Regulation of THI4(MOL1), a thiamine-biosynthetic gene of Saccharomyces cerevisiae

THI4, a Saccharomyces cerevisiae gene originally identified as a result of transient expression in molasses medium and named MOL1 is regulated by thiamine. Using a THI4 promoter-lacZ fusion on a centromeric yeast vector, we have shown that the THI4 is completely repressed throughout batch culture by thiamine at a concentration around 1 μM, but shows high level constitutive expression in thiamine-free medium. The transient expression pattern observed in molasses medium can be mimicked by the addition of 0·15 μM-thiamine to defined minimal medium. Cells grown in thiamine-free medium have an intracellular thiamine concentration of around 9 pmol/10⁷ cells. A low level (1 μM) of exogenous thiamine is completely sequestered from the medium within 30 min; intracellular thiamine concentrations rise rapidly, followed by a gradual decrease as a result of dilution during growth. A saturating extracellular level of thiamine leads to a maximal intracellular concentration of around 1600 pmol/10⁷ cells, at which point the transport system is shut down. After transfer from repressing to non-repressing medium, THI4 becomes induced when the intracellular concentration of thiamine falls to 20 pmol/10⁷ cells. A thi4::UARA3 disruption strain is auxotrophic for thiamine, but can grow in the presence of hydroxyethyl thiazole, indicating that the gene product is involved in the biosynthetic pathway leading to the formation of the thiazole precursor of thiamine.     

YEAST MUTANTS EXCRETING VITAMIN B1 AND THEIR USE IN THE PRODUCTION OF THIAMINE RICH BEERS*,†

In certain strains of Saccharomyces cerevisiae and Saccharomyces uvarum (carlsbergensis), high doses of ultraviolet radiation induced stable mutants excreting thiamine from their living cells during the ethanol production. In S. cerevisiae, the first mutagenesis step yielded mutants with the production of 0·3–0·5 mg extracellular thiamine. HCL/litre minimum medium without thiamine and the second one increased the production up to 2·0–2·1 mg thiamine. HCL/litre. In S. uvarum (carlsbergensis), mutants producing up to 0·76 mg thiamine. HCL/litre 10% Plato hopped wort were obtained in the first mutagenesis step combined with mitotic recombinations. The increase up to 0·92 mg/litre was achieved here by repeated selections. Both laboratory and pilot plant fermentations in 10 and 4% Plato hopped worts showed the suitability of selected mutants for the production of thiamine rich beers which fulfilled all quality requirements and contained 0·67–0·80 and 0·22–0·33 mg thiamine. HCI/litre, respectively.     

GENETIC CONTROL OF THIAMINE EXCRETION AND OF ITS SUPPRESSION IN SACCHAROMYCES CEREVISIAE

Three non-linked loci were found to be involved in thiamine excretion in Saccharomyces cerevisiae, i.e. thi2 which is complementary with thi3 and thi4, the latter two being substitutable mutually but acting also together. Mutation thi3 was present already in strain Nα, which produced relatively high frequencies of excreters after UV treatment. Non-excreting ‘revertants’ of unstable excreters derived from another haploid strain contained recessive mutation thi2, so that mutation thi3 can be substituted also by the metabolic or physiological disorders ruling after UV treatment. Mutations thi2 were found also in all 43 analysed stable excreting mutants obtained by UV treatment of strain Nα which indicates that they are either inevitable for the excretion or are specifically selected by the selection method used. At least one suppressor locus of thiamine excretion was present in that strain of S. cerevisiae, which did not produce thiamine excreters after UV treatment in spite of its ability to synthetise thiamine.     

