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

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.     

Ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> in the presence of orange-peel oil

Summary Two ethanologenic yeasts, Saccharomyces cerevisiae and Kluyveromyces marxianus, were used to ferment sugar solutions modeling hydrolyzed Valencia orange peel waste at 37°C. Orange stripper oil produced from orange peel was added in various amounts to determine its effect on ethanol production. The minimum peel oil concentration that inhibited ethanol production was determined after 24, 48 and 72 h and the two yeasts were compared to one another in terms of ethanol yield. Minimum inhibitory peel oil concentrations for ethanol production were 0.05% at 24 h, 0.10% at 48 h, and 0.15% at 72 h for both yeasts. S. cerevisiae produced more ethanol than K. marxianus at each time point.     

The effects of ethanol on growth rate and passive proton diffusion in yeasts

Summary When cell suspensions of Saccharomyces cerevisiae NRRL-Y132 and Kluyveromyces marxianus IGC-2771 were incubated in the presence of different concentrations of ethanol, the final stable pH values (pH _f ) reached in these suspensions increased with increasing ethanol concentration, indicating that ethanol enhanced passive proton diffusion into the cells. The plots of pH _f as a function of ethanol concentration were linear but biphasic, displaying different slopes below and above the transition ethanol concentrations. When S. cerevisiae NRRL-Y132 and K. marxianus IGC-2771 were grown in the presence of different concentrations of ethanol, the specific growth rate () similarly depended upon ethanol concentration in a linear, biphasic way. Plots of at each ethanol concentration against pH _f reached in cell suspensions at that ethanol concentration were linear and monophasic for S. cerevisiae NRRL-Y132 but biphasic for K. marxianus IGC-2771. Ethanol inhibition of growth and enhancement of proton diffusion are therefore correlated in these yeasts. Whereas ethanol inhibition of growth and enhancement of transmembrane proton diffusion were affected to the same degree by ethanol below and above the transition ethanol concentration in S. cerevisiae NRRL-Y132, these two parameters of ethanol inhibition were affected to different degrees below and above the transition in K. marxianus IGC-2771 as indicated by the inflection point in the plot of vs pH _f . Attempts to extent these findings to other yeasts showed that the correlation between the effects of ethanol on pH _f and is not a universal phenomenon among yeasts.Offprint requests to: S. G. Kilian     

Isolation and identification of killer yeasts from Agave sap (aguamiel) and pulque

Wild killer yeasts have been identified as inhibitory to strains used as starters in the production of alcoholic beverages such as beer and wine; therefore, killer or killer-resistant strains have been sought for use in alcoholic fermentations. In the current paper a total of 16 strains belonging to six species were isolated. From two samples of Agave sap (aguamiel) the following yeast strains were isolated: Candida lusitaneae (1), Kluyveromyces marxianus var. bulgaricus (2), and Saccharomyces cerevisiae (capensis) (1). Additionally, in seven samples of pulque (the fermented product), the species C. valida (six strains), S. cerevisiae (chevalieri) (4), S. cerevisiae (capensis) (1), and K. marxianus var. lactis (1) were found. The killer strains were C. valida and K. marxianus var. lactis from pulque and K. marxianus var. bulgaricus from aguamiel. One strain of S. cerevisiae (chevalieri) isolated from pulque which did not show killer activity was, on the other hand, resistant to other killer strains and it had a remarkable ethanol tolerance, suggesting that this strain could be used for alcohol production.     

Improved ethanol production from whey with Saccharomyces cerevisiae using permeabilized cells of Kluyveromyces marxianus

Permeabilized cells of Kluyveromyces marxianus CCY eSY2 were tested as the source of lactase in the ethanol fermentation of concentrated deproteinized whey (65–70 g/l lactose) by Saccharomyces cerevisiae CCY 10–13–14. Rapid lactose hydrolysis by small amounts of permeabilized cells following the fermentation of released glucose and galactose by S. cerevisiae resulted in a twofold enhancement of the overall volumetric productivity (1.03 g/l × h), compared to the fermentation in which the lactose was directly fermented by K. marxianus.     

Studies on metal resistance system inKluyveromyces marxianus

Through preliminary plate tests,Kluyveromyces marxianus was found to be much more resistant to toxic heavy metals compared to aCUP1 ^R strain ofSaccharomyces cerevisiae. Specific growth rate and maximum dry weights affected by increasing metal concentrations were determined to obtain precise patterns of resistance. Metal biosorption was also monitored during the course of growth in synthetic media containing respective metals at 0.5 mM final concentration. Although Zn- and Co-binding was negligible, as much as 90% of silver, 60% of copper, and 65% of cadmium were found to be absorbed by the end of active growth. Analysis of the protein profiles ofS. cerevisiae andK. marxianus on metal exposure suggested constitutive production of metallothionein inK. marxianus. Furthermore, a smaller protein synthesized byK. marxianus on induction by silver or cadmium accounts for the high resistance of the organism to these metals.     

