Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas

Saffron, the dry stigma of Crocus sativus L., is considered to be the world's most expensive spice. Three major apocarotenoids-ocrocin, crocetin and picrocrocin-are responsible for the colour and bitter taste of saffron. The final step in the biosynthesis of the 20-carbon esterified carotenoid crocin is the transformation of the insoluble crocetin into a soluble and stable storage form by glucosylation. These glucosylation reactions are catalysed by glucosyltransferases (GTases) that play a crucial role in natural-product biosynthesis. Using degenerate primers designed to match the plant secondary product GTase (PSPG) box we cloned two cDNAs, UGTCs2 and UGTCs3, from C. sativus stigmas that encode putative polypeptides of 460 and 475 amino acids, respectively. These genes were expressed differentially in saffron tissues. UGTCs2 was mainly expressed in fully developed stigmas, whereas UGTCs3 was mainly expressed in stamens. The UGTCs2 transcript was not detected in the stigma tissue of a Crocus species that does not synthesize crocin, while UGTCs3 and other structural genes for carotenoid biosynthesis were expressed in the stigma of all tested Crocus species. To identify the biochemical function of UGTCs2, the isolated cDNA was expressed in Escherichia coli cells. The recombinant protein UGTCs2 had glucosylation activity against crocetin, crocetin beta-D-glucosyl ester and crocetin beta-D-gentibiosyl ester. These results might suggest that the isolated clone UGTCs2 codes for a saffron crocetin GTase.