WO1991002803A1 - A method of suppressing chromosomal gene expression - Google Patents
A method of suppressing chromosomal gene expression Download PDFInfo
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- WO1991002803A1 WO1991002803A1 PCT/US1990/004713 US9004713W WO9102803A1 WO 1991002803 A1 WO1991002803 A1 WO 1991002803A1 US 9004713 W US9004713 W US 9004713W WO 9102803 A1 WO9102803 A1 WO 9102803A1
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- 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
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- 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
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- This invention relates to a method of suppressing the expression of a chromosomal gene in Bacillus by transforming a Bacillus host strain with a multicopy plasmid containing a DNA fragment which suppresses chromosomally encoded protease expression.
- the invention also relates to multicopy plasmids used for the transformation.
- proteases are usually produced. Very often at least one of these different proteases is an interfering protease in that it may not have the desired characteristics such as optimum pH, temperature stability, etc, and it may hydrolyze one or more of the other proteases or extracellular proteins produced and excreted by the cell. Interfering protease production is a particularly acute problem in the commercial production of proteases produced by genetically engineered bacteria such as proteases produced for use in laundry detergents.
- chromosomally encoded protease is produced and excreted by bacteria in addition to that produced * from the expression of plasmid genes which encode for a particularly desirable protease.
- These chromosomally expressed proteases often reduce the yield of the desired protease either through competition for transcription, translation, and/or secretion, or by hydrolyzing the plasmid-encoded protease. It would be advantagous to have a way of repressing or inhibiting chromosomal encoded protease expression in such instances. It would be particularly advantageous to suppress chromosomal gene expression in industrial bacterial strains because no such method exists other than gene inactivation or deletion.
- the present invention provides a method of inhibiting the expression of a chromosomal protease gene comprising transforming a Bacillus host with an multicopy plasmid containing a DNA fragment which suppresses chromosomally encoded protease expression.
- the present invention also provides multicopy plasmids capable of replicating in Bacillus which comprise a DNA fragment comprising a functional gene linked to the carboxyl terminal portion of the Bacillus licheniformis ATCC 53926 protease gene.
- Figure 1 is a restriction map of plasmid pC50.
- Figure 2 is a restriction map of plasmid pKLl.
- Figure 3 is a restriction map of plasmid pKL2
- Figure 4 is a restriction map of plasmid pK07
- Figure 5 is a restriction map of plasmid pKL2/SS.
- Figure 6 is a schematic representation of the various deletions in the B. licheniformis ATCC 53926 gene.
- Figure 7 is a schematic diagram for the construction of the plasmids pC50, pKLl, pKL2, pK07, and pKL2/SS.
- Figure 8. is a restriction map of plasmid pH70B.
- Figure 9. is a restriction map of plasmid pH70AMY Figure 10. is a restriction map of plasmid pC51AMY DESCRIPTION OF THE PREFERRED EMBODIMENTS Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about".
- the present invention provides a general method of inhibiting the expression of a chromosomally encoded protease gene in Bacilli comprising transforming a Bacillus host with an multicopy plasmid containing a DNA fragment which suppresses chromosomally encoded protease expression.
- the DNA fragment which suppresses the chromosomally encoded protease expression is the carboxyl terminal portion of the functional protease gene.
- the carboxyl terminal portion of a protease gene is defined as approximately the distal third of the gene. In the case of B. licheniformis ATCC 53926 the distal third is a DNA sequence between approximately the Sail and approximately the Sstl restriction sites.
- Multicopy plasmids which are useful in the method of the present invention are those which contain DNA sequences which comprise the carboxyl terminal portion of the chromosomally encoded protease gene.
- the amount of the interfering protease can be substantially reduced by transforming the host organism with a plasmid which contains a DNA fragment comprising the carboxyl terminal portion of the chromosomally encoded interfering protease gene.
- the presence of the approximately distal third of the gene in the plasmid suppresses a large percentage of the chromosomally encoded protease production.
- the production protease can be different from the chromosomally encoded protease.
- the production protease can be a Bacillus lentus alkaline protease produced by a transformed Bacillus 1icheniformis mutant strain as is disclosed in a copending U.S. patent application.
- a multicopy plasmid which encodes for the Bacillus lentus alkaline protease comprised of a DNA fragment which contains in the direction of transcription a Bacillus lentus alkaline protease functional gene linked to the Sall/Sstl restriction fragment of the B. licheniformis ATCC 53926 protease gene.
- the production enzyme can be something other than a protease.
- the production enzyme can be an ct-amylase produced by a transformed Bacillus licheniformis ATCC 53926 strain.
- a multicopy plasmid comprised of a DNA fragment which contains in the direction of transcription a functional gene which encodes for an cc-amylase is linked to the Sall/Sstl restriction fragment of the B. licheniformis ATCC 53926 protease gene.
