WO1992003561A1 - HYBRID α-AMYLASE PROMOTERS - Google Patents
HYBRID α-AMYLASE PROMOTERS Download PDFInfo
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- WO1992003561A1 WO1992003561A1 PCT/FI1991/000244 FI9100244W WO9203561A1 WO 1992003561 A1 WO1992003561 A1 WO 1992003561A1 FI 9100244 W FI9100244 W FI 9100244W WO 9203561 A1 WO9203561 A1 WO 9203561A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
Definitions
- 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.
- Bacillus strains offer many potential advantages in the production of cloned gene products, as compared with Escherichia coll .
- 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.
- B. subtilis the number of vector systems available 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.
- Enhancer genes directly or indirectly affect the transcription rate of exoenzyme genes. Examples of these genes include: sacQ from B. subtilis , (j. Bacteriol . 116 : 113 (1986)) ; sacQ from B . amyloliquefaciens (J. Bacteriol . 116 : 113 (1986)) ; sacQ from B . licheniformis (J . Bacteriol . 169 : 324 (1987)) ; prtR from B. natto or B. subtilis (J.
- SacU h (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 B. subtilis. (The superscript "h” denotes hyperproduction.) The production of exoenzymes is decreased in sacu ⁇ mutants.
- enhancer genes encode a protein/peptide which directly or indirectly affects the transcription (probably the rate of initiation) of the target gene.
- 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 affect either the enhancer protein's structural gene or the enhancer gene's promoter region (Msadek et al., J. Bacteriol. 172:824-834 (1990) ; Yang et al., J. Bacteriol. 156:113-116 (1986); Biochimie 56:1481 (1974)).
- Bacillus enhancer genes are especially evident with respect to protease and levansucrase gene expression.
- the increase of enzyme production may be as great as 10-100-fold compared to the wild-type cell.
- exoenzy es such as, for example, amylases, phosphatases, and ribonucleases
- the increase is usually no more than 2-3-fold.
- alkaline protease (apr) and levansucrase (lvs) proteins of B . subtilis are the best studied cases.
- target regions for sa ⁇ U, sacQ and ⁇ pr-mutations has been established in the upstream region of the promoter for each of these genes.
- 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.
- 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)) .
- the proposed apr target site of sacU and sacQ mutations contains a DNA sequence very similar to the DNA sequence in the upstream region of the levansucrase-gene (J. Bact . 270:296-300 (1988)).
- the upstream sequence similarity between the apr and lvs genes is shown in Figure one and the complete upstream sequence of apr in Figure two.
- 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 context. It is not known whether a specific position regarding the apr promoter (-35 and -10 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 in that region. It is not known whether these same factors may also effect the enhancement function in a heterologous environment.
- genetic elements of the ⁇ -amylase promoter which provide functional transcriptional modules for RNA polymerase binding and/or initiation.
- enhancer protein target modules that is, genetic elements of prokaryotic promoters which provide functional transcriptional modules necessary for enhancer protein action.
- 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.
- Figure 1 is a comparison of aprE and sac ⁇ upstream regions containing the target for sacU32(Hy) ; and sacQ36 ( ⁇ ) stimulation.
- the sacB sequence is from Henner, D.J. et al . , J. Bacteriol . 170 : 296 (1988) and Shimotsu, H. et al . , J. Bacteriol . 168 : 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. Bacteriol .
- Figure 3 is the DNA and amino acid sequence of the NH 2 region of the B . subtilis ⁇ -a ylase gene.
- the NH 2 -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 cial site and the 5' end of the ⁇ -amylase promoter constructs, upstream of which the new cial 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.
- 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.
- rDNA recombinant DNA
- RNA 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 gene is intended to refer to a gene which encodes a protein which directly or indirectly increases production of another protein.
- genetic sequences is intended to refer to a nucleic acid molecule (preferably DNA) .
- promoter 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 refers to a genetic transcriptional 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., 5' to) the transcriptional start site, although such modules may also be found downstream of (i.e., 3' to) the start site.
- target module 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.
- enhancer gene activity i.e., such as the protein or peptide encoded by an enhancer gene
- initiation module refers to a promoter module which is required to initiate transcription of operably linked genes with RNA polymerase. In prokaryotic promoters, initiation modules are usually located at about -10 and -35 nucleotides from the start site of transcription.
- hybrid promoter is meant a promoter 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.
- 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) .
- 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 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.
- a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
- 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 vec or. Markers, for example, are erythromycin and kanamycin resistance.
