Bottom-fermenting Yeast Strains Research Articles

Applications of the Third-Generation DNA Sequencing Technology to the Detection of Hop Tolerance Genes and Discrimination of Saccharomyces Yeast Strains

To expand the applicability of the third-generation DNA sequencing technologies for the brewing industry, the new experimental protocols for quality control (QC) brewery laboratories have been developed, using the MinION system. Firstly, the hop tolerance genes, horA and horC, were amplified with multiplex PCR, and the PCR amplicons were sequenced by the MinION system. As a result, all of the beer spoilage lactic acid bacteria (LAB) strains carrying horA and/or horC were positively identified. Secondly, STA1, SbKEX2 and GEX1, were evaluated as diagnostic marker genes (DMGs) for discriminating Saccharomyces cerevisiae and S. pastorianus strains that possess essentially indistinguishable nucleotide sequences of the rDNA and internal transcribed spacer (ITS) regions. As a consequence of multiplex PCR specific to STA1, SbKEX2 and GEX1, these DMGs were shown to separate these hard-to-discriminate strains into four groups, namely top-fermenting ale yeast strains (S. cerevisiae), bottom-fermenting lager yeast strains (S. pastorianus), non-brewing S. cerevisiae wild yeast strains, and S. cerevisiae var. diastaticus. This study also demonstrated that the multiplexed PCR amplicons are independently recognized and correctly identified by the MinION system. Taken together, the newly developed methods using the MinION system are applicable to the identification and discrimination of important brewery microorganisms for microbiological QC. It was also shown that the concurrent applications of the rDNA-sequencing-based species identification and DMG approaches by the MinION system are useful for the breweries that now manufacture both traditional beers and non-traditional beverages. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2021.1939606 .

Generation of new hybrids by crossbreeding between bottom-fermenting yeast strains

The genetic diversity of bottom-fermenting yeast classified as Saccharomyces pastorianus is poor because strains are restricted to a few genetically distinct groups. Crossbreeding is an effective approach to construct novel yeast strains, but it is difficult because of inefficiency to obtain mating-competent cells (MCCs) of bottom-fermenting yeast. By using mating pheromone-supersensitive mutants, we previously isolated several mating-competent meiotic segregants from two bottom-fermenting yeast strains: high isoamyl acetate-producing KY1247, and low diacetyl-producing KY2645. Here, we constructed novel non-GM hybrids carrying preferable characteristics from both parents by crossbreeding these bottom-fermenting strains for the first time. Sixteen a/a-type meiotic segregants from KY2645 and 12 α/α-type meiotic segregants from KY1247 were mixed, and cells resembling zygotes were isolated via micromanipulation. In total, 149 hybrids were obtained and verified by examining known single-nucleotide polymorphisms (SNPs) between the parental strains. A sporulation test showed that some of the hybrids were able to sporulate. Moreover, fermentation tests on a test-tube and pilot-plant scale identified two hybrids with production levels of isoamyl acetate and diacetyl that were almost the same as KY1247 and KY2645, respectively. Both of these hybrids produced satisfactory beer in terms of taste, flavor, and overall quality, comparable to that produced by the parental strains. Collectively, our results suggest that crossbreeding between bottom-fermenting yeast strains has the potential to increase the diversity of yeast strains available for brewing, and our method of isolating MCCs provides a huge advance for crossbreeding of bottom-fermenting yeast without using DNA recombination techniques.

