Bottom-Up Creation of an Enzyme for the Morita-Baylis-Hillman Reaction

计算设计和定向进化的结合是创造具有新功能的酶的一个有吸引力的策略，到目前为止已经为一小部分经过充分研究的转化提供了从头开始的生物催化剂。为了建立“自下而上”的酶设计作为实际应用的生物催化剂的可靠来源，我们必须超越简单的模型反应领域，转向具有复杂机制和高能量障碍的苛刻的化学转化。在这项提议中，我们将结合计算设计、实验室进化和遗传密码扩展中的最先进方法，为双分子Morita-Baylis-Hillman(MBH)反应创造高效和选择性的酶，这是一种在自然界中没有观察到的合成价值的碳-碳键形成反应。该研究计划的成功实施将扩大用于C-C键形成的生物催化剂的范围，并将突出“自下而上”设计作为为复杂的多步骤过程创造酶的可行策略。这项研究将与葛兰素史克(GSK)密切合作进行，并与BBSRC在工业生物技术方面的战略重点完全一致。这是一个高度跨学科的项目，处于酶设计和工程研究的前沿，在最先进的多学科环境中为研究生提供多样化的培训机会。学生将在有机合成、分子和结构生物学、蛋白质表达和纯化、实验室进化、生化分析和计算生物学方面获得广泛的专业知识。综合起来，培训计划将使学生掌握必要的技能，以追求未来的职业生涯在化学工业。

The combination of computational design and directed evolution is an attractive strategy to create enzymes with new function, that to date has delivered de novo biocatalysts for a small set of well-studied transformations. To establish 'bottom up' enzyme design as a reliable source of biocatalysts for practical applications, it is essential that we move beyond the realm of simple model reactions to demanding chemical transformations with complex mechanisms and high energy barriers. In this proposal, we will combine state-of-the-art methods in computational design, laboratory evolution and genetic code expansion to create efficient and selective enzymes for a bimolecular Morita-Baylis-Hillman (MBH) reaction, a synthetically valuable carbon-carbon bond forming reaction not observed in Nature. Successful implementation of the research program will expand the repertoire of biocatalysts for C-C bond formations and will highlight 'bottom-up' design as a viable strategy for creating enzymes for complex multi-step processes. The research will be carried out in close collaboration with GlaxoSmithKline (GSK) and aligns perfectly with the BBSRC strategic priorities in Industrial Biotechnology. This is a highly interdisciplinary project at the cutting edge of enzyme design and engineering research, offering diverse training opportunities for a post-graduate student in a state-of-the-art multidisciplinary setting. The student will gain broad expertise in organic synthesis, molecular and structural biology, protein expression and purification, laboratory evolution, biochemical assays and computational biology. Combined, the training program will equip the student with the necessary skills to pursue a future career in the chemical industry.