Abstract
The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus.
Basidiomycetous fungus Lentinula edodes (Berk.) Pegler is the second most widely cultivated mushroom in the world. The cultivation of this fungus makes use of significant amounts of woody polysaccharides, and utilization of the complex polysaccharides is dependent on its ability to synthesize hydrolytic and oxidative enzymes which convert woody polysaccharides into low-molecular-weight compounds that can be absorbed and assimilated for nutrition. Starch is a polymer of glucose and is perhaps, next to cellulose, the most widely available polymeric glucoside made by plants (16, 18). Starch is, therefore, available to fungi growing on plants or plant residues. Digestion of starch requires a complex of enzymes. Glucoamylases (1,4-α-d-glucan glucohydrolases; EC 3.2.1.3) are enzymes that, among others, are believed to be important in the utilization of starch by the basidiomycetous fungus. Glucoamylases are exohydrolases, which catalyze the release of β-d-glucose units from the nonreducing ends of amylose, amylopectin, and other polysaccharides (18). Glucoamylase-encoding genes have been cloned from several fungi including Aspergillus awamori (6, 15), Aspergillus niger (1, 3), Aspergillus oryzae (8), Aspergillus terreus (5, 23), and Neurospora crassa (22).
Although there have been many reports on glucoamylases in fungi, few studies on the production and regulation of glucoamylases in basidiomycetous fungi have been carried out. El-Zalaki and Hamza (2) studied five basidiomycetous fungus species for their ability to hydrolyze starch. L. edodes was found to be the most promising strain for amylase production. It was also reported that the glucoamylase from L. edodes hydrolyzed starch and glycogen, converting them almost completely into glucose (27). Although these studies have provided valuable information on mushroom glucoamylase physiology, molecular studies on this enzyme have not been initiated. We aimed to address this present void, first by cloning and characterizing an L. edodes glucoamylase gene (gla1), second by comparing the levels of glucoamylase activity and gene expression in L. edodes strains grown on a variety of substrates, and third by assessing gla1 expression in various developmental stages of mushroom development.
MATERIALS AND METHODS
Organisms and culture conditions.
L. edodes L54-A (monokaryon) and L54-B (monokaryon) and their mated product, L54 (dikaryon), were cultured on a high-nitrogen (HN) medium as previously described (30, 31). Fruit body primordia and mature mushrooms were obtained during a 6-week inoculation. The fruiting process was carried out in the HN medium supplemented with 1% (wt/vol) potato extract (PE) and 5% (wt/vol) sawdust (32).
Enzyme assays.
Glucoamylase activity was measured in 0.1 M sodium acetate (pH 5.0) as the release of reducing sugars from 1% soluble starch (Sigma) (20, 26). One unit of enzyme activity is defined as the amount of enzyme required to release 1 μmol of reducing sugar per min. In some cases, 1% glycogen (Sigma) was also used as a substrate to detect glucoamylase activity. All the reactions were performed at 30°C. A sample without enzyme was used as a control (30).
Preparation of genomic libraries.
L. edodes genomic DNA was prepared as previously described (32). After digestion, the DNA was ligated with DASHII arms (Stratagene) and packaged in vitro with a Gigapack II kit (Stratagene) as described by the supplier.
First-strand cDNA synthesis.
Total RNA (5.0 μg) was prepared as previously described (32) and used to synthesize cDNA. Reverse transcriptions were carried out in 20-μl reaction mixtures containing 50 U of Moloney murine leukemia virus reverse transcriptase (GIBCO), 15 pmol of oligo(dT)15, and 20 U of RNasin (Promega). Reactions were performed at 25°C for 10 min, at 45°C for 45 min, and at 75°C for 5 min.
Isolation of differentially expressed genes during L. edodes development.
