Prout, Luba; (2023) Natural and synthetic transaminase fusions and their use in biocatalysis. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text PROUT_PhD_THESIS_WithCorrections.pdf - Accepted Version Download (80MB) | Preview

Abstract

Amines are nitrogen-containing compounds that have wide-ranging applications such as in the manufacturing of dyes, polymers, personal care products, drugs, or as solvents, crop protection chemicals, and surfactants. Of relevance to this work, many single isomer amines also serve as building blocks for drugs and chemicals in the pharmaceutical, chemical, and agrochemical industries. The cost, toxicity, and complexity of classical chemical routes for amine production has been driving the acceleration of biocatalytic approaches. Earlier methods entailed single-step enzymatic conversions using, for example, transaminases or imine reductases. More recent work has involved multi-step enzyme cascades and de novo pathway engineering, for use both in vitro and in vivo. Despite these advances, there are still problems associated with enzyme catalysis, including low yields due to unfavourable reaction equilibria, the need for co-factor recycling in vitro, loss of intermediates to cellular enzymes, toxicity and insufficient uptake in whole cell biocatalysis or excessive substrate or intermediate efflux in vivo. Synthetic biology approaches that attempt to tackle these issues include host engineering, enzyme immobilisation and enzyme fusion engineering. The aim of this project was to investigate a natural transaminase enzyme fusion and use this information to design and engineer novel enzyme fusions. Naturally occurring multifunctional enzymes contain two or more catalytic modules that enable stepwise substrate transformation in situ. In vivo, the presence of multifunctional enzymes in metabolic pathways is thought to confer selective advantage via increased reaction rates and chemical stability or prevention of toxicity from reactive intermediates. This investigation focused on the characterisation of a putative natural fusion between a transaminase and an oxidoreductase, protein PP_2782 from Pseudomonas putida KT2440, following its discovery through previous research at UCL. Its three thermophilic homologs – from Thermaerobacter marianensis DSM 12885, Thermaerobacter subterraneus DSM 13965, and Thermincola ferriacetica Z-0001 – were also investigated for the first time. Using these fusions as a template, an attempt has been made to design, assemble and test synthetic fusions comprising (1) Chromobacterium violaceum DSM 30191 ω-transaminase, and (2) Equus caballus alcohol dehydrogenase, (3) Geobacillus stearothermophilus 22 alcohol dehydrogenase, and (4) Thalictrum flavum subsp. glaucum (S)-norcoclaurine synthase enzymes. The initial analysis of their activity is presented. Additionally, the role and activity of a gene cluster, PP_2777-PP_2787 loci in P. putida KT2440, that was found to contain the natural transaminase fusion, were also explored in this work.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as JSONXMLCSV

Archive Staff Only

View Item