Miura S, Zou W, Ueda M, Tanaka A
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Appl Environ Microbiol. 2000 Nov;66(11):4883-9. doi: 10.1128/AEM.66.11.4883-4889.2000.
A Saccharomyces cerevisiae strain, KK-211, isolated by the long-term bioprocess of stereoselective reduction in isooctane, showed extremely high tolerance to the solvent, which is toxic to yeast cells, but, in comparison with its wild-type parent, DY-1, showed low tolerance to hydrophilic organic solvents, such as dimethyl sulfoxide and ethanol. In order to detect the isooctane tolerance-associated genes, mRNA differential display (DD) was employed using mRNAs isolated from strains DY-1 and KK-211 cultivated without isooctane, and from strain KK-211 cultivated with isooctane. Thirty genes were identified as being differentially expressed in these three types of cells and were classified into three groups according to their expression patterns. These patterns were further confirmed and quantified by Northern blot analysis. On the DD fingerprints, the expression of 14 genes, including MUQ1, PRY2, HAC1, AGT1, GAC1, and ICT1 (YLR099c) was induced, while the expression of the remaining 16 genes, including JEN1, PRY1, PRY3, and KRE1, was decreased, in strain KK-211 cultivated with isooctane. The genes represented by HAC1, PRY1, and ICT1 have been reported to be associated with cell stress, and AGT1 and GAC1 have been reported to be involved in the uptake of trehalose and the production of glycogen, respectively. MUQ1 and KRE1, encoding proteins associated with cell surface maintenance, were also detected. Based on these results, we concluded that alteration of expression levels of multiple genes, not of a single gene, might be the critical determinant for isooctane tolerance in strain KK-211.
通过在异辛烷中进行长期立体选择性还原生物过程分离得到的酿酒酵母菌株KK - 211，对这种对酵母细胞有毒的溶剂表现出极高的耐受性，但与它的野生型亲本DY - 1相比，对亲水性有机溶剂，如二甲基亚砜和乙醇的耐受性较低。为了检测与异辛烷耐受性相关的基因，利用从在无异辛烷条件下培养的DY - 1和KK - 211菌株以及在有异辛烷条件下培养的KK - 211菌株中分离得到的mRNA，采用mRNA差异显示（DD）技术。鉴定出30个基因在这三种类型的细胞中差异表达，并根据它们的表达模式分为三组。这些模式通过Northern印迹分析进一步得到证实和定量。在DD指纹图谱上，在用异辛烷培养的KK - 211菌株中，包括MUQ1、PRY2、HAC1、AGT1、GAC1和ICT1（YLR099c）在内的14个基因的表达被诱导，而包括JEN1、PRY1、PRY3和KRE1在内的其余16个基因的表达则降低。据报道，由HAC1、PRY1和ICT1代表的基因与细胞应激相关，而AGT1和GAC1分别与海藻糖的摄取和糖原的产生有关。还检测到了编码与细胞表面维持相关蛋白质的MUQ1和KRE1。基于这些结果，我们得出结论，多个基因而非单个基因表达水平的改变可能是KK - 211菌株异辛烷耐受性的关键决定因素。
