Production of fatty acids & derivatives thereof

Abstract

Genetically engineered microorganisms are provided that produce products from the fatty acid biosynthetic pathway (fatty acid derivatives), as well as methods of their use.

1 . A microorganism comprising:
 (a) one or more exogenous nucleic acid sequences encoding at least one peptide selected from accA (EC 6.4.1.2), accB (EC 6.4.1.2), accC (EC 6.4.1.2), accD (EC 6.4.1.2), aceE (EC 1.2.4.1, 2.3.1.61, 2.3.1.12), aceF (EC 1.2.4.1, 2.3.4.16, 2.3.1.12), acpP (AAC74178), fadD (EC 2.3.1.86), cer1 (EC 4.1.99.5), fabA (EC4.2.1.60), fabB (EC 2.3.1.41), fabD (EC 2.3.1.39), fabG (EC 1.1.1.100), fabH (EC 2.3.1.180), fabI (EC 1.3.1.9), fabZ(EC 4.2.1.-), lipase (EC 3.1.1.3), malonyl-CoA decarboxylase (EC 4.1.1.9, 4.1.1.41), panD (EC 4.1.1.11), panK (EC 2.7.1.33), pdh (EC 1.2.4.1), and udhA (EC 1.6.1.1);   (b) a nucleic acid sequence encoding a wax synthase (EC 2.3.1.75) or an alcohol acetyltransferase (EC 2.3.1.84).   
     
     
         2 - 4 . (canceled) 
     
     
         5 . The microorganism of  claim 1 , wherein ackA (EC 2.7.2.1), ackB (EC 2.7.2.1), adhE (EC 1.1.1.1, 1.2.1.10), fabF (EC 2.3.1.179), fabR (accession NP — 418398), fadE (EC 1.3.99.3, 1.3.99.-), GST (EC 6.3.2.3), gpsA (EC 1.1.1.94), ldhA (EC 1.1.1.28), pflB (EC 2.3.1.54), plsB (EC 2.3.1.15), poxB (EC 1.2.2.2), pta (EC 2.3.1.8), or glutathione synthase (EC 6.3.2.3) is attenuated. 
     
     
         6 - 10 . (canceled) 
     
     
         11 . The microorganism of  claim 1 , wherein the microorganism additionally comprises:
 (a) an exogenous nucleic acid sequence encoding a thioesterase (EC 3.1.2.-, 3.1.1.-);   (b) at least one exogenous nucleic acid sequence encoding an enzyme selected from one or more components of the branch chain keto acid dehydrogenase complex (EC 1.2.4.4), llve (EC 2.6.1.42), lpd (EC 1.8.1.4), Ccr (EC1.1.19), IcmA (EC5.4.99.2), IcmB (5.4.99.13), fabH (EC 2.3.1.180), fabF (EC 2.3.1.179), fabH3 (EC 2.3.1.180), fabC3(NP 823468), beta-ketoacyl-ACP synthase II (EC 2.3.1.180), enoyl-CoA reductase (EC 1.3.1.34), enoyl-CoA isomerase (EC 4.2.1.-), and combinations thereof;   (c) at least one exogenous nucleic acid sequence encoding an enzyme selected from FabB (EC 2.3.1.41), FabK (EC 1.2.1.9), FabL (EC 1.2.1.9), FabM (EC 5.3.3.14), FadE (EC 1.3.99.3, 1.3.99.-), and combinations thereof;   (d) an exogenous nucleic acid sequence encoding ACP, Sfa, or combinations thereof; or   (e) an exogenous nucleic acid sequence encoding an enzyme selected from FadA (EC 2.3.1.16), FadI (EC 2.3.1.16), FadB (EC 1.1.1.35), FadJ (EC 4.2.1.17, EC 5.1.2.3, EC 5.3.3.8, EC 1.1.1.35), and combinations thereof.   
     
     
         12 - 15 . (canceled) 
     
     
         16 . The microorganism of  claims 1 , wherein accA, accB, accC, accD, or fadD is over-expressed. 
     
