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Molecular breeding of genes, pathways and genomes by DNA shuffling

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Abstract

Existing methods for optimization of sequences by random mutagenesis generate libraries with a small number of mostly deleterious mutations, resulting in libraries containing a large fraction of non-functional clones that explore only a small part of squence space. Large numbers of clones need to be screened to find the rare mutants with improvements. Library display formats are useful to screen very large libraries but impose screening limitations that limit the value of this approach for most commercial applications. By contrast, in both classical breeding and in DNA shuffling, natural diversity is permutated by homologous recombination, generating libraries of very high quality, from which improved clones can be identified with a small number of complex screens. Given that this small number of screens can be performed under the conditions of actual use of the product, commercially relevant improvements can be reliably obtained.

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References

  1. Stemmer, W. P. C. (1995) Searching sequence space.Bio/Technology 13: 549–553.

    Article  CAS  Google Scholar 

  2. Stemmer, W. P. C. (1994) DNA shuffling by random fragmentation and reassembly:In vitro recombination for molecular evolution.Proc. Natl. Acad. Sci. USA 91: 10747–10751.

    Article  CAS  Google Scholar 

  3. Stemmer, W. P. C. (1994) Rapid evolution of a proteinin vitro by DNA shuffling.Nature 370: 389–391.

    Article  CAS  Google Scholar 

  4. Crameri, A., E. Whitehorn, E. Tate, and W. P. C. Stemmer (1996) Improved green fluorescent protein by molecular evolution using DNA shuffling.Nature Biotechnol. 14: 315–319.

    Article  CAS  Google Scholar 

  5. Zhang, J., G. Dawes, and W. P. C. Stemmer (1997) Evolution of an effective fucosidase from a galactosidase by DNA shuffling and screening.Proc. Natl. Acad. Sci. USA 94: 4504–4509.

    Article  CAS  Google Scholar 

  6. Crameri, A., G. Dawes, E. Rodriguez, S. Silver, and W. P. C. Stemmer (1997) Molecular evolution of an arsenate detoxification pathway by DNA shuffling.Nature Biotechnol. 15: 436–438.

    Article  CAS  Google Scholar 

  7. Crameri, A., S.-A. Raillard, E. Bermudez, and W. P. C. Stemmer (1998) DNA shuffling of genes from diverse species accelerates directed evolution.Nature 391: 288–291.

    Article  CAS  Google Scholar 

  8. Stemmer, W. P. C., A. Crameri, K. D. Ha, T. M. Brennan, and H. L. Heyneker (1995) Single-step PCR assembly of a gene and a whole plasmid from large numbers of oligonucleotides.Gene 164: 49–53.

    Article  CAS  Google Scholar 

  9. Christians, F. C., L. Scapozza, A. Crameri, G. Folkers, and W. P. C. Stemmer (1999) Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling.Nature Biotechnol. 17: 259–264.

    Article  CAS  Google Scholar 

  10. Ness, J., M. Welch, L. Giver, M. Bueno, J. Cherry, T. Borchert, W. P. C. Stemmer, and J. Minshull (1999) Creation of a functionally diverse enzyme library by DNA family shuffling.Nature Biotechnol. 17: 893–896.

    Article  CAS  Google Scholar 

  11. Chang, C.-C., T. T. Chen, B. W. Cox, G. N. Dawes, W. P. C. Stemmer, J. Punnonen, and P. A. Patten (1999) Rapid evolution of a cytokine using molecular breeding.Nature Biotechnol. 17: 793–797.

    Article  CAS  Google Scholar 

  12. Soong, N.-W., L. Nomura, K. Pekrun, M. Reed, L. Sheppard, G. Dawes, and W. P. C. Stemmer (2000) Mole-cular breeding of viruses.Nature Genetics 25: 436–439.

    Article  CAS  Google Scholar 

  13. Powell, S. K., M. A. Kaloss, A. Pinkstaff, R. McKee, I. Burimski, M. Pensiero, E. Otto, W. P. C. Stemmer, and N.-W. Soong (2000) Breeding of retroviruses by DNA shuffling for improved stability and processing yields.Nature Biotechnol. 18: 1279–1282.

    Article  CAS  Google Scholar 

  14. Stemmer, W. P. C. and N.-W. Soong (1999) Molecular breeding of viruses for targeting and other clinical properties.Tumor Targeting 4: 59–62.

    Google Scholar 

  15. Zhang, Y.-X., K. Perry, V. A. Vinci, K. Powell, W. P. C. Stemmer, and S. B. del Cardayre (2002) Genome shuffling leads to rapid phenotypic improvement in bacteria.Nature 415: 644–646.

    Article  CAS  Google Scholar 

  16. Raillard, S., A. Krebber, Y. Chen, J. E. Ness, E. Bermudez, R. Trinidad, R. Fullem, C. Davis, M. Welch, J. Seffernick, L. P. Wackett, W. P. C. Stemmer, and J. Minshull (2001) Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes.Chemistry Biol. 125: 1–9.

    Google Scholar 

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  1. Maxygen, Inc., 515 Galveston Drive, 940663, Redwood City, CA, USA

    Willem P. C. Stemmer

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  1. Willem P. C. Stemmer
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Correspondence to Willem P. C. Stemmer.

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Stemmer, W.P.C. Molecular breeding of genes, pathways and genomes by DNA shuffling. Biotechnol. Bioprocess Eng. 7, 121–129 (2002). https://doi.org/10.1007/BF02932909

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  • DOI: https://doi.org/10.1007/BF02932909

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