Development of carboxyl methyltransferases for sustainable synthesis

这项研究提出的目的是为利用自然界的催化剂、酶生产化学品提供新的可持续的过程。我们将特别关注一种能够催化酯类化学物质生产的酶，酯类化学物质在药品等化学产品中无处不在，也是合成过程中重要的化学中间体。从酸中生成酯通常需要消耗能量或使用有毒化学物质，而且这些反应必须在没有水的情况下进行。该项目中正在开发的酶将允许在水条件下形成酯，使其与其他酶催化剂兼容，并允许开发用于化学合成或级联的生物合成途径。在这个项目中，我们将开发高效的酯形成酶突变体，能够催化合成生物塑料和药物的关键中间体的形成。我们将使用实验室中已经使用的最先进的筛选技术来识别突变体。然后，我们将把新酶与我们已经开发的其他较小的酶级联结合起来，用于从纤维素和木质素等生物可再生材料合成可生物降解的生物塑料。我们还将开发新的级联，通过与我们最近发现的其他新酶偶联来合成药物，这些酶惊人地能够催化水中酰胺键的形成。酰胺键是药物中重要的键。酯形成与酰胺形成的结合将取代传统酰胺键合成中使用的昂贵的偶联剂。酯形成酶需要在细胞中发现一种叫做SAM的特殊分子作为催化剂。我们将开发优化的细胞宿主（所谓的微生物细胞工厂）来生产所有所需的酶和SAM，这样细胞发酵就可以用来进行目标化学物质的合成。我们将演示将废塑料PET转化为药物的发酵过程。我们还将开发能够代谢甲醇的细胞，以驱动合成，并开发一种新型菌株，可以回收在酰胺键形成反应中产生的甲醇，该反应被酯形成酶使用，使整个过程能够在非常少量的甲醇下发挥作用。

The research proposed aims to provide new sustainable processes for the production of chemicals using nature's catalysts, enzymes. In particular we will focus on an enzyme that is able to catalyse the production of chemicals called esters, which are ubiquitous in chemical products such as pharmaceuticals and also important as chemical intermediates for synthetic processes. The formation of an ester from an acid often involves energy demanding processes or the use of toxic chemicals and the reactions must be done in the absence of water. The enzyme under development in this project will allow the formation of esters under aqueous conditions, making it compatible with other enzyme catalysts and allowing the development of biological synthetic pathways for chemical synthesis, or cascades. In this project, we will develop highly efficient mutants of the ester forming enzyme that are able to catalyse the formation of key intermediates for the syntheis of bioplastics and pharmaceuticals. We will use state-of-the-art screening technology that is already in use in our laboratories to identify the mutants. We will then couple the new enzymes with other smaller enzyme cascades that we have already developed for the synthesis of biodegradable bioplastics from biorenewable materials such as cellulose and lignin. We will also develop new cascades for the synthesis of pharmaceuticals by coupling with other new enzymes we have discovered recently that are surprisingly able to catalyse the formation of amide bonds in water. Amide bonds are important bonds within pharmaceuticals. The combination of the ester formation followed by the amide formation will enable replacement of expensive coupling reagents used in conventional amide bond synthesis. The ester forming enzyme needs a specific molecule found in cells called SAM to act as a catalyst. We will develop optimised cell hosts (so-called microbial cell factories) to produce all the required enzymes and the SAM, such that cell fermentations can be used to carry out the synthesis of the target chemicals. We will demonstrate a fermentation for the conversion of waste plastic, PET, into a pharmaceutical drug. We also will develop cells able to metabolise methanol that will drive the synthesis and a novel strain that can recycle the methanol produced in the amide bond forming reaction which is used by the ester forming enzyme making the whole process able to function with very small amounts of methanol.