Metabolic engineering of apple callus and tobacco leaf for astaxanthin production

Astaxanthin, a ketocarotenoid with superior antioxidant properties compared with other carotenoids, is increasingly demanded in feed, food, and pharmaceutical industries. This study aimed to engineer apple ( Malus domestica Borkh.) callus and tobacco ( Nicotiana benthamiana Domin) leaf for sustainable production of natural (3S,3'S)-astaxanthin. A plant expression vector, pRI101-Flag-BHY-BKT-PSY1-CrtI (BBPC), was constructed to deliver astaxanthin biosynthetic genes PSY1 , CrtI , BHY and BKT genes into ‘Orin’ apple callus and N. benthamiana leaf. Transgenic apple calli synthesized 40.4 μg/g dry weight (DW) astaxanthin, representing 53.5 % of total carotenoids. Transiently transformed N. benthamiana leaves produced 732.8 μg/g DW astaxanthin,accounting for about 18.2 % of total carotenoids. Furthermore, supplementing tomato juice (20 mL/L) and carrot juice (30 mg/L) increased astaxanthin levels in calli by 1.7-fold. Moreover, astaxanthin-rich tissues exhibited 2–4 fold higher antioxidant activity ( P < 0.05) than controls. Acute toxicity assays in mice revealed no adverse effects. These findings demonstrate the potential of engineered apple callus and N. benthamiana leaf as scalable sources of natural (3S,3'S)-astaxanthin for industrial applications. Engineered apple callus and tobacco leaf as scalable sources of natural (3S,3'S)-astaxanthin for industrial applications. • Astaxanthin-rich materials were generated by overexpression of biosynthetic genes. • Astaxanthin-rich calli and leaves show a stronger antioxidant activity. • Astaxanthin-rich materials produce natural high-bioactive (3S,3’S)-astaxanthin. • Astaxanthin-rich apple calli exhibit no acute toxicity in mice. • Tomato juice and carrot juice consumption increase astaxanthin levels.