| Lignin is second to cellulose as the abundant renewable biomass resource on earth and the most abundant natural phenolic polymer.Since that lignin has complex structures and difficulty to degrade,a large amount of lignin is discharged,resulting in waste of resources and environmental pollution.Microbial degradation technology provides a new idea for lignin utilization.A strain with ability of lignin degrading,identified as Bacillus amyloliquefaciens,was obtained in our previous research,in which,CotA laccase and dyedecolorizing peroxidase(BaDyP)could depolymerize β-O-4 type lignin to generate small molecule phenolic compounds such as vanillin and guaiacol.Gene catE encoding catechol dioxygenase was subsequently cloned from DNA of MN-13 strain.All these findings clarified that the key genes involved in the lignin degradation pathway could be found in DNA of B.amyloliquefaciens MN-13.However,part of lignin-derived phenolics need to convert into catechol via aromatic O-demethylation before cleavage of aromatic ring mediated by CatE and targeting to the central carbon metabolic process.To date,there is no reports regarding aromatic O-demethylase from Bacillus amyloliquefaciens have been reported.In order to screen and validate the possible aromatic O-demethylases in MN-13 strain and further refine the degradation pathway of lignin mediated by Bacillus amyloliquefaciens,the present study was carried out as follows:(1)On the basis of whole genome sequencing and assembly,genes of B.amyloliquefaciens MN-13 were annotated through Nr,GO,KEGG,COG,etc.The metabolic pathways of lignin were analyzed using bioinformatics to screen the key enzymes involved in lignin degradation and explore the pathway of lignin degradation mediated by B.amyloliquefaciens MN-13.Some key lignin degradation genes,including genes efeN,cotA,sodF,cypD and catE etc.,were identified.Based on these genes,lignin degradation pathways of benzoic acid and phenylacetic acid were deduced.(2)Based on whole genome sequences and annotated results,cypD,the gene encoding a cytochrome P450 enzyme with possible aromatic O-demethylation activity,was screened.Homologous recombination was used to knock down cypD and construct mutant strains to verify the aromatic O-demethylation of P450 enzymes.The results showed that the MN-13 strain could remove the aromatic O-methyl from guaiacol and convert it into catechol with growing on MSMA containing guaiacol,but exhibit no effect on vanillin and 4hydroxyphenylacetic acid.However,mutant 13-1 could neither grow on MSMA nor convert guaiacol,vanillin and 4-hydroxyphenylacetic acid.This is well documented that the P450 enzymes encoded by the cypD genes can catalyze the aromatic O-demethylation and exhibit an obligate property towards the lignin-derived product guaiacol.Since cytochrome P450 enzyme play an important role in maintaining cellular homeostasis,after knockdown of cypD gene,the colonial and bacterial morphology of strain 13-1 were indistinguishable from that of wild type strain MN-13,and the growth was better than the wild strain.Furthermore,knockdown of cypD gene affected the expression of key genes involved in lignin degradation.The above results confirmed that P450 enzymes encoded by cypD gene could catalyze aromatic O-methylation of lignin-derived phenols,which further refined the pathway of lignin degradation mediated by B.amyloliquefaciens MN-13.(3)BaDyP,a key enzyme of strain MN-13 for lignin depolymerizing,was combined with cellulase to treat straw,in order to evaluate the lignin depolymerization ability and increasing cellulose utilization of BaDyP.Except guaiacol,the purified BaDyP can oxidize ABTS,RB5,RB19,MnSO4 and VA,and the corresponding enzyme activities were 0.49 U/mL,0.0043 U/mL,0.248 U/mL,2.667 U/mL and 0.672 U/mL respectively.Besides,BaDyP could catalyze oxidative breakdown of β-O-4 bond and Cα-Cβ bond of lignin model compound GGE.Treatment of straw with BaDyP in combination with cellulase significantly increased the dextrose equivalent of straw;by analysis of FT-IR,SEM,XRD and XPS,it could be found that BaDyP can effectively degrade lignin and destroy its shielding effect on straw cellulose,and the structure of straw lignocellulose was effectively destroyed after treating with BaDyP and cellulase.The optimum process of treating straw with BaDyP and cellulase resulted in the highest percentage of straw glycation at pH 2,12 h and 20℃ with the addition of BaDyP at 4 U/g and cellulase at 10 U/g,and at this condition,62.44 mg/g straw with reducing sugars and 178.69 mg/g straw with soluble sugars were achieved.All these above findings provided a comprehensive and in-depth understanding of the mechanism of lignin degradation mediated by Bacillus amyloliquefaciens,and supplied important references for the use of Bacillus amyloliquefaciens in high-valued utilization of lignin. |
