Overview
Synthetic Biology Ph.D. Student.
Project title: Synthetic biology for green chemistry: Building in vivo enzymatic cascades using Carboxylic acid reductases (CARs).
Qualifications
The University of Exeter.
Doctor of Philosophy - Ph.D.
Biological Sciences.
Dates attended or expected graduation: 2017 – 2021.
MPhil/Ph.D. in Synthetic biology for green chemistry: building in vivo enzymatic cascades to perform example biotransformations using carboxylic acid reductases (CARs).
BBSRC funded studentship in partnership with GlaxoSmithKline.
The University of Portsmouth.
Masters of Research (MRes).
Biological Sciences.
Grade: Distinction.
Dates attended: 2016 – 2017.
Accreditation in the following modules:
Level 7 FHEQ:
Research Preparation Unit.
Research Unit.
Laboratory Project Entitled: Expression of the Dengue serotype 2 & Zika Envelope Proteins and purification of potential inhibitor compounds.
The University of Portsmouth.
Bachelor of Science (B.Sc.).
Biochemistry.
Grade: Upper second class with honors.
Dates attended: 2013 – 2016.
Accreditation in the following modules:
Level 4 FHEQ: Experimental biology, Biodiversity & Evolution, Introduction to Cell Biology & Biochemistry, Microbiology & Molecular Biology, Perspectives in Biochemistry and, Graduate Skills Level 1.
Level 5 FHEQ: Microbiology, Cell Biology, Biochemistry, Genetic Engineering, Development: How Form & Function Changes and, Macromolecules.
Level 6 FHEQ: Gene Organization and Expression, Genomics in Molecular Medicine, Biomolecular Science, Genes & Development.
Honors Project Entitled 'The Co-Crystallization of Human Serum Amyloid P Component (SAP) with Hairpin and Self-complementary Oligonucleotides.
Research
Research interests
- Analytical Biochemistry.
- Computational Biology.
- Drug Discovery.
- Enzymology.
- Protein Engineering.
- Structural Determination of Biomolecules.
- Synthetic Biology.
- Tropical Diseases & their Pathology.
Research projects
Synthetic biology for green chemistry: Building in vivo enzymatic cascades using Carboxylic acid reductases (CARs).
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 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 utilizing 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 characterizing a range of CARs (Finnigan et al. (2017) ChemCatChem, 9, 1005-17) have resulted from this, with a third describing modeling 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 characterized biochemically in vitro. A robust model of this cascade has been developed.
Publications
Key publications | Publications by category | Publications by year
Publications by category
Journal articles
Cutlan R, De Rose S, Isupov MN, Littlechild JA, Harmer NJ (2020). Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1868(2), 140322-140322.
Finnigan W, Cutlan R, Snajdrova R, Adams J, Littlechild J, Harmer NJ (2019). Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts. ChemCatChem
Thomas A, Cutlan R, Finnigan W, van der Giezen M, Harmer N (2019). Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction.
Communications Biology,
2(1).
Abstract:
Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction
AbstractCarboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.
Abstract.
Thomas A, Cutlan R, Finnigan W, van der Giezen M, Harmer N (2019). Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction.
Commun Biol,
2(1).
Abstract:
Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction.
Carboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) - a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.
Abstract.
Author URL.
Publications by year
2022
Cutlan R (2022). Synthetic Biology for Green Chemistry: Building in Vivo Enzymatic Cascades Using Carboxylic Acid Reductases (CARs).
Abstract:
Synthetic Biology for Green Chemistry: Building in Vivo Enzymatic Cascades Using Carboxylic Acid Reductases (CARs)
Biocatalysis has a proven track record of offering replacements for individual chemical reactions with a lower environmental impact. Cascade reactions are an extension of biocatalysis; coupling a series of reactions to provide replacement for more than one chemical step. Herein, this thesis describes the engineering of a multi-enzyme cascade reaction for the production of phenylacetylcarbinol (PAC). Using a carboxylic acid reductase (CAR) from Mycobacterium phlei and a pyruvate decarboxylase from Acetobacter pasteurianus this thesis demonstrated a biocatalytic cascade reaction in which benzoic acid and pyruvate are converted into PAC. This cascade was combined with several other enzymes to recycle spent cofactors and deplete inhibitor by-products.
Furthermore, this thesis has highlighted the discovery of five putative enzymes; three ancestral CARs (AncCARs) and two thiamine diphosphate (ThDP) dependent enzymes. CARs typically have poor stability and thus limited tractability in industrial reactions. Within this study ancestral sequence reconstruction was performed on type I CARs. This is a developing engineering tool that can identify stabilizing and enzymatically neutral mutations throughout a protein. A combined algorithm approach was used to reconstruct functional ancestors of the Mycobacterial and Nocardial Type I CAR orthologues. Carboxylic acid reduction by Ancestral CARs was confirmed. Each showed a preference for aromatic carboxylic acids. AncCARs also showed improved tolerance to solvents, pH and in vivo-like salt-like conditions. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher. Two ThDP enzymes were discovered using metagenomics. These were assessed in silico through homology modelling and docking simulations. Furthermore, this study has demonstrated the importance of each tool in the discovery of new enzymes from within the ThDP family. Our homology models were used in docking simulations with unique carboligation-like intermediates allowing a rationalization of the reactions and stereoisomerism of the products. Two functional enzymes ThDP enzymes were identified that are capable of producing PAC; one from Thermus thermophilus and another from bacterium HR16.
Abstract.
2020
Cutlan R, De Rose S, Isupov MN, Littlechild JA, Harmer NJ (2020). Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1868(2), 140322-140322.
2019
Finnigan W, Cutlan R, Snajdrova R, Adams J, Littlechild J, Harmer NJ (2019). Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts. ChemCatChem
finnigan W, Cutlan R, Snajdrova R, Adams J, Littlechild J, Harmer NJ (2019). Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts (dataset).
Thomas A, Cutlan R, Finnigan W, van der Giezen M, Harmer N (2019). Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction.
Communications Biology,
2(1).
Abstract:
Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction
AbstractCarboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.
Abstract.
Thomas A, Cutlan R, Finnigan W, Van Der Giezen M, Harmer N (2019). Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction.
Abstract:
Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction
Carboxylic Acid Reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.
Abstract.
Thomas A, Cutlan R, Finnigan W, van der Giezen M, Harmer N (2019). Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction.
Commun Biol,
2(1).
Abstract:
Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction.
Carboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) - a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.
Abstract.
Author URL.
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