Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

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     

Isolation of Meiotic Segregants from a Bottom Fermenting Yeast

Viable meiotic segregants were isolated from a bottom fermenting brewing yeast transformed by IME1 using a high copy number plasmid. These transformants sporulated under sporulation conditions and viable meiotic segregants were obtained from the spore asci. Flow cytometry showed that the meiotic segregants had a lower DNA content per cell than the parental strains, indicating meiosis had proceeded normally. Chromosomal observation by pulsed‐field gel electrophoresis and array comparative genomic hybridization (array‐CGH) showed that meiotic segregants, like the parent strains, contained two types of chromosome: the S. cerevisiae type and the S. bayanus type. In certain meiotic segregants, some chromosomes were missing, and the chromosome copy number changed. Isolation of meiotic segregants resulted in a ploidy reduction, which can be applied in the breeding of yeast strains.     

Nitrogen starvation induces expression of Lg‐FLO1 and flocculation in bottom‐fermenting yeast

When exponentially growing cells of bottom‐fermenting yeast were starved for nitrogen or were grown on proline (a non‐preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg‐FLO1, which is required for flocculation of bottom‐fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom‐fermenting yeast is under the control of a nitrogen catabolite repression (NCR)‐like mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

Pilot‐scale brewing using self‐cloning bottom‐fermenting yeast with high SSU1 expression

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     

Evaluating potential applications of indigenous yeasts and their β‐glucosidases

The potential applications of wild yeast strains with β‐glucosidase activity were investigated by assaying their enzymatic production under simulated oenological conditions, coupled with the exploration of the potential applications of the β‐glucosidases by studying the enzymatic activity and stability under similar oenological conditions. The assay of enzymatic locations revealed that the β‐glucosidase activities from these wild strains occurred in the extracellular fraction, and in whole and permeabilized cells. The effects of different oenological factors on β‐glucosidase production indicated that the F6 Trichosporon asahii strain had higher β‐glucosidase production than the other strains under low pH conditions. However, the F35 Hanseniaspora uvarum strain and the F30 Saccharomyces cerevisiae strain showed higher β‐glucosidase production under high‐sugar conditions. Furthermore, the influence of oenological factors on the activity and stability of the β‐glucosidases revealed that the enzyme from the F6 T. asahii strain had a stronger low‐pH‐value resistance than the other yeast β‐glucosidases. These results suggest that the F35 H. uvarum, F30 S. cerevisiae and the F6 T. asahii β‐glucosidases may have some potentially applicable values in the fermentation industry. Copyright © 2015 The Institute of Brewing & Distilling     

Use of PCR‐DHPLC (Polymerase Chain Reaction—Denaturing High Performance Liquid Chromatography) for the Rapid Differentiation of Industrial Saccharomyces pastorianus and Saccharomyces cerevisiae Strains

Strain specific detection and control of Saccharomyces pastorianus and Saccharomyces cerevisiae starter cultures is of great importance for the fermentation industry. The preconditions of strain specific fermentation characteristics can be ensured by periodic analysis and confirmation of the strain identity. With regard to industrial S. pastorianus and S. cerevisiae strains and a focus on brewing strains, the differentiation methods most available are time‐consuming and not very discriminative. In this work PCR‐DHPLC analysis was investigated as a novel approach for the differentiation of industrially used S. pastorianus and S. cerevisiae strains. The PCR‐DHPLC‐system was specific for S. cerevisiae strains and S. pastorianus hybrid strains that contain IGS2 rDNA, which originates from the S. cerevisiae ancestor. For the DNA of 177 strains of 41 non‐target species, which are typical for beverage and fermentation surroundings, the absence of PCR‐amplificates could be confirmed by DHPLC analysis. It was shown that single strains of S. cerevisiae and S. pastorianus could be differentiated. A strain specific differentiation within the group of top‐fermenting Saccharomyces cerevisiae strains could also be performed. For the group of bottom fermenting S. pastorianus brewing strains, strain‐to‐strain specific differences in the DHPLC chromatograms could be observed which can be used to differentiate and to compare two single strains with each other, although the comparison of chromatograms of an unknown S. pastorianus strain with a set of known S. pastorianus chromatograms could only reveal tendencies towards grouping into types. The differential DHPLC chromatogram characteristics (fluorescence intensities, number of peaks/side‐peaks/peak‐shoulders) within S. pastorianus are present, but not as distinctive as for S. cerevisiae. Additionally PCR‐DHPLC has advantages compared to other differentiation methods, such as species specificity, speed (2.5 h for one sample) and precision with the described limits.     

