Synthesis of L‐Tryptophan Analogs by Ancestral L‐Tryptophan Synthase β‐Subunit with High Organic Solvent Tolerance

Abstract Organic solvent‐tolerant enzymes expand their applicability in chemical reactions involving poorly water‐soluble substrates. The development and discovery of such enzymes remains challenging, even with advanced protein engineering approaches or screening from natural sources. In this study, we explored ancestral sequence reconstruction (ASR) as an alternative approach to identifying organic solvent‐tolerant enzymes. Using L‐tryptophan synthase β‐subunit as a model, an ASR approach on vast sequence data successfully identified AncTrpB1, an ancestral L‐tryptophan synthase β‐subunit exhibiting high organic solvent tolerance in 50% v/v dimethyl sulfoxide. Furthermore, AncTrpB1 also showed thermostability, high soluble expression levels, comparable catalytic activity to extant TrpBs, and broad substrate scope. X‐ray crystallographic analysis of AncTrpB1 suggests the formation of salt bridges at the dimer interface as a plausible factor for its organic solvent tolerance. The excellent properties of AncTrpB1, especially its organic solvent tolerance, enabled the high‐yield synthesis (∼1 g/10 mL) of L‐tryptophan analogs. Our findings demonstrate the broader applicability of ASR for developing practical enzymes.