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 . A process for the production of butadiene comprising:
 (a) culturing by fermentation in a sufficient amount of nutrients and media a non-naturally occurring microbial organism that produces crotyl alcohol; and   (b) converting crotyl alcohol produced by culturing said non-naturally occurring microbial organism to butadiene.   
     
     
         2 . The process of  claim 1 , wherein step (b) is performed by chemical dehydration in the presence of a catalyst. 
     
     
         3 . The process of  claim 1 , wherein said non-naturally occurring microbial organism comprises a crotyl alcohol pathway comprising at least one exogenous nucleic acid encoding a crotyl alcohol pathway enzyme expressed in a sufficient amount to produce crotyl alcohol, said crotyl alcohol pathway comprising an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase, or transferase, a crotonate reductase, a crotonyl-CoA reductase (alcohol forming), a glutaconyl-CoA decarboxylase, a glutaryl-CoA dehydrogenase, a 3-aminobutyryl-CoA deaminase, or a 4-hydroxybutyryl-CoA dehydratase. 
     
     
         4 . The process of  claim 3 , wherein said microbial organism comprises two, three or four exogenous nucleic acids each encoding a crotyl alcohol pathway enzyme. 
     
     
         5 - 6 . (canceled) 
     
     
         7 . The process of  claim 3 , wherein said crotyl alcohol pathway comprises a pathway selected from the group consisting of:
 an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, and a crotonyl-CoA reductase (alcohol forming);   an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase or transferase and a crotonate reductase;   a glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a glutaconyl-CoA decarboxylase and a crotonyl-CoA reductase (alcohol forming);   a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase;   a glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a glutaryl-CoA dehydrogenase and a crotonyl-CoA reductase (alcohol forming);   a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase;   a 3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a 3-aminobutyryl-CoA deaminase and a crotonyl-CoA reductase (alcohol forming);   a 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase or transferase and a crotonate reductase;   a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a 4-hydroxybutyryl-CoA dehydratase and a crotonyl-CoA reductase (alcohol forming); and   a 4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase or transferase and a crotonate reductase.   
     
     
         8 - 21 . (canceled) 
     
     
         22 . The process of  claim 3 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         23 . The process of  claim 3 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium. 
     
     
         24 . A non-naturally occurring microbial organism comprising a crotyl alcohol pathway, said crotyl alcohol pathway comprising at least one exogenous nucleic acid encoding a crotyl alcohol pathway enzyme expressed in a sufficient amount to produce crotyl alcohol, said crotyl alcohol pathway comprising an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase, or transferase, a crotonate reductase, a crotonyl-CoA reductase (alcohol forming), a glutaconyl-CoA decarboxylase, a glutaryl-CoA dehydrogenase, a 3-aminobutyryl-CoA deaminase, or a 4-hydroxybutyryl-CoA dehydratase. 
     
     
         25 . The microbial organism of  claim 24 , wherein said microbial organism comprises two, three or four exogenous nucleic acids each encoding a crotyl alcohol pathway enzyme. 
     
     
         26 - 27 . (canceled) 
     
     
         28 . The microbial organism of  claim 24 , wherein said crotyl alcohol pathway comprises a pathway selected from the group consisting of:
 an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, and a crotonyl-CoA reductase (alcohol forming);   an acetyl-CoA:acetyl-CoA acyltransferase, an acetoacetyl-CoA reductase, a 3-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase or transferase and a crotonate reductase;   a glutaconyl-CoA decarboxylase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a glutaconyl-CoA decarboxylase and a crotonyl-CoA reductase (alcohol forming);   a glutaconyl-CoA decarboxylase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase;   a glutaryl-CoA dehydrogenase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a glutaryl-CoA dehydrogenase and a crotonyl-CoA reductase (alcohol forming);   a glutaryl-CoA dehydrogenase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase, or transferase and a crotonate reductase;   a 3-aminobutyryl-CoA deaminase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a 3-aminobutyryl-CoA deaminase and a crotonyl-CoA reductase (alcohol forming);   a 3-aminobutyryl-CoA deaminase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase or transferase and a crotonate reductase;   a 4-hydroxybutyryl-CoA dehydratase, a crotonyl-CoA reductase (aldehyde forming), and a crotonaldehyde reductase (alcohol forming);   a 4-hydroxybutyryl-CoA dehydratase and a crotonyl-CoA reductase (alcohol forming); and   a 4-hydroxybutyryl-CoA dehydratase, a crotonaldehyde reductase (alcohol forming), a crotonyl-CoA hydrolase, synthetase or transferase and a crotonate reductase.   
     
     
         29 - 42 . (canceled) 
     
     
         43 . The microbial organism of  claim 24 , wherein said at least one exogenous nucleic acid is a heterologous nucleic acid. 
     
     
         44 . The microbial organism of  claim 24 , wherein said non-naturally occurring microbial organism is in a substantially anaerobic culture medium.