Mutation and Deletion of PAD1 and/or FDC1 and Absence of Phenolic Off-Flavor Production in Top- and Bottom-Fermenting Yeasts

To convert ferulic acid into 4-vinylguaiacol (4-VG), Saccharomyces cerevisiae must have intact PAD1 and FDC1 genes. British-type top-fermenting yeast strains have nonsense mutations in both of these genes; whereas, bottom-fermenting yeast strains have a nonsense mutation in their S. cerevisiae-type FDC1, and have lost their S. eubayanus-type FDC1 and PAD1 genes. Here, top-fermenting yeast transformants in which wild-type PAD1 and FDC1 derived from the laboratory yeast S. cerevisiae S288C were inserted in the genome, exhibited ferulic acid decarboxylation activity. Similarly, bottom-fermenting yeast transformants expressing wild-type S. cerevisiae-type FDC1 or S. eubayanus-type FDC1 also exhibited ferulic acid decarboxylation activity. Thus, the lack of ferulic acid decarboxylation activity in bottom-fermenting yeast is due to mutation of the S. cerevisiae-type FDC1 gene, coupled with absence of the S. eubayanus-type FDC1 gene.

An efficient method for isolating mating-competent cells from bottom-fermenting yeast using mating pheromone-supersensitive mutants.

Crossbreeding is an effective approach to construct novel yeast strains with preferred characteristics; however, it is difficult to crossbreed strains of brewer's yeast, especially the bottom-fermenting yeast Saccharomyces pastorianus, because of the relative inefficiency of the available methods to obtain mating-competent cells (MCCs). Here, we describe a productive method for the isolation of MCCs without artificial genetic modification. We focused on the characteristics of two mating pheromone-supersensitive mutants, Δbar1 and Δsst2, that show a growth defect in the presence of the mating pheromone. When MCCs secreting α-factor and a-factor were spotted on to a lawn of MATa Δbar1 and MATα Δsst2, a halo was observed around the respective MCCs. This plate assay was successful in identifying MCCs from bottom-fermenting yeast strains. Furthermore, by selecting for cells that caused the growth defect in pheromone-supersensitive cells on cultures plates, 40 α/α-type and six a/a-type meiotic segregants of bottom-fermenting yeast strains were successfully isolated and crossed with tester strains to verify their mating type. This method of isolation is expected to be applicable to other industrial yeast strains, including wine, sake and distiller's yeasts, and will enable MCCs without genetic modifications to be obtained. As a result, it will be a useful tool for more convenient and efficient crossbreeding of industrial yeast strains that can be applied to practical brewing. Copyright © 2017 John Wiley & Sons, Ltd.

Part I: the influence of serial repitching ofSaccharomyces pastorianuson the uptake dynamics of metal ions and fermentable carbohydrates during the fermentation of barley and gluten-free buckwheat and quinoa wort

Recently, research has been focusing on the use of alternative raw materials for brewing purposes and gluten-free beer-like beverages from malted buckwheat and quinoa are of commercial interest. A common commercial process involves the serial repitching of the yeast biomass, but this has not been described using buckwheat and quinoa wort fermentations. Our research studies (Parts I–III) explored the serial repitching of the yeast strain Saccharomyces pastorianus TUM 34/70 on the composition of a barley, buckwheat and quinoa fermentation medium. The present paper focuses on the fermentation performance and the uptake dynamics of metal ions and fermentable carbohydrates. Both pseudocereals showed high variations in all of the attributes examined during successive fermentations. In buckwheat the differences between successive fermentations were similar to those observed with barley, whereas differences in quinoa varied quite significantly from those observed with barley and showed a directional trend, suggesting a general weakening of the yeast from the sixth successive fermentation onward. In particular, the assimilation of the fermentable carbohydrates lessened and metal ion uptake appeared poorly controlled. It was concluded that buckwheat showed good potential for serial repitching of S. pastorianus TUM 34/70, whereas serial repitching of a quinoa wort appeared to be limited to five or six fermentations. Copyright © 2015 The Institute of Brewing & Distilling

Single nucleotide polymorphisms of PAD1 and FDC1 show a positive relationship with ferulic acid decarboxylation ability among industrial yeasts used in alcoholic beverage production

