MICROORGANISMS FOR PRODUCING 1,4-BUTANEDIOL AND METHODS RELATED THERETO
First Claim
1. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,4-butanediol pathway comprising at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol;
- said non-naturally occurring microbial organism further comprising;
(i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, and an alpha-ketoglutarate;
ferredoxin oxidoreductase;
(ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate;
ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof;
wherein said 1,4-butanediol pathway comprises a pathway selected from;
(a) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, and α
-ketoglutarate decarboxylase;
(b) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate;
CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α
-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase and an aldehyde/alcohol dehydrogenase;
(c) (i) an α
-ketoglutarate decarboxylase, or an α
-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase, or a glutamate;
succinate semialdehyde transaminase and a glutamate decarboxylase;
(ii) a 4-hydroxybutanoate dehydrogenase;
(iii) a 4-hydroxybutyryl-CoA;
acetyl-CoA transferase, or a butyrate kinase and a phosphotransbutyrylase;
(iv) an aldehyde dehydrogenase; and
(v) an alcohol dehydrogenase;
(d) 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA transaminase, and 4-hydroxybutyryl-CoA dehydrogenase;
(e) 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) and 4-aminobutan-1-ol transaminase;
(f) 4-aminobutyrate kinase, 4-aminobutyraldehyde dehydrogenase (phosphorylating), 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating), 4-aminobutan-1-ol transaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase, 4-hydroxybutyryl-phosphate dehydrogenase, and 4-hydroxybutyraldehyde dehydrogenase (phosphorylating);
(g) alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming), 5-hydroxy-2-oxopentanoic acid decarboxylase, and 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(h) glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase (phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde reductase, glutamyl-CoA reductase (alcohol forming), 2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating), 2-amino-5-hydroxypentanoic acid transaminase, 5-hydroxy-2-oxopentanoic acid decarboxylase, 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(i) 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ
-isomerase, or 4-hydroxybutyryl-CoA dehydratase;
(j) homoserine deaminase, homoserine CoA transferase, homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoA deaminase, 4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA reductase;
(k) succinyl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating);
(l) glutamate dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating);
(m) 4-aminobutyrate kinase;
4-aminobutyraldehyde dehydrogenase (phosphorylating);
4-aminobutan-1-ol dehydrogenase; and
4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase;
(n) 4-aminobutyrate kinase;
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating) or [(4-aminobutanolyl)oxy]phosphonic acid transaminase;
4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating);
(o) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid decarboxylase;
(p) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(q) alpha-ketoglutarate 5-kinase;
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);
2,5-dioxopentanoic acid reductase; and
5-hydroxy-2-oxopentanoic acid decarboxylase;
(r) alpha-ketoglutarate 5-kinase;
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);
2,5-dioxopentanoic acid reductase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(s) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase;
5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid decarboxylase;
(t) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase;
5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(u) glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;
glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(v) glutamate 5-kinase;
glutamate-5-semialdehyde dehydrogenase (phosphorylating);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(w) glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;
glutamyl-CoA reductase;
glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(x) glutamate 5-kinase;
glutamate-5-semialdehyde dehydrogenase (phosphorylating);
glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);
(y) homoserine deaminase;
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase;
4-hydroxybut-2-enoyl-CoA reductase;
(z) homoserine CoA transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and
4-hydroxybut-2-enoyl-CoA reductase;
(aa) homoserine deaminase;
4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, or 4-hydroxybutyryl-CoA ligase;
(bb) (i) alpha-ketoglutarate decarboxylase;
or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;
or glutamate;
succinate semialdehyde transaminase and glutamate decarboxylase;
(ii) 4-hydroxybutyrate dehydrogenase;
(iii) 4-hydroxybutyryl-CoA transferase;
