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Applied and Environmental Microbiology, July 1999, p. 2934-2941, Vol. 65, No. 7
Department of Genetics, Groningen
Biomolecular Sciences and Biotechnology Institute, University of
Groningen, 9751 NN Haren, The Netherlands
Received 8 February 1999/Accepted 13 April 1999
Despite a high capacity for secretion of homologous proteins, the
secretion of heterologous proteins by Bacillus subtilis is
frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis Bacillus subtilis and
related bacilli are attractive hosts for the production and secretion
of heterologous proteins. First, these gram-positive eubacteria secrete
proteins directly into the growth medium, which greatly facilitates
their downstream processing. Second, these organisms have a huge
capacity for protein secretion. For example, B. subtilis has
been reported to secrete the Bacillus amyloliquefaciens
Various bottlenecks for protein secretion in B. subtilis
have been identified in recent years. Such bottlenecks are related to
both the properties of the secreted protein and the machinery for
protein secretion. Most secreted proteins are synthesized as precursors
with an amino-terminal signal peptide, which is required for their
targeting to the preprotein translocase in the cytoplasmic membrane
(7, 26, 41). During or shortly after translocation of the
preprotein across the membrane, the signal peptide is removed by signal
peptidases (SPases), which is a prerequisite for release of the mature
protein from the membrane (for a recent review, see reference
5). Five paralogous chromosomally encoded type I
SPases have been identified in B. subtilis, two of which,
designated SipS and SipT, are of major importance for the processing of
secretory preproteins (35, 36).
Thus far, five potential bottlenecks in the secretion pathway of
B. subtilis have been documented. First, heterologous
proteins may form insoluble aggregates in the cytoplasm due to limited activity of chaperones (43). Second, the SPase SipS can be a limiting factor in preprotein processing (2, 40). Third, it
has been shown that the folding catalyst PrsA, which is attached to the
extracytoplasmic side of the membrane by lipid modification, sets a
limit to the high-level secretion of certain secretory proteins
(17). Fourth, it has been suggested that the cell wall forms
a barrier for at least one secreted heterologous protein, human serum
albumin (28). Fifth, it has been known for a long time that
B. subtilis secretes large amounts of proteases into the
medium, which can degrade secreted heterologous proteins (22, 30). Recent studies suggest that not only the secreted proteases but also cell-associated proteases are responsible for the degradation of secreted heterologous proteins (21, 34).
Secretion bottlenecks relating to the secreted protein are presently
poorly defined. Therefore, in the present studies, we have compared
secretion bottlenecks of four different heterologous reporter proteins
from eubacteria and eukaryotes (Bacillus licheniformis Plasmids, bacterial strains, and media.
Table
1 lists the plasmids and bacterial
strains used. Tryptone-yeast extract medium contained Bacto tryptone
(1%), Bacto yeast extract (0.5%), and NaCl (1%). Minimal medium for
B. subtilis was prepared as previously described
(36). S7 media 1 and 3, used for labeling of B. subtilis proteins with [35S]methionine (Amersham),
were prepared as described by van Dijl et al. (38). When
required, media for E. coli were supplemented with
ampicillin (100 µg/ml), kanamycin (20 µg/ml), or erythromycin (100 µg/ml); media for B. subtilis were supplemented with
erythromycin (1 µg/ml) or kanamycin (10 µg/ml).
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Evaluation of Bottlenecks in the Late Stages of
Protein Secretion in Bacillus subtilis
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-amylase (AmyL),
Escherichia coli TEM 
-lactamase (Bla), human pancreatic
-amylase (HPA), and a lysozyme-specific single-chain antibody. The
same expression and secretion signals were used for all four of these
proteins. Notably, all identified bottlenecks relate to late stages in
secretion, following translocation of the preproteins across the
cytoplasmic membrane. These bottlenecks include processing by signal
peptidase, passage through the cell wall, and degradation in the wall
and growth medium. Strikingly, all translocated HPA was misfolded, its
stability depending on the formation of disulfide bonds. This suggests
that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is
anticipated that the efficient secretion of particular groups of
heterologous proteins with the same secretion bottlenecks will require
the engineering of specifically optimized host strains.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-amylase (23) or the Staphylococcus aureus
protein A (9) to gram-per-liter concentrations in the growth
medium. Third, B. subtilis is a genetically highly amenable host organisms for which a large variety of genetic tools have been
developed (11) and which is well known with respect to fermentation technology. Fourth, B. subtilis has a
transparent genome, because its complete sequence is known
(18). Finally, B. subtilis is nonpathogenic and
free of endotoxins. Notwithstanding these advantages, the secretion of
various heterologous proteins by bacilli, in particular proteins of
eukaryotic origin, is frequently inefficient, which limits the
application potential of these organisms (for reviews, see references
22 and 30).
-amylase [AmyL], Escherichia coli TEM -lactamase
[Bla], human pancreatic -amylase [HPA], and a single-chain
antibody against lysozyme [SCA-Lys]), using the same expression and
secretion signals. The results show that different stages in secretion,
following translocation across the membrane, determine the secretion
efficiency of each of these reporter proteins.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Plasmids and bacterial strains
DNA techniques. Procedures for DNA purification, restriction, ligation, agarose gel electrophoresis, and transformation of E. coli were carried out as described by Sambrook et al. (27). Enzymes were from Boehringer Mannheim. B. subtilis was transformed as described by Tjalsma et al. (36). Residues 28 to 64 of signal sequence A2 were deleted by the gapped duplex DNA method for site-directed mutagenesis with the pMa/c phasmid vectors developed by Stanssens et al. (33) and the mutagenic primer IA2 (5'-TGCCGCCGCTGCGGTCTGACTCAGTTTTACTTGTAAATGGGA-3'). This resulted in signal sequence A2d. To obtain an HPA reporter gene cassette flanked by SmaI and SalI restriction sites, a SmaI site was introduced at the 3' end of the HPA signal sequence on M13mp10HPA by using the pMa/c phasmid vectors and the mutagenic primer HAI (5'-CCCTTATGACCCGGGTCGTCT-3'). A SCA-Lys reporter gene cassette, flanked by SalI and HindIII restriction sites, was obtained by PCR with the oligonucleotides HT1 (5'-CTAGAGTCGACCGCCCAAGCCCAGGTGCAGCTGC-3') and HT2 (5'-ATTGATAAGCTTACCGTTTGA TCTCGAGC-3'), using plasmid pUR4129 as a template.
Protein labeling, immunoprecipitation, SDS-PAGE, and fluorography. Pulse-chase labeling of B. subtilis, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and fluorography were performed as described previously (38). To measure the kinetics of protein release into the medium, samples withdrawn at various intervals after the chase were immediately diluted in ice-cold medium. Next, cells and growth medium were separated by centrifugation. To inhibit the translocation ATPase activity of SecA, sodium azide (1.5 mM) was added to the cells 5 min prior to labeling (15). Experiments were repeated at least two times, with the variation in the observed percentages of preprotein processing or release being in the 10% range.
Western blot analysis. Western blotting was performed as described by Towbin et al. (37). Samples for SDS-PAGE were prepared as described by van Dijl et al. (38). After separation by SDS-PAGE, proteins were transferred to Immobilon polyvinylidene difluoride (Millipore Corporation) or nitrocellulose (BA 85; Schleicher and Schuell) membranes. The presence of SCA-Lys in the media of overnight cultures was monitored with antibodies raised against Fv-antilysozyme (Unilever), and subsequent visualization of the bound antibodies was achieved with anti-rabbit immunoglobulin G conjugates (Promega).
Enzyme activity assays.
-Amylase activity in culture
supernatant was measured by using Starch Azure (Sigma) as described by
Smith et al. (32). The presence of biologically active
SCA-Lys in supernatants of overnight cultures was measured with a
pin-enzyme-linked immunosorbent assay as recommended by the supplier
(Collworth). Before the assay, 2 mM phenylmethylsulfonyl fluoride and
10 mM EDTA were added to the culture supernatants.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Processing, release into the medium, and degradation of YvcE fusion proteins. Signal sequence A2, which was randomly selected from the B. subtilis genome with a signal sequence selection vector based on AmyL (Fig. 1A) (31), was previously shown to direct the efficient secretion of AmyL and Bla by B. subtilis (32). With the availability of the complete sequence of the B. subtilis genome (18), signal sequence A2 was shown to correspond to the 5' region of the yvcE gene, which is located at 305.3° on the genetic map (11a). yvcE is predicted to encode a protein of 473 residues that shows a high degree of similarity to the cell wall-attached protein P54 of Enterococcus faecium, a protein with unknown function that is very rich in serine and asparagine residues (10). Signal sequence A2 specifies the first 60 residues of YvcE (Fig. 1B), including a typical tripartite signal peptide, with a putative type I SPase cleavage site between residues 30 and 31 (Fig. 1B). SPase cleavage at this site was confirmed by amino-terminal sequencing of mature Bla and AmyL that were exported to the periplasm of E. coli or the growth medium of B. subtilis, respectively, by using signal sequence A2 (data not shown). Consequently, the mature parts of secretory proteins fused to signal peptide A2 contain an amino-terminal extension which corresponds to the first 30 residues of mature YvcE. Below we refer to the latter mature fusion proteins as A2-Bla and A2-AmyL.
