One-Pot Efficient\nBioconversion of Crocetin from Zeaxanthin\nvia a Dual-Enzyme System

Saffron contains pharmacologic active compounds and a\nlarge number\nof flavor compounds. Both originate from carotenoids by biocatalyzed\noxidation. Crocetin in saffron exhibits various attractive bioactivities\nand could be considered in the treatment of various diseases including\ncancer, coronary heart disease, and hypertension. However, powerful\nstrategies for the efficient and sustainable source of crocetin are\nlacking. To address this issue, we demonstrated a one-pot approach\nto bioconversion of crocetin from zeaxanthin via a dual-enzyme system\nconsisting of carotenoid cleavage dioxygenase 2 (<i>Cs</i>CCD2-M1 (R192F/S323A)) and aldehyde dehydrogenase (<i>Syn</i>ALD) with optimized protein expression and reaction system. Meanwhile,\nit was observed that the byproduct 3-hydroxy-β-apo-8′-carotenoic\nacid (3-HACA) severely accumulated due to the imbalance in the activity\nof the <i>Cs</i>CCD2-M1 and <i>Syn</i>ALD. A suitable\nmolar ratio of 6:1 and the optimized dual-enzyme reaction conditions\nwere developed to eliminate 3-HACA completely. Consequently, the efficient\nbiosynthesis of crocetin (1.48 mg/L/h) and an 80.8% conversion rate\nof crocetin within 2 h were accomplished after scaling up the optimized\nsystem by 10 000 times. Moreover, over 34% of enzyme activity\nwas still retained in the residual enzyme system after three times\nof recycling. This study not only provides a sustainable means of\ncrocetin production <i>in vitro</i> but also presents a\nnew insight into the rapid construction of one-pot synthesis of high-value\ncompounds with complex structures.