A universal tool for rapid functional characterisation of antibiotic production genes in the bacterial genus, Streptomyces

在这个项目中，我们将开发一种基因工具，它将加速和简化由细菌链霉菌产生的新抗生素的发现。自从20世纪中叶抗生素的黄金时代以来，链霉菌提供了最丰富的新型抗菌化合物的来源。这些天然产物包括临床上重要的抗生素(四环素、链霉素和青霉素)、免疫抑制剂(FK506/520和雷帕霉素)和抗癌药物(阿霉素)。也许21世纪的主要医学挑战是开发新的抗生素来对抗细菌的耐药性。链霉菌的基因组测序，再加上抗生素生物合成基因的挖掘，揭示了该属尚未发现的巨大生物合成潜力，并突显了它们开发抗生素的巨大可能性。然而，需要开发新的遗传工具来快速表征抗生素生物合成基因的功能，并促进这种生化潜力的开发。目前，链霉菌的基因破坏是一个费力的过程。我们需要加快这一过程，以更快地表征抗生素生物合成基因，从而简化抗生素开发流程。生物合成基因的快速突变将允许通过基因工程操纵开发新化合物的途径。或者，对生产菌株的基因操作可以用来提高产量，从而提高所需抗生素的商业可行性。利用基因合成技术，我们将开发一种通用于所有测序链霉菌以及一些相关细菌的遗传工具。这一工具将消除目前表征抗生素生物合成所必需的繁琐的基因操作。在第一个实例中，我们将合成诱变盒，目标是破坏一些特有的抗生素生物合成基因簇。我们将通过破坏和删除已知的抗生素生物合成基因，在模式链霉菌天蓝色链霉菌和工业菌株土霉素生产链霉菌rimosus中证明这一系统的原理。将我们的工具应用于后一种生物体，将证明我们的系统在非模型、工业生物体中的实用性。我们将评估基因破坏发生的效率，并使用一系列分子生物学技术确定基因破坏的位置和稳定性。最后，我们将在开放获取期刊上发布结果后，通过生物资源库将在该项目期间开发的基因工具提供给学术和工业科学界，以实现我们的系统在开发新型抗生素方面的最大可能吸收。

During this project we will develop a genetic tool that will accelerate and simplify the discovery of new antibiotics produced by the bacterial genus Streptomyces. Ever since the golden age of antibiotics in the mid-20th century, streptomycetes have provided the richest source of novel antimicrobial compounds. These natural products include clinically-important antibiotics (tetracyclines, streptomycins, & penicillins), immunosuppressants (FK506/520 & rapamycin) and anti-cancer drugs (doxorubicin). Perhaps the major medical challenge in the 21st century is to develop new antibiotics to combat bacterial antibiotic resistance. Genome sequencing of streptomycetes, coupled with mining for antibiotic biosynthetic genes has revealed great undiscovered biosynthetic potential in this genus and highlights their enormous possibilities for antibiotic development. However, there is a need to develop new genetic tools that will allow the rapid characterization of antibiotic biosynthetic gene function and facilitate exploitation of this biochemical potential. Currently, gene disruption in streptomycetes is a laborious process. We need to speed this process to more rapidly characterise antibiotic biosynthetic genes and so simplify the antibiotic development pipeline. Rapid mutagenesis of biosynthetic genes will allow manipulation of pathways to develop new compounds through genetic engineering. Alternatively gene manipulation of a producing-strain can be used to improve yield and thus commercial viability of desired antibiotics. Taking advantage of gene synthesis technology, we will develop a genetic tool that is universally applicable to all sequenced streptomycetes as well as some related bacteria. This tool will remove the need for laborious gene manipulations that is necessary to characterise antibiotic biosynthesis at the present time. In the first instance, we will synthesise mutagenesis cassettes targeted at the disruption of a number of characterised antibiotic biosynthetic gene clusters. We will demonstrate proof-of-principle of this system in both a model streptomycete, Streptomyces coelicolor, and an industrial strain, the oxytetracycline producer Streptomyces rimosus through the disruption and deletion of known antibiotic biosynthetic genes. Application of our tool to the latter organism will demonstrate the utility of our system in non-model, industrial organisms. We will assess the efficiency with which gene disruption takes place and identify the location and stability of the gene disruption using a range of molecular biological techniques. Finally, we will make the genetic tools developed during this project available to the academic and industrial scientific communities through a biological resource repository following dissemination of results in open access journals so as to achieve the greatest possible uptake of our system for the development of novel antibiotics.