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，并在生产生物燃料分子以及其他在工业中应用的化学品。将产生一类新的含血红素酶的新代表，并详细研究其结构和催化特性。这些酶由于其将过氧化氢用作底物的能力而称为“过氧酶”。此处研究的酶（称为P450 KR和P450 OLET）使用过氧化酶将脂肪酸转化为有价值的烃分子烯烃。（适当尺寸）的烯烃可用于汽车发动机作为燃料，并在化学工业中还具有多种应用。例如制作塑料（和其他聚合物）和醇。在基于此应用的工作中，我们开发了产生KR和OLET P450酶的方法（使用自然产生酶的不同细菌克隆的基因）并纯化P450。这使我们能够确定OLET和KR识别的脂肪酸长度范围不同，因此KR P450产生的一组较短的链烯烃与OLET相比。因此，这些酶是互补的，并且能够使用廉价的脂肪酸作为底物产生各种不同的烷烃。在这个项目中，我们将分析这些酶如何将脂肪酸转化为烷烃。这将使用计算/建模程序（了解涉及的化学和哪些部分对于烯烃生产过程至关重要）以及结构，光谱和快速反应方法的组合（确定酶如何折叠并结合其底物，烯烃生产反应的快速反应以及了解机制）。这些研究对于使我们能够合理化脂肪酸对烷烃的这种重要生物化学转化至关重要，并且对于允许蛋白质工程（即以靶向方式突变酶）也至关重要形成不同产物（羟化脂肪酸）的地方。设计了KR和OLET酶以优化其反应性后，将探索不同的驱动其功能的途径 - 因为推动其反应的另一种方法是为它们提供不同类别的P450酶使用的不同蛋白质（“ Redox伴侣”）（例如，与人类药物代谢和类固醇相关的蛋白质相关）。一旦确定了最有效的驱动这些酶的方法，就可以使用将OLET/KR P450高水平的细菌细胞产生所需的短至中链烯烃。将进行烷烃的量化，以确定生产水平并确定在工业型发酵过程中不同链长烷烃产生的效率。在平行研究中，还将确定来自不同类型的脂肪酸（包括多不饱和和分支链脂质）的橄榄/KR P450的天然和工程形式的能力，以建立多种类型的脂质可以评估这些enzymes的替代产品，并确定各种类型的脂质，以使这些enzymes的替代品是否构建，也将确定各种类型的产品，并确定这些enzymes的潜在产品，并能够确定这些产品的各种产品。因此，该项目既具有基本和应用方面：首先可以详细了解生物技术重要的酶催化剂类别的结构/机制（使我们能够以合理的效果来设计olet和kr酶，以改善性能），其次是为生物学而产生的，用于生成的生物学，最大程度地用于构成生物学的产品 - 大多数化学生物学，并在合成的生物学中使用 - 大多数化学性能 - 申请。