Engineering macrolactam antimicrobial agents (EMLA)

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Microorganisms in our environment (e.g. soil bacteria) produce molecules, natural products (NP), that are used to develop important pharmaceuticals, such as antibiotics required to combat antimicrobial resistance (AMR), treat neglected diseases and tackle future pandemics. NP are also used as crop protection agents to boost crop yields and help feed the growing population. Many NP are assembled by nonribosomal peptide synthetase (NRPS) enzymes that couple amino acid building blocks into peptide products, and polyketide synthase (PKS) enzymes that condense malonic acid and other precursors to create polyketides. These huge 'megasynthase' (NRPS & PKS) possess thioesterase (TE) domains that cyclise peptide or polyketide chains to create cyclic structures (macrolactones). Although macrolactones possess exquisite bioactivity, they are prone to hydrolysis cleaving the ring which abolishes their activity. For example, daptomycin and erythromycin are clinically important macrolactone antibiotics from NRPS and PKS respectively, but pathogens have evolved hydrolase enzymes (esterases) which can cleave and deactivate these macrolactones leading to antimicrobial resistance (AMR). The emergence of antibiotic-resistant pathogens is one of the biggest threats we face today. Our government estimate that AMR causes 700,000 deaths each year globally, which is predicted to rise to 10 million, costing the global economy $100 trillion, by 2050. Chemical synthesis can be used to prepare more effective macrolactam derivatives, where the labile lactone is replaced by a more stable lactam bond. Although macrolactams have superior properties, and can evade AMR, their synthesis is very costly, polluting and unsustainable. We will address problems of AMR and food security by developing new methods for bioengineering megasynthase, creating sustainable routes to superior macrolactam antimicrobial agents for medical and agricultural use. The project builds on our recent success developing a new gene editing approach for NRPS reprogramming. Engineering NRPS and PKS, which are amongst the largest and most complex enzymes in nature, is extremely challenging and has met with limited success. However, we showed that gene editing can be used to introduce targeted changes to complex NRPS, enabling alternative amino acids precursors to be incorporated into peptide antibiotics. We envisage our approach could be used to engineer many different megasynthase. Initially, we will use gene editing and other methods to engineer NRPS derived from Actinobacteria (prolific antibiotic producers) delivering more stable lactam variants of the macrolactone antibiotics enduracidin (END) and ramoplanin (RAM), which entered phase III clinical trials for the treatment of vancomycin-resistant Enterococcus. RAM lactam variants have been prepared by chemical synthesis, and shown to be superior antibiotics, but their synthesis took >40 steps, using expensive and toxic reagents, and is not viable for drug development. We will generate improved END/RAM lactams in a clean, cheap, single-step fermentation, making more stable and effective antibiotics widely available. A similar approach will be developed to produce improved lactam variants of DAPT which could be used to treat MRSA and other life-threatening infections caused by antibiotic resistant pathogens. We will also explore bioengineering NRPS and hybrid PKS-NRPS enzymes from Bacillus (another soil bacteria) to produce improved lactam derivatives of cyclic lipopeptide antifungal agents (fengycin & surfactin). The Bacillus strains and lactam products can be used as crop protection agents to kill fungal plant pathogens that damage food crops, including rice which feeds half of the world's population. In addition to reprogramming NRPS/PKS to introduce different precursors, leading to lactam rather than lactone rings, we will also explore structure-guided engineering (fine tuning) of TE domains for more efficient macrolactam formation.