The genusSaccharomyces (Meyen) Reess

The authors submit a taxonomic evaluation of an intermediate group of strains between the speciesSaccharomyces carlsbergensis Hansen andSaccharomyces cerevisiae Hansen. The material consisted of atypical strains of “bottom” brewer’s yeasts and the synonymous strainsSaccharomyces monacensis Hansen andSaccharomyces mandshuricus Saito. It was found that there were two different serological types in the speciesSaccharomyces carlsbergensis, one of which was characterized by the presence of antigen “C” and was typical for this species, while the other possessed antigen “M” and was grouped roundSaccharomyces monacensis. This second serological type merges with a group of strains which gives only one third fermentation of raffinose, so that it is actually an intermediate betweenSaccharomyces cerevisiae Hansen andSaccharomyces carlsbergensis Hansen and indicates the course of progressive development from the former species to the latter. No close similarity was found betweenSaccharomyces mandshuricus Saito and some of the strains of the transitional group or typical representatives of the two main species, and the authors therefore consider that there is some obscurity as to its synonymity withSaccharomyces carlsbergensis.     

Course of transformation of benzaldehyde bySaccharomyces cerevisiae

The course of fermentation of benzaldehyde bySaccharomyces cerevisiae simultaneously fermenting a sugar substrate was studied by a polarographic method and gas chromatography. In single-dose experiments, 0.2% benzaldehyde was fermented. It was fermented within 90–120 minutes, giving rise to 23.1 to 39.2% phenylacetyl-carbinol and 40.5 to 24% benzyl alcohol. The addition of acetaldehyde to the fermentation medium retarded the transformation of benzaldehyde, lowered the formation of benzyl alcohol and raised the yield of phenyl-acetylcarbinol. In four-dose experiments the utilization of the individual doses of benzaldehyde for the formation of phenyl-acetylcarbinol is described.     

The genusSaccharomyces (Meyen) reess

One hundred and forty strains of yeasts originally determined as wine yeasts were reidentified on the basis of a wide range of characters. A hypothetical average type was established on the basis of morphological, physiological, biochemical and serological characters and the test strains were arranged according to the degree of their resemblance to this average. Strain 28–8 displayed 100% similarity to the theoretical average and can, thus be regarded as typical. Some other groups formed transitions to other related species likeSaccharomyces oviformis Osterwalder orSaccharomyces willianus Saccardo. The classification of certain synonymous species such asSaccharomyces tokyo Nakazawa,Saccharomyces yedo Nakazawa,Saccharomyces paradoxus Batshinskaya,Saccharomyces turbidans Hansen,Saccharomyces marchalianus Kufferath, etc. is discussed. The results of this experimental study indicate that there are insufficient grounds, according to the present principles of taxonomy, for regardingSaccharomyces ellipsoideus Hansen andSaccharomyces vini (Muntz) Kudryavtsev as separate species and that, on the contrary, wine yeasts will come to be included in the range of polymorphism of the speciesSaccharomyces cerevisiae Hansen.     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.     

Genetic relationship and biological status of the industrially important yeast <Emphasis Type="Italic">Saccharomyces eubayanus</Emphasis> Sampaio et al.

The genomes of the recently discovered yeast Saccharomyces eubayanus and traditional S. cerevisiae are known to be found in the yeast S. pastorianus (syn. S. carlsbergensis), which are essential for brewing. The cryotolerant yeast S. bayanus var. uvarum is of great importance for production of some wines. Based on ascospore viability and meiotic recombination of the control parental markers in hybrids, we have shown that there is no complete interspecies post-zygotic isolation between the yeasts S. eubayanus, S. bayanus var. bayanus and S. bayanus var. uvarum. The genetic data presented indicate that all of the three taxa belong to the same species.     

