Carole Camarasa

Evolutionary engineered Saccharomyces cerevisiae wine yeast strains with increased in vivo flux through the pentose phosphate pathway

Amplification of the flux toward the pentose phosphate (PP) pathway might be of interest for various S. cerevisiae based industrial applications. We report an evolutionary engineering strategy based on a long-term batch culture on gluconate, a substrate that is poorly assimilated by S. cerevisiae cells and is metabolized by the PP pathway. After adaptation for various periods of time, we selected strains that had evolved a greater consumption capacity for gluconate. (13)C metabolic flux analysis on glucose revealed a redirection of carbon flux from glycolysis towards the PP pathway and a greater synthesis of lipids. The relative flux into the PP pathway was 17% for the evolved strain (ECA5) versus 11% for the parental strain (EC1118). During wine fermentation, the evolved strains displayed major metabolic changes, such as lower levels of acetate production, higher fermentation rates and enhanced production of aroma compounds. These represent a combination of novel traits, which are of great interest in the context of modern winemaking.

A comparative transcriptomic, fluxomic and metabolomic analysis of the response of Saccharomyces cerevisiae to increases in NADPH oxidation

BackgroundRedox homeostasis is essential to sustain metabolism and growth. We recently reported that yeast cells meet a gradual increase in imposed NADPH demand by progressively increasing flux through the pentose phosphate (PP) and acetate pathways and by exchanging NADH for NADPH in the cytosol, via a transhydrogenase-like cycle. Here, we studied the mechanisms underlying this metabolic response, through a combination of gene expression profiling and analyses of extracellular and intracellular metabolites and 13u2009C-flux analysis.ResultsNADPH oxidation was increased by reducing acetoin to 2,3-butanediol in a strain overexpressing an engineered NADPH-dependent butanediol dehydrogenase cultured in the presence of acetoin. An increase in NADPH demand to 22 times the anabolic requirement for NADPH was accompanied by the intracellular accumulation of PP pathway metabolites consistent with an increase in flux through this pathway. Increases in NADPH demand were accompanied by the successive induction of several genes of the PP pathway. NADPH-consuming pathways, such as amino-acid biosynthesis, were upregulated as an indirect effect of the decrease in NADPH availability. Metabolomic analysis showed that the most extreme modification of NADPH demand resulted in an energetic problem. Our results also highlight the influence of redox status on aroma production.ConclusionsCombined 13u2009C-flux, intracellular metabolite levels and microarrays analyses revealed that NADPH homeostasis, in response to a progressive increase in NADPH demand, was achieved by the regulation, at several levels, of the PP pathway. This pathway is principally under metabolic control, but regulation of the transcription of PP pathway genes can exert a stronger effect, by redirecting larger amounts of carbon to this pathway to satisfy the demand for NADPH. No coordinated response of genes involved in NADPH metabolism was observed, suggesting that yeast has no system for sensing NADPH/NADP+ ratio. Instead, the induction of NADPH-consuming amino-acid pathways in conditions of NADPH limitation may indirectly trigger the transcription of a set of PP pathway genes.

Combined effects of nutrients and temperature on the production of fermentative aromas by Saccharomyces cerevisiae during wine fermentation

Volatile compounds produced by yeast during fermentation greatly influence the organoleptic qualities of wine. We developed a model to predict the combined effects of initial nitrogen and phytosterol content and fermentation temperature on the production of volatile compounds. We used a Box–Behnken design and response surface modeling to study the response of Lalvin EC1118® to these environmental conditions. Initial nitrogen content had the greatest influence on most compounds; however, there were differences in the value of fermentation parameters required for the maximal production of the various compounds. Fermentation parameters affected differently the production of isobutanol and isoamyl alcohol, although their synthesis involve the same enzymes and intermediate. We found differences in regulation of the synthesis of acetates of higher alcohols and ethyl esters, suggesting that fatty acid availability is the main factor influencing the synthesis of ethyl esters whereas the production of acetates depends on the activity of alcohol acetyltransferases. We also evaluated the effect of temperature on the total production of three esters by determining gas–liquid balances. Evaporation largely accounted for the effect of temperature on the accumulation of esters in liquid. Nonetheless, the metabolism of isoamyl acetate and ethyl octanoate was significantly affected by this parameter. We extended this study to other strains. Environmental parameters had a similar effect on aroma production in most strains. Nevertheless, the regulation of the synthesis of fermentative aromas was atypical in two strains: Lalvin K1M® and Affinity™ ECA5, which produces a high amount of aromatic compounds and was obtained by experimental evolution.

