Unravelling fungal biosynthetic potential by heterologous expression of silent gene clusters in Aspergillus nidulans and LC-MS-based metabolomic analysis

天然产物（NPs），主要是作为次级代谢产物（SM），对药物发现和开发做出了重要贡献。微生物如放线菌和子囊菌是这种NP的重要生产者。真菌在药物研究中对于NP生产变得越来越重要。用于给定SM的生物合成的基因通常在基因组中组织为所谓的生物合成基因簇（BGC）。SM BGC具有共同的结构，通常由骨架和不同的剪裁酶组成。在一些BGC中还包括用于调节和转运的其他基因。挖掘测序的微生物基因组揭示了大部分未开发的生物合成潜力。这些BGC中的大多数是沉默的或非常低表达的，如本项目中的两种菌株所观察到的。本项目旨在建立一个新的平台，将基因组学和合成生物学的力量与一个整合的代谢组学分析工具箱结合起来，联合收割机来表达和阐明沉默/隐藏基因的SM途径。对于最初的合理设计，11个来自壳青霉的SM BGC和9个来自无花果拟盘多毛孢的SM BGC将在特殊的构巢曲霉菌株中表达。将BGC的结构基因克隆到E. coli-Saccharomyces-Aspergillus穿梭载体的剪接的帮助下，通过基于PCR的突出端延伸酵母重组。为了激活基因，我们将BGC中转录因子的启动子替换为人工启动子。nkuA和8个主要SM途径缺失的nidulans，其最小化非同源重组事件并提供降低的宿主SM背景。将选择编码非必需PKS衍生色素的wA基因座作为BGC的整合位点。通过LC-HRMS对SM进行监测，我们可以检测SM配置文件中的微小变化，并提供有关成功激活BGC的信息。它还将帮助我们通过与公共和内部NP数据库相结合的去复制来评估结构新奇。新SM的结构将通过光谱方法来阐明。如有必要，还将使用同位素标记前体进料和X射线分析进行结构解析。簇中基因的重要性和功能将随后通过基因缺失、修饰的构建体的表达和中间体的检测来证明。Yin建立了该项目中使用的遗传工具，Li的团队在真菌SM的分析和结构阐明方面拥有长期经验。各小组之间存在着良好的合作关系，保证了项目的顺利实现。沉默/隐蔽BGC的表达将大大加速从基因组到NP和药物的过程。

Natural products (NPs), mostly as secondary metabolites (SMs), contribute significantly to the drug discovery and development. Microorganisms like Actinobacteria and Ascomycota are important producers of such NPs. Fungi become increasing important for NP production in pharmaceutical research. The genes for the biosynthesis of a given SM are usually organized in the genome as so-called biosynthetic gene cluster (BGC). The SM BGCs share a common set-up, usually consisting of a backbone and different tailoring enzymes. Additional genes for regulation and transport are also included in some BGCs. Mining the sequenced microbial genomes revealed the largely unexploited biosynthetic potential. Most of these BGCs are silent or very low expressed, as observed for the two strains in this project. This prohibits novel NP finding and should be exploited.This project is to build a novel platform and to combine the power of genomics and synthetic biology with an integrated metabolomic profiling tool box to express and elucidate SM pathways of silent/cryptic genes. For initial rational design, 11 SM BGCs from Penicillium crustosum and 9 from Pestalotiopsis fici will be expressed in a special Aspergillus nidulans strain. The structure genes of the BGCs will be cloned into E. coli-Saccharomyces-Aspergillus shuttle vectors with the help of the splicing by overhang extension PCR-based yeast recombination. For activation of the genes, the promoter of the transcription factor in BGC will be replaced by an artificial one.For heterologous expression, we use A. nidulans with deletions of nkuA and 8 major SM pathways, which minimizes non-homologous recombination events and provides a reduced host SM background. The wA locus encoding a non-essential PKS derived pigment will be chosen as the integration site of BGCs. This allowed the facile and initial screening of correct integration.Monitoring of SMs by LC-HRMS allows us to detect even minor changes in the SM profile and provide information on the successful BGC activation. It will also help us to assess structural novelty by dereplication in combination with public and in-house NP databases. Structures of novel SMs will be elucidated by spectroscopic methods. If necessary, feeding with isotope-labelled precursors and X-ray analysis will also be used for structure elucidation. The importance and function of the genes in clusters will be proven afterwards by gene deletion, expression of the modified constructs, and detection of intermediates.Both applicants work on the biosynthesis of fungal SM with complementary experiences. Yin established the genetic tools used in this project and Li´s group has long-time experience on analysis and structure elucidation of fungal SMs. It exists an excellent cooperation between the groups, which guarantees the successful realization of the project. Expression of silent/cryptic BGCs will strongly accelerate the process, from genome to NPs and to drugs.