| Buffalo has the advantage of being resistant to roughage compared to yellow cattle.Lignin is an important factor hindering the full utilization of hemicellulose and cellulose of roughage in ruminants.Previous studies have shown that microbes in the buffalo rumen have a high degradation rate of lignin.Therefore,studying the lignin-degradating pathway of rumen microorganisms can further clarify the reasons for roughage resistance of buffalo.In this study,a fungus has been isolated from the rumen contents of buffalo,initially identified as Aspergillus fumigatus.The enzyme activity of the main lignin-degrading enzymes,functional gene screening and lignin-degrading products were analyzed.The pathways of lignin degradation by this strain was explored,and the mechanism of resistance to rough feeding of buffalo was further revealed.(1)Study on the verification of lignin-degrading enzymes of Aspergillus fumigatus.In this experiment,the strain was further identified as Aspergillus fumigatus from the morphological characteristics,physiological and biochemical characteristics,ITS rRNA(Internal Transcribed Spacer)sequence and 28 S rRNA sequence.The degradation efficiency of sodium lignosulfonate was detected,and the degradation efficiency was up to 15.12%.The main lignin degrading enzymes were detected,while only the activity of ferulic acid esterase was detected,reaching a maximum value of 0.033 U/mL at 120 h.(2)Analysis of functional genes and pathways related with lignin degradation by Aspergillus fumigatus.To further investigate the types of lignin-degrading enzymes and the mechanism of lignin degradation by Aspergillus fumigatus,the third-generation PacBio RS II platform was used to detect the whole genome sequence of Aspergillus fumigatus,and the lignin-degradating genes were identified and analyzed.In the CAZyme database,the results of the auxiliary enzyme(AA)showed that a total of 29 lignin degrading enzyme genes were screened.These genes include the multi-copper oxidase,lignin-modified peroxidase,and 1,4-benzoquinone reductase.Specific primers were designed and PCR was performed to successfully obtain fragments of these genes.Twenty-nine genes were analyzed for KEGG metabolic pathways,and a total of 12 genes were annotated into the metabolic pathways.Among these genes,one gene is enriched in the galactose metabolic pathway,one gene is enriched in the phenylalanine metabolic pathway,and four gene are enriched in the pyruvate metabolism.These metabolic pathways are closely related to the lignin degradation by Aspergillus fumigatus.(3)The main intermediates and metabolic pathways of lignin degradation by Aspergillus fumigatus.GC-MS analysis of lignin degradation products,identified 65 acid substances,38 kinds of amino acid substances,23 kinds of carbohydrate,16 kinds of benzene ring substances,15 kinds of alcohols,12 kinds of amines,5 kinds of esters,4kinds ketones,4 pyrimidines,3 phenolics,2 salts,2 pyridines,and 1 aldehyde.There were75 different metabolites.Among them,12 new intermediates such as 5-aminovaleric acid,aconitic acid,guanidine and shikimic acid were found in the experimental group.Among the substances in the control group and the experimental group,there were 52 kinds of differential metabolites such as 3,4-dihydroxybenzoic acid,2-hydroxyacetophenone,3-hydroxybenzoic acid,thymol,fumaric acid and pyruvic acid showed an increasing trend with the prolongation of degradation time;Four differential metabolites of galactoside,isopropyl-β-d-thiogalactoside,glycine and 3-hydroxy-L-proline 2 showed a downward trend with the prolongation of degradation time and other benzoic acid and pyruvic acid.There were no significant changes in the seven differential metabolites of lactobionic acid,mannitol,N-methyl DL-alanine,4-hydroxybutyrate and xylose.Among them,fumaric acid is enriched in the pyruvic acid and phenylalanine metabolic pathways,and3-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid are enriched in the benzoate and toluene metabolic pathways,3,4-dihydroxyl Benzoic acid is also enriched in the aminobenzoic acid metabolic pathway,and other differential metabolites of benzoic acid enrichment pathways include phenylalanine metabolism,benzoate degradation,toluene degradation,and aminobenzoic acid degradation.The results showed that Aspergillus fumigatus had the ability to degrade lignin.Combined with enzymatic analysis,functional gene screening,GC-MS analysis of degradation products and metabolic pathway analysis,it was indicated that there were six metabolic pathways for lignin-degrading by Aspergillus fumigatus,were galactose metabolism,phenylalanine metabolism,pyruvate metabolism,benzoate degradation,toluene degradation and aminobenzoic acid degradation,respectively. |
