A Cell-Free Synthetic Biology Strategy for Targeted Antibiotic Discovery and Resistance Studies

Antimicrobial resistance (AMR), predicted to cause 10 million annual deaths by 2050, is a public health crisis faced with a limited pipeline of new antibiotics. Addressing this global pandemic requires measures to enhance antibiotic discovery, as well as study evolving AMR mechanisms in high-priority pathogens. Klebsiella pneumoniae, notoriously known for AMR, has a "phalanx" of membrane-associated defences that restrict intracellular access to antibiotics. In this thesis, we leverage cell-free synthetic biology in this non-model organism to study antibiotic sensitivity and resistance from an intracellular perspective. Additionally, we explore further synthetic biology tools for alternative antibiotic discovery.

In the first chapter, I have established a cell-free gene expression (CFE) system for K. pneumoniae. I optimise the assay for high-throughput screening, and as a proof-of-concept, test an FDA-approved drug library. Antibiotics with distinctive profiles between cell-free and conventional cell-based assays were identified during hit expansion. This data helps to address potential limitations in quantifying how individual antibiotics function inside the cell of a pathogen. Hence, our CFE platform provides a safe, low-cost, high-throughput, sensitive, and pathogen-targeted screening platform.

In the second chapter, I investigate genomic and proteomic differences between K. pneumoniae CFE system and the model E. coli. I also adapt CFE to study how laboratory-adapted AMR mutants of K. pneumoniae influence CFE and resistance to antibiotics, providing an in vitro model to interrogate AMR mechanisms. To the best of our knowledge, this is the first study that exploits CFE to study pathogen-specific AMR variants. As a key example, an RNA polymerase β-subunit H526L variant conferred strong CFE resistance to rifampicin by displaying a 58-fold IC50 increase. I highlight the adaptability of CFE to clinically relevant K. pneumoniae isolates to profile their intracellular resistance, complemented with LC-MS proteomics to characterise their extract proteome.

The third chapter takes a discovery-based approach to look for alternative antimicrobials using genome mining. Here I focus on isolating ribosomal peptides from marine bacteria, where there are several advantages to using cell-free biosynthesis for antimicrobial discovery. Ribosomal peptides, particularly lanthipeptides, are a promising source of novel antibiotics. These natural products are encoded by modular biosynthetic gene clusters (BGCs), which facilitate rapid genetic engineering. Overall, I target the expression of six lanthipeptide BGCs identified from obligate marine Streptomyces strains, which I characterise in silico, and attempt the heterologous expression in model Streptomyces hosts.

Taken together, these findings contribute to antibiotic discovery using cell-free synthetic biology, while also laying the foundation for the novel application of cell-free systems for studying pathogen-specific AMR from an intracellular perspective. We provide this new AMR research tool with promising avenues to explore infectious diseases and for targeted structure-activity relationship studies.