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A Customizable Approach for the Enzymatic Production and Purification of Diterpenoid Natural Products
Published on: October 4, 2019
Engineering cotton (+)-delta-cadinene synthase to an altered function: germacrene D-4-ol synthase
Yasuo Yoshikuni1, Vincent J J Martin, Thomas E Ferrin
1UCSF/UCB Joint Graduate Group in Bioengineering, University of California, Berkeley, 94720, USA.
Chemistry & Biology
|January 24, 2006
Summary
Researchers engineered a sesquiterpene synthase enzyme using combined rational design and random mutagenesis. A novel dual-activity screen enabled the isolation of mutants with altered product ratios, including one with 93% selectivity for germacrene D-4-ol.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzyme Engineering
Background:
- Sesquiterpene synthases are crucial enzymes in natural product biosynthesis.
- Engineering sesquiterpene synthases for altered activity and selectivity is challenging due to limited screening methods.
- A dual-activity screen combining sesquiterpene synthase function with chloramphenicol acetyltransferase (CAT) activity was developed.
Purpose of the Study:
- To generate a sesquiterpene synthase with modified activity and product selectivity.
- To identify key amino acid residues influencing (+)-delta-cadinene synthase product formation.
- To engineer a mutant enzyme with enhanced selectivity for germacrene D-4-ol.
Main Methods:
- Combined rational design and random mutagenesis of (+)-delta-cadinene synthase.
- Development and application of a high-throughput dual-activity screen using a fusion protein with chloramphenicol acetyltransferase (CAT).
- Error-prone PCR for gene mutagenesis, followed by screening for altered sesquiterpene selectivity.
- Homology modeling to analyze the structural role of the G helix.
- Site-directed and saturation mutagenesis to reconstruct the G helix.
Main Results:
- Successfully generated a library of sesquiterpene synthase mutants with altered (+)-delta-cadinene and germacrene D-4-ol ratios.
- Identified 21 clones with distinct product profiles.
- Homology modeling indicated the G helix is critical for (+)-delta-cadinene formation.
- Engineered a specific mutant (N403P/L405H) in the G helix that retained specific activity and achieved 93% selectivity for germacrene D-4-ol in vivo.
Conclusions:
- The developed dual-activity screen is effective for engineering sesquiterpene synthases.
- The G helix of (+)-delta-cadinene synthase is a key determinant of product selectivity.
- Site-directed mutagenesis of the G helix can yield enzymes with significantly altered and improved catalytic properties, such as enhanced germacrene D-4-ol production.
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