A novel Thraustochytrid strain for producing biomaterials containing long-chain polyunsaturated fatty acids

Abstract

Provided herein are Thraustochytrid microorganisms and methods for preparing bio-oils using the microorganisms, and more specifically, Schizochytrium sp. PB31 (PTA-123692) having bio-oil productivity, as well as methods for preparing bio-oils, particularly bio-oils having an omega-3 and / or 6 long chain polyunsaturated fatty acid content of more than 30% by weight based on total fatty acids, characterized by culturing such microorganisms.

Description

[Technical field]

[0001] Technical Field CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 300,101, filed January 17, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention describes a microorganism capable of producing bio-oil and a method for producing bio-oil using the microorganism, more specifically, the present invention relates to a method for producing oil having high added value omega-3 and / or omega-6 oils such as ARA, EPA, DPA and DHA. [Background technology]

[0003] Background technology Omega-3 fatty acids such as EPA and DHA have important biological roles in human cognitive, ocular and cardiovascular health. Fish such as salmon, sardines and mackerel are high in these fatty acids. Demand for such fatty acids, coupled with the depletion of natural sources due to climate change, overfishing and marine environmental pollution, will exceed supply in the near future, necessitating the development of safe, sustainable and alternative sources of such omega-3 oils and long-chain polyunsaturated fatty acids for human consumption. Thraustochytrids, single-celled marine protists found in coastal brackish waters, exploit the abundance of omega-3 fatty acids, mainly DHA, with some additional amounts of the omega-6 fatty acid, DPA, contributing to the high total PUFA content. Strains of Thraustochytrids, specifically the following genera, Schizochytrium, Aurantiochytrium and Thraustochytrium, have been cultivated in closed fermenters for the production of high omega-3 oils as an alternative source of fish omega-3 oils for human consumption and cell biomass for animal feed.

[0004] Disclosure of the Invention technical issues Disclosed herein is a novel Thraustochytrid strain, designated PB31, isolated from Hawaiian waters for commercialization of biomaterials containing DHA, and a process for its production, which produces approximately 70-80% lipids, with DHA comprising 30-50% by weight in commercial-scale fermenters.

[0005] The process involves cells being grown in low intensity light resulting in a higher growth rate compared to conventional heterotrophic growth conditions. The applicant's U.S. Patent No. 9,932,554 describes the above process including a signature culture condition called PSP, which combines conventional heterotrophic growth with the use of light (5 μmol photons / m 2 ) as a signal to activate photoreceptors within the cells. 2 The process combines low illumination (less than 100 nm, 100 s) with light intensity that does not activate the photosynthetic machinery within the cells and is therefore distinct from mixotrophic growth. The process also includes culturing, harvesting, drying, extraction and purification of the biological products obtained from this novel strain of Thraustochytrid.

[0006] The inventors of the present invention have also developed PB31 as a bioengineering platform to produce a variety of high-value polyunsaturated omega-3 and / or 6 lipids. Efficient nuclear transformation and easy gene targeting by homologous recombination are crucial features of this platform. The inventors of the present invention describe promising experiments to establish a reproducible transformation system of PB31 at various genomic loci. Targeted knockdown, knockout, knockin and point mutations in fatty acid or polyketide synthesis enzyme lipid biosynthesis pathway genes enable the synthesis of regulated polyunsaturated fatty acids in PB31.

[0007] Thraustochytrid PB31 can be utilized as a versatile platform for producing valuable biomolecules, including high-value omega-3 and / or 6 oils such as ARA, EPA, DPA and DHA. Here, the inventors demonstrate the utility of the system for the biosynthesis of modified omega-3 and / or 6 fatty acids. Summary of the Invention [Problem to be solved by the invention]

[0008] Resolving the problem The object of the present invention is to provide a microorganism with high productivity of polyunsaturated fatty acids, improving the efficiency of the cultivation process, thereby allowing the economical preparation of bio-oil.

[0009] Another object of the present invention is to provide a method for preparing bio-oil using the present microorganism. [Means for solving the problem]

[0010] In order to achieve the above object, the present invention provides a microorganism PB31 having bio-oil productivity, Schizochytrium sp. PB31 (PTA-123692), also referred to herein simply as PTA-123692.

[0011] Further, the present invention provides a method for preparing a bio-oil, the method comprising: (1) culturing a microorganism PB31; and (2) extracting and isolating a bio-oil containing omega-3 and / or 6 polyunsaturated fatty acids from the cultured microorganism. Effect of the Invention

