Exploiting Adaptive Laboratory Evolution of Streptomyces clavuligerus for Antibiotic Discovery and Overproduction

Abstract

Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition‐based adaptive laboratory evolution scheme is presented whereby an antibiotic‐producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces clavuligerus replicates were adaptively evolved against methicillin‐resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the wild‐type strain. Moreover, genome re‐sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid. This and other strains also acquired numerous single nucleotide polymorphisms, some of which are located within genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition‐based adaptive laboratory evolution can constitute a platform to create mutants that overproduce known antibiotics and possibly to discover new compounds as well.

The Fish Pathogen Yersinia ruckeri Produces Holomycin and Uses an RNA Methyltransferase for Self-resistance

“…No obvious ORF in this gene cluster can be assigned to the disulfide oxidase responsible for the formation of a reduced dithiolopyrrolone intermediate to holomycin. A similar absence was also observed in the thiomarinol gene cluster from the marine Gram-negative bacterium Pseudoalteromonas sp SANK 73390 (6), suggesting that the underlying chemical logic of disulfide formation is different in the Gram-negative bacteria.…”

“…Dithiolopyrrolone antibiotics possess broad-spectrum antibacterial activities against Gram-positive and Gram-negative bacteria (3,4). Holomycin appeared to be active against rifamycin-resistant bacteria (5) and also to inhibit the growth of the clinical pathogen methicillin-resistant Staphylococcus aureus N315 (6). Its mode of action is believed to inhibit RNA synthesis, although the exact mechanism has yet to be established in vitro.…”

Thermal and Solvent Stress Cross-Tolerance Conferred to Corynebacterium glutamicum by Adaptive Laboratory Evolution

“…The numbers of mutations acquired by each evolved strain during 65 days of serial passage, corresponding to ϳ370 generations, were much higher than we expected from the earlier ALE studies on other bacterial species (22,47). We speculated whether the evolved strains turned into mutators at some points in their evolution and carried out a fluctuation assay (38,39) to estimate the mutation rates of the parental and evolved strains.…”

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum

“…Identification of beneficial mutations, therefore, remains extremely laborious due to the lack of a selective pressure as imposed during biosensor-driven evolution. In contrast to random mutagenesis, adaptive evolution strategies have been shown to lead to a considerably low number of genomic modifications by selecting against detrimental mutations at the same time (Charusanti et al, 2012;Herring et al, 2006). Our biosensor-driven evolution of the ΔaceE strain resulted in a total of seven SNPs accumulated within the genome, which enabled the prompt identification of promising candidates for further analysis ( Fig.…”