Arabinase induction and carbon catabolite repression in Aspergillus niger and Aspergillus nidulans

The first aim of this thesis was to get a better understanding of the properties and the induction features of arabinan degrading enzymes and enzymes involved in the intracellular L-arabinose catabolic pathway in Aspergillus niger. The second aim was to understand the which role carbon catabolite repression plays in the induction process and to inventarize what phusiological consequences of an extreme carbon catabolite derepressed mutant are. The first part of this thesis the deals with the induction and characterisation of the arabinolytic enzyme system in Aspergillus niger. In the second part results of detailed studies on carbon catabolite repression of arabinase biosynthesis in Aspergillus nidulans are reported.In Chapter 2 several carbon sources were tested for their ability to induce arabinan degrading enzymes in A.niger N400. It was found that sugar beet pulp was the best inducing substrate for the induction of three arabinolytic enzymes. These three arabinan degrading enzymes were purified from the culture filtrate after growth on sugar beet pulp as the carbon source. The physico-chemical and kinetic properties of these three enzymes were characterised and found to correspond with those of the three arabinolytic enzymes purified from a commercial A . niger enzyme preparation by Rombouts et al. (1988). Also, the α-L-arabinofuranosidase B was found to correspond with an α-L-arabinofuranosidase B already characterised by Kaji and Tagawa (1970). Cloning and genetic analysis of the three genes by Flipphi et al (1993a; 1993b; 1993c) has shown that there are some discrepancies between the amino acid composition found by HPLC analysis and the one derived from the sequence data. This could be due to inaccuracies in HPLC analysis.Because of the finding in Chapter 2 that the monomeric sugar Larabinose induces arabinolytic activity, this sugar together with L-arabitol as an intermediate of the metabolic pathway of L-arabinose, was tested for inducing capacity in Chapter 3. In this case also an A.niger N572 xylulose kinase negative mutant strain was used which is disturbed in the last step of the metabolic pathway of L-arabinose, the conversion of D-xylulose to xylulose-5-phosphate (for a Scheme see Chapter 1 Fig. 2). This strain was characterised by Witteveen et al. (1989) by 13C- NMR analysis. Xylitol and L-arabitol were found to accumulate upon mycelial transfer to L-arabinose or D-xylose containing media.Using the sugars present in the metabolic pathway of L-arabinose and D-xylose it was found that within the wild type strain, L-arabinose induces α-L-arabinofuranosidase B but L-arabitol induces all three known arabinolytic enzymes. However, using the xylulose kinase negative mutant the level of induction on these two substrates was remarkable higher (up to 2 times). Moreover, in this mutant induction was also found using D-xylose and xylitol as a carbon source in transfer experiments. Of these two strains also the enzymes present in the catabolic pathway of L-arabinose were investigated. It was found in all cases that the activities in the xylulose kinase mutant were higher. The finding that L-arabitol dehydrogenase activity was almost 3 to 4 times higher on D-xylose and xylitol in comparison with the wild type was remarkable. Also induction of PNPA hydrolysing activity could already be detected using low amounts of L-arabitol (1.5 mM). Furthermore the induction of arabinases was found to be under the control of carbon catabolite repression. Induction on L-arabitol in the presence of 0.2% (w/v) D- glucose could only be detected when the concentration of D-glucose in the media became very low.These experiments lead to the conclusion that L-arabitol plays an important role in the induction of the arabinases in A. niger and that α-L-arabinofuranosidase B may trigger the biosynthesis of the other arabinases. It is known that the arabinolytic complex releases monomeric L-arabinose (Rombouts et al. 1988). This release of L-arabinose and its subsequent uptake by A. niger will convert the monomeric substrate into L-arabitol and thus induce all three arabinan degrading enzymes.Chapter 2 and 3 gave us information about the arabinan degrading enzyme system existing in A. niger and its induction, which could be activated by using simple low molecular weight substrates like L-arabitol an intracellular intermediate of the L-arabinose catabolic pathway. This induction system which involves both extra- and intracellular enzymes provides an excellent tool to study carbon catabolite repression of these enzymes in more detail. Chapter 4 and 5 describe experiments using Aspergillus nidulans, a fungus which has become a model organism for molecular genetic studies also because of the availability of a large collection of well defined mutant strains. In this case A. nidulans WGO96, which is used as the wild type strain, was first investigated for the presence of arabinolytic enzymes (Chapter 4). It was found that A. nidulans WG096 only produces 2 of the known arabinases, namely α-L-arabinofuranosidase B and endo-arabinase. α-L-arabinofuranosidase A could not be detected by Western blotting and