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

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

doi:10.3389/fmicb.2021.734780

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.

Highlights

Echinocandins are a family of cyclic lipohexapeptides with the excellent activity of anti-Candida and anti-Aspergillus, which are considered to be the most potential antifungal agents clinically (Denning, 2003; Patil and Majumdar, 2017)
The transformants harboring PgpdA, PgpdAt, and Ppgk evaluation cassettes displayed significant green fluorescent, while the parental strain did not show any visible fluorescence (Figure 4). These results demonstrated that promoters PgpdA, PgpdAt, and Ppgk successfully drove sgfp expression in C. empetri MEFC009
C. empetri was applied in the industrial production of FR901379, the precursor compound of antifungal agent micafungin

Summary

Introduction

Echinocandins are a family of cyclic lipohexapeptides with the excellent activity of anti-Candida and anti-Aspergillus, which are considered to be the most potential antifungal agents clinically (Denning, 2003; Patil and Majumdar, 2017). Three semisynthetic echinocandins, caspofungin, micafungin, and anidulafungin have been approved for the treatment of invasive fungal infections (IFIs). Genetic Manipulation for Coleophoma empetri (Hashimoto, 2009). The biosynthetic pathway of the lead compounds of caspofungin and anidulafungin have been identified in 2012 and 2013, respectively (Cacho et al, 2012; Chen et al, 2013; Hüttel, 2017). Compound FR901379 produced by filamentous fungi Coleophoma empetri is the lead compound of micafungin. The biosynthesis pathway of FR901379 is still cryptic, because of the difficulty of genetic manipulation of C. empetri. In 2009, Masato Yamada developed Agrobacterium tumefaciens-mediated transformation method (ATMT) in the C. empetri F11899, only the fragment of hygromycin B resistant gene was successfully integrated into the genome by random insertion (Yamada et al, 2009). It is still necessary to explore more effective transformation methods and available genetic elements

Methods

Results

Conclusion