Biocatalytic Oxyfunctionalisation using Unspecific Peroxygenases (UPOs)

使用非特异性过氧化酶 (UPO) 进行生物催化氧功能化

• 批准号: 2602946
• 金额: --
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2602946
• 关键词: Biocatalytic; Oxyfunctionalisation; using; Unspecific; Peroxygenases

The oxygenation of organic compounds presents an ongoing challenge to synthetic chemistry as conventional reagents present difficulties with toxicity, harsh reaction conditions and a lack of selectivity. Enzymatic oxygenations, by contrast, present many advantages with respect to both sustainability and selectivity. Recently, the discovery of unspecific peroxygenases (UPOs) has identified new possibilities for scalable biocatalytic oxygenations. UPOs are secreted enzymes from fungi that contribute to the degradation of lignin. UPOs present major advantages over the better-studied cytochromes P450 as they catalyze selective oxygenations at the expense of only hydrogen peroxide, without the addition of cofactors or auxiliary proteins. They also exhibit higher turnovers, superior stability and can be produced in large amounts amenable to lyophilization, resulting in an easy-to-use biocatalysts for selective oxygenation reactions. In a previous PhD project at York, we applied a robust system for the expression of AaeUPO from Agrocybe aegerita in the yeast Pichia pastoris and used the enzyme for the oxygenation of a preliminary series of compound classes. We now aim to explore the potential of UPOs with further studies on natural homologs and engineered enzymes, with a focus on the generation of agrochemical metabolites and the transformation of biomass-derived building blocks (BDBBs). The following major objectives will be pursued: 1) To clone genes encoding native UPOs and express in Pichia pastoris, to expand the library of enzymes we can use to oxygenate organic molecules.2) To characterise the activities of the new UPOs through substrate synthesis and biotransformation studies using substrates including agrochemicals and biomass-derived building blocks.3) To engineer reaction conditions for UPO-catalysed biotransformations using Design-of-Experiment (DoE) approaches4) To rational engineer UPOs to improve their catalytic properties, based on structure determination using X-ray crystallography.While UPOs have been investigated for the oxygenation of model substrates, their application to selective synthesis of agrochemical metabolites or BDBBs has not been explored. Neither has the optimization of process conditions particular to these targets, nor the engineering of the enzymes to address performance optimization. Selective oxyfunctionalisations are major reactions in industrial chemistry requiring urgent green and sustainable alternatives and many industrial collaborators highlight the need for biocatalytic alternatives. An increased understanding of UPOs, their mechanisms and process requirements is timely, as it will help to enable their industrial application.

有机化合物的氧化对合成化学提出了持续的挑战，因为常规试剂存在毒性、苛刻的反应条件和缺乏选择性的困难。相比之下，酶促氧化在可持续性和选择性方面具有许多优点。最近，非特异性过氧化物酶（UPO）的发现已经确定了可扩展的生物催化氧化的新可能性。UPO是真菌分泌的酶，有助于木质素的降解。UPO目前的主要优势，更好地研究细胞色素P450，因为它们催化选择性氧化的代价只有过氧化氢，没有添加辅因子或辅助蛋白质。它们还表现出更高的周转率、上级稳定性，并且可以大量生产，适于冻干，从而产生易于使用的用于选择性氧化反应的生物催化剂。在约克以前的博士项目中，我们应用了一个强大的系统，用于在酵母毕赤酵母中表达来自茶树菇的AaeUPO，并使用该酶对一系列初步的化合物进行氧化。我们现在的目标是探索UPO的潜力，进一步研究天然同系物和工程酶，重点是农用化学品代谢产物的产生和生物质衍生的构建单元（BDBBs）的转化。将努力实现以下主要目标：1）克隆编码天然UPO的基因并在毕赤酵母中表达，2）通过底物合成和生物转化研究，使用包括农用化学品和生物质衍生的构建块的底物，来验证新的UPO的活性。3）设计UPO的反应条件，4）基于使用X射线晶体学的结构测定，合理地设计UPO以改善其催化性能。虽然已经研究了UPO用于模型底物的氧化，但尚未探索其在选择性合成农用化学代谢物或BDBBs中的应用。既没有这些目标所特有的工艺条件的优化，也没有酶的工程化以解决性能优化。选择性氧官能化是工业化学中的主要反应，迫切需要绿色和可持续的替代品，许多工业合作者强调需要生物催化替代品。提高对UPO、其机制和工艺要求的了解是及时的，因为这将有助于使其能够在工业上应用。