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DNA shuffling method for generating highly recombined genes and evolved enzymes

Nature Biotechnology volume 19, pages 354–359 (2001)Cite this article

Abstract

We introduce a method of in vitro recombination or “DNA shuffling” to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated “sibling” chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.

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Figure 1: Random chimeragenesis on transient templates (RACHITT).
Figure 2: RACHITT generates highly mosaic shuffled proteins.
Figure 3: RACHITT-derived chimeric genes exhibit high-frequency recombination at closely spaced parental sequence polymorphisms (alleles).
Figure 4: Rate screen of the dszC chimeric library reveals improved clones.
Figure 5: Clones simultaneously improved for both rate and extent of substrate oxidation are identified by sequential selection and screening.
Figure 6: Evolution of substrate range. (A) A high level of phenotypic diversity is generated from the chimeric library.

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Acknowledgements

We thank the Enchira high-throughput screening and analytical chemistry groups for expert technical assistance; G. Mrachko, B. Blattmann, J. Arensdorf, E. Lange, J. Blanton, B. Folsom, U. Coco, J. Bryson, and L. Encell for developing additional assays, assistance with figures, statistical analyses and critical comments on the manuscript.

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  1. Charles H. Squires

    Present address: The Dow Chemical Company, 5501 Oberlin Dr., San Diego, CA, 92121

Authors and Affiliations

  1. Enchira Biotechnology Corporation, 4200 Research Forest Drive, The Woodlands, 77381, TX

    Wayne M. Coco, William E. Levinson, Michael J. Crist, Harm J. Hektor, Aldis Darzins, Philip T. Pienkos, Charles H. Squires & Daniel J. Monticello

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Correspondence to Wayne M. Coco.

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Coco, W., Levinson, W., Crist, M. et al. DNA shuffling method for generating highly recombined genes and evolved enzymes. Nat Biotechnol 19, 354–359 (2001). https://doi.org/10.1038/86744

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