Evaluation and application of constitutive promoters for cutinase production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cutinase as a promising biocatalyst has been intensively studied and applied in processes targeted for industrial scale. In this work, the cutinase gene tfu from Thermobifida fusca was artificially synthesized according to codon usage bias of Saccharomyces cerevisiae and investigated in Saccharomyces cerevisiae. Using the α-factor signal peptide, the T. fusca cutinase was successfully overexpressed and secreted with the GAL1 expression system. To increase the cutinase level and overcome some of the drawbacks of induction, four different strong promoters (ADH1, HXT1, TEF1, and TDH3) were comparatively evaluated for cutinase production. By comparison, promoter TEF1 exhibited an outstanding property and significantly increased the expression level. By fed-batch fermentation with a constant feeding approach, the activity of cutinase was increased to 29.7 U/ml. The result will contribute to apply constitutive promoter TEF1 as a tool for targeted cutinase production in S. cerevisiae cell factory.     

Employing oxygen pulses to modulate <Emphasis Type="Italic">Lachancea thermotolerans</Emphasis>–<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Chardonnay fermentations

Oxygen is sometimes deliberately introduced in winemaking at various stages to enhance yeast biomass formation and prevent stuck fermentation. However, there is limited information on how such interventions affect the dynamics of yeast populations. Our previous study in synthetic grape juice showed that oxygen supply enhances the persistence of Lachancea thermotolerans, Torulaspora delbrueckii and Metschnikowia pulcherrima. The three non-Saccharomyces yeasts showed differences in growth as a function of oxygen. The present study focused on evaluating the influence of short oxygen pulses on population dynamics and the aroma profile of Chardonnay wine inoculated with L. thermotolerans and Saccharomyces cerevisiae. The results confirmed a positive effect of oxygen on the relative performance of L. thermotolerans. The mixed culture fermentation with L. thermotolerans with S. cerevisiae developed a distinct aroma profile when compared to monoculture S. cerevisiae. Specifically, a high concentration of esters, medium chain fatty acids and higher alcohols was detected in the mixed culture fermentation. The data also showed that the longer persistence of L. thermotolerans due to addition of oxygen pulses influenced the formation of major volatile compounds such as ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl caprylate, ethyl caprate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, propanol, isobutanol, butanol, isoamyl alcohol, hexanol, isobutyric acid, butyric acid, iso-valeric acid, hexanoic acid, octanoic acid, and decanoic acid.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Interaction of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>–<Emphasis Type="Italic">Lactobacillus fermentum</Emphasis>–<Emphasis Type="Italic">Dekkera bruxellensis</Emphasis> and feedstock on fuel ethanol fermentation

The alcoholic fermentation for fuel ethanol production in Brazil occurs in the presence of several microorganisms present with the starter strain of Saccharomyces cerevisiae in sugarcane musts. It is expected that a multitude of microbial interactions may exist and impact on the fermentation yield. The yeast Dekkera bruxellensis and the bacterium Lactobacillus fermentum are important and frequent contaminants of industrial processes, although reports on the effects of both microorganisms simultaneously in ethanolic fermentation are scarce. The aim of this work was to determine the effects and interactions of both contaminants on the ethanolic fermentation carried out by the industrial yeast S. cerevisiae PE-2 in two different feedstocks (sugarcane juice and molasses) by running multiple batch fermentations with the starter yeast in pure or co-cultures with D. bruxellensis and/or L. fermentum. The fermentations contaminated with D. bruxellensis or L. fermentum or both together resulted in a lower average yield of ethanol, but it was higher in molasses than that of sugarcane juice. The decrease in the CFU number of S. cerevisiae was verified only in co-cultures with both D. bruxellensis and L. fermentum concomitant with higher residual sucrose concentration, lower glycerol and organic acid production in spite of a high reduction in the medium pH in both feedstocks. The growth of D. bruxellensis was stimulated in the presence of L. fermentum resulting in a more pronounced effect on the fermentation parameters than the effects of contamination by each microorganism individually.     

