Abstract

Background: Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plants – like raspberries and roses – by the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid ß-ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP. Results: Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) produced low amounts of ß-ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene in a high copy number plasmid in this former strain increased ß-ionone concentration 5-fold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of ß-ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain result in a final specific concentration of 1 mg/g at 50 hours, which represent a 15-fold increase. Conclusions: An efficient ß-ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the gene BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 gene - the highest ß-ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and economical process that could replace current chemical methods.