Engineered microorganisms & methods for improved crotyl alcohol production

Abstract

Disclosed are methods and engineered microorganisms that enhance or improve the production of crotyl alcohol. The engineered microorganisms include genetic modifications in alcohol dehydrogenase, alkene reductase or both enzymatic activities. By such genetic modifications, a crotyl alcohol production pathway is provided or improved.

1 - 33 . (canceled) 
     
     
         34 . A method for producing crotyl alcohol using an engineered microorganism, where crotyl alcohol is a bioproduct, or crotyl alcohol is an intermediate in the bioproduct pathway, the method comprising:
 culturing the engineered microorganism under conditions resulting in crotyl alcohol or use of crotyl alcohol as the intermediate in the bioproduct pathway resulting in bioproduct, wherein the engineered microorganism comprises a genetic modification causing partial or complete loss of activity in an alkene reductase that converts crotonaldehyde to butyraldehyde and at least one exogenous nucleic acid encoding an enzyme of the bioproduct pathway, where the bioproduct pathway is a crotyl alcohol pathway or uses a crotyl alcohol as an intermediate in the bioproduct pathway.   
     
     
         35 . The method of  claim 34 , wherein the engineered microorganism further comprises at least one genetic modification causing partial or complete loss of activity in (a) an alcohol dehydrogenase (ADH) that converts crotyl alcohol to crotonaldehyde, or (b) an ADH that converts butyraldehyde to butanol, or (c) both combination of (a) and (b). 
     
     
         36 . The method of  claim 35 , wherein the ADH activity is encoded by a gene selected from adhE, adhP, yqhD and yahK or any combination thereof. 
     
     
         37 . The method of  claim 34 , wherein the alkene reductase activity is selected from EC Number: 1.3.1.-class. 
     
     
         38 . The method of  claim 34 , wherein the alkene reductase activity is encoded by a nemA gene or yqjM gene or both. 
     
     
         39 . The method of  claim 34 , wherein the partial or complete loss of alkene reductase reduces the accumulation or conversion of butyraldehyde or butanol or both butyraldehyde and butanol. 
     
     
         40 . The method of  claim 34 , wherein the crotyl alcohol bioproduct pathway comprises 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. 
     
     
         41 . The method of  claim 34 , wherein the engineered microorganism comprises an enzyme converting crotyl alcohol to 1,3-butadiene, or where the enzyme is a linalool dehydratase, and/or wherein the crotyl alcohol produced is converted to 3-buten-2-ol and/or butadiene. 
     
     
         42 . The method of  claim 34 , wherein the engineered microorganism comprises a crotyl alcohol to butadiene pathway comprising one or more of the following exogenous nucleic acids encoding enzymes: crotyl alcohol kinase, 2-butenyl-4-phosphate kinase, butadiene synthase, 3-buten-2-ol synthase, 3-buten-2-ol synthase, crotyl alcohol isomerase, 3-buten-2-ol dehydratase, crotyl alcohol dehydratase, crotyl alcohol diphosphokinase, crotyl alcohol dehydratase/vinyl isomerase. 
     
     
         43 . The method of  claim 34 , wherein the crotyl alcohol produced is converted to 3-buten-2-ol and/or butadiene. 
     
     
         44 . The method of  claim 34 , wherein the engineered microorganism exhibits an increased growth on crotyl alcohol compared to a parental host microorganism that does not have the genetic modifications. 
     
     
         45 . The method of  claim 34 , wherein the engineered microorganism grows on medium containing about 0.138 mM to about 138 mM crotyl alcohol. 
     
     
         46 . A cell culture composition comprising crotyl alcohol or butadiene produced by the method of  claim 34 . 
     
     
         47 . An engineered microorganism comprising at least one genetic modification causing partial or complete loss of activity in an alkene reductase that converts crotonaldehyde to butyraldehyde and an exogenous nucleic acid encoding an enzyme of a bioproduct pathway, where the bioproduct pathway is a crotyl alcohol pathway or uses a crotyl alcohol as an intermediate in the bioproduct pathway. 
     
     
         40 . The engineered microorganism of  claim 47 , wherein the engineered microorganism further comprises at least one genetic modification causing partial or complete loss of activity in (a) an alcohol dehydrogenase (ADH) that converts crotyl alcohol to crotonaldehyde, or (b) an ADH that converts butyraldehyde to butanol, or (c) both combination of (a) and (b). 
     
     
         48 . The engineered microorganism of  claim 47 , wherein the ADH activity is encoded by a gene selected from adhE, adhP, yqhD and yahK or any combination thereof. 
     
     
         50 . The engineered microorganism of  claim 47 , wherein the alkene reductase activity is encoded by a nemA gene or yqjM gene or both. 
     
     
         51 . The engineered microorganism of  claim 47 , wherein the engineered microorganism exhibits an increased growth on crotyl alcohol compared to a parental host microorganism that does not have the genetic modifications. 
     
     
         52 . The engineered microorganism of  claim 47 , wherein the engineered microorganism grows on medium containing about 0.138 mM to about 138 mM crotyl alcohol. 
     
     
         53 . The engineered microorganism of  claim 47 , wherein the crotyl alcohol bioproduct pathway comprises 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.