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

Abstract

White-rot fungi efficiently degrade wood lignin; however, the mechanisms involved remain largely unknown. Recently, a forward genetics approach to identify several genes in Pleurotus ostreatus (Agaricales) in which mutations cause defects in wood lignin degradation was used. For example, pex1 encodes a peroxisome biogenesis factor and gat1 encodes a putative Agaricomycetes-specific DNA-binding transcription factor. In this study, we examined the effects of single-gene mutations in pex1 or gat1 on wood lignin degradation in another white-rot fungus, Gelatoporia (Ceriporiopsis) subvermispora (Polyporales), to investigate conserved and derived degradation mechanisms in white-rot fungi. G. subvermispora pex1 and gat1 single-gene mutant strains were generated from a monokaryotic wild-type strain, FP-90031-Sp/1, using plasmid-based CRISPR/Cas9. As in P. ostreatus, Gsgat1 mutants were nearly unable to degrade lignin sourced from beech wood sawdust medium (BWS), while Gspex1 mutants exhibited a delay in lignin degradation. We also found that the transcripts of ligninmodifying enzyme-encoding genes, mnp4, mnp5, mnp6, mnp7, and mnp11, which predominantly accumulate in FP-90031-Sp/1 cultured with BWS, were greatly downregulated in Gsgat1 mutants. Taken together, the results suggest that Gat1 may be a conserved regulator of the ligninolytic system of white-rot fungi and that the contribution of peroxisomes to the ligninolytic system may differ among species.

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris

“…It was reported that in genome-edited strains of P. ostreatus and Gelatoporia subvermispora, , part of the plasmid fragment had been integrated into the target site. Currently, off-target events are often reduced by using computational tools and re-editing the CRISPR components such as Cas proteins and gRNA .…”

Experimental evidence that lignin‐modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9

“…Genomic DNA was extracted from each strain via the cetyl trimethyl ammonium bromide method as described by Muraguchi et al (2003) and Zolan and Pukkila (1986). The extracted DNA ($3 μg) was subject to Southern blot analysis as described by Nakazawa et al (2023). Restriction enzymes to digest genomic DNA and primers to amplify a genomic fragment for a probe were XhoI and TN1022/TN1023 (vp2), PstI and TN1422/TN1423 (vp3), HindIII and BamHI, and TN1426/TN1427 (mnp2), XhoI and TN1430/TN1431 (mnp3), XhoI and TN1424/TN1425 (mnp6), XhoI and TN1428/TN1429 (lac2), respectively.…”

A Molecular Genetics Approach to Elucidating the Mechanisms Underlying Lignin Degradation by White-Rot Fungi

“…Future studies are required to utilize this technique in functional analyses. Recently, we used a DNA (plasmid)-based method to conduct a functional analysis of genes involved in the sexual development of P. ostreatus 46) and lignin degradation by G. subvermispora 47) . In these cases, the introduced plasmid(s) were randomly integrated into the host chromosomes.…”