Proteomics Analysis Of The Lignin Degradation Mechanism By Bacillus Amyloliquefaciens MN-13

Bacillus has broad application prospects in the degradation and utilization of lignin biomass.The current research on the lignin degradation mechanism of Bacillus is still incomplete,and needed to further investigation.In the early stage of our research,Bacillus amyloliquefaciens MN-13,which can degrade lignin,was isolated and screened from the fresh manure of beef cattle,and the laccase(CotA)and dye decoloring peroxidase(BaDyP)of the MN-13 strain could degrade ?-O-4 lignin(GGE).On this basis,the study used Label free quantitative proteomics technology to research the expression of protein in the MN-13 strain when cultured in an inorganic salt medium with alkali lignin or lignin model compounds as the sole carbon source for 48 h.Furthermore,the metabolic process of the strain on lignin was revealed,and the key genes for lignin degradation were screened for heterologous expression and functional verification.Compared with the inorganic salt medium control group:(?)The proteomic analysis results of alkali lignin experimental group showed that there were 831 differentially expressed proteins,404 proteins were up-regulated,and 427 proteins were down-regulated.When alkali lignin was used as the sole carbon source,the enzymes were all significantly differentially expressed of peroxidase,dioxygenase,superoxide dismutase,oxidoreductase and enzymes related to lipid synthesis and metabolism.In order to clarify the degradation process of alkaline lignin by MN-13 strain,the functional classification,functional enrichment and cluster analysis of differentially expressed proteins were carried out.The results showed that differential proteins were mostly involved in biosynthesis and metabolic processes,and were enriched in biosynthesis regulation and amino acid metabolism pathways.The KEGG pathway enrichment analysis found the benzoic acid degradation pathway and the tricarboxylic acid cycle pathway.Further analysis of the proteomics results found that the MN-13 strain can degrade alkali lignin through the?-ketoadipate pathway,benzoate degradation pathway and 4-hydroxyphenylacetic acid pathway.The degradation product acetyl-CoA could enter the tricarboxylic acid cycle and lipid synthesis process.(?)The lignin model compound A(2-phenoxy-1-phenyl-propane-1,3-diol)experiment group had 338 differentially expressed proteins,228 proteins were up-regulated,and 110 proteins were down-regulated;the compound B(1-(3,4-dimethoxypheny l)-2-(2-methoxyphenoxy)-propane-1,3-diol)experimental group had 296 differentially expressed proteins,139 proteins were up-regulated,and 157 proteins were down-regulated.In group A,ring-cleaving dioxygenase,heme-dependent peroxidase and superoxide dismutase were all differentially expressed.In group B,cytochrome P450 enzyme,acetyl-CoA dehydrogenase,transferase,carboxylase,synthetase and ring-cleaving dioxygenase were differentially expressed.There was a significant up-regulation of ring-cleaving dioxygenase in both groups,indicating that there was a ?-ketoadipate degradation pathway in both groups.Because acetyl-CoA dehydrogenase,transferase,and carboxylase were involved in lipid synthesis,it was speculated that the MN-13 strain is easy to use compound B for the synthesis and metabolism of intracellular lipids.This difference may be due to the difference in the substituents of compound A and B,leading to differential expression of the enzymes involved in the degradation of the two compounds.The stress protein and pyruvate dehydrogenase complex were significantly up-regulated in the two groups,indicating that when compound A or B was used as the carbon source,the MN-13 strain stimulated the expression of stress protein and promoted pyruvate.The pyruvate could be converted into acetyl-CoA and entered the TCA cycle to produce energy.By analyzing the differential expression protein of MN-13 strain under different carbon sources,it was found that cytochrome P450 participated in a variety of metabolic processes,and the expression of ring-cleaving dioxygenase was significantly up-regulated under different carbon sources.In this paper,using the MN-13 strain genome as the template,the yetO gene with a molecular weight of 3 186 bp was successfully cloned,which encoded a cytochrome P450 enzyme(CYP102A2)with a molecular weight of approximately 120 kDa.The degradation effect of recombinant CYP102A2 enzyme on lignin model compounds was analyzed by HPLC-MS,and the function of the ring-cleaving dioxygenase obtained in the previous stage was verified by spectrophotometer.The results showed that the recombinant CYP102A2 enzyme can catalyze the aryl-O-demethylation of isoferulic acid and GGE;The ring-cleaving dioxygenase can catalyze hydroquinone and a variety of catechol compounds,and the enzyme have high catalyze activity to catechol compounds.In summary,this study clarified the lignin degradation mechanism of B.amyloliquefaciens MN-13,and provided theoretical support for the application of Bacillus in the degradation of lignin.