Process for producing biopterin

Abstract

The present invention provides a transformed cell which is transformed by at least one gene of enzymes participating in biosynthesis of tetrahydrobiopterin and a process for the production of a biopterin compound using the same. In accordance with the present invention, the biopterin compound can be produced in large quantities in an industrial advantageous manner from less expensive materials.

1 . In a process for production of a biopterin compound by a transformed cell, a process for production of the biopterin compound which is characterized in that (a) a host cell is transformed by at least one gene of enzyme participating in biosynthesis of tetrahydrobiopterin, (b) the resulting transformed cell is cultured to produce tetrahydrobiopterin, (c) the resulting tetrahydrobiopterin is oxidized if necessary and (d) one or more biopterin compound(s) selected from the resulting tetrahydrobiopterin and dihydrobiopterin and biopterin where the said tetrahydrobiopterin is oxidized is/are collected.  
     
     
         2 . The process for production of the biopterin compound according to  claim 1 , wherein the biopterin compound in the culture broth or in a processed product thereof is oxidized and biopterin is collected therefrom.  
     
     
         3 . The process for production of the biopterin compound according to  claim 1  or  2 , wherein the collected dihydrobiopterin and/or biopterin are/is reduced to produce tetrahydrobiopterin.  
     
     
         4 . The process for production of the biopterin compound according to  claims 1  to  3 , wherein the enzyme(s) participating in the biosynthesis of tetrahydrobiopterin is/are one to three kind(s) of enzyme(s) selected from a group consisting of GTP cyclohydrase 1,6-pyruvoyltetrahydropterin synthase and sepiapterin reductase.  
     
     
         5 . The process for production of the biopterin compound according to  claims 1  to  3 , wherein the transformation is carried out by an expression vector having gene of 6-pyruvoyltetrahydropterin synthase and gene of sepiapterin reductase.  
     
     
         6 . The process for production of the biopterin compound according to  claim 5 , wherein the host cell is a mutant cell having a GTP cyclohydrase I activity which is not less than the GTP cyclohydrase I activity inherent to the cell of a wild type.  
     
     
         7 . The process for production of the biopterin compound according to  claim 4 , wherein the GTP cyclohydrase I gene to be introduced into the host cell is mtrA gene derived from  Bacillus subtilis.    
     
     
         8 . The process for production of the biopterin compound according to  claims 1  to  7 , wherein the host cell is a prokaryotic cell.  
     
     
         9 . The process for production of the biopterin compound according to  claim 8 , wherein the prokaryote is  Escherichia coli, Bacillus subtilis  or Actinomyces.  
     
     
         10 . The process for production of the biopterin compound according to  claims 1  to  7 , wherein the host cell is an eukaryotic cell.  
     
     
         11 . The process for production of the biopterin compound according to  claim 10 , wherein the eukaryote is yeast or filamentous fungi.  
     
     
         12 . The process for production of the biopterin compound according to  claim 11 , wherein the yeast is methanol assimilating yeast or fission yeast.  
     
     
         13 . The process for production of the biopterin compound according to  claim 11 , wherein the yeast is Saccharomyces yeast.  
     
     
         14 . The process for production of the biopterin compound according to  claims 1  to  13 , wherein the host cell is a mutant cell having a GTP synthesizing ability of not less than the GTP synthesizing ability of the cell of a wild type.  
     
     
         15 . The process for production of the biopterin compound according to  claim 14 , wherein the host cell is a mutant cell having a 8-azaguanine resistance of not less than the 8-azaguanine resistance of the cell of a wild type.  
     
     
         16 . The process for production of the biopterin compound according to  claim 14  or  15 , wherein the host cell is a genetically recombinant cell where guaBA gene coding for IMP dehydrogenase and GMP synthase is introduced thereinto.  
     
     
         17 . A transformed cell used for the production of the biopterin compound mentioned in  claims 1  to  16  in which gene of an enzyme participating in biosynthesis of tetrahydrobiopterin is introduced into a host cell.  
     
     
         18 . The transformed cell according to  claim 17 , wherein the host is a mutant cell having a GTP synthesizing ability of not less than the GTP synthesizing ability of the cell of a wild type.  
     
     
         19 . The transformed cell according to  claim 18 , wherein the host cell is a mutant cell having a 8-azaguanine resistance of not less than the 8-azaguanine resistance of the cell of a wild type.  
     
     
         20 . The transformed cell according to  claim 18  to  19 , wherein the host cell is a genetically recombinant cell where guaBA gene coding for IMP dehydrogenase and GMP synthase is introduced thereinto.  
     
     
         21 . The transformed cell according to  claim 18 , wherein the host cell is a mutant cell having a GTP cyclohydrase I activity which is not less than the GTP cyclohydrase I activity inherent to the cell of a wild type and gene of 6-pyruvoyltetrahydropterin synthase and gene of sepiapterin reductase are introduced into the said host cell.  
     
     
         22 . The transformed cell according to  claims 17  to  21 , wherein the GTP cyclohydrase I gene to be introduced into the host cell is mtrA gene derived from  Bacillus subtilis.