CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapC gene, encoding a protein with significant similarity to Hap3p of Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the genes hapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae, Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB, hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression of amdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on ␥-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdS and gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.

The regulation of the Aspergillus nidulans penicillin biosynthesis gene aat (penDE), which encodes acyl coenzyme A:6-aminopenicillanic acid acyltransferase (AAT), was analysed. Major transcriptional start sites map within 100 nucleotides upstream from the aat initiation codon. To study the regulation of aat expression, various aat-lacZ gene fusions were constructed, in which the aat promoter region was fused in frame with the Escherichia coli lacZ reporter gene. A. nidulans strains carrying recombinant plasmids integrated as single copies at the chromosomal argB locus were identified. In both fermentation and minimal media, aat-lacZ expression was maximal during the first 24 h of a fermentation run. Compared with minimal medium, aat-lacZ expression was increased two-fold in fermentation medium. Although AAT specific activity was reduced in mycelia grown on glucose instead of lactose, expression of aat-lacZ gene fusions was not repressed on glucose, suggesting that the glucose effect is mediated posttranscriptionally. The effect of glucose on AAT activity was reversed by further incubation of glucose-grown mycelia on lactose. Neither the inclusion of the first intron of the aat gene in the aat-lacZ fusion integrated at the chromosomal argB locus, nor the disruption of the acvA gene had any regulatory effect on aat-lacZ expression. In the heterologous, nonpenicillin producer A. niger, basal expression of aat-lacZ gene fusions was observed at about the same level as in A. nidulans.

The ␤-lactam antibiotic penicillin is produced as a secondary metabolite by some filamentous fungi. In this study, the molecular regulation of the Aspergillus (Emericella) nidulans penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) was analyzed. acvA and ipnA are divergently oriented and separated by an intergenic region of 872 bp. Translational fusions of acvA and ipnA with the two Escherichia coli reporter genes lacZ and uidA enabled us to measure the regulation of both genes simultaneously. A moving-window analysis of the 872-bp intergenic region indicated that the divergently oriented promoters are, at least in part, overlapping and share common regulatory elements. Removal of nucleotides ؊353 to ؊432 upstream of the acvA gene led to a 10-fold increase of acvA-uidA expression and simultaneously to a reduction of ipnA-lacZ expression to about 30%. Band shift assays and methyl interference analysis using partially purified protein extracts revealed that a CCAAT-containing DNA element within this region was specifically bound by a protein (complex), which we designated PENR1, for penicillin regulator. Deletion of 4 bp within the identified protein binding site caused the same contrary effects on acvA and ipnA expression as observed for all of the deletion clones which lacked nucleotides ؊353 to ؊432. The PENR1 binding site thus represents a major cis-acting DNA element. The intergenic regions of the corresponding genes of the ␤-lactam-producing fungi Penicillium chrysogenum and Acremonium chrysogenum also diluted the complex formed between the A. nidulans probe and PENR1 in vitro, suggesting that these ␤-lactam biosynthesis genes are regulated by analogous DNA elements and proteins.

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...

In Aspergillus nidulans, a DNA-binding complex, PENR1, was shown to bind to two CCAAT-boxcontaining DNA elements located in the promoter regions of the bidirectionally oriented penicillin biosynthesis genes acvA and ipnA, and of the aat promoter. Here, partial purification of PENR1 and western blotting using anti-HAPC sera indicated that the previously identified HAPC protein, which was suggested to be part of the CCAAT-binding complex AnCF, is also part of PENR1. This was confirmed by band shift assays using protein extracts of a ∆hapC strain which exhibited no PENR1 DNA-binding activity. Supershift assays and immunoprecipitation analysis using anti-HAPC sera provided evidence that HAPC is part of the PENR1 complex. In ∆hapC strains, penicillin production was reduced, as was expression of both an ipnA-lacZ and aat-lacZ gene fusion. Hence, HAPC-containing PENR1 appears to act as an activator on ipnA and aat expression. However, deletion of hapC had little effect on acvA expression during a fermentation run in fermentation medium. Previous results which had shown that specific deletion of the PENR1-binding site between acvA and ipnA resulted in a strong increase of expression of an acvA-uidA gene fusion, together with the present data, suggest the possibility of the existence of a repressor protein that binds close to or overlaps the PENR1-binding site. It is also shown that binding of PENR1 induced bending of a DNA fragment spanning the PENR1-binding site between acvA and ipnA in vitro.