Note: Descriptions are shown in the official language in which they were submitted.
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WO 94119471 PCTIFI94100072
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Method and System for Enhanced Production of
Commercially Important Exoproteins in Gram-Positive Bacteria
Field of the Invention
This invention relates to a method and expression system for
enhanced production of industrially and medically important exoproteins in
gram-positive bacteria, especially species of the genus Bacillus.
Background
In gram-positive bacteria secreted proteins are exported across
a cell membrane and a cell wall, and then are subsequently released into the
external medium. On the other hand, gram-negative bacteria are surrounded
by two cell (or surface) membranes; they have no cell wall. In gram-negative
bacteria, most exported proteins are not released from the cell but stay in
the
inter-membrane periplasmic space and in the cuter membrane.
Two types of components of the secretion machinery have been
identified in E. coli: soluble cytoplasmic proteins and membrane associated
proteins (see for review, Wickner et al., (1991 ) Annu. Rev. Biochem.,
60:101-124). Soluble cytoplasmic proteins, including SecB and heat shock
proteins, all prevent the folding of precursors of secreted proteins into a
conformation incompatible with secretion. The set of membrane-associated
proteins includes the peripheral membrane protein SecA, integral membrane
proteins Sect, SecE, SecD, SecF and the signal peptidases Lep and Lsp
(reviewed in Hayashi, S. and Wu, H.C. (1990) J. Bioenerg. Biomembr.,
22:451-471; Dalbey, R.E. (1991 ) Mol. MicrobioL, 5:2855-2860). These
membrane-associated proteins are involved in binding of the precursor and in
its translocation across the cytoplasmic membrane, followed by cleavage of
the signal peptide and release of the protein.
Knowledge on the secretion machinery of gram-positive bacteria
is more limited. The available data on B. subtilis, the genetically and
physiologically well characterized model organism of the genus, suggest an
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overall similarity with that of E, coli, but also differences in the structure
and
specificity of individual components, possibly reflecting demands set by the
very different composition and architecture of the respective cell envelopes.
Gram-positive bacteria such as B. subtilis, B. amyloliquefaciens,
B. licheniformis have a very high capacity for~secreting proteins, and indeed,
many bacillar extracellular enzymes are utilized industrially. Since secreted
proteins in gram-positive bacteria are so important commercially, and since
the gram-positive secreted proteins traverse through a cell envelope with a
very different structure from that of E. coli, the molecular mechanisms of
protein secretion in gram-positive bacteria is of considerable academic and
industrial importance.
In this regard we recently discovered a novel component, the
PrsA protein, of the secretion machinery of B, subtilis (Kontinen, V.P. and
Sarvas, M., (1988) J. Gen. Microbiol., 134:2333-2344; Kontinen, V.P., et al.,
(1991 ) Mol. Microbiol. 5:1273-1283). The prsA gene, which encodes the
PrsA protein, was initially defined by nonlethal mutations that decreased the
secretion of several exoproteins (Kontinen, V.P. and Sarvas, M., (1988) J.
Gen. Microbiol., 134:2333-2344). Based on the DNA sequence of the cloned
prsA gene and our subsequent work with this gene and protein, we assert
that prsA encodes a protein (PrsA) that acts as a chaperone, and is
translocated across the cytoplasmic membrane (for the initial work, see
Kontinen, V.P., et al., ( ~ 991 ) Mol. Microbiol. 5:1273-1283). The PrsA
protein
has been found to possess a limited amount of sequence homology (about
30%) with the PrtM protein of Lactococcus lactis, a protein proposed to assist
the maturation of an exported serine protease (Haandrikman, A.J., et al,
(1989) J. BacterioL, 171:2789-2794; Vos, P., et al., (1989) J. BacterioL,
171:2795-2802). A similar function has not been associated with other known
proteins of the secretion machinery of bacteria, suggesting that PrsA protein
is a novel type of component in the pathway of protein secretion facilitating
the release and probably folding of secreted proteins after their
translocation
across the cytoplasmic membrane in gram-positive bacteria.
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It is advantageous to produce proteins of interest in bacteria in
secreted form, since exported proteins usually maintain their native
conformation, in contrast to intracellular production which, in many cases,
results in aggregation of the produced protein. Another advantage of
producing industrially and medically important proteins in bacteria in
secreted
form is that secretion facilitates purification of the protein product.
Additionally, unlike E. coli, gram-positive bacteria such as Bacillus sp. do
not
contain toxic compounds like lipopolysaccharide, making them especially
appropriate hosts for production of medical and pharmaceutical proteins.
Increased yield of secreted proteins would be of great
significance for improving the gram-positive bacillar strains used in the
industrial production of a number of exoenzymes, such as alpha-amylases,
proteases and lipases. The strategy thus far has been to overexpress the
appropriate gene. There are known and readily available methods for doing
this, such as increasing gene expression by using multicopy plasmids or
enhancing the activity of the gene by modifying its regulatory elements, e.g.,
by using strong promoters, or multiple promoters. Dramatic increases of the
synthesis of exoproteins have been achieved this way, up to a level at which
increasing the synthesis is of no further benefit because of bottlenecks in
the
secretion machinery. It would be desirable to increase the capacity of
secretion in parallel with increased synthesis. However, to date this has not
been possible.
It is an object of the present invention to alleviate the bottleneck
of the secretion mechanism in gram-positive bacteria, and to provide a
method and a system whereby the levels of proteins normally secreted from
gram-positive bacteria such as Bacillus can be enhanced when the
expression of a given homologous or heterologous protein of interest has
been elevated over the amount normally produced in unmodified or wild type
organisms.
It is a further object of the present invention to describe bacterial
hosts and plasmids which can be used to enhance the production of a variety
of commercially important exoproteins.
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Summary
The invention provides a method and expression system for
enhancing the levels of homologous or hete,~ologous proteins) normally
secreted from gram-positive bacteria (such as Bacillus sp.) when expression
5 of the homologous or heterologous proteins) has been elevated over
<.
unmodified or wild type amounts produced'by unmodified or wild type
organisms.
The method and system of our invention comprise
overproduction of PrsA protein, or a functional homologue thereof, in a gram
10 positive bacterial host capable of also overproducing at least one
homologous
or heterologous exoprotein of interest. According to the teaching of the
invention, overproduction means an amount greater than wild-type, i.e., more
than the amount of the protein (PrsA or a functional homologue thereof, or
exoprotein of interest) normally produced by wild type bacteria. Also
15 according to the invention, overproduction is accomplished by standard
means known to the art, e.g., use of multicopy plasmids, multiple copies of
the genes encoding PrsA, or a functional homologues) thereof, and/or the
exoprotein of interest, in the chromosome of the host, combined with altering
the regulatory elements to increase expression, e.g., use of strong
20 promoter(s), use of multiple promoters, use of enhancers, and so forth. Use
of the method and system of the invention results in greatly enhanced
secretion, e.g., as much as five to ten fold over controls, of synthesized
exoproteins into the growth medium. Once in the growth medium these
secreted exoproteins can be recovered and purified in a straightforward
25 manner.
The expression system of the invention comprises a host gram-
positive bacteria, e.g., species of Bacillus, capable of expressing greater
than
wild-type amounts of PrsA protein, or a functional homologue thereof, and
greater than wild-type amounts of an exoprotein of interest, e.g., alpha
amylase, subtilisin, pneumolysin, lipases, or other exoproteases of
commercial interest. The method of the invention comprises using this
expression system to enhance production of commercially important
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exoproteins in gram-positive bacteria. According to the method, at least one
exoprotein of interest is overexpressed in a host gram-positive bacteria which
also overexpresses (i.e., expresses greater than the amounts produced by
wild type bacteria) PrsA protein, or a functional homologue thereof.
