Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 °C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l⁻¹. Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.     

Global Analysis of the Genes Involved in the Thermotolerance Mechanism of Thermotolerant Acetobacter tropicalis SKU1100

Acetobacter tropicalis SKU1100 is a thermotolerant acetic acid bacterium that grows even at 42 °C, a much higher temperature than the limit for the growth of mesophilic strains. To elucidate the mechanism underlying the thermotolerance of this strain, we attempted to identify the genes essential for growth at high temperature by transposon (Tn10) mutagenesis followed by gene or genome analysis. Among the 4,000 Tn10-inserted mutants obtained, 32 exhibited a growth phenotype comparable to that of the parent strain at 30 °C but not at higher temperatures. We identified the insertion site of Tn10 on the chromosomes of all the mutant strains by TAIL (Thermal Asymmetric Interlaced)-PCR, and found 24 genes responsible for thermotolerance. The results also revealed a partial overlap between the genes required for thermotolerance and those required for acetic acid resistance. In addition, the origin and role of these thermotolerant genes are discussed.     

Characterization of thermotolerant Acetobacter pasteurianus strains and their quinoprotein alcohol dehydrogenases

We isolated several thermotolerant Acetobacter species of which MSU10 strain, identified as Acetobacter pasteurianus, could grow well on agar plates at 41°C, tolerate to 1.5% acetic acid or 4% ethanol at 39°C, similarly seen with A. pasteurianus SKU1108 previously isolated. The MSU10 strain showed higher acetic acid productivity in a medium containing 6% ethanol at 37°C than SKU1108 while SKU1108 strain could accumulate more acetic acid in a medium supplemented with 4–5% ethanol at the same temperature. The fermentation ability at 37°C of these thermotolerant strains was superior to that of mesophilic A. pasteurianus IFO3191 strain having weak growth and very delayed acetic acid production at 37°C even at 4% ethanol. Alcohol dehydrogenases (ADHs) were purified from MSU10, SKU1108, and IFO3191 strains, and their properties were compared related to the thermotolerance. ADH of the thermotolerant strains had a little higher optimal temperature and heat stability than that of mesophilic IFO3191. More critically, ADHs from MSU10 and SKU1108 strains exhibited a higher resistance to ethanol and acetic acid than IFO3191 enzyme at elevated temperature. Furthermore, in this study, the ADH genes were cloned, and the amino acid sequences of ADH subunit I, subunit II, and subunit III were compared. The difference in the amino acid residues could be seen, seemingly related to the thermotolerance, between MSU10 or SKU1108 ADH and IFO 3191 ADH.     

Ethanol production in solid substrate fermentation using thermotolerant yeast

A novel solid substrate fermentation system was used to produce fuel ethanol from sweet sorghum and sweet potato using a thermotolerant Saccharomyces cerevisiae strain (VS3) and a local isolate of amylolytic Bacilllus sps. (VB9). The process was carried out on a laboratory scale using broth cultures. Alcohol produced was estimated by gas chromatography after an incubation time of 72 h at 37 and 42°C. More ethanol was produced in co-culture with a mixed substrate than with the thermotolerant yeast (VS3) alone. The maximum amount of ethanol produced in co-culture with a mixed substrate was 5 g/100 g of substrate at 37°C and 3·5 g/100 g of substrate at 42°C.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

Four species of the unicellular green alga Chlorella, C. vulgaris, C. luteoviridis, C. minutissima, and C. zofingiensis, were characterized with respect to DNA similarities as determined by quantitative DNA hybridization procedures. In contrast to previous DNA hybridization procedures. In contrast to previous results, C. vulgaris turned out to be a homogeneous species with the exception of strain 211-11c of the Göttingen collection, which was shown to belong to C. kessleri. Similary, C. luteoviridis and C. minutissima represent well defined species in terms of phenotypic and genotypic features. Whitin C. zofingiensis on strain is clearly different with respect to DNA base composition and DNA hybridization data even though it shares phenotypic characteristics with the other strains of C. zofingiensis.     

Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus

Kluyveromyces marxianus is a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. Unlike Saccharomyces cerevisiae, K. marxianus grows on low-cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluated K. marxianus for the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2-pyrone synthase (2-PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high-stability 2-PS mutant and a promoter substitution increased titer four-fold. 2-PS expression from a multi-copy pKD1-based plasmid improved TAL titers a further five-fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed-batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.     

Electron transport chain in a thermotolerant yeast

Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨ_m), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca²⁺ ions (Ca²⁺ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30^o, while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.     

