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Heterologous expression of the Aspergillus nidulans alcR–alcA system in Aspergillus niger
Fungal Genetics and Biology 37 (2002) 89–97 www.academicpress.com
Heterologous expression of the Aspergillus nidulans alcR–alcA system in Aspergillus niger I. Nikolaev,a M. Mathieu,a P.J.I. van de Vondervoort,b J. Visser,c and B. Felenboka,* a
Institut de G en etique et Microbiologie, Universit e Paris-Sud, UMR 8621 CNRS, B^atiment 409, Centre d’Orsay, F-91405 Orsay Cedex, France b Department of Phytopathology, Wageningen University, P.O. Box 8025, 6700 EE Wageningen, The Netherlands c FGT Consultancy, P.O. Box 396, 6700 AJ Wageningen, The Netherlands Received 27 February 2002; accepted 21 May 2002
Abstract The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitutive lowly expressed alcohol dehydrogenase activity. The overall induced expression of the alcA gene was of the same order in both fungi, as monitored by alcA transcription, alcohol dehydrogenase activity and heterologous expression of the reporter enzyme, b-glucuronidase. However, important differences in the pattern of alcA regulation were observed between the two fungi. A high basal level of alcA transcription was observed in A. niger resulting in a lower ratio of alcA inducibility. This may be due to higher levels of the physiological inducer of the alc regulon, acetaldehyde, from general metabolism in A. niger which differs from that of A. nidulans. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Heterologous expression; Aspergilli; Ethanol utilization genes; alcA gene regulation
1. Introduction Filamentous fungi have been successfully exploited for many years to produce heterologous proteins. Knowledge of the fungal model system, Aspergillus nidulans, is essential to enable efficient transfer to industrially relevant fungi. Among those Aspergillus niger is of particular interest. In A. nidulans, the ethanol utilization pathway encoded by the alc regulon namely the alcR–alcA genes, is widely used as an inducible expression system to express heterologous proteins at a high level for both fundamental and applied purposes (reviews: Felenbok, 1991; Felenbok and Sealy-Lewis, 1994; Felenbok et al., 2001). Notably, the system appears to be easily transposable to other organisms than fungi, for example in tobacco, a high level of induced yeast invertase was achieved when the corresponding gene was driven by the alcA promoter
*
Corresponding author. Fax: +33-1-69-15-78-08. E-mail address: [email protected] (B. Felenbok).
(Caddick et al., 1998). The reasons for this are the very high induction ratio, resulting from a high rate of mRNA and protein synthesis, and the capacity for tight control of induction parameters by varying the carbon sources in the medium. The key control factor in the pathway is the specific activator AlcR, which belongs to the zinc binuclear cluster protein family (Cahuzac et al., 2001; Lenouvel et al., 1997; Nikolaev et al., 1999; review: Felenbok et al., 2001). The alcR gene is positively autoregulated (Kulmburg et al., 1992; Lockington et al., 1987; Mathieu et al., 2000) and the level of AlcR in the cell correlates with the transcriptional activation of the alc genes among which the alcA and the aldA genes (encoding alcohol dehydrogenase I, ADHI, and aldehyde dehydrogenase, ALDH, respectively) have the highest transcriptional levels found for inducible genes in filamentous fungi (Felenbok et al., 1988; Flipphi et al., 2001; Panozzo et al., 1997). The strength of the inducible alcA and aldA promoters depends on different parameters including, for the alcA gene, the number of AlcR targets, their position, the synergistic mode of transcriptional activation (Panozzo et al., 1997). In addition, complete repression occurs when a rich
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carbon source such as glucose is provided. This repression is mediated by the general repressor CreA (reviews: Felenbok and Kelly, 1996; Felenbok et al., 2001), governing carbon catabolite repression in A. nidulans which acts independently on alcR and alcA transcription. Moreover, CreA exerts a permanent repressive effect under all physiological growth conditions, including the non-induced one (Fillinger et al., 1995; Mathieu and Felenbok, 1994). Two important mechanisms explain the total carbon catabolite repression of the alc regulon involving a direct competition between the AlcR and the CreA proteins in the same promoter regions of the alcR and the alcA genes (Mathieu and Felenbok, 1994; Mathieu et al., 2000; Panozzo et al., 1998) and a direct repression of alcR via a possible interaction with the transcriptional machinery (Mathieu et al., 2000). Another key element of the alc system is the inducer. We have shown that the physiological inducer is an intermediate catabolite in the pathway, acetaldehyde. Ethanol or other related carbon catabolite compounds (i.e. ethylamine, L -threonine) are converted by ADHI to produce acetaldehyde. This latter compound is metabolized into acetate by aldehyde dehydrogenase. This enzyme maintains the balance between the accumulation of acetaldehyde, which induces the alc system and its oxidation, to avoid intoxification of the cells (see Flipphi et al., 2001, 2002). Undoubtedly the alcR–alcA expression system has found its principal application in fundamental research in A. nidulans. In particular, important biological processes have been analysed such as development (Adams and Timberlake, 1990; Marhoul and Adams, 1995), cell division (Lu and Means, 1994; Waring et al., 1989), growth (Lu et al., 1990; McGoldrick et al., 1995), chromatin organization (Ram on Pacheco, 2000), secondary metabolism pathway (Fernandez-Canon and Pe~ nalva, 1995; Kennedy and Turner, 1996). The alcA promoter was used for the production of mammalian proteins in A. nidulans such as interferon, interleukin, growth hormone, epidermal growth factor, superoxide dismutase or lactoferrin and resulted in biologically active recombinant proteins (Davies, 1991; Ward et al., 1992). Production of the heterologous gene fused to the alcA promoter could be increased by several ways which consist to introduce additional copies of the alcR gene (Hintz et al., 1995), to place it under the control of the strong constitutive gpdA promoter (Mathieu and Felenbok, 1994), or to release the alcA gene from carbon catabolite repression (Panozzo et al., 1998). While A. nidulans is clearly the model fungal organism for fundamental research, A. niger is the fungus of industrial importance. It is used in biotechnology for the expression and the production of heterologous proteins. A number of general regulatory mechanisms are conserved between both species of aspergilli, such as carbon catabolite repression (CreA), nitrogen metabolic re-
pression (AreA), pH regulation (PacC) (review: see van den Homberg et al., 1997). A. niger possesses alcohol and aldehyde dehydrogenase activities that exhibit a different pattern of regulation from that seen in A. nidulans (Kelly et al., 1990; O’Connell and Kelly, 1988). For example, the endogenous alcohol dehydrogenase (ADH)1 activity is expressed at a low level and does not allow the fungus to grow on ethanol as a sole carbon source (Kelly et al., 1990). It was therefore of great interest to determine whether the A. nidulans alcR–alcA system could function as an inducible system in A. niger. In this study, we have shown that the alc system found in A. nidulans does not exist in A. niger. It is directly transposable to A. niger and the resulting level of expression of the alcA transcript in A. niger is comparable to that in A nidulans. Expressing heterologous protein, like b-glucuronidase (GUS), under control of the alcA promoter results in similar induction patterns in both A. nidulans and A. niger. However, remarkable differences in the induction ratio between the two species were observed, which are discussed taking into account the physiology of both fungi.
