This programme concerns two wide domain gene regulatory systems in the model filamentous fungus Aspergillus nidulans and related, industrially relevant species - Ph regulation of synthesis of secreted enzymes, permeases and exported metabolites and carbon catabolite repression of synthesis of enzymes involved in carbon nutrition - and their manipulation to improve production of penicillin and the secreted xylanolytic complex. Specific goals include characterisation of the mechanism signalling ambient Ph, analysis of target site interactions for the two regulatory systems, understanding of the relationship between pathway-specific induction and carbon catabolite repression in the ethanol regulon and characterisation of xylanase structural genes and their regulation with a view to improving production.
Ph regulation (London, Madrid): The PacC transcription factor is synthesised as a 678 residue protein whose three zinc fingers enable it to bind a GCCARG core sequence. Conversion of PacC to a functional form able to activate expression of alkaline-expressed genes and prevent transcription of acid-expressed genes is ambient Ph-sensitive, requiring signal transduction by the pal gene pathway and involves proteolytic removal of C-terminal approximately 60% of PacC. Mutations removing 100-214 C-terminal residues obviate the need for ambient Ph signal transduction and mimic growth at alkaline Ph whereas more severe truncations and a null allele mimic acidic growth Ph. The promoter of the alkaline-expressed, penicillin biosynthetic gene ipnA contains three physiological PacC binding sites essential for alkaline Ph-mediated transcriptional activation.The homologous 638 residue Penicillium PacC also binds GCCARG, complements a null A. nidulans pacC mutation and, like its A. nidulans homologue, is alkaline-expressed. The divergent bifunctional penicillin biosynthesis promoter in P. chrysogenum contains a high density of PacC binding sites. Two new Ph-signalling pal genes have been identified. palB encodes a calpain family cysteine protease, albeit not responsible for final conversion of PacC to its functional form. The palA and palI sequences identify yeast homologues and palA has a homologue in Caenorhabditis elegans, raising the possibility of conservation of Ph signalling between fungal and animal kingdoms.
Carbon catabolite repression (Orsay): Using a combination of classical and reverse genetic mutations in the alcA and alcR promoters and the divergent bifunctional promoter between prnD and prnB, it has been shown that the carbon catabolite repressor protein CreA acts by directly competing with the transcriptional activator protein AlcR in the alcA and alcR promoters but by indirectly and at a distance preventing the binding of an as yet unidentified positive-acting transcription factor in the prnD-prnB intergenic region. Consistent with these conclusions, a creA promoter mutation substantially diminishing creA transcription derepresses alc but not prn expression whereas a frameshift deleting the C-terminus of CreA derepresses prn but not alc expression.
The xylanolytic system (Wageningen, Valencia, Bagsvaerd): The three A. nidulans endo-xylanases and one beta-xylosidase have been biochemically characterised, their genes characterised molecularly and multicopy transformants obtained. A conserved promoter sequence has been shown to be involved in coordinate xylan/xylose induction. A severe creA mutation results in precocious induction and derepression of the four transcripts, consistent with the presence of in vitro CreA binding sites in the promoters.The xlnA and xlnB endo-xylanase genes are alkaline- and acid-expressed, respectively, as judged both by varying growth Ph and using Ph regulatory mutants. Genes encoding endo-xylanases in A. tubigensis (xlnC) and A. niger (xlnB and xlnC) and beta-xylosidases (xlnD) in both organisms have been molecularly characterised. A. niger xlnD multicopy transformants and disruptants have been used to show that beta-xylosidase is essential for complete xylan hydrolysis but not for induction. Non-inducible and constitutive A. niger regulatory mutants were obtained and the xlnR transcriptional activator gene mediating xylan induction was cloned and sequenced. xlnR is constitutively expressed and encodes a zinc binuclear cluster DNA binding protein. Four xlnR mutations were also sequenced. Comparisons in batch and fed batch fermentations of A. niger single copy transformants of xlnA from A. tubigensis with and without CreA promoter binding sites showed that lack of CreA binding sites result in a two-fold increase in expression (by western blots and assay) in conditions of carbon source excess but not in carbon-limited conditions. No effect of ambient Ph on transcript levels of A. niger/tubigensis xln genes was detected in batch fermentations. Increasing the xlnR copy number gave a major improvement in xylanase production in wild type and in xlnA and xlnD multicopy transformants.
MAJOR SCIENTIFIC BREAKTHROUGHS:
A. nidulans continues to be the leading organism for characterisation of Ph regulation and the characterisation of two forms of PacC confirms an important prediction of our new model. The characterisation of contrasting modes of CreA action has greatly advanced our understanding of and ability to manipulate carbon catabolite repression. Characterisation of the Ph regulatory system has been extended to P. chrysogenum and A. niger. The demonstration that manipulating both carbon catabolite repression and induction of the xylanolytic complex improves fermentation yields is allowing construction of industrial strains less influenced by fermentation conditions, enabling further optimisation for secondary parameters such as byproduct formation.
Funding SchemeCSC - Cost-sharing contracts
6703 HA Wageningen
91405 Gometz La Ville