Clavispora,a new yeast genus of the Saccharomycetales

In Candida lusitaniae van Uden et do Carmo-Sousa (1959), strains of opposite sex have been found. Cells of the opposite mating types conjugate and form asci with one to four clavate spores. These are easily liberated from the ascus. The type strain of Candida obtusa (Dietrichson) van Uden et do Carmo-Sousa ex van Uden et Buckley (1970) also produces ascospores after mating with one of the strains of Candida lusitaniae. As clavate ascospores are unknow in yeasts, a new genus, Clavispora, is proposed with Clavispora lusitaniae spec. nov. as type species.     

Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification–fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 °C and 37 °C, while the activity of cellulolytic enzymes is highest at around 50 °C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus β-glucosidase on the cell surface, which successfully converts a cellulosic β-glucan to ethanol directly at 48 °C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of β-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.     

Kluyveromyces fragilis SS-437: An associatively-profiled thermotolerant yeast

The lactose-utilizing Kluyveromyces fragilis SS-437 was found to have an associative temperature profile, but a thermotolerant growth yield behaviour. Cardinal growth temperatures were: 3°C minimum for growth; 41.5°C optimum; 44.5°C final maximum (growth and death rates equalize); 46.1°C initial maximum (maximum limit for growth).     

Studies on the growth of a thermotolerant yeast strain,Kluyveromyces marxianus IMB3, on sucrose containing media

A thermotolerant alcohol-producing yeast strain, Kluyveromyces marxianus IMB3 was shown to grow on sucrose (10% [w/v]) containing media at 45 °C. Under such conditions the organism reached stationary phase within 20 hours and yielded ethanol concentrations in the region of 33g/L. During growth on sucrose containing media the organism was found to produce a cell- associated activity capable of hydrolysing sucrose. This activity was shown to have a Km of 5.0mM when sucrose was used as the substrate. In addition the enzyme was shown to have a pH optimum of 5.0 and a temperature optimum of 50–55 °C and under those conditions the enzyme was shown to be relatively thermostable.     

Monoacylglycerols: glycolipid biosurfactants produced by a thermotolerant yeast, Candida ishiwadae

AIMS: To isolate and characterize biosurfactants produced by a thermotolerant yeast isolated in Thailand. MATERIALS AND RESULTS: Yeast strains isolated from plant material in Thailand were first screened for the ability to produce lipase and biosurfactant. A strain Y12, identified as Candida ishiwadae by physiological tests, survived at 45 degrees C and produced relatively large amounts of biosurfactants. From the culture filtrate of this strain, two glycolipid biosurfactants, a and b, were purified by solvent fractionation, silica gel and ODS column chromatographies. Compounds a and b were determined to be monoacylglycerols; 1-linoleylglycerol and 1-oleylglycerol, respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as sodium dodecyl sulphate. CONCLUSIONS: Glycolipid biosurfactants produced by a thermotolerant yeast, C. ishiwadae were characterized to be monoacylglycerols which exhibited high surfactant activities. SIGNIFICANCE AND IMPACT OF THE STUDY: A thermotolerant yeast strain, C. ishiwadae, could be a potential candidate for producing monoacylglycerols which are useful in industrial applications.     

Isolation of a thermotolerant bacterium producing medium-chain-length polyhydroxyalkanoate

Aims:  The aim of this study was to isolate a thermotolerant micro‐organism that produces polyhydroxyalkanoates (PHAs) composed of medium‐chain‐length (mcl) HA units from a biodiesel fuel (BDF) by‐product as a carbon source. Methods and Results:  We successfully isolated a thermotolerant micro‐organism, strain SG4502, capable to accumulate mcl‐PHA from a BDF by‐product as a carbon source at a cultivation temperature of 45°C. The strain could also produce mcl‐PHA from acetate, octanoate and dodecanoate as sole carbon sources at cultivation temperatures up to 55°C. Taxonomic studies and 16S rRNA gene sequence analysis revealed that strain SG4502 was phylogenetically affiliated with species of the genus Pseudomonas. This study is the first report of PHA synthesis by a thermotolerant Pseudomonas. Conclusions:  A novel thermotolerant bacterium capable to accumulate mcl‐PHA from a BDF by‐product was successfully isolated. Significance and Impact of the Study:  A major issue regarding industrial production of microbial PHAs is their much higher production cost compared with conventional petrochemical‐based plastic materials. Especially significant are the cost of a fermentative substrate and the running cost to maintain a temperature suitable for microbial growth. Thus, strain SG4502, isolated in this study, which assimilates BDF by‐product and produces PHA at high temperature, would be very useful for practical application in industry.     

