . coli which has preference for molecules longer than C ( 3 ) sucD P38947 . 1 172046062 Clostridium kluyveri (Sulzenbacher et al. , supra , ( 2004 ) ), and bdh I and bdh II sucD NP _ 904963 . 1 34540484 Porphyromonas gingivalis from C . acetobutylicum which converts butyryaldehyde into bphG BAA03892 . 1 425213 Pseudomonas sp adhE AAV66076 . 1 55818563 Leuconostoc mesenteroides butanol (Walter et al. , supra , ( 1992 ) ) . The gene product of bld AAP42563 . 1 31075383 Clostridium yqhD catalyzes the reduction of acetaldehyde , malondialde saccharoperbutylacetonicum hyde , propionaldehyde , butyraldehyde , and acrolein using NADPH as the cofactor (Perez et al. , 283 :7346 -7353 ( 2008 ) ; An additional enzyme type that converts an acyl- CoA to Perez et al . , J Biol. Chem . 283 :7346 -7353 ( 2008 ) ) . The adhA its corresponding aldehyde is malonyl- CoA reductase which gene product from Zymomonas mobilis has been demon transforms malonyl- CoA to malonic semialdehyde . Malo strated to have activity on a number of aldehydes including nyl- CoA reductase is a key enzyme in autotrophic carbon 65 formaldehyde , acetaldehyde , propionaldehyde, butyralde fixation via the 3 -hydroxypropionate cycle in thermoaci - hyde, and acrolein (Kinoshita et al. , Appl Microbiol Bio dophilic archael bacteria (Berg et al ., supra , ( 2007b ) ; technol 22: 249 - 254 ( 1985 ) ) . US 10 ,006 ,055 B2 103 104 NADPH - dependentmalonate semialdehyde reductase cata Protein GenBank ID GI Number Organism lyzes the reverse reaction in autotrophic CO2 - fixing bacte alrA BAB12273. 1 9967138 Acinetobacter sp . Strain M - 1 ria . Although the enzyme activity has been detected in ADH2 NP 014032 . 1 6323961 Saccharomyces cerevisiae Metallosphaera sedula , the identity of the gene is not known yqhD NP 417484 . 1 16130909 Escherichia coli 5 ( Alber et al ., supra , (2006 )) . bdh I NP 349892 . 1 15896543 Clostridium acetobutylicum bdh II NP 349891 . 1 15896542 Clostridium acetobutylicum 3, 5 -Dihydroxypentanoate Kinase ( FIG . 4 , Step E ) adha YP _ 162971 . 1 56552132 Zymomonas mobilis This enzyme phosphorylates 3 ,5 -dihydroxypentanoate in FIG . 4 (Step E ) to form 3 -hydroxy - 5 -phosphonatooxypen Enzymes exhibiting 4 -hydroxybutyrate dehydrogenase 10 tanoate (3H5PP ) . This transformation can be catalyzed by activity ( EC 1 . 1 . 1 .61 ) also fall into this category . Such enzymes in the EC class 2 . 7 .1 that enable the ATP - depen enzymes have been characterized in Ralstonia eutropha dent transfer of a phosphate group to an alcohol. (Bravo et al. , supra, ( 2004 )) , Clostridium kluyveri (Wolff and A good candidate for this step is mevalonate kinase (EC Kenealy , supra , ( 1995 ) ) and Arabidopsis thaliana (Breit 2 .7 . 1 .36 ) that phosphorylates the terminal hydroxyl group of kreuz et al. , supra , ( 2003 ) ) . The A . thaliana enzyme was 15 the methyl analog , mevalonate , of 3 , 5 - dihydroxypentanote . cloned and characterized in yeast [ 12882961] . Yet another Some gene candidates for this step are erg12 from S . gene is the alcohol dehydrogenase adhl from Geobacillus cerevisiae , mvk from Methanocaldococcus jannaschi, MVK thermoglucosidasius (Jeon et al. , J Biotechnol 135: 127 -133 from Homo sapeins, and mvk from Arabidopsis thaliana ( 2008 ) . col. 20 Protein GenBank ID GI Number Organism Protein GenBank ID GINumber Organism 4hbd YP _ 726053 . 1 113867564 Ralstonia eutropha H16 erg12 CAA39359. 1 3684 Sachharomyces cerevisiae 4hbd EDK35022. 1 146348486 Clostridium kluyveri mvk Q58487. 1 2497517 Methanocaldococcus jannaschii 4hbd 094B07 75249805 Arabidopsis thaliana 25 mvk AAH16140 . 1 16359371 Homo sapiens adhi AAR91477 . 1 40795502 Geobacillus M \mvk NP _ 851084 . 1 30690651 Arabidopsis thaliana thermoglucosidasius Glycerol kinase also phosphorylates the terminal Another exemplary enzyme is 3 - hydroxyisobutyrate hydroxyl group in glycerol to form glycerol- 3 - phosphate . dehydrogenase ( EC 1 . 1 . 1 .31 ) which catalyzes the reversible 30 This reaction occurs in several species, including Escheri oxidation of 3 - hydroxyisobutyrate to methylmalonate semi- chia coli , Saccharomyces cerevisiae , and Thermotoga mar aldehyde . This enzyme participates in valine , leucine and itima . The E . coli glycerol kinase has been shown to accept isoleucine degradation and has been identified in bacteria , eukaryotes, and mammals . The enzyme encoded by P84067 alternate substrates such as dihydroxyacetone and glyceral from Thermus thermophilus HB8 has been structurally char- 35 dehyde (Hayashi and Lin , supra, (1967 )) . T, maritime has acterized (Lokanath et al. , J Mol Biol 352 : 905 - 17 (2005 ) ) . two glycerol kinases (Nelson et al ., supra , (1999 )) . Glycerol The reversibility of the human 3 - hydroxyisobutyrate dehy kinases have been shown to have a wide range of substrate drogenase was demonstrated using isotopically - labeled sub specificity . Crans and Whiteside studied glycerol kinases strate (Manning et al . , Biochem J 231 : 481 - 4 ( 1985 ) ) . Addi from four different organisms ( Escherichia coli, S . cerevi tional genes encoding this enzyme include 3hidh in Homo 40 side , Bacillus stearothermophilus, and Candida mycod sapiens (Hawes et al ., Methods Enzymol 324 : 218 - 228 erma ) (Crans and Whitesides, supra , ( 2010 ) ; Nelson et al. , ( 2000 ) ) and Oryctolagus cuniculus (Hawes et al ., supra , supra , ( 1999) ) . They studied 66 different analogs of glycerol ( 2000 ) ; Chowdhury et al. , Biosci. Biotechnol Biochem . and concluded that the enzyme could accept a range of 60 :2043 - 2047 (1996 )) , mmsb in Pseudomonas aeruginosa , substituents in place of one terminal hydroxyl group and that and dhat in Pseudomonas putida ( Aberhart et al . , J Chem . 45 the hydrogen atom at C2 could be replaced by a methyl Soc. [ Perkin 1 ] 6 : 1404 - 1406 ( 1979 ) ; Chowdhury et al . , group . Interestingly , the kinetic constants of the enzyme supra , (1996 ) ; Chowdhury et al. , Biosci. Biotechnol Bio from all four organisms were very similar. The gene candi chem . 67 : 438 -441 (2003 )) . dates are : 50 Protein GenBank ID GI Number Organism Protein GenBank ID GI Number Organism P84067 P84067 75345323 Thermus thermophilus glpK AP _ 003883. 1 89110103 Escherichia coli K12 mmsb P28811 . 1 127211 Pseudomonas aeruginosa glpK1 NP _ 228760 . 1 15642775 Thermotoga maritime MSBS dhat Q59477 . 1 2842618 Pseudomonas putida glpK2 NP 229230 . 1 15642775 Thermotoga maritime MSB8 3hidh P31937 . 2 12643395 Homo sapiens Gut1 NP _ 011831. 1 82795252 Saccharomyces cerevisiae 3hidh P32185 . 1 416872 Oryctolagus cuniculus Homoserine kinase is another possible candidate that can The conversion ofmalonic semialdehyde to 3 -HP can also lead to the phosphorylation of 3 , 5 - dihydroxypentanoate . be accomplished by two other enzymes : NADH -dependent This enzyme is also present in a number of organisms 3 -hydroxypropionate dehydrogenase and NADPH -depen - 60 including E . coli , Streptomyces sp , and S . cerevisiae . Homo dent malonate semialdehyde reductase . An NADH - depen - serine kinase from E . coli has been shown to have activity dent 3 -hydroxypropionate dehydrogenase is thought to par on numerous substrates , including , L - 2- amino , 1 ,4 -butane ticipate in beta - alanine biosynthesis pathways from diol, aspartate semialdehyde , and 2 -amino - 5 -hydroxyvaler propionate in bacteria and plants (Rathinasabapathi B . , ate (Huo and Viola , supra , ( 1996 ) ; Huo and Viola , supra , Journal of Plant Pathology 159 :671 -674 ( 2002) ; Stadtman , 65 ( 1996 ) ) . This enzyme can act on substrates where the J . Am . Chem . Soc. 77 :5765 - 5766 ( 1955 ) ) . This enzyme has carboxyl group at the alpha position has been replaced by an not been associated with a gene in any organism to date . ester or by a hydroxymethyl group. The gene candidates are : US 10 ,006 ,055 B2 105 106 Chem . 281: 17864- 17869 (2006 )) . Isopentenyl diphosphate Protein GenBank ID G I Number Organism isomerase enzymes from Saccharomyces cerevisiae , Bacil thrB BAB96580 . 2 85674277 Escherichia lus subtilis and Haematococcus pluvialis have been heter coli K12 ologously expressed in E . coli (Laupitz et al ., Eur . J Bio SACTIDRAFT _ 4809 ZP _ 06280784 .1 282871792 Streptomyces 5 chem . 271: 2658 - 2669 ( 2004 ) ; Kajiwara et al. , Biochem . J sp . ACT- 1 Thr1 AAA35154 . 1 172978 Saccharomyces 324 (Pt 2 ): 421 - 426 ( 1997 )) . serevisiae Protein GenBank ID GI Number Organism 3H5PP Kinase (FIG . 4 , Step F ) 10 Idi NP _ 417365 . 1 16130791 Escherichia coli Phosphorylation of 3H5PP to 3H5PDP is catalyzed by IDI1 NP _ 015208 . 1 6325140 Saccharomyces cerevisiae 3H5PP kinase (FIG . 4 , Step F ) . Phosphomevalonate kinase Idi BAC82424 . 1 34327946 Sulfolobus shibatae ( EC 2 . 7 . 4 . 2 ) catalyzes the analogous transformation in the Idi AAC32209 . 1 3421423 Haematococcus pluvialis mevalonate pathway . This enzyme is encoded by erg8 in Idi BAB32625 . 1 12862826 Bacillus subtilis Saccharomyces cerevisiae ( Tsay et al. , Mol . Cell Biol. 15 11: 620 -631 ( 1991) ) and mvaK2 in Streptococcus pneumo Butadiene Synthase (FIG . 4 , Step I) niae, Staphylococcus aureus and Enterococcus faecalis Butadiene synthase catalyzes the conversion of 2 -butenyl (Doun et al. , Protein Sci. 14 :1134 -1139 (2005 ); Wilding et 4 -diphosphate to 1, 3 -butadiene . The enzymes described al. , J Bacteriol. 182 :4319 -4327 ( 2000 ) ) . The Streptococcus below naturally possess such activity or can be engineered pneumoniae and Enterococcus faecalis enzymes were < 0 to exhibit this activity . Isoprene synthase naturally catalyzes cloned and characterized in E . coli (Pilloff et al. , J Biol. the conversion of dimethylallyl diphosphate to isoprene, but Chem . 278 :4510 - 4515 (2003 ) ; Doun et al . , Protein Sci. can also catalyze the synthesis of 1, 3 -butadiene from 2 - bute 14 : 1134 - 1139 (2005 ) ) . nyl - 4 - diphosphate . Isoprene synthases can be found in sev 25 eral organisms including Populus alba (Sasaki et al ., FEBS Letters, 2005 , 579 ( 11) , 2514 - 2518 ), Pueraria montana Protein GenBank ID GI Number Organism (Lindberg et al. , Metabolic Eng, 12 ( 1 ) :70 - 79 ( 2010 ) ; Shar Erg8 AAA34596 . 1 171479 Saccharomyces cerevisiae key et al. , Plant Physiol. , 137 ( 2 ) : 700 -712 ( 2005 ) ), and mvaK2 AAGO2426 . 