RNA-seq based transcriptomic analysis of the non-conventional yeast Spathaspora passalidarum during Melle-boinot cell recycle in xylose-glucose mixtures. RNA-seq based transcriptomic analysis of the non-conventional yeast Spathaspora passalidarum during Melle-boinot cell recycle in xylose-glucose mixtures

The Mellet-boinot is a promising process to be applied for second-generation ethanol production by wild yeasts. However, the impact of this process on the physiology and fermentative performance of the xylose-fermenting yeast Spathaspora passalidarum during second-generation ethanol production, remains elusive. Therefore, we have conducted a deep transcriptomic analysis of S. passalidarum during five consecutive fed-batches and cell recycles, to determine the differences and global responses of differentially expressed genes (DEGs) during the Melle-Boinot process. A physiological adaptation was observed resulting in an increase on ethanol yield, ethanol volumetric productivity and ethanol titer. Furthermore, a decrease of the subproduct xylitol was also observed. A transcriptional regulation was achieved from the third cell recycle onwards and this regulation was maintained afterwards. Analysing the DEGs during the recycles showed an up-regulation of genes involved in ATP synthesis, N-Glycan biosynthesis, oxidative phosphorylation and purine metabolism, indicating as important mechanisms for adaptation throughout recycles due increased ethanol concentration. Moreover, the TCA cycle anabolic pathway, gluconeogenesis, glycogen and trehalose biosynthesis, fatty acid and sterol biosynthesis were also affected mainly due ethanol concentration and osmotic pressure implying that cell energy was generated towards the production of cell wall components in order to S. passalidarum cells to thrive in consecutive recycles. Taken together these results demonstrate that the Melle-Boinot process is a worthy strategy to be applied on 2G ethanol fermentation by native yeasts. Furthermore, we highlight major microbial molecular strategies for xylose conversion providing relevant insights for further metabolic engineering aiming to improve 2G bioethanol production. Overall design: We have conducted a deep transcriptomic analysis of S. passalidarum during five consecutive fed-batches and cell recycles, to determine the differences and global responses of differentially expressed genes (DEGs) during the Melle-Boinot fermentation process.

梅勒-博伊诺特（Melle-Boinot）工艺是一种极具应用前景的工艺，可用于野生酵母（wild yeasts）发酵生产第二代乙醇（second-generation ethanol）。然而，该工艺对木糖发酵酵母（xylose-fermenting yeast）帕拉达鲁姆匙囊酵母（Spathaspora passalidarum）在第二代乙醇生产过程中的生理特性与发酵性能的影响，目前仍未明确。为此，本研究对连续开展5次补料分批发酵（fed-batches）与细胞循环（cell recycles）的帕拉达鲁姆匙囊酵母进行了深度转录组分析（transcriptomic analysis），以明确梅勒-博伊诺特工艺过程中差异表达基因（differentially expressed genes, DEGs）的表达差异与全局响应特征。研究观察到，该酵母通过生理适应性进化，提升了乙醇得率（ethanol yield）、乙醇体积产率（ethanol volumetric productivity）与乙醇效价（ethanol titer）；同时还降低了副产物木糖醇（subproduct xylitol）的生成量。自第三次细胞循环起，转录调控（transcriptional regulation）机制开始发挥作用，并在此后持续维持。对循环过程中的差异表达基因进行分析后发现，参与ATP合成（ATP synthesis）、N-糖基化生物合成（N-Glycan biosynthesis）、氧化磷酸化（oxidative phosphorylation）及嘌呤代谢（purine metabolism）的基因呈现上调表达，提示上述通路是应对乙醇浓度升高带来的环境压力、实现连续循环过程中适应性存活的关键机制。此外，三羧酸循环（TCA cycle）合成代谢途径、糖异生（gluconeogenesis）、糖原（glycogen）与海藻糖生物合成（trehalose biosynthesis）、脂肪酸及固醇生物合成（sterol biosynthesis）也均受到显著调控，这主要源于乙醇浓度与渗透压（osmotic pressure）的变化，表明细胞能量被优先导向细胞壁组分的合成，以帮助帕拉达鲁姆匙囊酵母在连续细胞循环过程中存活与增殖。综上，本研究结果证实梅勒-博伊诺特工艺是一种适用于野生酵母发酵生产第二代乙醇的可行策略。此外，本研究还阐明了木糖转化过程中的核心微生物分子策略，为后续通过代谢工程（metabolic engineering）优化第二代生物乙醇（2G bioethanol）生产提供了重要的理论参考。实验设计：本研究对连续开展5次补料分批发酵与细胞循环的帕拉达鲁姆匙囊酵母进行了深度转录组分析，旨在明确梅勒-博伊诺特发酵过程中差异表达基因的表达差异与全局响应特征。