Protein, polynucleotide, expression vector, transformant, composition containing plasmalogen and preparation method of plasmalogen

Abstract

A protein comprising an amino acid sequence selected from the group consisting of (a), (b), (c), and (d) described below, and having a plasmalogen synthesis activity in a bacterium expressing the protein, (a) an amino acid sequence as shown in SEQ ID NO.1, (b) an amino acid sequence with more than 80% of sequence identity relative to the amino acid sequence as shown in SEQ ID NO.1, (c) an amino acid sequence as shown in SEQ ID NO.2, and (d) an amino acid sequence with more than 80% of sequence identity relative to the amino acid sequence as shown in SEQ ID NO.2.

Description

Technical Field

[0001] The present invention relates to a protein, a polynucleotide, an expression vector, a transformant, a composition containing plasmalogen and a method for preparing the plasmalogen. Background Art

[0002] Plasmalogen is a type of phospholipid with antioxidant effect and one of the glycerophospholipids. Plasmalogen exists in all tissues of mammals and accounts for about 18% of human phospholipids. It is well known that plasmalogens are particularly abundant in cranial nerves, myocardium, skeletal muscle, white blood cells and sperm.

[0003] Since many plasmalogens are combined with polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid, they participate in the storage of polyunsaturated fatty acids and the release of second messengers of intercellular signals such as prostaglandins and leukotrienes produced by these polyunsaturated fatty acids. In addition, plasmalogens are also involved in cell fusion and ion transfer. Since the vinyl ether bond (alkenyl bond) of plasmalogens is particularly sensitive to oxidative stress, plasmalogens also play an antioxidant function of cells.

[0004] In addition, it is known that plasmalogens have effects on promoting neurogenesis, suppressing neuroinflammation caused by lipopolysaccharide (LPS), and suppressing accumulation of amyloid β (Aβ) protein in the brain, etc. For example, Patent Document 1 discloses an anti-central nervous system inflammatory agent containing plasmalogens.

[0005] As is well known, except for cranial nerve diseases such as dementia, Parkinson's disease, depression and schizophrenia, plasmalogen decreases in diabetes, metabolic syndrome, ischemic heart disease, infectious diseases and immune abnormalities. For example, in non-patent literature 1, it is reported that ethanolamine type plasmalogen significantly decreases in the frontal lobe and hippocampus of the brain suffering from Alzheimer's disease. In addition, in non-patent literature 2, it is reported that plasmalogen decreases in the serum of Alzheimer's patients. In non-patent literature 3, it is reported that choline type plasmalogen decreases compared with a normal control group in an ischemic heart disease patient group.

[0006] It is considered that the prevention and improvement effects of these diseases can be expected by externally supplementing the reduced plasmalogens. Therefore, in the future, the demand for plasmalogens for preventing and treating diseases or maintaining health is expected to increase.

[0007] Plasmalogens are widely present in invertebrates and vertebrates, but have not yet been found in fungi and plant cells. In bacteria, it is reported that some obligate anaerobes such as Enterococcus faecalis disclosed in Non-Patent Document 4 and Bifidobacterium longum disclosed in Patent Document 2 synthesize plasmalogens, but plasmalogens have not been found in facultative anaerobic bacteria and aerobic bacteria (see Non-Patent Document 5).

[0008] Furthermore, in Non-Patent Document 4, two plasmalogen synthase genes (plsA, plsR) were identified, and it was reported that no plasmalogen was detected when expression of plasmalogen synthase of Clostridium perfringens was induced in aerobic cultured Escherichia coli.

[0009] Prior art literature

[0010] Patent Literature

[0011] (Patent Document 1) International Publication No. 2012 / 039472

[0012] (Patent Document 2) International Publication No. 2021 / 024872

[0013] (Non-patent document 1) Z.Guan et al, Decrease and Structural Modifications ofPhosphatidylethanolamine Plasmalogen in the Brain with Alzheimer Disease, JNeuropathol Exp Neurol, 58(7),740-747,1999

[0014] (Non-patent document 2) DB Goodenowe et al, Peripheral ethanolamine plasm alogendeficiency: a logical causative factor in Alzheimer's disease and dementia, JLipid Res, 48, 2485-2498, 2007

[0015] (Non-patent document 3) Takeo Sanada et al., Dynamics of serum plasmalogen in ischemic heart disease, Arteriosclerosis, 11, 535-539, 1983

[0016] (Non-patent document 4) David R Jackson et al, Plasmalogen Biosynthesis by Anaerobic Bacteria: Identification of a Two-Gene Operon Responsible for Plasmalogen Production in Clostridium perfringens, ACS Chemical Biology, 11, 12, 3421-3430, 2021

[0017] (Non-patent document 5) Howard Goldfine, The appearance, disappearance and reappearance of plasmalogens in evolution, Progress in Lipid Research, 49, 4, 493-498, 2010 Summary of the invention

[0018] Technical problem to be solved by the invention

[0019] If plasmalogens can be biosynthesized by bacteria, then plasmalogens can be easily and in large quantities obtained by the culture of bacteria. However, as mentioned above, when plasmalogens are biosynthesized by obligate anaerobic bacteria, it is necessary to culture bacteria under anaerobic conditions. Cultivation under anaerobic conditions requires a device that does not allow oxygen to enter, and when the culture scale becomes large, it becomes particularly difficult and complicated to maintain anaerobic conditions.

[0020] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a protein, a polynucleotide, an expression vector, a transformant, a composition containing a plasmalogen, and a method for producing a plasmalogen for biosynthesis of a plasmalogen by aerobic culturable bacteria.

[0021] Invent solutions to problems

[0022] The present inventors have found that plasmalogen can be synthesized under aerobic conditions by introducing a plasmalogen synthase gene (plsA) derived from anaerobic bacteria into aerobic bacteria, thereby completing the present invention.

