Antibiotic K16: Elucidation and Engineering Pathways to New Anti-infective Agents.

There is an urgent need for new anti-infective agents to fight viral pandemics and combat antimicrobial resistance (AMR) arising from bacterial and fungal pathogens such as MRSA and Candida auris. Anti-infectives are also needed to tackle neglected tropical diseases (NTD), particularly malaria, leishmaniasis and Chagas disease, which are all caused by single cell parasites. These NTD cause over 500,000 deaths each year, effecting the lives of more than 200 million of the poorest people in the less developed regions of the world. In the longer term, it is likely that NTD, if untreated, will account for more loss of lives than the current COVID-19 pandemic. Moreover, co-infections (comorbidity) are highly prevalent in the developing world. For example, mortality rates for individuals with NTD caused by parasitic infections increases significantly if they also become infected with HIV and other common viruses. Consequently, as new viral pandemics (e.g. COVID-19) emerge, it is increasingly important that effective treatments for common NTD are made available in the developing world.
Many of the anti-infective drugs used in the clinic today, including the majority of antibiotics, are derived from natural compounds produced by bacteria and fungi (microorganisms). These natural products are often highly complex structures and require further synthetic modifications to deliver the final drug molecule. The synthesis and manufacture of such compounds is difficult and expensive, providing little incentive for pharmaceutical companies to develop new drugs based on natural product structures. This is particularly problematic in the development of antibiotics to combat AMR and treatments for NTD, which generate little profit. An alternative approach for producing optimised natural product derivatives, is to manipulate the biosynthetic pathways (enzymes) in microorganisms that generate the natural products. By engineering the enzymes that catalyse the assembly of natural products it is possible to deliver variants, with improved properties, via a more efficient and cost-effective fermentation process.
Recently we discovered a new biosynthetic pathway that delivers antibiotic K16, a highly unusual natural product, with promising activity against fungal pathogens and parasites that cause NTD including leishmaniasis and Chagas disease. Initially we will sequence the genomes of microbes that produce K16 related natural products, to identify the genes encoding the biosynthetic enzymes that assemble the amino acids and other building blocks into the complex final product (K16). In many microorganisms, the genes required for natural product assembly are not switched on and consequently the biosynthetic enzymes and products are not present. To address this, we will establish methods to activate (switch on) the expression of the biosynthesis genes, so that we can isolate K16 related compounds which may have improved biological activity. We will determine the structures of the new K16-like compounds and test them against various fungal and parasitic pathogens. We will also characterise some of the key enzymes required for K16 biosynthesis including the enzymes that assemble the amino acids and other precursors (NRPS-PKS). We will also study a highly unusual enzyme that adds carbon dioxide in the final step to generate the bioactive K16 compound. With knowledge of how K16 and related natural products are assembled, we will proceed to manipulate (engineer) the biosynthetic enzymes to generate new K16-like products. For example, we will mutate the NRPS-PKS assembly lines, so that different amino acid precursors are accepted. In this way we aim to generate a library of new K16 variants with different structural modifications and potentially improved properties for future drug development.

Novel methods are urgently needed for the discovery and development of new anti-infectives required to overcome viral infections, antimicrobial resistance (AMR) and to treat neglected tropical diseases (NTD). Natural products (NP) have provided the inspiration for many of the anti-infectives in the clinic today. Synthetic optimisation of complex NP scaffolds is challenging, highly expensive and offers little return or incentive for commercial development of new drugs. Bioengineering approaches offer a more attractive alternative, providing a more sustainable and cheaper route to optimised NP variants via a single-step fermentation. Advances in genome sequencing, structural biology and synthetic biology have provided new insights and tools to enable more rapid discovery, engineering and optimisation of NP scaffolds. We recently used a genomics approach to discover the pathway to antibiotic K16, which has promising antifungal and antiprotozoal activity. K16 is assembled by NRPS-PKS enzymes with an unprecedented vitamin K dependent carboxylase (VKDC) catalysed carboxylation reaction, in the final step, delivering the bioactive product. Here we aim to mine genomes to discover gene clusters that generate other K16-like NP, developing methods to activate silent gene clusters. We will elucidate key steps in the biosynthesis of K16 including the NRPS initiation steps, an unusual Dieckmann cyclisation and the VKDC carboxylation reaction. The knowledge acquired will be used to develop new engineering approaches, reprogramming the NRPS-PKS to deliver K16 variants with improved properties. A novel MS-imaging technology will be deployed for rapid detection of products directly from colonies on plates. Finally, the structure and activity of new K16 variants will be determined to guide future drug development. The methods developed are generic and can be used in the discovery and engineering of a wide range of other NP scaffolds for pharma, agro and other important applications.