The 5?-sequence of the isopenicillin N-synthetase gene (pcbC) from Cephalosporium acremonium directs the expression of the prokaryotic hygromycin B phosphotransferase gene (hph) in Aspergillus niger

Kück, Ulrich; Walz, Markus; Mohr, Georg; Mracek, M

doi:10.1007/bf00257605

Cited by 33 publications

(10 citation statements)

References 29 publications

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“…After 7 (Ac) or 2 (Sm) days, transformants were transferred on solid rich medium with the above mentioned nourseothricin concentrations. We obtained with vector pD-NAT1 frequencies of about 20 to 40 transformants per 10 µg of DNA, which is comparable to the rather low frequencies that are obtained with the hph gene (Kück et al 1989, Walz andKück 1995). Most importantly, the transformants did not show cross-resistance neither to hygromycin B nor to phleomycin.…”

Section: Application Of the Nourseothricin Acetyltransferase Gene (Nasupporting

confidence: 57%

“…In this vector, the nat1 gene is flanked on both sites by multiple cloning sites that can be used for directed insertion of fungal genomic sequences to construct gene disruption strains by homologous recombination. Using previously reported transformation procedures for A. chrysogenum and S. macrospora, the transformants were kept without selection for 24 hours (Kück et al 1989, Walz andKück 1995). The germinated protoplasts were subsequently overlaid with top agar containing nourseothricin concentrations of 25 µg/ml (Ac) and 50 µg/ml (Sm).…”

Section: Application Of the Nourseothricin Acetyltransferase Gene (Namentioning

confidence: 99%

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Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi

Kück¹,

Hoff²,

Allgemeine³

2006

Fungal Genetics Reports

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Section: Application Of the Nourseothricin Acetyltransferase Gene (Nasupporting

confidence: 57%

Section: Application Of the Nourseothricin Acetyltransferase Gene (Namentioning

confidence: 99%

Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi

Kück¹,

Hoff²,

Allgemeine³

2006

Fungal Genetics Reports

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“…Construction, maintenance, and isolation of recombinant plasmids were performed using standard techniques (37). The cephalosporin C producer strain A3/2 (36) from A. chrysogenum was used as a recipient, and all transformants (Table 1) were obtained by a procedure described previously (24,47). The transgenic strains were selected on media containing either 10 U/ml of hygromycin B or 25 g/ml nourseothricin.…”

Section: Methodsmentioning

confidence: 99%

A Homologue of the Aspergillus velvet Gene Regulates both Cephalosporin C Biosynthesis and Hyphal Fragmentation in Acremonium chrysogenum

Dreyer

Eichhorn²,

Friedlin³

et al. 2007

Appl Environ Microbiol

127

102

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The Aspergillus nidulans velvet (veA) gene encodes a global regulator of gene expression controlling sexual development as well as secondary metabolism. We have identified the veA homologue AcveA from Acremonium chrysogenum, the major producer of the ␤-lactam antibiotic cephalosporin C. Two different disruption strains as well as the corresponding complements were generated as a prelude to detailed functional analysis. Northern hybridization and quantitative real-time PCR clearly indicate that the nucleus-localized AcVEA polypeptide controls the transcriptional expression of six cephalosporin C biosynthesis genes. The most drastic reduction in expression is seen for cefEF, encoding the deacetoxycephalosporine/deacetylcephalosporine synthetase. After 120 h of growth, the cefEF transcript level is below 15% in both disruption strains compared to the wild type. These transcriptional expression data are consistent with results from a comparative and time-dependent high-performance liquid chromatography analysis of cephalosporin C production. Compared to the recipient, both disruption strains have a cephalosporin C titer that is reduced by 80%. In addition to its role in cephalosporin C biosynthesis, AcveA is involved in the developmentally dependent hyphal fragmentation. In both disruption strains, hyphal fragmentation is already observed after 48 h of growth, whereas in the recipient strain, arthrospores are not even detected before 96 h of growth. Finally, the two mutant strains show hyperbranching of hyphal tips on osmotically nonstabilized media. Our findings will be significant for biotechnical processes that require a defined stage of cellular differentiation for optimal production of secondary metabolites.

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“…Cotransformation experiments were performed with A. chrysogenum producer strain A3/2 (27) by using vector pMW1 (hygromycin B resistance [20]) and derivatives of reporter gene plasmid pSIM9.9 (22) or plasmid pKSC1 (32) according to the methods of Walz and Kück (41). For the reintegration of the cpcR1 gene into the knockout strain T572⌬, a cotransformation experiment using plasmid pUT737 (phleomycin resistance [15]) together with pKSC1 was performed.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid pKSC1 (32), carrying 6.5 kb of genomic DNA containing the entire cpcR1 gene, was hydrolyzed with NcoI and EcoRI to remove a DNA fragment of about 2.5 kb. The hph cassette from plasmid pMW1 (20) was excised with PvuII and integrated into the cpcR1 gene at an AatII restriction site. The AatII site is located 1,112 bp downstream of the start codon.…”

Section: Methodsmentioning

confidence: 99%

Winged Helix Transcription Factor CPCR1 Is Involved in Regulation of β-Lactam Biosynthesis in the Fungus Acremonium chrysogenum

et al. 2004

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Winged helix transcription factors, including members of the forkhead and the RFX subclasses, are characteristic for the eukaryotic domains in animals and fungi but seem to be missing in plants. In this study, in vitro and in vivo approaches were used to determine the functional role of the RFX transcription factor CPCR1 from the filamentous fungus Acremonium chrysogenum in cephalosporin C biosynthesis. Gel retardation analyses were applied to identify new binding sites of the transcription factor in an intergenic promoter region of cephalosporin C biosynthesis genes. Here, we illustrate that CPCR1 recognizes and binds at least two sequences in the intergenic region between the pcbAB and pcbC genes. The in vivo relevance of the two sequences for gene activation was demonstrated by using pcbC promoter-lacZ fusions in A. chrysogenum. The deletion of both CPCR1 binding sites resulted in an extensive reduction of reporter gene activity in transgenic strains (to 12% of the activity level of the control). Furthermore, Acremonium transformants with multiple copies of the cpcR1 gene and knockout strains support the idea of CPCR1 being a regulator of cephalosporin C biosynthesis gene expression. Significant differences in pcbC gene transcript levels were obtained with the knockout transformants. More-than-twofold increases in the pcbC transcript level at 24 and 36 h of cultivation were followed by a reduction to approximately 80% from 48 to 96 h in the knockout strain. The overall levels of the production of cephalosporin C were identical in transformed and nontransformed strains; however, the knockout strains showed a striking reduction in the level of the biosynthesis of intermediate penicillin N to less than 20% of that of the recipient strain. We were able to show that the complementation of the cpcR1 gene in the knockout strains reverses pcbC transcript and penicillin N amounts to levels comparable to those in the control. These results clearly indicate the involvement of CPCR1 in the regulation of cephalosporin C biosynthesis. However, the complexity of the data points to a well-controlled or even functional redundant network of transcription factors, with CPCR1 being only one player within this process.

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The 5?-sequence of the isopenicillin N-synthetase gene (pcbC) from Cephalosporium acremonium directs the expression of the prokaryotic hygromycin B phosphotransferase gene (hph) in Aspergillus niger

Cited by 33 publications

References 29 publications

Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi

Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi

A Homologue of the Aspergillus velvet Gene Regulates both Cephalosporin C Biosynthesis and Hyphal Fragmentation in Acremonium chrysogenum

Winged Helix Transcription Factor CPCR1 Is Involved in Regulation of β-Lactam Biosynthesis in the Fungus Acremonium chrysogenum

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