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Genome Mining-based Studies on the Biosynthesis of Pyrroloindoline Diketopiperazines and p-Terphenyls in Aspergillus ustus
Filamentous fungi produce a multitude of secondary metabolites with crucial ecological functions to ensure their development, survival, and competitiveness in the ecosystem. These compounds exhibit a high structural diversity and play an important role in the discovery of novel drugs. Drug developme...
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| Format: | Doctoral Thesis |
| Language: | English |
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Philipps-Universität Marburg
2024
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| Online Access: | PDF Full Text |
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| Summary: | Filamentous fungi produce a multitude of secondary metabolites with crucial ecological functions to ensure their development, survival, and competitiveness in the ecosystem. These compounds exhibit a high structural diversity and play an important role in the discovery of novel drugs. Drug development is driven by various factors, including the need for new antimicrobial agents due to the growing threat of drug-resistant microorganisms. In this regard, nature's potential to provide a wide variety of products with versatile biological activities encourages scientists to search for new compounds and to understand their biosynthetic formation.
In recent decades, advances in bioinformatics have opened new possibilities to analyze genomic data of various organisms. The development of sequencing technologies and computational tools have revealed the potential of microorganisms, including filamentous fungi, to produce a variety of metabolites. Most of these secondary metabolites are products of polyketide synthases, non-ribosomal peptide synthetases, or terpene synthases, as well as tailoring enzymes such as oxidoreductases or methyl- and prenyltransferases. The genes coding for these biosynthetic enzymes are usually clustered in the genome, and their location and putative function can be predicted using appropriate bioinformatic tools. Genetic engineering methods have been developed with the objective of enhancing the production and diversity of biologically active fungal secondary metabolites, as well as the investigation of their biosynthesis.
Genome mining of Aspergillus ustus 3.3904 led to the identification of several putative biosynthetic gene clusters (BGCs). In cooperation with Haowen Wang, detailed analysis of a non-ribosomal peptide synthetase (NRPS) gene cluster suggested its involvement in the biosynthesis of the previously isolated diketopiperazine derivative protubonine B. Heterologous expression of the whole gene cluster in Aspergillus nidulans confirmed this hypothesis. Combination of further gene deletion experiments and structural elucidation of the intermediates allowed the elucidation of the protubonine biosynthetic pathway. The flavin-dependent monooxygenase PboD was identified as the key enzyme and catalyzes the hydroxylation at C-3 of the indole moiety followed by pyrrolidine ring formation. The stereospecific reaction was verified by in vitro experiments with the recombinant enzyme.
In a subsequent project in cooperation with Meiting Wu, the promiscuity of the flavin-dependent monooxygenase PboD towards various tryptophan-containing diketopiperazine derivatives was investigated. In vitro assays demonstrated that PboD exhibits high catalytic efficiency towards various indole diketopiperazines, especially those with a second aromatic moiety. Product isolation and elucidation of their structures demonstrated the expected stereospecific hydroxylation and pyrrolidine ring formation, thereby revealing two of them to be new products. Feeding of different indole diketopiperazines to an Aspergillus nidulans transformant bearing a truncated protubonine gene cluster with pboD and two acetyltransferase genes resulted in the formation of hydroxylated and acetylated products. These findings provide a deeper understanding of the formation of hydroxylated pyrroloindoline alkaloids in fungi. In addition, studies on the promiscuity of PboD open new possibilities for the chemoenzymatic synthesis of pyrroloindoline-derived natural products.
In a third project in collaboration with Dr. Jing Zhou, genome mining of A. ustus 3.3904 led to the identification of an NRPS-like gene cluster as a plausible candidate for the biosynthesis of p-terphenyl derivatives. To prove this, the BGC was activated by heterologous expression in A. nidulans LO8030, which resulted in the formation of the new p-terphenyls usterphenyllins A and B as well as uscandidusins A and B. As in the first project, gene deletion experiments and isolation of the pathway intermediates were conducted to confirm their proposed biosynthesis. The data indicated that enzymatic reduction and dehydration of the terphenylquinone atromentin represents a key step within the biosynthetic pathway. This results in the formation of a diphenylbenzene intermediate, containing a central benzene ring, which represents a characteristic structural feature of terphenyls. Subsequent modification reactions, including methylation, hydroxylation, and prenylation, are catalyzed by tailoring enzymes within the biosynthetic pathway and lead to the formation of usterphenyllins. Based on our findings, the last step in the biosynthesis of the dibenzofuran-containing final products uscandidusins A and B was proven to be a spontaneous, non-enzymatic oxidative reaction. Taken together, this study provides new insights into the previously unexplored p-terphenyl biosynthesis in fungi. |
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| DOI: | 10.17192/z2024.0229 |