5 According to the teaching of the invention, a functional homologue of PrsA
protein is a protein which when overexpressed is capable of enhancing the
secretory capability of a gram-positive bacteria with respect to secretion of
an
exoprotein of interest. Also according to the teaching of the invention, a
functional homologue of PrsA can be identified by several means including
sequence homology to prsA or PrsA, immunological reaction with anti-PrsA
antibodies) of high titer, and/or functionally, i.e., as a protein which when
overexpressed, is capable of enhancing the secretory capability of a gram-
positive bacteria with respect to secretion of an exoprotein of interest.
A preferred means for transforming host gram-positive bacteria,
such as species of Bacilli, so they produce greater than wild-type amounts of
PrsA protein is to transform the host with plasmid pKTH277 which carries the
prsA gene from Bacillus subtilis. Comparable plasmids can be constructed to
carry genes which encode functional homologues of the PrsA protein. These
plasmids can be used to transform host gram-positive bacteria so they
overproduce the functional homologues of PrsA. Once engineered to
overproduce PrsA homologues (which can also be referred to as PrsA-like
proteins), these host gram-positive strains can be used, according to the
teaching of the invention, for enhanced secretion of hyperproduced
exoproteins of interest.
The present invention also discloses, and includes, methods
and constructs related to our discovery that secretion in gram-positive
bacteria can be enhanced by increasing the amount of cellular PrsA protein,
or functional homologues) thereof, in gram-positive hosts that express
greater than wild-type amounts of exoproteins of interest.
In one aspect our invention includes an expression system for
enhancing secretion of exoproteins in gram-positive bacteria comprising a
gram-positive bacteria capable of expressing greater than wild-type amounts
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of PrsA protein, or functional homologues) thereof, and capable of
expressing greater than wild-type amounts of at least one exoprotein of
interest.
In another aspect our invention includes a gram-positive
bacteria able to express greater than wild-type amounts of at least one
exoprotein of interest further comprising pKTH277.
In yet another aspect, our invention includes a gram-positive
bacteria able to express greater than wild-type amounts of at least one
exoprotein of interest and further comprising at least one of the following:
at
least two copies of the prsA gene from Bacillus subfilis, or a functional
homologue thereof; the prsA gene from Bacillus subtilis, or a functional
homologue thereof, operatively linked to strong regulatory sequences which
result in overexpression of said prsA gene, or functional homologue thereof.
Our invention also includes a DNA construct comprising the
prsA gene from Bacillus subtilis, or a functional homologue thereof, under the
control of expression signals which cause overexpression of said prsA gene,
or functional homologue thereof, plus a vector further comprising the prsA
gene from Bacillus subtilis, or a functional homologue thereof, under the
control of expression signals which cause overexpression of said prsA gene,
or functional homologue thereof.
In yet another aspect our invention includes a method for
enhancing secretion of an exoprotein of interest in a gram-positive bacteria
comprising expressing greater than wild type amounts of PrsA protein from
Bacillus subtilis, or a functional homologue thereof in the gram-positive
bacteria, wherein the gram-positive bacteria is also capable of expressing
greater than wild type amounts of the exoprotein.
Still further the invention includes a method for creating an
improved non-Bacillus subtilis gram-positive host organism useful for
enhanced secretion of an exoprotein of interest that is overexpressed in the
host organism, the method comprising (a) identifying a gene from the host
organism that encodes a functional homologue of PrsA protein from Bacillus
subtilis, and (b) enhancing the expression of the gene identified in step (a)
by
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at least one of the following: introducing into the host organism at least one
additional copy of the gene; introducing into the host organism the gene
operatively linked to expression sequences which result in overexpression of
the gene.
The invention also includes a method for identifying a gene
which encodes a functional homologue of PrsA from Bacillus subtilis, the
method comprising identifying, by means of Southern blotting, DNA which
hybridizes with DNA probes) from the prsA gene from Bacillus subtilis, and
demonstrating that the gene encodes a protein which when overexpressed, is
capable of enhancing the secretory capability of a gram-positive bacteria with
respect to secretion of an exoprotein of interest.
Still further, the invention includes a method for identifiying a
gene which encodes a functional homologue of PrsA from Bacillus subtilis,
this method comprising identifying protein that -reacts with anti-PrsA
antibodies) of high titer, and demonstrating that when the protein is present
in greater than wild-type amounts in a gram-positive bacteria, the protein is
capable of enhancing the secretory capability of the gram-positive bacteria
with respect to secretion of an exoprotein of interest.
These and other features, aspects and advantages of the
invention will become better understood with reference to the following
description, examples, methods and materials, and appended claims.
Description
When the expression level (synthesis) of an exported protein is
high in gram-positive bacteria such as Bacillus sp., the capacity of the
secretion apparatus is a limiting factor in protein secretion and production
of
these proteins in secreted form. Our invention provides a system and method
for overcoming this limitation or bottleneck.
Our invention is based on our initial surprising discovery that
secretion in gram-positive bacteria such as species of Bacillus can be
enhanced by increasing the amount of only one component of the bacillar
export machinery, i.e., the amount of cellular PrsA protein, or functional
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homologues thereof, in gram-positive bacterial hosts that express greater
than wild-type amounts of exoproteins of interest. The method and system of
the invention are useful regardless of how the proteins of interest are
overproduced in the gram-positive bacterial host. Thus the method and
system can be used to improve a variety of overproducing commercial strains
now used in industrial applications.
The method and system of the invention can be used with any
gram-positive bacteria. Bacteria of the genus Bacillus are preferred.
Especially preferred are Bacillus alkalophilus, Bacillus amyloliquefaciens,
Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus,
Bacillus
lentus, Bacillus licheniformis, Bacillus stearothermophilus, Bacillus
subtilis,
and Bacillus thuringiensis.
The method and system of the invention can also be used with
any desired exoprotein of interest. Examples of exoproteins of interest that
75 may be produced according to the method and system of the invention are
listed below, where the exemplary exoproteins are presented by general
categories.
Antigenic proteins of microbes and protozoa: Capsule, outer
membrane and fimbria proteins from any gram negative bacteria, but
especially those from: Bacteroides fragilis, Fusobacterium spp., Bordetella
pertussis, Haemophilus influenzae, Yersinia entercolitica, Yersinia pestis,
Branhamellla catarrhalis, Escherichia coli, Klebsiella pneumonia, Vibrio
cholerae, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens,
Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitides,
Salmonella typhimurium, Salmonella typhi, Salmonella pararyphi 8.
Mycobacterium tyberculosis, Chlamydia trachomatis, and Shigella spp.
Protein toxins or secreted proteins from any bacteria, but
especially those from: Staphylococcus aureus, Pseudomonas aeruginosa,
Clostridium spp., Escherichia coli, Yersinia pestis, Vibrio cholerae,
Bordetella
pertussis, M-Protein of the Streptococcus pyogenes bacterium, Excreted
enzymes of Stretococcus mutans.
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Surface proteins of any microorganism, but especially those
from the following microorganisms (in all phases of development):
Plasmodium spp., Toxoplasma spp., Leishmania spp., Schistosoma spp.,
Trypanosama spp. Adhesion protein of Streptococcus sp., and adhesion
protein of Staphylococcus aureus.
