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US-12618088-B2 - Production of lipids and terpenoids in Auxenochlorella protothecoides

US12618088B2US 12618088 B2US12618088 B2US 12618088B2US-12618088-B2

Abstract

Methods to produce oils with modified profiles of fatty acid, carotenoids and/or terpenoids in microalgal mutants are provided. Microalgal mutants produce the oil containing fatty acids, carotenoids and/or terpenoids of a modified profile with a disruption or ablation of one or more alleles of an endogenous polynucleotide or comprising an exogeneous gene are also provided.

Inventors

  • Jeffrey Moseley
  • Byung-hee Lee
  • Chung-Soon Im
  • Jane Kim
  • Dayoung Kim
  • Riyaz Bhat

Assignees

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

Dates

Publication Date
20260505
Application Date
20240531

Claims (16)

  1. 1 . A mutant Auxenochlorella protothecoides to produce oil with modified profiles of omega-3 fatty acids, omega-6 fatty acids, lutein, zeaxanthin, astaxanthin, 4-keto-lutein, or squalene, comprising a knock-out of at least one allele of lycopene cyclase epsilon LCYE-1, lycopene cyclase epsilon LYCE-2, squalene epoxidase SQE-1, or squalene epoxidase SQE-2, or a replacement of a native FAD3 promoter by a FATA gene promoter from Auxenochlorella protothecoides or a stearoyl-ACP desaturase (SAD2) promoter from Auxenochlorella protothecoides.
  2. 2 . The mutant Auxenochlorella protothecoides of claim 1 , wherein the mutant Auxenochlorella protothecoides is characterized in that one or more of the alleles of exogenous beta-ketolase 1 gene (CrBKT1) is knocked in.
  3. 3 . An oil produced by the mutant Auxenochlorella protothecoides of claim 1 .
  4. 4 . Composition comprising a mutant Auxenochlorella protothecoides of claim 1 , a culture thereof, or an oil from the mutant Auxenochlorella protothecoides.
  5. 5 . The composition of claim 4 , wherein the composition is a cosmetic composition, a food composition, a composition for a food additive, a feed composition, a composition for a feed additive, a raw material composition for food, a raw material composition for feed, or a raw material composition for cosmetics.
  6. 6 . The mutant Auxenochlorella protothecoides of claim 1 , wherein a zeaxanthin has a percent (w/w) of zeaxanthin 2-3-fold higher compared to the wild-type microalgae and the zeaxanthin is present as a major carotenoid.
  7. 7 . The mutant Auxenochlorella protothecoides of claim 6 , wherein the percent (w/w) of zeaxanthin produced ranges between 40 to 90% of total identified carotenoids.
  8. 8 . The mutant Auxenochlorella protothecoides of claim 1 , wherein the oil contains a mixture of 4-keto lutein and astaxanthin, and wherein the astaxanthin is present as a major carotenoid.
  9. 9 . The mutant Auxenochlorella protothecoides of claim 8 , wherein the keto carotenoids produced have a range of between 20-90% (w/w) of total identified carotenoids.
  10. 10 . The mutant Auxenochlorella protothecoides of claim 1 , wherein the oil contains squalene.
  11. 11 . The mutant Auxenochlorella protothecoides of claim 1 , wherein an omega-6 fatty acids and an omega-3 fatty acids have a weight ratio of omega-6 fatty acids to omega-3 fatty acids in the oil that is low compared to the oil produced from wild type microalgae.
  12. 12 . The mutant Auxenochlorella protothecoides of claim 11 , wherein the weight ratio of omega-6 to omega-3 in the oil ranges from 1:1 to 5:1 compared to the oil produced from wild type microalgae which is 7:1.
  13. 13 . The mutant Auxenochlorella protothecoides of claim 1 , wherein the omega-3 fatty acids increased 3-5-fold and the overall polyunsaturated fatty acids increased 2-3-fold compared to the wild-type strain.
  14. 14 . The mutant Auxenochlorella protothecoides of claim 1 , wherein the microalgal mutant is characterized in that one or more of the alleles of the lycopene cyclase epsilon LCYE-1 gene, lycopene cyclase epsilon LYCE-2 gene are knocked out.
  15. 15 . The mutant Auxenochlorella protothecoides of claim 1 , wherein the microalgal mutant is characterized in that one or more of the alleles of the squalene epoxidase SQE-1 gene, or squalene epoxidase SQE-2 gene are knocked out.
