Genetic analysis of the metabolic pathways responsible for aroma metabolite production by Saccharomyces cerevisiae.

During alcoholic fermentation, higher alcohols, esters, and acids are formed from amino acids via the Ehrlich pathway by yeast, but many of the genes encoding the enzymes have not yet been identified. When the BAT1/2 genes, encoding transaminases that deaminate amino acids in the first step of the Ehrlich pathway are deleted, higher metabolite formation is significantly decreased. Screening yeast strains with deletions of genes encoding decarboxylases, dehydrogenases, and reductases revealed nine genes whose absence had the most significant impact on higher alcohol production. The seven most promising genes (AAD6, BAT2, HOM2, PAD1, PRO2, SPE1, and THI3) were further investigated by constructing double- and triple-deletion mutants. All double-deletion strains showed a greater decrease in isobutanol, isoamyl alcohol, isobutyric, and isovaleric acid production than the corresponding single deletion strains with the double-deletion strains in combination with ∆bat2 and the ∆hom2-∆aad6 strain revealing the greatest impact. BAT2 is the dominant gene in these deletion strains and this suggests the initial transaminase step of the Ehrlich pathway is rate-limiting. The triple-deletion strains in combination with BAT2 (∆bat2-∆thi3-∆aad6 and ∆bat2-∆thi3-∆hom2) had the greatest impact on the end metabolite production with the exception of isoamyl alcohol and isovaleric acid. The strain deleted for two dehydrogenases and a reductase (∆hom2-∆pro2-∆aad6) had a greater effect on the levels of these two compounds. This study contributes to the elucidation of the Ehrlich pathway and its significance for aroma production by fermenting yeast cells.

Identifying genes that impact on aroma profiles produced by Saccharomyces cerevisiae and the production of higher alcohols.

During alcoholic fermentation, many volatile aroma compounds are formed by Saccharomyces cerevisiae, including esters, fatty acids, and higher alcohols. While the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the regulation of the network, and the physiological roles of individual pathways within the network. Furthermore, different yeast strains tend to produce significantly different aroma profiles. These differences are of tremendous biotechnological interest, since producers of alcoholic beverages such as wine and beer are searching for means to diversify and improve their product range. Various factors such as the redox, energy, and nutritional balance of a cell have previously been suggested to directly or indirectly affect and regulate the network. To gain a better understanding of the regulations and physiological role of this network, we screened a subset of the EUROSCARF strain deletion library for genes that, when deleted, would impact most significantly on the aroma profile produced under fermentative conditions. The 10 genes whose deletion impacted most significantly on higher alcohol production were selected and further characterized to assess their mode of action within or on this metabolic network. This is the first description of a large-scale screening approach using aroma production as the primary selection criteria, and the data suggest that many of the identified genes indeed play central and direct roles within the aroma production network of S. cerevisiae.

Wine flavor and aroma.

The perception of wine flavor and aroma is the result of a multitude of interactions between a large number of chemical compounds and sensory receptors. Compounds interact and combine and show synergistic (i.e., the presence of one compound enhances the perception of another) and antagonistic (a compound suppresses the perception of another) interactions. The chemical profile of a wine is derived from the grape, the fermentation microflora (in particular the yeast Saccharomyces cerevisiae), secondary microbial fermentations that may occur, and the aging and storage conditions. Grape composition depends on the varietal and clonal genotype of the vine and on the interaction of the genotype and its phenotype with many environmental factors which, in wine terms, are usually grouped under the concept of "terroir" (macro, meso and microclimate, soil, topography). The microflora, and in particular the yeast responsible for fermentation, contributes to wine aroma by several mechanisms: firstly by utilizing grape juice constituents and biotransforming them into aroma- or flavor-impacting components, secondly by producing enzymes that transform neutral grape compounds into flavor-active compounds, and lastly by the de novo synthesis of many flavor-active primary (e.g., ethanol, glycerol, acetic acid, and acetaldehyde) and secondary metabolites (e.g., esters, higher alcohols, fatty acids). This review aims to present an overview of the formation of wine flavor and aroma-active components, including the varietal precursor molecules present in grapes and the chemical compounds produced during alcoholic fermentation by yeast, including compounds directly related to ethanol production or secondary metabolites. The contribution of malolactic fermentation, ageing, and maturation on the aroma and flavor of wine is also discussed.

