Back to soil: awakening the production of cryptic antibiotics in Streptomyces strains

抗生素耐药细菌病原体的发病率日益增加，导致迫切需要新的，临床有用的抗生素。医学上使用的大多数抗生素都是由放线菌产生的，尤其是一种被称为链霉菌的无害细菌，它在所有土壤环境中都很丰富。基于它们的基因组序列，每个链霉菌物种平均有潜力产生大约10-15种天然产物来源的抗菌剂。然而，当这些物种在实验室条件下生长时，通常只检测到一种或两种抗菌化合物。这是因为大多数抗生素基因簇在实验室条件下处于休眠状态（即不表达）。链霉菌物种在其基因组中保持这些完整的抗生素基因簇的事实表明，这些产物在自然界中是有用的，可能是在恶劣的土壤环境中攻击竞争微生物。这些休眠或隐藏的抗生素基因簇代表了一种未开发的新化学资源，它可能导致发现新的抗菌化合物，在临床中非常有用。这项研究计划的初步工作使我们能够首次在生态相关环境（包括无菌和非无菌土壤）中种植两种链霉菌。该项目旨在了解在这些链霉菌物种的基因组中编码的以前未表征的抗生素基因簇何时在更自然的环境中表达和产生。所产生的数据将为我们提供关键信息，使我们能够在实验室和工业发酵条件下产生可检测数量的这些化合物的转基因菌株。本研究的目的是：1。为了确定这些隐抗生素是如何由土壤中的链霉菌产生的。检测哪些隐抗生素是在生态相关条件下产生的。利用目标1和目标2中获得的信息，以产生转基因链霉菌变体，这些变体能够在实验室和发酵条件下产生可检测数量的这些新抗生素。为了实现这些目标，我们将使用RNA测序，这是一种用于识别在特定生长条件下表达哪些基因的技术。这将使我们能够将共表达的基因联系起来，并将提供识别隐性抗生素簇的全局和特定调节因子所需的信息，以及导致这些化合物产生的关键代谢途径。我们将使用色谱和质谱法来鉴定细菌细胞在无菌和非无菌土壤中生长时产生的新抗生素。我们将结合上述信息，以选择参与生产特定隐性抗生素的候选基因。然后，我们将一次一个地从细菌基因组中操纵这些候选基因。在某些情况下，我们将插入这些候选基因的额外副本，而在其他情况下，我们将从原始基因组中删除这些基因。每次实验结束后，将对这种特定抗生素的生产进行分析，看看这些基因变化是否会增加这种化合物的产量，或者是否会产生新的化合物。这将使我们能够产生这些链霉菌的变种，能够在实验室条件下产生可检测水平的新抗生素。这项工作的结果将在抗菌素耐药性（AMR）领域具有特别重要的意义，因为它旨在增加可用于测试的新型天然产物的数量，从而提供更多的候选先导物，以生产急需的新的临床相关抗菌素化合物。

The increasing incidence of antibiotic-resistant bacterial pathogens has resulted in an urgent need for new, clinically useful antibiotics. Most of the antibiotics used in medicine are produced by actinomycetes, and particularly a genus of harmless bacteria called Streptomyces, which is abundant in all soil environments. Based on their genome sequences, each Streptomyces species has the potential to produce on average around 10-15 antibacterial agents of natural product origin. However, when these species are grown under laboratory conditions, only one or two antimicrobial compounds are usually detected. This is because most of these antibiotic gene clusters appear dormant (i.e. are not expressed) under laboratory conditions. The fact Streptomyces species maintain these intact antibiotic gene clusters in their genomes suggest the products are useful in nature, probably to attack competitor microorganisms in the harsh soil environment. These dormant or cryptic antibiotic gene clusters represent an untapped resource in terms of novel chemistry which could lead to the discovery of new antimicrobial compounds that could be very useful in the clinic.Preliminary work leading to this research proposal has allowed us to grow two of these Streptomyces species, for the first time, in ecologically relevant environments including sterile and non-sterile soils. This project aims to understand when previously uncharacterised antibiotic gene clusters encoded within the genomes of these Streptomyces species are expressed and produced in more natural environments. The data generated will provide us with key information which will allow us to generate genetically modified strains with the ability to produce detectable amounts of these compounds under laboratory and industrial fermentation conditions. The aims of this research are:1. To identify how these cryptic antibiotics are made by Streptomyces species in soil.2. To detect which cryptic antibiotics are produced under ecologically relevant conditions.3. To use the information obtained in objectives 1 and 2 in order to generate genetically modified Streptomyces variants with the ability to produce detectable amounts of these new antibiotics under laboratory and fermentation conditions.To accomplish these aims we will use RNA sequencing, a technique used to identify which genes are being expressed under a particular growth condition. This will allow us to relate genes that are co-expressed and will provide the information required to identify global and specific regulators of cryptic antibiotic clusters, as well as key metabolic pathways leading to the production of these compounds. We will use chromatography and mass spectrometry to identify new antibiotics produced by the bacterial cells when grown in both sterile and non-sterile soils. We will combine the above information in order to select gene candidates involved in the production of particular cryptic antibiotics. We will then genetically manipulate these candidate genes from the bacterial genome one at a time. In some cases, we will insert extra copies of these candidate genes and in others we will delete these genes from the original genome. After each experiment, the production of that particular antibiotic will be analysed to see if these genetic changes increase production of the compound or if any new compounds are produced. This will allow us to generate variants of these Streptomyces species able to produce detectable levels of new antibiotics under laboratory conditions. The results from this work will have particular significance in the field of antimicrobial resistance (AMR) as it aims to increase the number of novel natural products available to test and therefore provide many more lead candidates to take forward in order to produce much needed new clinically relevant antimicrobial compounds.