Characterization of stress responses in pseudomonas putida using plasmid-based fluorescent reporter systems

Abstract

The rising interest in implementing microbial biosynthesis strategies for production of high-value compounds, as an alternative to traditional chemical manufacturing, has facilitated the development of microbial bioengineering. However, numerous valuable target substrates and products, such as C1 compounds and terpenoid-related molecules, exhibit considerable toxicity to microbial hosts, which limits their industrial application. Consequently, for the construction of more reliable strains the stress response pathways are aimed to be thoroughly studied. Nonetheless, well-characterized stress response reporter systems remain scarce for the recently emerged Pseudomonas putida as a model organism. This study aimed to evaluate the performance of engineered stress-response reporter constructs in P. putida and to investigate the stress responses induced by different valuable compounds. A series of fluorescence-based experiments was conducted in order to assess reporter systems’ performances in terms of sensitivity, response kinetics and strain stability during the chemical exposure to methanol, formate, isoprenol and lycopene. The results suggested that P. putida can tolerate relatively high concentrations of methanol, which mainly caused membrane and growth stress, formate that triggered mild stress from the DNA damage, and membrane and growth stress after the prolonged exposure, and lycopene, inducing the stress from the DNA integrity disruption. In contrast, even low concentrations of isoprenol induced moderate genotoxic effects and severe membrane and growth stresses.