Methods for increasing product yields
View Patent ↗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.
1. A non-naturally occurring microbial organism comprising:
(1) at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said at least one exogenous nucleic acid encodes an enzyme selected from the group consisting of:
i) an Acetyl-CoA synthase,
ii) a Pyruvate ferredoxin oxidoreductase or pyruvate dehydrogenase, and
iii) a pyruvate formate lyase;
(2) (a) an isopropanol pathway that converts acetyl-CoA to isopropanol, wherein the 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 1,4-butanediol pathway that converts acetyl-CoA to 1,4-butanediol; wherein the 1,4-butanediol pathway comprises at least five enzymes selected from the group consisting of: 1) an acetoacetyl-CoA thiolase; 2) a 3-hydroxybutyryl-CoA dehydrogenase; 3) a crotonase; 4) a crotonyl-CoA hydratase; 5) a 4-hydroxybutyryl-CoA reductase (alcohol forming); 6) a 4-hydroxybutyryl-CoA reductase (aldehyde forming); 7) a 1,4-butanediol dehydrogenase; 8) a 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA synthetase, 4-hydroxybutyryl-CoA hydrolase, or phosphotrans-4-hydroxybutyrylase/4-Hydroxybutyrate kinase; and 9) a 4-hydroxybutyrate reductase; or
(c) a 4-hydroxybutyrate pathway that converts acetyl-CoA to 4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises at least five enzymes selected from the group consisting of: 1) an acetoacetyl-CoA thiolase; 2) a 3-hydroxybutyryl-CoA dehydrogenase; 3) a crotonase; 4) a crotonyl-CoA hydratase; 5) a 4-hydroxybutyryl-CoA transferase, hydrolase or synthetase; 6) a phosphotrans-4-hydroxybutyrylase; and 7) a 4-hydroxybutyrate kinase; and
(3) a reductive TCA (rTCA) pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a rTCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said rTCA enzyme is selected from the group consisting of 1) an ATP-citrate lyase, 2) a citrate lyase, 3) a fumarate reductase, and 4) an alpha-ketoglutarate:ferredoxin oxidoreductase.
2. The organism of claim 1 , further comprising 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, an acetate kinase, a phosphotransacetylase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
3. The organism of claim 1 , further comprising an exogenous nucleic acid encoding an enzyme selected from the group consisting of a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
4. The organism of claim 3 , wherein said microbial organism utilizes a carbon feedstock selected from (i) CO, CO 2 , and H 2 , (ii) synthesis gas comprising CO and and (iii) synthesis gas comprising CO, CO 2 , and H 2 .
5. The non-naturally occurring microbial organism of claim 1 , wherein said organism comprises:
(1) said at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said at least one exogenous nucleic acid encodes an enzyme selected from the group consisting of i) an Acetyl-CoA synthase, ii) a Pyruvate ferredoxin oxidoreductase or pyruvate dehydrogenase, and iii) a pyruvate formate lyase;
(2) said isopropanol pathway that converts acetyl-CoA to isopropanol, wherein the 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; and
(3) said rTCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a rTCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said rTCA enzyme is selected from the group consisting of
1) an ATP-citrate lyase,
2) a citrate lyase,
3) a fumarate reductase, and
4) an alpha-ketoglutarate:ferredoxin oxidoreductase.
6. The organism of claim 5 , further comprising 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, an acetate kinase, a phosphotransacetylase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
7. The organism of claim 5 , further comprising an exogenous nucleic acid encoding an enzyme selected from the group consisting of a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
8. The organism of claim 7 , wherein said microbial organism utilizes a carbon feedstock selected from (i) CO, CO 2 , and H 2 , (ii) synthesis gas comprising CO and H 2 , and (iii) synthesis gas comprising CO, CO 2 , and H 2 .
9. The organism of claim 5 , wherein at least one of the isopropanol pathway enzymes is encoded by an exogenous nucleic acid.
