JP7061018B2 - A novel promoter and a method for producing a protein using the promoter - Google Patents

Description

The present invention is a promoter isolated from a Burkholderia bacterium, more preferably a constitutive promoter that does not require an inducible substrate, a recombinant DNA vector containing those promoters, and a host cell transformed with the vector. On how to produce a target protein.

A host vector system using a bacterium belonging to the genus Berkholderia has been developed (see Patent Document 1), and the promoter used in the expression vector for expressing a foreign protein is induced by arabinose or ram nose. Inducible promoters (see Non-Patent Documents 1 and 2) and the like are known. On the other hand, examples of the constitutive promoter that constitutively expresses a gene include a promoter obtained from a ribosomal protein gene (see Non-Patent Document 1) and a promoter obtained by modifying an inducible promoter into a constitutive form (see Patent Document 2). Is known, and when used in combination with the inducible type, complicated expression control becomes possible. However, their promoter activity is lower than that of the inducible promoter, and the expression level of the protein may be low. In fact, it has been confirmed that even in an expression system using a constitutive promoter obtained by modifying an inducible promoter, the expression level of the protein is about half that of the inducible promoter (Patent Document 3 and See Non-Patent Document 3).

When Burkholderia bacteria are used as a system of catalytic reaction for the production and decomposition of substances, it may not be possible to impart highly efficient activity to host cells simply by overexpressing the catalytic enzyme. In particular, when feedback is inhibited by a catalytic reaction product such as a metabolic enzyme, it is necessary to control the expression level of genes involved in the catalyst. In order to control the expression level to a constant level for a long period of time, it is not easy to use an inducible promoter, and it is preferable to use a constitutive promoter having a constant expression level. To control the gene expression level using a constitutive promoter, a gene insertion technique into chromosomal DNA using a transposon (see Non-Patent Document 4) is used, and an expression cassette of the target gene is inserted into the genome (see Non-Patent Document 4). This can be achieved by controlling the number of copies (see Non-Patent Document 5) and the number of copies thereof (see Non-Patent Document 6).

If a constitutive promoter showing an activity different from that of the constitutive promoter derived from the above ribosome protein gene can be obtained, not only the application to the genome insertion type expression cassette but also the transcriptional activity of multiple expression vectors having the same intracellular copy number can be obtained. By expressing the expression target gene under the control of different constitutive promoters, the expression level can be adjusted, and the expression level of the gene can be controlled in a more diverse manner.

Japanese Patent No. 6099627 Japanese Patent No. 5641297 Japanese Patent No. 3944577

Leftrell and Valvano, Appl. Environ. Microbiol. , 68 (12), 5965-5954 (2002) Cardona and Valvano, plasmid, 54 (3): 219-228 (2005) Nakashima and Tamura, Biotechnol. Bioeng. , 86: 136-148 (2004) Sallam et al. , J. Biotechol. , 121 (1), 13-22 (2006) Sallam et al. , Gene, 386 (1-2), 173-182 (2007) Sallam et al. , Apple. Environ. Microbiol. , 76 (8), 2531-3339 (2010)

An object of the present invention is to provide a new promoter so that various expression control can be performed when using an expression vector or a genome-inserted expression cassette in a Burkholderia bacterium. Another object of the present invention is to provide a new constitutive promoter. Furthermore, it is to provide a gene expression system utilizing the activity of these promoters and a method for producing a target protein.

The present inventors have a useful promoter having promoter activity because the newly acquired sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 express high levels of expression target proteins arranged downstream thereof. I confirmed that it was. Furthermore, since it expresses the expression target protein at a high level even without the addition of an expression inducer, it has been found that it is a constitutive promoter having a constitutive promoter activity having more preferable properties.

That is, the present application includes the following inventions.
[1] DNA characterized by having any of the following promoter activities (a) to (d):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing;
(B) DNA capable of hybridization under stringent conditions with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing;
(C) DNA consisting of a base sequence in which one or several bases are deleted, substituted, or added from the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing;
(D) A DNA consisting of a base sequence having at least 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence identity with SEQ ID NO: 1 or 2 in the sequence listing.
[2] The DNA according to [1], which is a constitutive promoter, preferably a constitutive promoter for transformation of a Burkholderia bacterial host.
[3] The DNA according to [1] or [2], wherein the gene encoding a protein that may contain a signal sequence is located downstream, preferably downstream of 30 bases or less.
[4] The gene encoding a protein is an enzyme, preferably an esterase (more preferably cholesterol esterase or lipase), or a peroxidase (more preferably an alkylhydroperoxidase reductase of SEQ ID NO: 3), according to [3]. DNA.
[5] The DNA according to [4], wherein the gene is a gene encoding an enzyme derived from a Burkholderia bacterium.
[6] A base having a sequence identity of at least 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more with the gene encoding the alkylhydroperoxide reductase of a bacterium belonging to the genus Berkholderia. The DNA according to [5], which comprises a part of an untranslated region upstream of the start codon of a gene consisting of a sequence.
[7] The DNA according to [6], wherein the alkylhydroperoxide reductase of a Burkholderia bacterium comprises the amino acid sequence represented by SEQ ID NO: 3.
[8] The DNA according to any one of [1] to [7], wherein the terminator sequence is arranged downstream.
[9] The DNA according to [8], wherein the terminator sequence consists of the base sequence represented by SEQ ID NO: 17, 18 or 19.
[10] The DNA according to [9], wherein the second terminator sequence is located upstream.
[11] The DNA according to [10], wherein the terminator sequence arranged downstream and the second terminator sequence arranged upstream are different from each other.
[12] The DNA according to [11], wherein the terminator sequence arranged downstream and the second terminator sequence arranged upstream consist of the base sequence represented by SEQ ID NO: 17 or SEQ ID NO: 18.
[13] The DNA according to any one of [6] to [12], wherein the gene encoding the chaperone required for the expression of the protein is arranged downstream of the gene encoding the protein.
[14] The DNA according to [13], wherein the chaperone is placed before the terminator sequence.
[15] The DNA according to [13] or [14], wherein the chaperone contains a signal sequence.
[16] The DNA according to [15], wherein the chaperone containing the signal sequence comprises the base sequence represented by SEQ ID NO: 15.
[17] An expression vector containing the DNA according to [1] to [16].
[18] An expression vector containing an insertion sequence or a transposon into which the DNA according to [1] to [16] has been inserted.
[19] A gene expression cassette in which an expression target gene is arranged downstream of the DNA according to [1] to [16].
[20] A transformant containing the DNA according to [1] to [16], the expression vector according to [17] or [18], or the expression cassette according to [19].
[21] A transformant prepared by transferring the DNA according to [1] to [16], the expression vector according to [17] or [18], or the expression cassette according to [19] into a host.
[22] The transformant according to [21], wherein the host is a microorganism classified into the genus Burkholderia.
[23] The transformant according to any one of [20] to [22], wherein the DNA transcribed as antisense RNA is arranged downstream of the DNA according to [1] to [16].
[24] A method for producing a protein encoded by an expression target gene, which comprises the step of culturing the transformant according to any one of [20] to [22].
[25] The method according to [24], wherein the expression target gene encodes esterase.
[26] A method for suppressing gene expression in a host cell using the transformant according to [23].

