Abstract In biotechnological applications, it is often necessary to introduce genes or entire pathways into a host cell, which can create a significant metabolic burden on the host, limiting productivity. In this study, we systematically investigated the physiological stress responses of Pseudomonas putida during heterologous protein production using a modular monitoring system consisting of a plasmid encoding a heterologous protein fused to eGFP and a chromosomally integrated capacity reporter. Our findings reveal that translation is the main bottleneck, with translational capacity becoming saturated under high expression loads. While increasing the strength of the RBS improved protein production for non-burdensome proteins, this effect was not observed for larger fusion proteins. Variations in fusion protein size suggested that translational demand, rather than the overall mass of protein produced, determines metabolic burden. We further evaluated how resource availability affects protein expression by modifying the metabolic regime or supplementing with amino acids. While the carbon source affected cellular capacity, it did not significantly alter heterologous protein production. Amino acid supplementation alleviated the growth defects of MBPeGFP-producing cells and modestly improved protein production rates. Together, these findings emphasize that metabolic burden is influenced not only by the size of the produced protein but also by transcript architecture, resource allocation, and the physiological state of the host. Therefore, successful optimization of heterologous protein production requires a holistic approach integrating construct design with host physiology and cultivation strategies.
Beentjes et al. (Thu,) studied this question.