Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Improvement of cytochrome P450 monooxygenase specificity by directed evolution

Axel Trefzer, Volker Jungmann, Istvan Molnar, Ajit Botejue, Dagmar Buckel, Gerhard Frey, D. Steven Hill, Mario Jörg, James M. Ligon, Dylan Mason, David Moore, J. Paul Pachlatko, Toby H. Richardson, Petra Spangenberg, Mark A. Wall, Ross Zirkle, Justin T. Stege

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4317-4325
Number of pages9
JournalApplied and Environmental Microbiology
Volume73
Issue number13
DOIs
StatePublished - Jul 2007

Fingerprint

avermectins
Mixed Function Oxygenases
cytochrome P-450
Cytochrome P-450 Enzyme System
cytochrome
enzyme
Synthetic Genes
synthetic genes
Molecular Evolution
site-directed mutagenesis
Electron Transport Complex IV
Enzymes
protein structure
Insecticides
enzymes
Site-Directed Mutagenesis
mutagenesis
Mutagenesis
Genetic Recombination
recombination

ASJC Scopus subject areas

  • Environmental Science(all)
  • Biotechnology
  • Microbiology

Cite this

Biocatalytic conversion of avermectin to 4″-oxo-avermectin : Improvement of cytochrome P450 monooxygenase specificity by directed evolution. / Trefzer, Axel; Jungmann, Volker; Molnar, Istvan; Botejue, Ajit; Buckel, Dagmar; Frey, Gerhard; Hill, D. Steven; Jörg, Mario; Ligon, James M.; Mason, Dylan; Moore, David; Pachlatko, J. Paul; Richardson, Toby H.; Spangenberg, Petra; Wall, Mark A.; Zirkle, Ross; Stege, Justin T.

In: Applied and Environmental Microbiology, Vol. 73, No. 13, 07.2007, p. 4317-4325.

Research output: Contribution to journalArticle

Trefzer, A, Jungmann, V, Molnar, I, Botejue, A, Buckel, D, Frey, G, Hill, DS, Jörg, M, Ligon, JM, Mason, D, Moore, D, Pachlatko, JP, Richardson, TH, Spangenberg, P, Wall, MA, Zirkle, R & Stege, JT 2007, 'Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Improvement of cytochrome P450 monooxygenase specificity by directed evolution', Applied and Environmental Microbiology, vol. 73, no. 13, pp. 4317-4325. https://doi.org/10.1128/AEM.02676-06
Trefzer, Axel ; Jungmann, Volker ; Molnar, Istvan ; Botejue, Ajit ; Buckel, Dagmar ; Frey, Gerhard ; Hill, D. Steven ; Jörg, Mario ; Ligon, James M. ; Mason, Dylan ; Moore, David ; Pachlatko, J. Paul ; Richardson, Toby H. ; Spangenberg, Petra ; Wall, Mark A. ; Zirkle, Ross ; Stege, Justin T. / Biocatalytic conversion of avermectin to 4″-oxo-avermectin : Improvement of cytochrome P450 monooxygenase specificity by directed evolution. In: Applied and Environmental Microbiology. 2007 ; Vol. 73, No. 13. pp. 4317-4325.
@article{bcb25d6d3e74493caf662525a2f4efdd,
title = "Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Improvement of cytochrome P450 monooxygenase specificity by directed evolution",
abstract = "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.",
author = "Axel Trefzer and Volker Jungmann and Istvan Molnar and Ajit Botejue and Dagmar Buckel and Gerhard Frey and Hill, {D. Steven} and Mario J{\"o}rg and Ligon, {James M.} and Dylan Mason and David Moore and Pachlatko, {J. Paul} and Richardson, {Toby H.} and Petra Spangenberg and Wall, {Mark A.} and Ross Zirkle and Stege, {Justin T.}",
year = "2007",
month = "7",
doi = "10.1128/AEM.02676-06",
language = "English (US)",
volume = "73",
pages = "4317--4325",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "13",

}

TY - JOUR

T1 - Biocatalytic conversion of avermectin to 4″-oxo-avermectin

T2 - Improvement of cytochrome P450 monooxygenase specificity by directed evolution

AU - Trefzer, Axel

AU - Jungmann, Volker

AU - Molnar, Istvan

AU - Botejue, Ajit

AU - Buckel, Dagmar

AU - Frey, Gerhard

AU - Hill, D. Steven

AU - Jörg, Mario

AU - Ligon, James M.

AU - Mason, Dylan

AU - Moore, David

AU - Pachlatko, J. Paul

AU - Richardson, Toby H.

AU - Spangenberg, Petra

AU - Wall, Mark A.

AU - Zirkle, Ross

AU - Stege, Justin T.

PY - 2007/7

Y1 - 2007/7

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=34447519310&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34447519310&partnerID=8YFLogxK

U2 - 10.1128/AEM.02676-06

DO - 10.1128/AEM.02676-06

M3 - Article

C2 - 17483257

AN - SCOPUS:34447519310

VL - 73

SP - 4317

EP - 4325

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 13

ER -