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Regulation of antibiotic formation in filamentous fungi

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As model system for metabolite production penicillin formation in filamentous fungi is investigated. The regulatory network controlling the biosynthesis of the key precursor ACV is studied with respect to genetic regulation, posttranslational events, enzyme localization, precursor supply and transport phenomena.
In the analysis of regulatory mechanisms that control the expression of penicillin biosynthesis genes upstream activating regions have been identified. Since the expression of the pcbAB gene is believed to be limiting, functional elements in the pcbAB upstream region have been analyzed by assaying beta-galactosidase activity in extracts from reombinant strains carrying sequential delections of the pcbAB promoter fused to the Escherichia coli lacZ as a reporter gene. Transformants with single copy constructs in the pyrG locus were selected. Two upstream regions (UAS I and II) important for expression of the pcbAB gene which also appear to be involved in glucose catabolite regulation of expression were detected. Both fragments were shifted in a gel retardation assay by DNA binding proteins. One protein strongly binds to UAS I in lactose grown cells but not in glucose grown cells. The nucleotide sequence of UAS I, that is recognized by the DNA-binding protein has been identified by trimming down the DNA fragment and protection analysis; it shows a GC rich sequence.
The mutant npe11 of P. chrysogenum lacks ACVS activity, but shows normal levels of isopenicillin N synthase (IPNS) and IPN acyltransferase (IAT). Southern and Northern analysis revealed that this mutant possesses a normal organization of the penicillin gene cluster although it is unable to transcribe the pcbAB gene. No mutation was identified in the promoter region of npe11, suggesting that the lack of transcription is due to an alteration of a trans-acting regulatory protein. This mutant is an excellent host for cloning this trans-acting regulator.
In another attempt to identify trans acting genes, a transformant of A. nidulans carrying a lacZ reporter gene fused the acvA promoter has been subjected to mutagenesis and screened for increased expression of beta-glactosidase. One such mutant, designated bol7, was found by genetic analysis to carry a mutation located on chromosome VIII. This mutant locus is now being subjected to further genetic analysis in order to try to locate its position. Fermentation studies have demonstrated that penicillin production is also enhanced in this strain.
To analyse the effects of overproduction of biosynthetic genes, four distinct groups of transformants were constructed through transformatrion of the P. chrysogenum strain Wis 54-1255 with DNA-fragments containing the penicillin biosynthetic genes. Selection of transformants was based on growth on acetamide as the sole N-source and penicilin V production in batch cultures. Subsequently specific rates of penicillin production and specific activities of penicillin biosynthetic enzymes were determined. Based on, amongst others the penicillin V production and the observed enzyme activities, it was concluded that the flux through the penicillin biosynthetic pathway is controlled by the isopenicillin N synthase in this particular strain. Elimination of this bottleneck resulted in an increase in penicillin production. A further increase in penicillin production could be obtained only through overexpression of the whole penicillin biosynthetic gene cluster. These findings indicate that the flux control is distributed over the penicillin biosynthetic pathway. For overexpression of ACV synthetase, a strain was constructed in which the promoter region of ACVS was deleted. This strain, based on the Wis 54-1255 strain, is currently used to test several strong promoters which are targeted in front of the pcbAB gene.
To investigate the possible role of pantothenate as a precursor of both 4'-phosphopantetheine as an essential cofactor in ACV synthetase and coenzyme A, required for acyltransferase activity, the final step, its biosynthesis is studied. The isolation of the pantoA and pantoB genes has been achieved by transformation and complementation of the corresponding mutants, using physical and genetic maps to locate cosmids containing these genes. The coding region of pantoB contains an open reading frame of 350 amino acids with a predicted Mr of 37.7 kDa, interrupted by a 60 bp intron. The pantoA gene was searched from a total of 586 cosmids covering most of chromosome 8 by restoration of a pantoA10 mutant phenotype to wild-type in absence of pantothenate supplementation. Further fragmentation of the identified cosmid lead to a 1.6 kb still complementing fragment, which is currently being sequenced.
