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Improved selectivity of an engineered multi-product terpene synthase.

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  • 1California Institute of Technology, Division of Chemistry & Chemical Engineering, 1200 E. California Blvd., Pasadena CA 91125, USA.

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|May 10, 2014
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Summary

Researchers engineered a sesquiterpene synthase, Cop2, to enhance germacrene D-4-ol production. A novel mutant, 17H2, achieved 77% selectivity for this valuable compound, improving upon the parent enzyme’s efficiency.

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Area of Science:

  • Enzyme Engineering
  • Biocatalysis
  • Natural Product Synthesis

Background:

  • Sesquiterpene synthases are crucial enzymes in natural product biosynthesis.
  • Cop2 is a sesquiterpene synthase with known cyclization activity.
  • Improving selectivity for specific sesquiterpene products is a key challenge.

Purpose of the Study:

  • To engineer the sesquiterpene synthase Cop2 for enhanced cyclization activity.
  • To identify mutants with improved selectivity for germacrene D-4-ol.
  • To characterize the catalytic properties of the engineered enzyme.

Main Methods:

  • High-throughput screening of Cop2 mutants using a non-natural substrate.
  • Characterization of a triple amino acid substitution mutant (17H2).
  • Substrate conversion analysis and kinetic comparisons (kcat/KM) of wild-type and mutant enzymes.
  • Homology modeling to predict the location of mutations relative to the active site.

Main Results:

  • A mutant, 17H2, was identified with three amino acid substitutions.
  • 17H2 converted farnesyl pyrophosphate (FPP) to germacrene D-4-ol with 77% selectivity.
  • This contrasts with the wild-type Cop2, producing 29% germacrene D-4-ol and 46% α-cadinol.
  • Mutations were confirmed to contribute to selectivity and are located near the active site.
  • The catalytic efficiency (kcat/KM) of 17H2 (0.62 mM⁻¹ s⁻¹) is similar to wild-type Cop2 (0.58 mM⁻¹ s⁻¹).

Conclusions:

  • Directed evolution and high-throughput screening can successfully engineer sesquiterpene synthases.
  • The 17H2 mutant demonstrates significantly improved selectivity for germacrene D-4-ol.
  • These mutations, discovered through screening, offer a non-rational approach to enzyme optimization.