Saccharomyces cerevisiae has been used for at least eight millennia in the production of alcoholic beverages (41). Along with ethanol and carbon dioxide, fermenting cultures of this yeast produce many low-molecular-weight flavor compounds. These alcohols, aldehydes, organic acids, esters, organic sulfides, and carbonyl compounds have a strong impact on product quality. Indeed, the subtle aroma balance of these compounds in fermented foods and beverages is often used as an organoleptic fingerprint for specific products and brands (42). Food fermentation by yeast and lactic acid bacteria is accompanied by the formation of the aliphatic and aromatic alcohols known as fusel alcohols. Fusel oil, which derives its name from the German word fusel (bad liquor), is obtained during the distillation of spirits and is enriched with these higher alcohols. While fusel alcohols at high concentrations impart off-flavors, low concentrations of these compounds and their esters make an essential contribution to the flavors and aromas of fermented foods and beverages. Fusel alcohols are derived from amino acid catabolism via a pathway that was first proposed a century ago by Ehrlich (13). Amino acids represent the major source of the assimilable nitrogen in wort and grape must, and these amino acids are taken up by yeast in a sequential manner (23, 32). Amino acids that are assimilated by the Ehrlich pathway (valine, leucine, isoleucine, methionine, and phenylalanine) are taken up slowly throughout the fermentation time (32). After the initial transamination reaction (Fig. ​(Fig.1),1), the resulting α-keto acid cannot be redirected into central carbon metabolism. Before α-keto acids are excreted into the growth medium, yeast cells convert them into fusel alcohols or acids via the Ehrlich pathway.



FIG. 1.

The Ehrlich pathway. Catabolism of branched-chain amino acids (leucine, valine, and isoleucine), aromatic amino acids (phenylalanine, tyrosine, and trytophan), and the sulfur-containing amino acid (methionine) leads to the formation of fusel acids and ...



Current scientific interest in the Ehrlich pathway is supported by increased demands for natural flavor compounds such as isoamyl alcohol and 2-phenylethanol, which can be produced from amino acids in yeast-based bioconversion processes (14), as well as by the need to control flavor profiles of fermented food products. The goal of this paper is to present a concise centenary overview of the biochemistry, molecular biology, and physiology of this important pathway in S. cerevisiae.

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The Ehrlich Pathway for Fusel Alcohol Production : a Century of Research on Saccharomyces cerevisiae Metabolism

Yeasts are predominant in the ancient and complex process of winemaking. In spontaneous fermentations, there is a progressive growth pattern of indigenous yeasts, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the ‘wine yeast’ and is widely preferred for initiating wine fermentations. The primary role of wine yeast is to catalyze the rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important, metabolites without the development of off-flavours. However, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialized wine yeast strains possessing a wide range of optimized, improved or novel oenological properties. This review highlights the wealth of untapped indigenous yeasts with oenological potential, the complexity of wine yeasts’ genetic features and the genetic techniques often used in strain development. The current status of genetically improved wine yeasts and potential targets for further strain development are outlined. In light of the limited knowledge of industrial wine yeasts’ complex genomes and the daunting challenges to comply with strict statutory regulations and consumer demands regarding the future use of genetically modified strains, this review cautions against unrealistic expectations over the short term. However, the staggering potential advantages of improved wine yeasts to both the winemaker and consumer in the third millennium are pointed out. Copyright # 2000 John Wiley & Sons, Ltd.

