Metabolically engineered plant cell cultures as biofactories for the production of high-value carotenoids astaxanthin and canthaxanthin

Abstract

Astaxanthin and canthaxanthin are high-value carotenoids with growing demand due to their antioxidant properties and applications in food, cosmetic, and pharmaceutical sectors. However, natural sources are limited and current production methods are often costly or unsustainable. In this study, we developed a plant-based platform for ketocarotenoid biosynthesis using metabolically engineered Nicotiana tabacum BY-2 cell suspension cultures. Specifically, we expressed a marine bacterial crtW gene (β-carotene ketolase) alone or in combination with overexpressed plant psy (phytoene synthase) and crtI (phytoene desaturase) genes. The resulting cell lines displayed visually distinct pigmentation and accumulated different ketocarotenoid profiles based on their genetic modifications. Single-gene transformants expressing crtW produced up to 50 µg g⁻¹ DW of canthaxanthin and 127 µg g⁻¹ DW of astaxanthin. Co-expression of all three genes significantly increased canthaxanthin accumulation to 788 µg g⁻¹ DW. Our results establish suspended undifferentiated plant cells as a scalable and sustainable system for ketocarotenoid production, offering a biological alternative to natural producers and chemical synthesis.

Keywords: Nicotiana tabacum BY-2 non-photosynthetic cells; Metabolic engineering; Molecular farming; Secondary metabolites; Sustainable bioproduction; Synthetic biology.

Fig. 1

Schematic overview of the carotenoid biosynthetic pathway in plants. The isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) building blocks can be produced from the methylerythritol phosphate (MEP) cytoplasmic and mevalonate (MVA) plastid pathways. The grey boxes indicate the main carotenoids and derivatives found in plant systems, while the red boxes indicate the heterologous ketocarotenoid pathway. Abbreviations of enzymes: GGPS (geranylgeranyl pyrophosphate synthase), PSY (phytoene synthase, CRTB for bacteria), PDS (phytoene desaturase, CRTI for bacteria), ZDS (ζ-carotene desaturase, CRTI for bacteria), Z-ISO (ζ-carotene isomerase, CRTI for bacteria), CRTISO (carotenoid isomerase, CRTI for bacteria), LCYE (lycopene ε-cyclase), LCYB (lycopene β-cyclase, CRTY for bacteria), CYP (cytochrome P450 enzyme), CHYB (β-carotene hydroxylase, BHY for algae, CRTZ for bacteria), CRTW (β-carotene ketolase, BKT for algae), VDE (violaxanthin de-epoxidase), ZEP (zeaxanthin epoxidase), NSY (neoxanthin synthase). This visual representation shows the stepwise process involved in ketocarotenoid synthesis.

Fig. 2

Schematic representation of the T-DNA cassette of (a) construct pY, (b) construct pI and (c) construct pW used for the transformation of BY-2 cells. Regions are represented as follows: left border (LB), nopaline synthase terminator (tNOS), kanamycin resistance marker (NeoR/KanR), nopaline synthase promoter (pNOS), matrix attachment regions (MAR), Cauliflower Mosaic Virus 35 S promoter (CaMV 35 S), site-specific recombination site (attB1 and attB2), transit peptide (TP), phytoene synthase coding sequence (AY324431), phytoene desaturase coding sequence (D90087), β-carotene ketolase coding sequence, 35 S terminator (T35S), right border (RB). All dark lines are nucleotide sequences without relevant features.

Fig. 3

Phenotype of Nicotiana tabacum BY-2 cell lines compared to the wild-type. (a) Classification of BY-2 cell lines containing the heterologous psy, crtI, and/or crtW genes resulting from the single and combinatorial transformations with the pY, pI, and/or pW vectors. (b) Pale, yellow or pink-orange pigmented tobacco BY-2 calli. (c) BY-2 cultured cells, the WT and the highest yielding cell lines for the production of xanthophylls, astaxanthin, and canthaxanthin.

Fig. 4

Carotenoid profile of tobacco BY-2 wild-type (WT) and the highest ketocarotenoid-producing line (YIW135). The resulting chromatograms were recorded at a wavelength of 445 nm, with the y-axis scaled to the highest peak to enhance clarity. The carotenoids identified were putative neoxanthin (*Neo), putative violaxanthin (*Vio), astaxanthin (Astx), canthaxanthin (Cx) and β-carotene (β-car).

Fig. 5

Carotenoid content in tobacco BY-2 cell lines is shown as µg g⁻¹ DW (micrograms per gram of dry weight). Quantification of (a) astaxanthin and (b) canthaxanthin in ketocarotenoid producing cell lines. Quantification of β-carotene in (c) xanthophyll and (d) ketocarotenoid producing cell lines. The presence (+) or absence (-) of the heterologous genes is indicated for each line. Each data point represents the mean result derived from three biological replicates from the HPLC run, with error bars indicating the standard deviation (SD) for each measurement. Different letters above the bars indicate statistically significant differences (p value (p) < 0.05), determined by one-way ANOVA followed by Tukey’s test with a single pooled variance.