Comparative transcriptome profiling analyses during the lag phase uncover YAP1, PDR1, PDR3, RPN4 and HSF1 as key regulatory genes in genomic adaptation to the lignocellulose derived inhitibor-stress for saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to adapt and in situ detoxify lignocellulose derived inhibitors such as furfural and HMF. The length of lag phase for cell growth in response to the inhibitor challenge has been used to measure tolerance of strain performance. Mechanisms of yeast tolerance at the genome level remain unknown. Using systems biology approache, this study investigated comparative transcriptome profiling, metabolic profiling, cell growth response and gene regulatory interactions of yeast strains and selective gene deletion mutations in response to HMF challenges during the lag phase of growth. Three hundred and sixty-five candidate genes were identified and found at least three significant components involving some of these genes that enable yeast adaptation and tolerance to HMF in yeast. First, functional enzyme coding genes such as ARI1, ADH6, ADH7 and OYE3, as well as gene interactions involved in the biotransformation and inhibitor detoxification were direct driving force to reduce HMF damages in cells. Expressions of these genes were regulated by YAP1 and its closely related regulons. Second, a large number of PDR genes, mainly regulated by PDR1 and PDR3, were induced during the lag phase and the PDR gene family-centered functions, including specific and multiple functions involving cellular transport such as TPO1, TPO4, RSB1, PDR5, PDR15, YOR1, and SNQ2, promoted cellular adaptation and survival in order to cope with the inhibitor stress. Third, expressed genes involving degradation of damaged proteins and protein modifications such as SHP1 and SSA4, regulated by RPN4, HSF1 and other co-regulators, were necessary for yeast cells to survive and adapt the HMF stress. A deletion mutation strainΔrpn4 was unable to recover the growth in the presence of HMF. Complex gene interactions and regulatory networks as well as co-regulations exist in yeast adaptation and tolerance to the lignocellulose derived inhibitor HMF. Both induced and repressed genes involving diversified functional categories are accountable for adaptation and energy rebalancing in yeast to survive and adapt the HMF stress during the lag phase of growth. Transcription factor genes YAP1, PDR1, PDR3, RPN4 and HSF1 appeared to play key regulatory rules for global adaptation in the yeast S. cerevisiae. Overall design: Cells were incubated on SC medium with HMF(30 mM) 6 h after pre-culture.Cultures grown under the same conditions without the HMF served as control. cells were harvested from 0, 10, 30, 60, 120 min, immediately frozen on dry ice, and then stored at -80 ℃. Two replicated experiments were carried out for each condition. Total RNA was isolated by hot phenol method and purified using RNeasy Mini Kit. Genome microarray of S. cerevisiae was fabricated with a version of 70-mer oligo set representing 6,388 genes. A mini-array consisting of quality control genes was designed on the top of the target array. Cy5 labeled RNA at 0 time point was designated as a reference and Cy3 was used to label test samples. An equal amount of at least 30 pmol Cy3 and Cy5 labeling reaction was applied for hybridization. Hybridization was performed based on Hegde et al (2000) with modifications using HS 4800 Hybridization station.

酿酒酵母（Saccharomyces cerevisiae）具有适应并原位解毒木质纤维素衍生的抑制剂，如糠醛和HMF的能力。针对抑制剂挑战引发的细胞生长滞后期的长度已被用作衡量菌株性能耐受度的指标。在基因组层面，酵母耐受的机制尚属未知。本研究采用系统生物学方法，对酵母菌株及其在生长滞后期对HMF挑战的响应中出现的选择性基因删除突变体，进行了比较转录组分析、代谢组分析、细胞生长反应和基因调控相互作用的探究。共确定了365个候选基因，并发现至少三个与这些基因相关的重要组分，这些组分使得酵母能够适应并对HMF产生耐受。首先，功能酶编码基因，如ARI1、ADH6、ADH7和OYE3，以及参与生物转化和抑制剂解毒的基因相互作用，是降低细胞内HMF损害的直接驱动力。这些基因的表达受到YAP1及其密切相关调控子的调控。其次，在滞后期大量PDR基因被诱导表达，主要由PDR1和PDR3调控，以PDR基因家族为中心的功能，包括涉及细胞运输的特定和多重功能，如TPO1、TPO4、RSB1、PDR5、PDR15、YOR1和SNQ2，促进了细胞对抑制剂压力的适应和生存。第三，涉及受损蛋白质降解和蛋白质修饰的基因，如SHP1和SSA4，受到RPN4、HSF1等共调节因子的调控，对于酵母细胞在HMF压力下的生存和适应至关重要。Δrpn4删除突变菌株在HMF存在的情况下无法恢复生长。在酵母对木质纤维素衍生的抑制剂HMF的适应和耐受中，存在复杂的基因相互作用和调控网络，以及共调控现象。涉及多样化功能类别的诱导和抑制基因，均对酵母在生长滞后期对HMF压力的适应和能量平衡负责。转录因子基因YAP1、PDR1、PDR3、RPN4和HSF1在酵母S. cerevisiae的全局适应中似乎发挥着关键的调控作用。总体设计：在预培养6小时后，将细胞在含有HMF（30 mM）的SC培养基上孵育。在相同条件下未添加HMF的培养物作为对照。在0、10、30、60、120分钟时收集细胞，立即在干冰上冷冻，并储存于-80℃。每个条件进行两次重复实验。通过热酚法提取总RNA，并使用RNeasy Mini Kit进行纯化。使用代表6,388个基因的70-mer寡核苷酸阵列构建S. cerevisiae基因组微阵列。在目标阵列顶部设计了一个包含质量控制基因的小型阵列。0时间点的Cy5标记RNA作为参考，Cy3用于标记测试样本。至少30 pmol的Cy3和Cy5标记反应用于杂交。杂交基于Hegde等（2000）的方法进行，并使用HS 4800杂交站进行了修改。