Bioengineering of next generation lipoglycopeptide antibiotics

卫生保健方面的主要挑战之一是提供能够对抗抗生素耐药病原体（超级细菌）的新型抗菌剂，这种病原体被广泛认为是一种主要的全球威胁。尽管如此，今天使用的大多数抗菌剂属于1970年以前发现的旧类抗生素。因此，迫切需要能够提供下一代抗生素的新方法。为了解决这一问题，我们建议研究ramoplanin和enduracidin家族脂糖肽类抗生素的生物合成和生物工程。脂糖肽是一种高效抗生素，具有相当大的临床潜力，拉莫planin已进入III期临床试验。然而，这些高度复杂的天然产物很难用传统的合成化学来修饰，并且无法通过药物开发所需的规模的全合成来实现。因此，产生改良和改进的第二代脂糖肽抗生素的努力受到严重限制。在这个项目中，我们将开发替代的生物合成工程方法，以实现这类抗生素的快速结构多样化，提供大量具有潜在改善抗菌活性的脂糖肽变体，以便与工业合作伙伴进行后续开发。首先，我们将对最高产的耐多酸素生产者S. fungicidicus的基因组进行测序，并利用该序列通过删除竞争途径和引入其他突变来进一步提高产品产量，从而优化脂糖肽的生产。我们还将探讨脂糖肽生物合成的关键步骤，包括糖基化、卤化和脂肪酸部分的组装。生物合成的见解将用于指导新的生物合成工程方法在优化的S.真菌宿主的发展。我们将开发生物工程方法来改变糖基化，卤化和脂化模式，以及杂化脂糖肽的肽序列。我们还将探索旨在用更稳定的内酰胺结构取代容易通过水解失活的内酯肽核的方法，以增加这些重要抗生素的体内寿命并提高其活性。我们开发的大量新的生物合成工程方法将是通用的，因此也可用于为其他有前途的抗生素类别（例如万古霉素和甘露肽霉素）以及具有治疗应用的其他天然产品变体（例如新的免疫抑制剂，抗病毒和抗癌剂）产生广泛的衍生物。此外，该方法还可用于生产新的农业除草剂、杀菌剂和杀虫剂。该项目还解决了关键的环境问题，通过提供方法，可以导致更环保和可持续的生物制造药物和其他生物活性产品，从可再生资源，通过发酵。考虑到我们对化石燃料的过度依赖，这一点尤其重要。化石燃料不仅用于能源，而且用于合成药物、农用化学品和其他我们今天生活中必不可少的产品的石化产品。用于合成的金属和其他元素的不可逆转的枯竭，以及传统化学制造过程中产生的有毒污染，也是主要的环境问题，因此需要发展替代办法，例如本建议中所述的办法。

One of the major challenges in healthcare is the provision of new antimicrobial agents that can combat antibiotic-resistant pathogens (superbugs), which are widely recognised as a major global threat. Despite this, the majority of antimicrobial agents used today belong to old classes of antibiotics discovered before 1970. Consequently, there is an urgent need for new approaches that can deliver the next generation of antibiotics. To address this, we propose to investigate the biosynthesis and bioengineering of lipoglycopeptide antibiotics of the ramoplanin and enduracidin family. The lipoglycopeptides are highly potent antibiotics which have considerable clinical potential, with ramoplanin having entered phase III clinical trials. However, these highly complex natural products are very difficult to modify using traditional synthetic chemistry, and are inaccessible through total synthesis on the scale required for drug development. Consequently, efforts to generate modified and improved second-generation lipoglycopeptide antibiotics are severely limited. In this project we will develop alternative biosynthetic engineering approaches to enable the rapid structural diversification of this class of antibiotics, providing access to large numbers of lipoglycopeptide variants with potentially improved antimicrobial activities, for subsequent development with industrial partners. Initially, we will sequence the genome of the most productive enduracidin producer, S. fungicidicus, and use this sequence to optimise production of lipoglycopeptides, by deleting competing pathways and introducing other mutations to further increase product yields. We will also explore key steps in the biosynthesis of lipoglycopeptides, including glycosylation, halogenation and assembly of the fatty acid moiety. The biosynthetic insights will then be used to guide the development of novel biosynthetic engineering methods in the optimised S. fungicidicus host. We will develop bioengineering methodology to alter the glycosylation, halogenation and lipidation patterns, as well as the peptide sequence of hybrid lipoglycopeptides. We will also explore approaches aimed at replacing the lactone peptide core, which is prone to inactivation through hydrolysis, with more stable lactam structures, to increase the longevity in vivo and improve the activity of these important antibiotics. The large repertoire of new biosynthetic engineering methodologies that we develop will be generic, and so can also be used to generate a wide range of derivatives for other promising classes of antibiotics (e.g. vancomycins and mannopeptimycins), as well as other natural product variants with therapeutic applications, for example new immunosuppressive, antiviral and anticancer agents. Moreover, the methodology could be adopted to produce new herbicides, fungicides and insecticides for agricultural use.This project also addresses key environmental issues, by providing methods that can lead to the more environmentally benign and sustainable biomanufacture of pharmaceuticals and other bioactive products from renewable resources, via fermentation. This is particularly important given our over reliance on fossil fuels, not just for energy, but also for the petrochemicals used in the synthesis of pharmaceuticals, agrochemicals, and other products that are essential in our lives today. The irreversible depletion of metals and other elements used in synthesis, along with the toxic pollution generated in traditional chemical manufacturing processes, are also major environmental concerns that necessitate the development of alternative approaches, such as those described in this proposal.