JP2005312426A - Esherichia coli presenting protein on cell surface - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide Esherichia coli presenting a protein in its active state on the cell surface. <P>SOLUTION: This invention relates to the Esherichia coli presenting the protein in its active state on the cell surface, wherein the Esherichia coli has a recombinant DNA containing a promoter sequence, a DNA encoding a pgsA which is a poly-γ-glutamine acid producing enzyme, and a DNA encoding the protein in this order. The promoter sequence comprises a base sequence or its fragment selected from the group consisting of (A) two kinds of base sequences disclosed in the specification, (B) a base sequence wherein at least one base is substituted, deleted or added for/from/to the prescribed sequence, (C) a base sequence of a DNA hybridizable to the prescribed sequence in a stringent condition, and (D) a base sequence having at least 80% sequence identity to the prescribed sequence, and exhibits an activity of a constitutional promoter in the Esherichia coli. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to Escherichia coli that presents a protein in an active state on the surface, and a method for using the Escherichia coli.

  In recent years, cell surface display technology that expresses useful proteins on the cell surface of yeast and bacteria has attracted attention.

  For example, various enzymes such as lipases, amylases, and cellulases are presented on the yeast surface using GPI anchors of cell surface localized proteins (Patent Documents 1 and 2). In yeast, a cell surface display technology using a sugar chain binding protein domain has also been developed (Patent Document 3).

  Bacterial surface expression was examined using an expression plasmid containing the gene for poly-γ-glutamic acid biosynthetic enzyme derived from Bacillus sp., And was expressed on the surface layer of gram-positive lactic acid bacteria or gram-negative bacterium E. coli. It is known that various proteins can be presented (Patent Document 4).

  Various studies have been conducted on the presentation of proteins on the surface of E. coli, but at present, there is a limit to the size of proteins that can be presented on the surface, and it is difficult to express proteins with a relatively large molecular weight. (Non-Patent Documents 1 to 3). For example, in the review of Non-Patent Document 3, it is described that the upper limit of the protein size that can be displayed on the surface layer was considered to be 60 to 70 amino acids, whereas a protein of 253 amino acids could be presented. However, there is a description that cells cannot survive if larger molecules are presented on the surface. The maximum size of the foreign protein displayed in the surface layer disclosed in Patent Document 4 is 442 amino acids, which is approximately the same size as the poly-γ-glutamate biosynthetic enzyme used for surface layer display. is there. A large foreign protein that has been actually presented on the surface of E. coli so far is an enzyme of about 47 kDa (Non-patent Document 4). However, this Non-Patent Document 4 also describes that there is a limit to the size that can be presented in the normal cell surface display technology. Thus, at present, no E. coli presents a relatively large protein of 50 kDa or more on the surface.

Until now, it has been difficult to present proteins such as enzymes having a catalytic function on the cell surface of E. coli. Therefore, in cell surface display in Escherichia coli, it was mainly to display heterologous peptides and fragments on the surface layer (Non-patent Documents 1 and 4). That is, there is a cell surface display technology that uses an outer membrane localized protein as an immobilized protein for surface layer display, can appropriately retain the three-dimensional structure of the target protein, and can fully exhibit the activity of this protein. It is desired.
JP-A-11-290078 International Publication No. 01/79483 Pamphlet International Publication No. 02/085935 Pamphlet International Publication No. 03/014360 Pamphlet International Publication No. 01/83787 Pamphlet Samuelson P, Gunneriusson E, Nygren PA, and Stahl S, “Display of proteins on bacteria REVIEW”, Journal of Biotechnology, 2002, 96, 129-154 Hoischen C, Fritsche C, Gumpert J, Westermann M, Gura K, and Fahnert B, “Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of Proteus mirabilis and Escherichia coli”, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2002 Month, pp. 525-531 Benhar I, “Biotechnological applications of phage and cell display”, Biotechnology Advances, February 1, 2001, Vol. 19, No. 1, pp. 1-33 Lee SY, Choi JH, and Xu Z, “Microbial cell-surface display”, Trends in Biotechnology, January 2003, Vol. 21, No. 1, pp. 45-52

  An object of the present invention is to provide Escherichia coli that presents proteins on the surface in an active state.

