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 are methods for the production of 4-HB and BDO.

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 . The non-naturally occurring microbial biocatalyst of  claim 1 , wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase. 
     
     
         4 . The non-naturally occurring microbial biocatalyst of  claim 3 , further comprising a nucleic acid encoding (a) an exogenous α-ketoglutarate decarboxylase or (b) an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase. 
     
     
         5 - 8 . (canceled) 
     
     
         9 . The non-naturally occurring microbial biocatalyst of  claim 1 , wherein said monomeric 4-HB is expressed at an intracellular concentration of at least about 5 mM. 
     
     
         10 . (canceled) 
     
     
         11 . The non-naturally occurring microbial biocatalyst of  claim 1 , further comprising a substantially anaerobic culture medium. 
     
     
         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 . The non-naturally occurring microbial biocatalyst of  claim 12 , wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase. 
     
     
         15 . The non-naturally occurring microbial biocatalyst of  claim 14 , further comprising a nucleic acid encoding (a) an exogenous α-ketoglutarate decarboxylase or (b) an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase. 
     
     
         16 - 19 . (canceled) 
     
     
         20 . The non-naturally occurring microbial biocatalyst of  claim 14  or  15 , wherein said exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid. 
     
     
         21 . The non-naturally occurring microbial biocatalyst of  claim 12 , wherein said BDO biosynthetic pathway comprises: (a) aldehyde dehydrogenase, and alcohol dehydrogenase or aldehyde/alcohol dehydrogenase, or (b) 4-hydroxybutyrate:CoA transferase or 4-butyrate kinase and phosphotransbutyrylase. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . The non-naturally occurring microbial biocatalyst of  claim 12 , further comprising a substantially anaerobic culture medium. 
     
     
         28 . The non-naturally occurring microbial biocatalyst of  claim 20 , wherein said monomeric BDO is expressed at an intracellular concentration of at least about 5 mM. 
     
     
         29 . (canceled) 
     
     
         30 . A method for the production of 4-HB, comprising culturing a non-naturally occurring 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 under substantially anaerobic conditions for a sufficient period of time to produce monomeric 4-hydroxybutanoic acid (4-HB). 
     
     
         31 . The method of  claim 30 , wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase or α-ketoglutarate decarboxylase. 
     
     
         32 . The method of  claim 30 , wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase. 
     
     
         33 . The method of  claim 32 , wherein said non-naturally occurring microbial organism further comprises a nucleic acid encoding (a) an exogenous α-ketoglutarate decarboxylase or (b) an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase. 
     
     
         34 - 37 . (canceled) 
     
     
         38 . The method of  claim 30 , wherein said monomeric 4-HB is expressed at an intracellular concentration of at least about 5 mM. 
     
     
         39 . (canceled) 
     
     
         40 . The method of  claim 30 , further comprising isolating 4-HB or γ-butyrolactone (GBL). 
     
     
         41 - 42 . (canceled) 
     
     
         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 30  or  44 , further comprising chemical hydrogenation of 4-HB, GBL or a mixture thereof to produce 1-4 butanediol (BDO) or tetrahydrofuran (THF). 
     
     
         46 . (canceled) 
     
     
         47 . A method for the production of BDO, comprising culturing 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 comprising 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 for a sufficient period of time to produce 1,4-butanediol (BDO). 
     
     
         48 . The method of  claim 47 , wherein said 4-HB biosynthetic pathway comprises 4-hydroxybutanoate dehydrogenase and succinyl-CoA synthetase and CoA-dependent succinic semialdehyde dehydrogenase or α-ketoglutarate decarboxylase. 
     
     
         49 . The method of  claim 47 , wherein said exogenous nucleic acid encodes 4-hydroxybutanoate dehydrogenase. 
     
     
         50 . The method of  claim 49 , further comprising a nucleic acid encoding (a) an exogenous α-ketoglutarate decarboxylase or (b) an exogenous succinyl-CoA synthetase, exogenous CoA-dependent succinic semialdehyde dehydrogenase or exogenous succinyl-CoA synthetase and exogenous CoA-dependent succinic semialdehyde dehydrogenase. 
     
     
         51 - 54 . (canceled) 
     
     
         55 . The method of  claim 49  or  50 , wherein said exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid. 
     
     
         56 . The method of  claim 47 , wherein said BDO biosynthetic pathway comprises: (a) aldehyde dehydrogenase and alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase or (b) 4-hydroxybutyrate:CoA transferase or 4-butyrate kinase and phosphotransbutyrylase. 
     
     
         57 - 60 . (canceled) 
     
     
         61 . The method of  claim 47 , further comprising a substantially anaerobic culture medium. 
     
     
         62 . The method of claim  59 , wherein said monomeric BDO is expressed at an intracellular concentration of at least about 5 mM. 
     
     
         63 . (canceled)