1 9937366 Staphylococcus aureus Populus tremulaxPopulus alba (Miller et al . , Planta , 213 mvaK2 AAG02457 . 1 9937409 Streptococcus pneumoniae 30 ( 3 ) :483 - 487 ( 2001 ) ) . Additional isoprene synthase enzymes mvaK2 AAG02442 . 1 9937388 Enterococcus faecalis are described in (Chotani et al. , WO /2010 /031079 , Systems Using Cell Culture for Production of Isoprene ; Cervin et al . , 3H5PDP Decarboxylase ( FIG . 4 , Step G ) US Patent Application 20100003716 , Isoprene Synthase Butenyl 4 -diphosphate is formed from the ATP - dependent Variants for Improved Microbial Production of Isoprene ) . decarboxylation of 3H5PDP by 3H5PDP decarboxylase 35 ( FIG . 4 , Step G ) . Although an enzyme with this activity has not been characterized to date a similar reaction is catalyzed Protein GenBank ID GI Number Organism by mevalonate diphosphate decarboxylase ( EC 4 . 1 . 1 . 33 ) , an isps BAD98243. 1 63108310 Populus alba enzyme participating in the mevalonate pathway for iso - ispS AAQ84170 . 1 35187004 Pueraria montana prenoid biosynthesis . This reaction is catalyzed by MVD1 in an ispS CAC35696. 1 13539551 Populus tremula x Populus alba Saccharomyces cerevisiae , MVD in Homo sapiens and MDD in Staphylococcus aureus and Trypsonoma brucei 3 -Hydroxyglutaryl - CoA Reductase ( Alcohol Forming ) ( Toth et al. , J Biol. Chem . 271 : 7895 - 7898 ( 1996 ); Byres et (FIG . 4 . Step J) al. , J Mol. Biol. 371 :540 - 553 ( 2007 ) ) . This step catalyzes the reduction of the acyl- CoA group in 45 3 -hydroxyglutaryl - CoA to the alcohol group . Exemplary bifunctional oxidoreductases that convert an acyl- CoA to Protein GenBank ID GI Number Organism alcohol include those that transform substrates such as MVD1 P32377 . 2 1706682 Saccharomyces cerevisiae acetyl- CoA to ethanol ( e . g ., adhE from E . coli (Kessler et al. , MVD NP _ 002452. 1 4505289 Homo sapiens supra , ( 1991 ) ) and butyryl- CoA to butanol (e . g . adhE2 from MDD ABO48418 . 1 147740120 Staphylococcus aureus 50 C . acetobutylicum (Fontaine et al. , supra , ( 2002 )) . In addi MDD EAN78728 . 1 70833224 Trypsonoma brucei tion to reducing acetyl- CoA to ethanol, the enzyme encoded by adhE in Leuconostoc mesenteroides has been shown to Butenyl 4 -Diphosphate Isomerase (FIG . 4 , Step H ) oxide the branched chain compound isobutyraldehyde to Butenyl 4 -diphosphate isomerase catalyzes the reversible isobutyryl -COA (Kazahaya et al. , supra , (1972 ) ; Koo et al. , interconversion of 2 -butenyl -4 -diphosphate and butenyl- 4 - 55 supra , (2005 )) . diphosphate . The following enzymes can naturally possess Another exemplary enzyme can convert malonyl - CoA to this activity or can be engineered to exhibit this activity . 3 -HP . An NADPH -dependent enzyme with this activity has Useful genes include those that encode enzymes that inter- characterized in Chloroflexus aurantiacus where it partici convert isopenenyl diphosphate and dimethylallyl diphos - pates in the 3 -hydroxypropionate cycle (Hugler et al. , supra , phate . These include isopentenyl diphosphate isomerase 60 ( 2002 ) ; Strauss and Fuchs , supra , ( 1993 ) ) . This enzyme, enzymes from Escherichia coli (Rodriguez - Concepción et with a mass of 300 kDa , is highly substrate -specific and al ., FEBS Lett , 473 ( 3 ) :328 - 332 ) , Saccharomyces cerevisiae shows little sequence similarity to other known oxidoreduc ( Anderson et al. , J Biol Chem , 1989 , 264 (32 ) ; 19169 -75 ) , tases ( Hugler et al. , supra, (2002 )) . No enzymes in other and Sulfolobus shibatae ( Yamashita et al, Eur J Biochem , organisms have been shown to catalyze this specific reac 2004, 271 (6 ) ; 1087 - 93 ) . The reaction mechanism of isomer - 65 tion ; however there is bioinformatic evidence that other ization , catalyzed by the Idi protein of E . coli , has been organisms can have similar pathways (Klatt et al. , supra , characterized in mechanistic detail ( de Ruyck et al. , J Biol. (2007 ) ) . Enzyme candidates in other organisms including US 10 , 006 , 055 B2 107 108 Roseiflexus castenholzii, Erythrobacter sp . NAP1 and such enzymes from E . coli are encoded by malate dehydro marine gamma proteobacterium HTCC2080 can be inferred genase (mdh ) and lactate dehydrogenase (ldhA ) . In addition , by sequence similarity . lactate dehydrogenase from Ralstonia eutropha has been shown to demonstrate high activities on 2 -ketoacids of various chain lengths including lactate , 2 -oxobutyrate , Protein GenBank ID GI Number Organism 2 - oxopentanoate and 2 - oxoglutarate ( Steinbuchel et al. , Eur. adhE NP _ 415757 . 1 16129202 Escherichia coli J. Biochem . 130 :329 - 334 (1983 )) . Conversion of alpha adhE2 AAK09379 . 1 12958626 Clostridium acetobutylicum ketoadipate into alpha -hydroxyadipate can be catalyzed by adhE AAV66076 .1 55818563 Leuconostoc 2 -ketoadipate reductase , an enzyme reported to be found in mesenteroides rat and in human placenta ( Suda et al. , Arch . Biochem . mcr AAS20429 . 1 42561982 Chloroflexus aurantiacus Biophys. 176 :610 -620 ( 1976 ) ; Suda et al. , Biochem . Bio Rcas_ 2929 YP _ 001433009 . 1 156742880 Roseiflexus phys . Res. Commun . 77 : 586 - 591 ( 1977 ) ) . An additional castenholzii candidate for this step is the mitochondrial 3 - hydroxybu NAP1 02720 ZP _ 01039179 . 1 85708113 Erythrobacter sp . NAP1 15 tyrate dehydrogenase (bdh ) from the human heart which has MGP2080 _ 00535 ZP _ 01626393. 1 119504313 marine gamma been cloned and characterized (Marks et al. , J . Biol. Chem . proteobacterium 267 : 15459 -15463 ( 1992 )) . This enzyme is a dehydrogenase HTCC2080 that operates on a 3 -hydroxyacid . Another exemplary alco hol dehydrogenase converts acetone to isopropanol as was Longer chain acyl- CoA molecules can be reduced to their 20 shown in C . beijerinckii (Ismaiel et al. , J . Bacteriol. 175 : corresponding alcohols by enzymes such as the jojoba 5097 -5105 ( 1993 )) and T. brockii (Lamed et al. , Biochem . J . (Simmondsia chinensis ) FAR which encodes an alcohol- 195: 183 -190 ( 1981 ); Peretz et al. , Biochemistry . 28 :6549 forming fatty acyl- CoA reductase. Its overexpression in E . 6555 (1989 )) . Methyl ethyl ketone reductase , or alterna coli resulted in FAR activity and the accumulation of fatty 25 tively , 2 - butanol dehydrogenase , catalyzes the reduction of alcohol (Metz et al ., Plant Physiology 122: 635 -644 (2000 ) ) . MEK to form 2 -butanol . Exemplary enzymes can be found in Rhodococcus ruber (Kosjek et al. , Biotechnol Bioeng . 86 : 55 - 62 ( 2004 ) ) and Pyrococcus furiosus ( van der et al. , Protein GenBank ID GI Number Organism Eur. J. Biochem . 268: 3062 - 3068 (2001 )) . FAR AAD38039. 1 5020215 Simmondsia chinensis 30 Protein GenBank ID GI Number Organism Another candidate for catalyzing this step is 3 - hydroxy 3 -methylglutaryl -CoA reductase (or HMG -CoA reductase ). mdh AAC76268. 1 1789632 Escherichia coli This enzyme reduces the CoA group in 3 -hydroxy -3 -meth - 35 IdhaIdh NPYP __ 415898725182 . 11 11386669316129341 RalstoniaEscherichia eutropha coli ylglutaryl -CoA to an alcohol forming mevalonate . Gene bdh AAA58352 . 1 177198 Homo sapiens adh AAA23199 . 2 60592974 Clostridium beijerinckii candidates for this step include : NRRL B593 adh P14941. 1 113443 Thermoanaerobacter brockii HTD4 Protein GenBank ID GI Number Organism adhA AAC25556 3288810 Pyrococcus furiosus 40 adh - A CAD36475 21615553 Rhodococcus ruber HMG1 CAA86503 . 1 587536 Saccharomyces cerevisiae HMG2 NP _ 013555 6323483 Saccharomyces cerevisiae HMG1 CAA70691. 1 1694976 Arabidopsis thaliana A number of organisms can catalyze the reduction of hmgA AAC45370 . 1 2130564 Sulfolobus solfataricus 4 -hydroxy - 2 -butanone to 1, 3 -butanediol , including those 45 belonging to the genus Bacillus, Brevibacterium , Candida , The hmgA gene of Sulfolobus solfataricus, encoding and Klebsiella among others , as described by Matsuyama et 3 -hydroxy - 3 -methylglutaryl -CoA reductase , has been al. U .S . Pat. No . 5 ,413 , 922 . A mutated Rhodococcus phe cloned , sequenced , and expressed in E . coli (Bochar et al . , nylacetaldehyde reductase (Sar268 ) and a Leifonia alcohol J Bacteriol. 179: 3632 -3638 ( 1997 )) . S . cerevisiae also has dehydrogenase have also been shown to catalyze this trans two HMG - CoA reductases in it (Basson et al. , Proc. Natl. 50 formation at high yields ( Itoh et al. , Appl. Microbiol . Bio Acad . Sci. U .S . A 83 :5563 -5567 (1986 ) ). The gene has also technol. 75 (6 ): 1249 - 1256 ). been isolated from Arabidopsis thaliana and has been shown Homoserine dehydrogenase (EC 1. 1 . 1. 13 ) catalyzes the to complement the HMG -COA reductase activity in S. NAD (P ) H - dependent reduction ofaspartate semialdehyde to cerevisiae ( Learned et al. , Proc. Natl. Acad . Sci . U . S . A homoserine . In many organisms, including E . coli, homos 86 : 2779 - 2783 ( 1989 ) ). erine dehydrogenase is a bifunctional enzyme that also 3 -Oxoglutaryl - CoA Reductase ( Aldehyde Forming ) ( FIG . 4 , catalyzes the ATP -dependent conversion of aspartate to Step K ) aspartyl- 4 -phosphate (Starnes et al. , Biochemistry 11 :677 Several acyl - CoA dehydrogenases are capable of reduc - 687 ( 1972 ) ) . The functional domains are catalytically inde ing an acyl- CoA to its corresponding aldehyde. Thus they 60 pendent and connected by a linker region ( Sibilli et al. , J can naturally reduce 3 -oxoglutaryl - CoA to 3, 5 -dioxopen Biol Chem 256 : 10228 - 10230 ( 1981 )) and both domains are tanoate or can be engineered to do so . Exemplary genes that subject to allosteric inhibition by threonine . The homoserine encode such enzymes were discussed in FIG . 4 , Step C . dehydrogenase domain of the E . coli enzyme, encoded by 3 , 5 - Dioxopentanoate Reductase (Ketone Reducing) (FIG . 