The present invention provides a novel promoter. In particular, by using the constitutive promoter of the present invention, constant high production of proteins can be expected, and by combining with promoters having different transcriptional activities, a highly efficient substance conversion system using a biocatalyst can be constructed. Is expected.

It is a figure which shows the expression vector for confirming the activity of the constitutive promoter and the expected sequence. T1 (Terminator1) and T2 (Terminator2) represent terminators, T1 represents SEQ ID NO: 27, and T2 represents SEQ ID NO: 26, respectively. It is a figure which shows the expression vector which introduced the gene encoding cholesterol esterase and its chaperone downstream of a constitutive promoter. Note that T1 and T2 are as described above. It is a figure which shows the cholesterol esterase activity which Burkholderia stabilis which transformed the cholesterol esterase expression vector shows. It is a figure which shows the cholesterol esterase produced by Burkholderia stabilis which transformed the cholesterol esterase expression vector. It is a figure which shows the cholesterol esterase activity when culturing in a different medium. It is a figure which shows that the cholesterol esterase of the Burkholderia stabilis wild strain is induced expression. It is a figure which shows the lipase activity when the lipase gene of Burkholderia plantari (Burkholderia plantarii) is arranged downstream of each promoter. It is a figure which shows the cholesterol esterase activity produced by each promoter in Burkholderia multiborans.

Hereinafter, the present invention will be described based on examples and the like, but the scope of the present invention is not construed as being limited to the following examples and the like. Genetic engineering techniques include, for example, the method of Maniatis et al. (Maniatis, T., et al. Molecular Cloning. Cold Spring Harbor Laboratory 1982, 1989), genetic engineering learned from the basics (2nd edition, Takaaki Tamura, 2017,). (Yodosha), Genetic Engineering (Hiroshi Nojima, 2013, Tokyo Chemical Co., Ltd.), Basic Experimental Methods for Proteins and Enzymes (Revised 2nd Edition, Takeichi Horio, Nankodo, 1994), WO2013065772A1, or various commercially available enzymes , It can be carried out according to the procedure manual etc. attached to the kits. In addition, RBS (ribosome-binding site), which is a ribosome binding site, may be present at an appropriate interval (usually several bases upstream of the start codon) between the promoter sequence and the start codon of the insert gene. .. Thereby, the RBS can be made into a consensus sequence to promote the start of translation.

In the first embodiment, the present invention relates to the DNA of any one of the following (a) to (d):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing;
(B) A DNA capable of hybridization under stringent conditions with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing, and having promoter activity;
(C) A DNA having a promoter activity consisting of a base sequence in which one or several bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing;
(D) A DNA having a promoter activity consisting of a base sequence having at least 80% or more sequence identity with SEQ ID NO: 1 or 2 in the sequence listing.
I will provide a.

The DNA of the present invention may have a base sequence substantially the same as the base sequence shown by SEQ ID NO: 1 or 2 in the sequence listing, as long as it has promoter activity. In the present specification, the promoters corresponding to SEQ ID NOs: 1 and 2 are also referred to as promoter 1 and promoter 2, respectively, for the sake of distinction. The "promoter activity" of the present invention causes genes located downstream of the promoter to be transcribed into mRNA.

In the present invention, the promoter activity is preferably determined based on whether or not protein expression is observed. That is, it is preferable to confirm by using an appropriate reporter gene. For example, a DNA in which a reporter gene is operably linked downstream of a promoter sequence can be introduced into a cell, and the amount of expressed protein can be quantified by an immunological method or a biochemical method. As the reporter gene, a β-galactosidase (lacZ) gene, a luciferase gene, a β-lactamase gene, a fluorescent protein gene such as a GFP (Green Fluorescent Protein) gene, a cholesterol esterase gene and the like can be used.

More specifically, as shown in this example, a DNA having a cholesterol esterase gene linked directly downstream of a promoter candidate (mutated sequence) and a chaperon and a terminator sequence downstream thereof is transferred to Berkholderia. -In the transformant obtained by transferring to Stabilis, the expression level of cholesterol esterase is actually increased as compared with the transformant in which DNA lacking the promoter candidate (mutated sequence) was transferred. By confirming, it can be determined that the promoter activity has increased. As the promoter activity, a constitutive promoter activity capable of expressing the expression target protein at a high level even without the addition of an expression inducer is preferable. Explaining the presence or absence of the constitutive promoter activity by taking the above-mentioned transformant as an example, when the transformant is cultured, the promoter expresses the target protein at a substantial level even in the medium without the inducer. You can judge whether it can be produced or not. The promoter activity of the base sequence represented by SEQ ID NO: 1 or 2 of the present invention is preferable in that it is a constitutive promoter activity.