The present invention provides E. coli that presents proteins on the surface in an active state,
The E. coli has a promoter sequence, a DNA encoding pgsA, a poly-γ-glutamate biosynthetic enzyme, and a recombinant DNA containing DNA encoding the protein in this order, and the promoter sequence is (A (B) a base sequence having at least one base substitution, deletion, or addition in the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, or (C) At least 80% sequence identity to the nucleotide sequence of DNA that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and (D) the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 A base sequence selected from the group consisting of a sexual sequence, or a fragment thereof, and a constitutive Exhibit Ta activity.

  In a preferred embodiment, the pgsA comprises Bacillus sp. Is from.

  In a more preferred embodiment, the DNA encoding the above pgsA is a base sequence represented by SEQ ID NO: 3; a base sequence having at least one base substitution, deletion, or addition in the base sequence represented by SEQ ID NO: 3; A base sequence of DNA that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 3; or a base sequence having at least 80% sequence identity to the base sequence set forth in SEQ ID NO: 3.

  In another preferred embodiment, the protein is an enzyme.

  In a further preferred embodiment, the enzyme is α-amylase.

  In yet another preferred embodiment, the enzyme is a lipase.

The present invention also provides a method for expressing a protein in an active state on the surface of E. coli, the method comprising:
(A) a step of constructing a promoter sequence, a DNA encoding pgsA which is a poly-γ-glutamate biosynthetic enzyme, and a recombinant DNA containing DNA encoding the protein in this order, the promoter sequence comprising: (A) the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, (B) the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, having at least one base substitution, deletion, or addition, C) at least 80% of the base sequence of DNA that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and (D) the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 A base sequence selected from the group consisting of a base sequence having sequence identity, or a fragment thereof, and constitutive in said E. coli Exhibit promoter activity, process;
(B) transforming E. coli with the recombinant DNA; and (c) culturing the transformed E. coli and expressing the protein on the surface of the E. coli.

  In a preferred embodiment, the protein is an enzyme.

  The present invention further provides a method for using any of the above-mentioned E. coli, wherein the method comprises a step of contacting the protein with a substrate and binding the substrate or converting the substrate into another substance. .

  In a preferred embodiment, the protein is α-amylase, the substrate is starch, and includes a step of aerobically culturing the E. coli to produce lactic acid.

  In another preferred embodiment, the protein is a lipase, the substrate is a racemic alcohol and an ester, and the E. coli is used as a catalyst for a transesterification reaction to enantioselectively produce an alcohol ester. including.

  The present invention also provides an enzyme agent comprising any of the above-mentioned E. coli.

  According to the present invention, there is provided Escherichia coli in which proteins are presented on the surface layer in an active state. In addition, according to the present invention, E. coli that efficiently displays a large protein on the surface layer, which has been considered impossible with E. coli, is provided. Such Escherichia coli can be cultivated in a large amount and can be industrially used depending on the use of the presented protein.

  The Escherichia coli of the present invention is Escherichia coli transformed so as to display the protein in an active state and encodes a promoter sequence, a DNA encoding polys-γ-glutamate biosynthetic enzyme pgsA, and the protein. Recombinant DNA containing DNA in this order is incorporated into the E. coli. Here, the “active state” means not only that the protein is presented on the surface layer but also that the protein can exhibit the desired activity. In the Escherichia coli of the present invention, the N-terminal side of the target protein is expressed as a fusion protein linked only to the C-terminal side of pgsA, and both ends of the protein are not connected to the surface layer. Therefore, the target protein can take a relatively stable three-dimensional structure and can sufficiently exhibit its activity.

  The protein that can be presented on the surface layer of E. coli of the present invention is not particularly limited, and is preferably a protein that is not originally localized on the cell surface layer of E. coli and is arranged for the purpose of fixing to the cell surface layer of E. coli. For example, secretory proteins, foreign proteins (antibodies, ligands, etc.) and the like can be mentioned. Examples of secreted proteins include amylases, cellulases, lipases and the like.

