Iterative precursor rebalancing improves phenazine-1-carboxylic acid production in engineered Pseudomonas...

Imbalanced precursor supply in nonlinear metabolic pathways limits flux toward target compounds. The shikimate pathway illustrates this challenge, as intracellular phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) concentrations typically differ by ca. tenfold, constraining production of shikimate-derived compounds. Here, we implemented a two-round Design-Build-Test-Learn (DBTL) framework to rebalance multi-precursor supply and improve shikimate-derived biosynthesis. Phenazine-1-carboxylic acid (PCA) production by the non-pathogenic soil bacterium Pseudomonas chlororaphis was used as a model system. The first DBTL cycle showed that E4P is mainly supplied through the non-oxidative pentose phosphate pathway and that an EDEMP cycle operates during glycerol catabolism, guiding interventions to rebalance precursor flux. The second DBTL cycle showed that P. chlororaphis can co-consume glycerol and glucose, enabling a synergistic co-feeding strategy. This strategy doubled PCA production and reduced the intracellular PEP/E4P ratio by ca. threefold, confirming improved precursor balance. A single fed-batch fermentation strategy further increased PCA titer to 3.6 g/L, a 4.2-fold improvement over the parental strain. This work demonstrates how iterative DBTL-guided flux rebalancing can alleviate multi-precursor imbalance in nonlinear pathways.