Browsing by Author "Swiegers, J. H."

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    Carnitine biosynthesis in Neurospora crassa: Identification of a cDNA coding for ε-N-trimethyllysine hydroxylase and its functional expression in Saccharomyces cerevisiae
    (2002) Swiegers, J. H.; Vaz, F.M.; Pretorius, I. S.; Wanders, R. J. A; Bauer, Florian
    The biosynthesis of L-carnitine in eukaryotic organisms was first elucidated in the ascomycete Neurospora crassa. The first step of the pathway is catalysed by ε-N-trimethyllysine hydroxylase (TMLH), which converts ε-N-trimethyllysine into β-hydroxy-N-ε-trimethyllysine in a reaction dependent on α-ketoglutarate, Fe2+ and oxygen. Here we report on the cloning of the N. crassa TMLH cDNA and its functional expression in Saccharomyces cerevisiae. The TMLH cDNA contains an open reading frame of 1413 base pairs encoding a predicted polypeptide of 471 amino acids. The Michaelis-Menten constants of the heterologously expressed enzyme were determined for ε-N-trimethyllysine, α-ketoglutarate, Fe2+ and correspond to 0.33 mM, 133 μM and 46 μM, respectively. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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    Carnitine-dependent metabolic activities in Saccharomyces cerevisiae: Three carnitine acetyltransferases are essential in a carnitine-dependent strain
    (2001) Swiegers, J. H.; Dippenaar, N.; Pretorius, I. S.; Bauer, Florian
    L-Carnitine is required for the transfer of activated acyl-groups across intracellular membranes in eukaryotic organisms. In Saccharomyces cerevisiae, peroxisomal membranes are impermeable to acetyl-CoA, which is produced in the peroxisome when cells are grown on fatty acids as carbon source. In a reversible reaction catalysed by carnitine acetyltransferases (CATs), activated acetyl groups are transferred to carnitine to form acetylcarnitine which can be shuttled across membranes. Here we describe a mutant selection strategy that specifically selects for mutants affected in carnitine-dependent metabolic activities. Complementation of three of these mutants resulted in the cloning of three CAT encoding genes: CAT2, coding for the carnitine acetyltransferase associated with the peroxisomes and the mitochondria; YAT1, coding for the carnitine acetyltransferase, which is presumably associated with the outer mitochondrial membrane, and YER024w (YAT2), which encodes a third, previously unidentified carnitine acetyltransferase. The data also show that (a) L-carnitine and all three CATs are essential for growth on non-fermentable carbon sources in a strain with a disrupted CIT2 gene; (b) Yat2p contributes significantly to total CAT activity when cells are grown on ethanol; and that (c) the carnitine-dependent transfer of activated acetyl groups plays a more important role in cellular processes than previously realised. Copyright © 2001 John Wiley & Sons, Ltd.
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    Regulation of respiratory growth by Ras: The glyoxylate cycle mutant, cit2Δ, is suppressed by RAS2
    (2006) Swiegers, J. H.; Pretorius, I. S.; Bauer, Florian
    In Saccharomyces cerevisiae the Ras/cAMP/PKA signalling pathway controls multiple metabolic pathways, and alterations in the intracellular concentrations of cAMP through modification of signalling pathway factors can be lethal or result in severe growth defects. In this work, the important role of Ras2p in metabolic regulation during growth on the non-fermentable carbon source glycerol is further investigated. The data show that the overexpression of RAS2 suppresses the growth defect of the glyoxylate cycle citrate synthase mutant, cit2Δ. The overexpression results in enhanced proliferation and biomass yield when cells are grown on glycerol as sole carbon source, and increases citrate synthase activity and intracellular citrate concentration. Interestingly, the suppression of cit2Δ and the enhanced proliferation and biomass yield are only observed when RAS2 is overexpressed and not in strains containing the constitutively active allele RAS2 val19. However, both RAS2 and RAS2 val19upregulated citrate synthase activity. We propose that the RAS2 overexpression results in a combination of general upregulation of respiratory growth capacity and an increase in mitochondrial citrate/ citrate synthases, which together, complement the metabolic requirements of the cit2Δ mutant. The data therefore provide new evidence for the role of Ras2p as a powerful modulator of metabolism during growth on a non-fermentable carbon source. © Springer-Verlag 2006.
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    The effect of increased yeast alchohol acetyltransferase and esterase activity on the flavour profiles of wine and distillates
    (2006) Lilly, M.; Bauer, Florian; Lambrechts, M. G.; Swiegers, J. H.; Cozzolino, D.; Pretorius, I. S.
    The fruity odours of wine are largely derived from the synthesis of esters and higher alcohols during yeast fermentation. The ATF1- and ATF2-encoded alcohol acetyltransferases of S. cerevisiae are responsible for the synthesis of ethyl acetate and isoamyl acetate esters, while the EHT1-encoded ethanol hexanoyl transferase is responsible for synthesizing ethyl caproate. However, esters such as these might be degraded by the IAH1-encoded esterase. The objectives of this study were: (a) to overexpress the genes encoding ester-synthesizing and ester-degrading enzymes in wine yeast; (b) to prepare Colombard table wines and base wines for distillation using these modified strains; and (c) to analyse and compare the ester concentrations and aroma profiles of these wines and distillates. The overexpression of ATF1 significantly increased the concentrations of ethyl acetate, isoamyl acetate, 2-phenylethyl acetate and ethyl caproate, while the overexpression of ATF2 affected the concentrations of ethyl acetate and isoamyl acetate to a lesser degree. The overexpression of IAH1 resulted in a significant decrease in ethyl acetate, isoamyl acetate, hexyl acetate and 2-phenylethyl acetate. The overexpression of EHT1 resulted in a marked increase in ethyl caproate, ethyl caprylate and ethyl caprate. The flavour profile of the wines and distillates prepared using the modified strains were also significantly altered as indicated by formal sensory analysis. This study offers prospects for the development of wine yeast starter strains with optimized ester-producing capability that could assist winemakers in their effort to consistently produce wine and distillates such as brandy to definable flavour specifications and styles. Copyright © 2006 John Wiley & Sons, Ltd.