JP4834900B2 - Promoter, vector having the same, recombinant microorganism, and protein production method - Google Patents

Description

【図面の簡単な説明】
【図１】本発明のプロモータを含む、ＰＬＤ生産用のＤＮＡの説明図である。
【図２】上記プロモータを含むベクターの説明図である。
【図３】上記ベクターを作成するための他の前駆体の説明図である。
【図４】比較のための、上記プロモーターを省いた、ＰＬＤ生産用のＤＮＡの説明図である。
【図５】上記ＰＬＤ生産用のＤＮＡを含むベクターの説明図である。
【図６】図２に示すベクターを組み込んだ、本発明の組換え微生物による、グルコース無添加における、ＰＬＤの生産能を示すグラフである。
【図７】図２に示すベクターを組み込んだ、本発明の組換え微生物による、グルコース濃度 5 g/Lにおける、ＰＬＤの生産能を示すグラフである。
【図８】図２に示すベクターを組み込んだ、本発明の組換え微生物による、グルコース濃度 15 g/L における、ＰＬＤの生産能を示すグラフである。
【図９】図２に示すベクターを組み込んだ、本発明の組換え微生物による、グルコース濃度 30 g/L における、ＰＬＤの生産能を示すグラフである。
【図１０】図２に示すベクターを組み込んだ、本発明の組換え微生物による、グルコース濃度 15 g/L における培養において、安定期での培養液中のＰＬＤを示すSDS-PAGEの図面代用写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a promoter capable of efficiently producing a protein of an enzyme such as phospholipase D (hereinafter referred to as PLD), a vector using the promoter, a recombinant microorganism incorporating the vector, and the recombination. The present invention relates to a method for producing a protein using a microorganism.
[0002]
[Prior art]
Since PLD has high transesterification activity, it can be applied to various useful phospholipid synthesis reactions. In order to broaden its application range, PLD is expected to be industrialized, that is, inexpensive due to mass production. ing.
[0003]
Due to the usefulness of PLD, screening of microorganisms that produce PLD in large quantities has been extensively performed. As a result of the screening, it is currently known that only the actinomycete group secretes and produces a large amount of PLD.
[0004]
Among these actinomycetes, Streptoverticillium cinnamoneum (IFO12852) (Streptoverticillium cinnamoneum, hereinafter abbreviated as Stv. Cinnamoneum (IFO12852)) has been found to be the strain with the highest secreted PLD activity at present. (Nakajima et al., Biotechnol. Bioeng., Vol. 44, 1193-1198 (1994)).
[0005]
The above IFO number is a catalog number of a microorganism that can be obtained from the Fermentation Research Institute (Osaka City), which is a domestic depositary organization for microorganisms. In addition, about Streptomycesium, on classification, it may be classified into Streptomyces (Streptomyces).
[0006]
In addition, for the wild strain Stv. Cinnamoneum, attempts have been made to maximize the expression activity of PLD by variously changing the medium composition and culture conditions using a medium based on meat extract. The maximum expression activity was about 2000 units / liter (hereinafter abbreviated as U / L). In this specification, L in the unit is 1000 cm unless otherwise specified. ^Three It shall be shown.
[0007]
In addition, when TSB medium with less contaminating protein, which is easy to purify the obtained PLD, was used, the expression activity of PLD in the wild strain Stv. Cinnamoneum was even lower, staying at around 1500 U / L. It was.
[0008]
[Problems to be solved by the invention]
However, in the above-mentioned conventional method, since the content ratio of PLD secreted into the medium is still low, the obtained PLD is expensive due to the time required to purify PLD from the medium and the production cost of PLD increases. Therefore, there is a problem that the applicable range of PLD is limited.
[0009]
[Means for Solving the Problems]
In order to solve the above problems, the promoter of the present invention is a base sequence derived from DNA of Streptoverticillium cinnamonium, and a protein base sequence (mat peptide) for protein production such as PLD It is characterized in that it has an activity to improve the protein production ability.
[0010]
The promoter has a base sequence from base number 38 to base number 1407 in the sequence listing of SEQ ID NO: 1, or a base in which one or several base sequences have been deleted, substituted or added in the base sequence It may be composed of a sequence, and may be bound to the upstream side of a protein base sequence for protein production and have an activity of improving the protein production ability.
[0011]
According to the above configuration, the ability to produce proteins such as PLD can be improved by having the above-described base sequence derived from DNA of Streptobacillus cinnamonium.
[0012]
In order to solve the above problems, the vector of the present invention is characterized by having the above promoter and a protein base sequence for protein production on the downstream side of the promoter.
[0013]
In the above vector, the protein base sequence may be used for PLD production, or may be derived from DNA of Streptobacillus cinnamonium.