Among industrial yeasts used for alcoholic beverage production, most wine and weizen beer yeasts decarboxylate ferulic acid to 4-vinylguaiacol, which has a smoke-like flavor, whereas sake, shochu, top-fermenting, and bottom-fermenting yeast strains lack this ability. However, the factors underlying this difference among industrial yeasts are not clear. We previously confirmed that both PAD1 (phenylacrylic acid decarboxylase gene, YDR538W) and FDC1 (ferulic acid decarboxylase gene, YDR539W) are essential for the decarboxylation of phenylacrylic acids in Saccharomyces cerevisiae. In the present study, single nucleotide polymorphisms (SNPs) of PAD1 and FDC1 in sake, shochu, wine, weizen, top-fermenting, bottom-fermenting, and laboratory yeast strains were examined to clarify the differences in ferulic acid decarboxylation ability between these types of yeast. For PAD1, a nonsense mutation was observed in the gene sequence of standard top-fermenting yeast. Gene sequence analysis of FDC1 revealed that sake, shochu, and standard top-fermenting yeasts contained a nonsense mutation, whereas a frameshift mutation was identified in the FDC1 gene of bottom-fermenting yeast. No nonsense or frameshift mutations were detected in laboratory, wine, or weizen beer yeast strains. When FDC1 was introduced into sake and shochu yeast strains, the transformants exhibited ferulic acid decarboxylation activity. Our findings indicate that a positive relationship exists between SNPs in PAD1 and FDC1 genes and the ferulic acid decarboxylation ability of industrial yeast strains.

Hydrogen sulphide production by bottom-fermenting yeast is related to nitrogen starvation signalling

Bottom-fermenting yeast subjected to nitrogen starvation during exponential growth were found to produce hydrogen sulphide. In order to investigate a possible relationship between nitrogen starvation and hydrogen sulphide production, bottom-fermenting yeast strains, with depressed nitrogen starvation responses, were constructed by disrupting GLN3 and GAT1, encoding transcription factors that activate the transcription of NCR (nitrogen catabolite repression)-sensitive genes. In order to construct additional bottom-fermenting yeast strains with depressed nitrogen starvation responses, DAL80-, GZF3- and URE2-overexpressing strains were also constructed as these genes are also involved in NCR, and repress transcription of NCR-sensitive genes. Bottom-fermenting yeast have two types of genes, those derived from Saccharomyces cerevisiae-type and S. bayanus-type genomes. Both types of genes were disrupted/overexpressed in the constructed strains. In wort fermentations, all of the constructed strains produced less hydrogen sulphide and less of the thiol off-flavour compounds, 3-methyl-2-butene-1-thiol and 2-mercapto-3-methyl-1-butanol than the parent strains. Copyright © 2013 The Institute of Brewing & Distilling

Pilot-scale brewing using self-cloning bottom-fermenting yeast with highSSU1expression

A pilot-scale fermentation was performed using SSU1-overexpressing bottom-fermenting yeast strains constructed by ‘self-cloning’. In these strains, the gene SSU1, encoding a plasma membrane protein that excretes sulphite, was highly expressed. The rate of fermentation of the two SSU1-overexpressing strains tested showed some reduction during the mid-fermentation phase as compared with the parental strain. These differences, however, did not affect overall fermentation and the final apparent extracts had decreased to a level normally obtained during brewing. The concentration of hydrogen sulphide in the wort remained low during fermentation in the case of the two self-cloning strains compared with the parent. The concentration of 2-mercapto-3-methyl-1-butanol, a sulphur compound that causes an ‘onion-like’ off-flavour, was also reduced in the case of the self-cloning strains, a result confirmed by sensory evaluation of the beer immediately after bottling. Furthermore, with these strains the anti-oxidation potential of bottled beer, as measured by electron spin resonance, was improved and the concentration of trans-2-nonenal in bottled beer after 7 days of accelerated aging at 37°C was decreased. These observations, together with the lower stale flavour score determined by sensory evaluation of bottled beer after a month of aging at 25°C, indicated that the flavour stability of the beer had been successfully improved. Copyright © 2013 The Institute of Brewing & Distilling