10. The non-naturally occurring microbial organism of claim 1 , wherein said organism comprises:
(1) said at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said at least one exogenous nucleic acid encodes an enzyme selected from the group consisting of: i) an Acetyl-CoA synthase, ii) a Pyruvate ferredoxin oxidoreductase or pyruvate dehydrogenase, and iii) a pyruvate formate lyase;
( 2 ) said 1,4-butanediol pathway that converts acetyl-CoA to 1,4-butanediol; wherein the 1,4-butanediol pathway comprises at least five enzymes selected from the group consisting of:
1) an acetoacetyl-CoA thiolase;
2) a 3-hydroxybutyryl-CoA dehydrogenase;
3) a crotonase;
4) a crotonyl-CoA hydratase;
5) a 4-hydroxybutyryl-CoA reductase (alcohol forming);
6) a 4-hydroxybutyryl-CoA reductase (aldehyde forming);
7) a 1,4-butanediol dehydrogenase;
8) a 4-hydroxybutyryl-CoA transferase, 4-hydroxybutyryl-CoA synthetase, 4-hydroxybutyryl-CoA hydrolase, or phosphotrans -4-hydroxybutyrylase / 4-Hydroxybutyrate kinase; and
9) a 4-hydroxybutyrate reductase; and
(3) said rTCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a rTCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said rTCA enzyme is selected from the group consisting of
1) an ATP-citrate lyase,
2) a citrate lyase,
3) a fumarate reductase, and
4) an alpha-ketoglutarate:ferredoxin oxidoreductase.
11. The organism of claim 10 , further comprising 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, an acetate kinase, a phosphotransacetylase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
12. The organism of claim 10 , further comprising an exogenous nucleic acid encoding an enzyme selected from the group consisting of a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
13. The organism of claim 12 , wherein said microbial organism utilizes a carbon feedstock selected from (i) CO, CO 2 , and H 2 , (ii) synthesis gas comprising CO and H 2 , and (iii) synthesis gas comprising CO, CO 2 , and H 2 .
14. The organism of claim 10 , wherein at least one of the 1,4-butanediol enzymes is encoded by an exogenous nucleic acid.
15. The non-naturally occurring microbial organism of claim 1 , wherein said organism comprises:
(1) said at least one exogenous nucleic acid encoding an enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said at least one exogenous nucleic acid encodes an enzyme selected from the group consisting of: i) an Acetyl-CoA synthase, ii) a Pyruvate ferredoxin oxidoreductase or pyruvate dehydrogenase, and iii) a pyruvate formate lyase;
(2) said 4-hydroxybutyrate pathway that converts acetyl-CoA to 4-hydroxybutyrate, wherein said 4-hydroxybutyrate pathway comprises at least five enzymes selected from the group consisting of:
1) an acetoacetyl-CoA thiolase;
2) a 3-hydroxybutyryl-CoA dehydrogenase;
3) a crotonase;
4) a crotonyl-CoA hydratase;
5) a 4-hydroxybutyryl-CoA transferase, hydrolase or synthetase;
6) a phosphotrans-4-hydroxybutyrylase; and
7 ) a 4-hydroxybutyrate kinase; and
(3) said rTCA pathway, wherein said microbial organism comprises at least one exogenous nucleic acid encoding a rTCA pathway enzyme expressed in a sufficient amount to enhance carbon flux through acetyl-CoA, wherein said rTCA enzyme is selected from the group consisting of
1) an ATP-citrate lyase,
2) a citrate lyase,
3) a fumarate reductase, and
4) an alpha-ketoglutarate:ferredoxin oxidoreductase.
16. The organism of claim 15 , further comprising 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, an acetate kinase, a phosphotransacetylase, an NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
17. The organism of claim 15 , further comprising an exogenous nucleic acid encoding an enzyme selected from the group consisting of a carbon monoxide dehydrogenase, a hydrogenase, a NAD(P)H:ferredoxin oxidoreductase, ferredoxin, and combinations thereof.
18. The organism of claim 17 , wherein said microbial organism utilizes a carbon feedstock selected from (i) CO, CO 2 , and H 2 , (ii) synthesis gas comprising CO and H 2 , and (iii) synthesis gas comprising CO, CO 2 , and H 2 .
19. The organism of claim 15 , wherein the at least one of the 4-hydroxybutyrate pathway enzymes is encoded by an exogenous nucleic acid.