Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.     

Mycocin sensitivity patterns of Kluyveromyces species: Kluyveromyces sensu lato vs. Kluyveromyces sensu stricto

The Kluyveromyces species reassigned to the genera Lachancea and Vanderwaltozyma are insensitive to five mycocins secreted by Pichia membranifaciens. The remaining Kluyveromyces species including species transferred to the genera Kazachstania, Nakaseomyces, and Tetrapisispora are sensitive to them. Only the neotype strain is insensitive to mycocins among Kluyveromyces lactis cultures.     

Split-Doa10: A Naturally Split Polytopic Eukaryotic Membrane Protein Generated by Fission of a Nuclear Gene

Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast Kluyveromyces lactis. In most species, Doa10 is encoded as a single polypeptide with 12–16 transmembrane helices (TMs), but split-KlDoa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The K. lactis DOA10 locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal KlDoa10 fragment. At least four additional Kluyveromyces species contain an IVS in the DOA10 locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream Kluyveromyces Doa10 fragment with its N-terminal RING-CH and two TMs resembles many metazoan MARCH (Membrane-Associated RING-CH) and related viral RING-CH proteins, suggesting that gene splitting may have contributed to MARCH enzyme diversification. Split-Doa10 is the first unequivocal case of a split membrane protein where fission occurred in a nuclear-encoded gene. Such a split may allow divergent functions for the individual protein segments.     

Yarrowia lipolytica: the novel and promising 2-phenylethanol producer

This is the first report on the ability of Yarrowia lipolytica strains to produce 2-phenylethanol (2-PE), which has not been identified for this species to date. 2-PE is a valuable aroma compound of rose-like odor. Its isolation from the other than microbial source—rose petals, is limited by the substrate availability. Thus, this chemical compound constitutes an attractive product for biotechnological conversions. To date, the ability to produce 2-PE has been described for such genera as Saccharomyces sp., Kluyveromyces sp., Geotrichum sp., and Pichia sp. This report provides evidence that Y. lipolytica is a novel 2-PE producer. Moreover, the titers of 2-PE obtained in Y. lipolytica NCYC3825 non-optimized cultures, nearly 2 g/l, are competitive to titers obtained by the other species.     

The emendation of the genusKluyveromyces v. d. Walt

The genusKluyveromyces has been emended to include species forming 1 – 4 crescentiform, reniform, prolate ellipsoidal or spheroidal ascospores.Fourteen species have been assigned to the emended genusKluyveromyces.     

Killer character in Kluyveromyces yeasts: Activity on Kloeckera apiculata

The screening for ‘killer’ character among 23 strains of the genus Kluyveromyces showed the presence of three ‘killer’ strains (K. phaffii; K. vanudenii and K. wikenii). Their ‘killer’ activity against yeasts of other genera is in agreement with the results of others studies. Particularly K. phaffii inhibited many strains of Kloeckera apiculata.     

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.     

Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected (“botrytized”) wine fermentations carried out at high (~30°C) and ambient (~20°C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by Saccharomyces. In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10⁶ cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of Candida. Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

Production of a key chiral intermediate of Betahistine with a newly isolated Kluyveromyces sp. in an aqueous two-phase system

(S)-(4-Chlorophenyl)-(pyridin-2-yl)methanol [(S)-CPMA] is an important chiral intermediate of anti-allergic drug Betahistine. Carbonyl reductase-producing microorganisms were isolated from soil samples for the stereoselective reduction of (4-chlorophenyl)-(pyridin-2-yl)methanone (CPMK) to (S)-CPMA. Among over 400 microorganisms isolated, one strain exhibiting the highest activity was selected and identified as Kluyveromyces sp. After optimization, the biotransformation reaction catalyzed by Kluyveromyces sp. CCTCC M2011385 whole-cell gave product (S)-CPMA in 81.5% ee and 87.8% yield at substrate concentration of 2 g/L in aqueous phase. Using an aqueous two-phase system (ATPs) consisted of PEG4000 (20%, w/w) and Na₂HPO₄ (14%, w/w), the product reached 86.7% ee and 92.1% yield at a higher substrate concentration of 6 g/L. The substrate tolerance and biocompatibility of microbial cells are greatly improved in ATPs by accumulating substrate/product in the upper PEG solution. This study, for the first time, reports the production of (S)-CPMA catalyzed by microbial cells.     

The K1 Toxin of Saccharomyces cerevisiae Kills Spheroplasts of Many Yeast Species

The Saccharomyces cerevisiae K1 toxin killed spheroplasts from the genera Candida, Kluyveromyces, and Schwanniomyces. Cells of these organisms were toxin insensitive. The toxin bound poorly to Kluyveromyces lactis cells. In contrast, Candida albicans bound the toxin to an extent similar to that seen with S. cerevisiae. Thus, wall receptors can define toxin specificity and are necessary but not sufficient for toxin action on intact cells.     

Yeast “Make-Accumulate-Consume” Life Strategy Evolved as a Multi-Step Process That Predates the Whole Genome Duplication

When fruits ripen, microbial communities start a fierce competition for the freely available fruit sugars. Three yeast lineages, including baker’s yeast Saccharomyces cerevisiae, have independently developed the metabolic activity to convert simple sugars into ethanol even under fully aerobic conditions. This fermentation capacity, named Crabtree effect, reduces the cell-biomass production but provides in nature a tool to out-compete other microorganisms. Here, we analyzed over forty Saccharomycetaceae yeasts, covering over 200 million years of the evolutionary history, for their carbon metabolism. The experiments were done under strictly controlled and uniform conditions, which has not been done before. We show that the origin of Crabtree effect in Saccharomycetaceae predates the whole genome duplication and became a settled metabolic trait after the split of the S. cerevisiae and Kluyveromyces lineages, and coincided with the origin of modern fruit bearing plants. Our results suggest that ethanol fermentation evolved progressively, involving several successive molecular events that have gradually remodeled the yeast carbon metabolism. While some of the final evolutionary events, like gene duplications of glucose transporters and glycolytic enzymes, have been deduced, the earliest molecular events initiating Crabtree effect are still to be determined.     

Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula

Phosphatase activities of yeasts belonging to the genera Saccharomyces, Kluyveromyces and Rhodotorula were studied. Rhodotorula rubra exhibited activities at acid, neutral and alkaline pH; the other yeasts only had activity at acid pH. Growing yeasts in a constant pH (4.5) medium decreased phosphatase activities in Saccharomyces and Kluyveromyces, while neutral activity was enhanced in Rhodotorula rubra which excreted more enzyme under these conditions. Washing cells with sucrose solutions lowered phosphatase activities in all yeasts, due to enzyme liberation. Acid phosphatase activities in isolated and purified cell walls were very small. Phosphatases thus appear not to be strongly bound to yeast cell walls.     

Sequencing-Based Analysis of the Bacterial and Fungal Composition of Kefir Grains and Milks from Multiple Sources

Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists.     

Phylogenetic Relationships of Species of the Genus Kluyveromyces van der Walt (Saccharomycetaceae) Deduced from the Partial Sequences of 18S and 26S Ribosomal RNAs

In order to clarify the phylogenetic relationships of the species classified in the genus Kluyveromyces (Saccharomycetaceae), three partial base sequences of 18S and 26S rRNAs of eighteen strains were determined. The regions determined of the strains corresponded to positions 1451 through 1618 (168 bases) of 18S rRNA and to positions 1611 through 1835 (225 bases) and 493 through 622 (130 bases) of a strain (IFO 2376) of Saccharomyces cerevisiae. The analyses of the partial base sequences suggested that the genus Kluyveromyces is phylogenetically heterogeneous, ranging from the strains that are quite close to the strain of S. cerevisiae to the strains that are distinct enough to be classified in genera separate from the genus Saccharomyces. From our sequence data, we concluded that the extent of the genus Kluyveromyces should be restricted to only one species, K. polysporus, the type species of the genus. Kluyveromyces phaffii was also distinct enough to deserve another genus. Kluyveromyces cellobiovorus was not close to any of the strains of Kluyveromyces species examined, and should be excluded from the genus. Most of the strains of the species examined were fairly close to the strain of S. cerevisiae.     

