The Biochemistry of Malic Acid Metabolism by Wine Yeasts – A Review

L-Malic acid is an essential intermediate of cell metabolism and the D,L-racemic mixture is used as an acidulant in a variety of foods and beverages. In the wine industry, it plays an important role during grape must fermentation, contributing to the “fixed acidity” that is important. The latter is important in defining the quality of wine. Genetic and biochemical characterisation of the L-malate utilising pathways in several yeast species has indicated that the physiological role and regulation of L-malate metabolism differ significantly between the K(-) and K(+) yeasts. A varietyof factors influence the ability of a yeast species to effectively degrade L-malate, including the conditions associated with wine fermentation and the yeast’s intrinsic ability to transport and effectively metabolise L-malate inside the cell. This paper reviews the ability of different yeast species associated with grapes and wine to degrade extracellular L-malate, and the underlying mechanisms in the differential utilisation of L-malate by different yeast species

Malic Acid in Wine: Origin, Function and Metabolism during Vinification

The production of quality wines requires a judicious balance between the sugar, acid and flavour components of wine.  L-Malic and tartaric acids are the most prominent organic acids in wine and play a crucial role in the winemaking process, including the organoleptic quality and the physical, biochemical and microbial stability of wine.  Deacidification of grape must and wine is often required for the production of well-balanced wines. Malolactic fermentation induced by the addition of malolactic starter cultures, regarded as the preferred method for naturally reducing wine acidity, efficiently decreases the acidic taste of wine, improves the microbial stability and modifies to some extent the organoleptic character of wine. However, the recurrent phenomenon of delayed or sluggish malolactic fermentation often causes interruption of cellar operations, while the malolactic fermentation is not always compatible with certain styles of wine. Commercial wine yeast strains of Saccharomyces are generally unableto degrade L-malic acid effectively in grape must during alcoholic fermentation, with relatively minor modifications in total acidity during vinification. Functional expression of the malolactic pathway genes, i.e. the malate transporter (mae1) of Schizosaccharomyces pombe and the malolactic enzyme (mleA) from Oenococcus oeni in wine yeasts, haspaved the way for the construction of malate-degrading strains of Saccharomyces for commercial winemaking

Malic Acid Distribution and Degradation in Grape Must During Skin Contact: The Influence of Recombinant Malo-Ethanolic Wine Yeast Strains

Wine acidity plays an important role in determining wine quality and ensuring physiochemical and microbiological stability. In high-acid wines, the L-malic acid concentration is usually reduced through bacterial malolactic fermentation, while acidulation in low-acidity wines is usually done during final blending of the wine before bottling.  This study showed that skin contact did not influence the relative concentration of L-malic acid in the pulp and juice fractions from Colombard, Ruby Cabernet and Cabernet Sauvignon grape musts, with 32%-44% of the L-malic acid present in the pulp fraction. Four recombinant malo-ethanolic (ME) Saccharomyces wine yeast strains containing the malic enzyme (mae2) and malate transporter (mael) genes of Schizasaccharomyces pombe, effectively degraded the L-malic acid in both the juice and pulp fractions of all three cultivars, with a complete degradation of malic acid in the juice fraction within 2 days

Genetic Engineering of an Industrial Strain of Saccharomyces cerevisiae for L-Malic Acid Degradation via an Efficient Malo-Ethanolic Pathway

The optimal ratio of L-malic and L-tartaric acid in relation to other wine components is one of the most important aspects that ultimately determine wine quality during winemaking. Winemakers routinely rely on the judicious use of malolactic fermentation (MLF) after alcoholic fermentation to deacidify and stabilise their wines. However, due to theunreliability of the process and unsuitable sensory modifications in some grape cultivars, especially for fruity-floral wines, MLF is often regarded as problematic and undesirable. Alternative methods for reducing the amounts of L-malic acid in wine will contribute to improving the production of quality wines in the future, especially in coolclimate regions. Most wine yeast strains of Saccharomyces are unable to effectively degrade L-malic acid, whereas the fission yeast Schizosaccharomyces pombe efficiently degrades high concentrations of L-malic acid by means of malo-ethanolic fermentation. However, strains of S. pombe are not suitable for vinification due to the production of undesirable off-flavours. Previously, the 5. pombe malate permease (mael) and malic enzyme (mae2) genes were  successfully expressed under the 3-phosphoglycerate kinase (PGK1) regulatory elements in 5. cerevisiae, resulting in a recombinant laboratory strain of S. cerevisiae with an efficient malo-ethanolic pathway. Stable integration of the S. pombe malo-ethanolic pathway genes has now been obtained through the construction of a unique integration strategy in a commercial wine yeast strain. Co-transformation of the linear integration cassette containing the mael and mae2 genes and PGK1 regulatory elements and a multi-copy plasmid containing the phleomycin-resistance marker into a commercial Saccharomyces cerevisiae strain resulted in the successful transformation and integration of the malo-ethanolic genes. The recombinant 5. cerevisiae strain was successfully cured of phleomycin-resistance plasmid DNA in order to obtain malo-ethanolic yeast containing only yeast-derived DNA. The integrated malo-ethanolic genes were stable in 5. cerevisiae and during synthetic and grape must fermentation, L-malic acid was completely fermented to ethanol without any negative effect on fermentation kinetics and wine quality

Factors involved in the regulation of the Schizosaccharomyces pombe malic enzyme gene

