|
|||||
|
|
Experimental evidence that lignin-modifying enzymes are essential for degrading plant cell wall lignin by Pleurotus ostreatus using CRISPR/Cas9 |
|
| Authors: | Takehito Nakazawa Iori Yamaguchi Yufan Zhang Chinami Saka Hongli Wu Keita Kayama Moriyuki Kawauchi Masahiro Sakamoto Yoichi Honda |
| Affiliation: | 1. Graduate School of Agriculture, Kyoto University, Kyoto, Japan;2. Graduate School of Agriculture, Kyoto University, Kyoto, Japan Contribution: Formal analysis (lead), Investigation (lead), Methodology (equal), Writing - original draft (supporting);3. Graduate School of Agriculture, Kyoto University, Kyoto, Japan Contribution: Investigation (supporting), Methodology (equal);4. Graduate School of Agriculture, Kyoto University, Kyoto, Japan Contribution: Data curation (supporting), Formal analysis (supporting), Investigation (supporting);5. Graduate School of Agriculture, Kyoto University, Kyoto, Japan Contribution: Formal analysis (supporting), Investigation (supporting), Validation (supporting);6. Graduate School of Agriculture, Kyoto University, Kyoto, Japan Contribution: Formal analysis (supporting), Validation (supporting);7. Graduate School of Agriculture, Kyoto University, Kyoto, Japan Contribution: Writing - review & editing (supporting) |
| 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. |
| Keywords: | |