Transcriptional response of ethanol-stressed vs. non-stressed culture. Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is well known for its high ethanol production performances. An original fermentation process that allows the yeast S. cerevisiae to produce in less than 45 h more than 150 g/l ethanol (i.e. 18.9°GL) was set up in our laboratory [1]. Under this condition, the yeast cells induce a dynamic process to adapt to increased ethanol concentration by a mechanism that is likely different to the stress response triggered by sudden ethanol addition to exponentially growing cells [2]. Kinetic analysis of the growth curve identified two main phases: a growth phase that ended up at 90 g/l ethanol and then an uncoupling phase during which non-growing cells kept producing ethanol. This latter phase is also characterized with an increased loss of viability. In order to investigate on a genome scale the expression changes occurring during this process, gene expression was quantified using DNA chips technology at six different time-points during fed-batch fermentation. [1] Alfenore et al, Appl. Microbiol. Biotechnol. 60 : 67-72, 2002. [2] Alexandre H. et al., FEBS Lett. 498(1) : 98-103, 2001. Overall design: We measure the changes in the gene expression of ethanol stressed culture at five different time-points during fed-batch fermentation compared to a common reference consisting of exponentially growing yeast cells ( sample number 1 : growing cells ; low ethanol concentration of 17 g/l ; specific growth rate of 0.3 h-1) . The sets corresponded to sample number 2 : growing cells/ethanol concentration of 60 g/l ; sample number 3 : before growth arrest/ethanol concentration of 90 g/l ; sample number 4 : growth arrest/ethanol concentration of 95 g/l ; sample number 5 : 1 hour after growth arrest/ethanol concentration of 100 g/l and sample number 6 : uncoupling phase/ethanol concentration of 125 g/l. For each sample, total RNAs from one yeast culture (no biological replicate) were extracted three times (technical replicates -extract). For labelling, we labelled the common reference with dCTP-Cy5 and labelled the sample with dCTP-Cy3 and hybridized cDNA on three independent microarrays, given rise to six data value per gene (each gene is duplicate in the slide).

酿酒酵母（Saccharomyces cerevisiae）以其高效的乙醇生产性能而闻名。本实验室建立了一套原创发酵工艺，可使酿酒酵母在45小时内合成超过150 g/L乙醇（即18.9°GL）[1]。在此培养条件下，酵母细胞会启动动态适应过程以应对乙醇浓度的升高，其机制可能与向指数生长期细胞中瞬时添加乙醇所触发的应激反应存在显著差异[2]。对生长曲线的动力学分析揭示了两个核心阶段：生长阶段终止于乙醇浓度达到90 g/L时，随后进入解偶联阶段，此时非增殖细胞仍可持续合成乙醇；该阶段同时伴随细胞存活率的显著降低。

为在基因组规模上解析该过程中的基因表达变化，本研究采用DNA芯片（DNA chips）技术，在补料分批发酵（fed-batch fermentation）过程中的6个不同时间点对基因表达水平进行定量检测。
[1] Alfenore 等, Appl. Microbiol. Biotechnol. 60: 67-72, 2002.
[2] Alexandre H. 等, FEBS Lett. 498(1): 98-103, 2001.

总体实验设计：本研究以指数生长期的酵母细胞作为通用对照样本（样本1：生长细胞，乙醇浓度17 g/L，比生长速率0.3 h⁻¹），对比分析补料分批发酵过程中5个不同时间点的乙醇胁迫培养物的基因表达变化。各待测样本信息如下：样本2：生长细胞，乙醇浓度60 g/L；样本3：生长停滞前，乙醇浓度90 g/L；样本4：生长停滞期，乙醇浓度95 g/L；样本5：生长停滞1小时后，乙醇浓度100 g/L；样本6：解偶联阶段，乙醇浓度125 g/L。
每个样本仅来自一次酵母培养物（无生物学重复），共进行三次总RNA提取（即技术重复-提取步骤）。标记实验中，将通用对照样本用dCTP-Cy5进行荧光标记，待测样本用dCTP-Cy3进行荧光标记，随后将标记后的cDNA与三张独立的微阵列芯片进行杂交，每个基因可获得六个有效数据值（芯片上每个基因设有两个重复位点）。