Mutants of Aspergillus nidulans Impaired in Penicillin Biosynthesis

Edwards, Giles; Holt, G.; Macdonald, K. D.

doi:10.1099/00221287-84-2-420

Cited by 32 publications

(16 citation statements)

References 8 publications

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

et al. 1988

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“…3A) and had wild-type morphology and conidiation. The mutation carried by this strain (described below) was designated npeE1 (npe, impaired in penicillin biosynthesis [14]; see Discussion). npeE1 is a temperature-sensitive mutation, the mutant phenotype being evident at 37ЊC but not at 25ЊC.…”

Section: Resultsmentioning

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.…”

Section: Discussionmentioning

confidence: 99%

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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“…In studies with Aspergillus nidulans where four complementation groups were identified (Edwards et al, 1974), the existence of a large group containing the majority of mutants was reported. This locus, designated npeA, has also been found to be affected in wild-type strains of Asp.…”

Section: Discussionmentioning

confidence: 99%

“…Complementation analysis with heterozygous diploids established the existence of two genetic loci. More recently, investigations of the genetics of penicillin production have been carried out in Aspergillus nidulans , and using mutants of this mould Edwards et al (1974) showed the presence of four genetic loci affecting penicillin synthesis.…”

Section: Introductionmentioning

confidence: 99%

Genetic and Biochemical Studies of Mutants of Penicillium chrysogenum Impaired in Penicillin Production

Normansell¹,

Normansell²,

Holt³

1979

Journal of General Microbiology

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Seventy-eight mutants of Penicillium chrysogenum strain NRRL 195 1, that were impaired in penicillin production, were isolated following treatment with various mutagens. Twelve that yielded about 10% of their parental penicillin titre were studied in detail. Analyses of heterozygous diploids formed between them revealed the existence of at least five complementation groups with respect to penicillin production -V, W, X, Y and Z. Most mutants belonged to group Y. A biochemical investigation of the intracellular peptides in strains representing the five groups demonstrated the absence of the tripeptide a-aminoadipoylcysteinyl-valine from mutants of groups X, Y and Z. Extracts of mutants of groups W, Y and Z were able to catalyse a penicillin acyl-exchange reaction, a mutant of group V showed only a trace of activity and a mutant from group X completely lacked this ability.

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Mutants of Aspergillus nidulans Impaired in Penicillin Biosynthesis

Cited by 32 publications

References 8 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

Genetic and Biochemical Studies of Mutants of Penicillium chrysogenum Impaired in Penicillin Production

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