Stojanovski, Gorjan; (2022) The development of chemoenzymatic methods toward novel aminoglycosides and a heterologous expression system for butirosin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The rise and spread of antimicrobial resistance has necessitated the development of novel antimicrobials which are e ffective against drug-resistant pathogens. Aminoglycoside antibiotics (AGAs) remain one of our most e ffective classes of bactericidal drugs yet are challenging molecules to derivatise by chemical synthesis, requiring the use of extensive protection and deprotection steps leading to long, atom- and step-ine fficient synthetic routes. Biocatalytic, chemoenzymatic and synthetic biology approaches for the generation of AGAs are of interest as they allow more concise and sustainable synthetic routes to novel compounds. This work presents a two-step chemoenzymatic route to allow regioselective modifications of the C-6’ position of AGAs. The route uses a transaminase enzyme to generate an aldehyde on the C-6’ position followed by reductive amination or a Pictet Spengler (PS) reaction to introduce substituents selectively on this position. Seven candidate transaminases were tested on a panel of aminoglycosides and the most promising enzyme, GenB4, was then coupled with a reductive amination or PS reaction to generate 12 novel 6’-gentamicin C1a analogues. Five of these compounds were active antimicrobials and four of these were potent against an aminoglycoside resistant Escherichia coli. Biocatalytic synthesis of vicinal 1,2-diols was also explored by characterising novel epoxide hydrolases (EHs). Eight α/β EHs and three limonene EHs were characterised in terms of their substrate scope. For the two most promising EHs, the e ffects of reaction time, temperature, substrate concentration and co-solvent were determined. Finally, a synthetic biology approach for the in vivo biosynthesis of butirosin (an AGA) was explored using Bacillus subtilis as a production host. A butirosin BGC was identified in Paenibacillus chitinolyticus, introduced into B. subtilis and the strain tested for the fermentative production of butirosin. While butirosin production was not realised, the expression of cluster specific genes was confirmed under the fermentation conditions.

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