Expressing <i>CaCCS</i> encoding capsanthin/capsorubin synthase in tomato increases fruit provitamin A content and yield

We developed a tomato (Solanum lycopersicum) biofortification strategy by engineering the simultaneous accumulation of β-carotene (provitamin A) and the pepper (Capsicum annuum)-specific ketocarotenoids capsanthin and capsorubin. Capsanthin/capsorubin synthase (CaCCS), an enzyme related to lycopene β-cyclase (LCYB) known for β-carotene biosynthesis, was investigated for its in vivo role in ripening pepper fruit (C. annuum). Silencing CaCCS by virus-induced gene silencing reduced flux from lycopene to β-carotene (the β-branch), altering carotenoid profiles and expression of carotenogenic genes. In a bacterial carotenogenic system, CaCCS was more effective than tomato LCYB1/2 in cyclizing lycopene to β-carotene and uniquely synthesized capsanthin. To evaluate its potential in tomato, we constitutively expressed CaCCS in "Micro-Tom" WT and the mutant pyp1-1(H7L), which is defective in xanthophyll esterification. CaCCS-transformed fruits accumulated higher levels of capsanthin, capsorubin, total carotenoids, β-carotene, and xanthophyll esters than controls, with weaker increases in pyp1-1(H7L). These results indicated that CaCCS enhanced flux through the β-branch and promoted xanthophyll ester formation, consistent with dual roles in lycopene cyclization and ketocarotenoid biosynthesis. Comparative analyses suggested that xanthophyll esterification facilitated total carotenoid accumulation. Beyond carotenoids, CaCCS expression increased fruit yield, β-carotene-derived volatiles, free amino acids, phenolics, and ascorbic acid, with a decline in reducing sugars. CaCCS was introduced into selected inbreds using controlled crosses to obtain CaCCS-derived hybrids, the fruit of which exhibited high provitamin A levels. Consumption of 37 to 131 g of such fruit could meet the recommended dietary allowance for provitamin A. These results establish CaCCS overexpression as a powerful metabolic engineering strategy to enhance provitamin A and enrich value-added phytochemicals in tomato.