Role in anaerobiosis of the isoenzymes for Saccharomyces cerevisiae fumarate reductase encoded by OSM1 and FRDS1

Saccharomyces cerevisiae possesses both a cytoplasmic and a mitochondrial fumarate reductase, encoded by FRDS1 and OSM1, respectively. While previous studies have shown that mutants lacking FRDS1 and OSM1 cannot grow under anaerobiosis (Arikawa et al., 1998 ), the physiological role of fumarate reductase (FR) remains poorly understood. Here, we report that an osm1 frds1 mutant is unable to grow anaerobically, even with glutamate as a sole nitrogen source, when succinate can be produced by the TCA oxidative branch. We also show that the growth of the mutant is not restored by adding acetoin, an alternative sink for NADH oxidation, but it is at least partly restored by the addition of oxygen or menadione, which can oxidize FADH2 in addition to NADH. These data indicate that the growth inhibition of the mutant is due to an inability to reoxidize FAD, rather than an indirect effect on NADH or an inability to produce succinate per se. During anaerobic growth, FRDS1 expression was two to eight times higher than that of OSM1, and fumarate reductase activity was higher in the osm1 mutant than in the frds1 mutant. FRDS1 expression was induced by anaerobiosis, and this induction was abolished in a rox1 mutant. We conclude that the formation of succinate is strictly required for the reoxidation of FADH2 during anaerobiosis, and that it is regulated through the control of FRDS1 expression when oxygen is limiting. Based on these data, we discuss the potential role of fumarate reductase in the regeneration of the FAD‐prosthetic group of essential flavoproteins. Copyright

Use of a continuous multistage bioreactor to mimic winemaking fermentation.

Continuous fermentation set-ups are of great interest for studying the physiology of microorganisms. In winemaking conditions, yeasts go through a growth phase and a stationary phase during which more than half of the sugar is fermented. A comprehensive study of wine-yeast physiology must therefore include yeasts in a non-growing phase. This condition is impossible to achieve within a chemostat, which led us to design a multi-stage fermentation device. In this study, we evaluated the ability of such a device to reproduce, in a series of steady states, the conditions of batch fermentation. Two-stage and four-stage fermentations were carried out with two different strains of Saccharomyces cerevisiae. The main characteristics of the fermentation process (biomass growth, by-product content of the medium) were compared with those observed in batch mode at the same stage of fermentation, which was defined by glucose uptake. The four-stage configuration showed a better ability to reproduce batch fermentation characteristics than the two-stage set-up. It also allowed to uncouple the variations of environmental parameters and proved to be a promising tool to gain new insights into yeast metabolism during alcoholic fermentation.

Metabolic Responses of Saccharomyces cerevisiae to Valine and Ammonium Pulses during Four-Stage Continuous Wine Fermentations

ABSTRACT Nitrogen supplementation, which is widely used in winemaking to improve fermentation kinetics, also affects the products of fermentation, including volatile compounds. However, the mechanisms underlying the metabolic response of yeast to nitrogen additions remain unclear. We studied the consequences for Saccharomyces cerevisiae metabolism of valine and ammonium pulses during the stationary phase of four-stage continuous fermentation (FSCF). This culture technique provides cells at steady state similar to that of the stationary phase of batch wine fermentation. Thus, the FSCF device is an appropriate and reliable tool for individual analysis of the metabolic rerouting associated with nutrient additions, in isolation from the continuous evolution of the environment in batch processes. Nitrogen additions, irrespective of the nitrogen-containing compound added, substantially modified the formation of fermentation metabolites, including glycerol, succinate, isoamyl alcohol, propanol, and ethyl esters. This flux redistribution, fulfilling the requirements for precursors of amino acids, was consistent with increased protein synthesis resulting from increased nitrogen availability. Valine pulses, less efficient than ammonium addition in increasing the fermentation rate, were followed by a massive conversion of this amino acid in isobutanol and isobutyl acetate through the Ehrlich pathway. However, additional routes were involved in valine assimilation when added in stationary phase. Overall, we found that particular metabolic changes may be triggered according to the nature of the amino acid supplied, in addition to the common response. Both these shared and specific modifications should be considered when designing strategies to modulate the production of volatile compounds, a current challenge for winemakers.

