Microorganisms for the production of 1,4-butanediol, 4-hydroxybutanal, 4-hydroxybutyryl-coa, putrescine and related compounds, and methods related thereto

Abstract

The invention provides non-naturally occurring microbial organisms comprising a 1,4-butanediol (BDO), 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine pathway comprising at least one exogenous nucleic acid encoding a BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine pathway enzyme expressed in a sufficient amount to produce BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine and further optimized for expression of BDO. The invention additionally provides methods of using such microbial organisms to produce BDO, 4-hydroxybutyryl-CoA, 4-hydroxybutanal or putrescine.

56 . A non-naturally occurring microbial organism, comprising a microbial organism having a 4-hydroxybutanal pathway comprising at least one exogenous nucleic acid encoding a 4-hydroxybutanal pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutanal, said 4-hydroxybutanal pathway comprising:
 succinyl-CoA reductase (aldehyde forming); 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase;   alpha-ketoglutarate decarboxylase; 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase;   succinate reductase; 4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate reductase;   alpha-ketoglutarate decarboxylase, or glutamate dehydrogenase or glutamate transaminase and glutamate decarboxylase and 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase; 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase; or   alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate dehydrogenase; and 5-hydroxy-2-oxopentanoate decarboxylase.   
     
     
         57 . The non-naturally occurring microbial organism of  claim 56 , wherein said microbial organism comprises exogenous nucleic acids encoding 4-hydroxybutanal pathway enzymes expressed in a sufficient amount to produce 4-hydroxybutanal, said 4-hydroxybutanal pathway comprising:
 4-hydroxybutyrate reductase; succinyl-CoA reductase (aldehyde forming); and 4-hydroxybutyrate dehydrogenase; or   4-hydroxybutyrate reductase; alpha-ketoglutarate decarboxylase; and 4-hydroxybutyrate dehydrogenase.   
     
     
         58 . The non-naturally occurring microbial organism of  claim 56 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         59 . A method for producing 4-hydroxybutanal, comprising culturing the non-naturally occurring microbial organism of  claim 56  under conditions and for a sufficient period of time to produce 4-hydroxybutanal. 
     
     
         60 . The method of  claim 59 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         61 . A non-naturally occurring microbial organism, comprising a microbial organism having a putrescine pathway comprising at least one exogenous nucleic acid encoding a putrescine pathway enzyme expressed in a sufficient amount to produce putrescine, said putrescine pathway comprising:
 succinate reductase; 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase;   alpha-ketoglutarate decarboxylase; 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase;   glutamate dehydrogenase or glutamate transaminase; glutamate decarboxylase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase;   alpha-ketoglutarate reductase; 5-amino-2-oxopentanoate dehydrogenase or 5-amino-2-oxopentanoate transaminase; 5-amino-2-oxopentanoate decarboxylase; and putrescine dehydrogenase or putrescine transaminase; or   alpha-ketoglutarate reductase; 5-amino-2-oxopentanoate dehydrogenase or 5-amino-2-oxopentanoate transaminase; ornithine dehydrogenase or ornithine transaminase; and ornithine decarboxylase;   
     
     
         62 . The non-naturally occurring microbial organism of  claim 61 , wherein said microbial organism comprises:
 four exogenous nucleic acids encoding succinate reductase; 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase;   four exogenous nucleic acids encoding alpha-ketoglutarate decarboxylase; 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase;   four exogenous nucleic acids encoding glutamate dehydrogenase or glutamate transaminase; glutamate decarboxylase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase;   four exogenous nucleic acids encoding alpha-ketoglutarate reductase; 5-amino-2-oxopentanoate dehydrogenase or 5-amino-2-oxopentanoate transaminase; 5-amino-2-oxopentanoate decarboxylase; and putrescine dehydrogenase or putrescine transaminase; or   four exogenous nucleic acids encoding alpha-ketoglutarate reductase; 5-amino-2-oxopentanoate dehydrogenase or 5-amino-2-oxopentanoate transaminase; ornithine dehydrogenase or ornithine transaminase; and ornithine decarboxylase.   
     
     
         63 . The non-naturally occurring microbial organism of  claim 61 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         64 . A method for producing putrescine, comprising culturing the non-naturally occurring microbial organism of  claim 61  under conditions and for a sufficient period of time to produce putrescine. 
     
     
         65 . The method of  claim 64 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         66 . A non-naturally occurring microbial organism, comprising a microbial organism having a 4-hydroxybutyryl-CoA (4-HBCoA) pathway comprising at least one exogenous nucleic acid encoding a 4-hydroxybutyryl-CoA pathway enzyme expressed in a sufficient amount to produce 4-hydroxybutyryl-CoA, said 4-hydroxybutyryl-CoA pathway comprising alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate dehydrogenase; and 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation). 
     
     
         67 . The non-naturally occurring microbial organism of  claim 66 , wherein said microbial organism comprises three exogenous nucleic acids encoding alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate dehydrogenase; and 5-hydroxy-2-oxopentanoate dehydrogenase (decarboxylation). 
     
     
         68 . The non-naturally occurring microbial organism of  claim 66 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         69 . A method for producing 4-hydroxybutyryl-CoA, comprising culturing the non-naturally occurring microbial organism of  claim 66  under conditions and for a sufficient period of time to produce 4-hydroxybutyryl-CoA. 
     
     
         70 . The method of  claim 69 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.