- the term “vehicle” is sometimes used for "vector.”
- 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.
- 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 gene to be expressed in a specific host.
- a desired structural gene 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 termination 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.
- a desired enhancer gene such enhancer gene would be the enhancer gene which encodes the protein which regulates the target module of the hybrid promoter.
- 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 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 “fragments,” “variants,” or “chemical derivatives” of a molecule.
- a molecule is said to be a "chemical 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 Pharmaceutical Sciences (1980) . Procedures for coupling such moieties to a molecule are well known in the art.
- 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.
- variants 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 fragment 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, tertiary, or quaternary structure of 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.
- 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.
- 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 B . subtilis apr promoter.
- the resulting promoter becomes highly responsive to hyperexpression and secretion in response to enhancer gene action or to mutations in such enhancer genes.
- 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 transcriptional promoter modules.
- Genetic sequences which are capable of providing promoter modules may be derived from genomic DNA, synthetic DNA, cloned DNA and combinations thereof.
- the preferred species source of the promoter modules of the invention is Bacillus, although any source may be used if the function of the module is preserved in the transformed host cell.
- 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 confers recognition of the sacU h , 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.
- hybrid promoters can be designed for any B . subtilis enhancer proteins in any manner which reveals the target activity.
- 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 which respond to the enhancer genes sacQ (especially sacQ from B . subtilis, B .
- amyloliquefaciens or B . licheni- formis prtR (especially prtR from B. natto or B . subtilis) , sacV (especially sacV from B . subtilis) , senH (especially senN from B. natto or B . subtilis) , sacU (especially sacU from B. subtilis) and degT (especially degT from B. stearothermophilus) may be designed in this manner.
- hybrid promoters can be designed which contain only one target module or which contain more than one target module operably 1inked together.
- Larger enhancer target regions such as, for example, the B region of the apr gene, carry target modules for several enhancer gene products.
- a hybrid 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.
- PCR polymerase chain reaction
- Prokaryote genomic DNA containing protein en ⁇ coding sequences will not contain introns, although it may contain spacers between transcriptional units.
- Eukaryote genomic DNA containing protein encoding eukaryotic sequences may or may not include naturally occurring introns.
- introns generally must be removed prior to cloning. Otherwise, either prokaryote or eukaryote encoding sequences may be expressed in the hosts of the invention.
- genomic DNA may be obtained in association with the 3* transcriptional termination regio .
- 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.
- 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-translated regions of the mRNA, may be retained and employed for transcriptional and translational regulation.
- 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.
- genomic DNA can be extracted and purified from any cell which expresses the protein of interest.
- genomic DNA can be performed by means well known in the art (for example, see Guide to Molecular Cloning Techniques , S.L. Berger et al . , eds. , Academic Press (1987)).
- 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 of interest and used to produce cDNA by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger et al., eds., Academic Press (1987)).
- 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 vitro, by techniques commonly used to enrich mRNA preparations for specific sequences, such as sucrose gradient centrifugation, or both.
- 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, 1982) , 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 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.
- a clone to a desired protein sequence 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 b) 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 desired transcriptional regulatory 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 desired 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 be on the left end whereas the carboxy terminus is intended to be at the right end.
- the genetic code is degenerate, more than one codon may be used to encode a particular amino acid (Watson, J.D. , In: Molecular Biology of the Gene , 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), pp. 356-357) .
- the peptide fragments are analyzed to identify 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.
- an amino acid sequence may be encoded by only a single oligonucleotide sequence
- 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.
- 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.
- 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 encoding 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. Molec.
- 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 identify 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 .
- the above-described DNA probe may be labeled with a detectable group.
- 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 32 P, 3 H, 14 C, 35 S, 125 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. Mol . Bioi . 113 : 231 (1977) and by T4 DNA polymerase replacement synthesis as described in, for example, Deen, K.C., et al . , Anal . Biochem. 135:456 (1983).
- 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.
- a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
- B . subtilis strain IH6064 may be used as a host.
- B. subtilis strain IH6064 is available frcm the Central Public Health Institute (CPHI), Helsinki, Finland.
- Strain IH6064 was constructed by transforming BGSC strain 1A289 (aroI906, metB5, sacA321, amyE) with DNA isolated from strain BGSC strain 1A46 (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 (arol marker) are selected and then screened for ⁇ -amylase positive phenotype.
- arol and amyE markers are known to be linked markers and therefore arol selection always yields a high percentage of amy + transformants.
- This transformation resulted in strain IH6064 (metB5 sacA321) .
