title: Biosynthesis of alkaloids from Penicillium palitans and malfilanol D from Aspergillus ustus
title: Biosynthese von Alkaloiden aus Penicillium palitans und Malfilanol D aus Aspergillus ustus
titleType: AlternativeTitle
Creators: name: Zhang, Zheng-Xi
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: Alkaloid
Biosynthesis
Natural product
Biosynthese
Viridicatol
Naturprodukt
Speradine F
Pharmacology &amp; therapeutics, prescription drugs
Pharmakologie, Therapeutik

description: Natural products (NPs), strict limitation to secondary metabolites (SMs), have been noted to exhibit high structural diversity and complexity including examples such as alkaloids, phenylpropanoids, polyketides, and terpenoids with their unique pharmacological or biological activities. Many potent antibiotics were successively discovered from ascomycetous fungi Penicillium, Cephalosporium, Aspergillus, etc. Driven by that, more and more discoveries of natural products were conducted, leading to the dereplication. The drawback is obvious. Traditional isolation from natural resources is unable to meet the demands of medicinal treatment because of technical barriers, low yield, and damage to natural organisms. The lead compounds with complex structures, e.g., functional groups with chiral centers and unstable molecules with rearrangement, are difficult to be synthesized due to lengthy and jumbled steps and low yield. With the help of genetic tools, cell factories become one of the approaches to address such challenges by de novo design of biosynthetic pathways. To elucidate the biosynthetic mechanism, advanced bioinformatics, biological technologies, and biochemical tools have been utilized to investigate the coding genes, which are usually located together as a biosynthetic gene cluster (BGC). BGC normally contains backbone enzyme(s) responsible for NPs’ scaffold, and tailoring enzymes responsible for skeleton’s post-modification. Enzyme-mediated reactions extremely increase the diversity of NP’s structures. Besides, nonenzymatic reactions also occur occasionally in the formation of NPs. 
In this thesis, the biosynthesis of alkaloids from Penicillium palitans was elucidated in cooperation with Zhanghai Li, and the formation mechanism of a sesquiterpenoid from Aspergillus ustus by isotopic labelling was carried out in cooperation with Marlies Peter. To investigate the alkaloid biosynthesis in P. palitans (projects 3.1 and 3.2), the SMs were uncovered first. Cultivation of P. palitans in mCDH medium at 25 °C for 15 days and LC-MS analysis revealed the presence of at least eight metabolites. Among them, alkaloids cyclopenol (1), viridicatol (6), and cyclopiazonic acid (7, CPA) as dominant products, and speradine F (8) as a minor product were identified from the wild type. Then, related genome mining was performed via bioinformatics analysis. NRPS- or PKS-NRPS-derived three alkaloids containing amino acids were further investigated due to the enzymatic potentials in their corresponding BGCs for the formation of the meta-hydroxylation at a monosubstituted benzene ring and highly oxygenated tetrahydrofuran. Genome mining, gene deletion, and heterologous expression led to the identification of two separate clusters, the four-gene vdo cluster responsible for cyclopenin (2) skeleton formation and the five-gene spe cluster for the CPA skeleton formation. Feeding experiments proved that the cytochrome P450 enzyme VdoD catalyzes the key step in the conversion of cyclopenin (2) to cyclopenol (1), demonstrating a less studied enzymatic meta-hydroxylation at a monoalkylated benzene ring. Heterologous expression (HE) established that spe cluster is responsible for the formation of CPA and its derivatives (7 – 12), and the highly oxygenated speradine F (8) was synthesized by multiple nonenzymatic hydroxylations. The study completes the biosynthesis of viridicatol and speradine F and illustrates two hydroxylation reactions in biosynthesis. For the identification of catalytic mechanism of the sesquiterpenoid malfilanol D (project 3.3), we carried out genome analysis at the very beginning, then proved the function by HE, characterization biochemically with recombinant protein, and isotopic labelling experiments. Sequence analysis of the genome of A. ustus led to the identification of an uncharacterized putative terpene cyclase, termed mfdS hereafter. HE in the model organism Aspergillus nidulans LO8030 was used to prove the gene function. An additional metabolite was detected by LC-MS and then isolated from the culture of the transformant. Interpretation of the NMR data, including 1H NMR, 13C NMR, 1H-1H COSY, HSQC, HMBC, and 1H-1H NOESY revealed that the conspicuous peak is a new compound malfilanol D (13). To prove that MfdS is solely responsible for the formation of malfilanol D, enzyme assay of farnesyl diphosphate (FPP, C15) with the recombinant protein was conducted. Additionally, based on sodium 13C-acetate labelling experiments, a novel sesquiterpene cyclase from A. ustus was demonstrated in the biosynthesis of the less investigated bicyclo[5.4.0]undecane sesquiterpenes. 1,2-alkyl shift of allylic cation leads to ring expansion and successive 1,2-hydride transfer, C-1 to C-6 ring closure, and capture of water, provides the final product malfilanol D.
The three cases, investigating products derived from NRPS, PKS-NRPS, and TC from ascomycetous fungus Penicillium palitans and Aspergillus ustus, illustrated that fungi are a promising pool to exploit bioactive SMs and interesting enzymes by elucidation of their complete biosynthesis.