Engineering membrane‐bound alkane monooxygenase from Marinobacter sp. for increased activity in the selective ω‐hy‐droxylation of linear and branched aliphatic esters

The regio‐ and stereoselective hydroxylation of unactivated C(sp 3 )‐H bonds is an important reaction in organic synthesis. While bacterial alkane monooxygenase AlkB catalyzes the terminal hydroxylation of aliphatic esters with excellent regioselectivity, the molecular principles of substrate recognition and selectivity of this integral membrane enzyme are still poorly understood. In this study, we investigated the substrate scope and engineered the medium‐chain alkane monooxygenase from Marinobacter sp. (M_AlkB) for the terminal hydroxylation of linear and branched esters of fatty acids and alcohols. For the first time, we demonstrated the stereoselectivity of AlkB toward prochiral substrates containing terminal gem ‐dimethyl groups, leading to the corresponding chiral β ‐methyl primary alcohols in good optical purity (51%–79% ee ). The hydroxylation products can be further derivatized to chiral diols and lactones. Substitution of the highly conserved active site residue F169 to leucine increased the activity toward short and medium‐chain esters up to two‐fold. While the wildtype enzyme displayed very low conversion of long‐chain substrates, activity toward n ‐dodecyl acetate could be increased 11‐fold by reducing the size of the tryptophan residue 60 situated in the putative substrate tunnel. Substitution of the peripheral I238 with valine increased activity regardless of the chain length of the substrate. Our results provide insight into the strong substrate dependency of the reported mutations and lay the groundwork for the establishment of a whole‐cell process for the regio‐ and stereoselective hydroxylation of linear and branched esters, leading to valuable bifunctionalized products. The deeper understanding gained from mutating key residues and the substrate acceptance of AlkB will guide future protein engineering campaigns.