| With the rapid development of society and the increasing demand for energy from human beings,people are inseparable from all aspects of life.Therefore,how to convert natural resources into energy has become one of the most indispensable problems for researchers in the present.Lignocellulose is a natural polymer compound with extremely rich reserves on the earth,and it is an extremely cheap raw material.Because of its wide source and reproducibility,it is inexhaustible.Lignocellulose is mainly composed of cellulose,hemicellulose and lignin.The composition of cellulose is mainly macromolecular polysaccharides such as glucose.Hemicellulose is a heterogeneous polymer composed of several different types of monosaccharides,most of which are pentoses and hexoses,including glucose,xylose,arabinose and galactose.At present,the industrial use of biotechnology to produce fuel ethanol mainly uses glucose in starch for industrial production of ethanol.In the study of lignocellulose hydrolysis and fermentation of ethanol,yeast can convert glucose into ethanol very well,and the xylose with the highest pentose content in hemicellulose is not well used.This is for lignocellulose ethanol Industrial production is a loss that cannot be ignored.The transportation of xylose is the main limiting factor in the process of xylose utilization in China.As the energy crisis is approaching,we have found a sugar transporter that efficiently and specifically transports xylose,improves the utilization of xylose by production strains,and uses lignocellulose to produce ethanol is one of the key means to ease energy problems.The previous research in this laboratory found that the Kluyveromyces marxianus GX-UN120 strain has 99% similarity to the K.marxianus DMKU3-1042 nucleic acid sequence,which has been reported by Japanese scholars.The gene of DMKU3-1042 strain located at the KLMA-70145 site has the highest transcriptional abundance when xylose is the sole carbon source compared to the transcriptional abundance of other single sugar sources.The role may be a xylose transporter.The Km＿SUT2 gene corresponding to the KLMA-70145 position in K.marxianus GX-UN120 strain has been studied earlier in this laboratory,so the laboratory has studied the Km＿SUT2 gene.At present,the function of the sugar transporter protein is generally studied by heterologous expression of the sugar transporter gene in the Saccharomyces cerevisiae EBY.VW4000 strain that has deleted all hexose transporters.Since S.cerevisiae cannot utilize xylose,it is necessary to introduce xylose metabolism pathways to study xylose transporters.In order to better study the ability of Km＿SUT2 sugar transporter gene to transport xylose,this study constructed and improved the xylose metabolism pathway(XR + XDH pathway)of S.cerevisiae engineering strain EBY.VW4000.The previous research found that the Km＿SUT2 transporter of K.marxianus GX-UN120 has low xylose transport capacity.In order to improve the xylose transport capacity of this sugar transport protein and explore the location of key amino acids related to transport,this study used methods of protein structure prediction,semi-flexible molecular docking,alanine scanning and site-directed mutation.Alanine scanning was performed on the six amino acid positions of the Km＿SUT2 gene of K.marxianus GX-UN120 to construct mutant recombinant S.cerevisiae EBY.VW4000-SUT2Q164A-XY,EBY.VW4000-SUT2E168A-XY,EBY.VW4000-SUT2Q293A-XY,EBY.VW4000-SUT2Q424A-XY,EBY.VW4000-SUT2F530A-XY,EBY.VW4000-SUT2L553A-XY.The solid medium experiment and liquid culture experiment were used.The results of the solid medium experiment showed that the growth rate of recombinant yeast strain EBY.The wild type is better.The growth rate of recombinant strain EBY.VW4000-SUT2L553A-XY on Sc-Trp solid medium with sorbose as the sole carbon source is worse than that of wild type.When sugar is the only carbon source,the growth is very weak and the growth of the wild type is better.The growth of the remaining recombinant yeast strain is not much different from that of the wild type.The results of liquid culture experiments showed that the recombinant S.cerevisiae strain EBY.VW4000-SUT2Q164A-XY,EBY.VW4000-SUT2E168A-XY,EBY.VW4000-SUT2Q293A-XY,EBY.VW4000-SUT2Q424A-XY,EBY.VW4000-SUT2F530A-XY,growth is better than wild type,and after significant difference analysis,the results are very significant differences.The growth of recombinant strain EBY.VW4000-SUT2L553A-XY on Sc-Trp liquid medium with glucose as the sole carbon source is almost the same as that of wild type,and the difference is not significant.Recombinant strain EBY.VW4000-SUT2Q164A-XY grows better than wild type on Sc-Trp liquid medium with xylose as the sole carbon source,but the difference is not significant.The growth of the recombinant strain EBY.VW4000-SUT2E168A-XY,EBY.VW4000-SUT2Q293A-XY,EBY.VW4000-SUT2F530A-XY,EBY.VW4000-SUT2L553A-XY is worse than the wild type,the difference is very significant.The growth of the recombinant strain EBY.VW4000-SUT2Q424A-XY on Sc-Trp liquid medium with xylose as the sole carbon source is almost the same as that of the wild type growth,and the difference is not significant.Combining the results of solid medium and liquid culture,the 553 th amino acid site on the Km＿SUT2 gene of the sugar transporter was replaced with alanine,and the growth of them all decreased and the difference was very significant,indicating that this site is a significant factor The sugar binding has a greater influence,and it is speculated that this site may be one of the key protein binding sites.Because the liquid culture experiment is closer to the ethanol fermentation process,it can reflect the effect of the replacement of the binding site on sugar transport to a certain extent.Determination of intracellular sugar of mutant recombinant S.cerevisiae EBY.VW4000-SUT2Q164A-XY,EBY.VW4000-SUT2E168A-XY,EBY.VW4000-SUT2Q293A-XY,EBY.VW4000-SUT2Q424A-XY,EBY.VW4000-SUT2F530A-XY,EBY.VW4000-SUT2L553A-XY and original S.cerevisiae Km＿SUT2-XY by pre-column derivatization.From the results of intracellular sugar liquid chromatography experiments,it is known that the mutation at position 553 has the greatest effect on the sugar transporter Km＿SUT2.Among them,the intracellular sugars of arabinose and galactose were only detected by this strain,the intracellular sugars of mannose were only detected by this strain,and the intracellular sugar content of glucose,ribose,and sorbose decreased significantly compared with the original strain.Obviously,lactose has the highest intracellular sugar content.These prove that mutation of the 553 site to alanine has the greatest influence on the sugar transport capacity of the Km＿SUT2 gene,and the site amino acid side chain group changes have the greatest impact.This site is the most researched among the six mutation sites.Combining with the experimental results of the previous experiment,the transcription abundance of the gene located at KLMA-70145 when xylose is the sole carbon source is the highest compared to the transcription abundance of other single sugar sources,positions 168,293,and 530.The amino acid changes at the point cause the growth on Sc-Trp liquid medium with xylose as the only carbon source to decrease and the difference is very significant,so these three sites may also be protein binding sites where the protein binds to xylose. |
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