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

点击翻译按钮获取中文摘要

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.