Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single‐stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice‐derived P. antarctica strain GB‐4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine‐5′‐phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB‐4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR‐amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil‐auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 μg DNA and a gene‐targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.     

Non‐homologous end joining‐mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination‐based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non‐homologous end‐joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C‐terminal‐truncated non‐functional ura3 selection marker and the truncated region were PCR‐amplified separately, mixed and directly used for the transformation. URA3⁺ transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C‐terminal encoding sequence that could restore the function of a truncated non‐functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR‐amplified target DNAs and C‐terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ‐based cloning and recombinant DNA construction. The one‐step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.     

YHp as a highly stable,hyper-copy,hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir⁰ strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3′ end of the RAF1 gene on the 2-μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir⁰ ura3^- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2-μm transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during nonselective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2-μm plasmid, and the red fluorescent protein expression was 54 fold compared with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.     

Aneuploidy,copy number variation and unique chromosomal structures in bottom‐fermenting yeast revealed by array‐CGH

DNA microarray for comparative genome hybridization (CGH) of bottom‐fermenting yeast was performed based on our in‐house DNA sequence data. Aneuploidy, copy number variation and unique chromosomal structures were observed among bottom‐fermenting yeast strains. Our array experiments revealed a correlation between copy number variation and mRNA expression levels. Chromosomal structures in a Saccharomyces carlsbergensis‐type strain and in a S. monacensis‐type strain that both belong to S. pastorianus phylogenetically differed greatly from those in contemporary industrial bottom‐fermenting yeast strains. The knowledge gained in this study contributes to a more precise genomic characterization of bottom‐fermenting yeast strains. Copyright © 2014 The Institute of Brewing & Distilling     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Saccharomyces pastorianus: genomic insights inspiring innovation for industry

A combination of biological and non‐biological factors has led to the interspecific hybrid yeast species Saccharomyces pastorianus becoming one of the world's most important industrial organisms. This yeast is used in the production of lager‐style beers, the fermentation of which requires very low temperatures compared to other industrial fermentation processes. This group of organisms has benefited from both the whole‐genome duplication in its ancestral lineage and the subsequent hybridization event between S. cerevisiae and S. eubayanus, resulting in strong fermentative ability. The hybrid has key traits, such as cold tolerance and good maltose‐ and maltotriose‐utilizing ability, inherited either from the parental species or originating from genetic interactions between the parent genomes. Instability in the nascent allopolyploid hybrid genome may have contributed to rapid evolution of the yeast to tolerate conditions prevalent in the brewing environment. The recent discovery of S. eubayanus has provided new insights into the evolutionary history of S. pastorianus and may offer new opportunities for generating novel industrially‐beneficial lager yeast strains. Copyright © 2014 John Wiley & Sons, Ltd.     

Disruption of URA7 and GAL6 improves the ethanol tolerance and fermentation capacity of Saccharomyces cerevisiae

Screening of the homozygous diploid yeast deletion pool of 4741 non-essential genes identified two null mutants (Deltaura7 and Deltagal6) that grew faster than the wild-type strain in medium containing 8% v/v ethanol. The survival rate of the gal6 disruptant in 10% ethanol was higher than that of the wild-type strain. On the other hand, the glucose consumption rate of the ura7 disruptant was better than that of the wild-type strain in buffer containing ethanol. Both disruptants were more resistant to zymolyase, a yeast lytic enzyme containing mainly beta-1,3-glucanase, indicating that the integrity of the cell wall became more resistance to ethanol stress. The gal6 disruptant was also more resistant to Calcofluor white, but the ura7 disruptant was more sensitive to Calcofluor white than the wild-type strain. Furthermore, the mutant strains had a higher content of oleic acid (C18 : 1) in the presence of ethanol compared to the wild-type strain, suggesting that the disruptants cope with ethanol stress not only by modifying the cell wall integrity but also the membrane fluidity. When the cells were grown in medium containing 5% ethanol at 15 degrees C, the gal6 and ura7 disruptants showed 40% and 14% increases in the glucose consumption rate, respectively.     

