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Applied and Environmental Microbiology, June 1999, p. 2382-2387, Vol. 65, No. 6
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Overexpression of the Saccharomyces cerevisiae
Mannosylphosphodolichol Synthase-Encoding Gene in Trichoderma
reesei Results in an Increased Level of Protein Secretion and
Abnormal Cell Ultrastructure
Joanna S.
Kruszewska,1
Arno H.
Butterweck,2
Wies
aw
Kurz
tkowski,3
Andrzej
Migdalski,1
Christian P.
Kubicek,2 and
Grazyna
Palamarczyk1,*
Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, 02-106 Warsaw,1
and State Institute of Hygiene, 00-791 Warsaw,3 Poland, and Institute of
Biochemical Technology and Microbiology, TU Wien, A-1060 Vienna,
Austria2
Received 21 September 1998/Accepted 2 April 1999
 |
ABSTRACT |
Production of extracellular proteins plays an important role in the
physiology of Trichoderma reesei and has potential
industrial application. To improve the efficiency of protein secretion,
we overexpressed in T. reesei the DPM1 gene of
Saccharomyces cerevisiae, encoding mannosylphosphodolichol
(MPD) synthase, under homologous, constitutively acting expression
signals. Four stable transformants, each with different copy numbers of
tandemly integrated DPM1, exhibited roughly double the
activity of MPD synthase in the respective endoplasmic reticulum
membrane fraction. On a dry-weight basis, they secreted up to
sevenfold-higher concentrations of extracellular proteins during growth
on lactose, a carbon source promoting formation of cellulases. Northern
blot analysis showed that the relative level of the transcript of
cbh1, which encodes the major cellulase (cellobiohydrolase
I [CBH I]), did not increase in the transformants. On the other hand,
the amount of secreted CBH I and, in all but one of the transformants,
intracellular CBH I was elevated. Our results suggest that
posttranscriptional processes are responsible for the increase in CBH I
production. The carbohydrate contents of the extracellular proteins
were comparable in the wild type and in the transformants, and no
hyperglycosylation was detected. Electron microscopy of the
DPM1-amplified strains revealed amorphous structure of the
cell wall and over three times as many mitochondria as in the control.
Our data indicate that molecular manipulation of glycan
biosynthesis in Trichoderma can result in improved
protein secretion.
| |
INTRODUCTION |
The saprophytic fungus
Trichoderma reesei secretes a wide range of enzymes
such as cellulases or hemicellulases, which are of considerable
biotechnical importance in the food, feed, and paper industries
(10). Despite the progress that has been made in studies of
the enzymology and molecular biology of Trichoderma hydrolytic proteins (37), the pathway of protein secretion
and its regulation are still poorly understood (1, 34). The
presence of cleavage sites for the signal peptidase and a Kex2-like
dipeptidyl peptidase in many cellulase prepropeptides (2,
7), the results of immunoelectron microscopy (6, 22),
and the recent cloning of a sar1 homologue (39)
support the hypothesis that the T. reesei secretory pathway
involves transport from the endoplasmic reticulum through the Golgi
complex to the plasma membrane.
Many extracellular proteins of fungi are glycosylated, and for the
cellulolytic enzymes, there is indirect evidence that O glycosylation
may be essential for secretion (19). O glycosylation in
T. reesei and other fungi occurs by the dolichol pathway
(14, 23, 38). This pathway differs from other eukaryotic
O-glycosylation by the involvement of phosphodolichol as a carrier
lipid and by the fact that transfer of the first mannosyl residue
occurs in the endoplasmic reticulum (11).
We have suggested previously (15) that
mannosylphosphodolichol synthase (MPD synthase;
EC 2.4.1.8.80) plays a key role in T. reesei O
glycosylation and is activated by cyclic AMP-dependent phosphorylation (16). The protein and the gene from
Saccharomyces cerevisiae (DPM1) have been
characterized elsewhere (12, 32). Loss of DPM1
expression is lethal (32). Successful attempts to clone
homologues of S. cerevisiae DPM1 from T. reesei
or other filamentous fungi have yet to be reported (29). In
an S. cerevisiae temperature-sensitive dpm1
mutant (33), the MPD synthase is required for O
mannosylation and participates in N glycosylation of proteins as a
donor of the last four mannosyl residues during the assembly of the
lipid-linked precursor oligosaccharide, i.e., dolichylpyrophosphate
GlcNac2Man9Glc3, and is required
for the biosynthesis of the glycosylphosphatidylinositol membrane
anchor (13). Cloned MPD synthases from human tissues and
Schizosaccharomyces pombe both encode a separate MPD
synthase class that lacks the C-terminal hydrophobic domain
(4), which is otherwise typical for S. cerevisiae, Ustilago maydis (42), and
Trypanosoma brucei. The major difference between the two
classes is that in the S. cerevisiae group a single
component (Dpm1p) has MPD synthase activity whereas in mammalian cells
MPD synthase activity is mediated by two proteins, i.e., catalytic Dpm1
and regulatory Dpm2 (28).
