Comparative Evaluation of Secreted Plant Carotenoid Cleavage Dioxygenase 1 (CCD1) Enzymes in Saccharomyces cerevisiae

Abstract

Enabling technologies in synthetic biology now present the opportunity to engineer wine yeast for enhanced novel aromas. In doing so, improved wine products will increase the desirability of wine for the consumer and add value to the winemaker. The action of the enzyme carotenoid cleavage dioxygenase 1 (CCD1) on β-carotene to produce β-ionone is of interest to improve the aroma and flavour of the wine. Engineering the yeast, Saccharomyces cerevisiae, to produce higher concentrations of CCD1 in grape-must presents an opportunity to increase the levels of this volatile organic compound, thus enhancing the organoleptic properties of wine. To this end, four phylogenetically diverse plant CCD1 genes were synthesised with a secretion signal peptide and transformed into S. cerevisiae. The relative ability of each enzyme secreted into the yeast supernatant to cleave the deep orange C40 β-carotene was determined by spectrophotometry; furthermore, the by-product of such cleavage, the highly aromatic C13 β-ionone, was assessed by head-space solid-phase micro-extraction, with analysis and detection by GCMS. Reduction in β-carotene levels and release of β-ionone from the supernatant were validated by LCMS detection of CCD1. These experiments demonstrated that expression in yeast of the CCD1s derived from Petunia hybrida and Vitis vinifera and their subsequent secretion into the medium provided superior efficacy in both β-carotene reduction and β-ionone liberation. We anticipate this knowledge being of benefit to future winemakers in producing a vinous product with enhanced organoleptic properties.