Biochemical and electron microscope autoradiographic studies of lipid synthesis in young and aging cultures of penicillium chrysogenum

The synthesis of lipids and long chain fatty acids in young and aging cultures of Penicillium chrysogenum was studied by identifying the intracellular location of radioactively labeled intermediates in the vegetative mycelium and identifying the lipids and fatty acids into which the label from 1-14C-palmitic acid was incorporated.Previous work has indicated that the pH of the growth medium of submerged cultures of Penicillium chrysogenum dropped from pH 7.4 in newly inoculated cultures to 3.2-3.8 during the 20-40 hour growth period. Young cultures were defined as those harvested before the pH began to drop and aging cultures were those harvested after the pH stabilized at near 3.4. Changes in the fatty acid composition of various cell fractions of the mycelium harvested as the pH of the medium declined suggested that a change in utilization of lipids employed in the synthesis of structural components in the cell may have occurred as a result of a shift in fatty acid metabolism as the culture aged.Young and aging cultures were incubated with l-14C-palmitic acid and harvested after 2, 10, 60, and 120 minutes. Samples of mycelia from each harvest were examined by light and electron microscopy and were prepared for autoradiography. In addition lipids were extracted from sonified mycelial samples, analyzed for total lipids, lipid classes, and phospholipid components by thin layer chromatography. Fatty acids were identified and quantified by gas liquid chromatography with percent distribution of label in fatty acids determined by liquid scintillation spectrometry. Results of biochemical analysis of some of the major lipid components were compared with electron microscope autoradiographs and related to changes in the location of labeled fatty acid in cell organelles or cell parts as the cells aged.In young cultures 70% of the radioactive label was recovered in phospholipids while about 15% was recovered in the free fatty acid component. By contrast, in aging cultures approximately 20% of the label was recovered in the phospholipids and 80f was recovered from the free fatty acid component. Electron autoradiographic data tend to support these biochemical findings in that numerous grains occur over the membranal components of the young cells and over cytoplasmic areas of lipid depots in aging cells. Phospholipids differed markedly also with large amounts of an unidentified phospholipid type found in aging cells not seen in young cells.Light and electron microscope observations of hyphal cells showed significant alterations in cell morphology over the forty hour growth period. Young cells were long, slender, with dense cytoplasm and thin cell walls. As the culture aged, cells became progressively shorter, thicker and more clubby in appearance with prominent lipid inclusions. The vacuolar lipid depots of aging cells were ,determined to be composed of free fatty acids with 20% of label being incorpoated into C20 and C21 long chain fatty acids. The synthesis of fatty acid chains longerthan C18 has not been previously reported in intact cultures of Penicillium chrysogenum.The lipid metabolism of young cells of Penicillium chrysogenum differed from that of aging cells in many aspects. Young cells incorporated label from precursor palmitic acid into membranes. However, as the cells aged, lipids were diverted to storage. Young and aging cells differed in the amounts and composition of total lipids, lipid classes, phospholipid components and fatty acids. Differences in morphology between young and aging cells could be demonstrated by light and electron microscopy. These structural changes paralleled the biochemical changes indicating a functional dissimilarity existed between the young and aging cells.Thesis (D. Ed.