Analysis Of The Lignocellulose Degradation System In Endophytic Bacterium Pantoea Ananatis Sd-1

With the increasing exhausted of energy resources,more and more severe challenges would appear in the resources and environment.The use of lignocellulosic biomass to transform of liquid fuel and valuable chemicals would produce enormous economic and social benefits and is helpful for sustainable development of human society.However,the complicated structure of lignocellulose affected its bioconversion and restricted its efficient utilization and development of biorefinery.In recent years,more and more studies showed that bacteria would play an increasingly pivotal role in the lignocellulose bioconversion process due to its wide range of environmental adaptability and versatile biological diversity.In this study,a rice endophytic bacterium Pantoea ananatis Sd-1 with high degradation capacity was used as the research object.Through systematical exploration of its characteristics,degradative system,and mechanisms,this study obtained the following innovative research achievements.(1)The rice straw degradation ability of P.ananatis Sd-1 was systematically characterized.After 6 days incubation of medium containing rice straw as a sole carbon resource with P.ananatis Sd-1,the percentage in the weight reduction of rice straw as well as its cellulose,hemicellulose and lignin components reached 62.7%,75.2%,78.8% and 35.6%,respectively.The maximum value of produced reducing sugar reached 0.1586 mg/ml.The highest values activities of the endoglucanase,exoglucanase,xylanase,lignin peroxidase and laccase were 1.55 U/ml,0.47 U/ml,25.29 U/ml,2.59 U/ml and 0.61 U/ml,respectively.(2)The genome of P.ananatis Sd-1 was accurately sequenced,and its database of lignocellulose degradation related genes were constructed and discussed through systematically bioinformatics analysis.The assembled genome of P.ananatis Sd-1 comprises a total of 4927,500 bp containing 4332 protein coding sequences(CDS),53.34% GC contents,65 tRNA coding sequences and 9 ribosomal RNA operons.After BLAST against with CAZy database by P.ananatis Sd-1 CDS,154 genes have multiple domains assigned to CAZy families,including 59 glycoside hydrolases(GHs),25 carbohydrate esterases(CEs),2 polysaccharide lyases(PLs),9 enzymes with auxillary activities(AAs)and 11 carbohydrate binding modules(CBMs).Genes encoding cellulase,hemicellulase and a part of ligninolytic enzymes were found in those CAZy genes.The number of CAZy genes in P.ananatis Sd-1 is higher than that of compared ligninolytic and cellulolytic bacteria strains as well as other P.ananatis genus strains.The amounts of CEs and AAs in P.ananatis Sd-1 are significantly higher than those in all compared strains.In addition,a lot of genes encoding ligninolytic enzymes including multicopper oxidase,catalase/hydroperoxidase,quinone oxidoreductase and glutathione S-transferase were found in the genome of P.ananatis Sd-1.The existence of quinone oxidoreductases,GMC family oxidoreductase and glutathione S-transferase possessed ?-aryl etherase activity suggested that non-enzymatic oxidation system i.e.Fenton chemistry pathway and ?-aryl ether degradation pathway might exist in the process of lignocellulose degradation by P.ananatis Sd-1.(3)The transcript levels of genes involved in lignocellulose degradation detection,enzymes activities assay and comparative proteome analysis of P.ananatis Sd-1 were performed.The results revealed that transcript levels of genes involved in lignocellulose degradation were significantly up-regulated in rice straw cultures compared to basic glucose cultures(at least P<0.05).Correspondingly,several lignocellulolytic enzymes,including endoglucanase,exoglucanase,?-glucanase,xylanase,lignin peroxidase and laccase,exhibited more active in rice straw relative to glucose substrates.The identification of secretome of P.ananatis Sd-1 by nano liquid chromatography-tandem mass spectrometry(nanoLC-MS/MS)showed that there were 108 and 52 proteins were identified in rice straw cultures and glucose cultures,respectively.The number of proteins involved in lignocellulose degradation was 15 and 3,respectively.These suggested that those lignocellulytic enzymes activities were regulated by substrates.(4)The existence of Fenton reaction-dependent(Fe2++H2O2+H+?Fe3++?OH+H2O)non-enzymatic oxidation system in the process of lignocellulose degradation by P.ananatis Sd-1 was confirmed.P.ananatis Sd-1 possesses unpretreated rice straw degradation capacity: after 3 days incubation with P.ananatis Sd-1,the percentage in the weight reduction of rice straw as well as its cellulose,hemicellulose and lignin components reached 46.7%,43.1%,42.9% and 37.9%,respectively.The addition of different hydroxyl radical scavengers resulted in a significant decline(P<0.001)in rice straw degradation,which suggested that its degradation pathway was hydroxyl radical-dependent.Pyrolysis gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy analysis revealed the consistency of chemical changes of rice straw components that exists between P.ananatis Sd-1 and Fenton reagent treatment.The Fe3+-reducing activity of P.ananatis Sd-1 in rice straw cultures was significantly higher than that of glucose cultures.According to the gas chromatography-mass spectrometry analysis,the Fe3+-reducing activity of hydroxyl phenolic groups containing aromatics in the metabolites from rice straw cultures was significantly higher than that of the secreted proteins.The transcript levels of the GMC family oxidoreductase related to hydrogen peroxide production and its enzyme activity in rice straw cultures were higher than that of glucose cultures,while the hydrogen peroxide concentration in rice straw cultures was lower than that of glucose cultures.These suggested that non-enzymatic oxidation system i.e.Fenton chemistry pathway might exist in lignocellulose degradation system of P.ananatis Sd-1.In conclusion,the degradative system of P.ananatis Sd-1 contained lignocellulolytic hydrolysis enzymes system and Fenton reaction-dependent non-enzymatic oxidation system.The degradation mechanism of P.ananatis Sd-1 is likely to be generating hydroxyl radical through Fenton reaction to oxidize the complete structure of lignocellulose at incipient stage.Then a series of secreted lignocellulolytic enzymes penetrated to the internal for hydrolysis and degraded lignocellulose.The present study provided sufficient scientific evidence for exploring the unclear mechanism of bacterial lignocellulose degradation,and had important scientific value.Meanwhile,it also laid a theoretical foundation for P.ananatis Sd-1 application in industrial lignocellulose materials biotransformation.