[0023] The protein according to the first aspect of the present invention is a protein comprising an amino acid sequence selected from the group consisting of (a), (b), (c) and (d) below, and has plasmalogen synthesis activity in bacteria expressing the protein.

[0024] (a) Amino acid sequence shown in SEQ ID NO.1

[0025] (b) an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO.1

[0026] (c) Amino acid sequence shown in SEQ ID NO.2

[0027] (d) an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO.2

[0028] The polynucleotide according to the second aspect of the present invention comprises a base sequence encoding the protein according to the first aspect of the present invention.

[0029] The expression vector according to the third aspect of the present invention comprises the polynucleotide according to the second aspect of the present invention.

[0030] The transformant according to the fourth aspect of the present invention comprises the above-mentioned expression vector according to the third aspect of the present invention.

[0031] The composition containing plasmalogen according to the fifth aspect of the present invention comprises the transformant according to the fourth aspect of the present invention or a processed product thereof.

[0032] The method for preparing plasmalogen according to the sixth aspect of the present invention comprises: a culturing step of culturing the transformant according to the fourth aspect of the present invention under aerobic conditions; and an obtaining step of obtaining plasmalogen from the transformant.

[0033] Effects of the Invention

[0034] According to the present invention, plasmalogen can be biosynthesized by aerobically culturable bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a diagram showing the structure of PlsA derived from Enterococcus faecalis K4 strain.

[0036] Figure 2A This is a diagram showing a CBB staining image in Example 1.

[0037] Figure 2B This is a diagram showing a Western blotting image in Example 1.

[0038] Figure 3A This is a diagram showing the chromatogram of the control Escherichia coli (BL21)-derived sample in Example 2.

[0039] Figure 3B This is a diagram showing a chromatogram of the transformed Escherichia coli-derived sample obtained in Example 1.

[0040] Figure 4A This is a diagram showing the chromatogram of the sample before the phospholipase A1 (PLA1) treatment in Example 3.

[0041] Figure 4B This is a diagram showing the chromatogram of the sample after the PLA1 treatment in Example 3.

[0042] Figure 4C This is a diagram showing the chromatogram of the sample treated with hydrochloric acid (HCl) after the PLA1 treatment in Example 3.

[0043] Figure 5A This is a diagram showing a Western blot image in Example 4.

[0044] Figure 5B This is a graph showing the relative activity of pAMPK / AMPK in Example 4.

[0045] Figure 6 This is a diagram showing cells after OilRed O staining in Example 5.

[0046] Figure 7 The figure shows the Western blot image in Example 6.

[0047] Figure 8 This is a diagram showing the total lipid and aldehyde-related chromatograms in Example 7. DETAILED DESCRIPTION

[0048] Embodiments according to the present invention are described with reference to the accompanying drawings. In addition, the present invention is not limited to the following embodiments and the accompanying drawings. In addition, in the following embodiments, expressions such as "having", "including" or "containing" also include the meaning of "consisting of" or "composed of".

[0049] (Implementation Method 1)

[0050] The protein according to the present embodiment consists of an amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, or an amino acid sequence having a sequence identity of 80% or more with respect to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2. The protein has a plasmalogen synthesis activity that mediates the biosynthesis of plasmalogen in bacteria expressing the protein, preferably in bacteria cultured aerobically. A protein having an amino acid sequence shown in SEQ ID NO.1 was identified in Enterococcus faecalis K4 strain. A protein having an amino acid sequence shown in SEQ ID NO.2 was identified in Bifidobacterium longum subspecies suis.

[0051] When two amino acid sequences are aligned to maximize their consistency, the ratio of the number of sites where the same amino acid exists in the two amino acid sequences to the number of full-length amino acids is called sequence identity. In addition, the ratio of the number of sites where the same amino acid exists and the number of sites where similar amino acids exist to the number of full-length amino acids is called sequence similarity.

[0052] "Similar amino acid" refers to another amino acid having a physicochemically similar side chain (conservative amino acid substitution). Amino acids having physicochemically similar side chains are classified, for example, as follows.

[0053] Aliphatic side chains: glycine (G), alanine (A), valine (V), leucine (L), and isoleucine (I)

[0054] Aliphatic-hydroxy side chain: serine (S) and threonine (T)

[0055] Contains amide side chains: asparagine (N) and glutamine (Q)

[0056] Aromatic side chains: phenylalanine (F), tyrosine (Y), and tryptophan (W)

[0057] Basic side chains: lysine (K), arginine (R), and histidine (H)

[0058] Acidic side chains: Aspartic acid (D) and Glutamic acid (E)

[0059] Sulfur-containing side chains: cysteine ​​(C) and methionine (M)

[0060] In addition, similar amino acids can be amino acids whose scores based on a matrix of similarity such as PAM250 used in sequence analysis tools such as Clustal W are greater than a predetermined threshold. When PAM250 is used, amino acids with strong similarity, such as amino acids whose scores between amino acids are greater than 0.5, are amino acids classified as the same group in the following groups 1 to 9.

[0061] Group 1 S, T and A

[0062] Group 2 N, E, Q and K

[0063] Group 3 N, H, Q and K

[0064] Group 4 N, D, E and Q

[0065] Group 5 Q, H, R and K

[0066] Group 6 M, I, L and V

[0067] Group 7M, I, L and F

[0068] Group 8 H and Y

[0069] Group 9 F, Y and W

[0070] Furthermore, as a matrix for defining similarity, a well-known matrix may be used, and in addition to PAM, BLOSUM or the like may also be used.

[0071] "80% or more sequence identity" with respect to an amino acid sequence means at least 80% sequence identity. The amino acid sequence of the protein may have 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95% or more, further more preferably 98% or more, and particularly preferably 99% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO.1, as long as it has plasmalogen synthesis activity.

