Studies on carbon metabolism in wild type and mutants of Aspergillus nidulans

This thesis deals with carbon metabolism in the lower eukaryote <em>Aspergillus nidulans.</em> This fungus is an attractive organism as a model to study genetics in relation to metabolism in lower eukaryotes.In chapter I the present state of affairs in this field is summarized for <em>A. nidulans</em> and some of the advantages of this fungus in particular for physiological and biochemical genetic studies are indicated. The picture of the metabolic abilities of <em>Aspergilli</em> is still far from complete notwithstanding the many studies in <em>A. nidulans</em> and <em>A.niger.</em> The latter is a related fungus used for the biosynthesis of industrial enzymes and for the large scale production of metabolites of commercial and economic interest.Ever since the early investigations into the physiology of <em>A.nidulans,</em> which started some 40 years ago, the use of mutants to study the effects of mutant genes on metabolism has become increasingly important. Therefore a brief review is given of the various ways in which mutants regarding to carbon metabolism in <em>A. nidulans</em> have been selected and isolated. An up to date list is added of mutations in carbon metabolism and, as far as these are known, of the metabolic defects involved.In chapter II a study was made on the metabolic features of mutants in carbon metabolism, since mutations may have effects on the level of a particular enzyme directly as well as on the regulation of other steps in metabolism ( <em>e.g.</em> inhibition, repression). The mutants used for this. purpose were defective in pyruvate kinase, pyruvate dehydrogenase complex, pyruvate carboxylase, transaldolase and in two non-specified lesions in glycerol metabolism. Growth tests with a variety of substrates and combinations of them were performed with the mutants and on the basis of the results obtained criteria were developed to distinguish the mutant phenotypes. Such growth tests can be used as a first step in the elucidation of unknown metabolic pathways and also to find a strategy to isolate particular mutants. From the carbon sources tested for growth, D- galacturonate emerged as a substrate useful to discriminate pyruvate kinase mutants, which showed good growth on this carbon source from mutants defective in the pyruvate dehydrogenase complex or pyruvate carboxylase, which grow badly or not at all. The differences observed between these mutants in their ability to use D-galacturonate as sole carbon source indicated that pyruvate is a metabolite formed during the degradation of D-galacturonate. Moreover, it was predicted that selection for a mutant phenotype unable to use this carbon source would lead, among others, to mutants unable to metabolize pyruvate, for instance pyruvate dehydrogenase complex mutants.In chapter III the results are described of an X-ray mutation experiment followed by enrichment on D-galacturonate. This led to a large number of mutants unable to grow on this carbon source. The criteria developed in chapter II were used to assign phenotypes to the mutants isolated. The majority of these were found to be defective in the pyruvate dehydrogenase complex. In addition mutants were isolated defective in pyruvate carboxylase and in glycerol metabolism, whereas a novel class of mutants was found blocked earlier in D-galacturonate metabolism. The mutants isolated were further analyzed by complementation tests with defined tester strains. It was found that all pyruvate dehydrogenase complex mutants studied belonged to one of the three genes already known. The pyruvate carboxylase mutants were novel with respect to their inability to complement in a heterokaryon with two pyruvate carboxylase tester strains which mutually complement. All mutants found in glycerol metabolism belonged to the same gene. Since mutants were obtained which were only defective in D-galacturonate metabolism, a preliminary study was started to analyze the metabolic route which this compound follows in wild type <em>A.nidulans.</em> A pathway for the degradation of D-galacturonate which leads to pyruvate as one of the products is known to occur in <em>Escherichia coli.</em> Although the presence of this pathway was easily demonstrated in the latter microorganism, no positive evidence has been found for a similar pathway operating in <em>A.nidulans.</em>In chapter IV the purification and some properties of the pyruvate dehydrogenase complex of <em>A.nidulans</em> are described. A purification scheme was developed using ultracentrifugation of the complex in a crude extract, polyethylene glycol precipitation of the concentrated enzyme complex and affinity chromatography on ethanol-Sepharose 2B. After a final sucrose density gradient centrifugation step, a pure multienzyme complex was obtained with a specific activity of 7.8 U mg <sup><font size="-1">-1</font></SUP>with a recovery of 16%. The preparation finally obtained still contained pyruvate dehydrogenase kinase, which enzyme was copurified. SDS-polyacrylamide gel electrophoresis showed that the purified pyruvate dehydrogenase complex is composed of 4 proteins which were assigned to the α <em></em> and βsubunit of pyruvate decarboxylase, lipoate acetyltransferase and of lipoamide dehydrogenase. The molecular weights of the complex enzymes resemble those of the complex of other eukaryotes and of <em>Bacilli.</em> The kinetic constants of the complex were listed and they were compared to those of the complexes of other sources.In chapter V a purification scheme for pyruvate kinase is described based mainly on the use of affinity chromatography with reactive dyes. Screening several of these dyes coupled to Sepharose resulted in two dyes which were used in sequence to purify pyruvate kinase: Mikacion Brilliant Yellow 6GS,and Cibacron Blue 3G-A as a dextran conjugate. The addition of ethylene glycol during these purification steps was necessary to warrant an optimal recovery of the enzyme activity. Although some pyruvate kinase activity could be recovered by biospecific elution, in the final purification scheme aspecific elution of the enzyme was preferred by enhancing the ionic strength of the elution buffer. A homogeneous enzyme preparation was obtained with specific activity of 67 U mg <sup><font size="-1">-1</font></SUP>after a final gel filtration on Sephacryl S-300. The overall yield was low due to instability of the enzyme. The purified pyruvate kinase was used to raise antibodies in a rabbit.In chapter VI the levels of pyruvate kinase specific activity were studied in wild type <em>A.nidulans</em> grown under different nutritional conditions. The specific activity of pyruvate kinase was high in mycelium grown on sucrose and low in mycelium grown on polygalacturonate or acetate. The latter two carbon sources allow growth of pyruvate kinase mutants. With antibodies against purified pyruvate kinase raised in a rabbit several pyruvate kinase mutants and wild type grown under pyruvate kinase inducing conditions were analyzed for the presence of a pyruvate kinase gene product in sucrose tolerant and sucrose sensitive pyruvate kinase mutant strains, by the use of immunological methods. Although pyruvate kinase could be detected easily in crude extracts of wild type by Ouchterlony double diffusion and tandem crossed immunoelectrophoresis, these methods failed when applied to extracts of pyruvate kinase mutants. Therefore Western blotting was introduced to screen for a pyruvate kinase gene product. Among 5 mutants, all at the same locus as found by non-complementation, only one was found, totally lacking imunological and enzymatic activity. Since a pyruvate kinase gene product was present in both a sucrose tolerant and a sucrose sensitive strain, the absence of such a gene product is not a prerequisite for sucrose tolerance observed with some pyruvate kinase mutants.