The regulation of ubiquinone-6 biosynthesis by Saccharomyces cerevisiae

Increasing concentrations of glucose (1-5%) in the growth medium depressed ubiquinone-6 biosynthesis in continuously cultured wild type Saccharomyces cerevisiae. In addition, an early intermediate in the pathway of ubiquinone-6 biosynthesis, i.e. 3,4-dihydroxy-5-hexaprenylbenzoate (3,4-DHHB), was found to accumulate. The increase in 3,4-DHHB levels varied inversely with the diminished levels of ubiquinone-6, suggesting that O-methylation of 3,4-DHHB is a regulated step in catabolite repression. Experiments using protoplasts demonstrated that the effect of catabolite repression on this pathway was reversible by 1.2 mM cAMP but not by other nucleotides and cyclic nucleotides. This response to cAMP was unaltered by the protein synthesis inhibitor cycloheximide, indicating that the regulatory control for this reaction must occur at the enzymatic level. Additional experiments demonstrated the presence of a heat-labile component of the cytoplasm, which was essential for this effect of cAMP. This observation suggests that this cytosolic effector may be translocated to the inner membrane of the mitochondria, the intracellular site for ubiquinone-6 biosynthesis.     

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1delta mutant

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase (meu1delta). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1delta cells. By comparing the results with this mutant with a meu1delta spe2delta mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1+ SPE2+ cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15delta mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.     

Coordinate regulation of phospholipid biosynthesis by serine in Saccharomyces cerevisiae.

The addition of L-serine to inositol-containing growth medium repressed membrane-associated CDPdiacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activities and subunit levels in wild-type Saccharomyces cerevisiae. Enzyme activities and subunit levels were not repressed when inositol was absent from the growth medium. The addition of L-serine to the growth medium did not affect the phospholipid composition of wild-type cells. CDPdiacylglycerol synthase and phosphatidylserine synthase were not regulated in the S. cerevisiae inositol biosynthesis ino2, ino4, and opi1 regulatory mutants, suggesting that regulation by inositol plus L-serine is coupled to inositol synthesis. Inositol and L-serine did not affect the activities of purified CDPdiacylglycerol synthase and phosphatidylserine synthase. The addition of compounds structurally related to L-serine to the growth medium of wild-type cells also resulted in a repression of CDPdiacylglycerol synthase and phosphatidylserine synthase but only in the presence of inositol. Phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was not regulated by inositol plus L-serine.     

Monoterpenoid biosynthesis in Saccharomyces cerevisiae

Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.     

Recent progress in understanding thiamin biosynthesis and its genetic regulation in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to synthesize thiamin pyrophosphate (TPP) de novo, which involves the independent formation of two ring structures, 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-β-hydroxyethylthiazole, in the early steps. In addition, this organism can efficiently utilize thiamin from the extracellular environment to produce TPP. Nineteen genes involved in the synthesis of TPP and the utilization of thiamin (THI genes) have been identified, and the function of several THI genes has been elucidated. All THI genes participating in the synthesis of the pyrimidine unit belong to multigene families. It is also intriguing that some thiamin biosynthetic proteins are composed of two distinct domains or form an enzyme complex. The expression of THI genes is coordinately induced in response to thiamin starvation. It is likely that the induction of THI genes is activated by a positive regulatory factor complex and that the protein–protein interaction among the factors is disturbed by TPP. Thiamin-hyperproducing yeast and fermented food containing a high content of thiamin are expected to be available in the future based on the progress in understanding thiamin biosynthesis and its genetic regulation in S. cerevisiae.     

Phosphatidylinositol biosynthesis: biochemistry and regulation

Phosphatidylinositol (PI) is a ubiquitous membrane lipid in eukaryotes. It is becoming increasingly obvious that PI and its metabolites play a myriad of very diverse roles in eukaryotic cells. The Saccharomyces cerevisiae PIS1 gene is essential and encodes PI synthase, which is required for the synthesis of PI. Recently, PIS1 expression was found to be regulated in response to carbon source and oxygen availability. It is particularly significant that the promoter elements required for these responses are conserved evolutionarily throughout the Saccharomyces genus. In addition, several genome-wide strategies coupled with more traditional screens suggest that several other factors regulate PIS1 expression. The impact of regulating PIS1 expression on PI synthesis will be discussed along with the possible role(s) that this may have on diseases such as cancer.     

Regulation of phosphatidylcholine biosynthesis in Saccharomyces cerevisiae

Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of Saccharomyces cerevisiae is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate (14)C-CH(3)-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 mm choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of (14)C-CH(3)-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium.