Although ASR has been successfully used for single domain proteins, less work has been carried out on multi-domain enzymes. CYP116B enzymes are multi-domain, Class VII P450s: electrons from NAD(P)H are transferred to the P450 haem via a fused reductase domain and an iron-sulfur ferredoxin. These are 'self-sufficient' enzymes, where the expressed proteins can be directly assayed for bioactivity without the need for additional redox partners . The proximity of the reductase and haem domains facilitates electron shuttling and enables a more effective transfer, enhancing their potential as biocatalysts . Therefore, we wanted to investigate whether ASR was also an effective engineering approach when applied to the three-domain protein of the CYP116B family, and whether the individual domains would retain folding and activity when inferred together.