Note: Descriptions are shown in the official language in which they were submitted.
2~8~82'1
WO9t/03561 PCT/FI91/002
TITLE OF T~E INVENTION
HYBRID ~-AMYLASE PROMOTERS
Field of the Invention
The present invention is directed to hybrid
bacterial promoters containing modules of the ~~
amylase promoter operably linked to modules of the B. -~ .
subtilis alkaline protease promoter. The invention is
also directed to the production of recombinant pro-
teins which are operably linked to such promoters.
Brief Description of the Backqround Art ;~
Bacillus strains offer many potential advantages in
the production of cloned gene products, as compared
with 2s~erichia coli. First, Bacilli are non-pathogenic
and do not synthesize endotoxins. Second, many of the
gene products are secreted into the growth medium, in
contrast to E. coli~ which retains most of the proteins
due to the presence of an outer membrane. Third, ~-
Bacilli have been widely used for production of ~-
industrial enzymes in large-scale fermentation
processes. `~
However, the number of vector systems available i-~
for expression of recombinant proteins in B. subtilis is ~-
limited. In addition, the understanding of
transcriptional regulatory control elements in B.
subtilis is not completely characterized.
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2 ~ 2 ~1
WO92/03561 PCT/Fl91/002
One important exoenzyme secreted in large amounts
by Bacilli and used in industrial production, is the ct-
amylase from B. amylolique~aciens (Ingle ~t al., Adv. Appl.
Microbiol. 24:257-278 (1987)). This enzyme has an Mr~
value of about 50,000 daltons and has been sequenced
(Takkinen et al., J. Biol. Chem 2s8: 1007-1013 (1983);
Chung et al., Biochem. J. 185:387--395 (1980)). The
expression of this enzyme în B. subtilis has been
reported ("Expression and Regulation of the Bacillus
arnyloliquefaci~ns ~-amylase gene in B. subtilis,l~ P.
Kallio, Ph.D. dissertation, University of Helsinki,
1987).
Obtainlng high level expression of foreign gene
products in Bacillus with an ~-amylase-based secretion
vector is technically complicated. Production of ~
amylase is saturated when there are 10-20 copies of
the ~-amylase gene/cell (Appl . ~icrobiol. Biotech. 27: 64-71
tl987). Thus, the kinetics of ~-amylase production
are no higher in a multicopy plasmid such as pKTH10
(which amplifies to 200 copies/cell in the stationary
phase of the host), than they are in a plasmid having
markedly lower copy numbers. In such multicopy
plasmid systems, s,ecretion of the protein product
becomes ra~e limiting and increasing the gene dosage
above this rate limitation merely results in
accumulation of the precursor within the host cell.
The use of multicopy plasmids thus is not
amenable to large scale industrial production in
Bacillus. There are several disadvantages in using
plasmids in Bacillus, the most important of which is
instability of the transformants. In addition to the
fact that plasmids are inherently unstable in these
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WO92/03561 PCT/F19~/002
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hosts, it appears that when the a~ility to express a
protein is substantially higher than the ability of
the host to secrete it, such overexpression results in
structural instability in the plasmid system.
Thus, an alternative to high copy number
extrachromosomal plasmid systems is needed for the
efficient expression of cloned proteins from Bac~llus in
industrial settings.
It is possible to stably integrate a desired gene
into the chromosome of a Bacillus hostO However, host
structural instability results if the chromosomal
integrates are amplified to 10-20 copies, due to the
repeated DNA sequences. Thus, to maintain stability
of thP host, optimal large scale production in
industrial fermentations is achieved when only a ~;
single copy (or a maximum of two copies at different
positions of the chromosome) is integrated into the
chromosome.
Accordingly, to compensate for low gene copy
number and to synthesize the maximum amount of
recombinant protein, strong, highly-efficient
promoters functional in Bacillus are needed. ;-
In Bacillus~ a large number of genes have been
described which either negatively or positively affect
the transcription frequency of ~acillus genes, and
especially of those genes which encode exoenzymes.
Such genes are termed enhancer genes. Enhancer genes
directly or indirectly affect the transcription rate
of exoenzyme genes. ExampIes of these genes include: ;~
sacQ from B. subtilis~ (J. Bacteriol . l I 6 : 113 (1986) ); sacQ ~,
from B. amyloliquefaci6~ns (J. B~cteriol. 116: 113 (1986));
sacQ from B. lichenifonnis (J. Bacteriol. 169- 324 (1987));
.. . ....... . . . . .. . . . . . . . . ..
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WV92/03561 PCT/FI91/002
-4-
prtR from 8. natto or B. subtilis (J. Bact~riol. 166: 20
tl986)); sacY from B. subtilis (FE~rs Letters 44: 39
(1987)); senN îrom B.natto or B. subtilis (J. Gen. Nicrob.
134:3269 (1988); sacU from B. subtilis (J. Bac~. 170:5093
(1988) and J. ~act. 170:5102 (1988)); and degT from
B.stearothermophilus (J. Bact. 172: 411--418(1990)).
SacUb (Kunst et al., WO 89/09264) is a chromosomal
mutation described in the early nineteen-seventies,
which enhances the production of proteases and
levansucrase in ~. subtilis. (The superscript "h"
denotes hyperproduction.) The produc-tion of
exoenzymes is decreased in SacU-- mutants.
All of the above enhancer genes encode a
protein/peptide whic.h directly or indirectly affects
the transcription (probably the rate of initiation) of
the target gene. For example, the wild type sacQ and
prtR genes enhance exoenzyme production ~mainly
proteases) in Bacillus when their own gene product (a
short peptide) is overexpressed. Overexpression of
the enhancer protein can be obtained by cloning the
enhancer gene into a multicopy plasmid.
Overexpression of the enhancer protein or
increased enhancement function can also by achieved by
mutations in the enhancer gene which affe~t either the
enhancer protein's structural gene or the enha~cer
gene's promoter region (Msadek et al., J. Bacteriol.
172:824--834(1990); Yang et al., J. Bacteriol. 166:113--116
(1986); Biochimie 56:1481 (1974)).
The effect of Bacillus enhancer genes is especially
evident with respect to protease and levansucrase gene
expression. In these cases, the increase of enzyme
production (and the amount of transcription) may be as
WO92~0356l 2 9 ~ ~ ~ 2 l~ PCT/Fl91/002~
great as 10-100-fold compared to the wild-type cell.
With other exoenzymes, such as, for example, amylases,
phosphatases, and ribonucleases, the increase is
usually no more than 2-3-fold. The alkaline protease
(ap~) and levansucrase (lvs) proteins of B. su~ilis are
the best studied cases.
The presence of target regions for sacU, sacQ and
hpr-mUtations has been established in the upstream ~;
region of the promoter for each of these genes. In the
B. subtilis alkaline protease ( aprE) promoter, the target
region for both sacU32 (Hy) and sacQ36(Hy) are found in
a region between -141 and -164 nucleotides upstream of
the transcriptional start site. However, stimulation
of the aprE promoter by the hpr-97 mutation required a
region upstream of base -200 (Henner, D.J., et al., J.
Bacteriol. 170:296--300~1988)). ~ -
Furthermore, it has been shown that the proposed -~
apr target site of s~cU and sacQ mutations contains a
DNA sequence very similar to the DNA sequence in the
upstream region of the levansucrase-gene (J. ~act.