Tulbaghia--A Southern African Phytomedicine.

The phytomedicine Tulbaghia consists of the fresh or dried subterranean organs of various Tulbaghia species. The genus is endemic to Southern Africa and includes about 20 species, of which only T. alliacea and T. capensis are naturally found in the winter rainfall climate area (the Western Cape). The genus forms part of the Alliaceae family and is a geophyte (plants with an underground perennation organ and leaves that die back annually). Their habitat can range from semi-desert to wet and boggy terrain. Wild garlic is most commonly prepared as an infusion or boiled in water and taken orally. Externally, as a medicated bath, wild garlic is used to treat paralysis and rheumatism and to reduce the temperature in a feverish patient. Internally, rhizome or bulb preparations are taken orally to treat fever; as a remedy for colds and influenza, asthma, tuberculosis, and stomach problems; as an antihypertensive; or to expel intestinal worms. It is also used as a prophylactic against winter infections. Rhizome pieces are often placed in castor oil to make eardrops. For fever and high blood pressure, a tea is made from the bulbs or rhizomes and a small cup taken three times daily. The leaves of the plant are used to treat esophageal cancer and may also be eaten as a vegetable. The demand for Tulbaghia in both formal and informal markets has grown exponentially. Sustainable harvesting focuses on only harvesting enough of the plant so that it still has the capacity for self-renewal. However, because both the above-ground and underground parts of Tulbaghia are commonly used in African traditional medicine, destructive harvesting of the whole plant is inevitable, thus necessitating the large-scale organized propagation of these plants. It is therefore important to establish a new strategy for the sustainable harvesting of these plants as commercial crops.

Expression of the mouse gonadotropin-releasing hormone receptor gene in alpha T3-1 gonadotrope cells is stimulated by cyclic 3',5'-adenosine monophosphate and protein kinase A, and is modulated by Steroidogenic factor-1 and Nur77.

Regulation of GnRH receptor (GnRHR) expression levels in the pituitary is a crucial control point in reproduction. The promoter of the mouse GnRHR gene contains nuclear receptor half-sites (NRS) at -244/-236 and -15/-7 relative to the translation start site. Although binding of steroidogenic factor-1 (SF-1) to the -244/-236NRS is implicated in mediating basal and gonadotrope-specific expression, no function or protein-DNA interactions have previously been described for the -15/-7NRS. We report that levels of the endogenous GnRHR mRNA in alpha T3-1 cells are stimulated by forskolin and 8-bromo-cAMP. We also show that the orphan nuclear receptor Nur77 is expressed in alpha T3-1 cells, and that both SF-1 and Nur77 bind to the -15/-7NRS and -244/-236NRS in vitro. We show that the activity of the proximal (-579/+1) mouse GnRHR promoter is up-regulated by protein kinase A, via a mechanism that is modulated by SF-1, both positively and negatively, through binding to the -244/-236NRS or the -15/-7NRS, respectively. Nur77 appears to be capable of acting as a negative regulator of this response, via the -15/-7NRS. Furthermore, we show that forskolin up-regulates SF-1 mRNA levels in alpha T3-1 cells, indicating that the levels of SF-1 play a role in modulating the protein kinase A response.

The effect of increased branched-chain amino acid transaminase activity in yeast on the production of higher alcohols and on the flavour profiles of wine and distillates.

In Saccharomyces cerevisiae, branched-chain amino acid transaminases (BCAATases) are encoded by the BAT1 and BAT2 genes. BCAATases catalyse the transfer of amino groups between those amino acids and alpha-keto-acids. alpha-Keto-acids are precursors for the biosynthesis of higher alcohols, which significantly influence the aroma and flavour of yeast-derived fermentation products. The objective of this study was to investigate the influence of BAT-gene expression on general yeast physiology, on aroma and flavour compound formation and on the sensory characteristics of wines and distillates. For this purpose, the genes were overexpressed and deleted in a laboratory strain, BY4742, and overexpressed in an industrial wine yeast strain, VIN13. The data show that, with the exception of a slow growth phenotype observed for the BAT1 deletion strain, the fermentation behaviour of the strains was unaffected by the modifications. The chemical and sensory analysis of fermentation products revealed a strong correction between BAT gene expression and the formation of many aroma compounds. The data suggest that the adjustment of BAT gene expression could play an important role in assisting winemakers in their endeavour to produce wines with specific flavour profiles.