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Applied and Environmental Microbiology, August 1998, p. 2794-2799, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Expression and Secretion of Defined Cutinase
Variants by Aspergillus awamori
I. A.
van
Gemeren,1,*
A.
Beijersbergen,1
C. A. M. J. J.
van den
Hondel,2 and
C. T.
Verrips1,3
Department of Biotechnology, Unilever
Research, 3133 AT Vlaardingen,1
Department of Molecular Genetics and Gene Technology, TNO
Nutrition and Food Research Institute, 3700 AJ
Zeist,2 and
Department of Molecular
and Cellular Biology, University of Utrecht, 3584 CH
Utrecht,3 The Netherlands
Received 17 March 1997/Accepted 15 May 1998
 |
ABSTRACT |
Several cutinase variants derived by molecular modelling and
site-directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae.
The majority of these variants are successfully produced by the
filamentous fungus Aspergillus awamori. However, the L51S
and T179Y mutations caused reductions in the levels of
extracellular production of two cutinase variants by A. awamori. Metabolic labelling studies were performed to
analyze the bottleneck in enzyme production by the fungus in detail.
These studies showed that because of the single L51S substitution,
rapid extracellular degradation of cutinase occurred. The T179Y
substitution did not result in enhanced sensitivity towards
extracellular proteases. Presumably, the delay in the extracellular
accumulation of this cutinase variant is caused by the enhanced
hydrophobicity of the molecule. Overexpression of the A. awamori gene encoding the chaperone BiP in the
cutinase-producing A. awamori strains had no
significant effect on the secretion efficiency of the cutinases. A
cutinase variant with the amino acid changes G28A, A85F, V184I, A185L,
and L189F that was known to aggregate in the endoplasmic
reticulum of S. cerevisiae, resulting in low extracellular
protein levels, was successfully produced by A. awamori. An initial bottleneck in secretion occurred before or during translocation into the endoplasmic reticulum but was rapidly
overcome by the fungus.
| |
INTRODUCTION |
Cutinases are produced by several
phytopathogenic fungi, including Fusarium solani pisi
(29), Magnaportha grisea (30), and
Colletotrichum gloeosporioides (7). These enzymes
can hydrolyze ester bonds in the cutin polymer, an insoluble
lipid-polyester matrix covering the surfaces of plant leaves
(18). Cutinase belongs to a class of esterases that are able
to hydrolyze fatty acid esters and emulsified triglycerides as
efficiently as lipases, without showing enhancement of activity in the
presence of a lipid-water interface (18). It has been found
that cutinase has an "in-the-wash" effect, making this protein
suitable for use in laundry detergents (31). The
three-dimensional X-ray structure of the cutinase from F. solani pisi (21) was used to design variants in a
protein-engineering study aimed at increasing knowledge of the
structure-function relationship of cutinase and improving its
performance in detergent formulations. The amino acid sequence was
modified in such a way that the hydrophobicity at the surface of the
enzyme was increased to form an enlarged lipid contact zone
(5) and decreased sensitivity towards anionic surfactants
present in detergents (6).
A synthetic copy of the cDNA encoding the cutinase from F. solani pisi has been expressed successfully in Saccharomyces
cerevisiae and Aspergillus awamori (32).
About 20% of cutinase variants expressed in S. cerevisiae
could not be produced at the same extracellular levels as the wild-type
cutinase is produced (unpublished results) (Table
1). In this study, we analyzed whether
these variants could be produced by the filamentous fungus
A. awamori.
Several studies have described the overproduction of both a molecular
chaperone and a heterologous protein with the aim of increasing the
yield of the heterologous protein (19, 25). Overexpression
of the BiP-encoding gene in S. cerevisiae resulted in an
increase in heterologous protein production (12). This suggests that overproduction of foreign proteins can result in saturation of the protein-folding machinery caused by limited availability of BiP in the endoplasmic reticulum of eukaryotes. A
similar situation may occur in filamentous fungi. Therefore, the gene
encoding the BiP protein from A. awamori,
bipA, was cloned (34) and subsequently
overexpressed in the cutinase-producing strains.
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MATERIALS AND METHODS |
Strains, media, and transformations.
Standard molecular
biology procedures were performed as described by Sambrook et al.
(27). Escherichia coli JM109 (37) and
1046 (F
met hsdS supE supF recA56) were used
as hosts for molecular cloning.