170:296-300 (1988)). The upstream sequence similarity
between the apr and lv5 genes is shown in Figure one
and the complete upstream sequence of apr in Figure
~wo.
When producing proteins which are heterologous to
Bacillus or heterologous to Gram-positive bacteria, the
level of protease which is present in the host may be
a concern. Enhancer genes substantially affect
protease production, and thus the increased amount of
protease activity in the culture supernatant may
drastically diminish the product yield. Even in the
case where the two major extracellular protease genes
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WO 92/03~61 ~ 0 ~ 6 8 ~ ~ -6- PCT/F191/OOZ44
(apr and npr accounting for 95% of the prbtease
activity in Bacillus) have been deletPd, as in most of
the production strains for foreign proteins in
Bacillus, the presence of the sacUHy or sacQ (pUBllO)
enhancer raises the amount of protease activity to
wild-type levels, due to the activation of several
minor proteases. Thus, a need exists for efficient
systems for the production of large scale amounts of
cloned proteins in Gram-positive bacteria, and
especially in Baclllus.
The target regions of enhancer gene activity are
not yet conclusively defined in the apr gene. No one
has demonstrated the interaction between the enhancer
protein and the DNA or the boundaries of this DNA and
it is not known whether the target region would
function out of its original cont~xt. It is not known
whether a specific position regarding the apr promoter
(-35 and -lO regions) is required, either at a certain
distance from the promoter or at a certain side of the
DNA strand. Further, it is not known whether a certain
DNA environment is required, for example, a specific
curvature or conformation of DNA i.n that region. It is
not known whether these same factors may also effect
the enhancement function iTI a heterologous
environment. Lastly, it is not known whether other apr
sequences, not in the actual target regions, function
together with the target site or whether lack of these
sequences may diminish the enhancement effect. These
and other concerns have been solved in a unique manner
arriving at the invention disclosed herein.
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WO 92/035~1 2 0 g ~ 8 2 4 PCr/~191/00244
SUMMARY OF THE INVENTION
Recognizing the potential importance that
enhancer proteins play in regulating gene expression,
and cognizant of the need for highly efficient recom-
binant gene expression systems for use in ~. subtilis,
the inventors have investigated the use of 8ac~11us
enhancer receptor modul2s in chimeric promoter
constructs.
These efforts have culminated in the development
of highly efficient hybrid promoters for recombinant
gene expression in ~acil l us .
According to the invention, there are first
provided genetic elements of the ~-amylase promoter
which provide functional transcriptional modules for
RNA polymerase binding and/or initiation.
According to the invention, there are also
provided enhancer protein target modules, that is,
genetic elements of prokaryotic promoters which
provide functional transcriptional modules necessary
for enhancer protein action.
According to the invention, there are further
provided highly efficient hybrid pr~moters containing
an enhancer protein target module operably linked to
a RNA polymerase initiation module, wherein such
target module is heterologous to such initiation
module.
According to the invention, there are further
provided expression vectors providing such hybrid
promoters, hosts transformed with such expression
vectors, and methods for producing the genetically
engineered or recombinant protein using such hosts.
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wo g~/0356 1 2 0 ~ 6 8 ~ ~ PCT/FI9l/D02~
-8-
DESCRIPTION OF~ THE FIGURES
Figure 1 is a comparison of aprE and sacB upstream
regions containing the target for sacU32(Hy); and
sac~36(Hy) stimulation. The sacB sequence is from
Henner, D.J. et al., J. 8acteriol. 170:296 (1988) and
Shimotsu, H. ~t al., J. sacteriol. 1~8:380-388 (1986).
The asterisks indicate identical nucleotides. The
positions with respect to the transcription start site -
are indicated.
Figure 2 shows the sequence of the promoter
region of the apr gene, Henner, D.J. et al., J. Bacteriol.
170:296 (1988) and Shimotsu, H. et al., J. ~acte~lol.
168:380-388 (1986). The 5' end of the transcript is
marked as "+1." The fragment B has been underlined.
Figure 3 is the DNA and amino acid sequence of
the NH2 region of the B. subtilis ct-amylase gene. The
NH2-terminal valine of exoamylase was taken as amino
acid 1. The cleavage between the signal sequence and
the exoamylase is indicated by a vertical bar. The
signal sequence (amino acids -1 to 31) is underlined.
The arrow shows the wild type claI site and the 5' end
of the ~-amylase promoter const:ructs, upstream o~ ~;
which the new ClaI sites have been added. From
constructs 302, 303 and 304 the DNA sequence between
the notation 1601-302, 1601-303 and 1601-304,
respectively, has been deleted. ~-
Such notation does not indicate the base number.
The bases deleted in construct 302 are bases 1-44. The
bases deleted in construct 303 are bases 1-69. The
bases deleted in construct 304 are bases 1-98. ~-
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W092/03561 PCT/FI91/002~
_9_ ~ -
BRIEF DESCRIPTIOI~ OF THE PREFERRED EMBODIMENTS
I. Definitions
In the description that follows, a number of
terms used in recombinant DNA (rDNA~ technology are
extensively utilized. In order to provide a clear and
consistent understanding of the specification and
claims, including the scope to be given such terms,
the following definitions are provided.
Gene. A DNA sequence containing a template for ;~
a RNA polymerase. The RNA transcribed from a gene may
or may not code for a protein. RNA that codes for a ;
protein is termed messenger RNA (mRNA).
A "complementary DNA" or "cDNA" gene includes
recombinant genes synthesized by reverse transcription
of mRNA and from which intervening sequences
(introns) have been removed.
Enhancer ~ene. The term "enhancer gene" is
intended to refer to a gene whic:h encodes a protein
which directly or indirectly increases production of
another protein.
Geneti Q sequence. As used herein, the term
"genetic sequences" is intended to refer to a nucleic
acid molecule (preferably DNA).
Promoter. The term "promoter" as used herein
refers to a module or group of modules which, at a
minimum, provides a binding site or initiation site
for RNA polymerase action. A promoter is generally
composed of multiple operably linked genetic elements
termed herein "modules."
Promoter Module. The term "module" as in
::
"promoter module" refers to a genetic transcriptional ~i
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WO92/03561 2 0 ~ PCT/F191/002~
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,
regulatory element which provides some measure of
control over the transcription of operably linked
coding sequences or other operably linked modules.
Each module in a promoter can convey a specific
piece of regulatory information to the host cell's
transcriptional machinery. At least one module in a
promoter functions to position the start site for RNA
synthesis. Other promoter modules~ regulate the
frequency of transcriptional initiation. Typically,
modules which regulate the frequency of
transcriptional initiation are located upstream of
(i.e., S' to) the transcriptional start site, although
such modules may also be found downstream of (i.e., 3'
to) the start site.
The term "target module," as used herein, refers
to a transcriptional regulatory element which confers
the ability to respond to enhancer gene activity
(i.e., such as the protein or peptide encoded by an
enhancer gene) on a promoter which otherwise would not
respond, or would respond less efficiently, to such
enhancer gene activity.
The term "initiation module" refers to a promoter
module which is reguired to initi.ate transcription of
operably linked genes with RNA polymerase. In
prokaryotic promoters, initiation modules are usually
located at about -l0 and -35 nucleotides from the
start site of transcription.
