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

• Published in: Applied and environmental microbiology Vol. 66; no. 11; pp. 4810 - 4816
• Main Authors: Zadra, Ivo, Abt, Beate, Parson, Walther, Haas, Hubertus
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.11.2000
• Subjects: agrB gene; antisense RNA; Aspergillus nidulans; Aspergillus nidulans - enzymology; Aspergillus nidulans - genetics; Base Sequence; beta-glucuronidase; Biological and medical sciences; Biotechnology; Down-Regulation; Endo-1,4-beta Xylanases; enzymology; Fundamental and applied biological sciences. Psychology; Fungal Proteins; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fungi; gene expression; Gene Expression Regulation, Fungal; Genes; Genes, Reporter; Genetic engineering; Genetic technics; genetics; glucose; Glucuronidase; Glucuronidase - genetics; Glucuronidase - metabolism; metabolism; Methods. Procedures. Technologies; Microbiology; Miscellaneous; Modification of gene expression level; Molecular Sequence Data; Mycology; niaD gene; nreB gene; pcbC gene; Penicillium chrysogenum; Penicillium chrysogenum - enzymology; Penicillium chrysogenum - genetics; promoter regions; Promoter Regions, Genetic; Promoter Regions, Genetic - genetics; recombinant DNA; regulator genes; reporter genes; RNA, Antisense; RNA, Antisense - genetics; RNA, Antisense - metabolism; Sequence Analysis, DNA; Synthetic digonucleotides and genes. Sequencing; transcription factors; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic; uidA gene; xylan; xylose; Xylosidases; Xylosidases - genetics; Xylosidases - metabolism; xylP gene; Nucleotide sequence; Enzyme; Induction; Transcription promoter; Xylan; Antisense RNA; Endo-1,4-β-xylanase; Regulator; Nitrogen; Gene expression; Metabolism; Penicillium chrysogenum; Fungi; Xylose; Regulation(control); Aspergillus nidulans; Glycosidases; Hydrolases; Fungi Imperfecti; O-Glycosidases; Thallophyta
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.66.11.4810-4816.2000

A highly inducible fungal promoter derived from the Penicillium chrysogenum endoxylanase ( xylP ) gene is described. Northern analysis and the use of a β-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.