2. Materials and methods 2.1. Strains and growth conditions The A. niger strains used in this work were NW219 (cspA1, pyrA6, leuA1, nicA1) and the wild-type strain N402 (cspA1). The A. nidulans wild-type strain WG096 pabaA1 ya2) was used as a control for the ADH activity assay. The transformant TgpdA:alcR obtained in the previous work (Flipphi et al., 2001) was used for Northern studies. To introduce the alcA:uidA expression construct into A. nidulans, an argB strain (yA2, pabaA1, argB2, uaZ11) (Flipphi et al., 2001) was used as a host for transformation. Media and supplements were as described by Cove (1966). For cultivation of A. niger the pH of growth media was adjusted to 6.0. Mycelium was pregrown for 16 h at 30 °C in minimal medium containing 1% of glucose and 10 mM NaNO3 as a carbon and nitrogen source, respectively, washed with sterile water and then transferred into minimal medium with the indicated carbon source. Induction was achieved after 5–7 h of additional growth on 0.1% of fructose with either 0.5% of ethanol or 50 mM of ethyl methyl ketone (EMK). For repression conditions 1% glucose was supplied simultaneously with the inducer to the medium. When indicated, yeast extract was added to the final concentration of 0.1%. Growth conditions for
1 Abbreviations used: ADH, alcohol dehydrogenase; GUS, b-glucuronidase; EMK, ethyl methyl ketone.
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A. nidulans were as described previously (Mathieu and Felenbok, 1994). Transformation of both aspergilli species was performed according to the standard procedure (Tilburn et al., 1983), except for the protoplasting of A. niger which was achieved at elevated concentrations (20 mg/ml) of Novozyme (Sigma–Aldrich, USA). 2.2. Plasmid construction To construct the plasmid containing both the alcR and the alcA genes, we used the previously made bAN7 vector (Panozzo et al., 1998) which carries the alcR encoding sequence under the control of the constitutive gpdA promoter. The 3.2-kb SpeI–NotI genomic fragment containing the entire alcA gene was cloned into the corresponding restriction sites of bAN7 resulting in the pAN10 construct. This plasmid was used to cotransform A. niger together with the pGW635 vector bearing the pyrA gene (Goosen et al., 1987). To generate the pGRP3 plasmid, the 3.9-kb XbaI– XbaI fragment of pGW635 containing the pyrA gene (Goosen et al., 1987) was first inserted into the XbaI site of pBluescript KSþ and then reisolated from the above vector via the NotI–EcoRI cut. The latter insert was cloned into bAN7 digested with the same enzymes. The pyrA gene appeared to be in the orientation opposite to gpdA:alcR. The alcA:uidA fusion in which the E. coli b-glucuronidase gene is driven by the alcA promoter was constructed as follows. The alcA promoter region starting from )423 bp to the ATG codon was amplified by PCR and was used to replace the gpdA sequence in the pAN52-1 vector (Punt et al., 1990) resulting in pAN52A. The required EcoRI and NcoI sites at the 50 and 30 termini of the PCR product were introduced during the amplification. The GUS coding sequence produced by PCR amplification from the plasmid pTRAN3 (Punt et al., 1991) was inserted in frame between the alcA promoter and the trpC terminator in the unique NcoI site of the pAN52-A vector. This alcA:uidA expressing plasmid was used in further experiments. 2.3. DNA and RNA analysis To screen for transformants that have integrated the alcA and alcR genes, a PCR approach with genomic DNA was used. Aspergillus DNA was isolated as described by Specht et al. (1982). Different portions of the alcA and alcR genes integrated into A. niger genome were amplified using the following pairs of oligos: GPD: 50 -CCCGCTTGAGCAGACATCACCATG0 3 and RB 2169: 50 -GCTATGTCTGCAGGAACATT-30 for pgpdA; RT 1598: 50 -CGGTGATTTCAGCATGGG30 and RB 3271: 50 -AGTGAAGAGCGCGCAAG-30 for alcR; AT 3119: 50 -TTCTCAATCTCAGGAAGCAA
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GC-30 and AB 3624: 50 -GCTTATTCTGGCATCTCTA GC-30 for alcA. Southern blot analysis was performed according to Sambrook et al. (1989). Total RNA was extracted as described by Lockington et al. (1985) and separated on glyoxal agarose gels according to Sambrook et al. (1989). The 32 P-labeled probes used were the entire alcA and alcR genes cloned into pBluescript. Ribosomal RNAs visualized by methylene blue staining served as an internal control to normalize the amounts of RNA loaded on a single blot. 2.4. Enzyme assays ADH and b-glucuronidase (GUS) activities were measured in crude protein extracts isolated from ground mycelia in the extraction buffer composed of 50 mM Naphosphate, pH 7.2, 100 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 1 mM PMSF. ADH activity was measured in 100 mM Na-phosphate buffer, pH 8.5, 2 mM MgCl2 , 4 mM NADþ , 0.2 M ethanol at 30 °C. In the absence of ethanol, the rate of NADþ reduction was negligible indicating that the specific dehydrogenase activity can be attributed to ADH. Specific activities were expressed in micromole of NADH formed per 1 min per mg of protein under conditions used. The GUS activity was measured as described by Jefferson (1987) using p-nitrophenyl-b-glucuronide (Sigma–Aldrich, USA) as a substrate. Specific activities were expressed in nmol of pnitrophenol formed per min per mg in crude protein extracts.
3. Results 3.1. No inducible alcohol dehydrogenase activity exists in A. niger A. niger grows very poorly on ethanol as a sole carbon source. Furthermore the fungus appears extremely resistant to high concentrations of allylalcohol (up to 25 mM, A. Dumay and F. Lenouvel, personal communication), suggesting that this compound is poorly metabolized by ADH into a highly toxic compound, acrolein (results not shown). This result could be explained by a low ADH activity. In fact, endogenous ADH activity was detected in crude extracts of A. niger mycelia grown under different physiological conditions: (i) non-induced (neutral carbon source); (ii) induced by ethanol: (iii) repressed by glucose in the presence of ethanol. ADH activity was compared with that in A. nidulans grown in the same conditions. Table 1 shows that in A. niger, the ADH activity did not change significantly in different growth conditions, whereas in A. nidulans, as expected, a strong induction of ADH was observed. Interestingly, comparable specific ADH
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Table 1 ADH activities in A. nidulans and A. niger wild-type strains under different growth conditions Fungus
Carbon source
ADH activity, U/mga
A. niger (WT)
1% Glucose 0.5% Ethanol + 0.1% fructose 0.5% Ethanol + 1% glucose
21 39 25
A. nidulans (WT)
1% Glucose 0.5% Ethanol + 0.1% fructose 0.5% Ethanol + 1% glucose
1 148 4
a
All mycelia were pregrown overnight on minimal medium with 1% glucose at 30 °C, washed and then transferred to minimal medium containing different carbon sources as indicated. The ADH activities were measured in cell extracts of mycelia after additional growth for 5 h in the presence of the inducer ethanol and /or glucose as indicated in Section 2 and expressed in U/mg.
