Az arabinóz és a laktóz metabolizmus kölcsönhatásainak vizsgálata Aspergillus nidulans-ban = Interactions between the arabinose and lactose metabolism in Aspergillus nidulans

Kutatásaink azon munkák folytatása volt, melynek keretében az Aspergillus nidulans fonalas gomba béta-galaktozidáz enzim aktivitásának képződését tanulmányoztuk. Jelen pályázatban a témát kiterjesztettük és elmélyítettük. Izoláltuk és klónoztuk a gombában előforduló összes (4 db) béta-galaktozidázt kódoló gént (BgaA-D), és jellemeztük kifejeződésük szabályozását. Ezek alapján A. nidulans-ban a BGAC a fő béta-galaktozidáz enzim. Elsőként izoláltunk fonalas gombafajból laktóz permeáz gént (LacA), melyre nézve hiánymutánst hoztunk létre, és ennek segítségével karakterizáltuk kifejeződésének szabályozását. Izotópos vizsgálatok révén a laktóz permeázt biokémiai szempontból is jellemeztük. Bebizonyítottuk, hogy mind a BgaC, mind a LacA kifejeződése CreA-függő karbon katabolit represszió alatt áll. Ugyancsak bebizonyítottuk, hogy az említett szabályozási mechanizmusnak determinánsa a tenyészet specifikus növekedési rátája, és egy adott specifikus növekedési ráta érték alatt derepresszió következik be. Talán legérdekesebb felfedezésünk egy új D-galaktóz lebontási útvonal volt, mely a Leloir-útvonallal párhuzamosan működik. Az első, aldóz reduktáz enzim(ek) által katalizált lépés alapján reduktívnak elkeresztelt útvonal a D-galaktóz dulcitollá (galaktitollá) történő, NADPH-függő redukcióját, majd ennek L-szorbózzá történő, NAD+-függő, L-arabitol dehidrogenáz katalizálta dehidrogénezését foglalja magába. A glikolízisig tartó további reakciókban a fruktokináz enzim is szerepel. | This research is based on those aimed to investigate the regulation of formation of the beta-galactosidase activity in the filamentous fungus Aspergillus nidulans. In this project, this topic was extended and included a more in-depth approach. We have isolated and cloned all the four beta-galactosidase-like genes of the fungus (BgaA-D), and characterized the regulation of their expression. We concluded that in A. nidulans, BGAC is the major beta-galactosidase. We have isolated and cloned a lactose permease (LacA), which was the first of its kind from a filamentous fungus. A LacA deletion mutant was created to study the regulation of gene expression. Using labelled lactose, LACA was also characterized from the biochemical point of view. Evidence was provided that the regulation of BgaC and LacA is under CreA-dependent carbon catabolite regulation. We have shown that this regulatory mechanism is determined by the specific growth rate, and that under a certain, 'critical' growth rate carbon derepression occurs. Arguably our most interesting discovery was a new D-galactose catabolic pathway that operates in parallel to the Leloir-pathway. The pathway is called reductive after the first step that involves the NADPH-dependent reduction of D-galactose into galactitol (dulcitol). Galactitol is subsequently reduced into L-sorbose by the NAD+-dependent L-arabinitol dehydrogenase. The subsequent reactions include fructokinase, which channels the carbon into the glycolytic pathway

D-galactose catabolism in Penicillium chrysogenum: Expression analysis of the structural genes of the Leloir pathway

In this study, we analyzed the expression of the structural genes encoding the five enzymes comprising the Leloir pathway of D-galactose catabolism in the industrial cell factory Penicillium chrysogenum on various carbon sources. The genome of P. chrysogenum contains a putative galactokinase gene at the annotated locus Pc13g10140, the product of which shows strong structural similarity to yeast galactokinase that was expressed on lactose and D-galactose only. The expression profile of the galactose-1-phosphate uridylyl transferase gene at annotated locus Pc15g00140 was essentially similar to that of galactokinase. This is in contrast to the results from other fungi such as Aspergillus nidulans, Trichoderma reesei and A. niger, where the ortholog galactokinase and galactose-1-phosphate uridylyl transferase genes were constitutively expressed. As for the UDP-galactose-4-epimerase encoding gene, five candidates were identified. We could not detect Pc16g12790, Pc21g12170 and Pc20g06140 expression on any of the carbon sources tested, while for the other two loci (Pc21g10370 and Pc18g01080) transcripts were clearly observed under all tested conditions. Like the 4-epimerase specified at locus Pc21g10370, the other two structural Leloir pathway genes – UDP-glucose pyrophosphorylase (Pc21g12790) and phosphoglucomutase (Pc18g01390) – were expressed constitutively at high levels as can be expected from their indispensable function in fungal cell wall formation

Regulation of the cyanide-resistant alternative respiratory pathway in the fungus Acremonium chrysogenum

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The Role of Metal Ions in Fungal Organic Acid Accumulation

Organic acid accumulation is probably the best-known example of primary metabolic overflow. Both bacteria and fungi are capable of producing various organic acids in large amounts under certain conditions, but in terms of productivity-and consequently, of commercial importance-fungal platforms are unparalleled. For high product yield, chemical composition of the growth medium is crucial in providing the necessary conditions, of which the concentrations of four of the first-row transition metal elements, manganese (Mn2+), iron (Fe2+), copper (Cu2+) and zinc (Zn2+) stand out. In this paper we critically review the biological roles of these ions, the possible biochemical and physiological consequences of their influence on the accumulation of the most important mono-, di- and tricarboxylic as well as sugar acids by fungi, and the metal ion-related aspects of submerged organic acid fermentations, including the necessary instrumental analytics. Since producing conditions are associated with a cell physiology that differs strongly to what is observed under “standard” growth conditions, here we consider papers and patents only in which organic acid accumulation levels achieved at least 60% of the theoretical maximum yield, and the actual trace metal ion concentrations were verified