Directed evolution of transketolase to broaden its substrate range for applications in chiral drug synthesis (deoTK)

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Transketolase is a powerful catalysts for asymmetric synthesis in that it controls the creation of up to two stereocenters, yet the natural substrate specificity and stereoselectivity are limiting. We aim to produce modified transketolases with new donor and acceptor substrate specificities by directed evolution, guided by information from protein structures of natural transketolases. Protein variants from focused and cassette random mutagenesis will be tested by enzymatic assays for their improved tolerance to non-natural screening probes. As nonnatural donor components, pyruvate and glyoxylate for acetyl and formyl transfer, respectively, are selected; non-natural acceptor specificities will be elicited for generic aliphatic and aromatic aldehydes, as well as for acyclic and cyclic ketones as novel substrates. If successful, such reactions catalyzed by novel mutant TKs will yield chiral linear or branched-chain aldehydes and methyl ketones that are characterized by a far lower density of polar functional groups than that of natural TK products. Mutant TK enzymes with novel catalytic abilities will be used in the synthesis of chiral bioactive compounds such as thiosugars, antibiotics, sympathomimetics, and steroids. This research should provide knowledge of general significance and broaden the current scope of biocatalysis.