The Cellobiose Degradation By Ruminiclostridium Thermocellum Based On Genomics And Transcriptomics

Ruminiclostridium thermocellum cellulosome is one of the most efficient cellulase systems in nature,but its catalytic efficiency is inhibited by cellobiose which is the cellulose degradation intermediate.Therefore,it is particularly important to seek several strains with a stable ability to degrade cellobiose and to explore the key genes and regulatory mechanisms about cellobiose metabolism,which can achieve high value use of cellulose biomass resource.R.thermocellum M3,inheriting the ability of resistance to cellobiose feedback inhibition,was used as the target in this research which aimed to elucidate its mechanism for stable degradation of cellobiose.The kinetic properties of cellobiose degradation by strain M3 were investigated in batch culture,on this basis,the core genes concerning cellobiose degradation by strain M3 were revealed by conjoined genetic and transcriptomic means.At the same time,the crucial metabolic pathway about the decomposition of cellobiose by R.thermocellum was clarified,with the following results:The kinetic characteristics of cellobiose degradation by strain M3 were investigated in this study.In contrast to other wild-type R.thermocellum,R.thermocellum M3 could still grow on cellobiose with 2% concentration.The optimum growth condition and glucose production was found at a cellobiose concentration of 12 g/L.Compared to the Avicel,the higher β-glucosidase activity was secreted by strain M3 when grown on cellobiose.It was also accompanied by higher glucose accumulation and the best production of glucose had reached 767.97mg/g cellobiose.The complete genome sequences of strain M3 were obtained using Pacbio sequencing platform and the combination of gene function annotation and comparative genomics was revealed to provide the information of genomic specificity about cellobiose degradation of strain M3.The identified total size of the M3 genome was about 3.6Mb with 39% GC.The number of encoding genes is 3077,most of which are involved in sugar metabolism and enzyme catalysis.Compared with other wild R.thermocellum,strain M3 is evolutionarily distinct with 192 specific genes.In addition,the genes encoding bgl X are reported for the first time in R.thermocellum.On the basis of discovering the specific genes for the degradation of cellobiose by strain M3,we further elucidated its metabolic regulation mechanism under different carbon sources and its response mechanism to cellobiose stress at the transcriptional level.The gene transcript levels of R.thermocellum M3 display significant differences under different substrates.In summary,the ability of strain M3 to resist feedback inhibition of cellobiose is closely correlated with the regulated expression of genes which encoding cellulosome structure,β-glucosidase,cellobiose phosphorylase,ABC transporter protein and others.Among them,secretion of β-glucosidase by strain M3 is mainly correlated with the expression of bgl X gene.Almost all differentially expressed genes enriching in the bacterial chemotaxis pathway were upregulated which suggests the strain M3 increases the perception and adaptation to cellobiose substrates through a biological strategy that enhances the expression of chemotactic genes.The above results indicate that R.thermocellum M3 can grow on 2% cellobiose.Cellobiose stress significantly changed the expression of genes related to the composition of cellulosome and sugar transport and genes involved in chemotaxis pathway.That information provides the solid foundation for new strategies and targets sites for engineering genetic modification of cellulosebased biomass saccharification by bacterial.