Study On Molecular Mechanism And Key Genes Identification Of Trametes Gibbosa CB-1 On Lignin

Lignin is an amorphous polymer composed of phenylpropane units connected by carbon-carbon and ether bonds.Lignin is the third largest biomass resource after cellulose and chitin in nature.Microbial degradation of wood can effectively avoid environmental pollution caused by chemicals.White rot fungi have been widely studied as the most effective microorganisms to degrade wood.Trametes gibbosa is a white rot fungus widely distributed in temperate and cold temperate regions.It has fast growth and strong wood decomposition ability.Additionally,the lignocellulose decomposing enzyme system is composed of laccase,manganese peroxidase and a series of synthetic cellulases.In this study,FTIR and GC-MS were used to study the process of lignin degradation,and the transcriptome and proteome were used to analyse the mechanism of lignin degradation in T.gibbosa and identify key genes.The main results are as follows:1.Analysis of Lignin degradation and depolymerization products by T.gibbosa.Lignin inhibited mycelium growth.After 10 days,lignin promoted the growth of mycelium,and the mycelium increased greatly and reached 1.00 g/L in 30 days.After 30 d,manganese peroxidase increased steadily by 9.44 times.The activities of lignin peroxidase and laccase increased in the early stage,peaked at 25 days,and then began to decrease.The total protein content of fungi increased with time,peaked at 25 days,and then began to decrease.The change trend was consistent with that of laccase but opposite to that of H₂O₂ content.The changes in total protein and H₂O₂ content were mainly affected by the oxidation process of lignin degraded by T.gibbosa.It was inferred that T.gibbosa attacked phenylpropane polymers and the side chain structure of benzene rings,and cleaved it into low molecular weight aromatic compounds in the early stage by FTIR and GC-MS.After 10 days,a large number of new functional groups were formed,and intermediate degradation products such as phenol,2,6-dimethoxyhydroquinone,butylsyringone,tyrosol,3,4-dimethoxybenzyl alcohol,benzoic acid,and phenylacetic acid were obtained.After 20 days,the side chains of low molecular weight aromatic compounds were oxidized,and long chain or cyclic carboxylic acids were formed to form hexadecanoic acid.Additionally,the absorption peak in the vibration region of the benzene ring skeleton becomes complex,and the structure of some benzene rings changes.T.gibbosa attacks the side chain structure of the benzene ring to depolymerize,and then the side chain is oxidized to low molecular weight compounds,which cleaves the benzene ring through the intracellular pathway and degrades fatty acids,alcohols and phenols at the same time.2.Study on molecular mechanism of lignin degradation by T.gibbosa.Lignin and T.gibbosa were cocultured for 25 days without lignin as the control group.The combined analysis of the proteome and transcriptome showed that most of the upregulated DEGs and DEPs were enriched in redox processes and carbohydrate metabolism processes.KEGG enrichment analysis showed that the expression of genes related to amino acid synthesis and metabolism decreased,while that of fatty acid degradation and carbohydrate-related metabolic pathways increased.Lignin promoted glucose metabolism.In response to the degradation of lignin,T.gibbosa produced oxaloacetic acid and acetic acid,however,the TCA cycle was generally inhibited.The genes of laccase,ethanol oxidase,pyranose oxidase,oligosaccharide oxidase,xylan,galactose and glycosyl hydrolase related to glucuronic acid and mannose were upregulated.Three new intracellular lignin degradation pathways of salicylic acid,pyrogallol and phenol were discovered.Catechol andβ-ketoadipate are produced by dioxygenase and monooxygenase,and then succinic acid,acetyl-Co A and succinate-coenzyme enter the TCA cycle.T.gibbosa responded to lignin degradation at different time points.Compared with 10days,the DEGs were enriched in biological processes and metabolic pathways such as cell growth and death,homologous recombination and DNA repair at 15 days.Due to the stimulation of lignin,the expression of fungal multidrug resistance protein genes increased,fungal mitosis was inhibited,autophagy intensified,and methylation abnormalities occurred.After 20 days,cell membrane movement was active,including sterol oxidase,heavy metal translocation protein and drug transporter,and the gene expression of cationic transmembrane transporters was significantly upregulated.On the 25th day,the differentially expressed genes were mainly enriched in the process of lignin metabolism and the biological process of oxidoreductase activity