Hygromycin‐resistance vectors for gene expression in Pichia pastoris

Pichia pastoris is a common host organism for heterologous protein expression and metabolic engineering. Zeocin‐, G418‐, nourseothricin‐ and blasticidin‐resistance genes are the only dominant selectable markers currently available for selecting P. pastoris transformants. We describe here new P. pastoris expression vectors that confer a hygromycin resistance base on the Klebsiella pneumoniae hph gene. To demonstrate the application of the vectors for intracellular and secreted protein expression, green fluorescent protein (GFP) and human serum albumin (HSA) were cloned into the vectors and transformed into P. pastoris cells. The resulting strains expressed GFP and HSA constitutively or inducibly. The hygromycin resistance marker was also suitable for post‐transformational vector amplication (PTVA) for obtaining strains with high plasmid copy numbers. A strain with multiple copies of the HSA expression cassette after PTVA had increased HSA expression compared with a strain with a single copy of the plasmid. To demonstrate compatibility of the new vectors with other vectors bearing antibiotic‐resistance genes, P. pastoris was transformed with the Saccharomyces cerevisiae genes GSH1, GSH2 or SAM2 on plasmids containing genes for resistance to Zeocin, G418 or hygromycin. The resulting strain produced glutathione and S‐adenosyl‐l ‐methionine at levels approximately twice those of the parent strain. The new hygromycin‐resistance vectors allow greater flexibility and potential applications in recombinant protein production and other research using P. pastoris. Copyright © 2014 John Wiley & Sons, Ltd.     

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

The bottom‐fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)‐type and two S. bayanus (Sb)‐type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p‐hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G‐418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p‐hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5‐fluoroorotic acid (5‐FOA) plates to generate the desired Ura^– homozygous disruptant (Scura3Δ::TDH3p‐hygro/Scura3Δ::TDH3p‐hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom‐fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1‐overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3? lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.     

PCR‐TTGE and RAPD‐PCR Techniques to Analyze Saccharomyces cerevisiae and Saccharomyces carlsbergensis Isolated from Craft Beers

In the Friuli Venezia Giulia region (North East of Italy) the production of craft beers has been increasing constantly. Usually microbreweries use yeasts supplied by Italian or foreign industrial breweries for beer production. Yeast species are often not known, moreover the vitality, the viability, the physiological state and the number of generation are not known. To improve the quality of the final product it is important to evaluate the quality of the yeast strain used and the lactic acid bacteria contamination. Various molecular methods have been developed to compare genetic characteristics of yeast strains used in beer and wine production. The methods proposed in this work, PCR‐TTGE and RAPD‐PCR techniques, allow the comparison of specific DNA sequences to identify and/or characterize yeast strains. The molecular methods are faster than traditional methods and they allowed the identification of the strains analysed as S. cerevisiae and the intraspecies differentiation among yeast strains tested within 8 h after cell growth.     

Impact of the Long‐Term Maintenance Method of Brewer's Yeast on Fermentation Course,Yeast Vitality and Beer Characteristics

The effect of the long‐term maintenance method used with a brewer's yeast on its technological properties was determined in laboratory fermentation trials with a 12°P all‐malt wort. The trials were performed at a constant temperature and under conditions of constant substrate concentration. Two cultures of a bottom fermenting yeast, Saccharomyces pastorianus RIBM 95, were tested — one culture was maintained by subculturing on wort agar slopes at 4°C and the other culture underwent a three year storage in liquid nitrogen at minus 196°C. Parameters under investigation included yeast vitality measured as acidification power (AP), fermentation time needed to reach an alcohol level of 4%, the yeast cell count, sedimentation of the yeast during the fermentation, and the production of beer flavour compounds in green beer. The yeast culture stored for three years in liquid nitrogen displayed a higher count of suspended cells, required a shorter time to attenuate the wort to produce 4% alcohol and produced a 1.5 to 2.5‐fold higher concentration of a number of flavour compounds. The long‐term storage method did not affect the sedimentation ability and vitality of the yeast strain tested.     