The promoter of the present invention has different activities depending on the type of medium for culturing the host and other conditions. Taking the case of producing cholesterol esterase as an example, for example, in the YS (yeast extract, sorbitol) medium, the promoter of sequence 2 may produce more than the sequence 1, and the LB (Lysogeny Broth) medium and NB may be produced. In (Nutrient Bros) medium, the promoter of sequence 1 may produce more than sequence 2.

The DNA of the present invention sets an expression target protein by arranging it upstream of a gene encoding a protein targeted for expression by a promoter (hereinafter referred to as “expression target protein”) (hereinafter referred to as “expression target gene”). If it can be expressed, its origin is not limited. The expression target protein is not particularly limited as long as it is a protein that can be expressed by the promoter of the present invention. Such proteins include enzymes such as esterase, lipase, amylase, glucoamylase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, mannosidase, isomerase, invertase, transferase, ribonuclease, deoxyribonuclease, kitinase. , Catalase, lacquerase, phenoloxidase, oxidase, oxidoreductase, cellulase, xylanase, peroxidase, hydrolase, cutinase, protease, phytase, lyase, pectinase, aminopeptidase, carboxypeptidase. Among the esterases, cholesterol esterase is preferable. The expression target protein may or may not contain a signal sequence. Usually, it is preferable that the expression target protein contains a signal sequence.

As a DNA having substantially the same base sequence, a DNA having a promoter activity by hybridizing with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing under stringent conditions. Can be mentioned. Here, the "stringent condition" is, for example, a condition of about "1xSSC, 0.1% SDS, 37 ° C.", and a stricter condition is "0.5xSSC, 0.1% SDS, 42 ° C.". The condition is about "0.2xSSC, 0.1% SDS, 65 ° C" as a stricter condition. As such, the stricter the hybridization conditions, the higher the homology with the probe sequence, preferably the isolation of DNA having high identity can be expected. However, the combination of SSC, SDS and temperature conditions described above is exemplary, and those skilled in the art will determine the stringency of hybridization above or other factors (eg, probe concentration, probe length, hybridization reaction). It is possible to realize the same stringency as above by appropriately combining (time, etc.).

The DNA of the present invention contains a base sequence in which a mutation such as deletion, substitution or addition occurs in one or more, preferably several nucleic acids in the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing. It is DNA and includes DNA having promoter activity. For example, in the base sequence represented by SEQ ID NO: 1 in the sequence listing, 1 to 52 base nucleic acids, preferably 1 to 26 base nucleic acids, more preferably 1 to 13 base nucleic acids, and even more preferably 1 to 5 base nucleic acids are missing. In the base sequence represented by SEQ ID NO: 2, such as DNA consisting of lost, substituted, or added base sequences, 1 to 54 base nucleic acids, preferably 1 to 27 base nucleic acids, more preferably 1 to 13 base nucleic acids, still more preferable. Examples thereof include DNA consisting of a base sequence in which 1 to 5 base nucleic acids are deleted, substituted, or added.

The DNA of the present invention has a full-length base sequence of at least 80 when calculated using the promoter base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing and BLAST or the like (for example, using the default or default parameters). % Or more, preferably 90% or more, more preferably 95% or more, still more preferably 98% or more sequence homology, preferably a promoter consisting of a base sequence having sequence identity.

The base sequence of the promoter of the present invention can be determined, for example, by the chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977)).

In the second embodiment, the present invention provides an expression vector containing the above DNA.

The expression vector is used to integrate the above DNA into an arbitrary location of a plasmid vector that autonomously replicates in a bacterium belonging to the genus Berkholderia, and to terminate a multicloning site or transcription that facilitates the introduction of an expression target gene downstream of the promoter sequence. It can be constructed by arranging a terminator. Further, the promoter region of the existing expression vector is composed of a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing, or a base sequence substantially the same as the base sequence represented by SEQ ID NO: 1 or 2. The expression vector of the present invention can also be prepared by substituting with DNA having promoter activity.

The plasmid vector is replicated in the host from the origin of replication. Therefore, it is preferable to use a plasmid vector having an origin of replication that can be replicated in the host. For Gram-negative bacteria such as Burkholderia, a plasmid vector having an origin of replication that can be replicated by Gram-negative bacteria is preferred. A plasmid vector having an origin of replication that can be replicated in Gram-negative bacteria includes the pBBR122 vector.

It can be expected that the plasmid vector into which the expression target gene is introduced downstream of the promoter of the constructed expression vector is transformed into a host cell, and the target protein is constitutively expressed in the transformant selected according to the selection marker of the vector. .. The shuttle vector, multi-cloning site, and terminator used here are not particularly limited. The present invention also includes a vector into which such an expression target gene has been introduced. The expression target gene is a gene encoding a protein to be expressed using the promoter of the present invention, and may be operably arranged downstream of the promoter of the present invention. The present invention also includes a plasmid vector in which the expression target gene is placed downstream of the promoter.

The expression target gene can be located downstream of SEQ ID NO: 1 or 2 to 30 bases or less, preferably 15 bases or less, more preferably 6 bases or less, particularly preferably directly linked. The expression target gene may have a signal sequence, for example, if the encoding protein is secretory.

It is preferable to place, for example, a (first) terminator sequence downstream of the expression target gene. A terminator sequence is a sequence that dissociates RNA polymerase from DNA and terminates transcription, and can usually be located downstream of a gene. As the terminator sequence of the present invention, various known terminator sequences can be used without particular limitation, and for example, any of SEQ ID NOs: 17, 18 or 19 disclosed in the sequence listing of the present specification may be used. When the expression target gene encodes cholesterol esterase (SEQ ID NO: 4), it is also convenient and preferable to use SEQ ID NO: 19, which is a natural terminator sequence originally attached to the gene.

In addition to the first terminator sequence, it is also preferred to place the second terminator sequence upstream of the promoter. This is expected to terminate the transcription of other upstream genes and avoid their effects. In this case, the first terminator sequence and the second terminator sequence described above may be the same, but it is usually more preferable to select a terminator sequence having a different sequence.

Depending on the protein targeted by the promoter of the present invention, chaperone may be expressed at the same time due to its expression or the like. This forms a polypeptide chain with the correct three-dimensional structure, which in turn produces a protein with the correct function. When a chaperone is required in producing a protein, it is preferable to express the protein and the chaperone at the same time.