4 , thrA , was separated from the domain , Step L ) 65 characterized , and found to exhibit high catalytic activity There exist several exemplary alcohol dehydrogenases and reduced inhibition by threonine (James et al. , Biochem that convert a ketone to a hydroxyl functional group . Two istry 41 :3720 -3725 ( 2002 )) . This can be applied to other US 10 ,006 ,055 B2 109 110 bifunctional threonine kinases including , for example , hom1 Example II of Lactobacillus plantarum ( Cahyanto et al. , Microbiology 152 : 105 -112 (2006 ) ) and Arabidopsis thaliana . The mono Exemplary Hydrogenase and CO Dehydrogenase functional homoserine dehydrogenases encoded by hom . in Enzymes for Extracting Reducing Equivalents from S . cerevisiae ( Jacques et al ., Biochim Biophys Acta 1544 : 5 Syngas and Exemplary Reductive TCA Cycle 28 -41 (2001 )) and hom2 in Lactobacillus plantarum (Cahy Enzymes anto et al ., supra , ( 2006 ) ) have been functionally expressed Enzymes of the reductive TCA cycle useful in the non and characterized in E . coli . naturally occurring microbial organisms of the present invention include one or more of ATP - citrate lyase and three 10 CO , - fixing enzymes : isocitrate dehydrogenase , alpha -keto Protein GenBank ID GI number Organism glutarate : ferredoxin oxidoreductase , pyruvate : ferredoxin thrA AAC73113 . 1 1786183 Escherichia coli K12 oxidoreductase . The presence of ATP - citrate lyase or citrate akthr2 O81852 75100442 Arabidopsis thaliana lyase and alpha - ketoglutarate :ferredoxin oxidoreductase homo CAA89671 1015880 Saccharomyces cerevisiae indicates the presence of an active reductive TCA cycle in an hom1 CAD64819 28271914 Lactobacillus plantarum organism . Enzymes for each step of the reductive TCA cycle hom2 CAD63186 28270285 Lactobacillus plantarum are shown below . ATP -citrate lyase (ACL , EC 2 .3 . 3 . 8 ) , also called ATP citrate synthase , catalyzes the ATP -dependent cleavage of 3 , 5 - Dioxopentanoate Reductase ( Aldehyde Reducing ) (FIG . citrate to oxaloacetate and acetyl -CoA . ACL is an enzyme of 4 , Step M ) 20 the RTCA cycle that has been studied in green sulfur bacteria Several aldehyde reducing reductases are capable of Chlorobium limicola and Chlorobium tepidum . The alpha reducing an aldehyde to its corresponding alcohol. Thus they (4 )beta ( 4 ) heteromeric enzyme from Chlorobium limicola can naturally reduce 3 , 5 - dioxopentanoate to 5 -hydroxy - 3 was cloned and characterized in E . coli (Kanao et al. , Eur. J . oxopentanoate or can be engineered to do so . Exemplary Biochem . 269: 3409 - 3416 (2002 ) . The C . limicola enzyme, genes that encode such enzymes were discussed in FIG . 4 , 25 encoded by aclAB , is irreversible and activity of the enzyme Step D . is regulated by the ratio of ADP/ ATP . A recombinant ACL 5 -Hydroxy - 3 - Oxopentanoate Reductase (FIG . 4 , Step N ) from Chlorobium tepidum was also expressed in E . coli and Several ketone reducing reductases are capable of reduc the holoenzyme was reconstituted in vitro , in a study elu ing a ketone to its corresponding hydroxyl group . Thus they cidating the role of the alpha and beta subunits in the can naturally reduce 5 -hydroxy - 3 -oxopentanoate to 3 , 5á 30 catalytic mechanism (Kim and Tabita , J . Bacteriol. 188 : 6544 -6552 ( 2006 ) . ACL enzymes have also been identified dihydroxypentanoate or can be engineered to do so . Exem in Balnearium lithotrophicum , Sulfurihydrogenibium sub plary genes that encode such enzymes were discussed in terraneum and other members of the bacterial phylum FIG . 4 , Step L . Aquificae (Hugler et al ., Environ . Microbiol. 9 :81 - 92 3 -Oxo -Glutaryl - CoA Reductase ( COA Reducing and Alco - 35 (2007 ) ) . This activity has been reported in some fungi as hol Forming) (FIG . 4 , Step 0 ) well . Exemplary organisms include Sordaria macrospora 3 -oxo - glutaryl- CoA reductase ( CoA reducing and alcohol (Nowrousian et al. , Curr . Genet. 37 : 189 - 93 (2000 ) , Asper forming ) enzymes catalyze the 2 reduction steps required to gillus nidulans, Yarrowia lipolytica (Hynes and Murray , form 5 -hydroxy - 3 -oxopentanoate from 3 -oxo -glutaryl - CoA . Eukaryotic Cell, July : 1039 - 1048 , (2010 ) and Aspergillus Exemplary 2 - step oxidoreductases that convert an acyl -CoA 40 niger (Meijer et al. J . Ind . Microbiol. Biotechnol. 36 : 1275 to an alcohol were provided for FIG . 4 , Step J . Such 1280 ( 2009 ) . Other candidates can be found based on enzymes can naturally convert 3 - oxo - glutaryl- CoA to 5 -hy sequence homology . Information related to these enzymes is droxy - 3 -oxopentanoate or can be engineered to do so . tabulated below :

Protein GenBank ID GINumber Organism aclA BAB21376 . 1 12407237 Chlorobium limicola aclB BAB21375 . 1 12407235 Chlorobium limicola aclA AAM72321 . 1 21647054 Chlorobium tepidum aclB AAM72322 . 1 21647055 Chlorobium tepidum aclA ABI50076 . 1 114054981 Balnearium lithotrophicum aclB AB150075 . 1 114054980 Balnearium lithotrophicum aclA AB150085 . 1 114055040 Sulfurihydrogenibium subterraneum aclB ABI50084 . 1 114055039 Sulfurihydrogenibium subterraneum acIA AAX76834 . 1 62199504 Sulfurimonas denitrificans aclB AAX76835 . 1 62199506 Sulfurimonas denitrificans acli XP 504787 . 1 50554757 Yarrowia lipolytica acl2 XP 503231 . 1 50551515 Yarrowia lipolytica SPBC1703. 07 NP _ 596202 . 1 19112994 Schizosaccharomyces pombe SPAC22A12 . 16 NP _ 593246 . 1 19114158 Schizosaccharomyces pombe acli CAB76165 . 1 7160185 Sordaria macrospora acl2 CAB76164 . 1 7160184 Sordaria macrospora aclA CBF86850 . 1 259487849 Aspergillus nidulans aclB CBF86848 259487848 Aspergillus nidulans US 10 ,006 , 055 B2 111 112 In some organisms the conversion of citrate to oxaloac 2984 ( 1985 ) ) . S . cerevisiae contains one copy of a fumarase etate and acetyl- CoA proceeds through a citryl- CoA inter encoding gene, FUM1, whose product localizes to both the mediate and is catalyzed by two separate enzymes , citryl cytosol and mitochondrion (Sass et al. , J . Biol. Chem . COA synthetase ( EC 6 .2 . 1. 18 ) and citryl- CoA lyase (EC 278 :45109 -45116 (2003 )) . Additional fumarase enzymes are 4 .1 . 3. 34 ) (Aoshima , M ., Appl. Microbiol. Biotechnol. found in Campylobacter jejuni (Smith et al ., Int. J . Biochem . 75 :249 -255 (2007 ). Citryl -CoA synthetase catalyzes the Cell. Biol. 31 : 961 - 975 ( 1999 )) , Thermus thermophilus activation of citrate to citryl- CoA . The Hydrogenobacter (Mizobata et al. , Arch . Biochem . Biophys. 355 :49 -55 ( 1998 ) ) thermophilus enzyme is composed of large and small sub and Rattus norvegicus (Kobayashi et al ., J . Biochem . units encoded by ccsA and ccsB , respectively (Aoshima et 10 89 : 1923 - 1931 ( 1981 )) . Similar enzymes with high sequence al. , Mol . Micrbiol. 52: 751 - 761 ( 2004 ) ) . The citryl- CoA homology include fuml from Arabidopsis thaliana and synthetase of Aquifex aeolicus is composed of alpha and fumC from Corynebacterium glutamicum . The MmcBC beta subunits encoded by sucC1 and sucD1 (Hugler et al ., fumarase from Pelotomaculum thermopropionicum is Environ . Microbiol. 9 :81 - 92 (2007 )) . Citryl- CoA lyase splits 15 another class of fumarase with two subunits (Shimoyama et citryl- CoA into oxaloacetate and acetyl- CoA . This enzyme al. , FEMS Microbiol. Lett. 270 :207 - 213 (2007 ) ) . is a homotrimer encoded by ccl in Hydrogenobacter ther mophilus (Aoshima et al ., Mol. Microbiol. 52 :763 -770 (2004 ) ) and aq _ 150 in Aquifex aeolicus (Hugler et al. , supra Protein GenBank ID GI Number Organism ( 2007 ) ) . The genes for this mechanism of converting citrate 20 fuma NP _ 416129 . 1 16129570 Escherichia coli to oxaloacetate and citryl- CoA have also been reported fumB NDNP _ 418546 . 1 16131948 Escherichia coli fumC NP 416128 . 1 16129569 Escherichia coli recently in Chlorobium tepidum (Eisen et al. , PNAS 99 ( 14 ) : FUM1 NP _ 015061 6324993 Saccharomyces cerevisiae 9509 - 14 ( 2002 ) . fumCQENRN8 . 1 39931596 Corynebacterium glutamicum fumc 069294 . 1 9789756 Campylobacter jejuni 25 fumC P84127 75427690 Thermus thermophilus fumH P14408 . 1 120605 Rattus norvegicus Protein GenBank ID GI Number Organism MmcB YP _ 001211906 147677691 Pelotomaculum CCSA BAD17844 . 1 46849514 Hydrogenobacter thermophilus thermopropionicum ccsB BAD17846 . 1 46849517 Hydrogenobacter thermophilus Mmcc YP _ 001211907 147677692 Pelotomaculum succi AAC07285 2983723 Aquifex aeolicus thermopropionic?? sucD1 AAC07686 2984152 Aquifex aeolicus 30 - cel BAD17841. 1 46849510 Hydrogenobacter thermophilus aq _ 150 AAC06486 2982866 Aquifex aeolicus Fumarate reductase catalyzes the reduction of fumarate to CTO380 NP _ 661284 21673219 Chlorobium tepidum CT0269 NP _ 661173 . 1 21673108 Chlorobium tepidum succinate . The fumarate reductase of E . coli , composed of CT1834 AAM73055 . 1 21647851 Chlorobium tepidum four subunits encoded by frdABCD , is membrane- bound 35 and active under anaerobic conditions . The electron donor Oxaloacetate is converted into malate by malate dehy for this reaction is menaquinone and the two protons pro drogenase (EC 1 . 1 . 1 . 37 ) , an enzyme which functions in both duced in this reaction do not contribute to the proton the forward and reverse direction . S . cerevisiae possesses gradient (Iverson et al. , Science 284 : 1961 - 1966 ( 1999 ) ) . The three copies of malate dehydrogenase , MDH1 (McAlister - 40 yeast genome encodes two soluble fumarate reductase Henn and Thompson , J. Bacteriol. 169 :5157 -5166 ( 1987) , isozymes encoded by FRDS1 (Enomoto et al ., DNA Res. MDH2 (Minard and McAlister -Henn , Mol. Cell. Biol. 3 :263 - 267 (1996 )) and FRDS2 (Muratsubaki et al. , Arch . 11 : 370 - 380 ( 1991 ) ; Gibson and McAlister -Henn , J . Biol. Biochem . Biophys . 352 : 175 - 181 ( 1998 ) ), which localize to Chem . 278 : 25628 - 25636 ( 2003 )) , and MDH3 (Steffan and as the cytosol and promitochondrion , respectively , and are used McAlister- Henn , J. Biol. Chem . 267 :24708 -24715 ( 1992 ) ), during anaerobic growth on glucose (Arikawa et al ., FEMS which localize to the mitochondrion , cytosol, and peroxi Microbiol. Lett. 165 : 111 -116 (1998 )) . some, respectively . E . coli is known to have an active malate dehydrogenase encoded by mdh . 50 Protein GenBank ID GI Number Organism FRDS1 P32614 418423 Saccharomyces cerevisiae Protein GenBank ID GI Number Organism FRDS2 NP 012585 6322511 Saccharomyces cerevisiae MDH1 NDNP _ 012838 6322765 Saccharomyces cerevisiae frdA NP _ 418578 . 1 16131979 Escherichia coli MDH2 NP _ 014515 116006499 Saccharomyces cerevisiae frdB NP _ 418577 . 1 16131978 Escherichia coli MDH3NP _ 010205 6320125 Saccharomyces cerevisiae 55 frdc NP _ 418576 . 1 16131977 Escherichia coli Mdh NP _ 417703 . 