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Applied and Environmental Microbiology, July 2007, p. 4317-4325, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.02676-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biocatalytic Conversion of Avermectin to 4''-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution ^,

Axel Trefzer,^1, Volker Jungmann,² István Molnár,^3, Ajit Botejue,¹ Dagmar Buckel,^2,¶ Gerhard Frey,¹ D. Steven Hill,^3,|| Mario Jörg,² James M. Ligon,^3,|| Dylan Mason,^1, David Moore,^1, J. Paul Pachlatko,^2, Toby H. Richardson,^1,¶¶ Petra Spangenberg,^2,|||| Mark A. Wall,¹ Ross Zirkle,^3, and Justin T. Stege^1*

Diversa Corporation, 4955 Directors Place, San Diego, California 92121,¹ Syngenta Crop Protection AG, Schwarzwaldallee 215, CH-4002 Basel, Switzerland,² Syngenta Biotechnology, Inc., 3054 Cornwallis Rd., Research Triangle Park, North Carolina 27709³

Received 16 November 2006/ Accepted 28 April 2007

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4''-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4''-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.

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* Corresponding author. Mailing address: Diversa Corporation, 4955 Directors Place, San Diego, CA 92121. Phone: (858) 526-5275. Fax: (585) 526-5775. E-mail: jstege{at}diversa.com

Published ahead of print on 4 May 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: DSM Anti-Infectives B.V., P.O. Box 425, 2600 AK Delft, The Netherlands.

Present address: University of Arizona, 250 E. Valencia Rd., Tucson, AZ 85706.

^¶ Present address: Bergfriedweg 1b, 79541 Lörrach, Germany.

^|| Present address: BASF Agricultural Products, 26 Davis Drive, Research Triangle Park, NC 27709.

Present address: 729 Oakbranch Drive, Encinitas, CA 92024.

Present address: 7696 Andasol St., San Diego, CA 92126.

Present address: Bölchenstrasse 21, CH-4411 Seltisberg, Switzerland.

^¶¶ Present address: Synthetic Genomics Inc., 11149 N. Torrey Pines Rd., La Jolla, CA 92037.

^|||| Present address: Haselbergstr. 2, 82377 Penzberg, Germany.

Present address: Martek Biosciences, 4909 Nautilus Ct. North, Boulder, CO 80301.

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Applied and Environmental Microbiology, July 2007, p. 4317-4325, Vol. 73, No. 13
0099-2240/07/$08.00+0     doi:10.1128/AEM.02676-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.