Method for Producing Recombinant 11-De-O-Methyltomaymycin

Abstract

The present invention provides a tomaymycin biosynthetic gene cluster of Streptomyces species FH6421, and its use for producing 11-de-O-methyltomaymycm.

1 . A nucleic acid molecule comprising at least one nucleic acid selected from the group consisting of:
 (a) a nucleic acid comprising at least one of the Open Reading Frames (ORFs) 1 to 19 of SEQ ID NO: 2 that encodes proteins of SEQ ID NOs: 4 to 22 or a variant or fragment thereof, whereby the variant or fragment encodes a functionally active variant or fragment of a protein of SEQ ID NOs: 4 to 22,   (b) a nucleic acid encoding at least one of the proteins of SEQ ID NOs: 4 to 22 or a functionally active variant or fragment thereof,   (c) a nucleic acid encoding a protein that is at least 70%, 80%, 90%, 95% or 97% identical in amino acid sequence to a protein or fragment thereof encoded by the nucleic acid of (a) or (b),   (d) a nucleic acid that hybridizes under stringent conditions with a nucleic acid of (a) to (c),   (e) a nucleic acid that is complementary to a nucleic acid of (a) to (d).   
     
     
         2 . The nucleic acid according to  claim 1  comprising the tomaymycin biosynthetic gene cluster of SEQ ID NO: 2 or having a variant or fragment sequence of SEQ ID NO: 2 harboring a variant or fragment of at least one of ORFs 1 to 19, whereby the variant or fragment encodes a functionally active variant or fragment of a protein of SEQ ID NOs: 4 to 22. 
     
     
         3 . An expression vector comprising a nucleic acid of  claim 1 . 
     
     
         4 . A cell comprising the expression vector according to  claim 3 . 
     
     
         5 . The cell of  claim 4 , wherein the cell is  Streptomyces  species FH6421. 
     
     
         6 . A method for producing a cell that harbors a tomaymycin biosynthetic gene cluster or a functionally active variant or fragment thereof. 
     
     
         7 . The method according to  claim 6 , wherein the cell harbors the tomaymycin biosynthetic gene cluster of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a functionally active variant or fragment thereof. 
     
     
         8 . The method according to  claim 7 , wherein the cell is a  Streptomyces  strain harboring the tomaymycin biosynthetic gene cluster of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or a functionally active variant or fragment thereof. 
     
     
         9 . The method according to  claim 8 , wherein the  Streptomyces  strain is selected from the group consisting of  Streptomyces achromogenes  var.  tomaymyceticus, Streptomyces  species FH6421, and  Streptomyces albus /pStW102tc. 
     
     
         10 . The method according to  claim 6 , wherein the cell harbors at least one ORF of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or functionally active variant or fragment thereof, and is capable of producing 11-de-O-methyltomaymycin.