[0014]
Furthermore, in the base sequence for protein, the base sequence from base number 1512 to base number 3030 in the sequence listing of SEQ ID NO: 1, or one or several base sequences in the base sequence are deleted, substituted or added A base sequence having protein-producing ability may be used.
[0015]
According to said structure, by having the said promoter, protein-producing ability by the base sequence for proteins, such as for the production | generation of phospholipase D derived from DNA of streptobacillus cinnamonium, for example can be improved. It becomes possible.
[0016]
The vector preferably has a palindromic sequence (terminator) for stopping protein translation downstream of the protein base sequence. According to the said structure, protein production ability, such as PLD, can be improved.
[0017]
In order to solve the above-mentioned problems, the recombinant microorganism of the present invention is characterized in that any of the vectors described above is incorporated into a host. In the above-mentioned recombinant microorganism, the host may be actinomycetes or Streptomyces lividans.
[0018]
In the above-described recombinant microorganism, the ability to produce a protein along the protein base sequence incorporated in the vector is improved by the incorporated promoter, and the protein is efficiently and highly purified in the medium. Can be produced.
[0019]
In order to solve the above problems, the method for producing a protein of the present invention comprises culturing the recombinant microorganism according to any of the above in a culture solution to produce a protein based on a protein base sequence. It is a feature. In the said manufacturing method, it is preferable to add glucose to a culture solution.
[0020]
According to the above method, since the promoter is incorporated into the vector, a protein such as PLD can be efficiently secreted and produced in the medium with high purity.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
The embodiment of the present invention will be described with reference to FIGS. 1 to 10 as follows.
[0022]
(1) Extraction of DNA (genome) from actinomycetes
DNA (for PLD generation) extraction from actinomycetes, “Genetic Manupulation of Streptomyces” (Hopwood, DA, Bibb, MJ, Chater, KF, Kieser, T., Bruton, CJ) , Kieser, HM, Lydiate, DJ, Smith, CP, Ward, JM, and Schremph, H. (1985) "Genetic Manupulation of Streptomyces: a Laboratory Manual", the John Innes Foundation, Norwich) .
[0023]
Actinomycetes (Stv. Cinnamoneum (IFO12852)) were cultured with shaking in sterile 100 mL Tryptic Soy Broth (hereinafter referred to as TSB medium) at a temperature of 30 ° C. for 48 hours. During the shaking culture, a stainless coil having a diameter of about 2-3 cm was added to the medium. This suppressed the formation of floc-like cells during shaking culture.
[0024]
The TSB medium includes pancreatic digest of casein 17.0 g / L, papaic digest of soybean meal 3.0 g / L, dextrose 2.5 g / L, sodium chloride 5.0 g / L, dipotassium phosphate 2.5 g / L manufactured by Difco. A thing was used. In this specification, sterilization is to kill microorganisms contained in a solution such as a medium by exposure at 121 ° C. for 20 minutes under saturated steam.
[0025]
After completion of the culture, the obtained cells were collected and washed three times with TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA sterilized by autoclave). After washing, suspend microbial cells in 5 mL of TEbuffer and add lysozyme and achromopeptidase to 2 mg / mL and 4 mg / mL, respectively, to promote lysis and liquid at 30 ° C. The mixture was kept warm until the viscosity became high (about 30 minutes).
[0026]
Thereafter, pronase, EDTA, and SDS were further added to the lysis solution, and the mixture was incubated at 37 ° C. for 2 hours. After the incubation, the chromosomal DNA of the actinomycetes used was extracted from the lysis solution by phenol / chloroform extraction method and polyethylene glycerol precipitation method with a glass rod. Finally, the extracted chromosomal DNA was subjected to RNase treatment, and the chromosomal DNA (genome) extraction operation was completed. In the present specification, a gene relating to the generation of PLD is referred to as a pld gene.
[0027]
Next, determination of the DNA sequences of the pld gene promoter region and the balindrom region will be described.
[0028]
After the chromosomal DNA (genome) taken out above is cleaved with the restriction enzyme Sau 3A1, the fragmented fragment DNAs are ligated to the cloning vector pUC8 and transformed into Escherichia coli Nova Blue (Novagen). A library was built.
[0029]
As a result of amino acid sequence analysis of PLD, about 25 amino acid sequences on the amino acid terminal side were confirmed, a DNA probe corresponding to the amino acid sequence was prepared, and the pld gene was identified by colony hybridization. As a result, two types of fragment DNA partially containing the pld gene were obtained.
[0030]
One is a fragment DNA containing a base sequence on the PLD amino acid terminal side and about 1500 bases (bases) upstream thereof, and the other is a base sequence on the terminal side of the PLD carboxyl group and about 1000 bases (bases) downstream thereof. It was a fragment DNA. The base sequence of each fragment DNA was analyzed by the Sanger method, and a map (base sequence) of the pld gene was constructed based on the overlapping base sequence.
