Flavocytochrome P450 BM3 engineering for scalable P450 reactions

具有氧原子的区域/立体选择性引入的氧官能化有机化学品的生成对于高价值化学品的工业制造是非常期望的，例如昂贵的中间体、药物代谢标准品、生物活性脂质、类固醇等。然而，传统方法在化学上具有挑战性，具有与缺乏位置选择性、使用苛刻试剂和加工相关的显著问题。酶化学提供了这种合成方法的替代方案，特别是细胞色素P450（P450）单加氧酶，可以以高度的区域和立体选择性将氧原子引入底物中。该CASE学生奖学金提案的主要目的是设计一种高活性P450-P450还原酶融合酶（flavocytochrome P450 BM 3; BM 3），以执行特定的氧化作用，从而产生高价值的合成中间体和含氧产品，用于生物技术开发，如下所述：BM 3是生物技术应用的理想P450系统。它是一种可溶性的单组分酶，其活性是多组分膜结合真核P450系统的约1000倍。我们对BM 3的结构和控制底物选择性/催化作用的残基有着深刻的理解。Munro和其他小组的先前研究已经证明了它的“延展性”以及通过合理诱变和半随机定向进化筛选所需活性来重新设计其底物选择性和催化性质的能力。该项目旨在分离催化活性药物成分和/或其中间体的对映体和区域选择性羟基化的BM 3变体。羟基化将集中在中等活化的位置，如苄基，烯丙基和叔碳中心。重要的目标包括末端烯烃，和选择性的BM 3突变体的环氧化或烯丙基羟基化的发展。在这些情况下，我们将使用具有或不具有F87 G突变的酶，其中Phe 87苯基侧链保护末端C=C双键并有利于在相邻碳上的羟基化。将使用以下方法：在曼彻斯特，学生将使用现有的BM 3点突变体面板研究底物结合和营业额，并在不同的P450区域中使用预先存在的突变体库（通常每个突变体改变1-2个残基），称为底物识别序列（SRS），其排列在活性位点并包含底物相互作用残基。将使用BBSRC赞助的曼彻斯特机器人设施，并使用表达细胞提取物和基于（i）底物依赖性NADPH氧化和（ii）通过“OxoPlate”技术的底物相关耗氧量的光学/荧光筛选文库，我们已经使用模型底物进行了验证。将使用底物结合（通过血红素吸光度，ITC）和转换试验验证具有所需活性的突变体。将组合有益突变（例如，与来自其他SRS区域的突变），并酌情使用二次筛选，以进一步优化底物识别/转换。相关突变体的血红素（P450）结构域将被纯化/结晶，使得它们能够以底物结合形式进行结构阐明，以使结合模式合理化，并允许通过合理诱变进一步“抛光”，以改善所需的反应。优化的突变体将在Pfizer使用体外（纯酶）和细胞培养系统分析产品形成的效率，从而研究产品概况、系统优化和规模扩大，以产生适合工业应用的量的所需氧产物。学生将接受蛋白质工程，酶生物化学，生物物理和结构分析方面的广泛培训;结合有机化学表征和工业放大技术，以促进目标含氧产品的生物技术应用。

Generation of oxyfunctionalized organic chemicals with regio/stereoselective introduction of oxygen atoms is highly desirable for industrial manufacture of high value chemicals, e.g. costly intermediates, drug metabolism standards, bioactive lipids, steroids etc. However, traditional methods are chemically challenging with significant problems associated with lack of positional selectivity, use of harsh reagents and processing. Enzyme chemistry offers alternatives to such synthetic approaches, and cytochrome P450 (P450) monooxygenases, in particular, can introduce oxygen atoms into substrates with a high degree of regio- and stereoselectivity. The major aim of this CASE studentship proposal is to engineer a high activity P450-P450 reductase fusion enzyme (flavocytochrome P450 BM3; BM3) to perform specific oxygenations leading to high value synthetic intermediates and oxygenated products for biotechnological exploitation, as described below: BM3 is an ideal P450 system for biotechnological applications. It is a soluble, single component enzyme with activity ~1000-fold that of multicomponent, membrane-bound eukaryotic P450 systems. We have a firm understanding of BM3's structure and residues controlling substrate selectivity/catalysis. Preceding studies from Munro and other groups have proven its 'malleability' and the ability to re-engineer its substrate selectivity and catalytic properties by both rational mutagenesis and semi-random directed evolution with screening for desired activities. This project aims to isolate BM3 variants that catalyse enantio- and regioselective hydroxylations of active pharmaceutical ingredients and/or their intermediates. Hydroxylations will be focused at positions of medium activation, such as benzylic, allylic and tertiary carbon centres. Important targets include terminal alkenes, and the development of selective BM3 mutants for either epoxidation or allylic hydroxylation. In these cases, we will use enzymes with or without F87G mutations, where the Phe87 phenyl side chain protects the terminal C=C double bond and favours hydroxylation at the adjacent carbon. The following approaches will be used: At Manchester, the student will investigate substrate-binding and turnover using an existing panel of BM3 point mutants, and employing pre-existing libraries of mutants (typically 1-2 residues changed per mutant) in different P450 regions known as substrate recognition sequences (SRS) that line the active site and contain substrate interacting residues. Libraries will be screened using BBSRC-sponsored robotic facilities at Manchester, and using expression cell extracts and optical/fluorescence screens based on (i) substrate-dependent NADPH oxidation and (ii) substrate-linked oxygen consumption by 'OxoPlate' technology, that we have already validated using model substrates. Mutants with desired activities will be validated using substrate binding (by heme absorbance, ITC) and turnover assays. Beneficial mutations will be combined (e.g. with ones from other SRS regions) and secondary screens used as appropriate to further optimize substrate recognition/turnover. Heme (P450) domains of relevant mutants will be purified/crystallized, enabling their structural elucidation in substrate-bound forms to rationalize binding mode and to allow further 'polishing' by rational mutagenesis to improve the desired reactions. Optimized mutants will be analyzed for efficiency of product formation at Pfizer, using both in vitro (pure enzyme) and cell culture systems, leading to studies of product profiles, system optimization and scale-up to generate quantities of desired oxy-products in amounts suited to industrial applications. The student will receive a broad training in protein engineering, enzyme biochemical, biophysical & structural analysis; combined with organic chemical characterization and industrial scale-up technology to facilitate biotechnological applications for the targeted oxygenated products.