A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis> ATCC 29253

Rapamycin is a macrocyclic polyketide with immunosuppressive, antifungal, and anticancer activity produced by Streptomyces hygroscopicus ATCC 29253. Rapamycin production by a mutant strain (UV2-2) induced by ultraviolet mutagenesis was improved by approximately 3.2-fold (23.6 mg/l) compared to that of the wild-type strain. The comparative analyses of gene expression and intracellular acyl-CoA pools between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes, but a depletion of intracellular methylmalonyl-CoA limited the rapamycin biosynthesis of the UV2-2 strain. Therefore, three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of rapamycin: propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase, and methylmalonyl-CoA ligase. Among them, only the PCC pathway along with supplementation of propionate was found to be effective for an increase in intracellular pool of methylmalonyl-CoA and rapamycin titers in UV2-2 strain (42.8 mg/l), indicating that the PCC pathway is a major methylmalonyl-CoA supply pathway in the rapamycin producer. These results demonstrated that the combined approach involving traditional mutagenesis and metabolic engineering could be successfully applied to the diagnosis of yield-limiting factors and the enhanced production of industrially and clinically important polyketide compounds.