Exploring the metabolic diversity of engineered fungal non-ribosomal peptide synthetase-like enzymes for the development of novel antibiotics

广泛的天然产物对人类具有重要的有益特性，其中许多是由真菌产生的。例如抗生素如青霉素，免疫抑制剂如环孢素或降胆固醇药物洛伐他汀。这些药物是由被称为聚酮合成酶（PKS）和非核糖体肽合成酶（NRPS）的蛋白质（酶）形成的，这些酶形成特定产物的方式已经得到了很好的研究。青霉素是近一个世纪前发现的，这种抗生素彻底改变了细菌感染的治疗方法。如今，由于耐多药细菌的出现，它的治疗用途受到威胁，迫切需要新的药物。该项目研究了一类具有生产新型抗生素潜力的真菌酶的开发。这些酶被称为nrpps类酶，似乎在蘑菇形成真菌和霉菌中非常广泛地分布。类似nrps的酶能产生多种天然产物，具有潜在的应用前景，如除草剂、抗病毒药物以及抗癌和糖尿病药物。此外，其中一些产品显示出有希望的抗生素活性。然而，它们的全部潜力尚未得到开发。造成这种情况的一个原因是对酶结构的了解有限，对这些酶形成其产物的机制缺乏了解。这使得很难设计出可以用来制造选择产品的酶版本。因此，本研究的一个主要目的是确定nrpps类酶关键部分的三维结构，以了解酶如何产生这些有价值的天然产物。我们发现，一种类似nrps的酶的具有抗生素潜力的产物之一可以有效抑制结核分枝杆菌，结核分枝杆菌是人类结核病的主要原因，并且经常产生极端的多药耐药性。随着nrps样酶的产物附着在结核分枝杆菌的临床相关靶标上，对该酶的更深入了解将有助于优化产生更有效的产物。此外，通过结合单个类nrpps酶，可以形成多种多样的新产品，具有广阔的潜力和谱供人类使用。对于这种酶组合，该项目将利用地衣中类似nrps的酶的特殊功能。地衣生产1000多种天然产品，但它们生长缓慢，缺乏基因改造工具，阻碍了它们的开发。我们已经成功地将这种地衣衍生的类似nrps的酶引入到其他更适合这种应用的真菌中。虽然迄今为止表征的所有类似nrpps的酶只能产生单一产物，但这种地衣衍生的酶可以产生多种与灭活分枝杆菌的产物相似的产物。因此，我们将描述这种地衣衍生酶的完整产品谱，以产生具有更高抗生素潜力的产品。此外，我们将利用其灵活性，通过蛋白质组合（融合）来产生自然界新颖的产品。

A wide range of natural products have important beneficial properties for humans and many of them are produced by fungi. Examples are antibiotics such as penicillin, immunosuppressants such as cyclosporine or the cholesterol-lowering drug lovastatin. These drugs are formed by proteins (enzymes) that are called polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) and the ways in which these enzymes form specific products has been well studied. Penicillin was discovered nearly a century ago and this antibiotic revolutionised the treatment of bacterial infections. Today, its therapeutic use is in jeopardy due to the emergence of multi-drug resistant bacteria and new drugs are urgently needed. This project investigates the exploitation of a new class of fungal enzymes with the potential to produce novel antibiotics. These enzymes are called NRPS-like enzymes and appear to be very widely distributed among mushroom-forming fungi and moulds.NRPS-like enzymes produce a variety of natural products with potential applications as herbicides and antiviral drugs as well as drugs against cancer and diabetes. Moreover, some of these products display promising antibiotic activity. However, their full potential is as yet unexplored. One reason for this is the limited understanding of the enzyme structure and the lack of knowledge of the mechanism by which these enzymes form their products. This makes it difficult to design enzyme versions that can be used to make products of choice. Therefore, one major aim of this study is to determine the three-dimensional structure of key parts of NRPS-like enzymes to understand how the enzymes make these valuable natural products.We have found that one of the products with antibiotic potential from an NRPS-like enzyme is effective at inhibiting Mycobacterium tuberculosis, a bacterium that is the main cause of human tuberculosis and that frequently develops extreme multi-drug resistance. As the product from the NRPS-like enzyme attaches to a clinically relevant target in M. tuberculosis, a deeper understanding of the enzyme will help to optimise the generation of even more effective products. Moreover, by combining individual NRPS-like enzymes, a great diversity of novel products can be formed, with widened potential and spectrum for human use. For such enzyme combinations, this project will harness the special features of an NRPS-like enzyme from a lichen. Lichens produce more than 1000 natural products, but their slow growth and the lack of tools for genetic modification hampers their exploitation. We have successfully introduced this lichen-derived NRPS-like enzyme into other fungi that are more suited to this application. While all NRPS-like enzymes characterised so far only produce a single product, this lichen-derived enzyme can generate a variety of products with similarity to the product that inactivates mycobacteria. Therefore, we will characterise the full product spectrum of this lichen-derived enzyme to generate products with increased antibiotic potential. In addition, we will capitalise on its flexibility by making protein combinations (fusions) to generate products that are novel to nature.