The protein encoded by the chaperone and the expression target gene may be located at distant locations on the sequence as long as they are expressed at the same time. However, since the promoter required to initiate transcription and the terminator to terminate transcription are required, the chaperone must be placed between the promoter and the terminator.

In order to express the chaperone at the same time as the expression target gene, the chaperone sequence may be inserted between the expression target gene and the terminator, and the chaperone may be expressed by the promoter of the expression target gene.

If the chaperone functions in a particular location, the chaperone sequence preferably comprises a signal sequence.

More specifically, in the case of cholesterol esterase having the amino acid sequence of SEQ ID NO: 5, the chaperone sequence is not particularly limited, and examples thereof include the amino acid sequence of SEQ ID NO: 12. This chaperone may include the signal sequence of SEQ ID NO: 14.

In the case of a lipase having the amino acid sequence of SEQ ID NO: 40, the chaperone sequence is not particularly limited, and examples thereof include the amino acid sequence of SEQ ID NO: 47. This chaperone may include the signal sequence of SEQ ID NO: 49.

In a third embodiment, the present invention provides a gene expression cassette containing the above DNA.

A DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing, or a DNA consisting of a base sequence substantially the same as the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing and having promoter activity. By preparing an expression cassette containing an expression target gene, it is possible to construct a recombinant protein expression system in a host cell containing the promoter of the present invention without constructing an expression plasmid vector. The present invention includes such an expression cassette in which an expression target gene is arranged downstream of a promoter.

It is known that in some bacteria including Burkholderia bacteria, the efficiency of homologous recombination is low in gene recombination experiments, and recombination at non-specific sites is likely to occur. In fact, for example, in a Bacterial genus Berkholderia, when a vector or linear DNA constructed with the intention of genetic recombination by homologous recombination is transformed into a bacterial cell of the genus Berkholderia, the DNA is transferred to the genome of the host cell. Non-specific incorporation can occur frequently. From this, for example, it consists of a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing, or a base sequence substantially equivalent to the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing, and is a promoter. A linear DNA in which an expression target gene is placed downstream of the active DNA and a selection marker gene such as a terminator or an antibiotic resistance gene is placed is constructed as an expression cassette, and the construct is transformed into a host cell. By inserting the gene into a non-specific site of the genome, the expression target gene can be expressed in the host cell by utilizing the promoter of the present invention. In this case, the linear DNA can be amplified by PCR after being constructed on a general cloning vector for E. coli. It is also possible to cut out the necessary part from the vector and prepare it.

As a method for inserting the expression system of the expression target gene into the genome, a known gene transfer method can be used, and a method of inserting using a transposon vector is preferable. The transposon vector is configured to contain a transposase gene encoding an enzyme required for sequence transfer and two repetitive sequences recognized by transposes. By incorporating a gene expression system between these recognition sequences, it can be used as a vector capable of constructing a genome-inserted constitutive expression system.

There are several known transposon vectors available in Burkholderia bacteria, such as the pUTminiTn5 vector (de Lorenzo et al., J. Bacteria, 172 (11), 6568-6571 (1990)). .. Therefore, the expression vector into which the DNA represented by SEQ ID NO: 1 or 2 in the sequence listing or the DNA having a promoter activity and having substantially the same base sequence as the base sequence represented by SEQ ID NO: 1 or 2 is introduced. To construct a recombinant cell having an expression cassette copy number from 1 copy to multiple copies by incorporating DNA containing up to the terminator downstream of the expression target gene into a transposon vector and transferring it to the genome of the host cell. Is possible. The genome insertion type expression system is not as high as the expression level of the multi-copy expression vector in terms of the number of copies of the expression cassette, but it is stably retained in the genome, so that the vector is selected and retained. It has the advantage that it is not necessary to add the antibiotics of.

The use of such a gene expression system can also be applied to the suppression of protein expression, which is the opposite of protein production. When antisense RNA containing the initiation codon of the intracellular expression target gene is overexpressed in the cell, the antisense mRNA expressed in the mRNA of the expression target gene hybridizes in the cell, and the mRNA is also impaired in translation and degraded. It is promoted and, as a result, it is possible to suppress the production of proteins derived from the expression target gene (Nakashima et al., Nucleic Acids Res., 34: e138 (2006); Nakashima and Tamura, Nucleic Acids Res., 37: (2009)). Therefore, a DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing, or a DNA having a base sequence substantially the same as the base sequence represented by the base sequence 1 or 2 in the sequence listing and having promoter activity. The antisense RNA of the expression target gene can be accumulated in the cell downstream of, and the production of the expression target gene-derived protein can be suppressed. Such a technique for utilizing antisense RNA or the like is an effective method for modifying the function of a host cell as well as gene disruption, and enables metabolic engineering for artificially modifying the metabolic pathway of the host cell.

In a fourth embodiment, the present invention provides a transformant comprising the expression vector or the gene expression cassette.

The host into which the vector containing the promoter of the present invention or the expression cassette is introduced is not limited, and examples thereof include microorganisms classified into the genus Burkholderia. Burkholderia bacteria are β-proteobacteria that are resident in various natural ecosystems such as the geosphere, hydrosphere, and plant surface. While there are strains with lipase-producing ability suitable for industrial production and strains with phytopathogenic filamentous fungal extermination ability, strains that cause opportunistic infections due to their pathogenic factor-producing ability, and phytopathogenic strains produced by the production of pectinase, etc. Various strains are included (Yoshiyuki Otsubo et al., Chemistry and Biology, Vol. 47 No. 1, pp. 35-42 (2009)).

The microorganisms classified into the genus Burkholderia include Burkholderia stabilis, Burkholderia multivorans, Burkholderia sepacia, and Burkholderia. (Burkholderia malleli), Burkholderia gurmae, Burkholderia ambifaria, Burkholderia Burkholderia Burkholderia (Burkholderia bullia) Burkholderia Burkholderia plantarii (AZEGAMI et al., Int J System Evol Microbiol, 37 (2): 144-152 (1987); Seo et al., BMC 16), et al., BMC 16).

