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J Bacteriol, March 1998, p. 1305-1310, Vol. 180, No. 5
Max-Planck-Institut für Molekulare
Pflanzenphysiologie, 14476 Golm, Germany
Received 9 October 1997/Accepted 19 December 1997
Sucrose-inducible secretory sucrose:sucrose 1-fructosyltransferase
(1-SST) from Aspergillus foetidus has been purified and subjected to N-terminal amino acid sequence determination. The enzyme
is extensively glycosylated, and the active form is probably represented by a dimer of identical subunits with an apparent molecular
mass of 180 kDa as judged from mobility in seminative acrylamide gels.
The enzyme catalyzes fructosyl transfer from sucrose to sucrose
producing glucose and 1-kestose. Oligosaccharides with a higher degree
of polymerization are not obtained with sucrose as the substrate. The
cDNA encoding the A. foetidus 1-SST has been cloned and
sequenced. Sequence homology was found to be highest to levanases, but
no hydrolytic activity was observed when levan was incubated with the
enzyme. Expression of the cloned gene in an invertase-deficient mutant
of Saccharomyces cerevisiae resulted in 1-kestose
production, with 6-kestose and neokestose being side products of the
reaction. Products were well distinguishable from those formed by yeast
transformants expressing a cytosolic invertase.
Fructans offer various benefits for
human health as nutrients and are also of industrial interest. Thus
there is considerable interest in the isolation and characterization of
enzymatic activities capable of fructosyl polymerization and also in
the genes encoding them. To date, genes of bacterial (9, 29, 32,
34) and plant (31, 38) origin have been described, and
there is also evidence for fructan production by fungi (13).
Whereas fructan polymerization in bacteria is performed by a single
enzyme starting from sucrose, a widely accepted model proposes
that sucrose:sucrose fructosyltransferase (SST) (EC 2.4.1.99) and
1,2- In vivo characterization of fructan-synthesizing enzymes is often
complicated by competitive reactions of fructan hydrolase and
invertases (EC 3.2.1.26), the latter of which is able to catalyze the
formation of all three kestose isomers when it is incubated with
sufficiently high concentrations of sucrose (3, 4). Whether
fructosyl transfer to water, which results in cleavage of sucrose, is a
side reaction catalyzed by SST or is due to possible contamination of
enzyme preparations by invertase can be answered only by expression of
sucrose:sucrose 1-fructosyltransferase (1-SST) genes in an
invertase-free background. Up to now, however, no successful
heterologous expression of SST in Saccharomyces cerevisiae has been described.
In this report we describe the purification, cloning, and
characterization of a 1-SST-like enzyme from the fungus
Aspergillus foetidus and its heterologous expression in
S. cerevisiae. The corresponding enzyme converts sucrose to
1-kestose with high efficiency but does not give rise to fructans with
higher degrees of polymerization. Therefore, it seems to resemble plant
1-SST enzymes rather than bacterial levansucrases or inulinsucrases.
Strains and plasmids.
A. foetidus (NRRL 337) was
obtained from the American Type Culture Collection (Rockville, Md.).
suc2 (invertase)-deficient S. cerevisiae YSH
2.64-1A (10) was used for expression of A. foetidus 1-SST cDNA. Escherichia coli XL1blue
(Stratagene, Heidelberg, Germany) was used for molecular methods.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Production of 1-Kestose in Transgenic Yeast
Expressing a Fructosyltransferase from Aspergillus
foetidus
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-fructan 1-fructosyltransferase (FFT) (EC 2.4.1.100) are the key
enzymes of fructan biosynthesis in plants (6).
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
ZAP II (Stratagene) was used for construction of the
cDNA library, and EMBL3 was used for construction of a genomic library.
Standard procedures. Standard molecular cloning techniques were performed as described by Sambrook et al. (28). Protein concentrations were determined with the Bio-Rad (Munich, Germany) protein assay kit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 12% polyacrylamide gels according to Laemmli (18), and staining was done with Coomassie brilliant blue R250. Rainbow high-molecular-weight markers (Amersham, Braunschweig, Germany) were used as standards.
