Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts (article)

This is the final version. Available on open access from Wiley via the DOI in this recordThe dataset associated with this article is located in ORE at: https://doi.org/10.24378/exe.1623Multi‐step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi‐step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi‐step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.Biotechnology & Biological Sciences Research Council (BBSRC)GlaxoSmithKlin

Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction (article)

This is the final version. Available on open access from Nature Research via the DOI in this recordThe research data supporting this publication are openly available in ORE at https://doi.org/10.24378/exe.2003Carboxylic 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.Glaxosmithkline Research & Development Lt

Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this recordBiocatalysis, the use of enzymes in chemical transformations, is an important green chemistry tool. Cascade reactions combine different enzyme activities in a sequential set of reactions. Cascades can occur within a living (usually bacterial) cell; in vitro in ‘one pot’ systems where the desired enzymes are mixed together to carry out the multi-enzyme reaction; or using microfluidic systems. Microfluidics offers particular advantages when the product of the reaction inhibits the enzyme(s). In vitro systems allow variation of different enzyme concentrations to optimise the metabolic ‘flux’, and the addition of enzyme cofactors as required. Cascades including cofactor recycling systems and modelling approaches are being developed to optimise cascades for wider industrial scale use. Two industrially important enzymes, transaminases and carboxylic acid reductases are used as examples regarding their applications in cascade reactions with other enzyme classes to obtain important synthons of pharmaceutical interest.Glaxosmithkline Research & Development LtdBiotechnology & Biological Sciences Research Council (BBSRC

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

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts (dataset)

The article associated with this dataset is located in ORE at: http://hdl.handle.net/10871/38080This is the dataset used for the Finnigan et al. (2019) article "Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts" published in ChemCatChem.Biotechnology and Biological Sciences Research Council (BBSRC)GlaxoSmithKlin

Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction (dataset)

Dataset underpinning a publication of the same name in Communications Biology. Curated data are provided in text, image, or .xlsx format. Unstructured data are provided in a wider range of formats - GraphPad Prism format is the most common.The article associated with this dataset is located in ORE at: http://hdl.handle.net/10871/39763Carboxylic 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.Glaxosmithkline Research & Development Lt

Diagnostic value of CD163 in cutaneous spindle cell lesions

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74045/1/j.1600-0560.2008.01179.x.pd