Unravelling the molecular biology of cheese ripening fungus, Penicillium camemberti

Kuria, Asaph

Unravelling the molecular biology of cheese ripening fungus, Penicillium camemberti

Publication Date

July 27, 2026

Creator

Kuria, Asaph

Abstract

The cheese ripening fungus Penicillium camemberti is used as a starter culture in the production of fermented (mould-ripened) cheeses such as Camembert and Brie. However, despite its significance in cheese production, the molecular genetic basis of how the species contributes to cheese ripening has mostly been a mystery. This formed the basis of the work presented in this thesis, which aimed at gaining insights into the molecular biology of P. camemberti and in particular the genes encoding enzymes involved with cheese ripening. To achieve this goal, work was undertaken in three work areas. Firstly, efforts were made to develop a CRISPR-Cas9 mediated genetic modification procedure for P. camemberti. As a proof of concept the niaD gene, whose activity mediates the breakdown of nitrate as a source of nitrogen, was targeted in P. camemberti. The gene was successfully deleted via CRISPR-Cas9 mediated manipulation using a homology directed DNA repair process involving replacement with a pyrithiamine resistance cassette. In addition the arp2 gene, which forms part of the DHN-melanin pigment biosynthesis pathway, was targeted in the closely related species Penicillium biforme and successfully manipulated via the same approaches. Results therefore supported the adoption of CRISPR-Cas9 mediated transformation for genetic manipulation of species in the P. camemberti group, and the development of this protocol paved the way for further molecular genetic studies of P.camemberti and related fungal species. In the case of P. camemberti, its enzymatic activity is linked to key quality characteristics of the outer mould-ripened cheeses associated with the species. Therefore, genes involved in production of enzymes were of great interest. In the second work area, genes encoding lipolytic enzymes were investigated. Lipolysis has been postulated to be involved in aroma and flavour formation of Camembert and Brie-type cheeses. The extracellular enzymes of the lipolytic system responsible for fat breakdown during the cheese ripening process were investigated using a combination of bioinformatic analysis, gene expression, proteomics and CRISPR-Cas9 mediated gene editing approaches. Bioinformatic analysis conducted using the FM013 reference genome revealed that P. camemberti has 20 genes encoding for putative secreted lipases (termed lip1-20). However, when the fungus was grown in liquid cultures, only five enzymes (Lip4, 6, 8, 9, and 20) were found to be part of the secretome via proteomics methods. Interestingly, studies of gene expression in media with and without olive oil did not show substantial differential expression of these genes. CRISPR-Cas9 mediated approaches were used successfully to delete three of the identified genes (lip4, 9, and 20) encoding enzymes that were part of the secretome. Analysis of the lipolytic activity of the three deletant strains showed a significant reduction in lipolysis for Δlip20 mutant compared to the parental strain. Furthermore, deletion of the lip20 gene led to a substantial disruption in production of flavour volatiles. However, deletion of the lip4 and lip9 genes had no significant impact and unfortunately it was not possible to generate lip6 and lip8 deletants. In the third work area, genes encoding proteolytic enzmyes were investigated. Extracellular proteolysis of P. camemberti has also been linked with the key characteristics of Camembert and Brie-type cheeses. The genes involved in this process were again investigated via a combination of bioinformatic analysis, gene expression, proteomics and CRISPR-Cas9 mediated genetic modification approaches. Bioinformatic analysis showed that P. camemberti has 49 genes encoding putative secreted enzymes with proteolytic activity. Intriguingly, the majority of these genes were shown to be expressed during growth in liquid media. Application of proteomics approaches was critical in the narrowing down of candidate genes, with only 16 enzymes found to be part of P. camemberti secretome. Differential expression of the 16 genes was examined by qRT-PCR in the presence of casein, the main milk protein in cheese. Results showed substantial expression of seven genes (prot4, 8, 9, 12, 24, 36, and 41). These were all successfully deleted via CRISPR-Cas9 mediated approaches. Proteolytic activity of the seven prot deletant mutants was compared to the parental strain, which showed a significant reduction in activity for Δprot4 and Δprot36 (an aspA homologue) strains. Interestingly, there were no differences in production of flavour volatiles between the parental strain and Δprot mutants except for Δprot9 and Δprot12. Deletion of prot9 and prot12 genes led to alterations in the composition of the flavour compounds in a pattern almost similar to the deletion of lip20 gene. In conclusion, this study has provided insights into the molecular functionality of P. camemberti regarding lipolytic and proteolytic activity, as well as contributing to the toolbox available for gene deletion studies. However, more work is needed to further reveal the functionality of this fungus during the ripening of cheese.

Item Type

ethesis

Thesis Type

PhD

Supervisors

Dyer, P.S.