Methods for bioengineering NRPS/PKS assembly lines delivering peptide natural products with electrophilic warheads.

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Natural products (NP) are molecules isolated from microorganisms and plants that inspired the development of many leading antibiotics, anticancer, immunosuppressive agents and other essential medicines that are widely used in the clinic today. Often the NP that are isolated from the native organism do not possess the prerequisite properties at the outset. Further synthetic modification is typically required to provide the final drug compound. However, NP are typically highly complex molecules requiring laborious multistep chemical synthesis, which is very expensive, polluting and increasingly unsustainable. The difficulty associated with the synthesis of optimised NP variants presents a major barrier to pharma companies undertaking drug development. This is particularly problematic in the manufacture of drugs required to treat diseases of developing world such as malaria, Leishmania and Chagas disease. These highly infectious diseases, caused by single celled (protozoan) parasites transmitted by insects, effect billions of the poorest people in the world and lead to over 500,000 deaths pa. Currently there are very few effective treatments available for these diseases. A NP artemisinin is used to treat malaria, but new strains of the malaria parasite (P. falciparum) have emerged which are resistant to artemisinin. Promising new NP leads have been identified for malaria and other related diseases, but the costs of synthesising derivatives have prevented new treatments being made available. An alternative for producing optimised NP derivatives, is to manipulate the biosynthetic assembly lines (enzymes) in the microorganisms that construct the parent NP. By reprogramming (engineering) the assembly line to accept different precursors, NP variants with improved properties can be delivered in a more efficient, cost-effective single-step fermentation process.In this project we aim to engineer biosynthetic pathways to produce NP derivatives with antiprotozoal activity that could be used to combat malaria or related diseases. The target NP are peptides, composed of amino acids with a reactive terminal functional group (warhead), produced by Streptomyces and other bacteria. These NP will be designed to bind to the proteasome of protozoa such as P. falciparum. Proteasomes are large multi-protein complexes responsible for degrading other proteins in the cell that are either damaged or no longer needed. The warhead of the peptide NP can cross-link with the proteasome inhibiting its function leading to cell death. We will use novel gene editing and other approaches to engineer the genes encoding the enzymes that assemble the warhead containing peptide NP. This will allow us to change the sequence of the peptides and also include different warheads, to improve their activity, selectivity and other properties for drug development. The biosynthetic assembly line includes nonribosomal peptide synthetase (NRPS) enzymes that condense amino acid precursors. By replacing domains, or subdomains, within the NRPS it is possible to change the sequence of the amino acids in the peptide products. Guided by earlier synthetic studies, we will create warhead containing peptides that are highly selective for the P. falciparum proteasome. Compounds that inhibit proteasomes in human as well as the parasite cell, would be toxic and unsuitable. We will also engineer assembly lines that deliver warhead containing peptides designed to inhibit the proteasomes in human cancer cells. This includes oprozomib a synthetic analogue, which is in clinical trials for treatment of multiple myeloma (bone marrow cancer). By developing an engineered pathway to this type compound, it may be possible to produce anticancer drugs, like oprozomib, in a single-step fermentation making them more widely available at lower costs. The methods we develop are generic and can be used to produce a range of warhead containing peptides for a number of other therapeutic applications.