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We have initiated heterologous expression studies of hydrophobin genes in Aspergillus niger. For these studies, we have chosen the ABH1 (hypA) gene, which encodes a class I hydrophobin expressed in the fruiting bodies of Agaricus bisporus, the white button mushroom (2, 3). Because of the characteristic self-assembly of these hydrophobins into sodium dodecyl sulfate (SDS)-insoluble amphipathic membranes, interest has arisen in possible medical and industrial applications (12), requiring an efficient method for production of hydrophobins.

An A. niger strain, AB4.1 (cspA1 pyrG1) (10), was transformed according to the method described in reference 8 with two types of constructs (Fig. 1), both containing the Escherichia coli hygromycin B phosphotransferase gene (7) allowing for selection of transformants: (i) pAN/ABH1HygB, a cDNA of ABH1, including nucleotides encoding the signal sequence for secretion (2, 3) cloned behind the gpd promoter and in front of the trpC terminator of Aspergillus nidulans (TAN1 transformants), and (ii) pKXABH1/pAN56-2, a cDNA of ABH1 encoding the mature hydrophobin fused in frame with a truncated A. niger glucoamylase gene (glaA-G2), separated by a KEX2 protease site and cloned between the gla promoter of A. niger and the trpC terminator of A. nidulans (TAN2 transformants). Sequence analysis of the ABH1 cDNA, including the cloning sites, verified the correctness of both constructs.

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Constructs pAN/ABH1HygB and pKXABH1/pAN56-2. (a) The cDNA of ABH1, including nucleotides encoding the signal sequence for secretion (2, 3) cloned into pAN52-1 NotI (9) behind the gpd promoter and in front of the trpC terminator of A. nidulans (TAN1 transformants). (b) The cDNA of ABH1 encoding the mature protein, fused in frame with a truncated A. niger glucoamylase gene (glaA-G2), separated by a KEX2 protease site and cloned into pAN56-2 (6) between the gla promoter of A. niger and the trpC terminator of A. nidulans (TAN2 transformants).

Our initial experiments analyzed the presence of ABH1 in the medium of 2-day-old shaken cultures of 10 TAN1 and 30 TAN2 transformants. The cultures were grown in minimal medium (13) at 150 rpm and 30°C with 5% maltodextrin replacing glucose for induction of the glucoamylase promoter in the case of the TAN2 transformants. ABH1 was detected by silver staining (5) and Western blot analysis (3) in 70% of both types of transformants analyzed, but at low concentrations. No less than the equivalent of 5 ml of medium per lane was necessary in an SDS-polyacrylamide gel electrophoresis (PAGE) gel to obtain a clear ABH1 band. Time course experiments showed the same low ABH1 protein level over a culture period of 5 days.

The reason for a low amount of a heterologous protein in the culture medium may be (i) low expression at RNA and/or protein level, (ii) malfunction of the secretory machinery, or (iii) instability of the protein in the culture medium. RNA analysis (exemplified for two individual TAN1 transformants [Fig. 2]) verified that the production of ABH1 was not hampered by a low expression level of the gene, locating the problem at a posttranscriptional level. To discriminate between the remaining possibilities, we used the so-called sandwiched-culture technique (Fig. 3a) previously described (13). A protein binding membrane (such as polyvinylidene difluoride [PVDF]) immobilizes proteins directly after secretion when placed under the colony. Specific antibodies allow for a detection of the newly secreted proteins.

View larger version (103K): [in this window] [in a new window]   FIG. 2.   Northern blot analysis. Lanes 1 and 2, RNA from two randomly chosen TAN1 transformants of A. niger. Lane 3, RNA from fruiting bodies of A. bisporus (strain HorstU1R). Lane 4, wild-type A. niger AB4.1. The upper panel shows hybridization signals with ABH1 cDNA probe; the lower panel shows an ethidium bromide staining of rRNA for loading control.

View larger version (49K): [in this window] [in a new window]   FIG. 3.   Sandwiched-culture technique. (a) Schematic view of a sandwiched culture. (b) Immunodetection of secreted ABH1 on PVDF membranes underlying sandwiched colonies of TAN1 transformant, TAN2 transformant, and wild type (WT), respectively, for 1 h.

A perforated polycarbonate membrane (diameter, 4.5 cm; pore size, 0.1 µm) was put onto the surface of 10 ml of perfectly level solidified minimal medium (13) containing 5% maltodextrin. The plate was heated to 70°C, and 1 ml of melted 1.25% agarose was poured over the surface. The temperature of the plate was brought to room temperature, and 5 µl of spore suspension (7 × 10⁷ spores ml¹) was added onto the center of the plate and allowed to soak into the agarose. A second perforated polycarbonate membrane was put on top of the agarose layer. In all cases, care was taken that no air bubbles were trapped. The sandwiched colonies of a wild-type strain (AB4.1) and a TAN1 and a TAN2 transformant were incubated for 3 days at 30°C, transferred onto PVDF membranes overlying fresh agar medium, and incubated for 1 h at 30°C. The immunosignals of ABH1 on the PVDF membranes (Fig. 3b) showed predominant secretion at the edge of the colonies, indicating active secretion in a growing zone of the colony (as shown previously for glucoamylase [13]) and a clear elevation of ABH1 secretion in a TAN2 transformant compared to that in a TAN1 transformant. No reaction was detected in the wild type. Therefore, we concluded that the TAN2 construct did result in a high level of ABH1 and effective secretion. Repeated experiments showed identical results when colonies were incubated in the presence of PVDF membranes for 15 min and 4, 24, and 48 h.