Based on our previous findings that MPD synthase activity in T. reesei is related to the level of protein secretion (15, 17), we determined whether overexpression of DPM1
resulted in enhanced cellulase secretion. Such an increase would
improve the secretion of other O-glycosylated proteins. We have
overexpressed S. cerevisiae DPM1 in T. reesei and
examined its effect on protein glycosylation and secretion.
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MATERIALS AND METHODS |
Strains and conditions for growth.
T. reesei TU-6, a
pyr4 mutant of T. reesei QM 9414 (10), was used as the recipient for transformation.
Escherichia coli JM 109 was used for plasmid propagation
(41).
T. reesei was cultivated in 2-liter shake flasks containing
1 liter of minimal medium, i.e., 1 g of MgSO4 · 7H2O, 6 g of
(NH4)2SO4, 10 g of
KH2PO4, 3 g of Na citrate · 2H2O, microelements (90 µM FeSO4, 20 µM
MnCl2, 20 µM ZnSO4, 100 µM
CaCl2, final concentrations in the medium), and 1% lactose
as a carbon source, as specified for the experiments, at 28°C on a
rotary shaker (250 rpm).
Preparation of cell extracts.
Mycelia were harvested by
filtration, washed with ice-cold tap water, dried, and suspended in 50 mM citrate buffer, pH 5.0 (10 ml/g [wet weight]). Cell extracts were
prepared by ultrasonication of the suspension. The homogenate was
centrifuged at 10,000 × g (15 min, 4°C), and the
supernatant (typically containing 2 to 4 mg of protein per ml) was used
for Western blot analysis.
Expression of S. cerevisiae DPM1 in T. reesei.
To amplify S. cerevisiae DPM1 in T. reesei, we introduced the gene into T. reesei fused to
the regulatory signals of the pki1 (pyruvate
kinase-encoding) gene (36). Thus, the complete coding sequence of the S. cerevisiae DPM1 gene was amplified by
PCR, with an Expand high-fidelity PCR system (Boehringer-Mannheim, Mannheim, Germany). The oligonucleotides Dpm1s
(5'-GCTCTAGAATGAGCATCGAATACTCTGTAA-3') and Dpm1r
(5'-GCTCTAGATTAAAAGACCAAATGGTATAGC-3') were used as forward
and reverse primers, respectively. Both primers had XbaI restriction sites attached to both ends to facilitate subsequent cloning. The reaction mixture for amplification contained 1 mM primers,
250 mM deoxynucleoside triphosphates, Expand high-fidelity reaction
buffer, 1.5 mM MgCl2, 2.6 U of enzyme mix, and
H2O to a total of 50 µl. Twenty nanograms of plasmid pDM8
(32) was used as a template. The temperature program was as
follows: denaturation at 96°C for 2 min; 30 cycles of 94°C for
30 s, 52°C for 30 s, and 72°C for 30 s; and a final
elongation at 72°C for 3 min. We obtained an 801-bp fragment that was
sequenced and cloned into the XbaI site of pUC19 to yield
pJSK1. Linearization of pJSK1 by SspI, partial digestion
with XbaI, and complete cleavage with SalI
generated an 0.8-kb XbaI/SalI fragment, which
was subcloned into pLMRS3 (26) previously digested
with SalI/XbaI to yield pDPMEX. This vector
was introduced into T. reesei TU-6 (10) by
cotransformation as described previously (20, 27), with 5 µg of pFG1 containing the T. reesei pyr4 gene
(8) and 15 µg of pDPMEX. Further isolation of
transformants was carried out as previously described (8).
Molecular biology methods.