  In this specification, lipase refers to a protein (enzyme) having an activity capable of liberating fatty acids from fats and oils. Here, the fats and oils are those in which fatty acids are bound to glycerol. If it has such activity, the origin of lipase will not be specifically limited, Generally, the lipase derived from microorganism, a plant, and an animal (for example, porcine pancreas) is used. The lipase may be 1,3-specific or non-specific. Lipases are also used industrially for transesterification, and examples of such lipases include lipase B derived from Candida antarctica CBS6678 strain, lipase derived from Risopus oryzae, and the like.

  In the present invention, a protein having a molecular weight of 60 kDa or more may be used. As described above, it was thought that it was difficult to display a large protein on the surface of E. coli, but in the present invention, surface display is possible even if the protein is 60 kDa or more.

  Examples of the protein having a molecular weight of 60 kDa or more used in the present invention include amylases (such as glucoamylase and α-amylase) and cellulases (such as endoglucanase, exocellobiohydrolase and β-glucosidase).

  In the present specification, amylases refer to enzymes that hydrolyze starch. Typical examples include glucoamylase and α-amylase, and also β-amylase and isoamylase.

  As used herein, glucoamylase refers to an exo-type hydrolase that cleaves glucose units from the non-reducing end of starch, and usually has a molecular weight of about 65.1 kDa. As long as it has such activity, its origin is not limited, but fungal glucoamylases such as Rhizopus and Aspergillus are preferred. For example, as described in Ueda et al. (Appl. Environ. Microbiol. 63: 1362-1366 (1997)), a glucoamylase derived from Rhizopus oryzae is preferably used.

  In the present invention, α-amylase refers to an endo-type enzyme that hydrolyzes the α1,4-glucoside bond of starch, and usually has a molecular weight of about 77.3 kDa. If it has this activity, the origin will not be limited, For example, (alpha) -amylase derived from an animal (saliva, pancreas, etc.), a plant (malt etc.), and microorganisms is used. Examples of the α-amylase derived from microorganisms include those derived from Bacillus stearothermophilus, Streptococcus bovis, and the like. For example, when non-cooked starch is used as the carbon source, α-amylase derived from Streptococcus bovis is preferable.

  In this specification, cellulases refer to enzymes that hydrolyze cellulose. Representative examples include endoglucanase and exocellobiohydrolase, and other examples include β-glucosidase.

  In this specification, endoglucanase refers to an endo-type enzyme that hydrolyzes the β1,4-glucoside bond of cellulose, and usually has a molecular weight of about 77.8 kDa. The origin is not limited as long as it has this activity, and insect (termite etc.), mold-derived and microorganism-derived endoglucanases are used. Examples of the endoglucanase derived from microorganisms include those derived from Clostridium cellulovorans, Clostridium thermocellum, and the like, and examples of mold derived endoglucanases derived from Trichoderma reesei and the like.

  In the present invention, exocellobiohydrolase refers to an exo-type hydrolase that cleaves cellobiose units from the non-reducing end of cellulose, and usually has a molecular weight of about 77.2 kDa. As long as it has such activity, its origin is not limited and mold-derived and microorganism-derived exo-cellobiohydrolase is used, but mold-derived exo-cellobiohydrolase is preferable. For example, as described in Fujita et al. (Appl. Environ. Microbiol. 70: 1207-1212 (2004)), an exocellobiohydrolase derived from Trichoderma reesei is preferably used.

  In the present specification, β-glucosidase refers to an enzyme that hydrolyzes cellooligosaccharide produced by hydrolysis of β1,4-glucoside bond of cellulose into glucose units, and its molecular weight is usually about 91.1 kDa. The origin is not limited as long as it has this activity, but β-glucosidase derived from insects (such as termites), plants (such as almonds), and fungi is used. Examples of mold-derived β-glucosidase include those derived from Aspergillus aculeatus.

  DNA encoding such a protein of interest is, for example, a method of isolating from a living organism; a method of selecting from genomic DNA of an organism using an appropriate probe or primer; or a chemical synthesis based on a known sequence It can be obtained by a method.