or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase;
(iv) 4-hydroxybutyryl-CoA reductase; and
(v) 4-hydroxybutyraldehyde reductase;
or aldehyde/alcohol dehydrogenase;
(cc) (i) alpha-ketoglutarate decarboxylase;
or succinyl-CoA synthetase and CoA-dependent succinate semialdehyde dehydrogenase;
(ii) 4-hydroxybutyrate dehydrogenase;
(iii) 4-hydroxybutyryl-CoA transferase;
or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and
(iv) aldehyde dehydrogenase; and
alcohol dehydrogenase;
or aldehyde/alcohol dehydrogenase;
(dd) (i) alpha-ketoglutarate decarboxylase;
or glutamate dehydrogenase;
glutamate decarboxylase; and
deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate transaminase;
or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;
(ii) 4-hydroxybutyrate dehydrogenase; and
(iii) 4-hydroxybutyrate kinase;
phosphotrans-4-hydroxybutyrylase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyrate kinase;
phosphorylating 4-hydroxybutanal dehydrogenase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyrate kinase;
phosphotrans-4-hydroxybutyrylase; and
alcohol forming 4-hydroxybutyryl-CoA reductase;
or 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase; and
alcohol forming 4-hydroxybutyryl-CoA reductase;
(ee) (i) glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase;
glutamyl-CoA reductase; and
glutamate-5-semialdehyde reductase;
or glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; and
alcohol forming glutamyl-CoA reductase;
or glutamate 5-kinase;
phosphorylating glutamate-5-semialdehyde dehydrogenase; and
glutamate-5-semialdehyde reductase;
(ii) deaminating 2-amino-5-hydroxypentanoic acid oxidoreductase or 2-amino-5-hydroxypentanoic acid transaminase; and
(iii) 5-hydroxy-2-oxopentanoic acid decarboxylase; and
4-hydroxybutyraldehyde reductase;
or decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming 4-hydroxybutyryl-CoA reductase;
(ff) succinyl-CoA reductase (aldehyde forming);
4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;
(gg) alpha-ketoglutarate decarboxylase;
4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;
(hh) succinate reductase;
4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;
(ii) alpha-ketoglutarate decarboxylase, or glutamate dehydrogenase or glutamate transaminase and glutamate decarboxylase and 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;
(jj) alpha-ketoglutarate reductase;
5-hydroxy-2-oxopentanoate dehydrogenase; and
5-hydroxy-2-oxopentanoate decarboxylase; and
optionally 1,4-butandiol dehydrogenase;
(kk) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase; and
4-hydroxybutyryl-CoA reductase (alcohol forming);
(ll) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase;
4-hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(mm) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase;
4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;
(nn) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylasel;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(oo) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;
(pp) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;
(qq) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);
(rr) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(ss) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);
(tt) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(uu) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;
(vv) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;
(ww) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);
(xx) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(yy) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);
(zz) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(aaa) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;
(bbb) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;
(ccc) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);
(ddd) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(eee) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);
(fff) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(ggg) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;
(hhh) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;
(iii) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);
(jjj) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;
(kkk) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming); and
(lll) any of the pathways that produce 1,4-butanediol as shown in any of FIG. 1, 8-13, 58, 62, 63 or 72-74.
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Abstract
The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO), 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine pathway comprising at least one exogenous nucleic acid encoding a BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine pathway enzyme expressed in a sufficient amount to produce BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine and further optimized for expression of BDO. The invention additionally provides methods of using such microbial organisms to produce BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine.
