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US-12618087-B2 - Thraustochytrid strain genetically engineered for the production of biomaterials including long-chain polyunsaturated fatty acids

US12618087B2US 12618087 B2US12618087 B2US 12618087B2US-12618087-B2

Abstract

Provided herein are microorganism of a Thraustochytrid and a method for preparing bio-oil using the same, and more particularly, Schizochytrium sp. PB31 (PTA-123692) having bio-oil producibility, and a method of preparing bio-oil, particularly bio-oil having a content of omega-3 and/or 6 long-chain polyunsaturated fatty acids of 30% or more by weight or more based on total fatty acids, characterized by culturing the microorganism.

Inventors

  • Jane Kim
  • Riyaz Bhat
  • Jeffrey Moseley
  • Chungsoon IM

Assignees

  • PHYCOIL BIOTECHNOLOGY INTERNATIONAL, INC.
  • PHYCOILBIOTECH KOREA, INC.

Dates

Publication Date
20260505
Application Date
20230117

Claims (20)

  1. 1 . A method of preparing a bio-oil, the method comprising the steps of: (1) culturing Schizochytrium sp. PB31 having ATCC accession number PTA-123692, wherein the Schizochytrium sp. PB31 having ATCC accession number PTA-123692 comprises a disruption of a Pfs B; and (2) collecting a cultured cell biomass and (3) extracting and separating the bio-oil containing an omega-3 polyunsaturated fatty acid or an omega-6 polyunsaturated fatty acids.
  2. 2 . The method of claim 1 , wherein the bio-oil comprises 30 to 55 wt % polyunsaturated fatty acid based on total fatty acids.
  3. 3 . The method of claim 1 , wherein the culturing of step (1) is a batchwise culturing, a fed-batchwise culturing, or a continuous culturing.
  4. 4 . The method of claim 1 , further comprising disrupting the cultured cell biomass.
  5. 5 . The method of claim 1 , further comprising the step of purifying the bio-oil containing the polyunsaturated fatty acid.
  6. 6 . The method of claim 2 , wherein the cultured cell biomass contains 70 to 80 wt % of lipid.
  7. 7 . The method of claim 1 , wherein the culturing is a heterotrophic fermentation.
  8. 8 . The method of claim 1 , wherein the culturing step includes use of a light having a light intensity of less than 5 μmol/m 2 /s.
  9. 9 . The method of claim 8 , wherein the light signal is generated by a continuous light or a discontinuous light.
  10. 10 . The method of claim 8 , wherein the light signal is generated by a light having a full spectrum or a light having a specific wavelength.
  11. 11 . The method of claim 1 , wherein the culturing is performed using a medium containing a carbon source and a nitrogen source.
  12. 12 . The method of claim 11 , wherein the carbon source is selected from the group consisting of a glucose, a fructose, a galactose, a mannose, a sucrose, an arabinose, a xylose, a sodium acetate, and a glycerol.
  13. 13 . The method of claim 1 , wherein the bio-oil contains an ARA, an EPA, a DPA, or a DHA.
  14. 14 . The method of claim 13 , wherein the bio-oil contains an ARA.
  15. 15 . The method of claim 13 , wherein the bio-oil contains an EPA.
  16. 16 . The method of claim 13 , wherein the bio-oil contains a DPA.
  17. 17 . The method of claim 13 , wherein the bio-oil contains a DHA.
  18. 18 . The method of claim 13 , wherein the cultured cell biomass contains 70 to 80 wt % of lipid.
  19. 19 . The method of claim 18 , wherein the bio-oil comprises 30 to 55 wt % polyunsaturated fatty acid based on total fatty acids.
  20. 20 . The method of claim 19 , further comprising the step of purifying the bio-oil containing the polyunsaturated fatty acid.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application No. 63/300,101 filed on Jan. 17, 2022, the disclosures of which are herein incorporated by reference in their entirety. TECHNICAL FIELD The present invention describes a microorganism having a bio oil-producing ability and a method of producing bio-oil using the microorganism. More specifically, it relates to a method for producing oil having high-value omega-3 and/or 6 oils like ARA, EPA, DPA, and DHA. BACKGROUND ART Omega-3 fatty acids such as EPA and DHA have important biological roles in human cognitive, eye, and cardiovascular health. Fish such as salmon, sardines, and mackerels contain high amounts