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A thermotolerant ethanol fermenting yeast strain is a key requirement for effective ethanol production at high temperature. This work aimed to select a thermotolerant yeast producing a high ethanol concentration from molasses and increasing its ethanol production by mutagenesis. Saccharomyces cerevisiae DMKU 3-S087 was selected from 168 ethanol producing strains because it produced the highest ethanol concentration from molasses at 40 °C. Optimization of molasses broth composition was performed by the response surface method using Box–Behnken design. In molasses broth containing optimal total fermentable sugars (TFS) of 200 g/L and optimal (NH4)2SO4 of 1 g/L, with an initial pH of 5.5 by shaking flask cultivation at 40 °C ethanol, productivity and yield were 58.4 ± 0.24 g/L, 1.39 g/L/h and 0.29 g/g, respectively. Batch fermentation in a 5 L stirred-tank fermenter with 3 L optimized molasses broth adjusted to an initial pH of 5.5 and fermentation controlled at 40 °C and 300 rpm agitation resulted in 72.4 g/L ethanol, 1.21 g/L/h productivity and 0.36 g/g yield at 60 h. Strain DMKU 3-S087 improvement was performed by mutagenesis using ultraviolet radiation and ethyl methane sulfonate (EMS). Six EMS mutants produced higher ethanol (65.2 ± 0.48–73.0 ± 0.54 g/L) in molasses broth containing 200 g/L TFS and 1 g/L (NH4)2SO4 by shake flask fermentation at 37 °C than the wild type (59.8 ± 0.25 g/L). Among these mutants, only mutant S087E100-265 produced higher ethanol (62.5 ± 0.26 g/L) than the wild type (59.5 ± 0.02 g/L) at 40 °C. In addition, mutant S087E100-265 showed better tolerance to high sugar concentration, furfural, hydroxymethylfurfural and acetic acid than the wild type.

This work aimed to evaluate in vitro antagonistic activities and mechanisms of endophytic yeasts against phytopathogenic fungi. A total of 407 yeast strains isolated from tissue of rice, corn, and sugarcane leaves were evaluated for their antagonistic activities against ten phytopathogenic fungi. Only strains of Wickerhamomyces anomalus and Kodamaea ohmeri were found to inhibit the growth of phytopathogenic fungi. Wickerhamomyces anomalus (seven strains) showed antagonistic activity against Curvularia lunata (cause of dirty panicle disease of rice), three Fusarium moniliforme strains (cause of bakanae disease of rice, stalk rot disease of corn, and red rot disease of sugarcane), and Rhizoctonia solani (cause of sheath blight disease of rice). Whereas four Kodamae ohmeri strains inhibited growth of F. moniliforme (cause of bakanae disease of rice). Antagonistic mechanisms of W. anomalus were based on the production of volatile organic compounds (VOCs) (mainly 3-methyl-1-butyl acetate and 3-methyl-1-butanol), fungal cell wall-degrading enzymes (β-1,3-glucanase and chitinase), and siderophores as well as phosphate and zinc oxide solubilization. As for K. ohmeri, the production of VOCs (mainly 3-methyl-1-butanol), β-1,3-glucanase and chitinase were hypothesized as possible mechanisms. The antagonistic activity of W. anomalus against these phytopathogenic fungi and of K. ohmeri against F. moniliforme, and the analysis of the VOCs produced by K. ohmeri are reported for the first time. Two W. anomalus strains, DMKU-RE13 and DMKU-CE52, were evaluated for controlling rice sheath blight disease caused by R. solani in rice plants in the greenhouse and found to suppress the disease 55.2–65.1%, whereas 3% validamycin suppressed this disease by 88.5%.

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.

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. [PUBLICATION ABSTRACT]

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 .

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.

The fermentation ability of thermotolerant Kluyveromyces marxianus BUNL-21 isolated in Laos was investigated. Comparison with thermotolerant K. marxianus DMKU3-1042 as one of the most thermotolerant yeasts isolated previously revealed that the strain possesses stronger ability for conversion of xylose to ethanol, resistance to 2-deoxyglucose in the case of pentose, and tolerance to various stresses including high temperature and hydrogen peroxide. K. marxianus BUNL-21 was found to have ethanol fermentation activity from xylose that is slightly lower and much higher than that of Scheffersomyces stipitis ( Pichia stipitis ) at 30 °C and at higher temperatures, respectively. The lower ethanol production seems to be due to large accumulation of acetic acid. The possible mechanism of acetic acid accumulation is discussed. In addition, it was found that both K. marxianus strains produced ethanol in the presence of 10 mM hydroxymethylfurfural or furfural, at a level almost equivalent to that in their absence. Therefore, K. marxianus BUNL-21 is a highly competent yeast for high-temperature ethanol fermentation with lignocellulosic biomass.

By random integrative mutagenesis with a kan MX4 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.

Zymomonas mobilis ZmCytC as a peroxidase bearing three heme c-binding motifs was investigated with ΔZmcytC constructed. The mutant exhibited filamentous shapes and reduction in growth under a shaking condition at a high temperature compared to the parental strain and became hypersensitive to exogenous H(2)O(2). Under the same condition, the mutation caused increased expression of genes for three other antioxidant enzymes. Peroxidase activity, which was detected in membrane fractions with ubiquinol-1 as a substrate but not with reduced horse heart cytochrome c, was almost abolished in ΔZmcytC. Peroxidase activity was also detected with NADH as a substrate, which was significantly inhibited by antimycin A. NADH oxidase activity of ΔZmcytC was found to be about 80% of that of the parental strain. The results suggest the involvement of ZmCytC in the aerobic respiratory chain via the cytochrome bc(1) complex in addition to the previously proposed direct interaction with ubiquinol and its contribution to protection against oxidative stress.

This study aimed to isolate and identify yeasts from peat in To Daeng peat swamp forest in southern of Thailand, and to investigate their ability to produce ethanol from glucose and xylose and to produce indole-3-acetic acid (IAA) and extracellular enzymes. A total of 65 yeast strains were obtained from 15 peat samples using an enrichment technique, and 61 strains were identified to be five species belonging to the phylum Ascomycota, namely Cyberlindnera subsufficiens , Debaryomyces fabryi , Meyerozyma guilliermondii , Saturnispora diversa and Schwanniomyces polymorphus var. africanus , and five species of the phylum Basidiomycota, namely Cryptococcus taiwanensis pro tem, Cutaneotrichosporon mucoides , Papiliotrema flavescens , Papiliotrema laurentii and Rhodotorula mucilaginosa . Four strains were unidentified and require further analysis. They differed from the type strain of P. flavescens by two nucleotide substitutions in the D1/D2 region of the LSU rRNA gene and nine nucleotide substitutions in the ITS region. R. mucilaginosa was the most prevalent yeast species, followed by S. polymorphus var. africanus , Cy. subsufficiens and D. fabryi . None of the yeast strains obtained in this study were able to ferment xylose to ethanol, but all ascomycetous yeast strains produced ethanol from glucose in a range of 9.0–58.0 g/L, with Cy. subsufficiens DMKU-YNB42-1 producing the highest ethanol concentration. A total of 62 strains produced IAA in a range of 9.0 to 66.9 mg/L, with the highest IAA produced by R. mucilaginosa DMKU-Y33-A. Investigation of the production of cellulases, xylanase, pectinase, amylase, protease and lipase revealed that all 65 yeast strains produced at least one extracellular enzyme, a lipase.