Physiological characterization of thermotolerant yeast for cellulosic ethanol production

The conversion of lignocellulose into fermentable sugars is considered a promising alternative for increasing ethanol production. Higher fermentation yield has been achieved through the process of simultaneous saccharification and fermentation (SSF). In this study, a comparison was performed between the yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus for their potential use in SSF process. Three strains of S. cerevisiae were evaluated: two are widely used in the Brazilian ethanol industry (CAT-1 and PE-2), and one has been isolated based on its capacity to grow and ferment at 42 °C (LBM-1). In addition, we used thermotolerant strains of K. marxianus. Two strains were obtained from biological collections, ATCC 8554 and CCT 4086, and one strain was isolated based on its fermentative capacity (UFV-3). SSF experiments revealed that S. cerevisiae industrial strains (CAT-1 and PE-2) have the potential to produce cellulosic ethanol once ethanol had presented yields similar to yields from thermotolerant strains. The industrial strains are more tolerant to ethanol and had already been adapted to industrial conditions. Moreover, the study shows that although the K. marxianus strains have fermentative capacities similar to strains of S. cerevisiae, they have low tolerance to ethanol. This characteristic is an important target for enhancing the performance of this yeast in ethanol production.     

Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress

During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H₂O₂ and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H₂O₂. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H₂O₂. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H₂O₂, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.     

Behaviour of the yeast Kluyveromyces marxianus var. marxianus during its autolysis

The lactic yeast Kluyveromyces marxianus var.marxianus (formerly K. fragilis) autolyzates at faster rate than Saccharomyces cerevisiae. During K. marxianus autolysis, quite similar release kinetics were observed for intracellular space markers (potassium ions, nucleotides), cell-wall components (polysaccharides, N-acetyl-D-Glucosamine) and non specific products (amino nitrogen). By Scanning Electronic Microscopy examination, no cell burst was observed, but a variation in cell shape (from ellipsoidal to cylindrical), as well as a 43% decrease in the internal volume were observed. The mechanism proposed for S. cerevisiae autolysis appeared also likely for K. marxianus.Abbreviations NacGlc N-acetyl-D-glucosamine - x total biomass (dry cellular weight) concentration     

Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.     

Improve carbon metabolic flux in Saccharomyces cerevisiae at high temperature by overexpressed TSL1 gene

This study describes a novel strategy to improve the glycolysis flux of Saccharomyces cerevisiae at high temperature. The TSL1 gene-encoding regulatory subunit of the trehalose synthase complex was overexpressed in S. cerevisiae Z-06, which increased levels of trehalose synthase activity in extracts, enhanced stress tolerance and glucose consuming rate of the yeast cells. As a consequence, the final ethanol concentration of 185.5 g/L was obtained at 38 °C for 36 h (with productivity up to 5.2 g/L/h) in 7-L fermentor, and the ethanol productivity was 92.7 % higher than that of the parent strain. The results presented here provide a novel way to enhance the carbon metabolic flux at high temperature, which will be available for the purposes of producing other primary metabolites of commercial interest using S. cerevisiae as a host.     

Differences in the flocculation mechanism ofKluyveromyces marxianus andSaccharomyces cerevisiae

Summary A study of the flocculation mechanism of aKluyveromyces marxianus strain, as compared with a strain ofSaccharomyces cerevisiae, is described. The involvement of cell wall proteins in the yeast's flocculation mechanism was studied by methyl-esterification or pepsin or acid phosphatase incubation of the cells. The influence of several ions on the flocculation mechanism was assayed. The obtained results indicated that the structure and/or the spatial arrangement of the cell wall groups involved in flocculation are not the same inK. marxianus as inS. cerevisiae.     

