Systematic approach for enhancing the production of secondary metabolites in streptomycetes

Abstract

With the rapid emergence of antibiotic microbial resistance (AMR) to all major classes of antibiotics and the decline in a number of potential candidates for new antibiotics, press an urgent need for the discovery of novel antibacterial compounds. Streptomyces, soil dwelling gram-positive bacteria, continue to be promising microorganisms for the production of clinically important secondary metabolites, including not only antibiotics, but also antiviral, antifungal, antiparasitic agents, antitumorals, and immunosuppressant compounds. Each Streptomyces species has the genetic potential to produce more than 30 secondary metabolites on average, which are diverse and differ between species. However, most secondary metabolite biosynthetic gene clusters (smBGC) are silent under laboratory culture conditions, limiting effective usage of Streptomyces. To overcome the limitation, the systematic understanding of the secondary metabolism and related regulatory mechanisms is required to reach the potential usage of the streptomycetes. In this study, I utilized three systematic approaches to discover novel smBGCs and elucidate multi-layered regulation on secondary metabolism of streptomycetes for enhancing secondary metabolite production. First, we present 30 high-quality assembled streptomycetes genome sequences by a hybrid strategy using both long-read and short-read genome sequencing methods. The assembled genomes have more than 97.4% completeness with total lengths ranging from 6.7 to 10.1 Mbp. In average, the annotation identified 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs. In silico prediction of smBGCs identified a total of 922 clusters, including many clusters unlinked to any known secondary metabolites production. In addition, smBGC analysis of the genomes revealed that S. venezuelae ATCC 14583, 14584 and 14585 possess the novel smBGC, producing anthelvencin. Through functional analysis of anthelvencin BGC encoded genes, I identified novel precursor synthesis gene and determined biosynthetic pathway for the anthelvencin. I anticipate that the availability of these genomes will accelerate a discovery of the novel secondary metabolites from Streptomyces and elucidation of complex smBGC regulation. Second, a microbial coculture, which widely utilized method to activate silent smBGCs, between S. coelicolor and M. xanthus was applied to determine hidden layer for antibiotic biosynthesis. Through transcriptome analysis, we found that iron competition triggered antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus. During coculture, M. xanthus enhanced the production of a siderophore, myxochelin, leading M. xanthus to dominate iron scavenging and S. coelicolor to experience iron-depleted conditions. This chemical communication, but not physical interaction, activated the actinorhodin biosynthetic gene cluster and the branched chain amino acid degradation pathway to supply a key biosynthetic precursor, acetyl-CoA, along with the activation of a novel actinorhodin export system. Furthermore, we found that the iron depletion increased the expression of 21 smBGCs in other Streptomyces species, as well. These findings suggested that the availability of key ions stimulated specific smBGCs, which had the potential to enhance secondary metabolite biosynthesis in Streptomyces. Third, I systematically comprehended multi-layered regulation of secondary metabolism of streptomycetes at a molecular level, via completing the 7.9 Mb linear genome sequence of immunosuppressant agent tacrolimus (FK506) producer, Streptomyces tsukubaensis, and integrating the multi-omics measurements. Along with accurate re-annotation of the FK506 gene cluster, a total of 2,389 transcription start sites (TSS) and 1,270 transcript 3’end positions (TEP) were determined via primary transcriptome analysis. The integrated analysis of transcriptome and translatome data revealed that secondary metabolite gene clusters, especially the FK506 cluster, undergo translational regulation with decreased translational efficiency in accordance with growth. Furthermore, we demonstrated that AT-rich codons delay translational elongation, especially strong ribosome pausing was observed at the rare TTA codon in the FK506 cluster. In order to validate the phenomena, CRISPR/Cas9 mediated codon replacement was performed, resolving the ribosome pausing at the TTA codon and increasing the FK506 production. This integrated genome-scale analysis provide insight into multi-layered regulation on secondary metabolism of streptomycetes. Overall, system biology approaches provides comprehensive understanding of secondary metabolism and potential targets for engineering the streptomycetes to optimize antibiotic production.