also a genomic blot probed with the A. niger abf A gene did not give any positive signal. Biosynthesis of both arabinases was also found to depend on pathway-specific induction and carbon catabolite repression. Like in A. niger L-arabitol was also found to act as an efficient inducer for both arabinases present in A. nidulans.The observations made by De Vries et al. (1994) confirm the postulated role of L-arabitol in the induction mechanism of arabinan degrading enzymes. They have characterised an A . nidulans L-arabinose non-utilising mutant strain, which was first isolated by Roberts (1963) and characterised by Clutterbuck (1981) as an L- arabinose non-utilising mutant. It was found to be a L-arabitol dehydrogenase negative mutant strain. They also investigated the formation of polyols when transferred to L-arabinose containing media and found a strong accumulation of intracellular L-arabitol This accumulation of L-arabitol resulted in this strain in a strong induction of the two A.nidulans arabinases, which was confirmed both by enzymatic analysis as by Western blotting.The existence of some genetically well defined carbon catabolite derepressed mutants (Arst and MacDonald 1975; Arst and Bailey 1977; Dowzer and Kelly 1989; 1991) gave us an excellent tool to investigate the carbon catabolite repressing phenomena in more detail with respect to the induction of the two arabinases present in A. nidulans. In this case also the response of the intracellular L- arabinose degrading enzymes to carbon catabolite repression was investigated together with intracellular xylitol and L-arabitol measurements (Chapter 5). The data found indicate a strong influence of creA on the biosynthesis of both α-L- arabinofuranosidase B and endo-arabinase. The effect of the creA mutations was found to be most marked under inducing conditions. Both the more extreme carbon catabolite derepressed mutants cre A d-30 and cre A d-4 show a remarkable induction of arabinase activities up to 6- to 10 fold the level reached in the wild type suggesting a considerable 'self' repression in the wild type. In the presence of D-glucose plus an inducer the creA mutations, particularly cre A d-30, result in carbon catabolite derepression although the level of activity found did not reach the level which was found under inducing conditions. The intracellular enzymes present in the L- arabinose catabolic pathway show a more on-hierarchical behaviour. For example, whereas L-arabitol is a better inducer than L-arabinose of L-arabinose reductase in the cre A d-4 strain, the reverse is true for the cre A d-2 strain. Furthermore, from the finding that intracellular xylitol and thus L-arabitol could be detected on a mixed carbon source (D-glucose/L-arabinose) we can conclude that uptake of L-arabinose and conversion takes places even in the presence of D-glucose as a repressing carbon source.Although the direct role of the CREA protein in carbon catabolite repression by acting as a repressor of gene expression has now been elucidated (Kulmburg et al 1993; Cubero and Scazzocchio 1994), the physiological consequences of this mutation have not yet been investigated. Therefore a comparison was made on the level of enzyme activities and metabolite concentrations present in the metabolic pathway of D-glucose and polyol concentrations which can be derived from this route (Chapter 6). The enzymatic data obtained suggest that only a few enzymes are, directly or indirectly, influenced by the CREA protein. These are hexokinase and fructose-6-phosphate reductase, which were found to have elevated activities and phosphofructokinase and pyruvate kinase, which were found to show decreased activities within the cre A d-30 mutant. Both the metabolites fructose-2,6-diphosphate and fructose-1,6-diphosphate, which are the respective activators for the last two enzymes, were found to have increased internal concentrations. The result of these changes in activity is that this leads to a higher flux through the side chains towards the formation of polyols and possibly to a decreased flux through the TCA cycle due to the decreased activity of pyruvate kinase. The accumulation of polyols in the cre A d-30 mutant strain is remarkable. Although the amount of polyols produced intracellularly remains at a constant level in both strains, the total amount (intra- an extracellular) produced by the cre A d-30 mutant strain is almost up to times the total polyol concentration formed by the wild type strain However the nature of the repressing signal remains unclear.The induction of the arabinolytic enzyme system in A.nidulans was studied together with its response to carbon catabolite repression by using different carbon catabolite derepressed mutant strains. A study of the carbon catabolite repression on extracellular and intracellular enzymes together with an an easy induction system of these enzymes is scarce. The high induction of arabinase activity on L- arabitol in the extreme mutant strain cre A d-30 has almost the same level as found in wild type A . niger under normal induction conditions. In this case it would be interestingly which level of arabinase activity could be reached if carbon catabolite derepressed mutants of A. niger become available.