Highly selective synthesis of t-crocin I via an in vitro four-enzyme cascade system

t-crocin I (t-crocin I) is the predominant and most extensively studied crocin congener, with antidepressant effects comparable to some first-line antidepressants. However, t-crocin I is still obtained mainly by plant extraction, leading to a high retail price and an unsustainable supply. Despite substantial efforts in heterologous production, most studies generate mixtures of multiple crocin congeners and their cis isomers. Approaches for selective t-crocin I production remain scarce.

This study aims to establish and optimize an in vitro multi-enzyme cascade for the selective, high-titer production of t-crocin I.

This research proposed an in vitro four-enzyme cascade system comprising the enzymes responsible for crocin biosynthesis in Gardenia jasminoides (GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13). At first, feasibility of the cascade for t-crocin I biosynthesis was verified stepwise using E. coli supernatants and purified proteins. Enzyme activity, reaction conditions, and cofactor usage were then optimized to improve the conversion rate of this system from zeaxanthin to t-crocin I. Finally, to achieve a higher titer of t-crocin I, cyclodextrin encapsulation was applied to overcome the low aqueous solubility of zeaxanthin and increase substrate loading.

Optimization of reaction conditions and determination of minimum enzyme loadings raised the t-crocin I titer from 5.02 to 8.98 μM at 10 μM zeaxanthin at lower enzyme cost. The GjUGT94E13-to-GjUGT74F8 (94:74) ratio was proved to be a principal determinant of the crocin product distribution: the t-crocin I proportion peaked at 95.78% at a 94:74 molar ratio of 1:4, whereas higher ratios diverted the t-crocin I towards a pentaglucosylated crocin derivative. Formulating zeaxanthin as a methyl-β-cyclodextrin inclusion complex raised its aqueous concentration to 1420 μM and allowed a substrate loading of 250 μM, at which the cascade produced 234.0 μM (228.6 mg L^-1) t-crocin I within 3 h, accounting for 96.7% of the total crocins.

The in vitro four-enzyme cascade developed here achieves selective, high-titer biosynthesis of t-crocin I directly from a carotenoid substrate, offering a route that preferentially produces t-crocin I while limiting the formation of other crocin congeners.