of these fatty acids. The demand for such fatty acids will exceed supply in the near future which combined with the natural source depletion due to climate change, overfishing, and environmental contaminants in the ocean is necessitating the development of alternative, safe, and sustainable sources of such omega-3 oils and long-chain polyunsaturated fatty acids for human consumption. Single-celled marine protists Thraustochytrids, found in coastal brackish waters, have been exploited for the abundance of omega-3 fatty acids, mainly DHA, and some additional amount of omega-6 fatty acids, DPA contributing to high total PUFA contents. Thraustochytrids especially strains in genera Schizoychytrium, Aurantiochytrium, and Thraustochytrium have been cultivated in enclosed fermenters for high omega-3 oil production as an alternative source to fish omega-3 oil for human consumption and cell biomass for animal feed. BRIEF SUMMARY OF THE INVENTION Disclosed in this application is a novel thraustochytrid strain named PB31 isolated from coastal waters in Hawaii for commercialization of biomaterials including DHA and its processes for production. The strain produces about 70-80% lipid and DHA comprises 30-50% by weight of that in the commercial scale fermenters. The processes include cells grown in low-intensity light resulting in a higher growth rate compared to the traditional heterotrophic growing conditions. U.S. Pat. No. 9,932,554 by the applicant of the present application describes the above process with signature culture condition called PSP. This condition combines traditional heterotrophic growth and low irradiance of light (less than 5 μmol photons/m2 s1) as a signal that activates photoreceptors in cells. The light intensity of said process does not activate photosynthesis machinery in the cells and is, therefore, differentiated from mixotrophic growth. The processes also include cultivation, harvesting, drying, extraction, and purification of bioproducts derived from this novel strain of thraustochytrid. The inventors of the present invention have also developed PB31 as a biotechnology platform to produce various high-value polyunsaturated omega-3 and/or -6 lipids. Efficient nuclear transformation and facile gene targeting by homologous recombination are critical features of this platform. Inventors of the present invention describe seminal experiments to establish a reproducible transformation system in PB31 at various genomic loci. Targeted knockdowns, knockouts, knock-ins, and point mutations in fatty acid or polyketide synthase lipid biosynthetic pathway genes enable the synthesis of tailored polyunsaturated fatty acids in PB31. Thraustochytrid PB31 can be employed as a general platform for the production of valuable biomolecules including high-value omega-3 and/or 6 oils like ARA EPA, DPA and DHA. Here, the inventors of the present invention demonstrate the utility of the system for the biosynthesis of modified omega-3 and/or 6 fatty acids. An object of the present invention is to provide microorganism having high polyunsaturated fatty acid productivity, and improving efficiency of a culture process, thereby allowing bio-oil to be prepared economically. Another object of the present invention is to provide a method of preparing bio-oil using the microorganism. In order to achieve the above objects, the present invention provides a microorganism, PB31, also designated as Schizochytrium sp. PB31 (PTA-123692), also just PTA-123692 herein, having bio-oil producibility. Further, the present invention provides a method of preparing bio-oil, the method comprising the steps of: (1) culturing the microorganism PB31, and (2) extracting and separating bio-oil containing omega-3 and/or 6 polyunsaturated fatty acids from the cultured microorganism. ADVANTAGEOUS EFFECTS The present invention shows that when using microorganism, oil having high-value omega-3 and/or 6 oils like-ARA, EPA, DPA and DHA can be effectively produced. DESCRIPTION OF THE DRAWING FIG. 1 shows the morphological features of PB31. FIG. 2 shows a diagram of Phylogenetic analysis placing PB31 within the lineage that includes Schizochytrium, Aurantiochytrium, and Thraustochtriidae. FIG. 3 is a graph showing biomass production under different grow