The total RNAs from each of the three developmental stages were fingerprinted by RNA arbitrarily primed PCR (RAP-PCR) (25). Ten microliters of RAP-PCR products from each of the three developmental stages was resolved on a 3% (wt/vol) Nusieve agarose gel (Promega). Differential bands that appeared in some stages but not in others were cut from the gel. The gel slice was put into 100 μl of 10 mM Tris-HCl (pH 8.0) and heated at 65°C for 5 min to extract the DNA. Five microliters of the eluate was used for reamplification with the same primers that generated the fingerprint, and reamplified PCR products were sequenced.
Library screening and product cloning.
Approximately 200 ng of DNA from a genomic library was added to a PCR mixture containing 2.0 U of Taq polymerase (Promega), 1× buffer (Promega), 2.5 mM MgCl2, 100 μM (each) deoxynucleoside triphosphate, and a 1.0 μM concentration of each of two primers. Four primers in four combinations were used for library screening: LeAMG5A (5′-TTCTTGCGAGTATTCACACG-3′), LeAMGL1 (5′-TCGAGCGATCCTTGACTATT-3′), T3 (5′-AATTAACCCTCACTAAAGGG-3′), and T7 (5′-GTAATACGACTCACTATAGGGC-3′). The following PCR cycle parameters were used: 4 min at 94°C for 1 cycle; 1 min at 94°C, 1 min at 58°C, and 5 min at 72°C for 35 cycles; and 10 min at 72°C for 1 cycle. Rapid amplification of cDNA ends was used to obtain full-length cDNA clones, allowing comparisons of genomic and cDNA sequences to be made (12). PCR products were cloned into PCRscript SK (Stratagene) or sequenced directly.
DNA sequencing.
The nucleotide sequences of PCR products were determined by using Taq polymerase cycle sequencing and an automated DNA sequencer (ABI 310; Perkin-Elmer Corp.). All DNAs were sequenced on both strands, and the encoded amino acid sequences were predicted by using Gene Jockey (Biosoft). Sequences were aligned by using SeqEd, version 2.0, software (Applied Biosystems).
Competitive PCR.
Relative transcript levels of L. edodes glucoamylase genes were determined by competitive PCR (32). Primers LeAMG3B (5′-TCTACGAATGAGGCTGTCCT-3′) and LeAMGL2 (5′-GCAGTGATCGTCGAGTCAAA-3′) were used to amplify gla1. The lengths of the PCR products for genomic DNA and cDNA were 247 and 194 bp, respectively. The competitive templates consisted of full-length genomic copies of the genes which had been amplified by PCR, and the concentrations of templates were estimated by gel electrophoresis (19). To determine the concentration of cDNA, serial titration tests including 20 to 40 cycles were performed. The optimized competitive PCR mixture contained (in a final volume of 20 μl) 0.2 U of Taq polymerase (Promega), 1× buffer (Promega), 2.5 mM MgCl2, 100 μM (each) deoxynucleoside triphosphate, and a 1.0 μM concentration of each of the primers. The following competitive PCR parameters were used: 4 min at 94°C for 1 cycle; 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C for 30 cycles; and 10 min at 72°C for 1 cycle. The PCR products were size fractionated in 2% (wt/vol) agarose, stained with ethidium bromide, and analyzed by using Molecular Analyst, version 1.5, software (Bio-Rad).
Nucleotide sequence accession number.
The nucleotide sequence of the L. edodes gla1 gene has been deposited in the GenBank database under accession no. AF220541.
RESULTS
Isolation of developmentally regulated genes in L. edodes.
One hundred RAP-PCR fragments were isolated, cloned, and sequenced. The putative functions of the fragments were identified by comparing their sequences with those in gene databases. One clone with a 390-bp fragment had significant homology with fungal glucoamylase genes in the databases. Based on the partial sequence of glucoamylase, we designed primers LeAMG5A and LeAMGL1 to amplify glucoamylase genes in dikaryotic strain L54.
Isolation and analysis of the glucoamylase genomic sequence by screening the library.
By using a previously described method (29), we successfully amplified 3,094 bp of the glucoamylase gene. A comparison of the sequence of the glucoamylase gene with the sequences of corresponding PCR products from monokaryotic parental strains L54-A and L54-B showed that the cloned gene originates from L54-A. The glucoamylase gene was designated gla1.
gla1 sequence analysis.
