Chassis Selection and Metabolic Fine-Tuning Enable Efficient <i>in planta</i> Betalain Production

Synthetic biology enables efficient production of valuable compounds in biological systems, including plants that capture atmospheric CO2 to synthesize and accumulate abundant and diverse specialized metabolites. Most plant synthetic biology studies to produce specialized metabolites have primarily used Nicotiana benthamiana as an underpinning metabolic chassis, due to its rapid agroinfiltration method, leaving much of the metabolic potential of other plant species underexplored. Here we engineered three distinct plant chassis-Arabidopsis, tobacco and soybean-by stably introducing an optimized betalain biosynthetic pathway and analyzed their metabolic impacts. Betalains are tyrosine-derived pigments, which are used as natural red and yellow food dyes with rapidly growing demand due to a recent regulatory shift. We fine-tuned metabolic balances by redesigning the RUBY betalain construct (RUBYv2), adding an extra DODA enzyme ("pull") and modulating tyrosine precursor supply ("push") using two different promoters. The "push-and-pull (push+pull)" lines produced higher betalain levels than the "pull" lines in all three species, even exceeding those of beet roots. While Arabidopsis and tobacco "push+pull" lines driven by a strong promoter showed dwarfism, corresponding soybean lines did not show severe growth defects, suggesting greater tolerance of soybean to the engineered pathway. This study demonstrates that careful plant chassis selection, coupled with precise control of pathway expression, is essential for maximizing the yield of target specialized metabolites, such as betalain pigments, without impairing overall plant growth.