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DNA shuffling method for generating highly recombined genes and evolved enzymes
W M Coco1, W E Levinson, M J Crist
1Enchira Biotechnology Corporation, 4200 Research Forest Drive, The Woodlands, TX 77381, USA. wcoco@enrichira.com
Nature Biotechnology
|April 3, 2001
Summary
We developed DNA shuffling, a novel in vitro recombination method to create diverse enzyme libraries. This technique achieves higher recombination rates and generates unique gene chimeras for improved enzyme function.
Area of Science:
- Biotechnology
- Molecular Biology
- Enzyme Engineering
Background:
- Enzyme evolution is crucial for developing biocatalysts with novel functions.
- Existing gene shuffling methods have limitations in recombination efficiency and chimera diversity.
Purpose of the Study:
- To introduce an improved in vitro recombination method, termed DNA shuffling, for generating diverse enzyme libraries.
- To demonstrate the efficacy of DNA shuffling in molecular breeding for enhanced enzyme performance.
Main Methods:
- DNA shuffling involves ordering, trimming, and joining randomly cleaved parental DNA fragments.
- A transient polynucleotide scaffold facilitates the annealing of DNA fragments.
- Chimeric libraries are generated with a high average of 14.0 crossovers per gene.
Main Results:
- The method achieved significantly higher recombination levels compared to other techniques.
- Unprecedented recombination rates were observed even in short regions of sequence identity.
- The engineered monooxygenase showed increased biodesulfurization and faster conversion of nonnatural substrates.
Conclusions:
- DNA shuffling is a powerful and distinct alternative to sexual PCR for gene family shuffling.
- This method enables the generation of unique enzyme chimeras with tailored properties.
- The approach has practical applications in directed evolution and biocatalyst development.
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