[0012] Advantageous Effects of the Invention The present invention shows that oils having high added value omega-3 and / or omega-6 oils such as ARA, EPA, DPA and DHA can be effectively produced when using microorganisms. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the morphological characteristics of PB31. [Diagram 2] FIG. 1 is a diagram of a phylogenetic analysis placing PB31 within a lineage including Schizochytrium, Aurantiochytrium, and Thraustochytriidae. [Diagram 3] 1 is a graph showing biomass production under different growth conditions. [Figure 4] 1 is a graph showing the effect of salinity on PB31 biomass production. [Diagram 5] FIG. 1 shows cells grown on a variable speed shaker. [Figure 6] 1 is a graph showing the productivity of PB31 on various feedstocks. [Figure 7] 1 is a graph showing glucose tolerance of PB31. [Figure 8] 1 is a graph showing the growth of PB31 in wastewater produced during raisin processing. [Figure 9] FIG. 1 shows the nucleotide sequence of the transforming DNA contained in plasmid pPB0128. [Figure 10] FIG. 1 shows the nucleotide sequence of the codon-optimized S. cerevisiae SUC2 gene (ScSUC2) of PB31 in plasmid pPB0136. [Figure 11] FIG. 1 shows the nucleotide sequence of the codon-optimized S. hindustanus bleomycin resistance gene of PB31 in plasmid pPB0240. [Figure 12A] FIG. 1 shows the nucleotide sequence of the transforming DNA contained in plasmid pPB0152. [Figure 12B] FIG. 1 shows the nucleotide sequence of the transforming DNA contained in plasmid pPB0152. [Figure 13] FIG. 1 shows the nucleotide sequence of the transforming DNA contained in plasmid pPB0218. [Figure 14] FIG. 1 shows the nucleotide sequence of the transforming DNA contained in plasmid pPB0258. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] BEST MODE FOR CARRYING OUT THE PRESENTINF Unless otherwise stated herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be all-inclusive in the manner of the term "comprising." The transitional terms / phrases (and any grammatical variations thereof) "comprising," "comprises," "comprise," "consisting essentially of," "consists essentially of," "consisting," and "consists of" may be used interchangeably.

[0015] The phrase "consisting essentially of" or "consists essentially of" indicates that the claim includes embodiments that contain the specified materials or steps as well as embodiments that do not materially affect the basic and novel characteristics of the claim.

[0016] The term "about" means within an acceptable error range of a value as determined by one of ordinary skill in the art, the range being determined in part by the constraints of how the value is measured or determined, i.e., the measurement system. When values ​​are described in the present application and claims, the term "about" meaning within an acceptable error range of the particular value should be assumed, unless otherwise specified. In the context of compositions containing amounts of ingredients, where the term "about" is used, these compositions contain the stated amount of ingredients with a variation (error range) of 0-10% (X±10%) around the value.

[0017] "Fatty acid" refers to a free fatty acid, a fatty acid salt, or a fatty acyl moiety in a glycerolipid. It is understood that the fatty acyl group of a glycerolipid can be described by the carboxylic acid or the anion of the carboxylic acid produced when the triglyceride is hydrolyzed or saponified.

[0018] A "fixed carbon source" is a molecule that contains carbon that can be utilized by the microorganisms cultured therein, generally an organic molecule that exists in the culture medium in solid or liquid form at ambient temperature and pressure. Thus, carbon dioxide is not a fixed carbon source.

[0019] Usage of the term "microorganism" in the patents that follow includes, but is not limited to, the following terms: "microalgae," "marine protists," "Thraustochytrids," and any other potential taxonomic classifications related to the PB31 microorganism, such as the genera Schizochytrium, Aurantiochytrium, Thraustochytrium, and Thraustochytriidae described herein. All taxonomic classifications used in this patent for the PB31 microorganism are based on current taxonomic classifications and available phylogenetic information, but are not intended to be limiting in the event that the standards of taxonomic classification used in this patent are revised after the filing date of this application.

[0020] "Microalgae" are eukaryotic microorganisms that contain chloroplasts or other plastids and can in some cases perform photosynthesis. Microalgae include obligate photoautotrophs that cannot metabolize fixed carbon sources for energy, and heterotrophs that can grow on fixed carbon sources. Microalgae include unicellular organisms that separate from sister cells immediately after cell division, such as Chlamydomonas, and microorganisms such as Volvox, which are simple multicellular photosynthetic microorganisms of two unique cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae include a group of unicellular microorganisms characterized by the presence of two flagella, or immature tripartite hair-like flagella, or unicellular or colonial microorganisms that have a flagellar stage in their life cycle, such as diatoms, dinoflagellates, and thraustochytrids.

[0021] With respect to recombinant cells, the term "knockdown" refers to a gene in which the production or activity of the protein encoded by the gene is partially suppressed (eg, about 1-95%).

[0022] An "oleaginous" cell is one that is capable of producing at least 20% lipid by dry cell weight, either naturally or by recombinant or classical strain improvement. An "oleagionous microbe" or "oleagionous microorganism" is a microorganism, including oleaginous microalgae, particularly eukaryotic microalgae that store lipids. Oleaginous cells also include cells from which some or all of the lipid or other content has been removed, and both living and dead cells.

[0023] With respect to functional oils, a "profile" is the distribution of types or triglycerides or fatty acyl groups within an oil. A "fatty acid profile" is the distribution of fatty acyl groups of the triglycerides of an oil, regardless of attachment to the glycerol backbone. Fatty acid profiles are typically determined by conversion to fatty acid methyl esters (FAMEs) followed by gas chromatography (GC) analysis with flame ionization detection (FID). A fatty acid profile may be expressed as 1 percent or more of fatty acids in the total fatty acid signal as determined from the area under the curve for fatty acids.

[0024] The terms "triglyceride," "triacylglyceride," and "TAG" are used interchangeably, as known to those of skill in the art.