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Citations
33 Claims
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1. A non-naturally occurring microbial organism, comprising a microbial organism having a 1,4-butanediol pathway comprising at least one exogenous nucleic acid encoding a 1,4-butanediol pathway enzyme expressed in a sufficient amount to produce 1,4-butanediol;
- said non-naturally occurring microbial organism further comprising;
(i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, a citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase, and an alpha-ketoglutarate;
ferredoxin oxidoreductase;(ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate;
ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; wherein said 1,4-butanediol pathway comprises a pathway selected from; (a) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, and α
-ketoglutarate decarboxylase;(b) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate;
CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α
-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase and an aldehyde/alcohol dehydrogenase;(c) (i) an α
-ketoglutarate decarboxylase, or an α
-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase, or a glutamate;
succinate semialdehyde transaminase and a glutamate decarboxylase;
(ii) a 4-hydroxybutanoate dehydrogenase;
(iii) a 4-hydroxybutyryl-CoA;
acetyl-CoA transferase, or a butyrate kinase and a phosphotransbutyrylase;
(iv) an aldehyde dehydrogenase; and
(v) an alcohol dehydrogenase;(d) 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA transaminase, and 4-hydroxybutyryl-CoA dehydrogenase; (e) 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) and 4-aminobutan-1-ol transaminase; (f) 4-aminobutyrate kinase, 4-aminobutyraldehyde dehydrogenase (phosphorylating), 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating), 4-aminobutan-1-ol transaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase, 4-hydroxybutyryl-phosphate dehydrogenase, and 4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (g) alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming), 5-hydroxy-2-oxopentanoic acid decarboxylase, and 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (h) glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase (phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde reductase, glutamyl-CoA reductase (alcohol forming), 2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating), 2-amino-5-hydroxypentanoic acid transaminase, 5-hydroxy-2-oxopentanoic acid decarboxylase, 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (i) 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ
-isomerase, or 4-hydroxybutyryl-CoA dehydratase;(j) homoserine deaminase, homoserine CoA transferase, homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoA deaminase, 4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA reductase; (k) succinyl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating); (l) glutamate dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating); (m) 4-aminobutyrate kinase;
4-aminobutyraldehyde dehydrogenase (phosphorylating);
4-aminobutan-1-ol dehydrogenase; and
4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase;(n) 4-aminobutyrate kinase;
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating) or [(4-aminobutanolyl)oxy]phosphonic acid transaminase;
4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating);(o) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid decarboxylase;(p) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(q) alpha-ketoglutarate 5-kinase;
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);
2,5-dioxopentanoic acid reductase; and
5-hydroxy-2-oxopentanoic acid decarboxylase;(r) alpha-ketoglutarate 5-kinase;
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);
2,5-dioxopentanoic acid reductase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(s) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase;
5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid decarboxylase;(t) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase;
5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(u) glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;
glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(v) glutamate 5-kinase;