1 16131126 Escherichia coli frdD NP _ 418475 . 1 16131877 Escherichia coli Fumarate hydratase ( EC 4 . 2 . 1 . 2 ) catalyzes the reversible The ATP -dependent acylation of succinate to succinyl hydration of fumarate to malate . The three fumarases of E . COA is catalyzed by succinyl -CoA synthetase ( EC 6 .2 . 1 . 5 ) . coli , encoded by fumA , fumB and fumC , are regulated under 60 The product of the LSC1 and LSC2 genes of S . cerevisiae different conditions of oxygen availability . FumB is oxygen and the succ and sucD genes of E . coli naturally form a sensitive and is active under anaerobic conditions. FumA is succinyl -CoA synthetase complex that catalyzes the forma active under microanaerobic conditions , and FumC is active tion of succinyl -CoA from succinate with the concomitant under aerobic growth conditions ( Tseng et al. , J. Bacteriol. 65 consumption of one ATP, a reaction which is reversible in 183: 461- 467 ( 2001) ; Woods et al ., Biochim . Biophys. Acta vivo (Buck et al ., Biochemistry 24 :6245 -6252 (1985 )) . 954: 14 - 26 ( 1988 ) ; Guest et al. , J . Gen . Microbiol. 131 : 2971 These proteins are identified below : US 10 ,006 ,055 B2 113 114 rubrum by sequence homology. A two subunit enzyme has Protein GenBank ID GI Number Organism also been identified in Chlorobium tepidum ( Eisen et al. , LSC1 NP _ 014785 6324716 Saccharomyces cerevisiae PNAS 99 ( 14 ) : 9509 - 14 ( 2002 ) ) . LSC2 NP _ 011760 6321683 Saccharomyces cerevisiae succ NP 415256 . 1 16128703 Escherichia coli sucD AAC73823 . 1 1786949 Escherichia coli Protein GenBank ID GI Number Organism korA BAB21494 12583691 Hydrogenobacter Alpha -ketoglutarate : ferredoxin oxidoreductase ( EC thermophilus 1 . 2 . 7 . 3 ) , also known as 2 - oxoglutarate synthase or 2 - oxo BAB21495 12583692 Hydrogenobacter glutarate: ferredoxin oxidoreductase (?FOR ), forms 10 korb thermophilus alpha - ketoglutarate from CO2 and succinyl- CoA with con ford BAB62132 . 1 14970994 Hydrogenobacter current consumption of two reduced ferredoxin equivalents . thermophilus OFOR and pyruvate : ferredoxin oxidoreductase (PFOR ) are forA BAB62133 . 1 14970995 Hydrogenobacter members of a diverse family of 2 - oxoacid : ferredoxin thermophilus 5 forB BAB62134 . 1 14970996 Hydrogenobacter ( flavodoxin ) oxidoreductases which utilize thiamine pyro thermophilus phosphate , COA and iron - sulfur clusters as cofactors and for BAB62135 . 1 14970997 Hydrogenobacter ferredoxin , flavodoxin and FAD as electron carriers thermophilus ( Adams et al. , Archaea . Adv . Protein Chem . 48 : 101 - 180 forE BAB62136 .1 14970998 Hydrogenobacter ( 1996 ) ) . Enzymes in this class are reversible and function in thermophilus the carboxylation direction in organisms that fix carbon by 20 Clim _ 0204 ACD89303. 1 189339900 Chlorobium limicola Clim _ 0205 ACD89302 . 1 189339899 Chlorobium limicola the RTCA cycle such as Hydrogenobacter thermophilus, Clim _ 1123 ACD90192 . 1 189340789 Chlorobium limicola Desulfobacter hydrogenophilus and Chlorobium species Clim 1124 ACD90193 . 1 189340790 Chlorobium limicola ( Shiba et al. 1985 ; Evans et al. , Proc . Natl. Acad . Sci . U . S . A . Moth _ 1984 YP _ 430825 . 1 83590816 Moorella thermoacetica 55 :92934 ( 1966 ); Buchanan , 1971) . The two- subunit Moth _ 1985 YP 430826 . 1 83590817 Moorella thermoacetica enzyme from H . thermophilus , encoded by korAB , has been 25 Moth _ 0034 YP _ 428917 . 1 83588908 Moorella thermoacetica ST2300 NP 378302 . 1 15922633 Sulfolobus sp . strain 7 cloned and expressed in E . coli ( Yun et al ., Biochem . Ape1472 BAA80470 . 1 5105156 Aeropyrum pernix Biophys. Res. Commun . 282 : 589- 594 (2001 ) ) . A five subunit Ape1473 BAA80471 . 2 116062794 Aeropyrum pernix OFOR from the same organism with strict substrate oord NP _ 207383 . 1 15645213 Helicobacter pylori specificity for succinyl -CoA , encoded by forDABGE , was oorA NP _ 207384 . 1 15645214 Helicobacter pylori recently identified and expressed in E . coli ( Yun et al. 30 oorB NP _ 207385 . 1 15645215 Helicobacter pylori 2002 ). The kinetics of CO2 fixation of both H . thermophilus oorC NP _ 207386 . 1 15645216 Helicobacter pylori OFOR enzymes have been characterized ( Yamamoto CT0163 NP _ 661069 . 1 21673004 Chlorobium tepidum et al ., Extremophiles 14 :79 - 85 ( 2010 ) ). A CO2 - fixing CT0162 NP _ 661068 . 1 21673003 Chlorobium tepidum OFOR from Chlorobium thiosulfatophilum has been purified korA CAA12243 . 2 19571179 Thauera aromatica and characterized but the genes encoding this enzyme 35 korB CAD27440 . 1 19571178 Thauera aromatica have not been identified to date . Enzyme candidates in » RruRm _ A2721 YP _ 427805 . 1 83594053 Rhodospirillum rubrum Chlorobium species can be inferred by sequence similarity Rru _ A2722 YP _ 427806 . 1 83594054 Rhodospirillum rubrum to the H . thermophilus genes . For example, the Chlorobium limicola genome encodes two similar proteins . Acetogenic Isocitrate dehydrogenase catalyzes the reversible decar bacteria such as Moorella thermoacetica are predicted to 40 encode two OFOR enzymes . The enzyme encoded by 40 boxylation of isocitrate to 2 - oxoglutarate coupled to the Moth _ 0034 is predicted to function in the CO2- assimilating reduction of NAD ( P ) * . IDH enzymes in Saccharomyces direction . The genes associated with this enzyme, cerevisiae and Escherichia coli are encoded by IDP1 and Moth _ 0034 have not been experimentally validated to date but can be inferred by sequence similarity to known OFOR icd , respectively (Haselbeck and McAlister - Henn , J. Biol. enzymes . - 45 Chem . 266 :2339 - 2345 ( 1991 ); Nimmo, H . G ., Biochem . J . OFOR enzymes that function in the decarboxylation 234 : 317 - 2332 ( 1986 ) ) . The reverse reaction in the reductive direction under physiological conditions can also catalyze TCA cycle , the reductive carboxylation of 2 -oxoglutarate to the reverse reaction . The OFOR from the thermoacidophilic isocitrate , is favored by the NADPH - dependent CO2- fixing archaeon Sulfolobus sp. strain 7 , encoded by ST2300 , has 50 IDH from Chlorobium limicola and was functionally been extensively studied ( Zhang et al . 1996 . A plasmid expressed in E . coli (Kanao et al. , Eur. J Biochem . 269 : based expression system has been developed for efficiently 1926 -1931 (2002 )) . A similar enzyme with 95 % sequence expressing this protein in E . coli ( Fukuda et al. , Eur. J . identity is found in the C . tepidum genome in addition to Biochem . 268: 5639 - 5646 ( 2001 ) ) and residues involved in some other candidates listed below . substrate specificity were determined ( Fukuda and Wakagi, Biochim . Biophys . Acta 1597: 74 - 80 ( 2002 ) ). The OFOR encoded by Ape147 2 / Ape147 3 from Aeropyrum pernix str. Protein GenBank ID GI Number Organism K1was recently cloned into E . coli , characterized , and found Icd AC184720 . 1 209772816 Escherichia coli to react with 2 - oxoglutarate and a broad range of 2 - oxoacids 60 IDP1 AAA34703 . 1 171749 Saccharomyces cerevisiae Idh BAC00856 . 1 21396513 Chlorobium limicola ( Nishizawa et al. , FEBS Lett. 579 : 2319 - 2322 (2005 ) ). Icd AAM71597 . 1 21646271 Chlorobium tepidum Another exemplary OFOR is encoded by oorDABC in icd NP _ 952516 . 1 39996565 Geobacter sulfurreducens Helicobacter pylori (Hughes et al . 1998 ). An enzyme spe icdicd YP _ 393560 . 78777245 Sulfurimonas denitrificans cific to alpha - ketoglutarate has been reported in ThaueraSu 6565 aromatica (Domer and Boll , J , Bacteriol. 184 ( 14 ), 3975 - 83 In H . thermophilus the reductive carboxylation of 2 -oxo (2002 ). A similar enzyme can be found in Rhodospirillum glutarate to isocitrate is catalyzed by two enzymes: 2 - oxo US 10 ,006 , 055 B2 115 116 glutarate carboxylase and oxalosuccinate reductase . 2 -Oxo Pyruvate : ferredoxin oxidoreductase (PFOR ) catalyzes the glutarate carboxylase ( EC 6 . 4 . 1. 7 ) catalyzes the ATP -depen reversible oxidation of pyruvate to form acetyl -CoA . The dent carboxylation of alpha -ketoglutarate to oxalosuccinate PFOR from Desulfovibrio africanus has been cloned and ( Aoshima and Igarashi, Mol . Microbiol. 62 : 748 -759 (2006 ) ) . This enzyme is a large complex composed of two subunits . expressed in E . coli resulting in an active recombinant Biotinylation of the large ( A ) subunit is required for enzyme enzyme that was stable for several days in the presence of function (Aoshima et al. , Mol. Microbiol. 51: 791 - 798 oxygen (Pieulle et al. , J . Bacteriol. 179 : 5684 - 5692 ( 1997 ) ) . (2004 ) ) . Oxalosuccinate reductase (EC 1 . 1 . 1 . - ) catalyzes the Oxygen stability is relatively uncommon in PFORs and is NAD - dependent conversion of oxalosuccinate to D - threo - 10 believed to be conferred by a 60 residue extension in the isocitrate . The enzyme is a homodimer encoded by icd in H . polypeptide chain of the D . africanus enzyme. Two cysteine thermophilus. The kinetic parameters of this enzyme indi- residues in this enzyme form a disulfide bond that protects cate that the enzyme only operates in the reductive carboxy lation direction in vivo , in contrast to isocitrate dehydroge it against inactivation in the form of oxygen . This disulfide nase enzymes in other organisms (Aoshima and Igarashi, J. 15 bond and the stability in the presence of oxygen has been Bacteriol . 190 : 2050 - 2055 ( 2008 ) ) . Based on sequence found in other Desulfovibrio species also (Vita et al. , Bio homology, gene candidates have also been found in Thio chemistry, 47 : 957 -64 ( 2008 )) . The M . thermoacetica PFOR bacillus denitrificans and Thermocrinis albus. is also well characterized (Menon and Ragsdale , Biochem 20 istry 36 : 8484 -8494 ( 1997 )) and was shown to have high activity in the direction of pyruvate synthesis during auto Protein GenBank ID GI Number Organism trophic growth (Furdui and Ragsdale , J. Biol. Chem . 275 : cfiA BAF34932 . 1 116234991 Hydrogenobacter thermophilus 28494- 28499 ( 2000 ) ). Further, E . coli possesses an unchar cifB BAF34931 .1 116234990 Hydrogenobacter 25 acterized open reading frame, ydbK , encoding a protein that thermophilus Icd BADO2487 . 