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.

We claim: 
     
         1 . A method of increasing, in a microorganism producing occidiofungin, production of occidiofungin diastereomers/conformers corresponding to the diastereomers/conformers having the total correlation spectroscopy (TOCSY) fingerprint set forth in  FIG. 5C  as the green NH correlations, the method comprising:
 genetically manipulating the microorganism to increase thioesterase activity of an occidiofungin gene N (ocfN) product (OcfN) within the microorganism, wherein the genetic manipulation of the microorganism comprises:
 increasing the OcfN thioesterase activity by expressing a gene encoding the ocfN thioesterase in a multicopy plasmid; 
 increasing the OcfN thioesterase activity by chromosomal integration of one or more additional copies of a gene encoding the OcfN thioesterase into the chromosome of the microorganism; and/or 
 substituting of the native promoter associated with the gene encoding the OcfN thioesterase with a promoter that increases the expression level of the occidiofungin gene N (ocfN) relative to the native promoter, 
 
 and wherein the increase in the OcfN thioesterase activity is as compared to a reference microorganism without the increase in OcfN thioesterase activity cultured under the same conditions. 
 
     
     
         2 . The method of  claim 1 , the method further comprising culturing the microorganism under conditions that permit the production of occidiofungin diastereomers/conformers corresponding to the diastereomers/conformers having the TOCSY fingerprint set forth in  FIG. 5C  as the green NH correlations and, optionally, purifying the occidiofungin diastereomers/conformers from the culture medium. 
     
     
         3 . The method of  claim 1  further comprising decreasing the thioesterase activity of an occidiofungin gene D (ocfD) product (OcfD) within the microorganism. 
     
     
         4 . A composition enriched for the occidiofungin diastereomers/conformers corresponding to the diastereomers/conformers having the TOCSY fingerprint set forth in  FIG. 5C  as the green NH correlations of  claim 1 . 
     
     
         5 . The composition of  claim 4  further comprising a pharmaceutically or agriculturally acceptable excipient or carrier. 
     
     
         6 . The composition of  claim 4 , wherein the occidiofungin diastereomers/conformers corresponding to the diastereomers/conformers having the TOCSY fingerprint set forth in  FIG. 5C  as the green NH correlations are present in the composition in an amount of at least 37% of the total amount of occidiofungin diastereomers/conformers within the composition. 
     
     
         7 . A method of treating a fungal infection comprising administering a fungicidally effective amount of the composition of  claim 5  to a subject in need thereof. 
     
     
         8 . The method of  claim 7 , 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. 
     
     
         9 . The method of  claim 7 , wherein the subject is a mammal. 
     
     
         10 . The method of  claim 7 , wherein the subject is a plant.