Methods for increasing product yields

Patent metadata

Field	Value
Publication number	US-2016145652-A1
Application number	US-201514806435-A
Country	US
Kind code	A1
Filing date	Jul 22, 2015
Priority date	Feb 23, 2010
Publication date	May 26, 2016
Grant date	—

Abstract

A non-naturally occurring microbial organism includes a microbial organism having a reductive TCA or Wood-Ljungdahl pathway in which at least one exogenous nucleic acid encoding these pathway enzymes is expressed in a sufficient amount to enhance carbon flux through acetyl-CoA. A method for enhancing carbon flux through acetyl-CoA includes culturing theses non-naturally occurring microbial organisms under conditions and for a sufficient period of time to produce a product having acetyl-CoA as a building block. Another non-naturally occurring microbial organism includes at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance the availability of reducing equivalents in the presence of carbon monoxide or hydrogen, thereby increasing the yield of redox-limited products via carbohydrate-based carbon feedstock. A method for enhancing the availability of reducing equivalents in the presence of carbon monoxide or hydrogen includes culturing this organism for a sufficient period of time to produce a product.

First claim

1 . A non-naturally occurring microbial organism having a reductive TCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a reductive TCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said at least one exogenous nucleic acid is selected from an ATP-citrate lyase, citrate lyase, a fumarate reductase, and an alpha-ketoglutarate:ferredoxin oxidoreductase.

2 .- 7 . (canceled)

8 . The non-naturally occurring microbial organism of claim 1 further comprising an exogenous nucleic acid encoding an enzyme selected from 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.

9 . The non-naturally occurring microbial organism of claim 1 further comprising
(a) an isopropanol pathway, said isopropanol pathway converting acetyl-CoA to isopropanol, wherein said isopropanol pathway comprises 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) an acetoacetate decarboxylase, and 4) an isopropanol dehydrogenase;
(b) a 1,3-butanediol pathway; said 1,3-butanediol pathway converting acetyl-CoA to 1,3-butanediol, wherein said 1,3-butanediol pathway comprises at least three enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4) 4-hydroxy, 2-butanone reductase, 5) Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase (ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8) Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoA reductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase (alcohol forming), 11) an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetate reductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyrate dehydrogenase;
(c) a 1,4-butanediol pathway, said 1,4-butanediol pathway converting acetyl-CoA to 1,4-butanediol, wherein said 1,4-butanediol pathway comprises at least five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5) 4-hydroxybutyryl-CoA reductase (alcohol forming), 6) 4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanediol dehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9) 4-Hydroxybutyrate reductase; and/or
(d) a 4-hydroxybutyrate pathway, said 4-hydroxybutyrate pathway converting acetyl-CoA to 4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises at least five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA transferase, hydrolase or synthetase, 6) Phosphotrans-4-hydroxybutyrylase, and 7) 4-Hydroxybutyrate kinase.

10 .- 29 . (canceled)

30 . The non-naturally occurring microbial organism of claim 1 further comprising an exogenous nucleic acid encoding an enzyme selected from carbon monoxide dehydrogenase, hydrogenase, NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.

31 . The non-naturally occurring microbial organism of claim 30 , wherein said microbial organism utilizes a carbon feedstock selected from CO, CO 2 , and H 2 , synthesis gas comprising CO and H 2 , and synthesis gas comprising CO, CO 2 , and H 2 .

32 . A method for enhancing carbon flux through acetyl-CoA, comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce a product having acetyl-CoA as a building block.

33 .- 62 . (canceled)

63 . A non-naturally occurring microbial organism having a Wood-Ljungdahl pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a Wood-Ljungdahl pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said at least one exogenous nucleic acid is selected from a) Formate dehydrogenase, b) Formyltetrahydrofolate synthetase, c) Methenyltetrahydrofolate cyclohydrolase, d) Methylenetetrahydrofolate dehydrogenase, e) Methylenetetrahydrofolate reductase, f) Methyltetrahydrofolate:corrinoid protein methyltransferase (AcsE), g) Corrinoid iron-sulfer protein (AcsD), h) Nickel-protein assembly protein (AcsF & CooC), i) Ferredoxin (Orf7), j) Acetyl-CoA synthase (AcsB & AcsC), k) Carbon monoxide dehydrogenase (AcsA), and l) Pyruvate ferredoxin oxidoreductase or pyruvate dehydrogenase, and m) pyruvate formate lyase.

64 .- 76 . (canceled)

77 . The non-naturally occurring microbial organism of claim 63 further comprising
(a) an isopropanol pathway, said isopropanol pathway converting acetyl-CoA to isopropanol, wherein said isopropanol pathway comprises 1) an acetoacetyl-CoA thiolase, 2) an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 3) an acetoacetate decarboxylase, and 4) an isopropanol dehydrogenase;
(b) a 1,3-butanediol pathway, said 1,3-butanediol pathway converting acetyl-CoA to 1,3-butanediol, wherein said 1,3-butanediol pathway comprises at least three enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2) Acetoacetyl-CoA reductase (CoA-dependent, alcohol forming), 3) 3-oxobutyraldehyde reductase (aldehyde reducing), 4) 4-hydroxy, 2-butanone reductase, 5) Acetoacetyl-CoA reductase (CoA-dependent, aldehyde forming), 6) 3-oxobutyraldehyde reductase (ketone reducing), 7) 3-hydroxybutyraldehyde reductase, 8) Acetoacetyl-CoA reductase (ketone reducing), 9) 3-hydroxybutyryl-CoA reductase (aldehyde forming), 10) 3-hydroxybutyryl-CoA reductase (alcohol forming), 11) an acetoacetyl-CoA transferase, an acetoacetyl-CoA hydrolase, an acetoacetyl-CoA synthetase, or a phosphotransacetoacetylase/acetoacetate kinase, 12) Acetoacetate reductase, 13) 3-hydroxybutyryl-CoA transferase, hydrolase, or synthetase, 14) 3-hydroxybutyrate reductase, and 15) 3-hydroxybutyrate dehydrogenase,
(c) a 1,4-butanediol pathway, said 1,4-butanediol pathway converting acetyl-CoA to 1,4-butanediol, wherein said 1,4-butanediol pathway comprises at least five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5) 4-hydroxybutyryl-CoA reductase (alcohol forming), 6) 4-hydroxybutyryl-CoA reductase (aldehyde forming), 7) 1,4-butanediol dehydrogenase, 8) 4-Hydroxybutyryl-CoA transferase, 4-Hydroxybutyryl-CoA synthetase, 4-Hydroxybutyryl-CoA hydrolase, or Phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase, and 9) 4-Hydroxybutyrate reductase, and/or
(d) a 4-hydroxybutyrate pathway, said 4-hydroxybutyrate pathway converting acetyl-CoA to 4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises at least five enzymes selected from 1) Acetoacetyl-CoA thiolase (AtoB), 2) 3-Hydroxybutyryl-CoA dehydrogenase (Hbd), 3) Crotonase (Crt), 4) Crotonyl-CoA hydratase (4-Budh), 5) 4-Hydroxybutyryl-CoA transf

Assignees

• Genomatica Inc

Inventors

• Burgard Anthony P
• Osterhout Robin E
• Sun Jun
• Pharkya Priti

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12P7/42 Primary

  Hydroxy-carboxylic acids · CPC title

• C12N9/0008 Primary

  acting on the aldehyde or oxo group of donors (1.2) · CPC title

• C12N9/0095

  acting on iron-sulfur proteins as donor (1.18) · CPC title

• C12P7/04

  acyclic · CPC title