[0072] When the protein according to the present embodiment comprises an amino acid sequence having a sequence identity of 80% or more with respect to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, as long as the protein has the above-mentioned plasmalogen synthesis activity, in addition to the amino acid sequence for improving translation efficiency, it may also have an amino acid sequence for protein purification and an amino acid sequence for improving the expression efficiency of a molecular chaperone, etc. As the amino acid sequence for protein purification, for example, a histidine tag, a SUMO tag, a glutathione-S-transferase, and a maltose binding protein can be cited. The amino acid sequence for improving translation efficiency, the amino acid sequence for purification, and the amino acid sequence for improving expression efficiency are added to at least one side of the N-terminus and the C-terminus. The amino acids added to the N-terminus or the C-terminus are, for example, several, preferably 1 to 9.

[0073] Next, the method for preparing the protein according to the present embodiment is described. The protein can be chemically synthesized by known methods such as solid phase method and liquid phase method according to its amino acid sequence. As shown below, the protein is preferably prepared by using genetic engineering and molecular biology methods.

[0074] First, determine the base sequence of the polynucleotide encoding the protein. The base sequence is determined by the codons corresponding to the amino acids. Based on the correspondence between amino acids and codons, many base sequences encoding the amino acid sequence are determined from an amino acid sequence. A computer can be used to determine the base sequence. If the codon usage frequency of the host used when expressing the gene encoding the amino acid sequence is taken into consideration, the appropriate base sequence encoding the protein can be determined. The polynucleotide can be synthesized by a known method based on the base sequence. In addition, the polynucleotide can be obtained as an amplification product obtained by PCR (Polymerase Chain Reaction) using the cDNA of Enterococcus faecalis (E.faecalis) K4 strain or Bifidobacterium longum (B.longum) suis subspecies as a template.

[0075] Then, an expression vector comprising a polynucleotide encoding a protein is prepared. As long as the expression vector can express the polynucleotide, there is no particular limitation. Preferably, the expression vector comprises a promoter sequence, an operator sequence, and a ribosome binding site (RBS) etc. to which the RNA polymerase expressing the added polynucleotide binds. In the expression vector, the above-mentioned polynucleotide can be configured under the control of the promoter sequence. As a promoter sequence, known promoter sequences such as the T7 promoter can be utilized. As required, a restriction enzyme site for cloning can be added to the 5' end and the 3' end of the polynucleotide encoding the protein, or a base sequence encoding a histidine tag, a SUMO tag etc. can be added to the 5' end of the polynucleotide encoding the protein. When a restriction enzyme is used, the base sequence of the restriction enzyme site contained in the polynucleotide encoding the protein can be replaced synonymously.

[0076] By introducing the above expression vector into a host capable of expressing an RNA polymerase bound to the above promoter sequence, for example, in the presence of an expression inducer, the above protein can be expressed in the host. The host is not particularly limited, but is preferably a prokaryotic cell that can be aerobically cultured. Specifically, Escherichia coli, Bacillus subtilis, yeast, filamentous fungi, plant cells and animal cells can be cited, but from the viewpoint of effectively expressing the above polynucleotide, Escherichia coli is preferably used.

[0077] The Escherichia coli used as a host is not particularly limited, and known Escherichia coli can be used, but it is preferably able to induce the expression of the protein encoded by the above polynucleotide in the presence of an expression inducer. Examples of expression inducers include IPTG, rhamnose, or a combination thereof. Examples of such Escherichia coli include BL21 (DE3) strains, which are lysogens of bacteriophage λDE3 and have, for example, an RNA polymerase gene that expresses the protein in the presence of an expression inducer. The expression vector is preferably able to induce the expression of the above polynucleotide by an RNA polymerase expressed in the presence of an expression inducer. Examples of expression vectors include pET vectors (e.g., pET-21a (+) and pET-24a (+)) and pGEX vectors.

[0078] Next, a transformant is prepared by introducing the expression vector prepared above into a host. The method for introducing the expression vector into the host is not particularly limited, including a method using competent cells prepared by a calcium chloride method and a rubidium chloride method, an electroporation method, and a protoplast method.

[0079] When the expression vector is pET-21a(+), Escherichia coli BL21(DE3) is preferably used as a transformant. The desired transformant can be obtained by confirming the colonies of the transformant by direct PCR or determining the base sequence of its DNA.

[0080] In addition, when the host bacterium colony cannot be obtained, as an expression vector, except for the promoter sequence used in the combination of RNA polymerase expressing protein in the presence of the above-mentioned expression inducing agent, an expression vector also having a gene encoding the RNA polymerase and a gene encoding the lysozyme of the RNA polymerase that suppresses expression in the absence of the expression inducing agent can be used. The gene encoding the RNA polymerase and the gene encoding the lysozyme may not be included in the expression vector, and may be included by the transformed host. At this time, the expression vector can be introduced into a host having a gene encoding the RNA polymerase that expresses in the presence of the expression inducing agent and a gene encoding the lysozyme of the RNA polymerase that suppresses expression in the absence of the expression inducing agent. As such a host, a host having a plasmid having a gene encoding the above-mentioned lysozyme is used, for example, BL21 (DE3) pLysS bacterial strain etc. That is, the transformant has an expression vector comprising a polynucleotide encoding the above-mentioned protein and a promoter sequence bound to an RNA polymerase that expresses the protein encoded by the polynucleotide, a gene encoding an RNA polymerase that is expressed in the presence of an expression inducer, and a gene encoding a lysozyme of the RNA polymerase whose expression is inhibited in the absence of an expression inducer.

[0081] The prepared transformant is preferably cultured under aerobic conditions to synthesize plasmalogen. Aerobic conditions refer to a state in which there is a certain amount of oxygen in the atmosphere. Aerobic conditions also include microaerobic conditions. In particular, aerobic conditions refer to microbial culture without using reagents and devices used in culture under anaerobic conditions.

