Molecular basis for the biosynthesis of the siderophore coprogen in the cheese-ripening fungus Penicillium roqueforti

Abstract

Background: Iron is an essential nutrient for microorganisms, including fungi, which have evolved strategies to acquire it. The most common strategy is the secretion of siderophores, low-molecular-weight compounds with a high affinity for ferric ions, which are involved in cellular iron uptake. Penicillium roqueforti, the fungus responsible for the ripening of blue-veined cheeses, produces coprogen, a hydroxamate-type siderophore. However, to date, the molecular basis for its biosynthesis remains elusive.

Results: In this study, we identified and characterized a biosynthetic gene cluster (BGC) responsible for coprogen biosynthesis in P. roqueforti, named the cop BGC. This BGC contains seven genes, three of which (copA, copB and copE) encode enzymes directly involved in coprogen biosynthesis from precursors molecules. Using CRISPR-Cas9, we targeted these three genes and analyzed the resulting mutants by Liquid Chromatography/High-Resolution Mass Spectrometry (LC/HRMS). Our results confirmed that all three genes are necessary for coprogen biosynthesis. Phenotypically, the mutants displayed growth differences under iron-deficient conditions, which correlated with their ability to either synthesize or fail to synthesize coprogen B and dimerumic acid, intermediates in the coprogen pathway with siderophore activity.

Conclusions: The results obtained in this work provide important insights into the molecular basis of coprogen biosynthesis in P. roqueforti, enhancing the understanding of how siderophores enable this fungus to thrive in iron-deficient environments.

Keywords: Penicillium roqueforti; Biosynthetic gene cluster; CRISPR-Cas9; Coprogen; Mass spectrometry; Siderophore.

Fig. 1

Schematic representation of the cop BGC identified in P. roqueforti. The genes are represented by red arrows in the 5′ to 3′ orientations. Details of the predicted proteins encoded by these genes are provided in Table 1

Fig. 2

Proposed biosynthetic pathway for coprogen production in P. roqueforti. The core biosynthetic route is highlighted within the black box. Dashed arrows indicate proposed degradation pathways. The black dashed arrow represents the putative origin of AMHO in the copB-12 mutant, potentially resulting from fusarinines degradation. Blue dashed arrows denote the proposed conversion of the intermediate coprogen B into AMHO and dimerumic acid. Green dashed arrows indicate the degradation of coprogen into dimerumic acid and N-acetyl fusarinine

Fig. 3

Comparison of the nucleotide sequences of the CRISPR-Cas9 target regions and the corresponding deduced amino acid sequences for P. roqueforti wild-type (WT) and the disrupted transformants copA-3 (A), copB-12 (B) and copE-30 (C). CRISPR-Cas9 target sites are marked in red, and induced insertions or deletions are highlighted in yellow. The amino end of each protein is shown, with regions unaffected by CRISPR-Cas9 highlighted in green. Subsequent regions exhibiting frameshifts are unmarked, and examples of premature stop codons are indicated by asterisks in pink

Fig. 4

Identification of compounds from the coprogen biosynthetic pathway using UHPLC/MS in P. roqueforti wild-type (WT) and disrupted transformants copE-30, copB-12, and copA-3. The top panels display the total ion current (TIC) of extracts of each strain, while the subsequent panels show the extracted ion chromatograms (EICs) for specific ions corresponding to each compound of interest. The m/z values for each identified ion are labeled in red above the respective peaks. In all cases, the mass error between the measured and theoretical mass was less than 5 ppm. Further details are provided in the main text

Fig. 5

A Growth of P. roqueforti wild-type (WT) and disrupted and mutants copA-3 copB-12 and copE-30 on MM9 plates supplemented with 100 µM BPS after 7 days. The same strains are also shown on CAS plates, where the orange halos around the colonies indicate siderophore activity. B Quantification of colony diameter (cm) for each strain grown on MM9 plates with 100 µM BPS after 7 days. Asterisks denote statistically significant differences in growth compared to WT strain (p < 0.05, Student’s t-test). C Siderophore index (SI) for P. roqueforti wild-type (WT) and the disrupted mutants copA-3 copB-12 and copE-30. Asterisks indicate statistically significant differences in SI compared to WT strain (p < 0.05, Student’s t-test)