Investigation of bacterial terpene natural product biosynthesis through heterologous expression

Diana Łomowska-Keehner, Tyler Alsup, Melvin Osei Opoku, Xiuting Wei, Wenbo Ning,
Caitlin McCadden, and Jeffrey D. Rudolf

University of Florida

Terpenoids are the largest class of natural products (NPs) with over 100,000 characterized compounds. These NPs show broad bioactivities including antibacterial, cytotoxic, anti-inflammatory, and antifeedant properties. Historically, plants have been known as the most prolific producers of terpenoids. However, genome mining revealed that bacteria, especially Streptomyces, have great terpenoid biosynthetic potential. We performed genome mining in search of uncharacterized bacterial terpene synthases (TSs) encoded in Streptomyces NP biosynthetic gene clusters (BGCs) that could generate novel terpene scaffolds. These bacterial TSs of unknown function were heterologously expressed in E. coli containing upregulated precursor genes and a kinase system under the control of a T7 inducible promoter to enable in vivo conversion of exogenously added isoprenol into terpene precursors. Notably, Streptomyces produce complex NP mixtures and their BGCs encoding TSs of interest are often silent (not expressed) under laboratory conditions. Thus, expressing TSs in E. coli with optimized terpene precursor genes decreases complexity of fermentation extracts and increases terpene titer. This system facilitated detection of TS products at small scale to rapidly validate TS activity and supports reliable terpene overproduction in large scale fermentations. With characterized TS products in hand, we can prioritize Streptomyces BGCs with promising tailoring enzymes clustered near TSs for heterologous expression to discover further functionalized bioactive terpenoid NPs. This presentation will showcase terpenes isolated thus far from previously uncharacterized bacterial TSs, our heterologous expression workflow, and BGCs under study.

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