The Genetic Location of Three Mutations Impairing Penicillin Production in Aspergillus nidulans

Makins, J. F.; Holt, G.; Macdonald, K. D.

doi:10.1099/00221287-129-10-3027

Cited by 14 publications

(21 citation statements)

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“…Also, mutant A. nidulans strains with altered ,-lactam-biosynthetic ability have been described (10) and characterized (20). The IPNS gene from a grampositive Streptomyces species is also of interest.…”

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Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans

et al. 1988

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,-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the ifiamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.The penicillin, cephalosporin, and cephamycin classes of P-lactam antibiotics share two common steps in their respective biosynthetic pathways: the joining of three amino acids to form 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) and the conversion of LLD-ACV to isopenicillin N. These two reactions are carried out in filamentous fungi and streptomycetes, including Penicillium chrysogenum and Aspergillus nidulans (producers of penicillin), Cephalosporium acremonium (producer of cephalosporin C), and Streptomyces lipmanii and Streptomyces clavuligerus (producers of cephamycins) (16). We have studied the biochemical and genetic basis of the conversion of LLD-ACV to isopenicillin N by cloning and characterizing the gene encoding the isopenicillin N synthetase (IPNS) enzyme from C. acremonium and P. chrysogenum (6, 23). The IPNS genes from these two fungi show a high degree of nucleotide and amino acid sequence identity. The 77% identity of the predicted amino acid sequences of the IPNSs from these fungi is so extensive that it is difficult to identify regions of potential functional importance. We therefore decided to extend the comparison to IPNS genes from other organisms which might be more distant evolutionarily.The IPNS gene from A. nidulans was of particular interest because of the well-developed genetic techniques available for this organism. Also, mutant A. nidulans strains with altered ,-lactam-biosynthetic ability have been described (10) and characterized (20

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confidence: 99%

“…Also, mutant A. nidulans strains with altered ,-lactam-biosynthetic ability have been described (10) and characterized (20 …”

mentioning

confidence: 99%

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans

et al. 1988

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“…npeE1 prevents penicillin biosynthesis and transcription of ipnA. Quantitative ␤-Gal assays with mycelial extracts from strains BPE-1 and BPE-2 corresponding to three different times of growth (12,24, and 36 h after inoculation) confirmed that the npeE1 mutation nearly abolished expression of the ipnAЈ::ЈlacZ fusions (Fig. 4A).…”

Section: Resultsmentioning

confidence: 73%

“…involving a number of auxotrophies. An example of the difficulty in genetically analyzing regulatory mutations in this pathway is provided by the penicillin-nonproducer mutations npeC007 and npeD0045 (11), which were incorrectly assigned to chromosomes IV and II, respectively, by haploidization and meiotic linkage analysis (24). However, using more sensitive scoring techniques available for pal mutations, Shah et al (28) showed them to be palA mutant (palA is located in chromosome III) and palF mutant (palF is in chromosome VII) alleles, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…npeE1 defines a positive-acting regulatory gene of ipnA transcription essential for penicillin production. Mutations impairing penicillin biosynthesis have been already described for A. nidulans (14) and belong to four different complementation groups, denoted npeA, npeB, npeC, and npeD (11), none of which is located in chromosome IV (24), to which the npeE1 mutation has been assigned by haploidization analysis. Therefore, npeE1 identifies a new npe gene.…”

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confidence: 99%

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A lacZ reporter fusion method for the genetic analysis of regulatory mutations in pathways of fungal secondary metabolism and its application to the Aspergillus nidulans penicillin pathway

