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Novel chassis for engineered pathways and automating biological experiments in the biotechnology industry

Asra, Aisha Mariam; (2019) Novel chassis for engineered pathways and automating biological experiments in the biotechnology industry. Doctoral thesis (Eng.D), UCL (University College London). Green open access

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Abstract

For the pharmaceutical industry, there has been a movement towards greener chemistry to reduce environmental impact. This movement has sparked interest from the pharmaceutical and chemicals industry in synthetic biology, which involves increasing the use of biological hosts and enzymatic processes to produce pharmaceutical products. This increase in biocatalysis and synthetic biology has sparked more of a need for diverse and versatile enzymes in order to customise the end product as well as diverse and robust host organisms to accommodate the reactions. Synthetic biology also opens opportunities for the discovery of complex molecules which have been inaccessible by traditional synthetic chemistry routes. Using enzymes from the toluene metacleavage pathway found in Pseudomonas putida and an omega-transaminase, an engineered metabolic pathway was constructed in Escherichia coli and in various P. putida strains for the production of novel amines. In this study the conversion of benzoate to 2-hydroxymuconic semialdehyde (2-HMSA) has been investigated in a whole cell biotransformation reaction to compare the feasibility of using P. putida to E. coli as an alternative host organism for industrial processes. P. putida KT2440 showed activity comparable to E. coli, indicating that this could be a suitable organism for use in industry. Nineteen omega-transaminases were screened to select a transaminase which was able to convert 2-HMSA to a novel amine. Results showed that (R) – selective transaminase appeared to have activity with 2-HMSA, with an arthrobacter mutant transaminase (ArRMut11) showing the highest conversion rates. The conversion of modified starting materials, p-toluic acid, m-toluic acid and 2,3- dimethylbenzoate were investigated with the engineered pathway. Identifying the amine products of the transaminase conversion of 2-HMSA and the modified starting substrates was attempted using a combination of HPLC and mass spectrometry, however the exact structures were not confirmed.

Type: Thesis (Doctoral)
Qualification: Eng.D
Title: Novel chassis for engineered pathways and automating biological experiments in the biotechnology industry
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10072295
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