A New Glycosyltransferase Enzyme from Family 91, UGT91P3, Is Responsible for the Final Glucosylation Step of Crocins in Saffron (Crocus sativus L.)

Abstract

Crocetin is an apocarotenoid formed from the oxidative cleavage of zeaxanthin, by the carotenoid cleavage enzymes CCD2 (in Crocus species) and specific CCD4 enzymes in Buddleja davidii and Gardenia jasminoides. Crocetin accumulates in the stigma of saffron in the form of glucosides and crocins, which contain one to five glucose molecules. Crocetin glycosylation was hypothesized to involve at least two enzymes from superfamily 1 UDP-sugar dependent glycosyltransferases. One of them, UGT74AD1, produces crocins with one and two glucose molecules, which are substrates for a second UGT, which could belong to the UGT79, 91, or 94 families. An in silico search of Crocus transcriptomes revealed six candidate UGT genes from family 91. The transcript profiles of one of them, UGT91P3, matched the metabolite profile of crocin accumulation, and were co-expressed with UGT74AD1. In addition, both UGTs interact in a two-hybrid assay. Recombinant UGT91P3 produced mostly crocins with four and five glucose molecules in vitro, and in a combined transient expression assay with CCD2 and UGT74AD1 enzymes in Nicotiana benthamiana. These results suggest a role of UGT91P3 in the biosynthesis of highly glucosylated crocins in saffron, and that it represents the last missing gene in crocins biosynthesis.

Molecular Cloning, Expression, and Functional Analysis of Glycosyltransferase (TbUGGT) Gene from Trapa bispinosa Roxb.

“…The conserved functional domain allows the genes to maintain a certain similarity in catalytic potency. Currently, the UGT crystal structure has been obtained mainly in plants, such as cassava [ 40 ], Saffron [ 41 ], Arabidopsis thaliana [ 42 , 43 ], etc. These UGTs only recognize UDPG as a sugar donor [ 44 ].…”

Terpenoid Transport in Plants: How Far from the Final Picture?

“…Crocins are glycosylated apocarotenoid pigments that originate from the oxidative cleavage of carotenoids, such as zeaxanthin, β-carotene, and lycopene. The main source of crocins is saffron, the dried stigmas of Crocus sativus , in which crocins originate in the stigmas from the cleavage of zeaxanthin by the CsCCD2 enzyme [ 187 ] and undergo subsequent dehydrogenation, glycosylation, and storage in the vacuole, where they can reach up to 10% of dried matter [ 77 , 100 , 188 , 189 , 190 ]. Crocins are also accumulated in the fruits of Gardenia jasminoides , where their synthesis starts from the carotenoids β-carotene, zeaxanthin, and lycopene by the action of GjCCD4a [ 191 ], and in the flowers of Buddleja davidii , in which the substrate is zeaxanthin and the enzymes involved are BdCCD4.1/BdCCD4.3 [ 192 ]; moreover, for these species, the whole pathway from the carotenoid precursor to the generation of crocins with different degree of sugar moieties have been fully revealed [ 191 , 193 ].…”

“…In C. sativus , the crocin biosynthetic pathway involves different compartments: the synthesis, indeed, starts in the chromoplast with the cleavage of zeaxanthin by the plastid-localized CsCCD2, leading to the formation of the C20 compound crocetin dialdehyde [ 77 , 194 ], continues in the ER with the dehydrogenation of crocetin dialdehyde into crocetin through the action of the ER-localized CsALDH3I1 enzyme [ 77 ], and ends in the cytosol with the action of the two steps of glucosylation, which is performed by CsUGT74AD1 [ 77 ] and CsUGT91P3 [ 188 ], although an additional UGT member named UGTCs4 has been recently reported [ 190 ]. Finally, crocins are transported in the vacuole, where they are stored at high concentration.…”

Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin

“…More specifically, CCD1 coding genes have been found in all the plant genomes studied so far (https://phytozome.jgi.doe.gov); the enzymes of this subfamily recognize and cleave a wide range of carotenoids at different positions (Garcia‐Limones et al., 2008; Huang et al., 2009; Ibdah et al., 2006; Ilg et al., 2014; Schmidt et al., 2006; Simkin et al., 2004), and, for this reason, their activities have been proposed to be associated to apocarotenoid degradation (Simkin, 2021). CCD2, closely related to CCD1, but with plastid localization, drives zeaxanthin catabolism at positions 7,8;7′,8′ in Crocus species, yielding crocetin‐dialdehyde (Ahrazem, Rubio‐Moraga, et al., 2016; Frusciante et al., 2014), further transformed into crocins (Gomez‐Gomez et al., 2017, 2018; López‐Jimenez et al., 2021; Moraga et al., 2004). Besides CCD2, in the CCD4 subfamily, specific chromoplastic localized CCD4 enzymes from Buddleja davidii and gardenia act on carotenoids at positions 7,8;7′,8′ to generate crocins as well (Ahrazem et al., 2017; Xu et al., 2020; Zheng et al., 2022).…”