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

转酮醇酶是一种强大的不对称合成催化剂，因为它控制着最多两个立体中心的产生，但天然的底物专一性和立体选择性是有限的。我们的目标是在天然转酮醇酶的蛋白质结构信息的指导下，通过定向进化产生具有新的供体和受体底物特异性的修饰的转酮醇酶。来自聚焦和盒式随机突变的蛋白质变体将通过酶分析进行测试，以改善它们对非自然筛选探针的耐受性。作为非天然给体组分，丙酮酸和乙醛分别用于乙酰基和甲酰基转移；非天然受体特异性将被诱导为普通脂肪醛和芳香醛，以及非环和环酮作为新的底物。如果成功，由新型突变体TKS催化的此类反应将产生手性直链或支链醛和甲基酮，其特征是极性官能团密度远低于天然TK产物。突变的TK酶具有新的催化能力，将用于合成手性生物活性化合物，如硫糖、抗生素、交感神经和类固醇。这项研究应该提供具有普遍意义的知识，并拓宽目前生物催化的范围。

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.