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92% of the total amount
of labeled A2-Bla was present in the mature form. Interestingly,
compared to the fast processing of the precursor, the release of mature A2-Bla into the medium was delayed, as only 61% of the mature protein was detected in the medium at time zero. As the rate of preprotein processing is temperature dependent, A2-Bla processing and
release were also studied at 25°C. Consistent with the results obtained at 37°C, the delay between processing and release was even
more pronounced when cells were incubated at 25°C; after 1 min of
chase, 58% of the labeled A2-Bla was in the mature form, whereas
only 8% of the mature A2-Bla was released into the growth medium
(Fig. 2C and D). Interestingly, a difference of about 3 min was
observed between the time point at which 50% of the pre-A2-Bla molecules were processed and the time point at which 50% of the mature
molecules were released into the medium. This suggests that cell wall
passage of a mature A2-Bla molecule takes about 3 min. Qualitatively
similar results were obtained for A2-AmyL. Also in this case,
processing of the precursor to the mature form was fast. In fact, no
pre-A2-AmyL could be detected in pulse-chase experiments performed at
37°C (Fig. 2E and F). Furthermore, the release of mature A2-AmyL was
slow compared to the processing of pre-A2-AmyL, in particular when
cells were labeled at 25°C (Fig. 2G and H); after 5 min of chase,
only 10% of the total amount of labeled A2-AmyL was detected in the
medium, whereas 85% was in the mature form. Interestingly, at
25°C, A2-AmyL was released into the medium at a much lower rate than
A2-Bla, whereas pre-A2-AmyL was processed much faster than pre-A2-Bla.
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) at each time point after the chase is
calculated as the amount of mature protein in both cellular and
supernatant fractions (including degradation products) divided by the
total amount of labeled protein (precursor form plus mature form in
cell and supernatant fractions). The percentage of release (
) is
calculated as the amount of mature protein in the supernatant fraction
divided by the total amount of labeled protein. (E to H) Processing and
release of A2-AmyL into the medium by B. subtilis 8G5(pS-A2)
were analyzed by pulse-chase labeling at 37°C (E and F) and 25°C (G
and H) as described for A2-Bla. c, cells; s, supernatant; p,
pre-A2-AmyL; m, mature A2-AmyL.
SPase limits the release of A2d-Bla into the medium.
As a
first approach to study the relationships between processing, release
into the medium, and degradation of hybrid preproteins containing
signal peptide A2, residues 28 to 64 of signal peptide A2 were deleted.
The resulting signal peptide, designated A2d, lacks the original SPase
cleavage site of YvcE plus the highly charged amino-terminal region of
YvcE, which contains 10 acidic and 6 basic residues (Fig. 1B).
Alternative SPase cleavage sites were specified by the sequences at the
point of fusion between signal sequence A2d and the bla gene
(Fig. 1C). As determined by -lactamase activity assays, the deletion
in signal peptide A2 had no effect on the total amount of -lactamase
secreted into the medium (data not shown). Next, the kinetics of
processing of pre-A2d-Bla by SPase and release of the mature form into
the medium were tested by pulse-chase labeling at 37 or 25°C.