Chromosomal DNA was isolated from
T. reesei by acid guanidinium thiocyanate-phenol-chloroform
extraction as described previously (8). Total RNA was
isolated by a single-step method described by Chomczynski and Sacchi
(3). Other molecular biological techniques were performed
according to standard protocols (35).
Biochemical techniques.
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, Western blotting, and
immunological detection of secreted cellobiohydrolase I (CBH I) were
carried out as described previously (31), by a
chemiluminescence assay (ECL Western blot analysis system; Amersham Life Sciences, Little Chalfont, Buckinghamshire, United Kingdom). When
intracellular CBH I was assayed, a secondary antibody was used to
remove nonspecific antigens. The secondary antibody was incubated (30 min, 4°C) with T. reesei cell walls prepared as described
elsewhere (30) at 1 mg/ml.
The presence of soluble polysaccharides and glycoproteins in the
culture filtrate was assayed by the phenol-sulfuric acid procedure
(5), with samples precipitated by 2 volumes of ethanol and
resuspended in distilled water. The calibration curve was prepared with
D-mannose. Protein was estimated according to the method of
Lowry et al. (25).
Enzyme activity assay.
MPD synthase activity was assayed in
a microsomal membrane fraction of T. reesei (14,
16).
Quantification of fungal dry weight.
Fungal dry weight was
quantified by filtering culture through a coarse (G1 grade)
sinter funnel, washing the fungal material with a threefold volume of
tap water, and drying the material to a constant weight at 110°C.
Electron microscopy.
Mycelia of T. reesei JSK97/3
(transformed strain) and T. reesei JSK97/C (control strain)
harvested after 200 h of cultivation were collected by
centrifugation and prepared for electron microscopy as described
previously (21) except that the procedure for
immunolocalization was omitted. Ultrathin sections were examined under
a JEM100C transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 80 kV.
| |
RESULTS |
Construction of T. reesei strains overexpressing
S. cerevisiae DPM1.
Our strategy to enhance the cellular MPD
synthase activity was to introduce multiple copies of the S. cerevisiae DPM1 gene into T. reesei. To this end, we
fused DPM1 under the 5' regulatory signals of the T. reesei pki1 (pyruvate kinase-encoding) (36) gene, which
allows a three- to fourfold increase in transcription (26).
The resulting vector was introduced into T. reesei TU-6 by
cotransformation, and prototrophic transformants were selected. Stable
transformants were isolated by three rounds of transfer from selective
to nonselective medium and screened by Southern hybridization for
genomic copies of DPM1. Four such transformants (JSK97/1,
-2, -3, and -6) were obtained. All of them carried integrated copies of
the yeast gene. T. reesei JSK97/C, the control, i.e., a
recipient TU-6 strain transformed with the pFG1 helper vector only,
gave no signal with the DPM1 probe.
To determine if the heterologous
DPM1 gene was correctly
expressed and translated, we carried out Northern blot analysis and
MPD
synthase activity assays of the transformants (Fig.
1). As
expected, no
DPM1
transcript was detected in the JSK97/C control,
but this transcript was
detected in all of the transformants.
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FIG. 1.
Transcription and function of the yeast DPM1
gene in T. reesei. (A) MPD synthase activity (given in
picomoles of [U-14C]mannose incorporated into the lipid
fraction per 5 min per milligram of protein) was measured in the
membrane (50,000 × g) fraction from the control
(strain C) and the transformants harboring the yeast DPM1
gene (strains 1, 2, 3, and 6). Values are means of at least five
independent measurements, with a standard deviation of <5%. (B)
Northern blot analysis of DPM1 mRNA. Equal amounts of total
RNA (20 µg) from the control (C) strain and from the transformants
(1, 2, 3, and 6) were loaded onto the gels, blotted, and probed with a
0.6-kb BanII fragment of the S. cerevisiae DPM1
gene. Loading controls, hybridized with a 1.9-kb KpnI
fragment of the T. reesei act1 (actin-encoding) gene, are
also shown.
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MPD synthase activity of the transformants increased 2- to 2.5-fold
over that of the control. This relatively low increase
is not due to
limiting amounts of GDP[
14C]Man in the assay, since the
use of a sixfold-higher concentration
of
GDP[
14C]Man in the assay did not result in a
higher ratio of activities
of the transformants and the control strain
(data not shown).
The assay was carried out in the presence of
exogenous dolichylphosphate
(5 nmol) to ensure that sufficient lipid
substrate was available
for the increased amount of MPD synthase in the
transformants.