Overexpression of vacuolar H+-ATPase-related genes in bottom-fermenting yeast enhances ethanol tolerance and fermentation rates during high-gravity fermentation

Vacuolar H+-ATPase (V-ATPase) is thought to play a role in stress tolerance. In this study it was found that bottom-fermenting yeast strains, in which the V-ATPase-related genes DBF2, VMA41/CYS4/NHS5 and RAV2 were overexpressed, exhibited stronger ethanol tolerance than the parent strain and showed increased fermentation rates in a high-sugar medium simulating high-gravity fermentation. Among the strains examined, the DBF2-overexpressing bottom-fermenting yeast strain exhibited the highest ethanol tolerance and fermentation rate in YPM20 medium. Using this strain, high-gravity fermentation was performed by adding sugar to the wort, which led to increased fermentation rates and yeast viability compared with the parent strain. These findings indicate that V-ATPase is a stress target in high-gravity fermentation and suggests that enhancing the V-ATPase activity increases the ethanol tolerance of bottom-fermenting yeast, thereby improving the fermentation rate and cell viability under high-gravity conditions. Copyright © 2012 The Institute of Brewing & Distilling

Immunosuppressive drug rapamycin restores sporulation competence in industrial yeasts

Industrial yeasts, including a sake yeast strain Kyokai no. 7 (K7), are generally unable to sporulate. Previously, we have reported that in K7 (Saccharomyces cerevisiae) cells, deletion of the G1 cyclin gene CLN3, a key activator of the cell cycle, allows the cells to induce IME1 transcription and sporulate under sporulation conditions. Here we show that treatment with the immunosuppressive drug rapamycin also restores sporulation competence in K7 cells. Moreover, sporulation was observed after rapamycin treatment in other industrial yeasts, namely bottom fermenting yeast strains and a wine yeast strain, which are not able to sporulate under normal sporulation conditions. These findings suggest that activation of TORC1 under sporulation conditions leads to sporulation incompetence in these yeasts. Thus, rapamycin treatment will be useful to restore sporulation competence in industrial yeasts.

Improved Flavor Stability by Aging Beer in the Presence of Yeast

Unwanted flavor changes occur during beer storage. Because yeast has a large reducing activity on aging compounds, the aim of this study was to examine the effect of refermentation on flavor stability. It was shown that the addition of yeast to aged beer removed aged flavor almost completely. Furthermore, the addition of yeast to fresh beer decelerated beer aging considerably. Both a top-fermenting and a bottom-fermenting yeast strain were able to exert this effect, and it was clear that initiation of a real refermentation process by adding sugar was not required. Finally, several yeast concentrations were tested, and it appeared that even adding an amount as low as 10,000 yeast cells/mL was sufficient to slow beer aging. Nonetheless, the effect was more pronounced when higher concentrations were used.

10-Hydroxy-2-decenoic Acid as an Antimicrobial Agent in Draft Keg-Conditioned Wheat Beer

In nature, 10-hydroxy-2-decenoic acid (10-HDA), an important unsaturated fatty acid, is only available in royal jelly. Natural 10-HDA exhibits several biological activities, including anticancer activity, and provides protection against acute radiation injury. In this study, we identified a method for extraction of 10-HDA from royal jelly. The antimicrobial effect of 10-HDA on beer-spoilage bacteria and yeast was investigated by flow cytometry and bacteriostatic circle (paper assay) experiments. We also determined the minimum inhibitory concentration of 10-HDA using the double-dilution method. Our results showed that 10-HDA could significantly inhibit common microorganisms, such as Lactobacillus brevis and Pediococcus damnosus, found in beer but had no inhibitory effect on yeast. A novel approach for preparing draft keg-conditioned wheat beer was developed using top-fermenting yeast strain 303 and bottom-fermenting yeast strain 308. The beer was then kegged for secondary fermentation. Because living yeasts were still in the beer, pasteurization could not be used, and it was difficult to keep everything clean and sanitary during brewing and kegging processes. In this study, 10-HDA was added to the green beer during kegging. We confirmed that 10-HDA, acting as an antimicrobial agent, increased the sanitary aspects of the kegged beer and improved the microbiological stability and shelf life of the beer.