A comparison of the genomic and cDNA sequences of gla1 indicated the presence of seven introns varying in size from 46 to 55 bp. All of the intron splice junctions conform to the GT--G rule. DNA sequencing shows that the gla1 open reading frame codes for a putative polypeptide of 571 amino acids. Based on the sequence comparison with fungal glycosyl hydrolases, the Gla1 protein can be assigned to glycosyl hydrolase family 15 (16, 18). The deduced molecular mass of the Gla1 protein is 61,167 Da (Fig. 1).
FIG. 1.
Alignment of the deduced Gla1 amino acid sequence with other fungal glucoamylase sequences. Accession numbers of sequences are as follows: C. rolfsii G2, D49448 (14); A. awamori GA1, K02465 (15); N. crassa GLA1, X67291 (22). Gaps introduced for optimal alignment are indicated by dashes. The consensus sequence is composed of residues shared by at least three of the mature proteins. The putative signal sequences are underlined. Five conserved sequences in catalytic domains are in boldface. The C-terminal SBDs are shaded. Alignment was done with SeqEd, version 2.0, software.
gla1 promoter and terminator sequence analysis.
We sequenced 482 bp upstream of the translation initiation codon and 534 bp downstream of the stop codon. There is a TATA box at position −52 with respect to the ATG codon. There are three potential CAAT boxes upstream of the ATG codon (−256, −261, −312). The 3′ end of the transcript has been located by comparing the cDNA sequence and the genomic sequence. There is a consensus ATAA polyadenylation region in the 142 bp downstream from the TAG stop codon. There is also an AT-rich region approximately 150 bp downstream from the stop codon, which may act as a polyadenylation signal.
Comparison of the deduced Gla1 amino acid sequence.
A comparison of the deduced Gla1 amino acid sequence with those of other fungal glucoamylases suggests the overall structure of the mature protein, which comprises an N-terminal catalytic domain containing regions that have been shown to be essential for enzyme activity, a C-terminal starch-binding domain (SBD), and heavily glycosylated hinge region (16, 17). In addition, the sequence shows a high degree of identity with the glucoamylase sequence from a basidiomycetous fungus Corticium rolfsii and lesser homologies with sequences of other fungal glucoamylases (Fig. 1). Alignment of the amino acid sequences in the N-terminal domain reveals the presence of five regions which are conserved in all fungal glucoamylases (Fig. 1). The SBD is also conserved in the L. edodes Gla1 protein. The similarities between the Gla1 protein and other fungal glucoamylases are from 45 to 61%. The region of highest conservation is found in the catalytic domain (65 to 75%). Based on the sequence alignment and the secondary structure prediction by using the self-optimized prediction method (4), the hinge region in Gla1 is estimated to comprise amino acid residues 446 to 467. Its length is similar to that of the hinge region of basidiomycetous fungus C. rolfsii G2 (amino acid residues 456 to 477) (14) but less than that of the hinge region of A. awamori GA1 (amino acid residues 470 to 514) (18, 21).
Effect of different media on the production of glucoamylase.
The production of glucoamylase by L. edodes strains 54-A, 54-B, and 54 under various conditions was studied (Table 1). There was no significant difference in enzyme activity between mycelia grown in liquid medium and those grown in solid medium (data not shown); therefore, only solid media were used in further studies. Effects of different carbon sources (glucose, starch, crystalline cellulose, and PE) on the production of glucoamylase were compared. In strain L54 grown in HN agar medium, peak glucoamylase activity appeared at day 18 (5.0 mU/g). The presence of 1% glucose decreased glucoamylase production to 3.3 mU/g (Table 1). The presence of 1% starch or 1% PE stimulated glucoamylase production by 2.7- and 2.4-fold, respectively. Furthermore, 1% cellulose did not increase the production of glucoamylase in this strain (Table 1). Monokaryotic strains L54-A and L54-B showed similar patterns of glucoamylase production in agar medium, but with lower activities than those of dikaryotic strain L54 (Table 1).