1 38602676 Hydrogenobacter is 51 % identical to the M . thermoacetica PFOR . Evidence thermophilus for pyruvate oxidoreductase activity in E . coli has been Tbd _ 1556 YP _ 315314 74317574 Thiobacillus denitrificans Tbd _ 1555 YP _ 315313 74317573 Thiobacillus denitrificans described (Blaschkowski et al. , Eur. J. Biochem . 123 : 563 Tbd _ 0854 YP _ 314612 74316872 Thiobacillus denitrificans Thal __ 0268 YP _ 003473030 289548042 Thermocrinis albus 30 569 ( 1982) ) . PFORs have also been described in other Thal _ 0267 YP _ 003473029289548041 Thermocrinis albus organisms, including Rhodobacter capsulatas ( Yakunin and Thal_ 0646 YP _ 003473406 289548418 Thermocrinis albus Hallenbeck , Biochimica et Biophysica Acta 1409 ( 1998 ) 39- 49 (1998 ) ) and Choloboum tepidum (Eisen et al. , PNAS Aconitase (EC 4 .2 . 1 .3 ) is an iron -sulfur - containing pro 35 99 ( 14 ): 9509 -14 ( 2002 )) . The five subunit PFOR from H . tein catalyzing the reversible isomerization of citrate and 3 . thermophilus, encoded by porEDABG , was cloned into E . iso -citrate via the intermediate cis -aconitate . Two aconitase enzymes are encoded in the E . coli genome by acnA and coli and shown to function in both the decarboxylating and acnB . AcnB is the main catabolic enzyme, while AcnA is CO2- assimilating directions ( Ikeda et al. 2006 ; Yamamoto et more stable and appears to be active under conditions of 40 al. , Extremophiles 14 :79 - 85 ( 2010 ) ) . Homologs also exist in oxidative or acid stress (Cunningham et al. , Microbiology C . carboxidivorans P7. Several additional PFOR enzymes 143 ( Pt 12 ) : 3795 - 3805 ( 1997 ) ). Two isozymes of aconitase are described in the following review (Ragsdale , S . W . , in Salmonella typhimurium are encoded by acnA and acnB Chem . Rev . 103: 2333 -2346 (2003 )) . Finally , flavodoxin (Horswill and Escalante - Semerena , Biochemistry 40 : 4703 reductases ( e . g . , fqrB from Helicobacter pylori or Campy 4713 ( 2001) ) . The S. cerevisiae aconitase, encoded by lobacter jejuni) (St Maurice et al. , J. Bacteriol. 189 :4764 ACO1, is localized to the mitochondria where it participates 4773 ( 2007 ) ) or Rnf- type proteins (Seedorf et al. , Proc . Natl . in the TCA cycle (Gangloff et al ., Mol. Cell . Biol. 10 :3551 Acad . Sci. USA . 105 :2128 - 2133 (2008 ) ; and Herrmann , J . 3561 ( 1990 ) ) and the cytosol where it participates in the Bacteriol 190 :784 - 791 (2008 )) provide a means to generate glyoxylate shunt (Regev -Rudzki et al. , Mol . Biol. Cell. 30 NADH or NADPH from the reduced ferredoxin generated 16 :4163 -4171 (2005 ) ) . by PFOR . These proteins are identified below .

Protein GenBank ID GI Number Organism acnA AAC7438 . 1 1787531 Escherichia coli acnB AAC73229 . 1 2367097 Escherichia coli HP0779 NP 207572 . 1 15645398 Helicobacter pylori 26695 H16 B0568 CAJ95365 . 1 113529018 Ralstonia eutropha DesfrDRAFT _ 3783 ZP _ 07335307 . 1 303249064 Desulfovibrio fructosovorans JJ Suden _ 1040 (acnB ) ABB44318 . 1 78497778 Sulfurimonas denitrificans Hydth _ 0755 ADO45152 . 1 308751669 Hydrogenobacter thermophilus CT0543 (acn ) AAM71785 . 1 21646475 Chlorobium tepidum Clim _ 2436 YP _ 001944436 . 1 189347907 Chlorobium limicola Clim _ 0515 ACD89607 . 1 189340204 Chlorobium limicola acnA NP _ 460671. 1 16765056 Salmonella typhimurium acnB NP 459163. 1 16763548 Salmonella typhimurium ACO1 AAA34389 . 1 170982 Saccharomyces cerevisiae US 10 ,006 ,055 B2 117 118 Protein GenBank ID GI Number Organism DesfrDRAFT _ 0121 ZP _ 07331646 . 1 303245362 Desulfovibrio fructosovorans JJ Por CAA70873 . 1 1770208 Desulfovibrio africanus por YP 012236 . 1 46581428 Desulfovibrio vulgaris str. Hildenborough Dde _ 3237 ABB40031. 1 78220682 Desulfo Vibrio desulfuricans G20 Ddes_ 0298 YP _ 002478891. 1 220903579 Desulfovibrio desulfuricans subsp . desulfuricans str. ATCC 27774 Por YP 428946 . 1 83588937 Moorella thermoacetica YdbK NP _ 415896 . 1 16129339 Escherichia coli nifT (CT1628 ) NP _ 662511 . 1 21674446 Chlorobium tepidum CJE1649 YP _ 179630 . 1 57238499 Campylobacter jejuni nify ADE85473 . 1 294476085 Rhodobacter capsulatus porE BAA95603 . 1 7768912 Hydrogenobacter thermophilus porD BAA95604 . 1 7768913 Hydrogenobacter thermophilus pora BAA95605 . 1 7768914 Hydrogenobacter thermophilus porB BAA95606 . 1 776891 Hydrogenobacter thermophilus porG BAA95607 . 1 7768916 Hydrogenobacter thermophilus FarB YP _ 001482096 . 1 157414840 Campylobacter jejuni HP1164 NP _ 207955 . 1 15645778 Helicobacter pylori Rnfc EDK333061 . 1 46346770 Clostridium kluyveri RnfD EDK33307 . 1 146346771 Clostridium kluyveri RnfG EDK33308 . 1 146346772 Clostridium kluyveri RnfE EDK33309 . 1 146346773 Clostridium kluyveri RnfA EDK33310 . 1 146346774 Clostridium kluyveri RnfB EDK33311. 1 146346775 Clostridium kluyveri

The conversion of pyruvate into acetyl- CoA can be cata - ( Takahashi- Abbe et al ., Oral . Microbiol Immunol. 18 : 293 lyzed by several other enzymes or their combinations 297 ( 2003 ) ) . E . coli possesses an additional pyruvate for thereof. For example , pyruvate dehydrogenase can trans mate lyase , encoded by tdcE , that catalyzes the conversion form pyruvate into acetyl- CoA with the concomitant reduc - 30 of pyruvate or 2 - oxobutanoate to acetyl- CoA or propionyl tion of a molecule of NAD into NADH . It is a multi - enzyme COA , respectively (Hesslinger et al. , Mol . Microbiol 27 :477 complex that catalyzes a series of partial reactions which 492 ( 1998 ) ) . Both pflB and tdcE from E . coli require the results in acylating oxidative decarboxylation of pyruvate . presence of pyruvate formate lyase activating enzyme, The enzyme comprises of three subunits : the pyruvate 35 encoded by pflA . Further , a short protein encoded by yfiD in decarboxylase (E1 ), dihydrolipoamide acyltransferase (E2 ) E . coli can associate with and restore activity to oxygen and dihydrolipoamide dehydrogenase ( E3 ). This enzyme is cleaved pyruvate formate lyase ( Vey et al ., Proc. Natl. Acad . naturally present in several organisms, including E . coli and Sci . U . S . A . 105: 16137 - 16141 ( 2008 ). Note that pflA and S . cerevisiae . In the E . coli enzyme, specific residues in the pfl? from E . coli were expressed in S . cerevisiae as a means El component are responsible for substrate specificity (Bis - to increase cytosolic acetyl- CoA for butanol production as swanger, H ., J . Biol. Chem . 256 :815 - 82 ( 1981 ) ; Bremer, J . , described in WO / 2008 / 080124 ) . Additional pyruvate for Eur. J . Biochem . 8 : 535 - 540 ( 1969) ; Gong et al. , J . Biol . mate lyase and activating enzyme candidates, encoded by pfl Chem . 275 : 13645 - 13653 ( 2000 ) ) . Enzyme engineering and act , respectively , are found in Clostridium pasteurianum efforts have improved the E . coli PDH enzyme activity 45 (Weidner et al. , J Bacteriol. 178 :2440 - 2444 ( 1996 ) ) . under anaerobic conditions (Kim et al. , J . Bacteriol. 190 : Further, different enzymes can be used in combination to 3851 - 3858 (2008 ) ; Kim et al. , Appl . Environ . Microbiol. convert pyruvate into acetyl- CoA . For example , in S . cer 73 :1766 - 1771 (2007 ) ; Zhou et al. , Biotechnol. Lett . 30 : 335 evisiae , acetyl- CoA is obtained in the cytosol by first decar 342 (2008 ) ) . In contrast to the E . coli PDH , the B . subtilis b oxylating pyruvate to form acetaldehyde ; the latter is complex is active and required for growth under anaerobic oxidized to acetate by acetaldehyde dehydrogenase and conditions (Nakano et al. , J . Bacteriol. 179 :6749 -6755 subsequently activated to form acetyl- COA by acetyl- CoA ( 1997) ) . The Klebsiella pneumoniae PDH , characterized synthetase . Acetyl- CoA synthetase is a native enzyme in during growth on glycerol, is also active under anaerobic several other organisms including E . coli (Kumari et al. , J . conditions ( 5 ). Crystal structures of the enzyme complex 55 Bacteriol . 177: 2878 - 2886 ( 1995 ) ), Salmonella enterica ( Sta from bovine kidney ( 18 ) and the E2 catalytic domain from rai et al. , Microbiology 151 :3793 - 3801 ( 2005 ) ; Starai et al. , Azotobacter vinelandii are available ( 4 ) . Yet another enzyme J . Biol . Chem . 280 :26200 -26205 (2005 ) ) , and Moorella that can catalyze this conversion is pyruvate formate lyase . thermoacetica ( described already ) . Alternatively, acetate can This enzyme catalyzes the conversion of pyruvate and CoA be activated to form acetyl- CoA by acetate kinase and into acetyl- CoA and formate . Pyruvate formate lyase is a ou phosphotransacetylase . Acetate kinase first converts acetate common enzyme in prokaryotic organisms that is used to into acetyl- phosphate with the accompanying use of an ATP help modulate anaerobic redox balance . Exemplary enzymes molecule . Acetyl- phosphate and CoA are next converted can be found in Escherichia coli encoded by pflB (Knappe into acetyl -CoA with the release of one phosphate by and Sawers , FEMS . Microbiol Rev. 6 : 383 -398 ( 1990 ) ) , 65 phosphotransacetylase . Both acetate kinase and phospho Lactococcus lactis (Melchiorsen et al ., ApplMicrobiol Bio transacetlyase are well -studied enzymes in several technol 58 :338 -344 ( 2002 )) , and Streptococcus mutans Clostridia and Methanosarcina thermophila . US 10 , 006 , 055 B2 119 120 Yet another way of converting pyruvate to acetyl- CoA is bacter thermophilus strain TK - 6 , although a gene with this via pyruvate oxidase . Pyruvate oxidase converts pyruvate activity has not yet been indicated (Yoon et al. 2006 ). into acetate , using ubiquione as the electron acceptor. In E . coli, this activity is encoded by poxB . PoxB has similarity to Finally , the energy - conserving membrane - associated Rnf pyruvate decarboxylase of S . cerevisiae and Zymomonas 5 type proteins (Seedorf et al. , Proc . Natl. Acad . Sci. U .S . A . mobilis . The enzyme has a thiamin pyrophosphate cofactor 105: 2128 -2133 ( 2008 ) ; Herrmann et al. , J . Bacteriol. 190 : (Koland and Gennis , Biochemistry 21 :4438 -4442 ( 1982 ) ); 784 - 791 ( 2008 )) provide a means to generate NADH or O ' Brien et al. , Biochemistry 16 :3105 - 3109 (1977 ) ; O 'Brien NADPH from reduced ferredoxin . Additional ferredoxin : and Gennis , J. Biol. Chem . 255 : 3302 - 3307 ( 1980 ) ) and a NAD ( P ) + oxidoreductases have been annotated in flavin adenine dinucleotide ( FAD ) cofactor. Acetate can then Clostridium carboxydivorans P7 .

