Crocetin and crocin production in plant and bacteria using a CCD4 enzyme fromNyctanthes arbor-tristis

Crocins are hydrophilic crocetin esters that are arising interest as cosmetics and pharmaceuticals. Crocetin dialdehyde, the precursor of crocetin, derives from a C7-C8(C7՛-C8՛) cleavage of carotenoids in a few plant species includingCrocus sativusorNyctanthes arbor-tristis. We investigated the genome ofN. arbor-tristisand identified an enzyme from the CCD4 subfamily catalyzing the cleavage of zeaxanthin to produce crocetin dialdehyde. This enzyme, NatCCD4.1, was used for the microbial production of crocetin dialdehyde in a two-phase culture system using direct extraction with n-dodecane, resulting in a titer of 109.2 ± 3.23 mg/L, which is the highest crocetin dialdehyde yield reported in bacteria so far. Further, a viral vector was used to express NatCCD4.1 inNicotiana benthamiana, triggering a crocin accumulation of 2.32 ± 0.69 mg/g DW. Our results provide new insights into crocin biosynthesis and demonstrate that NatCCD4.1 is a valuable tool for improving crocetin and crocin production in heterologous systems.