Table_4_Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri.DOCX

Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

米卡芬净（Micafungin）是一类用于治疗侵袭性真菌感染的重要棘白菌素类抗真菌剂。工业生产中，米卡芬净源自天然产物FR901379——一种由丝状真菌埃特筒囊霉（Coleophoma empetri）合成的非核糖体环六肽。该菌株的遗传操作难度限制了FR901379生物合成机制的解析。本研究针对工业生产FR901379的菌株埃特筒囊霉（Coleophoma empetri）MEFC009，构建了一套高效的遗传操作体系。首先，开发了一种便捷的原生质体介导转化（protoplast-mediated transformation, PMT）方法。其次，借助该转化方法验证了核心遗传元件：筛选标记hph、neo与nat均可用于该转化体系，且启动子Ppgk、PgpdA及PgpdAt在埃特筒囊霉MEFC009中均具有功能活性。第三，通过敲除ku80基因，将同源重组效率从4%提升至100%，获得了适用于基因靶向操作的优良底盘细胞。此外，本研究通过鉴定负责二羟萘（dihydroxynapthalene, DHN）-黑色素生物合成的聚酮合酶（polyketide synthase, PKS），证实了该遗传操作体系的应用优势。这套遗传操作体系将成为研究FR901379生物合成以及进一步挖掘埃特筒囊霉次级代谢产物的实用研究平台。