Microorganisms and methods for the biosynthesis of butadiene

Abstract

The invention provides non-naturally occurring microbial organisms having a butadiene pathway. The invention additionally provides methods of using such organisms to produce butadiene.

1 - 59 . (canceled) 
     
     
         60 . A non-naturally occurring microbial organism comprising a biosynthetic pathway for the production of crotyl alcohol wherein said biosynthetic pathway comprises at least one exogenous nucleic acid encoding at least one metabolic pathway enzyme expressed in a sufficient amount to produce crotyl alcohol, wherein said biosynthetic pathway comprises converting crotonyl-CoA to crotyl alcohol using one or more metabolic pathway enzymes, wherein said metabolic pathway enzymes are selected from the group consisting of:
 (i) a crotonyl-CoA reductase converting crotonyl-CoA to crotonaldehyde, and a crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol;   (ii) a crotonyl-CoA reductase converting crotonyl-CoA to crotyl alcohol; and   (iii) a crotonyl-CoA hydrolase, a crotonyl-CoA synthetase, or a crotonyl-CoA transferase converting crotonyl-CoA to crotonate, a crotonate reductase converting crotonate to crotonaldehyde, and a crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol, and   wherein said biosynthetic pathway further comprises:   (i) converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase;   (ii) converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase;   (iii) converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase;   (iv) converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase; or   (v) converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase.   
     
     
         61 . The non-naturally occurring microbial organism of  claim 60 , wherein said microbial organism comprises one, two, three, four, five or six exogenous nucleic acids each encoding a metabolic pathway enzyme. 
     
     
         62 . The non-naturally occurring microbial organism of  claim 60 , wherein said biosynthetic pathway comprises a crotonyl-CoA reductase converting crotonyl-CoA to crotonaldehyde and a crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol. 
     
     
         63 . The non-naturally occurring microbial organism of  claim 62 , wherein said biosynthetic pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase. 
     
     
         64 . The non-naturally occurring microbial organism of  claim 62 , wherein said biosynthetic pathway further comprises converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase. 
     
     
         65 . The non-naturally occurring microbial organism of  claim 62 , wherein said biosynthetic pathway further comprises converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase. 
     
     
         66 . The non-naturally occurring microbial organism of  claim 62 , wherein said biosynthetic pathway further comprises converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase. 
     
     
         67 . The non-naturally occurring microbial organism of  claim 62 , wherein said biosynthetic pathway further comprises converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase. 
     
     
         68 . The non-naturally occurring microbial organism of  claim 60 , wherein said biosynthetic pathway comprises a crotonyl-CoA reductase converting crotonaldehyde to crotyl alcohol. 
     
     
         69 . The non-naturally occurring microbial organism of  claim 68 , wherein said biosynthetic pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase. 
     
     
         70 . The non-naturally occurring microbial organism of  claim 68 , wherein said biosynthetic pathway further comprises converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase. 
     
     
         71 . The non-naturally occurring microbial organism of  claim 68 , wherein said biosynthetic pathway further comprises converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase. 
     
     
         72 . The non-naturally occurring microbial organism of  claim 68 , wherein said biosynthetic pathway further comprises converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase. 
     
     
         73 . The non-naturally occurring microbial organism of  claim 68 , wherein said biosynthetic pathway further comprises converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase. 
     
     
         74 . The non-naturally occurring microbial organism of  claim 60 , said biosynthetic pathway comprises a crotonyl-CoA hydrolase, crotonyl-CoA synthetase or crotonyl-CoA transferase converting crotonyl-CoA to crotonate, a crotonate reductase converting crotonate to crotonaldehyde, and crotonaldehyde reductase converting crotonaldehyde to crotyl alcohol. 
     
     
         75 . The non-naturally occurring microbial organism of  claim 74 , wherein said biosynthetic pathway further comprises converting acetyl-CoA to crotonyl-CoA using an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase and a 3-hydroxybutyryl-CoA dehydratase. 
     
     
         76 . The non-naturally occurring microbial organism of  claim 74 , wherein said biosynthetic pathway further comprises converting glutaconyl-CoA to crotonyl-CoA using a glutaconyl-CoA decarboxylase. 
     
     
         77 . The non-naturally occurring microbial organism of  claim 74 , wherein said biosynthetic pathway further comprises converting glutaryl-CoA to crotonyl-CoA using a glutaryl-CoA dehydrogenase. 
     
     
         78 . The non-naturally occurring microbial organism of  claim 74 , wherein said biosynthetic pathway further comprises converting 3-aminobutyryl-CoA to crotonyl-CoA using an 3-aminobutyryl-CoA deaminase. 
     
     
         79 . The non-naturally occurring microbial organism of  claim 74 , wherein said biosynthetic pathway further comprises converting 4-hydroxybutyryl-CoA to crotonyl-CoA using a 4-hydroxybutyryl-CoA dehydratase. 
     
     
         80 . A method for producing crotyl alcohol comprising culturing the non-naturally occurring microbial organism of  claim 60  under conditions and for a sufficient period of time to produce crotyl alcohol.