AW4-20, a pyrG (orotidine-5'-phosphate
decarboxylase)-deficient derivative of A. awamori
CBS 115.52 that contains a defined mutation at the BglII
site of the pyrG gene (9), was used as a
recipient strain for transformation experiments carried out as
described by Punt and Van den Hondel (23). Transformants of
the pyrG-deficient strain with cutinase vectors were
selected on the basis of uridine prototrophy. Transformants with
additional copies of the A. awamori bipA gene
were obtained by selection on medium containing 100 µg of hygromycin
per ml. All strains were cultivated in Aspergillus minimal
medium (3) supplemented with 0.5 or 0.1% (wt/vol) yeast
extract (Difco Laboratories, Detroit, Mich.).
Construction of plasmids.
Mutations in the cutinase gene
were obtained by site-directed mutagenesis via PCR (13). The
expression signals from the endoxylanase II (exlA) gene
(10) were used for inducible overexpression of the cutinase
alleles and bipA genes in A. awamori
strains. The mature cutinase genes were fused to the exlA
presequence, and the bipA gene was expressed with its
natural presequence. The coding regions were adapted for cloning into
the general fungal expression vector pAW14B12 (33). A
BspHI site was introduced at the start codon and an
AflII site was introduced at the stop codon of the genes,
which were subsequently cloned into the BbsI and
AflII sites of pAW14B12. Studies in one of our
laboratories have shown that the HindIII fragment
containing the exlA expression signals is sufficient for
high levels of gene expression in A. awamori
(unpublished results). Vectors containing four to eight HindIII fragments of cutinase cassettes in tandem at the
HindIII site of pUR7981 (Fig.
1), cosmid pJB8 (14)
containing a pyrG gene with a defined mutation at the
SalI site (9), were obtained via packaging by
using a Gigapack Gold kit from Stratagene (La Jolla, Calif.). Because
homologous integration of these large vectors at the pyrG
locus of A. awamori could not be accomplished in 30 separate transformation experiments, cotransformations were performed
with plasmid pAW4-1 (9). This plasmid contains the intact pyrG gene, and cotransformation with pAW4-1
resulted in several random multicopy transformants of cosmid
pUR7981-HX (Fig. 1). To obtain homologous integration at the
pyrG locus of AW4-20, two SalI cutinase cassettes
were cloned into the SalI site of pUR7981, resulting in
plasmid pUR7981-SX (Fig. 1). The A. awamori bipA gene, derived from plasmid pAWBiP
(34), was adapted and cloned into pAW14B12, resulting in
pUR7987 (35). For selection, pAWBiP and pUR7987
were provided with the hygromycin resistance expression cassette
derived from pAW15-7 (10), resulting in pUR7381 and
pUR7988, respectively (35).
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FIG. 1.
Schematic diagram of the construction of cutinase
variant gene (cutiX) integration vectors. pcutiX is a model expression
vector, in which cutiX is regulated by the exlA
(endoxylanase II) promoter (P) and terminator (T). pUR7981 is a
derivative of pJB8 containing the pyrG gene with a defined
SalI mutation, pyrG*. For random integration,
more than four cutiX expression cassettes were cloned into the
HindIII site of pUR7981, resulting in pUR7981-HX.
Targeted integration at the pyrG locus was performed with
pUR7981-SX, with pUR7981 containing two cutiX expression cassettes at
the SalI site.
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|
Protein analysis.
Screening of cutinase variant
transformants for cutinase activity was carried out with BYPO plates
containing an olive oil-arabic gum emulsion (8), with
previously described adjustments (33). The formation of
clear zones surrounding the colonies was used as an indication of
active enzyme production. Shake flask induction experiments were
performed as described by Gouka et al. (10) by using 5%
D-xylose as an inducer. In these analyses, the single-copy cutinase strain AW85 (33) containing a wild-type cutinase
expression cassette at the pyrG locus was used as a control
strain. The amount of cutinase produced extracellularly 42 h after
induction was measured spectrophotometrically by using
p-nitrophenyl butyrate (PNPB) (Sigma) as the substrate as
described by Van Gemeren et al. (32). In the PNPB
measurements, standard amounts of all of the purified variant cutinases
except L51S produced similar increases in absorbance, indicating that
the specific activities with this substrate were similar. L51S was not
produced in sufficient quantities to be tested in the purified form in
the PNPB assay, but the activities of crude preparations were
comparable to those of the other enzymes.
