Microorganisms for the production of 1,4-Butanediol
View Patent ↗The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO) pathway comprising at least one exogenous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO. The invention additionally provides methods of using such microbial organisms to produce BDO.
1. A non-naturally occurring microbial organism having nucleic acids encoding enzymes of a 1,4-butanediol (BDO) pathway, wherein said microbial organism is transformed with at least one heterologous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO, wherein said BDO pathway enzymes comprise:
(a) 4-aminobutyrate CoA transferase, said enzyme classified as EC 2.8.3.a and converting 4-aminobutyrate to 4-aminobutyryl-CoA; 4-aminobutyryl-CoA hydrolase, said enzyme classified as EC 3.1.2.a and converting 4-aminobutyrate to 4-aminobutyryl-CoA; or 4-aminobutyrate-CoA ligase, said enzyme classified as EC 6.2.1.a and converting 4-aminobutyrate to 4-aminobutyryl-CoA;
(b) 4-aminobutyryl-CoA oxidoreductase (deaminating), said enzyme classified as EC1.4.1.a and converting 4-aminobutyryl-CoA to 4-oxobutyryl-CoA; or 4-aminobutyryl-CoA transaminase, said enzyme classified as EC 2.6.1.a and converting 4-aminobutyryl-CoA to 4-oxobutyryl-CoA;
(c) 4-hydroxybutyryl-CoA dehydrogenase, said enzyme classified as EC 1.1.1.a and converting 4-oxobutyryl-CoA to 4-hydroxybutyryl-CoA, with the proviso that said non-naturally occurring microorganism is not a wild type microorganism inherently having any of the above genes; and
(d) 4-hydroxybutyryl-CoA reductase (alcohol forming), said enzyme classified as EC1.1.1.c and converting 4-hydroxybutyryl-CoA to 1,4-butanediol; or 4-hydroxybutyryl-CoA reductase, said enzyme classified as EC 1.2.1.b and converting 4-hydroxybutyryl-CoA to 4-hydroxybutanal, and 1,4-butanediol dehydrogenase, said enzyme classified as EC 1.1.1.a and converting 4-hydroxybutanal to 1,4-butanediol.
2. The non-naturally occurring microbial organism of claim 1 , wherein said microbial organism is transformed with heterologous nucleic acids encoding one enzyme of each step of:
(a) 4-aminobutyrate CoA transferase; 4-aminobutyryl-CoA hydrolase; or 4-aminobutyrate-CoA ligase;
(b) 4-aminobutyryl-CoA oxidoreductase (deaminating); or 4-aminobutyryl-CoA transaminase;
(c) 4-hydroxybutyryl-CoA dehydrogenase; and
(d) 4-hydroxybutyryl-CoA reductase (alcohol forming); or 4-hydroxybutyryl-CoA reductase and 1,4-butanediol dehydrogenase.
3. The non-naturally occurring microbial organism of claim 2 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyrate CoA transferase.
4. The non-naturally occurring microbial organism of claim 2 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyryl-CoA hydrolase.
5. The non-naturally occurring microbial organism of claim 2 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyrate-CoA ligase.
6. The non-naturally occurring microbial organism of claim 2 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyryl-CoA oxidoreductase (deaminating).
7. The non-naturally occurring microbial organism of claim 2 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyryl-CoA transaminase.
8. The non-naturally occurring microbial organism of claim 2 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-hydroxybutyryl-CoA reductase (alcohol forming).
9. The non-naturally occurring microbial organism of claim 2 , wherein said microbial is transformed with a heterologous nucleic acid encoding 4-hydroxybutyryl-CoA reductase and 1,4-butanediol dehydrogenase.
10. A method for producing BDO, comprising culturing the non-naturally occurring microbial organism of claim 1 under conditions and for a sufficient period of time to produce BDO.
11. The method of claim 10 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.
12. A non-naturally occurring microbial organism having nucleic acids encoding enzymes of a 1,4-butanediol (BDO) pathway, wherein said microbial organism is transformed with at least one heterologous nucleic acid encoding a BDO pathway enzyme expressed in a sufficient amount to produce BDO, wherein said BDO pathway enzymes comprise:
(a) 4-aminobutyrate CoA transferase, said enzyme classified as EC 2.8.3.a and converting 4-aminobutyrate to 4-aminobutyryl-CoA; 4-aminobutyryl-CoA hydrolase, said enzyme classified as EC 3.1.2.a and converting 4-aminobutyrate to 4-aminobutyryl-CoA; or 4-aminobutyrate-CoA ligase, said enzyme classified as EC 6.2.1.a and converting 4-aminobutyrate to 4-aminobutyryl-CoA;
(b) 4-aminobutyryl-CoA reductase (alcohol forming), said enzyme classified as EC 1.1.1.c and converting 4-aminobutyryl-CoA to 4-aminobutan-1-ol; or 4-aminobutyryl-CoA reductase, said enzyme classified as EC 1.2.1.b and converting 4-aminobutyryl-CoA to 4-aminobutanal, and 4-aminobutan-1-ol dehydrogenase, said enzyme classified as EC 1.1.1.a and converting 4-aminobutanal to 4-aminobutan-1-ol;
(c) 4-aminobutan-1-ol oxidoreductase (deaminating), said enzyme classified as EC 1.4.1.a and converting 4-aminobutan-1-ol to 4-hydroxybutanal; or 4-aminobutan-1-ol transaminase, said enzyme classified as EC 2.6.1.a and converting 4-aminobutan-1-ol to 4-hydroxybutanal; and
(d) 1,4-butanediol dehydrogenase, said enzyme classified as EC 1.1.1.a and converting 4-hydroxybutanal to 1,4-butanediol.
13. The non-naturally occurring microbial organism of claim 12 , wherein said microbial organism is transformed with heterologous nucleic acids encoding at least one enzyme of each step of:
(a) 4-aminobutyrate CoA transferase; 4-aminobutyryl-CoA hydrolase; or 4-aminobutyrate-CoA ligase;
(b) 4-aminobutyryl-CoA reductase (alcohol forming); or 4-aminobutyryl-CoA reductase and 4-aminobutan-1-ol dehydrogenase;
(c) 4-aminobutan-1-ol oxidoreductase (deaminating); or 4-aminobutan-1-ol transaminase; and
(d) 1,4-butanediol dehydrogenase.
14. The non-naturally occurring microbial organism of claim 13 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyrate CoA transferase.
15. The non-naturally occurring microbial organism of claim 13 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyryl-CoA hydrolase.
16. The non-naturally occurring microbial organism of claim 13 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyrate-CoA ligase.
17. The non-naturally occurring microbial organism of claim 13 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyryl-CoA reductase (alcohol forming).
18. The non-naturally occurring microbial organism of claim 13 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutyryl-CoA reductase and 4-aminobutan-1-ol dehydrogenase.
19. The non-naturally occurring microbial organism of claim 13 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutan-1-ol oxidoreductase (deaminating).
20. The non-naturally occurring microbial organism of claim 13 , wherein said microbial organism is transformed with a heterologous nucleic acid encoding 4-aminobutan-1-ol transaminase.
21. A method for producing BDO, comprising culturing the non-naturally occurring microbial organism of claim 12 under conditions and for a sufficient period of time to produce BDO.
22. The method of claim 21 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.