Cryptic metabolites and method for activating silent biosynthetic gene clusters in diverse microorganisms

Abstract

Microorganisms are prolific producers of natural products, a group of molecules that make up the majority of drugs approved by the FDA in the past 35 years. After decades of mining, the low-hanging fruit has been picked and so discovery of drug-like molecules from microorganisms has come to a near-halt. The reason for this lack of productivity is that most biosynthetic pathways that give rise to natural products are not active under typical laboratory growth conditions. These so-called ‘cryptic’ or ‘silent’ pathways are a major source of new bioactive molecules and methods that reliably activate them could have a profound impact on drug discovery. Disclosed herein is a rapid genetics-free method for eliciting and detecting cryptic metabolites using an imaging mass spectrometry-based approach. An organism of choice is challenged with elicitors from a small molecule library. The molecules elicited are then imaged by mass spec, which allows for rapid identification of cryptic metabolites. These are then isolated and characterized. Employing the disclosed approach activated production of cryptic glycopeptides from an actinomycete bacterium. The molecules that result, the keratinimicins and keratinicyclins, are metabolites with important structural features. At least two of these, keratinimicins B and C, are highly bioactive against several pathogenic strains. This approach will allow for rapid activation and identification of cryptic metabolites from diverse microorganisms in the future.

What is claimed is: 
     
         1 . A method for rapid eliciting cryptic metabolites, the method comprising the steps of:
 providing a first well comprising a first quantity of a medium and at least one microorganism from a first species;   providing a plurality of additional wells, each comprising the medium, at least one microorganism from the first species, and at least one compound;   growing microorganisms in each well;   imaging each well with mass spectrometry; and   identifying a difference in the mass spectrometry images between at least one of the additional wells and the first well, and identifying the at least one compound that elicited the difference.   
     
     
         2 . The method according to  claim 1 , further comprising isolating a molecule. 
     
     
         3 . The method according to  claim 2 , wherein isolating the molecule comprises:
 providing a larger culture, comprising second quantity of a medium and at least one microorganism from the first species, the second quantity being larger than the first quantity;   adding the identified at least one compound to the larger culture;   growing the microorganism; and   removing cells via centrifugation.   
     
     
         4 . The method according to  claim 3 , further comprising purifying via HPLC. 
     
     
         5 . The method according to  claim 2 , further comprising characterizing the molecule. 
     
     
         6 . The method according to  claim 2 , wherein the characterizing the molecule is accomplished via at least one method selected from the group consisting of: HPLC-ESI-MS or NMR. 
     
     
         7 . The method according to  claim 1 , wherein the mass spectrometry is laser-ablation electrospray ionization mass spectrometry (LAESI-MS). 
     
     
         8 . The method according to  claim 1 , wherein the plurality of wells is part of a plate containing at least 96 wells. 
     
     
         9 . The method according to  claim 1 , wherein the microorganism is a gram-negative bacterium, a gram-positive bacterium, or a fungus. 
     
     
         10 . The method according to  claim 9 , wherein the microorganism is  Amycolatopsis  sp. B24117. 
     
     
         11 . The method according to  claim 1 , wherein the genome sequence of the microorganism is not known. 
     
     
         12 . The method according to  claim 1 , wherein each of the at least one compounds is a subset of a small molecule library.