[0031]
As shown in the sequence table, it was confirmed from the analyzed base sequences that a palindromic sequence having a function of physically terminating protein translation exists downstream of the pld gene.
[0032]
In addition, as described later, from the high secretory expression activity level of PLD in actinomycetes incorporating the pld gene, it was assumed that a strong expression promoter sequence was present upstream of the pld gene. A region of about 1000 bases (base) of the base sequence was set as the promoter region. The promoter region is from base number 38 to base number 1407 in the sequence listing of SEQ ID NO: 1.
[0033]
(2) Construction of pUC702-promoter-pld plasmid
A pld secretion expression vector (pUC702-promoter-pld) for actinomycetes having a pld expression cassette was prepared by the following procedure (see FIGS. 1 to 3 and the sequence listing). The pld expression cassette refers to the promoter region (promoter and ribosome binding site) upstream of the pld gene, the pld gene (consisting of a signal sequence (sig peptide) and mature pld gene (mat peptide)), and the downstream of the pld gene A palindrome array.
[0034]
The ribosome binding site is from base number 1396 to base number 1403 in the sequence listing of SEQ ID NO: 1. The signal sequence is from base number 1408 to base number 1509 in the sequence listing of SEQ ID NO: 1. The mature pld gene is a portion corresponding to the PLD sequence after secretion that does not include a signal sequence, and is from base number 1510 to base number 3030 in the sequence listing of SEQ ID NO: 1. The palindromic sequence is from base number 3031 to base number 3213 in the sequence listing of SEQ ID NO: 1.
[0035]
In the sequence listing of SEQ ID NO: 2, amino acid numbers -34 to amino acid number -1 indicate a signal sequence. In the sequence listing of SEQ ID NO: 2, amino acid numbers 1 to 505 represent the amino acid sequence of PLD.
[0036]
In constructing the plasmid, gene manipulation was basically performed according to the Molecular cloning method. The restriction enzyme used was New England Biolabs, and the DNA ligation kit ver2 (Takara Shuzo) was used for ligation.
[0037]
1 to 3 show procedures for constructing a plasmid. First, PCR (Polymerase Chain (amplification) Reaction) method is used to obtain a pld expression cassette consisting of the pld gene promoter region derived from the actinomycete Stv. Cinnamoneum (IFO12852), the pld gene, and the palindromic sequence downstream of the pld gene. Amplified by In general, since actinomycetes have a high GC content, PCR amplification was carried out by dividing them into a promoter region and a pld gene-palindromic sequence.
[0038]
At that time, primers were designed so that TTG (leucine), which is the initiation codon in the signal sequence portion, becomes ATG (methionine). As PCR amplification conditions, the temperature and time of heat denaturation, annealing, and extension reaction were set to 94 ° C., 60 ° C., 68 ° C. for 15 seconds, 30 seconds, and 7 minutes, respectively, and this amplification was performed for 30 cycles.
[0039]
As a template, the chromosome of Stv. Cinnamoneum was used. For amplification, use KOD-plus-DNA-Polymerase (manufactured by Toyobo). In order to increase the amplification efficiency of PCR amplification, GC-Melt (manufactured by Clonetech) so that the final concentration in the reaction system is 10%. Was added.
[0040]
As a primer, 5′-GGGTACCACGTCATGGCGGGTCTCTCTCGTCCG-3 ′ (S-PLD-38-Kpn I) and 5′-TCTGCATGCTGCATCCTTAAACGAAGTAACGATTCCGCG-3 ′ (AS-Pro-Sph I) are used for amplification of the promoter region, and the pld gene -For amplification of palindromic sequences, 5'-ACAGCATGCTCCGCCACCGGCTCCGCCGTTTACACCGCT-3 '(S-Sig-Sph I) and 5'-GGTAAGCTTGGTACCATTTCCTCGCTGGTCGGTTCGGGGCCAGCGCAT-3' (AS-Hin dIII-Kpn I-3213) were used.
[0041]
The promoter region fragment amplified by the PCR method was cleaved with restriction enzyme Kpn I and restriction enzyme Sph I, ligated with pUC18 cleaved with the same enzyme, and then transformed into E. coli Nova Blue to obtain plasmid pUC18-promoter. .
[0042]
On the other hand, the pld gene-palindromic sequence fragment was cleaved with the restriction enzyme Sph I and Hin dIII sites, ligated with pUC19 cleaved with the same enzyme, and then transformed into E. coli Nova Blue to obtain plasmid pUC19-pld. It was.
[0043]
The gene sequences of plasmid pUC18-promoter and plasmid pUC19-pld were confirmed by a DNA sequencer (PE-biosystem gene analyzer 310).
