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.

A growing global population and the increasing prevalence of diet-related health issues such as “hidden hunger”, obesity, hypertension, and diabetes necessitate a fundamental rethinking of crop design and breeding. Synthetic metabolic engineering offers a method to modify and redesign metabolic pathways to increase the nutritional value of crops. We summarize recent advances in the biofortification of key nutrients including provitamin A, vitamin C, vitamin B9, iron, zinc, anthocyanins, flavonoids, and unsaturated fatty acids. We discuss the potential of multi-gene stacking, gene editing, enzyme engineering, and artificial intelligence in synthetic metabolic engineering. We propose future research directions and potential solutions centered on leveraging AI-driven systems biology, precision gene editing, enzyme engineering, agrobacterium-mediated genotype-independent transformation, and modular metabolic engineering strategies to develop next-generation nutritionally enhanced super crops and transform global food systems.

The canonical CRISPR/Cas editors are constrained by the requirement for specific PAMs, which substantially limits their editable target range. Rice (particularly indica rice) is sensitive to low temperature, which impacts the yield and restricts the geographic distribution of rice. In this study, we developed PAM-flexible multiplex genome editing tools based on SpG (recognising NGN-PAMs) and SpRY (recognising NNN-PAMs) variants. We then tested the feasibility of using a sweet potato leaf curl virus (SPLCV) replicon-based expression vector and single-stranded DNA-binding domain (DBD) to improve the editing efficiency of these PAM-flexible editors. Furthermore, we used SpG-mediated multiplex genome editing to achieve comprehensive improvement in cold tolerance in indica rice by editing WRKY transcription factors OsWRKY53 and OsWRKY63, to generate high cold-resistant indica rice lines. We concluded that these PAM-flexible multiplex genome editors are powerful tools for multi-gene editing for crop genetic improvement.