Kluyveromyces lactis and Saccharomyces cerevisiae, two potent deacidifying and volatile-sulphur-aroma-producing microorganisms of the cheese ecosystem

Cheese flavour is the result of complex biochemical transformations attributed to bacteria and yeasts grown on the curd of smear-ripened cheeses. Volatile sulphur compounds (VSCs) are responsible for the characteristic aromatic notes of several cheeses. In the present study, we have assessed the ability of Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae strains, which are frequently isolated from smear-ripened cheeses, to grow and deacidify a cheese medium and generate VSCs resulting from l-methionine degradation. The Kluyveromyces strains produced a wider variety and higher amounts of VSCs than the S. cerevisiae ones. We have shown that the pathway is likely to be proceeding differently in these two yeast genera. The VSCs are mainly generated through the degradation of 4-methylthio-oxobutyric acid in the Kluyveromyces strains, in contrast to the S. cerevisiae ones which have higher l-methionine demethiolating activity, resulting in a direct conversion of l-methionine to methanethiol. The deacidification activity which is of major importance in the early stages of cheese-ripening was also compared in S. cerevisiae and Kluyveromyces strains.     

Production of monoclonal antibody against protopectinase from Kluyveromyces wickerhamii

Four monoclonal antibodies (MCA 3–6, 4-2, 9-2, and 10-2) against protopectinase (an endo-polygalacturonase) from Kluyveromyces wickerhamii were prepared. MCAs 3-6 and 4-2 reacted to protopectinases produced by K. fragilis and K. marxianus as well as to the protopectinase produced by K. wickerhamii. However, 9-2 and 10-2 were specific for the protopectinase from K. wickerhamii. These MCAs did not inhibit the protopectinase reaction or the polygalacturonase reaction of protopectinases produced by these species. We used these MCAs to find protopectinases in the culture filtrates of Kluyveromyces yeasts.     

Kluyveromyces: systematics since 1970

The taxonomy ofKluyveromyces has been the object of intense study since van der Walt's (1970) monograph. This is an account of the major developments and the classification to be adopted in the 4th edition ofThe Yeasts, a Taxonomic Study. The guiding principles that will be followed in eventual revisions of the genus are presented.     

Heterologous Kluyveromyces lactis β-galactosidase secretion by Saccharomyces cerevisiae super-secreting mutants

Several Saccharomyces cerevisiae strains with a super-secreting phenotype have been transformed using a secretion plasmid containing the LAC4 gene and have proven to be effective in the secretion of Kluyveromyces lactis -galactosidase. The strain CGY1585 (ssc1-1) showed the highest secretion (1.7 EU ml^–1) in the culture medium. As far as we know, Kluyveromyces lactis -galactosidase is the largest sized protein and the only intracellular one among those secreted by these mutants hitherto. The recombinant strains all grew in lactose media.     

The yeastKluyveromyces africanus nov. spec. and its phylogenetic significance

Summary A new budding yeast species isolated from soil is described. Its outstanding features are, firstly, the formation of asci containing up to sixteen long oval to reniform ascospores and, secondly, a fermentative as well as oxidative metabolism. The assimilation of nitrate is absent and no pseudomycelium is formed. The taxonomic position of the yeast is discussed and it is pointed out that, due to its exceptional ascospore number (1–16), it cannot be classified in any of the existing fermentative genera of theEndomycetaceae (in sensu Lodder et Kreger-van Rij). The reniform shape of its ascospores indicates, however, its close relationship with the multispored genusKluyveromyces, on the one hand, and the newly proposed one to four-spored genusDekkeromyces on the other. The species is provisionally classified as aKluyveromyces species,Kluyveromyces africanus nov. spec., until further information regarding its sexual characteristics becomes available. By virtue of its more or less intermediate ascospore number, it establishes the direct derivation of the genusDekkeromyces fromDipodascus uninucleatus via the multispored yeast genusKluyveromyces.