Dokument

Summary:
Natural products (NPs), defined here as those derived from secondary metabolism, are produced by bacteria, fungi and plants. They are not essential for growth, development, and reproduction of an organism, but equip their producers with specific advantages. As a result, many secondary metabolites exhibit bioactivities that can benefit human health as drugs or drug leads. In context of the discovery of NPs, fungi serve as a prolific source. With advances in sequencing technologies and bioinformatics analysis tools the great potential deposited in genomes can be disclosed. Genetic engineering methods and improvements in analytical technologies provide the toolbox for exploiting that hidden potential of fungal genomes. The discovery of biosynthetic genes is facilitated by their cluster architecture. Based on the gene coding for the respective backbone enzyme, the produced secondary metabolites can be classified, e.g. polyketides, non-ribosomal peptides, and terpenoids. Genome mining of Aspergillus ustus 3.3904 unveiled several uncharacterized biosynthetic gene clusters (BGCs) with genes coding for different backbone enzymes and several tailoring enzymes. Among them, two putative terpene cyclases and a presumably twelve-gene BGC that are subject in this thesis. In the first project, a sesquiterpene cyclase, GdlS, was identified and confirmed as germacradienol synthase by heterologous expression and biochemical characterization including testing substrate specificity, ion dependence and determination of the kinetic parameters. Germacradienol is the key intermediate in the biosynthesis of the ‘earthy odor’ geosmin. The biosynthetic mechanism of a bifunctional germacradienol/geosmin synthase has been extensively studied in bacteria over the past 25 years. Only two reports on the biosynthesis of germacradienol and geosmin in fungi were described. Phylogenetic analysis of both N-termini und C-termini of the germacradienol/geosmin synthase SCO6073 from Streptomyces coelicolor with homologues from other bacteria and fungi revealed unequivocally the existence of distinct clades for bacteria, ascomycetous fungi, and basidiomycetous fungi. In particular, the existence of putative bifunctional enzymes in bacteria that catalyze both the conversion of FPP to germacradienol and its fragmentation to geosmin, and the existence of homologues for fungi that most likely code for two distinct enzymes catalyzing the two reactions independently. This led to the suggestion of a different strategy for germacradienol and geosmin production in bacteria and fungi. The workflow for the second project was the same as for the first project. Heterologous expression and in vitro reaction with recombinant protein unveiled MfdS as malfilanol D synthase. The enzyme’s promiscuity was tested by using different substrates and metal ions as additives. The kinetic parameters were determined. Malfilanol D is a sesquiterpenoid of the less investigated bicyclo[5.4.0]undecane class. The biosynthesis of other compounds belonging to this class, e.g. β-himachalene, is proposed via a C-1 to C-11 ring closure after isomerization of FPP to NPP. As a result of this cyclization procedure, the geminal dimethyl group is directly attached to the 6/7 fused ring. Malfilanol D and its congeners, malfilanol A – C, differ in that the geminal dimethyl group is separated from the fused ring by one CH2 group. Therefore, a different cyclization strategy is assumed. In order to get deep insights into the mechanism of MfdS, feeding with 13C labeled precursors was performed. Subsequent isolation and interpretation of the 13C NMR data provided evidence for a C-1 to C-10 cyclization with successive alkyl and hydride migrations and C-1 to C-6 ring closure to form the core skeleton of malfilanols. These results confirm a novel cyclization mechanism for sesquiterpenoids with a bicyclo[5.4.0]undecane skeleton. This work was done in collaboration with Zheng-Xi Zhang. In the third project, a putative twelve-gene psa BGC was identified with genes coding for a highly-reducing polyketide synthase (HR-PKS), several short-chain dehydrogenases / reductases (SDRs), and a cupin-domain containing protein, among other modifying enzymes. Because the formation of alkyl salicylaldehydes and derivatives requires the involvement of a HR-PKS, two SDRs, and a cupin-domain containing protein that acts as aromatase, similar cluster products were proposed. Indeed, heterologous expression of these genes confirmed this hypothesis, but with one major exception. More precisely, heterologous expression of merely psaPS (HR-PKS), psaOX1 (SDR), and psaOX2 (SDR) led to the accumulation of four compounds, 6-propyl salicylaldehyde, 6-propyl salicyl alcohol, 6-propyl salicylic acid, and a dihydro-γ-pyrone derivative. Single gene deletion proved the necessity of those three genes for the formation of the aromatic compounds. The introduction of psaCP (cupin-domain containing protein) does not increase the production of aromatic products by a water elimination catalyzed aromatization, as reported in a very recent publication. PsaCP directs the biosynthetic pathway towards the main products rather than shunt products and most likely acts as a reductase. This hypothesis will be the subject of further biochemical investigation.

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