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Engineering Transketolase for Industrial Biotechnology

Affaticati, PE; (2017) Engineering Transketolase for Industrial Biotechnology. Doctoral thesis , UCL (University College London).

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Abstract

Transketolase is a ubiquitous enzyme of the thiamine diphosphate-dependent (TPP) family involved in the Calvin cycle and the pentose phosphate pathway. Substrate-walking of E. coli transketolase progressively shifted the target acceptor substrate from phosphorylated aldehydes to non-polar aromatic aldehydes. However, its applicability as an industrial biocatalyst is limited by the lack of combination mutants exhibiting satisfactory substrate breadth and stability. The S385Y/D469T/R520Q variant, which had previously been thought to exhibit differential binding to aromatic substrates, was analysed. Three model substrates were docked into its active site thus revealing two binding pockets supporting π-π stacking interactions. Screening of this variant with other cyclic compounds revealed evolved activities towards valuable industrial building blocks including 4-(methylsulfonyl)benzaldehyde (4-MSBA), a precursor to thiamphenicol. A quadruple mutant was consequently engineered by recombining a stabilising mutation and used as a template for further evolution towards bulky aromatics. Site-directed mutagenesis of a key residue generated the H192P/S385Y/L466M/D469T/R520Q variant which exhibited 5.6-fold improved kinetics towards 4-MSBA compared to the triple mutant. The transition of TK from a model enzyme to a robust industrial biocatalyst however does not only rely on its ability to synthesise novel therapeutic molecules, but also on its thermo- and solvent-stability. 52 variants of TK across the tree of life were consequently aligned to engineer a consensus variant and reconstruct a common ancestor to TK speculated to have branched from proteobacteria, firmicutes and fungi. The resulting common ancestor exhibited trace levels of non-native activity towards non-phosphorylated sugars and provided an initial soluble enzyme to explore the stability/activity relationship of future de novo TKs.

Type: Thesis (Doctoral)
Title: Engineering Transketolase for Industrial Biotechnology
Event: University College London
Language: English
Keywords: Biocatalysis, transketolase, industrial biotechnology, protein engineering
UCL classification: UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
URI: http://discovery.ucl.ac.uk/id/eprint/1557943
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