FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W147803640> ?p ?o ?g. }

• W147803640 abstract "This thesis describes studies on the biochemical properties and regulation of L-arabinose metabolism and arabinan degrading enzymes of Aspergillus niger. We focused on the investigation of the catabolic pathway, firstly by isolating pathway specific regulatory mutants using a newly developed selection system and, secondly, by purifying the enzymes and characterising their kinetics for use in metabolic control analysis. Finally, by cloning genes encoding these enzymes we were able to analyse expression of these genes. Using a D-xylulose kinase deficient strain we developed a mutant selection system that identified genes involved in pentose catabolism and their regulation. The A. niger strain carrying the xkiA1 mutation lacks D-xylulose kinase and cannot grow on pentoses, such as D-xylose and L-arabinose, but these sugars still repress the use of other carbon sources such as D-gluconate. We used this genetic background to select for pentose-derepressed mutants on media containing combinations of gluconate with xylitol, L-arabinose or D-xylose. A subset of these mutants was further analysed and turned out to have rather interesting properties as described in chapters two and three. One of the mutants isolated using this method carried a mutation called xtlA36 and is described in chapter two. This mutation results in a severe decrease in xylitol consumption, suggesting that xtlA36 inactivates a xylitol transporter and opens the way of isolation of genes encoding the corresponding genes. Two other A. niger mutants, carrying araA and araB are specifically disturbed in the regulation of the arabinanase system in the presence of L-arabinose. Expression of three arabinanolytic genes, abfA, abfB and abnA , is substantially decreased or absent in the araA and araB , strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular enzyme activities of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. L-arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulates to a high intracellular concentration in the araA and araB mutants. This indicates that the decreased expression of the arabinolytic genes is not due to lack of inducer accumulation. Therefore, we propose that araA and araB are mutations in positively acting components of the regulatory system involved in the expression of the arabinanase encoding genes and the genes encoding the L-arabinose catabolic pathway. Chapter four describes cloning of the A. niger D-xylulose kinase encoding gene ( xkiA ) by direct complementation of the strain deficient in D-xylulose kinase activity. This enabled us to investigate the expression of xkiA in the presence of L-arabinose, L-arabitol, and D-xylose. Although XKI is part of the D-xylose catabolic pathway, expression of xkiA appeared not to be mediated by XLNR, the xylose-dependent positively acting xylanolytic regulator. Expression of xkiA is subject to carbon catabolite repression (ccr) but the wide domain regulator CREA is not directly involved. Using the araA and araB strains described in chapter three, we showed that xkiA is under control of the arabinanolytic regulatory system. Overexpression of xkiA enabled us to purify the encoded D-xylulose kinase enzyme. The molecular mass, determined using Electrospray Ionization Mass Spectrometry (ESI-MS) concurred with the calculated molecular mass of 62816.6 Da. The activity of D-xylulose kinase is highly specific for D-xylulose. Kinetic parameters were determined, including K m (D-xylulose), 0.76 mM and K m (ATP), 0.061 mM. In a D-xylulose kinase deficient strain a higher accumulation of intracellular arabitol and xylitol correlated to increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, respectively. This supports the suggestion that L-arabitol may be the specific low molecular weight inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and had no effect on expression of genes encoding xylan and arabinan degrading enzymes. This suggests that the enzymes preceding D-xylulose kinase in the L-arabinose/D-xylose catabolic pathway probably have more control on the flux through this pathway than D-xylulose kinase itself. In chapter five, we cloned the genes encoding A. niger L-arabitol dehydrogenase ( ladA ) and xylitol dehydrogenase ( xdhA ), and produced the enzymes in Escherichia coli . Analysis of the substrate specificity showed that LADA is most active on L-arabitol and also has significant activity on xylitol, but only low activity on D-sorbitol and galactitol. XDHA has the highest activity on xylitol, significant activity on D-sorbitol, but very low activity on L-arabitol. The higher activity on sorbitol for XDHA is in agreement with the amino acid similarity of the different enzyme classes, since a phylogenetic tree of L-arabitol dehydrogenases, xylitol dehydrogenases and sorbitol dehydrogenases (SDH) suggests that xylitol dehydrogenases are more similar to sorbitol dehydrogenases than L-arabitol dehydrogenases. Expression analysis of the pentose catabolic pathway genes confirmed the model in which an arabinose specific regulator activates the expression of all genes required for the conversion of L-arabinose to D-xylulose-5-phosphate. In addition, XLNR regulates the first step and, to a lesser extent, the other steps of the conversion of D-xylose into D-xylulose-5-phosphate. Using dye-affinity chromatography we isolated enzymes of the L-arabinose and D-xylose catabolic pathways that had not been described previously. Using the complete set of kinetic parameters a metabolic model was constructed which we used to perform steady state metabolic control analysis (chapter six). The metabolic model was used to analyse flux and metabolite concentration control of the L-arabinose catabolic pathway. The model predicts that flux control does not only reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14 respectively. This analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimisation of the L-arabinose catabolic pathway of A. niger . In chapter seven the results from chapters two through six are discussed in light of possible engineering applications and recent scientific developments such as genomics." @default.
