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

Lead Research Organisation: UNIVERSITY OF EXETER
Department Name: Biosciences

Abstract

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.

Publications

10 25 50
 
Description So far, we have engineered several new enzymes that can tolerate harsh conditions such as high temperature. These are currently being combined with another class of enzymes sourced from various thermophilic species leading to the production of high value pharmaceutical source material from inexpensive starting materials.
Exploitation Route The outcomes of this project will allow the use of Carboxylic acid reductases with C-C bond forming enzymes in a high temperature environment leading to the production of high value pharmaceutical precursors.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description Partnership with GSK 
Organisation GlaxoSmithKline (GSK)
Department Research and Development GSK
Country United Kingdom 
Sector Private 
PI Contribution GSK are interested in using biocatalytic reactions to generate pharmaceuticals and their precursors. Biocatalysts have several benefits that would aid their formulation processes such as a reduction in waste and operating under ambient conditions. Specifically, their interest is in the carboxylic acid reductase (CAR) enzyme family which is capable of reducing carboxylic acids to aldehydes. One of the key issues with CAR enzymes is their tolerance to physical conditions such as extremes of temperature, pH and solvents. Consequently, we used ancestral sequence reconstruction (ASR) to generate three Type 1 bacterial 'ancestral' CAR (AncCAR) enzymes. This work identified several structural regions of the CAR enzyme protein key to stability and reactant specificity. These enzymes have improved stability properties (particularly temperature and half-life) which are essential to their use in large-scale biocatalytic reactions. However, these enzymes have a limited rate of turnover and many of their reactions can be achieved more efficiently with known CAR enzymes. We also contributed to GSKs interests by achieving a complete enzyme cascade reaction using a CAR enzyme in combination with a pyruvate decarboxylase enzyme forming 1-hydroxy-1-phenyl-propan-2-one (a.k.a as phenylacetylcarbinol). This compound is a precursor to many ephedrine-based compounds commonly used as decongestants and appetite suppressors. During this reaction, we also demonstrated a refined cofactor recycling system for the CAR enzymes improving their use in vitro and expanded on the importance of using kinetic-based modelling to assess reactions. Furthermore, we demonstrated the importance of metagenomics in the assessment of genome libraries for the identification of novel thiamine diphosphate-dependent enzymes.
Collaborator Contribution Our partners at GSK have been invaluable in helping guide the project. They have aided in the critical evaluation and discussion of the results of experiments and have aided the workflow and goals of the project by adding a greater level of scrutiny of project progress. Furthermore, GSK has also provided access to equipment and materials essential to the project. Their liquid chromatography apparatus and expertise have allowed for the characterization of both cascade and enzymatic reactions. Moreover, their skills in chemical synthesis were invaluable in the generation of analytical standards required to assess 2-hydroxy ketone products. One aspect of working in the industrial partnership was the ability to experience research and development within an industrial setting which has helped to guide my career aspirations and research goals for the future.
Impact This has resulted in two publications related to this project in the field of biochemistry. One of which was a review of recent advances of specific enzyme classes & their use in cascades as well as an experimental paper relating to the creation of new CARs with novel properties. Furthermore, expertise and knowledge have invaluably been passed to all parties within this collaboration through the sharing of facilities, training opportunities and aspects of biocatalytic reactions.
Start Year 2017
 
Description Biotransformation conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact I provided a poster about the generation of ancestral CARs developed during the project and our aim to use these or other CAR enzymes with thiamine diphosphate-dependent enzymes for the generation of chemicals with the 2-hydroxy ketone motif. This allowed us to represent and discuss our proposed work with other parties with similar interests in the development of our proposed reaction scheme. This led to the exchange of materials essential to the generation of our 1-hydroxy-1-phenyl-propan-2-one cascade reaction.
Year(s) Of Engagement Activity 2019
URL https://biotrans2019.com/
 
Description Protein stability conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Attended the protein stability conference 2018. A poster presentation was given at this conference and was awarded the prize for the Young Scientist Award.
Year(s) Of Engagement Activity 2018