Abstract
The thermotolerant Kluyveromyces marxianus is a potential candidate for high-temperature ethanol fermentation. Although K. marxianus exhibited high ethanol productivity at 45 °C during the early fermentation stage, we observed a fermentation arrest due to the accumulated inhibitors. The stress responses of K. marxianus during high-temperature fermentation were revealed based on integration of RNA sequencing (RNA-Seq) and metabolite data. High temperature stimulated mitochondrial respiration but repressed the tricarboxylic acid (TCA) cycle, leading to increased generation of reactive oxygen species (ROS) and a lowered ratio of reduced nicotinamide adenine dinucleotide (NADH)/oxidized nicotinamide adenine dinucleotide (NAD+). Glycerol production was enhanced during the early fermentation stage, which might contribute to NADH reoxidation and ROS generation. Excess ROS could be neutralized by reduced nicotinamide adenine dinucleotide phosphate (NADPH) that might be reserved in the following ways: (1) decreased biosynthesis of branched-chain amino acids (BCAAs) reduced NADPH consumption; (2) enhanced acetic acid production increased NADPH regeneration. The degree of fatty acid unsaturation was also reduced to adapt to high temperature. In addition, stress responses were also observed after the fermentation arrest at 45 °C. Genes related to peroxidase activity, iron-sulfur cluster assembly, and flavin mononucleotide (FMN) binding were downregulated, while genes associated with DNA repair and lipid composition of the plasma were upregulated. The yeast also produced more ergosterol to deal with ethanol stress. This study gains comprehensive insights into the K. marxianus transcriptome under various stresses during high-temperature ethanol fermentation, providing rich information for further metabolic engineering towards improved stress tolerance and ethanol production.
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Acknowledgements
We are grateful to Mr. Shengkai Pan, Chinese Academy of Sciences, for his help in analyzing RNA-Seq data. We thank Dr. Savitree Limtong, Kasetsart University, Thailand, for her help in purchasing K. marxianus DMKU3-1042. We also thank Ms. Lan Summer Sun, China National Knowledge Infrastructure, for her generous help in the English editing.
Funding
This study was funded by the National Natural Science Foundation of China (grant numbers 31600067, 21506113, 31470229), the National Key R&D Program of China (grant number 2016YFE0108500), and the China Postdoctoral Science Foundation (grant numbers 2017T100064, 2015M581074).
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XF and PL contributed equally to this work. PL, SL, and LZ designed the experiments. XF performed the fermentation experiments, HPLC analysis, assays of NAD(H) and NADP(H), and RNA extraction. PL and XF performed the assays of ROS and pyruvate. PL analyzed the RNA-Seq data and wrote the manuscript. All authors read and approved the final manuscript.
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Fu, X., Li, P., Zhang, L. et al. Understanding the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation based on integration of RNA-Seq and metabolite data. Appl Microbiol Biotechnol 103, 2715–2729 (2019). https://doi.org/10.1007/s00253-019-09637-x
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DOI: https://doi.org/10.1007/s00253-019-09637-x