Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds

Abstract

The invention provides non-naturally occurring microbial organisms having a 4-hydroxybutyrate, gamma-butyrolactone, 1,4-butanediol, 4-hydroxybutanal, 4-hydroxybutyryl-CoA and/or putrescine pathway and being capable of producing 4-hydroxybutyrate, wherein the microbial organism comprises one or more genetic modifications. The invention additionally provides methods of producing 4-hydroxybutyrate, gamma-butyrolactone, 1,4-butanediol, 4-hydroxybutanal, 4-hydroxybutyryl-CoA and/or putrescine or related products using the microbial organisms.

1 . An isolated nucleic acid molecule selected from:
 (a) a nucleic acid molecule encoding an amino acid sequence referenced in Tables 53, 55 or 57, wherein said amino acid sequence comprises one or more variant amino acid positions set forth in Tables 54, 56 or 58 or Example XXXXIII;   (b) a nucleic acid molecule that hybridizes to the nucleic acid of (a) under highly stringent hybridization conditions and comprises a nucleic acid sequence that encodes one or more variant amino acid positions set forth in Tables 54, 56 or 58 or Example XXXXIII; and   (c) a nucleic acid molecule that is complementary to (a) or (b);   
       with the proviso that the encoded amino acid sequence is not the wild type parental sequence or a naturally occurring amino acid sequence. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein said amino acid sequence has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity, or is identical, to the amino acid sequence referenced in Table 53, 55 or 57. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein said amino acid sequence comprises at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more variants set forth in Tables 54, 56 or 58 or Example XXXXIII. 
     
     
         4 . A vector containing the nucleic acid molecule of  claim 1 . 
     
     
         5 . (canceled) 
     
     
         6 . A host cell comprising the vector of  claim 4 . 
     
     
         7 - 9 . (canceled) 
     
     
         10 . The host cell of  claim 6 , wherein said host cell is a non-naturally occurring microbial organism having a pathway that produces 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine. 
     
     
         11 - 12 . (canceled) 
     
     
         13 . Culture medium comprising the host cell of  claim 6 . 
     
     
         14 . An isolated polypeptide comprising an amino acid sequence referenced in Tables 53, 55 or 57, wherein said amino acid sequence comprises one or more variant amino acid positions set forth in Tables 54, 56 or 58 or Example XXXXIII. 
     
     
         15 - 19 . (canceled) 
     
     
         20 . A host cell expressing the polypeptide of  claim 14 . 
     
     
         21 . (canceled) 
     
     
         22 . A composition comprising the polypeptide of  claim 14  and at least one substrate for said polypeptide. 
     
     
         23 - 24 . (canceled) 
     
     
         25 . A method for producing 4-hydroxybutyrate (4-HB), gamma-butyrolactone (GBL), 4-hydroxybutyraldehyde (4-HBal), 4-hydroxybutyryl-CoA (4-HBCoA), 1,4-butanediol (BDO) and/or putrescine comprising culturing said host cell of  claim 6  under conditions and for a sufficient period of time to produce 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine. 
     
     
         26 . A method for producing 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine, comprising culturing a non-naturally occurring microbial organism having a pathway that produces 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine under conditions and for a sufficient period of time to produce 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine, wherein said microbial organism comprises a nucleic acid of  claim 1 . 
     
     
         27 . (canceled) 
     
     
         28 . A non-naturally occurring microbial organism, said microbial organism having a 4-hydroxybutyrate pathway and being capable of producing 4-hydroxybutyrate, wherein said microbial organism comprises a genetic modification, said genetic modification selected from:
 (A) a genetic modification that increases expression of acetate kinase and phosphotransacetylase;   (B) a genetic modification that inactivates expression of an aerobic respiratory control regulatory system;   (C) a genetic modification that truncates a glucose phosphotransferase system (PTS) permease;   (D) a genetic modification that increases expression of phosphoenolpyruvate carboxylase;   (E) a genetic modification that increases expression of alpha-ketoglutarate dehydrogenase;   (F) a genetic modification that increases expression of a non-phosphotransferase (PTS) glucose uptake system;   (G) a genetic modification that increases expression of a gamma-butyrolactone esterase;   (H) a genetic modification that decreases expression of succinyl-CoA synthetase;   (I) a genetic modification that decreases expression of an acyl coenzyme A thioesterase;   (J) a genetic modification that decreases expression of an alcohol dehydrogenase;   (K) a genetic modification that decreases expression of a non-energy-producing NADH dehydrogenase;   (L) a genetic modification that decreases expression of a cytochrome oxidase; and   (M) a combination of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all of the genetic modifications of parts (A)-(L).   
     
     
         29 - 34 . (canceled) 
     
     
         35 . A method for producing 4-hydroxybutyrate, comprising culturing the non-naturally occurring microbial organism of  claim 28  under conditions and for a sufficient period of time to produce 4-hydroxybutyrate. 
     
     
         36 . A method for producing 1,4-butanediol, comprising culturing the non-naturally occurring microbial organism of  claim 28  under conditions and for a sufficient period of time to produce 1,4-butanediol. 
     
     
         37 - 38 . (canceled) 
     
     
         39 . A method for producing isolated or purified 4-hydroxybutyrate or 1,4-butanediol, comprising culturing the non-naturally occurring microbial organism of  claim 28  under conditions and for a sufficient period of time to produce 4-hydroxybutyrate or 1,4-butanediol, and isolating or purifying the 4-hydroxybutyrate or 1,4-butanediol. 
     
     
         40 . (canceled) 
     
     
         41 . Culture medium comprising bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine, wherein said bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine has a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source, and wherein the bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine is produced by the host cell of  claim 6 . 
     
     
         42 . (canceled) 
     
     
         43 . Bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine having a carbon-12, carbon-13 and carbon-14 isotope ratio that reflects an atmospheric carbon dioxide uptake source, and wherein the bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine is produced by the host cell of  claim 6 . 
     
     
         44 . (canceled) 
     
     
         45 . A bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine produced according to the method of  claim 35 . 
     
     
         46 - 48 . (canceled) 
     
     
         49 . A biobased product comprising said bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine of  claim 43 , wherein said biobased product is a plastic, elastic fiber, polyurethane, polyester, polyhydroxyalkanoate, poly-4-hydroxybutyrate (P4HB), or a co-polymer thereof, poly(tetramethylene ether) glycol (PTMEG), polybutylene terephthalate (PBT), polyurethane-polyurea copolymer, or nylon. 
     
     
         50 - 51 . (canceled) 
     
     
         52 . A molded product obtained by molding a biobased product of  claim 49 . 
     
     
         53 . A process for producing a biobased product of  claim 49 , comprising chemically reacting said bioderived 4-HB, GBL, 4-HBal, 4-HBCoA, BDO and/or putrescine with itself or another compound in a reaction that produces said biobased product. 
     
     
         54 - 56 . (canceled)