     
         17 . The microorganism of  claims 1 , wherein the microorganism is in a vessel comprising a fermentation broth comprising at least 10 mg/L fatty acid ester or at least 10 mg/L wax. 
     
     
         18 . A fatty acid derivative produced by the microorganism of  claim 1 , wherein the fatty acid derivative comprises:
 (a) from about 1 to about 5 double bonds, a carbon chain length of from about 8 to about 30, about 1 to about 5 branch points, or between 1 and 10 cyclopropyl moieties:   (b) an A side and a B side, wherein the A side, the B side, or both the A side and the B side, are produced by the microorganism;   (c) an A side and a B side, wherein the A side, the B side, or both the A side and the B side, comprise from about 1 to about 5 double bonds;   (d) an A side and a B side, wherein the A side, the B side, or both the A side and the B side, comprise a carbon chain length of from about 1 to about 26;   (e) an A side and a B side, wherein the A side, the B side, or both the A side and the B side, comprise from about 1 to about 5 branch points; or   (f) an A side and a B side, wherein the A side, the B side, or both the A side and the B side, comprise between 1 and 5 cyclopropyl moieties.   
     
     
         19 - 26 . (canceled) 
     
     
         27 . The microorganism of  claims 1 , wherein the microorganism is an  E. coli, Arthrobacter  sp.,  Bacillus  sp.,  Botryococcus braunii, Chromatium  sp.,  Cladosporium resina  (ATCC22711),  Clostridium pasteurianum  VKM,  Clostridium tenanomorphum, Clostridium acidiurici, Corynebacterium  species,  cyanobacterial  species ( Nostoc muscorum, Anacystis  ( Synechococcus )  nidulans, Phormidium luridum, Chlorogloea fritschii, Trichodesmium erythaeum, Oscillatoria williamsii, Microcoleus chthonoplaseis, Coccochloris elabens, Agmenellum quadruplicatum, Plectonema terebrans, M vaginatus,  and  C. scopulorum ),  Desulfovibrio desulfuricans  (ATCC29577),  Kineococcus radiotolerans  (BAA-149),  Micrococcus luteus  (FD533, ATCC 272, 381, 382, ISU, 540, 4698, 7468, 27141),  Micrococcus  sp. (ATCC 146, 398, 401, 533),  Micrococcus roseus  (ATCC 412, 416, 516),  Micrococcus lysodeikticus, Mycobacterium  species,  Penicillium  sp.,  Aspergillus  sp.,  Trichoderma virida, Pullularia pullulans, Jeotgalicoccus  sp. ( M. candicans )(ATCC 8456),  Rhodopseudomonas spheroids Chlorobium  sp.,  Rhodospirillium rubrum  (ATCC11170),  Rhodomicrobium vannielii, Stenotrophomonas maltophilia  (ATCC 13637, 17444, 17445, 17666, 17668, 17673, 17674, 17679, 17677),  Saccharomycodes ludwigii  (ATCC 22711),  Saccharomyces  sp. ( oviformus,ludwiggi, tropicalis ),  Vibrio furnissii  M1 , Vibrio marinus  MP-1,  Vibrio ponticus, Serratia marinorubra, Ustilago maydis, Ustilago nuda, Urocystis agropyri, Sphacelotheca reiliana,  or  Tilletia  sp. ( foetida, caries, controversa ). 
     
     
         28 . A method of producing a fatty acid derivative comprising:
 (a) culturing the microorganism of  claim 1  under conditions sufficient to produce a fatty acid derivative; and   (b) separating the fatty acid derivative.   
     
