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EP-4737580-A1 - METHOD FOR PRODUCING, IN BIOREACTOR, RESOLVIN D5 FROM DOCOSAHEXAENOIC ACID-ENRICHED OIL BY USING LIPOXYGENASE

EP4737580A1EP 4737580 A1EP4737580 A1EP 4737580A1EP-4737580-A1

Abstract

The present invention relates to a composition and a method for producing resolvin D5, an endogenous substance in the human body, by means of a whole-cell reaction in flasks and bioreactors using a double 15-lipoxygenase derived from the bacterium Archangium violaceum, with polyunsaturated fatty acids obtained by hydrolysis and subsequent solvent extraction from a low-cost docosahexaenoic acid-enriched oil and high-purity docosahexaenoic acid reagents as substrates. More particularly, the present invention provides a method for obtaining docosahexaenoic acid at a purity of 90%, in which a docosahexaenoic acid-enriched oil is effectively hydrolyzed into polyunsaturated fatty acids comprising docosahexaenoic acid using a commercial lipase and an adsorption resin, and the resulting hydrolysate is then purified by solvent extraction, as well as a composition for use in such a method and a purified hydrolysate obtained thereby. In addition, the present invention provides a method for large-scale production of resolvin D5, an endogenous substance in the human body, in flasks and bioreactors through a whole-cell reaction using a double arachidonate 15-lipoxygenase derived from the bacterium Archangium violaceum, with a high-purity docosahexaenoic acid reagent as a substrate, as well as a composition for producing resolvin D5. According to the present invention, resolvin D5 can be produced in an environmentally friendly manner with high productivity and high yield, and thus can be usefully applied in various industrial fields, including pharmaceuticals, foods, and cosmetics.

Inventors

  • OH, DEOK-KUN
  • LEE, JIN

Assignees

  • Konkuk University Industrial Cooperation Corp

Dates

Publication Date
20260506
Application Date
20240731

Claims (20)

  1. A composition comprising a polyunsaturated fatty acid at a high purity derived from a DHA-containing oil obtained from fish or microalgae, for use in producing a dihydroxylated fatty acid having 20 to 22 carbon atoms.
  2. The composition of claim 1, wherein the polyunsaturated fatty acid is obtained by hydrolyzing the DHA-containing oil obtained from fish or microalgae.
  3. The composition of claim 2, wherein the hydrolysate of the DHA-containing oil obtained from fish or microalgae is obtained by hydrolyzing the DHA-containing oil using a lipase and an adsorption resin.
  4. The composition of claim 3, wherein the lipase is a lipase derived from Thermomyces lanuginosus, and the adsorption resin is SP207, HP20, HP2MG, SP825, SP850, or XAD761.
  5. The composition of claim 2, wherein the polyunsaturated fatty acid is obtained by purifying the hydrolysate of the DHA-containing oil obtained from fish or microalgae with a solvent in which hexane and acetonitrile are mixed at a volume ratio of 3 to 5:1.
  6. The composition of claim 1, wherein the polyunsaturated fatty acid comprises at least one fatty acid selected from the group consisting of docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, and arachidonic acid.
  7. The composition of claim 1, wherein the dihydroxylated fatty acid having 20 to 22 carbon atoms comprises resolvin D5.
  8. The composition of claim 1, wherein the composition comprises docosahexaenoic acid as the polyunsaturated fatty acid at a high purity of 70 to 99%.
  9. A method for producing resolvin D5, comprising: a) preparing a vector encoding a double 15-lipoxygenase gene derived from an Archangium violaceum; b) transforming a microorganism with the vector of step a); and c) reacting the transformed microorganism of step b) with a composition comprising a polyunsaturated fatty acid at a high purity derived from a DHA-containing oil obtained from fish or microalgae as a substrate, thereby producing resolvin D5.
  10. The method of claim 9, further comprising, after step c), a step d) of purification for producing resolvin D5 at a high yield.
  11. The method of claim 10, wherein the purification step d) is performed simultaneously or sequentially by ultrafiltration, column filtration, affinity chromatography, and reversed-phase chromatography.
  12. The method of claim 10, wherein the affinity chromatography is performed using an adsorption resin selected from HP20, HP2MG, SP207, SP825, SP850, or XAD761.
  13. The method of claim 10, wherein the reversed-phase chromatography is performed using a C1 (TMS) resin, a C4 (butyl) resin, a C8 (octyl) resin, or a C18 (octadecyl) resin.
  14. The method of claim 9, wherein the polyunsaturated fatty acid comprises at least one fatty acid selected from the group consisting of docosahexaenoic acid, eicosapentaenoic acid, docosapentaenoic acid, and arachidonic acid.
  15. The method of claim 9, wherein the composition of step c) comprises docosahexaenoic acid as the polyunsaturated fatty acid at a high purity of 70 to 99%, and is obtained by hydrolyzing the DHA-containing oil using a lipase and an adsorption resin.
  16. The method of claim 9, wherein the double 15-lipoxygenase of step a) comprises an amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80% sequence identity to SEQ ID NO: 1.
  17. The method of claim 15, wherein the polyunsaturated fatty acid is obtained by purifying a hydrolysate of the DHA-containing oil obtained from fish or microalgae with a solvent in which hexane and acetonitrile are mixed at a volume ratio of 3 to 5:1.
  18. The method of claim 15, wherein, in the hydrolysis step, when the concentration of the DHA-containing oil obtained from fish or microalgae is 5 to 15 g/L, the lipase is treated at a concentration of 800 to 1200 U/mL.
  19. The method of claim 9, wherein the reaction is performed at a pH in a range of 6 to 9.
  20. The method of claim 9, wherein the reaction is performed at a temperature in a range of 10 to 40 °C.

