Effects Of Ethanol Stress On Transcriptome Of Kluyveromyces Marxianus

Kluyveromyces marxianus,as a food-safe yeast,has the characteristics of heat resistance,fast growth and a wide range of available substrates.At present,the research on application prospect of ethanol production by Kluyveromyces marxianus has great potential.But with the increase of product ethanol,the metabolic capacity and product production capacity of yeast will be inhibited to varying degrees.Therefore,it is of great significance to study the effect of ethanol on fermentation.In this paper,the original strain is high temperature resistant yeast K.marxianus strain KM0.The ethanol tolerance limit concentration of strain KM0 under different temperature conditions was determined by stress culture in medium containing different concentrations of ethanol.After culture under extreme conditions,samples were taken for sample processing,then transcriptome sequencing technology was used to compare the stress treatment group with the control group,and the key differential expressed genes Adh4 and Ady2 related to ethanol tolerance were screened.The overexpression vector of differential gene Adh4 was constructed,which laid a foundation for the verification of functional genes.The main research is as follows:(1)By measuring the growth and viable number of K.marxianus strain KM0 cultured under different ethanol concentrations,it was determined that the optimum growth temperature of KM0 strain was 28℃,and the limit ethanol tolerance concentration was 6%.(2)KM0 strain cultured under 6% ethanol stress at 28℃ was used as the stress culture group,and the cells cultured with 0% ethanol were used as the control to prepare two groups of sample cells.The two groups of samples were subjected to transcriptome high-throughput sequencing.Through the analysis of transcriptome data,the gene differential expression between stress culture group and control group was determined.The results showed that the significant up regulation of Adh4(alcohol dehydrogenase)gene expression and the significant down regulation of Ady2(carboxylic acid transporter)gene were related to the process of ethanol tolerance of stressed growing yeast.(3)The overexpression vector of Adh4 gene with significantly upregulated expression was constructed by molecular biological methods which overexpressd the gene of Adh4 in KM0 strain,then several positive transformed strains were selected for stress culture test.However,its growth did not increase significantly when the ethanol concentration was 6%,8%and 10% at 28℃.Nevertheless,it laid a foundation for the subsequent study on improving the ethanol tolerance of K.marxianus KM0 strain.