By "hybrid promoter" is meant a promot~r in which
an initiation module is operably linked to a
heterologous target module. A target module which is
heterologous to an initiation module is a target
module which is not found naturally operably linked to
this initiation module in the host cell.
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WO92/03561 2 0 ~ ~ ~ 2 '1 PCT/~191/002~
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Operable linkaqe. An "operable linkage" is a
linkage in which a sequence is connected to another
sequence (or sequences) in such a way as to be capable
of altering the functioning of the sequence (or
sequences). For example, a protein encoding sequence
which is operably linked to the hybrid promoter of the
invention places expression of the protein encoding
sequence under the influence or control of the regula-
tory sequence. Two DNA sequences (such as a protein
encoding sequence and a promoter region sequence
linked to the 5' end of the encoding sequence) are
said to be operably linked if induction of promoter
function results in the transcription of the protein
encoding sequence mRNA and if the nature of the
linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift
mutation, (2) interfere with the ability of the
expression regulatory sequences to direct the expres-
sion of the mRNA or protein. Thus, a promoter region
would be operably linked to a DNA sequence if the
promoter were capable of effect:ing transcription of
that DNA sequence.
Cloninq vector. A "cloning vector" is a plasmid
or phage DNA or other DNA sequence which is able to
replicate autonomously in a host cell, and which is
characterized by one or a small number of endonuclease
recognition sites at which such DNA sequences may be
cut in a determinable fashion without loss of an
essential biological function of the vector, and into
which DNA may be spliced in order to bring about its
replication and cloning. The cloning vector may
further contain a marker suitable for use in the
identification of cells transformed with the cloning
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WO92/~3561 PCT/~91/002
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vector. Markers, for example, are erythromycin and
kanamycin resistance. The term "vehicle" is sometimes
used for "vector."
Ex~ression vector. An "expression vector" is a
vector similar to a cloning vector but is capable of
expressing a structural gene which has been cloned
into the expression vector, after transformation of
the expression vector into a host. In an expression
vector, the cloned structural gene (any coding
sequence of interest) is placed under the control of
(i.e., operably linked to) certain control sequences
which allow such genP to be expressed in a specific
host. In the expression vector of the invention, a
desired structural gane is operably linked to the
hybrid promoter of the invention. Expression control
sequences will vary, and may additionally contain
transcriptional elements such as termination sequences
and/or translational elements such as initiation and
tsrmination sites.
The expression vectors of the invention may
further provide, in an expression cassette other than
the one providing the hybrid promoters of the
invention, sequences encoding a desired enhancer gene.
In a preferred embodiment, such enhancer gene would be
the enhancer gene which encodes the protein which
regulates the target module of the hybrid promoter.
Functional Derivative. A "functional derivative"
of a molecule, such as a nucleic acid or protein, is
a molecule which has been derived from a native
molecule, and which possesses a biological activity
(either functional or structural) that is substan~
tially similar to a biological activity of the native
molecule, but not identical to the native molecule. A
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2~82~
W092/03561 PCT/F191/002
-13-
functional derivative of a protein may or may not
contain post-translational modifications, such as
covalently linked carbohydrate, depending on the
necessity of such modifications for the performance of
a specific function. The term "functional derivative"
is intended to include the 'Ifragments~ 1l l'variants, 1l or
'chemical derivativesll of a molecule.
As used herein, a molecule is said to be a
Ichemical derivative" of another molecule when it
contains additional chemical moieties not normally a
part of the molecule. Such moieties may improve the
molecule's solubility, absorption, biological half
life, etc. The moieties may alternatively decrease
the toxicity of the molecule, eliminate or attenuate
any undesirable side effect of the molecule, etc.
Moieties capable of mediating such effects are ~-
disclosed in Remington's Pharmac~LItical Sciences (1980).
Procedures for coupling such moieties to a molecule
are well known in the art.
Fragment. A "fragment" of a molecule such as a
nucleic acid or protein is meant to refer to a mole-
cule which contains a portion of the complete sequence
of the native molecule.
Variant. A "variant" of a molecule such as a
nucleic acid or protein is meant to refer to a mole~
cule substantially similar in structure and biological
activity to either the entire molecule, or to a
fra~ment thereof, but not identical to such molecule
or fragment thereof. A variant is not necessarily
derived from the native molecule itself. Thus,
provided that two molecules possess a similar
activity, they are considered variants as that term is
used herein even if the composition or secondary,
.
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W092/03s61 2 0 ~ ~ ~ 2 ~ PCT/Fl91/002~
-14-
tertiary, or quaternary structure o~ one of the
molecules is not identical to that found in the other,
or if the sequence of nucleic acid (or amino acid
residues) is not identical, or if the synthesis of one
of the variants did not derive from the other.
.:
; II. Identification of Promoter Modules
,;
The present invention provides hybrid promoters
providing heterologous regulatory promoter modules for
use in Bacillus expression systems. One module provided
by the invention is an initiation module. A second
module provided by the invention is a target module,
that is, a target element(s) for enhancer protein
action. Any hybrid promoter of the invention which
contains a target module of the invention is sensitive
to enhancer gene activity if such target module is a
target of the enhancer gene's protein.
Different promoter modules can be used in the
hybrid promoters of the invention so as to provide
targets for different enhancer genes and/or enhancer
gene mutations. In a preferred embodim2nt, the target
modules of the invention are those found 5' of
(upstream of), and operably linked to, the wild type
apr promoter, and especially, the ~. subtilis apr
promoter.
According to the invention, when such target
modules are operably linked to heterologous initiation
modules which provide an RNA polymerase initiation
site, the resulting promoter becomes highly responsive
to hyperexpression and secretion in response to
enhancer gene action or to mutations in such enhancer
genes.
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WV92/03561 P~T/~91/002
~15-
In the hybrld promoter of the invention, discrete
modules from different promoters have been assembled
such that the individual modules function cooperative-
ly or independently to activate transcription of the
operably linked encoded sequence. The modules which
are present in the hybrid promoters of the invention
can be further modified by mutation or by formation of
novel sequence junctions at the boundaries between
such modules. Such mutations may provide for the
deletion, addition or duplications of genetic
information. Spacing between modules in the hybrid
promoters of the invention is flexible, the only
limitation being that promoter function must be
preserved.
The process for genetically engineering the
hybrid promoters of the invention is facilitated
through the cloning of genetic sequences which are
capable of providing specific tra1lscriptional promoter
modules. Genetic sequences which are capable of
providing promoter modules may be derived from genomic
DNA, synthetic DNA, cloned D~TA and combinations
thereof. The preferred species source of the promoter
modules of the invention is Bacillus~ although any
source may be used if the funct:ion of the module is
preserved in the transformed host cell.
In the preferred hybrid promoter of the
invention, a target module from the B. subtilis alkaline
protease gene (apr) is operably linked to an initiation
module of the B. amyloliquefaciens ~-amylase gene. Any
module of the alkaline protease gene promoter which
provides a target sequence to facilitate enhancer
- activity may be used. Especially any module which
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W092tO3561 2 0 ~ ~ ~ 2 ~ PCT/F~91/002~
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confers recognition of the sacUh, sacQ or prtR
enhancers/mutations is preferred.
The identification of a target module is made by
testing a putative module's ability to transfer
sensitivity to enhancer gene action to a promoter
which is not normally responsive (or normally less
responsive) to such action.
