Bioengineering of next generation lipoglycopeptide antibiotics

医疗保健的主要挑战之一是提供新的抗菌剂，可以对抗耐药性病原体（超级细菌），这被广泛认为是一个主要的全球威胁。尽管如此，今天使用的大多数抗菌药物属于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。为了解决这个问题，我们建议研究雷莫普拉宁和雷帕霉素家族的脂糖肽抗生素的生物合成和生物工程。脂糖肽是具有相当大临床潜力的高效抗生素，雷莫拉宁已进入III期临床试验。然而，这些高度复杂的天然产物很难使用传统的合成化学进行修饰，并且无法通过药物开发所需规模的全合成进行修饰。因此，产生修饰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，使这类抗生素的快速结构多样化，提供大量的脂糖肽变体，具有潜在的改善抗菌活性，与工业合作伙伴的后续开发。首先，我们将对最高产的产黄单胞菌素的S.杀真菌剂，并使用该序列优化脂糖肽的生产，通过删除竞争途径和引入其他突变以进一步增加产物产率。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，然后将用于指导新的生物合成工程方法的优化S。杀真菌寄主我们将开发生物工程方法来改变糖基化，卤化和脂化模式，以及杂合脂糖肽的肽序列。我们还将探索旨在用更稳定的内酰胺结构取代易于通过水解失活的内酯肽核心的方法，以增加体内寿命并提高这些重要抗生素的活性。我们开发的大量新生物合成工程方法将是通用的，因此也可用于产生其他有前途的抗生素类（例如万古霉素和甘露肽霉素）的广泛衍生物，以及具有治疗应用的其他天然产物变体，例如新的免疫抑制剂，抗病毒剂和抗癌剂。此外，该方法还可用于生产农业用的新除草剂、杀真菌剂和杀虫剂，该项目还解决了关键的环境问题，提供了一些方法，可通过发酵，从可再生资源中生产对环境更友好和更可持续的药物和其他生物活性产品。考虑到我们对化石燃料的过度依赖，这一点尤为重要，不仅是能源，而且还包括用于合成药物，农用化学品和我们今天生活中必不可少的其他产品的石化产品。合成过程中使用的金属和其他元素的不可逆转的消耗，以及传统化学制造过程中产生的有毒污染，沿着也是主要的环境问题，因此有必要开发替代方法，例如本提案中所述的方法。

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.