  The promoter sequence used in the present invention is DNA homologous to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof, and exhibits constitutive promoter activity in the E. coli.

  Here, the “promoter sequence” is 20-30 base pairs upstream from the transcription start point (+1) and has a function of causing RNA polymerase to start transcription from an accurate position, or similar to the TATA box. It refers to the sequence of the region, and in addition to this sequence, it may contain a sequence necessary for protein other than RNA polymerase to associate for regulation of expression.

  Since the promoter sequence used in the present invention is localized upstream of the ORF of the D-amino acid aminotransferase derived from Bacillus sp. SK-1 strain as disclosed in Patent Document 5, and is constitutive. The target gene can be efficiently expressed without inducing gene expression with an inducer. Specifically, the promoter sequence is (A) a base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, and (B) a substitution or deletion of at least one base in the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. Or (C) the base sequence of DNA that hybridizes under stringent conditions to the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, and (D) the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2. One base sequence selected from the group consisting of base sequences having at least 80% sequence identity to the described base sequences. A fragment of the above DNA may be used as long as it exhibits constitutive promoter activity in the host E. coli.

  Here, “stringent conditions” refers to the following conditions, for example. For example, 6 × SSC (1 × SSC is 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhard't (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% Ficoll) 400), and 100 μg / mL salmon sperm DNA in a solution containing 50 ° C. overnight.

  “Sequence identity” refers to the sequence similarity of residues between two polynucleotides, and is determined by comparing two base sequences that are optimally aligned over the region of the base sequence being compared. obtain. The sequence identity number (percentage) determines the number of matching sites by determining the same nucleobase present in both sequences, and then calculating the number of matching sites by the number of bases in the sequence region to be compared. It can be calculated by dividing by the total number and multiplying the resulting value by 100. Algorithms for obtaining optimal alignment and homology include various algorithms that are usually available to those skilled in the art (eg, BLAST algorithm, FASTA algorithm, etc.). Sequence identity between nucleic acids is measured using, for example, sequence analysis software (for example, BLASTN, FASTA, etc.).

  Poly-γ-glutamic acid biosynthetic enzyme (pgsBCA) is a cell membrane protein involved in the synthesis of poly-γ-glutamic acid, which is a thread of natto, and consists of 393 amino acids pgsB, 149 amino acids pgsC, and 380 amino acids pgsA. It is an enzyme complex. Although the functions of each have not yet been elucidated, pgsA has hydrophilic amino acid sequences at the N-terminus and C-terminus, which are used as a secretion signal, targeting signal, and adhesion signal upon passage through the intracellular membrane. It is thought to work.

  It is known that a protein having a relatively small size can be expressed on the surface of Escherichia coli using an expression vector having DNA encoding pgsA (Patent Document 4). In the present invention, this pgsA-encoding DNA is used in order to present a larger size protein on the cell surface.

  In the present invention, the above pgsA is Bacillus sp. It is preferably derived from. More preferably, the DNA encoding pgsA has the base sequence set forth in SEQ ID NO: 3; the base sequence set forth in SEQ ID NO: 3; the base sequence having at least one base substitution, deletion, or addition; A base sequence of DNA that hybridizes to the base sequence described under stringent conditions; or a base sequence having at least 80% sequence identity to the base sequence set forth in SEQ ID NO: 3. Here, stringent conditions and sequence identity are as described above.

  The above-mentioned promoter sequence, DNA encoding pgsA, and recombinant DNA containing DNA encoding the protein of interest in this order may be introduced into the host in the form of an expression vector, or inserted into the host gene. Alternatively, homologous recombination with the host gene may occur and be incorporated into the chromosome. In the recombinant DNA, the DNA encoding pgsA and the DNA encoding the target protein are arranged so as to be expressed as a fusion protein in which the C-terminal side of pgsA and the N-terminal side of the target protein are linked. pgsA and the target protein may be directly linked or may be linked via a spacer consisting of several amino acids to several tens of amino acids. DNA encoding such a fusion protein is placed under the control of the promoter. These various types of DNA and recombinant DNA can be synthesized, combined, and inserted into an expression vector by techniques that can be commonly used by those skilled in the art.

