The Aspergillus nidulans Penicillin‐Biosynthesis Gene aat (penDE) is Controlled by a CCAAT‐Containing DNA Element

Abstract

Analysis of the promoter of the penicillin biosynthesis nut (penDE) gene of Aspergillus niduluns using band-shift assays led to the identification of a CCAAT-containing DNA element which was specifically bound by a protein (complex). The identified DNA element was localised about 250 bp upstream of the transcriptional-start sites of uat: Substitution of the CCAAT core sequence by GATCC led to a fourfold reduction of expression of an aat-1acZ gene fusion. The identified binding site thus was functional in vivo and positively influenced aat expression. Partial purification of the CCAAT binding protein and cross-competition experiments provided evidence that the binding protein is identical to the identified putative penicillin-regulatory protein PENRl , binding to the CCAAT element in the bidirectional intergenic promoter region between acvA (pcbAb) and ipnA (pcbC). Hence, PENRI seems to be involved in the regulation of all three penicillin-biosynthesis genes. Cross-competition experiments demonstrated that the promoter region of the corresponding aat (penDE) gene of Penicillium chrysogenum was capable to dilute the shift of the A. nidulans probe with PENRI, suggesting the presence of a similar regulatory mechanism in this fungus. Taken together with previous data, CCAAT-containing DNA elements thus seem to represent major cis-acting sites in the promoters of p-lactam-biosynthesis genes.Keywords: Aspergillus nidulans ; penicillin biosynthesis ; gene-regulatory protein ; CCAAT-binding protein; secondary metabolism.Aspergillus (Emericella) nidulans and Penicillium chrysogenum are filamentous fungi well known for their ability to produce the secondary metabolite penicillin. The entire biosynthesis pathway is catalysed by the three enzymes d+a-aminoadipyl)-L-cysteinyl-D-valine synthetase, isopenicillin N synthase and acyl-coenzyme A:isopenicillin N-acyltransferase (IAT), encoded by the genes acvA (JchAB), ipnA (pcbC) and aat (penDE) respectively. The genes are organised into a gene cluster and expressed from gene-specific promoters (Fig. 1, Fig. 6; Litzka et al., 1995 and reviewed in Demain, 1983;Kleinkauf and von Dohren, 1990;Queener, 1990;Skatrud, 1991 ;Aharonowitz et al., 1992;Brakhage and Turner, 1995). Because penicillin biosynthesis is one of the best studied secondary-metabolite-biosynthetic pathways in fungi, it represents an advanced model system to study regulation of secondary-metabolite biosynthesis in this class of organisms. In recent years, it has become evident that at the molecular level, regulation of penicillin biosynthesis is complex. Several protein factors seem to be involved (Brakhage and Van den Brulle, 1995;Chu et al., 1995;Feng et al., 1995;Haas and Marzluf, 1995; P6rez-Esteban et al., 1995;Tilburn et al., 1995;Then Bergh et al., 1996).For the studies presented here, A. nidu1an.s was used because this fungus has, in contrast to the deuteromycete P chrysogenum which is employed for industrial production of penicillin, a welldefined sexual cycle facilitating genetic analyses (MacDonald and Hol...

Relationship between Secondary Metabolism and Fungal Development

“…PENR1 positively regulates the expression of two penicillin biosynthetic genes, ipnA and aatA. Deletion of hapB, hapC, and hapE genes, which encode PENR1 complex components, resulted in identical phenotypes characterized by low levels of ipnA and aatA expression as well as a slow-growth phenotype and a reduction in conidiation (74,87,112).…”

“…The A. nidulans CCAAT binding protein complex PENR1 has been reported to regulate both development and penicillin production (74,112). PENR1 positively regulates the expression of two penicillin biosynthetic genes, ipnA and aatA.…”

Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae

“…In fact, the orthologuous HAP complex in mammals (CBF) has a wide range of target genes (Maity & de Crombrugghe, 1998). In fungi the HAP complex has been implicated in regulation of genes involved in the catabolism of carbon and nitrogen sources and secondary metabolism (Litzka et al, 1996). It is thus possible that different activator subunits can be associated with the core complex ('domain borrowing'), providing specificity for particular sets of genes.…”

Molecular Control of Expression of Penicillin Biosynthesis Genes in Fungi: Regulatory Proteins Interact with a Bidirectional Promoter Region

“…Sucrose (or glucose) repression of the ipnA (ϭpcbC) gene of A. nidulans is known (10,26,72), but Litzka and coworkers reported that expression of the A. nidulans pcbAB gene using reporter gene fusions was not repressed by the carbon source (53). The basic regulatory mechanisms of carbon and nitrogen regulation were initially believed to be similar in both fungi (28,47).…”