Savitree Limtong

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

ABSTRACT We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura+ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys− transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools.

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.

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

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.

Ogataea chonburiensis sp. nov. and Ogataea nakhonphanomensis sp. nov., thermotolerant, methylotrophic yeast species isolated in Thailand, and transfer of Pichia siamensis and Pichia thermomethanolica to the genus Ogataea

Two thermotolerant, methylotrophic yeast strains, PT44(T) and S051(T), were respectively isolated from a tree exudate and soil collected in Thailand. They were categorized as thermotolerant strains on the basis of their good growth below 20 degrees C and up to a relatively high temperature (37 degrees C). The major characteristics of the two strains that place them in the genus Ogataea are the formation of four helmet- or hat-shaped ascospores in a deliquescent ascus that may be produced parthenogenetically or by conjugation between a cell and its bud or between independent cells; multilateral budding; assimilation of nitrate; the presence of ubiquinone Q7; negative for Diazonium blue B colour and urease reactions; and the absence of arthroconidia and ballistoconidia. Analysis of the D1/D2 domains of the large-subunit rDNA sequence revealed that strain PT44(T) was differentiated from the strain S051(T) by 25 nucleotide substitutions and 1 gap in 554 nt, which was sufficient to justify the description of two separate species. The closest recognized species in terms of pairwise sequences similarity to PT44(T) was Pichia (Ogataea) dorogensis, with 13 nucleotide substitutions and 1 gap in 554 nt. Strain S051(T) was closest to Pichia thermomethanolica, with 7 nucleotide substitutions in 566 nt. Phenotypic characteristics of strains PT44(T) and S051(T) allowed them to be differentiated from each other and from the closest related species. On the basis of the above finding, the two strains represent two novel species of the genus Ogataea, for which the names Ogataea chonburiensis sp. nov. (type strain PT44(T) =BCC 21227(T) =NBRC 101965(T) =CBS 10363(T)) and Ogataea nakhonphanomensis sp. nov. (type strain S051(T) =BCC 21228(T) =NBRC 101966(T) =CBS 10362(T)) are proposed. We also propose the transfer of two thermotolerant methylotrophic members of the genus Pichia described previously to the genus Ogataea: Pichia siamensis is renamed Ogataea siamensis (Limtong, Srisuk, Yongmanitchai, Kawasaki, Yurimoto, Nakase & Kato) Limtong, Srisuk, Yongmanitchai, Yurimoto & Nakase comb. nov. (type strain JCM 12264(T) =TISTR 5818(T)) and Pichia thermomethanolica is renamed Ogataea thermomethanolica (Limtong, Srisuk, Yongmanitchai, Yurimoto, Nakase & Kato) Limtong, Srisuk, Yongmanitchai, Yurimoto & Nakase comb. nov. (type strain CBS 10098(T) =JCM 12984(T) =BCC 16875(T)).

Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand

A total of 1035 yeast isolates, obtained from rice and sugar cane leaves, were screened primarily for indole-3-acetic acid (IAA) production. Thirteen isolates were selected, due to their IAA production ranging from 1.2 to 29.3 mg g(-)(1) DCW. These isolates were investigated for their capabilities of calcium phosphate and ZnO(3) solubilisation, and also for production of NH(3), polyamine, and siderophore. Their 1-aminocyclopropane-1-carboxylate (ACC) deaminase, catalase and fungal cell wall-degrading enzyme activities were assessed. Their antagonism against rice fungal pathogens was also evaluated. Strain identification, based on molecular taxonomy, of the thirteen yeast isolates revealed that four yeast species - i.e. Hannaella sinensis (DMKU-RP45), Cryptococcus flavus (DMKU-RE12, DMKU-RE19, DMKU-RE67, and DMKU-RP128), Rhodosporidium paludigenum (DMKU-RP301) and Torulaspora globosa (DMKU-RP31) - were capable of high IAA production. Catalase activity was detected in all yeast strains tested. The yeast R. paludigenum DMKU-RP301 was the best IAA producer, yielding 29.3 mg g(-)(1) DCW, and showed the ability to produce NH3 and siderophore. Different levels of IAA production (7.2-9.7 mg g(-)(1) DCW) were found in four strains of C. flavus DMKU-RE12, DMKU-RE19, and DMKU-RE67, which are rice leaf endophytes, and strain DMKU-RP128, which is a rice leaf epiphyte. NH(3) production and carboxymethyl cellulase (CMCase) activity was also detected in these four strains. Antagonism to fungal plant pathogens and production of antifungal volatile compounds were exhibited in T. globosa DMKU-RP31, as well as a moderate level of IAA production (4.9 mg g(-)(1) DCW). The overall results indicated that T. globosa DMKU-RP31 might be used in two ways: enhancing plant growth and acting as a biocontrol agent. In addition, four C. flavus were also found to be strains of interest for optimal IAA production.

