Functional enhancement of Sake yeast strains to minimize the production of ethyl carbamate in Sake wine.

AIMS: In fermented alcoholic beverages and particularly in Japanese Sake wine, the ubiquitous presence of the probable human carcinogen ethyl carbamate (EC) is a topic of significant concern. This study aims to develop novel methods for the reduction of EC in Sake wine. METHODS AND RESULTS: To reduce the high levels of EC in Sake wine, urea-degrading and urea-importing yeast strains were created by integrating linear cassettes containing either the respective DUR1,2 or DUR3 genes, under the control of the constitutively active Saccharomyces cerevisiae PGK1 promoter, into the Sake yeast strains K7 and K9. The self-cloned, urea-degrading Sake strains K7(DUR1,2) and K9(DUR1,2) produced Sake wine with 87 and 68% less EC, respectively, while the urea-importing Sake yeast strain K7(DUR3) reduced EC by 15%. All functionally enhanced yeast strains were shown to be substantially equivalent to their parental strains in terms of fermentation rate, ethanol production, phenotype and transcriptome. CONCLUSIONS: Under the conditions tested, urea-degrading yeast (constitutive DUR1,2 expression) are superior to urea-importing yeast (constitutive DUR3 expression) for EC reduction in Sake wine, and constitutive co-expression of DUR1,2 and DUR3 does not yield synergistic EC reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: The self-cloned, substantially equivalent, urea-degrading Sake yeast strains K7(DUR1,2) and K9(DUR1,2), which contain the integrated DUR1,2 cassette, are capable of highly efficacious EC reduction during Sake brewing trials, are suitable for commercialization and are important tools for modern Sake makers in their efforts to reduce high EC levels in Sake wine.

Malo-ethanolic fermentation in Saccharomyces and Schizosaccharomyces.

Yeast species are divided into the K(+) or K(-) groups, based on their ability or inability to metabolise tricarboxylic acid (TCA) cycle intermediates as sole carbon or energy source. The K(-) group of yeasts includes strains of Saccharomyces, Schizosaccharomyces pombe and Zygosaccharomyces bailii, which is capable of utilising TCA cycle intermediates only in the presence of glucose or other assimilable carbon sources. Although grouped together, these yeasts have significant differences in their abilities to degrade malic acid. Typically, strains of Saccharomyces are regarded as inefficient metabolisers of extracellular malic acid, whereas strains of Sch. pombe and Z. bailii can effectively degrade high concentrations of malic acid. The ability of a yeast strain to degrade extracellular malic acid is dependent on both the efficient transport of the dicarboxylic acid and the efficacy of the intracellular malic enzyme. The malic enzyme converts malic acid into pyruvic acid, which is further metabolised to ethanol and carbon dioxide under fermentative conditions via the so-called malo-ethanolic (ME) pathway. This review focuses on the enzymes involved in the ME pathway in Sch. pombe and Saccharomyces species, with specific emphasis on the malate transporter and the intracellular malic enzyme.

Malo-ethanolic fermentation in grape must by recombinant strains of Saccharomyces cerevisiae.

Recombinant strains of Saccharomyces cerevisiae with the ability to reduce wine acidity could have a significant influence on the future production of quality wines, especially in cool climate regions. L-Malic acid and L-tartaric acid contribute largely to the acid content of grapes and wine. The wine yeast S. cerevisiae is unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of a malo-ethanolic fermentation. However, strains of Sz. pombe are not suitable for vinification due to the production of undesirable off-flavours. Heterologous expression of the Sz. pombe malate permease (mae1) and malic enzyme (mae2) genes on plasmids in S. cerevisiae resulted in a recombinant strain of S. cerevisiae that efficiently degraded up to 8 g/l L-malic acid in synthetic grape must and 6.75 g/l L-malic acid in Chardonnay grape must. Furthermore, a strain of S. cerevisiae containing the mae1 and mae2 genes integrated in the genome efficiently degraded 5 g/l of L-malic acid in synthetic and Chenin Blanc grape musts. Furthermore, the malo-alcoholic strains produced higher levels of ethanol during fermentation, which is important for the production of distilled beverages.

Cis-acting sites contributing to expression of divergently transcribed DAL1 and DAL4 genes in S. cerevisiae: a word of caution when correlating cis-acting sequences with genome-wide expression analyses.

