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.

Enumeration of fungi in barley.

Estimation of fungal contamination of barley grain is important as certain fungi can proliferate during the malting process. The following factors which may affect the enumeration of fungi were evaluated: dilution versus direct plating, presoaked versus unsoaked grain, five culture media: potato dextrose agar (PDA), acidified Czapek-Dox agar (ACA), pentachloronitrobenzene agar; (PCNB) dichloran rose bengal chloramphenicol agar (DRBC) and malt salt agar; two disinfectants' ethanol/water (80:20 v/v) and sodium hypochlorite (3.5% w/v in H2O). Two barley samples, one having a high incidence of storage fungi and one with a high incidence of field fungi were used and most fungi were identified to species level. Results showed that direct plating was superior to dilution plating for assessing the mycoflora of barley. Unsoaked grain gave significantly higher counts than presoaked grain in the case of Alternaria alternata, Rhizopus oryzae, Epicoccum nigrum and Mucor spp. Presoaked grain resulted in higher counts of Penicillium spp. Chlorine disinfection resulted in significantly higher counts of Aspergillus flavus, Eurotium spp. and Penicillium spp. Ethanol disinfection resulted in higher counts of Mucor spp., Phoma sorghina, Rhizopus oryzae and Aspergillus restrictus. PDA and ACA, in general gave some what better results than DRBC for both field and storage fungi. PCNB consistently gave the highest Fusarium counts. More than thiry fungal genera were found in the two samples.

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.