Burkholderia Plantary DSM 9509 can be purchased from the BioResource Center DSMZ.

Since Burkholderia bacteria exist in various natural ecosystems, the medium and conditions for culturing Burkholderia bacteria vary. For example, in order to culture Burkholderia sepacia, it may be cultured in PY medium (5% polypeptone, 5% yeast extract) at 28 ° C. (Japanese Patent Laid-Open No. 9-322776). For strains that can be purchased at DSMZ, the culture medium and conditions recommended by DSMZ are published as Cultivation conditions, which may be referred to.

In a fifth embodiment, the present invention provides a method for producing a protein encoded by an expression target gene located downstream of the promoter, which comprises a step of culturing the transformant.

More specifically, the target protein can be produced by culturing the transformant and isolating and purifying the protein encoded by the expression target gene from the culture. Here, the culture means a mixture of the cells and the culture solution obtained by culturing the bacteria, and the transformant uses a medium suitable for the host to be used, and is cultured by a static culture method or a roller bottle. It can be cultivated by a method or the like. The culture can be carried out by a known method, and the medium and culture conditions to be used can be appropriately determined depending on the type of host cell used.

When the target protein is produced in the cells or cells, the protein can be collected by disrupting the host cell. When the target protein is produced outside the host cell, the culture medium is used as it is, or the host cell is removed by centrifugation or the like. Then, the target protein can be isolated and purified from the above-mentioned culture by using a general biochemical method using various chromatographies used for isolation and production of the protein alone or in combination as appropriate. ..

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the scope of these Examples.

Example 1 Preparation of expression vector for measuring promoter activity Berkholderia stabilis (accession number: NITE BP-02704) was used in LB medium (1% Bacto ™ Tryptone, 0.5% Bacto ™ Yeast extract). , 0.5% sodium chloride) was shake-cultured at 30 ° C., 500 μL of the bacterial solution grown to the steady phase was added to 10 mL of LB medium, and the cells were cultured at 30 ° C. for 12 hours.

After culturing, the bacteria were collected by centrifugation (3,000 xg, 10 minutes, room temperature), resuspended in 500 μL Tris-EDTA (TE) buffer, 50 μL Proteinase K (20 mg / mL) and 25 μL 10% SDS. Was added and incubated at 55 ° C. for 30 minutes. After the reaction, 600 μL of a mixed solution of phenol, chloroform and isoamyl alcohol (50:49: 1) was added, and the mixture was stirred and then centrifuged (20,817 xg, 10 minutes, room temperature). The obtained supernatant is transferred to another tube and re-extracted with a mixed solution of phenol, chloroform and isoamyl alcohol, and then 50 μL of 3M sodium acetate and 350 μL of isopropanol are added to the obtained supernatant to precipitate DNA and recover it. The DNA was washed twice with 500 μL of 70% ethanol. The DNA was air dried at room temperature for 10 minutes, dissolved in 200 μL TE and stored at 4 ° C.

Using the purified DNA as a template, the polymerase chain reaction method (hereinafter abbreviated as PCR: abbreviated as PCR) using the synthetic oligodeoxyribonucleotide primers (hereinafter abbreviated as primers) described in SEQ ID NOs: 20 and 21, 22 and 23, 24 and 25. DNA was amplified by Saiki et al., Science, 239 487-491 (1988)). The PCR enzyme used was KOD FX Neo (manufactured by Toyobo). As a result, two types of terminators (SEQ ID NOs: 26 and 27) and multicloning sites (BglII, NheI, SnaBI, SacI, KpnI, SpeI, BamHI, XbaI, NdeI) fragments (SEQ ID NO: 28) were obtained. In the same manner as in the above method, DNA was amplified using the pBBR122 plasmid (manufactured by MoBiTech) as a template and the primers shown in SEQ ID NOs: 29 and 30 in the sequence listing. These DNA fragments amplified by the PCR reaction were subjected to agarose gel electrophoresis, excised from the gel, and purified using Wizard® SV Gel and PCR Clean-Up System (manufactured by Promega). This purified DNA fragment was ligated using In-Fusion® HD Cloning Kit (manufactured by TaKaRa), transformed into Escherichia coli DH5α, and then applied to LB agar medium containing 200 μg / mL kanamycin. After culturing at 28 ° C for 24 hours, the obtained colonies were subcultured in LB medium containing 200 μg / mL kanamycin sulfate, and after culturing at 28 ° C for 12 hours, Wizard (registered trademark) Plus MiniPreps DNA Preparation Systems ( The plasmid was extracted using Promega (manufactured by Promega). This plasmid was used as an expression vector for measuring promoter activity (Fig. 1).

Example 2 Preparation of Cholesterol Esterase Expression Vector Using the genomic DNA of Berkholderia stabilis purified in Example 1 as a template and using the primers set forth in SEQ ID NOs: 31 to 36 in the sequence listing, amplification of the DNA by PCR. Was done. As a result, a DNA fragment of a gene encoding cholesterol esterase and its chaperone and a DNA fragment of a promoter region were obtained (SEQ ID NOs: 1 and 2). Using the expression vector prepared in Example 1 as a template, DNA was amplified using the primers set forth in SEQ ID NOs: 37 and 38 in the sequence listing, and the DNA fragment of the expression vector was amplified. The promoter, cholesterol esterase and chaperone amplified by these PCR reactions, and the DNA fragment of the expression vector were purified and ligated in the same manner as in Example 1 to prepare an expression vector for cholesterol esterase (FIG. 2).

Example 3 Measurement of promoter activity using cholesterol esterase gene In order to introduce the expression vector of cholesterol esterase prepared in Example 2 into Berkholderia stabilis, the strain was subjected to 30 in 100 mL of LB medium until the logarithmic growth phase. After culturing with shaking at ° C, the cells were collected by centrifugation. 100 mL of ice-cold sterile water was added to the collected cells, and the cells were suspended and then centrifuged again to collect the cells. After repeating this operation once more, a 5 mL cooled 10% glycerin solution was added to the collected cells, the cells were well suspended, and the cells were collected by centrifugation.