Correlating genome-wide expression profiles with sequence searches of promoter regions is being used as a technique to identify putative binding sites for transacting factors or to refine consensus sequences of those already known. To evaluate the limitations of such an approach in our studies of GATA-mediated transcription in Saccharomyces cerevisiae, we identified the relative contributions made to DAL1 and DAL4 expression by each of five Gln3p-, and/or Gat1p-, and three Dal82p-binding site homologous sequences situated in the 829-bp intergenic region separating these highly related, divergently transcribed genes. Our data suggest that although the correlation of repeated sequences or sequence homologies appearing within promoter regions with expression profiles obtained from genome-wide transcription analyses can provide useful starting points for analyses of cis-acting sites, significant limitations and possibilities for misinterpretation also abound.

Ammonia regulates VID30 expression and Vid30p function shifts nitrogen metabolism toward glutamate formation especially when Saccharomyces cerevisiae is grown in low concentrations of ammonia.

The GATA family proteins Gln3p and Gat1p mediate nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae. When cells are cultured with a good nitrogen source (glutamine, ammonia), Gln3p and Gat1p are restricted to the cytoplasm, whereas with a poor nitrogen source (proline), they localize to the nucleus, bind to the GATA sequences of NCR-sensitive gene promoters, and activate transcription. The target of rapamycin-signaling cascade and Ure2p participate in regulating the cellular localization of Gln3p and Gat1p. Rapamycin, a Tor protein inhibitor, like growth with a poor nitrogen source, promotes nuclear localization of Gln3p and Gat1p. gln3 Delta and ure2 Delta mutants are partially resistant and hypersensitive to growth inhibition by rapamycin, respectively. We show that a vid30 Delta is more rapamycin-sensitive than wild type but less so than a ure2 Delta. VID30 expression is modestly NCR-sensitive, responsive to deletion of URE2, and greatly increases in low ammonia medium. Patterns of gene expression in a vid30 Delta suggest that the Vid30p function shifts the balance of nitrogen metabolism toward the production of glutamate, especially when cells are grown in low ammonia. CAN1, DAL4, DAL5, MEP2, DAL1, DAL80, and GDH3 transcription is down-regulated by Vid30p function with proline as the nitrogen source. An effect, however, that could easily be indirect.

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe.

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as L-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and L-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or L-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular L-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate.

Transcriptional regulation of the Schizosaccharomyces pombe malic enzyme gene, mae2.

The NAD-dependent malic enzyme from Schizosaccharomyces pombe catalyzes the oxidative decarboxylation of L-malate to pyruvate and CO2. Transcription of the S. pombe malic enzyme gene, mae2, was studied to elucidate the regulatory mechanisms involved in the expression of the gene. No evidence for substrate-induced expression of mae2 was observed in the presence of 0.2% L-malate. However, transcription of mae2 was induced when cells were grown in high concentrations of glucose or under anaerobic conditions. The increased levels of malic enzyme may provide additional pyruvate or assist in maintaining the redox potential under fermentative conditions. Deletion and mutation analyses of the 5'-flanking region of the mae2 gene revealed the presence of three novel negative cis-acting elements, URS1, URS2, and URS3, that seem to function cooperatively to repress transcription of the mae2 gene. URS1 and URS2 are also present in the promoter region of the S. pombe malate transporter gene, suggesting co-regulation of their expression. Furthermore, two positive cis-acting elements in the mae2 promoter, UAS1 and UAS2, show homology with the DNA recognition sites of the cAMP-dependent transcription factors ADR1, AP-2, and ATF (activating transcription factor)/CREB (cAMP response element binding).

Mutation of Gly-444 inactivates the S. pombe malic enzyme.

A mutant malic enzyme gene, mae2-, was cloned from a strain of Schizosaccharomyces pombe that displayed almost no malic enzyme activity. Sequence analysis revealed only one codon-altering mutation, a guanine to adenine at nucleotide 1331, changing the glycine residue at position 444 to an aspartate residue. Gly-444 is located in Region H, previously identified as one of eight highly conserved regions in malic enzymes. We found that Gly-444 is absolutely conserved in 27 malic enzymes from various prokaryotic and eukaryotic sources, as well as in three bacterial malolactic enzymes investigated. The evolutionary conservation of Gly-444 suggests that this residue is important for enzymatic function.

Engineering pathways for malate degradation in Saccharomyces cerevisiae.

