•Calculated ΔGbinding negatively correlate with reported sweetness intensities.•The ability of the receptor model to predict the sweetness intensity of sweeteners.
Natural sweeteners, such as stevia ...and thaumatin, exert their sweet taste by specifically binding to sweet taste receptors. However, the molecular basis of their sweetening power remains to be ascertained. In the present study, we built a comparative model of the hT1R2 and hT1R3 subunits in order to characterize their interactions with natural, non-caloric sweeteners – from glycosylated terpenoids to sweet proteins – at the molecular level. The binding free energy between hT1R2-hT1R3 and sweeteners of different families shows a strong correlation with their sweetness intensity for both, small sweeteners (r = −0.89) and sweet proteins (r = −0.97). The correlation is further improved and generalized throughout all families of sweeteners evaluated, when EC50 values are used instead of relative intensities (r = −0.91). Altogether, these results contribute to a better understanding of the sweetness perception of these sweeteners, and promote the use of docking for better prediction of resulting sweetness.
Tannin content and composition are critical quality components of red wines. No spectroscopic method assessing these phenols in wine has been described so far. We report here a new method using ...Fourier transform mid-infrared (FT-MIR) spectroscopy and chemometric techniques for the quantitative analysis of red wine tannins. Calibration models were developed using protein precipitation and phloroglucinolysis as analytical reference methods. After spectra preprocessing, six different predictive partial least-squares (PLS) models were evaluated, including the use of interval selection procedures such as iPLS and CSMWPLS. PLS regression with full-range (650−4000 cm-1), second derivative of the spectra and phloroglucinolysis as the reference method gave the most accurate determination for tannin concentration (RMSEC = 2.6%, RMSEP = 9.4%, r = 0.995). The prediction of the mean degree of polymerization (mDP) of the tannins also gave a reasonable prediction (RMSEC = 6.7%, RMSEP = 10.3%, r = 0.958). These results represent the first step in the development of a spectroscopic methodology for the quantification of several phenolic compounds that are critical for wine quality. Keywords: FT-MIR; PLS; tannins; red wine; SPE; Carménère
Microbial production of carotenoids has mainly focused towards a few products, such as β-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also ...shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and β-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a β-carotenogenic yeast strain, we first evaluated the performance of several β-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/gDCW of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/gDCW). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/gDCW in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date.
•First report on the engineering of Saccharomyces cerevisiae for the efficient production of violaxanthin.•Optimal violaxanthin pathway was constructed after screening several CrtZ and ZEP genes.•The impact of the extent of gene truncation on the catalytic activity of the translated enzymes was elucidated.•The data suggest that ZEP requires membrane association and accessory redox partners to efficiently epoxidate zeaxanthin.•The highest producing strain required the expression of 10 heterologous genes to reach a yield of 7.3 mg/gDCW of violaxanthin.
To assess olfactory function in children and to create and validate an odor identification test to diagnose olfactory dysfunction in children, which we called the Universal Sniff (U-Sniff) test.
This ...is a multicenter study involving 19 countries. The U-Sniff test was developed in 3 phases including 1760 children age 5-7 years. Phase 1: identification of potentially recognizable odors; phase 2: selection of odorants for the odor identification test; and phase 3: evaluation of the test and acquisition of normative data. Test—retest reliability was evaluated in a subgroup of children (n = 27), and the test was validated using children with congenital anosmia (n = 14).
Twelve odors were familiar to children and, therefore, included in the U-Sniff test. Children scored a mean ± SD of 9.88 ± 1.80 points out of 12. Normative data was obtained and reported for each country. The U-Sniff test demonstrated a high test—retest reliability (r27 = 0.83, P < .001) and enabled discrimination between normosmia and children with congenital anosmia with a sensitivity of 100% and specificity of 86%.
The U-Sniff is a valid and reliable method of testing olfaction in children and can be used internationally.
