Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity.

The enzyme CPI (cyclopropylsterol-cycloisomerase) from the plant sterol pathway catalyses the cleavage of the 9β,19-cyclopropane ring of the 4α-methyl-cyclopropylsterol cycloeucalenol to produce the Δ8-sterol obtusifoliol. Randomly mutated plasmids carrying the Arabidopsis thaliana cpi gene were screened for inactive CPI mutant enzymes on the basis of their ability to genetically complement a Saccharomyces cerevisiae erg7 (defective in oxidosqualene cyclase) ergosterol auxotroph grown in the presence of exogenous cycloeucalenol, and led to the identification of four catalytically important residues. Site-directed mutagenesis experiments confirmed the role of the identified residues, and demonstrated the importance of selected acidic residues and a conserved G108NYFWTHYFF117 motif.

Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals.

Genetic analysis of geraniol metabolism during fermentation.

Geraniol produced by grape is the main precursor of terpenols which play a key role in the floral aroma of white wines. We investigated the fate of geraniol during wine fermentation by Saccharomyces cerevisiae. The volatile compounds produced during fermentation of a medium enriched with geraniol were extracted by Stir-bar sorptive extraction and analysed by GC-MS.

QTL mapping of the production of wine aroma compounds by yeast.

Background: Wine aroma results from the combination of numerous volatile compounds, some produced by yeast and others produced in the grapes and further metabolized by yeast. However, little is known about the consequences of the genetic variation of yeast on the production of these volatile metabolites, or on the metabolic pathways involved in the metabolism of grape compounds. As a tool to decipher how wine aroma develops, we analyzed, under two experimental conditions, the production of 44 compounds by a population of 30 segregants from a cross between a laboratory strain and an industrial strain genotyped at high density.

Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems.

Numerous aromatic plant species produce high levels of monoterpenols, using geranyl diphosphate (GPP) as a precursor. Sweet basil (Ocimum basilicum) geraniol synthase (GES) was used to evaluate the monoterpenol profiles arising from heterologous expressions in various plant models. Grapevine (Vitis vinifera) calli were transformed using Agrobacterium tumefasciens and the plants were regenerated.

Identification of a lysine residue important for the catalytic activity of yeast farnesyl diphosphate synthase.

The Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) has been subjected to a set of mutations at the catalytic site, at position K254 to determine the in vivo impact. The mutated strains have been shown to exhibit various growth rates, sterol profiles and monoterpenol producing capacities. The results obtained suggest that K at position 254 helps to stabilize one of the three Mg(2+) forming a bridge between the enzyme and DMAPP, and demonstrate that destabilizing two of the three Mg(2+) ions, by introducing a double mutation at positions K197 and K254, results in a loss of FPPS activity and a lethal phenotype.

Metabolic engineering of monoterpene synthesis in yeast.

Terpenoids are one of the largest and most diverse families of natural compounds. They are heavily used in industry, and the trend is toward engineering modified microorganisms that produce high levels of specific terpenoids. Most studies have focused on creating specific heterologous pathways for sesquiterpenes in Escherichia coli or yeast.

The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae.

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p.

A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine.

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties.

Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin.

A putative 8,7SI (sterol 8,7-isomerase) from Zea mays, termed Zm8,7SI, has been isolated from an EST (expressed sequence tag) library and subcloned into the yeast erg2 mutant lacking 8,7SI activity. Zm8,7SI restored endogenous ergosterol synthesis. An in vitro enzymatic assay in the corresponding yeast microsomal extract indicated that the preferred Delta(8)-sterol substrate possesses a single C4alpha methyl group, in contrast with 8,7SIs from animals and fungi, thus reflecting the diversity in the structure of their active site in relation to the distinct sterol biosynthetic pathways.

Bread, beer and wine: Saccharomyces cerevisiae diversity reflects human history.

Fermented beverages and foods have played a significant role in most societies worldwide for millennia. To better understand how the yeast species Saccharomyces cerevisiae, the main fermenting agent, evolved along this historical and expansion process, we analysed the genetic diversity among 651 strains from 56 different geographical origins, worldwide. Their genotyping at 12 microsatellite loci revealed 575 distinct genotypes organized in subgroups of yeast types, i.

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.

Selection of hypervariable microsatellite loci for the characterization of Saccharomyces cerevisiae strains.

A survey of the genetic polyphormism that can be detected at different microsatellite loci in Saccharomyces cerevisiae strains was performed in order to develop an efficient microsatellite based typing technique. Using gel electrophoresis, we analyzed 41 microsatellite loci for 10 strains and observed that 29 loci displayed significant polymorphism. The measurement of the PCR amplicon size of 21 loci for 46 strains by capillary electrophoresis, allowed us to retain the six most variable sites for S.

Functional relationships between the Saccharomyces cerevisiae cis-prenyltransferases required for dolichol biosynthesis.

In the yeast Saccharomyces cerevisiae the RER2 and SRT1 genes encode Rer2 and Srt1 proteins with cis-prenyltransferase (cis-PT-ase) activity. Both cis-PT-ases utilize farnesyl diphosphate (FPP) as a starter for polyprenyl diphosphate (dolichol backbone) formation. The products of the Rer2 and Srt1 proteins consist of 14-17 and 18-23 isoprene units, respectively.

Farnesyl diphosphate synthase activity affects ergosterol level and proliferation of yeast Saccharomyces cerevisae.

The yeast farnesyl diphosphate synthase (FPPS) gene was engineered so as to construct allelic forms giving various activities of the enzyme. One of the substitutions was F96W in the chain length determination region. The other, K197, conserved within a consensus sequence found in the majority of FPP and GGPP synthases, was substituted by R, E and V.

Optimisation of interdelta analysis for Saccharomyces cerevisiae strain characterisation.

A new primer pair (delta12-delta21) for polymerase chain reaction-based yeast typing was designed using the yeast genome sequence. The specificity of this primer pair was checked by the comparison of the electrophoresis pattern with a virtual profile calculated from Blast data. The analysis of 53 commercial and laboratory Saccharomyces cerevisiae yeast strains showed a clear improvement of interdelta analysis using the newly designed primers.