- the method of the present invention can be used in any Bacillus strain it is preferred that the strain be Bacillus licheniformis. It is particularly preferred that the strain be mutant Bacillus licheniformis strains identified by the depository numbers ATCC 10716, ATCC 14580, and ATCC 53926, and DSM 641.
- the method of the present invention can be used in the production of any type of enzyme, it is preferred that the enzyme be a protease.
- Plasmid pK07 ( Figure 4) a derivative of plasmid pKLl ( Figure 2) is constructed. Plasmid pKLl is digested with Stul and Sail, and after subsequent phenolization and precipitation, treated with Klenow polymerase under the appropriate buffer conditions. This DNA is ligated with T4 ligase to form plasmid pK07. Plasmid pK07 is deleted for the proximal 2/3 of the structural protease gene including part of the promoter region. B. licheniformis ATCC 53926 transformed by plasmid pK07 produces only about 10% of the protease produced by the wild type strain.
- Plasmid pC50 was then digested with Aval and after phenolization and ethanol precipitation is religated with T4 ligase to give plasmid pKLl.
- Plasmid pKLl was digested with Aval and Stul and treated with Klenow polymerase in the presence of all four deoxyribonucletides in order to fill in the sticky ends generated by Aval. The reaction mixture was again treated with phenol and the DNA precipitated with ethanol, and religated with T4 ligase to give plasmid pKL2 ( Figure 3) .
- the distal third of plasmid pKL2 was deleted by digesting it with Sacl first and then with Sail under the appropriate buffers conditions as recommended by the manufacturer. After phenolization and ethanol precipitation, the digested DNA was redisolved in the appropriate buffer and Klenow polymerase added for conversion of the non-compatible protruding 3 1 (Sacl) and 5 1 (Sail) ends to blunt ends. After another phenolization and ethanol precipitation step, the DNA was redisolved in a buffer appropriate for the ligation reaction and T4 DNA ligase was added for the connection of the blunt ends of the DNA-fragments to give plasmid pKL2/SS ( Figure 5) .
- Table 1 The data for the relative protease production from the various deletion constructs appears in Table 1. Table 1
- the versatility of the method of the present invention can be shown in another preferred embodiment wherein chromosomal DNA expression is suppressed in a strain transformed to contain multiple copies of an ⁇ -amylase gene.
- B. licheniformis ATCC 53926 transformed by a plasmid containing a protease gene which has been inactivated by an ⁇ -amylase gene produces only about 20% of the protease produced by the wild type strain.
- the plasmid ( Figure 10) contains a structural amylase gene under the control of a B. licheniformis ATCC 53926 alkaline protease promoter and the Sail/Sstl restriction fragment of the B. licheniformis ATCC 53926 protease gene.
- the construction of the plasmid is described in Examples 6 and 7.
- plasmid DNAs except for pK07 were first transformed into B. subtilis using protoplast regeneration technique (Chang, S. & Cohen, S.N. (1979) Molec. Gen. Genet. 168.:111-115.) . After verifying the validity of the constructs, plasmid DNAs were purified using CsCl gradients. The plasmids were then transformed into B. licheniformis ATCC 53926. Plasmid pK07 is transformed diretly into B. licheniformis ATCC 53926 using the protoplast regenerative technique. Transformants were selected on tetracycline-containing plates and the desired constructs were identified by restriction analysis of mini lysate DNA with suitable restriction enzymes.
- Transformants were cultured in shake flasks at 39°C and the amount of alkaline protease activity produced was determined according to Norix, A., Bechet, J. J. & Roncons, C. (1970) Biochem. Biophys. Res. Commun. 4_1: 464 using N-CBZ-valin-p- nitrophenylester as substrate.
- Plasmid pC50 was digested with Aval and -after phenolization and ethanol precipitation- religated with T4 ligase.
- the ligation mixture was transformed into competent cells of B.subtilis SB202 (Marmur, J. , Seaman, E.S. & Levine, J. (1963) J. Bacteriol. 85:461-467). Selection for transformants was performed on nutrient plates containing 15 micrograms tetracycline/ml.
- Mini lysate plasmid DNA of transformants was isolated and restricted with EcoRl in order to identify the desired deletion construct missing the approximately 400 bp Aval fragment of pC50. For transformation of pKLl into B.
- licheniformis ATCC 53926 plasmid DNA was isolated on a large scale and purified using a CsCl-gradient. The purified DNA was then transformed into B. licheniformis ATCC 53926 protoplasts and the desired transformants selected on DM3-agar with 15 micrograms tetracycline and identified as described above for the transformation into B.subtilis.
- Example 2
- Plasmid pKL2 pKLl was digested with Aval and Stul. Restriction enzymes were removed by phenol treatment and subsequent ethanol precipitation. The digestion mixture was then treated with Klenow polymerase in the presence of all 4 deoxyribonucleotides in order to fill in the sticky ends generated by Aval. The reaction mixture was again treated with phenol and the DNA precipitated with ethanol. Ligation, transformation into B.subtilis SB202 and identification of transformants containing pKL2 ( Figure 3) was performed in the same way as described in example 1 for the construction of pKLl. Transformation of pKL2 into B. licheniformis ATCC 53926 also followed the procedure described in example 1.