  Examples of expression vectors into which the above recombinant DNA is inserted include plasmid vectors and phage vectors. Examples of plasmid vectors include pUC18, pUC19, pBluescript, pET and the like. Examples of phage vectors include λ phage vectors such as λgt10 and λgt11.

  Such an expression vector has an origin of replication and preferably has, for example, a selection marker for E. coli. Examples of selection markers include ampicillin resistance gene, kanamycin resistance gene, chloramphenicol resistance gene, and the like. Further, in order to efficiently express the structural gene of the protein, it is desirable to further include a so-called regulatory sequence such as an operator, terminator, enhancer, etc. that regulates the expression of this gene.

  The Escherichia coli of the present invention can be obtained by introducing the above recombinant DNA or expression vector. The introduction of DNA means that DNA is introduced into E. coli and expressed. Methods for introducing DNA include methods such as transformation, transduction, transfection, co-transfection, electroporation, and heat shock, and specifically include methods using lithium acetate and protoplast methods. .

  The type of E. coli used as a host in the present invention is not particularly limited, but is preferably a non-pathogenic strain, and more preferably a commercially available strain that is usually used in the field of genetic engineering. Examples include Escherichia coli K-12 strain HB101 strain, C600 strain, JM109 strain, DH5α strain, DH10B strain, XL-1BlueMRF ′ strain, TOP10F strain, and the like.

  E. coli into which the recombinant DNA has been introduced is selected by culturing in an appropriate medium, selecting with a selectable marker, and measuring the activity of the expressed protein. Whether the protein is immobilized on the cell surface can be confirmed by an immunoantibody method using an anti-protein antibody and a FITC-labeled anti-IgG antibody.

  The Escherichia coli of the present invention obtained as described above is used for the purpose of bringing the target protein displayed on the cell surface into contact with the substrate and binding the substrate or converting the substrate into another substance. obtain. For example, when α-amylase is displayed on the surface, starch can be liquefied and saccharified by anaerobically culturing the E. coli in a medium containing starch. After sufficiently saccharifying the starch in the medium, lactic acid fermentation can be carried out by culturing the E. coli aerobically. Alternatively, for example, when lipase is displayed on the surface, this E. coli cell can be used as a biocatalyst for a transesterification reaction.

  When the protein is an enzyme, the Escherichia coli of the present invention may be provided as an enzyme agent containing E. coli that displays the enzyme on the surface. Specifically, the enzyme agent of the present invention includes a suspension containing the Escherichia coli of the present invention and a medium capable of maintaining the Escherichia coli, or the Escherichia coli of the present invention which is cryopreserved or freeze-dried, and further dried at low temperature. Is mentioned.

  Furthermore, the E. coli of the present invention can be used as a combinatorial library by presenting various proteins (enzymes, antibodies, ligands, etc.) into which mutations are randomly introduced on the surface of different E. coli. From such a library, it is also possible to quickly and easily select a clone having the optimum properties according to the purpose. The combinatorial library herein refers to a collection of various mutants in which mutations are randomly introduced at the gene level and the diversity of proteins is artificially expanded.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this Example.

(Example 1: Preparation of E. coli displaying α-amylase on the cell surface)
(1) Construction of a vector for displaying amylase on the cell surface of Escherichia coli (see FIG. 1)
First, a plasmid pHCE1LB-pgaBCA (obtained from Bioleaders Corporation) containing the gene for pgsA (SEQ ID NO: 3), which is one of poly-γ-glutamate biosynthetic enzymes, was used as a template, and the primers of SEQ ID NOs: 4 and 5 were used. PCR was performed. The obtained fragment was digested with restriction enzymes PvuII and HindIII to obtain a PvuII-HindIII fragment.

  The PvuII-HindIII fragment of this pgsA gene was inserted into restriction sites EcoRV and HindIII of the plasmid pHCE1LB (obtained from Bioleaders Corporation) and ligated with a Ligation-Convenience Kit (manufactured by Nippon Gene Co., Ltd.). pHLA was obtained.