5 mL of ice-cold 10% glycerin solution was added to the collected cells, suspended, 40 μL each was dispensed, and the cells were instantly frozen in liquid nitrogen and stored at -80 ° C. The cells stored frozen were thawed on ice, and about 1 ng each of the two expression vectors prepared in Example 2 was added and mixed. This mixture was transferred to a 0.2 cm wide electroporation cuvette (manufactured by Bio-Rad), and an electric pulse was used with a gene transfer device GenePulsar II at an electric field strength of 12.5 kV / cm, a capacitance of 25 μF, and an external resistance of 200 Ω. Gave. A mixed solution of cells and DNA treated with electric pulse was mixed with 1 mL of LB medium, cultured at 30 ° C. for 1 hour, and then 200 μL of the bacterial solution was applied to an LB medium containing 200 μg / mL of canamycin sulfate. The cells were cultured at 30 ° C. for 1 day to obtain transformants of each expression vector.

Berkholderia stabilis introduced with an expression vector was inoculated into 5 mL of 3% yeast extract and 3% sorbitol medium (containing 100 μg / mL kanamycin sulfate) and cultured at 28 ° C. for 12 hours. This bacterial solution was added to 50 mL of 3% yeast extract and 3% sorbitol medium (containing 100 μg / mL kanamycin sulfate) so that the bacterial turbidity was OD = 0.2, and the temperature was 28 ° C. using a flask with a baffle. Was cultured for 36 hours. On the other hand, the wild strain of Berkholderia stabilis into which the expression vector was not introduced was similarly inoculated into 5 mL of 3% yeast extract and 3% sorbitol medium and cultured at 28 ° C. for 12 hours. This bacterial solution was added to 50 mL of 3% yeast extract and 3% sorbitol medium so that the bacterial turbidity was OD = 0.2, and cultured at 28 ° C. for 36 hours using a flask with a baffle. After culturing, the cholesterol esterase activity for p-nitrophenyl laurate was measured in order to evaluate the amount of cholesterol esterase produced by each strain.

After culturing, the culture broth was appropriately diluted and reacted with 1 mM p-nitrophenyl laurate at 37 ° C., and p-nitrophenol produced by the hydrolysis reaction was measured by absorbance at a wavelength of 400 nm. As a result, cholesterol esterase activity was hardly confirmed in the Berkholderia stabilis wild strain to which the expression vector was not introduced, but it was high in the transformant into which the promoter represented by the nucleotide sequence of SEQ ID NO: 1 or 2 was introduced. Cholesterol esterase activity was observed (Fig. 3). Further, the culture broth was diluted 3-fold with 3% yeast extract and 3% sorbitol medium (YS medium) and mixed, and the supernatant was recovered by centrifugation to obtain a crude protein extract. The crude protein extract was separated by SDS-PAGE using a 10-20% gradient gel and stained with Coomassie Brilliant Blue G250. The molecular weight of cholesterol esterase was 33.2 kDa, and a band of concentration corresponding to cholesterol esterase activity was observed at the expected size (Fig. 4). As described above, since cholesterol esterase was produced even under the condition that no fatty acid was added to the medium, it was found that the promoter represented by the base sequence of SEQ ID NO: 1 or 2 is a constitutive promoter.

Since cholesterol esterase is induced and expressed by certain fatty acids in the Berkholderia stabilis wild strain (see the reference example below), industrial production of cholesterol esterase produces cholesterol esterase in Berkholderia stabilis. It must be cultured in a medium containing those fatty acids. Therefore, cholesterol esterase must be separated and purified from a medium containing a water-insoluble fatty acid, which hinders stable production. The possibility of separating and purifying cholesterol esterase from a simple medium containing no fatty acid is an extremely significant achievement for producing a high-purity, stable-quality enzyme.

Example 4 Confirmation of expression in different media The transformants introduced with the promoter confirmed to be the constitutive promoter in Example 3 were inoculated into 5 mL of LB medium (containing 100 μg / mL kanamycin sulfate), respectively. The cells were cultured at 28 ° C. for 72 hours. On the other hand, the Burkholderia stabilis wild strain into which the expression vector was not introduced was also inoculated into 5 mL of LB medium and cultured at 28 ° C. for 72 hours. In addition, each of the transformants into which the promoter of the present invention was introduced is 5 mL of Defco (registered trademark) Nutrient Bros (manufactured by Becton Dickinson) (hereinafter abbreviated as NB medium) (including 100 μg / mL kanamycin sulfate). Inoculated into, and cultured at 28 ° C. for 72 hours. On the other hand, the Burkholderia stabilis wild strain into which the expression vector was not introduced was also inoculated into 5 mL of NB medium and cultured at 28 ° C. for 72 hours. After culturing, the cholesterol esterase activity for p-nitrophenyl laurate was measured in order to evaluate the amount of cholesterol esterase produced by each strain. After culturing, the culture broth was appropriately diluted and reacted with 1 mM p-nitrophenyl laurate at 37 ° C., and p-nitrophenol produced by the hydrolysis reaction was measured by absorbance at a wavelength of 400 nm (FIG. 5). As a result, cholesterol esterase activity was hardly confirmed in the Berkholderia stabilis wild strain to which the expression vector was not introduced, but cholesterol esterase activity was confirmed in the transformant into which the promoter of the present invention was introduced. From this result, it was confirmed that the promoter of the present invention can be constitutively expressed regardless of the medium.

Reference example Confirmation of cholesterol esterase induction expression in a wild strain of Berkholderia stabilis 100 mL of preculture medium (0.1% dipotassium hydrogen phosphate, 1% milk casein, 0.5% yeast extract, Precultured in a liquid medium (pH 7.0) containing 0.2% potassium chloride and 0.05% magnesium sulfate at 28 ° C. and 200 rpm for 24 hours. 2 mL of the obtained culture medium was sterilized with a 2 L jar fermenter (1.6 L 0.1% dipotassium hydrogen phosphate, 1.5% milk casein, 0.5% yeast extract, 0.2 L). It was added to a liquid medium (pH 6.5) containing% potassium chloride, 0.05% magnesium sulfate, 1% soybean flour, and 3% oleic acid, and cultured at 28 ° C. and 650 rpm for 48 hours.

