Discovery and development of new biocatalysts to efficiently access bioactive targets

采用跨学科的方法，我们的最终目标是通过利用大自然的生物合成机制，从简单的原料中生产出多种高价值的生物活性化合物。PKS/NRPS支架生物合成的模块化以及大量的组装后修饰酶为创造具有优化性能和生产的新型化合物提供了前景，本提议的重点是发现和开发新型生物催化剂。酶以极高的选择性催化反应，这在一般情况下是标准化学反应无法比拟的。虽然生物转化通常用于简单的拆分(例如，使用酰基酶)、还原以及最近在羟醛化学中的应用，但还有大量的酶催化转化尚未被用于清洁和高效地生产生物活性支架。该项目将从探索酶的生物催化潜力开始，我们最近分离了酶，以期在工业相关过程中利用它们独特的能力。例如，利用X射线晶体结构、酶分析和分子模拟的组合，我们已经提供了在抗生素abysSomicin(J.Am)的生物合成中存在Diels-Alderase(Abyu)的证据。化学。SoC.，2016)。我们将研究分子间和分子内的DA反应，以及使用可能形成不止一个环的底物(例如，使用参与抗生素四环素生物合成的类似物)来研究ABYU的选择性。对深山霉素途径上的更多酶进行了鉴定。还将探索这些酶的底物特异性，并通过分子建模来设计它们的活性部位，从而得到一系列具有潜在抗生素活性的非天然线性四硝酸酯。所有新化合物都将接受生物活性筛选。在含氧杂环、六元环四氢呋喃(THPS)和五元四氢呋喃(THFs)上组装了许多生物活性化合物。通过氧化复杂的线性底物中未活化的甲基来选择性地生成这些环将是非常困难的(可以说是不可能的)。然而，利用参与生物合成的加氧酶进行的令人兴奋的初步体外研究表明，酶确实可以用于选择性地产生THPS的四氢呋喃。这些和其他有趣的生物转化的机制将被阐明，底物的特异性将被探索。该项目将包括蛋白质化学(包括使用X射线结晶学、最先进的核磁共振和MS技术进行结构研究)、新化合物的分离和结构确定以及分子建模。

Using an interdisciplinary approach, our ultimate goal is to produce a multiplicity of high valuebiologically active compounds from simple feed-stocks by exploiting Nature's biosynthetic machinery.The modularity of the PKS/NRPS scaffold biosynthesis together with the plethora of post assemblymodifications of tailoring enzymes offer prospects of creating novel compounds with optimizedproperties and production and the focus of this proposal is the discovery and exploitation of novelbiocatalysts. Enzymes catalyze reactions with exquisite selectivity which, in general, simply cannot beemulated using standard chemical reactions. Whilst biotransformations are commonly used for simpleresolutions (e.g. using acylases), reductions and more recently in aldol chemistry, there are a plethoraof enzyme-catalyzed transformations which have yet to be exploited for the clean and efficientproduction of bioactive scaffolds. The project will begin by exploring the biocatalytic potential ofenzymes which we have recently isolated with a view to exploiting their unique capabilities inindustrially relevant processes. For example, using a combination of X-ray crystal structures, enzymeassays and molecular modeling, we have provided evidence for a Diels-Alderase (AbyU) in thebiosynthesis of the antibiotic abyssomicin (J. Am. Chem. Soc., 2016). We will investigate both interandintramolecular DA reactions as well as the selectivity of AbyU using substrates with the potentialof forming more than one ring (e.g. using analogues involved in the biosynthesis of the antibiotictetrodecamycin). Further enzymes on the abyssomicin pathway have been characterized. Thesubstrate specificities of these enzymes will also be explored and engineering of their active sites willbe informed by molecular modeling leading to a series of non-natural linear tetronates with potentialantibiotic activity. All new compounds will undergo screening for biological activity. Many bioactivecompounds are assembled on oxygen heterocycles, 6-membered ring tetrahydropyrans (THPs) and 5-membered tetrahydrofurans (THFs). The selective creation of these rings via oxidation of un-activatedmethyl groups in complex linear substrates would be very difficult (arguably impossible) to achievechemically. However exciting preliminary in vitro studies using oxygenases involved in the biosynthesisthe antibiotic mupirocin have shown that enzymes can indeed be used to selectively generate eitherTHFs of THPs. The mechanisms of these and other intriguing biotransformations will be elucidated andthe substrate specificities explored. The project will include protein chemistry (including structuralstudies using X-ray crystallography, state-of the-art NMR and MS techniques), isolation and structuredetermination of novel compounds and molecular modeling.