Correspondence: Fernando Torres, Laboratório de Biologia Molecular, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil 70910-900. Tel.: +55 61 3307 2423; fax: +55 61 3349 8411; e-mail: email@example.com
A Cryptococcus flavus gene (AMY1) encoding an extracellular α-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) 144DVVVNH149, (II) 235GLRIDSLQQ243, (III) 263GEVFN267, (IV) 327FLENQD332, placed the enzyme in the GH13 α-amylase family. Furthermore, sequence comparison suggests that the C. flavusα-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL−1) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.
α-Amylases (EC 188.8.131.52) are endo-glycosyl hydrolases that randomly cleave the α-1,4-glycosidic bonds present in starch. This reaction generates linear and branched oligosaccharides of various lengths. The α-amylase family consists of several enzymes that share common characteristics, such as a parallel (α/β)8 barrel structure and the catalytic mechanism (Van der Maarel et al., 2002). They also contain four highly conserved regions comprising the catalytic centre, the substrate and Ca+2-binding sites (Janeček, 1997; MacGregor et al., 2001). Only c. 10% of the known amylases are capable of binding and degrading raw starch and these enzymes possess a distinct sequence-structural module called a starch-binding domain (SBD) (Machovič & Janeček, 2006).
α-Amylases are among the most important industrial enzymes, with many applications in starch processing, brewing, alcohol production, textile and other industries (Gupta et al., 2003). Industrial processes for hydrolysis of starch to glucose rely on inorganic or enzymatic catalysis. The use of enzymes is preferred as it offers a number of advantages, including improved yields and cost savings (Satyanarayana et al., 2004). Enzyme hydrolysis also allows greater control over the specificity of the reaction and the stability of the generated products. The enzymes used in the industrial conversion of starch are estimated to account for 10–15% of the total world enzyme market (Satyanarayana et al., 2004).
Yeast species such as Schwanniomyces occidentalis, Lipomyces kononenkoae and Streptomyces fibuligera are known to produce highly active extracellular α-amylases and glucoamylases (Steyn & Pretorius, 1990). During the course of screening for amylolytic yeasts in the Brazilian biodiversity, a species classified as Cryptococcus flavus was isolated and its secreted amylase was biochemically characterized (Wanderley et al., 2004). The purified α-amylase (hereafter called Amy1) exhibited important properties for biotechnological applications such as a low Km (0.0056 mg mL−1) for soluble starch, high stability at pH 5.5 and optimal temperature at 50 °C. These features prompted the authors to explore the use of Amy1 in biotechnological processes such as starch conversion for the production of ethanol.
Although C. flavus is able to metabolize starch, it cannot produce high yields of ethanol and may be pathogenic to humans. On the other hand, the baker's yeast Saccharomyces cerevisiae is widely used for the industrial production of ethanol and has the Generally Regarded as Safe (GRAS) status. However, S. cerevisiae cannot degrade starch to fermentable sugars unless an external source of amylase is provided or the yeast is genetically modified to express these enzymes (Steyn & Pretorius, 1990).
The present study describes the isolation of an α-amylase gene (AMY1) from the yeast C. flavus. This gene was successfully expressed in S. cerevisiae, showing that this system may be used in starch-conversion processes using Amy1 as a source of α-amylase activity.
Materials and methods
Strains and plasmids
The C. flavus strain used in this work was maintained at the Molecular Biology Laboratory (Universidade de Brasília, Brazil). Escherichia coli strain DH5α and plasmid pGEM-T® (Promega) were used for general DNA manipulations. Plasmid YEp351PGK (Moraes et al., 1995) was used to express AMY1 in S. cerevisiae CENPK2 (MaTa/α, ura3-52/ura3-52, leu2-3,112, trp1-289/trp1-289, his3-1/his3-1).
Cryptococcus flavus was cultured at 25 °C in YPD (1% yeast extract, 2% peptone and 2% glucose) or synthetic starch medium (SSM: 0.67% YNB, 2% starch). For heterologous expression in S. cerevisiae, synthetic dextrose (SD) minimal medium containing 0.67% yeast nitrogen base (YNB), 2% glucose, 50 mM acetate buffer pH 5.5 and the appropriate amino acid supplements was used. SDA medium (0.67% YNB, 1% starch, 2% glucose, 50 mM acetate buffer, pH 5.5, 2% agar and appropriate amino acid supplements) was used for plate detection of amylolytic activity. Luria–Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.3) containing 100 μg mL−1 ampicillin was used for selection and growth of bacterial transformants.
Protein sequencing and molecular biology techniques
After growing C. flavus on SSM media for 60 h at 25 °C, α-amylase was purified from culture supernatants as described previously (Wanderley et al., 2004). The internal fragments were produced by proteolytic digestion using immobilized bovine trypsin (Pierce) in a solution of ammonium bicarbonate, pH 7.8. The mixture of amylase/immobilized trypsin was incubated at 37 °C for 4 h in an orbital shaker. The supernatant was collected after centrifugation and directly spotted onto a matrix-assisted laser desorption ionization (MALDI) plate with a saturated matrix solution of α-cyano-4-hydroxycinnamic acid. Peptide sequences of the native secreted enzyme were identified by monoisotopic mass analyses and sequenced using MS/MS data produced by past source decay (PSD) and collision induced dissociation (CID) fragmentation experiments obtained on a MALDI TOF-TOF Ultraflex® II spectrometer (Bruker Daltonics, Billerica). All DNA manipulations were carried out as described in Sambrook & Russel (2001). Restriction enzymes were obtained from New England Biolabs and Promega and used as instructed by the suppliers. All plasmid DNA was prepared with the QIAprep® Spin Miniprep Kit (Qiagen).
