Development of eRUBY maize with betalain-enriched endosperm using a push-and-pull synthetic metabolic engineering strategy

Betalain, an economically valuable water-soluble natural plant pigment, is prized for its strong antioxidant activity, making it popular as a dietary supplement and a visual marker for plant transformation. However, market demand significantly outstrips current production capacity. This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize (Zea mays L.) endosperm. We engineered a novel enhanced RUBY (eRUBY) system derived from the original polycistronic RUBY construct (CYP76AD1_P2ADODA1_P2ADOPA5GT unit, abbreviated CDG) by introducing arogenate dehydrogenase (ADHα) to increase the L-tyrosine substrate supply. All the genes were driven by the endosperm-specific promoter. Fusion of ADHα into a single polycistronic eRUBY construct (CDGA) produced significantly higher betanin (6.88 mg g⁻¹ dry weight) and isobetanin (1.81 mg g⁻¹ dry weight) levels than in CDG + A, which stacked the ADHα cassette independently with CDG. The high betalain accumulation in CDGA lines (which also exhibited higher transgene copy number) resulted in a 2.85–7.58-fold improvement in endosperm antioxidant capacity compared to WT (versus 2.48–2.80-fold in CDG + A). Importantly, transgenic plants maintained a normal phenotype. Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine, alanine, aspartate, and glutamate contributes to betalain production. Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety. Collectively, these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation.