Precursor Supply and Polyketide Antibiotic Biosynthesis in Oil-based Industrial Fermentations of Streptomyces Cinnamonensis

Author

Chaoxuan Li, Virginia Commonwealth University

Defense Date

2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Medicinal Chemistry

First Advisor

Dr. Kevin A. Reynolds

Abstract

Polyketides are a group of bioactive natural products synthesized by bacteria, fungi and plants with various acyl-CoA precursors, such as malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA. A sufficient supply of these precursors is a prerequisite for the high level production of polyketide products. A thorough understanding of relative roles of various metabolic pathways involved in precursor supply makes increased production by genetical manipulation, and thus rational strain improvement, a reality. Monensin A is a polyketide antibiotic assembled from one ethylmalonyl-CoA, seven methylmalonyl-CoA and five malonyl-CoA molecules by Streptomyces cinnamonensis. In the present work, the origin of these biosynthetic precursors was investigated using an industrially mutagenized monensin producer and industrial fermentation conditions. A hitherto disregarded metabolic pathway was discovered to play a significant role in providing methylmalonyl-CoA for monensin biosynthesis by gene disruption, isotope-labeling of monensin and analysis of in vivo acyl-CoA pools. This pathway starts from biosynthesis of butyryl-CoA from two molecules of acetyl-CoA, and goes through the intermediate of isobutyryl-CoA, and finally produces methylmalonyl-CoA by direct oxidation of the pro-S methyl group of isobutyryl-CoA.Industrial fermentation of the industrially mutagenized monensin producer yields significantly more monensin than the routine laboratory fermentation. This suggested the presence of abundant in vivo malonyl-CoA and methylmalonyl-CoA in this process and presented an opportunity to utilize it as a biological system for the high-titer production of heterologous polyketides derived from malonyl-CoA and/or methylmalonyl-CoA. The tetracenomycin C polyketide synthase (PKS) synthesizes tetracenomycin C, a polyketide with ten molecules of malonyl-CoA. In this work, the tetracenomycin C PKS gene cluster was introduced into two industrially mutagenized strains of Streptomyces cinnamonensis. Unprecedented multi-gram/liter of tetracenomycin production was observed in the resulting two strains, indicating the high potential of industrially mutagenized monensin production strains as efficient hosts for the production of malonyl-CoA-derived polyketides. For additional improvement in tetracenomycin yield, we attempted to increase malonyl-CoA supply to tetracenomycin C PKS by genetically manipulating metabolic pathways affecting production of malonyl-CoA and eliminating competition from monensin PKS for malonyl-CoA. However, only decreased tetracenomycin production was observed, demonstrating that the regulation of malonyl-CoA-related metabolic pathways is a complex process.

Comments

Part of Retrospective ETD Collection, restricted to VCU only.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

June 2008