In situ intein-mediated multiprotein assembly via engineered cross-species consortia

Inteins can connect flanking external proteins into a new protein fragment and excise themselves. Here, we report the <i>in situ</i> splicing of proteins by engineered microbial consortia. This study pioneers a programmable microbial consortia platform enabling <i>in situ</i> protein splicing through split intein-mediated assembly. Engineered <i>Escherichia coli</i> with the ePop autolysis system release intein-fused protein fragments via synchronized lysis, while <i>Pichia pastoris</i> secretes complementary domains, enabling extracellular reconstitution directly in culture. With the application of integrating quorum-sensing controls and eukaryotic secretion pathways, this approach bypasses <i>in vitro</i> purification, supporting scalable one-pot synthesis of multiple functional proteins. The platform's versatility in logic computation and antibiotic resistance engineering highlights its potential for adaptive biomanufacturing and context-aware biomaterial design.