Lycopene pathway rewiring by combinatorial co-expression of constituent enzymes in a cell-free protein synthesis...

We established a platform for reconstructing the lycopene pathway by combinatorial co-expression of five constituent enzymes using a crude Escherichia coli cell-free protein synthesis (CFPS) system. DNA templates were prepared in a modular format, and their concentrations were used as quantitative inputs to manipulate expression levels. Co-expression from mixed templates reconstructed a functional lycopene pathway directly in vitro and replaced the conventional mix-and-match of separately expressed enzymes. Using a plate-based colorimetric assay, we showed that lycopene production is highly sensitive to both enzyme identity and template dosage, reflecting resource competition, endogenous activities in the lysate, and protein solubility. One-factor-at-a-time and homolog-swap experiments revealed basic design rules but also emphasized that the results were small and biased compared to possible co-expression design space. To explore this space more efficiently, we combined CFPS experiments with a hybrid active learning (HAL) workflow that ranks candidate DNA combinations based on model-guided criteria. HAL-guided rounds suggested high-performing and even simplified lycopene pathway configurations, indicating that CFPS co-expression together with HAL can serve as a practical platform to design and refine metabolic pathways before refactoring them into cells.