Intracellular cutinase was analyzed after washing of the mycelium with
Triton X-100, disruption, and sodium dodecyl sulfate
(SDS) treatment of
the mycelium as described by Van Gemeren et
al. (
33). The
samples were subjected to Western blot analysis
(
27) by
using a cutinase-specific polyclonal antiserum raised
in rabbits. The
amounts of BiP protein present in the different
strains were determined
by a Western blot analysis of mycelial
extracts by using an
Aspergillus niger BiP-specific antiserum
raised in rabbits
(
35).
DNA and RNA procedures.
Mycelium samples were taken 15 or
22 h after induction with D-xylose in shake flask
experiments. A. awamori chromosomal DNA and total
RNA were isolated from mycelial powder as described by Kolar et al.
(17). A Northern blot analysis with a Hybond membrane
(Amersham International, Little Chalfont, Buckinghamshire, England) was
performed by using standard procedures (27). In the slot
blot analysis of DNA and RNA, twofold dilutions were blotted onto a
GeneScreen Plus membrane (Dupont NEN Products, Boston, Mass.) by using
a Milliblot-S apparatus (Millipore Corp., Bedford, Mass.). The blots
were hybridized with the ApaI-AflII cutinase
fragment or the XhoI bipA fragment
(34). The coding region of the glyceraldehyde-3-phosphate
dehydrogenase (gpdA) gene of A. niger
(1.4-kb HindIII fragment from pAB5-2
[22]) was used as an internal control for the amount
of nucleic acid blotted. The multiprime DNA labelling system (Amersham
International) was used to label the DNA probes with 32P.
Hybridization signals were quantified with an InstantImager (Packard,
Canberra, Australia). Transformants containing the bipA expression vectors were identified by colony hybridization
(16) by using the internal XhoI
bipA fragment as a probe.
Metabolic labelling and immunoprecipitation.
Cutinase-producing strains were cultured in shake flasks, and 5-ml
samples were taken 22 h after induction with 5%
D-xylose. Each mycelium sample was filtered over Miracloth
(Calbiochem, La Jolla, Calif.), washed with 5 ml of minimal medium
without yeast extract (MM-Y), and induced with 5 ml of this medium
containing 2.5% D-xylose. The mycelium was shaken for
1 h at 30°C and 125 rpm, and then 20 to 30 µCi of
35S-labelled methionine-cysteine (1,000 Ci/mmol; Pro-mix;
Amersham) per ml was added. After 20 min, the mycelium was washed again with MM-Y, suspended in MM-Y containing xylose, and 25 mM methionine and 25 mM cysteine were added. One-milliliter samples were taken at
intervals and kept on ice during further treatments. The medium was
removed by centrifugation, and the mycelium was washed with ice-cold
phosphate buffer (50 mM
Na2HPO4/NaH2PO4 [pH
7.0], 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF]). The
mycelium samples were disrupted in 1 ml of solubilization buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% SDS, 0.5% Triton X-100, 1 mM
EDTA, 1 mM PMSF) by adding 0.8 g of acid-washed glass beads and
vortexing three times for 30 s in glass vials. The disrupted mycelium samples were incubated for 5 min at 94°C in 2-ml Eppendorf tubes and centrifuged for 10 min at 14,000 × g and
4°C. The supernatant collected was designated the soluble
intracellular protein fraction.
Immunoprecipitation of the labelled cutinase was performed with a
solution containing 200 µl of the soluble intracellular
protein
fraction, 200 µl of immunoprecipitation buffer (50 mM
Tris-HCl [pH
7.5], 150 mM NaCl, 0.5% Tween 20, 1 mM EDTA, 1 mM
PMSF), and 25 µl
of cutinase-specific polyclonal antiserum. The
samples were rotated at
room temperature for 2 h, 30 µl of protein
A-Sepharose CL-4B
(0.14 g/ml; Sigma) was added, and the mixture
was incubated for an
additional 2 to 16 h. Subsequently, the samples
were centrifuged
for 10 min at 14,000 ×
g and 4°C. The
immunoprecipitates
were washed with immunoprecipitation buffer, and 10 µl of SDS
sample buffer was added. Extracellular proteins were
precipitated
by adding 15 µl of 50% (vol/vol) trichloroacetic acid
to 200 µl
of extracellular medium, incubating the preparation on ice
for
30 min, and centrifuging it for 10 min at 14,000 ×
g and 4°C.