  Next, an α-amylase gene (amyA; SEQ ID NO: 6) derived from Streptococcus bovis 148 strain (E. Satoh et al., Appl. Environ. Microbiol. 63: 4593-4596 (1997)) was inserted into plasmid pQE31 (Qiagen). PCR was performed using the plasmid pQE31-amyA as a template and the primers of SEQ ID NOs: 7 and 8. The obtained fragment was digested with restriction enzymes BamHI and HindIII to obtain a BamHI-HindIII fragment.

  The BamHI-HindIII fragment of this amyA gene is inserted into the cleavage sites of the restriction enzymes BamHI and HindIII of the plasmid pHLA, and ligated by a Ligation-Convenience Kit (manufactured by Nippon Gene Co., Ltd.), thereby having pgsA-amyA in this order. Thus, the target amylase display vector pHLAαAF was obtained. This vector was digested with restriction enzymes BamHI and HindIII, the introduction of the amyA gene was confirmed, and the base sequence was confirmed using ABI PRISM (registered trademark) 310 Genetic Analyzer (manufactured by Applide Biosystems).

(2) Preparation of E. coli presenting α-amylase on cell surface layer The vector pHLAαAF obtained in (1) above was transformed into E. coli NovaBlue (Novagen) competent cells by the heat shock method. The obtained transformant was added to Luria-Bertani (hereinafter referred to as LB) agar medium (pepton 1 w / v%, yeast extra) containing 100 μg / mL ampicillin and 0.25 w / v% remazole brilliant blue starch (Nacalai Tesque). 0.5 w / v%, sodium chloride 0.5 w / v%, and agar 1.5 w / v%) and cultured at 37 ° C. for 24 hours. A strain in which halo formed by the degradation of Remazole brilliant blue starch surrounding the colony was selected was selected.

  Subsequently, the selected transformant is cultured at 37 ° C. in a LB medium containing soluble starch 20 g / L using a microorganism culture apparatus BMJ-PI (manufactured by Able Co., Ltd. and Biot Co., Ltd.) for up to 48 hours. Sampled over time. The culture medium was separated into a medium and cells by centrifugation, and each α-amylase activity was measured. E. coli NovaBlue was used as a control. The α-amylase activity was performed according to a protocol using an α-amylase measurement kit (manufactured by Kikkoman Corporation).

The transformed E. coli had α-amylase activity. As shown in FIG. 2, the highest activity was exhibited 48 hours after culturing. In FIG. 2, 1U of α-amylase activity is N3-G5-β-CNP (synthetic substrate 2-chloro-4-nitrophenyl 6 ⁵ -azido-6 ⁵ -deoxy-β-maltopentaoside) per minute. Represents the titer of liberating 1 μmol of CNP (2-chloro-4-nitrophenol). On the other hand, almost no α-amylase activity was observed in the culture supernatant of the transformant.

(Example 2: Preparation of E. coli displaying lipase on cell surface)
(1-1) Construction of a vector for displaying lipase on the cell surface of E. coli First, a plasmid pWIFS-CALB containing a lipase B gene (CALB; SEQ ID NO: 9) derived from Candida antarctica CBS6678 strain was used as a template. PCR was performed using 11 primers. The obtained fragment was digested with restriction enzymes SalI and XhoI to obtain a SalI-XhoI fragment.

  By inserting the SalI-XhoI fragment of this CALB gene into the restriction sites SalI and XhoI of the plasmid pHLA and ligating them with a Ligation-Convenience Kit (manufactured by Nippon Gene Co., Ltd.), pgsA-CALB is obtained in this order. The target lipase display vector pHLACALB was obtained. This vector was digested with restriction enzymes SalI and XhoI, the introduction of the CALB gene was confirmed, and the base sequence was confirmed using ABI PRISM (registered trademark) 310 Genetic Analyzer (manufactured by Applide Biosystems).