• W147803640 created "2016-06-24" @default.
• W147803640 creator A5041297454 @default.
• W147803640 date "2005-01-01" @default.
• W147803640 modified "2023-09-27" @default.
• W147803640 title "Regulation and control of L-arabinose catabolism in Aspergillus niger" @default.
• W147803640 cites W1486567941 @default.
• W147803640 cites W1486899617 @default.
• W147803640 cites W1517356485 @default.
• W147803640 cites W1526050457 @default.
• W147803640 cites W1555583269 @default.
• W147803640 cites W1577900879 @default.
• W147803640 cites W1578262270 @default.
• W147803640 cites W1580400135 @default.
• W147803640 cites W158839569 @default.
• W147803640 cites W1589825327 @default.
• W147803640 cites W1603426439 @default.
• W147803640 cites W1606784130 @default.
• W147803640 cites W1625660792 @default.
• W147803640 cites W1642162696 @default.
• W147803640 cites W1650184556 @default.
• W147803640 cites W176207218 @default.
• W147803640 cites W1850754372 @default.
• W147803640 cites W1855933245 @default.
• W147803640 cites W1881761744 @default.
• W147803640 cites W1904167893 @default.
• W147803640 cites W1908662736 @default.
• W147803640 cites W1965129150 @default.
• W147803640 cites W1965373009 @default.
• W147803640 cites W1967161741 @default.
• W147803640 cites W1967976587 @default.
• W147803640 cites W1969853515 @default.
• W147803640 cites W1970113315 @default.
• W147803640 cites W1970314038 @default.
• W147803640 cites W1970572081 @default.
• W147803640 cites W1972686944 @default.
• W147803640 cites W1975354935 @default.
• W147803640 cites W1976798629 @default.
• W147803640 cites W1977440244 @default.
• W147803640 cites W1978165744 @default.
• W147803640 cites W1984860940 @default.
• W147803640 cites W1988497640 @default.
• W147803640 cites W1992184909 @default.
• W147803640 cites W1997090665 @default.
• W147803640 cites W2002711263 @default.
• W147803640 cites W2003996276 @default.
• W147803640 cites W2007052828 @default.
• W147803640 cites W2007694112 @default.
• W147803640 cites W2009310436 @default.
• W147803640 cites W2010483489 @default.
• W147803640 cites W2011291054 @default.
• W147803640 cites W2015297702 @default.
• W147803640 cites W2019662175 @default.
• W147803640 cites W2021231865 @default.
• W147803640 cites W2022074928 @default.
• W147803640 cites W2023096047 @default.
• W147803640 cites W2023764523 @default.
• W147803640 cites W2025309306 @default.
• W147803640 cites W2026553967 @default.
• W147803640 cites W2028622989 @default.
• W147803640 cites W2030590794 @default.
• W147803640 cites W2034194918 @default.
• W147803640 cites W2034711659 @default.
• W147803640 cites W2036323960 @default.
• W147803640 cites W2043990103 @default.
• W147803640 cites W2044623352 @default.
• W147803640 cites W2044747259 @default.
• W147803640 cites W2049113731 @default.
• W147803640 cites W2050674257 @default.
• W147803640 cites W2055043387 @default.
• W147803640 cites W2057281838 @default.
• W147803640 cites W2057282405 @default.
• W147803640 cites W2059235430 @default.
• W147803640 cites W2059936173 @default.
• W147803640 cites W2060297509 @default.
• W147803640 cites W2062644439 @default.
• W147803640 cites W2066488205 @default.
• W147803640 cites W2068372836 @default.
• W147803640 cites W2069983123 @default.
• W147803640 cites W2074122548 @default.
• W147803640 cites W2075147882 @default.
• W147803640 cites W2075814472 @default.
• W147803640 cites W2077572824 @default.
• W147803640 cites W2077797584 @default.
• W147803640 cites W2079358920 @default.
• W147803640 cites W2081494403 @default.
• W147803640 cites W2082136561 @default.
• W147803640 cites W2086081955 @default.
• W147803640 cites W2086421274 @default.
• W147803640 cites W2087096430 @default.
• W147803640 cites W2087468665 @default.
• W147803640 cites W2088433120 @default.
• W147803640 cites W2088962418 @default.
• W147803640 cites W2090727280 @default.
• W147803640 cites W2097890276 @default.
• W147803640 cites W2098805702 @default.
• W147803640 cites W2099830000 @default.
• W147803640 cites W2100837269 @default.
• W147803640 cites W2101076135 @default.
• W147803640 cites W2101245788 @default.

• next