Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

While many heterologous molecules can be produced at trace concentrations via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth coupling para -coumarate to indigoidine production and prototype two cut sets in Pseudomonas putida KT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth on p -CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY in para -coumarate minimal media. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields.