Experimental evidence that lignin‐modifying enzymes are essential for degrading plant cell wall lignin byPleurotus ostreatususingCRISPR/Cas9

Abstract

Lignin‐modifying enzymes (LMEs), which include laccases (Lacs), manganese peroxidases (MnPs), versatile peroxidases (VPs), and lignin peroxidases (LiPs), have been considered key factors in lignin degradation by white‐rot fungi because they oxidize lignin model compounds and depolymerize synthetic lignin in vitro. However, it remains unclear whether these enzymes are essential/important in the actual degradation of natural lignin in plant cell walls. To address this long‐standing issue, we examined the lignin‐degrading abilities of multiple mnp/vp/lac mutants of Pleurotus ostreatus. One vp2/vp3/mnp3/mnp6 quadruple‐gene mutant was generated from a monokaryotic wild‐type strain PC9 using plasmid‐based CRISPR/Cas9. Also, two vp2/vp3/mnp2/mnp3/mnp6, two vp2/vp3/mnp3/mnp6/lac2 quintuple‐gene mutants, and two vp2/vp3/mnp2/mnp3/mnp6/lac2 sextuple‐gene mutants were generated. The lignin‐degrading abilities of the sextuple and vp2/vp3/mnp2/mnp3/mnp6 quintuple‐gene mutants on the Beech wood sawdust medium reduced drastically, but not so much for those of the vp2/vp3/mnp3/mnp6/lac2 mutants and the quadruple mutant strain. The sextuple‐gene mutants also barely degraded lignin in Japanese Cedar wood sawdust and milled rice straw. Thus, this study presented evidence that the LMEs, especially MnPs and VPs, play a crucial role in the degradation of natural lignin by P. ostreatus for the first time.

Altered Expression of Two Small Secreted Proteins (ssp4 and ssp6) Affects the Degradation of a Natural Lignocellulosic Substrate by Pleurotus ostreatus

“…This is highly indicative of an adaptive, rather than inductive, response of the fungus to the growth niche. This concept, shown here along the three-week sampling period, is in high agreement with the recent analysis described by Nakazawa et al [38] and warrants a future emphasis on deciphering the kinetics of the substrate utilization process. In addition to the marked temporal changes observed in the measured transcriptional profiles, further analyses using WGCNA [43], which has been previously used to identify genes associated with sporulation processes in Ganoderma lingzhi [44] and for discovering the landmark genes involved in mushroom development and the evolution of complex multicellularity [45], we were able to identify key modules of genes highly associated with the ssp knock-down genotype (Figures 7 and 8).…”

“…Considering the recently proven redundancy of some components of the lignin-degrading machinery [37], it is likely that these are preferentially involved in lignin degradation under varying circumstances. Recently, using multiple mnp/vp/lac mutants of P. ostreatus, Nakazawa et al [38] demonstrated that MnPs and VPs have overlapping roles in the degradation of natural lignin.…”

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences

“…Multiple gene disruption with CRISPR/Cas9 may overcome this problem. For example, by creating a sextuple disruption strain for lignin-modifying enzyme (LME)-encoding genes, the crucial role of LMEs in lignin degradation by P. ostreatus was demonstrated (Nakazawa et al 2023b ; see below).…”

“…Additionally, one laccase-encoding gene, lac2 , was disrupted because its transcript accumulation was high when grown on beech wood sawdust medium supplemented with wheat bran (BWS; Wu et al 2020 ). Lignin-degrading abilities were mostly lost in the vp2 / vp3 / mnp3 / mnp6 / mnp2/lac2 sextuple-gene disruptants but not in the vp2 / vp3 / mnp3 / mnp6 / mnp2 and vp2 / vp3 / mnp3 / mnp6 / lac2 quintuple-gene disruptants grown on BWS, clearly showing their essential role (Nakazawa et al 2023b ). This is the first experimental evidence indicating that these LMEs play essential roles in wood lignin biodegradation by white-rot fungi.…”