Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO 2 is desirable, it is complicated by the fact that undesirable H 2 S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO 2 -producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO 2 and H 2 S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO 2 , resulting in high SO 2 and low H 2 S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO 2 than the parent but produced parental levels of H 2 S.The bottom-fermenting yeast Saccharomyces pastorianus is used to produce beer and has been proposed to be a natural hybrid between Saccharomyces cerevisiae and Saccharomyces bayanus (30). Bottom-fermenting yeasts have two types of genes, one set highly homologous (more than 90% identity) to those of S. cerevisiae and the other less so but highly homologous to S. bayanus (i.e., non-S. cerevisiae [Lg type]) (8,14,27,33). One way in which S. pastorianus differs from baker's yeast (S. cerevisiae) is its tendency to produce higher levels of both sulfite (SO 2 ) and hydrogen sulfide (H 2 S).It is well known that sulfur compounds in beer make significant contributions to flavor and aroma. SO 2 , for example, acts as an antioxidant, which slows the development of oxidation haze and staling of flavors in beer. In contrast, H 2 S has an aroma of rotten eggs and is also a precursor of other compounds with undesirable sensory characteristics. SO 2 and H 2 S are produced by yeast during reductive sulfate assimilation (Fig. 1). Inorganic sulfate is taken up through a sulfate permease and reduced to SO 2 by enzymes encoded by MET3, MET14, and MET16. SO 2 is then reduced to H 2 S by SO 2 reductase encoded by MET5 and MET10 (29). The next intermediate, homocysteine, which is synthesized from H 2 S and O-acetylhomoserine (OAH) by OAH sulfhydrylase encoded by MET17, leads to the formation of cysteine, methionine, and S-adenosylmethionine (SAM). SAM transcriptionally represses all of the genes involved in sulfate assimilation. Park and Bakalinsky previously reported that SSU1 encodes an SO 2 efflux pump that exports intracellular SO 2 through the plasma membrane (18).In the postgenomic era, systematic and high-throughput analyses of mRNA and proteins have become central to recent functional genomics initiatives. Metabolomics entails the analysis of all cellular metabolites and has become a powerful new tool for gaining insight into functional biology. Measurement of numerous metabolites within a cell and tracking concentration changes as a fu...
Sulfite (SO 2 ) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H 2 S) has an undesirable aroma. To discover the cellular processes that control SO 2 and H 2 S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H 2 S productivity. Ten of these genes are known to be involved in sulfur-containing amino acid metabolism, whereas UBI4 functions in the ubiquitin-proteasome system and SKP2 encodes an F-box-containing protein whose function is unknown. We found that the skp2 mutant accumulated H 2 S and SO 2 , because the adenosylphophosulfate kinase Met14p is a substrate of SCF Skp2 and more stable in the skp2 mutant than in the wild-type strain. Furthermore, the skp2 mutant grew more slowly than the wild-type strain under nutrient-limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild-type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild-type strain. Moreover, supplementation with methionine, S -adenosylmethionine, S -adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H 2 S/SO 2 production.
Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75 degrees C) with an apparent dissociation constant of 0.6 microM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.
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