Acetolactate synthase having 2-ketoisovalerate decarboxylase activity and uses thereof

Abstract

Provided herein are metabolically-modified microorganisms useful for producing biofuels. More specifically, provided herein are methods of producing high alcohols including isobutanol, 1-butanol, 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol from a suitable substrate and a recombinant acetolactate synthase having both synthase and decarboxylase activity.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 
     
         1 . A recombinant microorganism that produces isobutanol wherein the alcohol is produced from a metabolite comprising 2-keto acid and wherein the organism comprises (i) a heterologous mutant acetolactate synthase lacking 2-ketoacid decarboxylase activity and (ii) a heterologous 2-ketoacid decarboxylase. 
     
     
         2 . The recombinant microorganism of  claim 1 , wherein the microorganism is an  E. coli.    
     
     
         3 . The recombinant microorganism of  claim 1 , wherein the acetolactate synthase is derived from  B. subtilis.    
     
     
         4 . The recombinant microorganism of  claim 3 , wherein the acetolactate synthase comprises a sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:2. 
     
     
         5 . The recombinant microorganism of  claim 3 , wherein the acetolactate synthase comprises a sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:7. 
     
     
         6 . The recombinant microorganism of  claim 3 , wherein the acetolactate synthase comprises SEQ ID NO:2. 
     
     
         7 . The recombinant microorganism of  claim 3 , wherein the acetolactate synthase comprises SEQ ID NO:7. 
     
     
         8 . The recombinant microorganism of  claim 1 , wherein the microorganism is from a genus of  Corynebacterium, Lactobacillus, Lactococcus, Salmonella, Enterobacter, Enterococcus, Erwinia, Pantoea, Morganella, Pectobacterium, Proteus, Serratia, Shigella, Klebsiella, Citrobacter, Saccharomyces, Dekkera, Klyveromyces , or  Pichia.    
     
     
         9 . The recombinant microorganism of  claim 8 , wherein the microorganism is derived from  E. coli  or  S. cerevisiae.    
     
     
         10 . The recombinant microorganism of  claim 1 , wherein the biosynthetic pathway for the production of an amino acid in the organism is modified for production of isobutanol. 
     
     
         11 . The recombinant microorganism of  claim 1 , wherein the microorganism comprises reduced ethanol production capability compared to a parental microorganism. 
     
     
         12 . The recombinant microorganism of  claim 11 , wherein the recombinant microorganism comprises a reduction of an ethanol dehydrogenase, thereby providing a reduced ethanol production capability as compared to a parental microorganism. 
     
     
         13 . A method for producing isobutanol, the method comprising:
 viding a recombinant microorganism of  claim 1 ;   (b) culturing the microorganism of (a) in the presence of a suitable substrate or metabolic intermediate and under conditions suitable for the conversion of the substrate to isobutanol; and   (c) substantially purifying the isobutanol.