[0082] The transformant can be cultured by a known method suitable for the transformant. When Escherichia coli is used as the transformant, it can be cultured in LB agar medium or LB liquid medium, for example.

[0083] Plasmalogens belong to a subclass unique to glycerophospholipids, and are characterized in that they have a vinyl ether bond at the sn-1 position of the glycerol backbone and an ester bond at the sn-2 position. The plasmalogen in the present embodiment is not particularly limited as long as it is a glycerophospholipid that is generally classified as a plasmalogen. As plasmalogens, for example, ethanolamine-type plasmalogens (Pls), choline-type plasmalogens (PlsCho), inositol-type plasmalogens, and serine-type plasmalogens can be cited. In addition, Pls has a structure in which -CH2CH22NH2 is bonded to an oxygen atom bonded to phosphorus at the sn-3 position of the glycerol backbone. PlsCho has a structure in which -CH2CH2N(CH3)3 is bonded to an oxygen atom bonded to phosphorus at the sn-3 position of the glycerol backbone. The plasmalogens synthesized by the transformant according to the present embodiment have various numbers of carbon atoms constituting the carbon chain bonded to the oxygen atom at the sn-1 position, and contain plasmalogens having carbon numbers not found in mammals in addition to plasmalogens found in mammals.

[0084] As shown in the following examples, the transformant according to the present embodiment can synthesize plasmalogen even under aerobic conditions. Therefore, plasmalogen can be easily obtained without maintaining the culture atmosphere under anaerobic conditions.

[0085] (Implementation Method 2)

[0086] The plasmalogen-containing composition according to the present embodiment includes the transformant or a processed product thereof according to the above-mentioned embodiment 1. When the host of the transformant is a bacterium, the bacterial cells can be obtained as a precipitate by centrifuging the culture solution after the culture.

[0087] The processed products of the thalline are, for example, freeze-dried thalline, thalline dried with acetone, extracts obtained by subjecting the thalline to solvent extraction treatment, crushed thalline with ultrasound or the like, or broken products obtained by subjecting the thalline to a certain treatment, and compositions derived from the thalline or including a part of the thalline. As solvents for solvent extraction, water, organic solvents and mixtures thereof can be used. As organic solvents, ether, chloroform, benzene, hexane, methanol, ethanol, isopropanol and mixed solutions thereof can be cited. The extract extracted using the extraction solvent can be used as a liquid, or it can be used in the form of a liquid, gel or paste after being concentrated or diluted. In addition, dried products after drying them can also be used. Drying can be carried out by known methods such as spray drying, freeze drying, vacuum drying and fluidized drying.

[0088] The composition containing plasmalogen according to the present embodiment contains the plasmalogen synthesized by the transformant. Therefore, the composition containing plasmalogen is suitable for supplementing plasmalogen in the organism from the outside. For example, the composition containing plasmalogen is used to treat and prevent the disease of plasmalogen reduction. As the disease, for example, inflammatory diseases, inflammatory diseases of the central nervous system, dementia, Parkinson's disease, depression, schizophrenia, diabetes, metabolic syndrome, ischemic heart disease, infectious diseases and immune abnormalities can be enumerated.

[0089] In another embodiment, the composition containing plasmalogen is used as a therapeutic or preventive drug for the above-mentioned diseases in which plasmalogen is reduced. In addition, in other embodiments, the composition containing plasmalogen is used as a therapeutic or preventive drug for the above-mentioned diseases in which plasmalogen is reduced. In addition, in another embodiment, an oral composition for the treatment of a disease in which plasmalogen is reduced is provided, which comprises the bacterial cell of the transformant according to Embodiment 1 or a processed product thereof. As the oral composition, specifically, supplements, food compositions, foods and beverages, functional foods and food additives can be cited.

[0090] In addition, in another embodiment, a method for preparing plasmalogen is provided, which uses the transformant according to embodiment 1 described above. The method for preparing plasmalogen includes a culturing step and an obtaining step. In the culturing step, as described above, the transformant is cultured under aerobic conditions. In the obtaining step, plasmalogen is obtained from the transformant. In the obtaining step, as described above, plasmalogen can be obtained by treating the bacterial cells by solvent extraction and crushing by ultrasonic waves, etc. The plasmalogen obtained in the obtaining step can be purified or separated by a known method, or the purified plasmalogen can be concentrated.

[0091] (Example 1: Preparation of Escherichia coli transformed with a plasmalogen synthase gene derived from Enterococcus faecalis K4 strain)

[0092] [Clone K4 plsA]

[0093] The plsA gene derived from the Enterococcus faecalis K4 strain (hereinafter also referred to as "K4 plsA"), which is a facultative anaerobic lactic acid bacterium, was searched for by the following method.

[0094] The draft genome was analyzed by 250PE (paired-end sequencing) using HiSeq (registered trademark) 2500 (manufactured by Illumina) and Denovo analysis using MinION (registered trademark) (manufactured by Oxford Nanopore Technologies) to create a CDS list. By using the blast analysis, a gene (K4 plsA) that is presumed to be a hydroxyacyl-CoA dehydratase was found.

[0095] According to the above analysis, the amino acid sequence of the plasmalogen synthase protein derived from Enterococcus faecalis K4 strain (hereinafter also referred to as "K4 PlsA protein") is a sequence consisting of 1416 amino acids shown in SEQ ID NO.1. According to the prediction of NCBI Conserved Domains (NCBI, https: / / www.ncbi.nlm.nih.gov / Structure / cdd / cdd.shtml), it is believed that it contains, from the N-terminal side, an NBD sugar kinase HSP70 actin superfamily domain consisting of 257 amino acids, a YjiL domain consisting of 429 amino acids, a 2-hydroxyglutaryl-CoA dehydratase D component consisting of 355 amino acids and a 402 amino acid component ( Figure 1 ). In addition, the molecular weight estimated from GENETYX-MAC (manufactured by Genetics Corporation) was 156990, and the isoelectric point was estimated to be 5.77.