- pKLl was digested with Stul and Sail. Restriction enzymes were removed by phenol treatment and subsequent ethanol precipitation. The digestion mixture was treated with Klenow polymerase in the presence of all 4 deoxyribonucleotides in order to fill in the sticky ends generated by Sail. The reaction mixture was again treated with phenol and the DNA precipitated with ethanol. After ligation with T4 ligase, the reaction mixture was transformed into protoplasts of B. licheniformis ATCC 53926. Transformants were selected by growth on plates with DM3-agar with 15 micrograms tetracycline. The desired deletion construct was identified by restriction analysis of mini lysate DNA with EcoRl as described for pKLl in example 1. Example 4
- Plasmid pKL2 was digested with Sacl first and then with Sail under the appropriate buffers conditions as recommended by the manufacturer. After phenolization and ethanol precipitation, the digested DNA was redisolved in the appropriate buffer and Klenow polymerase added for conversion of the non-compatible protruding 3' (Sacl) and 5" (Sail) ends to blunt ends.
- Example 5 Expression of subtilisin in Derivatives of B.licheniformis ATCC 53926.
- B. licheniformis ATCC 53926 containing the deletion constructs pKLl, pKL2, pK07 or pKL2/SS respectively was cultured in shake flasks in the presence of 7.5 g tetracycline/ml in a complex medium suitable for protease expression (2.4 g/1 KH 2 P0 4 , 1 g/1 MgS0 4 x 7H 2 0, 0.5 g/1 MnS0 4 x 2H 2 0, 0.2 g/1 CaCl 2 x 2H 2 0, 3 g/1 soybean flour, 12 g/1 casein ⁇ Ha marsten ⁇ , 120 g/1 amylase-treated potatoe starch) .
- protease assay was performed using N-CBZ-valin-p-nitrophenylester as substrate and the rate of increase in absorbance at 340nm due to release of p-nitrophenol by the action of protease was determined.
- introduction of pC50 or pKLl into B. licheniformis ATCC 53926 led to overproduction of B. licheniformis ATCC 53926 protease as compared to B. licheniformis ATCC 53926 without plasmid
- productivity of the deletion constructs pKL2 and pK07 was less than 10% relative to the B. licheniformis ATCC 53926 parent.
- Table 1 Example 6 Construction of pH70 AMY
- the plasmid pJ06 was restricted with Bell, which cuts between the ribosome binding site and the translational start of the ⁇ -amylase gene, and at a second site located downstream of the structural ⁇ -amylase gene.
- the plasmid pH70B was restricted with Bell also, but under partial conditions. This plasmid is a derivative of pH70 and contains a Bell site subcloned from pMG56. Bell-cut pJ06 and pH70B were phenolized, ethanol precipitated, and ligated by the action of T4-DNA ligase.
- Competent cells of B. subtilis BD393 were transformed with the ligation mixture and selected on Kanamycin containing plates (10 microgram/ml) , with an overlay of 1% cornstarch. Transformants which produced clearing zones in the overlay were expected to contain the amylase gene cloned into pH70B. These transformants were verified by analysis of restriction digests of plasmid DNA prepared by a mini-lysate procedure. Those that contained the right construct were identified (amylase gene cloned downstream of the protease promoter, and under its control with the remainder of the protease gene downstream of the transcription putative transcription terminator of the amylase gene) . This construct was designated pH70 AMY. Plasmid-DNA was prepared from B. subtilis BD393 pH70 AMY.
- B. licheniformis ATCC 53926 containing the deletion construct PC51 AMY was cultured in shake flasks in a complex medium containing 7.5 ug tetracycline/ml.
- Plasmid pC51 AMY which carries the B. licheniformis ATCC 53926 alkaline protease gene inactivated by insertion of the promoterless ⁇ -amylase gene, caused a suppression of the protease of B. licheniformis ATCC 53926 to a level of 20% of the level characteristic of the wild type strain.
- Plasmid pJ06 is described in German Patent Application 3824827. Plasmid pC50 is described in U.S. patent application Serial Number 06/892,158 filed on July 30, 1986.
- Plasmid pH70B is a derivative of plasmid pH70 (DSM 5479) . Plasmid pMG56 was cut with EcoRl and then with BamHI. pH70 was cut with BamHI and with Ecol under partial cleavage conditions. Both digested plasmids were phenolized, mixed together, and precipitated with ethanol. After ligation with T4 DNA ligase, competant cells of Bacillus subtilis SB202 were transformed and selected on Kanamycin with 10 microgram/ml. Transformants were screened by Bell digests of mini lysate DNA. Clones exhibiting Bell site between the ribosome binding site and the translational start site were designated pH70B.