Comparing cytosolic expression to peroxisomal targeting of the chimeric L1/L2 (ChiΔH-L2) gene from human papillomavirus type 16 in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha

The chimeric ChiΔH‐L2 gene from human papillomavirus type 16, consisting of structural proteins L1 and L2, was successfully expressed in the cytosol of both Pichia pastoris and Hansenula polymorpha during methanol induction. In addition, a novel approach was employed whereby ChiΔH‐L2 was targeted to the peroxisome using peroxisomal targeting sequence 1 (PTS1) to compare ChiΔH‐L2 yields in the peroxisome vs the cytosol. The ChiΔH‐L2 gene was yeast‐optimized and cloned into plasmids aimed at genomic integration. Levels of intracellular ChiΔH‐L2 accumulation in the cytosol were highest in P. pastoris KM71 strain KMChiΔH‐L2 (1.43 mg/l), compared to the maximum production level of 0.72 mg/l obtained with H. polymorpha. ChiΔH‐L2 targeting to the peroxisome was successful; however, it appeared to negatively affect ChiΔH‐L2 production in both P. pastoris and H. polymorpha. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolic flux analysis for efficient pyruvate fermentation using vitamin-auxotrophic yeast of Torulopsis glabrata

The metabolism of a vitamin-auxotrophic pyruvate-producing microorganism, Torulopsis glabrata IFO 0005, was investigated by metabolic flux analysis. Particular attention was focused on the effect of culture conditions, such as dissolved oxygen (DO) concentration and thiamine concentration, on specific pathway activities. The results of metabolic flux analysis indicate that the thiamine concentration significantly affected pyruvate dehydrogenase and pyruvate decarboxylase activities, and plays an important role in cell growth and pyruvate production. Metabolic flux analysis was also utilized to clarify the metabolism of this strain during pyruvate fermentation under different oxygen supply conditions, and the reason for the enhanced pyruvate production under conditions of 30-40% DO concentration was clarified from the viewpoint of intracellular flux distributions. Based on the analysis of the effect of thiamine concentration on the metabolic fluxes, we conducted a fed-batch experiment where the initial thiamine concentration was reduced to 30 microg/l and thiamine was added at 10 microg/l during fermentation when the cell growth rate decreased to 0.2 h(-1). With separate addition of thiamine, the overall pyruvate yield could be improved by 15% due to the decrease of ethanol production.     

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.     

Functional interrelationships between carbohydrate and lipid storage,and mitochondrial activity during sporulation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae under conditions of nutrient stress, meiosis precedes the formation of spores. Although the molecular mechanisms that regulate meiosis, such as meiotic recombination and nuclear divisions, have been extensively studied, the metabolic factors that determine the efficiency of sporulation are less understood. Here, we have directly assessed the relationship between metabolic stores and sporulation in S. cerevisiae by genetically disrupting the synthetic pathways for the carbohydrate stores, glycogen (gsy1/2Δ cells), trehalose (tps1Δ cells), or both (gsy1/2Δ and tps1Δ cells). We show that storage carbohydrate-deficient strains are highly inefficient in sporulation. Although glycogen and trehalose stores can partially compensate for each other, they have differential effects on sporulation rate and spore number. Interestingly, deletion of the G₁ cyclin, CLN3, which resulted in an increase in cell size, mitochondria and lipid stores, partially rescued meiosis progression and spore ascus formation but not spore number in storage carbohydrate-deficient strains. Sporulation efficiency in the carbohydrate-deficient strain exhibited a greater dependency on mitochondrial activity and lipid stores than wild-type yeast. Taken together, our results provide new insights into the complex crosstalk between metabolic factors that support gametogenesis.     