glutamate-5-semialdehyde dehydrogenase (phosphorylating);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(w) glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;
glutamyl-CoA reductase;
glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(x) glutamate 5-kinase;
glutamate-5-semialdehyde dehydrogenase (phosphorylating);
glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(y) homoserine deaminase;
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase;
4-hydroxybut-2-enoyl-CoA reductase;(z) homoserine CoA transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and
4-hydroxybut-2-enoyl-CoA reductase;(aa) homoserine deaminase;
4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, or 4-hydroxybutyryl-CoA ligase;(bb) (i) alpha-ketoglutarate decarboxylase;
or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;
or glutamate;
succinate semialdehyde transaminase and glutamate decarboxylase;
(ii) 4-hydroxybutyrate dehydrogenase;
(iii) 4-hydroxybutyryl-CoA transferase;
or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase;
(iv) 4-hydroxybutyryl-CoA reductase; and
(v) 4-hydroxybutyraldehyde reductase;
or aldehyde/alcohol dehydrogenase;(cc) (i) alpha-ketoglutarate decarboxylase;
or succinyl-CoA synthetase and CoA-dependent succinate semialdehyde dehydrogenase;
(ii) 4-hydroxybutyrate dehydrogenase;
(iii) 4-hydroxybutyryl-CoA transferase;
or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and
(iv) aldehyde dehydrogenase; and
alcohol dehydrogenase;
or aldehyde/alcohol dehydrogenase;(dd) (i) alpha-ketoglutarate decarboxylase;
or glutamate dehydrogenase;
glutamate decarboxylase; and
deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate transaminase;
or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;
(ii) 4-hydroxybutyrate dehydrogenase; and
(iii) 4-hydroxybutyrate kinase;
phosphotrans-4-hydroxybutyrylase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyrate kinase;
phosphorylating 4-hydroxybutanal dehydrogenase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyrate kinase;
phosphotrans-4-hydroxybutyrylase; and
alcohol forming 4-hydroxybutyryl-CoA reductase;
or 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase; and
alcohol forming 4-hydroxybutyryl-CoA reductase;(ee) (i) glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase;
glutamyl-CoA reductase; and
glutamate-5-semialdehyde reductase;
or glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; and
alcohol forming glutamyl-CoA reductase;
or glutamate 5-kinase;
phosphorylating glutamate-5-semialdehyde dehydrogenase; and
glutamate-5-semialdehyde reductase;
(ii) deaminating 2-amino-5-hydroxypentanoic acid oxidoreductase or 2-amino-5-hydroxypentanoic acid transaminase; and
(iii) 5-hydroxy-2-oxopentanoic acid decarboxylase; and
4-hydroxybutyraldehyde reductase;
or decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming 4-hydroxybutyryl-CoA reductase;(ff) succinyl-CoA reductase (aldehyde forming);
4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;(gg) alpha-ketoglutarate decarboxylase;
4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;(hh) succinate reductase;
4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;(ii) alpha-ketoglutarate decarboxylase, or glutamate dehydrogenase or glutamate transaminase and glutamate decarboxylase and 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
4-hydroxybutyrate dehydrogenase; and
4-hydroxybutyrate reductase; and
optionally 1,4-butandiol dehydrogenase;(jj) alpha-ketoglutarate reductase;
5-hydroxy-2-oxopentanoate dehydrogenase; and
5-hydroxy-2-oxopentanoate decarboxylase; and
optionally 1,4-butandiol dehydrogenase;(kk) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase; and
4-hydroxybutyryl-CoA reductase (alcohol forming);(ll) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase;
4-hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(mm) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase;