[0044]
Next, as shown in FIG. 2, a pld gene-palindromic sequence fragment was excised from the pUC19-pld plasmid with restriction enzymes Sph I and Hin dIII, and pUC18-promoter cleaved with restriction enzymes Sph I and Hin dIII and After ligation, E. coli Nova Blue was transformed to obtain a plasmid pUC18-promoter-pld having a pld expression cassette consisting of promoter region-pld gene-palindromic sequence.
[0045]
On the other hand, as shown in FIG. 3, a shuttle vector pUC702 was prepared. pUC702 is an Escherichia coli vector pUC19 and an actinomycete vector pIJ702 linked at the Sac I and Kpn I sites, and was prepared according to the method described in the following report (title: Molnar et al., J. Ferment. Bioeng., 72 , 368-372 (1991)).
[0046]
Subsequently, a fragment containing the pld expression cassette was excised from the pUC18-promoter-pld plasmid with the restriction enzyme Kpn I, ligated with the shuttle vector pUC702, which was also cleaved with the restriction enzyme Kpn I, and transformed into E. coli Nova Blue. pUC702-promoter-pld was obtained. Whether or not the expression cassette of this plasmid pUC702-promoter-pld was inserted in the correct orientation was confirmed by a DNA sequencer.
[0047]
(3) Construction of pUC702-mature pld plasmid
A pld gene expression vector derived from actinomycetes (Stv. Cinnamoneum (IFO12852)) not having the above promoter region was constructed as shown in FIGS. 3 to 5 using a shuttle vector pUC702 as a comparative example.
[0048]
First, as shown in FIG. 4, the pld gene was amplified by a two-step PCR method. As conditions for the first PCR, the temperature and time of thermal denaturation, annealing, and extension reaction were, for example, 98 ° C., 52 ° C., and 72 ° C. for 45 seconds, 45 seconds, and 4 minutes, respectively, and this amplification was performed for 25 cycles. As a template, the chromosome of Stv. Cinnamoneum was used. For amplification, Pyrobest-DNA-polymerase (Takara Shuzo) was used, and GC-Melt (Clonetech) was added to the reaction system so that the final concentration was 10%.
[0049]
As the primers for amplification, 5′-CCCGGGAGCTGATAGCTTCTCCGCGTTGATCTTCC-3 ′ (Genome-S) and 5′-CCATGATTACGAATTCCCGGGGATCTTGGT-3 ′ (Genome2-AS) were used. The conditions for Second PCR were the product after First PCR as a template, and 5′-TCGGAATTCGAGGTACCATGCTCCGCCACCGGCTCCGC-3 ′ (Eco-Kpn-Sig-FW-PLD) and 5′-GCAGGTACCCCCCCTTGGCCGCGATTCCCG-3 ′ as primers for amplification. (Kpn-Palind-RV-PLD) was used. Other amplification conditions are the same as those for First PCR.
[0050]
Subsequently, after PCR, the amplified fragment (including signal sequence-mature pld gene-palindromic sequence) was cleaved with restriction enzyme Kpn I and ligated with pUC19 cleaved with the same enzyme, as shown in FIG. E. coli Nova Blue was transformed to obtain plasmid pUC19-pld. The sequence of the pld gene of this plasmid was confirmed with a DNA sequencer.
[0051]
The pUC19-pld plasmid was cleaved with KpnI to recover a fragment (fragment DNA) containing a signal sequence-mature pld gene-palindromic sequence. After ligation with the shuttle vector pUC702 shown in FIG. 3 cleaved with the same enzyme, the plasmid pUC702-pld was obtained by transformation into E. coli Nova Blue. Whether or not the pld gene portion of the pUC702-pld plasmid was inserted in the correct orientation was confirmed by a DNA sequencer.
[0052]
(4) Preparation of Actinomyces protoplasts
Preparation of actinomycete protoplasts and transformation described later were carried out according to the description of “Genetic Manupulation of Streptomyces” described above. Streptomyces lividans (Streptomyces lividans 1326 strain, hereinafter abbreviated as S. lividans) was used as actinomycetes. S. lividans was inoculated into a sterilized 5 mL TSB medium, and seed culture was performed at 30 ° C. for 2 to 3 days.
[0053]
Subsequently, 150 mL of YEME medium and the aforementioned stainless steel coil were placed in the Sakaguchi flask, 1 mL of the seed culture solution was inoculated, and main culture was performed at 30 ° C. for 36 to 40 hours. The above YEME medium contains Difco yeast extract 3 g / L, Difco bacto-peptone 5 g / L, Difco malt extract 3 g / L, glucose 10 g / L, sucrose 340 g / L, and sterilized in an autoclave. , MgCl ₂ ・ 6H ₂ O (2.5M) 2 mL / L added, glycine (20%) 25 mL / L included.
[0054]
After completion of the culture (before the S. lividans pigment turned red), the cultured medium containing the cells was taken out, and the above medium was centrifuged at 3000 rpm for 10 minutes. The supernatant was removed from the centrifuged medium, and the remaining cells were washed twice with 50 mL of 10.3% sucrose solution.