Irrespective of the temperature, processing of pre-A2d-Bla was slower
than that of pre-A2-Bla (Fig. 2 and 3),
but, interestingly, mature A2d-Bla was hardly detectable in the cells
(Fig. 3). Similarly, mature A2d-AmyL was detectable only in the medium
(data not shown). Thus, it seemed that translocation across the
membrane or processing by SPase was the bottleneck for the secretion of
A2d-Bla and A2d-AmyL. To discriminate between these possibilities, the
SPase SipS was overproduced at least 20-fold (data not shown) by using
plasmid pGDL51, which specifies both SipS and A2d-Bla. As shown in Fig. 4, the rate of processing of pre-A2d-Bla
was indeed significantly increased by SipS overproduction; after 2 min
of chase, 65% of the labeled A2d-Bla was mature in cells
overproducing SipS, whereas only 35% of the labeled A2d-Bla was
mature in cells producing wild-type levels of SipS. Even though the
rate of processing was greatly increased by SipS overproduction, hardly
any mature A2-Bla accumulated in the cell fraction (Fig. 4). Thus, by
deletion of the original SPase cleavage site and the 34 amino-terminal
residues of the mature YvcE from signal peptide A2, the bottleneck in
the secretion of A2-Bla was shifted from release into the growth medium to processing by SPase.
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The cell wall is a barrier for secretion of HPA. Secretion of HPA in an aprE nprE mutant B. subtilis strain was tested with signal peptide A2 or A13. Like A2, signal sequence A13 was randomly selected from the B. subtilis genome (31) and was shown to direct the efficient secretion of Bla and AmyL into the growth medium (32, 39). Genome sequencing has revealed that the signal sequence A13 is derived from the ydjM gene, which is located at 58.0° on the genetic map (11a). YdjM is predicted to contain a typical signal peptide, with putative type I SPase cleavage sites between residues 21 and 22 or residues 28 and 29. Signal sequence A13 specifies the first 15 residues of YdjM, and alternative SPase cleavage sites are specified by sequences at the fusion point between A13 and the reporter genes used in the present studies (Fig. 1B). When fused to signal peptide A13, both Bla and AmyL were efficiently processed by SPase and secreted into the growth medium (data not shown).
As determined by enzyme activity assays and Western blotting, only very low levels of A2-HPA or A13-HPA accumulated in the growth medium (data not shown), suggesting that these hybrid proteins were poorly secreted and/or degraded in the growth medium. The latter possibility was investigated by expressing A2-HPA or A13-HPA in B. subtilis WB600, a strain lacking six extracellular proteases (AprE, Bpr, Epr, Mpr, NprB, and NprE) (44). However, no increase in the amounts of secreted HPA was observed (data not shown), suggesting that degradation in the growth medium by any of these proteases did not represent the major bottleneck in HPA secretion. Pulse-chase labeling experiments were carried out to examine the synthesis and kinetics of processing of A2-HPA and A13-HPA. Both hybrid proteins were efficiently synthesized and processed to the mature form (Fig. 5A; only the results for A2-HPA are shown); immediately after the pulse (time zero),91% of the labeled
A2-HPA was mature. However, even after a chase of 1 h, no mature
A2-HPA or A13-HPA was released into the medium (Fig. 5B). To verify
that the observed maturation of A2-HPA was due to translocation across the membrane and subsequent processing by SPase, pulse-chase labeling experiments were carried out in which, prior to the labeling with [35S]methionine, sodium azide was added to the cells.
Thus, the translocation ATPase activity of the SecA protein, which is
the force generator for protein translocation across the membrane
(8, 29), was blocked. Indeed, the addition of sodium azide
strongly inhibited the processing of pre-A2-HPA (Fig. 5C), showing that
the observed processing in the absence of sodium azide is translocation
specific. Taken together, these findings suggested that translocated
mature A2-HPA accumulated in the cell wall. To test this hypothesis, cells were treated with lysozyme, and subsequently the resulting protoplasts were separated from released cell wall components by
centrifugation. As shown by Western blotting, 60% of the total amount of the A2-HPA was localized in the cell wall fraction (Fig. 5D).
In each fraction the presence of GroEL, which was used as a cytoplasmic
reporter for protoplast lysis, was monitored in parallel. From the
results we conclude that fewer than 30% of cells had lysed during
protoplasting (Fig. 5D), suggesting that a large fraction of
translocated mature A2-HPA accumulated in the cell wall. This
accumulation seems to depend on the formation of disulfide bonds, as no
mature A2-HPA was detectable in cells treated with the reducing agent
-mercaptoethanol (Fig. 5E) at 5 mM, a concentration that is not
inhibitory for growth of B. subtilis (data not shown).
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Degradation of single-chain antibodies in the growth medium.
To study the secretion of SCA-Lys by B. subtilis, the signal
peptides A2 and A13 were used, and the accumulation of A2-SCA-Lys or
A13-SCA-Lys in the medium of overnight cultures was monitored by
Western blotting. Similar to the case for A2-HPA and A13-HPA, no
SCA-Lys could be detected in the growth medium of B. subtilis 8G5, which is wild type with respect to the secretion of
proteases into the medium (Fig. 6A).