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Abstract
A method of inhibiting the expression of a chromosomal protease gene comprising transforming Bacillus host with a multicopy plasmid containing a DNA sequence comprising in the direction of transcription a functional gene operably linked to the carboxyl terminal portion of the protease gene.
Description
A METHOD OF SUPPRESSING CHROMOSOMAL GENE EXPRESSION BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method of suppressing the expression of a chromosomal gene in Bacillus by transforming a Bacillus host strain with a multicopy plasmid containing a DNA fragment which suppresses chromosomally encoded protease expression. The invention also relates to multicopy plasmids used for the transformation.
2. Description of the Related Art. One of the problems associated with the use of Bacillus as extracellular enzyme producers lies in the fact that a number of different proteases are usually produced. Very often at least one of these different proteases is an interfering protease in that it may not have the desired characteristics such as optimum pH, temperature stability, etc, and it may hydrolyze one or more of the other proteases or extracellular proteins produced and excreted by the cell. Interfering protease production is a particularly acute problem in the commercial production of proteases produced by genetically engineered bacteria such as proteases produced for use in laundry detergents. In these cases, chromosomally encoded protease is produced and excreted by bacteria in addition to that produced*from the expression of plasmid genes which encode for a
particularly desirable protease. These chromosomally expressed proteases often reduce the yield of the desired protease either through competition for transcription, translation, and/or secretion, or by hydrolyzing the plasmid-encoded protease. It would be advantagous to have a way of repressing or inhibiting chromosomal encoded protease expression in such instances. It would be particularly advantageous to suppress chromosomal gene expression in industrial bacterial strains because no such method exists other than gene inactivation or deletion. Attempts to utilize these methods in classical industrial Bacillus strains has not met with much success. There is a method that selectively enhances the expression of a selected gene disclosed in U.S. patent No. 4,792,523. The patent does not disclose any way of repressing gene expression.
SUMMARY OF THE INVENTION The present invention provides a method of inhibiting the expression of a chromosomal protease gene comprising transforming a Bacillus host with an multicopy plasmid containing a DNA fragment which suppresses chromosomally encoded protease expression. The present invention also provides multicopy plasmids capable of replicating in Bacillus which comprise a DNA fragment comprising a functional gene linked to the carboxyl terminal portion of the Bacillus licheniformis ATCC 53926 protease gene.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a restriction map of plasmid pC50. Figure 2 is a restriction map of plasmid pKLl. Figure 3 is a restriction map of plasmid pKL2 Figure 4 is a restriction map of plasmid pK07 Figure 5 is a restriction map of plasmid pKL2/SS. Figure 6 is a schematic representation of the various deletions in the B. licheniformis ATCC 53926 gene. Figure 7 is a schematic diagram for the construction of the plasmids pC50, pKLl, pKL2, pK07, and pKL2/SS. Figure 8. is a restriction map of plasmid pH70B.
Figure 9. is a restriction map of plasmid pH70AMY Figure 10. is a restriction map of plasmid pC51AMY DESCRIPTION OF THE PREFERRED EMBODIMENTS Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about".
The present invention provides a general method of inhibiting the expression of a chromosomally encoded protease gene in Bacilli comprising transforming a Bacillus host with an multicopy plasmid containing a DNA fragment which suppresses chromosomally encoded protease expression. The DNA fragment which suppresses the chromosomally encoded protease expression is the carboxyl terminal portion of the functional protease gene. The carboxyl terminal portion of a protease gene is defined as approximately the distal third of the gene. In the case of B. licheniformis ATCC 53926 the distal third is a DNA sequence between approximately the Sail and approximately the Sstl restriction sites.
Multicopy plasmids which are useful in the method of the present invention are those which contain DNA sequences which comprise the carboxyl terminal portion of the chromosomally encoded protease gene. For example, if a host organsism produces a chromosomally encoded interfering protease along with a production protease, the amount of the interfering protease can be substantially reduced by transforming the host organism with a plasmid which contains a DNA fragment comprising the carboxyl terminal portion of the chromosomally encoded interfering protease gene. The presence of the approximately distal third of the gene in the plasmid suppresses a large percentage of the chromosomally encoded protease production. The production protease can be different from the chromosomally encoded protease. For example, the production protease can be a Bacillus lentus alkaline
protease produced by a transformed Bacillus 1icheniformis mutant strain as is disclosed in a copending U.S. patent application. In this case, a multicopy plasmid which encodes for the Bacillus lentus alkaline protease comprised of a DNA fragment which contains in the direction of transcription a Bacillus lentus alkaline protease functional gene linked to the Sall/Sstl restriction fragment of the B. licheniformis ATCC 53926 protease gene. The production enzyme can be something other than a protease. For example, the production enzyme can be an ct-amylase produced by a transformed Bacillus licheniformis ATCC 53926 strain. In this case, a multicopy plasmid comprised of a DNA fragment which contains in the direction of transcription a functional gene which encodes for an cc-amylase is linked to the Sall/Sstl restriction fragment of the B. licheniformis ATCC 53926 protease gene.
