Construction Of ΔLg-cre2 Mutant Strain And Lg-CRE2 Functional Study Of Lenzites Gibbosa

Lenzites gibbosa is an important white rot fungus that can effectively degrade and utilize lignocellulose.Carbon catabolite repression(CCR)is a process which organisms preferentially use simple carbon sources and this phenomenon widespread in nature.It has been shown that in yeast and filamentous ascomycetes the carbon catabolite repressor CRE1 has a role in repressing holocellulase gene expression,and CRE2 is deubiquitinated and forms a complex with CRE3 that stabilizes ubiquitin-labeled CRE1.The relationship and function of CRE2 with CRE1 in white rot fungus has been little studied.This study was conducted to investigate the mechanism of CCR in L.gibbosa and to gain a deep understanding of the carbon catabolite process and the mechanism of white rot in wood.In this study,based on the cloning and bioinformatics analysis of Lg-cre2 and Lg-cre3 genes,molecular docking was used to analyze the binding effect of both;ΔLg-cre2 mutant strains were obtained by homologous recombination to verify the function of Lg-CRE2.The above studies aimed to reveal the mechanism of CCR in white rot fungus.The results of the study were as follows:1.Gene cloning and bioinformatics analysis of Lg-cre2 and Lg-cre3.Gene cloning results showed that Lg-cre2 was 3036 bp and Lg-cre3 was 2721 bp.Sequence analysis of Lg-CRE2 protein compared to C19 peptidase with deubiquitinase action.The Lg-CRE2 protein has four highly conserved motifs,DH-II,DH-III,DH-IV and DH-V,located at the α-helix structure.And five WD40 structural units predicted in the Lg-CRE3 protein sequence.Molecular docking simulations show that Lg-CRE2 and Lg-CRE3 can form stable complexes.2.Thin-walled hyphae of L.gibbosa were enzymatically digested to obtain protoplasts.When L.gibbosa mycelium was treated with 1.5% lyticase,a small amount of spherical protoplasts appeared at the tip of the mycelium at 1 h,and the diameter of protoplasts was larger than the width of the mycelium;when the treatment was 2 h,the mycelium was fragmented,and the number of protoplasts increased and arranged in a string;when the treatment was 3 h,the number of protoplasts increased significantly,and the state was rounded and full,and distributed.3.Vector construction and transformation of ΔLg-cre2 mutant strain.The p KOV-Δcre2::hpt knockout plasmid was used to obtain 11 transformants after homologous recombination.PCR results showed that transformants No.4,6,10 and 11 were positive knockout strains,and based on their growth on resistance plates,transformant No.4 was finally selected as the ΔLg-cre2 knockout strain for subsequent tests.4.Growth phenotypic studies of the ΔLg-cre2 mutant strain.The growth rate of ΔLg-cre2 knockout strain was slightly slower than that of wild-type strain at the beginning of germination on normal LNAS plates,but the difference was not significant.The colonies of the ΔLg-cre2 knockout mutant strain were white fluffy or cotton wool-like in texture,with radial colony edges,sparse mycelial density and lack of flocculent aerial mycelium.The microstructure was septate mycelium with a clamp connection and a few chlamydospores,and the apical branches of mycelium were few and single.5.Studies on growth phenotypes and cellulase activity under different carbon source treatments.The degradation of soluble starch and lignin by the knockout strain was significantly increased on the plates under different carbon sources treatment.The content of secreted protein and cellulase activity of the ΔLg-cre2 mutant strain increased in the liquid medium fermentation broth,but not significantly.However,the content of secreted protein and cellulase activity was significantly increased in both wild-type and ΔLg-cre2 mutant strains under complex carbon source treatment.6.Functional studies on the regulation of key genes expression by Lg-CRE2.The expression of Lg-cre2 in ΔLg-cre2 mutant strain decreased compared with the wild type under different carbon source treatments,but the expression of Lg-cre1 and Lg-cre3 genes increased,and the expression of cellulase genes was also significantly increased.Comparing the expression of key genes between simple and complex carbon sources,complex carbon sources induced a significant increase in key genes except for Lg-cre2.In summary,the Lg-cre2 gene of L.gibbosa influences cellulase genes expression by affecting the expression of CRE regulators.ΔLg-cre2 mutant strain significantly increased the degradation of lignocellulose with a decarbon catabolite repression effect while having less effect on the growth phenotype of the organism.