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

Axel Trefzer; Volker Jungmann; István Molnár; 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

doi:10.1128/AEM.02676-06

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

Axel Trefzer, Volker Jungmann, István Molnár, 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 (Corresponding Author)

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25 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 language	English
Pages (from-to)	4317-4325
Number of pages	9
Journal	Applied and Environmental Microbiology
Volume	73
Issue number	13
DOIs	https://doi.org/10.1128/AEM.02676-06
Publication status	Published - Jul 2007
MoE publication type	A1 Journal article-refereed

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Trefzer, A., Jungmann, V., Molnár, I., Botejue, A., Buckel, D., Frey, G., Hill, D. S., Jörg, M., Ligon, J. M., Mason, D., Moore, D., Pachlatko, J. P., Richardson, T. H., Spangenberg, P., Wall, M. A., Zirkle, R., & Stege, J. T. (2007). Biocatalytic conversion of avermectin to 4″-oxo-avermectin: Improvement of cytochrome P450 monooxygenase specificity by directed evolution. Applied and Environmental Microbiology, 73(13), 4317-4325. https://doi.org/10.1128/AEM.02676-06

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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 Istv{\'a}n Moln{\'a}r 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",

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doi = "10.1128/AEM.02676-06",

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Trefzer, A, Jungmann, V, Molnár, 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

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T2 - Improvement of cytochrome P450 monooxygenase specificity by directed evolution

AU - Trefzer, Axel

AU - Jungmann, Volker

AU - Molnár, István

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

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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.

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IS - 13

ER -

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

Abstract

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