Malolactic fermentation (MLF) is responsible for the decarboxylation of l-malic into lactic acid in most red wines and some white wines. It reduces the acidity of wine, improves flavor complexity and ...microbiological stability. Despite its industrial interest, the MLF mechanism is not fully understood. The objective of this study was to provide new insights into the role of pH on the binding of malic acid to the malolactic enzyme (MLE) of
To this end, sequence similarity networks and phylogenetic analysis were used to generate an MLE homology model, which was further refined by molecular dynamics simulations. The resulting model, together with quantum polarized ligand docking (QPLD), was used to describe the MLE binding pocket and pose of l-malic acid (MAL) and its l-malate (-1) and (-2) protonation states (MAL
and MAL
, respectively). MAL
has the lowest ∆G
, followed by MAL
and MAL, with values of -23.8, -19.6, and -14.6 kJ/mol, respectively, consistent with those obtained by isothermal calorimetry thermodynamic (ITC) assays. Furthermore, molecular dynamics and MM/GBSA results suggest that only MAL
displays an extended open conformation at the binding pocket, satisfying the geometrical requirements for Mn
coordination, a critical component of MLE activity. These results are consistent with the intracellular pH conditions of
cells-ranging from pH 5.8 to 6.1-where the enzymatic decarboxylation of malate occurs.
The effects of temperature (50–200 °C) and contact time (5–30 min) on the pressurized hot water extraction of deodorized thyme were explored for antioxidant activity, polyphenol profiles, and total ...antioxidants. Six not previously reported polyphenolic compounds were identified in thyme. An inverse correlation was found between the antioxidant activity and total antioxidants with the amount and diversity of polyphenols. The highest total extract yield and antioxidant activity were obtained at 200 °C, although maximum polyphenol extraction yields of hydroxycinnamic acids, flavones, flavonols/flavanones, and total polyphenols were detected at 100 °C and 5 min. Higher temperatures and longer exposure times reduced extract polyphenol diversity. Dihydroxyphenyllactic acid was the only phenolic compound for which extraction yield increased with temperature, probably as a product of the thermal degradation of rosmarinic acid. Consequently, for extracting phenolics from thyme, 100 °C and 5 min would be appropriate operating conditions, whereas antioxidant-active nonphenolic compounds were favored at higher temperatures and exposure times.
Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating ...light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast
Saccharomyces cerevisiae
. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.
The yeast Saccharomyces cerevisiae synthesises a variety of volatile aroma compounds during wine fermentation. In this study, the influence of fermentation temperature on (1) the production of ...yeast-derived aroma compounds and (2) the expression of genes involved in aroma compounds' metabolism (ADH1, PDC1, BAT1, BAT2, LEU2, ILV2, ATF1, ATF2, EHT1 and IAH1) was assessed, during the fermentation of a defined must at 15 and 28°C. Higher concentrations of compounds related to fresh and fruity aromas were found at 15°C, while higher concentrations of flowery related aroma compounds were found at 28°C. The formation rates of volatile aroma compounds varied according to growth stage. In addition, linear correlations between the increases in concentration of higher alcohol and their corresponding acetates were obtained. Genes presented different expression profiles at both temperatures, except ILV2, and those involved in common pathways were co-expressed (ADH1, PDC1 and BAT2; and ATF1, EHT1 and IAH1). These results demonstrate that the fermentation temperature plays an important role in the wine final aroma profile, and is therefore an important control parameter to fine-tune wine quality during winemaking.
Stevia rebaudiana is known for its sweet-tasting ent-kaurene diterpenoid glycosides. Several manufacturing strategies are currently employed to obtain Stevia sweeteners with the lowest possible ...off-flavors. The chemical composition of four commercial S. rebaudiana extracts, obtained by different technologies, was characterized using UHPLC-ESI-MS n . The composition of one of the ethanol-crystallized extracts (EC2) was entirely rebaudioside A, whereas the enzymatically modified (EM) extract contained the lowest concentration of this compound (2.7 mg/100 mg). The membrane-purified (MP) extract had the highest content of minor natural steviol glycosides (23.7 mg/100 mg total extract) versus an average of 2.4 mg/100 mg total extract for the EC samples. Thirteen trained panelists evaluated sweetness, bitterness, licorice, and metallic attributes of all four extracts. The highest licorice intensity (p ≤ 0.05) was found for MP. Both samples EC1 and EC2, despite their different chemical compositions, showed no significant differences in sensory perception.
Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized ...phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus
When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in
, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic
/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene
, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor
provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV's ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.
Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus
In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.
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