(1-2) Construction of vector for ROL display on cell surface of E. coli First, plasmid pWIFSProROL (T. Matsumoto et al., Appl. Environ. Microbiol. 68) containing Rhizopus oryzae lipase gene (ROL; SEQ ID NO: 12). : 4517-4522 (2002)) as a template, and PCR was performed using the primers of SEQ ID NOS: 13 and 14. The obtained fragment was digested with restriction enzymes BamHI and SalI to obtain a BamHI-SalI fragment.

  The BamHI-SalI fragment of this ROL gene is inserted into the restriction enzyme BamHI and SalI cleavage sites of the plasmid pHLA, and ligated with a Ligation-Convenience Kit (manufactured by Nippon Gene Co., Ltd.), thereby having pgsA-ROL in this order. The target lipase display vector pHLAROL was obtained. This vector was digested with restriction enzymes BamHI and SalI, the introduction of the ROL gene was confirmed, and the base sequence was confirmed using ABI PRISM (registered trademark) 310 Genetic Analyzer (Applide Biosystems).

(2) Preparation of E. coli presenting lipase B on the cell surface The vectors pHLACALB and pHLAROL obtained in (1-1) and (1-2) above were transferred to E. coli NovaBlue (Novagen) competent cells by the heat shock method. Transformed. The obtained transformant was spread on an LB agar medium containing 100 μg / mL ampicillin, 1 w / v% Gall powder (bile powder: Sigma), and 1 v / v% tri-n-butyrin, The cells were cultured at 37 ° C for 24-48 hours. A strain in which halo was confirmed around the colony was selected.

  The selected transformants were then cultured in LB liquid medium at 37 ° C. and sampled over time up to 48 hours. The culture medium was separated into a medium and cells by centrifugation, the cells were washed, frozen at −80 ° C., and then freeze-dried using a vacuum dryer. The obtained freeze-dried cells were crushed and the lipase activity of each was measured. E. coli NovaBlue was used as a control. The lipase activity was performed using a lipase kit S (manufactured by Marco Pharmaceutical Co., Ltd.) according to the protocol.

  The transformed E. coli had lipase activity. As shown in FIG. 3A, CALB increased in activity up to 48 hours after culture. In addition, as shown in FIG. 3B, ROL retained high activity until 48 hours after culturing. In FIG. 3, 1 IU of lipase activity is defined as the titer that liberates 1 μmol of SH groups from the substrate per minute at 30 ° C. On the other hand, almost no lipase activity was observed in the culture supernatant of this transformant.

(Example 3: Examination of optical resolution by Escherichia coli presenting lipase on the cell surface)
Since CALB is a lipase that is relatively frequently used for the synthesis of organic compounds (especially pharmaceutical intermediates, etc.), the optical resolution reaction was examined as follows. Escherichia coli presenting lipase B obtained in Example 2 (CALB surface-displaying E. coli) was cultured in an LB liquid medium at 37 ° C. for 24 hours. The culture medium was centrifuged to separate and wash the cells, frozen at −80 ° C., and then lyophilized using a vacuum dryer. The obtained freeze-dried microbial cells were crushed and used as whole-cell biocatalyst in the optical resolution reaction.

  The optical resolution reaction in this example is shown in the following formula. That is, (R) -1-phenylethyl acetate (R-PEA) is obtained from (RS) -1-phenylethanol (RS-1-PE) and vinyl acetate monomer (VA) by transesterification with lipase. It is a reaction.

  In 5 mL of n-hexane, 50 mg of (RS) -1-phenylethanol, 35.3 mg of vinyl acetate monomer, 0.5 g of molecular sieve 4A beads (manufactured by Nacalai Tesque), and 50 mg of crushed lyophilized cells The containing reaction solution was incubated at 37 ° C. with shaking at 150 rpm. The concentration of RS-1-PE and VA in the reaction solution was 80 mM. Sampling was performed over time up to 25 hours, and the production amount (yield) of R-form and S-form 1-phenylethyl acetate was measured by HPLC.

