Microorganisms and methods for the coproduction 1,4-butanediol and gamma-butyrolactone
First Claim
1. A method for producing 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL), comprising culturing a non-naturally occurring microbial organism for a sufficient period of time to produce 14-BDO and 4-hydroxybutyryl-CoA followed by spontaneous lactonization of 4-hydroxybutyryl-CoA to form GBL, wherein said 14-BDO and GBL are produced at a predetermined ratio of 14-BDO to GBL selected from the group consisting of 1:
- 1, 6;
1, 7;
1, 8;
1, 9;
1, 10;
1, 15;
1, 20;
1, 25;
1, 30;
1, 35;
1, 40;
1, 45;
1, 50;
1, 60;
1, 70;
1, 80;
1, 90;
1 and 100;
1, wherein said non-naturally occurring microbial organism comprises a 14-BDO pathway and a 4-hydroxybutyryl-CoA pathway, said 14-BDO pathway comprising at least one exogenous nucleic acid encoding a 14-BDO pathway enzyme expressed in a sufficient amount to produce 14-BDO, said 14-BDO pathway comprising;
a 4-hydroxybutyryl-CoA reductase (alcohol forming), a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase, a 1,4-butanediol dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, an 4-aminobutyrate dehydrogenase, an 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 4-hydroxybutyryl-phosphate reductase, a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hydroxy-2-oxopentanoate decarboxylase or a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation),said 4-hydroxybutyryl-CoA pathway comprising at least one exogenous nucleic acid encoding an 4-hydroxybutyryl-CoA pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA, said 4-hydroxybutyryl-CoA pathway comprising;
a 4-hydroxybutyryl-CoA transferase, a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, a 4-aminobutyrate dehydrogenase, a 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate dehydrogenase, or a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation).
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Accused Products
Abstract
The invention provides non-naturally occurring microbial organisms comprising 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL) pathways comprising at least one exogenous nucleic acid encoding a 14-BDO and GBL pathway enzyme expressed in a sufficient amount to produce 14-BDO and GBL. The invention additionally provides methods of using such microbial organisms to produce 14-BDO and GBL.
161 Citations
64 Claims
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1. A method for producing 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL), comprising culturing a non-naturally occurring microbial organism for a sufficient period of time to produce 14-BDO and 4-hydroxybutyryl-CoA followed by spontaneous lactonization of 4-hydroxybutyryl-CoA to form GBL, wherein said 14-BDO and GBL are produced at a predetermined ratio of 14-BDO to GBL selected from the group consisting of 1:
- 1, 6;
1, 7;
1, 8;
1, 9;
1, 10;
1, 15;
1, 20;
1, 25;
1, 30;
1, 35;
1, 40;
1, 45;
1, 50;
1, 60;
1, 70;
1, 80;
1, 90;
1 and 100;
1, wherein said non-naturally occurring microbial organism comprises a 14-BDO pathway and a 4-hydroxybutyryl-CoA pathway, said 14-BDO pathway comprising at least one exogenous nucleic acid encoding a 14-BDO pathway enzyme expressed in a sufficient amount to produce 14-BDO, said 14-BDO pathway comprising;
a 4-hydroxybutyryl-CoA reductase (alcohol forming), a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a 4-hydroxybutyrate reductase, a 1,4-butanediol dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, an 4-aminobutyrate dehydrogenase, an 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a 4-hydroxybutyryl-CoA reductase (aldehyde forming), a 4-hydroxybutyryl-phosphate reductase, a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate dehydrogenase, a 5-hydroxy-2-oxopentanoate decarboxylase or a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation),said 4-hydroxybutyryl-CoA pathway comprising at least one exogenous nucleic acid encoding an 4-hydroxybutyryl-CoA pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA, said 4-hydroxybutyryl-CoA pathway comprising; a 4-hydroxybutyryl-CoA transferase, a succinyl-CoA reductase (aldehyde forming), an alpha-ketoglutarate decarboxylase, a 4-hydroxybutyrate dehydrogenase, a succinate reductase, a succinyl-CoA transferase, a succinyl-CoA hydrolase, a succinyl-CoA synthetase, a glutamate dehydrogenase, a glutamate transaminase, a glutamate decarboxylase, a 4-aminobutyrate dehydrogenase, a 4-aminobutyrate transaminase, a 4-hydroxybutyrate kinase, a phosphotrans-4-hydroxybutyrylase, a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyryl-CoA hydrolase, a 4-hydroxybutyryl-CoA synthetase, an alpha-ketoglutarate reductase, a 5-hydroxy-2-oxopentanoate dehydrogenase, or a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64)
- 1, 6;
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18. A method for producing 1,4-butanediol (14-BDO) and gamma-butyrolactone (GBL), comprising culturing a non-naturally occurring microbial organism for a sufficient period of time to produce 14-BDO and 4-hydroxybutyryl-CoA followed by spontaneous lactonization of 4-hydroxybutyryl-CoA to form GBL, wherein said 14-BDO and GBL are produced at a predetermined ratio of 14-BDO to GBL selected from the group consisting of 1:
- 1, 6;
1, 7;
1, 8;
1, 9;
1, 10;
1, 15;
1, 20;
1, 25;
1, 30;
1, 35;
1, 40;
1, 45;
1, 50;
1, 60;
1, 70;
1, 80;
1, 90;
1 and 100;
1, wherein said non-naturally occurring microbial organism comprises a 14-BDO pathway and a 4-hydroxybutyryl-CoA pathway, said 14-BDO pathway comprising a first set of exogenous nucleic acids encoding 14-BDO pathway enzymes expressed in a sufficient amount to produce 14-BDO, said first set is selected from the group consisting of;i) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate reductase; and
a 1,4-butanediol dehydrogenase);ii) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;iii) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a 4-hydroxybutyryl-phosphate reductase; and