activity was found in A. niger in all three growth conditions, indicating that ADH was constitutively expressed in A. niger. In contrast, as expected, in A. nidulans ADH activity was low in non-induced conditions, drastically increased upon induction, and was fully repressed in the presence of glucose. Therefore, the pattern of regulation of ADH activity is completely different in the two aspergilli species. Next, it was important to determine if the alcR–alcA homologues were present or not in A. niger. Southern blots of A. niger genomic DNA were probed with the A. nidulans alcR and alcA genes. No cross-hybridization was observed under low stringency conditions suggesting that probably no homologues are present in the genome of A. niger (Fig. 1). Therefore, transformants of A. niger containing the alcR and alcA genes from A. nidulans should be easily identified and analyzed straightforward. 3.2. Truncated copies are often generated when A. niger is transformed with A. nidulans alcR and alcA genes To test the expression of an introduced alcA gene in A. niger, we decided to overexpress alcR under the constitutive derepressed gpdA promoter to avoid limiting amounts of AlcR in the cell. A. niger pyrA6 strain was co-transformed with a plasmid bearing the gpd:alcR–alcA gene construct (Mathieu et al., 2000) and a plasmid containing the pyrA gene (Goosen et al., 1987), used as a selective marker (see Fig. 1). Transformants were analyzed after selection for uridine prototrophy. Among 120 transformants, only 7 had integrated full length alcR and alcA copies whilst 73 transformants had integrated truncated copies of either the alcR or the alcA genes or both. Fig. 1 shows a typical Southern blot probed with alcR and alcA in which truncated copies (shown by arrows) were observed as compared to the expected size of the full-length plasmid integrated in the transformant
T2alc (lane 4). Chromosomal rearrangements were tested by PCR products as specified in Fig. 1. Transcriptional analysis of these transformants is shown in Fig. 2. No induced alcA transcript was observed in the A. niger transformant T1alc (lanes 1), as the result of a truncation of the gpd:alcR gene, rendering it non functional. These genomic rearrangements have not been described previously with other heterologous genes in A. niger. 3.3. The A. nidulans alcA gene is induced in A. niger transformants We examined the transcription of the A. nidulans alcA gene in the A. niger transformant, T2alc, containing the intact A. nidulans gpd:alcR gene. Two inducers, both known to induce the A. nidulans alc system, ethyl methyl ketone (EMK) and ethanol (Et), were tested. Northern blots are shown in Fig. 2. In the A. niger transformant T2alc, the alcA gene was inducible by both carbon compounds (lanes 2). The level of the alcA induced transcription was comparable in both organisms. Interestingly, ethanol was a better inducer than EMK in A. niger while the opposite situation occurs in A. nidulans (Flipphi et al., 2002). A partial derepression of the alcA gene was observed in both aspergilli, as expected in the constitutive derepressed gpd:alcR background as previously shown in A. nidulans (Panozzo et al., 1998; this work). The second feature that differentiates both aspergilli is the steady-state level of transcription of alcA under non-inducing conditions which is much higher in A. niger than in A. nidulans. To ascertain that the transcribed alcA gene from A. nidulans was correctly expressed in A. niger, ADH activity was measured in A. niger crude extracts after induction by ethanol. Fig. 3 shows that a clear induction of ADH activity was observed after 5 h of induction and this increase was 5-fold higher after 7 h. The non-induced level of ADH activity was high in agreement with the transcriptional analysis in T2alc shown in Fig. 2. 3.4. Heterologous expression of b-glucuronidase (GUS) driven by the alcA promoter is very efficient in A. niger Next, we wanted to test if the A. nidulans alcA promoter was able to drive the heterologous expression of a non-fungal protein in A. niger. For this we choose the GUS protein from E. coli which is an easily measurable reporter enzyme (Jefferson, 1987). Co-transformation in A. niger pyrA6 strain was carried out with the gpd:alcR–pyrA construct together with alcA:uidA plasmid. Transformants pyrþ were selected which harbored both constructs fully intact in single copies. Fig. 4A shows that in this gpd:alcR context, GUS activity (U/mg) in A. niger was highly inducible when driven by the alcA promoter. A 10-fold induction
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Fig. 1. Southern blots of A. niger transformants containing alcR and alcA genes from A. nidulans. Co-transformation of the A. niger pyrA6 strain was performed with the gpd:alcR–alcA plasmid (pAN10) together with the pyrA plasmid as described in Section 2. After selection for uridine prototrophy, DNA of transformants carrying both the alcR and alcA genes were submitted to Southern blot analysis after enzyme cleavage with the restriction enzymes SpeI and HindIII as indicated. SpeI site is unique and HindIII site is absent in pAN10. Left panel, alcR probe; right panel, alcA probe. Chromosomal rearrangements occurred resulting in truncated restriction fragments which are indicated by arrows in lanes 1, 2, and 3. The A. niger transformant, T2alc, (lane 4) shows a single integration event of the entire construct. Lane 2 contains 3-fold less amount of DNA. Lane R is the A. niger recipient strain. Differences in band intensities upon hybridization with the alcR or alcA probes (lanes 2 and 3) could be indicative of insertions of the truncated construct which lacks either the alcR or alcA full-length sequences. The map of the plasmid pAN10 is indicated at the bottom. Chromosomal rearrangements were identified using PCR products as probes, obtained with the corresponding oligonucleotides mapping in the alcA gene (AT 3119, AB 3624) and in the gpd:alcR gene (gpd, RB 2169, and RT 1598, RB 3271) as described in Section 2.
was observed after 7 h which increased even further after 12 h, showing remarkable stability. The induced production of GUS was only repressed partially by glucose as expected in the gpd:alcR context. Interestingly, when changing the medium composition of A. niger by adding a low concentration of yeast extract (0.1%), an almost constitutive expression of GUS was observed. We wanted to know if this constitutive expression was related only to the addition of yeast extract. Therefore, the same experiments with the same constructs were carried out in A. nidulans. Fig. 4B shows that in A. nidulans, GUS activity was strongly induced by ethanol. In addition, the presence of yeast extract did not lead to constitutive expression of GUS. Therefore, the differences observed upon addition of
yeast extract, likely reflect a different physiology between the two species of aspergilli. 4. Discussion This work aimed to study the expression in A. niger of both the A. nidulans alcR and alcA genes to apply this efficient expression system for heterologous protein production in an industrial fungal species. A. niger does not possess the equivalent of the A. nidulans alc system. There is no endogenous, inducible alcohol dehydrogenase activity even though there is a measurable constitutive ADH activity. Therefore, inducible heterologous protein expression was possible using the A. nidulans alc system in A. niger.
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Fig. 2. Northern blot analysis of A. niger transformants containing the alcR–alcA genes from A. nidulans grown under various physiological growth conditions. Two A. niger transformants T1alc and T2alc and one of A. nidulans containing the gpd:alcR–alcA construct were grown under the conditions described in Section 2. NI, non-induced growth conditions; Et, induction with 1% ethanol during 5 h; EMK, induction with 50 mM EMK (ethyl methyl ketone) during 5 h; EtG, 1% glucose repressed conditions in the presence of 1% ethanol during 5 h. The membranes were hybridized with the alcR and alcA 32 P-labeled probes as indicated on the right of the Northern blot. The amount of ribosomal RNA (rRNA) was visualized after staining the membrane with methylene blue. Hybridization with the alcR probe reveals an additional band of 1.6 kb in the A. nidulans RNA sample which results from the endogenous truncated transcript of the recipient strain.
Fig. 3. Alcohol dehydrogenase activity of the A. niger transformant T2alc containing the gpd:alcR–alcA genes. Alcohol dehydrogenase activity was measured during induction with 1% ethanol up to 7 h in the A. niger transformant T2alc (closed lozenges) and in the A. niger recipient strain R (open squares). ADH activity was expressed in U/mg as defined in Section 2 and under the legend to Table 1.
Our results show that the alc system is transferable to A. niger where it is as highly expressed as in A. nidulans. In the presence of non-limiting amounts of the AlcR protein (constitutively overexpressed from the gpdA promoter), a high level of the alcA gene transcription was observed. This leads to high expression of the encoded alcohol dehydrogenase or an even higher activity of the heterologous GUS protein driven by the alcA promoter. Moreover, in A. niger, the alcA promoter was found to be partially repressed in the presence of glucose
and ethanol. This repression is of the same order as that observed in A. nidulans in the gpd:alcR background (Panozzo et al., 1998). Therefore in A. niger, the equivalent of CreA homologue, is able to repress the heterologous alcA promoter. This result is expected since both proteins recognize the same GC rich targets and share almost 90% identical amino acid residues (Drysdale et al., 1993). Therefore, we can conclude that in A. niger, the transcriptional and translational machineries act efficiently with respect to expression of the heterologous A. nidulans alcA gene. However, some unexpected results were observed upon A. niger transformation with the alcR–alcA system. First, there was a high percentage of genomic rearrangements observed upon transformation with the alc genes. We do not know whether the observed deletions of the alc genes when integrated into A. niger DNA are specific to the alc system or reflects a general phenomenon since such an observation has not been described so far. Stability of the transformants was not monitored rigorously. However, a loss of the inducible ADH activity in a few cases was observed after several rounds of plating. More interesting are the differences observed between the two aspergilli species when submitted to the same growth conditions. Our results raise the important question of the nature of ethanol metabolism in A. niger. We know from the studies of the A. nidulans alc regulon that ethanol itself is not a direct inducer, but has to be metabolized into acetaldehyde, the physiological inducer (Flipphi et al., 2001, 2002). This compound is at a cross
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Fig. 4. b-Glucuronidase (GUS) activity of A. niger and A. nidulans transformants containing gpd:alcR and palcA:uidA construct. (A) Kinetics of GUS activity of A. niger transformant containing gpd:alcR and palcA:uidA genes grown under various growth conditions: noninduced conditions (NI, empty lozenges); induced by 50 mM EMK (closed squares); induced by 1% ethanol (Et, open triangles); repressed by 1% glucose in the presence of 1% ethanol (EtG, crosses). GUS activity was followed up to 12 h and expressed in U/mg as described in Section 2. (B) Kinetics of b-glucuronidase activity in A. niger and A. nidulans transformants containing gpd:alcR and alcA:uidA genes grown in the presence of 0.1% yeast extract and induced with 1% ethanol up to 12 h. Transformants in A. niger are shown as open lozenges and in A. nidulans as closed squares. GUS activity was expressed in U/mg as described in Section 2.
