Increasing sulphite formation in Saccharomyces cerevisiae by overexpression of MET14 and SSU1.
Saccharomyces cerevisiae produces sulphite as an intermediate product during the assimilatory reduction of sulphate to sulphide. Three genes, MET3, MET14 and MET16, are essential for this reduction. We investigated the level of transcription of these genes in strains of S. cerevisiae with high, medium and low sulphite formation. The level of MET14- and MET16-mRNA varied with sulphite production, whereas the level of MET3-mRNA was very weak in almost all strains. We also analysed the effect of overexpression of MET14 and MET16 on sulphite formation. Two strains with low sulphite production were transformed with high-copy plasmids containing either or both MET14 and MET16. The overexpression of these two genes leads to a two- to three-fold sulphite formation. In addition, inactivation of MET10, encoding a subunit of the sulphite reductase, also leads to a distinct increase in sulphite formation; however, the cells became methionine auxotroph. The overexpression of SSU1, a gene encoding a putative sulphite pump, yields a slight increase in sulphite accumulation, whereas overexpression of SSU1, together with MET14, increases sulphite formation up to 10-fold. Furthermore, sulphite formation strongly depends on growth conditions, e.g. yeast transformants growing in wort produce much higher amounts of sulphite when compared to growth in minimal media. The addition of glucose can also increase the sulphite formation in strains overexpressing MET14 and/or SSU1 under oxygen-limiting conditions, while the addition of glucose has no significant effect under aerobic conditions.
Donalies
Ute E B
UE
Technische Universität Berlin, Institut für Biotechnologie, Fachgebiet Mikrobiologie und Genetik, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany.
Stahl
Ulf
U
D027321:Anion Transport Proteins; D015152:Blotting, Northern; D003470:Culture Media; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D005800:Genes, Fungal; D005947:Glucose; D008565:Membrane Proteins / Q000096:biosynthesis*; D016366:Open Reading Frames; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013430:Sulfate Adenylyltransferase / Q000096:biosynthesis / Q000235:genetics; D013447:Sulfites / Q000032:analysis / Q000378:metabolism*; D014170:Transformation, Genetic
D027321:Anion Transport Proteins; D003470:Culture Media; D005656:Fungal Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C108311:Ssu1 protein, S cerevisiae; D013447:Sulfites; D010088:Oxidoreductases; D050862:Oxidoreductases Acting on Sulfur Group Donors; C052449:3'-phosphoadenylyl-5'-phosphosulfate reductase; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D013430:Sulfate Adenylyltransferase; D005947:Glucose