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

许多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，免疫抑制剂如环孢菌素或降胆固醇药物洛伐他汀。这些药物是由称为聚酮酶（PKS）和非核糖体肽合成酶（NRPS）的蛋白质（酶）形成的，并且这些酶形成特定产物的方式已经得到了很好的研究。青霉素是在近世纪前发现的，这种抗生素彻底改变了细菌感染的治疗方法。如今，由于多重耐药细菌的出现，其治疗用途处于危险之中，迫切需要新的药物。该项目研究开发一类新的真菌酶，具有生产新型抗生素的潜力。这些酶被称为NRPS-like酶，并且似乎在形成蘑菇的真菌和霉菌中非常广泛地分布。NRPS-like酶产生多种天然产物，这些天然产物具有潜在的应用，如除草剂和抗病毒药物以及抗癌症和糖尿病药物。此外，这些产品中的一些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。其中一个原因是对酶结构的了解有限，并且缺乏对这些酶形成其产物的机制的了解。这使得很难设计出可用于制造所选产品的酶版本。因此，本研究的一个主要目的是确定NRPS样酶的关键部分的三维结构，以了解这些酶如何产生这些有价值的天然产物。我们发现，NRPS样酶的一种具有抗生素潜力的产物可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。由于NRPS样酶的产物附着在M中的临床相关靶标上。结核病，对酶的更深入了解将有助于优化更有效产品的生产。此外，通过组合单个NRPS样酶，可以形成多种多样的新产品，具有更广泛的人类使用潜力和范围。对于这样的酶组合，该项目将利用地衣中NRPS样酶的特殊功能。地衣产生1000多种天然产物，但其生长缓慢和缺乏基因改造工具阻碍了其开发。我们已经成功地将这种地衣衍生的NRPS样酶引入到更适合这种应用的其他真菌中。虽然迄今为止所有的NRPS样酶仅产生单一产物，但这种地衣衍生的酶可以产生与灭活分枝杆菌的产物相似的多种产物。因此，我们将研究这种地衣衍生酶的全部产品谱，以产生具有增加的抗生素潜力的产品。此外，我们将利用其灵活性，通过蛋白质组合（融合）来产生对自然界新颖的产品。

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.