road between several catabolic pathways and its accumulation in A. nidulans depends mostly, as mentioned previously, on the fine tuning between the two structural enzymes (ADHI and ALDH) of the ethanol utilization pathway. In A. niger, it was observed that ethanol was an even better inducer of the alc genes than EMK, the best inducer in A. nidulans. This result implies that ethanol should be metabolized into acetadehyde by an endogenous non-inducible ADH and/or by another unknown activity. The ADH activity measured in crude extracts from A. niger was constitutive and was much lower than the inducible ADH in A. nidulans. This weaker ADH activity could explain the subsequent increased time required to obtain full induction in A. niger, 7 h, compared to the optimal 2 h induction in A. nidulans. We know from
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A. nidulans data that the induction process is directly related to the level of accumulation of acetaldehyde. Related to that feature, another important difference between the two fungi is the basal level of alcA expression. Our data clearly show that in A. niger transformants the non-induced basal level of alcA is much higher than that in A. nidulans. This effect is manifested at both the transcriptional and the enzyme activity levels. This result may be explained by an accumulation of acetaldehyde from the regular metabolism in A. niger and/or by a weaker ALDH activity. Interestingly, A. niger transformants containing the heterologous construct alcA:uidA in the gpd:alcR background, are substantially induced by ethanol and the steady-state level of activity is of the same order in both organisms: more than 10-fold after 10 h of induction. This indicates that in A. niger, the ADH activity is sufficient to metabolize ethanol into acetaldehyde which accumulates and induces the alcA promoter via AlcR. In the presence of yeast extract a large increase in the basal level of GUS is observed in A. niger but not in A. nidulans. After ethanol induction a constitutive expression is observed in A. niger whereas a very strong increase in GUS activity is observed only in A. nidulans. Therefore the alcA–alcR system itself responds correctly in both aspergilli, but the inducer metabolism differs. It has to be pointed out that yeast extract contains a lot of aminoacids and di-tripeptides. It is likely that in A. niger, deamination, decarboxylation, and subsequent oxidation of these compounds lead to inducer formation. These results emphasize the important differences in metabolism between A. niger and A. nidulans. Our data suggest that high levels of expression of heterologous proteins may be obtained in A. niger using the alcA promoter of A. nidulans. More generally, we can conclude that different parameters are involved in the level of heterologous induced protein accumulation, including the nature of the expressed heterologous protein and the metabolism of the fungal host.
Acknowledgments This work was supported in part by grants from the CNRS (UMR 8621), the University Paris-Sud and the European Communities contracts BIO4-CT96-0535 and QLK3-CT1999-00729. We thank Anne Dumay and Francßois Lenouvel who started the work, and Prof. Barry Holland for correcting the English, and Michel Flipphi and Christian Velot for helpful comments. References Adams, T.H., Timberlake, W.E., 1990. Developmental repression of growth and gene expression in Aspergillus. Proc. Natl. Acad. Sci. USA 87, 5405–5409.
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Caddick, M.X., Greenland, A.J., Jepson, J., Krause, K.-P., Qu, N., Riddell, K.V., Salter, M.G., Schuch, W., Sonnewald, U., Tomsett, B., 1998. An ethanol inducible gene switch for plants used to manipulate carbon metabolism. Nat. Biotechnol. 16, 177–180. Cahuzac, B., Cerdan, R., Felenbok, B., Guittet, E., 2001. Variation along a structural theme. The solution structure of an AlcR-DNA complex sheds light onto the unique tight and monomeric DNAbinding of a Zn2 Cys6 protein. Structure 9, 827–836. Cove, D.J., 1966. The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113, 51– 56. Davies, R.W., 1991. Molecular biology of a high-level recombinant protein production system in Aspergillus. In: Leong, S.A., Berka, R.M. (Eds.), Molecular Industrial Mycology Systems and Applications for Filamentous Fungi. Morris-Bekker Inc., New York, Basel, Hong Kong, pp. 45–58. Drysdale, M.R., Kolze, S.E., Kelly, J.M., 1993. The Aspergillus niger carbon catabolite repressor encoding gene, creA. Gene 130, 241– 245. Felenbok, B., 1991. The ethanol utilization regulon of Aspergillus nidulans: the alcA–alcR system as a tool for the expression of recombinant proteins. J. Biotechnol. 17, 11–18. Felenbok, B., Flipphi, M., Nikolaev, I., 2001. Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation. In: Moldave, K. (Ed.), Progress in Nucleic Acid Research and Molecular Biology, vol. 69. Academic Press, San Diego, pp. 149–204. Felenbok, B., Kelly, J., 1996. Regulation of Carbon Metabolism in Mycelial Fungi. In: Marzluf, G., Brambl, R. (Eds.), The Mycota III: Biochemistry and Molecular Biology. Springer, Berlin, pp. 369–380. Felenbok, B., Sealy-Lewis, H.M., 1994. Alcohol metabolism. In: Martinelli, S., Kinghorn, J.R. (Eds.), Aspergillus: 50 years on. Progress in Industrial Microbiology, vol. 29. Elsevier Science, Amsterdam, pp. 141–179, Chapter 6. Felenbok, B., Sequeval, D., Mathieu, M., Sibley, S., Gwynne, D.I., Davies, R.W., 1988. The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR. Gene 73, 385–396. Fernandez-Canon, J.M., Pe~ nalva, M.A., 1995. Overexpression of two penicillin structural genes in Aspergillus nidulans. Mol. Gen. Genet. 246, 110–118. Fillinger, S., Panozzo, C., Mathieu, M., Felenbok, B., 1995. The basal level of transcription of the alc genes in the ethanol regulon in Aspergillus nidulans is controlled both by the specific transactivator AlcR and the general carbon catabolite repressor CreA. FEBS Lett. 368, 547–550. Flipphi, M., Mathieu, M., Cirpus, I., Panozzo, C., Felenbok, B., 2001. Regulation of the aldehyde dehydrogenase gene (aldA) and its role in the control of the coinducer level necessary for induction of the ethanol utilization pathway in Aspergillus nidulans. J. Biol. Chem. 276, 6950–6958. Flipphi, M., Kocialkowska, J., Felenbok, B., 2002. Characteristics of physiological inducers of the ethanol utilization (alc) pathway in Aspergillus nidulans. Biochem. J. 364, 25–31. Goosen, T., Bloemheuvel, G., Gysler, C., Bie, D.A., de Broek, H.W., Swart, K., 1987. Transformation of Aspergillus niger using the homologous orotodine-50 -phosphate-decarboxylase gene. Curr. Genet. 11, 499–503. Hintz, W.E., Kalsner, I., Plawinski, E., Guo, Z., Lagosky, P.A., 1995. Improved gene expression in Aspergillus nidulans. Can. J. Bot. 73 (Suppl. 1), S876–S884. Jefferson, R.A., 1987. Assaying Chimeric genes in plants: The GUS gene fusion system. Plant Molec. Biol. Reporter 5, 387–405. Kelly, J.M., Drysdale, M.R., Sealy-Lewis, H.M., Jones, I.G., Lockington, R.A., 1990. Alcohol dehydrogenase III in Aspergillus nidulans is anaerobically induced and post-transcriptionally regulated. Mol. Gen. Genet. 222, 323–328.
