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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

  • Applied Genetics and Molecular Biotechnology
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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.

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Acknowledgments

This work was supported by the National Research Laboratory (NRL) program (R0A-2008-000-20030-0) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST), the Advanced Biomass R&D Center (ABC) funded by MEST (ABC-2010-0029800), and CKD Bio Advanced Technology Center funded by the Ministry of Knowledge Economy, Republic of Korea.

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Authors and Affiliations

  1. Department of Chemistry and Nano Science, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul, 120-750, South Korea

    Won Seok Jung, Young Ji Yoo, Sung Ryeol Park, Ah Reum Han, Yeon Hee Ban, Eun Ji Kim, Eunji Kim & Yeo Joon Yoon

  2. Department of Pharmaceutical Engineering, Sun Moon University, Chungnam, 336-708, South Korea

    Je Won Park

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  1. Won Seok Jung
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  2. Young Ji Yoo
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Correspondence to Yeo Joon Yoon.

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Won Seok Jung and Young Ji Yoo contributed equally to the work.

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Jung, W.S., Yoo, Y.J., Park, J.W. et al. 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. Appl Microbiol Biotechnol 91, 1389–1397 (2011). https://doi.org/10.1007/s00253-011-3348-6

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