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

Abstract

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.

The acetyl xylan esterase II gene from Penicillium purpurogenum is differentially expressed in several carbon sources, and tightly regulated by pH

“…For example, A. nidulans produces two endoxylanases, XlnA and XlnB, which are expressed at alkaline or acidic pH, respectively (14). The same organism has a β-xylosidase, the induction of which seems to be insensitive to pH changes (16), and similar results have been described for Penicillium chrysogenum, whose endoxylanase gene (xylP) is expressed independent of the environmental pH (23).…”

Mevalonate governs interdependency of ergosterol and siderophore biosyntheses in the fungal pathogen Aspergillus fumigatus

“…Overexpression of hmg1 by placing the gene under the control of the xylose-inducible xylP promoter (SI Appendix, Figs. S1B and S2C) (17,18) resulted in more than twofold increased TAFC production compared with WT in the absence of lovastatin (Table 1). Consistent with Hmg1 being the target of lovastatin toxicity, hmg1 overexpression increased lovastatin resistance and TAFC Cultures were grown for 48 h in minimal medium containing xylose as carbon source and lovastatin.…”

“…For high-iron conditions, FeSO 4 was added to a final concentration of 1.5 mM FeSO 4 ; addition of iron was omitted for iron-depleted medium. Xylose instead of glucose was used as the carbon source for induction of expression of hmg1 under the control of the xylP promoter (17,18). For radial growth assays, 50 conidia of respective strains were spotted onto described solid minimal medium.…”

Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum

“…We first constructed the inducible DsRedsilencing vector pREDi_xyl carrying the P. chrysogenum xylP promoter, which can be induced by xylan or xylose and repressed by glucose (Zadra et al, 2000). After transformation of vector pREDi_xyl into recipient strain P2red, we obtained 41 transformants, which were subsequently transferred onto solid MM medium supplemented with either xylose (inducing conditions) or glucose (repressing conditions) as the main carbon source.…”

“…This system is based on the previously described vector pREDi that carries the DsRed gene as a reporter for direct monitoring of gene silencing on solid media (Janus et al, 2007). The vector was modified by using the xylP promoter from P. chrysogenum, which is induced when xylose is the sole carbon source in the medium (Zadra et al, 2000). The applicability of this inducible RNAi system was further tested with the endogenous PcbrlA morphogene controlling conidiophore development, chosen as the specific phenotypic reporter.…”