[0055]
A cell suspension is prepared by adding a lysozyme solution (2 mg / mL in Lbuffer, filter sterilized) to the washed cells until the volume reaches 15 mL. For 30 minutes. The cell suspension was well stirred every time precipitation occurred.
[0056]
Thereafter, 15 mL of Pbuffer was added to the cell suspension and stirred well. Subsequently, the cell suspension was filtered through sterilized cotton wool. The filtrate after filtration was centrifuged at 3000 rpm for 7 minutes.
[0057]
Pbuffer was added to the bacterial cells obtained by removing the supernatant from the centrifuged filtrate. The amount of Pbuffer added is 4-5 × 10 in the measurement with a hemacytometer. ⁹ It adjusted so that it might become / mL. The cell protoplast suspension solution thus prepared was dispensed into a sterilized tube and stored at -70 ° C.
[0058]
L (Lysis) buffer is sucrose (10.3%) 100 mL, TES buffer (5.73%, pH 7.2) 10 mL, K ₂ SO _Four (2.5%) 1 mL, trace element solution 0.2 mL, KH ₂ PO _Four (0.5%) 1 mL, MgCl ₂ ・ 6H ₂ O (2.5M) 0.1 mL, CaCl ₂ (2.5M) 1 mL of distilled water was added to 1 mL, and lysozyme (manufactured by Nacalai) was added to 2 mg / mL immediately before use to the autoclave sterilized solution.
[0059]
P (protoplast) buffer is sucrose 10.3 g, K ₂ SO _Four 0.025 g, MgCl ₂ ・ 6H ₂ O 0.202 g, trace element solution 0.2 mL dissolved in distilled water and autoclaved as 80 mL, separately from this, each autoclaved KH ₂ PO _Four (0.5%) 1 mL, CaCl ₂ ・ 2H ₂ 10 mL of O (3.68%) and 10 mL of TES buffer (Nacalai, TES powder dissolved to 5.73% and adjusted to pH 7.2) were added.
[0060]
trace element solution is ZnCl ₂ 40 mg / L, FeCl _Three ・ 6H ₂ O 200 mg / L, CuCl ₂ ・ 2H ₂ O 10 mg / L, MnCl ₂ ・ 4H ₂ O 10 mg / L, Na ₂ B _Four O ₇ ・ 10H ₂ O 10 mg / L, (NH _Four ) ₆ Mo ₇ O _{twenty four} ・ 4H ₂ Each of them is dissolved in distilled water so as to be 10 mg / L.
[0061]
(5) Transformation of actinomycetes
As a host, S. lividans which is a protoprostrated actinomycete as described above was used. A frozen suspension of the S. lividans protoplast suspension prepared as described above was quickly thawed and centrifuged at 3000 rpm for 7 minutes at room temperature. Subsequently, 20 μL of the plasmid solution described in the above (2) was added, and then 0.5 mL of Tbuffer was added, followed by rapid pipetting.
[0062]
Within 3 minutes after adding Tbuffer, 5 mL of Pbuffer was added, and light centrifugation was performed (3000 rpm, 30 seconds). The supernatant was removed, 0.5 mL of Pbuffer was added to the precipitated residue, and 0.1 mL was applied to the surface of each R2YE plate almost uniformly and kept at 30 ° C.
[0063]
One to two days later, 0.7% soft agar (Nacalai) was added to each applied R2YE plate, with Costreptone (50 mg / mL in DMSO as a stock solution, manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 500 μg / mL. 3 mL each) was applied almost uniformly and kept at 30 ° C. Thereby, the transformation (recombinant microorganism) of the actinomycete S. lividans as a host was completed.
[0064]
This recombinant microorganism is deposited under the name Streptomyces lividans / pUC702-PLD (IFO 16465) with the Fermentation Research Institute (Osaka). Moreover, the plasmid solution described in the above (3) was similarly operated to obtain a transformed actinomycete S. lividans (recombinant microorganism).
[0065]
T (Transformation) buffer is a solution of SEPEG1000 in which 35.8 g PEG1000 is dissolved in 75 mL of distilled water, 25 mL of sucrose (10.3%), 0.2 mL of the aforementioned trace element solution, and K ₂ SO _Four (2.5%) 9.3 mL of autoclaved mixture mixed with 1 mL of CaCl ₂ (5M) 0.2 mL and Tris-maleic acid buffer 0.5 mL are added. The above Tris-maleic acid buffer is prepared by adjusting the pH of a 1 M Tris buffer solution to 8.0 with malic acid.
