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 . 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 a-ketoisovalerate;   (iv) a-ketoisovalerate to isobutyraldehyde; and   (v) isobutyraldehyde to isobutanol;   wherein the recombinant yeast microorganism comprises:   (a) an NADH-dependent ketol-acid reductoisomerase to catalyze the conversion of acetolactate to 2,3-dihydroxyisovalerate using NADH as a cofactor; and   (b) an NADH-dependent alcohol dehydrogenase to catalyze the conversion of isobutyraldehyde to isobutanol using NADH as a cofactor,   wherein the recombinant yeast microorganism has been engineered to disrupt, mutate, or delete one or more endogenous pyruvate decarboxylase (PDC) genes, and wherein said recombinant yeast microorganism has reduced endogenous PDC activity as compared to the corresponding yeast microorganism that has not been engineered to have reduce endogenous PDC activity.   
     
     
         2 . The recombinant yeast microorganism of  claim 1 , wherein said one or more endogenous PDC genes is selected from the group consisting of PDC1, PDC2, PDC5, and PDC6. 
     
     
         3 . The recombinant yeast microorganism of  claim 1 , wherein said recombinant yeast microorganism is engineered to disrupt, mutate, or delete one or more endogenous glycerol-3-phosphate dehydrogenase (GPD) genes. 
     
     
         4 . The recombinant yeast microorganism of  claim 3 , wherein said one or more endogenous GPD genes is selected from the group consisting of GPD1 and GPD2. 
     
     
         5 . The recombinant yeast microorganism of  claim 1 , wherein said recombinant yeast microorganism is engineered to disrupt an endogenous pyruvate dehydrogenase (PDH) gene. 
     
     
         6 . The recombinant yeast microorganism of  claim 1 , wherein said recombinant yeast microorganism is engineered or selected to grow on glucose independently of C2-compounds at a growth rate substantially equivalent to the growth rate of the corresponding yeast microorganism that has not been engineered to have reduced endogenous PDC activity. 
     
     
         7 . The recombinant yeast microorganism of  claim 1 , wherein the conversion of pyruvate to acetolactate is catalyzed by an acetolactate synthase. 
     
     
         8 . The recombinant yeast microorganism of  claim 7 , wherein said acetolactate synthase is a cytosolically-localized acetolactate synthase. 
     
     
         9 . The recombinant yeast microorganism of  claim 8 , wherein said cytosolically-localized acetolactate synthase is encoded by the  Lactococcus lactis  alsS gene. 
     
     
         10 . The recombinant yeast microorganism of  claim 8 , wherein said cytosolically-localized acetolactate synthase is encoded by the  Bacillus subtilis  alsS gene. 
     
     
         11 . The recombinant yeast microorganism of  claim 1 , wherein the conversion of 2,3-dihydroxyisovalerate to isobutyraldehyde is catalyzed by a dihydroxy acid dehydratase. 
     
     
         12 . The recombinant yeast microorganism of  claim 11 , said dihydroxy acid dehydratase is encoded by the  Lactococcus lactis  ilvD gene. 
     
     
         13 . The recombinant yeast microorganism of  claim 1 , wherein the conversion of α-ketoisovalerate to isobutyraldehyde is catalyzed by a 2-keto-acid decarboxylase. 
     
     
         14 . The recombinant yeast microorganism of  claim 13 , wherein said 2-keto-acid decarboxylase is encoded by the  Lactococcus lactis  kivD gene. 
     
     
         15 . The recombinant yeast microorganism of  claim 1 , wherein said recombinant yeast microorganism produces isobutanol at a yield which is at least 70% of the theoretical yield of isobutanol from glucose. 
     
     
         16 . The recombinant yeast microorganism of  claim 1 , wherein the recombinant yeast microorganism is a yeast of the  Saccharomyces clade.    
     
     
         17 . The recombinant yeast microorganism of  claim 16 , wherein said yeast of the  Saccharomyces clade  is  Saccharomyces cerevisiae.    
     
     
         18 . A method of producing isobutanol, comprising:
 (a) providing the recombinant yeast microorganism according to  claim 1 ; and   (b) cultivating the recombinant yeast microorganism in a culture medium containing a feedstock providing the carbon source, until the isobutanol is produced.   
     
     
         19 . The method of  claim 18 , wherein isobutanol is produced under anaerobic or microaerobic conditions. 
     
     
         20 . The method of  claim 18 , wherein isobutanol is produced at a yield which is at least 70% of the theoretical yield of isobutanol from glucose.