Isolation of a Histoplasma capsulatum cDNA that complements a mitochondrial NAD(+)‐isocitrate dehydrogenase subunit I‐deficient mutant of Saccharomyces cerevisiae

A cDNA library was prepared from Histoplasma capsulatum strain G‐217B yeast cells and an apparently full‐length cDNA for a subunit of the citric acid cycle enzyme NAD(+)‐isocitrate dehydrogenase was identified by sequence analysis. Its predicted amino acid sequence is more similar to the IDH1 regulatory subunit of S. cerevisiae NAD(+)‐isocitrate dehydrogenase than to the IDH2 catalytic subunit. After expression in S. cerevisiae from an S. cerevisiae promoter, it was shown to functionally complement an S. cerevisiae idh1 mutant, but not an idh2 mutant, for growth on acetate as a carbon source and for production of NAD(+)‐isocitrate dehydrogenase enzyme activity. These results confirm that the H. capsulatum cDNA encodes a homologue of subunit I of the S. cerevisiae mitochondrial isocitrate dehydrogenase isozyme that functions in the citric acid cycle. The HcIDH1 cDNA sequence is available in GenBank with Accession No. AF009036. Copyright © 1999 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.     

Bioconversion of heptanal to heptanol by Saccharomyces cerevisiae

Saccharomyces cerevisiae is widely known for its catalytic activity on substrates such as aldehyde and ketone. Interestingly, the activity of S. cerevisiae on heptanal (C₆H₁₃CHO), in spite of its being a very common aldehyde, has not been explored. The main objective of this study was therefore to investigate the bioconversion of heptanal, using a strain of the yeast S. cerevisiae. Bioconversion parameters such as incubation period, pH, concentration of substrate, yeast and maltose were also optimized. The study revealed heptanol as the major product. The optimum conditions for biotransformation were found to be: 3 days incubation; pH 7.0; heptanal concentration 0.15 ml/100 ml medium; and S. cerevisiae concentration of 0.15 g/100 ml medium. Reduction in maltose content (to 0.3 g maltose/100 ml medium) showed increased conversion of heptanal. Heptanoic acid and 2‐hydroxyheptanoic acid were obtained as two minor co‐products. The overall study showed that S. cerevisiae converted heptanal to heptanol by a yield of 68.9 ± 1.1% w/w under optimum conditions. Copyright © 2010 John Wiley & Sons, Ltd.     

Mutation of the CgPDR16 gene attenuates azole tolerance and biofilm production in pathogenic Candida glabrata

The PDR16 gene encodes the homologue of Sec14p, participating in protein secretion, regulation of lipid synthesis and turnover in vivo and acting as a phosphatidylinositol transfer protein in vitro. This gene is also involved in the regulation of multidrug resistance in Saccharomyces cerevisiae and pathogenic yeasts. Here we report the results of functional analysis of the CgPDR16 gene, whose mutation has been previously shown to enhance fluconazole sensitivity in Candida glabrata mutant cells. We have cloned the CgPDR16 gene, which was able to complement the pdr16Δ mutation in both C. glabrata and S. cerevisiae. Along with fluconazole, the pdr16Δ mutation resulted in increased susceptibility of mutant cells to several azole antifungals without changes in sensitivity to polyene antibiotics, cycloheximide, NQO, 5‐fluorocytosine and oxidants inducing the intracellular formation of reactive oxygen species. The susceptibility of the pdr16Δ mutant strain to itraconazole and 5‐fluorocytosine was enhanced by CTBT [7‐chlorotetrazolo(5,1‐c)benzo(1,2,4)triazine] inducing oxidative stress. The pdr16Δ mutation increased the accumulation of rhodamine 6G in mutant cells, decreased the level of itraconazole resistance caused by gain‐of‐function mutations in the CgPDR1 gene, and reduced cell surface hydrophobicity and biofilm production. These results point to the pleiotropic phenotype of the pdr16Δ mutant and support the role of the CgPDR16 gene in the control of drug susceptibility and virulence in the pathogenic C. glabrata. Copyright © 2013 John Wiley & Sons, Ltd.     

