Cloning, characterization of the acyl-CoA : 6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene

Abstract

The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5' region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39,240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, npe6 and npe8. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenum. Both genes show the same orientation and are separated by an intergenic region of 822 nucleotides.

Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli

“…Martin and his colleagues (17), by complementing AT mutants of P. chrysogenum, have cloned and sequenced the penDE from A. nidulans.…”

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

“…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 Holt, 1976;Timberlake and Marshall, 1988;Davis and Hynes, 1989; Clutterbuck, 1993).The A. nidulans nut @enDE) gene lies downstream of the ipnA gene, separated by an intergenic region of 825 bp (Ramon et al, 1987;Montenegro et al, 1990;Tobin et al, 1990; Fig. 1).…”

“…The A. nidulans nut @enDE) gene lies downstream of the ipnA gene, separated by an intergenic region of 825 bp (Ramon et al, 1987;Montenegro et al, 1990;Tobin et al, 1990;Fig. 1).…”

“…Oligonucleotides were supplied by MWG Biotech and are listed in Table 1. They were designed from published sequences (Kalnins et al, 1983;Ramon et al, 1987;Barredo et al, 1989;Montenegro et al, 1990;Tobin et al, 1990). Media and growth conditions.…”

Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum

“…The last step involves the exchange of the aL-aminoadipic acid side chain of isopenicillin N (IPN) by phenylacetic acid (activated as phenylacetylcoenzyme A [CoA] or phenylacetylglutathione) (2); this reaction is catalyzed by the enzyme acyl CoA:isopenicillin Nacyltransferase (IAT), encoded by the penDE gene, an enzyme that shows four other related activities (2). The IAT is a heterodimer (3,28,32) formed of two subunits of 29 and 11 kDa which are encoded by a single gene, penDE, both in Penicillium chrysogenum (6,29) and inAspergillus nidulans (21,29). Initial studies indicated that a single transcript of 1.15 kb encoding both subunits was formed, and a 40-kDa form of the IAT was observed in addition to the 29-and 11-kDa subunits, suggesting that a 40-kDa precursor protein is posttranslationally cleaved to form the two subunits (6,32).…”