The pantoD gene encoding panthotenate kinase is being searched by complementation of two bacterial temperature sensitive mutants. To accomplish this, a lambda-cDNA library has been prepared from mycelium grown in the absence of patothenate, transformed by electroporation, and analysed for lacZ-driven restoration at 42(. Isolated clones are currently analysed.
To identify CoA biosynthetic genes, a collection of 1100 A. nidulans temperature-sensitive mutants has been analysed, to identify two isolates with increased growth in the presence of CoA.
The postranslational modification of ACV synthetase is thought to be accomplished by a CoA-4'-phosphopantetheine-transferase or holo-ACVS synthase. This enzyme has been purified from extracts of A. nidulans employing apo-tyrocidine synthetase 1 and an apo-acyl carrier protein (nodF) as substrates. In the absence of CoA it binds tightly to these immobilized substrates, and can be released only by 6 M urea; however, renaturation attempts failed. The synthase is a protein of about 36 kDa, which is apparently N-terminally blocked. The activity has been shown to be abundant in other fungi like P. chrysogenum and Fusarium scirpi, also producing peptide antibiotics. The transfer reaction is completely inhibited by 3'5'ADP, the reaction product of 4'-phosphopantetheine transfer from CoA. The holo-acyl carrier protein synthase from E. coli, which has been identified in the meantime by Lambalot and Walsh proved to be inactive in modifying peptide synthetases.
In the investigation of substrate availability both lysine and cysteine biosynthesis are studied. In the lysine biosynthetic pathway the lysF mutant was found to accumulate homocitrate, and has been shown to encode homoaconitase activity studies and complementation. the gene has a size of 2397 bp with a single intron of 72 bp. The enzyme with a size of 775 amino acids (Mr 83,943) shows sequences characteristic for iron-sulfur clusters. The gene seems to be indirectly regulated by lysine.
The sD gene of A. nidulans, encoding APS kinase, a step in sulphate assimilation, has now been completely sequenced and its transcription start site determined. It carries a single intron, and is able to complement an sD mutant, confirming its identity. Northern hybridisation indicates that, though weak, the level of transcription is the same on methionine and sulphate containing media, indicating the absence of transcriptional regulation by sulphur source. sD gene comparisons reveal homology to the C-terminal region of the ATP sulphurylase (sC) gene, the preceding enzyme in the pathway. However, the ATP consensus binding site is altered in the sC gene, with aspartate replacing serine. This supports previous evidence for a nonfunctional APS kinase-like region, and a mode of regulation by allosteric inhibition of ATP sulphurylase by binding of phosphoadenosyl phosphosulphate (PAPS).
In order to study the involvement of the vacuolar membrane in penicillin biosynthesis by Penicillium chrysogenum - e.g. by regulating the activity of ACV synthetase which is tentatively attached to it, and/or by enclosing a compartment where penicillin precursors may be stored - a procedure for the isolation and purification of intact vacuoles and vacuolar vesicles from low and high penicillin producing Penicillium chrysogenum strains has been attempted. The first procedures yielded only functionally inactive vacuolar membranes, and the procedure had to be modified by replacing protoplastation by rapid breakage of mycelium with glass beads, the use of Iodixanol and/or Nycodenz instead of Percoll as density centrifugation medium, and the inclusion of various protective additives. The majority of ACVS was found in the soluble fraction, while only about 20% was retained in the membrane fraction comparable to the vacuolar membrane markers V-ATPase b-subunit and a-mannosidase, but also the cytosolic marker G6PDH. However, only ACVS remained tightly associated after treatment with Triton X-100 or 100 mM Na(2)CO(3) as could be shown by immunodetection.

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Technische Universität Berlin
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Letzte Aktualisierung: 31 Dezember 1997

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Permalink: https://cordis.europa.eu/project/id/BIO2942100/de

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