Microbial stem cells support productivity in dedicated factory cells in an asymmetrically dividing E. coli system

ABSTRACT A major challenge for many bio-manufacturing operations is that cells are burdened by the high fitness cost of product synthesis, limiting their growth and productivity. A potential solution is to decouple cell reproduction from product synthesis by dividing these conflicting tasks between two differentiated cell types. This work describes the use of an asymmetrically inherited protein cue to differentiate an E. coli culture into reproductive stem cells and fully dedicated factory cells. Cell differentiation is based on the ability to accumulate two factors: a variant of phage-derived T7 RNA polymerase (T7RNAP) and GP2, a peptide that inhibits native host cell RNA polymerase. Activating these two factors in factory cells inhibited growth and focused them on T7RNAP-driven product synthesis. Preventing their accumulation in stem cells allowed this cell type to grow and divide asymmetrically, generating new factory cells in the process. These differentiating cell cultures generated over eight-fold higher target protein titers compared to factory cell-only controls. Because they include a mechanism for preventing leaky T7RNAP-driven gene expression in stem cells and pre-induction cultures, it was possible to generate strains with multiple plasmid-based copies of a cytotoxic target gene, whereas leaky expression made the same plasmid inviable in conventional protein expression strains. This versatile genetic system could be useful for generating higher product titers, particularly in cases where product synthesis causes cytotoxicity.