Antigen proteins or viruses: HA and NA proteins of myxoviruses
(influenza A H1-H12, influenza B, influenza C): HN and F proteins of
paramyxoviruses (parainfluenze 1-4, Newcastle disease virus, Measles virus,
Respiratory syncytial virus, Parotitis virus, Distemper virus): G protein of
Rabies virus; E1 and E2 proteins of alfaviruses (Chikungunya, Western,
Easter, Venezuelan equine encephalitis virus, O'nyong-nyong virus, Semliki
Forest virsu, Sindbis virus); V1 and V3 proteins of flavin viruses (Dengue 1-
4,
Japanese encephalitis virus, Mite encephalitis viruses, Murray Valley
encephalitis virus, Kyasanur Forest disease virus, Looping ill virus, Omsk
hemorrhagic fever virus); surface proteins of German measles virus; surface
proteins of Hog Cholera virus; surface proteins of Equine arthritisvirus; G1
and G2 proteins of Bunya viruses (Rift Valley fever virus, Crimean
hemorrahagic fever virus, California encephalitis virus, Phlebotomus fever
virus); G1 and G2 proteins of arena viruses (Lassa fever virus, Lymphocytic
chorion meningitis virus); proteins V1-V4 of picorna viruses (polio 1-3,
Coxsackie A viruses 1-24, Coxsackie B viruses 1-6, ECHO viruses 1-8, 11-
34, hepatitis A virus, hepatitis B virus, hepatitis C virus, Human rhino
viruses
1-113); surface proteins of rota viruses; surface proteins of herpes viruses
(HSV 1, 2, Cytomegalo virus, Epstein-Barr virus, Equine abortion virus); VP1-
VP3 proteins of papova viruses (BK virus, Human wart virus); p proteins of
parvo viruses (mink enteritis virus, Bovine panro virus, Feline parvo virus,
Procine parvo virus); structure proteins of Human hepatitis B virus; surface
proteins of Ebola and Marburg viruses; and Hexone, pentone and fiber
proteins of adeno viruses, (Human adeno viruses 1-33).
Industrial enzymes: With regard to industrially important
enzymes, such enzymes may be amylolytic, lipolytic and proteolytic enzymes,
carbohydrases, transferases, isomerases, peroxidases, oxidoreductases,
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oxidases etc. More specifically, the enzyme of interest may be a protease, a
lipase, a cutinase, an amylase, a galactosidase, a pullulanase, a cellulase, a
glucose isomerase, a protein disuphide isomerase, a CGT'ase (cyclodextrin
gluconotransferase), a phytase, a glucose oxidase, a glucosyl transferase,
laccase, bilirubin oxidase, or a xylanase. Examples include, but are not
limited to: alpha-amylase, amino acid acylase, amyloglucosidase, bromelain,
phisine, beta-galactosidease, beta-gulcanase, glucose-ismorase,
glucoseoxidase, hemicellulase, invertase, catalase, collagenase, xsylanase,
lactase, lipase, naringinase, pancreatin, papain, pectinase,
penicillinamidase,
pepsin, protease, pullulanase, isoamylase, rennin, ribonuclease, cellulase,
streptokinase and trypsin.
Exoproteins of medical interest can also be produced. Such
proteins include diagnostic antigens, proteins that can be used as vaccines,
and pharmaceuticals.
According to the teaching of the invention, the exoproteins of
interest need not be native exoproteins, but instead can be novel proteins
that
have been designed and created to be exoproteins using genetic engineering
techniques. For example, a normally non-secreted protein from one species
(or an engineered non-native protein) can be engineered to be an "exo"
protein by adding a signal sequence to the sequence encoding the structural
protein. This engineered exoprotein can be expressed in a gram-positive
bacteria such as a species of Bacillus, which overexpresses PrsA protein, or
a functional homologue thereof. In this way the method of the invention can
be used to enhance secretion of these non-native or engineered proteins of
interest.
Turning now to aspects of our invention, to illustrate one
embodiment of our invention we show the effect overexpression of the prsA
gene from Bacillus subtilis has on the secretion of the two important
industrial
exoenzymes of Bacilli, alpha-amylase and subtilisin. For these studies, a 5.3
kB insert, containing the entire prsA gene from Bacillus subfilis, was cloned
into a low copy number shuttle plasmid (pICTH277), which was then used to
introduce additional copies of prsA into Bacillus subtilis. (The DNA and
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deduced amino acid sequences of the prsA gene from Bacillus subtilis
appear in the EMBUGenBank/DDBJ Nucleotide Sequence Data Libraries
under the accession number X57271.) (pKTH277 was obtained by ligating
the 5.3 kB EcoRl-BamHl fragment from pKTH268 with low copy number
shuttle plasmid pHPl3 linearized by digestion with respective restriction
enzymes and transforming into the E, colt strain TG1. The sizes of pKTH268
and pKTH277 are 8.5 kB and 10.2 k8, respectively. See also Kontinen, et al.,
(1991 ) Moi: Microbiol., 5:1273-1283).
The presence of pKTH277 in 8. subtilis increased the amount of the
protein corresponding to the PrsA protein by approximately 10-fold over the
wild type. When the genes for different secreted proteins are expressed in
strains of Bacillus containing these increased levels of PrsA protein, the
level
of protein secreted into the culture medium is increased substantially. For
example, the secretion of alpha-amylase of Bacillus amyloliquefaciens was
found to be increased by 2.5-fold in this system, the secreted level of the
thermoresistant alpha-amylase of Bacillus licheniformis was elevated by six-
fold, and subtilisin (alkaline protease) from Bacillus licheniformis was
secreted
at two times the control level.
In these studies the exoenzymes were overexpressed in host
strains in amounts likely to saturate the secretion machinery, either by
placing
the gene which encoded the exoprotein on a multicopy plasmid or inserting it
in the chromosome of the host. (In these studies, alt multicopy plasmids
coding for the exoenzymes were derivatives of pUB 110, which belongs to a
different incompatibility group than the shuttle plasmid pKTH277, allowing
their replication in the same host cell. The stability of these piasmids was
further increased in most cases by using a recE4 host strain, which prevents
efficient recombination between homologous sequences (Dubnau et ai.,
1973; Keggins et al., 1978).
The first exoenzyme studied to illustrate this aspect of our
invention was alpha-amylase of B. amyloliquefaciens (AmyE) encoded by
pKTH 10 (Palva, I. ( 1982a) Gene, 19:81-87; Palva, I., et al., { 1982b) Proc.
NatL Acad. Sci. USA, 79:5582-5586). We found that in the wild-type strain
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hyperproducing this alpha amylase, the presence of pKTH277 indeed
enhanced the secretion of alpha-amylase throughout the stationary phase of
growth, about 2.5-3 fold over the level of the control strain which did not
overexpressing PrsA. The highest concentration.of alpha amylase in the
culture supernatant (about 3400 microgramslml) was found after the growth
of 24 hours. In the absence of pKTH277, the strain secreted only 1200
micrograms/ml. 4ualitatively similar results were obtained when
alpha-amylase was expressed from one copy of the amyE gene, which was
inserted in the chromosome and transcribed at high level due to modified
regulation.