- IH6064 metalB5 sacA321
- subtilis Marburg strain such as those available from the BGSC
- 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.
- the actual identification of peptide sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
- an oligonucleotide complementary to this theoretical sequence or by constructing a set of oligonucleotides complementary to the set of "most probable" oligonucleotides
- a DNA mole ⁇ cule or set of DNA molecules, capable of functioning as a probe( ⁇ ) for the identification and isolation of clones containing a desired protein or DNA regulatory element.
- 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.
- transcriptional regulat ⁇ ory elements it is 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.
- Cloned protein encoding sequences obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and especially, operably linked to the hybrid promoters of the inven- tion. Such sequences may be introduced into a host cell to produce recombinant protein or a functional derivative thereof.
- 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.
- a Gram-positive bacterium is used as the host cell, such as, for example, a Bacillus or Clostridium perfringens, or C. tetanus .
- a member of the species Bacillus is used as a host cell.
- Such members include B . subtilis, B . lichen ⁇ formis , B . amyloliquefaciens, B . polymyxa, B . stearothermophilus , B . thermoproteolyticiis, B.
- the host cell is B . subtilis.
- a nucleic acid molecule such as DNA, is said to be "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.
- 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.
- 5' non-transcribing control sequences will include a region which contains the hybrid promoter of the invention for transcriptional control of the operably linked gene.
- prokaryo- tic hosts require 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.
- these regulatory signals are associated with a particular gene which is capable of a high level of expression in the host cell.
- a fusion product of the desired protein may be constructed.
- 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.
- signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable to subsequent removal.
- 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.
- sequences functional in the host cell may be substituted.
- 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.
- a vector which functionally inserts itself into the host chromosome.
- such vector may provide a DNA sequence element which promotes integration of DNA sequences in chromosomes.
- such DNA sequence element is 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 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 sequences to be expressed, or introduced into the same cell by co-transformation.
- a transformed sequence may also be incorporated into a plasmid or other vector capable of autonomous 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 useful when it is desired to cytoplasmically express a recombinant protein rather than secrete it.
- Factors of importance in selecting a particular vector include: 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.
- 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, recipient 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.
- 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 useful in the expression of heterologous or homologous genes which are operably linked to the hybrid promo ⁇ ters of the invention. Such proteins can be expressed either intracellularly or extracellularly in a Bacillus host.
- Plasmid pUBllO (Gryczan, T.J., et al . , J. Bacteriol . 134 : 318 (1978)) is freely available from the Bacillus Genetic Stock Center (BGSC) , The Ohio State University, Department of Biochemistry, 484 West 12th Avenue, Columbus, Ohio 43210, USA (strain 1E6) , and is fully described, with a restriction map, in the BGSC's Strains & Data: Fourth Edition (1989).
- BGSC Bacillus Genetic Stock Center
- new cial cloning sites were constructed upstream of the -35 region of the ⁇ - amylase promoter (downstream of the original ciaJ 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 of a ciaJ site at a required position upstream of the -35 region, and the 3' end was a Hindlll site within the structural part of the ⁇ - amylase gene.
- These 5 » end-truncated clai-Hindlll ⁇ - amylase fragments were used to replace the original wild type cial-Hindlll fragment of pKTH 1601.
- the new constructs were designated "302,” "303" and "304.”
- 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.
- DNA sequences that potentially could act as enhancer receptors, when inserted in the ⁇ -amylase promoter were derived from the alkaline protease gene (apr) of B . subtilis .
- Two sequences were used.
- the first sequence is a 48-bp fragment ( Figure 1) , suggested to be a sacQ and sacU receptor (J. Bact. 170 : 296-300 (1988)). It was made by oligonucleotide synthesis and flanked by ciai sites. This oligonucleotide was designated "receptor A.”
- the second sequence consisted of a -300 bp region upstream of the promoter (underlined in Figure 2) . The fragment was made by PCR from the chromosome of B .
- Receptor A was inserted as a single copy fragment in the ciaJ 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,” respectively.
- the receptor B was similarly joined to the cial site of plasmids pKTH 1601 and 302, transformed into E. coli DH5 ⁇ , characterized by restriction enzyme analysis and DNA sequencing, and designed "pKTH 1974" and "pKTH 1975,” respectively.
- the wild type B . subtilis ⁇ -a ylase gene (from pKTH 1601) and the modified ⁇ -amylase (1910-1913 and 1974-5) genes were integrated in the chromosome of B . subtilis . It is not necessary to use pKTH 1601 as the source of the wild- type B . anyloiiquefaciens ⁇ -a ylase gene, and any strain of B. amyloliquefaciens which does not contain a mutated ⁇ -amylase gene may be used.
