The Functional Analysis Of The Cleavage Polysaccharide Monooxygenase Family In Podospora Anserina In The Process Of Degradation Of Lignocellulose

Lignocellulose is a renewable resource that is extremely abundant.Degradation of lingocellulose into monosaccharides is the precondition for the production of biofuels and biobased chemicals.Lytic polysaccharide monooxygenases(LPMOs)are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism,making them interesting for the production of biofuel from cellulose.In this study,four degraded polysaccharide monooxygenase genes were knocked out by deletion of target genes,and their functions,effects on the growth and development of filamentous fungi Podospora anserina,and the degradation of lignocellulose and other materials were investigated.In this paper,the phylogenetic tree of LPMO was firstly constructed,and the homozygous recombination method was used to obtain P.anserina lytic polysaccharide monooxygenase gene deletion mutants.Two 1000 bp regions located upstream and downstream of the glucoamylase sequence were amplified from P.anserina genomic DNA.At the same time,the corresponding resistance marker genes were amplified from the plasmids pBC-Hygromycin,pBC-Phleomycin,and pBC-Geneticin,and gene knockout cassettes were constructed by fusion PCR,and transformants with resistance genes were obtained by transformation of protoplasts.Transformants were selected on medium containing the corresponding antibiotic concentration and screened for positive transformants further by PCR and Southern.Blot The resulting single mutants were then subjected to a pairwise hybridization using genetic cross to obtain multiple mutants.Phenotypic analysis of mutants of P.anserina lytic polysaccharide monooxygenase gene and its mutants,growth status comparison,identification of carboxymethylcellulose sodium enzyme activity,growth on lignocellulosic material Situation,growth conditions when xylan and xylose were used as sole carbon sources,and analysis of oxidative stress.According to the LPMO phylogenetic tree analysis,the relationship between P.anserina and N.crassa is most closely related to other fungi.From the comparison between the wildtype and the mutants,due to the deletion of the degraded polysaccharide monooxygenase gene,the enzyme activity decreased to some extent in all mutants compared to the wild type,indicating that LPMO was actually involved in theFracture of ?-1,4--glycosidic bond.When grown on standard M2 medium,the colony size of the mutant strain was smaller than that of the wild type,and the ascospora produced by the mutants was smaller and the number was less.The life cycle of all mutants is almost a half-day to two-day delay.When growing on different lignocellulosic materials as substrates,the number of ascospora of the mutants was reduced compared to the wild type,while the disperse of mature ascospores was delayed.When substrates such as cellulose,xylan,and xylose are grown as carbon sources,a mature ascaria ring is formed at the center of the cellulosic and xylan dishes,but the growth is slower on xylose,the time for the ascospores to disperse is delayed by about 4 days,while the mutants produce fewer mature fruiting bodies.Mutants are more sensitive to changes in oxidative stress than wild-type.In the paper,lytic polysaccharide monooxygenase geneswere deleted and mutant strains were obtained.The lpmo genes were knocked out,the growth of P.anserina was significantly affected,the diameter of the colony was reduced,the perithecium became smaller,and the number also decreased significantly.The life cycle of some mutant strains was delayed,Therefore,its ability to degrade lignocellulose and other materials also decreased.