Characterization of two Vitis vinifera carotenoid cleavage dioxygenases by heterologous expression in Saccharomyces cerevisiae
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Norisoprenoids are produced from carotenoids under oxidative degradation mediated by carotenoid cleavage dioxygenases (CCDs) and contribute to floral and fruity notes in grape berries and wine. The diversity of CCD substrates and products has been demonstrated by in vitro recombinant proteins extracted from Escherichia coli expressing CCD genes and of in vivo proteins in an E. coli system co-expressing genes for carotenoid synthesis and cleavage. In the current study, VvCCD1 and VvCCD4b were isolated from the cDNA library of Vitis vinifera L. cv. Cabernet Sauvignon and then transformed into carotenoid-accumulating recombinant Saccharomyces cerevisiae strains. The expression of the target genes was monitored during the yeast growth period, and the accumulation of carotenoids and norisoprenoids in the recombinant strains was measured. The results indicated that both of the VvCCDs cleaved β-carotene at the 7, 8 (7′, 8′) position into β-cyclocitral for the first time. Additionally, the two enzymes also degraded β-carotene at the 9, 10 (9′, 10′) position to generate β-ionone and cleaved lycopene at the 5, 6 (5′, 6′) position into 6-methyl-5-hepten-2-one. These findings suggested that the VvCCDs may possess more cleavage characteristics under the eukaryotic expression system in S. cerevisiae than the prokaryotic system in E. coli, which could better explain the biochemical functions of VvCCDs in grape berries.
KeywordsCarotenoid cleavage dioxygenase Vitis vinifera L cv. Cabernet Sauvignon Carotenoids Norisoprenoids Saccharomyces cerevisiae
Project administration, QHP; Formal analysis and writing - original draft, NM; Writing - review & editing, GLY, CQD and QHP; Methodology, DZ; Data curation, XYL.
This research was funded by National Natural Science Foundation of China, Grant Number 31272118, and the China Agriculture Research System (CARS-29). Qiu-Hong Pan received the funding. The funders had no role in study design, data collection and analysis, preparation of manuscript, and decision on publication.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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