Metabolic impact of heterologous protein production inPseudomonas putida: Insights into carbon and energy flux control

Abstract

AbstractFor engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism ofPseudomonas putidaat different levels of metabolic load. To this end, we constructed aP. putidaKT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature ofP. putida’s central metabolic network is solicited to sustain energy production.HighlightsHeterologous protein production inP. putidareshuffles the periplasmic metabolism.Increased protein production progressively decouples catabolism from anabolism.Protein production exerts a stronger control on energy than on carbon fluxes.Glucose is directed towards ATP production to meet the elevated energy demands.