Purification of 1-SST. Unless otherwise indicated, all purification steps were performed at 4°C. A. foetidus cultures were grown for 60 h at room temperature in sodium phosphate (200 mM, pH 7.5)-buffered medium containing 6% sucrose, 0.5% yeast nitrogen base (Difco, Detroit, Mich.), and M9 salts. Mycelia were separated from the supernatant by filtration through Miracloth. Proteins of the filtrate were precipitated with ammonium sulfate (90% saturation). The precipitate was recovered by centrifugation at 15,000 × g for 45 min, resuspended in 50 mM sodium acetate buffer (pH 5.6), and dialyzed against 50 mM Tris-HCl (pH 7.5)-500 mM NaCl. The dialyzed fraction had a final volume of about 50 ml per 3 liters of culture.
Adsorption to concanavalin A (ConA)-Sepharose was performed according to the supplier's instruction (Pharmacia Biotech, Uppsala, Sweden). ConA was equilibrated in 50 mM Tris-HCl (pH 7.5)-500 mM NaCl. Adsorbed protein was eluted with 0.25 M
-D-methylmannoside in 50 mM Tris-HCl (pH 7.5). The ConA eluate was loaded onto a Mono Q
anion-exchange column (Pharmacia HR 5/5) equilibrated with 50 mM
Tris-HCl (pH 7.5). Proteins were eluted with a linear KCl gradient (0 to 1 M) at a flow rate of 1 ml/min. Fractions of 1 ml were collected
and stored at 
20°C. Mono Q fractions showing sucrolytic activity
were applied to a Superdex 200 HR 10/30 column (bed volume, 24 ml
[Pharmacia]) equilibrated with 50 mM Tris-HCl (pH 7.5) at a
concentration of 8.3 µg of protein per ml and eluted with the same
buffer at a flow rate of 0.25 ml/min. Fractions containing the active
protein were pooled and concentrated in Centricon 30 concentrators
(Amicon, Beverley, Mass.).
Activity staining of seminative polyacrylamide gels.
Seminative PAGE gels were prepared according to Laemmli (18)
containing 0.1% SDS, but samples were loaded in a buffer containing the same amount of SDS but omitting -mercaptoethanol and the heat
step. After the electrophoresis, the gel was washed extensively with 50 mM sodium acetate (pH 5.6) containing 0.5% (vol/vol) Triton X-100 for
15 min to remove the SDS. Afterwards, the gel was incubated in 1 M
sucrose-50 mM sodium acetate (pH 5.6) for 30 min at room temperature.
After being washed repeatedly with water, reducing sugars were stained
with 1% (wt/vol) 2,3,5-triphenyltetrazolium chloride (TTC) in 0.25 M
NaOH, which forms red insoluble formazan. To localize glucose release,
TTC was heated to 100°C and then poured onto the gel. After a few
minutes the TTC solution was discarded, and the staining was stopped
with 5% (vol/vol) acetic acid.
Product identification by TLC. Soluble sugars were analyzed by thin-layer chromatography (TLC) according to the method of Wagner and Wiemken (39). F 1500 TLC-Ready-Foils (Schleicher & Schüll, Dassel, Germany) were used and developed at least two times with acetone-H2O (87:13 [vol/vol]). Sugars were visualized by applying urea phosphoric acid spray (25).
Photometric determination of glucose and fructose. Enzyme (1 µg) was incubated with sucrose for 1 h at 37°C. An aliquot of the incubation solution was added to 100 mM imidazol-HCl (pH 6.9)-5 mM MgCl2-2 mM NADP-1 mM ATP-2 U of glucose-6-phosphate-dehydrogenase (from yeast) per ml and used for photometric determination of glucose and fructose. Absorption differences at 334 nm were determined by sequential addition of 0.5 U of hexokinase (from yeast) and 2 U of phosphoglucoisomerase (from yeast) (33).
