Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora

Abstract

A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl 2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase ( hph ) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene ( gpd ) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.

A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum

“…4C ). Similar to the previous research in white rot fungi ( 22 , 42 ), it is not surprising that in our work these three promoters showed strong and similar strength. However, it has been challenging to accurately quantify their strength due to the variable expression levels among transformants.…”

The lignin‐degrading abilities of Gelatoporia subvermispora gat1 and pex1 mutants generated via CRISPR/Cas9

“…In this study, the strains were always grown at 28°C under continuous darkness. Finally, the hygromycin resistance transformation of G. subvermispora was carried out as described by Honda et al (2019) and Nguyen et al (2020).…”

Endophytes in Lignin Valorization: A Novel Approach

“…For example, Heterobasidium annosum ( Maijala et al, 2003 ), Phlebia spp. ( Arora and Sharma, 2009 ), Physisporinus rivulosus ( Hilden et al, 2007 ), Irpex lacteus ( Duan et al, 2018 ), Dichomitus squalens ( Marinović et al, 2018 ), Phanerochaete chrysosporium , Pleurotus ostreatus ( Kerem et al, 1992 ), Ceriporiopsis subvermispora ( Honda et al, 2019 ), and Trametes versicolor ( Bari et al, 2019 ). In comparison to fungal studies, research on potential of bacterial lignin deconstruction is scarce.…”