- 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 . subtilis cells with cm- selection (5 ⁇ g/ml) . This resulted in single crossing over, single copy, ⁇ -amylase positive, chromosomal integrates. To ensure that no ⁇ -amylase amplification took place, no cm-selection was applied after the primary transformation event.
- the B. subtilis strains carrying the integrated genes were then made competent for the addition of enhancer clones or mutations.
- 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 pKTH1743, which is a pUBllO derivative carrying a multilinker.
- a plasmid identical to pKTH1743 for the purposes of this invention may be constructed by replacing the ⁇ >vuII-£. ⁇ oRI fragment of pUBllO with the multilinker region of commercially available pUCl ⁇ .
- chromosomal mutation sacQ36Hy BGSCIA53
- sacUHy pap-9 from B . subtilis YY88
- Hosts providing the sacUHy mutations are available from the BGSC (for example, strains 1A95 (sacU(H)32), 1A165 (sacU(H)32), 1A159 (sacU(H)25), 1A199 (sacU(H)200) , and 1A200 (sac ⁇ (H)lOO) ) .
- the sacQ-pUBHO and prtR-pUBHO clones were directly transformed to competent B . subtilis strains carrying the integrated ⁇ -amylase genes by kanamycin (km) selection.
- the sacQHy and sacUHy mutations were transferred to the integration strains by congression (Molecular Biology of Bacillus, vol. I, Academic Press 1982, pp. 147-178) .
- DNA was isolated from B. subtilis strains carrying the above mutations and mixed with plasmid pE194 (strain 1E18 from the BGSC) .
- Chromosomal DNA and pE194 DNA were transformed together in competent B. subtilis by selecting the erythromycin resistance (em) marker of pE194 at permissive temperature (32°C) .
- the transformants were screened on skim milk plates for increased protease production which indicated the presence of either sacUHy or sacQHy mutations.
- elevated temperature 37°C
- the carrier plasmid was lost as monitored by the loss of em-marker.
- the ⁇ -amylase specific mRNA was assayed essentially by the method of Thomas (Proc. Nati . 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) method according to the manufacturer's instructions.
- the amounts of amylase specific RNA and the ⁇ - amylase activities are shown in Table 1.
- DNA construct no enhancer +sacQHy +prtR (pUBllO) +sacUHy mRNA 1 ⁇ -amy £ mRNA ⁇ -amy mRNA ⁇ -amy mRNA ⁇ -amy pKTH 1601 1 .8 16.2 26.5 0.9 0.7 18.9 29.5 pKTH 1910 16.4 1.3 48.5 48.5 4.2 1.3 15.6 28.5 pKTH 1974 9.0 1.8 43.0 35.1 7.2 1.7 15.9 28.9 pKTH 1911 4.7 .9 19.5 29.3 3.4 0.8 18.5 26.7 pKTH 1975 6.7 1.6 21.2 33.1 7.6 2.7 16.1 23.4 pKTH 1912 2 .9 19.3 29.8 7.7 1.8 28.5 38.8 pKTH 1913 2 1.5 0.8 2.2 0.8 2.3 3.2 B.S IH6064 3 B.S IH6064 [pKTHIO] 85.1 2.7
- B. amyloliquefaciens ⁇ -amylase specific mRNA The amount of mRNA produced by B. subtilis IH6064 carrying the construct pKTH1601 is taken as 1.
- the apr B. amyloliquefaciens ⁇ -amylase embodiment of the invention demonstrates several important advantages of the promoters, vectors, and methods of the present invention.
- the addition of enhancer receptors to the ⁇ -amylase promoter substantially increases production of cloned protein up to 20-fold.
- 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 as receptor A were equivalent to constructs providing the target module designated as receptor B with any of sacQ, sa ⁇ QHy or sacUHy enhancer genes when such target module was positioned as in the 302 constructs (compare pKTH 1911 and pKTH 1975 in Table 1) . This equivalency was also found with 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 effective at promoting ⁇ - amylase synthesis than constructs providing target module B when positioned as in the pKTH 1601 construct for enhancer genes sacQ and sacQHy (compare pKTH 1910 and pKTH 1974 in Table 1) .
- prtR gives the expected three-fold increase with the enhancer receptor B. Similar enhancement with prtR has been demonstrated with the apr promoter (J. Bacteriol . 265:3044-3050 (1987)) .
