title: A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in Streptomyces hygroscopicus ATCC 29253.
abstract: 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.
authors.affiliation: Department of Chemistry and Nano Science, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, South Korea.
authors.forename: Won Seok
authors.initials: WS
authors.lastname: Jung
authors.forename: Young Ji
authors.initials: YJ
authors.lastname: Yoo
authors.forename: Je Won
authors.initials: JW
authors.lastname: Park
authors.forename: Sung Ryeol
authors.initials: SR
authors.lastname: Park
authors.forename: Ah Reum
authors.initials: AR
authors.lastname: Han
authors.forename: Yeon Hee
authors.initials: YH
authors.lastname: Ban
authors.forename: Eun Ji
authors.initials: EJ
authors.lastname: Kim
authors.forename: Eunji
authors.initials: E
authors.lastname: Kim
authors.forename: Yeo Joon
authors.initials: YJ
authors.lastname: Yoon
mesh_terms: D000214:Acyl Coenzyme A / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000528:radiation effects; D015964:Gene Expression Regulation, Bacterial / Q000528:radiation effects; D005818:Genetic Engineering / Q000379:methods*; D043586:Methylmalonyl-CoA Decarboxylase / Q000235:genetics / Q000378:metabolism; D008765:Methylmalonyl-CoA Mutase / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis / Q000528:radiation effects; D020123:Sirolimus / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; C015357:methylmalonyl-coenzyme A; D008765:Methylmalonyl-CoA Mutase; D043586:Methylmalonyl-CoA Decarboxylase; D020123:Sirolimus