[0096] Enterococcus faecalis K4 strain was cultured in BD Difco (trademark) Lactobacilli MRS Broth (Nippon Becton Dickinson) at 37°C overnight to obtain genomic DNA. The obtained genomic DNA was used as a template, and PCR was performed using a forward primer consisting of the base sequence shown in SEQ ID NO.3 and a reverse primer consisting of the base sequence shown in SEQ ID NO.4. In the DNA from which the start codon and stop codon of the gene presumed to be plsA were removed, a histidine tag and a SUMO tag were added to the 5' end for amplification. The obtained PCR product was inserted into pETite N-HIS SUMO Expression Vector (LGC Biosearch Technologies) as an expression vector to obtain a recombinant plasmid (pETite N-HIS SUMO / plsA).

[0097] The obtained recombinant plasmid was introduced into Escherichia coli (HI-Control 10G Chemically Competent Cells (prepared by LGC Biosearch Technologies)) to obtain transformed Escherichia coli. The obtained transformed Escherichia coli was cultured overnight at 37°C in 100 mL of LB medium containing 30 μg / mL kanamycin. The obtained culture solution was centrifuged and the bacteria were collected. The bacteria were purified into plasmids using the QIAprep Spin Miniprep Kit (prepared by Qiagen). The purified plasmids were used to transform HI-Control BL21 (DE3) Chemically Competent Cells (prepared by LGC Biosearch Technologies).

[0098] [Expression of K4PlsA in transformed E. coli]

[0099] The obtained transformed E. coli was cultured overnight at 30°C in LB medium containing 30 μg / ml kanamycin. As a control, untransformed E. coli (BL21 strain) was cultured overnight at 30°C in LB medium without kanamycin. 300 μl of each culture solution was transferred to ZYM5052 medium (Studier, FW Protein Expr. Purif. 41, 207-234) and cultured at 30°C for 24 hours under anaerobic and aerobic conditions. The culture solution was centrifuged and subjected to CBB staining and protein blotting analysis using anti-histidine antibodies.

[0100] (result)

[0101] Figure 2A and 2B The results of CBB staining and Western blot analysis are shown respectively. "BL21" is untransformed E. coli, and "BL21+plsA" is the transformed E. coli obtained in this example. Figure 2B As indicated by the middle arrow, it was observed that the transformed E. coli cultured under anaerobic conditions and aerobic conditions expressed K4 PlsA protein.

[0102] (Example 2: Production of plasmalogen in K4 plsA transformed E. coli)

[0103] [Extraction of lipids from bacteria and detection of phospholipids]

[0104] The transformed E. coli obtained in Example 1 and the untransformed E. coli (BL21 strain) as a control were cultured under shaking in the presence of air for 18 hours. The cultured culture solution was centrifuged and the cells were collected. Physiological saline was added to the cells and mixed, centrifuged, and then the cells were washed by removing the supernatant. Physiological saline was further added to the cells and washed again. The supernatant was removed and stored at -30°C until use.

[0105] Measure the total weight of the thawed cells. Add 1 mL of water to the cells and mix, then add 3.75 mL of chloroform / methanol (1:2, v / v) and mix. Perform ultrasonic treatment for 10 minutes and leave at room temperature for 30 minutes. Add 1.25 mL of chloroform, followed by 1.25 mL of water. Centrifuge, then transfer the chloroform layer to a glass tube and re-extract the remaining water layer with 2 mL of chloroform. Dry the combined chloroform layers under nitrogen and resuspend them in hexane / isopropanol (3:2, v / v) until the cell concentration is 120 mg / mL and filter with a 0.45 μm filter. Inject 10 μL of the cell suspension into a high performance liquid chromatograph (HPLC) and detect with an evaporative light scattering detector (ELSD).

[0106] As a lipid standard sample, phosphatidylcholine (25 mg, 5 mg / mL in chloroform, A-29B, manufactured by SRL) was dried with nitrogen, and 1 mL of isopropanol / chloroform (2:1, v / v) was added and mixed. The sample was dissolved by ultrasound and stored at -30°C as a lipid reserve. The lipid standard solution was prepared by gradient dilution of the lipid reserve with HIP (hexane / isopropanol (3:2, v / v)). The lipid standard solution was passed through a 0.45 μm microporous filter, and 10 μL was taken as a sample for HPLC.

[0107] The HPLC conditions were as follows.

[0108] Chromatographic column: Lichrosphere DIOL (250×3mm, 5μm, manufactured by Merck)

[0109] Mobile phase A: Hexane / isopropanol / acetic acid (82:17:1) containing 0.08% triethylamine

[0110] Mobile phase B: isopropanol / water / acetic acid (85:14:1) containing 0.08% triethylamine

[0111] Flow rate: 0.8 mL / min

[0112] Column temperature: 50°C

[0113] gradient:

[0114] 0 min Mobile phase A 96% Mobile phase B 4%

[0115] 21 minutes Mobile phase A 63% Mobile phase B 37%

[0116] 25 min Mobile phase A 15% Mobile phase B 85%

[0117] 26 minutes Mobile phase A 15% Mobile phase B 85%

[0118] 29 minutes Mobile phase A 96% Mobile phase B 4%

[0119] 34 minutes Mobile phase A 96% Mobile phase B 4%

[0120] The ELSD conditions were as follows.

[0121] Evaporator temperature: 60℃

[0122] Atomizer temperature: 30℃

[0123] Nitrogen flow rate: 1.00SLM

[0124] The lipid standard solutions of different concentrations were also detected by HPLC-ELSD. A calibration curve was drawn based on the peak area of ​​each concentration, and the amount of phospholipids was calculated based on the peak area ratio corresponding to the phospholipids obtained from Escherichia coli with a retention time of 10 to 20 minutes.

