Indigo production identifies hotspots in cytochrome P450 BM3 for diversifying aromatic hydroxylation
Evolution of P450 BM3 is a topic of extensive research, but screening the various substrate/reaction combinations remains a time-consuming process. Indigo production has the potential to serve as a simple high-throughput method for reaction screening, as bacterial colonies expressing indigo (+) vari...
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Veröffentlicht in: | Faraday discussions 2024-09, Vol.252, p.29-51 |
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Sprache: | eng |
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Zusammenfassung: | Evolution of P450 BM3 is a topic of extensive research, but screening the various substrate/reaction combinations remains a time-consuming process. Indigo production has the potential to serve as a simple high-throughput method for reaction screening, as bacterial colonies expressing indigo (+) variants can be visually identified
via
their blue phenotype. Indigo (+) single variants, indigo (−) single variants and a combinatorial library, containing mutations that enable the blue phenotype, were screened for their ability to hydroxylate a panel of 12 aromatic compounds using the 4-aminoantipyrine colorimetric assay. Recombination of indigo (+) single variants to create a multiple-variant library is a particularly useful strategy, as all top performing P450 BM3 variants with high hydroxylation activity were either indigo (+) single variants or contained multiple substitutions. Furthermore, active variants, as determined using the 4-AAP assay, were further characterized and several variants were identified that gave more than 90% conversion with 1,3-dichlorobenzene and predominantly formed 2,6-dichlorophenol; other variants showed significant substrate selectivity. This supports the hypothesis that substitution at positions that enable the indigo (+) phenotype, or hotspot residues, is a general mechanism for increasing aromatic hydroxylation activity. Overall, this research demonstrates that indigo (+) single variants, identified
via
colorimetric colony-based screening, may be recombined to generate a multiply-substituted variant library containing many variants with high aromatic hydroxylation activity. The combination of colony-based screening and other screening assays greatly accelerates enzyme engineering, as readily-identified indigo (+) single variants can be recombined to create a library of active multiple variants without extensive screening of single variants.
Indigo (+) and indigo (−) single variants and a combinatorial library, with mutations that enable the blue phenotype, were screened for their ability to hydroxylate a panel of 12 aromatic compounds using the 4-aminoantipyrine colorimetric assay. |
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ISSN: | 1359-6640 1364-5498 1364-5498 |
DOI: | 10.1039/d4fd00017j |