Use of chlamydomonas insulata-derived 15s-lipoxygenase for simultaneously producing resolvin d5 and protectin dx

Abstract

The present invention relates to a Chlamydomonas insulata-derived 15S-lipoxygenase for simultaneously producing resolvin D5 and protectin Dx. More specifically, provided are: a composition for producing resolvin D5 and protectin Dx, the composition comprising, as an active ingredient, a Chlamydomonas insulata-derived 15S-lipoxygenase or a gene encoding same; a method for producing resolvin D5 and protectin Dx, the method comprising the step of treating a substrate with the composition; a recombinant expression vector for producing resolvin D5 and protectin Dx, the recombinant expression vector comprising the 15S-lipoxygenase gene; and a transformant in which a host cell is transformed with the recombinant expression vector.

Description

Use of 15S-lipolytic enzyme derived from Chlamydomonas insulta that simultaneously produces resolvin D5 and protectin DX

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0121046, filed September 5, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a 15S-lipooxidase derived from Chlamydomonas insulta that simultaneously produces resolvin D5 and protectin Dx, and more particularly, to a composition for producing resolvin D5 and protectin Dx comprising a 15S-lipooxidase derived from Chlamydomonas insulta or a gene encoding the same as an active ingredient; a method for producing resolvin D5 and protectin Dx comprising a step of treating the composition to a substrate; a recombinant expression vector for producing resolvin D5 and protectin Dx comprising the 15S-lipooxidase gene; and a transformant in which the recombinant expression vector is transformed into a host cell.

[0003] Dihydroxy fatty acids are substances that exist in the lipids of various organisms, are produced from lipids, and are contained in various organisms such as animals, plants, microorganisms, and insects. When fatty acids have a functional group called hydroxyl group, they have antibacterial, antifungal, and anti-inflammatory activities, and are used as raw materials for health functional foods, medicines, and cosmetics. Among these, omega-3 dihydroxy fatty acids (DiHFA) produced in animals are used as signaling substances and precursors, and are involved in various complex physiological activities. Omega-3 dihydroxy fatty acids belong to lipid-regulating agents, and lipid-regulating agents include inflammation-promoting mediators, and inflammation-promoting mediators among omega-3 dihydroxy fatty acids include protectin D (PD), resolvin (RV), and maresin (MaR).

[0004] Resolvins are endogenous lipid mediators produced by the combined reaction and double oxidation reaction of lipoxygenase (LOX), and are substances derived from omega-3 fatty acids eicosapentaenoic acid, n-3 docosapentaenoic acid, and docosahexaenoic acid. Lipoxygenase acts on the animal polyunsaturated fatty acids eicosapentaenoic acid, n-3 docosapentaenoic acid, and docosahexaenoic acid to convert them into resolvin E, resolvin T, and resolvin D, respectively. Recently, resolvin isomers and analogs have also been known to act as potent anti-inflammatory promoters. Resolvins and their analogs have the functions of resolving acute inflammation at the site of infection in the human body, blocking platelet aggregation, improving survival rate and reducing antibiotic requirements, and enhancing anti-inflammatory activity and phagocytosis.

[0005] Among protectins, protectin D1 (PD1, 10R,17S-dihydroxy-4Z,7Z,11E,13E,15Z,19Z-docosahexaenoic acid) is an endogenous stereoselective lipid mediator derived from the omega fatty acid docosahexaenoic acid. It is primarily found in tissues such as the retina, lungs, and nervous system. This substance has anti-inflammatory, anticancer, and neuroprotective properties. Studies in stroke patients and animal models of Alzheimer's disease have shown that treatment with this substance can potentially reduce inflammation caused by oxidative stress and prevent cell degeneration by inhibiting apoptosis. It is also known to have the ability to attenuate influenza virus replication, inhibit the infectivity of avian influenza viruses such as H5N1, and reduce inflammation. Considering the efficacy of Protectin treatment, which has been shown not to induce harmful effects on host RNA, which could cause harmful side effects in host cells, this substance with strong anti-inflammatory properties in viral or bacterial inflammation suggests that it could serve as a novel antiviral agent as well as a biomarker for viral infection.

