APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

June 2000, p. 2531–2535 Vol. 66, No. 6

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Molecular Cloning, Characterization, and Differential Expressionof a Glucoamylase Gene from the Basidiomycetous

Fungus Lentinula edodesJ. ZHAO,1 Y. H. CHEN,2 AND H. S. KWAN1*

Department of Biology, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong SAR,1 andDepartment of Microbiology, Nankai University, Tianjin,2 People’s Republic of China

Received 18 January 2000/Accepted 28 March 2000

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungusLentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which ispreceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes fora putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading framesequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodesa protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity betweenthe Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservationfound in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity andlevels 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, andfruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1gene expression shows that expression of gla1 was induced by starch and increased during the process offruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesisof the basidiomycetous fungus.

Basidiomycetous fungus Lentinula edodes (Berk.) Pegler isthe second most widely cultivated mushroom in the world. Thecultivation of this fungus makes use of significant amounts ofwoody polysaccharides, and utilization of the complex polysac-charides is dependent on its ability to synthesize hydrolytic andoxidative enzymes which convert woody polysaccharides intolow-molecular-weight compounds that can be absorbed andassimilated for nutrition. Starch is a polymer of glucose and isperhaps, next to cellulose, the most widely available polymericglucoside made by plants (16, 18). Starch is, therefore, avail-able to fungi growing on plants or plant residues. Digestion ofstarch requires a complex of enzymes. Glucoamylases (1,4-a-D-glucan glucohydrolases; EC 3.2.1.3) are enzymes that, amongothers, are believed to be important in the utilization of starchby the basidiomycetous fungus. Glucoamylases are exohydro-lases, which catalyze the release of b-D-glucose units from thenonreducing ends of amylose, amylopectin, and other polysac-charides (18). Glucoamylase-encoding genes have been clonedfrom 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 infungi, few studies on the production and regulation of glu-coamylases in basidiomycetous fungi have been carried out.El-Zalaki and Hamza (2) studied five basidiomycetous fungusspecies for their ability to hydrolyze starch. L. edodes wasfound to be the most promising strain for amylase production.It was also reported that the glucoamylase from L. edodeshydrolyzed starch and glycogen, converting them almost com-pletely into glucose (27). Although these studies have provided

valuable information on mushroom glucoamylase physiology,molecular studies on this enzyme have not been initiated. Weaimed to address this present void, first by cloning and char-acterizing an L. edodes glucoamylase gene (gla1), second bycomparing the levels of glucoamylase activity and gene expres-sion in L. edodes strains grown on a variety of substrates, andthird by assessing gla1 expression in various developmentalstages 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 ahigh-nitrogen (HN) medium as previously described (30, 31). Fruit body primor-dia and mature mushrooms were obtained during a 6-week inoculation. Thefruiting 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 torelease 1 mmol of reducing sugar per min. In some cases, 1% glycogen (Sigma)was also used as a substrate to detect glucoamylase activity. All the reactionswere performed at 30°C. A sample without enzyme was used as a control (30).

Preparation of genomic libraries. L. edodes genomic DNA was prepared aspreviously described (32). After digestion, the DNA was ligated with DASHIIarms (Stratagene) and packaged in vitro with a Gigapack II kit (Stratagene) asdescribed by the supplier.

First-strand cDNA synthesis. Total RNA (5.0 mg) was prepared as previouslydescribed (32) and used to synthesize cDNA. Reverse transcriptions were carriedout in 20-ml reaction mixtures containing 50 U of Moloney murine leukemiavirus reverse transcriptase (GIBCO), 15 pmol of oligo(dT)15, and 20 U ofRNasin (Promega). Reactions were performed at 25°C for 10 min, at 45°C for 45min, and at 75°C for 5 min.

Isolation of differentially expressed genes during L. edodes development. Thetotal RNAs from each of the three developmental stages were fingerprinted byRNA arbitrarily primed PCR (RAP-PCR) (25). Ten microliters of RAP-PCRproducts from each of the three developmental stages was resolved on a 3%(wt/vol) Nusieve agarose gel (Promega). Differential bands that appeared insome stages but not in others were cut from the gel. The gel slice was put into 100ml 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 primersthat generated the fingerprint, and reamplified PCR products were sequenced.

* Corresponding author. Mailing address: Department of Biology,The Chinese University of Hong Kong, Shatin, N. T., Hong KongSAR, People’s Republic of China. Phone: 852-26096285. Fax: 852-26035745. E-mail: hoishankwan@cuhk.edu.hk.