[0125] [Changes in plasmalogen caused by acid hydrolysis treatment]

[0126] As described above, 0.5 mL of 2,4-dinitrophenylhydrazine-hydrochloride solution (DNPH-HCl solution, prepared by Tokyo Chemical Industry Co., Ltd.) was added to the lipid extracted from the bacteria with chloroform / methanol and dried with nitrogen, and mixed. After emulsification by ultrasonic treatment, it was placed at room temperature for 30 minutes, and 0.5 mL of ultrapure water was added to the acid-hydrolyzed lipid and mixed, and then 1.9 mL of chloroform / methanol (1:2, v / v) was added and mixed. It was placed at room temperature for 10 minutes, and 0.625 mL of chloroform was added and mixed. Next, 0.625 mL of water was added and mixed. Centrifugation was performed at 3000 rpm at room temperature, and the chloroform layer was transferred to a glass tube and dried under nitrogen. The dried lipid was resuspended in 100 μL of acetonitrile and then passed through a 0.45 μm filter. 10 μL was taken as a sample for HPLC, injected into HPLC, and detected with UV (ultraviolet light) at a wavelength of 356 nm.

[0127] The aldehyde standard solution used as the standard was prepared as follows. 14 H 28 O, prepared by Tokyo Chemical Industry Co., Ltd., T2696), hexadecanol (C 16 H 32 O, prepared by Tokyo Chemical Industry Co., Ltd., H1296), heptadecanol (C 17 H 34 O, prepared by Tokyo Chemical Industry Co., Ltd., H1295) and octadecanol (C 18 H 36 O, prepared by Tokyo Chemical Industry Co., Ltd., O0368) were placed in Spitz tubes and adjusted to 1 mg / mL with acetonitrile. 200 μL of the prepared 1 mg / mL solution (only C 14 H 28 O is 100μL, 200μL and 400μL) are placed in Spitz tubes respectively, and 0.5mL DNPH-HCl solution is added to the sample dried with nitrogen and mixed. Emulsification is performed with ultrasound, and the sample is placed at room temperature for 30 minutes. 0.5mL ultrapure water and 1.9mL chloroform / methanol (1:2, v / v) are added to the sample and vortexed. Place at room temperature for 10 minutes, add 0.625mL chloroform, and vortexed. Add 0.625mL ultrapure water to the sample and vortexed. Centrifuge the sample at room temperature (3000rpm) for 5 minutes. Transfer the lower layer to another Spitz tube, dry it with nitrogen, dissolve it in 2mL acetonitrile, and store it at -30°C as an aldehyde standard stock solution.

[0128] Take each concentration of C 14 H 28120 μL of the aldehyde standard stock solution was filtered through a 0.45 μm filter, and 10 μL was injected into the HPLC. To prepare a mixed standard solution containing four aldehydes, 30 μL of each of the four aldehyde standard stock solutions was mixed. The obtained mixed aldehyde standard solution was filtered through a 0.45 μm filter, and 10 μL was injected into the HPLC.

[0129] The HPLC conditions were as follows.

[0130] Chromatographic column: XBridge BEAC18 (3.0×150, 2.5 μm, manufactured by Waters)

[0131] Column temperature: 40°C

[0132] Mobile phase A: acetonitrile

[0133] Mobile phase B: water

[0134] Flow rate: 0.3mL / min

[0135] Column temperature: 40°C

[0136] gradient:

[0137] 0 min Mobile phase A 30% Mobile phase B 70%

[0138] 25 min Mobile phase A 100% Mobile phase B 0%

[0139] 40 min Mobile phase A 100% Mobile phase B 0%

[0140] 40.1 min Mobile phase A 30% Mobile phase B 70%

[0141] 50 min Mobile phase A 30% Mobile phase B 70%

[0142] The amount of aldehydes was calculated by area ratio in the same manner as for phospholipids.

[0143] (result)

[0144] FIG. 3 shows chromatograms of each sample of the transformed E. coli according to Example 1 and the control E. coli detected by UV at a wavelength of 356 nm. Figure 3A and Figure 3B 1 and 2 are the results for the control E. coli (BL21) and the transformed E. coli according to Example 1. Aldehydes were not detected in the control E. coli sample, but were detected in the transformed E. coli sample. Since plasmalogens generate aldehydes by acid hydrolysis, it is shown that plasmalogens are produced in the transformed E. coli cultured aerobically.

[0145] (Example 3: Changes in plasmalogen caused by phospholipase A1 (PLA1) treatment and hydrochloric acid (HCl) treatment)

[0146] The transformed E. coli obtained in Example 1 was subjected to anaerobically static culture in ZYM5052 medium using an anaerobic gas generating bag (AnaeroPack). According to the procedure described in Example 2, lipids were extracted from the bacterial cells.

[0147] [Changes in phospholipids caused by total lipid hydrolysis treatment by phospholipase A1 (PLA1)]

[0148] A quantitative sample was prepared by hydrolyzing a portion of the total lipids with PLA1. 0.5 ml of the PLA1 solution purchased from SIGMA was prepared with 0.5 ml of 0.1 M citrate buffer (pH 4.5), and 1 ml of the enzyme solution was added to the total lipid extract. After emulsification with an ultrasonic bath, the suspension was incubated at 45°C for 60 minutes. The lipids were extracted with hexane / isopropanol, and the combined hexane layers were dried under nitrogen. The lipids were reconstituted with hexane / isopropanol (3:2, v / v) and injected into the HPLC-ELSD. In addition, as PLA1, PLA1 (Phospholipase A1: Phospholipase A1) derived from Aspergillus oryzae (prepared by SIGMA) was used. The minimum activity of the enzyme is 10 KLU / G liquid.