Description

TECHNICAL FIELD The present invention relates to a composition for producing high-purity resolvin D5 (RvD5) by a whole-cell reaction of a recombinant microorganism comprising a double-oxygenating arachidonate (ARA) 15-lipoxygenase (15-LOX) derived from Archangium violaceum in a bioreactor, and to a method for producing RvD5, an endogenous substance in the human body, using a hydrolysis extract of a docosahexaenoic acid (DHA)-containing oil as a substrate derived from a low-cost DHA-enriched fish (e.g., tuna) or microalgae. This invention was conducted with the support of a Collective Research Support Program funded by the Ministry of Science and ICT, entitled "Microbial engineering-based biosynthesis of human oxylipins based on metabolite profiling and elucidation of inflammation/obesity regulation mechanisms" (Project Identification Number: 1711154866; Performing Institution: Konkuk University). BACKGROUND ART Dihydroxylated fatty acids (DiHFAs) having 20 to 22 carbon atoms are lipid regulators containing two hydroxyl groups, which are mainly present in animal lipids, including those of the human body. In vivo, it is biosynthesized from animal-derived polyunsaturated fatty acids (PUFAs) through combined reactions of aspirin-treated cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX). DiHFAs generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) belong to lipid regulators and are classified as specialized pro-resolving mediators (SPMs), which are a type of human signaling molecules that promote the resolution of inflammation. SPMs are endogenous regulators of infection and inflammation and play important roles in various physiological activities, such as microbial clearance, pain relief, tissue regeneration, anti-inflammatory responses, and homeostasis, in animals including humans. Inflammation-promoting mediators include leukotrienes (LTs) and prostaglandins (PGs), whereasinflammation-resolving mediators include protectin (PDs), resolvins (Rvs), maresins (MaRs), and lipoxins (LXs). Among these, resolvin represents an endogenous lipid mediator class that is generated through combined reactions and double-oxygenation reactions catalyzed by lipoxygenase. Rvs ares derived from omega fatty acids having 20 or 22 carbon atoms, such as eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. Resolvin (Rvs) belongs to inflammation-resolving mediators that are mainly produced when lipoxygenases(LOXs) act on animal-derived unsaturated fatty acids, namely eicosapentaenoic acid and docosahexaenoic acid, thereby being converted into resolvin E and resolvin D, respectively. Rvs are known to be generated in trace amounts by macrophages during the resolution of inflammation and infection, in which they promote phagocytosis to remove viruses, bacteria, and inflammatory mediators, and facilitate the recovery of damaged tissues, thereby contributing to the resolution of inflammation and infection. Among these, resolvin D5 (RvD5) has been reported to exhibit diverse biological activities, including the ability to resolve inflammation even at trace levels in the human body, to reduce pain, and to decrease the required dosage of antibiotics in the treatment of infections, while also providing effects such as skin regeneration. In addition, resolvin D5 has been shown to reduce inflammation induced by intracellular oxidative stress, thereby exhibiting anti-inflammatory activity, and to suppress cell death through phagocytic activity, thereby contributing to the prevention of degeneration. Accordingly, if resolvin D5, which is an extremely high-cost specialized pro-resolving mediator (approximately 17,000,000 KRW per mg), can be mass-produced from a low-cost docosahexaenoic acid-enriched oil (80% (w/w), approximately 200,000 KRW per kg) through production optimization, industrialization thereof would be facilitated. Furthermore, resolvin D5 is suggested as a next-generation therapeutic material capable of addressing inflammation and infection, and is expected to be practically applicable in functional and efficacy studies in academic fields such as medicine and biotechnology. Lipoxygenase (LOX) catalyzes stereospecific and regioselective peroxidation through a dioxygenating reaction using polyunsaturated fatty acids having one or more cis,cis-1,4-pentadiene moieties as substrates. Based on stereospecificity, lipoxygenase is classified according to whether the resulting chirality or handedness exhibits an R-form or an S-form, and is referred to as R-lipoxygenases and S-lipoxygenases, respectively. Regioselectivity varies depending on the carbon chain length of the polyunsaturated fatty acid used as a substrate and the position of the pentadiene structure. Among these, lipoxygenase generates hydroxyl groups at the 5-, 8-, 9-, 11-, 12-, and 15-carbon positions of arachidonic acid, which is an animal-derived polyunsaturated fatty acid, and generates hydroxyl groups at the 7-, 10-, 11-,