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Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking

As the details of the chemical transformations in metabolism have become increasingly clear, and the enzymes catalysing many of the reactions have been characterized, it is understandable that biochemists should want to explain at the molecular level the metabolic homeostasis observed at the physiological level. How are the rates of synthesis and degradation of metabolites kept in close balance over a very wide range of external conditions without catastrophic rises or falls in the metabolite concentrations? The discoveries of feedback inhibition, co-operativity and covalent modification in enzymes, and of mechanisms for the control of enzyme synthesis and degradation, have disclosed a repertoire of molecular effects that potentially alter the fluxes in metabolic pathways. With such a range of effects to choose from, it is not surprising that disputes arise over explanations for the changes in flux through particular pathways under given circumstances. Since the explanations are usually verbal and qualitative, discrimination between different explanations, or assessment of their adequacy, is difficult. More recently, several groups have attempted theoretical analysis of the potential of these different molecular mechanisms to contribute to the control of metabolic flux. Since these theories can be given a mathematical formulation, they can be used in combination with appropriate experimental measurements to provide quantitative explanations and, potentially, predictions. The theories have often been controversial. Matters at issue have included the extent to which it is feasible to perform experiments to obtain the necessary data, the adequacy of the theories for making useful predictions, and the degree to which the quantitative measures of the theories do actually capture the relevant aspects of regulation and control. To some extent, this is a matter of semantics; a mathematical theory is more explicit about its underlying assumptions and the meaning of its statements, but regulation and control are two terms that have been used without strict adherence to any agreed definition in many different contexts (as noted in [1]). It is therefore inevitable that some will find that the use of these terms in the context of a mathematical theory places a narrower construction on them than they would like. These theories all include a form of sensitivity analysis; that is, the magnitude of the effect of some small change in a parameter (such as an enzyme activity) on a metabolic system property (such as the flux or the concentration of a metabolite) is mathematically related to the properties of the components of the system. Sensitivity analysis is widely used for analogous problems in other fields, including economics, ecology [2], engineering [3] and chemical kinetics [4-6]. Its application in biochemistry was pioneered by Higgins [7], but three variants subsequently arose: Metabolic Control Analysis, Biochemical Systems Theory and Crabtree and Newsholme's 'flux-oriented' theory (the term used in [8]). It is not possible to give a succinct account of the differences between the approaches, which is a controversial area [9-18] even though the underlying mathematics is equivalent to a considerable extent. One area of difference is the choice of the type of parameter that is changed for the determination of sensitivities. In Metabolic Control Analysis, enzyme concentration (or activity) is usually chosen; the response to an external modifier of a metabolic pathway is derived from the resulting sensitivities. In Biochemical Systems Theory [19-25], the primary parameters for the sensitivities are the 'rate constants' for synthesis and degradation of metabolite pools. Savageau has given many reasons for this choice of parameter; Cornish-Bowden has articulated some of the problems with it [15]. Although using these 'rate constants' simplifies the analysis procedures within Biochemical Systems Theory, there is not a one-to-one relationship between them and the enzymes of the system, which can create a slight complication in determining the sensitivity to variation of an enzyme activity. Savageau's theory is part of an integrated system for stability analysis and simulation, in addition to sensitivity analysis. Crabtree and Newsholme's theory [8,10,26-30] is intermediate between the two others, and the primary sensitivities are to an external modifier (a hypothetical one if necessary), but its mathematical development is less rigorous. In this review, I shall concentrate on Metabolic Control Analysis. This is because, apart from considerations of space, approximately two-thirds of the literature citations of theories of metabolic regulation in the past 5 years have been to Metabolic Control Analysis. This may relate to perceived ease of use, which has been compared using the different approaches on the same set of experimental results [31]. In the following review, I will not give a complete derivation and description of the basic concepts of Metabolic Control Analysis; clear accounts can be found in previous articles and reviews [9,32-39]. Instead I will try to indicate areas of disagreement, the scope of the basic theory and where it has been modified or extended, and recent approaches to experimental applications.

/pdf/metabolic-control-analysis-a-survey-of-its-theoretical-and-2f9dujatvt.pdf

Metabolic control analysis: a survey of its theoretical and experimental development.

