title: Exploration of the secondary metabolites in Aspergillus ustus and their biosynthesis
title: Erforschung der Sekundärmetaboliten in Aspergillus ustus und ihrer Biosynthese
titleType: AlternativeTitle
Creators: name: Wang，Haowen
Publisher: name: Philipps-Universität Marburg
Year: 2025
Subjects: nichtribosomalen Peptidsynthase
fungi
Biosynthese
filamentöse Pilze
natural products
Biotechnologie，Molekularbiologie，Biochemische Wege，Mykologie，Fungal Metaboliten，Naturstoffchemie
Aspergillus sp.
Life sciences
Biowissenschaften, Biologie
Biotechnology，Molecular Biology，Biochemical Pathways，Mycology，Fungal Metabolites，Natural Product Chemistry
genome mining

description: Natural products, also known as secondary metabolites, are highly abundant, especially those derived from bacteria and fungi. These secondary metabolites play pivotal roles in processes like signaling, habitat defense, and inhibiting competitors. These ecological functions give microbial natural products immense importance in drug discovery, uncovering new enzymatic mechanisms, and studying interspecies interactions. Between 1981 and 2020, 32% of approved small-molecule drugs by FDA were either natural products or their derivatives, with most sourced from microorganisms. Among the vast array of microbial secondary metabolites that have been isolated and identified, key categories include polyketides (PKs), nonribosomal peptides (NRPs), alkaloids, and terpenes. Advances in sequencing technology and bioinformatics have provided significant advantages for studying the biosynthesis of these compounds. Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs), which function in highly organized assembly lines, have been some of the most extensively researched enzymes in recent years. Alongside versatile tailoring enzymes such as prenyltransferases (PTs), flavin-dependent oxidoreductases, nonheme FeII/2-oxoglutarate-dependent monooxygenases, and cytochrome P450 enzymes, these systems drive the production of a diverse range of secondary metabolites. To unravel these biosynthetic mechanisms, researchers use advanced bioinformatics, cutting-edge biotechnologies, and sophisticated biochemical tools to study the genes coding for these enzymes, which are generally clustered together in the same biosynthetic gene cluster (BGC). 
In collaboration with Dr. Liujuan Zheng, Dr. Daniel Jonathan Janzen, and Dr. Yiling Yang, this thesis elucidates the biosynthetic pathways of four metabolites derived from Aspergillus ustus 3.3904. This PhD candidate was mainly responsible for chemical experiments. The metabolites oxepinamides D, E, and F were isolated and characterized from A. ustus 3.3904. Bioinformatics analysis identified two similar NRPS-containing gene clusters, named opa and opa2. The opa cluster is responsible for synthesizing oxepinamides E and F, while the opa2 cluster is involved in producing oxepinamide D.
In the biosynthesis of oxepinamide D, the NRPS OpaA2 from the opa2 cluster assembles a fused quinazolinone core structure using Ant, L-Phe, and L-Ala. The P450 enzyme OpaB2, analogous to OpaB, catalyzes the formation of the oxepin ring in a specific and stereoselective manner to produce the 1H-oxepin oxepinamide. Subsequently, OpaC2 adds a hydroxyl group at the C-3 position to complete the formation of oxepinamide D. Both OpaC2 and OpaC demonstrate high substrate specificity. Furthermore, feeding experiments with opaB and opaB2 transformants confirmed the absence of cross-talk between the two biosynthetic gene clusters. Additionally, our study shows that the E domains in OpaA and OpaA2 are crucial for the epimerization of phenylalanine and alanine, respectively. This epimerization is a critical step preceding the ring expansion catalyzed by the oxepinases OpaB and OpaB2. 
In addition to oxepinamides D, E, and F, (−)-protubonine B was also isolated and characterized from Aspergillus ustus. Detailed analysis of another NRPS gene cluster indicated its role in the biosynthesis of the diketopiperazine derivative (−)-protubonine B. This hypothesis was confirmed through heterologous expression of the entire gene cluster in Aspergillus nidulans. Subsequent gene deletion experiments, combined with structural elucidation of the intermediates, enabled the comprehensive mapping of the (−)-protubonine B biosynthetic pathway. Notably, the flavin-dependent monooxygenase PboD was identified as a crucial enzyme, responsible for hydroxylating the C-3 position of the indole moiety and facilitating the formation of the pyrrolidine ring. This stereospecific reaction was further validated through in vitro experiments using the recombinant enzyme. The intermediate, protubonine D, undergoes diacetylation by the acetyltransferases PboB and PboC, yielding the final product, (−)-protubonine B.
Besides the NRPS-derived compounds, ustethylin A, a novel polyketide, was also isolated and characterized from this fungus. This highly oxygenated aryl aldehyde contains a phenethyl group. Isotopic labeling experiments revealed that its backbone originates from malonyl-CoA, with the methyl group of the phenethyl residue, the phenyl methyl group, and the O-methyl group all derived from L-methionine. Transcriptomic analysis, coupled with gene deletion, expression studies, and further isotopic labeling, confirmed that ustethylin A is biosynthesized via a polyketide synthase (PKS)-related pathway. The key enzyme, polyketide synthase UttA, catalyzes the formation of the phenethyl backbone, with methylation being an essential modification step. This is followed by sequential and coordinated modifications by three oxidoreductases and an O-methyltransferase, culminating in the final production of ustethylin A. In this study, we employed chemical modification techniques to acetylate crude extracts of secondary metabolites, which enabled the determination of the chemical structure of the main product. This approach highlights an important role that chemical modification of natural products plays in advancing our understanding of secondary metabolite biosynthesis.