RESUMEN
BACKGROUND: There have been many successful strategies to implement xylose metabolism in Saccharomyces cerevisiae, but no effort has so far enabled xylose utilization at rates comparable to that of glucose (the preferred sugar of this yeast). Many studies have pointed towards the engineered yeast not sensing that xylose is a fermentable carbon source despite growing and fermenting on it, which is paradoxical. We have previously used fluorescent biosensor strains to in vivo monitor the sugar signalome in yeast engineered with xylose reductase and xylitol dehydrogenase (XR/XDH) and have established that S. cerevisiae senses high concentrations of xylose with the same signal as low concentration of glucose, which may explain the poor utilization. RESULTS: In the present study, we evaluated the effects of three deletions (ira2∆, isu1∆ and hog1∆) that have recently been shown to display epistatic effects on a xylose isomerase (XI) strain. Through aerobic and anaerobic characterization, we showed that the proposed effects in XI strains were for the most part also applicable in the XR/XDH background. The ira2∆isu1∆ double deletion led to strains with the highest specific xylose consumption- and ethanol production rates but also the lowest biomass titre. The signalling response revealed that ira2∆isu1∆ changed the low glucose-signal in the background strain to a simultaneous signalling of high and low glucose, suggesting that engineering of the signalome can improve xylose utilization. CONCLUSIONS: The study was able to correlate the previously proposed beneficial effects of ira2∆, isu1∆ and hog1∆ on S. cerevisiae xylose uptake, with a change in the sugar signalome. This is in line with our previous hypothesis that the key to resolve the xylose paradox lies in the sugar sensing and signalling networks. These results indicate that the future engineering targets for improved xylose utilization should probably be sought not in the metabolic networks, but in the signalling ones.
Asunto(s)
Glucosa , Redes y Vías Metabólicas/genética , Saccharomyces cerevisiae , Xilosa , Transporte Biológico , Fermentación , Eliminación de Gen , Glucosa/genética , Glucosa/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Plásmidos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Xilosa/genética , Xilosa/metabolismoRESUMEN
One of the challenges of establishing an industrially competitive process to ferment lignocellulose to value-added products using Saccharomyces cerevisiae is to get efficient mixed sugar fermentations. Despite successful metabolic engineering strategies, the xylose assimilation rates of recombinant S. cerevisiae remain significantly lower than for the preferred carbon source, glucose. Previously, we established a panel of in vivo biosensor strains (TMB371X) where different promoters (HXT1/2/4p; SUC2p, CAT8p; TPS1p/2p, TEF4p) from the main sugar signaling pathways were coupled with the yEGFP3 gene, and observed that wild-type S. cerevisiae cannot sense extracellular xylose. Here, we expand upon these strains by adding a mutated galactose transporter (GAL2-N376F) with improved xylose affinity (TMB372X), and both the transporter and an oxidoreductase xylose pathway (TMB375X). On xylose, the TMB372X strains displayed population heterogeneities, which disappeared when carbon starvation was relieved by the addition of the xylose assimilation pathway (TMB375X). Furthermore, the signal in the TMB375X strains on high xylose (50 g/L) was very similar to the signal recorded on low glucose (≤5 g/L). This suggests that intracellular xylose triggers a similar signal to carbon limitation in cells that are actively metabolizing xylose, in turn causing the low assimilation rates.
Asunto(s)
Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Azúcares/metabolismo , Xilosa/metabolismo , Transporte Biológico , Técnicas Biosensibles , Genotipo , Glucosa/metabolismo , Ingeniería Metabólica , Mutación , Plásmidos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
PURPOSE: The aim of this study was to examine the cytotoxic, genotoxic, antioxidant and antimicrobial activity of caffeic and rosmarinic acids and their salts with Li, Na and K with use of Escherichia coli K-12 recA:gfp strain as a model organism. METHODS: Cytotoxic potency of tested chemicals were calculated on the basis on the dose that confers inhibition percentage such as 20% for each concentrations of analysed chemicals. Genotoxic properties were calculated on the basis of the fold increase (FI) of SFI values normalized with control. Antioxidant potencies were established on the base of DPPH assay. Antimicrobial activity of chemicals were established on the value of minimal inhibitory concentration (MIC). RESULTS: Obtained results indicated that lower concentrations of tested compounds exhibited stronger GFP fluorescence response after rosmarinic acids and their salts treatment. Genotoxic effects seemed to be independent of the salt ions. The caffeic acid salts with Li, Na and K showed reduced genotoxic effect in comparison to the caffeic acid while increased cytotoxic effect than that of caffeic acid. Moreover, caffeinate salts exhibited better antimicrobial activity against E. coli (MIC=250µg/mL) than K caffeinate salt (MIC>500µg/mL). The MIC values of Li, Na and K rosmarinate salts were above 500µg/mL against all tested microorganisms. CONCLUSION: The results of the experiment show that there is no clear positive correlation between the antioxidant potency of caffeic and rosmarinic acids and their Li, Na and K salts and their cytotoxic effect. Used salts ions Li, Na and K do not significantly affect the antioxidant effect of natural phenolic compounds and they do not have a significant impact on the biological parameters such as cyto- and genotoxicity. Perhaps it is connected with the reaction environment including polarity of the solvent (water).