D.J. Coetzee

Production of 3R-hydroxy-polyenoic fatty acids by the yeast Dipodascopsis uninucleata

Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing a 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydroxylated metabolites of shorter chain length, e.g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), γ-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete β-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal β-oxidation as biosynthetic route to this new class of oxylipins.

A novel oxylipin-associated 'ghosting' phenomenon in yeast flocculation.

Research on the distribution of oxylipins (3-hydroxy fatty acids) in flocculant strains of the yeast Saccharomyces cerevisiae led to the uncovering of a novel ‘ghosting’ phenomenon observed during assumed lectin-mediated aggregation. We found that intracellular oxylipin-containing osmiophilic layers migrate through yeast cell walls in a ‘ghostlike’ fashion without visually affecting the cell wall structure or the layers. This migration resulted in the binding of these layers to cell walls of adjacent cells. Consequently, ‘ghosting’ seems a prerequisite for flocculation to occur. However, ‘ghosting’ alone may not be sufficient to ensure flocculation.

An acetylsalicylic acid-sensitive aggregation phenomenon in Dipodascopsis uninucleata

Aggregation of ascospores has been discovered in the yeast Dipodascopsis uninucleata. When this yeast is cultivated to reach the sexual reproductive stage, small ascospores are individually released from the tip of a sac-like ascus which then aggregate in orderly clusters. Acetylsalicylic acid (ASA) inhibited ascospore release and subsequent ordered aggregation process. We suggest that novel ASA-sensitive oxidised fatty acids (3 R-hydroxy-oxylipins) and small hooks located on the surface of these ascospores, are involved.

Yeast Eicosanoids II. The Influence of Non-Steroidal Anti-Inflammatory Drugs on the Life Cycle of Dipodascopsis

Summary The effect of the non-steroidal anti-inflammatory drugs acetylsalicylic acid and indomethacin on the life cycles of Dipodascopsis tothii and Dipodascopsis uninucleata is investigated. With ascosporogenesis as the most susceptible phase, it is found that these agents inhibit the completion of the life cycles of both species.

An isolation procedure for arachidonic acid producing Mortierella species

Malt extract agar and an incubation temperature of 5 °C were used to selectively isolate representatives of the genus Mortierella from soil. Fungi in a soil sample from mountain grassland able to grow under these conditions, amounted to a total of 2640 colony forming units per gram soil. Circa 94% of the total fungal isolates represented Mortierella subgenus Mortierella. The rest of the colony-forming units consisted of Mucor isolates (6.0%) and higher fungi (1.5%). All the Mortierella isolates produced arachidonic acid.

The value of orthogonal-field-alternation gel electrophoresis as a rapid identification process for some species representing the genus Saccharomyces

Abstract The chromosomal DNA band patterns for nine strains representing four species of the genus Saccharomyces were determined by an optimized orthogonal-field-alteration gel electrophoresis (OFAGE) method. These yeasts were grouped according to their similarities and then compared. Consequently the Saccharomyces strains were clustered into four groups on the basis of their chromosomal DNA band patterns. The band patterns of each of the four species proved to be unique.

The Production of Biologically Active 3-Hydroxy-5,8,11,14-Eicosa Tetraenoic Acid (3-HETE) and Linoleic Acid Metabolites by Dipodascopsis

Summary 3-Hydroxy-5,8,11,14-eicosatetraenoic acid (3-HETE) is a biologically active compound which has a signal transducing role in human neutrophils. When 12.5 mg/l arachidonic acid (AA) was fed to sexual reproductive cells of Dipodascopsis uninucleata and D. tothii the former transformed approximately 4% of AA to 3-HETE, while the latter produced only trace amounts of 3-HETE. The 3-HETE was only associated with cellular ethanol extracts of both species. D. uninucleata utilized 69% of the AA after 6h while D. tothii utilized 33%. The residual or unused AA was mainly present in the cells of D. tothii (96%) and D. uninucleata (98%) and only small amounts (4% and 2%) in their respective supernatants. Extraction with 80% aqueous ethanol succeeded in removing respectively 92% and 45% of AA present in the cells of D. tothii and D. uninucleata . The poor AA extraction from cells of D. uninucleata contributed to the presence of AA in the neutral lipid fraction which is insoluble in ethanol. If efficient AA extraction can be implemented, approximately 67% and 31% unused AA can be recovered from D. tothii and D. uninucleata respectively which can be recycled for biotransformation purposes. Although no traces of other 3-hydroxy fatty acids were detected, enzyme extracts from D. uninucleata metabolised exogenous 18:2 in a manner consistent with the presence of a prostaglandin endoperoxide synthase similar to that in fetal calf blood vessels.

The Phylogenetic Development of Species Representing the Genus Kluyveromyces

Summary The species representing the yeast genus Kluyveromyces are arranged in a new phylogenetic scheme evolving from the mycelioid hybridizing “primitive” ancestors associated with various habitats, towards the unicellular non-hybridizing “evolved” taxa which are nutritionally restricted and hence probably more dependent on specific habitats. During this evolutionary process a reduction in phenotypic and genetic characteristics generally occurred while an increase in the number of small chromosomes was observed in the OFAGE karyotypes.

The Value of Orthogonal-Field-Alternation Gel Electrophoresis and Other Criteria in the Taxonomy of the Genus Pichia Hansen emend. Kurtzman

Summary The relationships among one hundred and twenty-one Pichia strains were determined by orthogonal-field-alternation gel electrophoresis (OFAGE). In order to evaluate this genomic character, we compared the results obtained with other criteria, such as carbon source utilization patterns, coenzyme Q types and G+C contents. We found that similar DNA banding patterns do not necessarily coincide with similar physiological appearances, G+C values or coenzyme Q types. Strains within a species produced the same number of chromosomal bands, except for P. membranaefaciens . In the case of varieties of a species and species considered to be conspecific, similar OFAGE patterns were observed. The results obtained were also compared with Wickerhams (1970) phylogenetic scheme for nitrate positive Pichia species. It is interesting to note that the more “primitive” ancestors, P. finlandica, P. holstii and P. capsulata produced only two DNA bands, while species placed some distance from the origin of Wickerhams (1970) phylogenetic tree are mainly characterized by more than two chromosomal bands.

Ascospore aggregation and oxylipin distribution in the yeast Dipodascopsis tothii.

Upon cultivation of the yeast Dipodascopsis tothii in its sexual stage, small ascospores are released individually from the ascus tip, which then assemble in sheathed cluster balls. In contrast to Dipodascopsis uninucleata, this yeast produced smooth bean shaped ascospores with sheath-like appendages that assemble in a disordered sheathed ball of ascospores outside the ascus. Strikingly, upon release, the ascus tip contained 3-hydroxy oxylipins, while the released ascospore clusters contained little or no 3-hydroxy oxylipins as indicated by immunofluorescence microscopy. In D. uninucleata, these oxylipins are concentrated on the spore surface and interspore matrix, but not on the ascus tip.