Engineered microorganisms capable of producing target compounds under anaerobic conditions

Abstract

The present invention is generally provides recombinant microorganisms comprising engineered metabolic pathways capable of producing C3-C5 alcohols under aerobic and anaerobic conditions. The invention further provides ketol-acid reductoisomerase enzymes which have been mutated or modified to increase their NADH-dependent activity or to switch the cofactor preference from NADPH to NADH and are expressed in the modified microorganisms. In addition, the invention provides isobutyraldehyde dehydrogenase enzymes expressed in modified microorganisms. Also provided are methods of producing beneficial metabolites under aerobic and anaerobic conditions by contacting a suitable substrate with the modified microorganisms of the present invention.

What is claimed is: 
     
         1 . An isolated nucleic acid molecule encoding a modified ketol-acid reductoisomerase (KARI), wherein said modified KARI is a polypeptide which has been engineered to comprise two amino acid substitutions that alter the cofactor usage of the polypeptide, wherein said two amino acid substitutions correspond to amino acid residues Arg76 and Ser78 of the  E. coli  ketol-acid reductoisomerase (SEQ ID NO: 13), and wherein said modified KARI has NADH-dependent KARI activity. 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein said isolated nucleic acid molecule has been codon-optimized for expression in yeast. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the amino acid residues at positions corresponding to amino acid residues Arg76 and Ser78 of the  E. coli  ketol-acid reductoisomerase (SEQ ID NO: 13) are replaced with an amino acid residue selected from the group consisting of aspartic acid and glutamic acid. 
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein said polypeptide is a KARI polypeptide which has been derived from  Lactococcus, Shewanella , or  Salmonella.    
     
     
         5 . A recombinant microorganism comprising an isolated nucleic acid molecule of  claim 1 . 
     
     
         6 . The recombinant microorganism of  claim 5 , wherein said recombinant microorganism comprises an isobutanol producing metabolic pathway, said isobutanol producing metabolic pathway comprising the following substrate to product conversions:
 (a) pyruvate to acetolactate;   (b) acetolactate to 2,3-dihydroxyisovalerate;   (c) 2,3-dihydroxyisovalerate to α-ketoisovalerate;   (d) α-ketoisovalerate to isobutyraldehyde; and   (e) isobutyraldehyde to isobutanol,   
       and wherein said modified KARI catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate using NADH as an electron donor. 
     
     
         7 . The recombinant microorganism of  claim 6 , wherein the enzyme that catalyzes the conversion of pyruvate to acetolactate is an acetolactate synthase. 
     
     
         8 . The recombinant microorganism of  claim 7 , wherein said acetolactate synthase is derived from  Bacillus subtilis.    
     
     
         9 . The recombinant microorganism of  claim 6 , wherein the enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is a dihydroxy acid dehydratase. 
     
     
         10 . The recombinant microorganism of  claim 6 , wherein the enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde is a 2-keto acid decarboxylase. 
     
     
         11 . The recombinant microorganism of  claim 10 , wherein said 2-keto acid decarboxylase is derived from  Lactococcus lactis.    
     
     
         12 . The recombinant microorganism of  claim 6 , wherein the enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol is an alcohol dehydrogenase. 
     
     
         13 . The recombinant microorganism of  claim 12 , wherein said alcohol dehydrogenase is an NADH-dependent alcohol dehydrogenase. 
     
     
         14 . The recombinant microorganism of  claim 13 , wherein said NADH-dependent alcohol dehydrogenase is derived from  Lactococcus lactis.    
     
     
         15 . The recombinant microorganism of  claim 6 , wherein said recombinant microorganism is a recombinant yeast microorganism. 
     
     
         16 . The recombinant microorganism of  claim 15 , wherein said recombinant yeast microorganism has been engineered to reduce or eliminate pyruvate decarboxylase (PDC) activity. 
     
     
         17 . The recombinant microorganism of  claim 15 , wherein said recombinant yeast microorganism has been engineered to reduce or eliminate glycerol-3-phosphate dehydrogenase (GPD) activity. 
     
     
         18 . A method of producing isobutanol, said method comprising:
 (a) providing a recombinant microorganism according to  claim 6 ;   (b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing a carbon source until the isobutanol is produced.   
     
     
         19 . The method of  claim 18 , wherein said recombinant microorganism converts the carbon source to isobutanol under anaerobic conditions.