Mutant tyrosine repressor gene and utilization thereof

Abstract

A gene coding for a mutant tyrosine repressor having increased positive regulatory activity for expression of the tyrosine phenol lyase gene compared with a tyrosine repressor not having mutation is obtained by introducing a mutation into a tyrosine repressor gene of Erwinia herbicola , introducing the gene into which the mutation is introduced into Escherichia coli expressing a lactose operon under the control of a promoter and enhancer of the tyrosine phenol lyase gene derived from Erwinia herbicola , and selecting a transformed strain having increased β-galactosidase activity.

1 - 16 . (canceled)  
     
     
         17 . A DNA coding for the mutant tyrosine repressor having at least one or more mutations selected from replacement of valine residue at a position of 67 with an amino acid residue other than valine residue, replacement of tyrosine residue at a position of 72 with an amino acid residue other than tyrosine residue, replacement of aspartic acid residue at a position of 97 with an amino acid residue other than aspartic acid residue and replacement of isoleucine residue at a position of 402 with an amino acid residue other than isoleucine residue and having an increased positive regulatory activity for expression of tyrosine phenol lyase gene compared with a tyrosine repressor not having those mutations.  
     
     
         18 . The DNA according to  claim 17 , wherein the amino acid residue other than valine residue is an alanine residue, the amino acid residue other than tyrosine residue is a cysteine residue, the amino acid residue other than aspartic acid residue is a glycine residue and the amino acid residue other than isoleucine residue is a valine residue.  
     
     
         19 . The DNA according to  claim 18 , wherein the mutation consists of any of (a) replacement of valine residue at a position of 67 with an alanine residue, (b) replacement of tyrosine residue at a position of 72 with a cysteine residue, (c) replacement of valine residue at a position of 67 with an alanine residue and replacement of tyrosine residue at a position of 72 with a cysteine residue, or (d) replacement of valine residue with an alanine residueat a position of 67, replacement of tyrosine residue at a position of 72 with a cysteine residue and replacement of glutamic acid residue at a position of 201 with a glycine residue.  
     
     
         20 . The DNA according to  claim 17 , wherein a tyrosine repressor which has no mutation has the amino acid sequence shown as SEQ ID NO: 2.  
     
     
         21 . The DNA according to  claim 18 , wherein a tyrosine repressor which has no mutation has the amino acid sequence shown as SEQ ID NO: 2.  
     
     
         22 . The DNA according to  claim 19 , wherein a tyrosine repressor which has no mutation has the amino acid sequence shown as SEQ ID NO: 2.  
     
     
         23 . The DNA according to  claim 20 , wherein a tyrosine repressor which has no mutation has the amino acid sequence shown as SEQ ID NO: 2.  
     
     
         24 . The DNA according to  claim 21  which contains substitution, deletion, insertion, addition or inversion of one or several amino acids at a position other than the mutations, and in which positive regulatory activity for expression of tyrosine phenol lyase gene is increased compared with the tyrosine repressor having no mutation.  
     
     
         25 . The DNA according to  claim 22 , which contains substitution, deletion, insertion, addition or inversion of one or several amino acids at a position other than the mutations, and in which positive regulatory activity for expression of tyrosine phenol lyase gene is increased compared with the tyrosine repressor having no mutation.  
     
     
         26 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 17 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         27 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 18 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         28 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 19 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         29 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 20 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         30 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 21 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         31 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 22 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         32 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 23 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         33 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 24 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         34 . A bacterium belonging to the genus  Escherichia  or  Erwinia  harboring a structural gene of tyrosine phenol lyase operatively ligated to a promoter of the tyrosine phenol lyase gene having ability to produce a product of the structural gene, which further harbors the DNA according to  claim 25 , and in which expression of the structural gene is positively regulated by the mutant tyrosine repressor produced by the DNA.  
     
     
         35 . The bacteria belonging to the genus  Escherichia  or  Erwinia  according to  claim 26 , wherein the structural gene is a gene coding for tyrosine phenol lyase.  
     
     
         36 . A method or producing tyrosine phenol lyase, comprising the step of culturing the bacterium belonging to the genus  Escherichia  or  Erwinia  according to  claim 35  in a medium to produce tyrosine phenol lyase.  
     
     
         37 . The method for producing tyrosine phenol lyase according to  claim 36 , wherein an amount of tyrosine contained in the medium is less than an amount required to induce tyrosine phenol lyase in a wild-type strain of the bacterium belonging to the genus  Escherichia  or  Erwinia.    
     
     
         38 . A method for producing L-3, 4-dihydroxyphenylalanine, comprising the steps of producing tyrosine phenol lyase by culturing the bacterium belonging to the genus  Escherichia  or  Erwinia  according to  claim 35  in a medium to produce tyrosine phenol lyase and allowing the produced tyrosine phenol lyase to act on pyrocatechol and serine or pyrocatechol, pyruvic acid and ammonia to produce L-3, 4-dihydroxyphenylalanine.  
     
     
         39 . A method for producing L-3, 4-dihydroxyphenylalanine according to  claim 38 , wherein an amount of tyrosine contained in the medium is less than an amount required to induce tyrosine phenol lyase in a wild-type strain of the bacterium belonging to the genus  Escherichia  or  Erwinia.