Cloning genes from streptomyces cyaneogriseus subsp. noncyanogenus for biosynthesis of antibiotics and methods of use

Abstract

The present invention relates to the complete biosynthetic pathway for the formation of the LL-F28249 compounds and, most importantly, the major component LL-F28249α. The purified and isolated nucleic acid molecule encoding the proteins of the biosynthetic pathway, which is isolated from a wild-type or mutant Streptomyces , is fully described in FIG. 6 to FIG. 6 - 39 and SEQ ID NO:1. The DNA gene cluster and its expression in a suitable host enable the efficient production of the highly active natural metabolites and semisynthetic derivatives. The invention further concerns plasmids, vectors and host cells that contain and express the novel nucleic acid molecule. Of particular interest, the entire biosynthetic pathway fits compactly in three plasmids, Cos11, Cos36 and Cos40. The invention also concerns the purified and isolated biosynthesis proteins that are encoded by the whole DNA gene cluster. Additionally, the invention involves a new efficient, biochemical method of preparing moxidectin.

1 . A purified and isolated nucleic acid molecule encoding at least one protein of the biosynthetic pathway for producing an LL-F28249 compound, wherein said nucleic acid molecule is isolated from an antibiotic-producing wild-type or mutant  Streptomyces.    
     
     
         2 . The nucleic acid molecule according to  claim 1 , wherein the nucleic acid molecule is isolated from an antibiotic-producing wild-type or mutant  Streptomyces cyaneogriseus  subsp.  noncyanogenus.    
     
     
         3 . The nucleic acid molecule according to  claim 1 , wherein the LL-F28249 compound is LL-F28249α. 
     
     
         4 . The nucleic acid molecule according to  claim 1 , wherein the molecule has the nucleotide sequence set forth in SEQ ID NO:1 or its complementary strand. 
     
     
         5 . A nucleic acid sequence which hybridizes to the sequence of the nucleic acid molecule of  claim 4  and encodes a protein of the biosynthetic pathway for producing an LL-F28249 compound. 
     
     
         6 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 7697-10465 of SEQ ID NO:1. 
     
     
         7 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 10791-11570 of SEQ ID NO:1. 
     
     
         8 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 11659-12462 of SEQ ID NO:1. 
     
     
         9 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 12850-19875 of SEQ ID NO:1. 
     
     
         10 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 19865-31036 of SEQ ID NO:1. 
     
     
         11 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 31115-49246 of SEQ ID NO:1. 
     
     
         12 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 50449-51303 of SEQ ID NO:1. 
     
     
         13 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 51300-52706 of SEQ ID NO:1. 
     
     
         14 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 52809-69833 of SEQ ID NO:1. 
     
     
         15 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 69929-85429 of SEQ ID NO:1. 
     
     
         16 . The nucleic acid molecule of  claim 4 , wherein the molecule comprises nucleotides 85574-86338 of SEQ ID NO:1. 
     
     
         17 . A biologically functional plasmid or vector containing the nucleic acid molecule according to  claim 1 . 
     
     
         18 . The plasmid or vector according to  claim 17 , wherein the plasmid or vector comprises Cos11 having ATCC Designation Number PTA-4392, Cos36 having ATCC Designation Number PTA-4393 or Cos40 having ATCC Designation Number PTA-4394. 
     
     
         19 . A suitable host cell stably transformed or transfected by the plasmid or vector according to  claim 17 . 
     
     
         20 . The host cell according to  claim 19 , wherein the host is  Escherichia, Actinomycetales, Bacillus, Corynebacteria  or  Thermoactinomyces.    
     
     
         21 . The host cell according to  claim 20 , wherein the host is  Escherichia coli, Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseofuscus  or  Streptomyces ambofaciens.    
     
     
         22 . A biosynthesis protein encoded by the nucleic acid molecule according to  claim 1 . 
     
     
         23 . The protein according to  claim 22 , wherein the amino acid sequence is set forth in any one of SEQ ID NO:2 to SEQ ID NO:12, or a biologically active variant thereof. 
     
     
         24 . A process for the production of a protein involved in the biosynthesis of an LL-F28249 compound, said process comprising: growing, under suitable nutrient conditions, a prokaryotic or eukaryotic host cell transformed or transfected with a nucleic acid molecule according to  claim 1  in a manner allowing expression of the protein product, and isolating the desired protein product of the expression of the nucleic acid molecule. 
     
     
         25 . A protein product of the expression of the nucleic acid molecule in a prokaryotic or eukaryotic host cell according to  claim 24 . 
     
     
         26 . A plasmid or a combination of two or three plasmids for cloning the nucleic acid molecule which encodes the proteins of the biosynthetic pathway of an LL-F28249 compound, wherein said plasmid or combination contains the nucleic acid molecule that spans the entire biosynthetic gene cluster and encodes type I polyketide synthase that is responsible for producing the LL-F28249 compound. 
     
     
         27 . The combination according to  claim 26 , which comprises Cos11 having ATCC Designation Number PTA-4392, Cos36 having ATCC Designation Number PTA-4393 and Cos40 having ATCC Designation Number PTA-4394. 
     
     
         28 - 35 . (canceled)