Development of anti-diabetic living drugs via synthetic biology approach

Abstract

Type 2 Diabetes Mellitus (T2DM) is a prevalent metabolic disorder characterized by insulin resistance and impaired glucose regulation. Peptide-based drugs such as GLP-1 and its analog Exendin-4 are widely used in clinical treatment due to their ability to enhance insulin secretion and improve glycemic control. However, frequent injections, enzymatic degradation in the gastrointestinal tract, and short half-life limit their therapeutic efficiency and patient compliance. To address these challenges, this study aims to develop a living therapeutic system that enables the dynamic, gut-responsive production of anti-diabetic peptides using engineered Escherichia coli Nissle 1917. In the first part of the study, whole-cell biosensors responsive to physiologically relevant stimuli such as fatty acids, bile salts, and aspirin were constructed using synthetic regulatory elements. These biosensors were initially characterized through the expression of a fluorescent reporter gene (sfGFP) to determine their dose-response behavior and functionality. Following successful characterization, the reporter gene was replaced with either GLP-1 or Exendin-4 coding sequences, fused to various signal peptides (PhoA, MalE, TorA, DsbA, PelB) to promote extracellular secretion. Gibson Assembly and classical cloning techniques were used throughout the construct designs. The functional activity of secreted peptides was evaluated through ELISA-based quantification and in vitro bioassays using MIN6 insulin-secreting cells. MTT assays were performed to assess cell viability, and glucose-stimulated insulin secretion assays were conducted to determine the biological activity of the secreted peptides. Among the tested constructs, signal peptide–fused versions of GLP-1 and Exendin-4 showed significant effects on cell viability and insulin secretion, indicating successful expression and functionality of the therapeutic peptides. This study demonstrates the feasibility of combining probiotic bacteria with metabolite responsive gene circuits for targeted peptide delivery. The developed platform presents a promising strategy for designing next-generation living drugs capable of responding to the host environment and offering a self-regulated treatment for metabolic diseases like T2DM.