Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment.

UNLABELLED: Cell suspensions of four Dekkera bruxellensis strains (CBS 2499, CBS 2797, CBS 4459 and CBS 4601) were subjected to heat treatment in deionized water at four different temperatures (55·0, 57·5, 60·0 and 62·5°C) to investigate their thermal resistance. The decimal reduction times at a specific temperature were calculated from the resulting inactivation curves: the D-values at 55·0°C ranged from 63 to 79·4 s, at 57·5°C from 39·6 to 46·1 s, at 60·0°C from 19·5 to 20·7 s, at 62·5°C from 10·2 to 13·7 s. The z-values were between 9·2 and 10·2°C, confirming that heat resistance is a strain-dependent character. A protocol for the sanitization of 225 l casks by immersion in hot water was set up and applied to contaminated 3-year-old barrels. The heat penetration through the staves was evaluated for each investigated temperature by positioning a thermal probe at 8 mm deep. A treatment at 60°C for an exposure time of 19 min allowed to eliminate the yeast populations up to a log count reduction of 8. SIGNIFICANCE AND IMPACT OF THE STUDY: Brettanomyces/Dekkera bruxellensis is the main yeast involved in red wine spoilage that occurs during ageing in barrel, generating considerable economic losses. Current sanitization protocols, performed using different chemicals, are ineffective due to the porous nature of the wood. The thermal inactivation of D. bruxellensis cells by hot water treatment proves to be efficacious and easy to perform, provided that the holding time at the killing temperature takes into account the filling time of the vessel and the time for the heat penetration into the wood structure.

Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines.

Eighty four isolates of Brettanomyces bruxellensis, were collected during fermentation of Sangiovese grapes in several Tuscan wineries and characterized by restriction analysis of 5.8S-ITS and species-specific PCR. The isolates were subsequently analysed, at strain level, by the combined use of the RAPD-PCR assay with primer OPA-02 and the mtDNA restriction analysis with the HinfI endonuclease. This approach showed a high degree of polymorphism and allowed to identify seven haplotypes, one of them being the most represented and widely distributed (72 isolates, 85.7%). Physiological traits of the yeasts were investigated under a wine model condition. Haplotypes clustered into two groups according to their growth rates and kinetics of production of 4-ethylphenol and 4-ethylguaiacol. Hexylamine was the biogenic amine most produced (up to 3.92 mg l(-1)), followed by putrescine and phenylethylamine. Formation of octapamine was detected by some haplotypes, for the first time.

Catabolism of pyrimidines in yeast: a tool to understand degradation of anticancer drugs.

The pyrimidine catabolic pathway is of crucial importance in cancer patients because it is involved in degradation of several chemotherapeutic drugs, such as 5-fluorouracil; it also is important in plants, unicellular eukaryotes, and bacteria for the degradation of pyrimidine-based biocides/antibiotics. During the last decade we have developed a yeast species, Saccharomyces kluyveri, as a model and tool to study the genes and enzymes of the pyrimidine catabolic pathway. In this report, we studied degradation of uracil and its putative degradation products in 38 yeasts and showed that this pathway was present in the ancient yeasts but was lost approximately 100 million years ago in the S. cerevisiae lineage.

Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.

Alterations of the glucose metabolism in a triose phosphate isomerase-negative Saccharomyces cerevisiae mutant.

The absence of triose phosphate isomerase activity causes an accumulation of only one of the two trioses, dihydroxyacetone phosphate, and this produces a shift in the final product of glucose catabolism from ethanol to glycerol (Compagno et al., 1996). Alterations of glucose metabolism imposed by the deletion of the TPI1 gene in Saccharomyces cerevisiae were studied in batch and continuous cultures. The Deltatpi1 null mutant was unable to grow on glucose as the sole carbon source. The addition of ethanol or acetate in media containing glucose, but also raffinose or galactose, relieved this effect in batch cultivation, suggesting that the Crabtree effect is not the primary cause for the mutant's impaired growth on glucose. The addition of an energy source like formic acid restored glucose utilization, suggesting that a NADH/energy shortage in the Deltatpi1 mutant could be a cause of the impaired growth on glucose. The amount of glycerol production in the Deltatpi1 mutant could represent a good indicator of the fraction of carbon source channelled through glycolysis. Data obtained in continuous cultures on mixed substrates indicated that different contributions of glycolysis and gluconeogenesis, as well as of the HMP pathway, to glucose utilization by the Deltatpi1 mutant may occur in relation to the fraction of ethanol present in the media.

