Cytosolic isobutanol pathway localization for the production of isobutanol

Abstract

The present invention provides recombinant microorganisms comprising isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol, wherein said recombinant microorganism is selected to produce isobutanol from a carbon source. Methods of using said recombinant microorganisms to produce isobutanol are also provided. In various aspects of the invention, the recombinant microorganisms may comprise a cytosolically active isobutanol pathway enzymes. In some embodiments, the invention provides mutated, modified, and/or chimeric isobutanol pathway enzymes with cytosolic activity. 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.

1 . A recombinant yeast microorganism comprising an isobutanol producing metabolic pathway, wherein said isobutanol producing metabolic pathway comprises the following substrate to product conversions:
 (i) pyruvate to acetolactate;   (ii) acetolactate to 2,3-dihydroxyisovalerate;   (iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate;   (iv) α-ketoisovalerate to isobutyraldehyde; and   (v) isobutyraldehyde to isobutanol;   
       wherein
 a) the enzyme that catalyzes a substrate to product conversion of pyruvate to acetolactate is an acetolactate synthase; 
 b) the enzyme that catalyzes a substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate is a ketol-acid reductoisomerase derived from  Lactococcus lactis;    
 c) the enzyme that catalyzes a substrate to product conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is a dihydroxy acid dehydratase; 
 d) the enzyme that catalyzes a substrate to product conversion of a-ketoisovalerate to isobutyraldehyde is an α-ketoisovalerate decarboxylase; and 
 e) the enzyme that catalyzes a substrate to product conversion of isobutyraldehyde to isobutanol is an alcohol dehydrogenase. 
 
     
     
         2 . The recombinant yeast microorganism of  claim 1 , wherein said acetolactate synthase is derived from a bacterial organism. 
     
     
         3 . The recombinant yeast microorganism of  claim 2 , wherein said bacterial organism is  Bacillus subtilis.    
     
     
         4 . The recombinant yeast microorganism of  claim 1 , wherein said ketol-acid reductoisomerase is an NADH-dependent ketol-acid reductoisomerase. 
     
     
         5 . The recombinant yeast microorganism of  claim 1 , wherein said dihydroxy acid dehydratase comprises the amino acid sequence P(I/L)XXXGX(I/L)XIL (SEQ ID NO: 27), wherein X is any natural or non-natural amino acid. 
     
     
         6 . The recombinant yeast microorganism of  claim 5 , wherein said dihydroxy acid dehydratase enzyme is derived from a bacterial organism. 
     
     
         7 . The recombinant yeast microorganism of  claim 6 , wherein said bacterial organism is  Lactococcus lactis.    
     
     
         8 . The recombinant yeast microorganism of  claim 1 , wherein said a-ketoisovalerate decarboxylase is derived from a bacterial organism. 
     
     
         9 . The recombinant yeast microorganism of  claim 8 , wherein said bacterial organism is  Lactococcus lactis.    
     
     
         10 . The recombinant yeast microorganism of  claim 1 , wherein said alcohol dehydrogenase is derived from a bacterial organism. 
     
     
         11 . The recombinant yeast microorganism of  claim 10 , wherein said bacterial organism is  Lactococcus lactis.    
     
     
         12 . The recombinant yeast microorganism of  claim 1 , wherein the recombinant yeast microorganism has been engineered to inactivate one or more endogenous pyruvate decarboxylase (PDC) genes. 
     
     
         13 . The recombinant yeast microorganism of  claim 12 , wherein said one or more endogenous PDC genes is selected from PDC1, PDC5, and PDC6. 
     
     
         14 . The recombinant yeast microorganism of  claim 1 , wherein the recombinant yeast microorganism has been engineered to inactivate one or more endogenous glycerol-3-phosphate dehydrogenase (GPD) genes. 
     
     
         15 . The recombinant yeast microorganism of  claim 14 , wherein said one or more endogenous GPD genes is selected from GPD1 and GPD2. 
     
     
         16 . The recombinant yeast microorganism of  claim 1 , wherein the recombinant yeast microorganism is a yeast microorganism of the  Saccharomyces  clade. 
     
     
         17 . The recombinant yeast microorganism of  claim 16 , wherein said yeast microorganism of the  Saccharomyces  clade is  S. cerevisiae.    
     
     
         18 . A method of producing isobutanol, comprising:
 (a) providing a recombinant yeast microorganism according to  claim 1 ; and   (b) cultivating said recombinant yeast microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of the isobutanol is produced.