[0006] Lipoxygenase (LOX) is a dioxygenation enzyme that catalyzes the regio- and stereoselective peroxidation of animal-derived unsaturated fatty acids as substrates. It uses polyunsaturated fatty acids containing one or more cis, cis-1,4-pentadiene units as substrates, and catalyzes stereospecificity and reaction specificity through dioxygenation. Regiospecificity varies depending on the type of polyunsaturated fatty acid used as a substrate. Among them, lipoxygenase that generates hydroxyl groups at carbon positions 5, 8, 9, 11, 12, and 15 of arachidonic acid, an animal-derived polyunsaturated fatty acid, forms hydroxyl groups only at specific carbon positions in unsaturated fatty acids with 20 or more carbons, such as arachidonic acid. In the case of stereospecificity, they are divided into R-lipooxidase and S-lipooxidase due to differences in chirality or hand symmetry. In the case of arachidonic acid 15S-lipooxidase (hereinafter referred to as 15S-lipooxidase), it forms an S-hydroxyl group at the 15th carbon position in unsaturated fatty acids with 20 carbon atoms, such as arachidonic acid. In addition, it forms an S-hydroxyl group at the 17th carbon position in unsaturated fatty acids with 22 carbon atoms.

[0007] The most reported method for synthesizing dihydroxy fatty acids, including resolvin and protectin analogs, involves a stereoselective chemical synthesis of 20-30 steps, and is known to produce a yield of approximately 20%. However, this method has limitations in that benzene analogs and heavy metals are used and released during the synthetic process, and the pollutants and byproducts produced during this chemical synthesis are not biodegradable in nature, causing environmental pollution. In addition, dihydroxy fatty acids, including resolvin and protectin analogs, are biosynthesized in vivo through intercellular responses to tissue damage and infection, and are limited in that they are biosynthesized in small amounts of less than 250 pg / mL by the combined reaction of two or more lipolytic enzymes.

[0008] Recently, according to non-patent document 1, resolvin D5 was produced at the level of 4.2 mM (1.51 g / L) using a recombinant microorganism containing a double-oxygenating lipolytic enzyme through a whole-cell reaction. According to non-patent documents 2 and 3, in the case of protectin Dx and protectin Dx 10-epimer, 0.3 mM (108 mg / L) and 1.2 mM (433 mg / L) were produced, respectively, through a two-step bioconversion by a combined reaction of two types of single-oxygenating lipolytic enzyme-containing recombinant microorganisms. However, the production of protectin Dx by a double-oxygenating lipolytic enzyme has not been reported to date.

[0009] That is, to date, no 15S-lipolytic enzyme has been reported that catalyzes double oxygenation to quantitatively and simultaneously produce resolvin D5 and protectin Dx, and no method has been reported for producing resolvin D5 (RvD5) and protectin Dx (PDX) from docosahexaenoic acid and arachidonic acid using 15S-lipolytic enzyme.

[0010] [Prior Art Literature]

[0011] [Non-patent literature]

[0012] (Non-patent Document 1) DK Oh, et al. "Efficient biotransformation of docosahexaenoic acid-rich oils into the lipid mediator resolvin D5 by cells expressing 15S-lipoxygenase using a bioreactor", Bioresor. Technol. 388, 129750 (2023).

[0013] (Non-patent Document 2) DK Oh, et al. “Enzymatic formation of protectin Dx and its production by whole-cell reaction using recombinant lipoxygenases”, Catalysts, 12, 1145 (2022).

[0014] (Non-patent Document 3) DK Oh, et al. “Biocatalytic synthesis of protectin Dx 10-epimer by serial reactions of arachidonate 8R- and 15S-lipoxygenases”, World J. Microbiol. Biotechnology. 40, 219 (2024).

[0015] Against this backdrop, the inventors of the present invention have completed the present invention by confirming that resolvin D5 and protectin Dx can be simultaneously produced with high efficiency in a single step using 15S-lipid oxidase derived from Chlamydomonas insulfa, which catalyzes double oxygenation from polyunsaturated fatty acids.

[0016] Accordingly, the purpose of the present invention is to provide a composition for producing resolvin D5 and protectin Dx, which comprises as an active ingredient a 15S-lipooxidase comprising an amino acid sequence of sequence number 1 derived from Chlamydomonas incerta; or a gene comprising a base sequence of sequence number 2 encoding the same.

[0017] In addition, another object of the present invention is a method for producing Resolvin D5 and Protectin Dx, which comprises a step of treating the above-described composition on a substrate,

[0018] The above substrate provides a manufacturing method characterized in that it is docosahexanoic acid.

[0019] In addition, another object of the present invention is to provide a recombinant expression vector for producing resolvin D5 and protectin Dx, which comprises a 15S-lipid oxidase gene comprising the base sequence of SEQ ID NO: 2.

[0020] Another object of the present invention is to provide a transformant in which the above-described recombinant expression vector is transformed into a host cell.

[0021] In order to solve the above-described problem, a composition for producing resolvin D5 and protectin Dx is provided, which comprises as an active ingredient a 15S-lipid oxidase comprising an amino acid sequence of sequence number 1 derived from Chlamydomonas incerta; or a gene comprising a base sequence of sequence number 2 encoding the same.