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full‐length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l ‐cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l ‐cystine and some other cysteine conjugates, but not l ‐cystathionine or l ‐methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l ‐cysteine as a nitrogen source, and that overexpression of the gene results in increased H₂S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S‐ethyl‐l ‐cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

Construction of self‐cloning industrial brewer's yeast with SOD1 gene insertion into PEP4 prosequence locus by homologous recombination

Superoxide dismutase (SOD, encoded by SOD1), which can scavenge active oxygen free radicals, is an ideal endogenous antioxidase in beer. In this study, the SOD1 expression cassette was constructed, and this cassette contained the PGK1 promoter, the PGK1 terminator and the SOD1 gene fused to the signal sequence of the yeast mating pheromone α‐factor (MFα1_s). One of the prosequences of the PEP4 gene (encoding proteinase A, PrA) in Saccharomyces cerevisiae strain S‐6 was replaced by the SOD1 expression cassette via homologous recombination and the self‐cloning strain S54PS, which could improve the antioxidant capability and foam stability of beer, was successfully obtained. Fermentation results showed that the SOD activity of the final beer brewed with S54PS was increased by 21.06%. Accordingly, the DPPH‐radical scavenging activity of S54PS increased by 30.6% compared with that yielded by the parental strain S‐6. Furthermore, the PrA activity of S54PS was always lower than that of the parental strain at all stages of beer fermentation. The head retention of the beer (255 ± 4 s) was better than that of the parental strain (224 ± 1 s). Hence, this research implies that S54PS exhibits good brewing performance and can be applied to improve the industrial brewing process. Copyright © 2016 The Institute of Brewing & Distilling     

Investigating flavour characteristics of British ale yeasts: techniques,resources and opportunities for innovation

Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour‐related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 John Wiley & Sons, Ltd.     

The yeast Saccharomyces cerevisiae Pdr16p restricts changes in ergosterol biosynthesis caused by the presence of azole antifungals

Pdr16p belongs to the family of phosphatidylinositol transfer proteins in yeast. The absence of Pdr16p results in enhanced susceptibility to azole antifungals in Saccharomyces cerevisiae. In the major fungal human pathogen Candida albicans, CaPDR16 is a contributing factor to clinical azole resistance. The current study was aimed at better understanding the function of Pdr16p, especially in relation to azole resistance in S. cerevisiae. We show that deletion of the PDR16 gene increased susceptibility of S. cerevisiae to azole antifungals that are used in clinical medicine and agriculture. Significant differences in the inhibition of the sterol biosynthetic pathway were observed between the pdr16Δ strain and its corresponding wild‐type (wt) strain when yeast cells were challenged by sub‐inhibitory concentrations of the azoles miconazole or fluconazole. The increased susceptibility to azoles, and enhanced changes in sterol biosynthesis upon exposure to azoles of the pdr16Δ strain compared to wt strain, are not the results of increased intracellular concentration of azoles in the pdr16Δ cells. We also show that overexpression of PDR17 complemented the azole susceptible phenotype of the pdr16Δ strain and corrected the enhanced sterol alterations in pdr16Δ cells in the presence of azoles. Pdr17p was found previously to be an essential part of a complex required for intermembrane transport of phosphatidylserine at regions of membrane apposition. Based on these observations, we propose a hypothesis that Pdr16p assists in shuttling sterols or their intermediates between membranes or, alternatively, between sterol biosynthetic enzymes or complexes. Copyright © 2013 John Wiley & Sons, Ltd.     

The nucleotide sequence and initial characterization of pyruvate decarboxylase from the yeast Hanseniaspora uvarum

We have isolated a pyruvate decarboxylase (PDC) gene from the yeast Hanseniaspora uvarum using the Saccharomyces cerevisiae PDC1 gene as a probe. The nucleotide sequence of this gene was determined and compared to PDC genes from yeast and other organisms. The H. uvarum PDC gene is more than 70% identical to the S. cerevisiae PDC isozymes and possesses a putative thiamine diphosphate binding site. The PDC enzyme was purified and partially characterized. The H. uvarum PDC was very similar to other known PDCs; the K_m for pyruvate was 0·75 mM, and the enzyme is a homotetramer with subunits of M_r = 57 000. The sequence has been submitted to GenBank under Accession No. U13635.