Protein GenBank ID GI Number Organism HP1164 NP _ 207955 . 1 15645778 Helicobacter pylori RPA3954 CAE29395 . 1 39650872 Rhodopseudomonas palustris for BAH29712 . 1 225320633 Hydrogenobacter thermophilus yumc NP 391091. 2 255767736 Bacillus subtilis CJE0663 AAW35824 . 1 57167045 Campylobacter jejuni for P28861 . 4 399486 Escherichia coli hcad AAC75595 . 1 1788892 Escherichia coli LOC100282643 NP 001149023 . 1 226497434 Zea mays Rnfc EDK33306 . 1 146346770 Clostridium kluyveri RnfD EDK33307 . 1 146346771 Clostridium kluyveri RnfG EDK33308 . 1 146346772 Clostridium kluyveri RnfE EDK33309 . 1 146346773 Clostridium kluyveri RnfA EDK33310 . 1 146346774 Clostridium kluyveri RnfB EDK33311 . 1 146346775 Clostridium kluyveri Ccarb DRAFT 2639 ZP _ 05392639 . 1 255525707 Clostridium carboxidivorans P7 CcarbDRAFT _ 2638 ZP _ 05392638 . 1 255525706 Clostridium carboxidivorans P7 CcarbDRAFT _ 2636 ZP _ 05392636 . 1 255525704 Clostridium carboxidivorans P7 Ccarb DRAFT _ 5060 ZP _ 05395060 . 1 255528241 Clostridium carboxidivorans P7 CcarbDRAFT _ 2450 ZP _ 05392450 . 1 255525514 Clostridium carboxidivorans P7 Ccarb DRAFT _ 1084 ZP _ 05391084 . 1 255524124 Clostridium carboxidivorans P7 be converted into acetyl- CoA by either acetyl- CoA syn Ferredoxins are small acidic proteins containing one or thetase or by acetate kinase and phosphotransacetylase , as as more iron - sulfur clusters that function as intracellular elec described earlier. Some of these enzymes can also catalyze tron carriers with a low reduction potential. Reduced ferre the reverse reaction from acetyl -CoA to pyruvate . doxins donate electrons to Fe- dependent enzymes such as For enzymes that use reducing equivalents in the form of ferredoxin -NADP + oxidoreductase, pyruvate : ferredoxin NADH or NADPH , these reduced carriers can be generated oxidoreductase ( PFOR ) and 2 -oxoglutarate : ferredoxin oxi by transferring electrons from reduced ferredoxin . Two 40 doreductase (?FOR ). The H . thermophilus gene fdx1 enzymes catalyze the reversible transfer of electrons from encodes a [4Fe - 4S ] - type ferredoxin that is required for the reduced ferredoxins to NAD ( P ) " , ferredoxin :NAD + oxi reversible carboxylation of 2 - oxoglutarate and pyruvate by doreductase ( EC 1 . 18 . 1 . 3 ) and ferredoxin : NADP + oxi- OFOR and PFOR , respectively ( Yamamoto et al. , Extremo doreductase (FNR , EC 1 . 18. 1 . 2 ). Ferredoxin : NADP + oxi- 45 philes 14 : 79 - 85 ( 2010 ) ) . The ferredoxin associated with the doreductase ( FNR , EC 1 . 18 . 1 . 2 ) has a noncovalently bound Sulfolobus solfataricus 2 - oxoacid : ferredoxin reductase is a FAD cofactor that facilitates the reversible transfer of elec monomeric dicluster [ 3Fe- 4S ][ 4Fe - 4S ] type ferredoxin trons from NADPH to low -potential acceptors such as (Park et al . 2006 ) . While the gene associated with this ferredoxins or flavodoxins ( Blaschkowski et al. , Eur. J . protein has not been fully sequenced , the N - terminal domain Biochem . 123 : 563 - 569 ( 1982) ; Fujii et al. , 1977 ) . The Heli shares 93 % homology with the zfx ferredoxin from S . cobacter pylori FNR , encoded by HP1164 ( fqrB ) , is coupled acidocaldarius. The E . coli genome encodes a soluble to the activity of pyruvate: ferredoxin oxidoreductase ferredoxin of unknown physiological function , fdx . Some ( PFOR ) resulting in the pyruvate -dependent production of evidence indicates that this protein can function in iron NADPH ( St et al. 2007 ) . An analogous enzyme is found in 55 sulfur cluster assembly ( Takahashi and Nakamura , 1999 ) . Campylobacter jejuni (St et al . 2007) . A ferredoxin :NADP + Additional ferredoxin proteins have been characterized in oxidoreductase enzyme is encoded in the E . coli genome by Helicobacter pylori (Mukhopadhyay et al. 2003) and fpr (Bianchi et al . 1993 ). Ferredoxin :NAD + oxidoreductase utilizes reduced ferredoxin to generate NADH from NAD + . Campylobacter jejuni (van Vliet et al . 2001 ). A 2Fe -2S In several organisms, including E . coli, this enzyme is a 60 ferredoxin from Clostridium pasteurianum has been cloned component of multifunctional dioxygenase enzyme com and expressed in E. coli (Fujinaga and Meyer , Biochemical plexes . The ferredoxin :NAD + oxidoreductase of E . coli, and Biophysical Research Communications, 192 ( 3 ): encoded by hcaD , is a component of the 3 -phenylproppi - (( 1993 )) ) . Acetogenic bacteria such as Moorella thermo onate dioxygenase system involved in involved in aromatic os acetica , Clostridium carboxidivorans P7 and Rhodospiril acid utilization (Diaz et al. 1998 ) .NADH : ferredoxin reduc - lum rubrum are predicted to encode several ferredoxins, tase activity was detected in cell extracts of Hydrogeno - listed in the table below . US 10 ,006 ,055 B2 121 122 Protein GenBank ID GI Number Organism fdx1 BAE02673. 1 68163284 Hydrogenobacter thermophilus M11214 . 1 AAA83524 . 1 144806 Clostridium pasteurianum Zfx AAY79867 . 1 68566938 Sulfolobus acidocalarius Fdx AAC75578 . 1 1788874 Escherichia coli hp _ 0277 AAD07340 . 1 2313367 Helicobacter pylori fdxA CAL34484 . 1 112359698 Campylobacter jejuni Moth _ 0061 ABC18400 . 1 83571848 Moorella thermoacetica Moth 1200 ABC19514 . 1 83572962 Moorella thermoacetica Moth _ 1888 ABC20188 . 1 83573636 Moorella thermoacetica Moth _ 2112 ABC20404 . 1 83573852 Moorella thermoacetica Moth 1037 ABC19351 . 1 83572799 Moorella thermoacetica Ccarb DRAFT 4383 ZP _ 05394383 . 1 255527515 Clostridium carboxidivorans P7 Ccarb DRAFT 2958 ZP _ 05392958 . 1 255526034 Clostridium carboxidivorans P7 CcarbDRAFT _ 2281 ZP _ 05392281. 1 255525342 Clostridium carboxidivorans P7 CcarbDRAFT _ 5296 ZP 05395295 . 1 255528511 Clostridium carboxidivorans P7 Ccarb DRAFT _ 1615 ZP 05391615 . 1 255524662 Clostridium carboxidivorans P7 CcarbDRAFT _ 1304 ZP _ 05391304 . 1 255524347 Clostridium carboxidivorans P7 cooF AAG29808 . 1 11095245 Carboxydothermus hydrogenoformans fdxN CAA35699 . 1 46143 Rhodobacter capsulatus Rru _ A2264 ABC23064 . 1 83576513 Rhodospirillum rubrum Rru _ A1916 ABC22716 . 1 83576165 Rhodospirillum rubrum Rru _ A2026 ABC22826 . 1 83576275 Rhodospirillum rubrum cooF AAC45122 . 1 1498747 Rhodospirillum rubrum fdxN AAA26460 . 1 152605 Rhodospirillum rubrum Alvin _ 2884 ADC63789 . 1 288897953 Allochromatium vinosum DSM 180 fdx YP _ 002801146 . 1 226946073 Azotobacter vinelandii DJ CKL _ 3790 YP _ 001397146 . 1 153956381 Clostridium kluyveri DSM 555 fer1 NP _ 949965 . 1 39937689 Rhodopseudomonas palustris CGA009 fdx CAA12251 . 1 3724172 Thauera aromatica CHY _ 2405 YP _ 361202 . 1 78044690 Carboxydothermus hydrogenoformans fer YP _ 359966 . 1 78045103 Carboxydothermus hydrogenoformans fer AAC83945 . 1 1146198 Bacillus subtilis fdx1 NP 249053 . 1 15595559 Pseudomonas aeruginosa PA01 yfhL AP _ 003148. 1 89109368 Escherichia coli K - 12 35 Succinyl - CoA transferase catalyzes the conversion of succinyl -CoA to succinate while transferring the CoA moi- Protein GenBank ID GI Number Organism ety to a COA acceptor molecule . Many transferases have cat1 P38946 . 1 729048 Clostridium broad specificity and can utilize CoA acceptors as diverse as kluyveri acetate , succinate, propionate , butyrate , 2 -methylacetoac - " TVAG _ 395550 XP _ 001330176 123975034 Trichomonas vaginalis G3 etate , 3 -ketohexanoate , 3 -ketopentanoate , valerate , croto Tb11. 02 . 0290 XP _ 828352 71754875 Trypanosoma brucei nate , 3 -mercaptopropionate , propionate , vinylacetate , and pcal AAN69545 . 1 24985644 Pseudomonas putida butyrate , among others . pca ) NP _ 746082 . 1 26990657 Pseudomonas putida The conversion of succinate to succinyl- CoA can be 45 aarc- ACD85596 . 1 189233555 Acetobacter aceti carried by a transferase which does not require the direct consumption of an ATP or GTP. This type of reaction is An additional exemplary transferase that converts succi common in a number of organisms. The conversion of nate to succinyl- CoA while converting a 3 - ketoacyl- CoA to succinate to succinyl- CoA can also be catalyzed by succinyl a 3 -ketoacid is succinyl- CoA :3 :ketoacid -CoA transferase COA :Acetyl - CoA transferase . The gene product of cat1 of 50 (EC 2 . 8 .3 .5 ) . Exemplary succinyl- CoA : 3 :ketoacid -COA Clostridium kluyveri has been shown to exhibit succinyl transferases are present in Helicobacter pylori ( Corthesy COA : acetyl - CoA transferase activity ( Sohling and Theulaz et al . 1997 ) , Bacillus subtilis , and Homo sapiens Gottschalk . J . Bacteriol. 178 :871 - 880 ( 1996 ) ). In addition . (Fukao et al. 2000 ; Tanaka et al. 2002 ). The aforementioned the activity is present in Trichomonas vaginalis (van Grins - proteins are identified below . ven et al. 2008 ) and Trypanosoma brucei ( Riviere et al. 55 2004 ) . The succinyl- CoA :acetate CoA -transferase from Acetobacter aceti, encoded by aarC , replaces succinyl- CoA Protein GenBank ID GI Number Organism synthetase in a variant TCA cycle (Mullins et al. 2008 ) . HPAG1 _ 0676 YP _ 627417 108563101 Helicobacter pylori HPAG1_ _ 0677 YP _ 627418 108563102 Helicobacter pylori Similar succinyl -CoA transferase activities are also present 60 ScoA NP_ 391778 16080950 Bacillus subtilis in Trichomonas vaginalis (van Grinsven et al . 2008 ), Try ScoB NP _ 391777 16080949 Bacillus subtilis panosoma brucei (Riviere et al. 2004 ) and Clostridium OXCT1 NP _ 000427 4557817 Homo sapiens kluyveri ( Sohling and Gottschalk , 1996c ) . The beta -ketoa - OXCT2O NP _ 071403 11545841 Homo sapiens dipate : succinyl -CoA transferase encoded by pcaJ and pcaJ in Pseudomonas putida is yet another candidate (Kaschabek 65 Converting succinate to succinyl -CoA by succinyl- CoA : et al. 2002) . The aforementioned proteins are identified 3 :ketoacid - CoA transferase requires the simultaneous con below . version of a 3 -ketoacyl - CoA such as acetoacetyl- CoA to a US 10 ,006 ,055 B2 123 124 3 -ketoacid such as acetoacetate . Conversion of a 3 - ketoacid Citrate lyase (EC 4 . 