Novel plant cell suspension platforms for saffron apocarotenoid production and its impact on carotenoid and volatile profiles.

Saffron apocarotenoids, including crocins, picrocrocin and safranal, are valuable metabolites with pharmaceutical and cosmetic potential. However, their natural plant sources are difficult to cultivate, which limits large-scale production. The identification of carotenoid cleavage dioxygenases (CCDs), which catalyse the first and most critical step in their biosynthesis, has enabled the production of these apocarotenoids in heterologous plant systems. In this study, we aimed to generate plant cell suspensions expressing Crocus sativus CCD2 and Gardenia jasminoides CCD4a, along with a bacterial phytoene synthase to enhance carotenoid biosynthesis and CsUGT93P1, which improves crocin stability. Transgenic cell suspensions were established from Nicotiana benthamiana plants and Nicotiana tabacum cv. BY-2 cells. In BY-2 cells expressing GjCCD4a, crocin accumulation reached 770 μg/g DW, which further increased upon methyl jasmonate elicitation. Remarkably, the BY-2 transgenic cells exhibited an 18,000-fold increase in β-cyclocitral content compared to wild-type cells. The best-performing N. benthamiana and BY-2 lines were successfully cultivated in wave bioreactors, demonstrating their potential for saffron apocarotenoid production. In the BY-2 bioreactor, apart from saffron apocarotenoids, phytoene and notably high amounts of lycopene were produced, adding value to the platform and indicating a remodelling of the carotenoid pathway. This study establishes the viability and lays the foundation for the scalable production of saffron apocarotenoids and carotenoids in plant cell suspensions.