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Evolution of the C30 carotenoid synthase CrtM for function in a C40 pathway
Daisuke Umeno1, Alexander V Tobias, Frances H Arnold
1Department of Chemical Engineering, California Institute of Technology 210-41, Pasadena, California 91125, USA. umeno@cheme.caltech.edu
Journal of Bacteriology
|November 12, 2002
Summary
Researchers engineered carotene synthases to alter carotenoid production. Mutated CrtM successfully produced C40 carotenoids, demonstrating enzyme plasticity and potential for novel structures.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Metabolic Engineering
Background:
- Carotenoids are vital pigments with diverse biological roles.
- Carotene synthases (CrtM, CrtB) control carotenoid backbone synthesis.
- Understanding enzyme specificity is key for pathway engineering.
Purpose of the Study:
- To investigate the substrate specificity of C30 carotene synthase (CrtM) and C40 carotene synthase (CrtB).
- To engineer CrtM for C40 carotenoid synthesis.
- To explore the potential for creating novel carotenoid structures.
Main Methods:
- Heterologous expression of Staphylococcus aureus CrtM and Erwinia uredovora CrtB in Escherichia coli.
- Functional evaluation of wild-type and mutated enzymes in C30 and C40 biosynthetic pathways.
- Mutagenesis and screening to identify variants with altered specificity.
Main Results:
- Wild-type CrtM and CrtB showed negligible activity in the other's native pathway.
- 116 CrtM variants capable of C40 carotenoid synthesis were identified.
- Mutant CrtM enzymes exhibited broadened or shifted substrate specificities, with Phe 26 identified as the key determinant.
- Engineered CrtM variants performed comparably to CrtB in C40 pathways.
Conclusions:
- CrtM displays significant plasticity, enabling engineering for C40 carotenoid production.
- The specificity of CrtM is primarily determined by Phe 26.
- Enzyme engineering of carotene synthases offers potential for generating novel carotenoid structures.