Name | Diana Łomowska-Keehner |
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Organization or Institution | University of Florida |
Topic | Biochemistry / Chem Bio. |
Title | Investigation of Streptomycesnatural product biosynthesis through heterologous expression |
Author(s) | Diana Łomowska-Keehner, Jeffrey D. Rudolf |
Author Institution(s) | University of Florida |
Abstract | Natural products (NPs) are highly valued for their broad applications in medicine, agriculture, and consumer products. Notably, bacterial NPs from the genus Streptomyces provide approximately 70% of antibiotics used to date. Thus, it is critical to understand the function of enzymes encoded in biosynthetic gene clusters (BGCs) responsible for the production of NPs. Benditerpenoic acid (BND), produced by Streptomyces sp. CL12-4, has a 6,10-bicyclic eunicellane scaffold that is rare in bacteria. The cytochrome P450 enzymes responsible for each oxidation performed on this terpene scaffold and the order in which they act are unknown. In this work, heterologous expression of the bnd cluster is used to identify the function of each P450. Plasmids bearing different combinations of bnd genes under the control of a strong constitutive Streptomyces promoter were constructed. The constructs were conjugated into heterologous Streptomyces host species to increase production of BND and biosynthetic pathway intermediates. After fermentation, heterologous host cultures were extracted, analyzed by LCMS, and purified for structural elucidation. Studying key enzymes in biosynthetic pathways deepens our understanding of how nature constructs complex structures and improves bioinformatic predictions of gene function. Such data can be utilized for genome mining of related enzymes and discovery of novel chemistries. Furthermore, comprehensive knowledge of enzyme function in NP biosynthesis has implications for the fields of biocatalysis, green chemistry, and medicinal chemistry. |