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A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products
Published on: October 4, 2019
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Directed evolution of squalene synthase for dehydrosqualene biosynthesis
Maiko Furubayashi1, Ling Li1, Akinori Katabami1
1Department of Applied Chemistry and Biotechnology, Chiba University, 1-33 Yayoi-cho, Inage, Chiba 263-8522, Japan.
FEBS Letters
|August 6, 2014
Summary
Researchers engineered squalene synthase (SQS) into a dehydrosqualene synthase. This enzyme modification was achieved through directed evolution, altering SQS to produce carotenoid precursors.
Area of Science:
- Biochemistry and enzymology
- Metabolic engineering
- Molecular evolution
Background:
- Squalene synthase (SQS) is a key enzyme in sterol and hopanoid biosynthesis.
- SQSs share evolutionary origins with carotenoid synthases, but their product specificity is distinct.
- The mechanism governing SQS product selectivity remains poorly understood.
Purpose of the Study:
- To investigate the evolutionary relationship between squalene synthases and carotenoid synthases.
- To engineer SQS enzymes to produce dehydrosqualene, a C30 carotenoid backbone.
- To identify mutations responsible for altering SQS activity towards carotenoid synthesis.
Main Methods:
- Directed evolution of SQS from yeast, human, and bacterial sources.
- High-throughput screening using Pantoea ananatis phytoene desaturase for dehydrosqualene detection.
- Genetic analysis of evolved SQS mutants.
Main Results:
- Engineered SQS variants successfully produced dehydrosqualene.
- Mutations conferring dehydrosqualene synthase activity were identified.
- Key mutations were localized near proposed NADPH-binding residues.
Conclusions:
- SQS can be functionally redirected towards carotenoid backbone synthesis through targeted mutations.
- The NADPH-binding site plays a critical role in determining the product specificity of SQS.
- This study provides insights into the evolution and functional plasticity of terpene synthases.
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