[0149] [Changes in ether phospholipids (plasmalogens) caused by acid hydrolysis treatment]

[0150] Next, the ether phospholipids (plasmalogens) were acid hydrolyzed by 2,4-dinitrophenylhydrazine-hydrochloric acid solution (DNPH-HCl solution). After PLA1 hydrolysis, 0.5 mL of DNPH-HCl solution was added to the dried ether phospholipids. After standing at room temperature for 30 minutes, the hydrolyzed ether phospholipids were extracted using the above-mentioned chloroform-methanol method, and the chloroform layer was dried under nitrogen. The dried lipids were reconstituted with hexane / isopropanol (3:2 v / v) and an aliquot was injected into the HPLC-ELSD.

[0151] (result)

[0152] Figure 4A , Figure 4B and Figure 4C The chromatograms of the total lipids before PLA1 treatment, after PLA1 treatment, and after PLA1 treatment and hydrochloric acid treatment are shown respectively. Phospholipids other than ether phospholipids and sphingomyelin are decomposed by PLA1 treatment, but plasmalogens remain. When hydrochloric acid is treated after PLA1 treatment, plasmalogens are selectively decomposed. Therefore, these chromatograms show that plasmalogens are produced in the transformed E. coli cultured anaerobically.

[0153] (Example 4: Phosphorylation of AMP-activated protein kinase (AMPK) by purified plasmalogen)

[0154] 0.5 μg of plasmalogen purified from the transformed E. coli of Example 1 was suspended in OPTI-MEM (manufactured by Invitrogen) and added to NIH-3T3 culture medium (DMEM / 10% FCS), and the cells were collected after 3 hours. Then, Western blot analysis was performed using anti-pAMPK antibody (manufactured by Cell Signaling, product catalog number 2531) and anti-AMPK antibody (manufactured by Cell Signaling, catalog number 5831). In addition, the proteins of AMPK and phosphorylated AMPK (pAMPK) were quantified to determine the relative value of pAMPK / AMPK.

[0155] (result)

[0156] The results of Western blot analysis are shown in Figure 5A The relative activity of pAMPK / AMPK is shown in Figure 5B . The left side is a control sample without the addition of purified plasmalogen, and the right side is a sample with the addition of purified plasmalogen. The pAMPK / AMPK value of the sample with the addition of purified plasmalogen is statistically significantly larger (p<0.01). This indicates that the plasmalogen synthesized by the transformed Escherichia coli of Example 1 contributes to the phosphorylation of AMPK. It is well known that AMPK is activated by phosphorylation and is involved in sugar metabolism, lipid metabolism, etc. Therefore, it is suggested that the plasmalogen is effective in preventing and treating various diseases related to sugar metabolism or lipid metabolism, such as type 2 diabetes, dyslipidemia, obesity, etc.

[0157] (Example 5: Inhibition of lipid droplet formation caused by purified plasmalogen)

[0158] Oleic acid (manufactured by Cayman Chemical) was mixed with 4% defat-BSA solution in advance, and 0.5 μg / ml of plasmalogen purified from the transformed E. coli of Example 1 was added to DMEM / 10% FCS culture medium containing oleic acid at a final concentration of 150 μM, and mouse fetal fibroblasts (NIH-3T3 cells) were cultured for 18 hours. As a control, culture was performed in a culture medium without plasmalogen. The cells were collected, washed, and fixed with a 4% paraformaldehyde solution. Next, after being treated with a 60% isopropanol solution, they were stained with OilRed O staining solution (OilRed O (manufactured by Sigma) was diluted 1.7 times with water) for 15 minutes, washed with 60% isopropanol, stained with hematoxylin solution (hematoxylin (manufactured by Merk) was diluted 10 times with water) for 30 seconds, washed with water, embedded and observed.

[0159] (result)

[0160] The photographs of cells stained with OilRed O are shown in Figure 6 Compared to the control (plasmalogen-) cultured in a medium without purified plasmalogen, in which many lipid droplets were observed, almost no lipid droplets were observed in cells cultured in a medium containing purified plasmalogen (plasmalogen+). This suggests that the plasmalogen synthesized by the transformed Escherichia coli of Example 1 contributes to the inhibition of adipogenesis and lipolysis and is effective in the prevention and treatment of dyslipidemia and obesity.

[0161] (Example 6: Preparation of Escherichia coli transformed with artificial plsA gene derived from Bifidobacterium longum subsp. suis)

[0162] [Pig clone plsA]

[0163] The artificial plsA gene derived from Bifidobacterium longum subsp. suis, which is an obligate anaerobic bifidobacterium, was artificially synthesized by the following method.

[0164] The 65667-70586 region of the registered genome sequence of the benchmark strain of Bifidobacterium longum subsp. suis DSM 20211 (Bifidobacterium longum subsp. suis DSM 20211 contig3, Whole Genome Shotgun Sequencing: NZ_JDUC01000003.1) was selected, and the base sequence optimized for Escherichia coli expression was artificially synthesized using GeneArt (registered trademark) GeneOptimizer (registered trademark) (manufactured by Thermo Fisher Scientific), and it was ligated together with CACC (single-stranded DNA) to the cloning site of the pET100 Directional TOPO vector (manufactured by Thermo Fisher Scientific).

[0165] The amino acid sequence of the PlsA protein translated from this gene (hereinafter also referred to as "pig PlsA protein") is a sequence consisting of 1639 amino acids shown in SEQ ID NO. 2. According to the prediction of NCBI Conserved Domains (NCBI, https: / / www.ncbi.nlm.nih.gov / Structure / cdd / cdd.shtml), it is believed that from the N-terminus, there are NBD sugar kinase HS P70 actin superfamily domain consisting of 257 amino acids, YjiL domain consisting of 429 amino acids, COG3580 domain consisting of 370 amino acids, and 2-hydroxyglutaryl-CoA dehydratase D component consisting of 401 amino acids. In addition, the molecular weight estimated from GENETYX-MAC (manufactured by Genetics) is 178715, and the estimated isoelectric point is 5.90.

