Engineered Enterocins Expand Bactericidal Spectrum
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Solution Overview
Problem
Current Phage tail-like bacteriocins (PTLBs) have a limited binding and killing spectrum, making them impractical for broad applications against Enterococcus species, as they typically only target specific strains within the same species, and none have been reported to be produced by or active against Enterococci.
Innovation Solution
Identification, cloning, and expression of a genetic locus in Enterococcus faecalis that encodes a PTLB, termed an enterocin, with modifications to expand its bactericidal specificity by incorporating heterologous receptor binding proteins and adaptor proteins to target various Enterococcus species.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If PTLBs are used as antimicrobial agents, then they can kill specific bacterial strains, but their binding and killing spectrum is too narrow for practical use
Solution Approach 1:
The patent creates a universal PTLB platform where the core contractile structure serves multiple functions by accepting different heterologous RBPs. The base structure remains consistent while the RBP component varies to target different Enterococcus species, making the system universally applicable across multiple bacterial targets rather than limited to a single strain
Solution Approach 2:
The PTLB structure is segmented into distinct functional modules: the contractile tail structure (genes 1278-1289) that provides the killing mechanism, and the RBP (gene 1291) that provides target specificity. This segmentation allows independent optimization and replacement of the RBP component to expand the bactericidal spectrum while maintaining the reliable contractile killing function
2Adaptability or versatility
If heterologous RBPs are incorporated to expand bactericidal spectrum, then binding spectrum increases, but device complexity increases
Solution Approach 1:
The RBP gene (gene 1291) is extracted as a separate, replaceable component from the core PTLB gene cluster. This allows the RBP to be independently modified or replaced with heterologous RBPs to expand binding spectrum, while the core contractile structure remains unchanged and manageable in complexity
Solution Approach 2:
An adaptor protein (gene 1290) serves as an intermediary between the core PTLB structure and heterologous RBPs. This adaptor provides a standardized interface that simplifies the integration of different RBPs without requiring complex redesign of the entire PTLB system, thereby expanding binding spectrum while controlling complexity
3Ease of manufacture
If no PTLBs are produced by Enterococci, then there is no natural enterocin available, but this limits the development of Enterococcus-specific antimicrobials
Solution Approach 1:
Instead of relying on natural Enterococcus-produced PTLBs, the patent copies the PTLB gene cluster from another bacterium and expresses it in an Enterococcus host. This creates a synthetic enterocin that mimics natural PTLBs but is now produced by and specific to Enterococcus species, making the antimicrobial readily available and species-specific
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified enterocins demonstrate broad bactericidal activity against multiple Enterococcus strains, including E. faecalis and E. faecium, offering a potential therapeutic solution for infections and expanding their practical use as antimicrobials.
Implementation Method 1
Both R-type- and F-type PTLBs kill bacterial cells by first binding to specific targets ('receptors') on the bacterial cell surface via Receptor Binding Proteins (RBPs)
Data Source
AI summary
The present disclosure relates to the identification, cloning, and expression of a genetic locus within an Enterococcus genome that encodes a phage tail-like bacteriocin (PTLB), termed an enterocin. Also provided are non-natural enterocins, which have been engineered to have altered bactericidal specificity. Nucleic acid molecules encoding natural or non-natural enterocins, vector constructs containing such nucleic acids operably linked to a heterologous promoter, producer cells containing such vectors, the encoded enterocins, as well as methods of making and using such enterocins are described.