Directed evolution of transaldolases for novel specificities

醛缩酶是用于不对称合成的强有力催化剂，因为它们控制多达两个新的立体中心的产生，但它们的天然底物特异性和立体选择性是有限的。我们的目标是通过定向进化产生具有新的供体底物特异性和非天然立体特异性的修饰的转醛醇酶，由来自天然转醛醇酶的蛋白质结构的信息指导。由于机械的考虑，这样的蛋白质修饰必须涉及活性位点突变，这通常会对催化活性有害，因此需要通过额外的良性序列变化来调节催化残基。为了产生多样性，将应用DNA（家族）改组、epPCR和局部盒式诱变来取样足够大的功能序列空间。该项目还将开发用于新型立体选择性羟醛缩合反应的有效筛选方法，并系统地探索突变型转醛醇酶用于不对称合成对映体纯结构单元的能力。预期的机械的洞察力，以及功能工程，生物催化的C-C键形成反应的立体分化控制因素，应提供具有普遍意义的知识，并扩大目前的范围生物催化。

Aldolases are powerful catalysts for asymmetric synthesis in that they control the creation of up to two new stereocenters, yet their natural substrate specificities and stereoselectivities are limiting. We aim to produce modified transaldolases with new donor substrate specificities and unnatural stereo-specificities by directed evolution, guided by information from protein structures of natural transaldolases. Because of mechanistic considerations, such protein modifications must involve active-site mutations that usually will be deleterious to catalytic activity, and therefore will require adjustment of catalytic residues by additional benign sequence changes. For diversity generation, DNA (family) shuffling, epPCR and localized cassette mutagenesis will be applied to sample a sufficiently large functional sequence space. This project will also develop efficient screening assays for novel, stereoselective aldol reactions and explore systematically the capability of mutant transaldolases for the asymmetric synthesis of enantiopure building blocks. The anticipated mechanistic insight in, as well as the functional engineering of, factors controlling stereodifferentiation of biocatalytic C-C bond forming reactions should provide knowledge of general significance and broaden the current scope of biocatalysis.