IpO: plasmids and methods for simplified,PCR‐based DNA transplant in yeast

Many yeast experiments require strains modified by recombinant DNA methods. Some experiments require precise insertion of a DNA segment into the genome without a selectable marker remaining. For these applications, we developed a new PCR‐based method for marker‐free DNA transplant. The current PCR‐based method requires the labour‐intensive construction of a PCR template plasmid with repeats of the DNA segment flanking URA3. The design of a new vector, IpO, reduces the work in cloning a single copy of the DNA segment between overlapping URA3 fragments present in the vector. Two PCRs are performed that capture the DNA segment and one or the other URA3 fragment. When the PCR products are co‐transformed into yeast, recombination between the overlapping URA3 fragments restores URA3 and transposes the cloned DNA segment inside out, creating a repeat‐URA3‐repeat cassette. Sequences designed into the PCR primers target integration of the cassette into the genome. Subsequent selection with 5‐fluoro‐orotic acid yields strains that have 'popped out' URA3 via recombination between the DNA repeats, with the result being the precise insertion of the DNA segment minus the selectable marker. An additional advantage of the IpO method is that it eliminates PCR artifacts that can plague the current method's repeat‐containing templates. Copyright © 2014 John Wiley & Sons, Ltd.     

Construction of a brewing yeast expressing the glucoamylase gene STA1 by mating

Standard brewing yeast cannot utilize larger oligomers or dextrins, which represent about 25% of wort sugars. A brewing yeast strain that could ferment these additional sugars to ethanol would be useful for producing low‐carbohydrate diabetic or low‐calorie beers. In this study, a brewing yeast strain that secretes glucoamylase was constructed by mating. The resulting Saccharomyces cerevisiae 278/113371 yeast was MAT a/α diploid, but expressed the glucoamylase gene STA1 . At the early phase of the fermentation test in malt extract medium, the fermentation rate of the diploid STA1 strain was slower than those of both the parent strain S. cerevisiae MAFF113371 and the reference strain bottom‐fermenting yeast Weihenstephan 34/70. At the later phase of the fermentation test, however, the fermentation rate of the STA1 yeast strain was faster than those of the other strains. The concentration of ethanol in the culture supernatant of the STA1 yeast strain after the fermentation test was higher than those of the others. The concentration of all maltooligosaccharides in the culture supernatant of the STA1 yeast strain after the fermentation test was lower than those of the parent and reference strains, whereas the concentrations of flavour compounds in the culture supernatant were higher. These effects are due to the glucoamylase secreted by the constructed STA1 yeast strain. In summary, a glucoamylase‐secreting diploid yeast has been constructed by mating that will be useful for producing novel types of beer owing to its different fermentation pattern and concentrations of ethanol and flavour compounds. Copyright © 2017 The Institute of Brewing & Distilling     

Species identification of Wickerhamomyces anomalus and related taxa using β‐tubulin (β‐tub) DNA barcode marker

Wickerhamomyces anomalus is used in food and feed processing, although the species has been reported as an opportunistic human pathogen, predominantly in neonates. Neither phenotypic nor the most frequently applied genotypic marker (D1/D2 LSU ribosomal DNA) provide sufficient resolution for accurate identification of this yeast. In this study, the β‐tubulin gene was used for species identification by direct DNA sequencing and as marker in a species‐specific PCR assay. The results showed that all examined W. anomalus strains were clearly distinguished from the closely related species by comparative sequence analysis of the β‐tubulin gene. In addition, the species‐specific primers were also developed based on the β‐tubulin gene, which was employed for polymerase chain reaction with the template DNA of Wickerhamomyces strains. A single 218 bp species‐specific band was found only in W. anomalus. Our data indicate that the phylogenetic relationships between these strains are easily resolved by sequencing of the β‐tubulin gene and combined with species‐specific PCR assay. Copyright © 2012 John Wiley & Sons, Ltd.     

