Method for the production of 1-butanol

Abstract

A method for the production of 1-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 - 27 . (canceled) 
     
     
         28 . A recombinant microbial host cell comprising DNA molecules encoding polypeptides that catalyze each of the following substrate to product conversions:
 i) acetyl-CoA to acetoacetyl-CoA;   ii) acetoacetyl-CoA to 3-hydroxybutyryl-CoA;   iii) 3-hydroxybutyryl-CoA to crotonyl-CoA;   iv) crotonyl-CoA to butyryl-CoA;   v) butyryl-CoA to butyraldehyde; and   vi) butyraldehyde to 1-butanol;   
       wherein the polypeptide that catalyzes the substrate to product conversion of acetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase; the polypeptide that catalyzes the substrate to product conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoA dehydrogenase; the polypeptide that catalyzes the substrate to product conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA is crotonase; the polypeptide that catalyzes the substrate to product conversion of crotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase; the polypeptide that catalyzes the substrate to product conversion of butyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase; and the polypeptide that catalyzes the substrate to product conversion of butyraldehyde to 1-butanol is butanol dehydrogenase. 
     
     
         29 . The recombinant microbial host cell of  claim 28 , wherein the acetyl-CoA acetyltransferase 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: 4, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133 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 . The recombinant microbial host cell of  claim 28 , wherein the 3-hydroxybutyryl-CoA 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: 135, SEQ ID NO: 137, and SEQ ID NO: 139 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 . The recombinant microbial host cell of  claim 28 , wherein the crotonase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145 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 . The recombinant microbial host cell of  claim 28 , wherein the butyryl-CoA 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: 10, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, and SEQ ID NO: 187 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 . The recombinant microbial host cell of  claim 28 , wherein the butyraldehyde 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: 12, SEQ ID NO: 153, and SEQ ID NO: 189 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 . The recombinant microbial host cell of  claim 28 , 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: 16, SEQ ID NO: 153, SEQ ID NO: 155, and SEQ ID NO: 157 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. 
     
     
         35 . The recombinant microbial host cell of  claim 28 , wherein the acetyl-CoA acetyltransferase 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: 4, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133 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 3-hydroxybutyryl-CoA 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: 135, SEQ ID NO: 137, and SEQ ID NO: 139 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 crotonase has an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 141, SEQ ID NO: 143, and SEQ ID NO: 145 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 butyryl-CoA 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: 10, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, and SEQ ID NO: 187 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 butyraldehyde 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: 12, SEQ ID NO: 153, and SEQ ID NO: 189 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: 16, SEQ ID NO: 153, SEQ ID NO: 155, and SEQ ID NO: 157 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.   
     
     
         36 . The recombinant microbial host cell of  claim 28 , 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.    
     
     
         37 . The recombinant microbial host cell of  claim 36 , 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.    
     
     
         38 . A method to reduce the sensitivity of a recombinant microbial host cell to 1-butanol comprising:
 a) providing a recombinant microbial host cell which produces 1-butanol, wherein the recombinant microbial host cell comprises DNA molecules encoding polypeptides that catalyze each of the following substrate to product conversions:
 i) acetyl-CoA to acetoacetyl-CoA; 
 ii) acetoacetyl-CoA to 3-hydroxybutyryl-CoA; 
 iii) 3-hydroxybutyryl-CoA to crotonyl-CoA; 
 iv) crotonyl-CoA to butyryl-CoA; 
 v) butyryl-CoA to butyraldehyde; and 
 vi) butyraldehyde to 1-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. 
 
     
     
         39 . The method of  claim 38 , further comprising the step
 adjusting the one or more metabolic parameters to support the metabolic activity.   
     
     
         40 . The method of  claim 39 , wherein a decrease in one or more of the metabolic parameters indicates a decrease in metabolic activity. 
     
     
         41 . The method of  claim 40 , 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. 
     
     
         42 . The method of  claim 38 , wherein the polypeptide that catalyzes the substrate to product conversion of acetyl-CoA to acetoacetyl-CoA is acetyl-CoA acetyltransferase; the polypeptide that catalyzes the substrate to product conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA is 3-hydroxybutyryl-CoA dehydrogenase; the polypeptide that catalyzes the substrate to product conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA is crotonase; the polypeptide that catalyzes the substrate to product conversion of crotonyl-CoA to butyryl-CoA is butyryl-CoA dehydrogenase; the polypeptide that catalyzes the substrate to product conversion of butyryl-CoA to butyraldehyde is butyraldehyde dehydrogenase; and the polypeptide that catalyzes the substrate to product conversion of butyraldehyde to 1-butanol is butanol dehydrogenase.