Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor

[EN] Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with & alpha;- and & beta;-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2: agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2. The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates. Work supported by the European Commission, Horizon 2020 program (Project NEWCOTIANA, grant agreement 760331), and the Spanish Ministerio de Ciencia e Innovacion through Agencia Estatal de Investigacion (PID2020-114691RB-I00). MM was the recipient of a predoctoral contract (FPU16/05294) from the Ministerio de Educacion, Cultura y Deporte (Spain). Demurtas, OC.; Sulli, M.; Ferrante, P.; Mini, P.; Aragones, V.; Daròs, J.; Marti, M... (2023). Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor. Metabolites. 13(6). https://doi.org/10.3390/metabo13060729