Preliminary Studies On The Exploring Novel Lignin Degradation Mechanism With Distant Protoplast Fusion

Lignocellulose is the most abundant renewable resource on the earth.The depolymerization of lignin is the most critical step in the utilization of this resource.Lignin limits the accessibility of enzymes to cellulose and hemicellulose,resulting in reduced hydrolysis efficiency.Pretreatment is often carried out to remove or modify lignin to promote the decomposition of lignocellulose.Wood rot fungi can efficiently degrade lignin,and the reaction conditions are mild and pollution-free,which has attracted more and more people’s attention in pretreatment field.So far,it is generally believed that white-rot fungi use lignin-degrading enzymes to mineralize lignin,while brown-rot fungi use Fenton reaction to cleave lignocellulose.Recent reports mainly focus on the function and structure of important lignin degrading enzymes,but the existing lignin degrading enzymes cannot degrade lignin in in vitro reaction system.In addition,the investigation on the reaction mechanism of lignindegrading enzymes is mostly based on lignin model compounds rather than natural lignin.The immaturity of multi-gene knockout technology of filamentous fungi implies that the function of most of these enzymes cannot be validated by genetic means.Therefore,the existing mechanism research still needs to be improved,and there might also be new degradation mechanisms in these fungi.With the help of Pleurotus ostreatus and Saccharomyces cerevisiae distant protoplast fusion technology,a strain(D1-P)that does not contain the currently known lignin degrading enzymes of the same genus is obtained after solid culture with different carbon sources,enzyme activity determination and molecular verification,However,this strain can use corn stover to grow.The dry weight loss of lignin in corn straw treated by the strain was significantly different from that of the control group indicating that this strain can degrade ligin without the known enzymes.To futher prove this hypothesis and exploring novel mechanism,Nuclear magnetic resonance used to characterize the structure of the lignin degraded by D1-P.The nuclear magnetic resonance(NMR)spectrum showed a signal profile that does not exist in the degraded product by wild-type strain.There is no signal matching this chemical shift.This result indicates that D1-P might have a new lignin degradation mechanism.After semiquantitative integral analysis of different chemical bonds,it is speculated that the new degradation product is similar to dibenzodioxin,indicating that the fusant may use different mechanisms to degrade lignin.In order to further confirm that there is no classic lignin degrading enzyme gene in the fusion,we also verified this conclusion through genome scanning.This result will enrich the content of fungal lignin degradation mechanism and provide more information for a comprehensive understanding of fungal degradation of lignin.