[0022] Additionally, the composition may further comprise docosahexanoic acid as a substrate.

[0023] At this time, the concentration of the substrate may be 1 mM to 8 mM.

[0024] At this time, the concentration of whole cells containing the 15S-lipid oxidase in the composition may be 0.1 g / L to 1 g / L.

[0025] Additionally, the present invention provides a method for producing Resolvin D5 and Protectin Dx, comprising the step of treating the aforementioned composition on a substrate,

[0026] The present invention provides a manufacturing method characterized in that the substrate is docosahexanoic acid.

[0027] At this time, the above step may further include treating dimethyl sulfoxide as a solvent and polyvinyl pyrrolidone as a polymer.

[0028] At this time, the concentrations of the dimethyl sulfoxide and polyvinylpyrrolidone may be 1 (v / v)% to 5 (v / v)% and 1 (w / v)% to 5 (w / v)%, respectively.

[0029] At this time, the above step may be performed at pH 7.0 to 9.0.

[0030] At this time, the above step may be performed at a temperature of 15°C to 40°C.

[0031] At this time, the above manufacturing method may simultaneously produce resolvin D5 and protectin Dx in a single step.

[0032] Additionally, the present invention provides a recombinant expression vector for producing resolvin D5 and protectin Dx comprising a 15S-lipid oxidase gene comprising the base sequence of SEQ ID NO: 2.

[0033] Furthermore, the present invention provides a transformant in which the above-described recombinant expression vector is transformed into a host cell.

[0034] Dihydroxylated fatty acids including resolvin D5 and protectin Dx are signaling substances and are expected to be involved in various physiological activities in animals including humans. The 15S-lipooxidase provided in the present invention can produce dihydroxylated fatty acids including resolvin D5 and protectin Dx with high productivity and yield, and thus can be usefully used in various industrial fields such as medicine, food, and cosmetics, and thus the 15S-lipooxidase of the present invention has high utility value.

[0035] Figure 1 shows the results of confirming the primary product generated from arachidonic acid through HPLC chromatogram to determine the position selectivity of 15S-lipid oxidase derived from Chlamydomonas incerta.

[0036] Figure 2 shows the results of confirming the primary product generated from arachidonic acid through HPLC chromatogram to determine the stereoselectivity of 15S-lipid oxidase derived from Chlamydomonas incerta.

[0037] Figures 3a to 3c are the results of HPLC chromatograms to determine the stereoselectivity of 15S-lipid oxidase derived from Chlamydomonas incerta, which confirms the secondary products generated from arachidonic acid.

[0038] Figures 4a to 4d are chromatogram results obtained by identifying the reaction products of 15S-lipid oxidase derived from Chlamydomonas incerta through LC-MS / MS.

[0039] Figure 5 shows the results of analyzing the production of resolvin D5 and protectin Dx according to the concentration of dimethyl sulfoxide as a solvent and polyvinylpyrrolidone as a polymer in a whole-cell reaction using recombinant E. coli expressing 15S-lipid oxidase derived from Chlamydomonas incerta using docosahexaenoic acid as a substrate.

[0040] Figure 6 shows the results of analyzing the production of resolvin D5 and protectin Dx according to the concentration of whole cells or substrate (docosahexaenoic acid) in a whole-cell reaction using recombinant E. coli expressing 15S-lipid oxidase derived from Chlamydomonas incerta.

[0041] Figure 7 shows the results of analyzing the production of resolvin D5 and protectin Dx over time in a whole-cell reaction using recombinant E. coli expressing 15S-lipolytic enzyme derived from Chlamydomonas incerta.

[0042] Hereinafter, the present invention will be described in more detail.

[0043] Meanwhile, each description and embodiment disclosed herein can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the specific descriptions described below.

[0044] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described in this application. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0045] As described above, the present inventors more effectively produced 15S-hydroxylated fatty acids, 17S-hydroxylated fatty acids, dihydroxylated fatty acids including resolvin D5 and protectin Dx through a bioconversion process. Specifically, the present inventors cloned 15S-lipid oxidase derived from Chlamydomonas incerta, constructed a recombinant expression vector and a microorganism transformed therefrom, produced whole cells using the vector, and then treated the whole cells with a substrate to produce 17S-hydroxydocosahexaenoic acid (also called 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid, 17S-HDHA) and the final products, resolvin D5 (7S,17S-dihydroxy-4Z,8E,10Z,13Z,15E,19Z-docosahexaenoic acid) and protectin Dx (10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid), with high efficiency through an eco-friendly bioconversion method.

[0046] In a specific embodiment of the present invention, a recombinant E. coli expressing 15S-lipolytic enzyme derived from Chlamydomonas incerta was prepared for use in a whole-cell reaction.

