BackgroundHigh-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of cellulosic biomass to fuels or chemicals. Thermotolerant Kluyveromyces marxianus possesses intrinsic abilities to ferment and assimilate a wide variety of substrates including xylose and to efficiently produce proteins. These capabilities have been found to exceed those of the traditional ethanol producer Saccharomyces cerevisiae or lignocellulose-bioconvertible ethanologenic Scheffersomyces stipitis.ResultsThe complete genome sequence of K. marxianus DMKU 3-1042 as one of the most thermotolerant strains in the same species has been determined. A comparison of its genomic information with those of other yeasts and transcriptome analysis revealed that the yeast bears beneficial properties of temperature resistance, wide-range bioconversion ability, and production of recombinant proteins. The transcriptome analysis clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of reactive oxygen species, which tend to accumulate under all three conditions.ConclusionsThis study reveals many gene resources for the ability to assimilate various sugars in addition to species-specific genes in K. marxianus, and the molecular basis of its attractive traits for industrial applications including high-temperature fermentation. Especially, the thermotolerance trait may be achieved by an integrated mechanism consisting of various strategies. Gene resources and transcriptome data of the yeast are particularly useful for fundamental and applied researches for innovative applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0227-x) contains supplementary material, which is available to authorized users.

Ethanol fermentation ability of the thermotolerant yeast Kluyveromyces marxianus, which is able to utilize various sugars including glucose, mannose, galactose, xylose, and arabinose, was examined under shaking and static conditions at high temperatures. The yeast was found to produce ethanol from all of these sugars except for arabinose under a shaking condition but only from hexose sugars under a static condition. Growth and sugar utilization rate under a static condition were slower than those under a shaking condition, but maximum ethanol yield was slightly higher. Even at 40°C, a level of ethanol production similar to that at 30°C was observed except for galactose under a static condition. Glucose repression on utilization of other sugars was observed, and it was more evident at elevated temperatures. Consistent results were obtained by the addition of 2-deoxyglucose. The glucose effect was further examined at a transcription level, and it was found that KmGAL1 for galactokinase and KmXYL1 for xylose reductase for galactose and xylose/arabinose utilization, respectively, were repressed by glucose at low and high temperatures, but KmHXK2 for hexokinase was not repressed. We discuss the possible mechanism of glucose repression and the potential for utilization of K. marxianus in high-temperature fermentation with mixed sugars containing glucose.

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The respiratory chain of the ethanologenic bacterium Zymomonas mobilis was investigated, in which the pyruvate-to-ethanol pathway has been demonstrated to be mainly responsible for NADH oxidation and the tricarboxylic acid cycle is incomplete. Membranes from cells cultivated under aerobic or anaerobic growth conditions showed dehydrogenase and oxidase activities for NADH, D-lactate and D-glucose and ubiquinol oxidase activity. Intriguingly, the NADH oxidase activity level of membrane fractions from cells grown aerobically was found to be higher than that of membrane fractions from Escherichia coli or Pseudomonas putida grown aerobically, indicating a crucial role of the respiratory chain in NADH oxidation in the organism. Cyanide-resistant terminal oxidase activity was observed and appeared to be due to a bd-type ubiquinol oxidase as the only terminal oxidase encoded by the entire genome. The terminal oxidase with a relatively strong ubiquinol oxidase activity exhibited remarkably weak signals of cytochrome d. Considering these findings and the presence of a type-II NADH dehydrogenase but not a type-I, a simple respiratory chain that generates less energymay have evolved in Z. mobilis.

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Kluyveromyces marxianus possesses a useful potential to assimilate a wide variety of substrates at a high temperature, but the negative effect by coexisting glucose is critical for utilization of biomass containing various sugars. Such a negative effect on the activity of inulinase, which is the sole enzyme to hydrolyze sucrose, raffinose and inulin, has been demonstrated in K. marxianus without analysis at the gene level. To clarify the utilization capability of sucrose, raffinose and inulin and the glucose effect on inulinase in K. marxianus DMKU 3-1042, its growth and metabolite profiles on these sugars were examined with or without glucose under a static condition, in which glucose repression evidently occurs. Consumption of sucrose was not influenced by glucose or 2-deoxyglucose. On the other hand, raffinose and inulin consumption was hampered by glucose at 30°C but hardly hampered at 45°C. Unlike Saccharomyces cerevisiae, increase in glucose concentration had no effect on sucrose utilization. These sugar-specific glucose effects were consistent with the level of inulinase activity but not with that of the KmINU1 transcript, which was repressed in the presence of glucose via KmMig1p. This inconsistency may be due to sufficient activity of inulinase even when glucose is present. Our results encourage us to apply K. marxianus DMKU 3-1042 to high-temperature ethanol fermentation with biomass containing these sugars with glucose.

Considering its advantages including reduction of cooling cost and saving water during the fermentation process, which consequently cut down the total running cost, high-temperature fermentation with thermotolerant microbes is expected to be one of next-generation fermentation technologies. We focused on the establishment of high-temperature fermentation technology for ethanol production from biomass in Thailand, for which thermotolerant microbes suitable for various types of biomass were selected and advanced fermentation processes including a temperature-uncontrolled fermentation and a simultaneous fermentation and distillation under a low pressure were investigated.

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