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Applied and Environmental Microbiology, September 2001, p. 3882-3887, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.3882-3887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

Takeshi Sakamoto,dagger John M. Joern, Akira Arisawa,Dagger and Frances H. Arnold*

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125

Received 31 January 2001/Accepted 31 May 2001

We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ~4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ~20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.

* Corresponding author. Mailing address: Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology, Pasadena, CA 91125. Phone: (626)-395-4162. Fax: (626)-568-8743. E-mail: frances{at}cheme.caltech.edu.

dagger Present address: Biochemicals Laboratory, Yokohama Research Center, Mitsubishi Chemical Corp., Yokohama, Kanagawa 227-8502, Japan.

Dagger Present address: Central Research Laboratories, Mercian Corp., Fujisawa, Kanagawa 251-0057, Japan.

Applied and Environmental Microbiology, September 2001, p. 3882-3887, Vol. 67, No. 9
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.9.3882-3887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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