Kennedy, J., Turner, G., 1996. D-(L -a-aminoadipyl)-L -cysteinyl-D valine synthetase is a rate limiting enzyme for penicillin production in Aspergillus nidulans. Mol. Gen. Genet. 253, 189–197. Kulmburg, P., Sequeval, D., Lenouvel, F., Mathieu, M., Felenbok, B., 1992. Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans. Mol. Cell Biol. 12, 1932–1939. Lenouvel, F., Nikolaev, I., Felenbok, B., 1997. In vitro recognition of specific DNA targets by AlcR, a zinc binuclear cluster activator different from the other proteins of this class. J. Biol. Chem. 272, 15521–15526. Lockington, R.A., Sealy-Lewis, H.M., Scazzocchio, C., Davies, R.W., 1985. Cloning and characterization of the ethanol utilization in Aspergillus nidulans. Gene Amst. 33, 137–149. Lockington, R.A., Scazzocchio, C., Sequeval, D., Mathieu, M., Felenbok, B., 1987. Regulation of alcR, the positive regulatory gene of the ethanol utilization regulon of Aspergillus nidulans. Mol. Microbiol. 1, 275–281. Lu, K.P., Means, A.R., 1994. Expression of the noncatalytic domain of the NIMA kinase causes a G2 arrest in Aspergillus nidulans. EMBO J. 13, 2103–2113. Lu, K.P., Rasmussen, C.D., May, G.S., Means, A.R., 1990. Cooperative regulation of cell proliferation by calcium and calmodulin in Aspergillus nidulans. Mol. Endocrinol. 6, 365–374. Marhoul, J.F., Adams, T.H., 1995. Identification of developmental regulatory genes in Aspergillus nidulans by overexpression. Genetics 139, 537–547. Mathieu, M., Felenbok, B., 1994. The Aspergillus nidulans CREA protein mediates glucose repression of the ethanol regulon at various levels through competition with the ALCR-specific transactivator. EMBO J. 13, 4022–4027. Mathieu, M., Fillinger, S., Felenbok, B., 2000. In vivo studies of upstream regulatory cis-acting elements of the alcR gene encoding the transactivator of the ethanol regulon in Aspergillus nidulans. Mol. Microbiol. 36, 123–131. McGoldrick, C.A., Gruver, C., May, G.R., 1995. myoA of Aspergillus nidulans encodes an essential myosin I required for secretion and polarized growth. J. Cell Biol. 128, 577–587. Nikolaev, I., Lenouvel, F., Felenbok, B., 1999. Unique DNA binding specificity of the binuclear zinc AlcR activator of the ethanol utilization pathway in Aspergillus nidulans. J. Biol. Chem. 274, 9795–9802. O’Connell, M.J., Kelly, J.M., 1988. Differences in the regulation of aldehyde dehydrogenase genes in Aspergillus niger and Aspergillus nidulans. Curr. Genet. 14, 95–103. Panozzo, C., Capuano, V., Fillinger, S., Felenbok, B., 1997. The zinc binuclear cluster activator AlcR is able to bind to single sites, but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. J. Biol. Chem. 272, 22859– 22865. Panozzo, C., Cornillot, E., Felenbok, B., 1998. The CreA repressor is the sole DNA-binding protein responsible for carbon catabolite repression of the alcA gene in Aspergillus nidulans via its binding to a couple of specific sites. J. Biol. Chem. 273, 6367–6372. Punt, P.J., Dingemanse, M.A., Kuyvenhoven, A., Soede, R.D., Pouwels, P.H., van den Hondel, C.A., 1990. Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 93, 101–109. Punt, P.J., Greaves, P.A., Kuyvenhoven, A., van Deutekom, J.C.T., Kinghorn, J.R., Pouwels, P.H., van den Hondel, C.A., 1991. A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi. Gene 104, 119–122. Ram on Pacheco, A.C., 2000. Structure Chromatinienne et Expression Genique chez Aspergillus nidulans. PhD Thesis, University of ParisSud, Orsay, France.
I. Nikolaev et al. / Fungal Genetics and Biology 37 (2002) 89–97 Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Specht, C.A., Dirusso, C.C., Novotny, C.P., Ullrich, R.C., 1982. A method for extracting high molecular weight deoxyribonucleic acid from fungi. Anal. Biochem. 119, 158–163. Tilburn, J., Scazzocchio, C., Taylor, G.G., Zabicky-Zissman, J.H., Lockington, R.A., Davies, R.W., 1983. Transformation by integration in Aspergillus nidulans. Gene 26, 205–221.
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van den Homberg, J.P.W., vande Vandervoort, P.J.I., Fraissinet-Tachet, L., Visser, J., 1997. Aspergillus as a host for heterologous protein production: The problem of proteases. Tib. Tech. 15, 256–263. Ward, P.P., May, G.S., Headon, D.R., Conneely, O.M., 1992. An inducible expression system for the production of human lactoferrin in Aspergillus nidulans. Gene 122, 219–223. Waring, R.B., May, G.S., Morris, N.R., 1989. Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes. Gene 79, 119–130.
Heterologous expression of the Aspergillus nidulans alcR–alcA system in Aspergillus niger I. Nikolaev,a M. Mathieu,a P.J.I. van de Vondervoort,b J. Visser,c and B. Felenboka,* a
Institut de G en etique et Microbiologie, Universit e Paris-Sud, UMR 8621 CNRS, B^atiment 409, Centre d’Orsay, F-91405 Orsay Cedex, France b Department of Phytopathology, Wageningen University, P.O. Box 8025, 6700 EE Wageningen, The Netherlands c FGT Consultancy, P.O. Box 396, 6700 AJ Wageningen, The Netherlands Received 27 February 2002; accepted 21 May 2002
Abstract The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitutive lowly expressed alcohol dehydrogenase activity. The overall induced expression of the alcA gene was of the same order in both fungi, as monitored by alcA transcription, alcohol dehydrogenase activity and heterologous expression of the reporter enzyme, b-glucuronidase. However, important differences in the pattern of alcA regulation were observed between the two fungi. A high basal level of alcA transcription was observed in A. niger resulting in a lower ratio of alcA inducibility. This may be due to higher levels of the physiological inducer of the alc regulon, acetaldehyde, from general metabolism in A. niger which differs from that of A. nidulans. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Heterologous expression; Aspergilli; Ethanol utilization genes; alcA gene regulation
1. Introduction Filamentous fungi have been successfully exploited for many years to produce heterologous proteins. Knowledge of the fungal model system, Aspergillus nidulans, is essential to enable efficient transfer to industrially relevant fungi. Among those Aspergillus niger is of particular interest. In A. nidulans, the ethanol utilization pathway encoded by the alc regulon namely the alcR–alcA genes, is widely used as an inducible expression system to express heterologous proteins at a high level for both fundamental and applied purposes (reviews: Felenbok, 1991; Felenbok and Sealy-Lewis, 1994; Felenbok et al., 2001). Notably, the system appears to be easily transposable to other organisms than fungi, for example in tobacco, a high level of induced yeast invertase was achieved when the corresponding gene was driven by the alcA promoter
*
Corresponding author. Fax: +33-1-69-15-78-08. E-mail address: [email protected] (B. Felenbok).