The second exoprotein we tested was the thermoresistant
alpha-amylase of B. licheniformis (Amyl), the major liquefying alpha-amylase
of industrial importance (Ortlepp et al., 1983; Diderichsen, B., et al., (1991
)
Res. Microbiol., 142,. 793-796). Secretion of this enzyme in B. subtilis at
amounts comparable with those of the alpha amylase of 8. amyloliquefaciens
was achieved by expressing the appropriate gene from the secretion vector
based on the promoter and signal sequence of the gene of the latter enzyme
(Palva, I. (1982) Gene, 19:81-87; Palva, I., etal., (1982) Proc. Natl. Acad.
Sci.
USA, 79:5582-5586); Sibakov, M. (1986) Eur. J. Biochem., lsS, 577-581 ).
Introduction of pKTH277 into one such strain (to result in IH6760) increased
the amount of alpha-amylase in the culture medium about six fold, with the
same difference seen from late exponential stage to cultures of 45 hours.
Alkaline protease, subtilisin, is a different type of exoprotein,
whose precursor contains in addition to the signal sequence a further
extension, the prosequence (Wells, et al., (1983) Nucleic Acids Res.,
11:7911-7925; Wong, S.-L., etal., (1984) Proc. NatL Acad. Sci. USA,
81:1884-11188). The effect of an increased amount of PrsA on this secretion
was studied by comparing two strains, one with increased level of PrsA
(IH6789), another one with the wild type level. Both secreted the
heterologous subtilisin of 8. licheniformis (SubC, which is used as a laundry
powder and is an important industrial product) coded by the multicopy
plasmid pMJ57 (Hastrup, S. and Jacobs, M.F. (1990) In Zukowski, M.M., et
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aG, (eds.), Lethal phenotype conferred by xylose-induced overproduction of
apr-IacZ fusion protein, vol. 3. Academic Press, Inc., San Diego, California,
pp. 33-41 ). In this plasmid the subC gene is under the control of a xylose
inducible promoter. Comparison of the secretion of subtilisin from the two
strains, when fully induced; showed that its amount in the culture supernatant
of IH6789 (increased amount of PrsA) was about twofold higher than that of
the control IH6788 at all time points assayed.
We also studied the effect of pKTH277 on the natural low level
secretion of endogenous exoenzymes in a strain devoid of any plasmid
causing hypersecretion. The amount of secreted alpha amylase and total
proteases in the late exponential phase of growth or in overnight cultures was
the same in strains carrying pKTH277 or the cloning vector pHPl3. Based on
these results it appears that the increased amount of PrsA protein enhances
secretion of hyperproduced exoenzymes only.
In order to confirm the role of PrsA in the enhancement caused
by the 5.3 kb fragment in the above plasmids, we inactivated the prsA gene in
the plasmid pKTH277 by insertions in the EcoRV site of its prsA gene (at the
nucleotide 382). In pKTH3261 the insert was a 560 by fragment of the blaP
gene of 8. licheniformis flanked by translational stop codons, and in
pKTH3262 a 4.6 kb EcoRV fragment of phage lambda. SDS-PAGE analysis
of whole cell proteins of E. coli carrying these plasmids showed no full-size
PrsA protein expressed by either plasmid, and a putative truncated PrsA of
the expected size (14 kDa) expressed by pKTH3261 (data not shown). As a
control, we constructed pKTH3253 in which the 5.3 kbp fragment was
truncated for 1.9 kbp downstream of prsA, leaving this gene intact. In B.
subtilis (IH6624 carrying pKTHlO) the two plasmids with an insertion in prsA
did not enhance the secretion of alpha amylase, while pKTH3253 did.
Enhanced secretion obtained by overproduction of prsA is of
obvious advantage for large scale industrial production of exoenzymes. In
such applications it is sometimes desirable to avoid the use of potentially
unstable multicopy plasmids. One strategy is to insert one or few copies of
the structural gene of the exoenzyme in the chromosome, combined with
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altering its regulatory elements to increase expression. We therefore tested
the effect of PrsA overproduction on the secretion of alpha amylase in such a
system, where one copy of amyE was inserted in the chromosome fused to
the target sequence of the regulatory protein DegQ i~ a strain overexpressing
DegQ. Also in this strain the high level of
alpha-amylase secretion was enhanced abouttthree fold by increasing the
amount of PrsA protein (Table 1, strains BRB764 and IH67703). This
indicates that the enhancement of secretion is achieved when the starting
level of expression of exoprotein is high, regardless of the way the increased
expression of the target gene has been obtained.
Turning now to the presence of PrsA in gram-positive bacteria
other than Bacillus subtilis, we have confirmed the presence of PrsA or PrsA
homologues in other species, e.g., Bacillus amyloliquefaciens and Bacillus
licheniformis. The amount of PrsA protein in Bacillus amyloliquefaciens is
similar to the amount in Bacillus subtilis cells, while the amount of PrsA
protein in Bacillus licheniformis cells appears to be less. In addition, the
components of the secretion machinery in these gram-positive bacillar strains
are similar to that of Bacillus subtilis. Bacillus amyloliquefaciens and
Bacillus
licheniformis are two of the most widely used species of Bacillus in large
scale
industrial processes for the production of secreted proteases and amylases.
The method of the invention using overproduction of PrsA protein to increase
secretion of homologous and heterologous exoproteins of interest is
especially useful in these strains.
As part of our invention we also teach how PrsA protein and/or
the prsA gene can be identified in other gram-positive bacteria, and that
functional homologues of the PrsA protein from Bacillus subtilis exist and can
be used in the method and system of the invention. Functional homologues
of the PrsA protein from Bacillus subtilis can be identified in other gram-
positive species by using anti-PrsA antibody of high titer. Alternatively, the
prsA gene or homologous prsA-like genes which encode functional
homologues of the PrsA protein from Bacillus subtilis can be identified by
Southern blotting using probes) from the prsA gene, or a prsA-gene
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fragment. If the PrsA-like protein is found to exist, but there is
insufficient
homology to the prsA gene or PrsA protein to be detected unequivocally with
antibodies specific for the PrsA protein, or DNA probes containing sequences
homologous to the prsA gene, the homologous gene can be located and
5 cloned, so unequivocal identification can be made.
However, in most cases homologous proteins and genes can be
found using antibodies specific for the PrsA protein, or DNA probes
containing sequences homologous to the prsA gene. For immunological
identification, immunoblotting (Western blotting) can be used to detect the
10 PrsA protein with anti-PrsA antibody of high titer. The antiserum is
produced
by immunizing an appropriate animal (e.g., rabbit) with PrsA protein of B.
subtilis, or preferably with a PrsA protein homologue of another species more
closely related to the species of interest than B. subtilis. To identify the
PrsA,
the bacterium of interest can be grown on a number growth media, but
15 preferably, the bacterium is grown on a medium where there is minimal
induction of proteases. Bacterial cells are collected, again preferably at a
growth phase with minimal amount of proteases present, and broken with a
method appropriate for the species (usually a combination of enzymatic
treatment and mechanical disrupture with sonication, French pressure cell, or
shearing with glass beads). Samples of various sizes of broken cells and
particulate fraction of the disrupted whole cells, prepared with
ultracentrifugation, are electrophoresed in SDS-PAGE with standard
methods, proteins are transferred to membrane filters and detected with anti-
PrsA antiserum and the labeled second antibody. (Smaller amounts of PrsA
protein can be detected if the particulate fraction is prepared). In all these
steps standard methods and commercial reagents can be used.
