Gentamicin and d-serine selective pressure to improve the homogeneity of cells expressing target proteins

In this work, selective pressure was applied in E. coli to enrich the population expressing recombinant proteins and suppress the emergence of low-expression phenotypes. To this end, plasmids were constructed containing two reporter genes, a degradable green fluorescent protein (GFP) and a stable red fluorescent protein (RFP), positioned upstream of either a gentamicin acetyltransferase gene or a d-serine deaminase gene. Flow cytometry analysis revealed that gentamicin selection prevented the formation of bimodal populations and maintained predominantly homogeneous expression profiles, accompanied by up to 12-fold and 10-fold increases in RFP and GFP fluorescence, respectively. These results suggest that selective pressure favoured the enrichment and maintenance of highly expressing phenotypes while preserving operon functionality. Transcriptomic analysis indicated extensive physiological adaptation under gentamicin selection, including changes in translation-related functions and increased transcript abundance of the genes of interest. To establish an antibiotic-free strategy, d-serine was employed as an alternative selective agent. Detoxification of d-serine by d-serine deaminase similarly reduced population heterogeneity and resulted in predominantly single-expression populations with up to 4-fold and 6-fold higher RFP and GFP fluorescence, respectively. Furthermore, d-serine was evaluated as the sole nitrogen source, combining selective pressure with an auxotrophic strategy and yielding up to 6-fold and 15-fold increases in RFP and GFP fluorescence, respectively. These findings support the use of selective pressure to reduce recombinant expression heterogeneity and suppress low-expression subpopulations. Additionally, we highlighted the potential use of d-serine and similar toxic substrates to simultaneously function as selective agents, inducers, and sources of essential metabolites through detoxification.