     
         29 . The method of  claim 28 , wherein the microorganism further comprises:
 (a) an exogenous nucleic acid sequence encoding a thioesterase (EC 3.1.2.-, 3.1.1.-);   (b) at least one exogenous nucleic acid sequence encoding an enzyme selected from one or more components of the branch chain keto acid dehydrogenase complex (EC 1.2.4.4), llve (EC 2.6.1.42), lpd (EC 1.8.1.4), Ccr (EC1.1.19), IcmA (EC5.4.99.2), IcmB (5.4.99.13), fabH (EC 2.3.1.180), fabF (EC 2.3.1.179), fabH3 (EC 2.3.1.180), fabC3(NP 823468), beta-ketoacyl-ACP synthase II (EC 2.3.1.180), enoyl-CoA reductase (EC 1.3.1.34), enoyl-CoA isomerase (EC 4.2.1.-), and combinations thereof;   (c) at least one exogenous nucleic acid sequence encoding an enzyme selected from FabB (EC 2.3.1.41), FabK (EC 1.2.1.9), FabL (EC 1.2.1.9), FabM (EC 5.3.3.14), FadE (EC 1.3.99.3, 1.3.99.-), and combinations thereof;   (d) an exogenous nucleic acid sequence encoding ACP, Sfa, or combinations thereof; or   (e) an exogenous nucleic acid sequence encoding an enzyme selected from FadA (EC 2.3.1.16), FadI (EC 2.3.1.16), FadB (EC 1.1.1.35), FadJ (EC 4.2.1.17, EC 5.1.2.3, EC 5.3.3.8, EC 1.1.1.35), and combinations thereof.   
     
     
         30 . The method of  claim 28 , wherein ackA (EC 2.7.2.1), ackB (EC 2.7.2.1), adhE (EC 1.1.1.1, 1.2.1.10), fabF (EC 2.3.1.179), fabR (accession NP 418398), fadE (EC 1.3.99.3, 1.3.99.-), GST (EC 6.3.2.3), gpsA (EC 1.1.1.94), ldhA (EC 1.1.1.28), pflB (EC 2.3.1.54), plsB (EC 2.3.1.15), poxB (EC 1.2.2.2), pta (EC 2.3.1.8), or glutathione synthase (EC 6.3.2.3) of the microorganism is attenuated. 
     
     
         31 . The microorganism of  claim 1 , which produces an increased amount of fatty acid derivatives as compared to the wild-type microorganism. 
     
     
         32 - 36 . (canceled) 
     
     
         37 . The method of  claim 28 , wherein separating the fatty acid derivative comprises:
 allowing the fatty acid derivative to separate into an organic phase; and   purifying the fatty acid derivative from the organic phase.   
     
     
         38 . (canceled) 
     
     
         39 . A biofuel composition, comprising:
 at least about 11% or at least about 17% of a fatty acid derivative, wherein the fatty acid derivative comprises a carbon chain selected from the group consisting of 8:0, 10:0, 12:0, 14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 22:0, 22:1 or 22:3; and   at least about 80% conventional diesel fuel.   
     
     
         40 . The biofuel composition of  claim 39  wherein the-fatty acid derivative has δ 13 C of from about −10.9 to about −15.4, or a fraction of modern carbon of at least about 1.003. 
     
     
         41 . (canceled) 
     
     
         42 . The biofuel composition of  claim 40 , wherein the fatty acid derivative accounts for at least about 85% of biosourced fatty acid-derived material in the composition. 
     
     
         43 - 46 . (canceled) 
     
     
         47 . The biofuel composition of  claim 39 , further comprising a lower alcohol, a surfactant, or a microemulsion in the biofuel composition. 
     
     
         48 - 57 . (canceled) 
     
     
         58 . The biofuel composition of  claim 39 , wherein the biofuel comprises less than 0.1% glycerin, or less than 0.1% transesterification catalyst. 
     
     
         59 . (canceled) 
     
     
         60 . A fatty acid derivative produced by the method of  claim 28 . 
     
     
         61 . A biofuel composition comprising the fatty acid derivative of  claim 60 . 
     
     
         62 . The biofuel composition of  claim 61 , comprising at least about 11%, or at least about 17% of the fatty acid derivative, and at least about 80% of conventional diesel fuel.