[0025] In this disclosure, ranges are specified in abbreviated form to avoid detailing and describing each value within the range. Any suitable value within the range can be selected as the upper value, lower value, or end of the range, as necessary. For example, the range 0.1-1.0 represents the end values ​​of 0.1 and 1.0, as well as intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. When ranges are used herein, combinations and subcombinations of ranges (e.g., partial ranges within the disclosed ranges), and specific embodiments therein, are intended to be expressly included.

[0026] In the present invention, Thraustochytrid microorganisms were isolated from shallow Hawaiian waters to develop them as biotechnology platforms with various high-value polyunsaturated omega-3 and / or 6 lipid productivity.

[0027] Therefore, in one aspect, the present invention relates to PB31 having bio-oil productivity. The microorganism PB31 of the present invention is a microorganism classified as Thraustochytrid, and has productivity of omega-3 and / or omega-6 polyunsaturated fatty acids.

[0028] The microorganism of the present invention is a microorganism isolated from shallow waters in Hawaii, and may have the DNA sequence of the 18S rRNA gene shown in SEQ ID NO: 1. As a result of a search using NCBI (National Center for Biotechnology Information) BLAST, the microorganism was found to be a novel microorganism of the Thraustochytrium family, and was deposited with the American Type Culture Collection (ATCC) (Manassas, VA, USA) on December 1, 2016 as Schizochytrium sp. PB31, accession number PTA-123692.

[0029] The bio-oil produced by PTA-123692, in-house name PB31, according to the present invention is characterized in that it may contain 70% or more by weight of the dry biomass, and the content of polyunsaturated fatty acids contained in the bio-oil may be 30-55% by weight based on the weight of total fatty acids (TFA). The polyunsaturated fatty acids may be docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), or mixtures thereof, but preferably docosahexaenoic acid. Preferably, docosahexaenoic acid is characterized in that it is contained in an amount of 30% or more, preferably 30-50% by weight, based on the total fatty acid content. The bio-oil produced by PB31 may further contain saturated fatty acids such as palmitic acid or other fatty acid components in addition to the polyunsaturated fatty acids.

[0030] In addition, morphological and biochemical characteristics were analyzed to identify the microorganism of the present invention, and as a result, PB31 according to the present invention has the characteristics of the genus Schizochytrium, in which the cells divide successively into tetrads and octads before producing zoospores, resulting in irregularly shaped amoeboid cells (see FIG. 1 ).

[0031] The PB31 provided by the present invention has the advantage that the PB31 strain is a robust organism that grows rapidly and performs well under industrial / commercial-scale fermentation conditions such as high pressure and high shear force. PB31 does not produce significant amounts of other by-products (such as polysaccharides) during cultivation, which would prevent optimal cell biomass and oil productivity. Another advantage is that the cell wall of PB31 is strong enough to withstand high pressure in large-scale fermenters, and no pretreatment or additional steps are required to break the cells and extract oil during downstream processing. Another advantage is that the organism can be transformed simply and efficiently, and facile gene targeting by homologous recombination is reproducible by transformation at various genomic loci, allowing targeted knock-down, knock-out, knock-in and point mutations in fatty acid or polyketide synthesis enzyme lipid biosynthesis pathway genes, and thus the organism can be utilized as a versatile industrial biotechnology and metabolic engineering platform to produce valuable biomolecules, including high-value omega-3 and / or 6 oils such as ARA, EPA, DPA and DHA.

[0032] In the present invention, when the microorganism PB31 is cultured, the content of polyunsaturated fatty acids such as DHA in the biomass containing it and the bio-oil extracted therefrom is high, and the cells can be cultured at a high concentration. This has the advantage that bio-oil containing a high content of polyunsaturated fatty acids can be obtained with high productivity and high yield.

[0033] In another aspect of the invention, the invention provides a method of using PB31 as a platform for producing polyunsaturated fatty acids.

[0034] In another aspect of the present invention, the present invention provides an industrial biotechnology platform for producing polyunsaturated fatty acids, including PB31.

[0035] In an example of the present invention, PB31 can be efficiently and reproducibly transformed using a modified lithium acetate (LiAc) transformation method.

[0036] PB31 also undergoes homologous recombination and can be used as a tool to modulate various enzymatic activities by gene knockdown, knockout, and knockin.

[0037] Homologous recombination in PB31 can be used to enhance or suppress target gene activity by replacing endogenous regulatory elements with heterologous regulatory elements that are up- or down-regulated.

[0038] Specific point mutations can be introduced into endogenous enzymes to affect alterations in the catalytic activity of the targeted enzyme, resulting in enhanced product profiles or even novel products.

[0039] Optimized combinations of gene knockdown, knockout, and knock-in based on specific point mutations and / or homologous recombination coupled with modulation of endogenous regulatory elements will yield novel product profiles of PB31.

[0040] Yet another aspect of the present invention relates to the use of the PB31 of the present invention, a biomass containing it, or a concentrated or dried product of the biomass, or a bio-oil isolated from said microorganism in the production of food, beverages, nutritional preparations, pharmaceutical preparations, animal feed or cosmetics.

