Quantifying heterologous gene expression during ectopic MazF production in Escherichia coli

Abstract ObjectiveMazF is a sequence-specific endoribonuclease-toxin of the MazEF toxin-antitoxin system, which cleaves single-stranded RNA regions at ACA sequences in Escherichia coli. The MazEF system has been used in various biotechnology and synthetic biology applications. In this study, we infer how ectopic mazF overexpression affects production of heterologous proteins. To this end, we quantified the levels of fluorescent proteins expressed in E. coli from reporters translated from the ACA-containing or ACA-less mRNAs. Additionally, we addressed the impact of the 5’-untranslated region of these reporter mRNAs under the same conditions by comparing expression from mRNAs that comprise (canonical mRNA) or lack this region (leaderless mRNA).ResultsFlow cytometry analysis indicates that during mazF overexpression, fluorescent proteins are translated from the canonical mRNAs, as well as from the leaderless mRNAs. Our analysis further indicates that longer mazF overexpression generally increases the concentration of fluorescent proteins translated from ACA-less mRNAs, however it also substantially increases bacterial population heterogeneity. Finally, our results suggest that the strength and duration of mazF overexpression should be optimized for each experimental setup, to maximize the heterologous protein production and minimize the amount of phenotypic heterogeneity in bacterial populations, which is unfavorable in biotechnological processes.