Redefining Fermentation Parameters in Natural Products Drug Discovery

PROJECT SUMMARY
The discovery of antibiotics from soil microbes is widely regarded as one of the most significant achievements
in modern medicine, enabling many important medical procedures including surgery and cancer
chemotherapy. However, antibiotic resistance is approaching such a critical level that we are facing an eminent
public health disaster where many significant medical advancements may no longer be possible. There is an
urgent need to develop and/or discover novel classes of antibiotics, especially antibiotics that are active
against high-priority Gram-negative pathogens. Natural products have served as the scaffold for the vast
majority of our current antibiotics, and recent advances in genomics, metagenomics, and metabolomics clearly
indicate that there is still a vast wealth of biosynthetic potential encoded in bacterial genomes that could
produce novel antibiotics. Unfortunately, identifying a novel biosynthetic gene clusters (BGCs) in a genome
provides us with very little information about the chemical nature of the natural product it might produce. Thus,
the field of natural product discovery, and consequently the field of antibiotic discovery, faces two major
obstacles: how do we quickly and efficiently identify strains that have the potential to produce desirable novel
compounds from “silent” BGCs and then how do we consistently induce the expression of these BGCs to
characterize the compounds they produce. The induction of silent BGCs that produce antibiotics is likely
context or community dependent, especially given the self-harming effects of antimicrobial compounds. Our
previous work has validated a method for identifying microbes that produce antimicrobial compounds from
silent BGCs, and this proposal describes methods for optimizing single- and mixed-culture fermentation
conditions to consistently produce these compounds. In the research aims, we propose two complementary
approaches to develop reproducible and scalable fermentation conditions that can broadly stimulate silent
antibiotic production, which is often a rate limiting step in the field of natural products chemistry. Aim 1 will
expand on our observations that microbes increase the production of antimicrobial compounds when grown in
otherwise nutrient-limited media where complex polysaccharides are their dominant carbon source. Aim 2 will
use co-culture to manipulate microbial physiological prior to fermentation. We expect that our optimized culture
conditions will enable us to obtain natural product extracts that contain sufficient compound for feature-based
molecular networking (FBMN) analyses to dereplicate antimicrobial compounds prior to activity-guided
purification and structural elucidation of bioactive compounds (Aim 3).

Grant Number: 5R16AI167814-04
NIH Institute/Center: NIH
Principal Investigator: Anne Casper