Identification And Analysis Of Key Amino Acid Sites Of Sugar Transporters Km＿SUT1 In Kluyvermyces Marxianus

Lignocellulose is an ideal raw material for the production of bioethanol.Its hydrolysate is mainly glucose and xylose.Existing industrial microorganisms are inefficient in xylose utilization or are inhibited by glucose.Therefore,overcoming the inhibition of glucose and achieving efficient coutilization of pentose and hexose is one of the proble ms that need to be solved urgently.In Saccharomyces cerevisiae,the transport of sugar is a key ratelimiting step in sugar metabolism,and finding specific binding sites between transporters and sugar molecules is the key to understanding sugar transport.To study the relationship between the sugar transporter and the substrate,through the analysis and mutation of the key amino acid residues of the sugar transporter,to optimize the structure of the sugar transporter,thereby effectively improving the affinity between the substrate and the transporter.In this study,Xylose reductase gene XYL1 and Xylitol dehydrogenase gene XYL2 derived from Pichia stipits.The XYL1 and XYL2 genes were linked to the YEplac195 plasmid to construct the YEplac195-XYL1-XYL2 shuttle vector containing the xylose metabolism pathway,and the plasmid was electrotransformed into S.cerevisiae EBY.VW4000.A recombinant S.cerevisiae strain containing metabolizable xylose,glucose and other sugar substrates through the xylose metabolic pathway was successfully constructed,which can be used as a model yeast strain for studying sugar transporters.Cloning of Km＿SUT1 gene of K.marxianus GX-UN120.The gene is1809 bp and encodes 602 amino acids.Km＿SUT1 protein is localized in the plasma membrane by Prot Comp and does not contain a signal peptide by Signal P.The SMART functional domain prediction of the amino acid sequence shows that the Km＿SUT1 protein belongs to the facilitator superfamily 1(MFS1)and belongs to the Sugar＿tr subfamily.There were 12 possible transmembrane helices from inside to outside by TMpred.The three-dimensional structure model of Km＿SUT1 protein was established by Robetta.Using Auto Dock to simulate docking and Py Mol analysis of protein receptors and xylose ligands.The possible amino acid binding sites were determined by intermolecular interactions and hydrogen bonding positions,which were R186,M240,Y244,N378,W472,G499,respectively.The amino acid corresponding to Km＿SUT1 was mutated to alanine at site to obtain p RS424 vectors containing the mutated gene,which were electrotransformed into EBY.VW4000-XYL1-XYL2,respectively.The recombinant S.cerevisiae EBY.VW4000-Km＿SUT1-XYL1-XYL2 、 EBY.VW4000-R186A-XYL1-XYL2、EBY.VW4000-M240A-XYL1-XYL2、EBY.VW4000-Y244A-XYL1-XYL2、EBY.VW4000-N378A-XYL1-XYL2、EBY.VW4000-W472A-XYL1-XYL2、EBY.VW4000-G499A-XYL1-XYL2.The growth of recombinant yeast on different sugars was determined by dot plate assay and intracellular sugar transport was determined by HPLC.It was found that Km＿SUT1 has the function of transporting arabinose,galactose,ribose,xylose,lactose and sorbose.The R186 site did not alter the function of Km＿SUT1 protein and was not the key amino acid site for Km＿SUT1 receptor binding to sugar ligand.The mutation at Y244 makes Km＿SUT1 transport arabinose,xylose,glucose,sorbose and mannose in large quantities,which is significantly different from that of unmutated yeast,in which the transport of glucose and sorbose reaches 38.799 mg/g cell dry weight and 185.937 mg/g cell dry weight,it was speculated that Y244 changed the spatial conformation of Km＿SUT1 with sugar-binding domain,changed its affinity with sugar,and affected its transport of sugar.The mutation at N378 site increased the transport of xylose,glucose and sorbose by Km＿SUT1 to 0.197 mg/g,7.914 mg/g and50.302 mg/g,respectively,and decreased the transport of mannose.Mutation at W472 site increased the transport of Km＿SUT1 to glucose,sorbite and mannose,reaching 0.971 mg/g,39.918 mg/g and 1.007 mg/g,respectively.Mutations at the G499 site alter the transport of ribose,xylose,and sorbinose by Km＿SUT1.These key amino acid mutation sites alter the affinity of Km＿SUT1 to different sugars,thereby affecting its sugar transport capacity.