[0047] In another specific embodiment of the present invention, it was confirmed through FIGS. 1, 2, 3a to 3c and 4a to 4d that the 15S-lipid oxidase derived from Chlamydomonas incerta is a double oxygenated 15S-lipid oxidase.

[0048] Accordingly, the first aspect of the present invention relates to a composition for producing resolvin D5 and protectin Dx, which comprises as an active ingredient a 15S-lipooxidase comprising an amino acid sequence of sequence number 1 derived from Chlamydomonas incerta; or a gene comprising a base sequence of sequence number 2 encoding the same.

[0049] In the present invention, sequence numbers 1 and 2 are as described in the table below.

[0050]

[0051] In the present invention, the 15S-lipooxidase comprises the amino acid sequence of SEQ ID NO: 1, and all mutant enzymes that can achieve the effect intended by the present invention by one or more substitutions, deletions, additions, etc. to the sequence are included in the scope of the present invention. That is, the 15S-lipooxidase may comprise an amino acid sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1. In addition, the 15S-lipooxidase may be a product expressed from a gene comprising the base sequence of SEQ ID NO: 2.

[0052] In the present invention, the gene encoding the 15S-lipooxidase is not only composed of the base sequence of SEQ ID NO: 2, but also includes functional equivalents thereof, that is, all mutants that achieve the purpose of the present invention by inducing one or more mutations such as substitutions or deletions in the sequence. That is, the gene may include a base sequence having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%, or 100% identity to the base sequence of SEQ ID NO: 2.

[0053] In the present invention, the composition may further include docosahexanoic acid as a substrate.

[0054] In a specific embodiment of the present invention, in order to maximize the production effect of resolvin D5 and protectin Dx of the 15S-lipooxidase, the concentrations of solvent, polymer, whole cells, and substrate were optimized, and as a result, as shown in FIG. 5, the optimal concentrations of dimethyl sulfoxide as a solvent and polyvinylpyrrolidone as a polymer were found to be 2 (v / v)% and 2 (w / v)%, respectively. In addition, as shown in FIG. 6, it was confirmed that the production of resolvin D5 and protectin Dx was significantly increased at a whole cell concentration of 0.5 g / L and a substrate concentration of 6 mM.

[0055] Therefore, in the present invention, the concentration of the substrate may be 1 mM to 8 mM. Preferably, the concentration of the substrate may be 2 mM to 8 mM, more preferably 3 mM to 8 mM, more preferably 4 mM to 7 mM, more preferably 5 mM to 7 mM, and most preferably 6 mM.

[0056] In the present invention, the composition for producing resolvin D5 and protectin Dx comprises 15S-lipolytic enzyme derived from Chlamydomonas insulfa as an active ingredient, and may include whole cells comprising the 15S-lipolytic enzyme as an active ingredient.

[0057] Specifically, it is preferable to culture and use a transformant transformed with a recombinant expression vector containing a gene encoding the amino acid sequence of SEQ ID NO: 1 or a gene consisting of the base sequence of SEQ ID NO: 2, which contains the above 15S-lipid oxidase.

[0058] As the above recombinant expression vector, any plasmid vector used in the art for genetic recombination may be used, and specifically, the use of the pET-28(+)a plasmid is more preferable, but is not limited thereto.

[0059] In the present invention, the whole cells containing the 15S-lipid oxidase can be obtained by including the steps of: i) recovering primary whole cells by centrifuging the culture solution of the microorganism; ii) washing the recovered whole cells with a saline solution; iii) centrifuging the washed whole cells a second time to remove the supernatant and recover the whole cells; and iv) washing the secondarily recovered whole cells once again with a saline solution. Specifically, the recovery of the whole cells in step i) can be performed at a range of about 13,000 g using a device known in the art, such as a centrifuge, and the washing of the whole cells in step ii) is suitably performed with a sodium chloride solution of 0.85% or less.

[0060] In the present invention, the concentration of whole cells containing the 15S-lipooxidase in the composition may be 0.1 g / L to 1 g / L. Preferably, the concentration of whole cells may be 0.2 g / L to 0.8 g / L, more preferably 0.3 g / L to 0.7 g / L, more preferably 0.4 g / L to 0.6 g / L, and most preferably 0.5 g / L.

[0061] In a specific embodiment of the present invention, the amounts of resolvin D5 and protectin Dx produced by the recombinant E. coli expressing the 15S-lipooxidase were confirmed, and as shown in FIG. 7, it was confirmed that 2.29 mM (826 mg / L) resolvin D5 and 1.68 mM (606 mg / L) protectin Dx were produced.

