Improving furfural tolerance in a xylose-fermenting yeast Spathaspora passalidarum CMUWF1-2 via adaptive laboratory evolution.

BACKGROUND: Spathaspora passalidarum is a yeast with the highly effective capability of fermenting several monosaccharides in lignocellulosic hydrolysates, especially xylose. However, this yeast was shown to be sensitive to furfural released during pretreatment and hydrolysis processes of lignocellulose biomass. We aimed to improve furfural tolerance in a previously isolated S. passalidarum CMUWF1-2, which presented thermotolerance and no detectable glucose repression, via adaptive laboratory evolution (ALE). RESULTS: An adapted strain, AF2.5, was obtained from 17 sequential transfers of CMUWF1-2 in YPD broth with gradually increasing furfural concentration. Strain AF2.5 could tolerate higher concentrations of furfural, ethanol and 5-hydroxymethyl furfuraldehyde (HMF) compared with CMUWF1-2 while maintaining the ability to utilize glucose and other sugars simultaneously. Notably, the lag phase of AF2.5 was 2 times shorter than that of CMUWF1-2 in the presence of 2.0 g/l furfural, which allowed the highest ethanol titers to be reached in a shorter period. To investigate more in-depth effects of furfural, intracellular reactive oxygen species (ROS) accumulation was observed and, in the presence of 2.0 g/l furfural, AF2.5 exhibited 3.41 times less ROS accumulation than CMUWF1-2 consistent with the result from nuclear chromatins diffusion, which the cells number of AF2.5 with diffuse chromatins was also 1.41 and 1.24 times less than CMUWF1-2 at 24 and 36 h, respectively. CONCLUSIONS: An enhanced furfural tolerant strain of S. passalidarum was achieved via ALE techniques, which shows faster and higher ethanol productivity than that of the wild type. Not only furfural tolerance but also ethanol and HMF tolerances were improved.

Integration of comprehensive data and biotechnological tools for industrial applications of Kluyveromyces marxianus.

Among the so-called non-conventional yeasts, Kluyveromyces marxianus has extremely potent traits that are suitable for industrial applications. Indeed, it has been used for the production of various enzymes, chemicals, and macromolecules in addition to utilization of cell biomass as nutritional materials, feed and probiotics. The yeast is expected to be an efficient ethanol producer with advantages over Saccharomyces cerevisiae in terms of high growth rate, thermotolerance and a wide sugar assimilation spectrum. Results of comprehensive analyses of its genome and transcriptome may accelerate studies for applications of the yeast and may further increase its potential by combination with recent biotechnological tools including the CRISPR/Cas9 system. We thus review published studies by merging with information obtained from comprehensive data including genomic and transcriptomic data, which would be useful for future applications of K. marxianus.

Functional analysis of Mig1 and Rag5 as expressional regulators in thermotolerant yeast Kluyveromyces marxianus.

To analyze the glucose repression mechanism in the thermotolerant yeast Kluyveromyces marxianus, disrupted mutants of genes for Mig1 and Rag5 as orthologs of Mig1 and Hxk2, respectively, in Saccharomyces cerevisiae were constructed, and their characteristics were compared with those of the corresponding mutants of S. cerevisiae. MIG1 mutants of both yeasts exhibited more resistance than the corresponding parental strains to 2-deoxyglucose (2-DOG). Histidine was found to be essential for the growth of Kmmig1, but not that of Kmrag5, suggesting that MIG1 is required for histidine biosynthesis in K. marxianus. Moreover, Kmrag5 and Schxk2 were more resistant than the corresponding MIG1 mutant to 2-DOG, and only the latter increased the utilization speed of sucrose in the presence of glucose. Kmrag5 exhibited very low activities for gluco-hexokinase and hexokinase and, unlike Schxk2, showed very slow growth and a low level of ethanol production in a glucose medium. Furthermore, Kmrag5, but not Kmmig1, exhibited high inulinase activity in a glucose medium and exhibited greatly delayed utilization of accumulated fructose in the medium containing both glucose and sucrose. Transcription analysis revealed that the expression levels of INU1 for inulinase and GLK1 for glucokinase in Kmrag5 were higher than those in the parental strain; the expression level of INU1 in Kmmig1 was higher, but the expression levels of RAG1 for a low-affinity glucose transporter in Kmmig1 and Kmrag5 were lower. These findings suggest that except for regulation of histidine biosynthesis, Mig1 and Rag5 of K. marxianus play similar roles in the regulation of gene expression and share some functions with Mig1 and Hxk2, respectively, in S. cerevisiae.

Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1-2.

BACKGROUND: Efficient bioconversion of lignocellulosic biomass to bioethanol is one of key challenges in the situation of increasing bioethanol demand. The ethanologenic microbes for such conversion are required to possess abilities of utilization of various sugars including xylose and arabinose in lignocellulosic biomass. As required additional characteristics, there are a weak or no glucose repression that allows cells to simultaneously utilize various sugars together with glucose and thermotolerance for fermentation at high temperatures, which has several advantages including reduction of cooling cost. Spathaspora passalidarum ATCC MYA-4345, a type strains, isolated previously have mainly of these abilities or characteristics but its thermotolerance is not so strong and its glucose repression on xylose utilization is revealed. RESULTS: Newly isolated S. passalidarum CMUWF1-2 was found to have a high ability to produce ethanol from various sugars included in lignocellulosic biomass at high temperatures. The strain achieved ethanol yields of 0.43 g, 0.40 g and 0.20 g ethanol/g xylose at 30 °C, 37 °C and 40 °C, respectively. Interestingly, no significant glucose repression was observed in experiments with mixed sugars, being consistent with the strong resistance to 2-deoxyglucose, and antimycin A showed no effect on its growth in xylose medium. Moreover, the strain was tolerant to glucose and ethanol at concentrations up to 35.0% (w/v) and 8.0% (v/v), respectively. CONCLUSIONS: S. passalidarum CMUWF1-2 was shown to achieve efficient production of ethanol from various sugars and a high ethanol yield from xylose with little accumulation of xylitol. The strain also exhibited stress-resistance including thermotolerance and no detectable glucose repression as beneficial characteristics. Therefore, S. passalidarum CMUWF1-2 has remarkable potential for conversion of lignocellulosic biomass to bioethanol.

A Kluyveromyces marxianus 2-deoxyglucoseresistant mutant with enhanced activity of xylose utilization

Thermotolerant ethanologenic yeast Kluyveromyces marxianus is capable of fermenting various sugars including xylose but glucose represses to hamper the utilization of other sugars. To acquire glucose repression-defective strains, 33 isolates as 2-deoxyglucose (2-DOG)-resistant mutants were acquired from about 100 colonies grown on plates containing 2-DOG, which were derived from an efficient strain DMKU 3-1042. According to the characteristics of sugar consumption abilities and cell growth and ethanol accumulation along with cultivation time, they were classified into three groups. The first group (3 isolates) utilized glucose and xylose in similar patterns along with cultivation to those of the parental strain, presumably due to reduction of the uptake of 2-DOG or enhancement of its export. The second group (29 isolates) showed greatly delayed utilization of glucose, presumably by reduction of the uptake or initial catabolism of glucose. The last group, only one isolate, showed enhanced utilization ability of xylose in the presence of glucose. Further analysis revealed that the isolate had a single nucleotide mutation to cause amino acid substitution (G270S) in RAG5 encoding hexokinase and exhibited very low activity of the enzyme. The possible mechanism of defectiveness of glucose repression in the mutant is discussed in this paper (AU)

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Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses.

BACKGROUND: High-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. RESULTS: The 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. CONCLUSIONS: This 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.

A Kluyveromyces marxianus 2-deoxyglucose-resistant mutant with enhanced activity of xylose utilization.

