Microorganisms for Producing 1,3-Butanediol and Methods Related Thereto

Abstract

Provided herein is a non-naturally occurring microbial organism having a 1,3-butanediol (1,3-BDO) pathway and comprising at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme expressed in a sufficient amount to produce 1,3-BDO. In some embodiments, the pathway includes reducing equivalents from CO or hydrogen. In certain embodiments, a 1,3-BDO pathway proceeds by way of central metabolites pyruvate, succinate or alpha-ketoglutarate. Also provided herein is a method for producing 1,3-BDO, includes culturing such microbial organisms under conditions and for a sufficient period of time to produce 1,3-BDO.

1 . A non-naturally occurring microbial organism having a 1,3-butanediol pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a 1,3-butanediol pathway enzyme expressed in a sufficient amount to produce 1,3-butanediol; said non-naturally occurring microbial organism further comprising:
 (i) a reductive TCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme selected from the group consisting of an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase;   (ii) a reductive TCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme selected from the group consisting of a pyruvate:ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H 2  hydrogenase; or   (iii) at least one exogenous nucleic acid encoding an enzyme selected from the group consisting of a CO dehydrogenase, an H 2  hydrogenase, and combinations thereof;   
       wherein said 1,3-butanediol pathway comprises a pathway selected from the group consisting of:
 (a) (1) a 2-amino-4-ketopentanoate (AKP) thiolase; (2) an AKP dehydrogenase; (3) a 2-amino-4-hydroxypentanoate aminotransferase or oxidoreductase (deaminating); (4) a 2-oxo-4-hydroxypentanoate decarboxylase; and (5) a 3-hydroxybutyraldehyde reductase; 
 (b) (1) an AKP thiolase; (2) an AKP aminotransferase or oxidoreductase 
 (deaminating); (3) a 2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde reductase (ketone reducing); and (5) a 3-hydroxybutyraldehyde reductase; 
 (c) (1) an AKP thiolase; (2) an AKP aminotransferase or oxidoreductase 
 (deaminating); (3) a 2,4-dioxopentanoate decarboxylase; (4) a 3-oxobutyraldehyde reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone reductase; 
 (d) (1) an AKP thiolase; (2) an AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase (ketone reducing); and (5) a 3-hydroxybutyraldehyde reductase; 
 (e) (1) an AKP thiolase; (2) an AKP decarboxylase; (3) a 4-aminobutan-2-one aminotransferase or oxidoreductase (deaminating); (4) a 3-oxobutyraldehyde reductase (aldehyde reducing); and (5) a 4-hydroxy-2-butanone reductase; 
 (f) (1) an AKP thiolase; (2) an AKP decarboxylase; (3) a 4-aminobutan-2-one ammonia-lyase; (4) a butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase; 
 (g) (1) an AKP thiolase; (2) an AKP ammonia-lyase; (3) an acetylacrylate decarboxylase; (4) a butenone hydratase; and (5) a 4-hydroxy-2-butanone reductase; 
 (h) (1) an acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming); (2) a 3-oxobutyraldehyde reductase (ketone reducing); and (3) a 3-hydroxybutyraldehyde reductase; 
 (i) an acetoacetyl-CoA reductase (CoA dependent, alcohol forming) and (2) a 4-hydroxy-2-butanone reductase; 
 (j) (1) an acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming); (2) a 3-oxobutyraldehyde reductase (aldehyde reducing); and (3) a 4-hydroxy-2-butanone reductase; 
 (k) (1) an acetoacetyl-CoA reductase (ketone reducing) and (2) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (l) (1) an acetoacetyl-CoA reductase (ketone reducing); (2) a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and (3) a 3-hydroxybutyraldehyde reductase; 
 (m) (1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase; and (3) a 3-hydroxybutyryl-CoA reductase (alcohol forming); and 
 (n) (1) a 4-hydroxybutyryl-CoA dehydratase; (2) a crotonase; (3) a 3-hydroxybutyryl-CoA reductase (aldehyde forming); and (4) a 3-hydroxybutyraldehyde reductase; 
 (o) (1) a succinyl-CoA transferase, a succinyl-CoA synthetase or a succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyrate kinase, (5) a phosphotrans-4-hydroxybutyrylase, (6) a 4-hydroxybutyryl-CoA dehydratase, (7) a crotonase, (8) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), and (9) a 3-hydroxybutanal reductase; 
 (p) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a glutamate transaminase, (b) a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase; (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), and (8) a 3-hydroxybutanal reductase; 
 (q) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a glutamate transaminase, (b) a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), and (7) a 3-hydroxybutanal reductase; 
 (r) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a glutamate transaminase, (b) a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (s) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a glutamate transaminase, (b) a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (t) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a glutamate transaminase, (b) a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (8) a 3-hydroxybutyrate reductase; 
 (u) (1) (i) an alpha-ketoglutarate decarboxylase, or (ii) (a) a glutamate dehydrogenase and/or a glutamate transaminase, (b) a glutamate decarboxylase, and (c) a 4-aminobutyrate dehydrogenase and/or a 4-aminobutyrate transaminase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (7) a 3-hydroxybutyrate reductase. 
 (v) (1) a succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), and (8) a 3-hydroxybutanal reductase; 
 (w) (1) a succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), (7) a 3-hydroxybutanal reductase; 
 (x) (1) a succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (y) (1) a succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (z) (1) a succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (8) a 3-hydroxybutyrate reductase; 
 (aa) (1) a succinate reductase, (2) a 4-hydroxybutyrate dehydrogenase, (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (7) a 3-hydroxybutyrate reductase; 
 (bb) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (5) a 4-hydroxybutyrate kinase, (6) a pPhosphotrans-4-hydroxybutyrylase, (7) a 4-hydroxybutyryl-CoA dehydratase, (8) a crotonase, and (9) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (cc) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyrate kinase, (5) a phosphotrans-4-hydroxybutyrylase, (6) a 4-hydroxybutyryl-CoA dehydratase, (7) a crotonase, (8) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (9) a 3-hydroxybutyrate reductase; 
 (dd) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), and (8) a 3-hydroxybutanal reductase; 
 (ee) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (ff) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a 4-hydroxybutyrate kinase, (4) a phosphotrans-4-hydroxybutyrylase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (8) a 3-hydroxybutyrate reductase; 
 (gg) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyryl-CoA transferase, or 4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA reductase (aldehyde forming), and (8) a 3-hydroxybutanal reductase; 
 (hh) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, and (7) a 3-hydroxybutyryl-CoA reductase (alcohol forming); 
 (ii) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (aldehyde forming), (3) a 4-hydroxybutyrate dehydrogenase, (4) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, (8) a 3-hydroxybutyrate reductase; 
 (jj) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, (6) a 3-hydroxybutyryl-CoA reductase (aldehyde faulting), and (7) a 3-hydroxybutanal reductase; 
 (kk) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (4) a 4-hydroxybutyryl-CoA dehydratase, (5) a crotonase, and (6) a 3-hydroxybutyryl-CoA reductase (alcohol forming); and 
 (ll) (1) a succinyl-CoA transferase, succinyl-CoA synthetase or succinyl-CoA ligase, (2) a succinyl-CoA reductase (alcohol forming), (3) a 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA synthetase, (5) a 4-hydroxybutyryl-CoA dehydratase, (6) a crotonase, (7) a 3-hydroxybutyryl-CoA hydrolase, transferase or synthetase, and (8) a 3-hydroxybutyrate reductase. 
 
