Lasso Peptide Biosynthesis via Phage Display Screening
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Solution Overview
Problem
Current methods for developing peptide-based drugs face challenges due to undesirable physicochemical and pharmacokinetic properties, such as poor solubility, cell permeability, and instability, particularly for knotted peptides like cyclotides and conotoxins, which are difficult to produce and require costly extraction and fractionation processes.
Innovation Solution
The development of lasso peptides and related molecules, including fusion proteins with bacteriophage coat proteins and secretion signals, for optimized biosynthesis and high-throughput screening, allowing for genetic manipulation and cost-effective production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional extraction and fractionation methods are used for cyclotides and conotoxins, then the knotted peptide scaffolds can be obtained, but the process is slow and costly
Solution Approach 1:
The patent replaces traditional mechanical extraction and fractionation methods with a biological system based on phage display technology. The lasso peptide biosynthesis machinery is expressed in E. coli, and phage particles display the peptides for screening, substituting slow chemical/biological extraction with a controlled genetic expression and display system that enables high-throughput discovery
Solution Approach 2:
The patent utilizes the native lasso peptide biosynthesis machinery of Streptomyces coelicolor, expressed heterologously in E. coli, to automatically produce and process the peptide. The system self-organizes the complex biosynthetic pathway including NRPS enzyme assembly, substrate channeling, and post-translational modification without requiring manual intervention at each step
2Ease of manufacture
If solid phase peptide synthesis (SPPS) or expressed protein ligation (EPL) methods are used, then the circular peptide backbone can be generated, but the production is complex and costly
Solution Approach 1:
The patent replaces complex chemical synthesis methods (SPPS) or multi-step biochemical methods (EPL) with a single-step biological expression system. The lasso peptide biosynthesis machinery, when expressed in E. coli, automatically performs all necessary steps including peptide synthesis, cyclization, and lasso structure formation through native enzymatic activities, eliminating the need for complex external synthesis protocols
Solution Approach 2:
The patent incorporates the lasso peptide biosynthesis genes into the E. coli genome or plasmids beforehand, so that the complete biosynthetic pathway is pre-assembled and ready for expression. This preliminary genetic setup allows the system to automatically perform all biosynthetic steps upon induction, rather than requiring step-by-step manual intervention during the synthesis process
3Adaptability or versatility
If lasso peptides are produced through heterologous expression, then genetic manipulation is enabled, but the biosynthetic machinery must be successfully transferred to host cells
Solution Approach 1:
The patent uses E. coli as an intermediary host that can accept and express the Streptomyces coelicolor lasso peptide biosynthesis genes. The E. coli cellular machinery serves as a mediator that translates the heterologous genetic information into functional peptide production, bridging the gap between the native Streptomyces system and a more manipulatable host platform
Solution Approach 2:
The patent demonstrates that the lasso peptide biosynthesis machinery is universal enough to function across different bacterial species. The NRPS enzymes and associated biosynthetic components from Streptomyces coelicolor can be expressed and functionally active in E. coli, showing that the core biosynthetic pathway is conserved and adaptable across bacterial domains
Data Source
AI summary
Provided herein are lasso peptides libraries, and particularly phage display libraries of lasso peptides. Also provided herein are related methods and systems for producing the libraries and for screening the libraries to identify candidate lasso peptides having desirable properties.