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、头孢菌素和他汀类是最好的例子。最近在对真菌基因组测序的简便性方面的进展表明，真菌含有到目前为止出人意料的大量基因簇，这些基因簇似乎编码次生代谢物的途径，但它们的化学产物尚不清楚，也没有在药物发现计划中进行评估。这表明还有许多有益的产品有待发现和开发，这些产品可能包括新类别的抗生素，可以用来帮助对抗持续存在的抗生素耐药性问题。基于已有的选定目标真菌的基因组序列数据，以及其他选定感兴趣物种的此类数据的生成，我们将开发一条管道，快速对这些基因簇进行分类，然后设计质粒载体，允许它们在真菌米曲霉中表达，这是一个非常适合实验室和工业规模培养的物种。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；通常不是在实验室条件下由本地真菌表达的，以及仅凭基因组数据无法以任何程度的置信度预测的产物。根据其他真菌的典型情况，预测每个目标真菌都包含40-60个这样的基因簇。质粒载体将构建在我们已经开发和测试的一系列表达盒中，并将使用基于酵母的同源重组克隆的组合，在必要时通过Gibson Assembly进行扩充。这将使用直接来自基因组DNA的聚合酶链式反应产物来实现，或者在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，那么这些方法应该很容易扩展到高通量使用。我们的载体集将允许每个质粒最多四个基因的协调表达，有四个不同的可选标记可用，这意味着我们可以很容易地表达由16个基因组成的路径，并可以在证明有必要时升级这个系统以获得额外的基因，这对于真菌中遇到的大多数路径来说是足够的能力。然后将对米曲霉的转化子进行分析，以确定是否产生了新的产物，如果是，将通过反相高效液相色谱进行纯化，并通过MS和核磁共振分析来阐明结构。将提纯毫克量，以允许对一系列临床相关病原体进行抗菌检测，以确定每种化合物的抗菌效果。对于表现出抗菌特性的化合物，将根据一系列表现出对抗生素的特定抗药性的细菌对每种化合物进行评估，以快速消除任何具有已知作用模式或已经具有抗药性的化合物。对于通过评估的产品，我们的目标是全面描述生物合成途径，包括分离其生物合成的中间阶段，并确定适合进一步化学修饰的产品，以支持对这类化合物的结构-活性关系的研究。我们的目标是设计一条生产管道，使我们能够从最初的10种选定真菌中调查每一个候选基因簇。为这项研究选择的菌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。如果有时间，这将有助于在第二轮筛查中为更广泛的菌株选择提供信息。

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.