Simulation-led engineering of Diels-Alderase activity and selectivity for sustainable biosynthesis of new antibiotics

酶是一种重要的生物催化剂，在温和的条件下具有快速、选择性和高效的催化作用。生物技术利用这些天然生物催化剂的加速特性来制造高附加值的产品。目前特别感兴趣的是催化Diels-Alder反应的酶，这是一种[4+2]环加成反应，被广泛认为是20世纪合成有机反应的基石之一。它通常用于合成具有生物活性的天然产物，包括许多重要的药物。没有酶生物催化剂，通常必须采用苛刻的反应条件（高温，高压），并且对产品结果的精确控制是有限的。因此，催化这一反应的酶diels - alderase非常有吸引力。在这个项目中，重点将是diels - alderase参与四酸盐抗生素的生物合成，如asabyssomicins，具有非常有前景的抗生素活性的天然产物。我们已经详细地建立了其中两种的结构和机理，其中的计算模拟，包括对接，分子动力学和QM/MM反应模拟，具有很高的价值。在Diels-Alderase步骤之前和之后需要的关键剪裁酶也是如此，以达到活性聚酮基抗生素。该项目的目标是在新的计算预测方案中使用这些技术，该方案可以建议具有期望的活性和特异性变化的酶变体，例如获得具有改进的药用特性的四酸盐抗生素。为了测试和改进这些预测，将进行有前途的酶变体（产品结果，动力学和结构生物学）的实验表征。通过与我们的工业合作伙伴阿斯利康合作，工程酶的影响可以通过测试它们在药物发现中产生有价值的构建模块和支架的用途来实现。该跨学科项目结合了布里斯托尔酶计算模拟的专业知识和国际领先的学术团队的专业知识，以及聚酮类天然产物生物合成和相关实验技术（酶学，分子生物学，化学和结构生物学）的多学科专业知识。这种模拟与实验相结合的方法还在发展中，但将变得越来越重要；在布里斯托尔，我们处于这一发展的前沿。多学科环境确保学生将获得一系列技能，这些技能将武装他们未来在学术或工业生物科学（包括制药科学）的职业生涯。该学生将融入布里斯托尔充满活力的研究环境，包括计算化学中心和布里斯托尔生物设计研究所，确保广泛的互动，研讨会项目和课程。

Enzymes are remarkable biocatalysts that allow rapid, selective and efficient catalysis under mild conditions.Biotechnology exploits the rate-enhancing properties of these natural biocatalysts to manufacture high added-valueproducts. Of particular current interest are enzymes that catalyse the Diels-Alder reaction, a [4+2] cycloaddition reactionwidely recognised as one of the cornerstone synthetic organic reactions of the 20th century. It is commonly employed inthe synthesis of bioactive natural products, including numerous important pharmaceuticals. Without enzyme biocatalysts,harsh reaction conditions (high temperature, high pressure) often must be employed, and precise control over the productoutcome is limited. Enzymes that catalyse this reaction, Diels-Alderases, are therefore highly attractive. In this project, the focus will be on Diels-Alderases involved in the biosynthesis of tetronate antibiotics such asabyssomicins, natural products with highly promising antibiotic activity. We have already established the structure andmechanism of two of these in detail, for which computational simulation, including docking, molecular dynamicsand QM/MM reaction simulations, has proven highly valuable. The same is true for crucial tailoring enzymes that arerequired before and after the Diels-Alderase step to arrive at the active polyketide-based antibiotic. The project aims touse these techniques in new computational prediction protocols that can suggest enzyme variants with desired changesin activity and specifity, for example to obtain tetronate antiobiotics with improved characteristics for pharmaceuticaluse. To test and improve these predictions, experimental characterisation of promising enzyme variants (productoutcome, kinetics and structural biology) will be performed. By working with our industrial partner AstraZeneca, theimpact of engineered enzymes can be realised by testing their use for generating valuable building blocks and scaffolds forpharmaceutical drug discovery. This interdisciplinary project combines the expertise in computational simulation ofenzymes in Bristol and the expertise from an internationally leading academic team with multidisciplinary expertise ofpolyketide natural product biosynthesis and relevant experimental techniques (enzymology, molecular biology,chemistry and structural biology).Combining simulation and experiment in this way is still developing, but will become increasingly important; in Bristol, weare at the forefront of this development. The multidisciplinary environment ensures the student will acquire a range ofskills that will arm them for a future career in academic or industrial bioscience (including pharmaceutical science). Thestudent will be embedded in the vibrant research environment in Bristol, including the Centre for ComputationalChemistry and the Bristol BioDesign institute, ensuring a wide range of interactions, seminar programmes and courses.