A broader role for AmyR in Aspergillus niger: regulation of the utilisation of d-glucose or d-galactose containing oligo- and polysaccharides
- 1.4k Downloads
AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed.
KeywordsAspergillus niger AmyR Polysaccharide degradation Gene regulation
AmyR was the first transcriptional activator of the GAL4 type identified in filamentous fungi that is involved in the degradation of plant polysaccharides (Petersen et al. 1999). Its role in starch degradation has been studied in detail in different Aspergillus species, such as Aspergillus nidulans and Aspergillus oryzae, and AmyR was shown to activate expression of glucoamylase, α-amylase and α-glucosidase in the presence of starch or maltose (Gomi et al. 2000; Petersen et al. 1999; Tani et al. 2001). The amylolytic system has also been studied in Aspergillus niger, and a recent study compared the expression of genes related to starch degradation using micro-array analysis (Yuan et al. 2008) but did not study in detail other genes that may be under AmyR control.
Induction of the amylolytic system in Aspergillus is reported to be caused by the presence of starch or its disaccharide building block, maltose (Barton et al. 1972; Carlsen and Nielsen 2001; Pedersen et al. 2000; Santerre Henriksen et al. 1999), and the latter compound is often suggested to be the low-molecular-weight inducer of the system. However, the actual nature of the inducer has not been studied in detail. It has also been claimed that iso-maltose is the inducing compounds (Kato et al. 2002), but no conclusive evidence has been presented to verify this. d-glucose has been shown to repress the expression of the amylolytic genes (Tsukagoshi et al. 2001) but was also reported as an inducer for the amylolytic genes of A. oryzae (Carlsen and Nielsen 2001). However, no studies have been reported in which low levels of d-glucose were tested for induction of the amylolytic system in Aspergillus.
A role in both induction and repression has been observed previously for d-xylose and the xylanolytic system of A. niger (de Vries et al. 1999b). In this fungus, d-xylose induces the xylanolytic regulator XlnR that activates expression of xylanolytic and cellulolytic genes. The concentration of d-xylose in the medium affects the expression level of the genes. High d-xylose concentrations result in activation of the carbon catabolite repressor protein CreA that represses the expression of the XlnR-regulated genes.
Although XlnR was originally described as a xylanolytic activator, later studies indicated that it also controlled genes that were not involved in xylan degradation and utilization. These genes encoded enzymes involved in cellulose, xyloglucan and galactomannan degradation (de Vries et al. 1999a; Gielkens et al. 1999; Hasper et al. 2002).
Here, we present a study into the A. niger amyR gene in which we constructed deletion and multicopy strains. Using these strains, we have found that d-glucose induces the amylolytic system depending on the concentration in the medium. In addition, it became clear that AmyR has a broader physiological role than starch degradation in that it controls the production of d-glucose and d-galactose releasing enzymes.
Materials and methods
Strains and media
A. niger strains used in this study
Bos et al. (1988)
ΔargB, pyrA6, leuA1, nicA1
vanKuyk et al. (2004)
ΔargB, pyrA6, leuA1, nicA1, argB +
ΔargB, pyrA6, leuA1, nicA1, ΔamyR:: argB +
ΔargB, pyrA6, leuA1, nicA1, argB + , mcamyR
argBΔ, pyrA6, leuA1, nicA1, argB + , mcamyR
DNA manipulations and molecular biology techniques were performed using standard methods (Sambrook et al. 1989) and Escherichia coli DH5α. Gel extractions were performed using the QIAgen QIAquick gel extraction kit.
RNA analysis was performed as described previously (de Vries et al. 2002). Internal fragments of lacA (A00968, An01g12150; Kumar et al. 1992), aglC (AJ251873, An09g00260/270; Ademark et al. 2001), agdA (An04g06920; Pel et al. 2007), glaA (X00548, An03g06550; Boel et al. 1984), amyA (An04g06930) and amyR (An04g06910; Pel et al. 2007) were used as probes for expression analysis. A 0.7-kb EcoRI fragment from the gene encoding the 18S rRNA subunit (Melchers et al. 1994) was used as an RNA loading control.
Nucleotide sequences were analysed with computer programs based on those of Devereux et al. (Devereux et al. 1984). Sequence alignments were performed by using the Blast programs (Altschul et al. 1990) at the server of the National Center for Biotechnology Information, Bethesda, USA (http://www.ncbi.nlm.nih.gov/blast/). Synteny analysis was performed at the AspGD website (http://www.aspgd.org/) using the Sybil programme.
