Directed evolution of a tryptophan synthase alpha-subunit to engineer a C-C bond-forming enzyme for organic synthesis

立体选择性控制下的C-C键形成可能是有机合成中最重要的反应，因为所有有机化合物的基本结构都主要由碳原子组成。因此，开发用于该反应的新型催化剂一直是至关重要的。生物催化反应具有生产成本低、毒性小、反应条件温和等优点，是纯化学催化剂的有力替代品。然而，用于合成的C-C-键形成酶的种类仅限于特定的底物类别。因此，在本项目中，将建立一种新的酶，它首次催化环状不饱和化合物与醛的亲核加成反应。具体地说，这种酶是色氨酸合成酶的阿尔法亚单位(TRPA)，在自然界中，它催化吲哚-3-甘油磷酸可逆裂解成D-甘油醛-3-磷酸和吲哚(然后用作β-亚基的底物)，因此也允许合成方向的反向反应。虽然对TRPA进行了机械性的研究，但从未针对生物技术应用对其进行表征或优化。因此，本项目的目标是设计醛和吲哚衍生物的酶反应，以生成3-(1-羟基烷基)吲哚。这将使大底物类吲哚生物碱的手性构建块的合成路线变得容易，它代表了各种生物活性化合物的基础。起点将是来自嗜热生物体的TrPA，它们是耐热的，因此在修饰方面更加健壮。首先，独立酶的活性(不与β-亚基形成络合物)应因其自然反应而增加。下一步，我们的目标是扩大TRPA的底物光谱。在第一步中，我们将尝试增加它对非磷酸化醛的活性，因为磷酸基团在合成应用中有几个缺点(例如，由于它的不稳定性)。在此基础上，将对几种供体和受体分子的TRPA底物范围进行评估，并设计出可以转换更广泛底物谱的酶变体。在该项目结束时，将有一系列不同的TRPA变体，使吲哚与各种醛的立体选择性连接成为可能。为了实现上述目标，选择的方法是定向进化，它允许通过随机突变产生广泛的TRPA突变体，这可以进行测试，例如与新底物的转化率有关。将选出最好的变种进行新一轮的突变。作为支持，合理的方法应使用文献中的顺序和结构信息(例如，针对非磷酸化醛进行优化的相关酶)。

C-C bond formation under stereoselective control is probably the most important reaction in organic synthesis, as the basic structure of all organic compounds consists mainly of carbon atoms. Thus, the development of new catalysts for this reaction has always been of utmost importance. As powerful alternative to purely chemical catalysts, biocatalytic reactions have been established in recent years as they benefit from low production costs, little toxicity, and mild reaction conditions. However, the variety of C-C-bond-forming enzymes for synthetic applications is limited to specific substrate classes.Thus, in this project a new enzyme shall be established, which for the first time catalyzes a nucleophilic addition of a cyclic, unsaturated compound to an aldehyde. Specifically, this enzyme is the alpha-subunit (TrpA) of the tryptophan synthase which in nature catalyzes a reversible cleavage of indole-3-glycerol phosphate to D-glyceraldehyde-3-phosphate and indole (which is then used as substrate for the β-subunit) and thus also allows the reverse reaction in synthetic direction. While TrpA has been investigated mechanistically, it has never been characterized or optimized for biotechnological applications. Hence, the aim of this project is to engineer the enzymatic reaction of aldehydes and indole derivatives to form 3-(1-hydroxyalkyl) indoles. This will enable an easy synthetic route for chiral building blocks of the large substrate class of indole alkaloids, which represents the basis of various biologically active compounds.The starting point will be TrpAs from thermophilic organisms, which are thermostable and thus more robust with respect to modifications. At first, the activity of the stand-alone enzyme (without complex-formation with the beta-subunit) shall be increased for its natural reaction. Next, we aim at expanding the substrate spectrum of TrpA. In the first step, we will attempt to increase its activity for non-phosphorylated aldehydes as the phosphate group has several disadvantages in synthetic applications (e.g. due to its instability). Building on this, the substrate scope of TrpA for several donor and acceptor molecules will be evaluated and enzyme variants engineered, which can convert a broader substratespectrum. At the end of this project, a repertoire of different TrpA variants will be available that enables the stereoselective connection of indoles with various aldehydes.To reach the above-mentioned goals the method of choice is directed evolution that allows creating a wide range of TrpA mutants via random mutagenesis, which can be tested e.g. related to the conversion rate of new substrates. The best variants will be chosen for a new round of mutagenesis. As support, sequential and structural information from literature (e.g. related enzymes that were optimized for non-phosphorylated aldehydes) shall be used for a rational approach.