After 48 hours, the culture broth was collected and used as a culture broth in an oleic acid-added medium. After pre-culturing in the same pre-culture medium as above under the same conditions, the culture solution obtained by culturing under the same conditions in a medium in which only oleic acid in the main culture medium is replaced with palmitic acid is palmitin. The culture solution obtained by culturing under the same conditions in the culture solution of the acid-added medium and the medium in which only the oleic acid of the main culture medium was replaced with myristic acid was used as the culture solution of the myristic acid-added medium and the oleic acid of the main culture medium. The culture broth obtained by culturing under the same conditions on the medium excluding the above was used as the culture broth of the fatty acid-free medium.

The cholesterol esterase activity of each of the obtained culture broths was measured by the method described in JP-A-2008-86277. As a result, it was found that cholesterol esterase was not expressed in the non-added medium, but cholesterol esterase was expressed when fatty acids such as oleic acid, palmitic acid, and myristic acid were added to the medium (Fig. 6). From this, it is confirmed that cholesterol esterase is induced and expressed by the addition of these fatty acids.

Example 5 Measurement of promoter activity against other target genes Total synthesis of the sequence containing the lipase gene and chaperone gene derived from Berkholderia plantary (DSM 9509) set forth in SEQ ID NO: 45 was synthesized. The fully synthesized sequence containing the lipase gene and the chaperon gene was replaced with the sequence containing the cholesterol esterase gene and the chaperon gene of the expression vector prepared in Example 2 to obtain a lipase expression vector having SEQ ID NO: 1 or SEQ ID NO: 2 as a promoter. rice field. Using these plasmids, Burkholderia stabilis was transformed in the same manner as in Example 3 to obtain a transformant. This transformant was pre-cultured with 3% yeast extract and 3% sorbitol medium, and the pre-culture solution was diluted with the same medium so that the bacterial turbidity OD = 0.2 and main-cultured with 40 mL of medium. After culturing, the bacterial solution was centrifuged, and the activity of lipase contained in the supernatant on diacylglycerol was measured (FIG. 7). As a result, almost no lipase activity was confirmed in the Burkholderia stabilis wild strain, but high lipase activity was observed in the transformant into which the promoter of the present invention was introduced. From this result, it was confirmed that the promoter of the present invention can also be used for the expression of genes derived from bacteria other than Burkholderia stabilis.

Example 6 Confirmation of promoter activity in other Burkholderia genus In order to introduce the cholesterol esterase expression vector having the promoter of the present invention prepared in Example 3 into Burkholderia multibolans (NBRC102086), the strain was introduced. After culturing in 100 mL of LB medium at 30 ° C. until the logarithmic growth phase, the cells were collected by centrifugation. 100 mL of ice-cold sterile water was added to the collected cells, and the cells were suspended and then centrifuged again to collect the cells. After repeating this operation twice, a 5 mL-cooled 10% glycerin solution was added to the collected cells, the cells were well suspended, and the cells were collected by centrifugation. 5 mL of ice-cold 10% glycerin solution was added to the collected cells, suspended, 100 μL each was dispensed, and the mixture was stored at −80 ° C. The cryopreserved cells were thawed on ice, and about 1 ng of each of the cholesterol esterase expression vectors having the promoter of the present invention prepared in Example 3 was added and mixed. This mixture was transferred to a 0.1 cm wide electroporation cuvette (manufactured by Bio-Rad), and an electric pulse was used with a gene transfer device GenePulsar II at an electric field strength of 12.5 kV / cm, a capacitance of 25 μF, and an external resistance of 200 Ω. Gave. A mixture of cells and DNA treated with electric pulse was mixed with 1 mL of LB medium, cultured at 30 ° C. for 1 hour, and then 200 μL of the bacterial solution was applied to LB medium containing 30 μg / mL chloramphenicole. , 30 ° C. for 1 day to obtain transformants of each expression vector.

Berkholderia multiborans introduced with an expression vector was inoculated into 5 mL of 3% yeast extract and 3% sorbitol medium (containing 30 μg / mL chloramphenicol) and cultured at 28 ° C. for 48 hours. On the other hand, the wild strain of Berkholderia multiborans was also inoculated into 5 mL of 3% yeast extract and 3% sorbitol medium and cultured at 28 ° C. for 48 hours. After culturing, the cholesterol esterase activity for p-nitrophenyl laurate was measured in order to evaluate the amount of cholesterol esterase produced by each strain. After culturing, the culture solution was appropriately diluted and reacted with 1 mM p-nitrophenyl laurate at 37 ° C., and p-nitrophenol produced by the hydrolysis reaction was measured by absorbance at a wavelength of 400 nm (FIG. 8). As a result, the expression vector was obtained. Almost no cholesterol esterase activity was confirmed in the Berkholderia multibolans wild strain to which the promoter of the present invention was not introduced, but cholesterol esterase activity was confirmed in the transformant into which the cholesterol esterase expression vector having the promoter of the present invention was introduced. .. From this result, it was shown that the promoter of the present invention also acts as a constitutive promoter in other Burkholderia genera.

According to the present invention, the use of the promoter DNA of the present invention enables high production of protein, and more preferably, the use of constitutive promoter DNA enables constant high production of protein. It becomes possible to provide a highly efficient protein production method.