[0062] That is, it was confirmed that resolvin D5 and protectin Dx can be simultaneously produced with high efficiency through a single-step reaction using the 15S-lipolytic enzyme of the present invention.

[0063] Accordingly, the second aspect of the present invention is a method for producing Resolvin D5 and Protectin Dx, comprising the step of treating the composition on a substrate,

[0064] The present invention relates to a manufacturing method characterized in that the above substrate is docosahexanoic acid.

[0065] The composition and effect of the composition used in the manufacturing method of the present invention are the same as those described in the first aspect, and therefore, the description thereof is omitted.

[0066] In the present invention, the step may further include treating dimethyl sulfoxide as a solvent and polyvinyl pyrrolidone as a polymer.

[0067] In the present invention, the concentrations of the dimethyl sulfoxide and polyvinylpyrrolidone may be 1 (v / v)% to 5 (v / v)% and 1 (w / v)% to 5 (w / v)%, respectively. Preferably, when the concentration of the dimethyl sulfoxide is 1 (v / v)% to 5 (v / v)%, the concentration of the polyvinylpyrrolidone may be, for example, 1 (w / v)% to 4 (w / v)%, 1 (w / v)% to 3 (w / v)%, or 2 (w / v)%. Alternatively, when the concentration of the dimethyl sulfoxide is 1 (v / v)% to 4 (v / v)%, the concentration of the polyvinylpyrrolidone may be, for example, 1 (w / v)% to 5 (w / v)%, 1 (w / v)% to 4 (w / v)%, 1 (w / v)% to 3 (w / v)%, or 2 (w / v)%. Alternatively, when the concentration of the dimethyl sulfoxide is 1 (v / v)% to 3 (v / v)%, the concentration of the polyvinylpyrrolidone may be, for example, 1 (w / v)% to 5 (w / v)%, 1 (w / v)% to 4 (w / v)%, 1 (w / v)% to 3 (w / v)%, or 2 (w / v)%. Alternatively, when the concentration of the dimethyl sulfoxide is 2 (v / v)%, the concentration of the polyvinylpyrrolidone may be, for example, 1 (w / v)% to 5 (w / v)%, 1 (w / v)% to 4 (w / v)%, 1 (w / v)% to 3 (w / v)%, or 2 (w / v)%. Most preferably, the concentrations of dimethyl sulfoxide and polyvinylpyrrolidone may be 2 (v / v)% and 2 (w / v)%, respectively.

[0068] In the present invention, the above step may be performed at a pH of 7.0 to 9.0. Preferably, the reaction may be performed at around pH 8.0. To maintain these pH conditions, a HEPPS buffer solution may be used as a reaction solvent.

[0069] In the present invention, the above step may be performed at a temperature of 15°C to 40°C. Preferably, the reaction may be performed at a temperature of 17°C to 35°C, more preferably 20°C to 30°C, more preferably 22°C to 27°C, and most preferably around 25°C. By maintaining these conditions, the production activity of resolvin D5 and protectin Dx can be improved.

[0070] In the present invention, the above step may be performed through a whole-cell reaction.

[0071] In the present invention, the time of the whole cell reaction can be appropriately controlled according to a conventional method.

[0072] In the present invention, the manufacturing method may simultaneously produce resolvin D5 and protectin Dx in a single step.

[0073] In the present invention, the single reaction production amount of resolvin D5 and protectin Dx using the above manufacturing method may be 700 to 900 mg per liter and 500 to 700 mg per liter, respectively.

[0074] In addition, the third aspect of the present invention relates to a recombinant expression vector for producing resolvin D5 and protectin Dx comprising a 15S-lipid oxidase gene comprising the base sequence of SEQ ID NO: 2.

[0075] The term "recombinant expression vector" as used herein refers to a recombinant vector capable of expressing a target peptide in a suitable host cell, and a genetic construct containing essential regulatory elements operably linked to express the gene insert. The expression vector of the present invention includes expression regulatory elements such as a promoter, an operator, and an initiation codon, which are elements that suitable expression vectors generally have. The initiation and termination codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must be functional in a subject when the genetic construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

[0076] Additionally, a signal sequence for export of the fusion polypeptide may be included to facilitate protein release from cell culture media. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG initiation codon, must be provided. These exogenous translational control signals and initiation codons can come from a variety of natural and synthetic sources. Expression efficiency can be increased by introducing appropriate transcriptional or translational enhancing factors.

