Engineering the production of a conformational variant of occidiofungin that has enhanced inhibitory activity against fungal species

Abstract

Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity. This invention is directed to compositions enriched for particular occidiofungin diastereomers/conformers, methods of making compositions enriched for particular diastereomers/conformers and microorganisms suitable for producing enriched compositions of particular diastereomers/conformers. Methods of treating fungal infections or plants infected by fungi are also provided.

1 - 10 . (canceled) 
     
     
         11 . A bacterial mutant of bacterial strain  Burkholderia contaminans  MS14, wherein the ocfN gene in the bacterial strain  Burkholderia contaminans  MS14 is mutated such that the mutated ocfN gene in the bacterial mutant produces a decreased OcfN thioesterase activity in comparison with the ocfN gene in the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         12 . The bacterial mutant of  claim 11 , wherein the mutated ocfN gene of the bacterial mutant produces no OcfN thioesterase activity. 
     
     
         13 . The bacterial mutant of  claim 11 , wherein the mutated ocfN gene of the bacterial mutant is truncated compared with the ocfN gene in the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         14 . The bacterial mutant of  claim 11 , wherein the ocfN gene of the bacterial mutant has a frameshift compared with the ocfN gene in the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         15 . The bacterial mutant of  claim 11 , wherein the ocfN gene of the bacterial strain  Burkholderia contaminans  MS14 is deleted in the bacterial mutant. 
     
     
         16 . The bacterial mutant of  claim 11 , wherein the ocfN gene of the bacterial mutant has a point mutation of catalytic serine at position 73 of SEQ ID NO:3 of the OcfN thioesterase of the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         17 . The bacterial mutant of  claim 11 , wherein the ocfN gene of the bacterial mutant has an insertion mutation or a point mutation in the thioesterase motif of the OcfN thioesterase of the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         18 . The bacterial mutant of  claim 11 , wherein the ocfN gene of the bacterial mutant has a deletion of catalytic serine at position 73 of SEQ ID NO:3 of the OcfN thioesterase of the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         19 . The bacterial mutant of  claim 11 , wherein the ocfN gene of the bacterial mutant has a deletion of the thioesterase motif of the OcfN thioesterase of the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         20 . The bacterial mutant of  claim 11 , wherein the ocfD gene in the bacterial strain  Burkholderia contaminans  MS14 is mutated such that the mutated ocfD gene in the bacterial mutant produces an increased OcfD thioesterase activity in comparison with the ocfD gene in the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         21 . The bacterial mutant of  claim 11 , wherein the bacterial mutant has two or more copies of the ocfD gene of the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         22 . The bacterial mutant of  claim 11 , wherein the ocfD gene in the bacterial mutant has a promoter that increases expression of the ocfD gene compared with a promoter of the ocfD gene in the bacterial strain  Burkholderia contaminans  MS14. 
     
     
         23 . A composition comprising an occidiofungin produced by the bacterial mutant of  claim 11 . 
     
     
         24 . The composition of  claim 23 , further comprising a pharmaceutically or agriculturally acceptable excipient or carrier. 
     
     
         25 . The composition of  claim 23 , further comprising a pharmaceutically acceptable excipient or carrier selected from ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. 
     
     
         26 . The composition of  claim 23 , formulated to be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. 
     
     
         27 . A method for treating fungal infections in a subject comprising a step of administering the composition of  claim 23  to the subject. 
     
     
         28 . The method of  claim 27 , wherein the fungal infection is caused by at least one organism selected from the group consisting of  Candida albicans  LL,  Candida albicans  TE,  Candida glabrata  ATCC66032,  Candida parapsilosis  ATCC90018, and  Candida tropicalis  ATCC66029. 
     
     
         29 . The method of  claim 27 , wherein the subject is a mammal. 
     
     
         30 . The method of  claim 27 , wherein the subject is a plant.