Deacidification of grape musts is crucial for the production of well-balanced wines, especially in colder regions of the world. The major acids in wine are tartaric and malic acid. Saccharomyces cerevisiae cannot degrade malic acid efficiently due to the lack of a malate transporter and the low substrate affinity of its malic enzyme. We have introduced efficient pathways for malate degradation in S. cerevisiae by cloning and expressing the Schizosaccharomyces pombe malate permease (mae1) gene with either the S. pombe malic enzyme (mae2) or Lactococcus lactis malolactic (mleS) gene in this yeast. Under aerobic conditions, the recombinant strain expressing the mae1 and mae2 genes efficiently degraded 8 g/L of malate in a glycerol-ethanol medium within 7 days. The recombinant malolactic strain of S. cerevisiae (mae1 and mleS genes) fermented 4.5 g/L of malate in a synthetic grape must within 4 days.

The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids.

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein in similar to models proposed for other integral membrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1.5 kb. The mea1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5' to the MFml gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid.

A kinase-cyclin pair in the RNA polymerase II holoenzyme.

The RNA polymerase II holoenzyme consists of RNA polymerase II, a subset of general transcription factors, and regulatory proteins known as SRB proteins. The genes encoding SRB proteins were isolated as suppressors of mutations in the RNA polymerase II carboxy-terminal domain (CTD). The CTD and SRB proteins have been implicated in the response to transcriptional regulators. We report here the isolation of two new SRB genes, SRB10 and SRB11, which encode kinase- and cyclin-like proteins, respectively. Genetic and biochemical evidence indicates that the SRB10 and SRB11 proteins form a kinase-cyclin pair in the holoenzyme. The SRB10/11 kinase is essential for a normal transcriptional response to galactose induction in vivo. Holoenzymes lacking SRB10/11 kinase function are strikingly deficient in CTD phosphorylation. Although defects in the kinase substantially affect transcription in vivo, purified holoenzymes lacking SRB10/11 kinase function do not show defects in defined in vitro transcription systems, suggesting that the factors necessary to elicit the regulatory role of the SRB10/11 kinase are missing in these systems. These results indicate that the SRB10/11 kinase is involved in CTD phosphorylation and suggest that this modification has a role in the response to transcriptional regulators in vivo.

Cloning, sequence and in vitro transcription/translation analysis of a 3.2-kb EcoRI-HindIII fragment of Leuconostoc oenos bacteriophage L10.

A 3.2-kb EcoRI-HindIII DNA fragment of Leuconostoc oenos bacteriophage L10 was cloned and sequenced. Computer-assisted analysis of the sequence identified eleven possible open reading frames (ORFs) that were all on the same strand. In vitro transcription/translation analysis of the full-length DNA fragment yielded five prominent proteins that were correlated with ORFs by their sizes and expression from deleted clones. Only those ORFs containing recognizable Shine-Dalgarno sequences coded for proteins. Neither the nucleotide sequence, nor deduced amino-acid sequences showed significant homology with other known sequences.

Molecular analysis of the malic enzyme gene (mae2) of Schizosaccharomyces pombe.

Sequence analysis of a 4.6-kb HindIII fragment containing the malic enzyme gene (mae2) of Schizosaccharomyces pombe, revealed the presence of an open reading frame of 1695 nucleotides, coding for a 565 amino acid polypeptide. The mae2 gene is expressed constitutively and encodes a single mRNA transcript of 2.0 kb. The mae2 gene was mapped on chromosome III by chromoblotting. The coding region and inferred amino acid sequence showed significant homology with 12 malic enzyme genes and proteins from widely different origins. Eight highly homologous regions were found in these malic enzymes, suggesting that they contain functionally conserved amino acid sequences that are indispensable for activity of malic enzymes. Two of these regions have previously been reported to be NAD- and NADP-binding sites.

Analysis of the inducer-responsive CAR1 upstream activation sequence (UASI) and the factors required for its operation.

Induced production of arginase (CAR1) enzyme activity and steady-state CAR1 mRNA in Saccharomyces cerevisiae requires wild-type ARG80/ARGRI and ARG81/ARGRII gene products. We demonstrate here that these gene products, along with that of the MCM1 gene, are required for the inducer-dependent USAI-A, UASI-B and UASI-C elements to function but they are not required for operation of inducer-independent CAR1 UASC1 or UASC2. Through the use of single and multiple point mutations, the CAR1 UASI-B and UASI-C elements were demonstrated to be at least 23 bp in length. Moreover, simultaneous mutation of both ends of an elements gave stronger phenotypes than mutations at either end. The center of the element was more sensitive to mutation than were the ends.

Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae.

Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae. However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.

Tripartite structure of the Saccharomyces cerevisiae arginase (CAR1) gene inducer-responsive upstream activation sequence.

Arginase (CAR1) gene expression in Saccharomyces cerevisiae is induced by arginine. The 5' regulatory region of CAR1 contains four separable regulatory elements--two inducer-independent upstream activation sequences (UASs) (UASC1 and UASC2), an inducer-dependent UAS (UASI), and an upstream repression sequence (URS1) which negatively regulates CAR1 and many other yeast genes. Here we demonstrate that three homologous DNA sequences originally reported to be present in the inducer-responsive UASI are in fact three exchangeable elements (UASI-A, UASI-B, and UASI-C). Although two of these elements, either the same or different ones, are required for transcriptional activation to occur, all three are required for maximal levels of induction. The elements operate in all orientations relative to one another and to the TATA sequence. All three UASI elements bind protein(s); protein binding does not require arginine or overproduction of any of the putative arginine pathway regulatory proteins. The UASI-protein complex was also observed even when extracts were derived from arg80/argRI or arg81/argRII deletion mutants. Similar sequences situated upstream of ARG5,6 and ARG3 and reported to negatively regulate their expression are able to functionally substitute for the CAR1 UASI elements and mediate reporter gene expression.

Upstream induction sequence, the cis-acting element required for response to the allantoin pathway inducer and enhancement of operation of the nitrogen-regulated upstream activation sequence in Saccharomyces cerevisiae.

Expression of the DAL2, DAL4, DAL7, DUR1,2, and DUR3 genes in Saccharomyces cerevisiae is induced by the presence of allophanate, the last intermediate of the allantoin degradative pathway. Analysis of the DAL7 5'-flanking region identified an element, designated the DAL upstream induction sequence (DAL UIS), required for response to inducer. The operation of this cis-acting element requires functional DAL81 and DAL82 gene products. We determined the DAL UIS structure by using saturation mutagenesis. A specific dodecanucleotide sequence is the minimum required for response of reporter gene transcription to inducer. There are two copies of the sequence in the 5'-flanking region of the DAL7 gene. There are one or more copies of the sequence upstream of each allantoin pathway gene that responds to inducer. The sequence is also found 5' of the allophanate-inducible CAR2 gene as well. No such sequences were detected upstream of allantoin pathway genes that do not respond to the presence of inducer. We also demonstrated that the presence of a UIS element adjacent to the nitrogen-regulated upstream activation sequence significantly enhances its operation.

Rahnella aquatilis, a potential contaminant in lager beer breweries.

The species Rahnella aquatilis has been isolated mostly from water, soil, and, in a few cases, from human clinical specimens; little is known about its ecological role. The application of polyacrylamide gel electrophoresis of soluble proteins, DNA-DNA hybridizations and API 20 E systems has shown that Rahnella aquatilis might also be encountered as a contaminant in lager beer breweries.

A K2 neutral Saccharomyces cerevisiae strain contains a variant K2 M genome.

K2 neutral strain Saccharomyces cerevisiae USM12 was identified and characterized. This strain carried an M double-stranded RNA (dsRNA) genome encoding for resistance to K2 toxin. The M dsRNA was larger than the K2 killer yeast M dsRNA and homoduplex analysis of denatured and reannealed K2 neurtal M dsRNA revealed an inverted duplication. Heteroduplex analysis showed that two thirds of the K2 M genome had homology with the K2 neutral M genome. Hybridization showed that the USM12 M dsRNA had significant homology with the K2 M dsRNA. Protein profiles of extracellular proteins from USM12 and a cured strain indicated that USM12 did not secrete any toxin. This is the first time that a K2 neutral yeast strain has been characterized.

Plasmids in Leuconostoc oenos.

A new procedure was used to isolate 11 plasmids from eight Leuconostoc oenos strains. Plasmid DNA was not detected in 34 other strains of this species. Plasmid sizes ranged from 2.47 to 4.61 kilobase pairs. This is the first report of extrachromosomal elements in L. oenos.