Research progress on biosynthesis and synthetic biology of crocin

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Natural products are important resources for drug discovery and have marvelous roles in the treatment of various diseases, such as malaria, cancers, and viral infections. Crocins are the colored apocarotenoids derived from the stigmas of Crocus sativus and often used as a spice/colorant in the food industry. Pharmacologically, crocins confer protection to the central nervous system (especially in the treatment of Alzheimer’s disease) and prevent cardiovascular/cerebrovascular diseases. Unfortunately, the demand for crocins based on their pharmacological benefits combined with a complex harvesting process of C. sativus has driven the retail price of C. sativus stigmas up to 2000–7000 €/kg. Thus, their expense and limited supply greatly restrict the development and clinical application of crocin-based pharmaceuticals. It is therefore urgent to develop an alternative, sustainable way to bolster the supply of crocins. In recent years, synthetic biology has been widely used to overcome shortages of plant-based natural products, including artemisinic acid, etoposide aglycone, opioids, cannabinoids, breviscapine, and ginsenosides. As such, the genetic tools required for crocin biosynthesis have attracted a great deal of attention. The crocin biosynthetic pathway includes three stages: Stage (I), the precursor geranylgeranyl pyrophosphate (GGPP) is generated by the methylerythritol-4-phosphate (MEP) biosynthetic pathway; stage (II), C40-carotenoids are produced via the carotenoid biosynthetic pathway; stage (III), crocins are synthesized by three key catalytic steps, namely, carotenoid cleavage, aldehyde oxidation, and glucosyl group transfer. A series of studies have successfully decoded the crocin biosynthetic pathway and led to its reconstitution in Saccharomyces cerevisiae and Escherichia coli . Hereby, we summarized the chemical characterization of crocins using spectroscopy and mass spectrometry, and elucidated stage (III) of the crocin biosynthetic pathway. Carotenoid cleavage dioxygenases (CCDs) degrade carotenoids. CsCCD2, CaCCD2 ( Crocus ancyrensis ), BdCCD4.1 ( Buddleja davidii ), BdCCD4.3 and GjCCD4a ( Gardenia jasminoides ) can cleave the 7,8 (7′,8′) double bond of zeaxanthin to yield crocetin dialdehyde. Subsequently, crocetin dialdehyde was oxidized to crocetin by the catalysis of aldehyde dehydrogenases (ALDHs), including CsALDH54788, CsALDH3898, CsALDH20158, CsALDH11367, CsALDH3I1, CsALDH3, and GjALDH2C3. Finally, the biosynthesis of crocins from crocetin was catalyzed by UDP-glycosyltransferases (UGTs), including CsUGT74AD1, GjUGT74F8, GjUGT75L6, GjUGT94E5, GjUGT94E13, Bs-GT ( Bacillus subtilis 168), and Bc-GTA ( Bacillus cereus WQ9-2). And then, we displayed the research progress on the synthetic biology of crocins. With the rapid updating of genetic tools, we have the potential to generate more types of crocins. The in vivo production of crocins is highlighted in this review. The engineered E. coli with GjUGT74F8 and GjUGT94E13 can catalyze crocetin into five types of crocins. Furthermore, we put forward some suggestions that might help to improve the bioproduction of crocins, such as the exploration of evolutionary mechanisms of the key enzymes and the optimization of reaction conditions, etc. Taken together, this review will facilitate the industrial production of crocins and the development of crocin-based pharmaceuticals, providing a theoretical basis and method guidance for the sustainable development of crocin-based traditional Chinese medicine.