Engineering microbial cell factories for production of improved polyene antifungal agents

真菌感染是对人类健康日益严重的全球性威胁。由于新出现的多重耐药病原体（如烟曲霉、白色念珠菌和耳念珠菌），目前可用的少量抗真菌药物正变得越来越无效。尽管迫切需要替代治疗，但近年来出现的新候选药物很少，我们仍然依赖于一些已建立的抗真菌药物，特别是多基因，包括世卫组织基本药物两性霉素B （AmB）、制霉菌素（Nys）和匹马霉素（Pim）。多烯是由放线菌产生的复杂环状聚酮类大环内酯。由于多基因体具有广谱活性，且耐药率相对较低，因此是目前最有效、应用最广泛的抗真菌药物。AmB由结节链霉菌产生，是许多常见系统性真菌感染的一线治疗药物。最近，AmB被用于对抗与COVID-19相关的毛霉病（黑木耳）的爆发。AmB也是在发展中国家造成高死亡率的寄生虫病利什曼病的基本治疗方法。尽管多烯具有重要的医学意义，但却表现出严重的毒性，迫切需要更安全、毒性更小的替代品。SAR表明，AmB中的无外环c16 -羧酸，也存在于大多数其他多烯中，是毒性的关键决定因素。缺乏这一片段的AmB衍生物毒性显著降低，c16 -羧酸酯的化学修饰也提供了毒性较低的衍生物。除了c16修饰外，多烯与糖和其他基团的偶联可以提高溶解度，减少聚集，也可以降低毒性。然而，复杂多烯的化学合成非常具有挑战性，需要多个步骤，有害试剂和大量使用保护基团，这是昂贵的，不可持续的，难以扩大规模。在这个项目中，我们的目标是设计微生物细胞工厂，通过可持续的单步发酵方法，可以提供更安全、活性更高、毒性更低的多变异体。

Fungal infections are an escalating global threat to human health. The fewantifungal agents currently available are becoming increasingly ineffective due to emergingmultidrug resistant pathogens (e.g. Aspergillus fumigatus, Candida albicans & Candida auris).Despite the acute need for alternative treatments, very few new drug candidates have emerged inrecent years, and we remain reliant on a few established antifungals, particularly polyenesincluding WHO essential medicines amphotericin B (AmB), nystatin (Nys) and pimaricin (Pim).Polyenes are complex cyclic polyketide macrolides produced by Actinobacteria. Given polyeneshave potent broad-spectrum activity and the incidence of resistance to them is relatively low, theyremain the most effective and widely used antifungal agents. AmB, which is produced byStreptomyces nodosus, is the frontline treatment for many common systemic fungal infections.Recently, AmB was used to combat the outbreak of COVID-19 associated mucormycosis (blackfungus). AmB is also an essential treatment of the parasitic disease leishmaniasis which causeshigh mortality in the developing world. Despite their medical importance, polyenes exhibitsevere toxicity, and there is an urgent need for safer, less toxic alternatives. SAR shows that anexocyclic C16-carboxylate in AmB, which is also present in the majority of the otherpolyenes, is a key determinant of toxicity. AmB derivatives lacking this moiety are significantly lesstoxic and chemical modification of the C16-carboxylate also affords derivatives with loweredtoxicity.In addition to C16-modification, conjugation of polyenes with sugars and other moieties that improve solubility and reduce aggregation can also reduce toxicity. However,chemical synthesis of complex polyenes is very challenging, requiring multiple steps, deleteriousreagents and extensive use of protecting groups, which is expensive, unsustainable and difficult toscale-up. In this project we aim to engineer microbial cell factories, that can deliver safer polyenevariants with improved activity and lower toxicity via a sustainable single-step fermentationapproach.