Glutathione metabolism and dimorphism in Aureobasidium pullulans.

Yeast<-->mycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors. In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions. In accordance with this hypothesis, the specific GSH levels found in A. pullulans yeast cells were always significantly higher than those in mycelia. One the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology. The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols. Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g. glutathione reductase (GR), gamma-glutamyltranspeptidase (gamma GT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels. It is noteworthy that very similar specific gamma GT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology. On the other hand, the specific gamma GT and G6PD activities of A. pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences.

Effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae.

The effect of N-linked glycosylation on secretion, activity, and stability of alpha-amylase from Aspergillus oryzae grown as dispersed filaments was studied. In the presence of tunicamycin the fungus grew either as dispersed filaments or as one large pellet, whereas growth was as dispersed filaments in all control cultures. The presence of tunicamycin affected neither biomass, level of secreted alpha-amylase, nor total amount of secreted protein in cultures growing as dispersed filaments. In these cultures both glycosylated and nonglycosylated alpha-amylase appeared in the culture medium as well as in the cells, whereas in control cultures only the glycosylated form of alpha-amylase was found in the medium and in the cells. The presence of nonglycosylated alpha-amylase in the medium seemed to result from active secretion rather than from autolysis of the mycelium or extracellular deglycosylation. Deglycosylation with Endo H of crude alpha-amylase in culture filtrate did not affect its stability towards heat, acid pH, or proteolytic degradation.

Uptake of phenylacetic acid by two strains of Penicillium chrysogenum.

Uptake of phenylacetic acid, the side-chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54-1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high-yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid-derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250-3500 microM), and in the high-yielding strain at low phenylacetic acid concentrations (2. 8-100 microM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC-analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]-labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport-assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high-affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54-1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin.

Uptake of phenoxyacetic acid by Penicillium chrysogenum.

The uptake of phenoxyacetic acid by two different strains of Penicillium chrysogenum was studied. Phenoxyacetic acid (POA) was taken up by P. chrysogenum in a defined medium. Plots of initial velocity of POA uptake versus external substrate concentration, in the range 2-5000 microM, gave linear plots. Uptake of POA by induced and uninduced cells was identical. The initial velocity of POA uptake decreased as the pH of the suspension was increased from 5.4 to 7.2; the decrease closely paralleled the decline in the non-ionic form of the acid over this pH range. The initial velocity of POA uptake was not affected by the presence of phenylacetic acid. POA uptake proceeded until the cellular concentration was equal to the external concentration. It is concluded that POA is passively transported into P. chrysogenum by unmediated diffusion.

Utilization of side-chain precursors for penicillin biosynthesis in a high-producing strain of Penicillium chrysogenum.

Utilization of the side-chain precursors phenoxyacetic acid (POA) and phenylacetic acid (PA) for penicillin biosynthesis by Penicillium chrysogenum was studied in shake flasks. Precursor uptake and penicillin production were followed by HPLC analysis of precursors and products in the medium and in the cells. P. chrysogenum used both POA and PA as precursors, producing phenoxymethylpenicillin (penicillin V) and benzylpenicillin (penicillin G), respectively. If both precursors were present simultaneously, the formation of penicillin V was blocked and only penicillin G was produced. When PA was added at different times to cells that were induced initially for POA utilization and were producing penicillin V, the POA utilization and penicillin V formation were blocked, whereas the cells started utilizing PA and produced penicillin G. The blocking of the POA turnover lasted for as long as PA was present in the medium. If POA was added to cultures induced initially for PA utilization and producing penicillin G, this continued irrespective of the presence of POA. Utilization of POA increased concomitant with depletion of PA from the medium. Analysis of cellular pools from a growing cell system with POA as precursor to which PA was added after 48 h showed that the cellular concentration of POA was kept high without production of penicillin V and at a concentration comparable to the concentration in the medium. The cellular concentration of POA was higher than the concentration of PA that was utilized for penicillin G production.

Description of conidia from submerged cultivation of Thermomyces lanuginosus for use as a uniform inoculum.

Conidia produced by submerged cultivation of the thermophilic fungus Thermomyces lanuginosus were superior to conidia from agar plates when used as inoculum, due to a faster and more synchronous germination. With conidia derived from submerged liquid culture at 40-45 degrees C more than 90% germination was achieved at 50 degrees C within 3 h whereas the same percentage germination was only achieved after 5 h incubation of conidia produced on agar plates. The temperature during conidial formation, and conidial age at the time of harvesting, were factors influencing germination of the conidia.