Investigation of population dynamics in CO2-fixing bacteria associates plasmid expression loss to the expression of the genes of a heterologous production pathway

Introducing heterologous pathways into industrial hosts enables sustainable, cost-effective production of renewable chemicals. Plasmid-based expression ensures higher gene copy numbers than genomic integration and, greater production potential. However, imperfect plasmid inheritance makes the evaluation of production robustness essential for determining process viability. Stable strains are critical for maintaining consistent production, prolonging cultivations, ensuring reproducible titers, and minimizing byproduct formation. In this study we investigated plasmid expression stability in engineered C. necator Re2133 under plasmid-based expression of a heterologous isopropanol pathway. Through flow cytometry detecting plasmid-encoded eGFP, we monitored population dynamics of plasmid-based expression at single-cell level. Serial flask experiments revealed that the induction of the isopropanol pathways genes led to loss of plasmid-expressing, and potentially, plasmid-bearing cells within the culture. This instability was not caused by stationary phase or nutrient starvation. Additionally, although external isopropanol concentrations above 20 g.L-1 impaired growth, expression stability remained stable. Variations in carbon-to-nitrogen ratios showed no effect on expression stability, suggesting that intracellular isopropanol levels do not impact expression stability. Instead, we have found that the reduced expression stability was caused by increased metabolic burden on the cell through isopropanol production. Our work highlights the importance of single-cell evaluation for identifying production loss and engineering robust strains.