NRPS Library Construction via Non-Native Restriction Sites
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing non-ribosomal peptides (NRPs) using non-ribosomal peptide synthetases (NRPSs) are limited in efficiency and diversity, as they rely on native restriction enzyme recognition sequences that restrict modification and variation of NRPSs.
Innovation Solution
Introduction of non-native restriction enzyme recognition sequences into nucleic acids encoding NRPSs, allowing for the production of modified NRPSs and novel NRPs through controlled cleavage and ligation of nucleic acid fragments, enabling the creation of diverse NRPS modules and peptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If native restriction enzyme recognition sequences are used in NRPS nucleic acids, then the original NRPS function is maintained, but the ability to modify and create diverse NRPS variants is limited
Solution Approach 1:
The NRPS nucleic acid is divided into modular segments separated by restriction enzyme recognition sequences. Each module can be independently manipulated, allowing creation of diverse variants while preserving functional domains. The segmentation enables precise modification without compromising overall NRPS function.
Solution Approach 2:
Non-native restriction enzyme recognition sequences serve as intermediary elements inserted into the NRPS nucleic acid. These intermediary sequences facilitate controlled cleavage and recombination, enabling diverse NRPS variant creation while the modular design ensures original functional capability is maintained through proper domain preservation.
2Ease of manufacture
If restriction enzyme recognition sequences are introduced into NRPS nucleic acid, then controlled cleavage and ligation can be performed, but the complexity of nucleic acid manipulation increases
Solution Approach 1:
Restriction enzyme recognition sequences are pre-inserted into the NRPS nucleic acid at designated module boundaries before any modification is attempted. This preliminary action creates ready-to-use cleavage sites, simplifying subsequent manipulation steps and reducing the complexity of nucleic acid handling during actual variant creation.
Solution Approach 2:
The nucleic acid structure is modified by introducing specific recognition sequences that change its parameters (adding cleavage sites). However, the overall modular architecture and functional domain integrity are maintained, balancing increased manipulability with controlled structural complexity.
3Adaptability or versatility
If multiple NRPS modules are combined to create novel peptides, then peptide diversity increases, but the efficiency of production decreases
Solution Approach 1:
The NRPS system is segmented into standardized modules with uniform restriction enzyme recognition sequences. This segmentation allows rapid assembly of diverse peptide variants through controlled recombination of pre-characterized functional domains, maintaining production efficiency while increasing peptide diversity.
Solution Approach 2:
The modular NRPS design with universal restriction sites enables the same framework to produce multiple different peptide variants. Each module can be independently swapped or combined, allowing one NRPS construct to serve multiple functions and produce diverse peptides efficiently through standardized assembly procedures.
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
This approach enables efficient production of novel NRPSs and NRPs, expanding the diversity and range of peptides that can be synthesized, while maintaining the functional capability of the original NRPSs.
Implementation Method 1
a step of preparing a mixture of containing a plurality of resulting cleaved nucleic acid fragments by treating the set of starting plasmids with a restriction enzyme
Implementation Method 2
a step of forming a linked nucleic acid by ligating the plurality of cleaved nucleic acid fragments
Data Source
Figure 1A~1B
Figure 2
Figure 3
AI summary
The present disclosure pertains to produce a novel NRPS. In the present disclosure, a restriction enzyme recognition sequence was successfully introduced into a nucleic acid encoding an NRPS while maintaining a function of producing a target NRP. Various NRPSs can be produced using the introduced restriction enzyme recognition sequence. In one aspect, the present disclosure provides a nucleic acid containing a non-native restriction enzyme recognition sequence and encoding an NRPS. In another aspect, the present disclosure provides a method for efficiently producing a nucleic acid encoding a novel NRPS using a non-native restriction enzyme recognition sequence. In addition, the present disclosure also provides a novel NRPS and a novel NRP.