[0077] In the present invention, any conventional expression vector can be used. For example, plasmid DNA, phage DNA, etc. can be used. Specific examples of plasmid DNA include commercial plasmids such as pUC18 and pIDTSAMRT-AMP. Other examples of plasmids that can be used in the present invention include Escherichia coli-derived plasmids (pYG601BR322, pBR325, pUC118, and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24, and YCp50). Specific examples of phage DNA include λ-phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, and λZAP). Additionally, animal viruses such as retrovirus, adenovirus, or vaccinia virus, and insect viruses such as baculovirus may also be used. In the present invention, the use of pET-28a(+) is more preferred, but is not limited thereto. Since the amount of protein expression and the degree of modification of such expression vectors vary depending on the host cell, the host cell most suitable for the intended purpose can be selected and used.

[0078] Additionally, the fourth aspect of the present invention relates to a transformant in which the recombinant expression vector is transformed into a host cell.

[0079] The transformant of the present invention is obtained by introducing the recombinant expression vector of the present invention into a suitable host. The host cell types include Esherichia, Pseudomonas, Ralstonia, Alcaligenes, Comamonas, Burkholderia, Agrobacterium, Flabobacterium, Vibrio, Enterobacter, Rhizobium, Gluconobacter, Acinetobacter, Moraxella, Nitrosomonas, Aeromonas, Paracoccus, Bacillus, Examples of the host cells include various bacteria such as Clostridium, Lactobacillus, Corynebacterium, Arthrobacter, Achromobacter, Micrococcus, Mycobacterium, Streptococcus, Streptomyces, Actinomyces, Nocardia, and Methylobacterium. In addition to the above bacteria, yeasts such as Saccharomyces and Candida, and various molds can be used. Specifically, the Escherichia coli ER2566 strain can be used, but is not limited thereto.

[0080] As described above, when using bacteria such as E. coli as a host cell, it is preferable that the recombinant expression vector of the present invention be capable of autonomous replication within the host and have the necessary components for expression, such as a promoter, DNA containing a 15S-lipid oxidase gene, and a transcription termination sequence.

[0081] Methods for introducing recombinant DNA into bacteria include the calcium chloride method, electroporation method (Method Enzymol., 194, 182-187 (1990)), spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), and lithium acetate method (J. Bacteriol., 153, 163-168 (1983)).

[0082] In the present invention, the production of the 15S-lipooxidase can be carried out, for example, by transforming a host cell with a recombinant expression vector having a gene encoding it, culturing the resulting transformant, producing and accumulating the gene product, 15S-lipooxidase, in the culture (cultured cells or culture supernatant), and obtaining the enzyme from the culture.

[0083] The method for culturing the transformant of the present invention can be performed using a conventional method used for culturing host cells.

[0084] In the present invention, the above-mentioned culturing method may be any method commonly used for culturing microorganisms, such as batch, fluidized bed, continuous culture, or reactor type. As a medium for a transformant obtained using a bacterium such as Escherichia coli as a host, a complete medium or a synthetic medium, such as LB medium or M9 medium, may be exemplified. In addition, by culturing within the above-mentioned optimal temperature range, 15S-lipolytic enzyme can be accumulated and recovered within the cells.

[0085] Furthermore, a fifth aspect of the present invention comprises a 15S-lipid oxidase comprising an amino acid sequence of SEQ ID NO: 1 derived from Chlamydomonas incerta, a gene comprising a base sequence of SEQ ID NO: 2 encoding the 15S-lipid oxidase, a recombinant expression vector comprising the gene, or a transformant transformed with the recombinant expression vector;

[0086] solvent; and

[0087] It relates to a kit containing a polymer.

[0088] In addition, the sixth aspect of the present invention relates to a method for producing resolvin D5 and protectin Dx, comprising the step of treating the kit on a substrate.

[0089] In the present invention, the substrate may be docosahexaenoic acid.

[0090] In addition, the seventh aspect of the present invention relates to the use of a 15S-lipid oxidase comprising an amino acid sequence of SEQ ID NO: 1 derived from Chlamydomonas incerta for producing resolvin D5 and protectin Dx, or a gene comprising a base sequence of SEQ ID NO: 2 encoding the same.

[0091] In addition, the eighth aspect of the present invention relates to a composition for use in the production of resolvin D5 and protectin Dx, comprising a 15S-lipooxidase comprising an amino acid sequence of SEQ ID NO: 1 derived from Chlamydomonas incerta; or a gene comprising a base sequence of SEQ ID NO: 2 encoding the same.

[0092] As described above, according to the present invention, by using 15S-lipid oxidase derived from Chlamydomonas incerta strain, resolvin D5 and protectin Dx can be manufactured in high yield using dimethyl sulfoxide as a solvent and polyvinylpyrrolidone as a polymer for maximum production, and since it can be manufactured in an environmentally friendly manner compared to materials obtained by chemical synthesis using heavy metals and organic solvents, it is expected that it can be usefully used in various industrial fields such as medicine, food, and cosmetics.

