Carotenoids are an important group of natural and lipid-soluble pigments which humans cannot synthesize, thus acquiring them through diet. In humans, these molecules play multiple physiological and nutritional functions (Fiedor and Burda, 2014). Carotenoids are synthesized by plants, algae, fungi, and bacteria, displaying a yellow to red coloration, depending on the type of carotenoid and the concentration they reach in the different cells. Carotenoids are widely used as food colorants, nutraceuticals, animal feed additives, cosmetic ingredients, and health supplements (Fraser and Bramley, 2004). In all living organisms, carotenoids act as substrates to produce apocarotenoids (Ahrazem et al., 2016a). These metabolites are not simply breakdown products of carotenoids, as their functions include those of signaling and hormonal (Walter et al., 2010; Eroglu and Harrison, 2013; Jia et al., 2017). Apocarotenoids are present as volatile and soluble compounds. Crocins are water-soluble pigments of high commercial value, used mostly in food and to a lesser extent, in the pharmaceutical industries (Ahrazem et al., 2015). Crocins are glycosylated derivatives of the apocarotenoid crocetin and they exhibit a strong coloring capacity. In addition, crocins are powerful free radical quenchers, which is associated with the broad range of their health benefits (Nam et al., 2010; Georgiadou et al., 2012; Christodoulou et al., 2015; Bukhari et al., 2018). Interest in the therapeutic properties of crocins is increasing due to their analgesic and sedative properties (Amin and Hosseinzadeh, 2012), neurological protection, and anticancer activities (Finley and Gao, 2017; Skladnev and Johnstone, 2017). Furthermore, clinical trials indicate that crocins have a positive effect in the treatment of depression and dementia (Lopresti and Drummond, 2014; Mazidi et al., 2016). However, commercial utilization of crocins from saffron is limited due to elevated prices, a consequence of the high labor costs in harvesting and processing flower material (Ahrazem et al., 2015).

Crocus sativus is the main natural source of crocins, which accumulate at high levels during the development of the stigma and confer a dark red coloration (Moraga et al., 2009). Together with the safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde), the precursor of picrocrocin (β-D-glucopyranoside of hydroxyl-β-cyclocitral) is responsible for the flavor of the saffron spice (Tarantilis et al., 1995). In addition to Crocus, gardenia (Gardenia jasminoides) fruits are also a commercial source of crocins, but on a much lower scale, as they do not accumulate picrocrocin (Pfister et al., 1996; Moras et al., 2018). Among other plants that produce crocins, but which are not commercially exploited due to the low quantities accumulated, are Buddleja species (Liao et al., 1999). Therefore, only a few plant species can synthesize crocins, and this is because the carotenoid cleavage dioxygenases (CCDs) responsible for the production of crocins are not usually present in plants.

In Crocus, Gardenia, and Buddleja species, zeaxanthin is the precursor of crocetin (Figure 1). Cleavage of the zeaxanthin molecule at the 7,8 and 7′,8′ double bonds generates one molecule of crocetin dialdehyde and two molecules of 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) (Frusciante et al., 2014; Ahrazem et al., 2016c,2017). Crocetin dialdehyde is further converted to crocetin by the action of aldehyde dehydrogenase enzymes (ALDH), and crocetin is the substrate of glucosyltransferases (UGTs) that catalyze the formation of crocins, catalyzing the transfer of glucose molecules to both ends of the crocetin molecule (Moraga et al., 2004; Nagatoshi et al., 2012; Diretto et al., 2021). The HTCC molecule is also recognized by UGTs, resulting in the formation of picrocrocin, which is further metabolized to render safranal (Figure 1; López et al., 2021). In C. sativus the CCD2 enzyme catalyzed the cleavage of zeaxanthin at 7,8;7′,8′ double bonds (Frusciante et al., 2014). CsCCD2L is a plastidic enzyme that belongs to a new CCD subfamily only isolated from Crocus species (Ahrazem et al., 2016c), which is closely related to the CCD1 subfamily (Ahrazem et al., 2016a,b). In Buddleja davidii, and G. jasminoides, the CCD enzymes that catalyzed the same reaction belong to the CCD4 subfamily of CCDs (Ahrazem et al., 2016a; Xu et al., 2020). The Buddleja enzymes, BdCCD4.1 and BdCCD4.3, are as well plastidic enzymes expressed in flowers (Ahrazem et al., 2017).