1 . 3 .6 ) catalyzes a series of reactions back to a 3 -ketoacyl - CoA can be catalyzed by an resulting in the cleavage of citrate to acetate and oxaloac acetoacetyl- CoA : acetate : CoA transferase. Acetoacetyl etate . The enzyme is active under anaerobic conditions and COA : acetate : COA transferase converts acetoacetyl- CoA is composed of three subunits : an acyl- carrier protein (ACP , and acetate to acetoacetate and acetyl - CoA , or vice versa . 5 gamma) , an ACP transferase (alpha ), and a acyl lyase (beta ). Exemplary enzymes include the gene products of atoAD Enzyme activation uses covalent binding and acetylation of from E . coli (Hanai et al. , Appl Environ Microbiol 73 :7814 an unusual prosthetic group , 2 - (5 " -phosphoribosyl ) - 3 -' -de 7818 ( 2007 ), ctfAB from C . acetobutylicum ( Jojima et al ., phospho - CoA , which is similar in structure to acetyl- CoA . ApplMicrobiol Biotechnol 77 : 1219 - 1224 (2008 ), and ctfAB Acylation is catalyzed by CitC , a citrate lyase synthetase . from Clostridium saccharoperbutylacetonicum (Kosaka et 10 Two additional proteins , CitG and CitX , are used to convert al ., Biosci. Biotechnol Biochem . 71 :58 -68 ( 2007 ) ) are shown the apo enzyme into the active holo enzyme (Schneider et below . al. , Biochemistry 39 : 9438 - 9450 ( 2000 )) . Wild type E . coli does not have citrate lyase activity ; however, mutants defi cient in molybdenum cofactor synthesis have an active Protein GenBank ID GI Number Organism citrate lyase ( Clark , FEMS Microbiol. Lett . 55 :245 - 249 AtoA NP _ 416726 . 1 2492994 Escherichia coli ( 1990 ) ) . The E . coli enzyme is encoded by citEFD and the AtoD NP _ 416725 . 1 2492990 Escherichia coli citrate lyase synthetase is encoded by citC (Nilekani and CtfA NP 149326 . 1 15004866 Clostridium acetobutylicum CtfB NP _ 149327 . 1 15004867 Clostridium acetobutylicum SivaRaman , Biochemistry 22 : 4657 -4663 ( 1983 ) ) . The Leu CtfA AAP42564 . 1 31075384 Clostridium 20 conostoc mesenteroides citrate lyase has been cloned , char saccharoperbutylacetonicum acterized and expressed in E . coli (Bekal et al. , J . Bacteriol. CtfB AAP42565 . 1 31075385 Clostridium 180 :647 -654 ( 1998 ) ) . Citrate lyase enzymes have also been saccharoperbutylacetonicum identified in enterobacteria that utilize citrate as a carbon and energy source, including Salmonella typhimurium and Kleb Yet another possible COA acceptor is benzylsuccinate . 25 siella pneumoniae (Bott , Arch . Microbiol. 167 : 78 - 88 Succinyl - CoA : ( R ) - Benzylsuccinate CoA - Transferase func - ( 1997 ) ; Bott and Dimroth , Mol. Microbiol. 14 : 347 - 356 tions as part of an anaerobic degradation pathway for (1994 ) ) . The aforementioned proteins are tabulated below . toluene in organisms such as Thauera aromatica (Leutwein and Heider, J . Bact. 183 ( 14 ) 4288 - 4295 ( 2001 ) ) . Homologs can be found in Azoarcus sp . T , Aromatoleum aromaticum 30 Protein GenBank ID GI Number Organism EbN1, and Geobacter metallireducens GS- 15 . The afore citF AAC73716 . 1 1786832 Escherichia coli mentioned proteins are identified below . Cite AAC73717 . 2 87081764 Escherichia coli citD AAC73718 . 1 1786834 Escherichia coli citc AAC73719 . 2 87081765 Escherichia coli Protein GenBank ID GI Number Organism citG AAC73714 . 1 1786830 Escherichia coli citx AAC73715 . 1 1786831 Escherichia coli bbsE AAF89840 9622535 Thauera aromatica citF CAA71633 . 1 2842397 Leuconostoc mesenteroides Bbsf AAF89841 9622536 Thauera aromatica Cite CAA71632 . 1 2842396 Leuconostoc mesenteroides bbsE AAU45405 . 1 52421824 Azoarcus sp . T citD CAA71635 . 1 2842395 Leuconostoc mesenteroides bbsF AAU45406 . 1 52421825 Azoarcus sp . T citc CAA71636 . 1 3413797 Leuconostoc mesenteroides bbsE YP _ 158075 . 1 56476486 Aromatoleum aromaticum city CAA71634 . 1 2842398 Leuconostoc mesenteroides EbN1 citx CAA71634 . 1 2842398 Leuconostoc mesenteroides bbsF YP _ 158074 . 1 56476485 Aromatoleum aromaticum citF NP_ 459613 . 1 16763998 Salmonella typhimurium EbN1 cite AAL19573 . 1 16419133 Salmonella typhimurium Gmet_ 1521 YP _ 384480. 1 78222733 Geobacter metallireducens citD NP 459064 . 1 16763449 Salmonella typhimurium GS- 15 citc NP _ 459616 . 1 16764001 Salmonella typhimurium Gmet _ 1522 YP _ 384481. 1 78222734 Geobacter metallireducens cit NP 459611 . 1 16763996 Salmonella typhimurium GS - 15 45 citX NP _ 459612 . 1 16763997 Salmonella typhimurium citF CAA56217 . 1 565619 Klebsiella pneumoniae cite CAA56216 . 1 565618 Klebsiella pneumoniae Additionally , ygfH encodes a propionyl CoA : succinate citD CAA56215 . 1 565617 Klebsiella pneumoniae COA transferase in E . coli (Haller et al ., Biochemistry, cito BAH66541 . 1 238774045 Klebsiella pneumoniae 39 ( 16 ) 4622 - 4629 ) . Close homologs can be found in , forse city CAA56218 . 1 565620 Klebsiella pneumoniae example , Citrobacter youngae ATCC 29220 , Salmonella citX AAL60463. 1 18140907 Klebsiella pneumoniae enterica subsp . arizonae serovar, and Yersinia intermedia ATCC 29909 . The aforementioned proteins are identified below . Acetate kinase (EC 2 . 7 . 2 . 1 ) catalyzes the reversible ATP dependent phosphorylation of acetate to acetylphosphate . 55 EExemplary acetate kinase enzymes have been characterized Protein GenBank ID GI Number Organism in many organisms including E . coli, Clostridium acetobu ygfH NP 417395 . 1 16130821 Escherichia coli tylicum and Methanosarcina thermophila ( Ingram - Smith et str. K - 12 substr . al. , J . Bacteriol. 187 :2386 - 2394 ( 2005 ) ; Fox and Roseman , MG1655 J. Biol. Chem . 261: 13487 - 13497 ( 1986 ) ; Winzer et al. , CIT292 _ 04485 ZP _ 03838384 . 1 227334728 Citrobacter youngae ATCC 29220 Microbioloy 143 (Pt 10 ): 3279 -3286 ( 1997 )) . Acetate kinase SARI_ 04582 YP _ 001573497. 1 161506385 Salmonella enterica activity has also been demonstrated in the gene product of E . subsp . arizonae coli purt (Marolewski et al. , Biochemistry 33 : 2531 - 2537 serovar yinte0001_ 14430 ZP _ 046353641 . 238791727 Yersinia intermedia ( 1994 ) . Some butyrate kinase enzymes ( EC 2 . 7 . 2 . 7 ) , for ATCC 29909 " 65 example buk1 and buk2 from Clostridium acetobutylicum , also accept acetate as a substrate (Hartmanis , M . G . , J . Biol. Chem . 262 :617 -621 ( 1987 ) ) . US 10 ,006 ,055 B2 125 126 (Brasen and Schonheit , supra ( 2004 )) . Directed evolution or Protein GenBank ID GI Number Organism engineering can be used to modify this enzyme to operate at the physiological temperature of the host organism . The ackAAck AAB18301NP _ 416799 . 1. 1 161302311491790 EscherichiaClostridium acetobutylicumcoli enzymes from A . fulgidus, H . marismortui and P . aerophi Ack AAA72042. 1 349834 Methanosarcina thermophila 5 lum have all been cloned , functionally expressed , and char purT AAC74919 . 1 1788155 Escherichia coli buk1 NP _ 349675 15896326 Clostridium acetobutylicum acterized in E . coli (Brasen and Schonheit, supra (2004 ); buk2 Q97111 20137415 Clostridium acetobutylicum Musfeldt and Schonheit , supra (2002 )) . Additional candi dates include the succinyl- CoA synthetase encoded by The formation of acetyl- CoA from acetylphosphate is 10 sucCD in E . coli (Buck et al. , Biochemistry 24 :6245 -6252 catalyzed by phosphotransacetylase (EC 2 . 3 . 1 .8 ) . The pta (1985 )) and the acyl- CoA ligase from Pseudomonas putida gene from E . coli encodes an enzyme that reversibly con (Fernandez - Valverde et al. , Appl. Environ . Microbiol. verts acetyl- CoA into acetyl - phosphate (Suzuki , T ., Biochim . 59 : 1149 - 1154 ( 1993 ) ) . The aforementioned proteins are Biophys. Acta 191 :559 -569 ( 969 )) . Additional acetyltrans tabulated below . ferase enzymes have been characterized in Bacillus subtilis 15 (Rado and Hoch , Biochim . Biophys . Acta 321 : 114 - 125 Protein GenBank ID GI Number Organism ( 1973 ) , Clostridium kluyveri ( Stadtman , E ., Methods Enzy acs AAC77039 . 1 1790505 Escherichia coli mol. 1 : 5896 -599 (1955 ), and Thermotoga maritima (Bock et acoE AAA21945 . 1 141890 Ralstonia eutropha al. , J . Bacteriol. 181 : 1861- 1867 (1999 ) ) . This reaction is acs1 ABC87079 . 1 86169671 Methanothermobacter also catalyzed by some phosphotranbutyrylase enzymes ( EC _ 2 thermautotrophicus acs1 AAL23099. 1 16422835 Salmonella enterica 2 .3 . 1. 19 ) including the ptb gene products from Clostridium ACS1 001574 . 2 257050994 Saccharomyces cerevisiae acetobutylicum ( Wiesenborn et al. , App. Environ . Microbiol. AF1211 NP _ 070039 . 1 11498810 Archaeoglobus fulgidus 55 :317 -322 ( 1989 ) ; Walter et al ., Gene 134: 107 - 111 ( 1993 ) ) . AF1983 NP 070807 . 1 11499565 Archaeoglobus fulgidus Additional ptb genes are found in butyrate - producing bac SCS YP _ 135572 . 1 55377722 Haloarcula marismortui 25 PAE3250 NP _ 560604 . 1 18313937 Pyrobaculum aerophilum str . terium L2 - 50 (Louis et al. , J . Bacteriol. 186 : 2099 -2106 IM2 (2004 ) and Bacillus megaterium (Vazquez et al. , Curr. succ NP _ 415256 . 1 16128703 Escherichia coli Microbiol. 42 : 345 -349 ( 2001) . sucD AAC73823 . 1 1786949 Escherichia coli paar AAC24333. 2 22711873 Pseudomonas putida 30 Protein GenBank ID GI Number Organism The product yields per C -mol of substrate of microbial Pta NP _ 416800 . 1 71152910 Escherichia coli cells synthesizing reduced fermentation products such as Pta P39646 730415 Bacillus subtilis butadiene or crotyl alcohol, are limited by insufficientreduc Pta A5N801 146346896 Clostridium kluyveri Pta Q9XOL4 6685776 Thermotoga maritima ing equivalents in the carbohydrate feedstock . Reducing Ptb NP _ 349676 34540484 Clostridium acetobutylicum 35 equivalents , or electrons, can be extracted from synthesis Ptb AAR19757. 