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

色氨酸合酶α亚基的定向进化以设计用于有机合成的C-C键形成酶

• 批准号: 387150532
• 负责人: Dr. Markus Dick, Ph.D.
• 金额: --
• 依托单位: Division of Chemistry and Chemical Engineering
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/387150532?language=en
• 关键词: Directed; evolution; tryptophan; synthase; alpha

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.

在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主要由碳原子组成。因此，开发用于该反应的新催化剂一直是至关重要的。作为纯化学催化剂的强有力的替代品，生物催化反应近年来已经建立，因为它们具有生产成本低、毒性小和反应条件温和的优点。然而，用于合成应用的C-C键形成酶的种类仅限于特定的底物种类，因此，在本项目中将建立一种新的酶，其首次催化环状不饱和化合物与醛的亲核加成。具体地，该酶是色氨酸合酶的α-亚基（TrpA），其在自然界中催化吲哚-3-甘油磷酸可逆裂解为D-甘油醛-3-磷酸和吲哚（其然后用作β-亚基的底物），因此也允许在合成方向上的逆反应。虽然TrpA已被研究的机制，它从来没有被表征或优化的生物技术应用。因此，本项目的目的是设计醛和吲哚衍生物的酶促反应以形成3-（1-羟烷基）吲哚。这将使吲哚生物碱的大底物类别的手性构件的简单合成路线成为可能，吲哚生物碱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的TrpAs，其具有热稳定性，因此在修饰方面更稳健。首先，独立酶（不与β亚基形成复合物）的活性应因其自然反应而增加。接下来，我们的目标是扩大TrpA的底物谱。在第一步中，我们将尝试增加其对非磷酸化醛的活性，因为磷酸基团在合成应用中具有几个缺点（例如，由于其不稳定性）。在此基础上，将评估几种供体和受体分子的TrpA底物范围，并设计酶变体，其可以转化更广泛的底物谱。在这个项目的最后，一个不同的TrpA变体的库将是可用的，使吲哚与各种醛的立体选择性连接。为了达到上述目标，选择的方法是定向进化，允许通过随机诱变产生广泛的TrpA突变体，可以测试，例如与新底物的转化率相关。将选择最佳变体用于新一轮诱变。作为支持，应使用文献中的序列和结构信息（例如，针对非磷酸化醛优化的相关酶）进行合理的方法。