The precipitates were washed with 200 µl of
ice-cold acetone and
resuspended in 10 µl of sample buffer. Prior to
SDS-polyacrylamide
gel electrophoresis, the samples were heated at
94°C for 5 min
and centrifuged for 1 min. After Coomassie blue
staining and destaining,
the gels were treated for 15 min with Amplify
(Amersham), dried,
and exposed to X-ray film for 1 to 2 weeks.
| |
RESULTS |
Analysis of the production of cutinase variants by A. awamori.
Six cutinase variants that were poorly secreted by
S. cerevisiae were studied further with A. awamori (Table 1). The expression of the genes was
controlled by the inducible exlA promoter (10). In an earlier study, twofold more cutinase was produced by strains containing a cassette with the mature cutinase region fused directly to
the exlA presequence than by strains containing constructs with the cutinase prosequence in addition to the exlA
presequence (33). Similar cassettes expressing cutinase
variants were cloned in a tandem array into cosmids and were randomly
integrated into the genome of A. awamori. As a
result, the locus was unknown, but the cassettes were integrated
in a defined head-to-tail manner.
Transformants were prescreened for cutinase activity with a plate assay
and with shakeflask induction experiments. The transformants
with the
highest cutinase activities were selected for determinations
of the
numbers of expression cassettes and the cutinase-specific
mRNA levels
(Table
2). The extracellular cutinase
protein levels
determined by hydrolysis of PNPB were in agreement with
results
obtained by Western blotting (Fig.
2, lanes 1 through 4), indicating
similar
immunogenic reactivities.
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FIG. 2.
Western blot analysis of cutinase variant production by
A. awamori after 42 h of induction with 5%
D-xylose. For analysis of extracellular cutinase production
a fraction of the culture medium was used (lanes 1 through 4), and
intracellular fractions were obtained after SDS treatment of pulverized
mycelia (lanes 5 through 8). The following transformants were analyzed:
AW91 (lanes 1 and 5), AW23 (lanes 2 and 6), AW23-16 (lanes 3 and 7),
and AW48-15 (lanes 4 and 8).
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|
The production of four of the cutinase variants by
A. awamori transformants AW20-02/09 (E201K), AW28 (G82A,
A85F, V184I, A185L,
and L189F), AW34 (A29- and S30-), and
AW55-01/05 (W69Y) was comparable
to the production of the wild-type
cutinase by AW91-06. The protein
levels of cutinase variant T179Y
produced by transformants AW48-04
and AW48-15, however, were
considerably lower than the wild-type
protein level. Moreover, there
was almost no extracellular production
of cutinase variant L51S by
transformant AW23-16. To facilitate
a precise comparison of the
production of this cutinase variant
with wild-type cutinase production,
two cassettes containing the
wild-type gene or the L51S cutinase mutant
alleles were cloned
into a cosmid and integrated at the
pyrG
locus. The resulting
defined transformants, AW91 and AW23, exhibited
similar levels
of transcription of the cutinase cDNA (Table
2). The
level of
extracellular production of L51S cutinase by AW23 was more
than
10-fold lower than the level of wild-type cutinase production
by
AW91. This result is comparable to the results obtained with
the
randomly transformed counterparts AW91-6 and AW23-16 (Table
2). Western
blot analysis of the culture media from these strains
revealed patterns
similar to the activity measurement patterns
(Fig.
2, lanes 1 through
4). The amounts of intracellular cutinase
produced by AW91, AW23,
AW23-16, and AW48-15 were determined by
Western blot analysis of
SDS-treated, pulverized mycelia (Fig.
2, lanes 5 through 8). Assuming
that there were no differences
in translational efficiency, this
analysis revealed that there
is little intracellular accumulation of
the wild-type and variant
cutinases in
A. awamori.
Effect of overexpression of bipA.
To study
whether an increase in the level of the chaperone protein BiP could
improve the secretion of the cutinase variants, the A. awamori bipA gene (34) controlled either by its
own promoter or by the exlA expression signals was
introduced into the wild-type or cutinase variant-producing
transformants AW91, AW23, and AW48-15. Northern blot analysis
revealed a significant increase in the A. awamori
bipA mRNA in transformants of AW91, AW23, and AW48-15 containing multiple copies of the bipA gene (results not
shown). The A. awamori BiP protein level at 38 h after induction, however, was increased only in transformants of AW91
and AW23 containing extra bipA copies controlled
by the exlA promoter (Fig. 3A,
lanes 3 and 6, respectively). The 75-kDa band corresponds to the intact protein, which could have formed a dimer under nonreducing
conditions via one S-S bridge, resulting in the more slowly migrating
band. The more rapidly migrating bands presumably represent degradation products of BiPA (12). Strains containing extra copies of
the A. awamori bipA gene with its own promoter
did not contain increased BiPA levels (Fig. 3A, lanes 2 and 5),
presumably because of negative feedback regulation on the
transcription-translation level (35). The elevated
A. awamori BiP levels did not result in higher
cutinase production levels. Production of the wild-type cutinase even
decreased slightly (Fig. 3B).
