Study On The Transport And Metabolic Regulation Of Fermentable Sugars In Clostridium Tyrobutyricum And Its Application

Sugar transport systems are classified as three categories on the basis of energy coupling mechanism:phosphoenolpyruvate（PEP）-dependent phosphotransferase system（PTS）,proton symport system,and ABC（ATP-binding cassette）system.In bacteria,most of the carbohydrates,mainly hexose,are uptake into cell via PTS in the phosphorylated form,while other carbohydrates,mainly pentose,are uptake into cell by the proton cotransport and ABC system in the unphosphorylated form.It is well known that bacteria often use preferred carbon source（such as glucose）and inhibit the metabolism of other carbon sources to achieve rapid proliferation.This phenomenon is called carbon catabolite repression（CCR）,which is caused by the rapid adaptation of bacteria to the changes in the environment.The existence of CCR restrict the rapid utilization of mixed sugars,so relieving CCR to achieve substrate co-utilization has always been a research focus of bacterial fermentation.Clostridium tyrobutyricum ATCC 25755 is the dominant strain to produce butyric acid,but the uptake pathways of various carbon sources and related CCR mechanisms are rarely reported.To better utilize cheap biomass for butyric acid production,this study attempts to explore the carbon transport pathway and metabolic regulatory mechanism of C.tyrobutyricum,and then strengthen the substrate metabolism capacity through metabolic engineering strategy.Studies on the transport pathways of glucose,fructose,mannose and xylose in C.tyrobutyricum showed that glucose,fructose and mannose are mainly transported by PTS,while xylose is mainly absorbed by proton symporter（Xyl T-I,CTK＿C21040）.PTS^Fru-I（CTK＿C06320）is the main transporter permease for fructose,while PTS^Glc-I（CTK＿C20580,Glc G）is the main transporter permease for glucose and mannose.As C.tyrobutyricum has poor mannose metabolism ability,we overexpressed the mannose transport and/or metabolism genes from itself or C.acetobutylicum ATCC 824.Compared with the wild-type strain,the engineered strain ATCC 25755/glc G showed the best mannose metabolic ability,with 117%56%and 163%increasement in mannose consumption rate,butyric acid yield and productivity,respectively.It demonstrated that the rate-limiting step of mannose metabolism is mannose transport.In addition,the improvement of mannose metabolism was observed for ATCC 25755/glc G in the mixed carbon sources of glucose and mannose.The regulation of carbon metabolism in C tyrobutyricum was studied using glucose as a preferred carbon source.It was found that glucose and fructose could be co-utilized,while the metabolism of mannose and xylose were inhibited by glucose.Our data suggested that the inhibition between glucose and mannose metabolism was due to the competition of transport protein（Glc G）.Inactivation of the Glc G lead to co-utilization of glucose and mannose,although the metabolic capacities of both sugars were seriously impaired.Our results also demonstrated that carbon catabolic repression between glucose and xylose were controlled by the synergistic effect of Ccp A-and Xyl R-mediated CCR,in which the former played a dominant role.Therefore,the engineered strain ATCC 25755/Δhpr KΔxyl R could co-utilize glucose and xylose by destroying two regulation pathways of CCR simultaneously,resulting in 1.8-fold improvement in butyric acid productivity as compared to wild-type strain with mixed sugars as carbon source.In addition,ATCC 25755/Δhpr KΔxyl R exhibited excellent fermentation performance in the hydrolysates of corn cob,wheat straw and soybean straw.In summary,this study explored major transport pathways and metabolic regulatory mechanisms of fermentable sugars,enhanced the mannose metabolism and realized the co-utilization of glucose and xylose in C.tyrobutyricum by metabolic engineering modification,which lay the foundation for efficient butyric acid production from unpreferred carbon sources and cheap substrates by C.tyrobutyricum.