Overview

To enable the engineered bacteria to produce short peptides in the intestine, we have designed a secretion system. This system employs the Lpp'OmpA secretion system to expose and anchor the protein on the outer membrane of Escherichia coli. Subsequently, enterokinases in the small intestine cleave the short peptides at the DDDDK cleavage site near the C-terminal of the protein.

Lpp' OmpA secretion system

Lpp'OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins1. As a signal peptide, Lpp facilitates the modification and maturation of the protein in the periplasm. It then enables transport from the periplasm through the inner leaflet of the outer membrane via the type II secretion system, resulting in the formation of inner surface lipoproteins. Subsequently, OmpA transports the protein through the outer leaflet and anchors it on the cell surface.

Peptide synthesis

Following the Lpp'OmpA signal peptide, the protein contains a region for short peptide generation, including the DDDDK sequence and the target short peptide. The DDDDK sequence serves as a cleavage site for enterokinase, which cleaves the protein after the lysine residue (K). Enterokinase is an enzyme naturally secreted in the small intestine. Consequently, once the signal peptide has directed the protein to the outer membrane surface, the DDDDK-short peptide will be exposed outside the cell. Enterokinase in the small intestine will then cleave the peptide at the DDDDK site, releasing the target short peptide into the intestinal environment.

Overview

Upon reaching the intestines, the engineered bacteria are designed not to multiply indefinitely, thus preventing them from displacing the native gut microbiota. Additionally, they are programmed to die immediately upon exposure to the external environment. To enhance the controllability and safety of the engineered bacteria, we have designed a predator-prey quorum sensing system. This system ensures that the bacteria maintain a target quantity within the intestines and are programmed to die upon exposure to oxygen.

Basic circuit

In the LuxI/LuxR system, the LuxI protein synthesizes a small, diffusible signaling molecule known as acyl-homoserine lactone (AHL). As cell density increases, AHL accumulates both outside and inside the cells. When AHL reaches a critical concentration, it binds to and activates the LuxR transcriptional regulator, which in turn induces the expression of the killing gene (E) under the control of the luxI promoter (PluxI). Elevated levels of the killing protein ultimately result in cell death.

Prey-predator system

In this system, we employ two fundamental circuits: the LuxI/LuxR system and the LasI/LasR system. Our design designates the peptide producer as the predator and the controller as the prey.

When the prey density is low, predator cells undergo apoptosis due to the constitutive expression of the suicide gene (ccdB). In the prey cells, LuxI synthesizes an acyl-homoserine lactone (AHL) known as 3OC6HSL. As prey density increases, 3OC6HSL accumulates in the extracellular environment and, at sufficiently high concentrations, binds to the LuxR transcriptional regulator in predator cells. This binding leads to the increased expression of the antidote gene (ccdA), rescuing the predator cells from apoptosis. Conversely, LasI in the predator cells produces another AHL, 3OC12HSL, which diffuses into the prey cells and binds with LasR, activating the expression of the ccdB gene to initiate "hunting".

Therefore, when the controller density is low, the peptide producers will die. Only when the controller reaches a certain density can the peptide producers survive. If the density of the peptide producers becomes too high, the controllers are "hunted," reducing their density and causing the death of the peptide producers once again. This dynamic system ensures that our bacteria maintain a specific range of densities.

Oxygen-killing