Molecular Mechanism Of Lignocellulose-derived Xylose Utilization By Artificial Saccha-romyces Cerevisiae

Saccharomyces cerevisiae chassis cells face two key scientific issues in the process of lignocellulose xylose utilization,one is the utilization efficiency of chassis cells to xylose,the other is the tolerance of chassis cells to inhibitors.In this study,the molecular mechanisms underlying these two problems were investigated using synthetic biology techniques combined with various omics techniques,with the aim of improving the utilization of lignocellulosic xylose by S.cerevisiae.When S.cerevisiae was transferred from glucose to xylose medium,there was a brief arrest of cell growth,but when cells were transferred to xylose medium again,the arrest time of cell growth was significantly shortened,a phenomenon called xylose consumption memory(XCM).At the same time,it was found that XCM was affected by temperature,and this phenomenon was most significant when yeast was cultured at the optimum growth temperature(30℃).This phenomenon was not affected by the properties of the medium.The modification of histones,the structure of chromatin,and transcription factors all affect the adaptation of organisms to environmental fluctuations.In order to further explore the mechanism of "xylose consumption of memory".In this study,acetylomic assays were performed on cells at different stages.To explore the possible internal relationship between protein acetylation and XCM.Acetomic analysis showed that the H4K5 locus of histones was significantly associated with XCM.When mutating the H4K5 site,preventing its acetylation significantly enhanced "xylose-depleting memory".And it was found that when the acetylase HPA2,which interacts with the H4K5 site,was knocked out,the XCM was also enhanced.Again,it is confirmed that histone H4K5 locus plays an important role in the process of XCM.To further enhance the tolerance of S.cerevisiae to inhibitors in lignocellulose pretreatment.In this study,S.cerevisiae with four synthetic chromosomes was used as the chassis cell.The strain y YST167 with excellent phenotype was obtained by rapid evolution and screening using the SCRaMbLE system.Fermentation experiments were performed in xylose medium containing complex inhibitors.The growth rate of y YST167 was 2.16 times that of the starting strain.Whole-genome sequencing analysis revealed that y YST167 has a significant structural variant on the synthetic type Ⅱ chromosome,resulting in the deletion of the genes YSA1 and SUS1.The two genes were knocked out separately and in combination in the starting strain,and it was found that the knockout of both genes would increase the tolerance of the chassis cells to the inhibitor.To further explore the intrinsic link between YSA1,SUS1 and inhibitor tolerance,the transcriptome data of YSA1 and SUS1 knockout strains in the presence of compound inhibitors were analyzed.The results showed that the knockout of SUS1 gene significantly up regulated the xylose metabolic pathway and genes related to glycogen synthesis,providing more carbon skeleton and energy for cell growth and metabolism under adversity.In addition,the up regulation of related genes on the trehalose synthesis pathway provides a basis for cells to maintain a stable osmotic pressure.