1 38425288 butyrate - producing bacterium gas components such as CO and H , using carbon monoxide L2 - 50 dehydrogenase (CODH ) and hydrogenase enzymes , respec Ptb CAC07932. 1 10046659 Bacillus megaterium tively . The reducing equivalents are then passed to acceptors such as oxidized ferredoxins , oxidized quinones , oxidized The acylation of acetate to acetyl- CoA is catalyzed by 40 cytochromes, NAD ( P ) + , water, or hydrogen peroxide to enzymes with acetyl- CoA synthetase activity . Two enzymes form reduced ferredoxin , reduced quinones , reduced that catalyze this reaction are AMP - forming acetyl- CoA cytochromes , NAD ( P ) H , H2, or water, respectively . synthetase (EC 6 .2 . 1. 1 ) and ADP - forming acetyl -CoA syn - Reduced ferredoxin and NAD (P )H are particularly useful as thetase ( EC 6 . 2 . 1 . 13 ) . AMP - forming acetyl- CoA synthetase they can serve as redox carriers for various Wood - Ljungdahl ( ACS ) is the predominant enzyme for activation of acetate 45 pathway and reductive TCA cycle enzymes . to acetyl- CoA . Exemplary ACS enzymes are found in E . coli Here , we show specific examples of how additional redox (Brown et al. , J . Gen . Microbiol. 102 : 327 - 336 ( 1977 ) ) , availability from CO and /or H , can improve the yields of Ralstonia eutropha (Priefert and Steinbuchel , J. Bacteriol. reduced products such as butadiene or crotyl alcohol. 174 :6590 -6599 ( 1992 )) , Methanothermobacter thermauto - The maximum theoretical yield to produce butadiene trophicus ( Ingram - Smith and Smith , Archaea 2 : 95 - 107 50 from glucose is 1 mole /mole ( 0 . 3 g / g ) based on the pathway ( 2007 ) ) , Salmonella enterica (Gulick et al. , Biochemistry described in FIG . 2 . For the pathway described in FIG . 4 , the 42 : 2866 - 2873 ( 2003 )) and Saccharomyces cerevisiae (Jogl maximum theoretical yield under aerobic conditions is 0 . 28 and Tong , Biochemistry 43 : 1425 - 1431 ( 2004 ) ) . ADP - form - g / g . The maximum theoretical yield based on stoichiometry ing acetyl- CoA synthetases are reversible enzymes with a is 1 .09 mole /mole ( 0 .33 g / g ). Using rTCA and hydrogen , generally broad substrate range (Musfeldt and Schonheit , J . 55 this yield can be improved to 2 mole/ mole glucose ( 0 . 6 g / g ) . Bacteriol. 184 :636 -644 ( 2002 ) ) . Two isozymes of ADP - Similar yield improvements can be attained for crotyl alco forming acetyl- CoA synthetases are encoded in the Archaeo - hol via the proposed routes. globus fulgidus genome by are encoded by AF1211 and When both feedstocks of sugar and syngas are available , AF1983 (Musfeldt and Schonheit , supra (2002 )) . The the syngas components CO and H , can be utilized to enzyme from Haloarcula marismortui (annotated as a suc - 60 generate reducing equivalents by employing the hydroge cinyl- CoA synthetase ) also accepts acetate as a substrate and nase and CO dehydrogenase . The reducing equivalents reversibility of the enzyme was demonstrated (Brasen and generated from syngas components will be utilized to power Schonheit, Arch . Microbiol. 182 :277 -287 (2004 )) . The ACD the glucose to butadiene or crotyl alcohol production path encoded by PAE3250 from hyperthermophilic crenarchaeon ways. Pyrobaculum aerophilum showed the broadest substrate 65 As shown in above example , a combined feedstock strat range of all characterized ACDs, reacting with acetate , egy where syngas is combined with a sugar -based feedstock isobutyryl- CoA (preferred substrate ) and phenylacetyl- CoA or other carbon substrate can greatly improve the theoretical US 10 ,006 ,055 B2 127 128 yields. In this co - feeding approach , syngas components HQ Bacteriol. 183 :5134 -5144 (2001 ) ) . The crystal structure of and CO can be utilized by the hydrogenase and CO dehy - the CODH - II is also available ( Dobbek et al. , Science drogenase to generate reducing equivalents , that can be used 293 : 1281 - 1285 (2001 ) ). Similar ACS - free CODH enzymes to power chemical production pathways in which the car can be found in a diverse array of organisms including bons from sugar or other carbon substrates will be maxi- 5 mally conserved and the theoretical yields improved . In case Geobacter metallireducens GS - 15 , Chlorobium phaeobacte of butadiene or crotyl alcohol production from glucose or roides DSM 266 , Clostridium cellulolyticum H10 , Desulfo sugar, the theoretical yields improve from 1 .09 mol butadi vibrio desulfuricans subsp . desulfuricans str. ATCC 27774 , ene or crotyl alcohol per mol of glucose to 2 mol butadiene Pelobacter carbinolicus DSM 2380 , and Campylobacter or crotyl alcohol per mol of glucose . Such improvements curvus 525 . 92 .

Protein GenBank ID GI Number Organism CODH (putative ) YP _ 430813 83590804 Moorella thermoacetica CODH - II (COOS - II) YP _ 358957 78044574 Carboxydothermus hydrogenoformans CooF YP _ 358958 78045112 Carboxydothermus hydrogenoformans CODH (putative ) ZP _ 05390164 . 1 255523193 Clostridium carboxidivorans P7 CcarbDRAFT_ 0341 ZP 05390341 . 1 255523371 Clostridium carboxidivorans P7 CcarbDRAFT _ 1756 ZP _ 05391756 . 1 255524806 Clostridium carboxidivorans P7 CcarbDRAFT 2944 ZP _ 05392944 . 1 255526020 Clostridium carboxidivorans P7 CODH YP 384856 . 1 78223109 Geobacter metallireducens GS - 15 Cpha266 _ 0148 YP 910642. 1 119355998 Chlorobium phaeobacteroides (cytochrome c ) DSM 266 Cpha266 _ 0149 YP _ 910643. 1 119355999 Chlorobium phaeobacteroides (CODH ) DSM 266 Ccel _ 0438 YP _ 002504800 . 1 220927891 Clostridium cellulolyticum H10 Ddes _ 0382 (CODH ) YP _ 002478973 . 1 220903661 Desulfovibrio desulfuricans subsp . desulfuricans str. ATCC 27774 Ddes _ 0381 (CooC ) YP _ 002478972. 1 220903660 Desulfovibrio desulfuricans subsp . desulfuricans str. ATCC 27774 Pcar _ 0057 ( CODH ) YP _ 355490 . 1 7791767 Pelobacter carbinolicus DSM 2380 Pcar _ 0 YP 355491 . 1 7791766 Pelobacter carbinolicus DSM 2380 Pcar _ 0058 (HypA ) YP _ 355492. 1 7791765 Pelobacter carbinolicus DSM 2380 CooS (CODH ) YP _ 001407343 . 1 154175407 Campylobacter curvus 525 .92 provide environmental and economic benefits and greatly In some cases , hydrogenase encoding genes are located enhance sustainable chemical production . adjacent to a CODH . In Rhodospirillum rubrum , the Herein below the enzymes and the corresponding genes 35 encoded CODH /hydrogenase proteins form a membrane used for extracting redox from synags components are bound enzyme complex that has been indicated to be a site described . CODH is a reversible enzyme that interconverts where energy , in the form of a proton gradient, is generated CO and CO , at the expense or gain of electrons. The natural from the conversion of CO and H , O to CO , and H , ( Fox et physiological role of the CODH in ACS/ CODH complexes al. , J Bacteriol. 178 :6200 -6208 ( 1996 ) ). The CODH - I of C . is to convert CO2 to CO for incorporation into acetyl- CoA 40 hydrogenoformans and its adjacent genes have been pro by acetyl- CoA synthase . Nevertheless , such CODH posed to catalyze a similar functional role based on their enzymes are suitable for the extraction of reducing equiva - similarity to the R . rubrum CODH /hydrogenase gene cluster lents from CO due to the reversible nature of such enzymes . (Wu et al. , PLoS Genet. 1 : e65 ( 2005 ) ) . The C . hydrogeno Expressing such CODH enzymes in the absence of ACS formans CODH -I was also shown to exhibit intense CO allows them to operate in the direction opposite to their + oxidation and CO2 reduction activities when linked to an natural physiological role ( i. e ., CO oxidation ). electrode (Parkin et al. , J Am . Chem . Soc . 129 : 10328 - 10329 In M . thermoacetica , C . hydrogenoformans, C . carbox ( 2007 ) ) . The protein sequences of exemplary CODH and idivorans P7, and several other organisms, additional CODH hydrogenase genes can be identified by the following Gen encoding genes are located outside of the ACS/ CODH 50 Bank accession numbers. operons . These enzymes provide a means for extracting electrons ( or reducing equivalents ) from the conversion of carbon monoxide to carbon dioxide . The M . thermoacetica Protein GenBank ID GI Number Organism gene (Genbank Accession Number : YP _ 430813 ) is CODH - I YP _ 360644 78043418 Carboxydothermus expressed by itself in an operon and is believed to transfer 55 (CooS -I ) hydrogenoformans Coor YP _ 360645 78044791 Carboxydothermus electrons from CO to an external mediator like ferredoxin in hydrogenoformans a “ Ping -pong ” reaction . The reduced mediator then couples HypA YP _ 360646 78044340 Carboxydothermus to other reduced nicolinamide adenine dinucleotide phos hydrogenoformans phate (NAD ( P ) H ) carriers or ferredoxin - dependent cellular CooH YP _ 360647 78043871 Carboxydothermus hydrogenoformans processes (Ragsdale , Annals of the New York Academy of 60 cm YP _ 360648 78044023 Carboxydothermus Sciences 1125 : 129 - 136 ( 2008 ) ) . The genes encoding the C . hydrogenoformans hydrogenoformans CODH - II and CooF , a neighboring pro YP _ 360649 78043124 Carboxydothermus Coox hydrogenoformans tein , were cloned and sequenced (Gonzalez and Robb , CooL YP _ 360650 78043938 Carboxydothermus FEMS Microbiol Lett. 191: 243 - 247 (2000 ) ) . The resulting hydrogenoformans complex was membrane -bound , although cytoplasmic frac - 65 COOK YP 360651 78044700 Carboxydothermus tions of CODH - II were shown to catalyze the formation of hydrogenoformans NADPH suggesting an anabolic role (Svetlitchnyi et al. , J