Construction of cDNA and genomic libraries of A. foetidus.
Mycelia of A. foetidus cultivated for 48 h were isolated by filtration through Miracloth. RNA was isolated
following the method of Logemann et al. (20). Isolation of
mRNA was done with a poly(A)Tract poly(A)-RNA isolation kit (Promega,
Madison, Wis.). cDNA was synthesized by using a ZAP-cDNA synthesis
kit and Gigapack II gold packaging extract (Stratagene).
Cloning of A. foetidus 1-SST. Two degenerated, nested PCR primers were designed by using the N-terminal amino acid sequence. Primer AF1 (5'-GGAATTCAAYTAYGAYCARCCNTAYMGNGGNCARTAYCA), derived from the N terminus, was used in combination with an M13 universal primer (Stratagene) for PCR with the whole cDNA library as the template. The fragment amplified by the AF1-universal primer was eluted after agarose gel electrophoresis with the GeneClean kit (Bio 101 Inc.) and used as the template for the second PCR by using AF2 (5'-ATAGGATCCNCARAARAATGGATGAAYGA) in combination with the universal primer (Stratagene). The amplified PCR fragment was cloned into the pCRII vector (Invitrogen, Inc.). The insert, recovered by NotI-BamHI digestion, was used as the probe to screen cDNA and genomic libraries. The full-length cDNA clone obtained was named pCK14.
Transformation of S. cerevisiae. Yeast cells were transformed according to the method of Dohmen et al. (5). Construct 112CK14 contains the BamHI-Asp718 fragment of pCK14, which was blunt ended and cloned into the SmaI site of p112A1NE (Fig. 1A). Construct 112CK14L contains the 5' untranslated leader sequence of the spinach sucrose transporter (nucleotides 1 to 69 [24]) as a spacer between transcriptional and translational initiation sites, cloned into the PstI site of 112CK14 (Fig. 1B). Construct 112CK14S contains the vacuole-targeting sequence of the patatin gene B33 of Solanum tuberosum (nucleotides 724 to 1399 [28]), which was cloned into the blunted PstI site of 112CK14 (Fig. 1C).
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Nucleotide sequence accession number. The sequence of the open reading frame (ORF) of 1,611 bp encoding a 537-amino-acid protein (1-SST) with a deduced molecular mass of 59.1 kDa has been assigned EMBL accession no. AJ000493.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A. foetidus 1-SST production is dependent on growth conditions. When A. foetidus NRRL 337 was cultivated in rich liquid medium (2% glucose, 2% malt extract, 0.1% peptone), fructan-producing activity (degree of polymerization = 3) was secreted by the fungus, as demonstrated by TLC analysis of the culture supernatant incubated with sucrose. As expected, protein extracts of homogenized mycelia did not contain 1-kestose-producing activity. Furthermore, no other sucrolytic activities were detectable in the medium or in enzyme preparations of mycelia (data not shown). Different minimal media containing yeast nitrogen base and either glucose or sucrose as carbon source were tested. With both sugars, fungal growth was detectable within 2 days, but 1-SST activity was found only when sucrose was present in the culture medium (Fig. 2). Sucrose (0.2% [wt/vol]) added to a medium based on glucose as the main carbon source was sufficient to induce 1-SST production.
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Purification of 1-SST. A culture filtrate of a 60-h culture was used for the purification of the secreted protein. Protein in the culture filtrate was collected by ammonium sulfate precipitation at 90% saturation. About 3 mg of protein could be precipitated per liter of culture. After dialysis, protein from the ammonium sulfate precipitation was incubated with ConA-lectin at a concentration of 1 mg of protein per ml of ConA-Sepharose, as described in Materials and Methods.
