Journal
CHEMCATCHEM
Volume 9, Issue 17, Pages 3338-3348Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cctc.201700620
Keywords
alcohol dehydrogenases; biocatalysis; C-H activation; enzyme cascades; P450 monooxygenases
Categories
Funding
- European Union (EU) project ROBOX under EU's Horizon 2020 Programme Research and Innovation actions [635734, H2020-LEIT BIO-2014-1]
- BBSRC [BB/K00199X/1] Funding Source: UKRI
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The chemical synthesis of ketoisophorone, a valuable building block of vitamins and pharmaceuticals, suffers from several drawbacks in terms of reaction conditions and selectivity. Herein, the first biocatalytic one-pot double oxidation of the readily available -isophorone to ketoisophorone is described. Variants of the self-sufficient P450cam-RhFRed with improved activity have been identified to perform the first step of the designed cascade (regio- and enantioselective allylic oxidation of -isophorone to 4-hydroxy--isophorone). For the second step, the screening of a broad panel of alcohol dehydrogenases (ADHs) led to the identification of Cm-ADH10 from Candida magnoliae. The crystal structure of Cm-ADH10 was solved and docking experiments confirmed the preferred position and geometry of the substrate for catalysis. The synthesis of ketoisophorone was demonstrated both as a one-pot two-step process and as a cascade process employing designer cells co-expressing the two biocatalysts, with a productivity of up to 1.4gL(-1)d(-1).
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