Design and Evolution of Enzymes with Non-Canonical Catalytic Mechanisms

为任何所需的转化合理设计酶的能力将对制药和更广泛的化学部门产生重大影响，导致更有效和更可持续的合成路线获得高价值化学品。计算酶设计和定向进化的结合是目前实现这一雄心的最有吸引力的策略。然而，遗传密码提供的有限范围的官能团(即20个氨基酸)限制了可以安装到设计的活性中心的催化机制的范围，从而严重限制了可获得的化学转化的范围。我们实验室的先进蛋白质工程技术现在允许我们将非规范的“化学启发”氨基酸安装到酶活性部位，从而极大地扩展了利用自然遗传编码残基获得的有限功能范围。我们最近将这种遗传密码扩展技术与计算酶设计和实验室进化相结合，创造了第一种通过非规范有机催化机制运作的酶(自然2019,570,219)。在这次博士生学习中，我们现在将在这一早期成功的基础上雄心勃勃地扩展，以证明将非规范氨基酸集成到计算设计和定向进化工作流中可以导致创建具有新的催化机制和新的活性的酶。新功能氨基酸的设计将受到小分子有机催化剂的启发，这些催化剂能够促进自然界中没有观察到的大量有价值的转化。我们的方法融合了有机催化和生物催化的互补学科，将小分子系统的机械和功能的多功能性与可进化的蛋白质支架中实现的巨大的速度加速和反应选择性结合在一起。该项目牢牢嵌入化学、生物学和生物物理学的接口，这是BBSRC在“开发新的工作方式”议程中的关键驱动因素。它利用关键的生物科学技能，包括蛋白质工程、定向进化和酶特性，并具体利用多学科方法创造具有新功能的酶，这是工业生物技术部门的主要目标。

The ability to rationally design an enzyme for any desired transformation would have major impacts across the pharmaceutical and wider chemical sectors, leading to more efficient and sustainable synthetic routes to high value chemicals. The combination of computational enzyme design and directed evolution is currently the most attractive strategy to achieve this ambition. However, the limited range of functional groups presented by the genetic code (i.e. 20 amino acids) restricts the range of catalytic mechanisms which can be installed into designed active sites, thus severely limiting the repertoire of chemical transformations accessible. Advanced protein engineering technologies available in our laboratory now allow us to install non-canonical 'chemically inspired' amino acids into enzyme active sites, thus greatly expanding upon the limited range of functionality accessible with Nature's genetically encoded residues. We have recently combined this genetic code expansion technology with computational enzyme design and laboratory evolution to create the first enzyme that operates via a non-canonical organocatalytic mechanism (Nature 2019, 570, 219). In this PhD studentship, we will now expand ambitiously upon this early success, to demonstrate that the integration of non-canonical amino acids into computational design and directed evolution workflows can lead to the creation of enzymes with novel catalytic mechanisms and new activities. The design of new functional amino acids will be inspired by small molecule organocatalysts which are able to promote a plethora of valuable transformations not observed in Nature. Our approach merges the complementary disciplines of organocatalysis and biocatalysis, combining the mechanistic and functional versatility of small molecule systems with the enormous rate accelerations and reaction selectivities achievable within evolvable protein scaffolds.The project is firmly embedded at the interface of chemistry, biology and biophysics, a key driver for the BBSRC in the 'Exploiting new ways of working' agenda. It draws on key bioscience skills, including protein engineering, directed evolution and enzyme characterization, and specifically exploits a multi-disciplinary approach to the creation of enzymes with novel function, which is a major objective in the Industrial Biotechnology sector.