Microorganisms and methods for the co-production of isopropanol and 1,4-butanediol
The invention provides a non-naturally occurring microbial organism having n-propanol and isopropanol pathways, 1,4-butanediol (14-BDO) and isopropanol pathways, 1,3-butanediol (13-BDO) and isopropanol pathways or methylacrylic acid (MAA) and isopropanolpathways. The microbial organism contains at least one exogenous nucleic acid encoding an enzyme in each of the respective n-propanol, 14-BDO, 13-BDO or MAA and isopropanol pathways. The invention additionally provides a method for co-producing n-propanol and isopropanol, 14-BDO and isopropanol, 13-BDO and isopropanol or MAA and isopropanol. The method can include culturing an n-propanol and an isopropanol co-producing microbial organism, where the microbial organism expresses at least one exogenous nucleic acid encoding an n-propanol, an isopropanol, a 14-BDO, a 13-BDO and/or a MAA pathway enzyme in a sufficient amount to produce each of the respective products, under conditions and for a sufficient period of time to produce each of the respective products.
1. A non-naturally occurring microbial organism having a 1,4-butanediol pathway and an isopropanol 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 and at least one exogenous nucleic acid encoding an isopropanol pathway enzyme expressed in a sufficient amount to produce isopropanol, wherein said 1,4-butanediol pathway comprises:
(1) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase;
(2) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase;
(3) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase and a 4-hydroxybutyraldehyde reductase;
(4) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; or
(5) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase and a 4-hydroxybutyraldehyde reductase, and
wherein said isopropanol pathway comprises:
(6) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetate decarboxylase and an isopropanol dehydrogenase;
(7) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase and an isopropanol dehydrogenase; or
(8) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
2. The non-naturally occurring microbial organism of claim 1 , further comprising an acetyl-CoA pathway comprising at least one exogenous nucleic acid encoding an acetyl-CoA pathway enzyme expressed in a sufficient amount to produce acetyl-CoA, said acetyl-CoA pathway comprising:
a pyruvate kinase, a pyruvate dehydrogenase, or a pyruvate ferredoxin oxidoreductase.
3. The non-naturally occurring microbial organism of claim 1 , further comprising a succinyl-CoA pathway comprising at least one exogenous nucleic acid encoding a succinyl-CoA pathway enzyme expressed in a sufficient amount to produce succinyl-CoA, said succinyl-CoA pathway comprising:
a PEP carboxykinase, a PEP carboxylase, a malate dehydrogenase, a fumarase, a fumarate reductase, a succinyl-CoA transferase or a succinyl-CoA synthetase.
4. The non-naturally occurring microbial organism of claim 3 , wherein said succinyl-CoA pathway comprises one, two, three, four, five, six or seven exogenous nucleic acids each encoding a succinyl-CoA pathway enzyme.
5. The non-naturally occurring microbial organism of claim 1 , wherein said exogenous nucleic acid is a heterologous nucleic acid.
6. The non-naturally occurring microbial organism of claim 1 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
7. A method for producing 1,4-butanediol and isopropanol, comprising culturing a non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce 1,4-butanediol and isopropanol.
8. The method of claim 7 , wherein said conditions comprise substantially anaerobic culture conditions.
9. The method of claim 7 , wherein said exogenous nucleic acid is a heterologous nucleic acid.
10. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises one, two, three, four or five exogenous nucleic acids each encoding a 1,4-butanediol pathway enzyme.
11. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises one, two, three or four exogenous nucleic acids each encoding a isopropanol pathway enzyme.
12. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism comprises one, two, three, four or five exogenous nucleic acids each encoding a 1,4-butanediol pathway enzyme, and one, two, three or four exogenous nucleic acids each encoding a isopropanol pathway enzyme.
13. The non-naturally occurring microbial organism of claim 12 , wherein said microbial organism comprises exogenous nucleic acids encoding each of the enzymes of at least one of the 1,4-butanediol pathway comprising (1)-(5), and exogenous nucleic acids encoding each of the enzymes of at least one of the isopropanol pathway comprising (6)-(8).
14. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (1) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (6) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
15. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (1) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (7) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
16. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (1) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (8) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
17. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (2) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (6) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
18. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (2) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (7) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
19. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (2) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyryl-CoA synthetase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (8) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
20. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (3) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (6) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
21. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (3) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (7) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
22. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (3) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (8) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
23. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (4) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (6) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
24. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (4) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (7) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
25. The non-naturally occurring microbial organism of claim 24 , wherein said 1,4-butanediol pathway comprises (4) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde-forming) and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (8) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
26. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (5) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (6) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA transferase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
27. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (5) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (7) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA hydrolase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.
28. The non-naturally occurring microbial organism of claim 1 , wherein said 1,4-butanediol pathway comprises (5) a succinyl-CoA reductase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase and a 4-hydroxybutyraldehyde reductase; and said isopropanol pathway comprises (8) an acetyl-CoA acetyl thiolase, an acetoacetyl-CoA synthetase, an acetoacetate decarboxylase and an isopropanol dehydrogenase.