However, unlike A2-HPA and A13-HPA, upon pulse-chase labeling, mature
A2-SCA-Lys and A13-SCA-Lys could be detected in the medium (Fig. 6B).
These observations suggested that SCA-Lys was secreted into the growth
medium and subsequently degraded. To test this hypothesis, the
secretion of A13-SCA-Lys was monitored in B. subtilis
DB430, a strain lacking three secreted proteases
(AprE, Bpf, and NprE), and in B. subtilis WB600,
which lacks six secreted proteases (see above). As shown in Fig.
6A, secreted A13-SCA-Lys was detectable in the medium of B. subtilis DB430 (200 µg/liter), and the amount of this
secreted protein was slightly increased in B. subtilis
WB600 (250 µg/liter), showing that A13-SCA-Lys is subject to
degradation by secreted proteases of B. subtilis.
Interestingly, A2-SCA-Lys appeared to be more sensitive to
proteolysis than A13-SCA-Lys, as it was not detectable in the medium of
B. subtilis DB430 (results not shown). To test whether
the secreted A2-SCA-Lys or A13-SCA-Lys was biologically active, a
pin-enzyme-linked immunosorbent assay against lysozyme was
performed. The concentrations of biologically active A13-SCA-Lys matched well with the concentrations of A13-SCA-Lys as determined by
Western blotting. For example, the concentrations of active A13-SCA-Lys
in the media of B. subtilis 8G5, DB430, and WB600 were 89 (± 15), 202 (± 8), and 293 (± 14) µg/liter, respectively. From
these observations, we conclude that all secreted A13-SCA-Lys was
active.
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DISCUSSION |
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Materials and Methods
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In the present studies, we have investigated bottlenecks in the secretion of four different heterologous proteins, AmyL, Bla, HPA, and SCA-Lys, making use of the same expression and secretion signals. Interestingly, all identified secretion bottlenecks are at late stages in the secretion process, which follow translocation across the cytoplasmic membrane. Furthermore, the nature of the secretion bottleneck was determined largely by the secreted protein and, to a lesser extent, by the combination of the signal peptide and secretory protein.
The first step after preprotein translocation across the membrane is processing by SPases. We have previously shown that the rate of processing of a slowly processed hybrid preprotein (pre-A13i-Bla) could be increased by overproduction of SipS (40). Here we show that this is also true for pre-A2d-Bla. Taken together, these observations suggest that processing by SPase is a secretion bottleneck for those proteins whose mature forms reach the growth medium but whose precursors are slowly processed. Subsequent steps in the secretion process involve folding of mature proteins to their active and protease-resistant conformation. As shown for A2-Bla and A2-AmyL, passage through the cell wall is the major bottleneck in the secretion of proteins which are rapidly processed and folded into an active conformation. For the secretion of these proteins, proteolysis seems to be a major problem. In the case of AmyL, we show that proteolytic degradation takes place not only before its release into the medium, as recently reported by Stephenson and Harwood (34), but also after its release. The latter observation is unprecedented. This also applies to our novel observation that the site of AmyL degradation depended on the temperature, suggesting that the expression of activity of proteases involved in this process is temperature dependent or that reduced rates of translocation and release at 25°C make AmyL more vulnerable to cell-associated proteases. In contrast to the case for AmyL, no cell-associated degradation products of Bla were observed, suggesting that the degradation of this protein takes place largely in the medium. This is apparently also true for SCA-Lys, which confirms earlier observations by Wu et al. (45), who showed that a secreted antidigoxin SCA was sensitive to secreted proteases of B. subtilis. The stability of secreted SCA-Lys seems to depend also on the type of fusion protein that was constructed, because A2-SCA-Lys was less stable than A13-SCA-Lys. Finally, some heterologous proteins are unable to pass the cell wall, as examplified by HPA.
