Exploring Natures Silent Pharmacy

过去，真菌已被证明是生物活性化合物的重要来源，青霉素、头孢菌素和他汀类药物是最好的例子。最近的发展，我们可以轻松地测序真菌的基因组显示，真菌房子迄今意外大量的基因簇，似乎编码途径的次级代谢产物，但他们的化学产品是未知的，并没有在药物发现计划进行评估。这表明还有许多有益的产品有待发现和开发，其中可能包括新的抗生素类别，这些抗生素可以用于帮助解决抗生素耐药性的持续问题。基于选定目标真菌的基因组序列数据，加上其他选定物种的此类数据，我们将开发一个管道来快速地对这些基因簇进行分类，然后设计质粒载体，使它们能够在一种非常适合实验室和工业规模培养的真菌Aspergillusplasticum中表达。使用实验室友好的宿主真菌是必要的，因为我们的经验是，这些基因簇通常是神秘的;通常不会在实验室条件下由天然真菌表达，并且不能单独从基因组数据以任何置信度预测产物。根据其他真菌的典型情况，预计每个靶真菌含有40-60个这样的基因簇。质粒载体将在我们已经开发和测试的一系列表达盒中构建，并将使用基于酵母的同源重组克隆的组合，必要时通过吉布森组装进行扩增。这将使用直接来源于基因组DNA的PCR产物来实现，或者在这不容易实现的情况下，通过使用从基因组数据设计的合成DNA来实现。如果这些方法在我们的研究中被证明是成功的，那么这些方法应该容易扩展用于高通量使用。我们的载体组将允许每个质粒协调表达多达四个基因，具有四种不同的可选择标记，这意味着我们可以期望容易地表达包含16个基因的途径，并且如果这被证明是必要的，可以升级该系统用于额外的基因，这对于真菌中遇到的大多数途径来说是足够的容量。然后将对样品进行分析，以确定是否产生了新产物，如果是，则将通过反相HPLC纯化，并通过MS和NMR分析以阐明结构。将对毫克量进行纯化，以允许针对一系列临床相关病原体的抗菌试验，从而确定每种化合物的抗菌效力。对于显示抗菌特性的化合物，将针对一系列显示对抗生素的特征性抗性的细菌来评估每种化合物，以快速消除显示已知作用模式的任何化合物或那些已经普遍存在抗性的化合物。对于通过此评估的产品，我们的目标是充分验证生物合成途径，包括分离其生物合成的中间阶段，并鉴定适合进一步化学修饰的产物，以支持对结构的研究，我们的目标是设计一个生产流水线，使我们能够研究来自最初一组10种选定真菌的每个候选基因簇。本研究选择的分离株跨越了一系列不同的生活方式，包括昆虫，真菌和植物病原体，海洋真菌和土壤真菌。这将有助于为未来选择菌株提供信息，以便在第二轮筛选中进行更大规模的分析，如果有时间的话。

Fungi have proven to be an important source of bioactive compounds in the past, with penicillins, cephalosporins and statins amongst the best examples. Recent developments in the ease with which we can sequence the genomes of fungi have revealed that fungi house a hitherto unexpectedly large number of gene clusters which appear to encode pathways for secondary metabolites, yet their chemical products are unknown and have not been evaluated in drug-discovery programmes. This suggests that there are many beneficial products yet to be discovered and exploited and these may include new classes of antibiotic which could be deployed to help combat the ongoing problems with antibiotic resistance.Based on genome sequence data already available for selected target fungi, plus with generation of such data for other selected species of interest, we will develop a pipeline to quickly catalogue such gene clusters and to then design plasmid vectors to allow their expression in the fungus Aspergillus oryzae, a species which is very amenable to lab and industrial-scale cultivation. The use of a lab-friendly host fungus is necessary because our experience is that these gene clusters are usually cryptic; not usually expressed under laboratory conditions by the native fungus, and with products that cannot be predicted with any degree of confidence from genome data alone. The target fungi are each predicted to contain 40-60 such gene clusters based on what is typical for other fungi.The plasmid vectors will be constructed in a series of expression cassettes we have already developed and tested, and will be made using a combination of yeast-based homologous recombination cloning, augmented by Gibson Assembly where necessary. This will be achieved using PCR products derived directly from genomic DNA, or where this is not readily achievable, by use of synthetic DNA designed from genome data. Such approaches should be readily scalable for high throughput use if they prove to be successful in our studies.Our vector sets will allow coordinated expression of up to four genes per plasmid, with four different selectable markers available, meaning we can expect to readily express pathways comprising 16 genes, and could upgrade this system for additional genes should this prove necessary, which is ample capacity for the majority of pathways encountered in fungi.Transformants of A. oryzae will then be analysed to determine if a new product is produced, and if so, this will be purified by reverse-phase HPLC with analysis by MS and by nmr to elucidate the structure. Milligram quantities will be purified to allow antibacterial assays against a range of clinically-relevant pathogens to determine antibacterial efficacy for each compound. For compound displaying antibacterial properties, each compound will be evaluated against a range of bacteria displaying characterised resistance to antibiotics to quickly eliminate any compounds showing known modes of action or those where resistance is already prevalent.For products passing this evaluation, we aim to fully characterise the biosynthetic pathway, including isolation of the intermediate stages in their biosynthesis and to identify products suited to further chemical modification to support studies into structure-activity relationships in this group of compounds.Our aim is to design a production pipeline that will allow us to investigate every candidate gene cluster from an initial group of ten selected fungi. The isolates selected for this study have been chosen to span a range of differing lifestyles, including insect, fungal and plant pathogens, marine fungi and soil fungi. This would help to inform future choice of strains for a wider scale analysis in a second round of screening should there be time available.