Development and Implementation of Novel Genetic Tools for Investigation of FungalSecondary Metabolism

Dorte Koefoed Holm

    Research output: Book/ReportPh.D. thesisResearch

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    Abstract

    Filamentous fungi are eukaryotic multicellular organisms, which are essential in our ecosystem for degradationof organic matter. Additionally, lamentous fungi produce an arsenal of small, bioactive molecules,secondary metabolites, which may be exploited for commercial purposes e.g. as pigments, fungicides, insecticides,pharmaceuticals etc. In this study I have examined the possibilities for elucidating the biosyntheses offungal secondary metabolites, as well as identifying novel compounds, by application of genetic engineeringtechniques. I have focused on three levels of genetic engineering in three dierent lamentous fungi, all belongingto the genus Aspergillus: A. nidulans, A. niger, and A. aculeatus. A. nidulans is a well-characterizedeukaryotic model system for which many genetic tools have been developed. A. niger is a well-characterizedcell factory, however, as production strains have traditionally been constructed by random mutagenesis ordirected evolution approaches only few genetic tools are publicly available. A. aculeatus is an industrial enzyme producer, which was only recently genome-sequenced. No genetic tools exist for this organism, andonly very little is known about its potential to produce secondary metabolites.In this study I have examined parts of the secondary metabolism in all three fungi, using dierentapproaches. For A. nidulans we have constructed an entire genome-wide polyketide synthase (PKS) deletionlibrary, which has linked the known compounds austinol and dehydroaustinol to the corresponding PKS andwe have identied a new group of orsellinic acid-derived compounds. Also, I have successfully over-expressedindividual A. nidulans transcription factors and phosphatases in an eort to perturb regulation of secondarymetabolism and identify new compounds. Furthermore, A. nidulans was used as a host for examination ofsecondary metabolism by heterologous expression of secondary metabolite genes from A. niger, A. aculeatus,and A. terreus.In A. niger I have investigated the products of all 37 putative PKSs by heterologous expression of the genesin A. nidulans. This approach identied the products of three PKSs, which were subsequently examined indetail in A. niger. One of the products was 6-methylsalicylic acid (6-MSA), which proved to be the precursorto the meroterpenoid yanuthone D, a compound that was previously hypothesized to be of mixed terpenoidand shikimic acid origin. Individual gene deletions in this gene cluster mapped the entire biosynthetic pathwaytowards yanuthone D, meanwhile identifying six novel yanuthones, which were not previously described.The other two products both turned out to be the yellow pigment YWA1, which has previously beencharacterized from A. nidulans and A. fumigatus. YWA1 serves as the precursor to both naphtho--pyronesand black melanin (DHN-melanin), which is the pigment that provides the characteristic black color of A.niger. Intriguingly, only one of these PKS genes (napA) is placed in a typical secondary metabolite genecluster, but this PKS gene appears to be silenced.For investigation of secondary metabolism in fungi with no available genetic tools, I have applied twodierent approaches: heterologous expression of secondary metabolite genes in A. nidulans and/or plasmidbasedgene expression in the native fungus. Heterologous expression in A. nidulans was used for successfulreconstruction of the (+)-geodin pathway, by transfer of the entire (+)-geodin cluster from A. terreus. ForiiA. aculeatus, I have used a combination of the two approaches: I heterologously expressed a putative 6-MSA synthase from A. aculeatus in A. nidulans, verifying that the gene indeed encoded a 6-MSA synthase.Next, a transcription factor embedded in the putative gene cluster, was over-expressed from a plasmid-basedexpression platform, which was developed in this study. Expression of the transcription factor triggeredactivation of a biosynthetic pathway, which appear to be involved in production of ve novel compounds.Feeding experiments with fully 13C labelled 6-MSA, produced in this study, conrmed that 6-MSA wasincorporated into three of these compounds.Based on these results, I conclude that the combination of dierent interdisciplinary approaches is a verypowerful tool for investigating fungal secondary metabolism. The results of my studies also suggest thatcontinuing optimization of existing tools as well as development of new tools is equally important as mereapplication of existing tools.
    Original languageEnglish
    Place of PublicationKgs. Lyngby
    PublisherTechnical University of Denmark
    Number of pages398
    Publication statusPublished - 2013

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