According to the invention, hybrid promoters can
be designed for any s. subtilis enhancer proteins in any
manner which reveals the target activity. For
example, a strategy for such design may include (1)
the identification of a protein whose expression is
increased in response to the enhancer protein, (2) the
cloning of the 5' transcriptional regulatory region of
such protein, (3) the subcloning of fragments of such
5' region into a construct which operably links a
putative target module with an initiation module using
methods known in the art and (4) the selection of
those clones which reveal enhanced protein expression
of a reporter protein using hosts which provide the
enhancer gene or mutation thereof. Hybrid promoters
whic:h respond to the enhancer genes sacQ (especially
sacQ from B. subtilis, B. amyloliquefaciens or ~. licheni-
fcrmis)~ prtR (especially prtR from ~. natto or s.
subtilis), sacV (especially sacV from B. subtilis), senN
(especially senN from B. natto or B. subtilis), sacU
(especiallv sacU from s. subtilis) and degrr (especially
degT from ~. stearothermophilus) may be designed in this
manner.
According to the invention, hybrid promoters can
be designed which contain only one target module or
which contain more than one target module operably
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WO92/03~61 PCr/F191/00
-17-
linked together. Larger enhancer target regions, such
as, for example, the B region of the apr gene, carry
target modules for several enhancer gene products.
Thus a hybxid promoter may be designed which is
capable of responding to more than one enhancer gene
by operably linking desired target modules for each
enhancer to the initiation module.
Cloning of the modules of the invention is
facilitated by use of the polymerase chain reaction
(PCR) as such modules are generally of a size
consistent with a size capable of being amplified by
PCR. Once the sequence of the 5' region of a desired
gene is known, PCR primers tchemically synthesized by
means known in the art or commercially purchased) may
` be used to amplify specific sequences of the 5' region i~
for subsequent cloning and characterization as
described above.
:. .
III. Genetic Enqineerina_of Protein Encoding Sequences
The genetic engineering of protein encoding
sequences to be expressed under the c.ontrol of the
hybrid promoters ot the invention is also facilitated
through the cloning of those sequences.
Prokaryote yenomic DNA containing protein en-
coding sequences will not contain introns, although it
may contain spacers between transcriptional units.
EukaryotP genomic DNA containing protein encoding
eukaryotic sequences may or may not include naturally
occurring introns. For expression in the prokaryotic
hos~s of the invention, such introns generally must be
removed prior to cloning. Otherwise, either
prokaryote or eukaryote encoding sequences may be
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WO92/0356l 2 ~ 2 '1 PCT/FI91/002
-18-
expressed in the hosts of the invention. Moreover,
such genomic DNA may be obtained in association with
the 3' transcriptional termination region. Further,
such genomic DNA may be obtained in association with
the genetic sequences which encode a 5' non-translated
region of the desired mRNA and/or with the genetic
sequences which encode the 3' non-translated region.
To the extent that a host cell can recognize the
transcriptional and/or translational regulatory
signals associated with the expression of the mRNA and
protein, and to the extent that such signals do not
impede the hybrid promoters of the invention, then the
5' and/or 3' non-transcribed regions of the native
gene, and/or, the 5' and/or 3' non-transl~ted regions
of the ~RNA, may be retained and employed for
transcriptional and translational regulation.
To obtain coding sequences for proteins whose
genes are not rearranged prior to expression, genomic
DNA can be extracted and purified from any cell of any
host which carries the coding sequence, whether or not
the cell expresses the protein. To obtain coding
sequences for protains whose genes are rearranged
prior to expression, genomic DNA can be extracted and
purified from any cell which expresses the protein of
interest.
The extraction of genomic DNA can be performed by
means well known in the art (for example, see Guide to ~.
Molecula~ Cloning Techniques , S . L . Berger et al ., eds.,
Academic Press (1987)).
Alternatively, nucleic acid sequences which
encode a desired protein can be obtained by cloning
mRNA specific for that protein. mRNA can be isolated
from any cell which produces or expresses the protein
:
2~8~
WO92~03~61 PCr~FI91/002~
--19--
of interest and used to produce cDNA by means well
known in the art (for example, see Gr~ide to ~ol~cular
Cl~ g T~chniques~ S.L. Berger ~t al.~ eds., Academic
Press ~1987)). Preferably, the mRNA preparation used
will be enriched in mRNA coding for the desired
protein, either naturally, by isolation from a cells
which are producing large amounts of the protein, or
in vit~o, by techniques commonly used to enrich mRNA
preparations for specific sequences, such as sucrose
gradient centrifugation, or both.
To prepare DNA for cloning into a cloning vector
or an expression v~ector, a suitable DNA preparation
(either genomic DNA or cDNA) is randomly sheared or
enzymatically cleaved, respectively. Such DNA can then
be ligated into appropriate vectors to form a
recombinant gene (either genomic or cDNA) library.
A DNA sequence encoding a protein of interest or
its functional derivatives may be inserted into a
cloning vector or an expression vector in accordance
with conventional techniques, including blunt-ending
; or staggered-ending termini for ligation, restriction
enzyme digestion to provide appropriate termini,
filling in of cohesive ends as appropriate, alkaline
phosphatase treatment to avoid undesirable joining,
and ligation with appropriate ligases. Techniques for
such manipulations are disclosed by Maniatis, T., et
al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor
Laboratory, 19~2J~ and are well known in the art.
Libraries containing clones encoding a desired
protein or a desired transcriptional regulatory
element may be screened and a desired clone identified
by any means which specifically selects for the DNA of
interest. For example, as described above, if a clone
:. : : - : . .", "
:, .. . .,: :,
W092/03561 2 ~ 8 ~ 8 2 ~ PCT/FI91/002~
-20-
to a desired transcriptional regulatory element is
desired, such a clone can be identified by the ability
of the desired element to provide a function specific
for such element to a host cell transformed with the
cloned sequence. In a similar manner, if a clone to
a desired protein sequence is desired, such a clone
may be identified by any means used to identify such
protein or mRNA for such protein, including, for
example, a) by hybridization with an appropriate
nucleic acid probe(s) containing a sequence(s)
specific for the DNA of this protein, or b3 by
hybridization-selected translational analysis in which
native mRNA which hybridizes to the clone in question
is translated in vitro and the translation products are
further characterized, or, c) if the cloned genetic
sequences are themselves capable of expressing mRNA,
by immunoprecipitation of a translated protein product
produced by the host containing the clone.
Oligonucleotide probes specific for a desired
protein or specific for a desi'eed transcriptional
regula'cory element can be used to identify a desired
clone. Such probes can be designed from knowledge of
the nucleic acid sequence of the element or from the
amino acid sequence of the des:ired protein. The
sequence of amino acid residues in a peptide is
designated herein either through the use of the
commonly employed three-letter or single-letter
designations. A listing of these three-letter and
one-letter designations may be found in textbooks such
as Biochemistry , Lehninger, A., Worth Publishers, New
York, NY (1970). When the amino acid sequence is
listed horizontally, the amino terminus is intended to
. . . ~: . , :
,
:
:` . , . ' ' `
2 ~ 4
W092/03561 PCT/~9l/002
-21-
be on the left end whereas the carboxy terminus is
intended to be at the right end.
