Cloning and sequencing of pyruvate decarboxylase (PDC) genes from bacteria and uses therefor

Abstract

The invention provides isolated nucleic acids molecules which encode pyruvate decarboxylase enzymes having improved decarboxylase activity, substrate affinity, thermostability, and activity at different pH. The nucleic acids of the invention also have a codon usage which allows for high expression in a variety of host cells. Accordingly, the invention provides recombinant expression vectors containing such nucleic acid molecules, recombinant host cells comprising the expression vectors, host cells further comprising other ethanologenic enzymes, and methods for producing useful substances, e.g., acetaldehyde and ethanol, using such host cells.

1 - 9 . (canceled)  
     
     
         10 . An isolated polypeptide selected from the group consisting of: 
 a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6;    b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, under stringent conditions;    c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 50% homologous to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; and    d) a polypeptide comprising an amino acid sequence which is at least 30% homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.    
     
     
         11 . The isolated polypeptide of  claim 10  comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.  
     
     
         12 . The polypeptide of  claim 10 , wherein the polypeptide has improved decarboxylase activity.  
     
     
         13 . The polypeptide of  claim 10 , wherein the polypeptide has improved thermal stability.  
     
     
         14 . The polypeptide of  claim 10 , wherein the polypeptide has improved substrate affinity.  
     
     
         15 . The polypeptide of  claim 10 , further comprising heterologous amino acid sequences.  
     
     
         16 . An antibody which selectively binds to a polypeptide of  claim 10 .  
     
     
         17 . (canceled)  
     
     
         18 . A method for detecting the presence of a polypeptide of  claim 10  in a sample comprising: 
 a) contacting the sample with a compound which selectively binds to the polypeptide; and    b) determining whether the compound binds to the polypeptide in the sample to thereby detect the presence of a polypeptide of  claim 10  in the sample.    
     
     
         19 . The method of  claim 18 , wherein the compound which binds to the polypeptide is an antibody.  
     
     
         20 - 42 . (canceled)  
     
     
         43 . An enzyme extract comprising detectable levels of pyruvate decarboxylase derived from a host cell, which contains a nucleic acid molecule selected from the group consisting of: 
 a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% homologous to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a complement thereof;    b) a nucleic acid molecule comprising a fragment of at least 000 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or a complement thereof;    c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 50% homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6;    d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; wherein the fragment comprises at least 15 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; and    e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, under stringent conditions.    
     
     
         44 . The enzyme extract of claim  37 , wherein the pyruvate decarboxylase has improved decarboxylase activity.  
     
     
         45 . (canceled)  
     
     
         46 . The enzyme extract of  claim 43 , wherein the pyruvate decarboxylase has improved thermal stability.  
     
     
         47 . The enzyme extract of  claim 43 , wherein the pyruvate decarboxylase has improved substrate affinity.  
     
     
         48 . A method for selecting a pyruvate decarboxylase enzyme with improved decarboxylase activity comprising, 
 comparing the amino acid sequence of a pyruvate decarboxylase with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; and    altering at least one amino acid residue of said pyruvate decarboxylase to have identity with the corresponding amino acid residue of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, such that improved pyruvate decarboxylase activity is achieved.    
     
     
         49 . The method of  claim 48 , wherein the pyruvate decarboxylase activity comprises improved pyruvate decarboxylase affinity for pyruvate.  
     
     
         50 . The method of  claim 48 , wherein the pyruvate decarboxylase activity comprises improved thermal stability.  
     
     
         51 . A method for selecting a pyruvate decarboxylase enzyme with improved expression in a recipient host cell comprising, 
 comparing the nucleic acid sequence encoding a pyruvate decarboxylase with the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; and    altering at least one codon of said nucleic acid encoding said pyruvate decarboxylase enzyme to have identity with the corresponding codon of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, such that improved expression of said altered nucleic acid encoding pyruvate decarboxylase enzyme is achieved in said host cell.    
     
     
         52 . A method for selecting a pyruvate decarboxylase enzyme with improved expression in a recipient host cell comprising, 
 comparing the nucleic acid sequence encoding a pyruvate decarboxylase with the codon usage of said recipient host cell; and    altering at least one codon of said nucleic acid encoding said pyruvate decarboxylase enzyme to correspond with the codon usage of said recipient host cell such that improved expression of said altered nucleic acid encoding pyruvate decarboxylase enzyme is achieved in said host cell.    
     
     
         53 . A method for selecting a pyruvate decarboxylase enzyme with improved expression in a recipient host cell comprising, 
 comparing the nucleic acid sequence encoding a pyruvate decarboxylase with the codon usage of said recipient host cell; and    altering said recipient host cell to recombinantly produce at least one tRNA corresponding to a codon of said nucleic acid encoding said pyruvate decarboxylase enzyme such that improved expression of said nucleic acid encoding pyruvate decarboxylase enzyme is achieved in said altered host cell.    
     
     
         54 - 56 . (canceled)