Orthogonal Error-Prone DNA Polymerase for Bacterial Continuous Evolution
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Solution Overview
Problem
Current directed evolution methods in bacteria lack a system that can achieve all mutant types, long-DNA-fragment mutation, good continuity, and simple operation, which are essential for enzyme engineering and metabolic engineering applications.
Innovation Solution
A bacterial continuous evolution system utilizing an orthogonal linear gene expression vector combined with an orthogonal error-prone DNA polymerase, specifically designed for Bacillus thuringiensis, which includes a linear plasmid with a DNA replication and control gene cluster, a promoter, and a target gene, along with a mutant DNA polymerase with defined mutations, to enable efficient and controlled random mutation of target DNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional directed evolution method is used with in-vitro library construction, then library construction can be achieved, but throughput is low and time and cost consumption is large
Solution Approach 1:
The patent replaces the mechanical in-vitro library construction process with an in-vivo continuous evolution system using orthogonal DNA replication. The error-prone DNA polymerase performs random mutagenesis directly within the bacterial cell, eliminating the need for complex in-vitro library construction and significantly improving throughput while reducing time consumption.
Solution Approach 2:
The system enables self-service continuous evolution within the bacterial cell. The orthogonal DNA replication system with error-prone polymerase allows the cell to autonomously generate mutants of the target gene through random mutagenesis during DNA replication, eliminating the need for external library construction and enabling continuous evolution.
2Adaptability or versatility
If orthogonal DNA replication system is developed in yeast, then all mutant types and long-DNA-fragment mutation can be achieved, but application in bacteria remains a huge challenge
Solution Approach 1:
The patent segments the complex orthogonal DNA replication system into modular components: a linear plasmid vector with specific replication origin, an error-prone DNA polymerase with defined mutations, and a target gene insertion site. This modular segmentation allows the system to be constructed and optimized separately in bacteria, reducing overall system complexity while maintaining versatility.
Solution Approach 2:
The patent optimizes key parameters of the orthogonal replication system for bacterial application, including the mutation rate of the error-prone polymerase (achieved 6.7×10⁻⁶ mutations per base per generation), the structure of the linear plasmid vector, and the replication control mechanisms. These parameter adjustments enable the system to function effectively in bacteria with reduced complexity.
3Productivity
If error-prone DNA polymerase is used to increase mutation rate, then continuous evolution efficiency is improved, but genomic mutation rate may increase significantly
Solution Approach 1:
The patent extracts the error-prone DNA polymerase function into a separate, controllable element that operates only on the linear plasmid vector containing the target gene. The polymerase is introduced as an exogenous protein that specifically replicates the orthogonal plasmid, leaving the bacterial genomic DNA replication unaffected and maintaining genomic stability.
Solution Approach 2:
The linear plasmid vector serves as an intermediary between the error-prone polymerase and the target gene. The polymerase acts on the plasmid DNA as an intermediate substrate, generating mutants that are then expressed from the plasmid without directly mutating the bacterial genome. This intermediary mechanism isolates the mutagenesis process from genomic DNA.
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 system allows for the efficient continuous evolution of target DNA sequences, including all mutation types and long-DNA-fragment mutations, with a high mutation rate that is 6,700 times the genomic mutation rate, while maintaining operational simplicity and without significantly increasing the genomic mutation rate.
Implementation Method 1
The linear plasmid is replicated by the orthogonal DNA polymerase (the amino acid sequence of the wild-type polymerase is as shown in SEQ ID NO. 1) of GIL16
Implementation Method 2
The mutation rate of the optimal mutant reaches 6.82×10−7 per generation per cell per base, which is 6,700 times the genomic mutation rate
Data Source
AI summary
The present invention provides a bacterial continuous evolution system, an orthogonal error-prone DNA polymerase, and a continuous evolution method. In the present invention, by combining an orthogonal DNA replication system and an orthogonal error-prone DNA polymerase, a continuous evolution method that includes all mutant types, enables long-DNA-fragment mutation, and is good in continuity and simple and convenient to operate is obtained. By inducing the opening and closing of DNA polymerase expression, switching between a linear plasmid error-prone mutation process and a high-fidelity replication process is realized, so as to achieve the efficient and continuous evolution of a target DNA sequence.