  The results are shown in FIG. As shown in FIG. 4, the CALB surface-displayed E. coli catalyzed the optical resolution reaction. At 25 hours, the yield of R-PEA reached 76.3% and the enantiomeric excess reached 98.2%. CALB is a lipase that is difficult to express on the surface of yeast, but it was found from this example that it can be expressed on the surface with a practical level of activity in E. coli.

  According to the present invention, there is provided Escherichia coli in which proteins are presented on the surface layer in an active state. In addition, according to the present invention, there is provided Escherichia coli that presents a large foreign protein on the surface, which has been considered impossible with Escherichia coli. Such Escherichia coli can be cultivated in a large amount and can be industrially used depending on the use of the presented protein. In particular, since the protein is displayed on the surface in an active state, for example, when the protein is a lipase, it can be used very effectively as a catalyst for optical resolution in the production of a pharmaceutical raw material compound.

It is a schematic diagram of construction of a vector for displaying amylase on the cell surface of E. coli. It is a graph which shows the time-dependent change of the alpha-amylase activity of transformed Escherichia coli. It is a graph which shows a time-dependent change of the lipase activity of colon_bacillus | E._coli which displayed CALB on the surface. It is a graph which shows a time-dependent change of the lipase activity of colon_bacillus | E._coli which displayed ROL surface layer. It is a graph which shows a time-dependent change of the optical resolution reaction using colon_bacillus | E._coli which displayed CALB on the cell surface layer.

Claims (12)

E. coli that presents protein in an active state on the surface,
The E. coli has a promoter sequence, a DNA encoding pgsA, a poly-γ-glutamate biosynthetic enzyme, and a recombinant DNA comprising DNA encoding the protein in this order, and the promoter sequence is (A (B) a base sequence having at least one base substitution, deletion, or addition in the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, or (C) At least 80% sequence identity to the nucleotide sequence of DNA that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and (D) the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 A base sequence selected from the group consisting of a sexual sequence, or a fragment thereof, and a constitutive Exhibit Ta activity,
E. coli.   The pgsA is a Bacillus sp. The Escherichia coli according to claim 1, which is derived from Escherichia coli.   The DNA encoding pgsA is a base sequence shown in SEQ ID NO: 3; a base sequence having at least one base substitution, deletion, or addition in the base sequence shown in SEQ ID NO: 3; a base shown in SEQ ID NO: 3 The Escherichia coli according to claim 1 or 2, which is a base sequence of DNA that hybridizes to the sequence under stringent conditions; or a base sequence having at least 80% sequence identity to the base sequence set forth in SEQ ID NO: 3. .   The E. coli according to any one of claims 1 to 3, wherein the protein is an enzyme.   The E. coli according to claim 4, wherein the enzyme is α-amylase.   The E. coli according to claim 4, wherein the enzyme is a lipase. A method for expressing a protein in an active state on the surface of E. coli,
(A) a step of constructing a promoter sequence, a DNA encoding pgsA which is a poly-γ-glutamate biosynthetic enzyme, and a recombinant DNA containing DNA encoding the protein in this order, the promoter sequence comprising: (A) the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, (B) the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, having at least one base substitution, deletion, or addition, C) at least 80% of the base sequence of DNA that hybridizes under stringent conditions to the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and (D) the base sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 A base sequence selected from the group consisting of a base sequence having sequence identity, or a fragment thereof, and constitutive in said E. coli Exhibit promoter activity, process;
(B) transforming E. coli with the recombinant DNA; and (c) culturing the transformed E. coli and expressing the protein on the surface of the E. coli,
Including the method.   The method according to claim 7, wherein the protein is an enzyme.   The method for using Escherichia coli according to any one of claims 1 to 6, comprising a step of bringing the protein into contact with a substrate and binding the substrate or converting the substrate into another substance. ,Method.   The method according to claim 9, wherein the protein is α-amylase, the substrate is starch, and the bacterium is aerobically cultured to produce lactic acid.   10. The method according to claim 9, wherein the protein is a lipase, the substrate is a racemic alcohol and an ester, and enantioselectively produces an alcohol ester using the Escherichia coli as a catalyst for a transesterification reaction. the method of.   An enzyme agent comprising the Escherichia coli according to any one of claims 4 to 6.

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