Uncoupled glycerol distribution as the origin of the accumulation of 3-hydroxypropionaldehyde during the fermentation of glycerol by Enterobacter agglomerans CNCM 1210

Batch fermentation of glycerol to 1,3-propanediol (1,3PPD) by Enterobacter agglomerans CNCM 1210 showed the lethal accumulation of 3-hydroxypropionaldehyde (3-HPA) when performed under initial substrate content higher than 40 g/L. Assigned to the inhibition by the NAD/NADH ratio of the 3-HPA converting enzyme: 1,3PPD dehydrogenase, intracellular assays were conducted in an attempt to identify the metabolic mechanisms involved in the increase of that ratio. An overflow metabolism through the 1,3PPD formation pathway was established, while a catabolic limitation in the oxidative branch at the level of glyceraldehyde-3-phosphate dehydrogenase occurred. Uncoupled activities of synthesis and consumption of reducing equivalents are thus suspected to provoke the increase of the NAD/NADH ratio and the subsequent accumulation of 3-HPA. Copyright 1998 John Wiley & Sons, Inc.

Integrating transcriptomics and metabolomics for the analysis of the aroma profiles of Saccharomyces cerevisiae strains from diverse origins

BackgroundDuring must fermentation thousands of volatile aroma compounds are formed, with higher alcohols, acetate esters and ethyl esters being the main aromatic compounds contributing to floral and fruity aromas. The action of yeast, in particular Saccharomyces cerevisiae, on the must components will build the architecture of the wine flavour and its fermentation bouquet. The objective of the present work was to better understand the molecular and metabolic bases of aroma production during a fermentation process. For such, comparative transcriptomic and metabolic analysis was performed at two time points (5 and 50xa0g/L of CO2 released) in fermentations conducted by four yeast strains from different origins and/or technological applications (cachaça, sake, wine, and laboratory), and multivariate factorial analyses were used to rationally identify new targets for improving aroma production.ResultsResults showed that strains from cachaça, sake and wine produced higher amounts of acetate esters, ethyl esters, acids and higher alcohols, in comparison with the laboratory strain. At fermentation time T1 (5xa0g/L CO2 released), comparative transcriptomics of the three S. cerevisiae strains from different fermentative environments in comparison with the laboratory yeast S288c, showed an increased expression of genes related with tetracyclic and pentacyclic triterpenes metabolism, involved in sterol synthesis. Sake strain also showed upregulation of genes ADH7 and AAD6, involved in the formation of higher alcohols in the Ehrlich pathway. For fermentation time point T2 (50xa0g/L CO2 released), again sake strain, but also VL1 strain, showed an increased expression of genes involved in formation of higher alcohols in the Ehrlich pathway, namely ADH7, ADH6 and AAD6, which is in accordance with the higher levels of methionol, isobutanol, isoamyl alcohol and phenylethanol observed.ConclusionsOur approach revealed successful to integrate data from several technologies (HPLC, GC-MS, microarrays) and using different data analysis methods (PCA, MFA). The results obtained increased our knowledge on the production of wine aroma and flavour, identifying new gene in association to the formation of flavour active compounds, mainly in the production of fatty acids, and ethyl and acetate esters.

Fermentation performances and aroma production of non-conventional wine yeasts are influenced by nitrogen preferences

Saccharomyces cerevisiae is currently the most important yeast involved in food fermentations, particularly in oenology. However, several other yeast species occur naturally in grape must that are highly promising for diversifying and improving the aromatic profile of wines. If the nitrogen requirement of S. cerevisiae has been described in detail, those of non-Saccharomyces yeasts remain poorly studied despite their increasingly widespread use in winemaking. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we explored the fermentation performances, the utilisation of nitrogen sources and the volatile compound production of 10 strains of non-conventional yeasts in pure culture. Two different conditions were tested: one mimicking the grape juices nitrogen composition and one with all the nitrogen sources at the same level. We highlighted the diversity in terms of nitrogen preference and amount consumed among the yeast strains. Some nitrogen sources (arginine, glutamate, glycine, tryptophan and γ-aminobutyric acid) displayed the largest variations between strains throughout the fermentation. Several non-Saccharomyces strains produced important aroma compounds such as higher alcohols, acetate and ethyl esters in significantly higher quantities than S. cerevisiae.