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Updated: Dec 28, 2025
Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
Published on: August 8, 2016
Fluorescence-based high-throughput screening system for R-ω-transaminase engineering and its substrate scope
Feng Cheng1,2, Xiu-Ling Chen1,2, Chao Xiang1,2
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, People's Republic of China.
Researchers engineered an R-omega-transaminase (ω-TA) from Mycobacterium vanbaalenii (MvTA) to broaden its substrate scope for synthesizing R-amines. Directed evolution using a novel fluorescence assay yielded an improved MvTA variant with significantly enhanced activity and efficiency.
Area of Science:
- Biocatalysis
- Enzyme Engineering
Background:
- Omega-transaminases (ω-TAs) offer a stereoselective alternative to metal catalysts for prochiral ketone amination.
- The R-ω-transaminase from Mycobacterium vanbaalenii (MvTA) has a limited substrate scope, restricting its use in R-amine synthesis.
Purpose of the Study:
- To develop a sensitive fluorescence-based screening system to enhance the substrate scope of MvTA.
- To engineer MvTA variants with improved activity and efficiency for R-amine synthesis.
Main Methods:
- Development of a microtiter plate (MTP)-based fluorescence assay for ω-TA activity screening.
- Implementation of a KnowVolution campaign involving screening approximately 8000 MvTA clones.
- Directed evolution to identify beneficial substitutions (G68Y, F129A) and engineer improved variants (M3, M4, M5).
Main Results:
- The fluorescence assay demonstrated high sensitivity (μM), low background, and a wide dynamic range (ɀ-factor > 0.9).
- Engineered variants, particularly MvTA M5 (WT+G68Y+F129A), showed a 3.2-fold increase in catalytic efficiency toward NMA compared to wild-type (WT).
- MvTA M5 exhibited over 100-fold higher specific activity on acetonaphthone and significantly enhanced activity on six other prochiral ketones with >99% enantiomeric excess (e.e.) for R-amines.
Conclusions:
- A robust fluorescence screening system enables efficient enzyme evolution for ω-TAs.
- Engineered MvTA M5 demonstrates significantly improved catalytic performance and broadened substrate scope for R-amine synthesis.
- This work expands the utility of ω-TAs in stereoselective biocatalysis.
