Functionl Study Of Sugar Transporter Genes Km＿SUT26 And Km＿SUT28 Of Kluyveromyces Marxianus GX-UN120

Kluyveromyces marxianus has the characteristics of high temperature resistance,fast growth rate and a wide range of metabolic substrates.In the process of yeast fermentation,the first speed limiting step of substrate absorption and utilization is transmembrane transport.Therefore,it is particularly important to study the gene function of the sugar transporter in yeast Kluyveromyces marxianus.In this study,the function of two sugar transporter genes in saccharomyces cerevisiae EBY.VW4000 was studied by heterologous expression in saccharomyces cerevisiae EBY.VW4000.According to the different growth rates of the heterogenously expressed strains,the preference of two sugar transporters to different sugar transporters was found.The recombinant yeast EBY.VW400-Km＿SUT26 showed higher specificity in the utilization of sorbitose.utilization.The recombinant yeast EBY.v W400-Km＿SUT28 had better growth capacity on glucose,fructose,sucrose,mannoose,galactose,and xylose,ribose,arabinose,indicating that Km＿SUT28 gene may have better performance in promoting the fermentation of lignocellulosic sugar hydrolysate.The cloned vector of the above two genes was constructed and heterogenously expressed in a strain of Saccharomyces cerevisiae EBY.VW4000,which was deficient in sugar transporters.The protein was subcellular localization.Finally,the growth of the recombinant strain in different carbon sources was analyzed.（1）In this study,primers were designed according to the sequences of KLMA＿70003 gene locus and KLMA＿80389 gene locus annotated in the whole genome sequence of Kluyveromyces marxianus strain DMKU3-1042.The total DNA of Kluyveromyces marxianus GX-UN120 was used as the template for PCR amplification,and the DNA fragments of the two genes were obtained,which were named Km＿SUT26 and Km＿SUT28,respectively.The nucleic acid sequences of the two genes were compared on NCBI respectively,and it was found that the homology of the gene locus sequences corresponding to the sequences of Kluyveromyces marxianus DMKU3-1042 strain reached more than99%.Prediction of the structure of the two sugar transporters indicates that their structure conforms to the characteristics of the Major Facilitator Superfamily.They were compared with the known sugar transporters with high similarity by NCBI by the maximum quasi natural method for amino acid sequence alignment and phylogenetic tree was constructed,and protein structure and function were analyzed in combination with previous reports.（2）The recombinant S.cerevisiae with GFP green fluorescence gene fusion expression was observed by fluorescence microscope.It was found that green fluorescence was mainly concentrated in the cell periphery（on the cell membrane）,so the target gene may be encoding the membrane protein related to transport.（3）Explore the recombinant yeast in contained 2%Glucose,Fructose,Mannose,Sorbose,Xylose,Ribose,Arabinose,Galactose,Lactose,Sucrose 10different single carbon source on the solid medium growth situation,found that wine yeast EBY.VW4000 on 10 kinds of carbon sources were not grow,recombinant yeast Km＿SUT26 only grow in the medium sorbose,almost no growth in other medium.Recombinant yeast Km＿SUT28 can grow on all 10sugars.（4）The growth curve of recombinant yeast was measured in a liquid medium containing 2%Glucose,Fructose,Mannose,Sorbitose,Xylose,Ribose,Arabinose,Galactose,Lactose,Sucrose,10 different single carbon sources.The OD₆₀₀value of recombinant yeast EBY.VW4000-Km＿SUT26 was about 0.5when it reached the stable period in sorbitose liquid medium,and it did not grow significantly in other sugars.The recombinant strain EBY.VW4000-Km＿SUT28grew vigorously on all the tested six-carbon sugars and sucrose medium.When Glucose was the only carbon source,the OD₆₀₀value reached about 4 in the stable period.When Fructose or Sucrose is the only carbon source,the OD₆₀₀value can reach 6 in the stable period.When Mannitose or Galactose is a single carbon source,the OD₆₀₀value can reach 7 in the stable period.