     
     
         2 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, a ferredoxin, and combinations thereof. 
     
     
         3 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. 
     
     
         4 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism comprises two, three, four, five, six, seven, eight or nine exogenous nucleic acids, each encoding a 1,3-BDO pathway enzyme. 
     
     
         5 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the 1,3-butanediol pathways selected from the group consisting of (a)-(ll). 
     
     
         6 . The non-naturally occurring microbial organism of  claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         7 . The non-naturally occurring microbial organism of  claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         8 . A method for producing 1,3-BDO, comprising culturing a non-naturally occurring microbial organism according to  claim 1 , under conditions and for a sufficient period of time to produce 1,3-BDO. 
     
     
         9 . The method of  claim 8 , wherein said microbial organism further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of a pyruvate:ferredoxin oxidoreductase, an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof. 
     
     
         10 . The method of  claim 8 , wherein said microbial organism further comprises an exogenous nucleic acid encoding an enzyme selected from the group consisting of an aconitase, an isocitrate dehydrogenase, a succinyl-CoA synthetase, a succinyl-CoA transferase, a fumarase, a malate dehydrogenase, and combinations thereof. 
     
     
         11 . The method of  claim 8 , wherein said microbial organism comprises two, three, four, five, six, seven, eight or nine exogenous nucleic acids, each encoding a 1,3-BDO pathway enzyme. 
     
     
         12 . The method of  claim 8 , wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the 1,3-butanediol pathways selected from the group consisting of (a)-(ll). 
     
     
         13 . The method of  claim 8 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         14 . The method of  claim 8 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.