[0041] In particular, the PB31 provided in the present invention, the biomass containing it or concentrated or dried products of the biomass can be used in animal feed (particularly added or mixed for feed for cattle, pigs, chickens, goats, sheep and pet food) or aquatic animal feed without further purification or processing.

[0042] Therefore, in another aspect, the present invention provides a composition for use in a feed comprising the group consisting of PB31, a biomass obtainable by fermenting PB31, a concentrate or said biomass, and a dry product thereof.

[0043] On the other hand, in another aspect, the present invention relates to a method for producing biomass using PB31. The method for producing biomass of the present invention is characterized by comprising a step of culturing and fermenting PB31.

[0044] In the above production method, PB31 may be cultured using conventional heterotrophic fermentation, a closed system photobioreactor, or an open pond system.

[0045] In a particular embodiment, the production method according to the invention is characterized by culturing Schizochytrium sp. PB31 (PTA-123692) under conditions that provide a light signal with a particular range of light intensities in the culture. Preferably, light intensity refers to a very low light intensity that prevents the culture from being cultivated in an autotrophic or mixotrophic mode, in particular 0.1 μmol / m 2 s~5μmol / m 2 s, 0.1 μmol / m 2 s~4μmol / m 2 s, 0.1 μmol / m 2 s~3μmol / m 2 s, 0.5 μmol / m 2 s~5μmol / m 2 s, 0.5 μmol / m 2 s~4μmol / m 2 s, 0.5 μmol / m 2 s~3μmol / m 2 s, 1 μmol / m 2 s~5μmol / m 2 s, 1 μmol / m 2 s~4μmol / m 2 s, 1 μmol / m 2 s~3μmol / m 2 s, or 2 μmol / m 2 s~5μmol / m 2s. The light signal may be continuous or discontinuous light, may emit full spectrum light not limited to a particular range of wavelengths, or may provide light of a particular wavelength. When cultured by providing a light signal having such a particular range of light intensity, the microorganism according to the present invention may be cultured in a heterotrophic manner, but not in an autotrophic or mixotrophic manner.

[0046] In certain embodiments, blue light may be used in the production method. In particular, the wavelength of the light may be 400-500 nm, 450-500 nm or 450-490 nm.

[0047] In order to cultivate PB31, it is preferable to supply a carbon source together with the culture medium. The culture medium is not limited as long as it is a medium used to cultivate microorganisms, and may preferably include seawater. The carbon source can be any carbon source suitable for culturing microorganisms without limitation, and the carbon source may be, but is not limited to, monosaccharides such as glucose, fructose, galactose or mannose, disaccharides such as sucrose, and pentoses such as arabinose and xylose, sodium acetate, glycerol, crude glycerol, etc., and glucose is the most preferred.

[0048] It is preferable to supply the carbon sources in a continuous or semi-batch culture mode so that the appropriate concentrations are maintained. If necessary, methods such as pH stat or DO stat may be used, or the concentration of each carbon source may be measured in real time and supplied as needed.

[0049] Examples of such nutritional components include various nitrogen sources, phosphate sources and other components, which will be apparent to those skilled in the art. In addition, as an example of a medium, it will be apparent to those skilled in the art that a complex medium or a synthetic medium may be used.

[0050] Nitrogen sources can also include organic nitrogen sources such as yeast extract, corn steep liquor, beef extract, malt extract, peptone and tryptone, or inorganic sources such as ammonium acetate, ammonium nitrate, ammonium chloride, ammonium sulfate, sodium nitrate, urea or MSG.

[0051] During the cultivation of PB31 by the above-mentioned cultivation method, it is preferable to maintain the pH and / or temperature within a preset range. Methods well known in the art may be used to maintain a constant pH and / or temperature during cultivation. For example, but not limited to, a method using a cooling jacket using cooling water, a method using a pH controller to automatically supply acid or base, etc. may be used.

[0052] Preferably, the chloride concentration, sodium ion concentration and potassium ion concentration contained in the culture medium can be 0.6 to 0.8 g / L, 5.0 to 7.0 g / L and 0.4 to 0.6 g / L, respectively, and the culture can be carried out at a pH of 6 to 8. The acid used to adjust the pH can be preferably sulfuric acid, acetic acid, or a mixed acid thereof.

[0053] In a preferred embodiment, the microbial culture produced by the above production method can be harvested by methods such as centrifugation, flocculation, flotation and filtration. After harvesting the microbial product, several drying methods such as spray drying and drum drying can be used to obtain the culture powder.

[0054] In another aspect, the present invention provides a method for preparing a bio-oil, the method comprising the steps of (1) culturing PB31; and (2) extracting and isolating a bio-oil containing omega-3 and / or 6 polyunsaturated fatty acids from the cultured microorganism.

[0055] In the present invention, the culture in the above step (1) may be carried out in a mode selected from the group consisting of batch, semi-batch and continuous culture, and the above step (2) may further include a step of disrupting the cells.

[0056] The cell disruption may be cell disruption using an ultrasonic dispersion device, cell disruption using a pulsed electric field, cell disruption using enzymes, cell disruption using osmotic pressure, cell disruption using an electron beam, or cell disruption using an organic solvent.

