This technology uses engineered bacteria to continuously and safely release large proteins or nucleic acids, enabling controlled delivery for medicine, agriculture, and industry, with precise regulation and broad compatibility across different bacterial species.
Background
The field of bacterial protein secretion and delivery has seen significant advancements due to its broad applications in medicine, biotechnology, agriculture, and environmental science. Bacteria are attractive hosts for the production of valuable biomolecules, including therapeutic proteins, enzymes, and nucleic acids, because of their rapid growth and ease of genetic manipulation. However, the efficient and controlled release of these large biomolecules from living bacterial cells remains a major challenge. Traditional secretion pathways in Gram-negative bacteria are often limited by the size and complexity of the cargo, and many proteins of interest cannot be secreted efficiently or in an active form. There is a growing need for technologies that enable sustained, regulated, and safe delivery of large biomolecules directly from living bacteria, particularly for applications such as live bacterial therapeutics, targeted biocontrol in agriculture, and continuous protein production in industrial settings.
Current approaches to bacterial protein secretion and delivery face several significant limitations. Conventional secretion systems, such as Type I-VI secretion pathways, are often cargo-specific and inefficient for large or complex proteins, especially those exceeding 50-70 kDa. Many methods rely on cell lysis, which is typically explosive and results in the rapid death of the bacterial population, limiting the duration of protein release and raising safety concerns due to uncontrolled release of cellular contents. Additionally, phage-based lysis systems can trigger strong immune responses and are difficult to regulate precisely. Genetic instability and plasmid loss further complicate sustained protein production, and the inability to control the timing and rate of release restricts their utility in dynamic environments like the human gut or soil. These challenges underscore the need for new solutions that enable continuous, controlled, and safe release of large biomolecules from living bacteria without compromising cell viability or safety.
Technology Description
This technology is an engineered bacterial platform designed for the controlled, sustained production and release of large biomolecules—such as proteins and nucleic acids—from living Gram-negative bacteria. It leverages modified bacteriocin operons, particularly those encoding colicin lysis proteins, to achieve a process called "quasilysis," where the bacterial cell envelope is gently disrupted to allow continuous release of intracellular cargo without causing explosive cell death. The system incorporates a toxin-antitoxin module to stabilize the genetic construct and regulate bacterial viability, ensuring that the bacteria remain alive and functional for extended periods. Expression of the lysis protein and cargo biomolecules is tightly regulated by inducible or constitutive promoters, providing precise temporal and environmental control over the release process. The platform is compatible with a wide range of bacterial hosts, including both laboratory and probiotic strains, and supports the secretion of diverse cargos up to at least 100 kDa, with ongoing efforts to increase this limit.
What differentiates this technology is its ability to overcome the major limitations of conventional bacterial secretion systems and lytic delivery methods. Traditional secretion pathways often struggle with large or complex proteins, while lytic systems can cause rapid cell death, limiting sustained delivery and raising immunogenicity concerns. In contrast, this platform’s quasilysis approach enables prolonged, non-explosive release of large biomolecules, maintaining bacterial viability and allowing for continuous therapeutic or industrial protein delivery. Its broad host range, modular genetic architecture, and highly customizable regulatory controls make it adaptable for diverse applications, from live bacterial therapeutics targeting antibiotic-resistant pathogens and localized disease treatment, to agricultural biocontrol and industrial protein production. Additionally, built-in biocontainment features and compatibility with advanced formulation strategies further enhance its safety and commercial viability, positioning it as a foundational solution for next-generation biomolecule delivery across multiple sectors.
Benefits
- Enables sustained, controlled release of large biomolecules (proteins up to ~100 kDa and nucleic acids) from living Gram-negative bacteria without explosive cell death.
- Maintains bacterial viability over extended periods through quasilysis, allowing continuous protein delivery.
- Incorporates toxin-antitoxin modules for genetic stability and regulated bacterial survival.
- Offers broad host compatibility across multiple clinically and environmentally relevant Gram-negative bacteria, including probiotics and attenuated pathogens.
- Supports precise temporal and environmental control of protein release via diverse inducible and constitutive promoters.
- Facilitates applications in therapeutics, agriculture, environmental science, and industrial protein production.
- Reduces immunogenicity compared to phage-based lysis systems and enables biocontainment through auxotrophy and genetic safeguards.
- Allows delivery of diverse cargo types including therapeutic proteins, antimicrobial peptides, antibodies, enzymes, and genetic engineering tools like CRISPR-Cas.
Commercial Applications
- Live bacterial therapeutics delivery
- Continuous industrial protein production
- Targeted agricultural biocontrol
- CRISPR and gene editing delivery
- Probiotic-based antimicrobial treatments
Additional Information
Engineered bacterial platforms utilize modified bacteriocin operons to produce and continuously release large biomolecules. They employ colicin lysis proteins for controlled membrane permeabilization (quasilysis), enabling sustained cargo delivery from living Gram-negative bacteria. A toxin-antitoxin module ensures genetic stability and regulated viability, overcoming conventional secretion challenges.
Patent PCT/US2026/018028 filed 03/06/26