Because the genetic code is degenerate, more than
one codon may be used to encode a particular amino
acid (Watson, J.D., In: Molecular ~iology of the Gene, 3rd
Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp.
356-357). The peptide fragments are analyzed to
identi~y sequences of amino acids which may be encoded
by oligonucleotides having the lowest degree of
degeneracy. This is preferably accomplished by
identifying sequences that contain amino acids which
are encoded by only a single codon.
Although occasionally an amino acid sequence may
be encodecl by only a single oligonucleotide sequence,
frequently the amino acid sequence may be encoded by
any of a set of similar oligonucleotides. Impor-
tantly, whereas all of the members of this set contain
oligonucleotide sequences which are capable of en-
coding the same peptide fragment and, thus, poten-
tially contain the same oligonucleotide sequence as
the gene which encodes the peptide fragment, only one
member of the set contains the nucleotide sequence
that is identical to the exon coding sequence of the
gene. Because this member is present within the set,
and is capable of hybridizing to DNA even in the
presence of the other members of the set, it is
possible to employ the unfractionated set of oligo~
nucleotides in the same manner in which one would
employ a single oligonucleotide to clone the gene that
encodes the peptide.
Using the genetic code (Watson, J.D., In :
~olecul~r ~ology of the Gene~ 3rd Ed., W.A. Benjamin,
Inc., Menlo Park, CA (1977)), one or more different
WO92iO356l hQ~ PCT/FI91/002
-22-
.~
oligonucleotides can be identified from the amino acid
sequence, each of which would be capable of encoding
the desired protein. The probability that a par-
ticular oligonucleotide will, in fact, constitute the
actual protein's en~oding sequence can be estimated by
considering abnormal base pairing relationships and
the frequency with which a particular codon is
actually used (to encode a particular amino acid) in
eukaryotic cells. Such "codon usage rules" are
disclosed by Lathe, R., et al., J. Nolec. Biol. 1~3:1-12
(1985). Using the "codon usage rules" of Lathe, a
single oligonucleotide sequence, or a set of oligo-
nucleotide sequences, that contain a theoretical "most
probable" nucleotide sequence capable of encoding the
human secretory granule proteoglycan sequences is
identified.
The suitable oligonucleotide, or set of oligo-
nucleotides, which is capable of encoding a fragment
of the desired gene (or which is complementary to such
an oligonucleotide, or set of oligonucleotides) may be
synthesized by means well known in the art (see, for
example, Synthesis and Application of DNA and RNA, S.A.
Narang, ed., 1987, Academic Press, San Diego, CA) and
employed as a probe to ident~ify and isolate the cloned
gene by techniques known in the art. Techniques of
nucleic acid hybridization and clone identification
are disclosed by Maniatis, T., et al. (In: Molecular
Cloning, A Laooratory Nanual, Cold Spring Harbor Labora-
tories, Cold Spring Harbor, NY (1982)), and by Hames,
B.D., et al. (In: Nucleic Acid HyJ:Jridization, A Practical
Approach, IRL Press, Washington, DC (1985)), which
references are herein incorporated by reference.
Those members of the above-described gene library
' ~ -
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, . . . . . .
'~: , ' '' . ' ' ',: '
. ` . : : .
2~ h4
W092~03561 PCT/FI91/002
-23-
~ '
which are found to be capable of such hybridization
are then analyzed to determine the extent and nature
of the sequences which they contain.
To facilitate the detectio`n of the desired
encoding sequence, the above-described DNA probe may
be labeled with a detectable group. Such detectable
group can be any material having a detectable physical
or chemical property. Such materials have been well-
developed in the field of nucleic acid hybridization -~
and in general most any label useful in such methods
can be applied to the present invention. Particularly
useful are radioactive labels, such as 32p, 3H, 14C, 35S,
l~I, or the like. Any radioactive label may be
employed which provides for an adequate signal and has
a sufficient half-life. The oligonucleotide may be
radioactively labeled, for example, by "nick-transla-
tion" by well-known means, as described in, for ;
example, Rigby, P.J.W., et al., J. Nol. Biol. 113:237
(1977) and by T4 DNA polymerase replacement synthesis
as described in, for example, Deen, K.C., ~t al . , Anal .
Biochem. 135:456 (1983) .
Alternatively, polynucleotides are also useful as
nucleic acid hybridization probes when labeled with a
non-radioactive marker such as biotin, an enzyme or a
fluorescent group. See, for example, Leary, J.J., et
al., Proc. Natl. Acad. Sci. USA 80:4045 (1983); Renz, M., et
al., Nucl. Acids Res. 12:3435 (1984); and Renz, M., EMBO J.
6: 817 (1983) .
To clone a structural ~-amylase gene, B. subtilic
strain IH6064 may be used as a host. B. subtilis
strain IH6064 is avai~ble from the Central Public Health
Institute (CPHI), Helsinki, Finland. Strain IH6064 was
: ',~ ' .
. .
!
,:'. . . ' . .; .'. ' : . ' '
WO92/03561 ~ 8 2 ~ PCT/FI91/002
-2~
constructed by transforming BGSC strain lA289
(aroI906, metB5, sacA321, amyE) with DNA isolated from
strain BGSC strain lA46 (recE4, thr-5, trpC2). AmyE is
the abbreviation for the amylase structural gene.
Transformants which are able to grow on minimal plates
without aromatic amino acids (aroI marker) are
selected and then screened for ~-amylase positive
phenotype. The aroI and amyE markers are known to be
linked markers and therefore aroI selection always
yields a high percentage of amy+ transformants. This
transformation resulted in strain IH6064 (metB5
sacA321). To obtain ~-amylase wild type sequences for
use in the invention described herein, the use of the
particular strain, IH6064, is not necessary and any
wild-type B. subtilis Marburg strain (such as those
available from the BGSC) would give the same result.
In addition, the invention is not limited to ~-amylase
expression as other sequences of interest from Bacillus
or other prokaryotes may be cloned in a similar manner
to techniques disclosed herein or otherwise known in
the art.
Thus, in summary, the actual identification of
peptide sequences permits the identification of a
theoretical "most probable" DNA seq~ence, or a set of
such sequences, capable of encoding such a peptide.
By constructing an oligonucleotide complementary to
this theoretical se~uence (or by constructing a set of
oligonucleotides complementary to the set of "most
probable" oligonucleotides), one obtains a DNA mole-
cule (or set of DNA molecules), capable of functioning
as a probe(s) for the identification and isolation of
clones containing a desired protein or DNA regulatory
element.
., , . , : :
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,
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,
:, ~ ' .
2 ~
WO92/03561 PCTtF191/002
-25-
:
The above discussed methods are, therefore,
capable of identifying genetic sequences which are
capable of encoding a desired regulatory element,
protein, or fragments of such regulatory element or
protein. In order to further characterize protein
encoding genetic sequences, and especially, in order
to produce a recombinant protein, it is desirable to
express the proteins which these sequences encode. In
order to further characterize transcriptional regulat-
ory elements, it i5 desirable to utilize such elements
to regulate the transcription of a desired gene.
Such expression identifies those clones which
express proteins possessing characteristics of the
desired protein or which regulate protein expression
in a manner characteristic of the desired regulatory
element. Characteristics unique -to a protein may :
include the ability to specifically bind antibody
directed against such protein, the ability to elicit
the production of antibody which are capable of
binding to the protein, and the ability to provide an
protein-specific function to a recipient cell, among
others.
