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Background <p>Tag-based high-load expression in <i>Escherichia coli</i> can be accompanied by substantial drift of broth and intracellular pH, but mechanistic links between intracellular alkalinization, metabolism and process levers remain unclear.</p> Results <p>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&#xa0;mg/L and, in 3-L fed-batch, yields 662.1&#xa0;mg/L rhBNP while maintaining biomass.</p> Conclusions <p>Proton-economy-based process design stabilizes pH and productivity during production of rhBNP under the cSAT scheme in <i>E. coli</i>.</p>

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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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年2月1日

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  • Hang Nie,
  • Yajun Feng,
  • Guanghui Sun,
  • Haijie Li,
  • Jingwen Kang,
  • Yanqing Zhang,
  • Chenhui Zhu,
  • Yu Mi,
  • Yuan Guo,
  • Pei Ma

摘要

Background

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.

Results

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.

Conclusions

Proton-economy-based process design stabilizes pH and productivity during production of rhBNP under the cSAT scheme in E. coli.