Extracellular hydrolytic activities were assayed using 0.01% substrate, 20–40 μl sample and 25 mM sodium acetate pH 5.0 in a total volume of 100 μl. The mixtures were incubated for 1 h at 30°C after which the reaction was stopped by adding 100 μl 0.25 M Na2CO3. Absorbance was measured at 405 nm in a microtiter plate reader. The activity was calculated using a standard curve of p-nitrophenol. The substrates used for enzyme assays were all obtained from Sigma and were p-nitrophenol-α-arabinofuranoside, p-nitrophenol-α-xylopyranoside, p-nitrophenol-β-xylopyranoside, p-nitrophenol-α-galactopyranoside, p-nitrophenol-β-galactopyranoside, p-nitrophenol-α-glucopyranoside, p-nitrophenol-β-glucopyranoside, p-nitrophenol-α-fucopyranoside, p-nitrophenol-β-fucopyranoside, p-nitrophenol-β-glucuronoside and p-nitrophenol-β-mannopyranoside to measure α-arabinofuranosidase, α- and β-xylosidase, α- and β-galactosidase, α- and β-glucosidase, β-glucuronidase and β-mannosidase, respectively.
Identification and cloning of amyR
A lambda phage clone was isolated from an A. niger gDNA library using a fragment of the A. oryzae amyR gene as a probe. A 4.3-kb NsiI fragment was subcloned into pGEM-11 and designated pJG01. Sequence analysis showed that this fragment contained the coding region of A. niger amyR as well as the 5′-flanking region and part of the A. niger agdA gene. Further analysis using the Sybil tool at www.aspgd.org (Crabtree et al. 2007) demonstrated that the amylolytic cluster (amyA–aglA–amyR) is highly conserved in all eight sequenced Aspergilli, with only a small insertion downstream of amyR in Aspergillus terreus (Suppl. Fig. 1). In all these species except A. nidulans, the proteolytic regulator PrtT (Punt et al. 2008) lies directly upstream from the cluster.
Construction and analysis of disruption and multicopy amyR strains
A deletion construct (pAmyRd) was made by introducing a PstI site 800 bp downstream of the endogenous PstI site of pJG01. Plasmid pJG01 was then digested with BamHI/NsiI and the liberated fragment cloned into pGEM9. The resulting construct (pJG10) was digested with PstI/BamHI, and a BglII/PstI fragment containing the argB gene of A. niger was inserted. Insertion of this construct at the amyR locus would result in the removal of 1,600 bp, consisting of approximately 600 bp 5′-region and 1,000 bp coding region (including the zinc finger). This fragment was used to transform A. niger NW249. In addition, pJG01 was used in a co-transformation with pIM2101 (containing the A. niger argB gene (Lenouvel et al. 2002)) to obtain amyR multicopy strains in A. niger NW249. Transformants were screened for their ability to grow on starch. Selected transformants from the disruption transformation with reduced growth and from the multicopy transformation with improved growth on starch were used for Southern analysis and demonstrated that multiple disruption and multicopy transformants were obtained (data not shown). Based on this analysis, UU-A001.24 was selected as amyR multicopy (overexpression) transformant and UU-A101.1 was selected as an amyR disruptant.
The influence of AmyR extends beyond amylolytic genes
Based on recent A. niger genome annotations (Coutinho et al. 2009; Pel et al. 2007), genes encoding α-amylase (AMY), glucoamylase (GLA), α-glucosidase (AGD), β-glucosidase (BGL), α-galactosidase (AGL) and β-galactosidase (LAC) were analysed for their expression in an A. niger wild-type and amyR disruptant on maltose using previously published micro-array data (Yuan et al. 2008). This demonstrated that AmyR controls only a small subset of these genes (Suppl. Table 1), but this subset included two BGL-, two AMY-, two AGD-, two GLA-, two AGL and one LAC-encoding genes, supporting the activity assays performed in our study. All these genes contained at least one putative AmyR binding site in their promoter.
A similar expression pattern was observed for glaA, agdA and amyA on d-glucose, and expression was highest at the latest time point (Fig. 2). Expression of aglC and lacA was not reduced in the amyR disruptant compared to the reference on d-glucose but was increased in the amyR multicopy strain.
Influence of AmyR on growth of A. niger on oligo- and polysaccharides
No difference in growth was observed between the strains using sucrose, d-xylose or d-glucose as a carbon source and only a slight reduction in growth of the amyR disruptant on cellobiose and Guar gum. Furthermore, no difference in growth was observed on chitin, N-acetylglucosamine and inulin (data not shown).
