Fermentation and Biotechnology Laboratories, Astellas Pharma Inc, 156 Nakagawara, Kiyosu, Aichi 452-0915, Japan.
J Biosci Bioeng. 2011 Oct;112(4):409-14. doi: 10.1016/j.jbiosc.2011.06.002. Epub 2011 Jul 12.
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.
米卡芬净（FK463）是一种广泛用于治疗危及生命的深部真菌感染的药物。它是一种棘白菌素样脂肽，由去酰化 FR901379 的化学修饰而来，FR901379 是一种由 Coleophoma empetri F-11899 产生的脂肽抗生素。FR901379 的棕榈酰部分由 FR901379 酰基酶通过链霉菌 sp. no.6907 产生的 FR901379 酰基酶进行去酰化。在这项研究中，我们的目标是生成一种能够超量产生 FR901379 酰基酶的改良链霉菌 sp. no.6907 菌株。为了实现这一目标，使用紫外线诱变对链霉菌 sp. no.6907 进行了修饰菌株的生成，并使用琼脂平板筛选方法进行菌株选择，以有效地选择出一种产酰基酶的高产菌株。三个标记指数与提高的酰基酶产量相关：FR901379 的杀菌活性降低、脱脂牛奶的蛋白水解活性降低和表型特征。从高产突变体中克隆并随后测序酰基酶基因，结果显示该基因在结构基因或基因表达所需的 5' 侧翼区都没有突变。还对生长培养基进行了修改，以最大限度地提高酰基酶的产量。与原始生长条件（在原始未修饰培养基中培养的野生菌株）相比，我们成功地将酰基酶活性提高了约 65 倍。为了在发酵过程中最大限度地减少过量泡沫的形成，我们优化了搅拌速度参数，该参数是根据排出流量计算得出的。使用我们改进的菌株和优化的培养基和生长条件，我们开发了一种改进的、高度可重复的 FR901379 酰基酶稳定大规模生产方法。
