Molecular and classical genetic analysis of penicillin biosynthesis in 'Aspergillus nidulans'

Abstract

Clones of A. nidulans genomic DNA (pSTA200, pSTA201 and pSTA207) spanning 20 kb have been isolated and demonstrated by a combination of classical and molecular genetic means to represent the npeA locus, involved in the synthesis of penicillin, located on linkage group VI of A. nidulans. As well as containing, the gene encoding the second enzyme for penicillin biosynthesis, namely isopenicillin N synthetase (IPNS) (designated ipnA), results presented here show that these clones contain two additional genes to form a cluster of three contiguous penicillin biosynthetic genes. Our evidence suggests that these genes encode (L-a-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and acyltransferase (ACYT) (designated acvA and acyA respectively), the first and third enzymes reguired for penicillin biosynthesis, with the genetic order being acvA-ipnA-acyA. Transcripts have been identified for the three genes and their approximate sizes determined: acvA >9.5 kb, ipnA 1.4 kb and acyA 1.6 kb. Results indicate that the acvA and ipnA genes are divergently transcribed, whilst acyA is transcribed in the same orientation as ipnA. Northern blot and hybridisation analyses of the regulation of expression of the structural genes for penicillin biosynthesis indicated that all three genes are subject to some form of regulation, the mechanism of which is not clear from our studies. Hybridisation data appeared to suggest that if any regulatory genes exist for penicillin biosynthesis, they are not located immediately 5' of acvA or 3' of acyA at the A. nidulans npeA locus.