Methods of Increasing Dihydroxy Acid Dehydratase Activity to Improve Production of Fuels, Chemicals, and Amino Acids

Abstract

The present invention is directed to recombinant microorganisms comprising one or more dihydroxyacid dehydratase (DHAD)-requiring biosynthetic pathways and methods of using said recombinant microorganisms to produce beneficial metabolites derived from said DHAD-requiring biosynthetic pathways. In various aspects of the invention, the recombinant microorganisms may be engineered to overexpress one or more polynucleotides encoding one or more Aft proteins or homologs thereof. In some embodiments, the recombinant microorganisms may comprise a cytosolically localized DHAD enzyme. In additional embodiments, the recombinant microorganisms may comprise a mitochondrially localized DHAD enzyme. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

What is claimed is: 
     
         1 . A recombinant yeast microorganism comprising a recombinantly overexpressed polynucleotide encoding a dihydroxy acid dehydratase (DHAD), wherein said recombinant yeast microorganism is engineered to increase the expression or activity of one or more activator of ferrous transport (Aft) proteins, thereby increasing the dehydratase activity of DHAD. 
     
     
         2 . The recombinant yeast microorganism of  claim 1 , wherein the expression or activity of one or more Aft proteins is increased by engineering said recombinant yeast microorganism to overexpress one or more polynucleotides encoding one or more Aft proteins. 
     
     
         3 . The recombinant yeast microorganism of  claim 2 , wherein said one or more polynucleotides encoding one or more Aft proteins is a constitutively active Aft protein. 
     
     
         4 . The recombinant yeast microorganism of  claim 1 , wherein the expression or activity of one or more Aft proteins is increased by engineering said recombinant yeast microorganism to express one or more polynucleotides encoding one or more constitutively active Aft proteins. 
     
     
         5 . The recombinant yeast microorganism of  claim 1 , wherein the expression or activity of one or more Aft proteins is increased by engineering said recombinant yeast microorganism to delete or attenuate the activity or expression of an endogenous protein which regulates the activity of one or more Aft proteins. 
     
     
         6 . The recombinant yeast microorganism of  claim 5 , wherein said endogenous protein is a glutathione-dependent oxidoreductase selected from the group consisting of Grx3 and Grx4. 
     
     
         7 . The recombinant yeast microorganism of  claim 1 , 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-di hydroxyisovalerate;   (c) 2,3-dihydroxyisovalerate to α-ketoisovalerate;   (d) α-ketoisovalerate to isobutyraldehyde; and   (e) isobutyraldehyde to isobutanol;   
       and wherein said DHAD catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate. 
     
     
         8 . The recombinant yeast microorganism of  claim 7 , wherein the enzyme that catalyzes the conversion of pyruvate to acetolactate is an acetolactate synthase. 
     
     
         9 . The recombinant yeast microorganism of  claim 7 , wherein the enzyme that catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate is a ketol-acid reductoisomerase. 
     
     
         10 . The recombinant yeast microorganism of  claim 9 , wherein said ketol-acid reductoisomerase is an NADH-dependent ketol-acid reductoisomerase. 
     
     
         11 . The recombinant yeast microorganism of  claim 1 , wherein said DHAD is localized in the cytosol. 
     
     
         12 . The recombinant yeast microorganism of  claim 1 , wherein said DHAD is localized in the mitochondria. 
     
     
         13 . The recombinant yeast microorganism of  claim 1 , wherein said DHAD is derived from  Lactococcus lactis.    
     
     
         14 . The recombinant yeast microorganism of  claim 1 , wherein said DHAD is derived from  Streptococcus mutans.    
     
     
         15 . The recombinant yeast microorganism of  claim 7 , wherein the enzyme that catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde is a 2-keto acid decarboxylase. 
     
     
         16 . The recombinant yeast microorganism of  claim 7 , wherein the enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol is an alcohol dehydrogenase. 
     
     
         17 . The recombinant yeast microorganism of  claim 16 , wherein said alcohol dehydrogenase is an NADH-dependent alcohol dehydrogenase. 
     
     
         18 . The recombinant yeast microorganism of  claim 7 , wherein said recombinant yeast microorganism is engineered to inactivate one or more endogenous pyruvate decarboxylase (PDC) genes. 
     
     
         19 . The recombinant yeast microorganism of  claim 7 , wherein said recombinant yeast microorganism is engineered to inactivate one or more endogenous glycerol-3-phosphate dehydrogenase (GPD) genes. 
     
     
         20 . A method of producing isobutanol comprising: (a) providing the recombinant yeast microorganism of  claim 7 ; and (b) cultivating the recombinant yeast microorganism of  claim 7  in a culture medium containing a feedstock providing a carbon source, until a recoverable quantity of the isobutanol is produced.