Biocatalytic Oxyfunctionalisation using Unspecific Peroxygenases (UPOs)

有机化合物的氧化对合成化学提出了一个持续的挑战，因为传统试剂存在毒性，反应条件苛刻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都有许多优点。最近，非特异性过氧酶（UPOs）的发现为可扩展的生物催化氧化提供了新的可能性。UPOs是真菌分泌的酶，有助于木质素的降解。与研究得更好的细胞色素P450相比，UPOs具有主要优势，因为它们只需要过氧化氢就能催化选择性氧化，而不需要添加辅助因子或辅助蛋白。它们还表现出更高的周转率，优越的稳定性，并且可以大量生产，适合冻干，从而成为易于使用的选择性氧化反应生物催化剂。在约克大学之前的一个博士项目中，我们应用了一个强大的系统，在酵母毕赤酵母中表达来自aegercybe的AaeUPO，并使用该酶进行一系列化合物的初步氧化。我们现在的目标是通过对天然同源物和工程酶的进一步研究来探索UPOs的潜力，重点是农业化学代谢物的产生和生物质衍生构建块（BDBBs）的转化。主要目标如下：1)克隆原生UPOs编码基因并在毕赤酵母中表达，扩大酶库，用于有机分子的氧合。2)通过底物合成和使用包括农用化学品和生物质衍生构建块在内的底物进行生物转化研究来表征新型UPOs的活性。3)利用实验设计（Design-of-Experiment, DoE）方法设计upo催化生物转化的反应条件4)基于x射线晶体学的结构测定，合理设计upo以提高其催化性能。虽然人们已经研究了UPOs对模式底物的氧化作用，但它们在选择性合成农化代谢物或BDBBs方面的应用尚未得到探索。既没有针对这些目标的工艺条件优化，也没有酶的工程来解决性能优化问题。选择性氧化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了对生物催化替代品的需求。及时增加对upo及其机制和流程需求的了解，因为这将有助于实现其工业应用。

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.