LAUSR

Summary Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the colour and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the carotenoid cleavage dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison with CsCCD2L, in citrus callus engineered by Agrobacterium‐mediated supertransformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. We demonstrate that co‐expression of GjCCD4a with phytoene synthase and β‐carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved β‐carotene, in planta, and produced crocetin dialdehyde via C30 β‐apocarotenoid intermediate. GjCCD4a also cleaved C27 β‐apocarotenoids, providing a new route for C17‐dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast‐like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis‐9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non‐green crop tissues/organs.