[0057] The method for preparing the bio-oil of the present invention may further comprise a step (3) of purifying the bio-oil containing omega-3 and / or omega-6 fatty acids.

[0058] In the present invention, refining may include a step of collecting only the oil phase from the oil phase containing the bio-oil and the aqueous phase containing the cell parts, and may be carried out by including one or more steps of removing a solidified oil fraction, bleaching using bleaching earth or activated carbon, filtering, and deodorizing.

[0059] In the present invention, the deodorizing step may be carried out by a steam deodorization process under reduced pressure.

[0060] In one embodiment of the present invention, the method for preparing a bio-oil containing omega-3 and / or omega-6 fatty acids using PB31 includes: (1) culturing PB31; (2) harvesting the cultured cell biomass and extracting and isolating a bio-oil containing omega-3 and / or 6 polyunsaturated fatty acids.

[0061] The method comprises: (3) The method may further comprise refining the bio-oil containing the separated omega-3 and / or omega-6 polyunsaturated fatty acids.

[0062] Each step is described in detail below. The cultivation of PB31 in the above step (1) may proceed in a manner selected from batch, semi-batch and continuous cultivation, and it is preferable to use semi-batch or continuous cultivation.

[0063] In step (1), it is preferable to supply a carbon source for culturing PB31 by semi-batch or continuous culture. In this specification, the carbon source may be used without being limited to only the case of growth using PB31, and glucose, fructose, sucrose, galactose, glycerol, crude glycerol which is biodiesel waste liquid, etc. are preferable, but not limited thereto, and glucose is most preferable. The carbon source is preferably supplied in a continuous or semi-batch manner so as to maintain an appropriate concentration, and a method such as a pH stat or DO stat may be used as necessary, and a method of supplying a carbon source by measuring the concentration of each carbon source in real time as necessary, etc., may also be used. In addition, it is clear to those skilled in the art that nutrients necessary for the growth of PB31 may be contained in the medium, and various nitrogen sources, phosphate sources, other components, etc. may be contained, and complex media, synthetic media, etc. may be used. As nitrogen sources, organic nitrogen sources such as yeast extract, corn infusion, beef extract, malt extract, peptone, tryptone, etc., and inorganic nitrogen sources such as acetate, ammonium nitrate, ammonium sulfate, sodium nitrate, urea, etc. may be used.

[0064] In particular, it is preferable to set the salt concentration at an appropriate concentration level and carry out the culture within that range.

[0065] In step (1), it is preferable to maintain the pH and / or temperature within a predetermined range during the cultivation of PB31 by semi-batch or continuous cultivation. As a method for constantly maintaining the pH and / or temperature during the cultivation, a method known to those skilled in the art, such as a method using a cooling jacket with cooling water, a method using a pH controller to automatically supply acid or base, etc., can be used, but is not limited thereto.

[0066] Furthermore, it is preferable that the culture of PB31 by semi-batch or continuous culture is carried out under sufficient aeration and agitation. The aeration rate and agitation rate can be appropriately selected by those skilled in the art according to the process conditions. More specifically, since PB31 is aerotropic and has weak characteristics under shear stress caused by agitation, it is preferable that the agitation rate can be selected from 50 to 300 rpm, preferably 100 to 300 rpm, and the aeration rate can be 0.5 to 5 vvm, preferably 1 to 3 vvm.

[0067] The content of omega-3 and / or omega-6 polyunsaturated fatty acids in the bio-oil produced by the cultivation of step (1) according to the present invention is 30% by weight or more, preferably 40% by weight or more, and most preferably 50% by weight or more, based on the total fatty acids.

[0068] The step of harvesting the cultured PB31 and extracting and separating the bio-oil containing omega-3 and / or 6 polyunsaturated fatty acids by step (2) includes a step of disrupting cells after completing the culture of step (1). In the step of disrupting cells, the cell disruption can be induced by a method such as cell disruption using a pulsed electric field, cell disruption using an enzyme, cell disruption using an electron beam, etc., but is not limited thereto, and it is clear to those skilled in the art that a method using an organic solvent such as hexane may be used to disrupt cells and extract oil. In particular, when a disruption technique is used after cell disruption using osmotic pressure, the cell disruption effect can be enhanced.

[0069] As cell disruption progresses, phase separation occurs into an oil phase and an aqueous phase containing the cell debris, and only the oil phase is collected at this point, and the final bio-oil product may be obtained by the purification process in step (3).

[0070] The refining of the bio-oil by step (3) can be carried out by including one or more steps selected from the group consisting of leaving the oil phase at -5 to 0°C for 5 to 20 hours to extract the solidified oil fraction, bleaching the oil fraction using bleaching earth and / or activated carbon, filtering, and deodorizing, and preferably, these steps can be carried out sequentially.

[0071] Filtration is preferably carried out using a filter having a pore size of 0.5-1 μm, and deodorization is preferably carried out by a steam deodorization process under reduced pressure, but is not limited thereto.

