Research On Nonspecific Transmembrane Transport By Protein Transfer Channel SecY In Escherichia Coli

Sugar is the most important carbon and energy source of cells.Sugar can through the cell membrane into the cytoplasm by the specific sugar transport system in the living cells,which is particularly important physiological function in the cell life cycle.The mechanism of transport of these specific sugar transport systems are different,some systematic mechanism is active transport,and some systems are to promote the diffusion mechanism.Similar to sugar transport,protein transport also passes through a specific channel,which is a protein-conducting system that allows the polypeptide to be transferred across the membrane or integrated into the cell membrane.In bacteria and archaea,this channel is located on the cell membrane which formed by the SecY complex.It is proved that in its resting state,the SecY channel is sealed by a plug ring domain and an amino acid pore ring to prevent leakage of small molecule in the cytoplasm.The main purpose of this project is to realize the non-specific transport of ugars across the inner membrane of Escherichia coli.This research includes thefollowing key contents:Release of the SecY protein translocation channel fromfully preventing the permeation of the water-soluble substances by rationalmutation of its plug and pore ring residues,thus weakening the diffusion barrierof sugars across the inner membrane.Using of the mutant SecY channel,the SARSshell protein SCVE and the sugar kinase to construct a simple-diffusiontransmembrane system and a phosphorylation supplementary system.The replacementof the transport system of xylose,arabinose,lactose or cellobiose with thesimple-diffusion system will be tried,and the combination of the simple-diffusionsystem and the phosphorylation supplementary system will be employed to replacethe transport system of glucose,fructose or mannose.The recombinant strains withnew sugar transport systems will be used to produce xylitol in a mixed-sugarfermentation process.Moreover,the principles for designing,constructing andco-working of the simple-diffusion system and the phosphorylation supplementarysystem will be intensively studied.The realization mechanism of overcoming thesubstrate specificity,saturation phenomenon,inhibitor sensitivity,and inducerexclusion by using the simple-diffusion system will also be discussed.Thisproject is expected to provide a new sugar transport strategy for E.coli,and theresults will help to further expand the application prospect of E.coli inmetabolic engineering and synthetic biology.