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Assembly and engineering of BioBricks to develop an efficient NADH regeneration system
Feng Cheng1,2, Cheng-Jiao Wang1,2, Xiao-Xiao Gong1,2
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, China.
Applied and Environmental Microbiology
|December 11, 2024
Summary
This study engineered an efficient NADH regeneration system using alcohol dehydrogenase (ADH) and BioBricks assembly. The optimized system significantly enhanced catalytic efficiency and gene expression for biocatalysis, achieving over 95% yield in chiral herbicide synthesis.
Area of Science:
- Biocatalysis and synthetic biology
- Enzyme engineering and cofactor regeneration
Background:
- Cofactor regeneration is vital for redox biocatalysis in organic synthesis and pharmaceuticals.
- Alcohol dehydrogenase (ADH)-based systems offer in situ NAD(P)H regeneration but face challenges with low activity and expression in Escherichia coli.
- Previous efforts focused on ADH discovery and protein engineering, overlooking other crucial system components.
Purpose of the Study:
- To develop a highly efficient NADH regeneration system through BioBricks assembly and engineering.
- To enhance the catalytic efficiency of Geobacillus stearothermophilus ADH (GstADH) using semi-rational design.
- To optimize ribosome binding site (RBS) sequences for increased ADH gene expression.
Main Methods:
- BioBricks assembly (promoter, RBS, gene, terminator) for constructing the NADH regeneration system.
- Semi-rational design of GstADH to improve its catalytic efficiency.
- RBS library screening and optimization to enhance ADH translation rates.
- Application of the engineered system for asymmetric biosynthesis of l-phosphinothricin.
Main Results:
- A GstADH variant with a 2.1-fold increase in catalytic efficiency was generated.
- An optimized RBS led to a 3.2-fold increase in ADH gene translation rate.
- The engineered NADH regeneration system achieved a NADH generating velocity exceeding 2 s⁻¹ even at low NAD+ concentrations (0.1 mM).
- The system demonstrated a 6.7-fold overall performance enhancement compared to previous systems.
- Successful application in the asymmetric biosynthesis of l-phosphinothricin, yielding >95%.
Conclusions:
- The developed BioBricks-based NADH regeneration system is highly efficient and robust.
- This engineered system represents a significant advancement for NAD(P)H regeneration in biocatalysis.
- The system holds promise for various NADH-dependent biocatalytic processes, particularly in fine chemical synthesis.
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