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A novel approach for metabolic pathway optimization: Oligo-linker mediated assembly (OLMA) method
Shasha Zhang1,2, Xuejin Zhao1, Yong Tao1
1Chinese Academy of Sciences Key Laboratory of Microbial Physiological, and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101 China.
Journal of Biological Engineering
|December 25, 2015
Summary
A new DNA assembly method, oligo-linker mediated assembly (OLMA), enables simultaneous manipulation of multiple gene expression targets. This approach expands the combinatorial space and reduces inefficient mutants for improved product yield.
Area of Science:
- Synthetic biology
- Metabolic engineering
- Molecular biology
Background:
- Metabolic pathway gene expression imbalances limit desired product yields.
- Previous strategies focused on single targets like promoters or ribosome binding sites (RBS).
- Simultaneous manipulation of multiple gene expression targets remains underexplored.
Purpose of the Study:
- To introduce a novel DNA assembly method for concurrent manipulation of multiple gene expression regulatory targets.
- To demonstrate the method's capability in complex DNA assembly and metabolic pathway optimization.
Main Methods:
- Development of the oligo-linker mediated assembly (OLMA) method.
- Incorporation of up to 8 regulatory targets in a single assembly step.
- Application of OLMA to assemble lacZ expression cassettes and optimize lycopene biosynthesis enzymes.
Main Results:
- The OLMA method successfully assembled multiple DNA fragments and optimized enzyme combinations.
- Demonstrated the ability to explore a larger combinatorial design space.
- Reduced the generation of inefficient mutants compared to traditional methods.
Conclusions:
- The OLMA method utilizes a synthetic DNA oligo library for versatile target manipulation (promoters, RBSs, gene order).
- OLMA facilitates PCR-free and zipcode-free DNA assembly.
- This method offers a powerful tool for engineering complex genetic systems.