Cloning of AMY1
Based on the sequence of amy-CS2, the Cryptococcus sp. S-2 α-amylase gene described by Iefuji et al. (1996), primers CF5 and CF3 were designed (Table 1), which were used in a PCR reaction containing 20 ng of C. flavus genomic DNA, 10 pmol of each primer, dNTPs at 2.5 mM and 1 U of Taq DNA polymerase (Cenbiot, Brazil) in a final reaction volume of 50 μL. The system was submitted to 30 amplification cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1.5 min and a final elongation cycle (5 min for 72 °C). A c. 0.9-kb amplicon was cloned into the pGEM-T® vector and sequenced. Because the cloned fragment showed high sequence identity to amy-CS2, primers CFAMY5 and CFAMY3 were designed (Table 1) to clone the entire genomic coding region (c. 2.0 kb) by PCR using Tgo DNA polymerase (Roche Molecular Biochemicals) and the following conditions: 35 amplification cycles of 94 °C for 30 s, 55 °C for 60 s, 72 °C for 2 min, followed by a final elongation cycle (72 °C for 7 min). A reverse transcriptase (RT)-PCR procedure was used to clone the c. 1.9-kb AMY1 cDNA. Briefly, total RNA from C. flavus cells grown in SSM was isolated using the TRizol® method (Invitrogen) and cDNA was synthesized using the SuperScript® III First Strand Synthesis System for RT-PCR (Invitrogen) following the supplier's recommendations. PCR was carried out with Tgo DNA polymerase using primers CFAMY5 and CFAMY3 as described above. The nucleotide sequence for AMY1 was deposited at GenBank under accession number EU014874.
In order to express AMY1 in S. cerevisiae, the cDNA was amplified by RT-PCR using primers CFAMY5 and CFAMY3, which introduce Bgl II sites at amplicon ends (Table 1). The cloned cDNA was digested with Bgl II, and the c. 1.9-kb fragment was isolated and cloned into the yeast expression vector YEp351 PGK linearized with the same enzyme. The resulting vector was named YEpAMY1. Saccharomyces cerevisiae CENPK2 was transformed as reported previously (Chen et al., 1992). Transformants expressing α-amylase were selected by the production of hydrolysis haloes after 72 h of incubation at 30 °C in SSM and stained with iodine vapour as described by Moraes et al. (1995). A colony of S. cerevisiae harbouring YEpAMY1 was precultured in SD medium and 2 mL of this culture was transferred to a 1-L conical flask containing 200 mL of the same medium. Cells were incubated at 30 °C on a rotatory shaker at 200 r.p.m., and cell growth was monitored at 600 nm. At different time intervals, 5 mL samples were collected and centrifuged at 5000 g for 10 min and the supernatant was used for further analysis. α-Amylase activity was determined by monitoring starch hydrolysis as described by Moraes et al. (1995). One unit of amylase activity was defined as the amount of enzyme necessary to hydrolyse 0.1 mg of starch per minute. Protein deglycosylation was performed with PNGase F (New England Biolabs) according to the manufacturer's instructions.
Electrophoresis and enzymatic activities in gel
The apparent molecular mass of the recombinant enzyme was determined in a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970). Proteins were visualized after silver staining as described by Blum et al. (1987). Molecular mass markers were as follows: β-galactosidase (116 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), REase Bsp98l (25 kDa), β-lactoglobulin (18.4 kDa) and lysozyme (14.4 kDa) (Fermentas Life Sciences). Activity gels were prepared as described by Wanderley et al. (2004). Briefly, after samples were resolved on a 12% SDS-PAGE, the gel was washed with distilled water, incubated with 50 mM sodium acetate (pH 5.5) for 60 min and then incubated at 4 °C for 12 h in a solution containing 0.5% starch (in 50 mM sodium acetate, pH 5.5). After this incubation period, the gel was further incubated at 37 °C for 2 h and bands displaying amylase activity were detected after staining with iodine solution (1% I2 in 0.5 M KI).
