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

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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.