The Biosynthesis of Non-Endogenous Apocarotenoids in Transgenic Nicotiana glauca

Abstract

Crocins are high-value compounds with industrial and food applications. Saffron is currently the main source of these soluble pigments, but its high market price hinders its use by sectors, such as pharmaceutics. Enzymes involved in the production of these compounds have been identified in saffron, Buddleja, and gardenia. In this study, the enzyme from Buddleja, BdCCD4.1, was constitutively expressed in Nicotiana glauca, a tobacco species with carotenoid-pigmented petals. The transgenic lines produced significant levels of crocins in their leaves and petals. However, the accumulation of crocins was, in general, higher in the leaves than in the petals, reaching almost 302 µg/g DW. The production of crocins was associated with decreased levels of endogenous carotenoids, mainly β-carotene. The stability of crocins in leaf and petal tissues was evaluated after three years of storage, showing an average reduction of 58.06 ± 2.20% in the petals, and 78.37 ± 5.08% in the leaves. This study illustrates the use of BdCCD4.1 as an effective tool for crocin production in N. glauca and how the tissue has an important impact on the stability of produced high-value metabolites during storage.

Skin Protection by Carotenoid Pigments

“…Examples include crocin and apocarotenoids, which are responsible for yellow, orange, and red colors. The main source of these pigments is precious saffron; therefore, genetic engineering methods are used to increase their accumulation in plants [385]. However, apocarotenoids are beyond the scope of this review.…”

Research Progress in Heterologous Crocin Production

“…It may be a new direction to use genetic engineering technology to transform the key enzymes of the synthetic pathway of crocetin to produce crocetin in species with a high yield of β-carotene or the potential to synthesize β-carotene or crocetin (Figure 4). According to available reports, many microorganisms have been successfully transformed to synthesize crocetin or crocetin, including E. coli [7], yeast [62], Nicotiana glauca [108], Chlorella vulgaris [109], and Dunaliella salina [104]. Recently, the transient transformation of N. benthamiana to synthesize crocins was reported [102].…”

“…A related species, N. glauca, contains carotenoid pigments in its petals. Huang et al [108] expressed the Bd-CCD4.1 enzyme from B. davidii constitutively in its petals and leaves and obtained 321.6 ± 21.3 µg/g and 302.7 ± 25.6 µg/g DCW crocin, respectively. Interestingly, in their transgenic lines expressing CsCCD2L, the difference in the accumulation of crocin between leaves and petals may have been due to the relatively higher accumulation of zeaxanthin or the tissue specificity of CCD in leaves [108].…”

“…Huang et al [108] expressed the Bd-CCD4.1 enzyme from B. davidii constitutively in its petals and leaves and obtained 321.6 ± 21.3 µg/g and 302.7 ± 25.6 µg/g DCW crocin, respectively. Interestingly, in their transgenic lines expressing CsCCD2L, the difference in the accumulation of crocin between leaves and petals may have been due to the relatively higher accumulation of zeaxanthin or the tissue specificity of CCD in leaves [108]. Martí et al used tobacco etch virus to drive the expression of Cs-CCD2L and Bd-CCD4.1 in N. benthamiana and found that only Cs-CCD2L could produce 2.18 ± 0.23 mg of crocins and 8.24 ± 2.93 mg of picrocrocin per gram (DCW) over 13 days.…”

Production of Saffron Apocarotenoids in Nicotiana benthamiana Plants Genome-Edited to Accumulate Zeaxanthin Precursor

“…Interestingly, expression in planta of the above CCDs is sufficient for crocin accumulation, suggesting that the ALDH and UGT activities are endogenously expressed in plant tissues. Heterologous expression systems include maize endosperm [ 3 ], rice and citrus calli [ 12 , 13 ], Nicotiana benthamiana leaves [ 7 , 11 , 14 ], transplastomic tobacco leaves [ 11 ], N. tabacum and N. glauca leaves [ 15 , 16 ], and transgenic tomato fruits [ 11 , 17 ]. Since many of these tissues contain a variety of different carotenoids, but very low to null levels of zeaxanthin, the question arises about which are the in planta carotenoid substrates of CCDs with narrow substrate specificity, such as CsCCD2.…”