4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;(nn) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylasel;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(oo) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;(pp) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;(qq) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);(rr) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(ss) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);(tt) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(uu) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;(vv) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;(ww) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);(xx) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(yy) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);(zz) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(aaa) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;(bbb) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;(ccc) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);(ddd) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(eee) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);(fff) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(ggg) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase;
4-Hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;(hhh) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;(iii) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming);(jjj) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;(kkk) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA reductase (alcohol forming); and(lll) any of the pathways that produce 1,4-butanediol as shown in any of FIG. 1 , 8-13, 58, 62, 63 or 72-74. - View Dependent Claims (2, 3, 4, 7, 8, 9, 11)
- said non-naturally occurring microbial organism further comprising;
-
5-6. -6. (canceled)
-
10. (canceled)
-
12. A non-naturally occurring microbial organism, comprising a microbial organism having a 4-hydroxybutyrate pathway comprising at least one exogenous nucleic acid encoding a 4-hydroxybutyrate pathway enzyme 4-hydroxybutyrate expressed in a sufficient amount to produce 4-hydroxybutyrate;
- said non-naturally occurring microbial organism further comprising;
(i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase and an alpha-ketoglutarate;
ferredoxin oxidoreductase;(ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate;
ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; (a) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase; and
4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase;(b) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase; and
4-Hydroxybutyryl-CoA transferase, hydrolase or synthetase;(c) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyrate kinase;(d) Succinate reductase; and
4-Hydroxybutyrate dehydrogenase;(e) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming); and
4-Hydroxybutyrate dehydrogenase;(f) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase); and
4-Hydroxybutyrate dehydrogenase;(g) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase); and
Succinyl-CoA reductase (alcohol forming);(h) acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase, a 3-hydroxybutyryl-CoA dehydrogenase, a crotonase, a crotonyl-CoA hydratase, a 4-hydroxybutyryl-CoA transferase, a phosphotrans-4-hydroxybutyrylase, and a 4-hydroxybutyrate kinase; (i) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, and α
-ketoglutarate decarboxylase;(j) (i) an α
-ketoglutarate decarboxylase, or an α
-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase, or a glutamate;
succinate semialdehyde transaminase and a glutamate decarboxylase;
(ii) a 4-hydroxybutanoate dehydrogenase;(k) succinyl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating); (l) glutamate dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating); (m) homoserine deaminase;
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase;
4-hydroxybut-2-enoyl-CoA reductase;(n) homoserine CoA transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and
4-hydroxybut-2-enoyl-CoA reductase;(o) homoserine deaminase;
4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, or 4-hydroxybutyryl-CoA ligase;(p) succinyl-CoA reductase (aldehyde forming); and
4-hydroxybutyrate dehydrogenase;(q) alpha-ketoglutarate decarboxylase; and
4-hydroxybutyrate dehydrogenase;(r) succinate reductase; and
4-hydroxybutyrate dehydrogenase;(s) alpha-ketoglutarate decarboxylase, or glutamate dehydrogenase or glutamate transaminase and glutamate decarboxylase and 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase; and