Flocculation in industrial strains of Saccharomyces cerevisiae: role of cell wall polysaccharides and lectin‐like receptors

Yeast flocculation is the reversible aggregation of yeast cells promoted by the interaction between lectin‐like protein receptors with mannose side chains on adjacent cell walls. Flocculation is governed by several physiological factors, including the type of nutrient sugar available to yeast. We grew four industrial strains of Saccharomyces cerevisiae , representing applications in the brewing, winemaking and bioethanol sectors, to late stationary phase and quantified the cellular content of mannans, glucans and lectin‐like proteins on yeast cell surfaces. Results indicated that brewing and champagne strains showed moderate to high flocculation ability when grown with glucose, fructose, maltose or galactose, whereas winemaking and fuel alcohol strains only showed moderate flocculation when grown on maltose and galactose. All yeast strains studied were weakly flocculent when grown on mannose. With regard to lectin‐like receptors, their number played a more important role in governing yeast flocculation than the mannan and glucan contents in yeast cell walls. We conclude that all the industrial strains of S. cerevisiae belonged to New‐Flo type on the basis of their flocculation behaviour observed when cultured on different sugars. Quantification of yeast cell wall polysaccharides and receptor sites indicates that mannan and glucan levels remain almost constant, irrespective of the strain under investigation. The main difference in flocculation characteristics in industrial yeast strains appears to be due to variations in concentrations of lectin‐like cell surface receptors. Our findings may benefit brewers, winemakers and other yeast‐based technologies in design of media to prevent premature flocculation during fermentation. Copyright © 2017 The Institute of Brewing & Distilling     

Genetically Similar Isolates of Salmonella enterica Serotype Enteritidis Persistent in China for a Long‐Term Period

Salmonella enterica serotype Enteritidis (S. Enteritidis) is an important causative agent of nontyphoidal salmonellosis in human populations. In this study, we collected 72 S. Enteritidis strains from 2004 to 2014 in Ningbo, mid‐east China. Of the 72 strains, we identified a dominant clone of 58 strains recovered from patient's feces (n = 48), blood (n = 1), pleural effusion (n = 1), chickens (n = 3), and dessert cakes (n = 5) by pulsed‐field gel electrophoresis (PFGE) and variable‐number of tandem repeat analysis (MLVA). The profile arrangements of MLVA were SE1‐SE2‐SE3‐SE5‐SE6‐SE8‐SE9: 4‐4‐3‐11‐10‐1‐3. These dominant strains were susceptible to ampicillin, chloramphenicol, tetracycline, ciprofloxacin, gentamicin, cefotaxime and trimethoprim–sulfamethoxazole, and resistant to nalidixic acid. Additionally, all isolates harboured virulence genes invA, sipA, sopE, and spvB when tested by PCR. Our results reveal that genetically similar S. Enteritidis strains which accounted for several outbreaks as well as blood infection and pleural cavity infection are prevalent in China for a long‐term period. This situation calls for further attention in the prevention and control of foodborne disease caused by Salmonella species.     

Multi‐gene phylogenetic analysis reveals that shochu‐fermenting Saccharomyces cerevisiae strains form a distinct sub‐clade of the Japanese sake cluster

Shochu is a traditional Japanese distilled spirit. The formation of the distinguishing flavour of shochu produced in individual distilleries is attributed to putative indigenous yeast strains. In this study, we performed the first (to our knowledge) phylogenetic classification of shochu strains based on nucleotide gene sequences. We performed phylogenetic classification of 21 putative indigenous shochu yeast strains isolated from 11 distilleries. All of these strains were shown or confirmed to be Saccharomyces cerevisiae, sharing species identification with 34 known S. cerevisiae strains (including commonly used shochu, sake, ale, whisky, bakery, bioethanol and laboratory yeast strains and clinical isolate) that were tested in parallel. Our analysis used five genes that reflect genome‐level phylogeny for the strain‐level classification. In a first step, we demonstrated that partial regions of the ZAP1, THI7, PXL1, YRR1 and GLG1 genes were sufficient to reproduce previous sub‐species classifications. In a second step, these five analysed regions from each of 25 strains (four commonly used shochu strains and the 21 putative indigenous shochu strains) were concatenated and used to generate a phylogenetic tree. Further analysis revealed that the putative indigenous shochu yeast strains form a monophyletic group that includes both the shochu yeasts and a subset of the sake group strains; this cluster is a sister group to other sake yeast strains, together comprising a sake‐shochu group. Differences among shochu strains were small, suggesting that it may be possible to correlate subtle phenotypic differences among shochu flavours with specific differences in genome sequences. Copyright © 2017 John Wiley & Sons, Ltd.