Physiological and molecular characterization of yeast cultures pre-adapted for fermentation of lignocellulosic hydrolysate

To reach an economically feasible bioethanol process from lignocellulose, efficient fermentation by yeast of all sugars present in the hydrolysate has to be achieved. However, when exposed to lignocellulosic hydrolysate, Saccharomyces cerevisiae is challenged with a variety of inhibitors that reduce yeast viability, growth and fermentation rate, and in addition damage cellular structures. In order to evaluate the yeast capability to adapt to lignocellulosic hydrolysates and to investigate the yeast molecular response to inhibitors, fed-batch cultivation of an industrial S. cerevisiae strain was performed using either spruce hydrolysate or a sugar medium as feed. The physiological effects of cultivating yeast in spruce hydrolysate was comprehensively studied by assessment of yeast performance in simultaneous saccharification and fermentation (SSF), measurement of furaldehyde reduction activity, assessment of conversion of phenolic compounds and genome wide transcription analysis. The yeast cultivated in spruce hydrolysate developed a rapid adaptive response to lignocellulosic hydrolysate, which significantly improved its fermentation performance in subsequent SSF experiments. Yeast adaptation to hydrolysate was shown to involve induction of NADPH-dependent aldehyde reduction activity and conversion of phenolic compounds during the fed-batch cultivation and these properties were correlated to the expression of several genes encoding oxido-reductase activities, notably AAD4, ADH6, OYE2/3 and YML131w. The other most significant transcriptional changes involved genes involved in transport mechanisms, such as YHK8, FLR1 or ATR1. A large set of genes were found to be associated to transcription factors involved in stress response (Msn2p, Msn4p, Yap1p but also cell growth and division (Gcr4p, Ste12p, Sok2p) that were most likely activated at the post-transcriptional level.为实现木质纤维素基生物乙醇工艺的经济可行性，必须实现酵母对水解液中所有糖类的高效发酵。然而，当酿酒酵母（Saccharomyces cerevisiae）暴露于木质纤维素水解液时，会面临多种抑制剂的胁迫：这些抑制剂会降低酵母的存活率、生长速率与发酵效率，同时还会破坏细胞结构。为评估酿酒酵母对木质纤维素水解液的适应能力，并探究其对抑制剂的分子响应机制，本研究以工业酿酒酵母（S. cerevisiae）菌株为对象，分别以云杉水解液与糖基培养基为补料底物，开展补料分批培养实验。本研究通过同步糖化发酵（SSF）实验评估酵母发酵性能、检测糠醛还原酶活性、分析酚类化合物转化情况，并开展全基因组转录组分析，对云杉水解液培养酵母的生理效应进行了全面解析。经云杉水解液培养的酿酒酵母可快速产生对木质纤维素水解液的适应性响应，这一特性显著提升了其在后续同步糖化发酵实验中的发酵性能。研究表明，在补料分批培养过程中，酵母对水解液的适应性响应涉及NADPH依赖型醛还原酶活性的诱导与酚类化合物的转化，这些特性与多个编码氧化还原酶的基因表达相关，其中包括AAD4、ADH6、OYE2/3及YML131w。其余显著的转录组变化涉及转运机制相关基因，例如YHK8、FLR1与ATR1。此外，大量基因与应激响应相关转录因子（Msn2p、Msn4p、Yap1p）及细胞生长分裂相关转录因子（Gcr4p、Ste12p、Sok2p）相关，这些转录因子极有可能在转录后水平被激活。