[0066]
R2YE plates were sterilized by autoclaving 2.2 g of Difco Bacto agar to 100 mL of R2YE medium, and autoclaved separately before the agar solidified. ₂ PO _Four (0.5%) 1 mL, CaCl ₂ ・ 2H ₂ O (5M) 0.4 mL, L-proline (20%) 1.5 mL, NaOH (1 N) 0.7 mL were added, and a predetermined amount was injected into a sterilized petri dish to obtain an R2YE plate. R2YE medium consists of sucrose 103 g, K ₂ SO _Four 0.25 g, MgCl ₂ ・ 6H ₂ O 10.12 g, glucose 10 g, Difco casaminoacids 0.1 g, trace element solution 2 mL, Difco yeast extract 5 g and TES buffer 5.73 g were dissolved in 1 L of distilled water.
[0067]
(6) Culture of recombinant S. lividans (recombinant microorganism)
Recombinant S. lividans obtained by the method described above was cultured using an aeration and stirring type culture apparatus. First, R2YE plates were inoculated into a test tube containing 5 mL of TSB medium supplemented with the above-mentioned Costreptone (final concentration 5 μg / mL), and cultured at 30 ° C. for 2 to 3 days. Further, 100 mL of TSB medium and Tiostreptone (final concentration 5 μg / mL) were added to the Sakaguchi flask, and 1 mL of the seed culture solution of the test tube was inoculated, and further cultured at 30 ° C. for 2 to 3 days. .
[0068]
For the main culture, add 1 L TSB medium and 5 ostreptone (final concentration 5 μg / mL) to the aeration and agitation culture device (Able, 2 L jar fermenter (BMJ-02PI)), and seed culture of Sakaguchi flask 50 mL of the solution was inoculated. Culturing was carried out by controlling the culture temperature to 28 ° C., pH = 7.0, DO (dissolved oxygen concentration) = 2 ppm, and aeration volume 2 vvm.
[0069]
(7) PLD activity measurement
When choline oxidase, peroxidase and phenol are reacted with choline produced when PC (phosphatidylcholine or lecithin) is hydrolyzed by PLD, a red quinone dye is produced. The activity of the PLD was measured by measuring this dye with a spectrophotometer using Cholinesterase B-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). One unit (U) of enzyme activity is defined as liberating 1 μmol of choline per minute.
[0070]
First, a reaction substrate mixed solution is prepared. The mixed composition per tube in the reaction substrate mixed solution is H ₂ O 40 μL, 200 mM Tris-HCl (pH = 7.4) 20 μL, 10 mM CaCl ₂ 10 μL, 1% Triton X-100 10 μL, 50 mg / mL Lecithin Emulsion 10 μL. The above Lecithin Emulsion is a suspension of Lecithin 500 mg obtained from eggs dissolved in 1 mL of diethyl ether and made up to 10 mL with water.
[0071]
In the PLD activity measurement, 90 μL of the above reaction substrate mixed solution was mixed with 10 μL of the PLD enzyme solution, stirred well, and allowed to react at 37 ° C. for 10 minutes. After the reaction, 0.5 mM EDTA was added to stop the reaction. Next, 500 μL of a substrate enzyme agent (choline oxidase, peroxidase, phenol) was added and incubated at 37 ° C. for 5 minutes, and 500 μL of a reaction stop solution was added to stop the reaction.
[0072]
Next, a calibration curve is created. Mix water and cholinesterase B-Test Wako standard solution in 150 μL and 0 μL, 140 μL and 10 μL, 130 μL and 20 μL, 120 μL and 30 μL, respectively, and measure their absorbance (wavelength 505 nm). Each was measured with a photometer. Calibration curves were prepared assuming that the respective absorbance values corresponded to PLD activity with 0 U / L, 500 U / L, 1000 U / L, and 1500 U / L. This calibration curve was used to determine the PLD activity in the sample.
[0073]
(8) Electrophoresis (SDS-PAGE)
In order to confirm the secretory expression of PLD, SDS-PAGE electrophoresis was performed using a 12.5% separation gel. For electrophoresis, 20 μL of medium sample was used. Gel staining was performed using a dye (Coomassie Brilliant Blue). The electrophoresis is performed in the literature (Garfin et al., Methods Enzymol. 182, 425-441 (1990)).
[0074]
(9) Purification of PLD
The PLD secreted and produced in the medium was purified with a cation exchange resin (Macroprep high S (manufactured by Bio-Rad), etc.) at pH 6. Elution was performed by sodium chloride step elution (0.5 M) or linear concentration gradient method (0-0.5 M). Heparin affinity chromatography also showed the same effect.
[0075]
The inventors have cloned a pld gene derived from Stv. Cinnamoneum (IFO 12852) and found a highly efficient process for producing PLD by recombinant microorganisms using various gene expression systems.
