Functional recombinant expression of cytochrome P450s and nitrilases in Escherichia coli, and their use in biocatalysis

Zoupali, Migkena Artemis (2023) Functional recombinant expression of cytochrome P450s and nitrilases in Escherichia coli, and their use in biocatalysis. Doctoral thesis, Northumbria University.

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Abstract

The use of enzymes to perform biocatalytic reactions has emerged as a key area in industrial biotechnology due to the increased demand for a sustainable and economical method of producing a variety of chemicals, while eliminating the harmful biproducts formed and the harsh conditions used.

Two different types of enzymes were used throughout this work: the cytochrome P450s and the nitrilases. Cytochrome P450s are a superfamily of haem-containing enzymes that catalyse a vast array of oxidative reactions, including the synthesis of active pharmaceutical ingredients, with a high degree of regio- and stereoselectivity. However, they exhibit low recombinant expression in E. coli, poor stability, and low turnover number. Moreover, eight out of ten existing classes require the help of electron transport proteins for biocatalytic processes, with only class VII and VIII being self-sufficient systems which contain all the redox partner proteins needed for biocatalysis.

On the other hand, nitrilase enzymes catalyse the hydrolysis of nitrile compounds into the appropriate carboxylic acid and ammonia consequently have numerous commercial and biotechnological uses. Nitriles are intermediates in the synthesis of pharmaceuticals, and their chemical hydrolysis requires high temperatures and pH, and formation of toxic by-products. Nitrilases can hydrolyse the nitriles in a single step in environmentally friendly conditions.

This project involved the cloning and recombinant expression of already characterised human enzymes CYP2A6, CYP2B6, CYP3A4, CYP3A5 and CYP2D6 in E. coli strains, and their participation in 2S, 6S; 2R,6R hydroxynorketamine production from racemic norketamine in whole cell biotransformations; a biotransformation of particular interest to Quotient Sciences. CYP2B6 and CYP3A4, indeed, hydroxylated norketamine to hydroxynorketamine in whole cell biotransformations.

Moreover, after mining the Prozomix database two novel metagenome genes, 914 and 198 were identified with sequence identity up to 24% to human CYP3A5. Both 914 and 198 were cloned and recombinantly expressed. Metagenome gene 914 revealed activity against ketamine, which N-demethylated to norketamine, and hydroxylated to hydroxyketamine, while 198 did not hydroxylate ketamine or norketamine.

Furthermore, 20 candidate nitrilases taken from the private collection of Prof. Gary Black, were expressed, and assayed against a panel of six nitriles to determine their catalytic activity. Only one nitrilase enzyme showed good activity against two nitriles, benzyl (2-cyanoethyl) carbamate and 2-(4-cyanophenyl)-N-ethylacetamide, which are of interest to Quotient Sciences.

In summary, the development of comparative methods in different cloning strategies will allow future scientist to enhance the expression and catalytic activity of the P450s. Moreover, the development of hydroxynorketamine from CYP2B6 and CYP3A4, hydroxyketamine from metagenome genes, and the development of both nitriles from nitrilase enzyme Q2GR86 will permit their use to be applied in industrial processes.

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