A tailored cytochrome P450 monooxygenase from <i>Gordonia rubripertincta</i> CWB2 for selective aliphatic monooxygenation

Cytochrome P450 monooxygenases are recognized as versatile biocatalysts due to their broad reaction capabilities. One important reaction is the hydroxylation of non-activated C-H bonds. The subfamily CYP153A is known for terminal hydroxylation reactions, giving access to functionalized aliphatics. Whilst fatty derivatives may be converted by numerous enzyme classes, midchain aliphatics are seldomly accepted, a prime property of CYP153As. We report here on a new CYP153A member from the genome of the mesophilic actinobacterium <i>Gordonia rubripertincta</i> CWB2 as an efficient biocatalyst. The gene was overexpressed in <i>Escherichia coli</i> and fused with a surrogate electron transport system from <i>Acinetobacter</i> sp. OC4. This chimeric self-sufficient whole-cell system could perform hydroxylation and epoxidation reactions: conversions of C6-C14 alkanes, alkenes, alcohols and of cyclic compounds were observed, yielding production rates of, <i>e</i>.<i>g</i>., 2.69 mM h^-1 for 1-hexanol and 4.97 mM h^-1 for 1,2-epoxyhexane. Optimizing the linker compositions between the protein units led to significantly altered activity. Balancing linker length and flexibility with glycine-rich and helix-forming linker units increased 1-hexanol production activity to 350 % compared to the initial linker setup with entirely helical linkers. The study shows that strategic coupling of efficient electron supply and a selective enzyme enables previously challenging monooxygenation reactions of midchain aliphatics.