Dissecting host stress responses for predictable heterologous gene expression in E. coli

Predictable expression of heterologous genes remains a key challenge in biotechnology, largely due to cellular stresses imposed on the production host. Here, we systematically dissect stress responses in Escherichia coli MG1655 expressing diverse proteins under varying promoters and translation efficiencies. Using independent component analysis on new and existing large transcriptomic datasets, we identify distinct responses to transcriptional and translational stresses: excessive heterologous messenger RNA (mRNA) triggers a cold shock response that controls mRNA stability (cspA-I, deaD), while protein production activates a heat shock response involving proteolysis and chaperone functions. We further identify a broad adaptation response, consistently co-activated with the heat shock response during protein production, that provides stationary phase regulation (rspAB) and osmoregulation (betABIT). Targeting these latter functions through strain and media modifications significantly increases eGFP production. Other host stress responses depend on the protein being expressed; e.g. we find production of cysteine-rich proteins to uniquely activate functions regulating iron- and redox homeostasis and oxidative stress responses. This work demonstrates a holistic, systems-level view of cellular stresses to heterologous gene expression by considering transcriptional, translational, and product-specific contributions, paving the way toward predictable and optimized expression strategies.