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

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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.