Effect of thioredoxin tag, oxidizing environment, and temperature on the global metabolome of E. coli strains

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 its relation to 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 obtaining soluble 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. These results have implications for enhancing strain performance and bioprocess optimization for producing “difficult-to-express” proteins in E. coli .