ASR has been widely used for single domain proteins , but enzymes composed of multiple domains linked by highly linkers, present a particular challenge since these regions influence conformational dynamics (e.g., needed for efficient electron transfer) but are also inferred with least confidence in the ASR. Here we show that ASR can also be applied successfully to the three-domain family of self-sufficient CYP116B monooxygenases, generating self-sufficient single polypeptide ancestral proteins that are all properly folded, with electron transfer across the domains and enzymatic activity retained. Conducting ASR on the full length CYP116B system has several advantages, including that the activity of the P450 domain can be directly evaluated because all ancestors are self-sufficient and do not need to be reconstituted. Furthermore, one single series of experiments can yield new synthetic reductases as well as P450s, the activity of which is verified by the ability to support P450 activity in each ancestor. These synthetic reductases should be a useful addition to natural sequences available for engineering self-sufficient fused chimeric enzyme systems , an approach which has become a very popular for generating active P450s.