To identify the prsA gene, or a prsA-like gene that encodes a
functional homologue of PrsA in a species of interest, Southern blotting can
be used. In this method, appropriate DNA probes from the prsA gene are
hybridized to/with appropriately fragmented and electrophoresed
chromosomal DNA of the species of interest according to the standard
method of Southern hybridization. The hybridization probe may be any
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fragment of DNA containing the prsA gene of B. subtilis, or a fragment of this
gene, or a DNA fragment containing the prsA gene homologue of another
species or a fragment of that gene. Once identified, the prsA gene
homologue can be sequenced to further confirm;.ifs identity.
The teaching of the present invention includes not only
overexpression of the PrsA protein of Bacillus subtilis, but also
overexpression of a functional homologue of the PrsA protein from Bacillus
subtilis in a gram-positive species of interest. According to the teaching of
the
invention, either the prsA gene of B. subtilis or the prsA gene homologue from
another species, including the host species, is introduced into the host
species. The prsA gene or its homologue is brought under the control of
expression signals which are active in the species of interest in order to
result
in the high level (but not lethal) expression of the prsA gene. This can be
accomplished in a variety of ways, including:
(1 ) The transfer of the plasmid pKTH277 to the species of
interest. The transfer can take place with any method of transformation
applicable to that species, like transformation, transduction, protoplast
transformation, electroporation or conjugation. pKTH277 is maintained as
multiple copy plasmid in many other gram-positive species other than
Bacillus, and the expression signals of the prsA gene in that plasmid are
active in many gram-positive species.
(2) Inserting the 5.3 kb Sad fragment of pKTH277 into any
other plasmid compatible with the species of interest and maintaining it at
suitable copy number for high, but not lethal level of expression of prsA,
relative to the activity of the prsA gene of 8. subtilis in that species. The
plasmid is then transferred to that species using any method of transformation
applicable to that species. Alternatively, the fragment of DNA inserted into
the plasmid can contain a prsA gene homologue of another species with its
expression signals.
(3) Inserting the DNA fragment of pKTH277 encoding the signal
sequence and the mature part of the PrsA protein to an expression vector
suitable for the species of interest, under the control of expression signals
in
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that plasmid to achieve high level expression of prsA. As above, the
appropriate fragment may derive from a prsA gene homologue of another
species.
(4) The DNA constructions of paragraphs (2) and (3) above can
be inserted into the chromosome of the species of interest instead of a
plasmid. In that case, expression signals have to be chosen which are active
enough to ensure high level expression of PrsA although there is only one
copy of the gene per the genome.
As inventors who are also basic scientists, by design and of
necessity much of our work is done under laboratory or simulated industrial
co~~ditions. However, with the help of industrial collaborators, it has been
shown that the method and system of our invention work very well with
commercially useful bacterial stains, under industrial fermentation
conditions.
Methods and materials used in our studies, and examples of our
invention are included below to further aid those skilled in the art in
practicing
the method and system of our invention.
Materials and Methods
Bacterial strains and plasmids
prs mutants of Bacillus subtilis Marburg 168 and their parent
strains are shown in Table 1. Listed are also 8. subfilis and E. coli strains
overexpressing PrsA protein and B. subtilis strains with enhanced secretion of
exoenzymes due to increased cellular amount of PrsA protein and their
appropriate control strain. E. coli strains used as cloning hosts with plasmid
vectors were HB101, TG1 and DHScc (Sambrook J., et al., (1989) Molecular
cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York) and with lambda, Kw25 1 (Promega, Madison,
Wisconsin).
pHPl3 (Haima, P., et al., (1987) Mol. Gen. Genet.,
209:335-342, pJH101 (Ferrari, F.A., et al., (1983) J. Bacteriol.,
154:1513-1515), pGEM3zf(+) (Promega) and pDR540 (Pharmacia, Upsala,
Sweden) were used as cloning vectors for prsA gene and its fragments.
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Properties of these plasmid vectors and constructed derivative plasmids
carrying the prsA gene with a 5.3 kb (pKTH277 and pKTH268) or 3.4 kb
(pKTH3253) insert are shown in Table 2. The prsA gene in pKTH3253 was
disrupted by inserting a fragment either from B. licheniformis blaP gene (0.5
kb) or bacteriophage lambda genome (4.6 kb) in th~_unique EcoRV site in the
ORF of prsA (the resulted plasmids were pKTH~3261 and pKTH3262).
pKTHlO (Palva, I. (1982a) Gene, 19:81-87; Palva, I., etal., (1982b) Proc.
Natl. Acad. Sci. USA, 79:5582-5586) and pMJ57 (Hastrup, S. and Jacobs,
M.F. (1990) In Zukowski, M.M., et al., (eds.), Lethal phenotype conferred by
xylose-induced overproduction of apr-IacZ fusion protein, vol. 3. Academic
Press, Inc., San Diego, California, pp. 33-41 ) are multicopy plasmids
producing large amounts of B. amyloliquefaciens alpha-amylase (AmyE) and
B. licheniformis subtilisin (SubC) secreted into the external medium,
respectively. pKTH1582 has been constructed cloning the amyl gene from
B. licheniformis on a secretion vector system of B, subtilis (BRB360 in
Sibakov, M. (1986) Eur. J. Biochem., lsS, 577-581; Palva, I. (1982a) Gene,
19:81-87; Palva, I., et al., (1982b) Proc. Natl. Acad. Sci. USA, 79:5582-
5586).
Growth media and culture conditions
Bacteria were grown in modified L-broth (1 % tryptone, 0.5%
yeast extract, 0.5% NaCI) with shaking at 37 degrees C, or on L-plates
containing 1.5% agar (Difco, Detroit, Michigan) with appropriate antibiotics
at
+37 degrees C (Kontinen, V.P., et al., (1991 ) Mol. Microbiol. 5:1273-1283);
plates were modified for alpha amylase (5% starch and 2.5% agar) and
subtilisin overexpressing strains (0.2% xylose and 1 % milk powder). L-broth
was supplemented with 2% soluble starch (Merck, Darmstadt, Germany) or
used as 2-fold concentrated medium for production of exoenzymes. Strains
producing subtilisin were grown on L-plates containing 1 % milk powder. The
production of exoenzymes was studied in two-fold concentrated L-broth with
vigorous shaking. The growth was indicated by turbidity of the culture
measured with KIettSummerson colorimeter (Klett Manufacturing Co., Inc.
N.Y.) using a no. 66 filter.
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Enzyme assays
Alpha amylase was assayed with Phadebas tablets (Pharmacia)
as described in Kontinen, V.P. and Sarvas, M., (1988) J. Gen. Microbiol.,
134:2333-2344. For the plate assay bacteria were streaked on L-plate
containing 5% of starch, and the halo around colonies was measured after
incubation of the plates at 4 degrees C. Typically, there was no zone around
wild-type colonies producing endogenous alpha amylase, while that of strains
carrying pKTHlO was more than 2 mm depending on the age of plates.
Subtilisin of B. licheniformis and chromosomally-encoded proteases were
assayed in 1 ml of 0.1 M Tris-0.0 1 M CaCl2 (pH 8.0) with a chromogenic
peptide substrate succinyl-AIaAIa-Pro-Phe-p-nitroanilide (Del Mar, E.G., et
al.,
(1979) Anal. Biochem., 99:316-320). The rate of hydrolysis was measured on
a Hewlett Packard diode array spectrophotometer at 410 nm. To determine
the specific activities of alpha-amylases and subtilisin their amount in a
sample
of culture supernatant was estimated either as in ((Kontinen, V.P. and Sarvas,
M., (1988) J. Gen. Microbiol., 134:2333-2344) or with SDS-PAGE stained with
Coomassie Blue. The enzyme amounts were expressed as micrograms/ml.