1995

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Secondary metabolism, usually superfluous under laboratory conditions, is intrinsically elusive to genetic analysis of its regulation. We describe here a method of analyzing regulatory mutations affecting expression of secondary metabolic genes, with an Aspergillus nidulans penicillin structural gene (ipnA [encoding isopenicillin N-synthase]) as a model. The method is based on a targeted double integration of a lacZ fusion reporter gene in a chromosome different from that containing the penicillin gene cluster. The trans-acting regulatory mutations simultaneously affect lacZ expression and penicillin biosynthesis. One of these mutations (npeE1) has been analyzed in detail. This mutation is recessive, prevents penicillin production and ipnA::lacZ expression, and results in very low levels of the ipnA message at certain times of growth. This indicates that npeE positively controls ipnA transcription. We also show that this tandem reporter fusion allows genetic analysis of npeE1 by using the sexual and parasexual cycles and that lacZ expression is an easily scorable phenotype. Haploidization analysis established that npeE is located in chromosome IV, but npeE1 does not show meiotic linkage to a number of known chromosome IV markers. This method might be of general applicability to genetic analysis of regulation of other fungal secondary metabolic pathways.Secondary metabolism in microbes is often elusive to genetic analysis, because most, if not all, pathways classified in this category are dispensable under laboratory conditions. The penicillin biosynthetic pathway (22) is a prototype of such pathways in filamentous ascomycetes and has been extensively used as a model for at least four reasons. (i) It is a rather simple pathway, and only three enzymes are required to convert primary metabolites (three amino acids) into penicillin; (ii) the corresponding structural genes, which are clustered, have been cloned and characterized from several species; (iii) the end product can be sensitively detected with a bioassay; and (iv) it is of obvious biotechnological interest. As a consequence, a wealth of information about this pathway has been accumulated. In contrast, regulation of penicillin biosynthesis is largely unelucidated, possibly because the absence of a sexual cycle in Penicillium chrysogenum has hindered formal genetic analysis of the putative regulatory mechanisms. This problem has been circumvented by using Aspergillus nidulans (26), a closely related plectomycete amenable to formal genetic studies (9) and for which sophisticated molecular biology techniques are available (31).By using molecular techniques to analyze transcription of the A. nidulans ipnA gene (encoding isopenicillin N-synthase, a key enzyme catalyzing the central step in the pathway), we have described two modes of transcriptional regulation of a penicillin structural gene. Carbon regulation in response to the availability of a preferred carbon source modulates ipnA expression through the action of a yet undefined negative-acting regulatory ge...

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The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis.

et al. 1990

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Clones of Aspergillus nidulans genomic DNA spanning 20 kb have been isolated and shown by a combination of classical and molecular genetic means to represent the npeA locus, previously found to be one of four loci (npeA, npeB, npeC and npeD) involved in the synthesis of penicillin. As well as containing the gene encoding the second enzyme for penicillin biosynthesis, namely isopenicillin N synthetase (IPNS) (designated ipnA), our results show that these clones (pSTA200, pSTA201 and pSTA207) contain two more genes to form a cluster of three contiguous penicillin biosynthetic genes. Our evidence suggests that these genes encode delta (L‐alpha‐aminoadipyl)‐L‐cysteinyl‐D‐valine synthetase (ACVS) and acyl transferase (ACYT) (designated acvA and acyA respectively), the first and third enzymes required for penicillin biosynthesis, with the gene 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. All three mRNA species are observed in cells grown in fermentation medium but not in cells grown in minimal medium, suggesting that the control of penicillin biosynthesis is, in part, at the level of mRNA accumulation. Finally our results show that acvA and ipnA genes are divergently transcribed, whilst acyA is transcribed in the same orientation as ipnA.

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The Genetic Location of Three Mutations Impairing Penicillin Production in Aspergillus nidulans

Abstract: Three mutations impairing penicillin production in Aspergillus nidulans, npeB, npeC and npeD, have been located on linkage groups III, IV and II, respectively, and positioned relative to other loci on these chromosomes.

Cited by 14 publications

References 12 publications

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans

A lacZ reporter fusion method for the genetic analysis of regulatory mutations in pathways of fungal secondary metabolism and its application to the Aspergillus nidulans penicillin pathway

The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis.

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