A Versatile Transcription–Translation in One Approach for Activation of Cryptic Biosynthetic Gene Clusters

Abstract

The ever-growing drug resistance problem worldwide highlights the urgency to discover and develop new drugs. Microbial natural products are a prolific source of drugs. Genome sequencing has revealed a tremendous amount of uncharacterized natural product biosynthetic gene clusters (BGCs) encoded within microbial genomes, most of which are cryptic or express at very low levels under standard culture conditions. Therefore, developing effective strategies to awaken these cryptic BGCs is of great interest for natural product discovery. In this study, we designed and validated a Transcription− Translation in One (TTO) approach for activation of cryptic BGCs. This approach aims to alter the metabolite profiles of target strains by directly overexpressing exogenous rpsL (encoding ribosomal protein S12) and rpoB (encoding RNA polymerase β subunit) genes containing beneficial mutations for natural product production using a plug-and-play plasmid system. As a result, this approach bypasses the tedious screening work and overcomes the false positive problem in the traditional ribosome engineering approach. In this work, the TTO approach was successfully applied to activating cryptic BGCs in three Streptomyces strains, leading to the discovery of two aromatic polyketide antibiotics, piloquinone and homopiloquinone. We further identified a single BGC responsible for the biosynthesis of both piloquinone and homopiloquinone, which features an unusual starter unit incorporation step. This powerful strategy can be further exploited for BGC activation in strains even beyond streptomycetes, thus facilitating natural product discovery research in the future.

Investigation of the Molecular Landscape of Bacterial Aromatic Polyketides by Global Analysis of Type II Polyketide Synthases

“…Furthermore, we observed a starter unit‐dependent clade in the CLF trees. For example, murayaquinone [10] and piloquinone [11] have an identical polyketide scaffold, but the uncanonical isocaproyl‐CoA starter unit in piloquinone sets it apart from murayaquinone in the CLF tree (Figure S3). These results indicate that the phylogenetic cladogram of CLFs has a high correlation with the products’ chemical structures and their respective biosynthetic courses.…”

“…[27] B) HPLC analysis of the culture extract. C) Structure of WS-5995 D (9) and other known derivatives (10)(11)(12).…”

Double‐reporter‐guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527

“…Many methods were designed and employed to awaken expression of silent gene cluster (Alam et al, 2021;Ji et al, 2018;Luo et al, 2013;Wang et al, 2019;F. Xu, Nazari, et al, 2017;Q. Zhang et al, 2020).…”

Synthesis and Characterization of Polydopamine‐Modified Montmorillonite Loaded with Silver Nanoparticles for Antibacterial Functionalization

“…Although researches have shown that screening active ingredients from microbial metabolites [23] or translating antibacterial substances by transcription percentage of microbe cells, [24] is also effective ways to develop new antibiotics from natural microbial resources. However, compared with silver nanoparticles‐loaded polydopamine‐modified montmorillonite (AgNP/PDA−Mt), the preparation of the synthetic antimicrobial agent is simpler and more convenient.…”