Identification And Regulation Of Cellobiose Transport System Of Clostridium Acetobutylicum

Energy is the material basis for human survival and development.However,the pollution problems caused in all countries have become more and more serious with the exhaustion of energy,make the development of green and environmentally friendly renewable energy to be an urgent need.Biofuels is known as green energy sources that hardly cause environmental pollution and contribute to the banlance of ecological environment.Bio-butanol is considered as a promising biofuel with high energy and good fuel economy.It can be produced by solventogenic clostridia through ABE（Acetone-Butanol-Ethanol）fermentation pathway,such as Clostridium acetobutylicum and Clostridium beijerinckii.C.acetobutylicum is a typical representative,which can convert various monosaccharides,disaccharides and polysaccharides into butanol and other by-products such as acetone and ethanol.In this study,cellobiose transportion and regulation in C.acetobutylicum ATCC824 were determined.The main research results are as follows:First,we confirmed two gene clusters to encode putative PTS（phosphotransferase system）systems of cellobiose（CAC0383-0386 and CAC1407-1408）,which specifically and highly expressed under cellobiose.The results of gene mutation indicated that mutants inactivating CAC0383 or CAC1407 can still grow on cellobiose,while double mutant of CAC0383 and CAC1407 can not grow on cellobiose,suggesting that both gene clusters of CAC0383-0386 and CAC1407-1408 encode PTSs for transportion of cellobiose in C.acetobutylicum ATCC824(named PTS1^ceband PTS2^ceb,respectively).Second,the other potential sugar substrates transported by PTS1^ceband PTS2^cebwere screened from varous hexoses（galactose）,pentoses（arabinose,xylose）,disaccharides（sucrose,lactose）,and polysaccharides（xylan）.The results indicated that the mutant inactivating CAC1407 can grow well on these sugars,while the mutant inactivating CAC0383 did not grow on galactose,suggesting that PTS1^cebis also in charge of transportion of galactose.Finally,we successfully developed a counter selectable system based on upp（encoding uracil phosphoribosyl transferase）in C.acetobutylicum.C.acetobutylicumΔupp can indeed grow in the medium containing5-fluorouracil（5-Fu）,but wild-type strain can not grow.Therefore,the plasmid or fragment containing upp gene can be quickly lost by adding 5-Fu in upp mutant.At the same time,based on the ClosTron system,a gene knock-in method that inserts foreign genes into target locations in the genome is established.In sum,we not only identified the cellobiose transport system of C.acetobutylicum and analyzed its expression regulation mechanism,but also developed a new counter selectable system based on upp in this study.It will not only lay the foundation for the efficient utilization of lignocellulose by C.acetobutylicum,but also provide convenient tools for the genetic modification of Clostridia.