[0072] The bio-oil produced by the production method of the present invention may contain, but is not limited to, about 30-55% by weight of total fatty acids (TFA), preferably 30% or more polyunsaturated fatty acids. Preferably, the bio-oil may contain 30-55% by weight of docosahexaenoic acid. EXAMPLES

[0073] Mode of Invention The present invention will now be described in detail with reference to the following examples. These examples are only intended to specifically illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0074] I. Microbial Isolation PB31 was isolated from fallen leaves in shallow coastal seawater. After washing with sterile seawater, the collected leaves were cut into small rectangular pieces and soaked in the same seawater for 24 hours at room temperature. 1 ml of this seawater was transferred to each well of a 12-well plate containing 2 ml of seawater, 0.3% yeast extract, 2% glucose, and 300-600 μg / ml penicillin and streptomycin. A few pieces of washed leaves were placed on a seawater agar plate containing 0.3% yeast extract, 2% glucose, 600 μg / ml penicillin G, and 600 μg / ml streptomycin. After several dilutions into new 12-well plates and streaking onto new agar plates, the inventors were able to obtain pure single-cell cultures. Each colony isolated from the agar plate was transferred to a new agar plate and grown to confluence. Once the cells on the agar plate were checked for contamination, they were harvested and expanded into a 125 ml flask containing 50 ml of medium and grown under PSP conditions at room temperature on an orbital shaker at 150 rpm for 4 days. As the cells grew, the culture turned pale yellow, whitish yellow, or vanilla in color. After checking for contamination, a small amount of the cell culture was transferred to a new flask for maintenance, and the remaining cell culture was transferred to a clean 50 ml Falcon tube and centrifuged at maximum speed for 3 minutes to collect the cell pellet. The pellet was lyophilized and stored in a -20°C freezer for further analysis.

[0075] II. Microbial Identification PB31 cells grown for 4 days in basal growth medium containing 2% glucose were harvested by centrifugation. Basal growth medium consisted of: 13.5g Na2SO4, 1.5g NaCl, 2.57g MgSO47H2O, 0.3g CaCl2, 0.75g NaNO3, 3g yeast extract, 2.38g HEPES, 0.027g NH4Cl, 10ml trace metals stock solution, 1ml chelated iron solution, 80ml 0.1M phosphate solution (pH 7.8) and 1ml vitamin solution in 1L. Trace metals stock solution consisted of: 0.1g Na2EDTA 2H2O, 0.114g H3BO3, 4.9mg FeCl36H2O, 1.92mg MnCl24H2O, 2.2mg ZnSO47H2O and 0.48mg CoCl26H2O in 100ml. The chelated iron stock solution consisted of: 0.5 g Na2EDTA, 10 ml 1M HCl solution and 0.081 g FeCl36H2O in 100 ml. The sterile vitamin stock solution consisted of: 50 mM HEPES (pH 7.8), 0.44 g thiamine HCl, 0.005 g biotin, 0.027 g B12 and 0.619 g D-pantothenate hemicalcium in 200 ml. The phosphate stock solution consisted of: 90.8 ml 1M K2HPO4 and 9.2 ml 1M KH2PO4 in 1 L.

[0076] By microscopic observation and isolation of single colonies from agar plates, the isolated strain showed characteristics of a microorganism belonging to the genus Thraustochytrids.

[0077] Some zoospores with two flagella were observed, especially early in the growth period or when glucose was not present in the culture medium. The growing cells actively divided into bipartites, tetrads, and octads, and upon entering the stationary growth phase, oil droplets accumulated intracellularly (Figure 1). A literature search revealed that "Schizochytrium species are characterized by successive two cell divisions into tetrads and octads prior to the production of zoospores. Schizochytrium also produces 'irregularly shaped amoeboid cells' (Mycosphere, Cultural optimization of Thraustochytrids for Biomass and Fatty Acid Production, 2011)." These observations suggested that the novel PB31 strain described in this patent may be a Schizochytrium species.

[0078] To confirm the genetic identity of this strain, genomic DNA was extracted from PB31 using CTAB and phenol / chloroform / isoamyl alcohol protocols and the partial 18S rRNA gene was amplified using primers PB00020 and PB00021 (Honda et al. 1999) with Herculase II fusion DNA polymerase enzyme with dNTPs combo kit (Agilent, USA), followed by Sanger sequencing (Genewiz, USA) of the amplified PCR products. The sequences of the primers used to amplify 18S rRNA are shown below.

[0079] PB00020 Forward: 5'-AACCTGGTTGATCCTGCCAGTA-3' (SEQ ID NO: 3) PB00021 Reverse: 5'-CCTTGTTACGACTTCACCTTCCTCT-3' (SEQ ID NO: 4) The PCR protocol included the following steps.

[0080] 1) 95℃ for 2 minutes, 2) 95°C for 20 seconds, 3) 50°C for 20 seconds, 4) 72°C for 1 minute, repeat steps 2 to 4 30 times, 5) 72°C for 3 minutes and 6) 24℃ for 1 minute Pairwise comparison of the 18S ribosomal RNA gene sequence of PB31 (SEQ ID NO:1 and SEQ ID NO:2) showed that it is closely related to Schizochytrium species, Thraustochytriidae species and Aurantiochytrium species (Table 1 and FIG. 2).

