FEMS yeast research最新文献

D-xylose utilization by yeasts is an essential feature for improving second-generation ethanol production. However, industrial yeast strains are incapable of consuming D-xylose. Previous analyzes of D-xylose-consuming or fermenting yeast species reveal that the genomic features associated with this phenotype are complex and still not fully understood. Here we present a previously neglected yeast enzyme related to D-xylose metabolism, D-xylose dehydrogenase (XylDH), which is found in at least 105 yeast genomes. By analyzing the XylDH gene family, we brought evidence of gene evolution marked by purifying selection on codons and positive selection evidence in D-xylose-consuming and fermenting species, suggesting the importance of XylDH for D-xylose-related phenotypes in yeasts. Furthermore, although we found no putative metabolic pathway for XylDH in yeast genomes, namely the absence of three bacterial known pathways for this enzyme, we also provide its expression profile on D-xylose media following D-xylose reductase for two yeasts with publicly available transcriptomes. Based on these results, we suggest that XylDH plays an important role in D-xylose usage by yeasts, likely being involved in a cofactor regeneration system by reducing cofactor imbalance in the D-xylose reductase pathway.

The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.

A key driver of quality in wines is the microbial population that undertakes fermentation of grape must. Winemakers can utilise both indigenous and purposefully inoculated yeasts to undertake alcoholic fermentation, imparting wines with aromas, flavours and palate structure and in many cases contributing to complexity and uniqueness. Importantly, having a toolbox of microbes helps winemakers make best use of the grapes they are presented with, and tackle fermentation difficulties with flexibility and efficiency. Each year the number of strains available commercially expands and more recently, includes strains of non-Saccharomyces, strains that have been improved using both classical and modern yeast technology and mixed cultures. Here we review what is available commercially, and what may be in the future, by exploring recent advances in fermentation relevant strain improvement technologies. We also report on the current use of microbes in the Australian wine industry, as reported by winemakers, as well as regulations around, and sentiment about the potential use of genetically modified organisms in the future.

Yeasts are important microorganisms used in different fermentation processes. The cocoa beans must go through a correct fermentation process to obtain good-quality chocolate, which involves the action of yeasts and bacteria, and yeasts play a crucial role since they act in the first days of fermentation. In coffee, several studies have shown that the microbiota in the fruits is also a relevant factor. The fermentation process (regardless of the processing type) improves the beverage's quality. In this sense, studies using starter cultures in these two raw materials are important for better control of the process, and optimization of fermentation time, in addition to the improvement and diversification of volatile and non-volatile compounds produced by yeasts. Thus, this review discusses the importance and role of yeasts during fermentation, their metabolism, the produced compounds, and how yeast and the different chemical reactions help increase the quality of chocolate and coffee.

In this study, the effect of five different Torulaspora delbrueckii strains in combination with an ale type Saccharomyces cerevisiae on physical, chemical, microbiological, aroma composition, and sensory profiles of beer were examined. The ethyl alcohol content of produced beers ranged from 5.46% (v/v) to 5.93% (v/v), while the highest alcohol amount was obtained using a pure culture of S. cerevisiae. The major volatiles among beer aroma compounds was acetaldehyde, n-propanol, 3-methyl-butanol, 2-methyl-butanol, ethyl acetate, isoamyl acetate, 2,3-butanedione, and 2,3-pentanedione. It was ascertained that the total amount of higher alcohols was higher in the S. cerevisiae control beer compared to all mixed fermentations. Total ester levels were higher in all the mixed culture beers than the control beer. Sensory evaluation showed that all the mixed cultures of S. cerevisiae and T. delbrueckii positively influenced the sensory profile of the beers. Strain Y1031 was the most preferred and was characterized as rich in hop aroma and full bodied. It is therefore a suitable strategy to use T. delbrueckii in mixed fermentations with S. cerevisiae to produce beer with a distinctive flavor. The results demonstrate that, T. delbrueckii strains isolated or commercialized for winemaking can be equally employed as well in brewing.

Wine is composed of multitudinous flavour components and volatile organic compounds that provide this beverage with its attractive properties of taste and aroma. The perceived quality of a wine can be attributed to the absolute and relative concentrations of favourable aroma compounds; hence, increasing the detectable levels of an attractive aroma, such as β-ionone with its violet and berry notes, can improve the organoleptic qualities of given wine styles. We here describe the generation of a new grape-must fermenting strain of Saccharomyces cerevisiae that is capable of releasing β-ionone through the heterologous expression of both the enzyme carotenoid cleavage dioxygenase 1 (CCD1) and its substrate, β-carotene. Haploid laboratory strains of S. cerevisiae were constructed with and without integrated carotenogenic genes and transformed with a plasmid containing the genes of CCD1. These strains were then mated with a sporulated diploid wine industry yeast, VIN13, and four resultant crosses-designated MQ01-MQ04-which were capable of fermenting the must to dryness were compared for their ability to release β-ionone. Analyses of their fermentation products showed that the MQ01 strain produced a high level of β-ionone and offers a fermenting hybrid yeast with the potential to enhance the organoleptic qualities of wine.

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).

Yarrowia lipolytica has been considered one of the most promising platforms for the microbial production of fatty acids and derived products. The deletion of the faa1 gene coding for an acyl-CoA synthetase leads to the accumulation and secretion of free fatty acids (FFAs) into the extracellular space. The secretion of products is beneficial for the development of microbial cell factories to avoid intracellular inhibitory effects and reduce downstream processing costs. However, the mechanism behind the secretion of fatty acids is not well known. As a starting point, we compared the transcriptome of this mutant showing FFA secretion to a wildtype-like strain not showing this phenotype. The 12 most upregulated genes were evaluated for involvement in FFA secretion by the creation of deletion and overexpression mutants, among them MCH2, YMOH, three cell wall proteins CWP3, CWP4, and CWP11, M12B, and three proteins with unknown functions YUP1, YUP2, and YUP3. None of these proteins take a clear or isolated role in FFA export. As the transcriptomic data revealed an overrepresentation of cell wall-related proteins, some of them were further examined on a theoretical and experimental way. Surprisingly, overexpression of Ygpi led to the production of FFAs in the wildtype-like genetic background. Finally, some of the evaluated genes showed involvement in resistance to FFA toxicity.

The bacterial toxin-antitoxin systems are each composed of a toxin, which severely inhibits bacterial cells growth, and a specific neutralizing antitoxin. Some toxin-antitoxin systems are functional when expressed in the yeast Saccharomyces cerevisiae. For instance, the expression of the relE toxin gene leads to a strong growth defect in yeast, whereas the expression of the relB antitoxin gene restores growth. Nevertheless, there is no available data regarding the required expression levels of each component of the relBE system leading to these growth phenotypes, neither their effects on cell viability. Here we used a double inducible plasmid-based system to independently modulate the relative amounts of relB and relE, and performed growth and gene expression analyses. These results allow us to correlate growth phenotypes to the expression levels of the toxin and the antitoxin, and to determine the levels necessary to observe either a strong growth inhibition or a normal growth. We also showed that the relE expression produces cell cycle progression defect without affecting cell viability. These results provide a detailed characterization of the functioning of the relBE system in S. cerevisiae, and open applicative perspectives of yeast growth control by bacterial toxin-antitoxin systems.

Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima.