Efficient production and delivery of bioactive peptides via Gram-negative bacterial secretion system

Engineered Gram-negative bacteria use the microcin V secretion system to produce and release various active peptides efficiently. This versatile technology works across multiple bacterial species and preserves peptide functionality for research and therapeutic uses.

Background

The production and secretion of bioactive peptides are critical in various biotechnological and medical applications, including drug development, therapeutic delivery, and industrial enzyme production. Gram-negative bacteria are often employed as host systems for peptide expression due to their well-characterized genetics and ability to perform post-translational modifications. Efficient secretion systems are essential for recovering peptides in their active form and for minimizing intracellular accumulation, which can lead to toxicity and reduced yields. The ability to harness and optimize bacterial secretion mechanisms is thus critical for advancing peptide-based technologies and expanding their practical applications.

Current approaches to peptide secretion in Gram-negative bacteria face significant challenges, primarily related to limited secretion efficiency and substrate specificity. They also often struggle with the export of heterogeneous and larger peptides, resulting in low yields and compromised biological activity of the secreted products. There are also constraints on narrow substrate recognition, restricting their versatility across different peptide types and lengths. Environmental factors, such as temperature and host strain compatibility, further complicate the optimization process, making it difficult to achieve consistent and scalable production.

These limitations hinder the effective utilization of bacterial systems for diverse peptide research and therapeutic purposes, underscoring the need for more robust and adaptable secretion strategies.

Technology description

Gram-negative bacterial cells are engineered to express and secrete heterologous peptides using the microcin V type I secretion system (T1SS) derived from E. coli. This system utilizes a two-plasmid setup: one plasmid encodes a secretion signal sequence fused to the target peptide, while the other encodes both the C39 peptidase-containing ATP-binding cassette transporter (such as CvaB) and the membrane fusion protein (like CvaA). The engineered bacteria efficiently transport peptides ranging from 26 to 66 amino acids from the cytosol to the external environment. Technology was demonstrated across multiple Gram-negative species, all retaining their biological activity post-secretion.

This technology stands out due to its remarkable versatility and efficiency in peptide secretion, primarily influenced by peptide length rather than charge or hydrophobicity. The system exhibits vigorous substrate recognition, enabling the secretion of a wide array of peptides without extensive modification. Its ability to preserve the biological activity of peptides post-secretion ensures functionality across different applications. Also, the cross-species functionality broadens its applicability for peptide research, production, and in vivo delivery, particularly within the gut environment. Environmental conditions, such as lower temperatures and specific bacterial strains, further enhance secretion efficiency, making this system a highly adaptable and optimized platform for biotechnological and therapeutic applications.

Keywords: biocides, peptides, recombinant DNA