Genome-Wide Mutagenesis Links Multiple Metabolic Pathways with Actinorhodin Production in Streptomyces coelicolor.

Zhong Xu,Yemin Wang,Yuanyuan Li,Meifeng Tao,Zixin Deng,Robert M. Kelly

doi:10.1128/aem.03005-18

Abstract

Streptomyces species are important antibiotic-producing organisms that tightly regulate their antibiotic production. Actinorhodin is a typical antibiotic produced by the model actinomycete Streptomyces coelicolor To discover the regulators of actinorhodin production, we constructed a library of 50,000 independent mutants with hyperactive Tn5 transposase-based transposition systems. Five hundred fifty-one genes were found to influence actinorhodin production in 988 individual mutants. Genetic complementation suggested that most of the insertions (76%) were responsible for the changes in antibiotic production. Genes involved in diverse cellular processes such as amino acid biosynthesis, carbohydrate metabolism, cell wall homeostasis, and DNA metabolism affected actinorhodin production. Genome-wide mutagenesis can identify novel genes and pathways that impact antibiotic levels, potentially aiding in engineering strains to optimize the production of antibiotics in StreptomycesIMPORTANCE Previous studies have shown that various genes can influence antibiotic production in Streptomyces and that intercommunication between regulators can complicate antibiotic production. Therefore, to gain a better understanding of antibiotic regulation, a genome-wide perspective on genes that influence antibiotic production was needed. We searched for genes that affected production of the antibiotic actinorhodin using a genome-wide gene disruption system. We identified 551 genes that altered actinorhodin levels, and more than half of these genes were newly identified effectors. Some of these genes may be candidates for engineering Streptomyces strains to improve antibiotic production levels.

Highlights

Streptomyces species are important antibiotic-producing organisms that tightly regulate their antibiotic production
To identify genes that affect the production of ACT, which is synthesized from acetyl coenzyme A and malonyl-CoA (Fig. 1A), a library with approximately 50,000 mutants was constructed in S. coelicolor M145 via pHL734-mediated transposon mutagenesis [36]. pHL734 is a suicide plasmid; each mutant obtained by this method results from a single random transposition event [36]
The variations in ACT production of all mutants were quantified by measuring the absorbance at 633 nm (UV633) of alkaline extracts of cultures grown on yeast extract-beef extract-Bacto peptone (YBP) agar for 84 h

Summary

Introduction

Streptomyces species are important antibiotic-producing organisms that tightly regulate their antibiotic production. Many studies have been conducted to elucidate the regulation of antibiotic production in Streptomyces, predominantly in S. coelicolor, and such studies have used DNA microarrays to reveal gene expression patterns during metabolic switching [26, 27], allowed the construction of genome-wide metabolic models [28,29,30], and identified transcriptional regulators required for antibiotic production [31,32,33,34,35]. Some of these genes may play important roles for strain improvement

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Results

Conclusion