Expression of the yeast MPD synthase-encoding gene in T. reesei.
To determine if the doubled activity of MPD synthase
influenced the rate of protein secretion or the final concentration of the secreted protein, we cultivated the transformants and the control
strain on medium containing lactose as the sole carbon source and
measured protein production (Fig. 2). All
of the transformants secreted increased amounts of protein. The
differences were most pronounced following extended cultivation and
reached the maximum at 180 to 200 h. The effect was even more
drastic when calculated per unit of cell mass (gram of fungal dry
weight), reaching up to sevenfold-higher levels (Fig. 2). Note that the
parental strain had only negligible amounts of cellulase activity
within its cell wall (18) and that the increase in activity
in the transformants could not be due to a more efficient release from
or passage through the cell wall.
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FIG. 2.
Effect of DPM1 overexpression on protein
secretion by T. reesei. The figure shows the specific rate
of protein secretion (milligrams of protein per gram of fungal dry
weight, left axis) and the ratio of protein-bound carbohydrate to
protein concentration (right axis). Open symbols, secreted glycans;
solid symbols, secreted proteins; circles, control strain JSK97/C;
squares, JSK97/3 transformant. The data shown are from a single
experiment only; however, in at least five parallel experiments,
similar patterns were observed. DW, dry weight.
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Overexpression of MPD synthase might preferentially affect the amount
of the secreted glycoproteins or their degree of glycosylation.
We
determined the concentration of total protein-bound carbohydrate
in the
medium and normalized it to the amount of protein (Fig.
2). The
resulting values were comparable in the transformants
and in the
wild-type strain, leading us to conclude that overexpression
of
DPM1 does not affect the degree of glycosylation of the
secreted
proteins.
Cellulase formation in T. reesei strains overexpressing
DPM1.
We made Northern blots of mRNA from all the
transformants and hybridized them to a fragment of the cbh1
gene. CBH I accounts for more than 50% of total secreted protein in
T. reesei and can be used as a marker for total cellulase
formation. Despite the significant increase in cellulase formation,
cbh1 transcript levels were not increased in the
transformants (Fig. 3).
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FIG. 3.
Effect of DPM1 overexpression on
cbh1 gene expression in T. reesei. Total RNA was
isolated from the control (C) and the transformant (1, 2, 3, and 6)
strains, and equal amounts (20 µg) were loaded onto agarose gels,
blotted, and hybridized with a 1.5-kb BglI fragment of
cbh1, randomly primed with [ -32P]dATP.
Actin controls were used as described in the legend to Fig. 1.
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The results from Northern blotting suggested that a posttranscriptional
process may be responsible for cellulase overproduction
in the
transformants. We determined the intra- and extracellular
cellulase
contents. CBH I was again used as the model. All but
one of the
transformants contained clearly detectable intracellular
levels of CBH
I, which were absent in the wild-type strain (Fig.
4).
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FIG. 4.
Effect of DPM1 overexpression on intra- and
extracellular levels of CBH I. The presence of CBH I in the culture
fluid (A) or in cell extracts (B) was analyzed by Western blotting and
immunostaining. Equal volumes of the culture filtrate were loaded onto
the gels for panel A, whereas equal amounts of protein (25 µg) were
loaded onto the gels for panel B. Lane C, control strain; lanes 1, 2, 3, and 6, transformant strains.
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Cell ultrastructure of T. reesei strains overexpressing
DPM1.
The JSK97/3 transformant, showing the highest rate of
growth and protein secretion (per milliliter of medium), was subjected to electron microscopy and compared with the control (JSK97/C). Some
essential differences were observed. The cell wall of T. reesei JSK97/3 has a clearly flocculous structure (Fig.
5a to c). The cytoplasm of the
transformed cells was electron transparent, and the number of
mitochondria, but not of vacuoles and nuclei, was three times higher
than what we observed in the control (Table 1). The cell wall of the control JSK/97 C
strain (Fig. 5d and e) showed a compact cell wall layer, and the
cytoplasm was electron opaque and densely packed with ribosomes.
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FIG. 5.