Enhancing the tolerance of the <Emphasis Type="Italic">Starmerella bacillaris</Emphasis> wine strain to dehydration stress

A protocol was developed to obtain Starmerella bacillaris as an active dry wine yeast (ADWY), which will facilitate winemakers to realize sequential inoculations of grape must to improve wine complexity. In the present study, several compound solutions were analyzed during the dehydration–rehydration process for Starmerella bacillaris strains isolated from related environments of alcoholic beverages. The ADWY obtained from Starmerella bacillaris were evaluated in fermentations at laboratory scale, obtained by sequential- and co-inoculation with Saccharomyces cerevisiae; the fermentative and aromatic parameters were evaluated and discussed. Our results for one Starmerella bacillaris strain show that the enhancement of viability might lead to a 4-fold higher survival rate when cells are dried in the presence of 10% trehalose, followed by rehydration in 0.5% galactose solution. In co- and sequentially inoculated grape must fermentations with Saccharomyces cerevisiae, the laboratory-scale wines obtained with Starmerella bacillaris ADWY did not show major changes in terms of the main volatile compounds, but there was an improvement in the fermentation performance behavior. The present study paves the way to develop a protocol for developing Starmerella bacillaris as an ADWY.     

Incubation of <Emphasis Type="Italic">Aquilaria subintegra</Emphasis> with Microbial Culture Supernatants Enhances Production of Volatile Compounds and Improves Quality of Agarwood Oil

Incubation with microbial culture supernatants improved essential oil yield from Aquilaria subintegra woodchips. The harvested woodchips were incubated with de man, rogosa and sharpe (MRS) agar, yeast mold (YM) agar medium and six different microbial culture supernatants obtained from Lactobacillus bulgaricus, L. acidophilus, Streptococcus thermophilus, Lactococcus lactis, Saccharomyces carlsbergensis and S. cerevisiae prior to hydrodistillation. Incubation with lactic acid bacteria supernatants provided higher yield of agarwood oil (0.45% w/w) than that obtained from yeast (0.25% w/w), agar media (0.23% w/w) and water (0.22% w/w). The composition of agarwood oil from all media and microbial supernatant incubations was investigated by using gas chromatography-mass spectrometry. Overall, three major volatile profiles were obtained, which corresponded to water soaking (control), as well as, both YM and MRS media, lactic acid bacteria, and yeast supernatant incubations. Sesquiterpenes and their oxygenated derivatives were key components of agarwood oil. Fifty-two volatile components were tentatively identified in all samples. Beta-agarofuran, α-eudesmol, karanone, α-agarofuran and agarospirol were major components present in most of the incubated samples, while S. cerevisiae-incubated A. subintegra provided higher amount of phenyl acetaldehyde. Microbial culture supernatant incubation numerically provided the highest yield of agarwood oil compared to water soaking traditional method, possibly resulting from activity of extracellular enzymes produced by the microbes. Incubation of agarwood with lactic acid bacteria supernatant significantly enhanced oil yields without changing volatile profile/composition of agarwood essential oil, thus this is a promising method for future use.     

Identification of yeasts during alcoholic fermentation of <Emphasis Type="Italic">tchapalo</Emphasis>, a traditional sorghum beer from Côte d’Ivoire

This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d’Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d’Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and metabolic activators: HXT3 gene expression and fructose/glucose discrepancy in sluggish fermentation conditions

When exposed to mixtures of glucose and fructose, as occurs during the fermentation of grape juice into wine, Saccharomyces cerevisiae uses these sugars at different rates. Moreover, glucose and fructose are transported by the same hexose transporters (HXT), which present a greater affinity for glucose, so that late in fermentation, fructose becomes the predominant sugar. Only a few commercial fermentation activators are available to optimally solve the problems this entails. The aim of this study was to investigate the relation between HXT3 gene expression and fructose/glucose discrepancy in two different media inoculated with a commercial wine strain of S. cerevisiae in the presence of three metabolic activators. Fermentation kinetics, vitality and major metabolites were also measured. Rehydration with ergosterol improved the area under the curve and the growth rate (µ _max ) in both studied media. Also, the fructose/glucose discrepancy values were improved with all activator treatments, highlighting rehydration in the presence of ascorbic acid. The yeast rehydration process was demonstrated to influence HXT3 expression under the studied conditions. Tetrahydrofolic acid treatment greatly influenced HXT3 gene expression, especially on the 12th day of the fermentation process. To a lesser extent, ergosterol and ascorbic acid also improved this parameter.     

Engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase,xylosidase and a bacterial xylose isomerase

Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes.     

Gene expression profiles of the thermotolerant yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain KKU-VN8 during high-temperature ethanol fermentation using sweet sorghum juice

Objectives

To investigate gene expression profiles of the thermotolerant yeast Saccharomyces cerevisiae strain KKU-VN8, a potential high-ethanol producer, in response to various stresses during high-temperature ethanol fermentation using sweet sorghum juice (SSJ) under optimal conditions.

Results

The maximal ethanol concentration obtained by S. cerevisiae KKU-VN8 using SSJ at 40 °C was 66.6 g/l, with a productivity of 1.39 g/l/h and a theoretical ethanol yield of 81%. Quantitative RT-PCR assays were performed to investigate the gene expression profiles of S. cerevisiae KKU-VN8. Differential expression of genes encoding heat-shock proteins (HSP82, HSP104, SSA4), genes involved in trehalose metabolism (TPS1, TPS2, NTH1) and genes involved the glycolytic pathway (ADH1, ADH2, CDC19) at various time points during fermentation was observed. The expression levels of HSP82, HSP104, SSA4, ADH1 and CDC19 were significantly higher than those of the controls (10.2-, 4-, 8-, 8.9- and 5.9-fold higher, respectively). In contrast, the expression levels of TPS1, TPS2, NTH1 and ADH2 were approx. 2-fold less than those of the controls.

Conclusions

The highly expressed genes encoding heat-shock proteins, HSP82 and SSA4, potentially play an important role in helping S. cerevisiae KKU-VN8 cope with various stresses that occur during high-temperature fermentation, leading to higher ethanol production efficiency.

     

Antibiotic activity of fungi isolated from soil samples from Indonesia

Two hundred and thirteen fungal cultures were recovered from 88 soil samples from different parts of Indonesia; 39.4% belonged to the genusPenicillium, 19.7% to the genusAspergillus, 9.9% to the genusFusarium and the rest to different systematic groups. One hundred and fifty two cultures were antibiotically active; 80% of these were antagonists ofBacillus subtilis, 55% ofEscherichia coli, 20% ofSaccharomyces cerevisiae and 37% ofCandida pseudotropicalis. In agreement with previous observations it was found that the activity spectrum of antagonists was related to the altitude above sea level at which they were found. As the altitude increased, the incidence of antagonists with both antibacterial and antifungal activity decreased, but the incidence of antagonists with only antibacterial or only antifungal activity increased. Fungi of the generaPenicillium andAspergillus were the most frequent antibiotic producers. The incidence of penicillin producers was much lower than in collections of fungi isolated in higher latitudes (China, Bulgaria, Slovakia).     

The use of yeast for microbial degradation of some selected mycotoxins

Several biodegradation experiments were carried out using 10 different yeast strains.Saccharomyces spp., Kluyveromyces spp. andRhodotorula spp. were tested for biodegradation of selected mycotoxins (ochratoxin A, nivalenol, deoxynivalenol and fumonisin B₁) standardsin vitro. There was confirmed that some yeast strains are able to degrade some mycotoxins. However, great differences between individual strains were observed. Moreover, 12Saccharomyces cerevisiae strains were tested for their potential capability to degrade zearalenone and fumonisins in Sabouraud broth. Two strains were capable to degrade zearalenone totally, one strain decreased the mycotoxin concentration up to 25%, and one strain up to 75% of original amount. Two strains were capable to degrade fumonisins partially.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.