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FIG. 3.
Effect of overexpression of bipA in
A. awamori. (A) Western blot analysis of BiP
protein levels by using BiP-specific antiserum of mycelial extracts
from the following transformants: AW91 (lane 1), AW91 containing
bipA (under control of the natural promoter) (lane 2),
AW91 containing bipA-PexlA (under control of
the exlA promoter) (lane 3), AW23 (lane 4), AW23 containing
bipA (lane 5), and AW23 containing
bipA-PexlA (lane 6). (B) Effect of
overexpression of bipA on extracellular cutinase
production. Averages of the values from three independent shake flask
induction experiments are shown. wt, wild type.
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|
Metabolic labelling analysis of cutinase production.
Secretion
of wild-type and variant cutinases by A. awamori
was analyzed by metabolic labelling experiments performed with [35S] methionine-cysteine. The mycelial wet weights
and protein patterns on SDS gels were identical for all strains at
specific time points, indicating that the cultures grew equally and
induction of protein synthesis was the same in all cultures.
Analysis of cutinase production by AW91 expressing the wild-type
cutinase showed that the increase in extracellular cutinase
levels over
time was not reflected by a similar decrease in intracellular
cutinase
levels (Fig.
4). This suggests that
labelled cutinase
was synthesized even after removal of the
radiolabelled substrate,
probably by ongoing translation of
cutinase-specific mRNA with
labelled methionine and cysteine which had
formed an intracellular
storage pool. Cycloheximide was not added to
the cultures to arrest
translation because this treatment could also
have blocked the
production of necessary chaperones.
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FIG. 4.
Pulse-labelling analysis of strains producing cutinase
variants. Samples were taken at several time points during the chase.
These samples were subjected to immunoprecipitation with
cutinase-specific antiserum and separated by SDS-polyacrylamide gel
electrophoresis and autoradiography was performed. The following
transformants were analyzed: AW91, AW23, AW48-15, and AW28.
Representative results from one of three analyses are shown.
Abbreviations: I, intracellular fractions; E, extracellular fractions;
p, precutinase; m, mature cutinase.
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|
Transformant AW23 expressing cutinase variant L51S exhibited reduced
intra- and extracellular cutinase levels compared to
AW91 (Fig.
4). The
extracellular cutinase level of AW23 increased
slightly for 0.5 h
and then declined rapidly (Fig.
4). This result
is consistent with
extracellular proteolytic degradation of the
L51S variant. To analyze
the possibility that production of L51S
induced synthesis of
extracellular proteases, purified wild-type
cutinase was added to AW23
culture medium. The cutinase was not
degraded after 24 h of
incubation at 30°C, suggesting that there
was no increased induction
of extracellular protease production
and that this variant has
increased proteolytic susceptibility
compared with that of the
wild-type cutinase.
The cutinase variant T179Y production pattern of AW48-15 resembled that
of wild-type strain AW91 (Fig.
4). Nevertheless, the
extracellular
cutinase level was significantly lower than the
extracellular cutinase
level in AW91. The possibility that extracellular
proteolytic breakdown
occurred was excluded, as purified T179Y
was stable for 24 h at
30°C in the culture medium of wild-type
A. awamori or of AW48-15. This suggests that there was an
intracellular
bottleneck in the production of variant T179Y.
A surprising result was obtained with transformant AW28, which
contained a gene encoding a cutinase variant with multiple
mutations.
Although this variant was produced at wild-type levels
after 42 h
(Table
2), the pattern of intracellular cutinase production
differed
from that of the other cutinase variants (Fig.
4). The
intracellular fractions of AW91 and AW48 contained two forms of
cutinase, a 22.2-kDa precursor with a prepeptide and the mature
20.6-kDa form. In most cases, the precursor form was present at
a low
level (10%) compared to the mature cutinase and stayed visible
over
the entire 5-h experiment. In the intracellular fraction
of AW28, 66%
of the cutinase was present in the preform at zero
time (Fig.
