A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi

Punt, Peter J.; Greaves, P.; Kuyvenhoven, A.; Deutekom, J.C.T. van; Kinghorn, James R.; Pouwels, Peter H.; Hondel, Cees A. M. J. J. van den

doi:10.1016/0378-1119(91)90476-r

Cited by 34 publications

(29 citation statements)

References 17 publications

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“…pTRAN3-1A carries the uidA reporter gene under control of the A. nidulans niaD promoter and normally drives expression of nitrate reductase in a strictly nitrate-induced and nitrogen metabolite-repressed manner (8,43). Expression analysis indicated that the Penicillium xylP promoter yields about 23 times more GUS activity than the niaD promoter under fully induced conditions (with nitrate as the sole nitrogen source).…”

Section: A Nidulansmentioning

confidence: 99%

xylP Promoter-Based Expression System and Its Use for Antisense Downregulation of the Penicillium chrysogenum Nitrogen Regulator NRE

Zadra¹,

Abt²,

Parson

et al. 2000

Appl Environ Microbiol

117

124

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A highly inducible fungal promoter derived from the Penicillium chrysogenum endoxylanase (xylP) gene is described. Northern analysis and the use of a ␤-glucuronidase (uidA) reporter gene strategy showed that xylP expression is transcriptionally regulated. Xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Comparison of the same expression construct as a single copy at the niaD locus in P. chrysogenum and at the argB locus in Aspergillus nidulans demonstrated that the xylP promoter is regulated similarly in these two species but that the level of expression is about 80 times higher in the Aspergillus species. The xylP promoter was found to be 65-fold more efficient than the isopenicillin-N-synthetase (pcbC) promoter in Penicillium and 23-fold more efficient than the nitrate reductase (niaD) promoter in Aspergillus under induced conditions. Furthermore, the xylP promoter was used for controllable antisense RNA synthesis of the nre-encoded putative major nitrogen regulator of P. chrysogenum. This approach led to inducible downregulation of the steady-state mRNA level of nre and consequently to transcriptional repression of the genes responsible for nitrate assimilation. In addition, transcription of nreB, which encodes a negative-acting nitrogen regulatory GATA factor of Penicillium, was found to be subject to regulation by NRE. Our data are the first direct evidence that nre indeed encodes an activator in the nitrogen regulatory circuit in Penicillium and indicate that cross regulation of the controlling factors occurs.

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Section: A Nidulansmentioning

confidence: 99%

xylP Promoter-Based Expression System and Its Use for Antisense Downregulation of the Penicillium chrysogenum Nitrogen Regulator NRE

Zadra¹,

Abt²,

Parson

et al. 2000

Appl Environ Microbiol

117

124

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“…(37). Glucoamylase (1,4-␤-D-glucan glucohydrolase; EC 3.2.1.3) is one of the most prominent thermostable industrial enzymes (18) and is mainly produced by Aspergillus niger fermentation.…”

mentioning

confidence: 99%

N-Glycan Modification in Aspergillus Species

Kainz

Gallmetzer

Hatzl

et al. 2008

Appl Environ Microbiol

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The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional ␣-1,2-mannosidase producing increased amounts of N-glycans of the Man 5 GlcNAc 2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan 5 GlcNac 2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man 3 GlcNAc 2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi.

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“…2). All deletion clones contained ATG initiation codons which were fused in frame with both the E. coli lacZ and uidA reporter genes located on plasmid pTRAN2 (9,43). DNA sequence analysis of the intergenic regions of all recombinant plasmids confirmed the precise positions of the deletions and the generation of translational fusions (not shown).…”

Section: Resultsmentioning

confidence: 93%

“…In this study, we used a reporter gene system developed by Punt et al (43) which allowed measurement of the effects of deletions in the region between acvA and ipnA on the expression of both genes simultaneously. A major cis-acting DNA element which is involved in the regulation of both genes was characterized in detail by both genetic and biochemical methods.…”

mentioning

confidence: 99%

Identification of a major cis-acting DNA element controlling the bidirectionally transcribed penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) of Aspergillus nidulans

Bergh¹,

Litzka²,

Brakhage³

1996

J Bacteriol

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The ␤-lactam antibiotic penicillin is produced as a secondary metabolite by some filamentous fungi. In this study, the molecular regulation of the Aspergillus (Emericella) nidulans penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) was analyzed. acvA and ipnA are divergently oriented and separated by an intergenic region of 872 bp. Translational fusions of acvA and ipnA with the two Escherichia coli reporter genes lacZ and uidA enabled us to measure the regulation of both genes simultaneously. A moving-window analysis of the 872-bp intergenic region indicated that the divergently oriented promoters are, at least in part, overlapping and share common regulatory elements. Removal of nucleotides ؊353 to ؊432 upstream of the acvA gene led to a 10-fold increase of acvA-uidA expression and simultaneously to a reduction of ipnA-lacZ expression to about 30%. Band shift assays and methyl interference analysis using partially purified protein extracts revealed that a CCAAT-containing DNA element within this region was specifically bound by a protein (complex), which we designated PENR1, for penicillin regulator. Deletion of 4 bp within the identified protein binding site caused the same contrary effects on acvA and ipnA expression as observed for all of the deletion clones which lacked nucleotides ؊353 to ؊432. The PENR1 binding site thus represents a major cis-acting DNA element. The intergenic regions of the corresponding genes of the ␤-lactam-producing fungi Penicillium chrysogenum and Acremonium chrysogenum also diluted the complex formed between the A. nidulans probe and PENR1 in vitro, suggesting that these ␤-lactam biosynthesis genes are regulated by analogous DNA elements and proteins.

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A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi

Cited by 34 publications

References 17 publications

xylP Promoter-Based Expression System and Its Use for Antisense Downregulation of the Penicillium chrysogenum Nitrogen Regulator NRE

xylP Promoter-Based Expression System and Its Use for Antisense Downregulation of the Penicillium chrysogenum Nitrogen Regulator NRE

N-Glycan Modification in Aspergillus Species

Identification of a major cis-acting DNA element controlling the bidirectionally transcribed penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) of Aspergillus nidulans

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