Effect of DPM1 overexpression on cellular ultrastructure
of T. reesei. Ultrathin sections are shown from T. reesei transformant JSK97/3 (a to c) and the control strain
JSK97/C (d to f). (a to c) Filamentous material at the surface of the
cell wall; electron-transparent cytoplasm; mitochondria (b and c) and
endoplasmic reticulum and plasma membrane (b). The cell wall of the
control strain is composed of one compact layer, not covered by
filamentous material, and the cytoplasm is electron opaque, showing
cell wall, cross wall, nucleus, tonoplast, and vacuole. c, cross wall;
cw, cell wall; er, endoplasmic reticulum; fm, filamentous material; m,
plasma membrane; M, mitochondria; N, nucleus; Ne, nuclear envelope; t,
tonoplast; V, vacuoles.
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TABLE 1.
Ultrastructural differences between the T. reesei strain overexpressing the DPM1 gene, JSK97/3,
and the control strain, JSK97/Ca
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| |
DISCUSSION |
Secretory pathways of filamentous fungi are widely studied. So
far, however, no clear data on the regulation of secretion are
available (1, 34). We improved protein secretion in T. reesei by overexpressing the yeast DPM1 gene, involved
in biosynthesis of glycosylphosphatidylinositol anchor and O
mannosylation and N glycosylation of protein. CBH I contains both N-
and O-linked mannose, and dolichyl phosphate is involved in O
mannosylation in T. reesei (14, 34).
We have previously suggested that the O-mannosyl linkage is an
essential or a rate-limiting step for cellulase secretion (15, 19). The present data are consistent with this hypothesis since higher levels of activity of MPD synthase, the key enzyme in the O-mannosylation reaction, correspond to increased levels of protein production-secretion.
The mechanism of this process, however, is not obvious. The
extracellular proteins secreted by the parental strain and by the
transformants overexpressing MPD synthase contain the same percentages
of carbohydrate and are not overglycosylated. These results also are
consistent either with a limiting role of O mannosylation in protein
secretion or with tight control of the number of occupied sites in the
nascent protein and the glycan chain length. Alternatively, O
mannosylation of Aspergillus glucoamylase may limit the
conformational space available to the unfolded peptide and help
stabilize the folded protein (24, 40). If this explanation
applies to T. reesei cellulases, then an increase in O
mannosylation might increase protein production by slowing down the
turnover of the nascent protein. This hypothesis is supported by our
findings of higher intracellular levels of CBH I in the
DPM1-amplified strains. However, no hyperglycosylated,
secreted proteins were detected in the culture medium. The
DPM1-transformed strains secreted most of their protein at a
time of cultivation when the parent had already stopped protein secretion. Whether this behavior is related to the hypothesized effect
on protein turnover is not known, but it could be related to other
major physiological changes in the transformants.
We identified several such major changes in this study, e.g.,
alterations in cell wall architecture, increase in the number of
mitochondria, and changes of the electron density of the cytoplasm in
the DPM1-transformed strains (Table 1). The increase in the number of mitochondria was concomitant with a two- to threefold increase in activity of cytochrome c oxidase, a
mitochondrial marker enzyme. Hardwick et al. (9) observed
altered membrane organization in yeast strains overexpressing
DPM1. These transformants accumulated small vesicles that
were not observed in the present study with T. reesei.
Hardwick et al. (9) reasoned that overexpression of MPD
synthase deregulates dolichol metabolism and thereby alters the
properties of endoplasmic reticulum and Golgi membranes; if this
explanation is also true for T. reesei, it may give rise to
a number of pleiotropic effects.
Our results from this study clearly show that overexpression of yeast
DPM1 in T. reesei influences protein secretion.
While at the cellular level the effect is not yet defined, to the best of our knowledge this is the first report of protein secretion in a
filamentous fungus being increased by molecular genetic manipulation.
| |
ACKNOWLEDGMENTS |
This work was supported by grants (662699203 to J.S.K. and 6PO4B
01712 to G.P.) from the State Committee for Scientific Research (KBN),
Warsaw, Poland, and by grants from the Ministry of Science and Research
of Austria (GZ 49.694/3-II/A/4/1990) and from the Austrian Science
Foundation (P 7542, 1990-1991) to C.P.K.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biochemistry and Biophysics, Polish Academy of Sciences,
Pawi
skiego 5a, 02-106 Warsaw, Poland. Phone: 48 22 658 47 02. Fax: 48 39 121 623. E-mail: gp{at}ibbrain.ibb.waw.pl.
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0099-2240/99/$04.00+0
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Abstract
Introduction