(Caddick et al., 1998). The reasons for this are the very high induction ratio, resulting from a high rate of mRNA and protein synthesis, and the capacity for tight control of induction parameters by varying the carbon sources in the medium. The key control factor in the pathway is the specific activator AlcR, which belongs to the zinc binuclear cluster protein family (Cahuzac et al., 2001; Lenouvel et al., 1997; Nikolaev et al., 1999; review: Felenbok et al., 2001). The alcR gene is positively autoregulated (Kulmburg et al., 1992; Lockington et al., 1987; Mathieu et al., 2000) and the level of AlcR in the cell correlates with the transcriptional activation of the alc genes among which the alcA and the aldA genes (encoding alcohol dehydrogenase I, ADHI, and aldehyde dehydrogenase, ALDH, respectively) have the highest transcriptional levels found for inducible genes in filamentous fungi (Felenbok et al., 1988; Flipphi et al., 2001; Panozzo et al., 1997). The strength of the inducible alcA and aldA promoters depends on different parameters including, for the alcA gene, the number of AlcR targets, their position, the synergistic mode of transcriptional activation (Panozzo et al., 1997). In addition, complete repression occurs when a rich
1087-1845/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 7 - 1 8 4 5 ( 0 2 ) 0 0 0 3 7 - 3
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carbon source such as glucose is provided. This repression is mediated by the general repressor CreA (reviews: Felenbok and Kelly, 1996; Felenbok et al., 2001), governing carbon catabolite repression in A. nidulans which acts independently on alcR and alcA transcription. Moreover, CreA exerts a permanent repressive effect under all physiological growth conditions, including the non-induced one (Fillinger et al., 1995; Mathieu and Felenbok, 1994). Two important mechanisms explain the total carbon catabolite repression of the alc regulon involving a direct competition between the AlcR and the CreA proteins in the same promoter regions of the alcR and the alcA genes (Mathieu and Felenbok, 1994; Mathieu et al., 2000; Panozzo et al., 1998) and a direct repression of alcR via a possible interaction with the transcriptional machinery (Mathieu et al., 2000). Another key element of the alc system is the inducer. We have shown that the physiological inducer is an intermediate catabolite in the pathway, acetaldehyde. Ethanol or other related carbon catabolite compounds (i.e. ethylamine, L -threonine) are converted by ADHI to produce acetaldehyde. This latter compound is metabolized into acetate by aldehyde dehydrogenase. This enzyme maintains the balance between the accumulation of acetaldehyde, which induces the alc system and its oxidation, to avoid intoxification of the cells (see Flipphi et al., 2001, 2002). Undoubtedly the alcR–alcA expression system has found its principal application in fundamental research in A. nidulans. In particular, important biological processes have been analysed such as development (Adams and Timberlake, 1990; Marhoul and Adams, 1995), cell division (Lu and Means, 1994; Waring et al., 1989), growth (Lu et al., 1990; McGoldrick et al., 1995), chromatin organization (Ram on Pacheco, 2000), secondary metabolism pathway (Fernandez-Canon and Pe~ nalva, 1995; Kennedy and Turner, 1996). The alcA promoter was used for the production of mammalian proteins in A. nidulans such as interferon, interleukin, growth hormone, epidermal growth factor, superoxide dismutase or lactoferrin and resulted in biologically active recombinant proteins (Davies, 1991; Ward et al., 1992). Production of the heterologous gene fused to the alcA promoter could be increased by several ways which consist to introduce additional copies of the alcR gene (Hintz et al., 1995), to place it under the control of the strong constitutive gpdA promoter (Mathieu and Felenbok, 1994), or to release the alcA gene from carbon catabolite repression (Panozzo et al., 1998). While A. nidulans is clearly the model fungal organism for fundamental research, A. niger is the fungus of industrial importance. It is used in biotechnology for the expression and the production of heterologous proteins. A number of general regulatory mechanisms are conserved between both species of aspergilli, such as carbon catabolite repression (CreA), nitrogen metabolic re-
pression (AreA), pH regulation (PacC) (review: see van den Homberg et al., 1997). A. niger possesses alcohol and aldehyde dehydrogenase activities that exhibit a different pattern of regulation from that seen in A. nidulans (Kelly et al., 1990; O’Connell and Kelly, 1988). For example, the endogenous alcohol dehydrogenase (ADH)1 activity is expressed at a low level and does not allow the fungus to grow on ethanol as a sole carbon source (Kelly et al., 1990). It was therefore of great interest to determine whether the A. nidulans alcR–alcA system could function as an inducible system in A. niger. In this study, we have shown that the alc system found in A. nidulans does not exist in A. niger. It is directly transposable to A. niger and the resulting level of expression of the alcA transcript in A. niger is comparable to that in A nidulans. Expressing heterologous protein, like b-glucuronidase (GUS), under control of the alcA promoter results in similar induction patterns in both A. nidulans and A. niger. However, remarkable differences in the induction ratio between the two species were observed, which are discussed taking into account the physiology of both fungi.
2. Materials and methods 2.1. Strains and growth conditions The A. niger strains used in this work were NW219 (cspA1, pyrA6, leuA1, nicA1) and the wild-type strain N402 (cspA1). The A. nidulans wild-type strain WG096 pabaA1 ya2) was used as a control for the ADH activity assay. The transformant TgpdA:alcR obtained in the previous work (Flipphi et al., 2001) was used for Northern studies. To introduce the alcA:uidA expression construct into A. nidulans, an argB strain (yA2, pabaA1, argB2, uaZ11) (Flipphi et al., 2001) was used as a host for transformation. Media and supplements were as described by Cove (1966). For cultivation of A. niger the pH of growth media was adjusted to 6.0. Mycelium was pregrown for 16 h at 30 °C in minimal medium containing 1% of glucose and 10 mM NaNO3 as a carbon and nitrogen source, respectively, washed with sterile water and then transferred into minimal medium with the indicated carbon source. Induction was achieved after 5–7 h of additional growth on 0.1% of fructose with either 0.5% of ethanol or 50 mM of ethyl methyl ketone (EMK). For repression conditions 1% glucose was supplied simultaneously with the inducer to the medium. When indicated, yeast extract was added to the final concentration of 0.1%. Growth conditions for
1 Abbreviations used: ADH, alcohol dehydrogenase; GUS, b-glucuronidase; EMK, ethyl methyl ketone.
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A. nidulans were as described previously (Mathieu and Felenbok, 1994). Transformation of both aspergilli species was performed according to the standard procedure (Tilburn et al., 1983), except for the protoplasting of A. niger which was achieved at elevated concentrations (20 mg/ml) of Novozyme (Sigma–Aldrich, USA). 2.2. Plasmid construction To construct the plasmid containing both the alcR and the alcA genes, we used the previously made bAN7 vector (Panozzo et al., 1998) which carries the alcR encoding sequence under the control of the constitutive gpdA promoter. The 3.2-kb SpeI–NotI genomic fragment containing the entire alcA gene was cloned into the corresponding restriction sites of bAN7 resulting in the pAN10 construct. This plasmid was used to cotransform A. niger together with the pGW635 vector bearing the pyrA gene (Goosen et al., 1987). To generate the pGRP3 plasmid, the 3.9-kb XbaI– XbaI fragment of pGW635 containing the pyrA gene (Goosen et al., 1987) was first inserted into the XbaI site of pBluescript KSþ and then reisolated from the above vector via the NotI–EcoRI cut. The latter insert was cloned into bAN7 digested with the same enzymes. The pyrA gene appeared to be in the orientation opposite to gpdA:alcR. The alcA:uidA fusion in which the E. coli b-glucuronidase gene is driven by the alcA promoter was constructed as follows. The alcA promoter region starting from )423 bp to the ATG codon was amplified by PCR and was used to replace the gpdA sequence in the pAN52-1 vector (Punt et al., 1990) resulting in pAN52A. The required EcoRI and NcoI sites at the 50 and 30 termini of the PCR product were introduced during the amplification. The GUS coding sequence produced by PCR amplification from the plasmid pTRAN3 (Punt et al., 1991) was inserted in frame between the alcA promoter and the trpC terminator in the unique NcoI site of the pAN52-A vector. This alcA:uidA expressing plasmid was used in further experiments. 2.3. DNA and RNA analysis To screen for transformants that have integrated the alcA and alcR genes, a PCR approach with genomic DNA was used. Aspergillus DNA was isolated as described by Specht et al. (1982). Different portions of the alcA and alcR genes integrated into A. niger genome were amplified using the following pairs of oligos: GPD: 50 -CCCGCTTGAGCAGACATCACCATG0 3 and RB 2169: 50 -GCTATGTCTGCAGGAACATT-30 for pgpdA; RT 1598: 50 -CGGTGATTTCAGCATGGG30 and RB 3271: 50 -AGTGAAGAGCGCGCAAG-30 for alcR; AT 3119: 50 -TTCTCAATCTCAGGAAGCAA
91
GC-30 and AB 3624: 50 -GCTTATTCTGGCATCTCTA GC-30 for alcA. Southern blot analysis was performed according to Sambrook et al. (1989). Total RNA was extracted as described by Lockington et al. (1985) and separated on glyoxal agarose gels according to Sambrook et al. (1989). The 32 P-labeled probes used were the entire alcA and alcR genes cloned into pBluescript. Ribosomal RNAs visualized by methylene blue staining served as an internal control to normalize the amounts of RNA loaded on a single blot. 2.4. Enzyme assays ADH and b-glucuronidase (GUS) activities were measured in crude protein extracts isolated from ground mycelia in the extraction buffer composed of 50 mM Naphosphate, pH 7.2, 100 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 1 mM PMSF. ADH activity was measured in 100 mM Na-phosphate buffer, pH 8.5, 2 mM MgCl2 , 4 mM NADþ , 0.2 M ethanol at 30 °C. In the absence of ethanol, the rate of NADþ reduction was negligible indicating that the specific dehydrogenase activity can be attributed to ADH. Specific activities were expressed in micromole of NADH formed per 1 min per mg of protein under conditions used. The GUS activity was measured as described by Jefferson (1987) using p-nitrophenyl-b-glucuronide (Sigma–Aldrich, USA) as a substrate. Specific activities were expressed in nmol of pnitrophenol formed per min per mg in crude protein extracts.