While the method of the present invention can be used in any Bacillus strain it is preferred that the strain be Bacillus licheniformis. It is particularly preferred that the strain be mutant Bacillus licheniformis strains identified by the depository numbers ATCC 10716, ATCC 14580, and ATCC 53926, and DSM 641.
While the method of the present invention can be used in the production of any type of enzyme, it is preferred that the enzyme be a protease.
In a preferred embodiment, plasmid pK07 (Figure 4) a derivative of plasmid pKLl (Figure 2) is constructed. Plasmid pKLl is digested with Stul and Sail, and after subsequent phenolization and precipitation, treated with Klenow polymerase under the appropriate buffer conditions. This DNA is ligated with T4 ligase to form plasmid pK07. Plasmid pK07 is deleted for the proximal 2/3 of the structural protease gene including part of the promoter region. B. licheniformis ATCC 53926 transformed by plasmid pK07 produces only about 10% of the protease produced by the wild type strain.
In order to show that the distal third of the
functional gene suppresses chromosomally encoded protease production, various deletion constructs (Figures 6 & 7) were prepared and their relative protease production was compared. A plasmid containing the structural B. licheniformis ATCC 53926 gene in which the distal third of the gene has been deleted was constructed (Figure 7) . First, the alkaline protease gene from B. licheniformis ATCC 53926 was cloned into the vector pBC16 which encodes resistance to tetracycline (J. Bacteriol. 133;897-903, (1978)). The resulting construct was plasmid pC50 (Figure 1) which has been described in U.S. patent application Serial Number 06/892,158, filing date 7/30/86. Plasmid pC50 was then digested with Aval and after phenolization and ethanol precipitation is religated with T4 ligase to give plasmid pKLl. Plasmid pKLl was digested with Aval and Stul and treated with Klenow polymerase in the presence of all four deoxyribonucletides in order to fill in the sticky ends generated by Aval. The reaction mixture was again treated with phenol and the DNA precipitated with ethanol, and religated with T4 ligase to give plasmid pKL2 (Figure 3) . The distal third of plasmid pKL2 was deleted by digesting it with Sacl first and then with Sail under the appropriate buffers conditions as recommended by the manufacturer. After phenolization and ethanol precipitation, the digested DNA was redisolved in the appropriate buffer and Klenow polymerase added for conversion of the non-compatible protruding 31 (Sacl) and 51 (Sail) ends to blunt ends. After another phenolization and ethanol precipitation step, the DNA was redisolved in a buffer appropriate for the ligation reaction and T4 DNA ligase was added for the connection of the blunt ends of the DNA-fragments to give plasmid pKL2/SS (Figure 5) . The data for the relative protease production from the various deletion constructs appears in Table 1.
Table 1
STRAIN RELATIVE PROTEASE PRODUCTION
ATCC 53926 100%
ATCC 53926/pKL2 < 10%
ATCC 53926/pK07 < 10% ATCC 53926/pKL2/SS ca. 65%
ATCC 53926/pBC16 60-80%
The versatility of the method of the present invention can be shown in another preferred embodiment wherein chromosomal DNA expression is suppressed in a strain transformed to contain multiple copies of an α-amylase gene. Specifically, B. licheniformis ATCC 53926 transformed by a plasmid containing a protease gene which has been inactivated by an α-amylase gene produces only about 20% of the protease produced by the wild type strain. The plasmid (Figure 10) contains a structural amylase gene under the control of a B. licheniformis ATCC 53926 alkaline protease promoter and the Sail/Sstl restriction fragment of the B. licheniformis ATCC 53926 protease gene. The construction of the plasmid is described in Examples 6 and 7.
All of the above plasmid DNAs except for pK07 were first transformed into B. subtilis using protoplast regeneration technique (Chang, S. & Cohen, S.N. (1979) Molec. Gen. Genet. 168.:111-115.) . After verifying the validity of the constructs, plasmid DNAs were purified using CsCl gradients. The plasmids were then transformed into B. licheniformis ATCC 53926. Plasmid pK07 is transformed diretly into B. licheniformis ATCC 53926 using the protoplast regenerative technique. Transformants were selected on tetracycline-containing plates and the desired constructs were identified by restriction analysis of mini lysate DNA with suitable restriction enzymes. Transformants were cultured in shake flasks at 39°C and the amount of alkaline protease activity produced was determined according to Dupaix, A., Bechet, J. J. & Roncons, C. (1970)
Biochem. Biophys. Res. Commun. 4_1: 464 using N-CBZ-valin-p- nitrophenylester as substrate.