Enrichment of a continuous culture of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with the yeast <Emphasis Type="Italic">Issatchenkia orientalis</Emphasis> in the production of ethanol at increasing temperatures

A fermentation system was continuously fed with sugar-cane syrup and operated with recycling of Saccharomyces cerevisiae cells at temperatures varying from 30 to 47°C. The aim of the present work was to obtain and study the colonies of isolates showing elongated cells of yeasts which were sporadically observed at the end of this continuous process. Based on a sequence of assays involving methods of classical taxonomy and RAPD-PCR, two groups of isolates showing characteristics of non-Saccharomyces yeasts were identified in the yeast population where S. cerevisiae was the dominant yeast. The largest group of non-Saccharomyces yeasts, resulting from a slow proliferation over the 2 months, reached a final level of 29.6% at the end of the process. RAPD-PCR profiles obtained for the isolates of this dominant non-Saccharomyces yeast indicated that they were isolates of Issatchenkia orientalis. Pichia membranifaciens was the only species of non-Saccharomyces yeast detected together with I. orientalis but at a very low frequency. The optimum temperature for ethanol formation shown by the isolate 195B of I. orientalis was 42°C. This strain also showed a faster ethanol formation and biomass accumulation than the thermotolerant strain of S. cerevisiae used as the starter of this fermentation process. Some isolates of I. orientalis were also able to grow better at 40°C than at 30°C on plates containing glycerol as carbon source. Yeasts able to grow and produce ethanol at high temperatures can extend the fermentation process beyond the temperature limits tolerated by S. cerevisiae.     

Studies on the use of a thermotolerant strain of Kluyveromyces marxianus in simultaneous saccharification and ethanol formation from cellulose

 The thermotolerant yeast strain, Kluyveromyces marxianus IMB3, was found to be capable of ethanol production during growth at 45°C on media containing milled paper and exogenously added commercial cellulase. At maximum achievable cellulose concentrations in shake-flask cultures, ethanol production increased to 6.6 g/l at 45°C, representing an overall level of conversion of 21% of the maximum theoretical yield. Subsequent studies involving variations in added cellulase concentrations to the batch systems demonstrated that ethanol yields could be increased to 10 g/l at 45°C, which represented 39% of the maximum theoretical yield. As a result of ethanol production at 45°C in the systems examined, we suggest that the thermotolerant ethanol-producing yeast strain K. marxianus represents a novel candidate for use in simultaneous saccharification and conversion of the resulting substrates to ethanol. Received: 9 June 1994/Received revision: 8 August 1994/Accepted: 12 August 1994     

Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.     

Characterisation of a novel thermotolerant yeast, Kluyveromyces marxianus var marxianus: development of an ethanol fermentation process

The fermentation characteristics of the novel, thermotolerant, isolate Kluyveromyces marxianus var marxianus were determined to evaluate its aptitude for use in an ethanol production process. Sustainable growth was not observed under anaerobic conditions, even in the presence of unsaturated fatty acid and sterol. A maximum ethanol concentration of 40 g L⁻¹ was produced at 45°C, with an initial specific ethanol production rate of 1.7 g g⁻¹ h⁻¹. This was observed at ethanol concentrations below 8 g L⁻¹ and under oxygen-limited conditions. The low ethanol tolerance and low growth under oxygen-limited conditions required for ethanol production implied that a simple continuous process was not feasible with this yeast strain. Improved productivity was achieved through recycling biomass into the fermenter, indicating that utilising an effective cell retention method such as cell recycle or immobilisation, could lead to the development of a viable industrial process using this novel yeast strain. Received 14 February 1998/ Accepted in revised form 19 May 1998     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

The genetic relationships of nine strains of Chlorella saccharophila were determined by DNA hybridization techniques. Four strains are closely related to the type strain 211-9a and one strain seems to be moderately related, whereas the taxonomic position of the remaining three strains is not clear. C. saccharophila, like C. sorokiniana, is another species of Chlorella containing strains which are heterogeneous in their overall DNA base sequence and partly also in morphological, biochemical and physiological characters.     

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60‐1, was bred from a parental strain, MT8‐1, via stepwise adaptation. YK60‐1 grew at 40°C, a temperature at which MT8‐1 could not grow at all. YK60‐1 exhibited faster growth than MT8‐1 at 30°C. To investigate the mechanisms how MT8‐1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress‐responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60‐1. Furthermore, nontargeting metabolome analysis showed that YK60‐1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8‐1. In conclusion, S. cerevisiae MT8‐1 acquired thermotolerance by induction of specific stress‐responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1116–1123, 2013     

Rapid growth of a thermotolerant yeast on palm oil

A thermotolerant and rapidly-growing yeast for production of single cell protein from palm oil was isolated and identified as Candida tropicalis F129. The optimum temperature and pH for growth were 38°C and 6.0, respectively. The yeast grew with a high specific growth rate, of 0.92/h in 2% (v/v) palm oil medium, compared with other oil-assimilating yeasts or hydrocarbon-utilizing thermophilic yeasts. The overall cell yield was 1.01 g dry cells/g palm oil after 12 h.