3. Results 3.1. No inducible alcohol dehydrogenase activity exists in A. niger A. niger grows very poorly on ethanol as a sole carbon source. Furthermore the fungus appears extremely resistant to high concentrations of allylalcohol (up to 25 mM, A. Dumay and F. Lenouvel, personal communication), suggesting that this compound is poorly metabolized by ADH into a highly toxic compound, acrolein (results not shown). This result could be explained by a low ADH activity. In fact, endogenous ADH activity was detected in crude extracts of A. niger mycelia grown under different physiological conditions: (i) non-induced (neutral carbon source); (ii) induced by ethanol: (iii) repressed by glucose in the presence of ethanol. ADH activity was compared with that in A. nidulans grown in the same conditions. Table 1 shows that in A. niger, the ADH activity did not change significantly in different growth conditions, whereas in A. nidulans, as expected, a strong induction of ADH was observed. Interestingly, comparable specific ADH
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Table 1 ADH activities in A. nidulans and A. niger wild-type strains under different growth conditions Fungus
Carbon source
ADH activity, U/mga
A. niger (WT)
1% Glucose 0.5% Ethanol + 0.1% fructose 0.5% Ethanol + 1% glucose
21 39 25
A. nidulans (WT)
1% Glucose 0.5% Ethanol + 0.1% fructose 0.5% Ethanol + 1% glucose
1 148 4
a
All mycelia were pregrown overnight on minimal medium with 1% glucose at 30 °C, washed and then transferred to minimal medium containing different carbon sources as indicated. The ADH activities were measured in cell extracts of mycelia after additional growth for 5 h in the presence of the inducer ethanol and /or glucose as indicated in Section 2 and expressed in U/mg.
activity was found in A. niger in all three growth conditions, indicating that ADH was constitutively expressed in A. niger. In contrast, as expected, in A. nidulans ADH activity was low in non-induced conditions, drastically increased upon induction, and was fully repressed in the presence of glucose. Therefore, the pattern of regulation of ADH activity is completely different in the two aspergilli species. Next, it was important to determine if the alcR–alcA homologues were present or not in A. niger. Southern blots of A. niger genomic DNA were probed with the A. nidulans alcR and alcA genes. No cross-hybridization was observed under low stringency conditions suggesting that probably no homologues are present in the genome of A. niger (Fig. 1). Therefore, transformants of A. niger containing the alcR and alcA genes from A. nidulans should be easily identified and analyzed straightforward. 3.2. Truncated copies are often generated when A. niger is transformed with A. nidulans alcR and alcA genes To test the expression of an introduced alcA gene in A. niger, we decided to overexpress alcR under the constitutive derepressed gpdA promoter to avoid limiting amounts of AlcR in the cell. A. niger pyrA6 strain was co-transformed with a plasmid bearing the gpd:alcR–alcA gene construct (Mathieu et al., 2000) and a plasmid containing the pyrA gene (Goosen et al., 1987), used as a selective marker (see Fig. 1). Transformants were analyzed after selection for uridine prototrophy. Among 120 transformants, only 7 had integrated full length alcR and alcA copies whilst 73 transformants had integrated truncated copies of either the alcR or the alcA genes or both. Fig. 1 shows a typical Southern blot probed with alcR and alcA in which truncated copies (shown by arrows) were observed as compared to the expected size of the full-length plasmid integrated in the transformant
T2alc (lane 4). Chromosomal rearrangements were tested by PCR products as specified in Fig. 1. Transcriptional analysis of these transformants is shown in Fig. 2. No induced alcA transcript was observed in the A. niger transformant T1alc (lanes 1), as the result of a truncation of the gpd:alcR gene, rendering it non functional. These genomic rearrangements have not been described previously with other heterologous genes in A. niger. 3.3. The A. nidulans alcA gene is induced in A. niger transformants We examined the transcription of the A. nidulans alcA gene in the A. niger transformant, T2alc, containing the intact A. nidulans gpd:alcR gene. Two inducers, both known to induce the A. nidulans alc system, ethyl methyl ketone (EMK) and ethanol (Et), were tested. Northern blots are shown in Fig. 2. In the A. niger transformant T2alc, the alcA gene was inducible by both carbon compounds (lanes 2). The level of the alcA induced transcription was comparable in both organisms. Interestingly, ethanol was a better inducer than EMK in A. niger while the opposite situation occurs in A. nidulans (Flipphi et al., 2002). A partial derepression of the alcA gene was observed in both aspergilli, as expected in the constitutive derepressed gpd:alcR background as previously shown in A. nidulans (Panozzo et al., 1998; this work). The second feature that differentiates both aspergilli is the steady-state level of transcription of alcA under non-inducing conditions which is much higher in A. niger than in A. nidulans. To ascertain that the transcribed alcA gene from A. nidulans was correctly expressed in A. niger, ADH activity was measured in A. niger crude extracts after induction by ethanol. Fig. 3 shows that a clear induction of ADH activity was observed after 5 h of induction and this increase was 5-fold higher after 7 h. The non-induced level of ADH activity was high in agreement with the transcriptional analysis in T2alc shown in Fig. 2. 3.4. Heterologous expression of b-glucuronidase (GUS) driven by the alcA promoter is very efficient in A. niger Next, we wanted to test if the A. nidulans alcA promoter was able to drive the heterologous expression of a non-fungal protein in A. niger. For this we choose the GUS protein from E. coli which is an easily measurable reporter enzyme (Jefferson, 1987). Co-transformation in A. niger pyrA6 strain was carried out with the gpd:alcR–pyrA construct together with alcA:uidA plasmid. Transformants pyrþ were selected which harbored both constructs fully intact in single copies. Fig. 4A shows that in this gpd:alcR context, GUS activity (U/mg) in A. niger was highly inducible when driven by the alcA promoter. A 10-fold induction
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Fig. 1. Southern blots of A. niger transformants containing alcR and alcA genes from A. nidulans. Co-transformation of the A. niger pyrA6 strain was performed with the gpd:alcR–alcA plasmid (pAN10) together with the pyrA plasmid as described in Section 2. After selection for uridine prototrophy, DNA of transformants carrying both the alcR and alcA genes were submitted to Southern blot analysis after enzyme cleavage with the restriction enzymes SpeI and HindIII as indicated. SpeI site is unique and HindIII site is absent in pAN10. Left panel, alcR probe; right panel, alcA probe. Chromosomal rearrangements occurred resulting in truncated restriction fragments which are indicated by arrows in lanes 1, 2, and 3. The A. niger transformant, T2alc, (lane 4) shows a single integration event of the entire construct. Lane 2 contains 3-fold less amount of DNA. Lane R is the A. niger recipient strain. Differences in band intensities upon hybridization with the alcR or alcA probes (lanes 2 and 3) could be indicative of insertions of the truncated construct which lacks either the alcR or alcA full-length sequences. The map of the plasmid pAN10 is indicated at the bottom. Chromosomal rearrangements were identified using PCR products as probes, obtained with the corresponding oligonucleotides mapping in the alcA gene (AT 3119, AB 3624) and in the gpd:alcR gene (gpd, RB 2169, and RT 1598, RB 3271) as described in Section 2.