a 1,4-butanediol dehydrogenase;iv) a succinyl-CoA transferase, a succinyl-CoA hydrolase, or a succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;v) a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);vi) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);vii) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming);
a 4-hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;viii) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming);
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;ix) a succinyl-CoA transferase, a succinyl-CoA hydrolase or succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming), a 4-hydroxybutyrate kinase, a 4-hydroxybutyryl-phosphate reductase; and
a 1,4-butanediol dehydrogenase;x) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming);
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;xi) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming);
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);xii) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming);
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);xiii) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;xiv) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;xv) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);xvi) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a 4-hydroxybutyryl-phosphate reductase; and
1,4-butanediol dehydrogenase;xvii) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
1,4-butanediol dehydrogenase;xviii) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);xix) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate reductase; and
1,4-butanediol dehydrogenase;xx) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;xxi) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase and 4-hydroxybutyryl-CoA reductase (alcohol forming);xxii) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a 4-hydroxybutyryl-phosphate reductase; and
a 1,4-butanediol dehydrogenase;xxiii) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA synthetase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;xxiv) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and
4-hydroxybutyryl-CoA reductase (alcohol forming);xxv) a glutamate dehydrogenase or a glutamate transaminase;
a glutamate decarboxylase;
a 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate reductase; and
a 1,4-butanediol dehydrogenase;xxvi) a glutamate dehydrogenase or a glutamate transaminase;
a glutamate decarboxylase;
a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;xxvii) a glutamate dehydrogenase or glutamate transaminase;
a glutamate decarboxylase;
a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a phosphotrans-4-hydroxybutyrylase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);xxviii) a glutamate dehydrogenase or a glutamate transaminase;
a glutamate decarboxylase;
a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase;
a 4-hydroxybutyryl-phosphate reductase; and
a 1,4-butanediol dehydrogenase;xxix) a glutamate dehydrogenase or a glutamate transaminase;
a glutamate decarboxylase;
a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;xxx) a glutamate dehydrogenase or a glutamate transaminase;
a glutamate decarboxylase;
a 4-aminobutyrate dehydrogenase or a 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; and
a 4-hydroxybutyryl-CoA reductase (alcohol forming);xxxi) an alpha-ketoglutarate reductase;
a 5-hydroxy-2-oxopentanoate dehydrogenase;
a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation);
a 4-hydroxybutyryl-CoA reductase (aldehyde forming); and
a 1,4-butanediol dehydrogenase;xxxii) an alpha-ketoglutarate reductase;
a 5-hydroxy-2-oxopentanoate dehydrogenase;
a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation); and
a 4-hydroxybutyryl-CoA reductase (alcohol forming); andxxxiii) an alpha-ketoglutarate reductase;
a 5-hydroxy-2-oxopentanoate dehydrogenase;
a 5-hydroxy-2-oxopentanoate decarboxylase; and
a 1,4-butanediol dehydrogenase, said 4-hydroxybutyryl-CoA pathway comprising a second set of exogenous nucleic acids encoding 4-hydroxybutyryl-CoA pathway enzymes expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA, said second set selected from the group consisting of;i) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase; and
a phosphotrans-4-hydroxybutyrylase;ii) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (aldehyde forming);
a 4-hydroxybutyrate dehydrogenase; and
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;iii) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming);
a 4-hydroxybutyrate kinase; and
a phosphotrans-4-hydroxybutyrylase;iv) a succinyl-CoA transferase, a succinyl-CoA hydrolase or a succinyl-CoA synthetase;
a succinyl-CoA reductase (alcohol forming); and
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;v) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase and a phosphotrans-4-hydroxybutyrylase;vi) a succinate reductase;
a 4-hydroxybutyrate dehydrogenase; and
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;vii) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase; and
a phosphotrans-4-hydroxybutyrylase;viii) an alpha-ketoglutarate decarboxylase;
a 4-hydroxybutyrate dehydrogenase; and
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase;ix) a glutamate dehydrogenase or a glutamate transaminase;
a glutamate decarboxylase;
an 4-aminobutyrate dehydrogenase or an 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase;
a 4-hydroxybutyrate kinase; and
a phosphotrans-4-hydroxybutyrylase;x) a glutamate dehydrogenase or a glutamate transaminase;
a glutamate decarboxylase;
an 4-aminobutyrate dehydrogenase or an 4-aminobutyrate transaminase;
a 4-hydroxybutyrate dehydrogenase; and
a 4-hydroxybutyryl-CoA transferase, a 4-hydroxybutyryl-CoA hydrolase or a 4-hydroxybutyryl-CoA synthetase; andxi) an alpha-ketoglutarate reductase;
a 5-hydroxy-2-oxopentanoate dehydrogenase and a 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation).
- 1, 6;
Specification