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

越来越多的耐药细菌病原体导致了对新的、临床有用的抗生素的迫切需求。医学上使用的大多数抗生素都是由放线菌产生的，特别是一种名为链霉菌的无害细菌，它在所有土壤环境中都有大量存在。根据它们的基因组序列，每个链霉菌物种平均有潜力生产大约10-15种天然产品来源的抗菌剂。然而，当这些物种在实验室条件下生长时，通常只检测到一到两种抗菌化合物。这是因为这些抗生素基因簇中的大多数在实验室条件下似乎处于休眠状态(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，这些产品在自然界是有用的，可能会在恶劣的土壤环境中攻击竞争对手的微生物。这些休眠或隐蔽的抗生素基因簇在新的化学方面代表着一种未开发的资源，这可能导致发现新的抗菌化合物，这些化合物可能在临床上非常有用。导致这项研究计划的初步工作使我们第一次能够在生态相关的环境中种植其中两种链霉菌，包括无菌和非无菌土壤。该项目旨在了解这些链霉菌基因组中编码的以前未具体化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能够检测到数量的这些化合物的转基因菌株。本研究的目的是：1.鉴定这些隐蔽的抗生素是如何由链霉菌在土壤中制造的。检测在生态相关的条件下产生哪些隐蔽的抗生素。为了利用在目标1和2中获得的信息来产生转基因链霉菌变种，能够在实验室和发酵条件下产生可检测到的这些新抗生素的量。为了实现这些目标，我们将使用RNA测序，这是一种用于确定哪些基因在特定生长条件下表达的技术。这将使我们能够将共同表达的基因联系起来，并将提供所需的信息，以确定隐蔽抗生素簇的全球和特定调节因子，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仪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来选择与生产特定隐蔽抗生素有关的候选基因。然后，我们将对细菌基因组中的这些候选基因进行基因操作，一次一个。在某些情况下，我们会插入这些候选基因的额外副本，而在另一些情况下，我们会从原始基因组中删除这些基因。每次实验后，将对该特定抗生素的生产进行分析，以确定这些基因变化是否会增加化合物的生产，或者是否会产生任何新的化合物。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到的新抗生素水平。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，因为它旨在增加可供测试的新天然产品的数量，从而提供更多的领先候选对象，以便生产急需的新的临床相关抗菌化合物。

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.