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Library screening and product cloning. Approximately 200 ng of DNA froma genomic library was added to a PCR mixture containing 2.0 U of Taq poly-merase (Promega), 13 buffer (Promega), 2.5 mM MgCl2, 100 mM (each) de-oxynucleoside triphosphate, and a 1.0 mM concentration of each of two primers.Four primers in four combinations were used for library screening: LeAMG5A(59-TTCTTGCGAGTATTCACACG-39), LeAMGL1 (T3 (59-AATTAACCCTCACTAAAGGG-39), and T7 (59-GTAATACGACTCACTATAGGGC-39). Thefollowing PCR cycle parameters were used: 4 min at 94°C for 1 cycle; 1 min at94°C, 1 min at 58°C, and 5 min at 72°C for 35 cycles; and 10 min at 72°C for 1cycle. Rapid amplification of cDNA ends was used to obtain full-length cDNAclones, allowing comparisons of genomic and cDNA sequences to be made (12).PCR products were cloned into PCRscript SK (Stratagene) or sequenced di-rectly.

DNA sequencing. The nucleotide sequences of PCR products were determinedby using Taq polymerase cycle sequencing and an automated DNA sequencer(ABI 310; Perkin-Elmer Corp.). All DNAs were sequenced on both strands, andthe encoded amino acid sequences were predicted by using Gene Jockey (Bio-soft). Sequences were aligned by using SeqEd, version 2.0, software (AppliedBiosystems).

Competitive PCR. Relative transcript levels of L. edodes glucoamylase geneswere determined by competitive PCR (32). Primers LeAMG3B (59-TCTACGAATGAGGCTGTCCT-39) and LeAMGL2 (59-GCAGTGATCGTCGAGTCAAA-39) were used to amplify gla1. The lengths of the PCR products for genomicDNA and cDNA were 247 and 194 bp, respectively. The competitive templatesconsisted of full-length genomic copies of the genes which had been amplified byPCR, and the concentrations of templates were estimated by gel electrophoresis(19). To determine the concentration of cDNA, serial titration tests including 20to 40 cycles were performed. The optimized competitive PCR mixture contained(in a final volume of 20 ml) 0.2 U of Taq polymerase (Promega), 13 buffer(Promega), 2.5 mM MgCl2, 100 mM (each) deoxynucleoside triphosphate, and a1.0 mM concentration of each of the primers. The following competitive PCRparameters 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 productswere 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 accessionno. AF220541.

RESULTS

Isolation of developmentally regulated genes in L. edodes.One hundred RAP-PCR fragments were isolated, cloned, andsequenced. The putative functions of the fragments were iden-tified by comparing their sequences with those in gene data-bases. One clone with a 390-bp fragment had significant ho-mology with fungal glucoamylase genes in the databases. Basedon the partial sequence of glucoamylase, we designed primersLeAMG5A and LeAMGL1 to amplify glucoamylase genes indikaryotic strain L54.

Isolation and analysis of the glucoamylase genomic se-quence by screening the library. By using a previously de-scribed method (29), we successfully amplified 3,094 bp of theglucoamylase gene. A comparison of the sequence of the glu-coamylase gene with the sequences of corresponding PCRproducts from monokaryotic parental strains L54-A and L54-Bshowed that the cloned gene originates from L54-A. The glu-coamylase gene was designated gla1.

gla1 sequence analysis. A comparison of the genomic andcDNA sequences of gla1 indicated the presence of seven in-trons varying in size from 46 to 55 bp. All of the intron splicejunctions conform to the GT--G rule. DNA sequencing showsthat the gla1 open reading frame codes for a putative polypep-tide of 571 amino acids. Based on the sequence comparisonwith fungal glycosyl hydrolases, the Gla1 protein can be as-signed to glycosyl hydrolase family 15 (16, 18). The deducedmolecular mass of the Gla1 protein is 61,167 Da (Fig. 1).

gla1 promoter and terminator sequence analysis. We se-quenced 482 bp upstream of the translation initiation codonand 534 bp downstream of the stop codon. There is a TATAbox at position 252 with respect to the ATG codon. There arethree potential CAAT boxes upstream of the ATG codon(2256, 2261, 2312). The 39 end of the transcript has been

located by comparing the cDNA sequence and the genomicsequence. There is a consensus ATAA polyadenylation regionin the 142 bp downstream from the TAG stop codon. There isalso an AT-rich region approximately 150 bp downstream fromthe stop codon, which may act as a polyadenylation signal.

Comparison of the deduced Gla1 amino acid sequence. Acomparison of the deduced Gla1 amino acid sequence withthose of other fungal glucoamylases suggests the overall struc-ture of the mature protein, which comprises an N-terminalcatalytic domain containing regions that have been shown to beessential for enzyme activity, a C-terminal starch-binding do-main (SBD), and heavily glycosylated hinge region (16, 17). Inaddition, the sequence shows a high degree of identity with theglucoamylase sequence from a basidiomycetous fungus Corti-cium rolfsii and lesser homologies with sequences of otherfungal glucoamylases (Fig. 1). Alignment of the amino acidsequences in the N-terminal domain reveals the presence offive regions which are conserved in all fungal glucoamylases(Fig. 1). The SBD is also conserved in the L. edodes Gla1protein. The similarities between the Gla1 protein and otherfungal glucoamylases are from 45 to 61%. The region of high-est conservation is found in the catalytic domain (65 to 75%).Based on the sequence alignment and the secondary structureprediction by using the self-optimized prediction method (4),the hinge region in Gla1 is estimated to comprise amino acidresidues 446 to 467. Its length is similar to that of the hingeregion of basidiomycetous fungus C. rolfsii G2 (amino acidresidues 456 to 477) (14) but less than that of the hinge regionof 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 betweenmycelia grown in liquid medium and those grown in solidmedium (data not shown); therefore, only solid media wereused in further studies. Effects of different carbon sources(glucose, starch, crystalline cellulose, and PE) on the produc-tion of glucoamylase were compared. In strain L54 grown inHN agar medium, peak glucoamylase activity appeared at day18 (5.0 mU/g). The presence of 1% glucose decreased glu-coamylase production to 3.3 mU/g (Table 1). The presence of1% starch or 1% PE stimulated glucoamylase production by2.7- and 2.4-fold, respectively. Furthermore, 1% cellulose didnot increase the production of glucoamylase in this strain (Ta-ble 1). Monokaryotic strains L54-A and L54-B showed similarpatterns of glucoamylase production in agar medium, but withlower activities than those of dikaryotic strain L54 (Table 1).

The production of glucoamylase at various developmentalstages (mycelium growth, primordium appearance, and fruit-ing body formation) in the fruiting HN-PE-sawdust mediumwas studied. In the mycelium growth stage, the peak glucoamy-lase activity was 5.1 mU/g. However, the activities of glucoamy-lase were increased to 20.0 and 26.4 mU/g in primordia and infruiting bodies, respectively (Table 1). Enzyme activities usingglycogen as the assay substrate were similar to those withstarch as the assay substrate (data not shown).

Reverse transcription-PCR analysis of gla1 gene expression.The results of competitive PCR analysis of gla1 expression withvarious substrates and at different developmental stages areshown in Fig. 2 and Table 1. In HN-starch medium, levels ofgla1 mRNA were 3.3- and 14.1-fold higher than those of gla1mRNA in HN and HN-glucose media, respectively. The lowestlevel obtained for gla1 mRNA was 0.092 pmol/g of total RNAin HN-glucose medium, whereas the highest level was achievedin the fruiting body. PE (1%) increased the level of gla1mRNA, but the addition of 1% cellulose decreased the pro-

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duction of gla1. The low level of gla1 mRNA was found in themonokaryotic strain L54-A even though no glucoamylase ac-tivity was detected (data not shown). In the fruiting medium(HN-PE-sawdust), the gla1 mRNA level was lower in the my-celium growth stage. However, the fruiting process increasedthe gla1 mRNA levels to 2.6 and 9.9 pmol/g of total RNA in theprimordia and fruiting body, respectively (Table 1).

DISCUSSION

Although there have been many reports on the cloning offungal glucoamylase genes, only one glucoamylase cDNA se-quence, from basidiomycetous fungus C. rolfsii, has been re-ported (14). Here we report for the first time a basidiomycet-ous fungus glucoamylase gene with a potential 59 promoter

region. The 482-bp sequence upstream of the translation ini-tiation site in gla1 would be useful in the further study of generegulation and modification.

Both catalytic domains and SBDs in L. edodes Gla1 arehomologous to those from other fungi, which indicates that theGla1 domains may perform functions similar to those per-formed by these domains in a variety of fungal glucoamylases.The primary sequences of the hinge regions of the fungalglucoamylases are not really conserved among species. Twobasidiomycetous glucoamylases, L. edodes Gla1 and C. rolfsiiG2, show a strong homology in the hinge regions: both regionscontain fewer serine and threonine residues than those fromother fungal glucoamylases (14, 16). There are several rolessuggested for the hinge region of fungal glucoamylases: con-tributing to starch degradation (7), extending the peptide back-

FIG. 1. Alignment of the deduced Gla1 amino acid sequence with other fungal glucoamylase sequences. Accession numbers of sequences are as follows: C. rolfsiiG2, D49448 (14); A. awamori GA1, K02465 (15); N. crassa GLA1, X67291 (22). Gaps introduced for optimal alignment are indicated by dashes. The consensus sequenceis composed of residues shared by at least three of the mature proteins. The putative signal sequences are underlined. Five conserved sequences in catalytic domainsare in boldface. The C-terminal SBDs are shaded. Alignment was done with SeqEd, version 2.0, software.

