Heterologous expression of Bixa orellana cleavage dioxygenase 4-3 drives crocin but not bixin biosynthesis

Abstract

Annatto (Bixa orellana) is a perennial shrub native to the Americas, and bixin, derived from its seeds, is a methoxylated apocarotenoid used as a food and cosmetic colorant. Two previous reports claimed to have isolated the carotenoid cleavage dioxygenase (CCD) responsible for the production of the putative precursor of bixin, the C24 apocarotenal bixin dialdehyde. We re-assessed the activity of six Bixa CCDs and found that none of them produced substantial amounts of bixin dialdehyde in Escherichia coli. Unexpectedly, BoCCD4-3 cleaved different carotenoids (lycopene, β-carotene, and zeaxanthin) to yield the C20 apocarotenal crocetin dialdehyde, the known precursor of crocins, which are glycosylated apocarotenoids accumulated in saffron stigmas. BoCCD4-3 lacks a recognizable transit peptide but localized to plastids, the main site of carotenoid accumulation in plant cells. Expression of BoCCD4-3 in Nicotiana benthamiana leaves (transient expression), tobacco (Nicotiana tabacum) leaves (chloroplast transformation, under the control of a synthetic riboswitch), and in conjunction with a saffron crocetin glycosyl transferase, in tomato (Solanum lycopersicum) fruits (nuclear transformation) led to high levels of crocin accumulation, reaching the highest levels (>100 µg/g dry weight) in tomato fruits, which also showed a crocin profile similar to that found in saffron, with highly glycosylated crocins as major compounds. Thus, while the bixin biosynthesis pathway remains unresolved, BoCCD4-3 can be used for the metabolic engineering of crocins in a wide range of different plant tissues.

Figure 1

Apocarotenoid biosynthesis pathways in different plant species. A, Crocin biosynthesis pathways in C. sativus, B. davidii, and G. jasminoides. B, Proposed bixin biosynthesis pathway in B. orellana. C, Maximum-likelihood phylogenetic tree of CCDs from A. thaliana (At), B. orellana (Bo), B. davidii (Bd), C. clementina (Cc), C. sativus (Cs), C. ancyrensis (Ca), G. jasminoides (Gj), S. lycopersicum (Sl), O. sativa (Os), and Synechocystis sp. (Syn). The bootstrap consensus tree inferred from 500 replicates is shown. The percentage of replicate trees in which the associated CCDs clustered together in the bootstrap test is shown next to the branches and branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The carotenoid substrates, position of cleavage, and bootstrap values are indicated. Protein sequences are shown in Supplemental Figure S1.

Figure 2

Lack of bixin dialdehyde production in bacterio by different BoCCDs. HPLC–PDA–HRMS profiles of E. coli cells producing lycopene (L) and expressing various Bixa carotenoid dioxygenases, induced for 16 h at 20°C with arabinose (0.2%, w/v). Cells expressing the pTHIO-DAN empty vector are indicated with C-. A, Maximum absorbance UV–Vis chromatogram in the 250–700 nm range. Online spectra of the main peaks (L, lycopene and CD?, putative crocetin dialdehyde) are shown in the insets. µAU, Micro-absorbance units. B, Ion chromatograms of lycopene (L, M+H+: 537.4442 ± 2 ppm) extracted with the Xcalibur Software (Thermo Fisher Scientific). C, Ion chromatograms of Bixin Dialdehyde (BD, M+H+: 349.2162 ± 2 ppm) were extracted with the Xcalibur Software (Thermo Fisher Scientific).

Figure 3

BoCCD4-3 cleaves completely lycopene, b-carotene, and zeaxanthin to produce crocetin dialdehyde in E. coli. A–C, LC–HRMS chromatograms of E. coli extracts accumulating lycopene (A), β-carotene (B), and zeaxanthin (C) after induction with 0.2% of arabinose (w/v) for 16 h at 20°C. The ion of crocetin dialdehyde CD (M+H)+: 297.1846 was extracted. Cells expressing BoCCD4-3 accumulate a compound that has both the accurate mass and the chromatographic mobility of the crocetin dialdehyde standard. D, MS spectrum and (inset) MS/MS spectrum of the crocetin dialdehyde peak produced by E. coli cells expressing BoCCD4-3. E, Cleavage reactions catalyzed by BoCCD4-3.

Figure 4

Subcellular localization of BoCCD4-3 in N. benthamiana leaves. A, Confocal images of N. benthamiana leaves expressing (top to bottom): GFP, BoCCD4c:GFP, TP:BoCCD4c:GFP (BoCCD4c fused to the CsCCD2 transit peptide), and CsCCD2:GFP fusion proteins. Red (chlorophyll fluorescence), green (GFP fluorescence), and merged (overlap of chlorophyll and GFP fluorescence) are shown. The unfused GFP protein shows the typical cytoplasmic and nuclear localization. Both BoCCD4-3:GFP and CsCCD2:GFP localize to plastids. Scale bars: 7 μm. Arrows in the BoCCD4c:GFP composite image point at green fluorescent protrusions of the plastid stroma (stromules). B, 3D reconstruction of red and green fluorescence in plastids expressing BoCCD4-3:GFP, TP:BoCCD4-3:GFP, and CsCCD2:GFP. The latter localizes to plastid-associated speckles (Demurtas et al., 2018). Scale bars: 7 μm.

Figure 5

Crocetin/crocin production in N. benthamiana leaves transiently expressing CsCCD2 or BoCCD4-3. A, Representative LC–HRMS chromatograms of the extracted accurate mass of crocetin (M+H⁺ 329.1747), generated from crocin fragmentation, in leaves expressing BoCCD4-3, TP:BoCCD4-3, CsCCD2, and CsCCD2short (CsCCD2 lacking the TP) and C- (empty vector). B, Quantification of crocetin and crocins in agroinfiltrated leaves. Data are the avg ± sd of three independent pools of agroinfiltrated plants. See Supplemental Table S3 for quantitative data. HPLC–PDA chromatograms are shown in Supplemental Figure S6, A.

Figure 6

Crocetin/crocin production in transplastomic tobacco plants expressing BoCCD4-3. A, Visual phenotype of transplastomic lines #2 and #11 cultured in vitro. B, Quantitation of crocetin and crocins in young leaves from lines #2 and #11. Data are the avg ± sd of three biological replicates. See Supplemental Table S4 for quantitative data. HPLC–PDA chromatograms are shown in Supplemental Figure S6, B.

Figure 7

Crocetin/crocin production in transgenic Microtom fruits expressing TP:BoCCD4-3 and CsUGT74AD1. A, Phenotypes of Microtom T1 transgenic fruits expressing TP:BoCCD4-3 + CsUGT74AD1. B, Quantification of crocetin and crocins in ripe fruits. Data are the avg ± sd of three biological replicates (fruits) from each line. HPLC–PDA chromatograms are shown in Supplemental Figure S6, C.