Circular Bacteriocin Production Using Split Intein Cyclization
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Solution Overview
Problem
Existing methods for producing and characterizing circular bacteriocins are laborious, expensive, and time-consuming, and often require the native bacteriocin-producing bacteria, limiting the efficiency and versatility of industrial applications.
Innovation Solution
A method using split intein circular ligation of peptides and proteins (SICLOPPS) system for in vitro and in vivo production of circular bacteriocins, involving fusion polypeptides with split inteins that circularize bacteriocins without additional native context proteins, enabling high-throughput expression and characterization of characterized and novel circular bacteriocins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional methods are used to produce and characterize circular bacteriocins, then the native bacteriocin-producing bacteria are required, but the process becomes laborious, expensive, and time-consuming
Solution Approach 1:
The patent introduces an intein-based circular ligation system as an intermediary mechanism that enables bacteriocin circularization without requiring the native bacterial host. The intein acts as a self-processing mediator that facilitates head-to-tail cyclization of the bacteriocin precursor through spontaneous protein splicing, thereby eliminating the need for complex native bacterial systems while maintaining production reliability
Solution Approach 2:
The invention extracts the essential circularization function from the native bacterial system by isolating and engineering the intein component. This extracted intein-based circular ligation system can then be applied to any bacteriocin precursor in vitro, separating the circularization capability from the native bacterial context and enabling streamlined, rapid production without the laborious steps of traditional methods
2Reliability
If traditional methods are used to produce circular bacteriocins, then additional native context proteins are required, but the device complexity and cost increase
Solution Approach 1:
The invention extracts and utilizes only the essential intein component responsible for circularization, eliminating the need for other native context proteins such as leader peptidases and transport proteins. This extracted intein-based system achieves correct circularization with minimal components, dramatically reducing device complexity while maintaining reliability
Solution Approach 2:
The intein-based system is self-sufficient for circularization, requiring no additional native bacterial proteins or complex cellular machinery. The intein autonomously catalyzes its own splicing and the bacteriocin's circularization through self-processing, eliminating dependencies on other proteins and simplifying the overall production system
3Ease of manufacture
If linear bacteriocins are produced, then the production process is simpler, but the stability against temperature, pH, and proteolytic enzymes is reduced
Solution Approach 1:
The invention implements preliminary circularization action during the in vitro production phase itself, rather than requiring post-production circularization steps. The intein-mediated circular ligation occurs concurrently with protein synthesis and purification, ensuring the bacteriocin is circularized before application. This preliminary circularization maintains both manufacturing simplicity and enhanced stability, as the circular structure is established during the already-necessary production workflow
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
Facilitates fast and efficient production of circular bacteriocins with maintained or enhanced antimicrobial activity, allowing for high-throughput screening and expression of variants, including those with non-natural amino acids, and improving stability against heat, pH, and protease degradation.
Implementation Method 1
a method of making a circular bacteriocin, comprising expressing in a cell-free protein system or in E. coli a fusion polypeptide comprising an amino acid sequence of a bacteriocin flanked at both the N- and C-termini by a split intein that circularizes the bacteriocin
Data Source
AI summary
Provided herein are fusion polypeptides that include an amino acid sequence of a bacteriocin flanked at both the N- and C-termini by a split intein that circularizes the bacteriocin. Also provided are nucleic acids and genetic vectors encoding the fusion polypeptide, and microbial cells genetically engineered with the nucleic acids or genetic vectors. Further provided are methods of making a circular bacteriocin, methods of screening using a library of nucleic acids or genetic vectors encoding the fusion polypeptide, and methods of controlling the growth of an organism using circular bacteriocins made by the methods provided herein.