Method for the production of 2-butanol

Abstract

A method for the production of 2-butanol by fermentation using a microbial production host is disclosed. The method employs a reduction in temperature during the fermentation process that results in a more robust tolerance of the production host to the butanol product.

1 - 38 . (canceled) 
     
     
         39 . A recombinant microbial host cell comprising DNA molecules encoding polypeptides that catalyze each of the following substrate to product conversions:
 i) pyruvate to alpha-acetolactate,   ii) alpha-acetolactate to acetoin,   iii) acetoin to 2,3-butanediol,   iv) 2,3-butanediol to 2-butanone, and   v) 2-butanone to 2-butanol;   
       wherein the polypeptide that catalyzes the substrate to product conversion of pyruvate to alpha-acetolactate is acetolactate synthase, the polypeptide that catalyzes the substrate to product conversion of alpha-acetolactate to acetoin is acetolactate decarboxylase, the polypeptide that catalyzes the substrate to product conversion of acetoin to 2,3-butanediol is butanediol dehydrogenase, the polypeptide that catalyzes the substrate to product conversion of 2,3-butanediol to 2-butanone is diol dehydratase or glycerol dehydratase, and the polypeptide that catalyzes the substrate to product conversion of 2-butanone to 2-butanol is butanol dehydrogenase. 
     
     
         40 . The recombinant microbial host cell of  claim 39 , 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. 
     
     
         41 . The recombinant microbial host cell of  claim 39 , 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. 
     
     
         42 . The recombinant microbial host cell of  claim 39 , wherein the butanediol 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: 6, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89 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. 
     
     
         43 . The recombinant microbial host cell of  claim 39 , wherein the diol dehydratase or glycerol dehydratase comprises fused large, medium, and small subunits and has at least 95% identity to an amino acid sequence comprising all three of the amino acid sequences encoding large, medium, and small subunits, selected from the group consisting of:
 a) SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12;   b) SEQ ID NO: 93, SEQ ID NO: 95, and SEQ ID NO: 97;   c) SEQ ID NO: 99, SEQ ID NO: 101, and SEQ ID NO: 103;   d) SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109;   e) SEQ ID NO: 135, SEQ ID NO: 136, and SEQ ID NO: 137;   f) SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140;   g) SEQ ID NO: 146, SEQ ID NO: 148, and SEQ ID NO: 150;   h) SEQ ID NO: 141, SEQ ID NO: 142, and SEQ ID NO: 143; and   i) SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166;   
       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. 
     
     
         44 . The recombinant microbial host cell of  claim 39 , 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. 
     
     
         45 . The recombinant microbial host cell of  claim 39 , 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; 
       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; 
       wherein the butanediol 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: 6, SEQ ID NO: 85, SEQ ID NO: 87, and SEQ ID NO: 89 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; 
       wherein the diol dehydratase or glycerol dehydratase comprises fused large, medium, and small subunits and has at least 95% identity to an amino acid sequence comprising all three of the amino acid sequences encoding large, medium, and small subunits, selected from the group consisting of:
 a) SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12; 
 b) SEQ ID NO: 93, SEQ ID NO: 95, and SEQ ID NO: 97; 
 c) SEQ ID NO: 99, SEQ ID NO: 101, and SEQ ID NO: 103; 
 d) SEQ ID NO: 105, SEQ ID NO: 107, and SEQ ID NO: 109; 
 e) SEQ ID NO: 135, SEQ ID NO: 136, and SEQ ID NO: 137; 
 f) SEQ ID NO: 138, SEQ ID NO: 139, and SEQ ID NO: 140; 
 g) SEQ ID NO: 146, SEQ ID NO: 148, and SEQ ID NO: 150; 
 h) SEQ ID NO: 141, SEQ ID NO: 142, and SEQ ID NO: 143; and 
 i) SEQ ID NO: 164, SEQ ID NO: 165, and SEQ ID NO: 166; 
 
       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; and 
       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. 
     
     
         46 . The recombinant microbial host cell of  claim 39 , wherein the recombinant microbial host cell is selected from the group consisting of  Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Saccharomyces, Pichia, Candida , and  Hansenula.    
     
     
         47 . The recombinant microbial host cell of  claim 46 , wherein the recombinant microbial host cell is selected from the group consisting of  Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis , and  Saccharomyces cerevisiae.    
     
