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Antimicrobial Agents and Chemotherapy, September 2002, p. 3035-3038, Vol. 46, No. 9
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.9.3035-3038.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

Barry G. Hall*

Biology Department, University of Rochester, Rochester, New York 14627-0211

Received 18 October 2001/ Returned for modification 9 March 2002/ Accepted 16 May 2002

In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 ß-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

* Mailing address: Biology Department, Hutchison Hall, University of Rochester, Rochester, NY 14627-0211. Phone: (585) 275-0721. Fax: (585) 275-2070. E-mail: drbh{at}mail.rochester.edu.

Antimicrobial Agents and Chemotherapy, September 2002, p. 3035-3038, Vol. 46, No. 9
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.9.3035-3038.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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