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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.Recently, thermotolerant microorganisms were found among mesophiles with optimum growth temperatures that are 5-10°C higher than those of the typical mesophilic strains Fuel Ethanol Production from Sugarcane 122 Potential of Thermotolerant Ethanologenic Yeasts Isolated from ASEAN Countries… http://dx.doi.org/10.5772/intechopen.79144 123Recently, there have been several reports on ethanol production at high temperatures using P. kudriavzevii (formerly known as I. orientalis). Several P. kudriavzevii strains were reported to grow and produce high levels of ethanol at high temperatures. The strain DMKU 3-ET15 was isolated from traditional fermented pork sausage in Thailand by an enrichment technique in a medium supplemented with 4% ethanol at 40°C. The strain produced 78.6 g/L ethanol from 180 g/L glucose at 40°C [20]. The strain KVMP10 that was isolated from soil located beneath apple trees for ethanol production from orange peel achieved 54 g/L ethanol at 42°C [48]. Strain RZ8-1 that was recently isolated from various samples collected from plant orchards in Thailand produced 33.8 g/L ethanol from 160 g/L glucose at 40°C [49].

Candida tropicalis, a xylose-fermenting yeast, has the potential for converting cellulosic biomass to ethanol. Thermotolerant C. tropicalis X-17, which was isolated in Laos, was subjected to repetitive long-term cultivation with a gradual increase in temperature (RLCGT) in the presence of a high concentration of glucose, which exposed cells to various stresses in addition to the high concentration of glucose and high temperatures. The resultant adapted strain demonstrated increased tolerance to ethanol, furfural and hydroxymethylfurfural at high temperatures and displayed improvement in fermentation ability at high glucose concentrations and xylose-fermenting ability. Transcriptome analysis revealed the up-regulation of a gene for a glucose transporter of the major facilitator superfamily and genes for stress response and cell wall proteins. Additionally, hydropathy analysis revealed that three genes for putative membrane proteins with multiple membrane-spanning segments were also up-regulated. From these findings, it can be inferred that the up-regulation of genes, including the gene for a glucose transporter, is responsible for the phenotype of the adaptive strain. This study revealed part of the mechanisms of fermentability at high glucose concentrations in C. tropicalis and the results of this study suggest that RLCGT is an effective procedure for improving multistress tolerance.

Kmmig1 as a disrupted mutant of MIG1 encoding a regulator for glucose repression in Kluyveromyces marxianus exhibits a histidine-auxotrophic phenotype. Genome-wide expression analysis revealed that only HIS4 in seven HIS genes for histidine biosynthesis was down-regulated in Kmmig1 . Consistently, introduction of HIS4 into Kmmig1 suppressed the requirement of histidine. Considering the fact that His4 catalyzes four of ten steps in histidine biosynthesis, K . marxianus has evolved a novel and effective regulation mechanism via Mig1 for the control of histidine biosynthesis. Moreover, RNA-Seq analysis revealed that there were more than 1,000 differentially expressed genes in Kmmig1 , suggesting that Mig1 is directly or indirectly involved in the regulation of their expression as a global regulator.

is one of the most important enzymes involved in degradation of lignocelullose biomass. Two genes encoding α-L-Arabinofuranosidase (abfA), each from Bacillus subtilis DB104 (abfAa1) and an indigenous Indonesian B. licheniformis CW1 (abfAb3), were cloned by the PCR approach and expressed in Escherichia coli. Sequences analysis of abfAa1 and abfAb3 revealed that each consists of 1721 and 1739 base pairs long DNA, respectively. Each clone contains a hypothetical open reading frame of 1503 and 1509 bp that encode an Abfa protein of 500 and 502 amino acids for abfAa1 and abfAb3, respectively. The deduced amino acid sequence of AbfaB3 shares 75% identity to that of AbfaA1. The recombinant enzymes were expressed constitutively in E. coli. Partial characterization of those enzymes revealed that the AbfaA1 and AbfaB3 were optimally active at 50 ºC and 60 ºC at pH 6, respectively. Thermostability studies of the recombinant enzymes with p-nitrophenyl α-L-arabinofuranoside at their optimal conditions showed that up to 50% AbfaA1 activity was lost after 5 h incubation at 50 ºC, whereas the AbfaB3 retained its activity over 75% after 12 h pre-incubation o at 60 C. This thermostability study of recombinant AbfaB3 showed for the first time that the arabinofuranosidase from B. licheniformis is a thermostable enzyme. The recombinant enzyme showed a higher optimal reaction temperature (60 ºC) in comparison to the previously reported thermostable arabinofuranosidase. The thermostable AbfaB3 has a potential to be applied to the degradation of lignocellulose biomass synergistically with thermostable xylanases, for instance in the production of xylo-oligosaccharides.

Kluyveromyces marxianus has the attractive potential of utilization capability of various sugars in addition to thermal tolerance and protein productivity. The yeast, however, has an intrinsic system for catabolite repression, by which cells downregulate the metabolism of alternative sugars when glucose coexists. To acquire glucose-repression-free mutants, we isolated and characterized 2-deoxyglucose-resistant mutants from kanMX4-inserted mutants. The insertion site was determined by TAIL-PCR followed by nucleotide sequencing, indicating that the kanMX4 cassette was intragenically or intergenically inserted. Further analysis of the sugar utilization ability allowed to classify the intragenically inserted mutants including the mig1 mutant into two categories. One group showed enhanced utilization of xylose in the presense of glucose, presumably due to a defect in the glucoserepression mechanism, and the other group showed delayed utilization of glucose, probably by reduction of the uptake or initial catabolism of glucose. Considering the possible functions of the disrupted genes in these mutants, it is assumed that K. marxianus has undiscovered mechanisms for glucose repression and complex regulation for the uptake or initial catabolism of glucose.This is an open access article licensed under the terms of the Creative Commons Attribution-Non-Commercial 4.0 International Public License (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/legalcode), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.