Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

Ping Men; Min Wang; Jinda Li; Ce Geng; Xuenian Huang; Xuefeng Lu

doi:10.3389/fmicb.2021.734780

Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

Front Microbiol. 2021 Aug 20:12:734780. doi: 10.3389/fmicb.2021.734780. eCollection 2021.

Authors

Ping Men^{1

2

3

4}, Min Wang^{1

2

3}, Jinda Li^{1

2

3}, Ce Geng^{1

2

3}, Xuenian Huang^{1

2

3}, Xuefeng Lu^{1

2

3

4

5

6}

Affiliations

¹ Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
² Shandong Energy Institute, Qingdao, China.
³ Qingdao New Energy Shandong Laboratory, Qingdao, China.
⁴ College of Life Science, University of Chinese Academy of Sciences, Beijing, China.
⁵ Marine Biology and Biotechnology Laboratory, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
⁶ Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.

Abstract

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.

Keywords: Coleophoma empetri; marker; melanin biosynthesis; micafungin; nonhomologous end-joining; promoter; protoplast transformation.