Engineering macrolactam antimicrobial agents (EMLA)

我们环境中的微生物（例如土壤细菌）产生分子，天然产物（NP），用于开发重要的药物，例如对抗抗菌素耐药性（AMR）所需的抗生素，治疗被忽视的疾病和应对未来的流行病。NP也被用作作物保护剂，以提高作物产量，帮助养活不断增长的人口。许多NP是由非核糖体肽合成酶（NRPS）和聚酮合成酶（PKS）组装的，前者将氨基酸构建块偶联成肽产物，后者浓缩丙二酸和其他前体生成聚酮。这些巨大的“大合成酶”（NRPS和PKS）具有硫酯酶（TE）结构域，可以环化肽链或聚酮链以产生环状结构（大内酯）。虽然大内酯具有良好的生物活性，但它们容易被水解裂解环，从而使其活性丧失。例如，达托霉素和红霉素分别来自NRPS和PKS，是临床上重要的大内酯类抗生素，但病原体已经进化出水解酶（酯酶），可以裂解和失活这些大内酯类抗生素，导致抗微生物药物耐药性（AMR）。耐抗生素病原体的出现是我们今天面临的最大威胁之一。我国政府估计，抗生素耐药性每年在全球造成70万人死亡，预计到2050年将增加到1 000万人，给全球经济造成100万亿美元的损失。化学合成可用于制备更有效的大内酰胺衍生物，其中不稳定的内酯被更稳定的内酰胺键取代。虽然大内酰胺具有优异的性能，可以避免抗菌素耐药性，但其合成成本高，污染严重，不可持续。我们将通过开发生物工程巨合酶的新方法来解决抗菌素耐药性和粮食安全问题，为医疗和农业使用的优质大内酰胺抗菌剂创造可持续的途径。该项目建立在我们最近成功开发一种用于NRPS重编程的新基因编辑方法的基础上。NRPS和PKS是自然界中最大、最复杂的酶，其工程设计极具挑战性，并且取得了有限的成功。然而，我们发现基因编辑可用于引入复杂NRPS的靶向改变，使替代氨基酸前体被纳入肽抗生素中。我们设想我们的方法可以用于设计许多不同的巨合酶。最初，我们将使用基因编辑和其他方法来设计来自放线菌（多产的抗生素生产者）的NRPS，提供更稳定的内酯类抗生素enduracidin （END）和ramoplanin （RAM）的内酰胺变体，这两种抗生素已进入治疗万古霉素耐药肠球菌的III期临床试验。RAM内酰胺变体已通过化学合成制备，并被证明是优良的抗生素，但它们的合成需要40到40个步骤，使用昂贵且有毒的试剂，并且不适合药物开发。我们将通过清洁、廉价的单步发酵生产改进的END/RAM内酰胺，使更稳定、更有效的抗生素广泛使用。将开发一种类似的方法来生产改进的DAPT内酰胺变体，可用于治疗MRSA和其他由抗生素耐药病原体引起的危及生命的感染。我们还将探索从芽孢杆菌（另一种土壤细菌）中提取的生物工程NRPS和杂交PKS-NRPS酶，以生产环脂肽抗真菌剂（fengycin和surfactin）的改良内酰胺衍生物。芽孢杆菌菌株和内酰胺产品可用作作物保护剂，杀死破坏粮食作物的植物真菌病原体，包括养活世界一半人口的水稻。除了重新编程NRPS/PKS以引入不同的前体，导致内酰胺而不是内酯环，我们还将探索TE结构域的结构引导工程（微调），以更有效地形成内酰胺。

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.