[0076]
First, the present inventors previously tried to construct an expression system using E. coli or yeast (Pichia pastoris) as an expression system for PLD using a recombinant vector. In contrast to Stv. Cinnamoneum, only an expression activity level of 1/20 in the case of Escherichia coli and 1/4 in the case of yeast could be confirmed. However, in each of the above cases, since the production amount of PLD as a protein itself could be confirmed in large quantities, in these expression systems, it was inhibited that PLD derived from actinomycetes formed a correct natural three-dimensional structure. Therefore, it was thought that the amount of expression activity was low.
[0077]
Yamane et al. Have also developed a secretory production system in Escherichia coli using PLD derived from Streptomyces antibioticus, but the maximum expression activity was about 3500 U / L (Iwasaki et al.). al., J. Ferment. Bioeng., 79, 417-421 (1995)). Therefore, it was considered that development of a host-vector system for actinomycetes was extremely important for the development of a high secretion production system for actinomycete PLD.
[0078]
Therefore, the present inventors used S. lividans which is a kind of actinomycetes as a host, an expression system incorporating a pld gene derived from Stv. Cinnamoneum (pUC702-pld, shown in FIGS. 3 to 5), and a pld gene. In contrast, an expression system further incorporating a promoter region (pUC702-promoter-pld, shown in FIGS. 1 to 3) was constructed. Each constructed recombinant plasmid was transformed into actinomycetes S. lividans, and each transformed S. lividans was cultured in a test tube.
[0079]
As a result, in the expression system of S.lividans / pUC702-pld, only an expression level equivalent to that of the wild strain Stv. Cinnamoneum could be confirmed, but the expression system of S.lividans / pUC702-promoter-pld ( In the test tube culture, at 20000 U / L, a remarkably large expression level (about 15 times the amount) was confirmed with respect to the wild strain Stv. Cinnamoneum (1300 U / L in the test tube culture).
[0080]
Therefore, the promoter according to the present invention was considered to be extremely strong in the extracellular production of a desired protein such as PLD and extremely effective in the expression of various recombinant proteins including PLD.
[0081]
Then, the examination about the optimization of the culture condition of this expression system S.lividans / pUC702-promoter-pld was performed using the aeration stirring type culture tank. The conditions were as follows: the initial added glucose concentrations in the TSB medium described above were 0 g / l, 5 g / l, 15 g / l, 30 g / l, culture temperature 28 ° C., pH = 7.0, and DO (dissolved). Cultivation was performed by controlling the oxygen concentration to 2 ppm.
[0082]
As shown in FIG. 6, when glucose was not added, the activity of secreted PLD in the medium was about 20000 U / L, but as shown in FIGS. The production amount is increased, and it can be seen that about 30 000 U / L of PLD activity can be secreted and produced in the medium by adding 30 g / l of glucose. This PLD activity is about 20 times higher than the PLD activity of the wild strain Stv. Cinnamoneum (1500 U / L in the same TSB medium), and the PLD activity of the wild strain Stv. Cinnamoneum (the medium is different). In the optimized case, the expression level is about 15 times that of 2000 U / L).
[0083]
However, even when the initial glucose concentration was increased beyond 30 g / l, the amount of bacterial cells increased, but the amount of PLD produced did not increase. This was thought to be due to the effect of catabolite repression due to the presence of excess glucose. The catabolite repression means catabolic product suppression, that is, the synthesis of many enzymes is inhibited by metabolites such as glucose.
[0084]
Further, for the initial glucose concentration of 15 g / l, the purity of PLD secreted and produced in the medium was confirmed by electrophoresis (SDS-PAGE) as shown in FIG. In the CBB dye staining, almost no contaminating protein was observed, and almost PLD could be confirmed as a single band.
[0085]
The amount of PLD protein secreted and produced in this medium was quantified from the SDS-PAGE band and found to be about 75 mg / L. When the activity of PLD was calculated from this quantitative value, it was about 400 U / L. Previously, the present inventors found that the activity of natural PLD purified from Stv. Cinnamoneum was 468 U / L (Ogino et wl., J. Biochem., 125, 263-269 (1999)). From this, the PLD obtained in the present invention had a value very close to that of a natural type PLD.
[0086]
Therefore, in the method for producing PLD (protein) according to the present invention, the PLD in the medium has a high concentration and high purity, and the activity of the obtained PLD is extremely close to that of the natural PLD. From the above, it can be seen that all the problems in various conventional expression systems including Escherichia coli have been solved.
[0087]
In addition, since the PLD production method according to the present invention has high PLD purity secreted into the medium, separation and recovery of PLD can be performed by ion exchange chromatography using a single-stage ion exchange column after removing the cells. Therefore, the PLD can be purified very quickly and inexpensively, and the production cost of the PLD can be reduced.
[0088]
As described above, the PLD expression system derived from actinomycetes (recombinant microorganisms) in which a promoter derived from actinomycetes found by the present inventors is incorporated into a vector, a highly active PLD very close to the natural type is obtained from a medium (that is, bacteria Since it can be mass-produced in a high purity and high concentration form in vitro, it can be seen that it is a very effective PLD expression system.
