Construction of Penicillium crustosum as an expression platform for fungal secondary metabolite biosynthesis
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Philipps-Universität Marburg
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Secondary metabolites are organic compounds that are formed by living organisms. Due to their wide range of pharmacological activities, secondary metabolites have been used in the treatment of diseases for centuries. The primary producers of secondary metabolites are fungi, bacteria and plants. In the case of filamentous fungi, the enzymes for secondary metabolite biosynthesis are typically encoded in biosynthetic gene clusters within their genomes. In recent decades, substantial advances in sequencing technologies and bioinformatic tools have facilitated the mining of microbial genomes for biosynthetic gene clusters and the linkage to corresponding products. However, studies have shown that most gene clusters are cryptic or silent under laboratory conditions. The activation of these silent clusters may result in the discovery of new bioactive secondary metabolites with potential therapeutic applications. In this thesis, a Penicillium crustosum host strain was constructed to provide a new platform for the heterologous expression of fungal genes. In previous studies, the biosynthesis of a number of secondary metabolites in P. crustosum was investigated, including terrestric acid, clavatol, hydroxyclavatol, and their adducts peniphenones and penilactones. In addition, the P. crustosum pcr4401 pigment locus was demonstrated to be a suitable integration site for heterologous genes.
In the first project, a novel split marker-based marker recycling strategy was developed for the genetic manipulation of P. crustosum PRB-2. This approach involved the use of the bidirectional pyrG selection marker for gene deletion and subsequent marker recycling in the resulting transformants. Furthermore, the biosynthesis of terrestric acid, one of the main metabolites in PRB 2, was completely elucidated by proving the function of the cytochrome P450 enzyme TraB. In the course of this project, the split marker strategy was used for the deletion of the DNA repair gene ku70 in a P. crustosum ΔpyrG mutant to generate a strain with improved gene targeting efficiencies. Subsequently, the pyrG marker was removed and reused for the deletion of traB in the obtained Δku70 mutant. In parallel, traB was heterologously expressed in the frequently used Aspergillus nidulans host strain LO8030. Both experiments confirmed the key role of TraB in the conversion of viridicatic acid to crustosic acid via hydroxylation and spontaneous ring formation.
The second project mainly focused on the construction of a new expression platform for fungal genes in P. crustosum. To optimize P. crustosum as a heterologous host, the aforementioned split marker-based marker recycling strategy was used for multiple genetic manipulation steps in a single strain. First, the DNA repair gene ligD was deleted to provide a second P. crustosum strain with an enhanced gene targeting background. Furthermore, the two highly expressed biosynthetic pathways for terrestric acid (tra) and clavatol (cla) were inactivated by gene deletion to generate a minimized secondary metabolite profile. Subsequently, the P. crustosum pcr4401 pigment gene was replaced by the A. nidulans wA locus, which comprises the naphthopyrone synthase gene wA including its flanking regions. This enables the use of constructs originally designed for the commonly used A. nidulans LO8030 host strain also in our P. crustosum expression platform. Moreover, transformants can be easily detected by an albino phenotype. Finally, the pcribo gene, which is involved in the biosynthesis of riboflavin, was disrupted to provide an additional selection marker for convenient genetic manipulation in P. crustosum. The successful expression of a single polyketide synthase (PKS) gene and an entire gene cluster in our P. crustosum host strain JZ52 confirmed its suitability as a valuable alternative to the existing heterologous expression systems.
In the third project in cooperation with Dr. Wen Li and Yu Dai, genome mining led to the identification of an uncharacterized polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid gene cluster (pem) in P. crustosum. Gene overexpression, deletion, and feeding experiments proved its involvement in the biosynthesis of the new tetramates penilactams A–C. Moreover, the distinct cla cluster was demonstrated to provide the reactive ortho-quinone methide intermediate, which couples with the tetramic acids derived from the pem cluster to form penilactams A–C. In this project, the doctoral candidate was mainly involved in conducting gene deletion experiments in P. crustosum. This study provides another example of complex natural product formation by the crosstalk of two independent biosynthetic pathways in fungi.
The fourth project in cooperation with David Breyer and Leyao Chen focused on the heterologous expression of a PKS gene derived from the plant pathogen Colletotrichum higginsianum in two distinct host systems. Heterologous expression in the P. crustosum host strain JZ52 led to the production of the anticipated melanin precursor 1,3,6,8-tetrahydroxynaphthalene. However, the expression of the same PKS gene in A. nidulans LO8030 resulted in the formation of the new compounds higginidulans A and B. Furthermore, the distinct cichorine gene cluster in host strain LO8030 was demonstrated to be involved in the biosynthesis of higginidulans A and B. In this project, the doctoral candidate was primarily responsible for the construction of the P. crustosum strains. The findings of this study provide an example of the involvement of two separate gene clusters from different fungal species in product formation. Moreover, they highlight the importance of employing alternative host systems for heterologous expression.
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Created: 2024Issued: 2025-08-06Updated: 2025-08-06
Faculty
Fachbereich Pharmazie
Publisher
Philipps-Universität Marburg
Language
eng
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DoctoralThesis
Keywords
Expression PlatformHeterologous Expression
DFG-subjects
Heterologe ExpressionSekundärmetaboliteExpressionsplattform
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615
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Zhou, Jenny: Construction of Penicillium crustosum as an expression platform for fungal secondary metabolite biosynthesis. : Philipps-Universität Marburg 2025-08-06. DOI: https://doi.org/10.17192/z2024.0514.
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This item has been published with the following license: In Copyright