The following examples are meant to illustrate but not limit the invention. Example l
Construction of Plasmid pKLl
Plasmid pC50 was digested with Aval and -after phenolization and ethanol precipitation- religated with T4 ligase. The ligation mixture was transformed into competent cells of B.subtilis SB202 (Marmur, J. , Seaman, E.S. & Levine, J. (1963) J. Bacteriol. 85:461-467). Selection for transformants was performed on nutrient plates containing 15 micrograms tetracycline/ml. Mini lysate plasmid DNA of transformants was isolated and restricted with EcoRl in order to identify the desired deletion construct missing the approximately 400 bp Aval fragment of pC50. For transformation of pKLl into B. licheniformis ATCC 53926 plasmid DNA was isolated on a large scale and purified using a CsCl-gradient. The purified DNA was then transformed into B. licheniformis ATCC 53926 protoplasts and the desired transformants selected on DM3-agar with 15 micrograms tetracycline and identified as described above for the transformation into B.subtilis. Example 2
Construction of Plasmid pKL2 pKLl was digested with Aval and Stul. Restriction enzymes were removed by phenol treatment and subsequent ethanol precipitation. The digestion mixture was then treated with Klenow polymerase in the presence of all 4 deoxyribonucleotides in order to fill in the sticky ends generated by Aval. The reaction mixture was again treated with phenol and the DNA precipitated with ethanol. Ligation, transformation into B.subtilis SB202 and identification of transformants containing pKL2 (Figure 3) was performed in the same way as described in example 1 for the construction of pKLl. Transformation of pKL2 into B.
licheniformis ATCC 53926 also followed the procedure described in example 1.
Example 3 Construction of pK07
For the construction of pK07 (Fig.4) pKLl was digested with Stul and Sail. Restriction enzymes were removed by phenol treatment and subsequent ethanol precipitation. The digestion mixture was treated with Klenow polymerase in the presence of all 4 deoxyribonucleotides in order to fill in the sticky ends generated by Sail. The reaction mixture was again treated with phenol and the DNA precipitated with ethanol. After ligation with T4 ligase, the reaction mixture was transformed into protoplasts of B. licheniformis ATCC 53926. Transformants were selected by growth on plates with DM3-agar with 15 micrograms tetracycline. The desired deletion construct was identified by restriction analysis of mini lysate DNA with EcoRl as described for pKLl in example 1. Example 4
Construction of pKL2/SS
The plasmid pKL2, which was deleted for an upstream portion of the alkaline protease gene including part of the putative promoter, was used to construct a second deletion in the distal third of the structural gene between restriction sites Sail and Sacl. Plasmid pKL2 was digested with Sacl first and then with Sail under the appropriate buffers conditions as recommended by the manufacturer. After phenolization and ethanol precipitation, the digested DNA was redisolved in the appropriate buffer and Klenow polymerase added for conversion of the non-compatible protruding 3' (Sacl) and 5" (Sail) ends to blunt ends. After another phenolization and ethanol precipitation step, the DNA was redisolved in a buffer appropriate for the ligation reaction and T4 DNA ligase was added for the connection of the blunt ends of the DNA-fragments. Protoplasts of Bacillus subtilis BC92 were transformed with
the ligation mixture and transformants were selected on DM3 regeneration agar with 15 micrograms/ml tetracycline and identified by restriction analysis of mini lysates. The construct was designated pKL2SS. For transformation of pKL2/SS into B. licheniformis ATCC 53926, plasmid DNA was isolated on a large scale and purified using a CsCl- gradient. The purified DNA was then transformed into B. licheniformis ATCC 53926 protoplasts and the desired transformants selected on DM3-agar with 15 micrograms tetracycline and identified as described above for the transformation into B.subtilis.
Example 5 Expression of subtilisin in Derivatives of B.licheniformis ATCC 53926. B. licheniformis ATCC 53926 containing the deletion constructs pKLl, pKL2, pK07 or pKL2/SS respectively was cultured in shake flasks in the presence of 7.5 g tetracycline/ml in a complex medium suitable for protease expression (2.4 g/1 KH2P04, 1 g/1 MgS04 x 7H20, 0.5 g/1 MnS04 x 2H20, 0.2 g/1 CaCl2 x 2H20, 3 g/1 soybean flour, 12 g/1 casein {Ha marsten} , 120 g/1 amylase-treated potatoe starch) . At time intervals aliquots were taken for measurement of protease activity. The protease assay was performed using N-CBZ-valin-p-nitrophenylester as substrate and the rate of increase in absorbance at 340nm due to release of p-nitrophenol by the action of protease was determined. Whereas introduction of pC50 or pKLl into B. licheniformis ATCC 53926 led to overproduction of B. licheniformis ATCC 53926 protease as compared to B. licheniformis ATCC 53926 without plasmid, the productivity of the deletion constructs pKL2 and pK07 was less than 10% relative to the B. licheniformis ATCC 53926 parent. Plasmid pKL2/SS missing the distal sequences between the Sail and Sstl sites caused expression of approximately the same level of protease as the plasmid vector without insert DNA (pBC16) . (Table 1)
Example 6 Construction of pH70 AMY The plasmid pJ06 was restricted with Bell, which cuts between the ribosome binding site and the translational start of the α-amylase gene, and at a second site located downstream of the structural α-amylase gene. The plasmid pH70B was restricted with Bell also, but under partial conditions. This plasmid is a derivative of pH70 and contains a Bell site subcloned from pMG56. Bell-cut pJ06 and pH70B were phenolized, ethanol precipitated, and ligated by the action of T4-DNA ligase.
