Microcin-MGE Overexpression and Purification for MDR Enterobacteriaceae
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Solution Overview
Problem
Current methods for purifying and utilizing microcin H47 and microcin I47 are inefficient, leading to conflicting reports on their efficacy against bacterial infections, and there is a need for a controlled, scalable production of antimicrobial peptides to address multi-drug resistant (MDR) and extensively drug resistant (XDR) bacteria.
Innovation Solution
Genetically engineered microorganisms are developed to overexpress microcins MccH47 and MccI47, post-translationally modified with mono-glycosylated cyclic enterobactin (MGE) to form MccH47-MGE and MccI47-MGE compositions, which are purified and formulated for oral administration, enabling effective treatment of bacterial infections and dysbiosis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If microcin H47 and microcin I47 are produced by live bacteria, then antimicrobial activity can be demonstrated, but purification is extremely difficult and conflicting reports on efficacy arise
Solution Approach 1:
The patent extracts the microcin production pathway genes (mch operon for MccH47 and mci operon for MccI47) from their native bacterial context and transfers them into E. coli BL21(DE3) expression systems. This allows the microcins to be produced as purified products rather than requiring complex live bacterial cultures, directly resolving the purification difficulty while maintaining reliable antimicrobial efficacy through controlled expression.
Solution Approach 2:
The patent uses pLysS lysogen and T7 RNA polymerase as intermediaries to control microcin expression. The pLysS lysogen provides stable background expression control, while T7 RNA polymerase enables inducible high-level production. This intermediary system allows precise control over microcin production, enabling both reliable efficacy assessment and straightforward purification by inducing expression only when needed.
2Reliability
If microcin production is increased to address MDR/XDR infections, then therapeutic effectiveness improves, but production control and scalability become challenging
Solution Approach 1:
The patent implements dynamic control of microcin production through inducible T7 promoters (pLysS and pT7). Expression levels can be adjusted by controlling IPTG induction timing and concentration, allowing optimization between low-level background expression (for cell viability) and high-level induced expression (for therapeutic production). This dynamic system enables scalable production while maintaining therapeutic effectiveness.
Solution Approach 2:
The patent changes key production parameters including induction timing (OD600 monitoring), inducer concentration (IPTG), and growth conditions to optimize microcin yield. By systematically adjusting these parameters, the system achieves scalable production that maintains reliable therapeutic effectiveness across different production scales.
3Ease of operation
If genetically modified bacteria are used to produce microcins, then production control improves, but administration complexity increases due to genetic modification requirements
Solution Approach 1:
The patent extracts the microcin production capability from complex live bacterial systems and transfers it to a controlled recombinant expression system in E. coli BL21(DE3). This extraction simplifies administration by allowing purified microcin peptides to be administered directly without requiring live genetically modified bacteria, reducing administrative complexity while maintaining production control through the recombinant system.
Solution Approach 2:
The patent uses T7 RNA polymerase and IPTG as intermediaries to control microcin expression, providing a simple on/off switch mechanism. This intermediary control system simplifies administration compared to regulating gene expression in live probiotic bacteria, as it requires only small molecule induction rather than complex bacterial physiology management.
Data Source
AI summary
This disclosure relates to genetically engineered microorganisms for overexpressing microcin compositions, e.g., MccH47 compositions and MccI47 compositions, which are post-translationally modified with a covalent linkage at the C-terminus to a siderophore, such as mono-glycosylated cyclic enterobactin (MGE), to form microcin-MGE compositions, e.g., MccH47-MGE and MccI47-MGE compositions, the purified compositions themselves, methods of making the purified compositions, and methods of using the purified compositions to treat or reduce the risk of bacterial infections or dysbiosis.