CA2086824A1 - Hybrid.alpha.-amylase promoters - Google Patents

Abstract

This invention relates to the identification of highly efficient hybrid promoters for recombinant gene expression in prokaryotes.
The hybrid promoters of the invention provide highly efficient promoters containing (1) an enhancer protein target module and (2) an RNA polymerase initiation module. According to the invention, there is further provided expression vectors providing such hybrid promoters, hosts stably transformed with such expression vectors, and methods for producing the genetically engineered or recombinant protein using such hosts.

Description

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 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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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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3~61 ~ 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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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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_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 : . ,, ' . . ,,., ~ ~

WO92/03561 2 0 ~ PCT/F191/002~

, 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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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 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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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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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.
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; 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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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 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.
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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 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 :

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WO92~03~61 PCr~FI91/002~

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 :. : : - : . .", "
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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 . . . ~: . , :
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2 ~ 4 W092/03561 PCT/~9l/002 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 .~
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 ' ~ -~,:

, . . . . . .
'~: , ' '' . ' ' ',: ' . ` . : : .

2~ h4 W092~03561 PCT/FI91/002 ~ ' 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 : ',~ ' .

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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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WO92/03561 PCTtF191/002 :
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-~, , .

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~V092/03561 PCT/~91/002 , 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 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.

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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 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~

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 , ~.

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203~2ll W~92/03561 PCT/~91/002 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."
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, 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 ~::

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2~!o8 W092J03561 PCT/~91/~2 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 , ~

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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 .'',' ' .
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W092/0356l PCT/~91/002 .
(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.

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W092/03561 ~CT/~91/002 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.
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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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Claims (29)

WHAT IS CLAIMED IS:

1. A hybrid promoter for expression of recombinant genes in a prokaryotic host, wherein said hybrid promoter comprises a target module of an enhancer gene operably linked to an initiation module for RNA polymerase, and wherein said target module is heterologous to said initiation module.

2. The hybrid promoter of claim 1, wherein said target module is a target module found in the 5' regulatory region of the apr gene.

3. The hybrid promoter of claim 2, wherein said apr gene is the 8. subtilis apr gene.

4. The hybrid promoter of claim 1, wherein said target module is a target module of a protein product of an enhancer gene and wherein said enhancer gene is selected from the group consisting of sacQ, prtR, sacV, senN, sacU, and degT.

5. The hybrid promoter of claim 4, wherein said target module is a target module for the sacU enhancer gene.

6. The hybrid promoter of claim 5, wherein said sacU enhancer gene is the B. subtilis sacU gene.

7. The hybrid promoter of claim 4, wherein said target module is a target module for the sacQ enhancer gene.

8. The hybrid promoter of claim 7, wherein said sacQ enhancer is selected from the group consisting of the sacQ enhancer of B. subtilis, B. amyloliquefaciens and B.
licheniformis.

9. The hybrid promoter of claim 4, wherein said target module is a target module for the prtR enhancer gene.

10. The hybrid promoter of claim 9, wherein said prtR enhancer gene is the B. natto or the B. subtilis prtR
enhancer gene.

11. The hybrid promoter of claim 1, wherein said initiation module is the initiation module of a gene which encodes a prokaryote exoenzyme.

12. The hybrid promoter of claim 11, wherein said exoenzyme is .alpha.-amylase.

13. The hybrid promoter of claim 12, wherein said .alpha.-amylase is B. amyloliquefaciens .alpha.-amylase.

14. The hybrid promoter of claim 1, wherein said hybrid promoter is operably linked to a bacterial secretion signal.

15. The hybrid promoter of claim 14, wherein said bacterial secretion signal is a Bacillus secretion signal.

16. The hybrid promoter of claim 14, wherein said secretion signal is an amylase secretion signal.

17. The hybrid promoter of claim 14, wherein said secretion signal is the secretion signal of levansucrase.

18. An expression vector for expression of recombinant genes in a prokaryotic host, wherein said expression vector comprises the hybrid promoter of any one of claims 1-17.

19. The expression vector of claim 18, wherein said vector further comprises a structural gene operably linked to said hybrid promoter.

20. The expression vector of claim 19, wherein said structural gene is .alpha.-amylase.

21. The expression vector of claim 18, wherein said vector further comprises a structural gene encoding said enhancer gene, said enhancer gene not operably linked to said hybrid promoter and wherein said gene encoding said enhancer gene is expressible in said host cell.

22. The expression vector of claim 18, wherein said expression vector further comprises sequences WO 92/0356l PCT/FI91/00244 which promote the integration of said vector into the genome of said host cell.

23. Expression vector pKTH1910.

24. Expression vector pKTH1975.

25. Expression vector pKTH1912.

26. A host cell transformed with the expression vector of claim 18.

27. The host cell of claim 26, wherein said host cell is selected from the group consisting of B.
subtilis, B. licheniformis, B. amyloliquefaciens, B. polymyxa, B. stearothermophilus, B. thermoproteolyticus, 8. coagulans, B.
thuringiensis, B. megaterium, B. cereus, B. natto, B. acido-caldarius, Clostridium perfringens, and c. tetanus.

28. The host cell of claim 27, wherein said host cell is B. subtilis.

29. A method of expressing a protein in a prokaryotic host, wherein said method comprises the steps of:
(1) transforming said host with the expression vector of claim 18, wherein said expression vector comprises a gene providing a desired coding sequence operably linked to said hybrid promoter, and wherein said host cell provides the enhancer gene activity required by said target module on said hybrid promoter; and (2) expressing said protein.