| Biodegradation of lignin is a worldwide research hotspot and problem.Clarifying the biodegradation mechanism of lignin is a key scientific problem that needs to be clarified first,and it is also a theoretical foun dation urgently needed to solve practical application problems.This study combined proteomics and metabolomics techniques to ferment different lignin model compound substrates with Aspergillus fumigatus G-13.The purpose of this study was to elucidate the expression of degrading enzymes and their related metabolic pathways during the degradation of lignin by strains,and to clarify the mechanism of enzyme production and degradation.First of all,three lignin model compound substrates(ferulic acid,p-coumaric acid and sinapic acid)were fermented with A.fumigatus G-13 to select the optimal substrate concentration for the growth of A.fumigatus G-13.The results showed that the optimal substrate concentrations were 1 mol/L sinapic acid,1 mol/L p-coumaric acid,and 2 mol/L ferulic acid.The difference of enzyme activity between p-coumaric acid and sinapic acid substrate was the biggest.Therefore,p-coumaric acid and ferulic acid were selected as substrates for A.fumigatus G-13 fermentation for subsequent proteomics and metabolomics analysis.Secondly,through Label-free proteomics analysis,a total of 1447 peptides were identified and 208 proteins were identified.Among them,134 proteins had significant changes,73 proteins were up-regulated and 61 proteins were down-regulated.After screening,the key proteins of A.fumigatus G-13 to degrade lignin model compounds were catechol dioxygenase,glutathione reductase,glucanase,isoamyl alcohol oxidase,glyceraldehyde-3-phosphate dehydrogenase,superoxide dismutase.LC/MS based non-targeted metabonomics technology was used to detect the intermediate metabolites of this strain in different stages of degradation of lignin model compounds.Positive and negative ion modes obtained 8832 and 4223 metabolites,respectively,and 4173 and 1596 were annotated.The types of metabolites are mainly amino acids,lipids,carbohydrates,organic acids and their derivatives,benzene ring compounds and organic heterocyclic compounds.Enrichment analysis of differential metabolite functions found that there were several pathways related to the degradation of lignin in A.fumigatus G-13.Among them,starch and sucrose metabolism,pentose phosphate pathway,glutathione metabolism and ortho-cleavage pathway of dihydroxylated aromatic ring were closely related to lignin degradation.In summary,combined proteomics and metabolomics,the biological pathways of A.fumigatus G-13 degradation of lignin model compounds were explored.The study found that the fermentation of sinapic acid by th is strain produced higher ligninase activity than p-coumaric acid substrate,which was related to the high aryl ether content in the syringyl structure of sinapic acid.A.fumigatus G-13 degrades lignin mainly by breaking C?-C? chemical bonds,oxidizing side chains and catalyzing benzene ring cleavage in the lignin structure.Then,lignin is depolymerized to form oligomers such as diarylpropane and aryl ether,and then dioxygenase acts on the benzene ring to promote its clea vage.Sinapic acid substrates can provide an important diversion pathway to the pentose phosphate pathway to balance metabolic flux,which effectively prevent metabolic inhibition and increase enzyme production.Partially up-regulated enzymes are conducive to initiating multiple reactions to coordinate with each other and accelerate the methylation process in the body,and further promote the energy metabolism of the strain to assist the degradation of lignin by A.fumigatus G-13.This study analyzes key enzymes and metabolic fluxes through different bioinformatics methods.The key proteins and metabolites were screened for verification and analysis,which laid the foundation for future research on the degradation or depolymerization of natural lignocellulose substrates. |
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