was observed after 7 h which increased even further after 12 h, showing remarkable stability. The induced production of GUS was only repressed partially by glucose as expected in the gpd:alcR context. Interestingly, when changing the medium composition of A. niger by adding a low concentration of yeast extract (0.1%), an almost constitutive expression of GUS was observed. We wanted to know if this constitutive expression was related only to the addition of yeast extract. Therefore, the same experiments with the same constructs were carried out in A. nidulans. Fig. 4B shows that in A. nidulans, GUS activity was strongly induced by ethanol. In addition, the presence of yeast extract did not lead to constitutive expression of GUS. Therefore, the differences observed upon addition of
yeast extract, likely reflect a different physiology between the two species of aspergilli. 4. Discussion This work aimed to study the expression in A. niger of both the A. nidulans alcR and alcA genes to apply this efficient expression system for heterologous protein production in an industrial fungal species. A. niger does not possess the equivalent of the A. nidulans alc system. There is no endogenous, inducible alcohol dehydrogenase activity even though there is a measurable constitutive ADH activity. Therefore, inducible heterologous protein expression was possible using the A. nidulans alc system in A. niger.
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Fig. 2. Northern blot analysis of A. niger transformants containing the alcR–alcA genes from A. nidulans grown under various physiological growth conditions. Two A. niger transformants T1alc and T2alc and one of A. nidulans containing the gpd:alcR–alcA construct were grown under the conditions described in Section 2. NI, non-induced growth conditions; Et, induction with 1% ethanol during 5 h; EMK, induction with 50 mM EMK (ethyl methyl ketone) during 5 h; EtG, 1% glucose repressed conditions in the presence of 1% ethanol during 5 h. The membranes were hybridized with the alcR and alcA 32 P-labeled probes as indicated on the right of the Northern blot. The amount of ribosomal RNA (rRNA) was visualized after staining the membrane with methylene blue. Hybridization with the alcR probe reveals an additional band of 1.6 kb in the A. nidulans RNA sample which results from the endogenous truncated transcript of the recipient strain.
Fig. 3. Alcohol dehydrogenase activity of the A. niger transformant T2alc containing the gpd:alcR–alcA genes. Alcohol dehydrogenase activity was measured during induction with 1% ethanol up to 7 h in the A. niger transformant T2alc (closed lozenges) and in the A. niger recipient strain R (open squares). ADH activity was expressed in U/mg as defined in Section 2 and under the legend to Table 1.
Our results show that the alc system is transferable to A. niger where it is as highly expressed as in A. nidulans. In the presence of non-limiting amounts of the AlcR protein (constitutively overexpressed from the gpdA promoter), a high level of the alcA gene transcription was observed. This leads to high expression of the encoded alcohol dehydrogenase or an even higher activity of the heterologous GUS protein driven by the alcA promoter. Moreover, in A. niger, the alcA promoter was found to be partially repressed in the presence of glucose
and ethanol. This repression is of the same order as that observed in A. nidulans in the gpd:alcR background (Panozzo et al., 1998). Therefore in A. niger, the equivalent of CreA homologue, is able to repress the heterologous alcA promoter. This result is expected since both proteins recognize the same GC rich targets and share almost 90% identical amino acid residues (Drysdale et al., 1993). Therefore, we can conclude that in A. niger, the transcriptional and translational machineries act efficiently with respect to expression of the heterologous A. nidulans alcA gene. However, some unexpected results were observed upon A. niger transformation with the alcR–alcA system. First, there was a high percentage of genomic rearrangements observed upon transformation with the alc genes. We do not know whether the observed deletions of the alc genes when integrated into A. niger DNA are specific to the alc system or reflects a general phenomenon since such an observation has not been described so far. Stability of the transformants was not monitored rigorously. However, a loss of the inducible ADH activity in a few cases was observed after several rounds of plating. More interesting are the differences observed between the two aspergilli species when submitted to the same growth conditions. Our results raise the important question of the nature of ethanol metabolism in A. niger. We know from the studies of the A. nidulans alc regulon that ethanol itself is not a direct inducer, but has to be metabolized into acetaldehyde, the physiological inducer (Flipphi et al., 2001, 2002). This compound is at a cross
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Fig. 4. b-Glucuronidase (GUS) activity of A. niger and A. nidulans transformants containing gpd:alcR and palcA:uidA construct. (A) Kinetics of GUS activity of A. niger transformant containing gpd:alcR and palcA:uidA genes grown under various growth conditions: noninduced conditions (NI, empty lozenges); induced by 50 mM EMK (closed squares); induced by 1% ethanol (Et, open triangles); repressed by 1% glucose in the presence of 1% ethanol (EtG, crosses). GUS activity was followed up to 12 h and expressed in U/mg as described in Section 2. (B) Kinetics of b-glucuronidase activity in A. niger and A. nidulans transformants containing gpd:alcR and alcA:uidA genes grown in the presence of 0.1% yeast extract and induced with 1% ethanol up to 12 h. Transformants in A. niger are shown as open lozenges and in A. nidulans as closed squares. GUS activity was expressed in U/mg as described in Section 2.
road between several catabolic pathways and its accumulation in A. nidulans depends mostly, as mentioned previously, on the fine tuning between the two structural enzymes (ADHI and ALDH) of the ethanol utilization pathway. In A. niger, it was observed that ethanol was an even better inducer of the alc genes than EMK, the best inducer in A. nidulans. This result implies that ethanol should be metabolized into acetadehyde by an endogenous non-inducible ADH and/or by another unknown activity. The ADH activity measured in crude extracts from A. niger was constitutive and was much lower than the inducible ADH in A. nidulans. This weaker ADH activity could explain the subsequent increased time required to obtain full induction in A. niger, 7 h, compared to the optimal 2 h induction in A. nidulans. We know from
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A. nidulans data that the induction process is directly related to the level of accumulation of acetaldehyde. Related to that feature, another important difference between the two fungi is the basal level of alcA expression. Our data clearly show that in A. niger transformants the non-induced basal level of alcA is much higher than that in A. nidulans. This effect is manifested at both the transcriptional and the enzyme activity levels. This result may be explained by an accumulation of acetaldehyde from the regular metabolism in A. niger and/or by a weaker ALDH activity. Interestingly, A. niger transformants containing the heterologous construct alcA:uidA in the gpd:alcR background, are substantially induced by ethanol and the steady-state level of activity is of the same order in both organisms: more than 10-fold after 10 h of induction. This indicates that in A. niger, the ADH activity is sufficient to metabolize ethanol into acetaldehyde which accumulates and induces the alcA promoter via AlcR. In the presence of yeast extract a large increase in the basal level of GUS is observed in A. niger but not in A. nidulans. After ethanol induction a constitutive expression is observed in A. niger whereas a very strong increase in GUS activity is observed only in A. nidulans. Therefore the alcA–alcR system itself responds correctly in both aspergilli, but the inducer metabolism differs. It has to be pointed out that yeast extract contains a lot of aminoacids and di-tripeptides. It is likely that in A. niger, deamination, decarboxylation, and subsequent oxidation of these compounds lead to inducer formation. These results emphasize the important differences in metabolism between A. niger and A. nidulans. Our data suggest that high levels of expression of heterologous proteins may be obtained in A. niger using the alcA promoter of A. nidulans. More generally, we can conclude that different parameters are involved in the level of heterologous induced protein accumulation, including the nature of the expressed heterologous protein and the metabolism of the fungal host.
Acknowledgments This work was supported in part by grants from the CNRS (UMR 8621), the University Paris-Sud and the European Communities contracts BIO4-CT96-0535 and QLK3-CT1999-00729. We thank Anne Dumay and Francßois Lenouvel who started the work, and Prof. Barry Holland for correcting the English, and Michel Flipphi and Christian Velot for helpful comments. References Adams, T.H., Timberlake, W.E., 1990. Developmental repression of growth and gene expression in Aspergillus. Proc. Natl. Acad. Sci. USA 87, 5405–5409.
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