| With the development and progress of society,the demand for energy is becoming more and more intense.as an environmentally friendly renewable energy,fuel ethanol has attracted more and more attention.Cellulosic ethanol based on lignocellulose such as corn and wheat straw has become the inevitable development trend of fuel ethanol because of its low price and a wide range of raw materials.After complete hydrolysis of lignocellulose,the mixture of many kinds of sugars is obtained,in which the content of pentose,especially xylose,can not be used effectively.Sugar transport is one of the rate-limiting steps in the process of sugar metabolism.The discovery of effective pentose transporters will help to improve the total sugar utilization of cellulose hydrolysates and accelerate the process of commercial production of cellulose ethanol industry.In this study,the function of four sugar transporter genes,Km＿SUT1,Km＿SUT6,Km＿SUT10 and Km＿SUT11,was investigated by using Kluyveromyces marxianus GX-UN120 as the starting strain.Firstly,four target genes Km＿SUT1,Km＿SUT6,Km＿SUT10 and Km＿SUT11 were cloned,and the resistance screening marker G418 was inserted into the Km＿SUT1,Km＿SUT6,Km＿SUT10 and Km＿SUT11 genes by fusion PCR to obtain the gene knockout box.The knockout box was successfully integrated into the genome of GX-UN120 by homologous double exchange,and single gene knockout mutants△Km＿SUT1,△Km＿SUT6,△Km＿SUT10 and △Km＿SUT11 were obtained.In order to study the effect of deletions of genes Km SUT1,Km＿SUT6,Km＿SUT10 and Km＿SUT11 on the sugar transport ability of K.marxianus GX-UN120,the mutants were inoculated in solid and liquid media with xylose,ribose,arabinose,lactose,sorbose,glucose,fructose,mannose and galactose as sole carbon sources,and GX-UN120 as control.The specific growth rate and substrate specific consumption rate of the original strain and mutant in various sugars were calculated to analyze the transport ability of sugar transporters Km＿SUT1,Km＿SUT6,Km＿SUT10 and Km＿SUT11 to various sugars.The results of spot plate test showed that the growth status of mutant △Km＿SUT1 on nine kinds of sugars was basically the same as that of the original strain on nine sugars;the growth of mutant △ Km＿SUT6 on lactose was significantly better than that of the original strain on lactose and on the other eight sugars;the growth of mutant △ Km＿SUT10 on glucose,fructose and mannose was worse than that of the original strain,and its growth on the other six sugars was basically the same.The growth status of the mutant △Km＿SUT11 and the original strain on nine kinds of sugars were basically the same.The results of liquid culture showed that the deletion of Km＿SUT1 gene reduced the specific matrix consumption rate of GX-UN120 on glucose,lactose and xylose by 13.28%,47.13% and 31.03%,respectively,indicating that the sugar transporter encoded by Km＿SUT1 gene may be an important sugar transporter in the transmembrane transport of glucose,lactose and xylose.The deletion of Km＿SUT6 gene reduced the specific consumption rate of GX-UN120 on galactose,lactose and arabinose by 19.34%,59.34% and 37.66%,respectively.The deletion of Km＿SUT10 gene reduced the specific matrix consumption rate of GX-UN120 on galactose,lactose and arabinose by 26.57%,27.15% and 30.71%,respectively.The deletion of Km＿SUT11 gene reduced the specific matrix consumption rate of GX-UN120 on galactose,lactose and arabinose by 48.81%,27.47% and 60.79%,respectively,indicating that the sugar transporters encoded by genes Km＿SUT6、Km＿SUT10 and Km＿SUT11may be important sugar transporters in the transmembrane transport of galactose,lactose and arabinose.In addition,the deletion of Km＿SUT11 gene increased the specific matrix consumption rate of GX-UN120 on sorbose and fructose by31.82% and 20.37%,respectively,indicating that the existence of sugar transporter encoded by Km＿SUT11 gene may affect the transport of sorbose and fructose by other sugar transporters. |
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