Genome comparison and evolutionary analysis of different industrial lager yeasts (Saccharomyces pastorianus)

Fermented beverages, especially beer, have accompanied human civilizations throughout our history. The yeast strains used for lager beer fermentation have long been recognized as hybrids between two Saccharomyces species. For hundreds of years, lager yeast (Saccharomyces pastorianus) has been subjected to multiple rounds of domestication owing to artificial selection during beer production. As a result, this species comprises a genetically diverse collection of strains that are used in different breweries. However, the scope of genetic diversity captured during the domesticated evolution of this species remains to be determined. To begin to address this, we collected the genome information of the only four lager strains that had been whole‐genome sequenced. For the first time, genome comparison was conducted between lager yeasts and clear signatures were found that defined each industrial yeast strain. The genetic variation comprises both single nucleotide polymorphisms and insertions and deletions. In addition, the core–pan genome was introduced for the first time to the genomic analysis of lager yeasts, detecting numerous strain‐specific and species‐shared genes. Furthermore, phylogenetic tree and synteny analysis results obtained in this study revealed information regarding the evolutionary relationship and group differentiation of studied strains. Genome comparison of the lager strains will, therefore, enable the characterization of the overall genetic diversity of this species, assist in the identification of genomic loci that play important roles in regulating key industrial phenotypes, and highlight the understanding of the hybrid nature and evolutionary details. Copyright © 2016 The Institute of Brewing & Distilling     

Co-existence of two types of chromosome in the bottom fermenting yeast,Saccharomyces pastorianus

The bottom fermenting yeasts in our collection were classified as Saccharomyces pastorianus on the basis of their DNA relatedness. The genomic organization of bottom fermenting yeast was analysed by Southern hybridization using eleven genes on chromosome IV, six genes on chromosome II and five genes on chromosome XV of S. cerevisiae as probes. Gene probes constructed from S. cerevisiae chromosomes II and IV hybridized strongly to the 820-kb chromosome and the 1500-kb chromosome of the bottom fermenting yeast, respectively. Five gene probes constructed from segments of chromosome XV hybridized strongly to the 1050-kb and the 1000-kb chromosomes. These chromosomes are thought to be S. cerevisiae-type chromosomes. In addition, these probes also hybridized weakly to the 1100-kb, 1350-kb, 850-kb and 700-kb chromosome. Gene probes constructed from segments including the left arm to TRP1 of chromosome IV and the right arm of chromosome II hybridized to the 1100-kb chromosome of S. pastorianus. Gene probes constructed using the right arm of chromosome IV and the left arm of chromosome II hybridized to the 1350-kb chromosome of S. pastorianus. These results suggested that the 1100-kb and 1350-kb chromosomes were generated by reciprocal translocation between chromosome II and IV in S. pastorianus. Three gene probes constructed using the right arm of chromosome XV hybridized weakly to the 850-kb chromosome, and two gene probes from the left arm hybridized weakly to the 700-kb chromosome. These results suggested that chromosome XV of S. cerevisiae was rearranged into the 850-kb and 700-kb chromosomes in S. pastorianus. These weak hybridization patterns were identical to those obtained with S. bayanus. Therefore, two types of chromosome co-exist independently in bottom fermenting yeast: one set which originated from S. bayanus and another set from S. cerevisiae. This result supports the hypothesis that S. pastorianus is a hybrid of S. cerevisiae and S. bayanus. © 1998 John Wiley & Sons, Ltd.     

Nucleotide sequences of alcohol acetyltransferase genes from lager brewing yeast,Saccharomyces carlsbergensis

The nucleotide sequences of alcohol acetyltransferase genes isolated from lager brewing yeast, Saccharomyces carlsbergensis have been determined. S. carlsbergensis has one ATF1 gene and another homologous gene, the Lg-ATF1 gene. There was a high degree of homology between the amino acid sequences deduced for the ATF1 protein and the Lg-ATF1 protein (75·7%), but the N-terminal region has a relatively low degree of homology. Southern analysis and contour-clamped homogeneous electric field analysis of Saccharomyces strains suggest that the ATF1 gene is located on chromosome XV in S. cerevisiae and that the Lg-ATF1 gene might originate from the ‘non-S. cerevisiae’ genome of S. carlsbergensis, which is similar to that of S. bayanus and S. pastorianus. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank data banks with the Accession Numbers D63449 (ATF1) and D63450 (Lg-ATF1).     

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     

A two-step cloning-free PCR-based method for the deletion of genes in the opportunistic pathogenic yeast Candida lusitaniae

We describe a new cloning-free strategy to delete genes in the opportunistic pathogenic yeast Candida lusitaniae. We first constructed two ura3 Δ strains in C. lusitaniae for their use in transformation experiments. One was deleted for the entire URA3 coding sequence; the other possessed a partial deletion within the coding region, which was used to determine the minimum amount of homology required for efficient homologous recombination by double crossing-over of a linear DNA fragment restoring URA3 expression. This amount was estimated to 200 bp on each side of the DNA fragment. These data constituted the basis of the development of a strategy to construct DNA cassettes for gene deletion by a cloning-free overlapping PCR method. Two cassettes were necessary in two successive transformation steps for the complete removal of a gene of interest. As an example, we report here the deletion of the LEU2 gene. The first cassette was constituted by the URA3 gene flanked by two large fragments (500 bp) homologous to the 5' and 3' non-coding regions of LEU2. After transformation of an ura3 Δ recipient strain and integration of the cassette at the LEU2 locus, the URA3 gene was removed by a second transformation round with a DNA cassette made by the fusion between the 5' and 3' non-coding regions of the LEU2 gene. The overall procedure takes less than 2 weeks and allows the creation of a clean null mutant that retains no foreign DNA sequence integrated in its genome.     

GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria

GABA transaminase (GABA‐T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA‐T from Saccharomyces cerevisiae (ScGABA‐TKG) is an α‐ketoglutarate‐dependent enzyme encoded by the UGA1 gene, while higher plant GABA‐T is a pyruvate/glyoxylate‐dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA‐T). The GABA‐T from A. thaliana is localized in mitochondria and mediated by an 18‐amino acid N‐terminal mitochondrial targeting peptide predicated by both web‐based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA‐TKG and AtGABA‐T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA‐TKG and AtGABA‐T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA‐TKG deletion mutant Δuga1 with AtGABA‐T or ScGABA‐TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat‐induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA‐T is functionally interchangeable with ScGABA‐TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA‐T is about half as efficient in doing so as ScGABA‐TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate‐limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae

To examine the effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae, we constructed deletion strains of all combinations of the trehalase genes ATH1, NTH1 and NTH2 and examined their growth behaviour and intracellular trehalose accumulation under non‐stress and saline‐stress conditions. Saline stress was induced in yeast cells by NaCl addition at the exponential growth phase. All deletion strains showed similar specific growth rates and trehalose accumulation to their parent strain under non‐stress conditions. However, under the saline stress condition, one single deletion strain, nth1Δ, two double deletion strains, nth1Δ ath1Δ and nth1Δ nth2Δ, and the triple deletion strain nth1Δnth2Δ ath1Δ, all of which carry the nth1Δ deletion, showed increased trehalose accumulation as compared to the parent and other deletion strains. In particular, our statistical analysis revealed that the triple deletion strain showed a higher growth rate under the saline stress condition than the parent strain. Moreover, some deletion strains showed further trehalose accumulation under non‐stress conditions by overexpression of the TPS1 or TPS2 genes encoding the enzymes related to trehalose biosynthesis at the mid‐exponential phase. Such increased trehalose accumulation prior to NaCl addition could improve the growth of these strains under saline stress. Our results indicate that high trehalose accumulation prior to NaCl addition, rather than after NaCl addition, is necessary to achieve high growth activity under stress conditions. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of deletion of different PP2C protein phosphatase genes on stress responses in Saccharomyces cerevisiae

A key mechanism of signal transduction in eukaryotes is reversible protein phosphorylation, mediated through protein kinases and protein phosphatases (PPases). Modulation of signal transduction by this means regulates many biological processes. Saccharomyces cerevisiae has 40 PPases, including seven protein phosphatase 2C (PP2C PPase) genes (PTC1–PTC7). However, their precise functions remain poorly understood. To elucidate their cellular functions and to identify those that are redundant, we constructed 127 strains with deletions of all possible combinations of the seven PP2C PPase genes. All 127 disruptants were viable under nutrient‐rich conditions, demonstrating that none of the combinations induced synthetic lethality under these conditions. However, several combinations exhibited novel phenotypes, e.g. the Δptc5Δptc7 double disruptant and the Δptc2Δptc3Δptc5Δptc7 quadruple disruptant exhibited low (13°C) and high (37°C) temperature‐sensitive growth, respectively. Interestingly, the septuple disruptant Δptc1Δptc2Δptc3Δptc4Δptc5Δptc6Δptc7 showed an essentially normal growth phenotype at 37°C. The Δptc2Δptc3Δptc5Δptc7 quadruple disruptant was sensitive to LiCl (0.4 m ). Two double disruptants, Δptc1Δptc2 and Δptc1Δptc4, displayed slow growth and Δptc1Δptc2Δptc4 could not grow on medium containing 1.5 m NaCl. The Δptc1Δptc6 double disruptant showed increased sensitivity to caffeine, congo red and calcofluor white compared to each single deletion. Our observations indicate that S. cerevisiae PP2C PPases have a shared and important role in responses to environmental stresses. These disruptants also provide a means for exploring the molecular mechanisms of redundant PTC gene functions under defined conditions. Copyright © 2014 John Wiley & Sons, Ltd.