[0166] The obtained recombinant plasmid was introduced into Escherichia coli BL21 (DE3) competent cells (ChampionTM21; manufactured by SMOBIO Technology) to obtain transformed Escherichia coli.

[0167] The obtained transformed Escherichia coli was cultured overnight at 37°C in 10 mL of LB medium containing 50 μg / mL ampicillin. 3 mL of the same culture solution was cultured overnight at 37°C in 100 mL of Overnight Express (registered trademark) Instant TB medium (Merck). The obtained culture solution was centrifuged and the cells were collected. After the cells were suspended in 20 mM Tris-HCl buffer (pH 7.0), an extract derived from the transformed Escherichia coli was obtained by ultrasonic disruption. In addition, the cell extract was centrifuged at 14,000 rpm for 15 minutes to obtain a supernatant.

[0168] [Expression of porcine PlsA protein caused by transformation of Escherichia coli]

[0169] In order to confirm whether the transformed E. coli obtained expressed the desired porcine PlsA protein under aerobic conditions, samples cultured by the following three methods were prepared.

[0170] Culture method 1: Pre-culture the transformed E. coli in LB medium at 37°C and 150 rpm with shaking overnight. Then, take 3% of the cells and inoculate them in 10 ml of fresh LB medium and culture them at 37°C and 150 rpm with shaking overnight.

[0171] Culture method 2: Pre-culture the transformed E. coli in LB medium at 37°C, 150 rpm with shaking overnight. Then, take 3% of the cells and inoculate them into 10 ml of fresh LB medium, shake and culture at 37°C, 150 rpm, and when OD660 reaches 0.4, induce with IPTG (0.5 mM), and then shake and culture at 37°C, 150 rpm for 3 hours.

[0172] Cultivation method 3: Pre-culture the transformed E. coli in LB medium at 37°C and 150 rpm with shaking overnight. Then, take 3% of the cells and inoculate into 10 mL Overnight Express (registered trademark) Instant TB Medium (Merck) and culture at 37°C and 150 rpm with shaking overnight.

[0173] The obtained culture solution was centrifuged and the cells were collected. After the cells were suspended in 20 mM Tris-HCl buffer (pH 7.0), they were broken by ultrasonic waves to obtain an extract derived from transformed Escherichia coli. In addition, the cell extract was centrifuged at 14,000 rpm for 15 minutes to obtain a supernatant. Each sample was dissolved in an electrophoresis buffer to make the cell weight per volume the same, and after equal amounts of electrophoresis, an anti-His antibody (manufactured by Proteintech, 66005-1-Ig) was used for Western blotting analysis.

[0174] (result)

[0175] The results of Western blot analysis are shown in Figure 7 Sample numbers 2 and 5 are samples of culture method 1, sample numbers 1 and 4 are samples of culture method 2, and sample numbers 3 and 6 are samples of culture method 3. In addition, sample numbers 1, 2, and 3 were subjected to electrophoresis at 10 μl, and sample numbers 4, 5, and 6 were subjected to electrophoresis at 20 μl. As shown by the arrows in the figure, bands of the desired size were confirmed in sample numbers 3 and 6, confirming that the transformed E. coli expressed the porcine PlsA protein.

[0176] (Example 7: Production of plasmalogen in Escherichia coli transformed with porcine PlsA)

[0177] The transformed E. coli obtained in Example 6 was statically cultured or shaken, and untransformed E. coli (BL21 strain) was used as a control. According to the procedures described in Example 2, lipid extraction from the cells, phospholipid detection, acid hydrolysis treatment, and aldehyde detection were performed.

[0178] (result)

[0179] Figure 8The chromatograms of each sample detected by ELSD and each sample treated with DNPH-HCl solution are shown. Aldehydes were detected in both the static culture and the shaking culture of the transformed E. coli. On the other hand, no aldehydes were detected in the untransformed E. coli. Since plasmalogens produce aldehydes by acid hydrolysis, it is shown that plasmalogens are produced in the transformed E. coli. Therefore, it is shown that the porcine PlsA protein has plasmalogen synthesis activity in the aerobic cultured E. coli.

[0180] Table 1 shows the weights of phospholipids and aldehydes per 1 mg of wet weight of Escherichia coli cells, which were determined based on the calibration curve.

[0181] [Table 1]

[0182]

[0183] Without departing from the broad spirit and scope of the present invention, the present invention can have various embodiments and variations. In addition, the above-mentioned embodiments are used to explain the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is represented by the scope of the claims rather than the embodiments. Various modifications made within the scope of the claims and the scope of the invention meaning equivalent thereto are deemed to be within the scope of the present invention.

[0184] This application is based on Japanese Patent Application No. 2022-139676 filed on September 2, 2022. The entire specification, claims and drawings of Japanese Patent Application No. 2022-139676 are incorporated herein by reference.

Claims

1. A protein comprising an amino acid sequence selected from the group consisting of (a), (b), (c) and (d) below, and having plasmalogen synthesis activity in a bacterium expressing the protein, (a) the amino acid sequence shown in SEQ ID NO.1, (b) an amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO.1, (c) the amino acid sequence shown in SEQ ID NO.2, (d) An amino acid sequence having a sequence identity of 80% or more to the amino acid sequence shown in SEQ ID NO.

2.

2. A polynucleotide comprising a base sequence encoding the protein according to claim 1.

3. An expression vector comprising the polynucleotide according to claim 2. A transformant comprising the expression vector according to claim 3.

5. A composition containing plasmalogen, comprising the transformant according to claim 4 or a processed product thereof.

6. A method for preparing a plasmalogen, comprising: A step of culturing the transformant according to claim 4 under aerobic conditions; and A step of obtaining plasmalogen from the transformant.

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