Abstract Description

Escherichia coli (E. coli) is a preferred host for protein expression; however, it necessitates cell lysis. Conventional lysis methods are widely used for recovering intracellular proteins; however, they remain costly, with an increased risk of product contamination and protein denaturation. Programmed cell autolysis, inspired by bacteriophage-derived holins and endolysins, offers an alternative by triggering cell lysis from within the host cell upon external induction. Chemical and physical induction methods pose a metabolic burden and are inefficient for large-scale production. Therefore, an optogenetically programmed cell autolysis system was engineered to simplify protein recovery and reduce metabolic burden from the E. coli. Optogenetic autolytic vectors pDusk-SRRz and pET28a-pD-SRRz were constructed and transformed into E. coli T7 Express lysY. Expression was achieved by using dark and blue light induction, respectively. After 16 hours of optogenetic induction, cell autolysis was triggered, with cell death rates of 90.33 ± 3.51% and 72.00 ± 2.09%, and released proteins reaching 358.89 ± 15.65 µg/ml and 374.28 ± 12.38 µg/ml for pDusk-SRRz and pET28a-pD-SRRz, respectively. Scanning electron microscopy showed that there were structural changes on the cell surface after autolysis with pET28a-pD-SRRz, resulting in more cell death than pDusk-SRRz. This study introduced a non-toxic, reversible optogenetic cell-autolytic system that simplifies protein expression and recovery, demonstrating great potential for facile extracellular secretion of proteins in industrial applications.