Transcription of the Schizosaccharomyces pombe malic enzyme gene, mae2, is induced when cells are grown on high glucose concentrations or under non-aerated conditions. Two cis-acting elements in the mae2 promoter, upstream activator sequences UAS1 and UAS2, are required for basal expression, whilst three negative-acting, upstream repressor sequences are involved in general derepression of mae2. Both the Pka1 and Sty1 signal transduction pathways are involved in the induced expression of mae2 under fermentative conditions. Expression of mae2 seems to be regulated in response to the carbon source, lack of oxygen and osmotic stress conditions, probably to assist in maintaining the intracellular redox balance.Articl

The biochemistry of malic acid metabolism by wine yeasts – a review

CITATION: Saayman, M. & Viljoen-Bloom, M. 2006. The biochemistry of malic acid metabolism by wine yeasts – a review. South African Journal of Enology & Viticulture, 27(2):113-122, doi:10.21548/27-2-1612.The original publication is available at http://www.journals.ac.za/index.php/sajevL-Malic acid is an essential intermediate of cell metabolism and the D,L-racemic mixture is used as an acidulant in a variety of foods and beverages. In the wine industry, it plays an important role during grape must fermentation, contributing to the “fixed acidity” that is important. The latter is important in defining the quality of wine. Genetic and biochemical characterisation of the L-malate utilising pathways in several yeast species has indicated that the physiological role and regulation of L-malate metabolism differ significantly between the K(-) and K(+) yeasts. A variety of factors influence the ability of a yeast species to effectively degrade L-malate, including the conditions associated with wine fermentation and the yeast’s intrinsic ability to transport and effectively metabolise L-malate inside the cell. This paper reviews the ability of different yeast species associated with grapes and wine to degrade extracellular L-malate, and the underlying mechanisms in the differential utilisation of L-malate by different yeast species.http://www.journals.ac.za/index.php/sajev/article/view/1612Publisher's versio

The Biochemistry of Malic Acid Metabolism by Wine Yeasts – A Review

L-Malic acid is an essential intermediate of cell metabolism and the D,L-racemic mixture is used as an acidulant in a variety of foods and beverages. In the wine industry, it plays an important role during grape must fermentation, contributing to the “fixed acidity” that is important. The latter is important in defining the quality of wine. Genetic and biochemical characterisation of the L-malate utilising pathways in several yeast species has indicated that the physiological role and regulation of L-malate metabolism differ significantly between the K(-) and K(+) yeasts. A varietyof factors influence the ability of a yeast species to effectively degrade L-malate, including the conditions associated with wine fermentation and the yeast’s intrinsic ability to transport and effectively metabolise L-malate inside the cell. This paper reviews the ability of different yeast species associated with grapes and wine to degrade extracellular L-malate, and the underlying mechanisms in the differential utilisation of L-malate by different yeast species

Cloning, characterisation, and heterologous expression of the Candida utilis malic enzyme gene

The Candida utilis malic enzyme gene, CME1, was isolated from a cDNA library and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1,926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of approximately 70.2 kDa. The inferred amino acid sequence suggested a cytosolic localisation for the malic enzyme, as well as 37 and 68% homologies with the malic enzymes of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Expression of the CME1 gene was subject to carbon catabolite repression and substrate induction, similar to the regulatory mechanisms observed for the C. utilis dicarboxylic acid permease. The CME1 gene was successfully expressed in S. cerevisiae under control of the S. cerevisiae PGK1 promoter and terminator. When coexpressed with the S. pombe malate permease gene (mae1), it resulted in a recombinant S. cerevisiae strain able to completely degrade 90% of the extracellular L-malate within 24 h. © Springer-Verlag 2006.Articl

Utilising Grape Pomace for Ethanol Production

Chemical analyses of grape pomace revealed the presence of significant amounts of fermentable sugars that are retained in the pomace after pressing of the grapes. Furthermore, treatment of the pomace with purified hydrolases indicated that the enzymatic biodegradation of the pomace could release additional fermentable sugars. We isolated and evaluated yeast strains associated with grape pomace for their ability to hydrolyse the complex polysaccharides found in grape pomace and to utilise the fermentable sugars for the production of ethanol. Two Pichia rhodanensis isolates were able to partially hydrolyse the pomace polysaccharides, but fermentation of the pomace resulted only in a small increase in the amount of ethanol produced. The study revealed that significant amounts of ethanol could be obtained from the residual sugars associated with grape pomace. However, the complex structure of the pomace polysaccharides apparently renders it unsusceptible to efficient hydrolysis under fermentative conditions

Cloning, characterisation, and heterologous expression of the Candida utilis malic enzyme gene

The Candida utilis malic enzyme gene, CME1, was isolated from a cDNA library and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1,926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of approximately 70.2 kDa. The inferred amino acid sequence suggested a cytosolic localisation for the malic enzyme, as well as 37 and 68% homologies with the malic enzymes of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Expression of the CME1 gene was subject to carbon catabolite repression and substrate induction, similar to the regulatory mechanisms observed for the C. utilis dicarboxylic acid permease. The CME1 gene was successfully expressed in S. cerevisiae under control of the S. cerevisiae PGK1 promoter and terminator. When coexpressed with the S. pombe malate permease gene (mae1), it resulted in a recombinant S. cerevisiae strain able to completely degrade 90% of the extracellular L-malate within 24 h. © Springer-Verlag 2006.Articl