IV. ExPression of Proteins Usinq the Expression
Vectors of the Invent:Lon ;
.
~ To express a desired protein, transcriptional and
translational signals recognizable by an appropriate
-i host are necessary. Cloned protein encoding sequences,
obtained through the methods described above, and
preferably in a double-stranded form, may be opérably
linked to sequences controlling transcriptional
expression in an expression vector, and especially,
a, operably linked to the hybrid promoters of the inven-
~, , .
., .
:: . . - , . .,: . . ................... . .
.. . .
2 ~ 2 ~
~V092/03561 PCT/~91/002
-26-
,
tion. Such sequences may be introduced into a host
cell to produce recombinant protein or a functional
derivative thereof.
According to the invention, any prokaryote host
which is capable of providing a desired enhancer gene
and in which the hybrid promoters of the invention are
capable of responding to such enhancer gene may be
utilized. In a preferred embodime~t, a Gram-positive
bacterium is used as the host cell, such as, for
- example, a Bacillus or Clostri~ium perfringens, or c.
teta~us. In a highly preferred embodiment, a member of
the species Bacillus is used as a host cell. Such
members include B. subtilis, B. l~chen~.~ormis, ~.
amyloliqu~faciens, B. polymyxa, R. ~tearo~h~rmopAilus, B.
thermoproteolyti~us. B. coagulans, B. tAuringiensis, B.
mega~erium, B. cereus, B. natto, or B. acidocaldarius. In an
especially highly preferred embodiment, the host cell
is B. subtilis.
A nucleic acid molecule, such as DNA, is said to ~ -
`j be i'capable of expressing" a polypeptide if it con-
tains expression control sequences which contain
transcriptional regulatory information and such
. sequences are "operably linked" to the nucleotide
sequence which encodes the polypeptide.
The precise nature of the regulatory regions
needed for gene expression may vary between species or
cell types, but shall in general include, as neces-
sary, 5' non-transcribing and 5' non-translating (non-
coding) sequences involved with initiation of tran-
scription and translation respectively. Especially,
~d such 5' non-transcxibing control sequences will
include a reyion which contains the hybrid promoter of
,:'-' , ~ : , , . , ~ ,
,':~ . ' ' "
~ W092/03561 2 ~ ~ ~ 8 2 1
27
the invention for transcriptional control of the
operably linked gene.
Expression of a recombinant protein in prokaryo-
tic hosts requires the use of regulatory regions
functional in such hosts, and preferably prokaryotic
regulatory systems. A wide variety of transcrip-
tional and translational regulatory sequences can be
employed, depending upon the nature of the prokaryotic
:
host. Preferably, these regulatory signals are
associated with a particular gene which is capable of
a high level of expression in the host cell.
If desired, a fusion product of the desired
protein may be constructed. For example, the sequence
encoding the desired protein may be linked to a signal
sequence which will allow secretion of the protein
from, or the compartmentalization of the protein in,
the host cell. Such signal sequences may be designed
with or without specific protease sites such that the
signal peptide sequence is amenable to subsequent
removal. Alternatively, the native signal sequence
for a protein may be used, or a combination of vector
and native signal sequences.
Transcriptional initiation regulatory signals
which can be operably linked to the hybrid promoters
of the invention can be selected which allow for
repression or activation, so that expression of the
operably linked genes can be modulated in a specific
manner.
Where the native expression control sequences
signals do not function satisfactorily in a desired
host cell, then sequences functional in the host cell
may be substituted.
A :
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".''" , ~ ,,' '"' ' .' . ' ' ' ' '
. ' '' . : .
wo 92/03s61 2 ~ ~ ~ g ~ ~ PCT/F191/002~
-2~
To transform a host cell with the DNA constructs
of the invention many vector systems are available,
depending upon whether it is desired to insert the
genetic DNA construct into the host cell chromosomal
DNA, or to allow it to exist in an extrachromosomal
form.
Genetically stable transformants may also be
constructed with vector systems, or transformation
systems, whereby a desired protein's DNA is integrated
into the host chromosome. Such integration may occur
de novo within the cell or~ in a most preferred embodi-
ment, be assisted by transformation with a vector
which functionally inserts itself into the host
chromosome. For example, such vector may provide a
DNA sequence element which promotes integration of DNA
sequences in chromosomes. In a preferred embodiment,
such DNA sequence element i5 a sequence homologous to
a sequence present in the host chromosome such that
the integration is targeted to the locus of the
genomic sequence and targets integration at that locus
, in the host chromosome.
'! `' Cells which have stably integrated the introduced
DNA into their chromosomes are selected by also
~3; introducing one or more markers which allow for
selection of host cells which contain the expression
vector in the chromosome, for example the marker may
provide biocide resistance, e.g., resistance to
antibiotics, or the like. The selectable marker gene
can either be directly linked to the DNA gene
seque.nces to be expressed, or introduced into the same
cell by co-transformation.
A transformed sequence may also be incorporated
into a plasmid or other vPctor capable of autonomous
~ .
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W092/0356] ~ ~ 6 8 2 ~ PCT/F191/00
-29-
replication in the recipient host. Any of a wide
variety of vectors for Bacillus may be employed for this
purpose. A plasmid vector is especially usaful when it
is desired to cytoplasmically express a recombinant
protein rather than secrete it.
Factors of importance in selecting a particular
vector includa: the ease with which recipient cells
that contain the vector may be recognized and selected
from those recipient cells which do not contain the
vector; the number of copies of the vector which are
desired in a particular host; and whether it is
desirable to be able to "shuttle" the vector between
host cells of different species. -~
Once the vector or DNA sequence containing the
construct(s) is prepared for expression, the DNA con-
struct(s) is introduced into an appropriate host cell
by any of a variety of suitable means. After the
introduction of the vector, resipient cells are grown
in a selective medium, which selects for the growth of
vector-containing cells. Expression of the cloned
gene sequence(s) results in the production of the
desired protein. This expression can take place in a -~
continuous manner in the transformed cells, or in a
controlled manner.
The expressed protein is isolated and purified in
accordance with conventional conditions, such as
extraction, precipitation, chromatography, affinity
chromatography, electrophoresis, or the like.
The hybrid promoters, vectors and methods of the
invention are useful in identifying those genes which
respond to a specific enhancer gene and in identifying
desired mutations in such genes. The hybrid promo-;~
ters, vectors and methods of the invention are also
: .
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.
" : . ..
WO92/03~61 2 ~ g ~ 8 ~ ~ PCT/~t/0~2~
-30-
useful in the expression of heterologous or homologous
genes which axe operably linked to the hybrid proms-
ters of the invention. Such proteins can be expressed
either intracellularly or extracell~larly in a Bacillus
~ host.
:'-
~; The examples below are for illustrative purposes
only and are not deemed to limit the scope o~ the
invention.
~."
EXAMPLES
:
Example 1
Construction of ~-am~lase Inteqration Vectors
?
.. . .
