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