Increased concentrations of d-glucose results in altered morphology in amyR multicopy strains
AmyR has been studied in detail in several Aspergilli, with emphasis on genes involved in starch and maltose hydrolysis, although a recent study (Yuan et al. 2008) already indicated that other genes may also be controlled by AmyR. Analysis of enzyme activity in our study provided clear indications for a broader role of this regulator, in that we observed strongly elevated levels of both α- and β-glucosidase as well as α- and β-galactosidase in the amyR multicopy strain compared with the reference. The reduction in enzyme activity in the amyR deletion strain was less obvious, possibly due to the fact that the activities in the reference were already very low. Analysis of publicly available micro-array data of an A. niger wild-type and amyR disruptant during growth on maltose (Yuan et al. 2008) demonstrated that some, but not all genes encoding these enzymes (Coutinho et al. 2009), were downregulated in the disruptant strain, suggesting regulation by AmyR. All the downregulated genes contained at least one putative AmyR binding site, which would be a pre-requisite for direct AmyR regulation. For one putative α-galactosidase encoding gene (An04g02700), no putative AmyR binding site was identified in the DSM genome strain (CBS 513.88) (Coutinho et al. 2009). However, a putative binding site was detected in the genome of A. niger ATCC 1015 that was sequenced by JGI (Andersen et al. 2011). As we used the laboratory strain N402, this result suggests that this strain resembles ATCC 1015 with respect to the promoter of this gene. A similar difference was observed previously for the acetyl xylan esterase encoding gene axeA (R.P. de Vries, unpublished data). In both N402 and ATCC 1015, the promoter of this gene contains an XlnR consensus sequence, while CBS 513.88 does not. Also, several of the genes whose expression is not affected in the amyR disruption strain contain putative AmyR binding sites in their promoter. This confirms that the presence of such a sequence is no direct indication for regulation by AmyR, as was concluded previously for AmyR, XlnR and other regulators of Aspergillus (Coutinho et al. 2009). However, the absence of such binding sites strongly suggests that the gene is not (directly) regulated.
These data were confirmed for one α-galactosidase encoding gene (aglC) and one β-galactosidase encoding gene (lacA) using Northern analysis. On maltose and starch these two genes followed a similar pattern as the established AmyR regulated genes glaA, amyA and agdA. However, some differences were observed. The expression of lacA was only observed at late time points (8 h on maltose and 24 h on starch), while aglC expression peaked at 2 h, and the other genes were expressed on all time points. The reduced expression of aglC at later time points is not due to a reduction in expression compared with the pre-culture, as micro-array data from another study (R.P. de Vries, unpublished results) demonstrates lower aglC expression for the pre-culture than after 2 h on maltose or glucose. The expression of aglC in the amyR deletion strain was higher than that observed for the other genes tested, suggesting an AmyR-independent induction of aglC. Expression of aglC on d-glucose was observed previously (Ademark et al. 2001) and was shown to be independent of the major carbon catabolite repression system CreA (Ruijter and Visser 1997) while d-galactose induction of this gene was not observed (Ademark et al. 2001).
Expression of agdA in the reference strain on glucose was only observed after prolonged incubation, which correlates well with detectable α-glucosidase activity during growth of this strain on glucose as these activities were measured after 6 h. The activity level is higher than expected based on the expression of agdA, but this is likely due to the presence of additional α-glucosidases in the medium that are also produced under this condition.
Regulation of α- and β-glucosidase and α- and β-galactosidase activity by AmyR was further supported by growth profiling. Reduced growth of the amyR deletion strain compared with the reference was observed on substrates containing α- (starch, maltose, melibiose, melezitose, raffinose, sucrose) and β-linked d-glucose (cellobiose) as well as α- (melibiose, raffinose, carrageenan) and β-linked d-galactose (lactose, carrageenan), while improved growth on several of these substrates was observed for the amyR multicopy strain. This provides in vivo evidence for a function for AmyR in utilization of these oligo- and polysaccharides by A. niger. This situation is similar to that for another Aspergillus transcriptional regulator related to polysaccharide degradation: XlnR. This protein was first described as a xylanolytic regulator (van Peij et al. 1998b) but was later shown to also regulate genes involved in cellulose degradation (van Peij et al. 1998a) and galactomannan degradation (de Vries et al. 1999a).
