InDel mutagenesis in active site loops to engineer enzymes

酶工程领域发展到一个成熟的技术，影响到各个领域，如工业生物催化，生物医学和合成生物学。蛋白质工程师主要依靠氨基酸替换来优化蛋白质，而不依赖于定向进化、理性设计甚至计算从头酶设计等酶工程策略。这与natures方法形成鲜明对比，在natures方法中，除了氨基酸取代之外，包括插入和缺失（InDel）在内的骨架修饰是关键因素。虽然一些个别研究成功地应用氨基酸InDel来改善酶，但我们缺乏对这种方法的系统探索。鉴于InDel在自然进化中的成功，不难相信InDel工程有潜力成为一项跨越式技术。我建议系统地探讨InDel工程的活性位点循环。我们专注于Rieske非血红素双加氧酶作为新兴的一类生物催化剂，因为我们发现特别重要的InDel在其活性位点循环。拟议的研究将通过使用不同的策略来整合InDel，并通过解决酶工程中的三个不同挑战来验证InDel工程，即产品选择性，底物范围和非生理化学。所有目标反应都具有高合成价值和/或环境意义。我们的目标是解释所产生的InDels的酶的结构和功能的后果。

The field of enzyme engineering developed to a mature technology that impacts various areas such as industrial biocatalysis, biomedicine and synthetic biology. Independent of the enzyme engineering strategy, such as directed evolution, rational design or even computational de novo enzyme design, protein engineers mainly rely on amino acid substitutions to optimize proteins. This is in stark contrast to natures approach, where next to amino acid substitutions, backbone modifications including insertions and deletions (InDels) are key players. While a few individual studies successfully applied amino acid InDels to improve enzymes, we lack a systematic exploration of this approach. Given the success of InDels in natural evolution, it is not difficult to believe that InDel engineering has the potential to become a leapfrog technology. I propose to systematically explore InDel engineering of active site loops. We focus on Rieske non-heme dioxygenases as emerging class of biocatalysts since we found particularly significant InDels in their active site loops. The proposed research will validate InDel engineering by using different strategies to incorporate InDels and by addressing three different challenges in enzyme engineering, namely product selectivity, substrate scope and access to non-physiological chemistry. All target reactions are of high synthetic value and/or of environmental significance. We aim to explain the consequences of the generated InDels for enzyme structure and function.