# Escherichia coli BL21 adapts its central carbon metabolism to recombinant protein production and oxygen limitation

## TL;DR

This study explores how E. coli BL21 adapts its metabolism under stress from protein production and low oxygen, revealing how it maintains stability.

## Contribution

The study reveals how E. coli BL21 adapts its central metabolism to both recombinant protein production and oxygen limitation.

## Key findings

- Strains with higher plasmid copy numbers showed more pronounced growth retardation and metabolic changes.
- Oxygen limitation caused lower metabolic activity but maintained energy and redox balance without affecting mCherry production.
- Both strains showed similar responses to oxygen limitation, with significant central metabolite pool adaptations.

## Abstract

High-yielding recombinant protein expression systems often face challenges due to the metabolic burden caused by the competition for cellular resources, resulting in reduced growth and, hence, limiting their industrial applicability. Furthermore, industrial recombinant protein production is also affected by the occurrence of oxygen gradients, which is a prevalent issue in large-scale bioreactors. These gradients create a heterogeneous environment in the bioreactor, which affects cell growth and metabolism, having severe consequences on the process performance. Both these factors alter cellular physiology and metabolism, thereby affecting recombinant protein yields. Understanding metabolic adaptations to these stress conditions is crucial for uncovering the underlying cellular mechanisms, which can direct further optimization of the recombinant strains. In this study, we aimed to explore the combined response of the central metabolism of Escherichia coli to metabolic burden and microaerobic conditions. Two recombinant protein-producing E. coli BL21 strains carrying XylS/Pm vectors with low (A2-mCh) and medium plasmid copy numbers (A3-mCh), and producing mCherry protein, were studied by introducing oxygen limitation. Central metabolite pools were analyzed by three targeted LC–MS/MS methods, using the isotope dilution strategy for absolute quantification.

Both recombinant strains exhibited different levels of metabolic burden, with the strain possessing a higher plasmid copy number showing more pronounced growth retardation and a stronger impact on metabolite pools. Both strains, however, showed a similar response to oxygen limitation, with significant adaptations in the central metabolite pools. The low plasmid copy number strain showed an increase in the concentration of lower glycolytic and tricarboxylic acid cycle metabolites, while the pools of upper glycolytic and pentose phosphate pathways and nucleoside phosphates were mostly unaffected. However, a more extreme response was seen in A3-mCh, where the majority of the metabolite pools were increased. Oxygen limitation caused lower metabolic activity, but the energy charge and redox balance were maintained, and no negative effect was observed on mCherry production rates.

This study provides insights into metabolic adaptations in E. coli BL21 recombinant strains, having quite robust mechanisms to maintain intracellular metabolic homeostasis during both internal and external perturbations.

The online version contains supplementary material available at 10.1186/s12934-026-02924-z.