Table 1. Bacterial strains
Strain Relevant genotype and Parent strain,
properties source or
reference
B.subtilis
IH6064 metB5 sacA32l (Sibakov et al.,
1983)
IH6090 his metBS sac32) IH6064
IH6157 IH6090 (pKTHlO) IH6090
IH6160 IH6064 (pKTHlO) IH6064
IH6480 prs-3 metBS sacA321 (pKTHlO) IH6157
IH8482 prs-29 metBS sacA321 (pKTH10) IH6157
IH6483 prs-33 metBS sacA321 (pKTH10) IH6 157
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IH6484 prs-40 metBS sacA32i (pKTH10}IH6157
IH6485 prs-3 metBS sacA321 IH6480
IH6487 prs40 metB5 sacA321 IH6484
IH6489 prs-II mefBS sacA321 (pKTHlO)' ~ IH6160
~
5 IH6491 prs-13 metBS sacA321 (pKTH:~~O)IH6160
IH6494 prs-33 metB5 sacA321 IH6483
IH6497 prs-26 metBS sacA321 (pKTH10)IH6157
IH6498 prs-13 metBS sacA321 IH6491
IH6501 prs-26 metB5 sacA321 IH6497
10 IH6504 prs-II metBS sacA321 IH6489
IH6513 QB917 (pKTHlO) QB917
IH6521 prs-13 hisAl trpC2 (pKTHlO) IH6513
IH6523 prs-Il metBS sacA32J (pKTHlO)IH6513
IH6622 IH6624 ,(pKTH277) IH6624
15 IH6623 IH6624 (pHPl3) IH6624
IH6624 PSLI (pKTHlO) PSLI
IH6654 prs-29 metBS sacA321 IH6482
IH6752 PSLI (pMJ57) PSLI
IH6755 PSLI (pHPl3) PSLI
20 IH6757 PSLI (pKTH1582) PSLI
IH6759 IH6755 (pHPl3) IH6755
IH6760 IH6757 (pKTH277) IH6757
IH6770 BRB764 (pKTH277) BRB764
IH6774 IH6160 (pKTH277) IH6160
IH6788 IH6752 (pKTH3229) IH6752
IH6789 IH6752 (pKTH3230) IH6752
PSLI arg(GH) 15, IeuAB, rm-, recE4,IA510 in BGSC
stp, thrA
QB917 hisAl thr-5 trpC2 IAIO in BGSC
BRB764 ::~('PS"bE'-amyE) (pKTH1743} A. Palva, University
of Helsinki
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B.subtilis
IH6559 prsA29, recE4, trpC2 (::pKTH1601Kontinen et al,
) 1991
IH6799 IH6064 (::pKTH3200) IH6064
IH6811 IH6624 (pKTH3253) IH6624
IH6812 IH6624 (pKTH3261 ) IH6624
IH6813 IH6624 (pKTH3262) IH6624
E. coli
EH1568 HBIOI (pKTH268) HBIOI
EH1581 DH5a (pKTH3101) DHSa
EH1631 TGI (pKTH3180) TGI
EH1639 TGI (pGEM3zf(+)) TGI
EH 1640 TGI (pDR540) TGI
EH1674 TGI (pKTH277) TGI
EH1675 TGI (pKTH3253) TGI
EH1678 TGI (pKTH3261) TG1
EH 1679 TGI (pKTH3262) TGI
1 ) pKTH1743 is a dreivative of pUB110 carrying a 0.3 kbp insert with an
ORF for B. subtilis deco gene
2) The Bacillus Genetic Stock Center, The Ohio State University, Ohio
Table 2. Plasmids
Plasmid Cloned genes and Derivative of
number resistance markers (Source and/or references)
pKTH10 amyE neo pUB110 (Palva, 1982;
Gryczan et al., 1978)
pKTH268 prsA bia pGEM3zf(+) (Promega;
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Sambrook et al., 1989)
pKTH277 prsA cat ermC pHPl3 (Haima et al.,
1987)
pKTH1582 amyl neo pUB110 (I. ~Palva:
Sibakov; 1986)
pKTH1743 degQ neo pUB110 (A. Palva)
pKTH1786 bla cat neo pAM~il (M. Simonen;
Leblanc and Lee, 1984)
pKTH3101 'prsA bla pKTH268
pKTH3180 OIP~e~-'prsA bia pDR540 (Pharmacia)
pKTH3229 ermC pHPl3
pKTH3230 prsA ermC pKTH3229
pKTH3253 prsA cat ermC pHPl3
pKTH3261 0.5 kb insert in pKTH3253
prsA
cat ermC
pKTH3262 4.5 kb insert in pKTH3253
prsA
cat ermC
pJH101 bla cat tet (Ferrari et al., 1983)
pMJ57 OIPX~,-'subC cat pUB110 (Hastrup and
Jacobs, 1990
Examples
Example 1
Enhancement of alpha amylase secretion in B. subtilis
when there is an increased amount of PrsA protein
The following table (Table 3) illustrates enhancement of a-
amylase secretion in Bacillus subtilis under various conditions when the host
gram-positive bacteria also overexpresses PrsA protein.
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Table 3. Enhancement of a-amylase secretion in B. subtilis by
overexpression of PrsA protein
The plasmid PrsA was
expressed from a-
Strain No. a-amylase amylase
Plasmid No. prsA gene
in
expressed from secreted
a
the plasmid
gene in pg~ml
IH6160 --- 1000
Multicopy plasmide~
IH6774 KTH277 Intact 3100
BRB764 --- 630
Chromosome~
IH6770 KTH277 Intact 2000
IH6811 pKTH3253 Intact 3700
IH6812 Multicopy plasmid~pKTH3261 Disrupted 1300
IH6813 KTH3262 Disru ted 1400
a) pKTH10 with amyE of 8. amyloliquefaciens
b) One copy of amyE under a promoter of increased activity
c) The a-amylase activity of culture supernatant was assayed in
the late stationary phase of growth
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Example 2
Enhancement of alpha amylase secretion in B:' amyloiiquefaciens
when there is hyperexpression of-PrsA protein
This example demonstrates the_effect of overproduction of PrsA
protein of B. subtilis in B. amyloliquefaciens when the host gram-positive
bacteria also hyperexpresses PrsA protein.
ALK02732 is a derivative of ALK02100 and contains the
multicopy plasmid pKTHlO encoding the a-amylase of B. amyloliquefaciens.
The strain thus contains tens of copies of a-amylase gene, and secretes
about 20 fold more a-amylase than the wild type strain ALK02100
(Vehmaanpera et al., J. Biotechnol. 1991,19,221-240). Immunoblotting of
cells of ALK02732 with anti PrsA antiserum showed that the amount of PrsA
protein was similarly.small in ALK02732 as in ALK02100, which is referred to
below. Thus PrsA is rate limiting for protein secretion in ALK02732.
The plasmid pKTH277 was transferred with electroporation to
the B. amyioliquefaciens strain ALK02732 to make ALK02732(pKTH277).
The amount of PrsA in ALK02732(pKTH277) was many folds higher than in
ALK02732, as determined with immunoblotting. This is in good agreement
with the similar increase of PrsA proteins in B. subtilis strains transformed
with PKTH277.
