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.