Utjecaj koncentracije otopljenog kisika na intracelularni pH i na brzinu rasta Aspergillus niger

With the results presented we would like to emphasize the influence of dissolved oxygen concentration in a medium on intracellular pH values and consequently on overall metabolism of microorganisms. Intracellular pH of mycelium grown under different concentrations of dissolved oxygen in the medium was determined by recently developed cryo 31P-NMR method. Sensitivity of proteins toward proton concentration was well recognised, therefore pH influences on the activities of some key regulatory enzymes of Aspergillus niger were determined at pH values similar to those detected in the cells grown under lower dissolved oxygen concentrations. The results have shown significantly reduced specific activities of hexokinase, 6 phosphofructokinase and glucose-6-phosphate dehydrogenase in more acidic environment, while pyruvate kinase was found to be relatively insensitive toward higher proton concentration. As expected, due to reduced specific activities of some key regulatory enzymes under more acidic conditions, overall metabolism should be hindered in media with lower dissolved oxygen concentration, as confirmed by detecting reduced specific growth rates.Prikazanim rezultatima istaknut je utjecaj koncentracije otopljenog kisika u mediju na intracelularne pH-vrijednosti i s tim u vezi na ukupni metabolizam mikroorganizama. Intracelularni pH micelija, uzgajanog pri različitim koncentracijama otopljenog kisika u mediju, određen je nedavno razvijenim postupkom krio-31P-NMR. Poznata je osjetljivost proteina na koncentracije protona. Stoga je određen utjecaj pH na aktivnosti nekih ključnih regulatornih enzima u A. niger pri pH-vrijednostima sličnim onima otkrivenim u stanica što su rasle pri smanjenim koncentracijama otopljenog kisika. Rezultati su pokazali bitno smanjenu specifičnu aktivnost heksokinaze, 6-fosfofruktokinaze i glukoza-6-fosfat dehidrogenaze u jače kiseloj sredini, dok je piruvat kinaza relativno neosjetljiva na ve}e koncentracije protona. Očekivalo bi se da će u medijima sa smanjenom koncentracijom otopljenog kisika cjelokupni metabolizam biti ometen zbog smanjenih specifičnih aktivnosti nekih ključnih regulatornih enzima u kiselijim uvjetima. To je i potvrđeno jer su utvrđene smanjene brzine specifičnog rasta A. niger

Identification of a Transcription Factor Controlling pH-Dependent Organic Acid Response in Aspergillus niger.

Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis

Influence of Dissolved Oxygen Concentration on Intracellular pH and Consequently on Growth Rate of Aspergillus niger

With the results presented we would like to emphasize the influence of dissolved oxygen concentration in a medium on intracellular pH values and consequently on overall metabolism of microorganisms. Intracellular pH of mycelium grown under different concentrations of dissolved oxygen in the medium was determined by recently developed cryo 31P-NMR method. Sensitivity of proteins toward proton concentration was well recognised, therefore pH influences on the activities of some key regulatory enzymes of Aspergillus niger were determined at pH values similar to those detected in the cells grown under lower dissolved oxygen concentrations. The results have shown significantly reduced specific activities of hexokinase, 6 phosphofructokinase and glucose-6-phosphate dehydrogenase in more acidic environment, while pyruvate kinase was found to be relatively insensitive toward higher proton concentration. As expected, due to reduced specific activities of some key regulatory enzymes under more acidic conditions, overall metabolism should be hindered in media with lower dissolved oxygen concentration, as confirmed by detecting reduced specific growth rates

Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium. © 2010 Springer-Verlag

Crystal structure of pure-silica ZSM-12 with tetraethylammonium cations from X-ray powder diffraction data

Pure-silica ZSM-12, containing tetraethylammonium cations (TEA) was synthesized using a ‘two-silica’ source strategy and its structure was determined from laboratory X-ray powder diffraction data. This is the first known zeolite structure in which the positions of the TEA cations have been established. The ordered arrangement of TEA cations in the straight nonintersecting channels of the host material, which is reflected in several weak superstructural diffraction peaks, was determined by using the combination of simulated annealing and Rietveld refinement. In accordance with Raman spectroscopy, the calculations confirmed the presence of a tt.tt form of the TEA cations and the absence of the other stable form (tg.tg) which was also consistent with the dimensions and elliptical cross-section of the channels. Therefore in ZSM-12 structure pore (channels) geometry is directly connected with the conformation of TEA cations. The compound with a moiety formula C16H40N2O84Si42 (Mr = 2784.28, Z = 4) crystallizes in the monoclinic Cc space group with unit cell parameters a = 25.2177(9), b = 15.1903(5), c = 24.4754(7) Å, β = 108.516(2)°. The final structure, which resulted in a good agreement between calculated and observed powder diffraction pattern (Rwp = 10.17%), was additionally confirmed by results from thermogravimetric measurements and FTIR spectroscopy

Changes in primary metabolism leading to citric acid overflow in aspergillus niger

For citric acid-accumulating Aspergillus niger cells, the enhancement of anaplerotic reactions replenishing tricarboxylic acid cycle intermediates predisposes the cells to form the product. However, there is no increased citrate level in germinating spores and a complex sequence of developmental events is needed to change the metabolism in a way that leads to an increased level of tricarboxylic acid cycle intermediates in mycelia. A review of physiological events that cause such intracellular conditions, with the special emphasis on the discussion of hexose transport into the cells and regulation of primary metabolism, predominantly of glycolytic flux during the process, is presented