The amount of a-amylase secreted to the growth medium (Luria
broth of double strength with 2% of soluble starch) by ALK02732 and
ALK02732(pKTH277) were determined during logarithmic and early stationary
phases of growth (shake flask cultures). The results are shown in Tables 4-1
(Exp. 1 ) and 4-2 (Exp. 2). It can be seen that in two separate experiments
the
amount of a-amylase in the culture medium of ALK02732(pKTH277) was 1.5
to 2.5 fold the amount in the growth medium of ALK02732.
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Tables 4-1 and 4-2. The Effect of pKTH277 on a-amylase secretion of B.
amyloliquefaciens (ALK02732).
5 Table 4-1, Experiment 1.
ALK02732 ALK02732 (pKTH277)
TIME(h) GROWTH a-AMYLASE GROWTH' a-AMYLASE
(mg/I) (mg/I)
10 3 106 1,1 100 1,1
4 241 8,8 240 8,8
5 426 47 430 72
6 520 160 525 224
7 576 480 580 650
15 8 625 800 630 1050
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Table 4-2, Experiment 2.
ALK02732 ALK02732(pKTH277)
GROWTH a-AMYLASE GROWTH a-AMYLASE
~
TIME(h)
r
(mg~l) (mg~l)
3 100 0,9 112 1,5
4 250 6 272 9
4,5 350 24 363 38
5 425 50 437 80
6,5 545 190 577 450
8 585 320 637 1300
12 702 1600
12,5 700 900
The density of the culture as determined with the Klett-Summerson
colorimeter using filter no. 66, indicated with Klett units.
Materials and Methods Used in this Example
Bacterial strains and plasmids: B. amyloliquefaciens strain
ALK02732 (described by Vehmaanpera et al., 1991 ) was used in
transformation and growth experiments. ALK02732 (pKTH277) was made by
transforming plasmid pKTH277 into ALK02732 (this study) and used in
growth experiments. Plasmid pKTH277 carrying prsA gene is described by
Kontinen et al. (1991 ).
Transformation of pKTll277 into ALK02732 by electroporation:
Plasmid pKTH277 was isolated using the alkaline lysis method and
methylated with BamHl methylase (New England Biolabs) according to
manufacturers instructions. About 0.5 Ng of methylated plasmid DNA was
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used for electroporation. Electroporation was done as described by
Vehmaanpera (1989). Cells were pulsed in 0.2cm sample cuvettes (Bio-Rad
Laboratories) with Gene PuIserT"~ apparatus (Bio-Rad Laboratories) set at 1.5
kV, 25NF and 400i~. Transformants were screened for chloramphenicol
resistance on Luria-Kanamycin(lONg/ml)-Chloramphenicol(5Ng/ml) plates.
(Kanamycin was also on the plates to avoid loss of pKTHlO, since ALK02732
contains pKTHlO, conferring kanamycin resistance).
Growth experiments and sample collection: First ALK02732 and
ALK02732 (pKTH277) were grown over night on Luria plates. From the
plates bacteria was added to 10 ml Luria and they were grown to logarithmic
phase (Klett 100). Then 1 ml Glycerol (1/10 of cultivation volume) was added
and cell suspension was frozen and stored in -70C. Growth experiments
were started by diluting Klett 100 cells 1:100 or 1:200 in 2x Luria + 2%
starch.
The Luria used in growth experiments contained no salt. Cultivation volume
was 20 ml and growth was in bottles in 37C with vigorous shaking. For both
strains Kanamycin (lONg/ml) and for ALK02732 (pKTH277) also
Chloramphenicol (5Ng/ml) was supplemented to growth medium. The growth
was indicated by measurements with Klett-Summerson colorimeter (Klett
Manufacturing Co., Inc. N.Y.) using a number 66 filter. 0.5 ml samples were
taken during growth, samples were centrifugated and culture supernatants
were stored in -20C for a-amylase assays.
a-amylase assays: a-amylases in culture supernatants were
determined using Phadebas tablets (Pharmacia). Samples were incubated
for 1 h at 37C in 1 ml buffer (50mM MES pH 6.8, 50mM NaCI, 1 OONM CaCl2
containing 1/4 dispersed phadebas tablet, after which 50N1 5M NaOH was
added to stop the reaction. After filtration through lAlhatman no.1 filter
paper,
absorbency of the filtrate was measured using 616-624nm as an analytical
wave length range and 800-804nm as a reference wave length range.
Commercially available a-amylase of B. amyloliquefaciens (Sigma) was used
as a standard and results were expressed as mg enzyme per I.
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References: Kontinen V., Saris P. and Sarvas M. (1991 ): A
gene (prsA) of Bacillus subtilis involved in a novel, late stage of protein
export. MoL Microbiol. 5:1273-1283. Vehmaanpera, J. (1989):
Transformation of Bacillus Amyloliquefacien~ by electroporation. FEMS
Microbiol. Lett 61:165-170. Vehmaanpe:rav;J., Steinborn G. and Hofemeister
J.(1991 ): Genetic manipulation of Bacillus amyloliquefaciens. J.
Biotechnol.i 9:221-240.
Example 3
Enhancement of secretion of subtilis from B. lentus
when there is hyperexpression of PrsA protein
This example demonstrates the method and system of the
invention in enhancing secretion of overexpressed exoproteins in B. lentus
when the host gram-positive bacteria also hyperexpresses PrsA protein.
The effect of hyperexpression of prsA has been tested with
respect to the secretion of subtilisin from B. lentus, commercially available
as
ExperaseT"' and described in WO 89/06279 (in the name of Novo Nordisk
A/S). The subtilisin is transcribed from the plasmid pPL1800 which is based
on the expression vector pPL1759 (Hansen, C., Thesis, 1992, The Technical
University of Denmark) with a pUB110 origin and the promoter and signal
peptide from the alpha-amylase of B. licheniformis (amyL). The plasmid
pSX94 is described in WO 89; 06279. The B, subtilis strain SHa273 used for
production is a protease weak derivative of DN1885 (Jorgensen, P.L. et aL,
(1991 ) FEMS Microbiol. Lett., 77:271-276), in which two additional proteases
apr and npr have been inactivated. The secretion of the B. lentus subtilisin
was measured from strains either with (MOL253) or without (MOL252) the
prsA plasmid pKTH277, and growth was performed at 30°C in soya broth
BPX supplemented with kanamycin and chloramphenicol.
Measurements of subtilisin levels from the two strains after five
days show that the strain with the prsA plasmid has a four fold higher
secretion of the 8. lentus subtilisin (160 microgramslml) compared to the
strain without this plasmid (40 micrograms/ml).
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The BPX medium used has the following composition:
BPX: Potato starch 100 g/I
Barley flour 50 g/I
BAN 5000 SKB 0.1 g/I
Sodium caseinate 10 g/I
Soy Bean Meal 20 g/I
Na2HP04, 12 H20 9 g/I
Pluronic 0.1 g/I
Strain List:
B. subtilis Genotype and properties Parent strain
DN 1885 amyE, amyR2 RUB200
PL1801 amyE, amyR2, apr-, npr- DN1885
MOL252 PL1801 (pPL1800) PL1801
MOL253 PL1801 (pPL1800, pKTH277) PL1801
The RUB 200 strain is described by Yoneda et al., 1979, Biochem
Biophys. Res. Common. Vol. 91, 1556-64.
