Ancestral Sequence Reconstruction to Accelerate Non-heme Iron-dependent Biocatalyst Engineering.

Nature provides access to biological catalysts that can expand the chemical transformations accessible to synthetic chemists. Among these, α-ketoglutarate, non-heme iron-dependent (NHI) enzymes stand out as scalable biocatalysts for catalyzing selective oxidation reactions. Many NHI enzymes require protein engineering to improve their activity, selectivity, or stability. However, the reliance of this strategy on the innate stability of the enzyme can thwart the success of the engineering campaign. Harnessing innately stable enzymes can overcome these challenges and accelerate biocatalyst engineering. Herein, we highlight the use of ancestral sequence reconstruction (ASR) to mine for thermostable enzymes that can serve as superior starting points for protein engineering. In our effort to develop a biocatalytic route to tropolones, we identified an NHI enzyme that demonstrated poor stability, diminished activity at high substrate concentrations, and a limited substrate scope. We compared the in-lab evolution of the modern NHI enzyme and its ancestor, demonstrating the improved evolvability profile of the latter. By engineering the ancestral protein, we accessed variants with enhanced thermostability and expression, increased rates, and a substrate scope broader than those of their modern counterparts. Altogether, this work provides a strategy to rapidly access enzyme backbones that can accelerate engineering of more robust and synthetically useful NHI enzymes.