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FI930531A FI930531A0 (en) | 1990-08-13 | 1993-02-08 | HYBRID A-AMYLASPROMOTORER |
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US56584790A | 1990-08-13 | 1990-08-13 | |
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JP (1) | JPH06500689A (en) |
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CN111808834A (en) * | 2020-07-12 | 2020-10-23 | 广东溢多利生物科技股份有限公司 | Method for efficiently expressing high-temperature-resistant alpha-amylase in bacillus subtilis, recombinant promoter and application |
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JPWO2010082619A1 (en) | 2009-01-16 | 2012-07-05 | 大塚化学株式会社 | High expression promoter and gene product production method using the same |
CN114181858B (en) * | 2021-12-09 | 2023-10-10 | 禾丰食品股份有限公司 | Functional feed additive for preventing and treating necrotic enteritis of birds and preparation method thereof |
Citations (3)
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EP0130074A1 (en) * | 1983-06-27 | 1985-01-02 | Genentech, Inc. | Portable inducible control system, expression vectors containing them, microorganisms transformed with them, and their use in expressing exogenous protein |
EP0227260A1 (en) * | 1985-10-25 | 1987-07-01 | Omnigene Inc | Enhancing the expression of genes in microorganisms |
EP0349353A1 (en) * | 1988-03-22 | 1990-01-03 | Institut Pasteur | DNA sequences comprising the bacillus subtilis functional part of locus sacU and sacUh, vectors containing these sequences and their use in processes for the production of proteins |
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- 1991-08-12 CA CA 2086824 patent/CA2086824A1/en not_active Abandoned
- 1991-08-12 EP EP19910914179 patent/EP0544707A1/en not_active Withdrawn
- 1991-08-12 AU AU83125/91A patent/AU8312591A/en not_active Abandoned
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EP0130074A1 (en) * | 1983-06-27 | 1985-01-02 | Genentech, Inc. | Portable inducible control system, expression vectors containing them, microorganisms transformed with them, and their use in expressing exogenous protein |
EP0227260A1 (en) * | 1985-10-25 | 1987-07-01 | Omnigene Inc | Enhancing the expression of genes in microorganisms |
EP0349353A1 (en) * | 1988-03-22 | 1990-01-03 | Institut Pasteur | DNA sequences comprising the bacillus subtilis functional part of locus sacU and sacUh, vectors containing these sequences and their use in processes for the production of proteins |
Non-Patent Citations (5)
Title |
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GENE, Vol. 46, No. 2/3, 1986, Amsterdam, NL., ZUKOWSKI M.M. & MILLER L., "Hyperproduction of an Intracellular Heterologous Protein in a SacUh Mutant of Bacillus Subtilis", pages 247-255. * |
GENE, Vol. 83, No. 2, 30 November 1989, Amsterdam, NL., WONG S.-L., "Development of an Inducible and Enhancible Expression and Secretion System in Bacillus Subtilis", pages 215-223. * |
JOURNAL OF BACTERIOLOGY, Vol. 169, No. 1, January 1987, AMERICAN SOCIETY FOR MICROBIOLOGY, AMORY A. et al., "Characterization of the sacQ Genes from Bacillus Licheniformis and Bacillus Subtilis", pages 324-333. * |
JOURNAL OF BACTERIOLOGY, Vol. 169, No. 7, July 1987, AMERICAN SOCIETY FOR MICROBIOLOGY, TANAKA T. et al., "prtR Enhances the mRNA Level of the Bacillus Subtilis Extracellular Proteases", pages 3044-3050. * |
JOURNAL OF BACTERIOLOGY, Vol. 170, No. 1, January 1988, AMERICAN SOCIETY FOR MICROBIOLOGY, HENNER D. et al., "Location of the Targets of the hpr-97, sacU32(Hy) and sacQ36(Hy) Mutations in Upstream Regions of the Subtilisin Promoter", pages 296-300. * |
Cited By (2)
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CN111808834A (en) * | 2020-07-12 | 2020-10-23 | 广东溢多利生物科技股份有限公司 | Method for efficiently expressing high-temperature-resistant alpha-amylase in bacillus subtilis, recombinant promoter and application |
CN111808834B (en) * | 2020-07-12 | 2023-08-04 | 广东溢多利生物科技股份有限公司 | Method for efficiently expressing high-temperature resistant alpha-amylase in bacillus subtilis, recombinant promoter and application |
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JPH06500689A (en) | 1994-01-27 |
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CA2086824A1 (en) | 1992-02-14 |
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