Several reports have provided evidence for different inducing compounds of the amylolytic system in Aspergillus. Most commonly, maltose is suggested to be the inducer of AmyR (Barton et al. 1972; Carlsen and Nielsen 2001; Pedersen et al. 2000; Santerre Henriksen et al. 1999), but also glucose (Carlsen and Nielsen 2001). Another study claimed that the inducer is iso-maltose (Kato et al. 2002), but a conclusive study has so far not been published. A. niger has been shown to produce high levels of glucoamylase in the presence of maltose or starch (Barton et al. 1972; Gouka et al. 1997a, b; Pedersen et al. 2000; Schrickx et al. 1995). In natural biotopes, high levels of maltose are uncommon, and it is therefore unlikely that the maltose liberated by A. niger during hydrolysis of starch is able to avoid the high levels of glucoamylase and get imported into the cell. It is more likely that (nearly) all maltose is hydrolysed extracellularly to d-glucose. Our study demonstrates that AmyR-regulated genes in A. niger are induced during growth on low levels of d-glucose, as their expression increases during the cultivation, which is similar to what was shown previously in A. oryzae (Carlsen and Nielsen 2001). As d-glucose has been shown to act as a repressor through the carbon catabolite repressor protein CreA (Ruijter and Visser 1997), the expression levels are likely a balance between induction through AmyR and repression through CreA. A previous study showed a similar effect for d-xylose and XlnR, in which higher d-xylose concentrations resulted in reduced expression of xylanolytic genes, mediated by CreA (de Vries et al. 1999b). In most studies reported previously (reviewed in Tsukagoshi et al. 2001), d-glucose is considered a repressing rather than an inducing carbon source. However, usually 1–3% (67–200 mM) of d-glucose is used in these studies, which are concentrations at which the repression through CreA probably overrules induction through AmyR. These data suggests that in A. niger d-glucose or a metabolic product thereof is the inducer of AmyR as was shown previously for A. oryzae (Carlsen and Nielsen 2001). Higher induction of the AmyR-regulated genes on maltose and starch than on d-glucose supports this hypothesis, as growth on these substrates would give a gradual release of d-glucose, resulting in very low steady-state levels. Based on studies in other Aspergilli, differences in the possible inducing compounds may exist between species of this genus. The presence of the recently discovered mal-cluster (Hasegawa et al. 2010; Vongsangnak et al. 2009) in A. oryzae that consists of a putative second maltose-responsive regulator, a maltose transporter and a maltase, suggests that, in this species, at least part of the maltose is transported into the fungal cell. This cluster is also present in Aspergillus flavus, Aspergillus clavatus, Neosartorya fischeri and Aspergillus fumigatus but not in A. niger, A. nidulans and A. terreus (Suppl. Fig. 2), suggesting a different approach to starch utilisation in these two groups of Aspergilli.
The increased hydrolysis of maltose to d-glucose in the amyR multicopy strains is supported by similar morphology of these strains on maltose and on high concentrations of d-glucose. This morphology is likely due to the presence of a high intracellular concentration of d-glucose or a metabolite thereof as it is not observed in the reference strain when it is exposed to high extracellular d-glucose concentrations. This implies that d-glucose transport is upregulated in the multicopy strains, resulting in this higher intracellular concentration of d-glucose or a metabolic product of d-glucose. Analysis of the expression of the three A. niger d-glucose transporters reported previously (Jorgensen et al. 2007; vanKuyk et al. 2004) in the published AmyR micro array data set (Yuan et al. 2008) demonstrated that two of these genes (both encoding high affinity d-glucose transporters) are downregulated in the amyR deletion strain, suggesting control by AmyR. However, considering the presence of multiple putative d-glucose transporters in the A. niger genome (Pel et al. 2007), more functional data is needed on these other putative d-glucose transporters before any firm conclusion can be drawn. A similar growth phenotype was observed for strains under secretion stress (Carvalho et al. 2011), but it is unlikely that this can explain our results. It has been well-documented that AmyR-regulated genes are subject to carbon catabolite repression (Tsukagoshi et al. 2001), indicating that an increase in glucose concentration would reduce expression of these genes, resulting in lower protein production and likely also reduced rather than increased secretion stress
In conclusion, our study has shown that the influence of AmyR in A. niger extends beyond starch hydrolysis and suggests a role for d-glucose or a metabolic product thereof as the inducer of the AmyR system in this fungus.
