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.

1 . A non-naturally occurring microbial organism, comprising 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;   succinyl-CoA reductase (aldehyde forming); 4-hydroxybutyrate dehydrogenase; 4-hydroxybutyryl-CoA transferase or 4-hydroxybutyryl-CoA hydrolase or 4-hydroxybutyryl-CoA synthetase; and 4-hydroxybutyryl-CoA reductase (aldehyde forming);   succinyl-CoA reductase (aldehyde forming); 4-hydroxybutyrate dehydrogenase; 4-hydroxybutyrate kinase; phosphotrans-4-hydroxybutyrylase; and 4-hydroxybutyryl-CoA reductase (aldehyde forming); or   succinyl-CoA reductase (aldehyde forming); 4-hydroxybutyrate dehydrogenase; 4-hydroxybutyrate kinase; and 4-hydroxybutyryl-phosphate reductase.   
     
     
         2 . The non-naturally occurring microbial organism of  claim 1 , wherein said microbial organism comprises three exogenous nucleic acids encoding succinyl-CoA reductase (aldehyde forming); 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase. 
     
     
         3 . The non-naturally occurring microbial organism of  claim 1 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         4 . A method for producing 4-hydroxybutanal, comprising culturing the non-naturally occurring microbial organism of  claim 1  under conditions and for a sufficient period of time to produce 4-hydroxybutanal. 
     
     
         5 . The method of  claim 4 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         6 . The non-naturally occurring microbial organism of  claim 1 , said 4-hydroxybutanal pathway comprising alpha-ketoglutarate decarboxylase; 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase. 
     
     
         7 . The non-naturally occurring microbial organism of  claim 6 , wherein said microbial organism comprises three exogenous nucleic acids encoding alpha-ketoglutarate decarboxylase; 4-hydroxybutyrate dehydrogenase; and 4-hydroxybutyrate reductase. 
     
     
         8 . The non-naturally occurring microbial organism of  claim 6 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         9 - 10 . (canceled) 
     
     
         11 . The non-naturally occurring microbial organism of  claim 1 , said 4-hydroxybutanal pathway comprising succinate reductase; 4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate reductase. 
     
     
         12 . The non-naturally occurring microbial organism of  claim 11 , wherein said microbial organism comprises three exogenous nucleic acids encoding succinate reductase; 4-hydroxybutyrate dehydrogenase, and 4-hydroxybutyrate reductase. 
     
     
         13 . The non-naturally occurring microbial organism of  claim 11 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . The non-naturally occurring microbial organism of  claim 1 , said 4-hydroxybutanal pathway comprising 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.   
     
     
         17 . The non-naturally occurring microbial organism of  claim 1 , said 4-hydroxybutanal pathway comprising alpha-ketoglutarate reductase; 5-hydroxy-2-oxopentanoate dehydrogenase; and 5-hydroxy-2-oxopentanoate decarboxylase. 
     
     
         18 . 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; or   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.   
     
     
         19 . The non-naturally occurring microbial organism of  claim 18 , said putrescine pathway comprising alpha-ketoglutarate decarboxylase; 4-aminobutyrate dehydrogenase or 4-aminobutyrate transaminase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase. 
     
     
         20 . The non-naturally occurring microbial organism of  claim 18 , said putrescine pathway comprising glutamate dehydrogenase or glutamate transaminase; glutamate decarboxylase; 4-aminobutyrate reductase; and putrescine dehydrogenase or putrescine transaminase. 
     
     
         21 . The non-naturally occurring microbial organism of  claim 18 , said putrescine pathway comprising 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.