Chimeric types of chromosome X in bottom-fermenting yeasts

To determine the structure of the chimeric chromosome X of bottom-fermenting yeasts. Eight cosmid clones carrying DNA from chromosome X of bottom-fermenting yeasts were selected by end-sequencing. Four of the cosmid clones had Saccharomyces cerevisiae (SC)-type and Saccharomyces bayanus (SB)-type chimeric ends, two had SC-type ends and two had SB-type ends. Sequencing revealed that the bottom-fermenting yeast strains in this study had four types of chromosome X: SC-SC, SC-SB, SB-SC and SB-SB. The translocation site in the chimeric chromosome is conserved among bottom-fermenting yeast strains, and was created by homologous recombination within a region of high sequence identity between the SC-type sequence and the SB-type sequence. Existing bottom-fermenting yeast strains share a common ancestor in which the chimeric chromosome X was generated. The knowledge gained in this study sheds light on the evolution of bottom-fermenting yeasts.

Development of Bottom-Fermenting Saccharomyces Strains That Produce High SO 2 Levels, Using Integrated Metabolome and Transcriptome Analysis

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO(2) is desirable, it is complicated by the fact that undesirable H(2)S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO(2)-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO(2) and H(2)S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO(2), resulting in high SO(2) and low H(2)S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO(2) than the parent but produced parental levels of H(2)S.

Identification and characterization of amidase‐ homologous AMI1 genes of bottom‐fermenting yeast

It has been proposed that a bottom-fermenting yeast strain of Saccharomyces pastorianus is a natural hybrid between S. cerevisiae and S. bayanus and possesses at least two types of genome. In the process of conducting expressed sequence tag (EST) analysis, we isolated bottom-fermenting yeast-specific (BFY) genes that have no significant homology with sequences in the S288C database. One of the BFY genes, AMI1, encodes a protein with homology to an amidase conserved among plants, Bacillus subtilis, Neurospora crassa, Schizosaccharomyces pombe and Saccharomyces species, with the exception of S. cerevisiae S288C. In the bottom-fermenting yeast, three alleles of AMI1 (one AMI1-A and two AMI1-B alleles) were found on different chromosomes. AMI1-A on chromosome XIII is most homologous to the S. bayanus AMI1 gene, while AMI1-B on chromosome X is most homologous to the Saccharomyces paradoxus AMI1 gene. Overproduction of AMI1 in S. cerevisiae resulted in a slow-growth phenotype. Although a hydropathy plot shows that Ami1p has a putative signal sequence, it was located in the cell, not secreted into the medium. By metabolome analysis of intracellular compounds, the amount of histidine and arginine is increased, and the amount of threonine, lysine and nicotinic acid is decreased in the Ami1p-overproducing strain as compared with the control, suggesting that Ami1p may hydrolyse some amides related to amino acid and niacin metabolism in the cell.

Identification of bottom‐fermenting yeast genes expressed during lager beer fermentation

It has been proposed that bottom-fermenting yeast strains of Saccharomyces pastorianus possess at least two types of genomes. Sequences of genes of one genome [S. cerevisiae (Sc)-type] have been found to be highly homologous (more than 90% identity) to S. cerevisiae S288C sequences, while those of the other [Lager (Lg)-type] are less so. To identify and discriminate Lg-type from Sc-type genes expressed during lager beer fermentation, normalized cDNA libraries were constructed and analysed. From approximately 22 000 ESTs, 3892 Sc-type and 2695 Lg-type ORFs were identified. Expression patterns of Sc- and Lg-type genes did not correlate with particular cell functions in KEGG classification system. Moreover, 405 independent clones were isolated that have no significant homology with sequences in the S288C database, suggesting that they include the bottom-fermenting yeast-specific (BFY) genes. Most of BFY genes have significant homology with the S. bayanus genome.