Results and discussion
Isolation of the AMY1 gene
A secreted α-amylase from C. flavus was purified from culture supernatants and sequence analysis of two tryptic fragments revealed 100% identity to positions 21–38 and 420–433 of AMY-CS2, the α-amylase isolated from Cryptococcus sp. S-2 (Iefuji et al., 1996). Because the two proteins shared high sequence identity, primers were designed based on the sequence from amy-CS2 and an c. 2.0-kb amplicon corresponding to the genomic version of AMY1 was obtained. AMY1 contains two introns at positions 867–912 and 1024–1060. These introns begin with GT(G/A)AGT and end with AG, which are general intron signatures. The first intron in AMY1 is of a size equal to the first intron present in amy-CS2 (46 bp) while the second intron (37 bp) is 16 bp smaller. The AMY1 cDNA was cloned by RT-PCR using mRNA isolated from cells grown in the presence of starch. The cDNA sequence showed the highest identities with the Cryptococcus sp. S-2 and Aspergillus nigerα-amylase genes: 92% and 89%, respectively. The deduced protein, Amy1, has 631 amino acid residues and its sequence exhibits homology to α-amylases from Cryptococcus sp. S-2 (97%), Aspergillus terreus (46%), Aspergillus fumigatus (45%), Aspergillus clavatus (44%) and Aspergillus kawachii (44%). Sequence comparison with other amylases places Amy1 in the GH13 family because it has the four conserved signature regions (I, II, III, IV) present at positions 144–149, 235–243, 263–267 and 327–332 and the highly conserved catalytic residues Asp-239, Glu-264 and Asp-332 present in regions II, III and IV, respectively (Fig. 1). Furthermore, the five amino acid residues His-149, Asp-239, Glu-264, Gln-331 and Asp-332 in Amy1 correspond to residues His-122, Asp-206, Glu-230, His-296 and Asp-297 in Taka-amylase (Janeček, 1997). Asp-206, Glu-230 and Asp-297 were found to play a role in catalysis while His-122, His-210 and His-296 have been shown to be involved in substrate recognition in the active site in mammalian pancreatic α-amylases (Ishikawa et al., 1992, 1993) and in the transition state stabilization, but not directly in catalysis of barley α-amylase (Søgaard et al., 1993b). Interestingly, regions II and IV in Amy1 contain uncharged Gln (Gln-241 and Gln-331) in place of the highly conserved His residues (His-210 and His-296 in Taka-amylase A, respectively). A similar substitution was also observed in region II of the Lipomyces kononenkoaeα-amylase (Kang et al., 2004). Janeček et al. (1999) observed that a glycine replacing the histidine at the end of region II was a general feature of archaeal as well as plant α-amylases. It has been proposed that in Thermococcus profundus, these residues may be involved in the catalytic mechanism (Lee et al., 1996). The histidine residue of the Bacillus stearothermophilusα-amylase that is equivalent to His-210 in Taka-amylase may control the specificity and thermal stability of the enzyme (Vihinen et al., 1990). A similar function has been reported for the same residue in the enzyme from Bacillus subtilis (Takase, 1994). In CGTases, histidine residues are probably responsible for both cycling and amylolytic activities (Mattsson et al., 1995). However, activity as well as substrate specificity of these enzymes could be modified by mutation of nonessential amino acid residues adjacent to or near the catalytic residues (Takase, 1992; Inohara-Ochiai et al., 1997).
The AMY1 cDNA was cloned into YEp351PGK, which allows constitutive expression of foreign genes under the control of the yeast PGK1 promoter. The resulting plasmid, YEpAMY1, was used to transform the S. cerevisiae CENPK2 strain and successful α-amylase expression was observed after the formation of distinct hydrolysis haloes around colonies (Fig. 2a–d). No starch hydrolysis halo was observed in either untransformed cells or cells transformed with YEp351PGK (Fig. 2e and f). The time course of growth and secreted amylase production by a selected S. cerevisiae transformant during batch culture is shown in Fig. 3. The extracellular amylolytic activity increased during growth and reached a maximal value (3.93 U mL−1) at 60 h of incubation (Fig. 3a). Cell growth was equivalent to that of the clone transformed with the vector alone (data not shown), indicating that the amylase production did not impair cell growth, and resulted in constitutive and cumulative production of α-amylase. Amylolytic activity was correlated to a single band with an apparent molecular mass of c. 67 kDa, which is similar to that from the native enzyme (Fig. 3b and c). After deglycosylation with PNGase F, which removes the glycan portion of N-glycoproteins, both native and recombinant enzymes showed a molecular mass of c. 66 kDa, which is in good agreement with the predicted size of the mature protein (65 865 Da) (Fig. 4a and b). Three potential N-glycosylation sites were identified in Amy1 (61NGT, 190NRT and 269NPS) but more experimental data are needed to confirm the precise location of these sites. Although Amy1 has 97% sequence identity with AMY-CS2, the latter could only be efficiently secreted in yeast when its C-terminal was deleted (Iefuji et al., 1996). The only striking differences in the C-terminal region of both enzymes are three substitutions at positions 606 (Pro→Thr), 608 (Asn→Ser) and 623 (Ala→Thr). It is unclear whether these substitutions alone are enough to promote efficient secretion of Amy1 in S. cerevisiae.
This work represents the first report of the cloning and secretion of a C. flavusα-amylase gene in S. cerevisiae. The successful expression of Amy1 in yeast shows that this enzyme may be used in biotechnological processes. In fact, work is under way to add a glucoamylase gene to system, which should enable the resulting strain to ferment starch to ethanol directly.
This project was supported by FAP/DF, CNPq and CAPES (Brazil). The authors are indebted to Hugo Costa Paes for manuscript revision.