Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors

Abstract

The invention provides a non-naturally occurring microbial biocatalyst including a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway having at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid (4-HB). Also provided is a non-naturally occurring microbial biocatalyst including a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways include at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). Additionally provided is a method for the production of 4-HB. The method includes culturing a non-naturally occurring microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway including at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase or α-ketoglutarate decarboxylase under substantially anaerobic conditions for a sufficient period of time to produce monomeric 4-hydroxybutanoic acid (4-HB). Further provided is a method for the production of BDO. The method includes culturing a non-naturally occurring microbial biocatalyst, comprising a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways including at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-hydroxybutyrate kinase, phosphotranshydroxybutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase for a sufficient period of time to produce 1,4-butanediol (BDO). The 4-HB and/or BDO products can be secreted into the culture medium.

1 . A non-naturally occurring microbial biocatalyst, comprising a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway comprising at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase, wherein said exogenous nucleic acid is expressed in sufficient amounts to secrete monomeric 4-hydroxybutanoic acid (4-HB). 
     
     
         2 . The non-naturally occurring microbial biocatalyst of  claim 1 , wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase. 
     
     
         3 - 5 . (canceled) 
     
     
         6 . The non-naturally occurring microbial biocatalyst of  claim 1 , further comprising a nucleic acid encoding an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase. 
     
     
         7 . The non-naturally occurring microbial biocatalyst of  claim 1 , wherein said microbial organism lacks an endogenous 4-HB biosynthetic activity selected from 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, and α-ketoglutarate decarboxylase. 
     
     
         8 - 11 . (canceled) 
     
     
         12 . A non-naturally occurring microbial biocatalyst, comprising a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, said pathways comprise at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase, wherein said exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). 
     
     
         13 . The non-naturally occurring microbial biocatalyst of  claim 12 , wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase or α-ketoglutarate decarboxylase. 
     
     
         14 - 16 . (canceled) 
     
     
         17 . The non-naturally occurring microbial biocatalyst of  claim 12 , further comprising a nucleic acid encoding an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase. 
     
     
         18 . The non-naturally occurring microbial biocatalyst of  claim 12 , wherein said microbial organism lacks an endogenous 4-HB biosynthetic activity selected from 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase CoA-dependent succinic semialdehyde dehydrogenase and α-ketoglutarate decarboxylase. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The non-naturally occurring microbial biocatalyst of  claim 12 , wherein said BDO biosynthetic pathway comprises aldehyde dehydrogenase, and alcohol dehydrogenase or aldehyde/alcohol dehydrogenase. 
     
     
         22 . The non-naturally occurring microbial biocatalyst of  claim 21 , wherein said BDO biosynthetic pathway further comprises 4-hydroxybutyrate:CoA transferase or 4-butyrate kinase and phosphotransbutyrylase. 
     
     
         23 - 29 . (canceled) 
     
     
         30 . A method for the production of 4-HB, comprising culturing a non-naturally occurring microbial biocatalyst of  claim 1  for a sufficient period of time to produce monomeric 4-hydroxybutanoic acid (4-HB). 
     
     
         31 - 39 . (canceled) 
     
     
         40 . The method of  claim 30 , further comprising isolating 4-HB. 
     
     
         41 . The method of  claim 30 , further comprising a culture medium having a pH of about 7.5 or less. 
     
     
         42 . The method of  claim 41 , further comprising isolating γ-butyrolactone (GBL). 
     
     
         43 . The method of  claim 42 , wherein said isolation of GBL comprises separation of GBL. 
     
     
         44 . The method of  claim 43 , further comprising distillation to produce substantially pure GBL. 
     
     
         45 . The method of  claim 44 , further comprising chemical hydrogenation of 4-HB, GBL or a mixture thereof to produce 1-4 butanediol (BDO). 
     
     
         46 . The method of  claim 44 , further comprising chemical hydrogenation of 4-HB, GBL or a mixture thereof to produce tetrahydrofuran (THF). 
     
     
         47 . A method for the production of BDO, comprising culturing a non-naturally occurring microbial biocatalyst of  claim 12  for a sufficient period of time to produce 1,4-butanediol (BDO). 
     
     
         48 - 61 . (canceled) 
     
     
         62 . The method of  claim 47 , wherein said monomeric BDO is expressed at an intracellular concentration of at least about 5 mM. 
     
     
         63 . (canceled)