Abstract
BackgroundMonoterpenes are important contributors to grape and wine aroma. Moreover, certain monoterpenes have been shown to display health benefits with antimicrobial, anti-inflammatory, anticancer or hypotensive properties amongst others. The aim of this study was to construct self-aromatizing wine yeasts to overproduce de novo these plant metabolites in wines.ResultsExpression of the Ocimum basilicum (sweet basil) geraniol synthase (GES) gene in a Saccharomyces cerevisiae wine strain substantially changed the terpene profile of wine produced from a non-aromatic grape variety. Under microvinification conditions, and without compromising other fermentative traits, the recombinant yeast excreted geraniol de novo at an amount (~750 μg/L) well exceeding (>10-fold) its threshold for olfactory perception and also exceeding the quantities present in wines obtained from highly aromatic Muscat grapes. Interestingly, geraniol was further metabolized by yeast enzymes to additional monoterpenes and esters: citronellol, linalool, nerol, citronellyl acetate and geranyl acetate, resulting in a total monoterpene concentration (~1,558 μg/L) 230-fold greater than that of the control. We also found that monoterpene profiles of wines derived from mixed fermentations were found to be determined by the composition of the initial yeast inocula suggesting the feasibility of producing ‘à la carte’ wines having predetermined monoterpene contents.ConclusionsGeraniol synthase-engineered yeasts demonstrate potential in the development of monoterpene enhanced wines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0306-5) contains supplementary material, which is available to authorized users.
Highlights
Introduction of theC. breweri linalool synthase (LIS) gene into wine yeast strain T73-4 (T73Lis) under the control of the TDH3 yeast promoter was our first attempt to construct a self-aromatizing wine yeast [17]
gas chromatography (GC) and gas chromatography–mass spectrometry (GC–mass spectrometry (MS)) analyses of these culture media showed similar extraordinarily high geraniol yields (8,017.85 ± 1,245.81 and 7,859.12 ± 1,614.62 μg/L after 32 h) (Fig. 1b). These levels are about 16-fold higher than those produced by recombinant S. cerevisiae laboratory strains expressing the same geraniol synthase (GES) gene, about 1.6-fold the amount produced by laboratory yeasts co-expressing GES and an optimized farnesyl diphosphate synthase [25, 26], and about 120-fold the amount of linalool excreted by engineered T73-4 wine strains expressing LIS [17, 21]
484.51 ± 79.92 175.71 ± 16.52 178.66 ± 23.60 56.46 ± 6.60 1,558.48 ± 195.13 nd, not detected. a Numbers between brackets refer to the GC peaks in Fig. 3. b Aroma descriptors according to Feranoli’s [35]. c Odor detection threshold values (OTVs) in water were taken from the lists of Leffingwell & Associates. d Values represent the means and standard deviation of two different microvinifications, three technical replications and two analytical replications
Summary
Introduction of theC. breweri LIS gene into wine yeast strain T73-4 (T73Lis) under the control of the TDH3 yeast promoter was our first attempt to construct a self-aromatizing wine yeast [17]. The amount of geraniol-derived linalool produced by YR377 (T73Ges) was about 7.5 times greater than that obtained with T73Lis (~141 versus ~19 μg/L) and the total de novo terpene concentration is more than 80-times greater, illustrating the importance of the MTPS employed in engineering strain T73. These results justify the strategy of engineering the wine yeast isoprenoid pathway as a means of achieving efficient plant-derived aromatic monoterpene production during alcoholic fermentation.
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