Francis Karst

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.e. bread, beer, wine, sake. Some of these groups presented unexpected relatedness: Bread strains displayed a combination of alleles intermediate between beer and wine strains, and strains used for rice wine and sake were most closely related to beer and bread strains. However, up to 28% of genetic diversity between these technological groups was associated with geographical differences which suggests local domestications. Focusing on wine yeasts, a group of Lebanese strains were basal in an FST tree, suggesting a Mesopotamia‐based origin of most wine strains. In Europe, migration of wine strains occurred through the Danube Valley, and around the Mediterranean Sea. An approximate Bayesian computation approach suggested a postglacial divergence (most probable period 10 000–12 000 bp). As our results suggest intimate association between man and wine yeast across centuries, we hypothesize that yeast followed man and vine migrations as a commensal member of grapevine flora.

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.

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. We subjected the Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) to a set of amino acid mutations in the catalytic site at position K197. Mutated strains have been shown to exhibit various growth rate, sterol amount, and monoterpenol‐producing capacities. These results are discussed in the context of the potential use of these mutated strains for heterologous expression of monoterpenoid synthases, which was investigated using Ocimum basilicum geraniol synthase. The results obtained with up to 5 mg/L geraniol suggest a major improvement compared with previous available expression systems like Escherichia coli or yeast strains with an unmodified ERG20 gene that respectively delivered amounts in the 10 and 500 µg/L range or even a previously characterized K197E mutation that delivered amounts in the 1 mg/L range. Biotechnol. Bioeng. 2011; 108:1883–1892.

Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance.

Abstract 7-aminocholesterol has been described as being a strong inhibitor of yeast and of Gram+-bacteria proliferation. In order to determine the precise molecular target of the toxicity of this compound, we searched for yeast resistance linked to gene over-expression. We named the new yeast gene that was isolated RTA1 (EMBL X84736). This gene led to strong resistance to the inhibitor. Gene sequencing revealed that RTA1 is adjacent to the NAB1 gene which is orientated in an opposite direction and localized on chromosome VII. The RTA1 gene, which encodes a putative protein with seven potential membrane-spanning segments, was shown to be a non-essential gene in yeast.

Sterol metabolism and ERG2 gene regulation in the yeast Saccharomyces cerevisiae

Certain exogenously‐supplied sterols, like ergost‐8‐enol, are efficiently converted into ergosterol in yeast. We have taken advantage of this property to study the regulation of the Δ8‐Δ7‐sterol isomerase‐encoding ERG2 gene in an ergosterol auxotrophic mutant devoid of squalene‐synthase activity. Ergosterol starvation leads to an 8–16‐fold increase in ERG2 gene expression. Such an increase was also observed in wild‐type cells either grown anaerobically or treated with SR31747A a sterol isomerase inhibitor. Exogenously‐supplied zymosterol is entirely transformed into ergosterol, which represses ERG2 transcription. By contrast, exogenously‐supplied ergosterol has little or no effect on ERG2 transcription.

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. Thale cress (Arabidopsis thaliana) was transformed using the floral dip method. Tobacco (Nicotiana benthamiana) leaves were agro-infiltrated for transient expression. Although, as expected, geraniol was the main product detected in the leaves, different minor products were observed in these plants (V. vinifera: citronellol and nerol; N. benthamiana: linalool and nerol; A. thaliana: none). O. basilicum GES expression was also carried out with microbial system yeasts (Saccharomyces cerevisiae) and Escherichia coli. These results suggest that the functional properties of a monoterpenol synthase depend not only on the enzymes amino-acidic sequence, but also on the cellular background. They also suggest that some plant species or microbial expression systems could induce the simultaneous formation of several carbocations, and could thus have a natural tendency to produce a wider spectrum of monoterpenols.

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. An intricate correlation has been found between the FPPS activity, the amount of ergosterol synthesized and cell growth of a mutant strain defective in FPPS. About 40% of wt FPPS activity was sufficient to support normal growth of the mutant. With further decline of FPPS activity (20 down to 3%) the amount of ergosterol remained unchanged at ∼0.16% (vs dry weight), whereas growth yield decreased and lag times increased. We postulate that, in addition to ergosterol initiating and maintaining growth of yeast cells, FPP and/or its derivatives participate in these processes.

The role of ERG20 gene (encoding yeast farnesyl diphosphate synthase) mutation in long dolichol formation. Molecular modeling of FPP synthase.

The yeast Saccharomyces cerevisiae strain LB332 bearing a mutation in the ERG20 gene encoding farnesyl diphosphate synthase (FPPS) synthesizes significantly longer dolichols than the wild type strain FL100 (14-31 and 14-19 isoprene units, respectively). The measurement of the short chain prenyl alcohols excreted into the medium shows that increased amounts of geraniol, dimethylallyl and isopentenyl alcohols but not farnesol are synthesized by the mutant strain. The wild type FPPS synthesizes farnesyl diphosphate (FPP) as the only product. The K197E substitution, as opposed to F112A/F113S in avian FPPS, does not change product specificity. Consequently, the possibility that mutated yeast FPPS synthesizes longer polyprenols is unlikely. This is supported by additional evidence such as in vitro analysis of the mutated FPPS products and molecular modeling. We suggest that formation of longer dolichols in vivo is the result of a change in the isopentenyl diphosphate/farnesyl diphosphate ratio caused by the erg20 mutation which in turn affects the activity of cis-prenyltransferase.

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. In accordance with the proposed catalytic mechanism, a series of lipophilic ammonium-ion-containing derivatives possessing a variety of structures and biological properties, potently inhibited the Zm8,7SI in vitro. To evaluate the importance of a series of conserved acidic and tryptophan residues which could be involved in the Zm8,7SI catalytic mechanism, 20 mutants of Zm8,7SI were constructed as well as a number of corresponding mutants of the Saccharomyces cerevisiae 8,7SI. The mutated isomerases were assayed in vivo by sterol analysis and quantification of Delta(5,7)-sterols and directly in vitro by examination of the activities of the recombinant Zm8,7SI mutants. These studies have identified His(74), Glu(78), Asp(107), Glu(121), Trp(66) and Trp(193) that are required for Zm8,7SI activity and show that binding of the enzyme-substrate complex is impaired in the mutant T124I. They underline the functional homology between the plant and animal 8,7SIs on one hand, in contrast with the yeast 8,7SI on the other hand, in accordance with their molecular diversity and distinct mechanisms.

Identification of a Lysine Residue Important for the Catalytic Activity of Yeast Farnesyl Diphosphate Synthase

The Saccharomyces cerevisiaeERG20 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 Mg2+ forming a bridge between the enzyme and DMAPP, and demonstrate that destabilizing two of the three Mg2+ ions, by introducing a double mutation at positions K197 and K254, results in a loss of FPPS activity and a lethal phenotype.