[0093] The dihydroxy fatty acids produced according to the present invention are expected to be involved in various physiological functions in animals, including humans, as signaling substances.

[0094] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0095] <Example 1>

[0096] Preparation of whole-cell reactions with 1.15S-lipolytic enzyme

[0097] In order to produce 15S-lipolytic enzyme derived from Chlamydomonas incerta strain, DNA synthesis was requested from LNC Bio based on the lipolytic enzyme DNA base sequence of SEQ ID NO: 2 derived from Chlamydomonas incerta, the sequence of which was already known. The synthesized lipolytic enzyme DNA of SEQ ID NO: 2 was provided in the form of a pET28 vector.

[0098] Microorganisms transformed with the above vector were cultured in 1 L of LB (Luria-Bertani; Difco, Sparks, MD, USA) medium containing 20 μg / mL kanamycin at 37°C under aeration conditions of 200 rev / min in a shaking Erlenmeyer flask. When the whole-cell absorbance reached 0.6 to 0.8 at 600 nm, a final concentration of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression of enzymes, and the culture was cultured for 16 h at 16°C with shaking at 150 rev / min. In addition, the dual 15-hydroxy fatty acid enzyme produced by overexpression as described above was centrifuged at 6,000× gravitational acceleration (g) at 4℃ for 30 minutes, washed twice with 0.85% sodium chloride (NaCl), and then used as a recombinant cell for producing resolvin D5 and protectin Dx. The transformed microorganism was cultured for the production of 15S-hydroxy fatty acid, 17S-hydroxy fatty acid, resolvin D5, and protectin Dx by adding 20% ​​glycerine solution, and then stored frozen at -70℃ before use.

[0099] <Example 2>

[0100] 2. Identification of 15S-lipolytic enzyme

[0101] To identify the lipooxidizing enzyme, the primary product, hydroxylated fatty acid, generated from arachidonic acid was identified as 15-hydroxylated arachidonic acid using HPLC (Fig. 1), and the hydroxylated fatty acid was identified as 15S-hydroxylated fatty acid (Fig. 2). The secondary product, dihydroxylated fatty acid, generated from arachidonic acid was identified as 5S,15S-dihydroxylated arachidonic acid (5S,15S-dihydroxyeicosatetraenoic acid, 5S,15S-DiHETE) and 8S,15S-dihydroxylated arachidonic acid (8S,15S-DiHETE) using HPLC (Figs. 3a to 3c). The chemical structures of all products generated by the reaction of 15S-lipoxygenase with arachidonic acid were identified by LC-MS / MS (Figs. 4a to 4d). As a result, the lipooxidase derived from the microbial Chlamydomonas incerta was identified as a double-oxygenating 15S-lipoxygenase.

[0102] <Example 3>

[0103] Optimization of solvent, polymer, cell, and substrate concentrations in the production of resolvin D5 and protectin Dx using 3.15S-lipooxidase.

[0104] In order to determine the optimal concentration of dimethyl sulfoxide, a solvent, and polyvinylpyrrolidone, a polymer, to increase the production of the above-mentioned resolvin D5 and protectin Dx, whole-cell reactions were performed at various concentrations of dimethyl sulfoxide and polyvinylpyrrolidone for 60 minutes at pH 8.0 and 25°C in 1 mM docosahexaenoic acid. As a result, it was confirmed that the optimal concentration was 2% for each (Fig. 5).

[0105] And in order to find out the optimal concentration of cells and substrate under the optimal concentration of dimethyl sulfoxide as a solvent and polyvinylpyrrolidone as a polymer, when the whole-cell reaction was performed for 60 minutes at pH 8.0 and 25℃ for docosahexaenoic acid as a substrate in a concentration range of 1 mM to 8 mM, it was confirmed that the optimal concentration of cells was 0.5 g / L and the optimal concentration of substrate was 6 mM (Fig. 6).

[0106] <Example 4>

[0107] 4. Confirmation of simultaneous production of resolvin D5 and protectin Dx using 15S-lipooxidase.

[0108] The amounts of resolvin D5 and protectin Dx, each produced using docosahexaenoic acid, an unsaturated fatty acid, as a substrate by recombinant E. coli expressing the above 15S-lipid oxidase, were confirmed. Whole-cell reaction was performed for 90 minutes at pH 8.0 and 25°C using 6 mM docosahexaenoic acid as a substrate, and the production levels of resolvin D5 and protectin Dx were confirmed for each reaction time.