4-hydroxybutyrate dehydrogenase; and(t) a 4-hydroxybutyrate pathway selected from any of the pathways that produce 4-hydroxybutyrate as shown in any of FIG. 1 , 8-13, 58, 62, 63 or 72-74. - View Dependent Claims (13, 14, 15, 21)
- said non-naturally occurring microbial organism further comprising;
-
16-18. -18. (canceled)
-
20. (canceled)
-
22. A non-naturally occurring microbial organism, comprising a microbial organism having a gamma-butyrolactone pathway comprising at least one exogenous nucleic acid encoding a gamma-butyrolactone pathway enzyme expressed in a sufficient amount to produce gamma-butyrolactone;
- said non-naturally occurring microbial organism further comprising;
(i) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a citryl-CoA synthetase, a citryl-CoA lyase, a fumarate reductase, isocitrate dehydrogenase and an alpha-ketoglutarate;
ferredoxin oxidoreductase;(ii) a reductive TCA pathway comprising at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme, wherein said at least one exogenous nucleic acid is selected from a pyruvate;
ferredoxin oxidoreductase, a phosphoenolpyruvate carboxylase, a phosphoenolpyruvate carboxykinase, a CO dehydrogenase, and an H2 hydrogenase;
or(iii) at least one exogenous nucleic acid encodes an enzyme selected from a CO dehydrogenase, an H2 hydrogenase, and combinations thereof; (a) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase; and
spontaneous or enzyme catalyzed;(b) Acetoacetyl-CoA thiolase or acetoacetyl-CoA synthase;
3-Hydroxybutyryl-CoA dehydrogenase;
Crotonase;
Crotonyl-CoA hydratase;
Phosphotrans-4-hydroxybutyrylase;
amd spontaneous or enzyme catalyzed;(c) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(d) Succinate reductase;
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(e) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(f) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (aldehyde forming);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase;
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(g) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(h) Alpha-ketoglutarate decarboxylase or (Glutamate dehydrogenase and/or Glutamate transaminase;
Glutamate decarboxylase;
4-aminobutyrate dehydrogenase and/or 4-aminobutyrate transaminase);
4-Hydroxybutyrate dehydrogenase;
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(i) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyrate kinase;
Phosphotrans-4-hydroxybutyrylase;
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(j) Succinyl-CoA transferase, or Succinyl-CoA synthetase (or succinyl-CoA ligase);
Succinyl-CoA reductase (alcohol forming);
4-Hydroxybutyryl-CoA transferase, or 4-Hydroxybutyryl-CoA synthetase; and
4-Hydroxybutyryl-CoA hydrolase or spontaneous;(k) alpha-ketoglutarate reductase;
5-hydroxy-2-oxopentanoate dehydrogenase; and
5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation)(l) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, and α
-ketoglutarate decarboxylase;(m) 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate;
CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α
-ketoglutarate decarboxylase;(n) (i) an α
-ketoglutarate decarboxylase, or an α
-ketoglutarate dehydrogenase and a CoA-dependent succinic semialdehyde dehydrogenase, or a glutamate;
succinate semialdehyde transaminase and a glutamate decarboxylase;
(ii) a 4-hydroxybutanoate dehydrogenase;
(iii) a 4-hydroxybutyryl-CoA;
acetyl-CoA transferase, or a butyrate kinase and a phosphotransbutyrylase;(o) 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA oxidoreductase (deaminating), 4-aminobutyryl-CoA transaminase, and 4-hydroxybutyryl-CoA dehydrogenase; (p) 4-aminobutyrate CoA transferase, 4-aminobutyryl-CoA hydrolase, 4-aminobutyrate-CoA ligase, 4-aminobutyryl-CoA reductase (alcohol forming), 4-aminobutyryl-CoA reductase, 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating) and 4-aminobutan-1-ol transaminase; (q) 4-aminobutyrate kinase, 4-aminobutyraldehyde dehydrogenase (phosphorylating), 4-aminobutan-1-ol dehydrogenase, 4-aminobutan-1-ol oxidoreductase (deaminating), 4-aminobutan-1-ol transaminase, [(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating), [(4-aminobutanolyl)oxy]phosphonic acid transaminase, 4-hydroxybutyryl-phosphate dehydrogenase, and 4-hydroxybutyraldehyde dehydrogenase (phosphorylating); (r) alpha-ketoglutarate 5-kinase, 