UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus)

Abstract

Summary Saffron, a spice derived from the dried red stigmas of Crocus sativus, is one of the oldest natural food additives. The flowers have long red stigmas, which store significant quantities of the glycosylated apocarotenoids crocins and picrocrocin. The apocarotenoid biosynthetic pathway in saffron starts with the oxidative cleavage of zeaxanthin, from which crocins and picrocrocin are derived. In the processed stigmas, picrocrocin is converted to safranal, giving saffron its typical aroma. By a targeted search for differentially expressed uridine diphosphate glycosyltransferases (UGTs) in Crocus transcriptomes, a novel apocarotenoid glucosyltransferase (UGT709G1) from saffron was identified. Biochemical analyses revealed that UGT709G1 showed a high catalytic efficiency toward 2,6,6‐trimethyl‐4‐hydroxy‐1‐carboxaldehyde‐1‐cyclohexene (HTCC), making it suited for the biosynthesis of picrocrocin, the precursor of safranal. The role of UGT709G1 in picrocrocin/safranal biosynthesis was supported by the absence or presence of gene expression in a screening for HTCC and picrocrocin production in different Crocus species and by a combined transient expression assay with CsCCD2L in Nicotiana benthamiana leaves. The identification of UGT709G1 completes one of the most highly valued specialized metabolic biosynthetic pathways in plants and provides novel perspectives on the industrial production of picrocrocin to be used as a flavor additive or as a pharmacological constituent.

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system

“…In this virus-driven system, the expression of an appropriate CCD in N. benthamiana is sufficient to trigger the apocarotenoid pathway in this plant. Recently, it was observed the same situation in a transient expression system (Diretto et al, 2019a). These observations indicate that the zeaxanthin cleavage is the limiting step of apocarotenoid biosynthesis in this species, while aldehyde dehydrogenation and glycosylation steps can be efficiently complemented by endogenous orthologue enzymes.…”

“…Chlorophyll a and b were determined by measuring absorbance at 645 nm and 663 nm as previously described (Richardson et al, 2002). Mass spectrometry carotenoid, chlorophyll derivative and apocarotenoid identification and quantification was carried out as previously described (Ahrazem et al, 2018; Diretto et al, 2019a; Rambla et al, 2016). Mass spectrometry analysis of other isoprenoids (tocochromanols and quinones) were performed as reported before (Sulli et al, 2017).…”

“…The HTCC molecule is also recognized by UGTs, resulting in production of picrocrocin (Fig. 1) (Diretto et al, 2019a). In C. sativus , the plastidic CsCCD2L enzyme catalyzes the cleavage of zeaxanthin at 7,8;7′,8′ double bonds (Ahrazem et al, 2016c; Frusciante et al, 2014), which is closely related to the CCD1 subfamily (Ahrazem et al, 2016b, 2016a).…”

“…Due to its high scientific and commercial interest, crocetin and crocins biosynthesis has arisen great attention, and attempts to metabolically engineer their pathway in microbial systems have been reported, although with limited success (Chai et al, 2017; Diretto et al, 2019a; Tan et al, 2019; Wang et al, 2019). More in detail, the use of the saffron CCD2L enzyme resulted in a maximum accumulation of 1.22 mg/l, 15.70 mg/l, and 4.42 mg/l of crocetin in, respectively, Saccharomyces cerevisiae (Chai et al, 2017; Tan et al, 2019) and Escherichia coli (Wang et al, 2019).…”

“…More in detail, the use of the saffron CCD2L enzyme resulted in a maximum accumulation of 1.22 mg/l, 15.70 mg/l, and 4.42 mg/l of crocetin in, respectively, Saccharomyces cerevisiae (Chai et al, 2017; Tan et al, 2019) and Escherichia coli (Wang et al, 2019). In a subsequent study aimed to confirm, in planta , the role of a novel UGT in picrocrocin biosynthesis (Diretto et al, 2019a), Nicotiana benthamiana leaves were transiently transformed, via Agrobacterium tumefaciens , with CCD2L, alone or in combination with UGT709G1, which led to the production of 30.5 μg/g DW of crocins, with a glycosylation degree ranging from 1 to 4 (crocin 1-4) (unpublished data). Although promising, these results cannot be considered efficient and sustainable in the context of the development of an industrial system for the production of the saffron high-value apocarotenoids.…”