Optimization of Protoplast Preparation and Establishment of Genetic Transformation System of an Arctic-Derived Fungus Eutypella sp.

Abstract

Arctic-derived fungus Eutypella sp. D-1 has attracted wide attention due to its huge ability to synthesize secondary metabolites. However, current studies only focus on stimulating its production of new secondary metabolites by OSMAC strategies, and the relationship between secondary metabolites and biosynthetic gene clusters (BGCs) has not been explored. In this study, the preparation and regeneration conditions of Eutypella sp. D-1 protoplasts were explored to lay a foundation for the study of genetic transformation of this fungus. Orthogonal experiment showed that the optimal preparation conditions were 0.75 M NaCl, 20 g/L of lysing enzyme, and 20 g/L of driselase, 28°C for 6 h. The maximum yield of Eutypella sp. D-1 protoplasts could reach 6.15 × 106 cells·ml−1, and the concentration of osmotic stabilizer NaCl was the most important factor for Eutypella sp. D-1 protoplasts. The results of FDA staining showed that the prepared protoplasts had good activity. Besides, the best protoplasts regeneration medium was YEPS, whose maximum regeneration rate is 36%. The mediums with nitrogen sources, such as SR and RM, also had good effects on the Eutypella sp. D-1 protoplast regeneration, indicating that nitrogen sources played an important role on the Eutypella sp. D-1 protoplast regeneration. Subsequent transformation experiments showed that hygromycin resistance genes (hrg) could be successfully transferred into the genome of Eutypella sp. D-1, indicating that the prepared protoplasts could meet the needs of subsequent gene manipulation and research. This study lays a foundation for the genetic transformation of Eutypella sp. D-1.

CRISPR/Cas9 RNP-assisted validation of palmarumycin biosynthetic gene cluster in Lophiotrema sp. F6932

“…Thus, we used several existing protoplast isolation methods as the basis for developing a simple yet efficient method for protoplast generation from Lophiotrema sp. F6932 ( Abdallah et al, 2017 ; Roth and Chilvers, 2019 ; Ning et al, 2022 ). Preliminary studies had shown that potato dextrose broth (PDB) was the best media for generating mycelia for protoplast isolation from this fungus compared with two other media; malt extract broth and Sabouraud dextrose broth ( Supplementary Figure S5 ).…”

“…Moreover, selection of an appropriate type and concentration of an osmotic stabilizer in the protoplasting medium is critical in preventing lysis or shriveling of protoplast due to osmotic stress ( Ren et al, 2018 ; De Wu and Chou, 2019 ). Other than the enzymes and osmotic stabilizers, other factors such as enzymatic hydrolysis temperature, duration of incubation, fungal age and mycelium status need to be optimized for each strain ( Jin et al, 2020 ; Ning et al, 2022 ). For example, depending on whether the fungal strain is slow or fast-growing, very short culture time may yield less mycelium and thus low yield of protoplast.…”

“…For example, depending on whether the fungal strain is slow or fast-growing, very short culture time may yield less mycelium and thus low yield of protoplast. On the other hand, prolonged culture time may result in aged mycelia whose thick cell wall may be resistant to enzymatic hydrolysis ( Wei et al, 2012 ; Ning et al, 2022 ).…”

“…F6932. Therefore, through the optimization of various growth parameters and the used of different types and concentrations of commercial enzymes and osmotic stabilizers, and borrowing from existing protoplast isolation protocols ( Abdallah et al, 2017 ; Roth and Chilvers, 2019 ; Ning et al, 2022 ), we designed a simple yet efficient method for protoplast isolation from Lophiotrema sp. F6932.…”

“…Generally, different fungal strains will vary in their sensitivity to different enzymes because of the differences in the cell wall composition and structure ( Jin et al, 2020 ). Therefore, depending on the nature of the fungus cell wall, different cell-wall digesting enzymes will act on different sites of the cell wall, resulting in variations in the level of cell wall hydrolysis and consequently the quantity of protoplasts released ( Ning et al, 2022 ). Thus, the observed difference in the yield of protoplasts in the current study is indicative of differences in the sensitivity of Lophiotrema sp.…”