The authors thank Ec Agbo, Joep Geerlings and Dirk Blom for technical assistance.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Google Scholar
- Andersen MR, Salazar MP, Schaap PJ, van de Vondervoort PJ, Culley D, Thykaer J, Frisvad JC, Nielsen KF, Albang R, Albermann K, Berka RM, Braus GH, Braus-Stromeyer SA, Corrochano LM, Dai Z, van Dijck PW, Hofmann G, Lasure LL, Magnuson JK, Menke H, Meijer M, Meijer SL, Nielsen JB, Nielsen ML, van Ooyen AJ, Pel HJ, Poulsen L, Samson RA, Stam H, Tsang A, van den Brink JM, Atkins A, Aerts A, Shapiro H, Pangilinan J, Salamov A, Lou Y, Lindquist E, Lucas S, Grimwood J, Grigoriev IV, Kubicek CP, Martinez D, van Peij NN, Roubos JA, Nielsen J, Baker SE (2011) Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88. Genome Res 21:885–987CrossRefGoogle Scholar
- Barton LL, Georgi CE, Lineback DR (1972) Effect of maltose on glucoamylase formation by Aspergillus niger. J Bacteriol 111:771–777Google Scholar
- Boel E, Hjort I, Svensson B, Norris F, Norris KE, Fiil NP (1984) Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs. EMBO J 3:1097–1102Google Scholar
- Carlsen M, Nielsen J (2001) Influence of carbon source on α-amylase production by Aspergillus oryzae. Appl Microbiol Biotechnol 57:346–349Google Scholar
- Coutinho PM, Andersen MR, Kolenova K, vanKuyk PA, Benoit I, Gruben BS, Trejo-Aguilar B, Visser H, van Solingen P, Pakula T, Seiboth B, Battaglia E, Aguilar-Osorio G, de Jong JF, Ohm RA, Aguilar M, Henrissat B, Nielsen J, Stalbrand H, de Vries RP (2009) Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae. Fungal Genet Biol 46(Suppl 1):S161–S169CrossRefGoogle Scholar
- Crabtree J, Angiuoli SV, Wortman JR, White OR (2007) Sybil: methods and software for multiple genome comparison and visualization. Methods Mol Biol 408:93–108Google Scholar
- de Vries RP, van den Broeck HC, Dekkers E, Manzanares P, de Graaff LH, Visser J (1999a) Differential expression of three α-galactosidase genes and a single β-galactosidase gene from Apergillus niger. Appl Environ Microbiol 65:2453–2460Google Scholar
- de Vries RP, van de Vondervoort PJI, Hendriks L, van de Belt M, Visser J (2002) Regulation of the α-glucuronidase encoding gene (aguA) from Aspergillus niger. Mol Gen Genet 268:96–102Google Scholar
- Gielkens MM, Dekkers E, Visser J, de Graaff LH (1999) Two cellobiohydrolase-encoding genes from Aspergillus niger require d-xylose and the xylanolytic transcriptional activator XlnR for their expression. Appl Environ Microbiol 65:4340–4345Google Scholar
- Gouka RJ, Punt PJ, van den Hondel CA (1997b) Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels. Appl Environ Microbiol 63:488–497Google Scholar
- Melchers WJG, Verweij PE, van den Hurk P, van Belkum A, de Pauw BE, Hoogkamp-Korstanje AA, Meis JFGM (1994) General primer-mediated PCR for detection of Aspergillus species. J Clin Microbiol 32:1710–1717Google Scholar
- Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, Turner G, de Vries RP, Albang R, Albermann K, Andersen MR, Bendtsen JD, Benen JA, van den Berg M, Breestraat S, Caddick MX, Contreras R, Cornell M, Coutinho PM, Danchin EG, Debets AJ, Dekker P, van Dijck PW, van Dijk A, Dijkhuizen L, Driessen AJ, d’Enfert C, Geysens S, Goosen C, Groot GS, de Groot PW, Guillemette T, Henrissat B, Herweijer M, van den Hombergh JP, van den Hondel CA, van der Heijden RT, van der Kaaij RM, Klis FM, Kools HJ, Kubicek CP, van Kuyk PA, Lauber J, Lu X, van der Maarel MJ, Meulenberg R, Menke H, Mortimer MA, Nielsen J, Oliver SG, Olsthoorn M, Pal K, van Peij NN, Ram AF, Rinas U, Roubos JA, Sagt CM, Schmoll M, Sun J, Ussery D, Varga J, Vervecken W, van de Vondervoort PJ, Wedler H, Wosten HA, Zeng AP, van Ooyen AJ, Visser J, Stam H (2007) Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 25:221–231CrossRefGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning—a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory, Cold Spring HarbourGoogle Scholar
- van Peij N, Gielkens MMC, de Vries RP, Visser J, de Graaff LH (1998a) The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl Environ Microbiol 64(10):3615–3619Google Scholar