Mono Q anion-exchange chromatography allowed enrichment of A. foetidus 1-SST by a factor of 28. Subsequent size exclusion chromatography on Superdex 200 yielded a single protein band with a size of around 180 kDa in seminative SDS-PAGE (Fig. 3, left panel, lane 4). This protein fraction, showing an 87.5-fold increase of specific activity of A. foetidus 1-SST (expressed as micromoles of hexose released from sucrose per minute and milligrams of protein at a temperature of 37°C [Table 1]), was used to determine the N-terminal amino acid sequence of A. foetidus 1-SST and also to characterize its enzymatic properties.
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Cloning of the 1-SST gene. On the basis of a 22-amino-acid N-terminal peptide sequence obtained by protein sequencing, two nested oligonucleotides were designed and used as primers for consecutive PCRs, each in combination with the M13 universal primer, which hybridized to a vector sequence that was ligated to cDNA of A. foetidus mycelia during construction of a cDNA library in the lambda ZapII phage vector. The amplified fragment was approximately 1.6 kb in length and was cloned into the pCRII vector. The 1.6-kb BamHI-NotI fragment was prepared from the recombinant plasmid and used to probe the A. foetidus cDNA library to screen for full-length cDNA clones.
Phage particles corresponding to 23 hybridizing plaques were isolated, and plasmids were rescued by in vivo excision. Sequence analysis of three clones containing the largest inserts showed that they contained the same cDNA insertion. One of the clones, termed CK14, was completely sequenced on both strands. The sequence comprises an ORF of 1,611 bp encoding a protein of 537 amino acids with a deduced molecular mass of 59.1 kDa. Part of the ORF is a putative signal sequence encoding 19 amino acids. These amino acids were not present at the N terminus of the mature peptide that was purified from culture filtrate, indicating that the signal peptide for extracellular location is cleaved during secretion. A genomic clone, GK1, isolated by using the 1.6-kb PCR fragment as a probe, was used for further investigation of the 5' end of the A. foetidus 1-SST gene. An in-frame stop codon 75 bp upstream of the putative ATG start codon of CK14 was identified in GK1, indicating that nucleotide 682 most probably represents the translational start of the A. foetidus 1-SST gene. Comparison of A. foetidus 1-SST with other-fructofuranosidases reveals that the six domains identified by
Gunasekaran et al. (12), which are well conserved among
bacterial sucrases and yeast invertase, are also present in A. foetidus 1-SST and other fructosyltransferases of different
origins. We compared sucrolytic enzymes, e.g., cell wall invertase of
Nicotiana tabacum (11), suc2 invertase
of S. cerevisiae (36), and levanase of Bacillus subtilis (22) with transfructosylases
such as 1-SST from artichoke (14) and Jerusalem artichoke
(PCT/NL961/00012) and sucrose:fructan 6-fructosyltransferase from
barley (31). Plant enzymes show an insertion of a threonine
in box 3, whereas they miss the less-conserved amino acid 4 in box 6 (Fig. 4). An asparagine-to-serine
exchange in box 1 seems to be characteristic for the plant
fructosyltransferases shown but not for fructosyltransferases in
general as it is not present in the ftf gene of
Streptococcus mutans. In A. foetidus 1-SST, it is
also missing.
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Expression of A. foetidus 1-SST in S. cerevisiae. To prove the identity of CK14 as a cDNA for the A. foetidus 1-SST gene, we expressed it in S. cerevisiae. The yeast system was chosen in preference to bacterial expression systems because of its competence for protein glycosylation.
For yeast transformation, the insert of pCK14 and two chimeric gene constructs were cloned in the shuttle vector p112A1NE (24). Transformants containing the cDNA insert of pCK14 without modifications were not able to synthesize 1-kestose (Fig. 5, lane 4).
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Enzymatic properties of A. foetidus 1-SST are dependent
on sucrose concentration.