[0109] As a result, as shown in Fig. 7, it was confirmed that omega-3 dihydroxy fatty acids including about 4.0 mM (1.44 g / L) resolvin D5 and protectin Dx were produced by the whole-cell reaction in which 15S-lipid oxidase was expressed. At this time, 2.29 mM (826 mg / L) resolvin D5 and 1.68 mM (606 mg / L) protectin Dx were produced, respectively, and in particular, the concentration of protectin Dx obtained in the present invention is a result of a 5.6-fold increase compared to 0.3 mM (108 mg / L) produced by a two-step bioconversion by a combination reaction of recombinant microorganisms containing single-oxygenating lipooxidase reported in Non-Patent Document 2.

[0110] The present invention is an invention carried out through the following tasks.

[0111] [National Research and Development Project Supporting This Invention]

[0112] [Project ID] 00398879

[0113] [Project Number] RS-2024-00398879

[0114] [Ministry Name] Ministry of Agriculture, Food and Rural Affairs

[0115] [Name of project management (specialized) organization] National Institute of Agricultural and Food Technology Planning and Evaluation

[0116] [Research Project Name] Technology Commercialization Support (R&D)

[0117] [Research Project Name] Microbial Fermentation of Omega-3 Fatty Acids to Produce Food Materials and Development of Next-Generation Omega-3 Fatty Acid Products

[0118] [Name of the project performing organization] Konkuk University Industry-Academic Cooperation Foundation

[0119] Research Period: April 1, 2024 - December 31, 2026

Claims

1. A composition for producing resolvin D5 and protectin Dx, comprising as an active ingredient a 15S-lipid oxidase comprising an amino acid sequence of sequence number 1 derived from Chlamydomonas incerta; or a gene comprising a base sequence of sequence number 2 encoding the same.

2. A composition according to claim 1, characterized in that the composition further comprises docosahexanoic acid as a substrate.

3. A composition according to claim 2, characterized in that the concentration of the substrate is 1 mM to 8 mM.

4. A composition according to claim 1, characterized in that the concentration of whole cells containing the 15S-lipolytic enzyme in the composition is 0.1 g / L to 1 g / L.

5. A method for producing resolvin D5 and protectin Dx, comprising the step of treating a substrate with a composition of any one of claims 1 to 4, A manufacturing method characterized in that the above substrate is docosahexanoic acid.

6. A manufacturing method according to claim 5, characterized in that the step further comprises treating dimethyl sulfoxide as a solvent and further treating polyvinyl pyrrolidone as a polymer.

7. A manufacturing method according to claim 6, wherein the concentrations of dimethyl sulfoxide and polyvinylpyrrolidone are 1 (v / v)% to 5 (v / v)% and 1 (w / v)% to 5 (w / v)%, respectively.

8. A manufacturing method according to claim 5, characterized in that the step is performed at a pH of 7.0 to 9.

0.

9. A manufacturing method according to claim 5, characterized in that the step is performed at a temperature of 15°C to 40°C.

10. In the fifth paragraph, the manufacturing method is characterized in that the manufacturing method simultaneously produces resolvin D5 and protectin Dx in a single step.

11. A manufacturing method according to claim 5, characterized in that the concentration of docosahexaenoic acid is 1 mM to 8 mM.

12. A recombinant expression vector for producing resolvin D5 and protectin Dx comprising a 15S-lipid oxidase gene having a base sequence of sequence number 2.

13. A transformant in which a recombinant expression vector according to Article 12 is transformed into a host cell.

14. A 15S-lipooxidase comprising an amino acid sequence of sequence number 1 derived from Chlamydomonas incerta, a gene comprising a base sequence of sequence number 2 encoding the 15S-lipooxidase, a recombinant expression vector comprising the gene, or a transformant transformed with the recombinant expression vector; solvent; and A kit containing polymers.

15. A kit according to claim 14, characterized in that the concentration of the transformant is 0.1 g / L to 1 g / L.

16. A kit according to claim 14, wherein the solvent is dimethyl sulfoxide and the polymer is polyvinylpyrrolidone.

17. A kit according to claim 16, wherein the concentrations of dimethyl sulfoxide and polyvinylpyrrolidone are 1 (v / v)% to 5 (v / v)% and 1 (w / v)% to 5 (w / v)%, respectively.

18. A method for producing resolvin D5 and protectin Dx, comprising a step of treating a substrate with a kit according to any one of claims 14 to 17.

19. Use of a 15S-lipid oxidase comprising an amino acid sequence of SEQ ID NO: 1 derived from Chlamydomonas incerta for producing resolvin D5 and protectin Dx or a gene comprising a base sequence of SEQ ID NO: 2 encoding the same.

20. A composition for use in the production of resolvin D5 and protectin Dx, comprising a 15S-lipooxidase comprising an amino acid sequence of sequence number 1 derived from Chlamydomonas incerta; or a gene comprising a base sequence of sequence number 2 encoding the same.

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