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)主要作为次生代谢物(Sms)，对药物的发现和开发具有重要意义。像放线杆菌和子囊菌这样的微生物是这种NPs的重要生产者。在药学研究中，真菌对NP的生产变得越来越重要。特定SM的生物合成基因通常被组织在基因组中，即所谓的生物合成基因簇(BGC)。SM BGC共享一个共同的设置，通常由一个主干和不同的剪裁酶组成。一些BGC中还包括其他调节和运输的基因。挖掘已测序的微生物基因组揭示了很大程度上尚未开发的生物合成潜力。正如本项目中的两个菌株所观察到的那样，这些BGC大多是沉默的或非常低表达的。这个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结合起来，建立一个集成的代谢组谱工具箱来表达和阐明沉默/隐藏基因的SM通路。为了初步的合理设计，11个来自壳青霉的SM BGC和9个来自无花果拟青霉的SM BGC将在一种特殊的尼杜拉曲霉菌株中表达。利用悬垂延伸聚合酶链式反应进行酵母重组，通过剪接将BGC的结构基因克隆到大肠杆菌-酵母-曲霉穿梭载体中。对于异源表达，我们使用了缺失nkuA和8条主要SM途径的根癌农杆菌，从而最大限度地减少了非同源重组事件，并提供了更少的宿主SM背景。编码非必需的PKS衍生色素的WA基因座将被选为BGC的整合位点。LC-HRMS对短信的监控使我们能够检测到SM配置文件中的微小变化，并提供有关BGC成功激活的信息。它还将帮助我们结合公共和内部的NP数据库来评估结构的新颖性。通过光谱学方法对新型SM的结构进行了研究。如有必要，还将使用同位素标记的前体进样和X射线分析来确定结构。簇中基因的重要性和功能将在随后通过基因缺失、修饰构建体的表达和中间体的检测来证明。两位申请者都以互补的经验致力于真菌SM的生物合成。殷建立了这个项目中使用的遗传工具，李S团队在真菌短信的分析和结构解析方面拥有长期的经验。小组之间存在着良好的合作关系，这是项目成功实现的保证。沉默/隐蔽的BGC的表达将有力地加速这一过程，从基因组到NPs再到药物。

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.