There are at least three major reasons why the cell wall of B. subtilis can form a bottleneck in the secretion of heterologous proteins. First, the wall is relatively thick (10 to 50 nm), with a high concentration of immobilized negative charge (e.g., teichoic or teichuronic acids) (1). This negative charge might affect the release of proteins with surface-exposed positively charged residues into the growth medium. Second, translocated proteins have to fold in the wall environment to become active and protease resistant. This requires the presence of folding catalysts, such as PrsA (12, 16, 17), thiol-disulfide oxidoreductases (our unpublished observations), and cations (e.g., Ca2+ and Fe3+) (25). Third, the membrane-cell wall interface contains numerous proteases, such as the wprA-encoded CWBP52 (20, 34) and homologues of the E. coli HtrA and Tsp proteases (our unpublished observations), which appear to play a role in the quality control of secreted proteins.
In the case of A2-Bla, it is not entirely clear why passage through the
cell wall is a bottleneck in secretion. Notably, A2-AmyL passes the
wall at a much lower rate than A2-Bla, which may be related to
differences in the physicochemical properties (e.g., molecular mass,
rate of folding, shape, and surface charge) of these proteins. The
observed degradation of A2-AmyL in the wall most likely is related to a
combination of relatively slow folding of A2-AmyL and the presence of
wall-associated protease activity, as shown for AmyL secreted with its
authentic signal peptide (34). The reason for the failure of
A2-HPA to pass the wall most likely is related to its misfolding. This
view is based on the observation that no A2-HPA activity could be
demonstrated in wall fractions that contained substantial amounts of
the A2-HPA protein as shown by Western blotting (data not shown). As
judged from the amounts of A2-HPA in these wall fractions, we should
have been able to show its activity if this material has been correctly
folded. Thus, it seems that incorrectly folded A2-HPA is unable to pass the wall, which could be due to electrostatic interactions with wall
components. This would be consistent with the hypothesis that the rate
of cell wall passage is determined by the unfolding-folding transition
of proteins during or shortly after their translocation across the
cytoplasmic membrane (4, 19, 25). Alternatively, the
misfolded A2-HPA could form aggregates in the wall which cannot be
released into the growth medium. As all A2-HPA accumulating in the wall
was proteolyzed upon the addition of the reducing agent
-mercaptoethanol, even in a strain lacking six secreted proteases
(data not shown), it seems that the formation of disulfide bonds is
required to stabilize (misfolded) A2-HPA. Notably, these disulfide
bonds are probably incorrectly formed, as the A2-HPA accumulating in
the wall is not active (see above). The view that disulfide bonds are
required to stabilize A2-HPA is consistent with our unpublished
observation that at least two of the three putative disulfide bond
oxidoreductases of B. subtilis, which we have recently
identified, are required for the productive secretion of proteins with
a disulfide bond required for stability. Taken together, these
observations suggest that (correct) formation of disulfide bonds is a
major bottleneck for the secretion of heterologous proteins with
multiple disulfide bonds in B. subtilis.
In conclusion, this comparative analysis has identified various bottlenecks in the late stages of the secretion of heterologous proteins by B. subtilis. Some of these bottlenecks, such as SPase limitation and proteolysis of the secreted protein in the wall and growth medium, have been identified previously. A novel bottleneck concerns the catalysis of disulfide bond formation. Most importantly, the present results clearly point out that the optimization of the secretion of individual heterologous proteins is likely to require engineered B. subtilis strains in which specific secretion bottlenecks have been removed. We are confident that the engineering of dedicated B. subtilis host strains for the secretion of a wide range of heterologous proteins at commercially significant levels will soon be possible, because of our increasing knowledge of the secretion apparatus, the availability of the complete genome sequence, and the genetic amenability of this organism.
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ACKNOWLEDGMENTS |
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The first two authors contributed equally to this work.
We thank J. D. H. Jongbloed, M. L. van Roosmalen, L. Frencken, C. R. Harwood, and members of the European Bacillus Secretion Group for stimulating discussions, M. Sulter for expert technical assistance, and H. Mulder for preparing the figures.
A. B. was supported by a grant (BIO2-CT93-0254) from the European Union. H.T was supported by Unilever, Gist-Brocades, and Genencor Int. J.M.v.D and S.B. were supported, in part, by grants (BIO2-CT93-0254 and BIO2-CT96-0097) from the European Union.
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FOOTNOTES |
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* Corresponding author. Present address: Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Phone: 31 50 3633079. Fax: 31 50 3632348. E-mail: J.M.van.Dijl{at}farm.rug.nl.
Present address: ID-DLO, 8200 AB, Lelystad, The Netherlands.
Present address: Department of Chemical Engineering, University of
Washington, Seattle WA 98195-1750.
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Abstract
Introduction
Materials and Methods
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Discussion
References
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