Polynucleotides for production of farnesyl dibenzodiazepinones

Abstract

This invention provides genes and their encoded proteins, involved in the biosynthesis of farnesyl dibenzodiazepinones, including ECO-04601. The invention relates to expression vectors comprising the genes and to host cells transformed with these vectors. The invention further relates to methods of producing farnesyl dibenzodiazepinone compounds using the genes and proteins of the invention, for example, involving expression of biosynthetic pathway genes in transformed host cells.

1 . An isolated polynucleotide comprising a polynucleotide sequence, or a polynucleotide sequence complementary thereto, selected from the group consisting of:
 a) a polynucleotide encoding a polypeptide having at least 90% sequence identity to a polypeptide consisting of amino acids 1-438 of SEQ ID NO: 48 and having adenylating amide synthetase activity;   b) a polynucleotide encoding a polypeptide having at least 90% sequence identity to a polypeptide consisting of amino acids 1-290 of SEQ ID NO: 22 and having isoprenyl transferase activity;   c) a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:47; and   d) a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:23.   
     
     
         2 . An isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:47. 
     
     
         3 . An isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:23. 
     
     
         4 . The isolated polynucleotide of  claim 1 , wherein said polypeptide of a) has at least 95% sequence identity to a polypeptide consisting of amino acids 1-438 of SEQ ID NO: 48. 
     
     
         5 . The isolated polynucleotide of  claim 1 , wherein said polypeptide of a) has at least 99% sequence identity to a polypeptide consisting of amino acids 1-438 of SEQ ID NO: 48. 
     
     
         6 . The isolated polynucleotide of  claim 1 , wherein said polypeptide of b) has at least 95% sequence identity to a polypeptide consisting of amino acids 1-290 of SEQ ID NO: 22. 
     
     
         7 . The isolated polynucleotide of  claim 1 , wherein said polypeptide of b) has at least 99% sequence identity to a polypeptide consisting of amino acids 1-290 of SEQ ID NO: 22. 
     
     
         8 . A purified polypeptide selected from the group consisting of:
 a) a polypeptide comprising amino acids 1-290 of SEQ ID NO: 22; and   b) a polypeptide having at least 90% sequence identity to a polypeptide comprising amino acids 1-290 of SEQ ID NO: 22 and having an isoprenyl transferase activity; and   c) a polypeptide encoded by a polynucleotide, the complement of which hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising amino acids 1-290 of SEQ ID NO: 22, and having an isoprenyl transferase activity.   
     
     
         9 . A purified polypeptide comprising amino acids 1-290 of SEQ ID NO: 22. 
     
     
         10 . The purified polypeptide of  claim 8 , wherein said polypeptide of b) has at least 95% identity to a polypeptide comprising amino acids 1-290 of SEQ ID NO: 22. 
     
     
         11 . An expression vector comprising a polynucleotide of  claim 1 . 
     
     
         12 . The expression vector of  claim 11 , wherein said polynucleotide encodes a polypeptide having at least 90% sequence identity to a polypeptide comprising amino acids 1-438 of SEQ ID NO: 48 and having adenylating amide synthetase activity. 
     
     
         13 . The expression vector of  claim 11 , wherein said polynucleotide encodes a polypeptide having at least 90% sequence identity to a polypeptide comprising amino acids 1-290 of SEQ ID NO: 22, and having isoprenyl transferase activity. 
     
     
         14 . An isolated host cell transformed with an expression vector of  claim 11 . 
     
     
         15 . The isolated host cell of  claim 14 , wherein said host cell is a bacterial host cell. 
     
     
         16 . A method for producing a farnesyl dibenzodiazepinone compound, comprising:
 a) providing a prokaryote transformed with an expression vector of  claim 11 ; and   b) culturing the prokaryote under conditions such that (i) an adenylating amide synthetase or an isoprenyl transferase is expressed, and (ii) a farnesyl dibenzodiazepinone compound is synthesized.   
     
     
         17 . The method of  claim 16 , wherein said prokaryote is  E. coli.    
     
     
         18 . The method of  claim 16 , wherein said prokaryote is an actinomycete. 
     
     
         19 . An isolated polynucleotide encoding:
 a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 70, 71, 74, 76, 78, 80, 82, 84, 86 and 88; or   b) a polypeptide having at least 85% sequence identity to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 70, 71, 74, 76, 78, 80, 82, 84, 86 and 88, and having the same biological function as the corresponding polypeptide.   
     
     
         20 . A cosmid selected from the group consisting of cosmid 046KM deposited under IDAC accession no. 250203-06 and cosmid 046KQ deposited under IDAC accession no. 250203-07. 
     
     
         21 . The cosmid of  claim 20 , wherein said cosmid is inserted into a prokaryotic host for expressing a product.