Yeast Organism Producing Isobutanol at a High Yield

Abstract

The present invention provides recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise a modification resulting in the reduction of pyruvate decarboxylase and/or glycerol-3-phosphate dehydrogenase 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 .- 20 . (canceled) 
     
     
         21 . A recombinant yeast microorganism for producing an acetolactate intermediate, wherein the recombinant yeast microorganism has been engineered to:
 (a) express a heterologous acetolactate synthase (ALS);   (b) disrupt, mutate, or delete one or more endogenous pyruvate decarboxylase (PDC) genes, wherein said recombinant yeast microorganism has reduced endogenous PDC activity as compared to the corresponding yeast microorganism that has not been engineered to have reduced endogenous PDC activity; and   (c) disrupt, mutate, or delete one or more endogenous glycerol-3-phosphate dehydrogenase (GPD) genes, wherein said recombinant yeast microorganism has reduced endogenous GPD activity as compared to the corresponding yeast microorganism that has not been engineered to have reduced endogenous GPD activity;   
       and wherein said heterologous ALS catalyzes the conversion of pyruvate to said acetolactate intermediate within said recombinant yeast microorganism. 
     
     
         22 . The recombinant yeast microorganism of claim  1 , wherein all endogenous PDC genes and all endogenous GPD genes are disrupted, mutated, or deleted. 
     
     
         23 . The recombinant yeast microorganism of claim  1 , wherein said one or more endogenous PDC genes is selected from the group consisting of PDC 1, PDC2, PDC5, and PDC6. 
     
     
         24 . The recombinant yeast microorganism of claim  1 , wherein said one or more endogenous GPD genes is selected from the group consisting of GPD1 and GPD2. 
     
     
         25 . The recombinant yeast microorganism of claim  1 , wherein said ALS is a cytosolically-localized ALS. 
     
     
         26 . The recombinant yeast microorganism of claim  5 , wherein said cytosolically-localized ALS is encoded by the  Lactococcus lactis  alsS gene. 
     
     
         27 . The recombinant yeast microorganism of claim  5 , wherein said cytosolically-localized ALS is encoded by the  Bacillus subtilis  alsS gene. 
     
     
         28 . The recombinant yeast microorganism of claim  1 , wherein said recombinant yeast microorganism has been engineered to disrupt or delete an endogenous pyruvate dehydrogenase (PDH) gene. 
     
     
         29 . The recombinant yeast microorganism of claim  1 , wherein said recombinant yeast microorganism further expresses a heterologous gene encoding a ketol-acid reductoisomerase. 
     
     
         30 . The recombinant yeast microorganism of claim  1 , wherein said recombinant yeast microorganism further expresses a heterologous gene encoding a dihydroxy acid dehydratase. 
     
     
         31 . The recombinant yeast microorganism of claim  1 , wherein said recombinant yeast microorganism further expresses a heterologous gene encoding an α-ketoisovalerate decarboxylase. 
     
     
         32 . The recombinant yeast microorganism of claim  11 , wherein said heterologous gene encoding an α-ketoisovalerate decarboxylase is obtained from  Lactococcus lactis.    
     
     
         33 . The recombinant yeast microorganism of claim  1 , wherein said recombinant yeast microorganism expresses a heterologous gene encoding an alcohol dehydrogenase. 
     
     
         34 . The recombinant yeast microorganism of claim  1 , wherein said recombinant yeast microorganism expresses
 (a) a heterologous ketol-acid reductoisomerase to catalyze the conversion of acetolactate to 2,3-dihydroxyisovalerate;   (b) a heterologous dihydroxy acid dehydratase to catalyze the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate;   (c) a heterologous α-ketoisovalerate decarboxylase to catalyze the conversion of α-ketoisovalerate to isobutyraldehyde; and   (d) an alcohol dehydrogenase to catalyze the conversion of isobutyraldehyde to isobutanol.   
     
     
         35 . The recombinant yeast microorganism of claim  1 , wherein said recombinant yeast microorganism is a yeast of a genus selected from the group consisting of  Saccharomyces, Kluyveromyces, Candida, Pichia, Hansenula , and  Schizosaccharomyces.    
     
     
         36 . The recombinant yeast microorganism of claim  14 , wherein said recombinant yeast microorganism is a yeast of a genus selected from the group consisting of  Saccharomyces, Kluyveromyces, Candida, Pichia, Hansenula , and  Schizosaccharomyces.    
     
     
         37 . A method of producing isobutanol, comprising:
 (a) providing a recombinant yeast microorganism according to claim  14 ;   (b) cultivating the microorganism in a culture medium containing a feedstock providing the carbon source, until the isobutanol is produced; and   (c) recovering the isobutanol.