Thermotolerant ethanologenic yeast Kluyveromyces marxianus is capable of fermenting various sugars including xylose but glucose represses to hamper the utilization of other sugars. To acquire glucose repression-defective strains, 33 isolates as 2-deoxyglucose (2-DOG)-resistant mutants were acquired from about 100 colonies grown on plates containing 2-DOG, which were derived from an efficient strain DMKU 3-1042. According to the characteristics of sugar consumption abilities and cell growth and ethanol accumulation along with cultivation time, they were classified into three groups. The first group (3 isolates) utilized glucose and xylose in similar patterns along with cultivation to those of the parental strain, presumably due to reduction of the uptake of 2-DOG or enhancement of its export. The second group (29 isolates) showed greatly delayed utilization of glucose, presumably by reduction of the uptake or initial catabolism of glucose. The last group, only one isolate, showed enhanced utilization ability of xylose in the presence of glucose. Further analysis revealed that the isolate had a single nucleotide mutation to cause amino acid substitution (G270S) in RAG5 encoding hexokinase and exhibited very low activity of the enzyme. The possible mechanism of defectiveness of glucose repression in the mutant is discussed in this paper. [Int Microbiol 18(4):235-244 (2015)].

Essentiality of respiratory activity for pentose utilization in thermotolerant yeast Kluyveromyces marxianus DMKU 3-1042.

By random integrative mutagenesis with a kanMX4 cassette in Kluyveromyces marxianus DMKU 3-1042, we obtained three mutants of COX15, ATP25 and CYC3 encoding a cytochrome oxidase assembly factor (singleton), a transcription factor required for assembly of the Atp9p subunit of mitochondrial ATP synthase and cytochrome c heme lyase, respectively, as mutants lacking growth capability on xylose and/or arabinose. They exhibited incapability of growth on non-fermentable carbon sources, such as acetate or glycerol, and thermosensitiveness. Their biomass formation in glucose medium was reduced, but ethanol yields were increased with a high ethanol level in the medium, compared to those of the parental strain. Experiments with respiratory inhibitors showed that cox15 and cyc3, but not atp25, were able to grow in glucose medium containing antimycin A and that the atp25 mutant was KCN-resistant. Activities of NADH and ubiquinol oxidases in membrane fractions of each mutant became a half of that of the parent and negligible, respectively, and their remaining NADH oxidase activities were found to be resistant to KCN. Absolute absorption spectral analysis revealed that the peak corresponding to a + a 3 was very small in atp25 and negligible in cox15 and cyc3. These findings suggest that the K. marxianus strain possesses an alternative KCN-resistant oxidase that is located between primary dehydrogenases and the ubiquinone pool and that the respiratory activity is essential for utilization of pentoses.

Utilization capability of sucrose, raffinose and inulin and its less-sensitiveness to glucose repression in thermotolerant yeast Kluyveromyces marxianus DMKU 3-1042.

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.

Growth and ethanol fermentation ability on hexose and pentose sugars and glucose effect under various conditions in thermotolerant yeast Kluyveromyces marxianus.

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.

The crucial role of alcohol dehydrogenase Adh3 in Kluyveromyces marxianus mitochondrial metabolism.

The function of mitochondrial Adh3 in the thermotolerant yeast Kluyveromyces marxianus was investigated. An ADH3-disrupted mutant exhibited growth retardation on non-fermentable carbon sources, except for ethanol, and this was suppressed by supplementation with antioxidants. Detailed analysis of the phenotype revealed that the mutant showed an increase in the activity of NADH dehydrogenase, sensitivity to H(2)O(2), and accumulation of reactive oxygen species (ROS), and that these carbon sources increased the activity of succinate dehydrogenase. The increase in both activities may reflect enhanced expression of both dehydrogenases by elevation of their substrate levels. The ROS level became low when antioxidants were added. These findings suggest that the ADH3 mutation and such carbon sources cause an elevation of the substrate level of the respiratory chain and eventually of the ROS level via increased expression of primary dehydrogenases, which in turn causes cell growth retardation. Adh3 might thus play a crucial role in the control of the NADH/NAD(+) balance in mitochondria.