[0089]
As described above, there are few expression systems capable of secreting and producing recombinant proteins in a medium with high purity, and application to production of various useful proteins other than the PLD is expected. That is, the expression cassette (promoter-signal sequence-palindromic sequence) of the vector (plasmid) according to the present invention includes, for example, secretory production of useful proteins derived from various actinomycetes such as cholesterol oxidase. It was assumed to be extremely effective in secretory production.
[0090]
Moreover, S. lividans / pUC702-promoter-pld which is the recombinant microorganism of the present invention is similar to Stv. Cinnamoneum (Fukuda et al., Biotechnol. Lett., 18 951-956 (1996)), and can be efficiently immobilized on microorganism-retaining particles (BSPs), enabling the continuous production of useful proteins such as PLD using immobilized cells. In addition, the productivity of useful proteins can be dramatically improved, and the cost of the useful proteins can be reduced.
[0091]
【The invention's effect】
The promoter of the present invention, as described above, is a base sequence derived from DNA of Streptobacillus cinnamonium, and is linked to the upstream side of the protein base sequence for protein production to improve the protein production ability. It is the structure which has activity.
[0092]
As described above, the vector of the present invention has the above promoter and a protein base sequence for protein production such as PLD on the downstream side of the promoter.
[0093]
As described above, the recombinant microorganism of the present invention has a configuration in which the vector is incorporated in a host such as actinomycetes.
[0094]
As described above, the method for producing a protein of the present invention is a method for producing a protein based on a protein base sequence by culturing the above-described recombinant microorganism in a culture solution.
[0095]
Therefore, the above-described configuration and method can improve the productivity of proteins such as PLD by using the promoter, and can reduce the cost of the protein by industrialization, thereby expanding the application range. Play.
[0096]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is an explanatory diagram of DNA for PLD production containing a promoter of the present invention.
FIG. 2 is an explanatory diagram of a vector containing the promoter.
FIG. 3 is an explanatory view of another precursor for preparing the vector.
FIG. 4 is an explanatory diagram of DNA for PLD production, omitting the promoter, for comparison.
FIG. 5 is an explanatory diagram of a vector containing DNA for producing the PLD.
6 is a graph showing the ability to produce PLD in the absence of glucose by the recombinant microorganism of the present invention incorporating the vector shown in FIG.
FIG. 7 is a graph showing the ability to produce PLD at a glucose concentration of 5 g / L by the recombinant microorganism of the present invention incorporating the vector shown in FIG.
FIG. 8 is a graph showing the ability to produce PLD at a glucose concentration of 15 g / L by the recombinant microorganism of the present invention incorporating the vector shown in FIG.
FIG. 9 is a graph showing the ability to produce PLD at a glucose concentration of 30 g / L by the recombinant microorganism of the present invention incorporating the vector shown in FIG.
FIG. 10 is a drawing-substituting photograph of SDS-PAGE showing PLD in a culture solution at a stable period in culture at a glucose concentration of 15 g / L by the recombinant microorganism of the present invention incorporating the vector shown in FIG. is there.

Claims (10)

  It consists of a base sequence having the base sequence from base number 38 to base number 1407 in the sequence listing of SEQ ID NO: 1, or one or several base sequences deleted, substituted or added in the above base sequence, A promoter that is bound to the upstream side of a protein base sequence for producing a protein derived from a fungus and has an activity of improving the protein-producing ability.   A vector comprising the promoter according to claim 1 and a protein base sequence for protein production downstream of the promoter.   The vector according to claim 2, wherein the protein base sequence is used for producing phospholipase D.   The vector according to claim 2, wherein the protein base sequence is derived from DNA of Streptobacillus cinnamonium.   The protein base sequence is a protein obtained by deleting, substituting, or adding a base sequence from base number 1512 to base number 3030 in the sequence listing of SEQ ID NO: 1, or one or several base sequences in the base sequence 3. The vector according to claim 2, wherein the vector has a production ability.   6. The vector according to any one of claims 2 to 5, which has a palindromic sequence for stopping protein translation downstream of the protein base sequence.   The palindromic sequence is a protein sequence in which the base sequence from base number 3031 to base number 3213 in the sequence listing of SEQ ID NO: 1, or one or several base sequences in the base sequence is deleted, substituted or added The vector according to claim 6, wherein the vector is a base sequence having a function of stopping translation. A recombinant microorganism characterized in that the vector according to any one of claims 2 to 7 is incorporated into Streptomyces lividans as a host. A method for producing a protein, comprising culturing the recombinant microorganism according to claim 8 in a culture solution to produce a protein based on the protein base sequence. The method for producing a protein according to claim 9 , wherein glucose is added to the culture solution.

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