Synthetic Biology for Green Chemistry: Building In vivo Enzymatic Cascades Using Carboxylic Acid Reductases (CARs)

概述：酶级联是非常有吸引力的化学转化的生物催化方法。这些方法设想使用一系列酶在单个过程中进行多个协调反应步骤。这具有许多优点，包括消除对中间体纯化的需要，并且通常允许副产物被回收。酶级联反应已在体外和体内得到证实。在体内使用级联是非常有吸引力的，因为不需要添加昂贵的辅因子。一个特别的优点是在嗜热菌中进行酶级联反应。这些可以更容易地集成到混合的化学-生物催化管道中，并且高温通常会消除竞争微生物。一种特别有吸引力的方法是利用羧酸还原酶（汽车）实施体内级联。这些酶将羧酸还原成醛，这在化学上是具有挑战性的，无法在不还原成醇的情况下实现，并且产生大量的化学废物。汽车还充当有吸引力的桥反应，因为它们的底物通过许多其他步骤产生，并且它们的产物是许多后续反应的底物。GSK/BBSRC CASE项目最近得出结论，清楚地表明了这项研究的潜力。描述酯酶的出版物（Sayer等人（2016）Sci.代表：6，25542）和表征一系列汽车（Finnigan et al.（2017）ChemCatChem，9，1005-17），第三个描述模型目前正在修订中。本项目旨在进一步发展第一个项目中进行的原理验证工作，并将其用于GSK的实际应用。本项目旨在以汽车为例开发体内嗜热级联，用于工业用途。该项目旨在通过以下方式开发这些内容：1.证明基于CAR的酶级联在E.杆菌2.使用酶级联在体外和体内使用酶级联将前体化合物转化为工业相关的醇。扩大可以耦合利用CAR酶的反应范围。初步研究表明，这些步骤都是可行的。具体而言，基于CAR的酶级联已经在体外进行了彻底的生物化学表征。已经开发了一个强大的模型，这个级联。

Overview: Enzyme cascades are highly attractive biocatalytic approaches for chemical transformations. These approaches envisage using a series of enzymes to perform multiple coordinated reaction steps in a single process. This has many advantages, including removing the need for intermediate purification, and often allowing side products to be recycled. Enzyme cascades have been demonstrated in vitro and in vivo. Using cascades in vivo is highly attractive as there is no need to add in expensive cofactors. A particular advantage would be to perform enzyme cascades in thermophiles. These can be more easily integrated into mixed chemical-biocatalytic pipelines, and the high temperatures generally eliminate competitor microbes. A particularly attractive approach would be to implement in vivo cascades utilising carboxylic acid reductases (CARs). These enzymes reduce carboxylic acids to aldehydes, which is chemically challenging to achieve without reducing to the alcohol, and which produces large quantities of chemical waste. CARs also act as an attractive bridge reaction, as their substrates are produced by many other steps, and their products are substrates for many following reactions. A GSK/BBSRC CASE project recently concluded that has demonstrated clearly the potential of this research. Publications describing an esterase (Sayer et al. (2016) Sci. Rep., 6, 25542) and characterising a range of CARs (Finnigan et al. (2017) ChemCatChem, 9, 1005-17) have resulted from this, with a third describing modelling currently in revision. This project aims to further develop the proof-of-principle work performed in the first project, and exploit it for practical use by GSK.This project aims to develop in vivo thermophilic cascades using CARs as examples for industrial use. The project will aim to develop these by:1. Demonstrating that CAR-based enzyme cascades are effective in E. coli. 2. Using an enzyme cascade to convert a precursor compound to an industrially relevant alcohol using an enzyme cascade in vitro and in vivo.3. Extending the range of reactions that can be coupled to exploit the CAR enzymes.Pilot studies have shown that these steps are all feasible. Specifically, a CAR based enzyme cascade has been thoroughly characterised biochemically in vitro. A robust model of this cascade has been developed.