The ~-amylase gene from Bacillus amyloliquefaciens has
been cloned in plasmid pUB110, resulting in plasmid
pKTH10 (aena 19:81-87 (~982)). Plasmid pUB110 (Gryczan,
T.J., et al.~ J. Baateriol. 134:318 (1978)) is freely
available from the B~cillus Genetic Stock Center (BGSC),
The Ohio State University, Department of Biochemistry,
484 West 12th Avenue, Columbus;, Ohio 43210, USA
(strain lE6), and is fully described, with a
restriction map, in the BGSC~s Strains ~ Data: Fourth
E:dition (1989). From pKTH10, the complete ~-amylase
gene, together with its upstream and downstream
transcription termination signals, was released with
ClaI and samHI digestions and joined to the equivalent
sites of pBR322. The hybrid plasmid was trans~ormed in
E. coli DH5~ (J. ~ol. 8iol. ~66:557 (1983)) and the
transformants plated on L-1% w/v soluble starch-
ampicillin (ap, 25 ~g/ml) plates. (Luria-agar plates,
Miller, J.H., Experiments in Molecular Biology, Cold Spring
, ~.
, .
,. ~.
. .
, ......... .
1 . : . . . . ~ ::
... . . .. . . .
., : ~ ::: : :
203~2ll
W~92/03561 PCT/~91/002
-31-
Harbor, NY, 1972; the starch is from Sigma Chemical
Co.) The colonies containing the ~-amylase plasmid
were readily detected from a "halo" around an ~-
amylase expressing E. coli colony on the plate. To an
~-amylase pBR322 plasmid, at the ~arhHI site downstr~am
of the ~-amylase gene, a 2 kb fragment from the
chromosomal DNA of B. sub~711s IH6064 was inserted (it
has been earlier characterized that this fragment can
be used for a recombination site in chromosomal
integration in ~. sub~ilis). This approach for cloning `
chromosomal fragments for integration has been
described in detail in Appl. ~icrobiol. ~iotech. 27: 64-71
(1987). Furthermore, the chloramphenicol resistance
gene (cm) from the plasmid pC194 ~available as strain
lE17 from the BGSC) was joined to the PvuII of pBR322.
The hybrid pBR322 plasmid, containing the ~-amylase
gene, the s:hromosomal fragment of B. subtilis and the
cm-gene from pC194 was characterized by restriction
enzyme analysis and designated "pKTH 1601."
As a next step, new ClaI cloning sites were
constructed upstream of the -:35 region of the ~-
.~ j,
amylase promoter (downstream of the original ClaI site,used for the cloning of the ~-amylase gene). The new
ClaI sites (3 positions) were generated by using PCR
fragments as described herein. The 5' end of these
fragments consisted s~f a ClaI site at a required
position upstream of the -35 region, and the 3' end
was a HindIII site within the structural part of the ~-
amylase gene. These 5' end-truncated ClaI-HindIII ~-
amylase fragments were used to replace the original
wild type ClaI-HindIII fragment of pKTH 1601. The new
constructs were designated "302," "303" and "304."
,^ ~
., ;,
....... . . . . . . .. . . . ...
.:. . ,.: . : : : - ~ , . ~ :: , ..... , :
,
WO9~/0356~ 2 ~ PCT/~91/~2
-3~-
The primer sequence which was used for the PCR
for construct 302 was:
5'-TTCTATCGATCATCAGACAGGGTATTTTTTATG.
The PCR primer for construct 303 was:
5'~TTCTATCGATGTCCAGACTGTCCGCTGTGTA.
The PCR primer for construct 304 was:
5'-TTCTATCGATGGAATAAAGGGGGGTTGTTATT. ;
For the 3'end of constructs 302, 30 and 304, the
following 3' primer was used:
5'-CACGGATTGATTAAAGCTTGTT.
In pXTH 1601, and in the 302, 303 and 304
constructs, the DNA sequence upstream of the Cl aI site
j is thus identical (pBR322 sequence~. The positions of
the new CZaI sites are depicted in Figure 3. ;'
.
Example 2
Additio~ Potential Enhancer Receptors at Different
Positions of the ~-amylase Inteqration Vector
- ~ !
DNA sequences that potentially could act as
enhancer receptors, when inserted in the ~-amylase
promoter, were derived ~rom the alkaline protease gene
(apr) of ~. subtilis. Two sequencels were used. The first
seqUence is a 48-bp fragment (Figure 1), suggested to
be a sacQ and sacU receptor (J. ~act. 170:296-300
(1988)). It was made by oligonucleotide synthesis and
flanked by ClaI sites. This oligonucleotide was
designated "receptor A." The second sequence
consisted of a -300 bp region upstream of the promoter
(underlined in Figure 2j. The fragment was made by PCR
from the chromosome of ~. subtilis IH 6064, flanked by
ClaI sites and designated "receptor B." Receptor A was
~::
. ,. .. ,. .,. , ~ : ,:
. .
. .
:
2~!o8
W092J03561 PCT/~91/~2
-33-
inserted as a single copy fragment in the ClaI sites of
pKTH 1601 and of constructs 302, 303 and 304. The
hybrid vectors were transformed into E. coli DH5~, the
hybridization positive clones were characterized by
DNA sequencing and designated "pKTH 1910," "1911,"
~` "1912" and "1913, 1I respectively. The receptor B was
similarly joined to the ClaI site of plasmids pKTH 1601
:~ .
and 302, tran~formed into E. coli DH5~, characteri~ed
by restriction enzyme analysis and DNA sequencing, and
designed "pKTH 1974" and "pKTH 1975," respectively.
Ex~mple 3
;~ Inte~ration of the Wild T~ype and Modified ~-amylase
;~` Genes into the Chr~omosome of ~. subtllis IH6064
:t;
'~r~ To test the enhancer effects, the wild type B.
.'. subtilis ~-amylase gene (from pKTH 1601) and the
`q modified ~-amylase (1910-1913 and 1974~5) genes were
integrated in the chromosome of ~. subtilis. It is not
necessary to use pKTH 1601 as th~e source of the wild-
~ type ~. anyloliq~afaciens ~-amylase gene, and any strain
,~ of ~. amyloliquefa~iens which does not contain a mutated
`~ ~-amylase gene may be used. In addition, the sequence
of ~-amylase is known, and desired fragments of this
sequence may be constructed using techniques well
known in the art, such as PCR.
~ The plasmids were isolated from E. coli and
`` transformed into competent B . suotilis cells with cm-
selection ~5 ~g/ml). This resulted in single crossing
over, single copy, ~-amylase positive, chromosomal
-j integrates. To ensure that no ~-amylase amplification
` took place, no cm-selection was applied after the
''i
, ~
:.
.
' ' ` ':: . '
:'' ' :
''~": '' : ''. . '
,
W092/03561 2 ~ & & 8 ~ ~ PCT/F191tO02~
-34-
primary transformation event. The s . subtilis strains
carrying the integrated genes were then made competent ~;
for the addition of enhancer clones or mutations.
'
Example 4
Addition_of the Enhancer Genes or Mutations to
. subtilis Strains carryinq the Inteqrated
~-amylase_Genes
The enhancer genes sacU, sacQ and prtR were tested.
The sacQ and prtR genes were isolated from the
chromosome of B. subtilis IH6064 by PCR according to the
known sequences and the primers described below.
PCR fragments flanked by suitable restriction
enzyme sites were cloned in the plasmid pKTHl743,
i~ which is a pUBllO derivative carrying a multilinker.