Example 4
The presence of PrsA protein in
Bacillus amyloliquefaciens and Bacillus subtilis
The presence of PrsA protein has been demonstrated in two
strains of 8. amyloliquefaciens, strain ALK089 and strain ALK02100. Strain
ALK089 is an industrial strain used for the production a-amylase. Strain
ALK089 is an overproducer described in (Bailey, M.J. and Markkanen P.H. J.
Appl. Chem. Biotechnol. 1975,25,73-79). Strain ALK02100 is a derivative of
ATCC23843 (J. Vehmaanpera, FEMS Microbiology Letters 49(1988)
101-105).
The presence of PrsA protein has also been demonstrated in
two strains of Bacillus licheniformis, strain 749/C (Pollock, M.R. (1965)
Biochem. J. 94,666-6/5), and ATCC 14580.
The PrsA protein in these non Bacillus subtilis gram positive
bacteria was identified using immunoblotting techniques. More specifically,
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cells collected at late exponential phase of growth (to minimize the amount of
proteases - PrsA protein is protease sensitive) were immunoblotted, treated
with lysozyme shortly (to make cell wall leaky, but again avoiding long
treatment to minimize proteolysis), and solubilized at 100°C with
sample
5 buffer containing 2% SDS. PrsA protein was detected with rabbit antiserum
(KH1283) raised against PrsA protein of B~ subfilis produced in Escherichia
coli (thus there was minimal antigenic doss reaction with any bacillar protein
except PrsA). The antiserum detects nanogram amounts of B. subtilis like
PrsA in the immunoblots.
10 In addition, all four strains were found to contain a protein of the
size of PrsA of B. subtilis, and specifically identified by the above
antiserum.
The intensity of the staining of the band was approximately similar in both B.
amyloliquefaciens strains and similar to that of PrsA protein in wild type B.
subfilis. Coomassie,Blue staining of parallel SDS-PAGE of cellular proteins of
15 B. amyloliquefaciens showed only a very weak band at the position of PrsA
protein like in the case of B. subtilis and consistent with PrsA protein of 8.
amyloliquefaciens being a minor cellular protein as it is in 8. subtilis. The
intensity of staining of the PrsA protein of the two B. licheniformis strains
was
weaker than in B. subtilis, suggesting a somewhat smaller amount of PrsA
20 protein. However, it cannot be excluded that the weaker staining is due to
less efficient binding of the antiserum of PrsA protein of B. licheniformis
than
to that of B. subtilis. Table 5 is a summary of the roughly estimated amount
of PrsA in the different strains.
25 Table 5. Approximate amounts of PrsA protein found in cells of
Bacillus strains during early stationary growth phase (at a cell density of
Klett
400). Estimates are based on Western blots and are preliminary.
Strain PrsA in cells (arbitrary units)
30 B. amyloliquefaciens
RH2078 4
RH2079 4'~
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B, licheniformis
RH2080 1
' RH 305 1
B. subfilis
IH6064 5
IH67742~ 100'
1 ) Cells were collected from mid-logarithmic phase (at a density
of Klett 100) of growth and the PrsA values estimated to correspond a density
of Klett 400.
2) This is an overproducer of PrsA, due to the content of
pKTH277.
Materials and Methods Used in this Example
Strains: Bacillus amyloliquefaciens RH2078 = ALKOB9 =
VTTI97 = E18. This is an a-amylase overproducer. A gift from
J.Vehmaanpera, ALKO. Bacillus amyloliquefaciens RH2079 = ALK02100,
derived from ALK02099 (pE194/pC194). A gift from J. Vehmaanpera, ALKO.
Bacillus licheniformis RH2080 = BRAS = ATCC14580. This is a producer of
thermoresistent a-amylase. A gift from P.Saris, BI, H:ki. Bacillus
licheniformis RH305 = 749/c. This is a penicillinase constitutive strain,
originally derived from J.O.Lampen. Bacillus subtilis IH6074. This is metB5
sacA321.Ref. M.Sibakov et.al.1983. Bacillus subtilis IH6774. This is derived
from IH6064, contains plasmids pKTHlO (carrying the a-amylase gene) and
pKTH277(carrying the gene for coding PrsA).
Growth media: Luria-agar plates (L-plates); twice concentrated
Luria-broth (2x L)
Purified PrsA protein: PrsA was purified from pKTH277
containing B. subtilis by M. Lauraeus.
Immune serum: E. coli-produced PrsA of B. subtilis, which was
run in and cut out from a SDS-gel.
Chemicals: Phenylmethylsulfonylfluoride, Sigma P-7626.
100mM in ethanol at -20°C. EDTA, TitriplexRlll p.a. Merck 8418. As a
0.5 M
solution, pH 8. Lysozyme, Sigma L-6876. This was used as 1 mg/ml in the
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following solution: 20mM potassium phosphate pH 7,15 mM MgCl2, 20%
sucrose. TCA 100% BCAt Protein Assay Reagent by Pierce.
SDS-PAGE and Western Blot equipment and chemicals according to BioRad.
Blots were stained with 4-chloro-1-naphtol.
Culture conditions and sample preparation for gel
electrophoresis: Bacteria were grown on L-.plates overnight at 37°C.
Colonies were picked with a glass rod into a preweighed Eppendorf tube, and
weighed. Sample buffer was added to get either 10 or 100 mg cells(ww)/ml.
Samples were heated 10 min at 100°C.
Bacteria were grown in 2xL broth with agitation at 37°C. To
minimize the protease effect bacteria (from -20°C) were first grown to
Klett
100 (corresponding to about 1-2 mg cells ww/ml, or about 1 O9cells/ml). This
was used as an inoculum at 10'2 dilution. 20 ml of bacteria were grown in
Klett flasks and 4 ml samples were taken at Klett 100, at Klett 100+2h (Klett
appr.400), and at Klett 100+4h (Klett appr.550).
Samples were immediately transferred into an ice bath, PMSF
was added to 1 mM, and EDTA to 10 mM. Cells were separated from culture
supernatant by centrifugation at 12 OOOxg 10 min., and treated with
lysozyme, 15 min at 37°C in a 1 /20 volume. An equal volume of sample
buffer
was addad. Culture supernatant was precipitated in 10% TCA at 4°C and
concentrated 20-fold in sample buffer.
The samples were run in 12% SDS-PA gels, stained with
Coomassie Brilliant Blue R, or blotted onto PVDF filters according to BioRad.
PrsA was detected with the specific anti-PrsA rabbit antiserum
KH 1283.
Example 5
Enhanced Secretion of Lipase from Pseudomonas mendocina
in Bacillus subtilis that Overproduces PrsA Protein
Using the prsA gene in pKTH277, scientists at Genencor
International, South San Francisco, CA, USA, have shown that when Bacillus
subtilis overexpresses both PrsA protein and lipase (from Pseudomonas
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mendocina, a gram-negative bacteria), the amount of lipase secreted into the
medium is about 3.5 times greater than it is in controls that do not
overexpress the prsA gene. The Genencor International scientists used
industrial strains of Bacillus, and industrial fermentation conditions. (Data
not
shown).
Conclusion
Thus it can be seen that the present invention discloses a
method and system for enhancing the production of industrially and medically
important exoproteins in gram-positive bacteria. Without departing from the
spirit and scope of this invention, one of ordinary skill can make various
changes and modifications to the invention to adapt it to various usages and
conditions. As such, these changes and modifications are properly, equitably,
and intended to be, within the full range of equivalence of the following
claims. Various features of the invention are also evident from the following
claims.
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