In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase Special attention is devoted to physiological studies on S cerevisiae strains in which structural genes encoding these key enzymes have been inactivated by gene disruption

/pdf/pyruvate-metabolism-in-saccharomyces-cerevisiae-3p6vin11c5.pdf

Pyruvate Metabolism in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae responds to osmotic stress, i.e., an increase in osmolarity of the growth medium, by enhanced production and intracellular accumulation of glycerol as a compatible solute. We have cloned a gene encoding the key enzyme of glycerol synthesis, the NADH-dependent cytosolic glycerol-3-phosphate dehydrogenase, and we named it GPD1. gpd1 delta mutants produced very little glycerol, and they were sensitive to osmotic stress. Thus, glycerol production is indeed essential for the growth of yeast cells during reduced water availability. hog1 delta mutants lacking a protein kinase involved in osmostress-induced signal transduction (the high-osmolarity glycerol response [HOG] pathway) failed to increase glycerol-3-phosphate dehydrogenase activity and mRNA levels when osmotic stress was imposed. Thus, expression of GPD1 is regulated through the HOG pathway. However, there may be Hog1-independent mechanisms mediating osmostress-induced glycerol accumulation, since a hog1 delta strain could still enhance its glycerol content, although less than the wild type. hog1 delta mutants are more sensitive to osmotic stress than isogenic gpd1 delta strains, and gpd1 delta hog1 delta double mutants are even more sensitive than either single mutant. Thus, the HOG pathway most probably has additional targets in the mechanism of adaptation to hypertonic medium.

GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway.

Eight different enzymes for glycolysis and alcoholic fermentation were overproduced in a common Saccharomyces cerevisiae strain by placing their genes on multicopy vectors. The specific enzyme activities were increased between 3.7- and 13.9-fold above the wild-type level. The overproduction of the different glycolytic enzymes had no effect on the rate of ethanol formation, even with those enzymes that catalyse irreversible steps: hexokinase, phosphofructokinase and pyruvate kinase. Also the simultaneous increase in the activities of pairs of enzymes such as pyruvate kinase and phosphofructokinase or pyruvate decarboxylase and alcohol dehydrogenase, did not increase the rate of ethanol production. The levels of key glycolytic metabolites were also normal, compared to the reference strain.

Overproduction of glycolytic enzymes in yeast.

We deleted most of the pyruvate decarboxylase structural gene PDC1 from the genome of Saccharomyces cerevisiae. Surprisingly, mutants carrying this deletion allele showed a completely different phenotype than previously described point mutations. They were able to ferment glucose and their specific pyruvate decarboxylase activity was only reduced to 45% of the wild type level. Northern blot analysis revealed that a sequence in the yeast genome homologous to PDC1 and formerly designated as a possible pseudogene is expressed and may code for a different but closely related pyruvate decarboxylase. The products of the two PDC genes seem to form hybrid oligomers, however both homooligomers have enzyme activity. Thus, the product of the PDC1 gene is not absolutely neccessary for glucose fermentation in yeast.

A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations

We have cloned the structural gene for yeast transaldolase Transformants carrying the TAL1 gene on a multicopy plasmid over-produced transaldolase A deletion mutant which was constructed using the cloned gene did not show any detectable transaldolase activity in vitro Furthermore, both transaldolase isoenzymes which were detected in wild-type crude extracts by immunoblotting were missing in the deletion mutants Thus, TAL1 is the only transaldolase structural gene in yeast TAL1 is not an essential gene Deletion of the transaldolase gene did not affect growth on complete media with different carbon sources or on synthetic media However, the transaldolase-deficient strains accumulated sedoheptulose 7-phosphate, an intermediate of the pentose-phosphate pathway Mutants lacking both transaldolase and phosphoglucose isomerase grew more slowly than the single mutants They accumulated more sedoheptulose 7-phosphate on medium containing fructose than on glucose medium This shows that fructose 6-phosphate and glyceraldehyde 3-phosphate, metabolites of glycolysis, can enter the nonoxidative part of the pentose-phosphate pathway

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb15440.x

Ine Schaaff

Papers

Overproduction of glycolytic enzymes in yeast.

A deletion of the PDC1 gene for pyruvate decarboxylase of yeast causes a different phenotype than previously isolated point mutations

Molecular analysis of the structural gene for yeast transaldolase.