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.

What is claimed is: 
     
         1 . A method of producing isobutanol, comprising:
 a) providing a recombinant microorganism comprising an isobutanol producing metabolic pathway, wherein the recombinant microorganism has been engineered to contain one or more modifications in a transcriptional regulator of a PDC gene;   b) cultivating the microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of the isobutanol is produced; and   c) recovering the isobutanol.   
     
     
         2 . The method of  claim 1 , wherein the microorganism comprises an isobutanol producing metabolic pathway comprising 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.   
     
     
         3 . The method of  claim 1 , wherein the microorganism expresses
 (a) an acetolactate synthase to catalyze the conversion of pyruvate to acetolactate;   (b) a ketol-acid reductoisomerase to catalyze the conversion of acetolactate to 2,3-dihydroxyisovalerate;   (c) a dihydroxyacid dehydratase to catalyze the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate;   (d) an α-ketoisovalerate decarboxylase to catalyze the conversion of α-ketoisovalerate to isobutyraldehyde; and   (e) an alcohol dehydrogenase to catalyze the conversion of isobutyraldehyde to isobutanol.   
     
     
         4 . The method of  claim 1 , wherein the microorganism is selected to produce isobutanol at a yield of greater than about 10 percent theoretical. 
     
     
         5 . The method of  claim 1 , wherein the microorganism is selected to produce isobutanol at a yield of greater than about 20 percent theoretical. 
     
     
         6 . The method of  claim 1 , wherein the microorganism is selected to produce isobutanol at a yield of greater than about 50 percent theoretical. 
     
     
         7 . The method of  claim 1 , wherein the PDC gene is PDC1 or PDC5. 
     
     
         8 . The method of  claim 1 , wherein the PDC gene is PDC1 and PDC5. 
     
     
         9 . The method of  claim 1 , wherein the one or more modifications of the transcriptional regulator of a PDC gene result in a reduction of pyruvate decarboxylase gene transcription. 
     
     
         10 . The method of  claim 1 , wherein the transcriptional regulator of a PDC gene is PDC2. 
     
     
         11 . The method of  claim 10 , wherein the one or more modifications of PDC2 result in a decreased PDC2 activity. 
     
     
         12 . The method of  claim 11 , wherein the one or more modifications in PDC2 result in a loss of function mutation. 
     
     
         13 . The method of  claim 10 , wherein the one or more modifications in PDC2 decrease expression from a PDC1 or a PDC5 promoter. 
     
     
         14 . The method of  claim 13 , wherein the one or more modifications in PDC2 decrease expression from a PDC1 and a PDC5 promoter. 
     
     
         15 . The method of  claim 10 , wherein the one or more modifications in PDC2 decrease expression of PDC1 or PDC5. 
     
     
         16 . The method of  claim 15 , wherein the one or more modifications in PDC2 decrease expression of PDC1 and PDC5. 
     
     
         17 . The method of  claim 10 , wherein the one or more modifications in PDC2 inhibit expression of PDC1 or PDC5. 
     
     
         18 . The method of  claim 17 , wherein the one or more modifications in PDC2 inhibit expression of PDC1 and PDC5. 
     
     
         19 . The method of  claim 1 , wherein the recombinant microorganism comprises one or more complete deletions of pyruvate decarboxylase genes resulting in a reduction of pyruvate decarboxylase activity of a polypeptide encoded by said gene. 
     
     
         20 . The method of  claim 1 , wherein said recombinant microorganism has reduced endogenous PDC activity as compared to the corresponding recombinant microorganism that has not been engineered to have reduced endogenous PDC activity. 
     
     
         21 . A method in accordance with  claim 1 , wherein the microorganism is a yeast microorganism of the  Saccharomyces  clade. 
     
     
         22 . The method of  claim 1 , wherein the recombinant microorganism grows on glucose independently of C2-compounds at a growth rate substantially equivalent to the growth rate of a parental microorganism without altered PDC activity. 
     
     
         23 . The method of  claim 1 , wherein the microorganism is a  Saccharomyces  sensu stricto yeast microorganism. 
     
     
         24 . The method of  claim 23 , wherein the  Saccharomyces  sensu stricto yeast microorganism is selected from one of the species:  S. cerevisiae, S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis  or hybrids thereof.