Purified 1-SST from A. foetidus was used to analyze enzyme specificity. The protein was
purified from culture filtrate as described above, and homogeneity was
confirmed by SDS-PAGE. The specific activity of the enzyme was 257.2 µmol of hexose µg
1 min1 when it was
incubated with 1 M sucrose.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In this report, we describe the purification of 1-SST from A. foetidus NRRL 337 and the isolation of a cDNA and a genomic DNA of the corresponding gene. Twenty-two amino acids of the N terminus of the purified protein were determined. This sequence could also be found in the deduced amino acid sequence of the cloned cDNA, and furthermore, the cDNA encoded an enzyme with the same catalytic properties as the purified 1-SST when it was expressed in an invertase-deficient yeast strain under the control of the ADH promoter. This is convincing evidence that the cloned cDNA and genomic clones represent the 1-SST gene of A. foetidus.
The A. foetidus 1-SST gene is a low-copy-number gene as revealed by a DNA blot experiment with digested genomic DNA that was hybridized to the CK14 sequence (data not shown). A single band hybridized to CK14 even under low-stringency conditions, suggesting that no additional copies or homologous genes are present in the genome of A. foetidus. This finding is in agreement with our finding that no sucrolytic activity other than that of 1-SST was detectable in culture filtrate or protein extracts of fungal mycelia. The ability of A. foetidus to grow on sucrose as a sole carbon source therefore seems to be entirely dependent on the 1-SST activity.
Growth experiments with the fungus demonstrated that secretion of 1-SST seems to be regulated through the physiological status of the culture. When the medium was not buffered or barely buffered and the sucrose concentration was low, only small amounts of 1-SST were secreted and the pH of the medium quickly dropped to pH 2. Higher concentrations of sucrose stimulated production of 1-SST.
An in-frame stop codon is present in the A. foetidus 1-SST gene 75 bp upstream of the 1,611-bp ORF of the cDNA clone CK14. We therefore believe that the cDNA encodes the entire 1-SST protein. The encoded protein consists of 537 amino acids and thus has a calculated mass of 59,150 Da, including a 1,900-Da signal peptide.
Due to glycosylation, the apparent molecular mass of the protein by SDS-PAGE was much higher than that calculated for the ORF of CK14. Enzymatic deglycosylation revealed two protein species with molecular masses of around 60 kDa. Currently, we have no explanation for the double band that was obtained for the deglycosylated protein. Compared to the sizes of other secreted proteins of fungi from the genus Aspergillus, an increase in mass of about 40 kDa seems extraordinarily high given that only eight possible glycosylation sites could be identified within the sequence of the mature peptide. Glucose oxidase of Aspergillus niger also contains eight consensus motifs for glycosylation, but the increase in molecular mass caused by posttranslational modification of the protein is only around 10 kDa (7).
Preparations of active A. foetidus 1-SST show a single protein band of 180 kDa by seminative PAGE. This band disappears when gels are run under denaturing conditions (2% SDS, 100 mM dithiothreitol). As only one peptide species with a molecular mass of 90 kDa results from reductive denaturation, we believe that the active A. foetidus 1-SST is a dimer with identical subunits.
Dimerization has also been shown for secreted inulinase (Inu1) of Kluyveromyces marxianus CBS 6556 (27), which shows 36% amino acid identity to A. foetidus 1-SST. Both proteins belong to a large family of sucrose-cleaving enzymes that share extensive homology in certain protein domains. For example, amino acids 38 to 45 are 100% identical to amino acids 50 to 57 of Inu1 of K. marxianus (19) or amino acids 39 to 46 of invertase (Suc2) from S. cerevisiae (35). The highest degree of homology to sequences in the database was found between A. foetidus 1-SST and the fructanase gene levJ of Actinomyces naeslundii T14V (23). Despite an identity of 41% at the amino acid level, these proteins do not share enzymatic specificity. The levJ gene product hydrolyzes sucrose, raffinose, inulin, and levan (23) but does not synthesize fructan, whereas A. foetidus 1-SST does not hydrolyze levan or inulin.