Current awareness.

In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on yeasts. Each bibliography is divided into 10 sections. 1 Books, Reviews & Symposia; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals - search completed 7th Mar. 2001)

Isolation, nucleotide sequence, and physiological relevance of the gene encoding triose phosphate isomerase from Kluyveromyces lactis.

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae DeltaTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess.

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.

Glycerol production in a triose phosphate isomerase deficient mutant of Saccharomyces cerevisiae.

Interesting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemicals. In this paper, we show that engineered yeast cells deficient in the triose phosphate isomerase activity are able to produce glycerol without the use of steering agents. High yields of conversion of glucose into glycerol (80-90% of the theoretical yield) and productivity (1.5 g L-1 h-1) have been obtained by a bioconversion process carried out in a poor and clean medium. We obtained indications that the growth phase at which the biomass was collected affect the process. The best results were obtained using cells collected at the end of exponential phase of growth. In perspective, the strategies and the information about the physiology of the cells described here could be useful for the developing of new biotechnological processes for glycerol production, outflanking the problems related to the use of high level of steering agents.

Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system.

Autoselection systems allow the selection of a genetically engineered population independently of the growth medium composition. The structure of a Saccharomyces cerevisiae population transformed with an autoselection plasmid, in which a carbon-source-dependent modulation of the plasmid copy number occurs, was analysed. By means of flow cytometric procedures we tested the cell viability, dynamics of growth and heterologous protein production at single cell level. Such analyses allow the identification and the tracking of a specific cellular sub-population with a higher plasmid copy number which arises after the carbon source shift. The effects of the cellular plasmid distribution on the dynamics of growth are also discussed.

Fermentation of whey and starch by transformed Saccharomyces cerevisiae cells.

Among the main agro-industrial wastes, whey and starch are of prime importance. In previous work we showed that strains of Saccharomyces cerevisiae transformed with the episomal plasmid pM1 allow production of yeast biomass and ethanol from whey/lactose. Ethanol production from whey and derivatives has been improved in computer-controlled bioreactors, while fermentation studies showed that the composition of the medium greatly modulates the productivity (g ethanol produced/l in 1 h of fermentation). A yeast strain for the simultaneous utilization of lactose and starch has also been developed. Biotechnological perspectives are discussed.

Copy number modulation in an autoselection system for stable plasmid maintenance in Saccharomyces cerevisiae.

Efficient expression of a foreign gene requires a stable vector present at a high number of copies per cell. We have constructed an autoselection system for the stable maintenance of expression vector in the yeast Saccharomyces cerevisiae that uses the fructose 1,6-bisphosphate aldolase gene (FBA1) to stabilize plasmids in cells bearing a disruption of the chromosomal FBA1 gene. This system allowed us to obtain stable production of a reporter heterologous enzyme (Escherichia coli beta-galactosidase) in rich media. By using an inducible promoter to regulate the expression of FBA1 gene, we have also obtained the modulation of plasmid copy number by carbon source.

Bioconversion of lactose/whey to fructose diphosphate with recombinant saccharomyces cerevisiae cells.

Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli beta-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. We showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

The promoter of Saccharomyces cerevisiae FBA1 gene contains a single positive upstream regulatory element.

