Metabolically independent and accurately adjustable Aspergillus sp. expression system

Abstract

Filamentous fungi are well-established expression hosts often used to produce extracellular proteins of use in the food and pharmaceutical industries. The expression systems presently used in Aspergillus species rely on either strong constitutive promoters, e.g., that for glyceraldehyde-3-phosphate dehydrogenase, or inducible systems derived from metabolic pathways, e.g., glaA (glucoamylase) or alc (alcohol dehydrogenase). We describe for Aspergillus nidulans and Aspergillus niger a novel expression system that utilizes the transcriptional activation of the human estrogen receptor by estrogenic substances. The system functions independently from metabolic signals and therefore can be used with low-cost, complex media. A combination of positive and negative regulatory elements in the promoter drives the expression of a reporter gene, yielding a linear dose response to the inducer. The off status is completely tight, yet the system responds within minutes to induction and reaches a level of expression of up to 15% of total cell protein after 8 h. Both Aspergillus species are very sensitive to estrogenic substances, and low-cost inducers function in the picomolar concentration range, at which estrogenic substances also can be found in the environment. Given this high sensitivity to estrogens, Aspergillus cells carrying estrogen-responsive units could be used to detect xenoestrogens in food or in the environment.

FIG. 1.

(A) β-Galactosidase activities of the A. nidulans ERE-URA-RS reporter strain at various final concentrations of DES. The strain was grown and shifted to induction medium, and the enzyme activity of the reporter was determined after 8 h. bI, before induction. Error bars indicate standard deviations of three independent experiments. (B) β-Galactosidase activities of the A. nidulans ERE-URA-nirA (triangles) and ERE-RS-nirA (circles) reporter strains at various final concentrations of DES. The strains were induced for 8 h, and the enzyme activities were measured.

FIG. 2.

Comparison of β-galactosidase activities of A. niger (triangles) and A. nidulans (squares). Both species carry the pERE-URA-nirA reporter construct. The inducer (DES) was added to a concentration of 1 nM (0-h control), and incubation proceeded for 2, 4, 6, 8, and 24 h before the determination of reporter enzyme activities. Error bars indicate standard deviations of three independent experiments.

FIG. 3.

Northern blot analysis of the A. nidulans ERE-URA-nirA reporter strain induced with 1 nM DES. RNA was prepared at the indicated time points. The blot was hybridized once with the lacZ gene, stripped, and reprobed with the A.nidulans acnA (actin-encoding) gene as a loading control.

FIG. 4.

Saturation of intracellular reporter enzyme activity. β-Galactosidase was measured after induction with 1 nM DES for various times in ERE construct strains ERE-URA-RS (triangles), ERE-URA-nirA (circles), and ERE-RS-nirA (squares). Error bars indicate standard deviations of three independent experiments.