Transcriptional control of expression of fungal beta-lactam biosynthesis genes.

The most commonly used beta-lactam antibiotics for the therapy of infectious diseases are penicillin and cephalosporin. Penicillin is produced as end product by some fungi most notably by Aspergillus (Emericella) nidulans and Penicillium chrysogenum. Cephalosporins are synthesised by several bacteria and fungi, e.g. by the fungus Acremonium chrysogenum (syn. Cephalosporium acremonium). The biosynthetic pathways leading to both secondary metabolites start from the same three amino acid precursors and have the first two enzymatic reactions in common. The penicillin biosynthesis is catalysed by three enzymes encoded by acvA (pcbAB), ipnA (pcbC) and aatA (penDE). The genes are organised into a cluster. In A. chrysogenum, in addition to acvA and ipnA, which are also clustered, a second cluster contains the genes for enzymes catalysing the reactions of the later steps of the cephalosporin pathway (cefEF, cefG). Transcription of biosynthesis genes is subject to sophisticated control by nutritional factors (e.g. glucose, nitrogen), amino acids such as lysine and methionine, and ambient pH. Some regulators have been identified such as the A. nidulans pH regulatory protein PACC and the transcriptional complex PENR1. PENR1 is a HAP-like transcriptional complex similar or identical to AnCF. Additional positive regulatory factors seem to be represented by recessive trans-acting mutations of A. nidulans (prgA1, prgB1, npeE1) and P. chrysogenum (carried by mutants Npe2 and Npe3). The GATA-binding factor NRE appears to be involved in the regulation of the penicillin biosynthesis genes by the nitrogen source in P. chrysogenum. Formal genetic evidence suggests the existence of transcriptional repressors as well.

Regulation of the Aspergillus nidulans penicillin biosynthesis gene acvA (pcbAB) by amino acids: implication for involvement of transcription factor PACC.

The beta-lactam antibiotic penicillin is produced as an end product by some filamentous fungi only. It is synthesized from the amino acid precursors L-alpha-aminoadipic acid, L-cysteine, and L-valine. Previous data suggested that certain amino acids play a role in the regulation of its biosynthesis. Therefore, in this study the effects of externally added amino acids on both Aspergillus (Emericella) nidulans penicillin production and expression of the bidirectionally oriented biosynthesis genes acvA (pcbAB) and ipnA (pcbC) were comprehensively investigated. Different effects caused by amino acids on the expression of penicillin biosynthesis genes and penicillin production were observed. Amino acids with a major negative effect on the expression of acvA-uidA and ipnA-lacZ gene fusions, i.e., histidine, valine, lysine, and methionine, led to a decreased ambient pH during cultivation of the fungus. An analysis of deletion clones lacking binding sites for the pH-dependent transcriptional factor PACC in the intergenic regions between acvA-uidA and ipnA-lacZ gene fusions and in a pacC5 mutant (PacC5-5) suggested that the negative effects of histidine and valine on acvA-uidA expression were due to reduced activation by PACC under acidic conditions. These data also implied that PACC regulates the expression of acvA, predominantly through PACC binding site ipnA3. The repressing effect caused by lysine and methionine on acvA expression, however, was even enhanced in one of the deletion clones and the pacC5 mutant strain, suggesting that regulators other than PACC are also involved.

The Aspergillus nidulans penicillin-biosynthesis gene aat (penDE) is controlled by a CCAAT-containing DNA element.

Analysis of the promoter of the penicillin biosynthesis aat (penDE) gene of Aspergillus nidulans 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 aat. Substitution of the CCAAT core sequence by GATCC led to a fourfold reduction of expression of an aat-lacZ gene fusion. The identified binding site thus was functional in vivo and positively influenced at 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 PENR1, binding to the CCAAT element in the bidirectional intergenic promoter region between acvA (pcbAb) and ipnA (pcbC). Hence, PENR1 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 PENR1, 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 beta-lactam-biosynthesis genes.