Competent cells of B. subtilis BD393 were transformed with the ligation mixture and selected on Kanamycin containing plates (10 microgram/ml) , with an overlay of 1% cornstarch. Transformants which produced clearing zones in the overlay were expected to contain the amylase gene cloned into pH70B. These transformants were verified by analysis of restriction digests of plasmid DNA prepared by a mini-lysate procedure. Those that contained the right construct were identified (amylase gene cloned downstream of the protease promoter, and under its control with the remainder of the protease gene downstream of the transcription putative transcription terminator of the amylase gene) . This construct was designated pH70 AMY. Plasmid-DNA was prepared from B. subtilis BD393 pH70 AMY.
Example 7 Construction of pC5i AMY Both pH70 Amy and pC51 were cut with BamHI and Sacl. phenolized, ethanol-precipitated and ligated with T4-DNA- ligase. Competent cells of B. subtilis BD393 were transformed with the ligation mixture and transformants selected on tetracycline containing plates (15 micrograms/ml) with a cornstarch overlay. Clones which produced clearing zones were identified, and verified, by restriction analysis of plasmid DNA from mini lysates. The
correct construct was designated as pC51 AMY. pC51 AMY DNA was purified and used to transform protoplasts of B. licheniformis ATCC 53926.
Example 8 Expression of Subtilisin in B. licheniformis ATCC 53926
B. licheniformis ATCC 53926 containing the deletion construct PC51 AMY was cultured in shake flasks in a complex medium containing 7.5 ug tetracycline/ml. In this complex medium, Plasmid pC51 AMY, which carries the B. licheniformis ATCC 53926 alkaline protease gene inactivated by insertion of the promoterless α-amylase gene, caused a suppression of the protease of B. licheniformis ATCC 53926 to a level of 20% of the level characteristic of the wild type strain. Experimental Methods
The following methods mentioned in the text were performed essentially as described by T. Maniatis, E.F. Fritsch & J. Sambrook et al. (1982) , Molecular Cloning, A Laboratory Manual: * phenol treatment
* ethanol precipitation
* Klenow polymerase treatment (fill-in reaction)
* ligation with T4 ligase
Mini lysate DNA was prepared according to Bimboi , H.C. & Doly, J. (1979) Nucl. Acids Res. 7:1513.
Large scale plasmid DNA preparation was accomplished essentially as described by Godson, G.N. & Vapnek, D. (1973) Biochem. Biophys. Acta 299:516.
Protoplast transformation was carried out as described by Chang, S. & Cohen, S.N. (1979) Molec. Gen. Genet. 168:111-115.
Preparation of competent cells and their subsequent transformation followed the protocol of Cahn, F.H. & Fox, M.S. (1968) J. Bacteriol. 95:867-875. Plasmids
Plasmid pJ06 is described in German Patent Application 3824827.
Plasmid pC50 is described in U.S. patent application Serial Number 06/892,158 filed on July 30, 1986.
Plasmid pH70B. Plasmid pH70B is a derivative of plasmid pH70 (DSM 5479) . Plasmid pMG56 was cut with EcoRl and then with BamHI. pH70 was cut with BamHI and with Ecol under partial cleavage conditions. Both digested plasmids were phenolized, mixed together, and precipitated with ethanol. After ligation with T4 DNA ligase, competant cells of Bacillus subtilis SB202 were transformed and selected on Kanamycin with 10 microgram/ml. Transformants were screened by Bell digests of mini lysate DNA. Clones exhibiting Bell site between the ribosome binding site and the translational start site were designated pH70B.
Deposit of Microorganisms A living culture of B. licheniformis ATCC 53926 containing plasmid pK07 has been accepted for deposit in Deutsche Sammlung Von Mikroorgnismen (DSM) , Grisebachstr. 8, 3400 Gottingen, Federal Republic of Germany, under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of patent procedure and has been assigned the identification number DSM 5472.
Claims
1. A method of inhibiting the expression of chromosomal protease gene comprising transforming a Bacillus host with an multicopy plasmid containing a DNA fragment which suppresses chromosomally encoded protease expression.
2. The method of claim 1 wherein said DNA fragment is a sequence between approximately the Sail and Sstl restriction sites of the B. licheniformis ATCC 53926 alkaline protease gene.
