Tackling antimicrobial resistance (AMR) with protein engineering to produce novel yanuthone-based antimicrobial agents

The aim of this project is to produce and characterise a few of the proteins that play key roles in the biosynthetic production of the antimicrobial yanuthone D. Yanuthones are a naturally occurring group of secondary metabolites with bioactive properties. One derivative, yanuthone D is a meroterpenoid antibiotic with a structure different from most current antibiotics (e.g., penicillin and subsequent generation of cephalosporins), with antimicrobial activity against human pathogens including the superbug MRSA and Candida albicans.

The biosynthetic production of yanuthone D involves 8 enzymatic steps. As is common in the production of antimicrobials, once the core structure of the compound is synthesised (the polyketide 6-methylsalicylic acid (6-MSA), in yanuthone production), several 'tailoring' enzymes add and remove smaller components such as hydroxylations, decarboxylation, epoxidation, prenylation and glycosylations.

Harnessing the reactions of tailoring enzymes in antimicrobial production has been shown to produce new compounds and can result in development of potent new antimicrobials. Furthermore, synthetic biological approaches at the genetic level of the production pathway such as gene mutations, deletions and replacements can generate novel compounds. These compounds, ultimately resulting from recombinant enzyme engineering can not only be used to develop medicinal chemicals and antimicrobials but can also serve as potential templates for greener organic chemical synthesis.

Key tailoring enzymes in the latter stages of yanuthone D production have been identified but are yet to be properly characterised. The project will focus initially on cytochrome P450 and decarboxylase tailoring enzymes. This will include the recombinant production and purification to allow their characterisation by multiple biophysical techniques, including UV-vis spectroscopy, and analysed for antimicrobial productions.

Crystallization of pure recombinant proteins will take place to generate crystal structures of these enzymes that coupled with an understanding of their biophysical properties will facilitate future drug and compound design and engineering. Once characterised, the tailoring enzymes will be engineered by targeted mutagenesis techniques to explore the full range of yanuthone derivatives that can be produced via protein engineering aiming to produce more potent antimicrobials as well as a range of chemical templates to link to chemical synthetic producing further active synthetic derivates.

Synthetic biology approaches will be employed to manipulate the antimicrobial production pathway in vivo using the existing and engineered enzyme variants and will be explored utilising LC/GC-MS analysis to monitor resulting compounds that are enzymatically produced. These link iteratively with synthetic organic approaches with resulting compounds and derivatives subsequently assessed for their chemical, medicinal, and antimicrobial properties.