We use the most powerful biological design process, evolution, to optimize existing enzymes and invent new ones, thereby circumventing our profound ignorance of how sequence encodes function. Starting with a subunit of the homodimeric tryptophan synthase enzyme, we evolved a set of variants able to synthesize a broad range of noncanonical tryptophan analogs, which is now used commercially at scale. The TrpS β-subunit (TrpB) leverages its pyridoxal 5’-phosphate (PLP) cofactor to perform a direct β-substitution reaction between serine and indole to make L-tryptophan (Trp), releasing water as the only byproduct. The activity of TrpB drops significantly in the absence of its allosteric activator, the α-subunit of TrpS (TrpA). Several years ago, we used directed evolution to uncover mutations that recover this lost activity and enable TrpB to be used as a standalone enzyme. This simplification gave us a platform for rapid evolution of TrpB variants for synthesis of a wide range of ncAAs, especially analogs of L-Trp. This TrpB platform is highly versatile and readily evolvable, which was not immediately apparent from TrpB’s important natural function and metabolic context. We recently discovered that a single substitution of a conserved residue in the enzyme active site unlocks activity on phenol and analogs of phenol for synthesis of derivatives of L-tyrosine. This enables us to access a whole new function space for the efficient TrpB platform, where analogs of both L-Trp and L-Tyr can be prepared in a single step from stoichiometric amounts of low-cost starting materials.

Tryptophan synthase platform for synthesis of noncanonical amino acids

ACS Spring 2022

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