  16. 16 . The mutant Auxenochlorella protothecoides of claim 1 , wherein the microalgal mutant is characterized in that the native FAD3 promoter is replaced with a stearoyl-ACP desaturase (SAD2) promoter or a promoter from the Auxenochlorella protothecoides FATA gene encoding acyl-ACP thioesterase.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional of U.S. patent application Ser. No. 17/519,854, filed on Nov. 5, 2021, which claims the benefit of U.S. Provisional Patent Application No. 63/109,901 filed Nov. 5, 2020, the entire disclosure of each of which is hereby incorporated by reference in its entirety for all purposes. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM The official copy of the Sequence Listing is submitted concurrently with the specification as an xml file, made with WIPO Sequence Version 2.1.0, via EFS-Web, with a file name of “PBI009.xml”, a creation date of May 24, 2024, and a size of 66 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is incorporated in its entirety by reference herein. TECHNICAL FIELD The present invention describes microalgae having a functional oil-producing ability comprising an altered profile of fatty acids, carotenoids and/or terpenoids and a method of extracting oil using the microalgae. More specifically, it relates to a method for producing oil having modified fatty acids, carotenoids and/or terpenoids content compared to wild-type microalgae, such as Auxenochlorella protothecoides with inhouse strain designation as PB5. BACKGROUND ART Industrial production of lipids and fats from oleaginous microorganisms has been investigated since the late 19th century, but the promise of these “single cell oils” has only been realized in the last 20-30 years1. Oleaginous microorganisms trigger biosynthesis of storage lipids in response to imbalances between carbon (C) supply and other major nutrients such as nitrogen (N) or phosphorus (P) that are required for growth. This response allows accumulation of excess C under conditions where limitation for other nutrients prevents cell growth and division. Microbial storage lipids are largely composed of triacylglycerides (TAGs), which can be mobilized and used for rapid growth when the limiting nutrient or nutrients become available. Subsets of yeasts, fungi, and algae, but very few bacteria are oleaginous1. In the case of some algae, oil production can be stimulated by photosynthesis or by heterotrophic fermentation of sugars, but there is little economic incentive to make lipids which have similar composition or properties to commodity plant oils. In the early 2000s there was a flurry of interest and investment in photosynthetic production of biofuels from algae, but this has now largely faded due to the unfavorable energy return and economics of fuel production from aquatic microbes2,3. Heterotrophic oil productivity is substantially higher than is the case for photoautotrophic production, but the price of common sugar feedstocks is seldom any less that one quarter of the price of typical commodity oils, and the lipid yield for most heterotrophic processes is less than 25%. The economics can improve if the carbon source comes from a low value waste stream (e.g., lactose from cheese production, cellulosic sugars from agricultural waste). On the other hand, heterotrophic production of very long chain polyunsaturated fatty acids (VLCPUFAs) from algae, thraustochytrids and yeasts provides a roadmap for successful commercialization of microbial oils. Docosahexaenoic acid (DHA) from the dinoflagellate algae Crypthecodinium cohnii and arachidonic acid (ARA) from the yeast Mortierella alpina are both important components of infant formula1. These fatty acids are important components of human breast milk and are vital for brain and nervous system development4. The microbial oils address a particular market need for which there are no appropriate substitutes, since the only other significant sources of VLCPUFAs come from fish oils which are subject to contamination with toxins and heavy metals. Other successful commodity products from aquatic microalgae follow the VLCPUFA model. Astaxanthin and beta-carotene are key carotenoids for human nutrition; the former is one of the most powerful antioxidants known, while beta-carotene also has strong antioxidant activity and is a pro-vitamin A supporting vision5. Synthetic astaxanthin derived from petrochemicals is used extensively as a colorant in fish farming but is not approved for human consumption in food or supplements. The major natural source of astaxanthin is the chlorophyte Haematococcus lacustris (also H. pluvalis), which is grown photoautotrophically in ponds or bioreactors. Haematococcus cultures can produce up to 5% of their biomass as astaxanthin when they are subjected to nutrient and high light stress. Astaxanthin is also produced commercially by the heterotrophic fermentation of the yeast Phaffia rhodozyma, but the carotenoid content of the biomass is much lower than Haematococcus6,7. Natural beta-carotene is extracted from another chlorophyte, the halophile Dunaliella salina, which is grown photoautotrophically in large salt ponds. Both Haematococcus and Dunaliella fetch high prices for the biomass, but p