Crocetin and crocin production in plant and bacteria using a CCD4 enzyme from Nyctanthes 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 including Crocus sativus or Nyctanthes arbor-tristis. We investigated the genome of N. arbor-tristis and 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 in Nicotiana 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.