Multi-enzymatic cascade reactions for oxyfunctionalization of alkanes employing P450 BM3 monooxygenases

Selective oxyfunctionalization of hydrocarbons is a major challenge in industrial organic synthesis. The biocatalytic activation of alkanes under mild reaction conditions, using molecular oxygen as benign oxidant, has emerged as an interesting alternative synthesis route. In this regard, cytochrome P450 monooxygenases represent a promising class of catalysts for the selective hydroxylation of non-activated carbon atoms. Successful development of biocatalysts that meet the process requirements can be achieved by combination of enzyme engineering, as well as reaction and process engineering. A novel process concept was developed based on the multi-enzymatic double oxidation of alkanes to alkanones in a one-pot synthesis, employing a cell free as well as a whole cell catalytic system that were benchmarked against each other. The enzymatic component of the catalytic cascade are a P450 BM3 monooxygenase and (S)- or (R)-selective alcohol dehydrogenases (RE-ADH and Lb-ADH). As model compounds n-heptane and cyclooctane were employed. Additionally, for n-heptane as substrate, a strategy for chiral resolution of (S)- or (R)-heptanols was developed. Initially, by means of protein engineering the monooxygenase variant P450 BM3 CM1 NADH was generated, showing a high coupling efficiency for NADH as cofactor (44%) and a high turnover frequency with the substrate n-heptane (600 1/min). The initial step of the biocatalytic oxidation cascade consists in the hydroxylation of the alkyl chain, catalyzed by a P450 BM3 monooxygenase under consumption of NAD(P)H to produce (R/S)-heptanols or cyclooctanol. In the second oxidation step and depending on the selectivity of the employed ADH, the oxidation of (S)- or (R)-heptanol or complete oxidation of cyclooctanol to the corresponding ketones is achieved. In addition, the ADH-catalyzed sequential oxidation allows the direct regeneration of the cofactor. As further extension, the complete oxidation of n-heptane employing both ADHs in a three enzyme system was achieved, allowing a shift of ...