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)]Keywords: Kluyveromyces marxianus · glucose repression · 2-deoxyglucose-resistant mutants · ethanol fermentation on xylose · thermotolerant yeas

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

Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries and Their Application in High- Temperature Fermentation

Thermotolerant ethanologenic yeasts receive attention as alternative bio-ethanol producers to traditionally used yeast, Saccharomyces cerevisiae. Their utilization is expected to provide several benefits for bio-ethanol production due to their characteristics and robustness. They have been isolated from a wide variety of environments in a number of ASEAN countries: Thailand, Vietnam, Laos, and Indonesia. One of these yeasts, Kluyveromyces marxianus has been investigated regarding characteristics. Some strains efficiently utilize xylose, which is a main component of the 2nd generation biomass. In addition, the genetic basis of K. marxianus has been revealed by genomic sequencing and is exploited for further improvement of the strains by thermal adaptation or gene engineering techniques. Moreover, the glucose repression of K. marxianus and its mechanisms has been investigated. Results suggest that K. marxianus is an alternative to S. cerevisiae in next-generation bio-ethanol production industry. Indeed, we have succeeded to apply K. marxianus for bio-ethanol production in a newly developed process, which combines high-temperature fermentation with simultaneous fermentation and distillation under low pressure. This chapter aims to provide valuable information on thermotolerant ethanologenic yeasts and their application, which may direct the economic bioproduction of ethanol and other useful materials in the future

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

Abstract 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

Additional file 2: of Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1–2

Figure S1. A phylogenetic tree of CMUWF1–2 isolated in this study and other strains published in databases was constructed. Numbers indicate percentages of bootstrap sampling, derived from 1000 samples. The numbers in parentheses are GenBank accession numbers. Schizosaccharomyces pombe NRRL Y-12796 was an outgroup in the analysis. (PDF 389 kb

Additional file 1: of Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1–2

Table S1. Xylose-utilizing yeasts isolated from natural samples and its sources. (PDF 357 kb

Additional file 6: of Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1–2

Figure S3. Effects of 2-DOG were examined on YPMan, YPGal, YPXyl, and YPAra plates with or without 0.01% 2-DOG. (PDF 367 kb

Additional file 5: of Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1–2

Figure S2. Effects of 2-DOG were examined on YPXyl plates containing various concentrations of 2-DOG. (PDF 411 kb