TABLE 1.
Effect of different media and conditions on the production of enzymes and transcript levels of the glucoamylase gene
Medium or stage | Glucoamylase activitya (mU/gc) for strain:
|
cDNA concentration (pmol/g of total RNA)b for strain L54 | ||
---|---|---|---|---|
L54 | L54-A | L54-B | ||
Growth medium | ||||
HN | 5.0 ± 0.2 (18) | 0.0 | 0.0 | 0.40 ± 0.03 |
HN + 1% glucose | 3.3 ± 0.2 (14) | 0.0 | 0.0 | 0.092 ± 0.004 |
HN + 1% starch | 13.4 ± 0.6 (16) | 2.5 ± 0.2 (24) | 2.6 ± 0.2 (28) | 1.3 ± 0.1 |
HN + 1% cellulose | 5.2 ± 0.2 (18) | 0.0 | 0.0 | 0.49 ± 0.03 |
HN + 1% PE | 12.0 ± 0.6 (14) | 2.2 ± 0.2 (24) | 2.1 ± 0.2 (28) | 0.82 ± 0.05 |
Fruiting medium | ||||
Mycelium | 5.1 ± 0.4 | 2.0 ± 0.2 | No growth | 0.11 ± 0.01 |
Primordia | 20.0 ± 0.7 | No primordium formation | No primordium formation | 2.6 ± 0.2 |
Fruiting body | 26.4 ± 1.2 | No fruiting | No fruiting | 9.9 ± 0.4 |
Mean ± standard deviation from triplicate cultures. Numbers in parentheses indicate the time required for maximum activity (in days).
Mean ± standard deviation from three independent cultures.
Milliunits per gram of medium for growth medium; milliunits per gram of fresh mycelium for fruiting medium.
The production of glucoamylase at various developmental stages (mycelium growth, primordium appearance, and fruiting body formation) in the fruiting HN-PE-sawdust medium was studied. In the mycelium growth stage, the peak glucoamylase activity was 5.1 mU/g. However, the activities of glucoamylase were increased to 20.0 and 26.4 mU/g in primordia and in fruiting bodies, respectively (Table 1). Enzyme activities using glycogen as the assay substrate were similar to those with starch as the assay substrate (data not shown).
Reverse transcription-PCR analysis of gla1 gene expression.
The results of competitive PCR analysis of gla1 expression with various substrates and at different developmental stages are shown in Fig. 2 and Table 1. In HN-starch medium, levels of gla1 mRNA were 3.3- and 14.1-fold higher than those of gla1 mRNA in HN and HN-glucose media, respectively. The lowest level obtained for gla1 mRNA was 0.092 pmol/g of total RNA in HN-glucose medium, whereas the highest level was achieved in the fruiting body. PE (1%) increased the level of gla1 mRNA, but the addition of 1% cellulose decreased the production of gla1. The low level of gla1 mRNA was found in the monokaryotic strain L54-A even though no glucoamylase activity was detected (data not shown). In the fruiting medium (HN-PE-sawdust), the gla1 mRNA level was lower in the mycelium growth stage. However, the fruiting process increased the gla1 mRNA levels to 2.6 and 9.9 pmol/g of total RNA in the primordia and fruiting body, respectively (Table 1).
FIG. 2.
Competitive reverse transcription-PCR analysis of gla1 gene expression in various substrates (A to E) and the fruiting medium (F to H). The amounts of the competitive templates are indicated above the gels as dilution factors. The levels of transcripts in samples were based on estimated equivalence points between competitive products and target cDNAs. The sizes of the PCR products are indicated on the left.
DISCUSSION
Although there have been many reports on the cloning of fungal glucoamylase genes, only one glucoamylase cDNA sequence, from basidiomycetous fungus C. rolfsii, has been reported (14). Here we report for the first time a basidiomycetous fungus glucoamylase gene with a potential 5′ promoter region. The 482-bp sequence upstream of the translation initiation site in gla1 would be useful in the further study of gene regulation and modification.
