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

Abstract

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.

FIG. 1

(A) Penicillin biosynthesis gene cluster of A. nidulans (reviewed in references and 6). (B) acvA-uidA and ipnA-lacZ gene fusions carrying the full-length intergenic region between acvA and ipnA (strain FLIRT), which was integrated in a single copy at the chromosomal argB gene locus (39). Roman numerals indicate the chromosomes on which the genes are located.

FIG. 2

Effects of various l-amino acids (10 mM) on the expression of the acvA-uidA and ipnA-lacZ gene fusions in strain FLIRT in minimal medium (AMM). Data obtained without the addition of amino acids (0) were set at 100. The relative values given represent the means of data obtained in single experiments. SD were less than 10%. See the text for details.

FIG. 3

Penicillin titers after the addition of amino acids to the medium. Cultures of A. nidulans FLIRT in FM were supplemented with 50 mM histidine (H), valine (V), lysine (K), or methionine (M). Control cultures did not contain amino acids (0). Data represent the means of results of a single experiment. SD were less than 15%.

FIG. 4

Determination of cis-acting DNA elements involved in the effects of exogenously added l-amino acids on the expression of acvA-uidA and ipnA-lacZ. Cultures were grown in AMM with a 10 mM concentration of the indicated l-amino acid. The expression of acvA-uidA and ipnA-lacZ gene fusions was determined as β-GLU and β-GAL specific activities, respectively. Data obtained without the addition of l-amino acids for each strain were set at 100. The relative values given for the deletion clones represent the means of results of three complete experiments. SD were less than 15%. Deletions are indicated by broken lines. The four PACC binding sites bound in vitro by PACC (42) were designated 1 (ipnA1), 2 (ipnA2), 3 (ipnA3), and 4AB (ipnA4AB) (17). Sites that were found to be functional in vivo for the expression of ipnA-lacZ (17) and/or for acvA-uidA expression are marked by hatched boxes. Site 3, which seems to be of major importance for both ipnA-lacZ expression (17) and acvA-uidA expression, is marked by a filled box. WT, wild-type strain FLIRT; nd, not determined.

FIG. 5

Effects of l-amino acids on acvA-uidA and ipnA-lacZ expression and extracellular pH in both mutant strain PacC5-5 and the wild type. A. nidulans FLIRT (wild type for pacC) (WT) and PacC5-5 were cultivated for 48 h with or without (0) different l-amino acids at a concentration of 10 mM in AMM. The expression of acvA-uidA and ipnA-lacZ gene fusions was determined as β-GLU and β-GAL specific activities, respectively. The values given represent the means of data obtained with single experiments. SD are shown. The experiments were repeated three times. Their results were essentially identical. SD of values calculated from the means of these independent experiments were less than 15%.