Study On The Mannitol Transport And Metabolic Regulation In Clostridium Tyrobutyricum Based On Transcriptome Sequencing

As the earth’s energy consumption accelerates,the environment continues to deteriorate.Therefore,the development and utilization of biomass resources is an important direction to meet the growing energy needs and achieve environmental protection and sustainable development.Bio-based chemicals production from biomass fermentation not only relieve the pressure caused by the exhaustion of fossil energy,but also reduce the emission of pollution.Clostridium tyrobutyricum is a gram-positive anaerobic bacterium and produces butyric acid as the major product,which is considered as a promising microbial cell factory.In previous study,mannitol was used as a cheap co-substrate for C.tyrobutyricum to increase the butyric selectivity because mannitol has a higher reduced state and is abundant in marine plants such as brown algae.Bio-based chemicals production from mannitol not only reduce energy consumption and economic cost,but also protect the environment.Therefore,the main purpose of this study was to study the metabolism and regulatory mechanism of mannitol in C.tyrobutyricum and lay a foundation for strengthening the mannitol metabolism and relieving carbon catabolite repression during mixed carbon source fermentation.Transcriptome sequencing was first performed on samples of late log growth stage under mannitol substrate,late log growth stage under xylose substrate,early and late log growth stage under glucose and mannitol mixed substrates.The overall quality of the samples was good,and the data were reliable.Through the analysis of differentially expressed genes(DEGs)under different carbon sources(Man10＿16h vs.Xyl10＿16h)in the transcriptome,17 genes related to carbon sources transport and metabolic were obtained.By analyzing DEGs from the mixed carbon source at different time points(Glu Man5＿8h vs.Glu Man5＿16h),7 genes related to carbon metabolism repression were obtained.In addition,the reliability of transcriptome was verified by RT-q PCR.Further experiments were performed based on the related DEGs obtained from transcriptomic data to explore the mannitol transport pathway in C.tyrobutyricum.The experiment results showed that mannitol is transported to the cytoplasm by phosphoenolpyruvate-dependent phosphotransferase system.Four genes on the mannitol operon: CTK＿C05280(mtl A),CTK＿C05290(mtl R),CTK＿C05300(mtl F)and CTK＿C05310(mtl D)were proved to be responsible for mannitol transport and metabolism in C.tyrobutyricum.Glucose,as an effective carbon source for C.tyrobutyricum,has an inhibitory effect on mannitol utilization.The underlying metabolic regulatory mechanism was explored based on the related DEGs which found from the transcriptome data.On the one hand,deletion of hpr K gene resulted in a co-utilization of glucose and mannitol,although a lag phase was observed,suggesting that the metabolic repression of mannitol is mediated by catabolite control protein A(Ccp A).On the other hand,it was found that the mutation of Mtl R at H397 D could alleviate the carbon catabolite repression(CCR)to some extent,demonstrating that the CCR is also mediated by transcription factor Mtl R.To sum up,various engineered strains were constructed to explore the mannitol transport and metabolic regulation mechanisms in C.tyrobutyricum.The CCR phenomenon was alleviated to some extent in the obtained strains,which lays the foundation for efficient butyric acid and butanol production from seaweed extract by C.tyrobutyricum.