The carotenoid biosynthetic pathway has been modified by genetic engineering in higher plants to increase the general amount of carotenoids and/or to produce specific carotenoids (Farre et al., 2014). In previous studies, CsCCD2L was transiently expressed alone and was sufficient to allow the significant crocins accumulation in tobacco plants (Diretto et al., 2019; Marti et al., 2020; López et al., 2021). In this study, we achieved a stable expression of the CsCCD2L gene alone or in combination with genes involved in the carotenoid biosynthesis and accumulation, like BrCrtZ and AtOrMut, to increase the availability of substrates for CsCCD2L activity. We chose the Nicotiana species as a heterologous host. The high metabolic versatility, non-food status, and high biomass production of the genus Nicotiana make this genus one of the most promising biofactories for the sustainable production of high-value metabolites (Molina-Hidalgo et al., 2021). Metabolic analyses showed the accumulation of these nutritional substances reaching in Nicotiana glauca almost 400 μg/g DW, which indicates that N. glauca is a better system than Nicotiana tabacum to produce crocins because the transgenic N. glauca plants expressing CsCCD2L alone could accumulate much higher amounts of crocins as compared with the transgenic N. tabacum plants coexpressing CsCCD2L, BrCrtZ, and AtOrMut genes.

Crocins are mainly known for being responsible for the color of the saffron spice. In addition to their pigment capacity, crocins show bioactive properties with several therapeutic and pharmacological applications (Mykhailenko et al., 2019). The saffron apocarotenoids had a global market size valued at 374.6 million USD in 2020 and is expected to reach USD 721.5 million by 2028, being the medical application segment the one projected to be the fastest-growing segment.¹ Between 120,000 and 200,000 flowers are needed to produce 1 kg of dried saffron stigma threads, which equates to 370–470 h of work. Consequently, the process is very labor-intensive and risky since it is highly dependent on environmental conditions, leading to high costs.

To overcome these limitations for obtaining saffron apocarotenoids while considering the advances of metabolic engineering, its progression into synthetic biology, and the elucidation of the saffron pathway, we were able to engineer plants from both N. glauca and N. tabacum expressing CsCCD2L. Several efforts have been made by the scientific community to transfer the synthesis of apocarotenoids from saffron to other hosts, looking for the low-cost production of these rare metabolites. It is known that the use of the saffron CsCCD2L enzyme resulted in a maximum accumulation of 1.22, 15.70, and 4.42 mg/l of crocetin in 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 UGTs in picrocrocin biosynthesis (Diretto et al., 2019), Nicotiana benthamiana leaves were transiently transformed, via A. tumefaciens, with CsCCD2L, 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). In addition, Marti et al. (2020), using a virus-driven expression of CsCCD2L in adult N. benthamiana plants demonstrated that CsCCD2L expression alone is sufficient for significant crocin accumulation in transiently transformed tobacco (up to 2.18 mg/g DW) (Marti et al., 2020).

The expression of CsCCD2L in N. glauca and N. tabacum allowed the accumulation of notable amounts of up to 400 and 36 μg/g DW of crocins, respectively. The underlying reason for the accumulated crocin variation observed among the analyzed N. glauca and N. tabacum lines could be due to the higher transcript levels of CsCCD2L N. glauca lines, and as well to the higher levels of zeaxanthin present in the leaves of N. glauca plants. The amount of crocins obtained is much higher in lines from N. glauca than in those from N. tabacum and the ones previously described using an A. tumefaciens-mediated transient expression of the CsCCD2L in N. benthamiana leaves, alone or in combination with UGT709G1 (30.5 μg/g DW of crocins). However, this accumulation is lower than the one reported in leaves of adult N. benthamiana using a recombinant virus that expressed CsCCD2L (2.18 mg/g DW of crocins) (Marti et al., 2020). However, crocin production in N. gluaca provides be more effective compared to the data obtained for the production of other carotenoids in this plant; expression of the β-carotene ketolase (CrtO) gene led to a total ketocarotenoid concentration in leaves of 136.6 (young) or 156.1 (older) μg/g dry weight and in petals of 165 μg/g dry weight (Zhu et al., 2007; Mortimer et al., 2017).

