Achieving soluble expression of disulfide bond-containing recombinant proteins in Escherichia coli is challenging. While strategies such as low post-induction temperature, fusion tags, and engineered strains have been employed to achieve soluble protein expression, their specific effects on E. coli metabolism and, crucially, how these metabolic shifts relate to the challenge of achieving soluble protein expression remain unclear. Here, we performed untargeted metabolomics to study the key metabolic changes associated with co-expression of fusion tags in E. coli strains at low and high cultivation temperatures. Using a mass spectrometry-based approach, we identified 121 differentially abundant metabolites. The metabolomes of BL21 (DE3) and SHuffle strains exhibited distinct intracellular pools of amino acids and redox regulators. We further studied the expression of platelet-derived growth factor (PDGF) as a model disulfide-rich protein that generally tends to aggregate when expressed in E. coli. A lower induction temperature and the addition of a thioredoxin tag were observed to be crucial for enhancing soluble expression of PDGF in both strains. However, SHuffle showed heightened metabolic stress during PDGF production compared to BL21. Soluble PDGF expression was associated with higher levels of peptides, nucleotides, and glycolysis and TCA cycle intermediates, while PDGF expression as inclusion bodies was associated with higher levels of amino acids, nucleobases, and pentose phosphate pathway intermediates. Our findings highlight the potential of LCMS-based metabolic profiling in understanding the capacities of different host strains for protein processing and guiding metabolic engineering for improved recombinant protein folding and expression in E. coli.
Keywords: E. coli BL21 (DE3); LCMS; PDGF; SHuffle; Thioredoxin tag; Untargeted metabolomics.
© 2025. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.