One-Pot Efficient Bioconversion of Crocetin from Zeaxanthin via a Dual-Enzyme System

Saffron contains pharmacologic active compounds and a large number of flavor compounds. Both originate from carotenoids by biocatalyzed oxidation. Crocetin in saffron exhibits various attractive bioactivities and could be considered in the treatment of various diseases including cancer, coronary heart disease, and hypertension. However, powerful strategies for the efficient and sustainable source of crocetin are lacking. To address this issue, we demonstrated a one-pot approach to bioconversion of crocetin from zeaxanthin via a dual-enzyme system consisting of carotenoid cleavage dioxygenase 2 (CsCCD2-M1 (R192F/S323A)) and aldehyde dehydrogenase (SynALD) with optimized protein expression and reaction system. Meanwhile, it was observed that the byproduct 3-hydroxy-β-apo-8′-carotenoic acid (3-HACA) severely accumulated due to the imbalance in the activity of the CsCCD2-M1 and SynALD. A suitable molar ratio of 6:1 and the optimized dual-enzyme reaction conditions were developed to eliminate 3-HACA completely. Consequently, the efficient biosynthesis of crocetin (1.48 mg/L/h) and an 80.8% conversion rate of crocetin within 2 h were accomplished after scaling up the optimized system by 10 000 times. Moreover, over 34% of enzyme activity was still retained in the residual enzyme system after three times of recycling. This study not only provides a sustainable means of crocetin production in vitro but also presents a new insight into the rapid construction of one-pot synthesis of high-value compounds with complex structures.