Ancestral diterpene cyclases show increased thermostability and substrate acceptance
Natalie M Hendrikse1,2,3, Gwenaëlle Charpentier1, Erik Nordling3
1School of Engineering Sciences in Chemistry, Biotechnology and Health, Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden.
The FEBS Journal
|October 29, 2018
Summary
Ancestral sequence reconstruction (ASR) revived bacterial diterpene cyclases with improved stability and activity. These ancestral enzymes, unlike modern ones, accepted new substrates, showcasing ASR
Area of Science:
- Biocatalysis and synthetic biology
- Enzyme engineering
- Molecular evolution
Background:
- Bacterial diterpene cyclases are key for producing complex molecules.
- Ancestral sequence reconstruction (ASR) is a powerful tool for enzyme engineering.
Purpose of the Study:
- To explore ASR for enhancing bacterial diterpene cyclases.
- To generate remodeled cyclases with improved stability, activity, and substrate promiscuity.
Main Methods:
- Ancestral sequence reconstruction (ASR) of spiroviolene synthase.
- Expression in Escherichia coli and protein characterization.
- Kinetic analyses at varying temperatures.
Main Results:
- ASR yielded ancestral cyclases with up to fourfold increased soluble protein.
- Resurrected enzymes showed enhanced thermostability (7-13 °C shift).
- Ancestral enzymes accepted both C20-GGPP and C15-FPP substrates, unlike the modern enzyme.
Conclusions:
- ASR can generate robust and promiscuous biocatalysts.
- Understanding enzyme evolution provides insights into catalytic mechanisms.
- ASR offers potential for sustainable production of valuable chemical building blocks.
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