3. The method of claim 1 wherein said Bacillus host is Bacillus licheniformis.
4. The method of claim 3 wherein said Bacillus host is Bacillus licheniformis ATCC 10716.
5. The method of claim 3 wherein said Bacillus host is Bacillus licheniformis ATCC 14580.
6. The method of claim 3 wherein said Bacillus host is Bacillus licheniformis ATCC 53926.
7. The method of claim 3 wherein said Bacillus host is Bacillus licheniformis DSM 641.
8. An multicopy plasmid capable of replicating in Bacillus containing a DNA fragment comprising a functional gene linked to the Sail/Sstl restriction fragment of the B. licheniformis ATCC 53926 protease gene..
9. The multicopy plasmid of claim 8 wherein said functional gene is the B. licheniformis ATCC 53926 protease gene.
10. The multicopy plasmid of claim 8 wherein said functional gene is the amylase gene from B. licheniformis ATCC 53926.
11. A multicopy plasmid having the cleavage map substantially as shown in Figure 4.
12. A transformed Bacillus licheniformis host cell comprising a hybrid plasmid of claim 11.
13. A transformed host cell of claim 8 wherein said host cell is Bacillus licheniformis ATCC 10716.
14. A transformed host cell of claim 8 wherein said host cell is Bacillus licheniformis ATCC 14580.
15. A transformed host cell of claim 8 wherein said host cell is Bacillus licheniformis ATCC 53926.
16. A transformed host cell of claim 8 wherein said host cell is Bacillus licheniformis DSM 641.
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Cited By (3)
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WO2003087149A2 (en) * | 2002-04-10 | 2003-10-23 | Novozymes A/S | Bacillus licheniformis mutant host cell |
WO2003093453A3 (en) * | 2002-04-10 | 2004-05-13 | Novozymes As | Bacillus licheniformis mutant host cell |
WO2022229574A1 (en) | 2021-04-30 | 2022-11-03 | IFP Energies Nouvelles | Insertion of multicopies of a gene of interest into the genome of a fungus |
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WO1986001825A1 (en) * | 1984-09-21 | 1986-03-27 | Genex Corporation | Bacillus strains with reduced extracellular protease levels |
EP0214435A2 (en) * | 1985-08-03 | 1987-03-18 | Henkel Kommanditgesellschaft auf Aktien | Alkaline protease, method for the production of hybrid vectors and genetically transformed microorganisms |
FR2604726A1 (en) * | 1986-10-02 | 1988-04-08 | Agency Ind Science Techn | BACILLUS SUBTILIS STRAIN WITH EXTRA-CELL PROTEASE ACTIVITIES, METHOD FOR OBTAINING THE STRAIN, AND METHOD FOR SECRETING PROTEINS USING THE STRAIN |
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Patent Citations (3)
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WO1986001825A1 (en) * | 1984-09-21 | 1986-03-27 | Genex Corporation | Bacillus strains with reduced extracellular protease levels |
EP0214435A2 (en) * | 1985-08-03 | 1987-03-18 | Henkel Kommanditgesellschaft auf Aktien | Alkaline protease, method for the production of hybrid vectors and genetically transformed microorganisms |
FR2604726A1 (en) * | 1986-10-02 | 1988-04-08 | Agency Ind Science Techn | BACILLUS SUBTILIS STRAIN WITH EXTRA-CELL PROTEASE ACTIVITIES, METHOD FOR OBTAINING THE STRAIN, AND METHOD FOR SECRETING PROTEINS USING THE STRAIN |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003087149A2 (en) * | 2002-04-10 | 2003-10-23 | Novozymes A/S | Bacillus licheniformis mutant host cell |
WO2003087149A3 (en) * | 2002-04-10 | 2004-03-18 | Novozymes As | Bacillus licheniformis mutant host cell |
WO2003093453A3 (en) * | 2002-04-10 | 2004-05-13 | Novozymes As | Bacillus licheniformis mutant host cell |
EP1696035A2 (en) * | 2002-04-10 | 2006-08-30 | Novozymes A/S | Improved bacillus host cell |
EP1696035A3 (en) * | 2002-04-10 | 2008-04-02 | Novozymes A/S | Improved bacillus host cell |
EP1995319A1 (en) * | 2002-04-10 | 2008-11-26 | Novozymes A/S | Bacillus licheniformis mutant host cell |
US7521204B2 (en) | 2002-04-10 | 2009-04-21 | Novozymes A/S | Bacillus host cell |
EP2213745A1 (en) * | 2002-04-10 | 2010-08-04 | Novozymes A/S | Improved bacillus host cell |
US8911969B2 (en) | 2002-04-10 | 2014-12-16 | Jens Tonne Andersen | Bacillus host cell |
WO2022229574A1 (en) | 2021-04-30 | 2022-11-03 | IFP Energies Nouvelles | Insertion of multicopies of a gene of interest into the genome of a fungus |
FR3122436A1 (en) | 2021-04-30 | 2022-11-04 | IFP Energies Nouvelles | Multicopy insertion of a gene of interest into the genome of a fungus |
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