2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating), 2,5-dioxopentanoic acid reductase, alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, alpha-ketoglutaryl-CoA ligase, alpha-ketoglutaryl-CoA reductase, 5-hydroxy-2-oxopentanoic acid dehydrogenase, alpha-ketoglutaryl-CoA reductase (alcohol forming), 5-hydroxy-2-oxopentanoic acid decarboxylase, and 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (s) glutamate CoA transferase, glutamyl-CoA hydrolase, glutamyl-CoA ligase, glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase (phosphorylating), glutamyl-CoA reductase, glutamate-5-semialdehyde reductase, glutamyl-CoA reductase (alcohol forming), 2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating), 2-amino-5-hydroxypentanoic acid transaminase, 5-hydroxy-2-oxopentanoic acid decarboxylase, 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation); (t) 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, vinylacetyl-CoA Δ
-isomerase, or 4-hydroxybutyryl-CoA dehydratase;(u) homoserine deaminase, homoserine CoA transferase, homoserine-CoA hydrolase, homoserine-CoA ligase, homoserine-CoA deaminase, 4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase, 4-hydroxybut-2-enoate reductase, 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybut-2-enoyl-CoA reductase; (v) succinyl-CoA reductase (alcohol forming), 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating); (w) glutamate dehydrogenase, 4-aminobutyrate oxidoreductase (deaminating), 4-aminobutyrate transaminase, glutamate decarboxylase, 4-hydroxybutyryl-CoA hydrolase, 4-hydroxybutyryl-CoA ligase, or 4-hydroxybutanal dehydrogenase (phosphorylating); (x) 4-aminobutyrate kinase;
4-aminobutyraldehyde dehydrogenase (phosphorylating);
4-aminobutan-1-ol dehydrogenase; and
4-aminobutan-1-ol oxidoreductase (deaminating) or 4-aminobutan-1-ol transaminase;(y) 4-aminobutyrate kinase;
[(4-aminobutanolyl)oxy]phosphonic acid oxidoreductase (deaminating) or [(4-aminobutanolyl)oxy]phosphonic acid transaminase;
4-hydroxybutyryl-phosphate dehydrogenase; and
4-hydroxybutyraldehyde dehydrogenase (phosphorylating);(z) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid decarboxylase;(aa) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase (alcohol forming); and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(bb) alpha-ketoglutarate 5-kinase;
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);
2,5-dioxopentanoic acid reductase; and
5-hydroxy-2-oxopentanoic acid decarboxylase;(cc) alpha-ketoglutarate 5-kinase;
2,5-dioxopentanoic semialdehyde dehydrogenase (phosphorylating);
2,5-dioxopentanoic acid reductase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(dd) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase;
5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid decarboxylase;(ee) alpha-ketoglutarate CoA transferase, alpha-ketoglutaryl-CoA hydrolase, or alpha-ketoglutaryl-CoA ligase;
alpha-ketoglutaryl-CoA reductase;
5-hydroxy-2-oxopentanoic acid dehydrogenase; and
5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(ff) glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;
glutamyl-CoA reductase (alcohol forming);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(gg) glutamate 5-kinase;
glutamate-5-semialdehyde dehydrogenase (phosphorylating);
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(hh) glutamate CoA transferase, glutamyl-CoA hydrolase, or glutamyl-CoA ligase;
glutamyl-CoA reductase;
glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(ii) glutamate 5-kinase;
glutamate-5-semialdehyde dehydrogenase (phosphorylating);
glutamate-5-semialdehyde reductase;
2-amino-5-hydroxypentanoic acid oxidoreductase (deaminating) or 2-amino-5-hydroxypentanoic acid transaminase; and
5-hydroxy-2-oxopentanoic acid decarboxylase or 5-hydroxy-2-oxopentanoic acid dehydrogenase (decarboxylation);(jj) homoserine deaminase;
4-hydroxybut-2-enoyl-CoA transferase, 4-hydroxybut-2-enoyl-CoA hydrolase, 4-hydroxybut-2-enoyl-CoA ligase;
4-hydroxybut-2-enoyl-CoA reductase;(kk) homoserine CoA transferase, homoserine-CoA hydrolase, or homoserine-CoA ligase;
homoserine-CoA deaminase; and
4-hydroxybut-2-enoyl-CoA reductase;(ll) homoserine deaminase;
4-hydroxybut-2-enoate reductase; and
4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA hydrolase, or 4-hydroxybutyryl-CoA ligase;(mm) (i) alpha-ketoglutarate decarboxylase;