4). After
0.5 and 2.5 h of chase, 11 and 1% of the
intracellular cutinase
contained a prepeptide, respectively. In
addition, the level of the
intracellular mature cutinase form
decreased more rapidly in AW28 than
in the other strains (Fig.
4).
| |
DISCUSSION |
Previous studies have shown that the cutinase cDNA from
F. solani pisi is efficiently expressed by the
industrial hosts S. cerevisiae and A. awamori (32, 33). Protein-engineering studies have
been performed to obtain a more stable enzyme with greater performance
in detergents. In these studies, about 20% of the cutinase variants
were not produced efficiently by S. cerevisiae (unpublished results) (Table 1). In this report, we describe the
production of several of these variants by A. awamori.
Most of the cutinase variants were produced efficiently by
A. awamori. However, two variants, L51S and T179Y,
were produced at low levels, and the bottleneck was shown to be located
after transcription. The possibility that there was less efficient
translation of the transcripts encoding these two variants cannot be
excluded. However, according to the general codon usage bias of
aspergilli (20; unpublished results), the
alterations of L51S (TCT) and T179Y (TAC) do not require the use of
rare tRNAs. The drastic effect of the substitution of one amino acid on
protein production has been described earlier for bacterial, yeast, and
mammalian cells (15, 24, 36). When A. awamori
glucoamylase was expressed in yeast, amino acid deletions resulted in
both intracellular accumulation and higher susceptibility to
proteolytic degradation of this protein (2). Protein
aggregation and intracellular accumulation often are observed during
heterologous protein production in S. cerevisiae
(26, 28). In general, filamentous fungi do not exhibit
pronounced accumulation (11, 33), and this was also the case
with the cutinase variants.
The extracellular level of variant L51S produced by A. awamori AW23 declined rapidly. The L51S mutation was designed
to obtain more compatibility with anionic surfactants by distortion of
the hydrophobic patch near the active site. A serine located at the surface of a molecule frequently introduces flexibility, resulting in a
more open structure. This could have rendered the cutinase molecule
more susceptible to extracellular proteases.
The slow extracellular accumulation of variant T179Y by strain AW48-15
was not due to extracellular proteolytic breakdown. The T179Y
substitution probably causes increased sensitivity to intracellular
proteolytic enzymes. As the rationale for generating this cutinase
variant was to increase hydrophobicity, it is conceivable that the
intracellular form of the protein aggregates transiently, is
recognized as foreign, and subsequently is rapidly degraded. The
variant protein showed a slight improvement in performance on
lipid substrates and enhanced stability with anionic detergents (unpublished results). Disruption of protease-encoding genes, which has
been shown to be a useful approach for optimization of protein
production by aspergilli (1, 4), could improve the
production of cutinase variants L51S and T179Y.
In contrast to S. cerevisiae (26),
A. awamori AW28 produced the cutinase variant with
five amino acid substitutions at a level comparable to that of
the wild type. The hydrophobicity of the surface around the active site
has been increased in this variant, resulting in greater
stability of the enzyme and better washing performance
(26). On the other hand, this increased hydrophobicity
could have caused reduced efficiency in the translocation process
across the endoplasmic reticulum membrane. This problem appears to be
solved by the fungus, possibly with the aid of chaperones. However,
overexpression and overproduction of the gene encoding the BiP protein
from A. awamori (34) did not stimulate
production of the wild-type cutinase or of variants L51S and T179Y,
indicating that the level of this chaperone is not a major limiting
factor in the secretion of these proteins. Further studies will focus on the effect of the BiP concentration on the production of other secretory proteins by Aspergillus strains.
| |
ACKNOWLEDGMENTS |
C. Hjort (Novo Nordisk, Bagsværd, Denmark) is acknowledged for
providing the preliminary methods used for metabolic labelling. C. Visser and L. van Schie (Unilever Research, Vlaardingen, The Netherlands) are thanked for providing cutinase PCR fragments. J. W. Kalhorn is thanked for construction of the strains with extra
bipA gene copies.
This project was supported by SENTER, a program of the Dutch Ministry
of Economical Affairs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Unilever Research, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands. Phone: 31 10 4605263. Fax: 31 10 4605383. E-mail: ingeborg-van.gemeren{at}unilever.com.
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Abstract
Introduction
