Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum.
Penicillium
/ genetics
Secondary Metabolism
/ genetics
CRISPR-Cas Systems
Aspergillus nidulans
/ genetics
Polyketide Synthases
/ genetics
Multigene Family
Gene Targeting
/ methods
Gene Expression Regulation, Fungal
Fungal Proteins
/ genetics
Biosynthetic Pathways
/ genetics
Metabolic Engineering
/ methods
Gene Expression
Penicillium crustosum
Fungal host
Genetic dereplication
Heterologous expression
Natural products
Secondary metabolite biosynthesis
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
24 Jul 2024
24 Jul 2024
Historique:
received:
17
05
2024
accepted:
16
07
2024
revised:
08
07
2024
medline:
24
7
2024
pubmed:
24
7
2024
entrez:
24
7
2024
Statut:
epublish
Résumé
Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.
Identifiants
pubmed: 39046587
doi: 10.1007/s00253-024-13259-3
pii: 10.1007/s00253-024-13259-3
doi:
Substances chimiques
Polyketide Synthases
79956-01-7
Fungal Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
427Subventions
Organisme : Deutsche Forschungsgemeinschaft
ID : Li844/11-1
Organisme : Deutsche Forschungsgemeinschaft
ID : INST 160/620-1
Organisme : Chinese Government Scholarship
ID : 202206170029
Informations de copyright
© 2024. The Author(s).
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