or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;
or glutamate;
succinate semialdehyde transaminase and glutamate decarboxylase;
(ii) 4-hydroxybutyrate dehydrogenase;
(iii) 4-hydroxybutyryl-CoA transferase;
or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase; and
(iv) 4-hydroxybutyryl-CoA reductase;(nn) (i) alpha-ketoglutarate decarboxylase;
or succinyl-CoA synthetase and CoA-dependent succinate semialdehyde dehydrogenase;
(ii) 4-hydroxybutyrate dehydrogenase;
(iii) 4-hydroxybutyryl-CoA transferase;
or 4-hydroxybutyrate kinase and phosphotrans-4-hydroxybutyrylase;(oo) (i) alpha-ketoglutarate decarboxylase;
or glutamate dehydrogenase;
glutamate decarboxylase; and
deaminating 4-aminobutyrate oxidoreductase or 4-aminobutyrate transaminase;
or alpha-ketoglutarate dehydrogenase and CoA-dependent succinate semialdehyde dehydrogenase;
(ii) 4-hydroxybutyrate dehydrogenase; and
(iii) 4-hydroxybutyrate kinase;
phosphotrans-4-hydroxybutyrylase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyrate kinase;
phosphorylating 4-hydroxybutanal dehydrogenase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyrate kinase;
phosphotrans-4-hydroxybutyrylase; and
alcohol forming 4-hydroxybutyryl-CoA reductase;
or 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA ligase; and
alcohol forming 4-hydroxybutyryl-CoA reductase;(pp) (i) glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase;
glutamyl-CoA reductase; and
glutamate-5-semialdehyde reductase;
or glutamate CoA transferase or glutamyl-CoA hydrolase or glutamyl-CoA ligase; and
alcohol forming glutamyl-CoA reductase;
or glutamate 5-kinase;
phosphorylating glutamate-5-semialdehyde dehydrogenase; and
glutamate-5-semialdehyde reductase;
(ii) deaminating 2-amino-5-hydroxypentanoic acid oxidoreductase or 2-amino-5-hydroxypentanoic acid transaminase; and
(iii) 5-hydroxy-2-oxopentanoic acid decarboxylase; and
4-hydroxybutyraldehyde reductase;
or decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase;
4-hydroxybutyryl-CoA reductase; and
4-hydroxybutyraldehyde reductase;
or decarboxylating 5-hydroxy-2-oxopentanoic acid dehydrogenase and alcohol forming 4-hydroxybutyryl-CoA reductase;(qq) a gamma-butyrolactone pathway comprising a pathway selected from any of the pathways that produce 4-hydroxybutyryl-CoA or 4-hydroxybutyryl phosphate as shown in FIG. 1 , 8-13, 58, 62-63 or 72-74, wherein gamma-butyrolactone is produced enzymatically or by spontaneous chemical conversion. - View Dependent Claims (23, 24, 25, 32)
- said non-naturally occurring microbial organism further comprising;
-
26-31. -31. (canceled)
-
33. A carboxylic acid reductase, comprising an amino acid sequence having an amino acid substitution selected from E16K;
- Q95L;
L100M;
A1011T;
K823E;
T941S;
H15Q;
D198E;
G446C;
S392N;
F699L;
V8831;
F467S;
T987S;
R12H;
V295G;
V295A;
V295S;
V295T;
V295G;
V295V;
V295L;
V2951;
V295M;
V295P;
V295F;
V295Y;
V295W;
V295D;
V295E;
V295N;
V295Q;
V295H;
V295K;
V295R;
M296G;
M296A;
M296S;
M296T;
M296C;
M296V;
M296L;
M2961;
M296M;
M296P;
M296F;
M296Y;
M296W;
M296D;
M296E;
M296N;
M296Q;
M296H;
M296K;
M296R;
G297G;
G297A;
G297S;
G297T;
G297C;
G297V;
G297L;
G2971;
G297M;
G297P;
G297F;
G297Y;
G297W;
G297D;
G297E;
G297N;
G297Q;
G297H;
G297K;
G297R;
G391G;
G391A;
G391S;
G391T;
G391C;
G391V;
G391L;
G391I;
G391M;
G391P;
G391F;
G391Y;
G391W;
G391D;
G391E;
G391N;
G391Q;
G391H;
G391K;
G391R;
G421G;
G421A;
G421S;
G421T;
G421C;
G421V;
G421L;
G421I;
G421M;
G421P;
G421F;
G421Y;
G421W;
G421D;
G421E;
G421N;
G421Q;
G421H;
G421K;
G421R;
D413G;
D413A;
D413S;
D413T;
D413C;
D413V;
D413L;
D413I;
D413M;
D413P;
D413F;
D413Y;
D413W;
D413D;
D413E;
D413N;
D413Q;
D413H;
D413K;
D413R;
G414G;
G414A;
G414S;
G414T;
G414C;
G414V;
G414L;
G414I;
G414M;
G414P;
G414F;
G414Y;
G414W;
G414D;
G414E;
G414N;
G414Q;
G414H;
G414K;
G414R;
Y415G;
Y415A;
Y415S;
Y415T;
Y415C;
Y415V;
Y415L;
Y415I;
Y415M;
Y415P;
Y415F;
Y415Y;
Y415W;
Y415D;
Y415E;
Y415N;
Y415Q;
Y415H;
Y415K;
Y415R;
G416G;
G416A;
G416S;
G416T;
G416C;
G416V;
G416L;
G416I;
G416M;
G416P;
G416F;
G416Y;
G416W;
G416D;
G416E;
G416N;
G416Q;
G416H;
G416K;
G416R;
S417G;
5417A;
S417S;
S417T;
S417C;
S417V S417L;
S417I;
S417M;
S417P;
S417F;
S417Y;
S417W;
S417D;
S417E;
S417N;
S417Q;
S417H;
S417K; and
S417R, or combinations thereof.
- Q95L;
Specification