Insight Into Metabolic Pathways Of Alkaliphilic Halotolerant Bacterium-Bacillus Ligniniphilus L1 For Lignin Depolymerization To Produce Vanillic Acid

Lignin is a polymer composed of phenylpropane units connected by different types of aryl ether bonds and carbon-carbon bonds,and its reserves are second only to cellulose as the second largest biomass resource in nature.There are a variety of organisms in nature that can efficiently degrade lignin,such as fungi and bacteria.Bacteria have good environmental adaptability,can withstand extreme environments and relatively easy genetic engineering,and other characteristics,and play an important role in the biodegradation of lignin.In recent years,studies have shown that some bacteria have a "biological funnel" metabolic pathway,that is,multiple lignin metabolic pathways can be used to aggregate different lignin depolymerization products into a common intermediate metabolite,thereby achieving a single compound from lignin Efficient conversion.Therefore,elucidating the metabolic pathways of bacterial depolymerization lignin will provide an important theoretical basis for the efficient biodegradation of lignin to achieve high-value applications.In this paper,a Bacillus ligniniphilus L1 that can degrade lignin and obtain a large amount of vanillic acid(vanillic acid accounts for more than 40% of the total amount of monocyclic aromatic compounds)as the research object,through bioinformatics analysis,molecular cloning,enzyme activity detection,Research methods such as high-performance liquid chromatography(HPLC)and gas chromatography/mass spectrometry(GC-MS)of metabolites are used to mine key enzyme genes in the metabolic pathways of lignin and its derived aromatic compounds,and heterologous key enzyme genes Expression and study of enzymatic properties.Finally,the metabolic pathway of vanillic acid produced by depolymerizing lignin in Bacillus ligniniphilus L1 was elucidated.The main research results are as follows:(1)Based on transcriptome and genomic data of Bacillus ligniniphilus L1,a total of 76 coding genes of lignase were identified,mainly including dehydrogenase,decarboxylase,monooxygenase,oxygenase,and 30 of them were successfully heterologously expressed in E.coli BL21.(2)Clarified the existence of four "biological funnel" metabolic pathways in Bacillus ligniniphilus L1 with β-aryl ether,biphenyl(5-5),p-hydroxybenzoic acid and syringaldehyde as the intermediate product of lignin degradation with vanillic acid as the intermediate.(3)Clarified the enzymatic properties of vanillin dehydrogenase,the key enzyme for vanillin conversion to vanillic acid.The optimal reaction condition for vanillin dehydrogenase is p H 7.5,and the optimal reaction temperature is 37.And it shows better thermal stability at the optimum temperature.