     
         48 . A recombinant microbial host cell comprising DNA molecules encoding polypeptides that catalyze each of the following substrate to product conversions:
 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 polypeptide that catalyzes the substrate to product conversion of pyruvate to alpha-acetolactate is acetolactate synthase, the polypeptide that catalyzes the substrate to product conversion of alpha-acetolactate to acetoin is acetolactate decarboxylase, the polypeptide that catalyzes the substrate to product conversion of acetoin to 3-amino-2-butanol is acetoin aminase, the polypeptide that catalyzes the substrate to product conversion of 3-amino-2-butanol to 3-amino-2-butanol phosphate is aminobutanol kinase, the polypeptide that catalyzes the substrate to product conversion of 3-amino-2-butanol phosphate to 2-butanone is aminobutanol phosphate phospho-lyase, and the polypeptide that catalyzes the substrate to product conversion of 2-butanone to 2-butanol is butanol dehydrogenase. 
     
     
         49 . The recombinant microbial host cell of  claim 48 , 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. 
     
     
         50 . The recombinant microbial host cell of  claim 48 , 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. 
     
     
         51 . The recombinant microbial host cell of  claim 48 , 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. 
     
     
         52 . The recombinant microbial host cell of  claim 48 , 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. 
     
     
         53 . The recombinant microbial host cell of  claim 48 , 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. 
     
     
         54 . The recombinant microbial host cell of  claim 48 , 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. 
     
     
         55 . The recombinant microbial host cell of  claim 48 , 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;
 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;   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;   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;   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; and   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.   
     
     
         56 . The recombinant microbial host cell of  claim 48 , wherein the recombinant microbial host cell is selected from the group consisting of  Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Saccharomyces, Pichia, Candida , and  Hansenula.    
     
     
         57 . The recombinant microbial host cell of  claim 56 , wherein the recombinant microbial host cell is selected from the group consisting of  Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis , and  Saccharomyces cerevisiae.    
     
     
         58 . A method to reduce the sensitivity of a recombinant microbial host cell to 2-butanol comprising:
 a) providing a recombinant microbial host cell which produces 2-butanol, wherein the recombinant microbial host cell comprises DNA molecules encoding polypeptides that catalyze each of the following substrate to product conversions:
 i) pyruvate to alpha-acetolactate, 
 ii) alpha-acetolactate to acetoin, 
 iii) acetoin to 2,3-butanediol, 
 iv) 2,3-butanediol to 2-butanone, and 
 v) 2-butanone to 2-butanol; 
   or DNA molecules encoding polypeptides that catalyze each of the following substrate to product conversions:
 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 at least one of the DNA molecules is heterologous to the recombinant microbial host cell;   b) growing the recombinant microbial host cell in a fermentation culture; and   c) determining the metabolic activity of the fermentation culture by monitoring one or more metabolic parameters selected from optical density, pH, respiratory quotient, fermentable carbon substrate utilization, CO 2  production, and 1-butanol production.   
     
     
         59 . The method of  claim 58 , further comprising the step adjusting the one or more metabolic parameters to support the metabolic activity. 
     
     
         60 . The method of  claim 59 , wherein a decrease in one or more of the metabolic parameters indicates a decrease in metabolic activity. 
     
     
         61 . The method of  claim 60 , wherein the adjusting the one or more metabolic parameters is lowering the temperature of the fermentation culture when a decrease in metabolic activity is detected. 
     
     
         62 . The method of  claim 58 , wherein the polypeptide that catalyzes the substrate to product conversion of pyruvate to alpha-acetolactate is acetolactate synthase, the polypeptide that catalyzes the substrate to product conversion of alpha-acetolactate to acetoin is acetolactate decarboxylase, the polypeptide that catalyzes the substrate to product conversion of acetoin to 2,3-butanediol is butanediol dehydrogenase, the polypeptide that catalyzes the substrate to product conversion of 2,3-butanediol to 2-butanone is diol dehydratase or glycerol dehydratase, the polypeptide that catalyzes the substrate to product conversion of acetoin to 3-amino-2-butanol is acetoin aminase, the polypeptide that catalyzes the substrate to product conversion of 3-amino-2-butanol to 3-amino-2-butanol phosphate is aminobutanol kinase, the polypeptide that catalyzes the substrate to product conversion of 3-amino-2-butanol phosphate to 2-butanone is aminobutanol phosphate phospho-lyase, and the polypeptide that catalyzes the substrate to product conversion of 2-butanone to 2-butanol is butanol dehydrogenase.