acting on diphenols and related substances as donors.The KEGG pathway mainly focuses on metabolic processes such as peroxisome（ko04146）,secondary metabolite biosynthesis（ko01110）,carbon metabolism（ko01200）,fatty acid degradation（ko00071）,ubiquinone and other terpenoid biosynthesis（ko00130）.The laccase genes lcc3 and lcc4,phenol 2-monooxygenase,3-hydroxybenzoate-6-hydroxylase,oxalate decarboxylase and acyl coenzyme A oxidase were significantly upregulated.After 30 days,the N-glycan biosynthesis pathway was significantly enriched in glycan biosynthesis and metabolism.At this stage,the utilization of polysaccharides and polysaccharides increased,and carboxylic acid metabolism increased significantly.The weighted correlation network analysis was constructed by combining 15 sample data.A total of 1452 genes were clustered in the coral1 module,which were most related to lignin degradation.GO annotation analysis showed that the genes were significantly enriched in oxidoreductase activity,peptidase activity,cell response to stimulation,signal transduction,lignin metabolism and phenylpropanoid metabolism,while the rest were concentrated in glucose metabolism.The core genes of the regulatory network of modular genes include two b ZIP genes（gene＿7794,gene＿6673）that are highly linked to two GROES proteins,one WD40 repeat protein and one disulfide isomerase.These genes are all related to protein folding.3.Identification and functional analysis of laccase gene Tglcc1.The length of the coding region of the Tglcc1 gene was 1563 bp,which contained two conserved domains,Cu RO＿3 and Cu RO＿1.The prokaryotic expression vector p ET-32a-lcc1was constructed and transformed into E.coli BL21.The Tg LCC1 protein was successfully expressed by IPTG induction.The overexpression vector p CAMBIA1301-gpd-lcc1 was constructed and transformed into A.tumefaciens LBA4404.Fifteen micrograms/m L was selected as the initial screening concentration of hygromycin,and 35μg/m L was selected as the second screening concentration.The best transformant,λlcc1-1,was obtained,in which gene expression was 2.84 times higher than that of the wild type.The density of mycelium was higher than that of the wild type,and the fibre layer increased.The laccase activity ofλlcc1-1was 3 times higher than that of the wild type,and the content of reactive oxygen species was0.96 of that of the wild type.After being cultured in LNAS medium containing lignin for 25days,the lignin content（10.80 mg/m L）ofλlcc1-1 fermentation broth was 0.5 of that of wild type（19.58 mg/m L）.The morphological mycelium of the mutant was dense,the degradation rate of lignin was increased,and the oxidation of aromatic compounds was accelerated.4.Identification and functional analysis of b ZIP transcription factor Tgb ZIP73The length of the coding region of the Tgb ZIP73 gene is 1680 bp,which contains a conserved b ZIP＿u1 domain.The overexpression vector p CAMBIA1301-gpd-Tgb ZIP73 was constructed and transferred into wild-type T.gibbosa mediated by A.tumefaciens.The expression ofλTgb ZIP73-2 was 2.21 times higher than that of the wild type.From the genomic alignment of Tgb ZIP73,the upstream segment（left arm）of 1880 bp and the downstream segment（right arm）of 1251 bp were selected as homologous arms.An expression vector for p KOV21-73U-73D inhibition was constructed.Protoplasts were prepared by lysozyme.High-quality protoplasts were obtained by enzymatic hydrolysis for 3-3.5 h.After linearizing the vector,PEG mediated protoplast transformation.After screening,the gene expression of the best transformant,△Tgb ZIP73＿2,was 0.08 of that of the wild type.Compared with the wild type,the mycelium ofλTgb ZIP73 grew faster,and the colony was round.Compared with the wild type,the mycelia of△Tgb ZIP73 grew slowly,sparsely and radially.After 25 days of culture with lignin,the lignin content of△Tgb ZIP73 was 1.25 times higher than that of the wild type.The lignin content of theλTgb ZIP73 fermentation broth was 0.38 that of the wild type.Tgb ZIP73 promoted the growth of mycelium and promoted the degradation of lignin.In conclusion,this study investigated the mechanism of the T.gibbosa response to lignin stress.Additionally,this is the first time that an overexpression laccase mutant improved the degradation rate of lignin at the T.gibbose genetic level.A PEG-mediated protoplast transformation system was established to explore the function of the b ZIP gene in promoting lignin degradation in basidiomycetes for the first time.It not only seeks to elucidate the mechanism of lignin degradation but also provides a theoretical basis for the efficient utilization of lignocellulose.Also,excellent mutants were constructed to accelerate the rate of lignin degradation.