The profiles of crocins were different from those reported in saffron stigma, where trans-crocin 4, followed by trans-crocin 3 were the major crocins detected. In contrast, in our transgenic plants, trans-crocin-3 was more abundant than trans-crocin-4. In N. benthamiana leaves expressing transiently only CsCCD2L, the major crocin species were the trans-crocin 3, and trans-crocin with two glucose molecules (Supplementary Figure 3). These data indicated that different endogenous UGTs belonging to Nicotiana species recognize crocetin and crocins and can transfer different glucose molecules by changing the qualitative pattern. However, other factors, such as the presence of hydrolase activities that affect the stability of the different crocins synthesized in tobacco cells, and the transport of these crocins to the vacuole and their stability therein, could be influencing the different profiles observed.

It is well-known that BCH overexpression resulted in increased zeaxanthin and xanthophyll contents, both in microbes and plants (Lagarde et al., 2000; Du et al., 2010; Arango et al., 2014). Therefore, overexpression of BCH could enhance the levels of zeaxanthin as the preferred substrate of CsCCD2L (Frusciante et al., 2014). On the other hand, the Orange gene has proven to play an important role in carotenoid accumulation by activating chromoplast differentiation in non-green tissues (Lu et al., 2006), although its expression in green tissues exerts no effect on carotenoid levels (Yuan et al., 2015), we decided to determine whether its introduction could increase the carotenoid content by promoting the formation of carotenoid-sequestering structures. In this context, BrCrtZ and AtOrMut were introduced in N. tabacum to boost the crocin content, since the accumulation of these metabolites was 10-fold lower than N. glauca. The introduction of the two transgenes together with CsCCD2L has most likely led to an increase in the carotenoid pool used as a substrate by the enzyme and, consequently, in a 3.5-fold increase in crocin compared to the sole expression of CsCCD2L.

By introducing CsCCD2L alone or in combination with BrCrtZ and AtOrMut, a decreased level of the major and other minor carotenoids normally present in N. glauca and N. tabacum was detected. Among all the carotenoids detected, lutein was one of the carotenoids showing the higher decrease, likely due to the ability of CsCCD2L to act over the β-ring of this molecule as reported in previous in vitro studies where lutein can act as a substrate of this cleavage activity (Frusciante et al., 2014). Zeaxanthin, the crocin precursor, is not a major carotenoid in Nicotiana plant leaves, although the production of the crocins in N. glauca and N. tabacum suggests that the expression of CsCCD2L can drag the leaf metabolic flux toward the production of these apocarotenoids. The level of β-carotene was also lower among the N. glauca transgenic plants, the same phenomenon has been shown in N. tabacum plants transformed with the three genes suggesting a hydroxylation of β-carotene to zeaxanthin under the influence of CsCCD2L in N. glauca and the combination of the exogenous BrCrtZ and CsCCD2L in N. tabacum. Gotz et al. (2002), previously described the increase of zeaxanthin in tobacco plants (N. tabacum L. cv. Samsun) by transformation with a heterologous carotenoid gene encoding β-carotene hydroxylase (CrtZ) from Erwinia uredovora under constitutive promoter control. The increase of zeaxanthin in transgenic plants is due to the catalytic activity of the additional β-carotene hydroxylase rather than being primarily caused by an elevation in the conversion of violaxanthin to zeaxanthin in high light (Gotz et al., 2002).

It has been reported that the expression of an appropriate CsCCD2L in N. benthamiana is sufficient to activate the apocarotenoid pathway in this plant (Diretto et al., 2019; Marti et al., 2020). It seems that both N. glauca and N. tabacum followed the same pattern as N. benthamiana. The crocins produced in this report, using metabolically engineered N. glauca (400 μg/g DW) and N. tabacum (136 μg/g DW), are lower than those obtained in N. benthamiana (2 mg/g DW) using a virus-driven system expressing transient CsCCD2L. However, the stable expression of CsCCD2L can compensate for the low apocarotenoid yields leading to higher apocarotenoid productivities by reducing costs and labor. Further increase of N. glauca and N. tabacum content in these apocarotenoids can also be achieved by combining different types of promoters and the expression of other carotenogenic genes to increase the content of the precursors of crocins, zeaxanthin, and lutein. Thus, an approach of focused metabolic engineering intended toward the production of crocins will establish N. glauca as a potential industrial host for the production of these apocarotenoids. N. glauca is not only a better system for crocin biosynthesis and accumulation than N. tabacum but also a nicotine-free species and perennial plant from which the leafy biomass can be harvested repeatedly for crocin production.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

CZ, OA, PC, and LG-G conceived, designed the research, and wrote the manuscript. CZ, OA, XH, AR-M, TC, and LG-G conducted the experiments. CZ, OA, XH, AR-M, TC, PC, and LG-G analyzed the data. All authors read and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.