SEQ ID NO: 1: Nucleotide sequence of promoter 1 SEQ ID NO: 2: Nucleotide sequence of promoter 2 SEQ ID NO: 3: Nucleotide sequence of alkylhydroperoxide reductase derived from Berkholderia stabilis SEQ ID NO: 4: Nucleotide sequence of mature cholesterol esterase SEQ ID NO: 5 : Amino acid sequence of a mature cholesterol esterase SEQ ID NO: 6: Nucleotide sequence of a signal sequence of cholesterol esterase SEQ ID NO: 7: Nucleotide sequence of a signal of cholesterol esterase SEQ ID NO: 8: Nucleotide sequence of a direct link between a signal sequence and a mature cholesterol esterase SEQ ID NO: 9: Nucleotide sequence in which a signal sequence and a mature cholesterol esterase are directly linked SEQ ID NO: 10: Nucleotide sequence in which a signal sequence, a mature cholesterol esterase and chaperon required for its expression are directly linked SEQ ID NO: 11: SEQ ID NO: 10 Nucleotide sequence of chaperon contained as a partial sequence in SEQ ID NO: 12: Nucleotide sequence of SEQ ID NO: 11 SEQ ID NO: 13: Nucleotide sequence of signal sequence of SEQ ID NO: 11 SEQ ID NO: 14: Nucleotide sequence of SEQ ID NO: 13 SEQ ID NO: 15: Signal of chaperon Nucleotide sequence sequence number 16 in which the sequence and the mature chaperon are directly linked: Amino acid sequence sequence number 17 in which the signal sequence of the chaperon and the mature chaperon are directly linked SEQ ID NO: 17: Nucleotide sequence sequence number 18: Nucleotide sequence sequence number 19 in the terminator sequence : Nucleotide sequence of the terminator sequence located downstream of cholesterol esterase in the natural state SEQ ID NO: 20: Synthetic oligodeoxyribonucleotide primer (Terminator1-pBBR-F)
SEQ ID NO: 21: Synthetic oligodeoxyribonucleotide primer (Terminator1-MCS-R)
SEQ ID NO: 22: Synthetic oligodeoxyribonucleotide primer (MCS-Terminator1-F)
SEQ ID NO: 23: Synthetic oligodeoxyribonucleotide primer (MCS-Terminator2-R)
SEQ ID NO: 24: Synthetic oligodeoxyribonucleotide primer (Terminator2-MCS-F)
SEQ ID NO: 25: Synthetic oligodeoxyribonucleotide primer (Terminator2-pBBR-R)
SEQ ID NO: 26: Terminator sequence (Terminator 2)
SEQ ID NO: 27: Terminator sequence (Terminator1)
SEQ ID NO: 28: Multicloning site SEQ ID NO: 29: Synthetic oligodeoxyribonucleotide primer (pBBR-Terminator2-F)
SEQ ID NO: 30: Synthetic oligodeoxyribonucleotide primer (pBBR-Terminator1-R)
SEQ ID NO: 31: Synthetic Oligodeoxyribonucleotide Primer (Cholesterol Esterase-F)
SEQ ID NO: 32: Synthetic Oligodeoxyribonucleotide Primer (Cholesterol Esterase-R)
SEQ ID NO: 33: Synthetic oligodeoxyribonucleotide primer (Promoter1-F) for amplifying the promoter of SEQ ID NO: 1.
SEQ ID NO: 34: Synthetic oligodeoxyribonucleotide primer (Promoter1-R) for amplifying the promoter of SEQ ID NO: 1.
SEQ ID NO: 35: Synthetic oligodeoxyribonucleotide primer for amplifying the promoter of SEQ ID NO: 2 (Promoter2-F)
SEQ ID NO: 36: Synthetic oligodeoxyribonucleotide primer (Promoter2-R) for amplifying the promoter of SEQ ID NO: 1.
SEQ ID NO: 37: Synthetic oligodeoxyribonucleotide primer for amplifying an expression vector (Terminator2-chaperon-F)
SEQ ID NO: 38: Synthetic oligodeoxyribonucleotide primer for amplifying an expression vector (Terminator1-R)
SEQ ID NO: 39: Nucleotide sequence of lipase derived from mature Berkholderia plantarii SEQ ID NO: 40: Nucleotide sequence of lipase derived from mature Berkholderia plantarii SEQ ID NO: 41: Signal sequence of lipase derived from Berkholderia plantarii Nucleotide sequence No. 42: Amino acid sequence of the signal of lipase derived from Berkholderia plantarii SEQ ID NO: 43: Nucleotide sequence of direct link between the signal sequence of lipase derived from Berkholderia plantarii and the mature lipase SEQ ID NO: 44: Nucleotide sequence No. 44: Berkhol Amino acid sequence in which the signal sequence of lipase derived from Delia plantarii and mature lipase are directly linked SEQ ID NO: 45: Included in the signal sequence, the base sequence in which mature cholesterol esterase and chaperon are directly linked SEQ ID NO: 46: SEQ ID NO: 45. Nucleotide sequence of chaperon SEQ ID NO: 47: Nucleotide sequence of SEQ ID NO: 46 SEQ ID NO: 48: Nucleotide sequence of the signal sequence contained in SEQ ID NO: 45 SEQ ID NO: 49: Amino acid sequence of SEQ ID NO: 48

Claims (10)

DNA of any one of the following (a) to ( c ):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 in the sequence listing;
( B ) The base sequence represented by SEQ ID NO: 1 in the sequence listing contains 1 to 26 bases deleted, substituted, or added, or the base sequence represented by SEQ ID NO: 2 contains 1 to 27 bases. A DNA consisting of a deleted, substituted, or added base sequence and having promoter activity;
( C ) A DNA having a promoter activity, consisting of a base sequence having at least 90 % or more sequence identity with SEQ ID NO: 1 or 2 in the sequence listing. The DNA according to claim 1, wherein the promoter activity is a constitutive activity. An expression vector containing the DNA according to claim 1 or 2. An expression vector containing an insertion sequence or a transposon into which the DNA according to claim 1 or 2 is inserted. A gene expression cassette in which an expression target gene is arranged downstream of the DNA according to claim 1 or 2. A gene cassette in which a DNA transcribed as antisense RNA is placed downstream of the DNA according to claim 1 or 2. A transformant comprising the expression vector according to claim 3 or 4 or the gene expression cassette according to claim 5. The transformant according to claim 7, wherein the host is a microorganism classified into the genus Burkholderia. A method for producing a protein encoded by an expression target gene, comprising the step of expressing the expression target gene using the DNA according to claim 1 or 2. 9. The method of claim 9, comprising culturing the transformant according to claim 7.

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