Untersuchungen zur Biosynthese von Flavoglaucin, Echinulin und ihren Analoga im Ascomyceten Aspergillus ruber

Naturstoffe, die aus dem Sekundärmetabolismus stammen werden von Organismen nicht unbedingt zum Überleben, der Reproduktion oder der Differenzierung benötigt, bieten aber dennoch einen signifikanten Selektionsvorteil in der Konkurrenz mit anderen Lebewesen. Daraus resultiert, dass viele dieser Natur...

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Bibliographic Details
Main Author: Nies, Jonas
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:German
Published: Philipps-Universität Marburg 2021
Subjects:
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Natural products which arise from an organism’s secondary metabolism are not necessarily re-quired for survival, reproduction and differentiation but offer a significant selective advantage in their respective environment. As a result, many of these natural products display significant biolog-ical activities in organisms. Due to the biological activity of many of these substances, the fungal secondary metabolism for example is an attractive source for the discovery and development of new drugs. So far, only a limited number of all existing fungi has been identified, which means that there is still an enormous unused potential for the production of pharmaceutically relevant natural products. This potential can be accessed through various bioinformatic, molecular biological and biochemical approaches. The discovery of biosynthetic genes for secondary metabolites in fungi is facilitated by the fact that relevant genes for the production of a given compound are located in close proximity to each other on the chromosome, i.e., they are clustered. These biosynthetic gene clusters (BGCs) contain one or more genes whose protein products are responsible for the formation of the carbon backbone of the respective substance. This backbone is structurally diversified by tailoring enzymes encoded by other genes in the cluster. In this thesis, BGCs for two different natural products and their conge-ners from the ascomycete Aspergillus ruber were identified and confirmed. In addition, individual steps in these metabolic pathways were examined in more detail. The prenylated salicylaldehyde derivative flavoglaucin and its congeners differ from each other only in the number and position of the double bonds of the heptyl side chain. Previous studies have shown that some of these substances are biologically active, for example by acting as antioxi-dants, showing anti-inflammatory effects, or binding to human opioid and cannabinoid receptors. Although flavoglaucin and its congeners have been known since 1934, not much was known about their biosynthesis other than that they are products of polyketide metabolism. This thesis describes the discovery of a gene cluster (fog cluster) in the genome of A. ruber through genome mining, which contains all the genes necessary for the biosynthesis of these substances. Heterologous ex-pression of this BGC in the model ascomycete Aspergillus nidulans confirmed that the fog cluster is responsible for the formation of flavoglaucin and its congeners. Gene deletions in the heterologous expression strain, the overexpression of the gene for the highly reducing polyketide synthase (HR-PKS) FogA in A. nidulans, in vitro enzyme assays with the prenyltransferase FogH and feeding exper-iments to investigate the FAD-dependent oxidoreductase FogF showed an unusual route to the final aldehyde product. The polyketide skeleton is reductively released as a salicyl alcohol by FogA in cooperation with the cupin protein FogC and the short-chain dehydrogenases/reductases FogB and FogD, only to be reoxidized to the salicylaldehyde by FogF after C3-hydroxylation by the cyto-chrome P450 (CYP) FogE and C5-prenylation by FogH. This reoxidation is remarkable, as product release from an HR-PKS usually occurs hydrolytically as a carboxylic acid or reductively as an alde-hyde. Modification of the intermediates apparently requires the hydroxymethyl group of the salicyl alcohol, because prenylation of a synthesized aldehyde substrate analog by FogH does not occur. This work was carried out in cooperation with Dr. Huomiao Ran. The biosynthesis of flavoglaucin shows some parallels to that of sordarial in Neurospora crassa and trichoxide in Trichoderma virens, but differs significantly in terms of prenylation, the final reoxidation of an alcoholic intermediate to an aldehyde and the variability of the saturation pattern in the heptyl side chain. Furthermore, the biosynthesis of indole alkaloids of the echinulin family was investigated in more detail. A previous study showed that two prenyltransferases EchPT1 and EchPT2 catalyze the re-verse C2 prenylation and several consecutive prenylations at different positions of the indole ring of the cyclic dipeptide cyclo-L-tryptophan-L-alanine. It was speculated that a CYP encoded in the identified BGC introduces two exo double bonds on the substituents of the 2,5-diketopiperazine ring. By heterologous expression of the postulated ech BGC in A. nidulans, the formation of echinulin family indole alkaloids could be clearly assigned to this cluster. However, only substances without a double bond (echinulin series) and with a (∆10) double bond on the tryptophan substitu-ent (neoechinulin A series) were formed. The enzyme for the introduction of the second (∆14) dou-ble bond on the alanine substituent (neoechinulin B series) does not seem to be encoded by the genes of the ech cluster. The expansion of the expressed BGC by three additional potential candi-date genes downstream of the ech cluster did not yield the products with both double bonds. Feeding experiments confirmed the introduction of the ∆10 double bond by the CYP EchP450 en-coded in the ech cluster but refuted the hypothesis that EchP450 also catalyzes the formation of the ∆14 double bond. Further in vitro enzyme assays with the prenyltransferase EchPT2 showed its clear preference for substrates without an exo double bond, which contributes to some extent to the explanation of the different product ratios between the various echinulin family alkaloids in A. ruber.