Sornsiri Pattanakittivorakul

Laboratory evolution is an effective means of understanding microbial adaptation to the environment. We previously isolated four thermoadapted Zymomonas mobilis mutants, which showed a 2 °C rise in the critical high temperature (CHT), by performing multiple-parallel adaptation experiments. In the present study, the individual mutations in these mutants were intensively analyzed. Two mutations in each adapted mutant were found to primarily contribute to the increase in the upper temperature limit. RNA sequencing (RNA-seq) analysis revealed that the two mutations led to the upregulation of 79–185 genes and the downregulation of 242–311 genes. The findings from transcriptomic and physiological experiments suggest two common and primary mechanisms for thermal resistance: a decrease in the activity of diacylglycerol kinase, which may change the structure of lipopolysaccharide (LPS) probably to strengthen the membrane structure, and an increase in the expression of genes for GroEL/GroES or cell wall hydrolase to repair the protein or membrane damage that occurs at such critical temperatures. Additionally, transporters including efflux pumps may contribute to intracellular homeostasis by expelling toxic compounds such as ethanol and acetate or by maintaining the K⁺ concentration. The results of this study on four independently thermoadapted mutants led to the conclusion that the mutants have almost the same thermal adaptation strategies and thus their molecular diversity is limited.

High-temperature fermentation (HTF) of ethanol can reduce costs of cooling, sterilization, and related equipment compared to the costs of general ethanol fermentation. To realize HTF, however, there are various issues to be considered, such as the fermentation temperature upper limit for ethanol-producing thermotolerant yeast, the size of a fermenter that does not require cooling, and the effective temperature for suppressing microbial contamination. This study focused on these issues and also on downstream processes that exploit the advantages of HTF at temperatures exceeding 40 °C. The permissible size of a fermenter without cooling was estimated by simulating heat generation and heat dissipation. Fermentation productivity at high temperatures when using the thermotolerant yeast Kluyveromyces marxianus and the inhibitory effect of high temperatures on the growth of contaminant microorganisms were examined. After fermentation, the recovery and concentration of ethanol were performed by reduced-pressure distillation (RPD) and membrane separation. These experiments demonstrate that efficient HTF can reduce the amount of saccharifying enzymes in simultaneous saccharification and fermentation and can shorten the transition time from the saccharification step to the fermentation step in separate saccharification and fermentation, that RPD at fermentation temperatures enables a smooth connection to the HTF step and can be performed with a relatively weak vacuum, and that membrane separation can reduce the running cost compared to the cost of general distillation on a compact scale.

Kluyveromyces marxianus is an attractive thermotolerant yeast species for ethanol production because of its ability to utilize various carbon sources as a fermentation substrate. The use of thermotolerant microorganisms enables the performance of high-temperature ethanol fermentation, which has several advantages, including the reduction of cooling costs and minimization of contamination risks. To improve K. marxianus for ethanol fermentation under stress conditions, two strains, DMKU 3-1042 and DMKU 3-118, were adapted for heat resistance and resistance to toxic substances in pulp wastewater from a paper mill, respectively, resulting in the generation of KMR1042 and KMR118, respectively. Both adapted mutants exhibited clumpy clusters of cells as pseudo-hyphae and altered colony morphology, and their sedimentation speeds were much faster than those of the corresponding parent strains. The two mutants showed stronger tolerance to various stresses and higher performance for ethanol production than those of the corresponding parent strains at high temperatures or in the presence of toxic substances. Genome sequencing analysis revealed that both mutants had disruption of the same gene, SWI5, despite adaptation under different stress conditions, suggesting that the formation of pseudo-hyphae is a common strategy of K. marxianus for coping with stresses.

Kluyveromyces marxianus is an attractive thermotolerant yeast species for ethanol production because of its ability to utilize various carbon sources as a fermentation substrate. The use of thermotolerant microbes enables performance of high-temperature ethanol fermentation, which has several advantages including reduction of cooling costs and minimization of contamination risk. To improve K. marxianus for ethanol fermentation under stress conditions, two strains, DMKU 3-1042 and DMKU 3-118, were adapted for heat resistance and toxic substances in pulp wastewater from a paper mill, respectively, resulting in the generation of KMR1042 and KMR118, respectively. Both adapted mutants exhibited clumpy clusters of cells as pseudohyphae and altered colony morphology, and their sedimentation speeds were much faster than those of the corresponding parent strains. The two mutants showed stronger tolerance to various stresses and higher performance for ethanol production than those of the corresponding parent strains at high temperatures or in the presence of toxic substances. Genome sequencing analysis revealed that both mutants had disruption of the same gene, SWI5, despite adaptation under different stress conditions, suggesting that the formation of pseudohyphae is a common strategy of K. marxianus for coping with stresses.

Several cellulose-hydrolysis enzymes are required for eco-friendly utilization of cellulose as renewable biomass, and it would therefore be beneficial if fermenting microbes can provide such enzymes without genetic engineering. Thermotolerant and multisugar-fermenting Kluyveromyces marxianus is one of the promising yeasts for high-temperature fermentation and has genes for putative oligosaccharide-degradation enzymes. Mutants obtained after multiple mutagenesis showed significantly higher activity than that of the parental strain for cellobiose fermentation. The efficient strains were found to have amino acid substitutions and frame-shift mutations in 26-28 genes including 3 genes for glucose transporters. These strains grown in a cellobiose medium showed higher β-glucosidase than that of the parental strain and greatly reduced glucose utilization. The introduction of KTH2 for a glucose transporter into one of the efficient mutants reduced the cellobiose fermentation activity of the mutant. The results suggest that release from glucose repression significantly promotes the uptake of cellobiose. Co-culture of one efficient strain and the parental strain allowed good fermentation of both glucose and cellobiose, suggesting that the efficient strains are useful for conversion of cellulosic biomass to ethanol.

During ethanol fermentation, yeast cells are exposed to various stresses that have negative effects on cell growth, cell survival, and fermentation ability. This study, therefore, aims to develop Kluyveromyces marxianus-adapted strains that are multi-stress tolerant and to increase ethanol production at high temperatures through a novel evolutionary adaptation procedure. K. marxianus DMKU 3-1042 was subjected to repetitive long-term cultivation with gradual increases in temperature (RLCGT), which exposed cells to various stresses, including high temperatures. In each cultivation step, 1% of the previous culture was inoculated into a medium containing 1% yeast extract, 2% peptone, and 2% glucose, and cultivation was performed under a shaking condition. Four adapted strains showed increased tolerance to ethanol, furfural, hydroxymethylfurfural, and vanillin, and they also showed higher production of ethanol in a medium containing 16% glucose at high temperatures. One showed stronger ethanol tolerance. Others had similar phenotypes, including acetic acid tolerance, though genome analysis revealed that they had different mutations. Based on genome and transcriptome analyses, we discuss possible mechanisms of stress tolerance in adapted strains. All adapted strains gained a useful capacity for ethanol fermentation at high temperatures and improved tolerance to multi-stress. This suggests that RLCGT is a simple and efficient procedure for the development of robust strains.