Antibiotic K16: Elucidation and Engineering Pathways to New Anti-infective Agents.

迫切需要新的抗感染药物来抗击病毒大流行和对抗由MRSA和金黄色念珠菌等细菌和真菌病原体引起的抗菌素耐药性(AMR)。还需要抗感染药物来应对被忽视的热带疾病，特别是疟疾、利什曼病和恰加斯病，这些疾病都是由单细胞寄生虫引起的。这些非传染性疾病每年造成50多万人死亡，影响到世界欠发达地区2亿多最贫困人口的生活。从长远来看，如果不及时治疗，新城疫造成的生命损失很可能会超过当前的新冠肺炎疫情。此外，合并感染(共病)在发展中国家非常普遍。例如，如果由寄生虫感染引起的NTD患者也感染了艾滋病毒和其他常见病毒，那么他们的死亡率会显著增加。因此，随着新的病毒大流行(例如新冠肺炎)的出现，在发展中国家提供针对常见NTD的有效治疗方法变得越来越重要。今天临床上使用的许多抗感染药物，包括大多数抗生素，都来自细菌和真菌(微生物)产生的天然化合物。这些天然产物往往是高度复杂的结构，需要进一步的合成修饰才能提供最终的药物分子。这类化合物的合成和制造既困难又昂贵，对制药公司开发基于天然产品结构的新药几乎没有激励作用。这在开发对抗AMR的抗生素和治疗NTD方面尤其有问题，因为这些药物产生的利润很少。生产优化天然产物衍生物的另一种方法是操纵产生天然产物的微生物中的生物合成途径(酶)。通过设计催化天然产物组装的酶，有可能通过更有效和更具成本效益的发酵过程来提供具有更好性能的变种。最近，我们发现了一种新的生物合成途径，可以传递抗生素K16，这是一种非常不寻常的天然产物，对引起NTD的真菌病原体和寄生虫具有良好的活性，包括利什曼病和恰加斯病。首先，我们将对产生K16相关天然产品的微生物的基因组进行排序，以确定编码生物合成酶的基因，这些酶将氨基酸和其他构建块组装成复杂的最终产品(K16)。在许多微生物中，天然产物组装所需的基因没有被激活，因此生物合成酶和产物不存在。为了解决这个问题，我们将建立激活(开启)生物合成基因表达的方法，以便我们能够分离出可能具有改善生物活性的K16相关化合物。我们将确定新的K16类化合物的结构，并测试它们对各种真菌和寄生虫的抑制作用。我们还将描述K16生物合成所需的一些关键酶的特征，包括组装氨基酸和其他前体的酶(NRPS-PKS)。我们还将研究一种非常不寻常的酶，它在最后一步中添加二氧化碳，生成具有生物活性的K16化合物。了解了K16和相关天然产物是如何组装的，我们将继续操纵(工程)生物合成酶来产生新的类似K16的产品。例如，我们将对NRPS-PKS装配线进行突变，以便接受不同的氨基酸前体。通过这种方式，我们的目标是为未来的药物开发生成一个具有不同结构修改和潜在改进特性的新K16变异体的文库。

There is an urgent need for new anti-infective agents to fight viral pandemics and combat antimicrobial resistance (AMR) arising from bacterial and fungal pathogens such as MRSA and Candida auris. Anti-infectives are also needed to tackle neglected tropical diseases (NTD), particularly malaria, leishmaniasis and Chagas disease, which are all caused by single cell parasites. These NTD cause over 500,000 deaths each year, effecting the lives of more than 200 million of the poorest people in the less developed regions of the world. In the longer term, it is likely that NTD, if untreated, will account for more loss of lives than the current COVID-19 pandemic. Moreover, co-infections (comorbidity) are highly prevalent in the developing world. For example, mortality rates for individuals with NTD caused by parasitic infections increases significantly if they also become infected with HIV and other common viruses. Consequently, as new viral pandemics (e.g. COVID-19) emerge, it is increasingly important that effective treatments for common NTD are made available in the developing world.Many of the anti-infective drugs used in the clinic today, including the majority of antibiotics, are derived from natural compounds produced by bacteria and fungi (microorganisms). These natural products are often highly complex structures and require further synthetic modifications to deliver the final drug molecule. The synthesis and manufacture of such compounds is difficult and expensive, providing little incentive for pharmaceutical companies to develop new drugs based on natural product structures. This is particularly problematic in the development of antibiotics to combat AMR and treatments for NTD, which generate little profit. An alternative approach for producing optimised natural product derivatives, is to manipulate the biosynthetic pathways (enzymes) in microorganisms that generate the natural products. By engineering the enzymes that catalyse the assembly of natural products it is possible to deliver variants, with improved properties, via a more efficient and cost-effective fermentation process. Recently we discovered a new biosynthetic pathway that delivers antibiotic K16, a highly unusual natural product, with promising activity against fungal pathogens and parasites that cause NTD including leishmaniasis and Chagas disease. Initially we will sequence the genomes of microbes that produce K16 related natural products, to identify the genes encoding the biosynthetic enzymes that assemble the amino acids and other building blocks into the complex final product (K16). In many microorganisms, the genes required for natural product assembly are not switched on and consequently the biosynthetic enzymes and products are not present. To address this, we will establish methods to activate (switch on) the expression of the biosynthesis genes, so that we can isolate K16 related compounds which may have improved biological activity. We will determine the structures of the new K16-like compounds and test them against various fungal and parasitic pathogens. We will also characterise some of the key enzymes required for K16 biosynthesis including the enzymes that assemble the amino acids and other precursors (NRPS-PKS). We will also study a highly unusual enzyme that adds carbon dioxide in the final step to generate the bioactive K16 compound. With knowledge of how K16 and related natural products are assembled, we will proceed to manipulate (engineer) the biosynthetic enzymes to generate new K16-like products. For example, we will mutate the NRPS-PKS assembly lines, so that different amino acid precursors are accepted. In this way we aim to generate a library of new K16 variants with different structural modifications and potentially improved properties for future drug development.