Ancestral sequence reconstruction for protein engineers

In addition to its value in the study of molecular evolution, ancestral sequence reconstruction (ASR) has emerged as a useful methodology for engineering proteins with enhanced properties. Proteins generated by ASR often exhibit unique or improved activity, stability, and/or promiscuity, all of which are properties that are valued by protein engineers. Comparison between extant proteins and evolutionary intermediates generated by ASR also allows protein engineers to identify substitutions that have contributed to functional innovation or diversification within protein families. As ASR becomes more widely adopted as a protein engineering approach, it is important to understand the applications, limitations, and recent developments of this technique. This review highlights recent exemplifications of ASR, as well as technical aspects of the reconstruction process that are relevant to protein engineering. [Display omitted] •Proteins produced by ancestral sequence reconstruction (ASR) often exhibit high activity, stability, and evolvability.•ASR studies guide engineering by highlighting function-shifting substitutions.•ASR emphasizes fundamental drivers of, and constraints to, functional innovation.•Recent developments in ASR are making it more accessible to protein engineers.