[0081] [Table 1]

[0082] III. Growth experiments and medium optimization The initial inoculum in a 250 ml Erlenmeyer flask containing 100 ml of seawater, 0.3% yeast extract, and 2% glucose was incubated for 1 h at 4 °C for 1 h in a PSP shaker (light intensity 0.1–1 μmol photons / m 2 The cells were grown at room temperature in a shaker fitted with 6-12 custom-made white or blue LED bulbs for 48 h. After 48 h of growth in the initial flask, 5 ml of cell culture was transferred to three new flasks containing 95 ml of basal growth medium and 2% glucose. The incubator shaker was set up as follows: one flask was designated as a dark shaker, one was set up in a shaker fitted with blue LED bulbs for the PSP experimental condition, and the last one was set up in a shaker fitted with 35 μmol photons / m 2 The cells were kept under white light conditions at 400 nm and 1000 nm for 1 h. The cell cultures were grown for 4-5 days until all the glucose supplied was consumed. One ml of cell culture was harvested from each flask to measure the biomass and glucose consumption daily. The biomass production of PB31 under different conditions is shown in Figure 3. Growth under PSP conditions yielded the highest biomass compared to dark conditions. Based on the lipid data, blue light conditions gave the best productivity and higher DHA production (Table 2).

[0083] [Table 2]

[0084] Unless otherwise stated, the following set of experiments was carried out at room temperature at 150 rpm in a PSP shaker to optimize the medium for optimal PB31 growth.

[0085] 1. Salinity The initial inoculum was prepared as described above. 5 ml of the initial inoculum was transferred to four 250 ml baffled flasks set up with 95 ml of basal growth medium containing different Na2SO4 concentrations (12, 17, 22 and 27 g / L), 30 mM MSG and 4% glucose. Cell growth was measured by taking 1 ml samples from the flasks daily and measuring biomass and glucose consumption for 4-6 days or until all glucose was consumed. Based on the experimental data, 17 g / L Na2SO4 gave the best growth (Figure 4).

[0086] 2. Dissolved oxygen level Two incubator shakers were set: one shaking at 150 rpm and the other at 190 rpm. Flasks containing 100 ml of cell culture were incubated for 3 days. Cell proliferation in the different conditions was measured in the same way as above. With the faster shaker speed, the inventors were able to reduce proliferation by almost 24 hours when compared to 150 rpm (Figure 5).

[0087] 3. Carbon Source Monosaccharides (glucose, fructose, galactose and mannose), disaccharides (sucrose) and pentoses (arabinose and xylose) were used to test the growth of PB31. Other commonly used carbon sources such as sodium acetate and glycerol were also tested in combination with glucose or separately. Cells were incubated with 1% of the various carbon sources in 6-well plates. Xylose and arabinose were tested in combination with 1% glucose. Different ratios of glucose and glycerol mixtures were tested with working total carbon source concentrations reaching 2%-4% (w / v) in cell cultures in 250 ml flasks.

[0088] Compared to other feedstocks tested, glucose gave the best productivity in 6-well plate experiments. Glycerol was the next best carbon source used by the cells. Other carbon sources such as galactose, fructose and mannose also gave good growth. Using a mixture of glucose with a 5-carbon ring sugar (arabinose or xylose) did not provide any additional benefit compared to the glucose-only sample (Figure 6).

[0089] PB31 was grown in media containing different concentrations of glucose (20 g / L, 40 g / L, 60 g / L, 80 g / L and 100 g / L) to determine its sugar tolerance level (FIG. 7). The experiment was carried out for 5 days. The best biomass productivity was achieved at 20 g / L glucose, but higher concentrations of glucose (up to 100 g / L) were well tolerated by PB31 without significant effects on its growth characteristics, suggesting that PB31 can grow efficiently in highly concentrated glucose solutions.

[0090] Flask data for PB31 showed higher growth and higher DHA (C22:6n3) productivity at 2 (w / v)% glycerol (16.7 mg / ml, 51.68%) compared to 2 (w / v)% glucose (12.8 mg / ml, 40.33%) (Table 3). When compared to results reported by others (US Patent Publication No. 20090117194; Burja et al. data), we achieved higher biomass, total fatty acid and DHA productivity. 4% (w / v) glycerol provided further increases in DHA, TFA and biomass. Glucose-glycerol mixtures at various ratios also performed better than glucose alone, with improvements in TFA, DHA and biomass depending on the increasing concentration of glycerol.

[0091] [Table 3]

[0092] 4. Nitrogen Source Different ratios of inorganic and organic nitrogen sources were tested for the growth of PB31. For inorganic nitrogen, the inventors varied the sodium nitrate, ammonium chloride and MSG concentrations in the medium to determine their effect on biomass and DHA productivity. For organic nitrogen sources, the inventors varied the percentage of yeast extract in the medium from 0.1% to 0.3% (w / v). The best biomass productivity and DHA percentage was achieved with 20 mM NaNO3 + 0.5 mM NH4Cl + 0.3% yeast extract + 30 mM MSG. However, this condition resulted in the lowest total fatty acid percentage due to the higher amount of total nitrogen in the medium used (Table 4).

