Strain selection and scale-up fermentation for FR901379 acylase production by Streptomyces sp. no. 6907(METHOD)

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概要

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• Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandinlike lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5'-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.

著者

• Isogai Yasuhiro

  Department Of Biotechnology Toyama Prefectural University

• Hashimoto Michizane

  Exploratory Research Laboratories Fujisawa Pharmaceutical Co. Ltd.

• Tsuboi Masaru

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.

• Shimizu Shiho

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.

• Yamada Masato

  Department Of Biotechnology Toyama Prefectural University

• Hashimoto Seiji

  Department Of Nephrology Division Sapporo General City Hospital

• Hino Motohiro

  Exploratory Research Laboratories Fujisawa Pharmaceutical Co. Ltd.

• Hashimoto Seiji

  Department Of Biotechnology Toyama Prefectural University

• Hino Motohiro

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.

• Tanaka Fumihiro

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.

• Yamada Masato

  Biotechnology Research Center Faculty Of Engineering Toyama Prefectural University

• Ueda Satoshi

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.

• Kinoshita Masayoshi

  Fermentation and Biotechnology Laboratories, Astellas Pharma Inc.

• Oohata Nobutaka

  Fermentation and Biotechnology Laboratories, Astellas Pharma Inc.

• Isogai Yasuhiro

  Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University

• Hashimoto Seiji

  Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University

• Isogai Yasuhiro

  Biotechnology Research Center Faculty Of Engineering Toyama Prefectural University

• Kinoshita Masayoshi

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.

• Shimizu Shiho

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.:(present Office)project And Quality

• Tsuboi Masaru

  Fermentation And Biotechnology Laboratories Astellas Pharma Inc.:(present Office)toyama Technology C

• Hashimoto Seiji

  Biotechnology Research Center Faculty Of Engineering Toyama Prefectural University

• Ueda Satoshi

  Fermentation And Biotechnology Laboratories

• Oohata Nobutaka

  Fermentation And Biotechnology Laboratories

• Hashimoto Seiji

  Exploratory Research Laboratories Fujisawa Pharmaceutical Co. Ltd.

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