Cleere, Matthew
(2017)
Pigment biosynthesis genes in the blue cheese fungus Penicillium roqueforti.
MRes thesis, University of Nottingham.
Abstract
The fungus Penicillium roqueforti is used in the production of blue cheese worldwide. It confers a distinct flavour and texture due to a wide range of volatiles and enzymes produced. Blue veins that develop during the culturing process add a uniquely recognizable characteristic to the final product. The resulting colour comes from the pigments that make up melanin covering the spore surface. The process by which P. roqueforti produces these melanin pigments has yet to be identified. New strains have been produced using UV mutagenesis to generate spore colour mutants whose conidia range from brown, pink, green, intense blue, and white in appearance. This research may have commercial potential because it might facilitate the production of novel cheeses with a range of vein colours.
In this study, the genetic controls of pigment biosynthesis in P. roqueforti were investigated using a combination of bioinformatic, biochemical, and gene manipulation approaches. Initially, bioinformatics analysis was performed to determine whether a DHN-Melanin pathway, involved with pigment biosynthesis in the phylogenetically related Aspergillus fumigatus, was present in P. roqueforti. This identified a set of six genes, some of which were clustered in the genome. The genes identified were homologues of alb1 (Albino 1), ayg1 (Aspergillus yellow-green 1), arp2 (Aspergillus reddish-pink 2), arp1 (Aspergillus reddish-pink 1), abr1 (Aspergillus brownish-red 1), and abr2 (Aspergillus brownish-red 2). To determine whether the DHN-Melanin pathway was functional in P. roqueforti, first biochemical inhibitor work was performed. Second, gene sequencing of a series of UV colour mutants was undertaken to assess any correlation between change in spore colour and mutation in genes of the DHN-Melanin pathway. Third, deletion strains were produced targeting each of the six genes believed to be involved in pigment biosynthesis. Finally, experimental work was conducted to gain insights into the ecological importance of spore pigment formation in P. roqueforti.
As a result of the combined bioinformatic, biochemical and gene manipulation work, a definitive DHN-Melanin pathway for the fungus P. roqueforti is proposed. This information will be of importance to both the scientific community who study Penicillium roqueforti and related organisms and the industrial cheese producers who use them.
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