Engineering macrolactam antimicrobial agents (EMLA)

工程大环内酰胺抗菌剂（EMLA）

• 批准号: BB/X002241/1
• 负责人: Jason Micklefield
• 金额: $ 66.45万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX002241%2F1
• 关键词: Engineering; macrolactam; antimicrobial; agents; EMLA

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.

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），治疗被忽视的疾病和应对未来流行病所需的抗生素。NP还用作作物保护剂，以提高作物产量并帮助养活不断增长的人口。许多NP通过非核糖体肽合成酶（NRPS）和聚酮化合物合成酶（PKS）组装，所述非核糖体肽合成酶（NRPS）将氨基酸结构单元偶联成肽产物，所述聚酮化合物合成酶（PKS）缩合丙二酸和其他前体以产生聚酮化合物。这些巨大的“大合成酶”（NRPS & PKS）具有硫酯酶（TE）结构域，其使肽或聚酮链环化以产生环状结构（大环内酯）。虽然大环内酯具有精致的生物活性，但它们易于水解裂解环，从而消除它们的活性。例如，达托霉素和红霉素分别是来自NRPS和PKS的临床上重要的大环内酯抗生素，但是病原体已经进化出水解酶（酯酶），其可以切割和灭活这些大环内酯，导致抗微生物剂抗性（AMR）。抗药性病原体的出现是我们今天面临的最大威胁之一。我们的政府估计，AMR每年在全球造成70万人死亡，预计到2050年将增加到1000万人，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环内酰胺衍生物，其中不稳定的内酯被更稳定的内酰胺键取代。虽然大环内酰胺具有上级性质，并且可以避免AMR，但是它们的合成非常昂贵、污染和不可持续。我们将通过开发生物工程大合成酶的新方法来解决AMR和食品安全问题，为医疗和农业用途的上级大环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NRPS重编程新基因编辑方法的基础上。NRPS和PKS是自然界中最大和最复杂的酶之一，其工程化极具挑战性，并且取得了有限的成功。然而，我们发现，基因编辑可以用于向复杂的NRPS引入靶向改变，使替代氨基酸前体能够被纳入肽抗生素中。我们设想我们的方法可以用于工程许多不同的megasynthase。最初，我们将使用基因编辑和其他方法来工程化来自放线菌（多产抗生素生产者）的NRPS，提供更稳定的大环内酯抗生素雷帕霉素（END）和雷莫拉宁（RAM）的内酰胺变体，这些变体进入了治疗万古霉素耐药肠球菌的III期临床试验。RAM内酰胺变体已经通过化学合成制备，并且显示为上级抗生素，但是它们的合成需要>40个步骤，使用昂贵且有毒的试剂，并且对于药物开发不可行。我们将在清洁，廉价，单步发酵中产生改进的END/RAM内酰胺，使更稳定，更有效的抗生素广泛使用。将开发类似的方法来生产DAPT的改进内酰胺变体，其可用于治疗MRSA和由抗生素耐药病原体引起的其他危及生命的感染。我们还将探索来自芽孢杆菌（另一种土壤细菌）的生物工程NRPS和混合PKS-NRPS酶，以生产改进的环脂肽抗真菌剂（芬枯草菌素和表面活性剂）的内酰胺衍生物。芽孢杆菌菌株和内酰胺产品可用作作物保护剂，以杀死损害粮食作物的真菌植物病原体，包括养活世界一半人口的水稻。除了重新编程NRPS/PKS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结构域的结构导向工程（微调），以更有效地形成大环内酰胺。