Research Progress in Area 4 方向 ( 四 ) 課題進展 175 New Bioactive Diterpenoid Production via Marine Microbial Factories Prof. Wenjun Zhang University of California, Berkeley Abstract Diterpenoids are an important family of pharmaceutical compounds known to mankind, but their limited and unsustainable supply as well as challenging organic synthesis impede the diterpenoid-based new drug discovery. This project aims to develop and implement a novel platform for new diterpenoid production using microbial fermentation, with an ultimate goal of discovery of new bioactive natural products. The central principle of this new platform is to supply marine microbial cultures with a diterpenoid scaffold of interest and screen for the microbe(s) that can efficiently convert the fed precursor to a substantially modified final product. The enabling technology is the engineered biosynthesis of an isotopically labeled diterpenoid scaffold of interest, which facilitates the molecular tracking and modified metabolite identification in microbial cultures via mass spectrometry analysis. In addition, the scaffold can be selected to maximize the possibility of producing bioactive compounds and design the relevant bioassays based on the known activity of natural compounds derived from this scaffold. Research Activities and Progress • Purified isotopically labelled diterpenoid scaffold using mass spectrometry-based HPLC; • Performed an initial validation trial of 7 marine strains and controls in order to parameterize high-throughput platform design; • Performed an initial screening trial of 40 marine strains. Key Findings • ~1 mg of purified scaffold compound was purified from ~10 L of cell culture using XAD4 resin, LH20 size exclusion chromatography and RP-HPLC; • Validation experiments established key parameters such as microbial growth time, compound supplementation concentration, organic extraction method, and scale of cultures needed for LC-MS analysis; • Initial screening trial illustrated consumption and derivatization of diterpenoid scaffold, however mostly of masses smaller than the scaffold; • Poor extraction efficiency may explain absence of larger derivatized scaffold products. Research Output Publication 1 Trained personnel 2 Fig.1 Screening of engineered labeled diterpenoid scaffold through marine library
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