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bone (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 enzymesfrom Saccharomyces cerevisiae host cells. However, Libby et al.(13) reported that deletions of the hinge region of A. awamoriglucoamylase did not affect its ability to degrade starch andthat the deletions had a negative effect on its thermostability. It

remains to be determined what roles the hinge region in Gla1can play in the enzyme function.

The major function of the fungal glucoamylase is to degradepolymeric starch and thereby to provide a soluble simple car-bon source for nutrition. It is more efficient for the fungi to usereadily available carbon sources. Therefore, the production ofthe 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 wasobserved when the fungus was grown on glucose. The enzymewas strongly induced when the fungus was grown on starch asthe carbon source, even in the presence of glucose. In anotherfungus, A. terreus, no glucoamylase mRNA was detected afterthe transfer to medium with 1% (wt/vol) starch plus 1% (wt/vol) glucose, indicating carbon catabolite repression of thesynthesis of glucoamylase (23). Our results on the effect ofsingle or mixed carbon sources in the regulation of gla1 indi-cate that such carbon catabolite regulation is also working in L.edodes. A low, constitutive level of glucoamylase was found inthe 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 asa carbon source strongly induced the enzyme. These resultsagree with those previous studies of L. edodes cultures supple-mented with different sugars (27) and thereby indicate that theregulation of glucoamylase in L. edodes may be controlled by acomplex regulatory system.

In addition to their activities for starch degradation, a fewfungal glucoamylases have been studied for their role in thedevelopmental process. It was shown that the STA1 to STA3genes encoding three glucoamylase isozymes responsible forstarch hydrolysis in S. cerevisiae are coregulated with geneMUC1, which is essential for pseudohyphal and invasivegrowth (24). During the development of fruiting bodies ofbasidiomycetous fungus Schizophyllum commune, there is a 10-to 15-fold increase in glucoamylase activity, whereas little or noactivity was found in homokaryons or dikaryons (20). Inhibi-tion studies with CO2 indicated that the glucoamylase activityis directly associated with fruiting, as a change from fruiting tovegetative growth of the dikaryotic mycelium leads to a loss inactivity, whereas the already-formed fruiting bodies show noloss (20). Furthermore, the cell-bound location of glucoamy-lases and the fruiting-specific increase of enzyme activity makethe glucoamylases in S. commune comparable to the intracel-lular sporulation-specific glucoamylases of S. cerevisiae, whichdegrades the stored glycogen during spore formation (28). In

FIG. 2. Competitive reverse transcription-PCR analysis of gla1 gene expres-sion in various substrates (A to E) and the fruiting medium (F to H). Theamounts of the competitive templates are indicated above the gels as dilutionfactors. The levels of transcripts in samples were based on estimated equivalencepoints between competitive products and target cDNAs. The sizes of the PCRproducts are indicated on the left.

TABLE 1. Effect of different media and conditions on the production of enzymes and transcript levels of the glucoamylase gene

Medium or stageGlucoamylase activitya (mU/gc) for strain: cDNA concentration

(pmol/g of total RNA)b

for strain L54L54 L54-A L54-B

Growth mediumHN 5.0 6 0.2 (18) 0.0 0.0 0.40 6 0.03HN 1 1% glucose 3.3 6 0.2 (14) 0.0 0.0 0.092 6 0.004HN 1 1% starch 13.4 6 0.6 (16) 2.5 6 0.2 (24) 2.6 6 0.2 (28) 1.3 6 0.1HN 1 1% cellulose 5.2 6 0.2 (18) 0.0 0.0 0.49 6 0.03HN 1 1% PE 12.0 6 0.6 (14) 2.2 6 0.2 (24) 2.1 6 0.2 (28) 0.82 6 0.05

Fruiting mediumMycelium 5.1 6 0.4 2.0 6 0.2 No growth 0.11 6 0.01Primordia 20.0 6 0.7 No primordium formation No primordium formation 2.6 6 0.2Fruiting body 26.4 6 1.2 No fruiting No fruiting 9.9 6 0.4

a Mean 6 standard deviation from triplicate cultures. Numbers in parentheses indicate the time required for maximum activity (in days).b Mean 6 standard deviation from three independent cultures.c Milliunits per gram of medium for growth medium; milliunits per gram of fresh mycelium for fruiting medium.

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L. edodes, fruit body formation may be accompanied by theglucoamylase degradation of cellular glycogen (11, 20). Weshow here a strong glucoamylase activity in the fruiting stage,indicating that glucoamylase releases glucose from glycogen toprovide nutrients for fruiting and therefore plays an importantrole in the morphogenesis of the basidiomycetous fungus.

ACKNOWLEDGMENTS

Y. H. Chen was supported by the Resident Fellow Scheme of UnitedCollege. This work was partially supported by grants from the Re-search Grants Council of the Hong Kong Special Administrative Re-gion, China (RGC no. CUHK189/94M and CUHK364/95M).

We thank Eddie Deane for his critical review.

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