Both catalytic domains and SBDs in L. edodes Gla1 are homologous to those from other fungi, which indicates that the Gla1 domains may perform functions similar to those performed by these domains in a variety of fungal glucoamylases. The primary sequences of the hinge regions of the fungal glucoamylases are not really conserved among species. Two basidiomycetous glucoamylases, L. edodes Gla1 and C. rolfsii G2, show a strong homology in the hinge regions: both regions contain fewer serine and threonine residues than those from other fungal glucoamylases (14, 16). There are several roles suggested for the hinge region of fungal glucoamylases: contributing to starch degradation (7), extending the peptide backbone (16), and increasing thermostability (16). Semimaru et al. (21) reported that the partial deletion of the hinge region of A. awamori glucoamylase prevented the secretion of enzymes from Saccharomyces cerevisiae host cells. However, Libby et al. (13) reported that deletions of the hinge region of A. awamori glucoamylase did not affect its ability to degrade starch and that the deletions had a negative effect on its thermostability. It remains to be determined what roles the hinge region in Gla1 can play in the enzyme function.
The major function of the fungal glucoamylase is to degrade polymeric starch and thereby to provide a soluble simple carbon source for nutrition. It is more efficient for the fungi to use readily available carbon sources. Therefore, the production of the fungal glucoamylase is under carbon catabolite regulation (9, 10). In a study on regulation of the glucoamylase from A. niger, Fowler et al. (3) found that glucoamylase activity was observed when the fungus was grown on glucose. The enzyme was strongly induced when the fungus was grown on starch as the carbon source, even in the presence of glucose. In another fungus, A. terreus, no glucoamylase mRNA was detected after the transfer to medium with 1% (wt/vol) starch plus 1% (wt/vol) glucose, indicating carbon catabolite repression of the synthesis of glucoamylase (23). Our results on the effect of single or mixed carbon sources in the regulation of gla1 indicate that such carbon catabolite regulation is also working in L. edodes. A low, constitutive level of glucoamylase was found in the mycelium grown in the medium without glucose or starch. The presence of glucose repressed production of the enzyme, presumably by carbon catabolite repression, whereas starch as a carbon source strongly induced the enzyme. These results agree with those previous studies of L. edodes cultures supplemented with different sugars (27) and thereby indicate that the regulation of glucoamylase in L. edodes may be controlled by a complex regulatory system.
In addition to their activities for starch degradation, a few fungal glucoamylases have been studied for their role in the developmental process. It was shown that the STA1 to STA3 genes encoding three glucoamylase isozymes responsible for starch hydrolysis in S. cerevisiae are coregulated with gene MUC1, which is essential for pseudohyphal and invasive growth (24). During the development of fruiting bodies of basidiomycetous fungus Schizophyllum commune, there is a 10- to 15-fold increase in glucoamylase activity, whereas little or no activity was found in homokaryons or dikaryons (20). Inhibition studies with CO2 indicated that the glucoamylase activity is directly associated with fruiting, as a change from fruiting to vegetative growth of the dikaryotic mycelium leads to a loss in activity, whereas the already-formed fruiting bodies show no loss (20). Furthermore, the cell-bound location of glucoamylases and the fruiting-specific increase of enzyme activity make the glucoamylases in S. commune comparable to the intracellular sporulation-specific glucoamylases of S. cerevisiae, which degrades the stored glycogen during spore formation (28). In L. edodes, fruit body formation may be accompanied by the glucoamylase degradation of cellular glycogen (11, 20). We show here a strong glucoamylase activity in the fruiting stage, indicating that glucoamylase releases glucose from glycogen to provide nutrients for fruiting and therefore plays an important role in the morphogenesis of the basidiomycetous fungus.
ACKNOWLEDGMENTS
Y. H. Chen was supported by the Resident Fellow Scheme of United College. This work was partially supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (RGC no. CUHK189/94M and CUHK364/95M).
We thank Eddie Deane for his critical review.
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