Research And Application Of The SecY Protein-translocation Channel In Escherichia Coli For Sugar Transport

Microbial fermentation plays an important role in green bio-manufacturing,in which the construction of efficient cell factories is known as the major point.Escherichia coli has become a widely-used bacterial host due to its clear genetic background,simple manupilation,rapid growth,and easy large-scale fermentation.Rational design of intracellular pathways through metabolic engineering is one of the ways to improve the performance of cell factories,and sugar transport is a prerequisite for its further metabolism inside cells.The uptake of sugars by E.coli relies on a variety of specific transporters on its cytoplasmic membrane,which function in either an active transport or facilitated diffusion manner,leading to narrow sugar spectrum,transport saturation phenomenon,competitive/noncompetitive inhibition,and carbon catabolite repression（CCR）.The ideal solution is to develop an efficient simple diffusion channel without excessively interfering with cell growth,allowing sugar molecules to enter the cytoplasm under the concentration difference between the two sides of the membrane.The SecY protein-translocation channel（SecYEG）of E.coli is located on the cytoplasmic membrane.We have previously reported that co-expression of the SecY（ΔP）channel（a mutant without plug domain）and the outer membrane porin（SCVE）could form a passageway for monosaccharides to freely diffuse into the cytoplasm.However,the advantages of the SecY（ΔP）channel for transporting monosaccharides when compared with the specific transporters are still unclear,and its applications have not yet been fully explored.Moreover,the transport ability of the SecY（ΔP）channel for disaccharides,such as lactose,is quite weak.In this work,we evaluated the advantages of SecY（ΔP）channel for uptake of monosaccharides.Then,the SecY（ΔP）channel was applied to automatically control the carbon flux in cells for efficient synthesis of xylitol.After that,fermentative production of D-allulose was accomplished with a SecY（ΔP）channel-engineered cell factory.Finally,site-directed mutagenesis of the pore ring of the SecY（ΔP）channel was investigated to improve the uptake performance for lactose.The detailed contents are as follows.1.Evaluation of the advantages of SecY（ΔP）channel for transport of monosaccharides.We constructed the SecY（ΔP）channel in E.coli,and then demonstrated its ability to transport monosaccharides using C¹⁴-labeled glucose.The engineered E.coli with the SecY（ΔP）channel rather than the glucose PTS could maintain a specific growth rate of 0.228 h^-1 on 100 g/L glucose,which was 80%higher than that of the wild type E.coli,suggesting that the glucose transport saturation has been broken.The growth of mutant cells was not affected when competitive inhibitors of glucose were present in the medium.In fermentation of xylose-glucose mixtures,the engineered E.coli achieved simultaneous utilization of sugars,and the specific growth rate was increased from 0.174 h^-1 to 0.309 h^-1.Therefore,it can be seen that the SecY（ΔP）channel has obvious superiority in uptake of monosaccharides,which provides an important theoretical basis for subsequent applications.2.Intelligent self-control of carbon metabolic flux for xylitol synthesis by use of SecY（ΔP）channel.Fermentation experiments of xylose showed that xylitol-phosphate could no longer inhibit the uptake of xylose by cells with the help of SecY（ΔP）channel.After introduing the xylitol synthesis pathway,we used mixed sugar（xylose and glucose）for fermentation under the synergistic effect of SecY（ΔP）channel and CCR.In the presence of glucose,the instantaneous consumption rate of xylose was≈0.81 m M·h^-1·OD^-1,and the instantaneous yield of xylitol was≈0.72 mol/mol.After glucose was exhausted,the instantaneous consumption rate of xylose was≈1.51 m M·h^-1·OD^-1,and the instantaneous yield of xylitol was≈0.25 mol/mol.These results suggest that cells were able to automatically regulate the metabolic flux of xylose.The novel cell factory for xylitol synthesis not only realized the simultaneous transport of glucose and xylose,but also could automatically adjust the distribution of xylose flux according to the changes of glucose level,thus ensuring a high product yield.3.Transport of substrate without phosphorylation by the SecY（ΔP）channel for D-allulose synthesis.A synthetic pathway for D-allulose was constructed using the SecY（ΔP）channel and D-psicose 3-epimerase（DPEase）,resulting in a yield of≈0.05 g/g through fermentation.Then,knockout of PTS,fructokinase（Mak）and by-product pathways was carried out,increasing the yield of D-allulose to≈0.59 g/g.Further regulation of the carbon metabolic flux of the Embden-Meyerhof-Parnas（EMP）pathway got a final D-allulose yield of≈0.95 g/g.After medium optimization,the D-allulose titer reached≈23.3 g/L via fed-batch fermentation,in which the productivity throughout the logarithmic growth phase was≈1.03 g/（L·h）.4.Tageted mutation of the pore ring of SecY（ΔP）channel and its functionality in lactose transport.The experiment of green fluorescent protein（GFP）expression demonstrated that the SecY（ΔP）channel allowed slow passage of lactose.In order to increase the diffusion rate of lactose,mutation of its pore ring domain was carried out.First,a site-directed non-random mutagenesis method was developed.When all of the six isoleucines（Ile）of the pore ring were substituted with valine（Val）,the growth of the mutant E.coli（ΔLac Y,SecY[ΔP,Val],SCVE）on lactose was obviously accelerated.Next,we further performed site-directed random mutagenesis and obtained a mutant strain named E.coli 2RM53,whose specific growth rate during the logarithmic growth phase was increased by 13.1%when compared with E.coli（ΔLac Y,SecY[ΔP,Val],SCVE）.The results indicate that appreopriate adjustment of the amino acids of the SecY（ΔP）pore ring would effectively improve the lactose transport performance of the channel,thus laying a foundation for the use of the SecY（ΔP）channel of E.coli to take up other disaccharides.In summary,a free diffusion system established in E.coli using the SecY（ΔP）channel was able to overcome the defects of the native sugar transport systems,and it was successfully applied to the cell factories for production of xylitol and D-allulose.Mutation of the pore ring of the SecY（ΔP）channel could obviously improve its lactose transport efficiency,indicating that the SecY（ΔP）channel might be used in E.coli as a non-specific transporter for uptake of disaccharides.