Enabling Unspecific Peroxygenases for Applications in Industrial Biocatalysis

由微生物催化的氧合反应在工业上具有重要价值，可用于对类固醇等复杂药物的选择性官能化。在这些情况下，羟基化是由细胞色素P450(P450)催化的，通常与用于反应的真菌的细胞膜有关。因此，P450在工业羟化反应中的潜力导致了大量研究，目的是使这些酶能够在一系列工业相关底物上进行许多其他选择性反应。然而，许多设想的应用需要在方便的实验室宿主(如大肠杆菌)中异源表达P450，但与这种表达策略相关的复杂性阻碍了提供可用催化剂的努力。其原因包括溶解性差(特别是真核细胞P450)和稳定性差，催化剂转化率很低，但特别是P450对烟酰胺辅因子和氧化还原转移蛋白活性的要求。这些困难现在刺激了对P450表达的替代宿主的寻找，也刺激了对催化同等过程的更稳定和更有活性的酶的寻找。最近，“非特异性过氧酶”(UPO)的发现为生物催化氧化领域注入了活力。UPO是由丝状真菌分泌的，是它们降解顽固植物物质的催化库的一部分。UPO吸引了人们的极大兴趣，因为尽管它们是“非特定的”--这意味着大量的底物被转化--但它们在区域和对映体选择性方面也具有高度的选择性。然而，与P450相比，UPO最引人注目的优势是它们的稳定性--作为分泌蛋白，它们已经进化成在具有挑战性的pH、温度和有机溶剂条件下都是稳定的；可以实现200 S-1的活性转换，并且它们的全部活性只依赖于过氧化氢，而不需要昂贵的烟酰胺辅因子或氧化还原转移蛋白。由于UPO的工作还处于初级阶段，大多数组织都依赖于种植野生类型的真菌Agrocybe aegerita来生产它，随之而来的是真菌发酵和从复杂的分泌蛋白混合物中纯化酶的复杂性。在最近的工作中，我们在一个异源系统--毕赤酵母中表达了AaUPO。这使该酶的发酵生产具有更好的控制优势，并且在产量和纯化简单性方面也得到了极大的提高。我们目前正在对重组UPO进行表征，并评估其对可扩展的不对称氧化苯基碳、烯烃和硫化物的适用性。在这个学生项目中，我们将寻求利用重组UPO在一系列合成相关的有机底物转化中的优越活性。

Oxygenation reactions catalysed by microorganisms are valued in industry for the selective functionalisation of complex pharmaceuticals such as steroids. In these cases, the hydroxylations are catalysed by cytochromes P450 (P450), usually associated with the cell-membranes of the fungi that are employed for the reactions. The potential of P450 for industrial hydroxylations has thus resulted in a large volume of research on the enabling of these enzymes for many other selective reactions on a range of industrially relevant substrates. However, many of the envisaged applications require the heterologous expression of P450 in a convenient laboratory host, such as E. coli, but the complexities associated with this expression strategy have frustrated efforts to provide usable catalysts. The reasons include poor solubility (especially of eukaryotic P450) and stability, very low catalyst turnover, but especially the requirements of P450 for nicotinamide cofactors and redox transfer proteins for activity. These difficulties have now stimulated the search both for alternative hosts for P450 expression, but also for more stable and active enzymes that catalyse equivalent processes. The field of biocatalytic oxygenations was recently energised by the discovery of 'unspecific peroxygenases' (UPOs), which are secreted by filamentous fungi as part of their catalytic arsenal for the degradation of recalcitrant plant matter. UPOs have attracted a great deal of interest as, although they are 'unspecific' - meaning that a wide range of substrates is transformed - they are also highly selective in terms of both regio and enantioselectivity. However, the most compelling advantages of UPOs over P450s are their stability - as secreted proteins they have evolved to be stable under challenging conditions of pH, temperature, and also organic solvents; activity - turnovers of 200 s-1 can be achieved, and their dependence only on hydrogen peroxide for full activity, removing the need for either expensive nicotinamide cofactors or redox transfer proteins. As UPO work is in its infancy, most groups have dependent on growing the wild-type strain of fungus Agrocybe aegerita for its production, with the attendant complexities of fungal fermentation and purification of the enzyme from the complex mix of secreted proteins. In recent work, we have expressed AaUPO in a heterologous system - the yeast Pichia pastoris. This gives the advantage of superior control of fermentative production of the enzyme, and also huge improvements in yield and simplicity of purification. We are currently characterising the recombinant UPO and assessing its suitability for scaleable asymmetric oxygenations of benzylic carbons, alkenes, and sulfides. In this studentship project, we will look to exploit the superior activity of recombinant UPOs in the transformation of a range of synthetically relevant organic substrates.