Dioxygenation of polyunsaturated fatty acids in fungi

Abstract

Polyunsaturated fatty acids play a central role in all biological systems. They are constituents of the plasma membrane and serve as precursors to signaling molecules generated in response to external events. The conversion of polyunsaturated fatty acids into signaling molecules starts by the hydrolysis of fatty acids from phospholipid molecules and the free fatty acids are subsequently oxygenated. The products of this reaction are called oxylipins. Many of the oxylipins in animals, plants and fungi are produced by the dioxygenase-type of oxygenation and this type of oxygenation is the focus of this thesis. Animal and plant dioxygenases have been extensively studied. The dioxygenases of the third eukaryotic kingdom, the fungi, have received comparatively little attention. A few of the fungal dioxygenases have been described in detail, but most studies have either just shown the presence of a catalytic activity or the presence of oxylipins. Many fungal oxylipins differ from their animal and plant counterparts with respect to their molecular structures. Their formation is likely catalyzed either by similar enzymes with specificities different from their plant and animal homologs, or by a novel group of dioxygenases. In depth analysis of dioxygenation pathways in fungi will lead to a better understanding of the complex fungal life cycle, and will add to the knowledge on their animal and plant equivalents. In Chapter 2 the characterization is described of two previously unknown oxylipins in the mushroom Agaricus bisporus. These oxylipins are potential products of a dioxygenase with unknown specificity or they could be produced by a new sort of dioxygenase. To relate fungal oxylipins to a possible function in growth and development, in Chapter 3 their occurrence was studied in different developmental stages of Schizophyllum commune, a model fungus closely related to A. bisporus. Oxylipin analysis of S. commune vegetative mycelium and fruiting bodies showed that their oxylipin profiles are similar. Potentially, oxylipins play a constant role during the S. commune life cycle and it is likely that, in analogy with plants and animals, oxylipins serve as signaling molecules in fungi. In Chapter 4 the cloning of a putative dioxygenase gene from S. commune is described. A phylogenetic tree was constructed, which showed that the fungal fatty acid heme dioxygenase (FAHD) family has diverged into six different gene sub-groups. Since complete studies connecting fungal FAHD genes, proteins, oxylipins and functions are currently not available, it remains unclear if proteins belonging to the same FAHD gene sub-group also form similar oxylipins or have similar functions. Analysis of the A. niger genome revealed that this fungus contains three putative dioxygenase genes and the result of their disruption an over-expression is described in Chapter 5. In A. nidulans putative dioxygenase genes are related to oxylipin formation and to the balance between sexual and asexual sporulation. In contrast, A. niger transformants were not altered in their ability to produce oxylipins or sporulation. Since A. niger and A. nidulans produce the same oxylipins and also have similar dioxygenase genes, it is likely that oxylipins not only influence the balance between asexual and sexual reproduction, but that they play a more general role in the fungal life cycle.