Fermentive production of four carbon alcohols

Abstract

Methods for the fermentive production of four carbon alcohols are provided. Specifically, butanol, preferably 2-butanol is produced by the fermentive growth of a recombinant bacteria expressing a 2-butanol biosynthetic pathway. The recombinant microorganisms and methods of the invention can also be adapted to produce 2-butanone, an intermediate in the 2-butanol biosynthetic pathways disclosed herein.

1 - 16 . (canceled) 
     
     
         17 . A method for the production of 2-butanol comprising:
 1) providing a recombinant microbial host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of:
 i) pyruvate to alpha-acetolactate; 
 ii) alpha-acetolactate to acetoin; 
 iii) acetoin to 3-amino-2-butanol; 
 iv) 3-amino-2-butanol to 3-amino-2-butanol phosphate; 
 v) 3-amino-2-butanol phosphate to 2-butanone; and 
 vi) 2-butanone to 2-butanol;
 wherein the at least one DNA molecule is heterologous to said microbial host cell; and 
 
   2) contacting the host cell of (1) with a fermentable carbon substrate in a fermentation medium under conditions whereby 2-butanol is produced.   
     
     
         18 . (canceled) 
     
     
         19 . A method according to  claim 17  wherein the fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides. 
     
     
         20 . A method according to  claim 17  wherein the polypeptide that catalyzes a substrate to product conversion of pyruvate to alpha-acetolactate is acetolactate synthase. 
     
     
         21 . A method according to  claim 17  wherein the polypeptide that catalyzes a substrate to product conversion of alpha-acetolactate to acetoin is acetolactate decarboxylase. 
     
     
         22 . A method according to  claim 17  wherein the polypeptide that catalyzes a substrate to product conversion of acetoin to 3-amino-2-butanol is acetoin aminase. 
     
     
         23 . A method according to  claim 17  wherein the polypeptide that catalyzes a substrate to product conversion of 3-amino-2-butanol to 3-amino-2-butanol phosphate is aminobutanol kinase. 
     
     
         24 . A method according to  claim 17  wherein the polypeptide that catalyzes a substrate to product conversion of 3-amino-2-butanol phosphate to 2-butanone is aminobutanol phosphate phospho-lyase. 
     
     
         25 . A method according to  claim 17  wherein the polypeptide that catalyzes a substrate to product conversion of 2-butanone to 2-butanol is butanol dehydrogenase. 
     
     
         26 . A method according to  claim 17  wherein the cell is selected from the group consisting of: a bacterium, a cyanobacterium, a filamentous fungus, and a yeast. 
     
     
         27 . A method according to  claim 26  wherein the cell is a member of a genus selected from the group consisting of  Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula  and  Saccharomyces.    
     
     
         28 . A method according to  claim 20  wherein the acetolactate synthase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:77, and SEQ ID NO:79 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         29 . A method according to  claim 21  wherein the acetolactate decarboxylase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 81, and SEQ ID NO:83 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         30 . A method according to  claim 22  wherein the acetoin aminase has an amino acid sequence having at least 95% identity to an amino acid sequence as set forth in SEQ ID NO:122 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         31 . A method according to  claim 23  wherein the aminobutanol kinase has an amino acid sequence having at least 95% identity to an amino acid sequence as set forth in SEQ ID NO:124 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         32 . A method according to  claim 24  wherein the aminobutanol phosphate phospho-lyase has an amino acid sequence having at least 95% identity to an amino acid sequence as set forth in SEQ ID NO:126 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         33 . A method according to  claim 25  wherein the butanol dehydrogenase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:72, SEQ ID NO:75, and SEQ ID NO:91 based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix. 
     
     
         34 - 35 . (canceled)