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

• Published in: Applied microbiology and biotechnology Vol. 91; no. 5; pp. 1389 - 1397
• Main Authors: Jung, Won Seok, Yoo, Young Ji, Park, Je Won, Park, Sung Ryeol, Han, Ah Reum, Ban, Yeon Hee, Kim, Eun Ji, Kim, Eunji, Yoon, Yeo Joon
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2011; Springer; Springer Nature B.V
• Subjects: Acyl Coenzyme A - genetics; Acyl Coenzyme A - metabolism; Analysis; Antifungal agents; Antitumor activity; Applied Genetics and Molecular Biotechnology; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biosynthesis; Biosynthetic Pathways - radiation effects; Biotechnology; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Bacterial - radiation effects; Genetic Engineering - methods; Genetic research; Life Sciences; Metabolism; Methylmalonyl-CoA Decarboxylase - genetics; Methylmalonyl-CoA Decarboxylase - metabolism; Methylmalonyl-CoA Mutase - genetics; Methylmalonyl-CoA Mutase - metabolism; Microbial Genetics and Genomics; Microbiology; Mutagenesis; Mutagenesis - radiation effects; Sirolimus - metabolism; Streptomyces - enzymology; Streptomyces - genetics; Streptomyces - metabolism; Streptomyces - radiation effects; Streptomyces hygroscopicus; Studies; Metabolic engineering; Rapamycin; Methylmalonyl-CoA; Propionyl-CoA carboxylase; Random mutagenesis; Carbon-carbon ligases; Streptomyces hygroscopicus; Streptomycetaceae; Sirolimus; Enzyme; Biosynthesis; Precursor; Optimization; Actinomycetales; Mutagenesis; Ligases; Bacteria; Actinomycetes
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3348-6

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.