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Molecular mechanism and engineering of P450 peroxygenases for synthetic biology applications

用于合成生物学应用的 P450 过氧化酶的分子机制和工程

基本信息

批准号：
BB/N006275/1
负责人：
Andrew Munro
金额：
$ 58.88万
依托单位：
University of Manchester
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2016
资助国家：
英国
起止时间：
2016 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FN006275%2F1
关键词：
Molecular mechanism engineering P450 peroxygenases

项目摘要

The proposed project will characterize an important new type of enzyme catalyst with uses in the production of biofuel molecules, as well as other chemicals with applications in industry. Two representatives of a new class of heme-containing enzymes will be produced and their structural and catalytic properties studied in detail. These enzymes are termed "peroxygenases" due to their ability to use hydrogen peroxide as a substrate; and the enzymes studied here (named P450 KR and P450 OleT) use peroxide to convert fatty acids into the valuable hydrocarbon molecules alkenes. The alkenes (of appropriate size) can be used in car engines as fuel, and have multiple other applications in the chemicals industry; e.g. in making plastics (and other polymers) and alcohols. In work underpinning this application, we have developed methods to produce the KR and OleT P450 enzymes (using genes cloned from different bacteria that naturally produce the enzymes) and for purifying the P450s. This has enabled us to establish that the ranges of lengths of fatty acids recognized by OleT and KR are different, such that the KR P450 produces a group of shorter chain alkenes than can OleT. These enzymes are thus complementary and together are able to produce a wide range of different alkenes using cheap fatty acids as substrates. In this project, we will analyse how these enzymes function to convert fatty acids into alkenes. This will be done using both computational/modelling procedures (to understand the chemistry involved and which parts of the enzymes are crucial for the alkene production process) and through a combination of structural, spectroscopic and fast reaction methods (to determine how the enzymes fold and bind their substrates, how fast the alkene production reaction occurs, and to understand the mechanism involved). These studies are essential to enable us to rationalize how this important biochemical transformation of fatty acids to alkenes occurs, and will also be crucial to allow protein engineering (i.e. mutating enzymes in a targeted way) to be done to improve binding of selected fatty acids (particularly short chain lipids that generate more volatile alkenes with better properties as biofuels) and to disfavour unwanted side reactions where a different product (hydroxylated fatty acid) is formed. Having engineered the KR and OleT enzymes to optimize their reactivity, different routes to driving their function will be explored - since another way of driving their reactions is by providing them with different proteins ("redox partners") that are used by other classes of P450 enzymes (e.g. those involved in human drug metabolism and steroid synthesis). Once the most efficient means of driving these enzymes is identified, work will be done to produce the desired short- to mid-chain alkenes using bacterial cells that make the OleT/KR P450s at high levels. Quantification of alkenes will be done to determine production levels and to establish the efficiency of generation of different chain length alkenes in an industrial-type fermentation process. In parallel studies, the ability of native and engineered forms of the OleT/KR P450s to produce alkene or hydroxylated products from different types of fatty acids (including polyunsaturated and branched chain lipids) will also be determined, in order to establish whether diverse types of lipids can be substrates for these enzymes, and to evaluate their potential to make distinct types of products with industrial applications. This project thus has both fundamental and applied aspects: first to enable a detailed understanding of the structure/mechanism of two members of a biotechnologically important class of enzyme catalyst (enabling us to engineer the OleT and KR enzymes rationally for improved performance), and second to demonstrate their versatility and uses in synthetic biology for industrial exploitation - most notably in generating alkenes for biofuel and chemical products applications.

拟议的项目将描述一种重要的新型酶催化剂，用于生产生物燃料分子以及其他工业应用化学品。将产生一类新的含血红素的酶的两个代表，并详细研究它们的结构和催化性质。这些酶被称为“过氧酶”，因为它们能够使用过氧化氢作为底物;这里研究的酶（命名为P450 KR和P450 OleT）使用过氧化物将脂肪酸转化为有价值的烃分子烯烃。烯烃（适当大小）可用于汽车发动机作为燃料，并在化学工业中有多种其他应用;例如制造塑料（和其他聚合物）和酒精。在支持这一应用的工作中，我们开发了生产KR和OleT P450酶（使用从天然产生酶的不同细菌克隆的基因）和纯化P450的方法。这使我们能够确定OleT和KR识别的脂肪酸长度范围是不同的，因此KR P450比OleT产生一组链更短的烯烃。因此，这些酶是互补的，并且一起能够使用廉价的脂肪酸作为底物来生产各种不同的烯烃。在这个项目中，我们将分析这些酶如何将脂肪酸转化为烯烃。这将使用计算/建模程序（以了解所涉及的化学以及酶的哪些部分对烯烃生产过程至关重要）并通过结构，光谱和快速反应方法的组合（以确定酶如何折叠和结合其底物，烯烃生产反应发生的速度以及了解所涉及的机制）来完成。这些研究对于使我们能够合理化脂肪酸到烯烃的这一重要生物化学转化是如何发生的至关重要，同时也是蛋白质工程的关键（即以靶向方式突变酶）以改善所选脂肪酸的结合（特别是短链脂质，其产生更易挥发的烯烃，具有更好的生物燃料特性）并避免形成不同产物（羟基化脂肪酸）的不希望的副反应。在对KR和OleT酶进行工程改造以优化其反应性之后，将探索驱动其功能的不同途径-因为驱动其反应的另一种方式是通过为它们提供由其他类别的P450酶（例如参与人类药物代谢和类固醇合成的那些酶）使用的不同蛋白质（“氧化还原配偶体”）。一旦确定了驱动这些酶的最有效方法，将使用高水平生产OleT/KR P450的细菌细胞来生产所需的短链至中链烯烃。将对烯烃进行定量，以确定生产水平，并确定工业型发酵工艺中不同链长烯烃的生成效率。在平行研究中，还将确定OleT/KR P450的天然和工程化形式从不同类型的脂肪酸（包括多不饱和和支链脂质）产生烯烃或羟基化产物的能力，以确定不同类型的脂质是否可以作为这些酶的底物，并评估它们制造具有工业应用的不同类型的产品的潜力。因此，该项目既有基本方面，也有应用方面：首先能够详细了解生物技术上重要的酶催化剂类别的两个成员的结构/机制（使我们能够合理地设计OleT和KR酶以改善性能），第二，展示它们的多功能性和在合成生物学中的用途，以供工业开发-最显著的是用于生产用于生物燃料和化学产品应用的烯烃。