Continuous ethanol production using immobilized yeast cells entrapped in loofa-reinforced alginate carriers

A culture of Saccharomyces cerevisiae M30 entrapped in loofa-reinforced alginate was used for continuous ethanol fermentation in a packed-bed reactor with initial sugar concentrations of 200-248 g/L. Maximum ethanol productivity of 11.5 g/(L·h) was obtained at an ethanol concentration of 57.4 g/L, an initial sugar concentration of 220 g/L and a dilution rate (D) of 0.2 h-1. However, a maximum ethanol concentration of 82.1 g/L (productivity of 9.0 g/(L·h)) was obtained at a D of 0.11 h-1. Ethanol productivity in the continuous culture was 6-8-fold higher than that in the batch culture. Due to the developed carriers high biocompatibility, high porosity, and good mechanical strength, advantages such as cell regeneration, reusability, altered mechanical strength, and high capacity to trap active cells in the reactor were achieved in this study. The immobilized cell reactor was successfully operated for 30 days without any loss in ethanol productivity. The average conversion yield was 0.43-0.45 throughout the entire operation, with an immobilization yield of 47.5%. The final total cell concentration in the reactor was 37.3 g/L (17.7 g/L immobilized cells and 19.6 g/L suspended cells). The concentration of suspended cells in the effluent was 0.8 g/L.

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.

Candida phangngensis sp. nov., an anamorphic yeast species in the Yarrowia clade, isolated from water in mangrove forests in Phang-Nga Province, Thailand

Two yeast strains (TM2-16 and PT1-17(T)) were isolated by membrane filtration from samples of estuarine water collected from two mangrove forests, in Khao Lumpee-Haad Thaimueang National Park and Mu Ko Ra-Ko Prathong National Park, Phang-Nga Province, Thailand. Analysis of the D1/D2 domain of the large-subunit rDNA sequences revealed that the sequences of the two strains were identical. The closest species in terms of pairwise sequence similarity was Candida galli, but the level of nucleotide substitutions (13.2 %) was sufficient to justify the description of a separate species. Phylogenetic analysis demonstrated that the two strains occupy a basal position with respect to Yarrowia lipolytica and C. galli of the Yarrowia clade, supported by a high bootstrap value. The two strains showed identical phenotypic characteristics, including proliferation by multilateral budding, absence of ascospores and ballistoconidia and negative Diazonium blue B and urease reactions. The major ubiquinone was Q-9. On the basis of the above findings, these two strains were assigned to a single novel species of the genus Candida, for which the name Candida phangngensis sp. nov. is proposed. The type strain is PT1-17(T) (=BCC 21306(T) =NBRC 101970(T) [corrected] =CBS 10407(T)).

Comparison of the Gene Expression Patterns of Alcohol Dehydrogenase Isozymes in the Thermotolerant Yeast Kluyveromyces marxianus and Their Physiological Functions

Four genes encoding alcohol dehydrogenase (Adh) isozymes in the thermotolerant yeast Kluyveromyces marxianus, a potent candidate for ethanol production at high temperatures, were investigated. Of these, KmADH3 and KmADH4 were cloned and sequenced, and their deduced amino acid sequences were compared with those of KmAdh1 and KmAdh2 and other Adhs of Kluyveromyces lactis and Saccharomyces cerevisiae. The four KmAdhs had high sequence similarity, though KmAdh3 and KmAdh4 possessed an amino-terminal extension as a mitochondrial targeting sequence, and appear to belong to the zinc-containing Adh family. These results and the results of Southern blot experiments suggest that there are at least four Adh isozymes in K. marxianus, two cytoplasmic enzymes and two mitochondrial enzymes. The expression profile revealed that KmADH genes are differently expressed depending on growth phase and carbon source, suggesting that these highly homologous Adhs play distinctive roles in cells.

High-temperature ethanol fermentation by immobilized coculture of Kluyveromyces marxianus and Saccharomyces cerevisiae.

Suspended and immobilized cocultures of the thermotolerant yeast, Kluyveromyces marxianus DMKU 3-1042 and the mesophilic flocculent yeast, Saccharomyces cerevisiae M30 were studied for their abilities to improve production and stability of ethanol fermentation. Sugarcane juice and blackstrap molasses, at initial sugar concentrations of 220 g/L, were used as carbon sources. The results indicated that the coculture system could improve ethanol production from both sugarcane juice and blackstrap molasses when the operating temperature ranged between 33 °C and 45 °C. High temperature tolerances were achieved when the coculture was immobilized. The immobilized coculture was more effective in high-temperature ethanol fermentation than the suspended cultures. The coculture immobilized on thin-shell silk cocoon and fermented at 37 °C and 40 °C generated maximal ethanol concentrations of 81.4 and 77.3 g/L, respectively, which were 5.9-8.7% and 16.8-39.0% higher than those of the suspended cultures, respectively.