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

Abstract

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.

Research Progress in Heterologous Crocin Production

“…Crocetin and crocin V were successfully produced in engineered E. coli and Saccharomyces cerevisiae, but these strains showed low productivity [7,[61][62][63]. Crocin has also been successfully synthesized in vitro using an enzyme cascade [64]. The authors demonstrated an 80.8% conversion rate of crocetin within 2 h with a yield of 1.48 mg/L/h by optimizing the ratio of enzymes in the system and reducing the accumulation of intermediate byproducts [64].…”

“…Crocin has also been successfully synthesized in vitro using an enzyme cascade [64]. The authors demonstrated an 80.8% conversion rate of crocetin within 2 h with a yield of 1.48 mg/L/h by optimizing the ratio of enzymes in the system and reducing the accumulation of intermediate byproducts [64]. Clearly, to address the high demand for crocins, there is a need for alternative methods.…”

“…Zeaxanthin is a direct precursor in the synthesis of crocins [64]. The downstream pathway mainly consists of three steps.…”

Efficient One-Pot Synthesis of Uridine Diphosphate Galactose Employing a Trienzyme System

“…With new advances in in vitro multienzyme cascade reactions and modular combinations of enzyme reactions, − numerous researchers have begun to pay close attention to in vitro enzymatic technology for the efficient and accurate synthesis of UDP-Gal. Artificially designed in vitro enzyme cascade platforms have shown significant advantages, including high product yields, shorter mass transfer, faster reaction rates, and higher tolerance to toxic products …”

“…Artificially designed in vitro enzyme cascade platforms have shown significant advantages, including high product yields, shorter mass transfer, faster reaction rates, and higher tolerance to toxic products. 16 In the in vitro multienzyme cascade reaction, three main pathways can be employed to synthesize UDP-Gal. 17 The salvage pathway (Scheme 1a) involves the conversion of Gal-1P to UDP-Gal by uridine diphosphate sugar pyrophosphorylase (GalU) in the presence of uridine triphosphate (UTP).…”