InDel mutagenesis in active site loops to engineer enzymes

活性位点环中的 InDel 诱变以改造酶

• 批准号: 445351264
• 负责人: Professor Dr. Bernhard Hauer
• 金额: --
• 依托单位: Institut für Biotechnologie und Wirkstoff-Forschung gGmbH (ibwf)
• 依托单位国家: 德国
• 项目类别: Reinhart Koselleck Projects
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/445351264?language=en
• 关键词: InDel; mutagenesis; active; site; loops

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.

酶工程已发展成为一项成熟的技术，影响着工业生物催化、生物医学和合成生物学等各个领域。与定向进化、理性设计甚至计算从头设计等酶工程策略不同，蛋白质工程师主要依靠氨基酸取代来优化蛋白质。这与自然方法形成鲜明对比，在自然方法中，除了氨基酸取代之外，包括插入和删除（InDels）在内的主干修饰是关键因素。虽然一些单独的研究成功地应用氨基酸InDels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，我们不难相信InDel工程有潜力成为一项跨越式技术。我建议系统地探索活性位点环的InDel工程。我们将Rieske非血红素双加氧酶作为一类新兴的生物催化剂，因为我们在其活性位点环中发现了特别重要的InDels。拟议的研究将通过使用不同的策略来整合InDel工程，并通过解决酶工程中的三个不同挑战，即产品选择性，底物范围和非生理化学的获取，来验证InDel工程。所有的靶反应都具有很高的合成价值和/或环境意义。我们的目的是解释所产生的indel对酶的结构和功能的影响。