Application of Shotgun DNA Microarray Technology to Gene Expression Analysis in Lager Yeast

We used a shotgun DNA microarray strategy for gene expression analysis in brewing lager yeast for the first time. We constructed a random genomic library from a bottom-fermenting (lager) yeast strain, Weihenstephan 34/70, which is widely used in lager beer brewing. A DNA fragment size of ≈2.5 kbp was chosen, and 20,160 clones from the genomic library were printed on the shotgun DNA microarray. The gene expression analysis was carried out with total RNA extracted from lager yeast strains for a series of fermentation trials. Among the 7,636 spots that showed more than the limit of detection, 207 were selected as showing significant expression changes. In all, 37 spots were first repressed and then induced, and 62 spots were first induced and then repressed. Those spots were sequenced, annotated, and categorized into four clusters composed of the following sets of genes: 1) significantly induced, 2) significantly repressed, 3) first repressed and then induced, and 4) first induced and then repressed. These different patterns of expression for genes related to the fermentation process have been discussed. Therefore, we have shown that the shotgun DNA microarray technology is a very useful tool for accomplishing gene expression studies with lager yeast strains.

Modeling of Saccharide Utilization in Primary Beer Fermentation with Yeasts Immobilized in Calcium Alginate

Immobilized beer fermentation was studied using an industrial bottom-fermenting yeast strain Saccharomyces cerevisiae. The yeast cells were immobilized in 2.5% calcium alginate gel and used for brewing in a five-vessel cascade reactor. The fermentation was performed at 15 degrees C at various flow rates. A nonstructured mathematical model was developed to simulate the performance of continuous primary fermentation of lager beer. The model was based on the following variables: maltose, maltotriose, glucose, fructose, ethanol, and cell concentration. Experimental values of these variables were determined in samples taken at regular intervals. For experimental data fitting a nonlinear regression was used. Substrate consumption was characterized by specific substrate consumption rate and saturation constant. The values of these two parameters were optimized for all four substrates. Inhibition effects of substrates and product were analyzed using various inhibition patterns. Only the inhibition effect of maltose on maltose consumption was clearly identified. A good-fitting relationship for maltose inhibition was found, and inhibition constants were calculated.

Region of FLO1 proteins responsible for sugar recognition.

Yeast flocculation is a phenomenon which is believed to result from an interaction between a lectin-like protein and a mannose chain located on the yeast cell surface. The FLO1 gene, which encodes a cell wall protein, is considered to play an important role in yeast flocculation, which is inhibited by mannose but not by glucose (mannose-specific flocculation). A new homologue of FLO1, named Lg-FLO1, was isolated from a flocculent bottom-fermenting yeast strain in which flocculation is inhibited by both mannose and glucose (mannose/glucose-specific flocculation). In order to confirm that both FLO1 and Lg-FLO1 are involved in the yeast flocculation phenomenon, the FLO1 gene in the mannose-specific flocculation strain was replaced by the Lg-FLO1 gene. The transformant in which the Lg-FLO1 gene was incorporated showed the same flocculation phenotype as the mannose/glucose-specific flocculation strain, suggesting that the FLO1 and Lg-FLO1 genes encode mannose-specific and mannose/glucose-specific lectin-like proteins, respectively. Moreover, the sugar recognition sites for these sugars were identified by expressing chimeric FLO1 and Lg-FLO1 genes. It was found that the region from amino acid 196 to amino acid 240 of both gene products is important for flocculation phenotypes. Further mutational analysis of this region suggested that Thr-202 in the Lg-Flo1 protein and Trp-228 in the Flo1 protein are involved in sugar recognition.