Mechanism-guided control of intracellular alkalinization and process pH for production of recombinant human B-type natriuretic peptide under the cSAT scheme in Escherichia coli

Tag-based high-load expression in Escherichia coli can be accompanied by substantial drift of broth and intracellular pH, but mechanistic links between intracellular alkalinization, metabolism and process levers remain unclear. Using a cleavable self-aggregating tag (cSAT) to produce recombinant human B-type natriuretic peptide (rhBNP), we combine broth pH tracking, AF-C ratiometric intracellular pH imaging and multi-omics, which together suggest that ammoniagenic amino-acid catabolism, glyoxylate-centred carbon rerouting and respiratory shifts are major contributors to intracellular alkalinization. Guided by this proton-economy model, stepwise medium engineering (complex nitrogen, phosphate, glucose and ammonium sulfate with a tuned carbon to nitrogen ratio) lowers shake-flask broth pH from values above 8.5 to about 7.0, increases titers 2.9-fold over LB medium to 115.4 mg/L and, in 3-L fed-batch, yields 662.1 mg/L rhBNP while maintaining biomass. Proton-economy-based process design stabilizes pH and productivity during production of rhBNP under the cSAT scheme in E. coli.