Physiological and metabolic diversity in the yeast <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Kluyveromyces marxianus is homothallic hemiascomycete yeast frequently isolated from dairy environments. It possesses phenotypic traits such as enhanced thermotolerance, inulinase production, and rapid growth rate that distinguish it from its closest relative Kluyveromyces lactis. Certain of these traits, notably fermentation of lactose and inulin to ethanol, make this yeast attractive for industrial production of ethanol from inexpensive substrates. There is relatively little known, however, about the diversity in this species, at the genetic, metabolic or physiological levels. This study compared phenotypic traits of 13 K. marxianus strains sourced from two European Culture Collections. A wide variety of responses to thermo, osmotic, and cell wall stress were observed, with some strains showing multi-stress resistance. These traits generally appeared unlinked indicating that, as with other yeasts, multiple resistance/adaptation pathways are present in K. marxianus. The data indicate that it should be possible to identify the molecular basis of traits to facilitate selection or engineering of strains adapted for industrial environments. The loci responsible for mating were also identified by genome sequencing and PCR analysis. It was found that K. marxianus can exist as stable haploid or diploid cells, opening up additional prospects for future strain engineering.     

<Superscript>13</Superscript>C metabolite profiling to compare the central metabolic flux in two yeast strains

¹³C metabolite profiling to quantify the dynamic changes of central carbon metabolites was attempted using mass isotopomer distribution analysis in two yeast strains, Saccharomyces cerevisiae and Kluyveromyces marxianus. Mass and isotopomer balances of the intermediates were examined and calculated in both yeast species and central carbon metabolic fluxes were successfully determined. Metabolic fluxes of pentose phosphate pathway in K. marxianus were 1.66 times higher than S. cerevisiae. The flux difference was also supported by relatively high abundance of partially labeled fructose 6-phosphate and 3-phosphoglycerate as well as an increased concentration of labeled L-valine in K. marxianus. Metabolic flux analysis combined with dynamic metabolite profiling has provided better understanding in the central carbon metabolic pathways of two model organisms and can be applied as a method to analyze more complicated metabolic networks in other organisms.     

Expression of exoinulinase genes in Saccharomyces cerevisiae to improve ethanol production from inulin sources

To improve inulin utilization and ethanol fermentation, exoinulinase genes from the yeast Kluyveromyces marxianus and the recently identified yeast, Candida kutaonensis, were expressed in Saccharomyces cerevisiae. S. cerevisiae harboring the exoinulinase gene from C. kutaonensis gave higher ethanol yield and productivity from both inulin (0.38 vs. 0.34 g/g and 1.35 vs. 1.22 g l^?1 h^?1) and Jerusalem artichoke tuber flour (0.47 vs. 0.46 g/g and 1.62 vs. 1.54 g l^?1 h^?1) compared with the strain expressing the exoinulinase gene from K. marxianus. Thus, the exoinulinase gene from C. kutaonensis is advantageous for engineering S. cerevisiae to improve ethanol fermentation from inulin sources.     

Evaluation of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> as a source of yeast autolysates

Cells of Kluyveromyces marxianus FII 510700 and Saccharomyces cerevisiae CBS 1907 were autolysed in phosphate buffer, pH 4.5, for a maximum of 10 days to compare chemical changes that occur in the carbohydrate, protein, amino acid and nucleic acid content. Approximately 2.2–3% carbohydrate, 9.5–12% protein, 0.6–1.0% DNA and 6–7% RNA were recovered in the autolysates. The main amino acids were β-alanine, phenylalanine, cysteine, methionine, glutamic acid and isoleucine. No significant differences in the yeast autolysates of K. marxianus and S. cerevisiae were observed. Consequently, K. marxianus produced from lactose-based media has potential as a source of yeast autolysates used in the food industry. Electronic Publication     

Lipid content of Saccharomyces cerevisiae strains with different degrees of ethanol tolerance

Summary We analysed the fatty acid and sterol compositions of various Saccharomyces cerevisiae strains with ethanol tolerance varying from 4% to 12% (v/v) ethanol and at different concentrations of ethanol. The results we obtained agree with the existence of a relationship between membrane fluidity and ethanol tolerance but they do not support a direct role of unsaturated fatty acids in this tolerance. On the other hand, they support the importance of ergosterol in this phenomenon.     

Sexual forms of yeasts in clinical samples

The sexual or teleomorphic state of yeasts has only been described in a few clinically involved species, mainly those of the Saccharomycetaceae family. With the aim of gathering information on their incidence in human pathology, a study has been made of a total of 2,135 strains isolated from clinical samples and cultivated in McClary agar. From these, 8 strains in teleomorphic state were identified: Kluyveromyces marxianus [1], Pichia anomala [2], Pichia farinosa [1], Pichia membranaefaciens [1] and Saccharomyces cerevisiae [3]. The two strains of P. anomala were responsible for fungemia; K. marxianus and the two strains of S. cerevisiae produced vaginitis; the other strains were oral cavity colonizers.