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Abstract Maximizing protein secretion is an important target in the design of engineered living systems. In this paper, we characterize a trade-off between cell growth and per-cell protein secretion in the curli biofilm secretion system of Escherichia coli Nissle 1917. Initial characterization using 24-h continuous growth and protein production monitoring confirms decreased growth rates at high induction, leading to a local maximum in total protein production at intermediate induction. Propidium iodide (PI) staining at the endpoint indicates that cellular death is a dominant cause of growth reduction. Assaying variants with combinatorial constructs of inner and outer membrane secretion tags, we find that diminished growth at high production is specific to secretory variants associated with periplasmic stress mediated by outer membrane secretion and periplasmic accumulation of protein containing the outer membrane transport tag. RNA sequencing experiments indicate upregulation of known periplasmic stress response genes in the highly secreting variant, further implicating periplasmic stress in the growth–secretion trade-off. Overall, these results motivate additional strategies for optimizing total protein production and longevity of secretory engineered living systems Graphical Abstract 

Abstract Bacterial cellulose (BC) has excellent material properties and can be produced sustainably through simple bacterial culture, but BC‐producing bacteria lack the extensive genetic toolkits of model organisms such asEscherichia coli(E. coli). Here, a simple approach is reported for producing highly programmable BC materials through incorporation of engineeredE. coli. The acetic acid bacteriumGluconacetobacter hanseniiis cocultured with engineeredE. coliin droplets of glucose‐rich media to produce robust cellulose capsules, which are then colonized by theE. coliupon transfer to selective lysogeny broth media. It is shown that the encapsulatedE. colican produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, capsules are produced which can alter their own bulk physical properties through enzyme‐induced biomineralization. This novel system uses a simple fabrication process, based on the autonomous activity of two bacteria, to significantly expand the functionality of BC‐based living materials.