Ensemble and iterative engineering for maximal 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. Strong growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods however require multi-gene edits for implementation which may be initially perceived as impractical. Here, we evaluated 4,114 potential solutions to pair growth on para-coumarate to indigoidine production and prototype two cutsets in Pseudomonas putida KT2440. The implemented design was enhanced using adaptive lab evolution and the resulting strains were characterized for emergent phenotypes. Using x-ray tomography we revealed increased cell density in this strain. Proteomics identified two upregulated peroxidases that mitigate increased reactive oxygen species formation. Nine additional stepwise modifications informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% yield in minimal salt media. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields.