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Engineering a beta-carotene ketolase for astaxanthin production
Luan Tao1, Jolanta Wilczek, J Martin Odom
1Biological and Chemical Sciences and Engineering, Central Research and Development, E. I. DuPont de Nemours Inc., Wilmington, DE 19880-0328, USA.
Metabolic Engineering
|August 8, 2006
Summary
Protein engineering of the beta-carotene ketolase (crtW) gene enhanced astaxanthin production. Mutations improved enzyme activity, with combined mutations yielding the highest yields for this valuable carotenoid.
Area of Science:
- Biotechnology
- Enzyme Engineering
- Microbial Biochemistry
Background:
- Astaxanthin is a valuable carotenoid with significant commercial applications.
- Beta-carotene ketolase (crtW) is a key enzyme in astaxanthin biosynthesis.
- Improving crtW enzyme activity is crucial for efficient astaxanthin production.
Purpose of the Study:
- To engineer the beta-carotene ketolase (crtW) gene for enhanced astaxanthin production.
- To develop a high-throughput screening method for identifying improved crtW variants.
- To investigate the effects of specific mutations on enzyme activity and carotenoid production.
Main Methods:
- Cloning of the crtW gene from Sphingomonas sp. DC18.
- Development of a colorimetric screening assay for enzyme activity.
- Localized random mutagenesis of the crtW gene and its ribosomal binding site (RBS).
- Characterization of mutant enzymes and their impact on carotenoid profiles.
Main Results:
- Six beneficial mutations within the crtW gene were identified (H96L, R203W, A205V, A208V, F213L, A215T).
- A combination of R203W/F213L mutations significantly boosted astaxanthin production.
- A mutation in the RBS (a438t) further enhanced astaxanthin yields.
- Mutants showed varying substrate specificities, with some exhibiting reduced activity on beta-carotene for canthaxanthin production.
Conclusions:
- Protein engineering strategies, including targeted mutagenesis and RBS optimization, can effectively enhance astaxanthin production via crtW.
- Specific mutations near conserved histidine motifs are critical for improving enzyme function.
- The developed screening method provides a robust platform for directed evolution of carotenoid biosynthetic enzymes.
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