In the deduced peptide sequence of A. foetidus 1-SST,
we could recognize the six domains which are conserved among bacterial and fungal -fructofuranosidases (12). As these boxes can
be identified in fructosyltransferases of plant and bacterial origin also, it was interesting to investigate whether it would be possible to
discriminate sucrolytic enzymes from transfructosylating enzymes. The
fructosyltransferase of S. mutans as an example of a
bacterial fructosyltransferase and the plant 1-SST from artichoke
(14) and Jerusalem artichoke (PCT/NL961/00012) and
sucrose:fructan 6-fructosyltransferase from barley (31) were
compared to sucrose-cleaving enzymes, e.g., cell wall invertase of
N. tabacum (11), suc2 invertase of
S. cerevisiae (35), and levanase of B. subtilis (22). The sequence comparison does not reveal
fructosyltransferase-specific motifs, as the similarity is higher for
sequences of similar origins (e.g., plant versus bacterium) than for
sequences of similar functions. Interestingly, the invertase of
A. niger contains only one of these domains
(2) and shares a sequence homology of only 38% with
A. foetidus 1-SST, clearly demonstrating that these two
enzymes are barely related. Enzymatic specificity of A. foetidus 1-SST is more similar to that of plant 1-SST, which is a
key enzyme in fructan synthesis (6, 21, 36). The
irreversible fructosyl transfer between two sucrose units to form
1-kestose and glucose provides the substrate for synthesis of higher
fructans of the inulin series but probably also provides a substrate
for levan synthesis (16, 30). A plant 1-SST has been
partially purified from barley and analyzed in vitro (30).
The main reaction catalyzed by the enzyme is fructosyl transfer with
sucrose as donor and acceptor, but cleavage of sucrose was also
detectable. As the enzyme was purified from plant extracts which also
contain different types of invertases, it cannot be ruled out that
contaminating invertases gave rise to the sucrose cleavage. In
contrast, Koops and Jonker (17) purified 1-SST from
Helianthus tuberosus and found no hydrolysis of sucrose, at
least for short incubation periods. The question of whether 1-SST can
catalyze fructosyl transfer from sucrose to water could probably be
answered by expression of a 1-SST gene in an invertase-free background.
To allow discrimination of the A. foetidus 1-SST-encoded fructosyltransferase activity from invertase, we decided to produce the 1-SST in the invertase-deficient yeast strain YSH 2.64-1A (10). The cDNA insert of pCK14 could be functionally expressed in yeast when the 5' untranslated region was extended by 70 bp taken from the sucrose transporter gene of spinach (24). This insertion readjusts the structure of yeast genes, which usually contain long 5' leader sequences separating the upstream activating sequence and the ATG start codon (8).
The main product of A. foetidus 1-SST reactions is 1-kestose, the smallest of the inulin-type fructans. Other kestose isomers are also synthesized by A. foetidus 1-SST but occur only in trace amounts at the pH optimum of the enzyme. Hydrolysis of sucrose by A. foetidus 1-SST shows saturation at a substrate concentration of about 500 mM. In contrast, yeast invertase does not seem to saturate even at 1 M sucrose. Furthermore, kestose production is a side reaction of invertase, never exceeding 20% of the sucrose turnover.
In addition to the extracellular 1-SST protein, intracellular localization could be obtained by fusing an N-terminal signal sequence of the patatin gene B33 from Solanum tuberosum (26) to the A. foetidus 1-SST coding region. Products formed by intracellular and extracellular A. foetidus 1-SST did not differ. This allows the production of fructo-oligosaccharides in the baker's yeast S. cerevisiae. As oligofructans are well-known for their health benefits in human nutrition (15), this finding is also of considerable biotechnological interest.
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FOOTNOTES |
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* Corresponding author. Mailing address: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Karl-Liebknecht-Str. 25, 14476 Golm, Germany. Phone: 49-331-9772786. Fax: 49-331-9772301. E-mail: heyer{at}mpimp-golm.mpg.de.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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J Bacteriol, March 1998, p. 1305-1310, Vol. 180, No. 5
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