Castellano Garrido, Daniel;
(2025)
Biocatalytic Routes to Tetrahydroisoquinoline and Amaryllidaceae Alkaloids.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Tetrahydroisoquinolines (THIQs) and Amaryllidaceae alkaloids (AAs) are pharmaceutically important natural products, yet their chemical synthesis remains challenging due to complex stereochemistry and limited process scalability. Biocatalysis offers a sustainable alternative; however, many of the enzymes from these biosynthetic pathways are still poorly understood or difficult to engineer. This thesis explores a range of key natural plant enzymes and engineered biocatalysts to harness and expand access to bioactive alkaloids. Chapter 1 reviews existing biocatalytic strategies with a Responsible Research and Innovation perspective, identifying opportunities for greener and more efficient synthesis. Chapter 2 reports an in silico–inspired mutagenesis study of Thalictrum flavum norcoclaurine synthase (TfNCS) active site residues to probe enantioselectivity. Selected mutants displayed improved yields and reversal of stereoselectivity for some of the tetrahydroisoquinolines assayed. A one-pot/two-enzyme cascade also enabled norcoclaurine production at 5 mL scale. Chapter 3 demonstrates the catalytic versatility of norbelladine synthase (NBS) and salsolinol synthase (SalSyn), providing evidence for their dual Pictet–Spenglerase activity by employing a high-throughput cell free protein expression platform to select for conditions that improved the solubility and activity of plant enzymes while avoiding formation of inclusion bodies. Chapter 4 introduces a modular biocatalytic framework employing imine reductases (IREDs) integrated with cofactor recycling strategies, hydroxylation and methylation cascades. This approach enabled access to both natural and non-natural Amaryllidaceae derivatives, with optimisation of individual modules paving the way for future one-pot cascades. The novelty of this work lies in applying an in silico mutagenesis tool to guide the stereoselective control of TfNCS, implementing high-throughput screening methodologies to reveal new functionalities of NBS and SalSyn as Pictet-Spenglerases and establishing a modular IRED-based platform that expands alkaloid biosynthesis beyond the natural plant enzymes. Collectively, these findings advance enzymatic strategies for sustainable alkaloid production and broaden the synthetic biology toolkit for accessing complex natural products.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Biocatalytic Routes to Tetrahydroisoquinoline and Amaryllidaceae Alkaloids |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10215878 |
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