A plasmid identical to pKTHl743 for the purposes of
this inventlon may be constructed by replacing the
Pv~ EcoRI fragment of pUBllO with the multilinker
, region of commercially available pUCl8. In addition
to cloned sacQ and prtR genes, chromosomal mutation
sacQ36Hy (BGSCIA53) and sacUHy (pap-9 from B. subtilis
YY88) also were used. Hosts providing the sacUHy
mutations are available from the BGSC (for example,
strains lA95 (sacU(H) 32), lA165 (sacU(H) 32), lA159
(sacU(H) 25), lA199 (sacU(H) 200), and lA200
(sacU (H) 100) ) . :~
The sacQ-pUBllO and prtR-pUB110 clones were
directly transformed to competent B. subtilis strains
carrying the inteyrated ~-amylase genes by kanamycin ;
(km) selection. The sacQHy and sacUHy mutations were
transferred to the integration strains by congression
.'',' ' .
~-
., .
: - : . : .
. ~ ,.' ' ,
. . ~ . , , : : ~ , ~, : .
2 0 ~
W092/0356l PCT/~91/002
-35-
.
(Mol~cular Bi~logy of ~acillus~ vol. I, Academic Press
1982, pp. 147 178). DNA was isolated from ~. suhtilis
strains carrying the above mutations and mixed with
plasmid pE194 tstrain lE18 from the BGSC). Chromosomal ~,
DNA and pE194 DNA were transformed together in -
competent ~. subtil 7s by selecting the erythromycin ~--
resistance (em) marker of pE194 at permissive
temperature (32C). The transformants were screened on
` skim milk plates for increased protease production
; which indicated the presence of either sacUHy or sacQHy
mutations. By growing the correct transformants at
elevated temperature (37C) the carrier plasmid was
lost as monitored by the loss of em-marker.
~ .
Example 5
;j Assay of the ~-amylase S~ecific mRNA and the ;~
Produced ~-Amylase Activity
To test the effect of the enhancer recPptors on
amylase expression in a wild type ~. subtilis strain
or in strain~ carrying different enhancer genes or
')r7' mutations, the strains were grown in L-broth starch
~7 media in a rotary shaker at 37~C (10 ~g/ml km was used
~, when appropriate). Samples were withdrawn up to 6
hours after cell turbidity Klett = 100.
From these samples, the ~-amylase specific ~RNA
7~ was assayed essentially by the method of Thomas (Pro~
.` ~atl . Acad. Sci. USA 77:5201-5205 (1980)) using the Zeta-
Probe nylon membrane according to the manufacturer's
suggestions. The ~-amylase activity was determined
from the supernatant using the Phadebas~ (Pharmacia)
~7~ ' method according to the manufacturer's instructions.
,
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:, .
WO92/03561 2 ~ 8 ~ PCT/~91/002
The amounts of amylase specific RNA and the ~-
amylase activities are shown in Tabl- 1.
.. ''
~ .
'.
'. ~'~`",
. ' ' ~
2 ~ 8 2 ll , '
WO 92/0356~ -37_ P~/F191/00244
~ O ~ ~
a 0 ~ o
:I: ~ ~ N ~ 1 N S' ) H ~
~'1 ~Y ut
~1 ~ ~
+ ~ D ~1 ~
, ~ ~ S ~ ~
~ o
ô . a: 1:
~q i3~ eo ~, ~ ,o '
:~ ~ o _i _i o ~ ~ o
. _ g P. ~3 '.
P~ ~ ,~
+ ~ ~ ~t`1` ~ 0 c~
~ :~ 0 ~, ~
u~ ~n ,I r ~1 g ~
~, . . . o CD X ~ ~ a~
~ ~D co m a~ . . . o .C t~ .
:r: ~ O ~ ~ ~ `.`:
o ~ ~
m o ~ ~ o h
~q ~ O ~ tn
+ z ~ a~
. ,C
. .
c~ u~
,~ ~ . . ,.
. ~ ~3 ~ ,~ o o 3 o
_ U 1~ ~n ~o
l Z ~ ~ q co u~ co ~1 ~ O
$ ~ .
~ ¢U h ~ - ~:
0 N 1~
t~ ~ 1 I 1
_ a~ ,
o ~ V ~ U .~ ~o o o
. ~ --~ tO S ~ _ H U ~: ~
~ ,~ x t~
~ O ~
O o 1~ rl 1~ ~1 --I O ~ h ~ ~2. ;~ o
O t) C 5~ C H H E-l m ~ m
R :Z; ~X X E~ n ~ - N "~
" ` , .................. " ' ` :
2~&~
W092/03561 ~CT/~91/002
-38-
The apr B. amyloliquefaciens ~-amylase embodiment of
the invention, described herein, demonstrates several
important advantages of the promoters, vectors, and
; methods of the present invention. First, the addition
of enhancer receptors to the ~-amylase promoter
substantially increases production of cloned protein
up to 20-fold. For example, production of ~-amylase
was 20-fold higher than that already present in the
wild-type cell. The increase in ~-amylase
transcription seen above might be due to the action of
wild-type sacU gene. This is a very useful
construction, because no additional protease activity
is induced and production of foreign proteins is thus
. unaffected.
~. Constructs providing the target module designated
i as receptor A were equivalent to constructs providing
`~ the target module designated as receptor B with any of
sacQ, sacQHy or sacUHy enhancer genes when such target
-`. module was positioned as in the 302 constructs
(compare pKTH 1911 and pKTX 1975 in Table 1). This
equivalency was also found wi1:h the sacUHy enhancer
gene when the targets were positioned as in the 1601
constructs (compare pKTH 1601 and pKTH 1910, last
column in Table 1).
constructs providing the target module designated
as receptor A were more ef~ective at promoting ~-
amylase synthesis than constructs providing target
module B when positioned as in the pKTH 1601 construct
~or enhancer genes sacQ and sacQHy (compare pKTH 1910
and pKTH 1974 in Table 1).
Second, the enhancement of the wild-type ~-
amylase promoter o~ B. amyloliquefaciens (unlike the B.
:, .
~ . , . '
.~
,,
. . .
: `, : , : . : .. .:, , .. :: ,-:
'.: , ''''' ''' . ,:',' , , :
~' . ' , ' :: '
2~3~)2~
WOg2/035~1 PCT/~91/002
39
. , .
subtills ~-amylase promo~er)is very good with sacQ
overexpression or with modified sacU protein (sacUHy).
There is no response to overexpressed pr~R. The
expression of wild-type ~-amylase with sacQ (pUB110) of -
sacUHy approaches the saturation level (compared with
production with multicopy plasmid pKTHlO). However,
with sa~Q (pUBllO) combined with the ~-amylase/receptor
A construct, even higher production (greater than 60-
fold) can be demonstrated.
Third,-the use of p~tR (pUBllO) gives the expected
three-fold increase with the enhancer receptor B.
Similar enha~cement with prtR has been dem~nstrated
with the apr promoter (~. Bacteri~l. 169: 3044-3059
(1987))~
All references cited herein are incorporated
herein by reference. While this invention has been
described in detail and with reference to specific
embodiments thereof, it will be apparent to one ` :
skilled in the art that various changes and ~
modifications could be made therein without departing .:
from the spirit and 6cope thereof.
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,
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., ~ . .
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: :. . : . :
::