Proteins are tightly bound to PVDF membranes, but a small part of ABH1 (no decrease in immunosignal was detected on the PVDF membranes after extraction) could be dissociated from the membrane by an incubation with 100% trifluoroacetic acid (TFA) for 30 min at room temperature. After removal of TFA by an N₂ stream, the proteins were taken up in 1 ml of SDS sample buffer, and 25 µl was subjected to SDS-PAGE and Western blot analysis (Fig. 4, lane 1). ABH1 antibodies reacted with a protein band running at a 14-kDa position, representing monomeric ABH1 (3) successfully cleaved from glucoamylase at the KEX2 protease site. A 10-times dilution of the sample still gave a positive signal with anti-ABH1 antibodies (data not shown).

View larger version (48K): [in this window] [in a new window]   FIG. 4.   Western blot analysis of extracellular ABH1 produced in sandwiched colonies of TAN2 transformants. Lane 1, ABH1 dissociated from PVDF membrane by TFA incubation. Lane 2, no ABH1 was detected in static liquid medium. Lane 3, ABH1, dissociated from PVDF as in lane 1, was not detectable after 24 h of incubation at 30°C in a 3-day-old TAN2 culture medium. Lane 4, ABH1, dissociated from PVDF as in lane 1, was intact after 24 h of incubation at 30°C in H2O. Lane 5, partially increased stability of ABH1 was obtained when the pH and medium components were adjusted.

To analyze whether the enhanced excretion of ABH1 in sandwiched cultures compared to shaken liquid cultures was due to different culture conditions, a control experiment was carried out. A sandwiched TAN2 colony was grown for 3 days as described for the previous experiment, transferred to the surface of 10 ml of static liquid medium, and incubated for 24 h at 30°C. The medium was harvested, and a sample of 25 µl was subjected to SDS-PAGE. As in shaken cultures, no ABH1 was detected in the surface-grown liquid culture (lane 2). Also, no ABH1 was detected when the cultures were grown on 1 ml instead of 10 ml of medium nor when the 10 ml of liquid medium was concentrated 8 times prior to gel electrophoresis (data not shown). This suggested to us that ABH1 was being degraded both in static and in shaken cultures and that degradation could be diminished by immobilizing the proteins (i.e., both ABH1 and proteases) on a PVDF membrane immediately after secretion.

We analyzed the stability of ABH1 after dissociating it from the PVDF membrane. After incubation for 24 h at 30°C with the culture medium of a 3-day-old TAN2 transformant, ABH1 was not detected (lane 3), whereas its level was not reduced after incubation in H₂O (lane 4). Therefore, it is probable that the low amount of ABH1 in the culture medium of the transformed strains is due to extracellular proteolysis.

The A. niger strain (AB4.1) used as a recipient for transformation experiments is known to contain extracellular proteases, most notably aspergillopepsin A and aspergillopepsin B, both belonging to the class of aspartyl proteases (1, 4). However, the use of a protease-deficient strain, AB1.18 (UV mutated in the pepA gene, encoding aspergillopepsin A) (4), did not yield more secreted ABH1 (data not shown). Therefore, other proteases must be responsible for the degradation of ABH1. Van Noort et al. (11) demonstrated a single pH optimum for proteolytic activity of A. niger AB4.1 at pH 4.0, whereas no myoglobin degradation was detected at pH 6.5 or higher. We analyzed the influence of increased pH on the proteolysis of ABH1. The initial pH was increased from 6.0 to 7.0, and the buffer capacity of the medium was increased by raising the phosphate concentration to four times the amount indicated by Wösten et al. (13) and adding 2% CaCO₃ to the medium. This resulted in a final pH of the medium of 6.1 instead of pH 3 to 4 after 24 h of growth of the TAN2 transformant. The adjustment of the pH and medium components only partially restored the yield of ABH1 (lane 5, 25-µl sample of static culture medium). We concluded that efficient expression of ABH1 and some other heterologous proteins in A. niger awaits a protease-free strain.

We have also found the sandwiched-culture technique a reliable method to detect the production of engineered proteins which could not be detected in a culture medium of Schizophyllum commune. This method represents a rapid way to assess expression levels of a heterologous protein in filamentous fungi without interference by protein degradation.