The glycolytic enzyme fructose 1,6-bisphosphate aldolase is encoded by the FBA1 gene of Saccharomyces cerevisiae. Transcription of aldolase gene is not regulated by glucose and high levels of expression have been observed also during growth on nonfermentable carbon source. A FBA1::lacZ gene fusion was constructed and a deletion analysis demonstrated the presence of a unique cis-acting positive upstream element (UAS) required for high levels of FBA1 expression. This element is located between positions -550 and -440 upstream of the aldolase open reading frame and it contains sequences known to constitute the binding sites for the multifunctional proteins RAP1 and ABFI and two TTCC motifs.

Yeast 2 micron vectors replicate and undergo recombination in Torulaspora delbrueckii.

In order to develop a procedure for transformation of the industrial yeast Torulaspora delbrueckii, we have constructed a set of recombinant plasmids carrying Saccharomyces cerevisiae ARS and 2 microns origin of replication and kanamycin-G418 resistance gene of Tn903(601) as a selective marker. In this paper we show that S. cerevisiae ARS vectors can replicate autonomously and that vectors bearing the whole S. cerevisiae 2 microns sequence yield stable transformants. We also present evidence to show that 2 microns vectors undergo an FLP-mediated inter- and intramolecular recombination, which suggests that T. delbrueckii can support the amplification and partition mechanisms of these plasmids.

Translational regulation of the expression of zein cloned in yeast under an inducible GAL promoter.

Yeast cells transformed by a plasmid containing a zein sequence fused to an hybrid yeast promoter GAL1-10/CYC1 accumulate, during a batch growth in galactose minimal medium, large amounts of zein only during a growth-limited phase that precede the entering into the stationary phase. We found that zein is fairly stable in yeast cells and the increased accumulation of zein polypeptide depends mainly upon a marked increase of its rate of synthesis. The increase of the rate of heterologous protein synthesis is not dependent on variation in the plasmid copy number and it is not related to the relative level of zein mRNA, indicating the existence of a postranscriptional regulation that modulates the translatability of this messenger RNA in function of the growth conditions. A possible explanation of this modulation is discussed in terms of a codon bias effect that slow-down the translation of heterologous mRNAs during the exponential phase of growth.

Transcription and expression of zein sequences in yeast under natural plant or yeast promoters.

Maize genomic fragments containing the regulatory and coding regions of a zein gene for a low size class 23-kd protein have been inserted in an interspecific Escherichia coli-Saccharomyces cerevisiae expression vector in different constructions. The presence of the inducible GAL1-10 upstream activation site (UAS) allows us to regulate differentially by carbon sources the transcription of the zein gene both under the plant promoter and under the yeast CYC-1 promoter. We found that the zein promoter region is properly recognized at the correct transcription start, while different termination points occur during transcription. The yeast UAS was also shown to function as a typical eukaryotic enhancer regardless of its distance or orientation with respect to the plant promoter. Yeast cells transformed by a plasmid containing a zein sequence fused to a short piece of the CYC-1 gene produced a fused polypeptide, of expected mol. wt, in variable amount from 0.2 to 5% depending on the growth phase conditions.

Analysis of protein and cell volume distribution in glucose-limited continuous cultures of budding yeast.

Method of flow cytometric analysis have recently been developed that make it possible to obtain segregated data on a single cell basis. In particular, it has been previously demonstrated that protein distributions obtained by flow cytometry give information about the law of growth of the cell population and the law of growth of the single cell; thus these distribution show how the microbial population is actually growing at the moment of the analysis and may yield more accurate and predictive information. We have extended the analysis of protein distribution and cell volume distribution to continuous cultures of Saccharomyces cerevisiae growing in a glucose-limited chemostat. We have found that: (1) to each dilution rate corresponds a given protein and volume distribution that does not change with time in steady state cultures; (2) there is a good proportionality between the average cell volume and the average protein content; (3) the protein distribution obtained can be easily analyzed with the model of growth of yeast previously developed in our laboratory; (4) the analysis of perturbed states shows that both protein distribution and volume distribution change very quickly; thus they are very sensitive parameters and can be used for monitoring and controlling industrial fermentation.