Phenotypic heterogeneity in bioprocesses is suspected to reduce performances, even in case of monoclonal cultures. Here, robustness of an engineered isopropanol-overproducing strain and heterogeneity of its plasmid expression level were evaluated in fed-batch cultures. Previously, eGFP was identified as a promising plasmid expression reporter for C. necator. Here, the behavior of 3 engineered strains (isopropanol overproducer, eGFP producer, and isopropanol/eGFP co-producers) was compared at the single-cell and population levels. Production yields and rates have been shown to be dependent on isopropanol/acetone tolerance. A link could be established between the variations in the fluorescence intensity distribution and isopropanol / acetone production using the eGFP-biosensor. Co-production of isopropanol and eGFP exhibited cumulative metabolic burden compared to single overexpression (isopropanol or eGFP). Expression of eGFP during isopropanol production resulted in lower isopropanol tolerance with a loss of membrane integrity resulting in protein leakage and reduced plasmid expression. The co-expression of heterologous isopropanol pathway and eGFP-biosensor enabled to demonstrate the heterogeneity of robustness and plasmid expression at the single cell level of C. necator. It highlighed the conflicting interactions between isopropanol overproduction and eGFP reporter system. Fluorescent reporter strains, a crucial tool for monitoring subpopulation heterogeneity although biases have to be considered. •Co-expression of heterologous isopropanol pathway and eGFP-biosensor to evaluate plasmid expression stability at single-cell level.•The co-production of eGFP and Isopropanol impacts strain robustness and performances comparing to the single overexpression (Isopropanol or eGFP).•Increased cell permeabilization and plasmid loss with increasing isopropanol and acetone concentration.