Dna encoding a protein having d-lactate dehydrogenase activity and uses thereof

Abstract

This invention provides a polynucleotide that encodes a protein having lactate dehydrogenase activity and such protein that can be used for producing D-lactic acid. This polynucleotide has the nucleotide sequence as shown in SEQ ID NO: 1 (a), and it hybridizes under stringent conditions with a probe comprising all or part of the nucleotide sequence as shown in SEQ ID NO: 1 or a complementary strand thereof and encodes a protein having D-lactate dehydrogenase activity (b).

1 . A polynucleotide as described in any of the following (a) to (g): 
 (a) a polynucleotide comprising the nucleotide sequence as shown in SEQ ID NO: 1;    (b) a polynucleotide that hybridizes under stringent conditions with a probe comprising all or part of the nucleotide sequence as shown in SEQ ID NO: 1 or a complementary strand thereof and that encodes a protein having D-lactate dehydrogenase activity;    (c) a polynucleotide that encodes a protein consisting of the amino acid sequence as shown in SEQ ID NO: 2;    (d) a polynucleotide that encodes a protein consisting of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 by substitution, deletion, insertion, or addition of one or several amino acid residues and having D-lactate dehydrogenase activity;    (e) a polynucleotide that encodes a protein having 70% or higher homology to the amino acid sequence as shown in SEQ ID NO: 2 and having D-lactate dehydrogenase activity;    (f) a polynucleotide that encodes a protein having an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 4 by substitution of one or more amino acid residues selected from the list of amino acid residue substitutions shown in Table 1 and having D-lactate dehydrogenase activity:                TABLE 1                 List of amino acid residue substitutions                   Substitution type   Position of substitution   Amino acid substituent                         1   40   Valine (Val)     2   112   Isoleucine (Ile)     3   131   Histidine (His)     4   139   Isoleucine (Ile)     5   181   Glutamic acid (Glu)     6   266   Glycine (Gly)     7   267   Leucine (Leu)     8   268   Phenylalanine (Phe)     9   269   Asparagine (Asn)     10   270   Glutamic acid (Glu)     11   271   Aspartic acid (Asp)     12   272   Tryptophan (Trp)     13   273   Serine (Ser)     14   274   Glycine (Gly)     15   276   Glutamic acid (Glu)     16   277   Phenylalanine (Phe)     17   287   Serine (Ser)     18   292   Leucine (Leu)     19   293   Valine (Val)                                                                     wherein positions of substitution are indicated as the positions from methionine, which corresponds to the initiation codon; or    (g) a polynucleotide that encodes a protein having an amino acid sequence containing at least amino acid residues 78 and 79, 152 to 175, 235, and 296 of the amino acid sequence as shown in SEQ ID NO: 2 and having D-lactate dehydrogenase activity.    
   
   
       2 . A protein as described in any of the following (h) to (1): 
 (h) a protein that consists of the amino acid sequence as shown in SEQ ID NO: 2 of the Sequence Listing;    (i) a protein that consists of an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 2 of the Sequence Listing by substitution, deletion, insertion, or addition of one or several amino acid residues and that has D-lactate dehydrogenase activity;    (j) a protein that has 70% or higher homology to the amino acid sequence as shown in SEQ ID NO: 2 and that has D-lactate dehydrogenase activity;    (k) a protein that has an amino acid sequence derived from the amino acid sequence as shown in SEQ ID NO: 4 by substitution of one or more amino acid residues selected from the list of amino acid residue substitutions shown in Table 1 and that has D-lactate dehydrogenase activity; or    (l) a protein that has an amino acid sequence containing at least amino acid residues 78 and 79, 152 to 175, 235, and 296 of the amino acid sequence as shown in SEQ ID NO: 2 and that has D-lactate dehydrogenase activity.    
   
   
       3 . A DNA construct that comprises a DNA segment having DNA according to  claim 1  and a DNA segment having DNA that encodes a promoter or a homolog thereof  
   
   
       4 . The DNA construct according to  claim 3 , wherein the promoter is of the pyruvate decarboxylase gene.  
   
   
       5 . The DNA construct according to  claim 3  or  4 , wherein the promoter is of the pyruvate decarboxylase 1 gene of  Saccharomyces.    
   
   
       6 . The DNA construct according to  claim 5 , wherein the promoter is of the pyruvate decarboxylase 1 gene of  Saccharomyces cerevisae.    
   
   
       7 . A transformant that carries the DNA according to  claim 1  in an expressible manner in a host.  
   
   
       8 . The transformant according to  claim 7 , wherein the host is a microorganism selected from the group consisting of eukaryotic microorganisms including yeast and fungi and prokaryotic microorganisms including lactic acid bacteria,  Escherichia  bacteria, and  Bacillus  bacteria.  
   
   
       9 . The transformant according to  claim 7  or  8 , wherein the DNA is carried in an expressible manner under the control of a promoter or a homolog thereof  
   
   
       10 . The transformant according to  claim 9 , wherein the promoter is of the pyruvate decarboxylase gene.  
   
   
       11 . The transformant according to  claim 9  or  10 , wherein the promoter is of the pyruvate decarboxylase 1 gene of  Saccharomyces.    
   
   
       12 . The transformant according to  claim 11 , wherein the DNA is carried in an expressible manner under the control of the promoter of the host pyruvate decarboxylase 1 gene.  
   
   
       13 . The transformant according to any one of  claims 7  to  12 , wherein the host microorganism is  Saccharomyces cerevisae.    
   
   
       14 . A method for producing D-lactic acid comprising steps of: 
 culturing the transformant according to any one of  claims 7  to  13 ; and    recovering at least one member selected from the group consisting of D-lactic acid, a salt thereof, and a derivative thereof from the culture product.    
   
   
       15 . A method for producing a lactic acid polymer comprising steps of: 
 culturing the transformant according to any one of  claims 7  to  13 ;    recovering at least one member selected from the group consisting of D-lactic acid, a salt thereof, and a derivative thereof from the culture product; and    producing a lactic acid polymer using the recovered D-lactic acid or a derivative thereof as at least one polymerization material.