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Contenuto archiviato il 2024-04-16

Wide domain control of primary and secondary metabolism in aspergili


To elucidate the mechanisms of carbon catabolite repression and pH regulation in controlling expression of genes involved in both primary and secondary metabolism in two Aspergillus species for both scientific and practical benefit.
Current studies involve:
sequencing of Aspergillus nidulans (A nidulans) pacC;
analysis of iso-penicillin N synthetase (IPNS) transcription with respect to carbon catabolite repression (CCR);
making constructs to produce regulatory proteins;
preliminary identification of regulatory proteins on prn, alc and IPNS genes;
selection and mapping of Aspergillus niger (A niger) mutations affecting carbon catabolite repression and potential of hydrogen (pH) regulation;
metabolic analysis of A nidulans and A niger mutant and wild type strains, potential of hydrogen.

A number of significant results have been achieved. The ALCR and CREA binding sites have been identified, the first to be performed with A nidulans transcriptional regulators. The transactivator ALCR seems to share properties of the 3 main groups of deoxyribonucleic acid (DNA) binding proteins. The molecular mechanism of induction and repression of the ethanol regulon is now well understood, as is the mechanism of double repression of the prn cluster.
IPNS expression has been shown to be under CCR. As creA alleles do not fully derepress IPNS transcription, 3 possibilities arise: a gene different from creA controls CCR of secondary metabolism; very little creA function is required for IPNS repression. The combination of a repressing C source and a creAd 30 allele might result in a derepressed but noninduced situation. Experiments are in progress to distinguish these possibilities, as each is of intrinsic biotechnological interest.
Through the use A nidulans probes isolated from genes of wide domain function, namely creA and pacC, it has been possible to isolate the equivalent sequences from the industrially valuable organism A niger, thus providing a means by which metabolic regulation in this organism can be analysed at the molecular level.
In vivo phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy has been found to be a powerful method to measure the cytoplasmic pH in young Aspergillus hyphae . pH sensitive fluorescent dyes can be used to monitor selectively the intracellular pH of the vacuolar compartment of the fungal cells which play an important role in cellular pH regulation.
The presence of zinc fingers and a high frequency of S/TPXX motifs in the derived amino acid sequence of pacC support the hypothesis that it is a transcriptional regulator.

The identification of the ALCR targets on the alcR and alcA promotors will facilitate the construction of superinducible promotors able to express higher amounts of heterologous protein. Disruption of the CREA binding sites should result in complete glucose derepressed alcR-alcA expression, allowing growth of these A nidulans strains on a glucose medium. This feature is of valuable interest for industrial purposes.
Two wide domain regulatory systems, carbon catabolite repression and pH regulation, will be investigated in the filamentous fungus Aspergillus nidulans and the results extended to more industrially important species Aspergillus niger and from primary to secondary metabolism. A powerful combination of physiological and non-invasive spectroscopic studies and classical and molecular genetics will be used to investigate regulatory genes mediating each of these important forms of regulation and the receptor sites for their products. The interaction of carbon catabolite repression with pathway-specific induction mechanisms, a frequent obstacle to using inexpensive substrates for industrial fermentation, will also be investigated. The choice of these two wide domain regulatory systems is based on their involvement in regulation of industrially important products such as pharmaceuticals, fine chemicals, primary metabolites and heterologous and homologous proteins. A. nidulans is an ideal organism for an in depth investigation of molecular interactions underlying carbon catabolite repression and pH regulation and accompanying pathway-specific regulation. These fundamental studies pave the Way for exploitation of corresponding regulatory systems in the most industrially important fungi such as A. niger and Penicillium chrysogenum where their molecular and physiological analysis will greatly contribute to strain improvement.


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