title: Strain selection and scale-up fermentation for FR901379 acylase production by Streptomyces sp. no. 6907.
abstract: Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like 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.
authors.affiliation: Fermentation and Biotechnology Laboratories, Astellas Pharma Inc, 156 Nakagawara, Kiyosu, Aichi 452-0915, Japan. satoshi-ueda@jp.astellas.com
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ueda
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Kinoshita
authors.forename: Fumihiro
authors.initials: F
authors.lastname: Tanaka
authors.forename: Masaru
authors.initials: M
authors.lastname: Tsuboi
authors.forename: Shiho
authors.initials: S
authors.lastname: Shimizu
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Oohata
authors.forename: Motohiro
authors.initials: M
authors.lastname: Hino
authors.forename: Masato
authors.initials: M
authors.lastname: Yamada
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Isogai
authors.forename: Seiji
authors.initials: S
authors.lastname: Hashimoto
mesh_terms: D000581:Amidohydrolases / Q000096:biosynthesis* / Q000378:metabolism; D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D054714:Echinocandins / Q000737:chemistry / Q000378:metabolism; D005285:Fermentation; D055666:Lipopeptides / Q000737:chemistry / Q000378:metabolism; D000077551:Micafungin; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D013302:Streptomyces / Q000145:classification / Q000201:enzymology*
chemical_list: D000935:Antifungal Agents; D003470:Culture Media; D054714:Echinocandins; C110368:FR 901379; D055666:Lipopeptides; D010456:Peptides, Cyclic; D000581:Amidohydrolases; C019665:amidase; D000077551:Micafungin