[0093] [Table 4]

[0094] 5. Wastewater as a carbon source Two liters of wastewater, derived from water used to wash dried grapes and containing approximately 2-4% glucose, was obtained from Lion Raisins (Selma, CA). PB31 was grown under different conditions using the raisin wastewater to compare its growth against a basal growth medium. The test conditions were as follows:

[0095] 1) Basal growth cell medium + 2% (w / v) glucose 2) Wastewater + NaNO3 (same concentration as #1) 3) Wastewater (RW) only (raisin wastewater) 4) 25% (v / v) wastewater (25% wastewater + 75% basal growth medium) 5) 50% (v / v) wastewater (50% wastewater + 50% basal growth medium) 6) 75% (v / v) wastewater (75% wastewater + 25% basal growth medium) Cell cultures were grown in 6-well plates for 4 days and growth was monitored by measuring the optical density of the cell cultures on the 3rd and 4th days.

[0096] The growth of PB31 in raisin wastewater was comparable to that in normal medium. A mixture of basal growth medium and 25–50% (v / v) raisin wastewater yielded the highest growth, indicating the potential for wastewater utilization in large-scale fermentation of PB31 (Figure 8).

[0097] III. Mutagenesis PB31 cells were subjected to random mutagenesis using ultraviolet light, followed by triclosan treatment to improve growth rate and lipid production. Four 10 ml cultures of logarithmic-phase PB31 were placed on petri dishes and placed 10–15 cm away from a UV bulb in a biological safety hood for 14–60 min. One ml from each condition was diluted 1:10,000 in basal growth medium and spread onto agar plates containing 1–10 mM triclosan. Approximately 3–5 colonies were picked from each plate and grown on 6-well plates. Growth rate was monitored by measuring optical density at 750 nm using a UV spectrophotometer, and glucose consumption was monitored by measuring optical density at 340 nm.

[0098] Three samples showed faster glucose consumption and growth rates. These strains were named PB31-2, PB31-4 and PB31-5. The mutated strains also produced higher amounts of lipids and DHA compared to wild-type cells in flask and 5L fermenter experiments (Tables 5 and 6).

[0099] [Table 5]

[0100] [Table 6]

[0101] Semi-batch 5000L fermentation and optimization of PB31-4 PB31-4 was grown for 5 days in a custom-built 5000 L commercial-scale fermenter equipped with a low-irradiance light strip to obtain PSP conditions. The medium for the large-scale fermenter consisted of the following in 1 L of water: 13-17 g Na2SO4, 1.5 g NaCl, 3-6 g MgSO4 7H2O, 0.3 g CaCl2, 1.7 g NaNO3, 3-5 g yeast extract, 12-20 g MSG, 0.027 g NH4Cl, 10-20 ml of trace metals stock solution, 1-2 ml of chelated iron solution, 80 ml of 0.1 M phosphate solution (pH 7.8), and 4 ml of vitamin solution. The trace metals stock solution consisted of: 0.1 g Na2EDTA 2H2O, 0.114 g H3BO3, 4.9 mg FeCl3 6H2O, 1.92 mg MnCl2 4H2O, 2.2 mg ZnSO4 7H2O, and 0.48 mg CoCl2 6H2O in 100 ml. The chelated iron stock solution consisted of: 0.5 g Na2EDTA, 10 ml 1 M HCl solution, and 0.081 g FeCl3 6H2O in 100 ml. The sterile vitamin stock solution consisted of: 50 mM HEPES (pH 7.8), 0.44 g thiamine HCl, 0.005 g biotin, 0.027 g B12, and 0.619 g D-pantothenate hemicalcium in 200 ml. The stock phosphate solution consisted of: 90.8ml 1M K2HPO4 and 9.2ml 1M KH2PO4 in 1 L. pH was adjusted using 8% NaOH, 8% KOH and 10% H2SO4 solutions. 50ppm antifoam was used to control foam and bubbles formed during fermentation. Dissolved oxygen levels were kept below 1 during the lipid production step.

[0102] Results from 18 large scale fermentor runs of PB31-4 shown in Table 7 demonstrate consistent production of over 70% total oil and 45% DHA oil by weight in the biomass.

[0103] [Table 7]

[0104] Total lipids were extracted from freeze-dried cell pellets grown in 250 ml flasks. Approximately 2 g of sample in a 50 ml Falcon tube was treated with 5 ml HCl in a water bath at 80°C for approximately 1 hour with occasional shaking. After the sample was cooled, 35-40 ml of ethyl ether was added to the Falcon tube followed by vortexing and centrifugation at maximum rpm. The upper solvent layer was collected in a round-bottom flask. Extraction with ethyl ether was performed two more times. 30 ml of petroleum ether was added to the Falcon tube followed by vortexing and centrifugation. The upper solvent layer was collected and the ether solvent in the round-bottom flask was evaporated using a Rotavapor.

[0105] 25 mg of extracted oil was transferred to a 5 ml glass vial. 1 ml of isooctane and 2 ml of 14% boron trifluoride (BF3) were added to the sample followed by incubation on a 100°C heat block for 1 hour. The cooled methylated oil sample was transferred to a 15 ml Falcon tube after adding 5 ml of saturated NaCl solution and 1 ml of isooctane. After vortexing and centrifugation, the upper oil layer was collected in a microtube for GC chromatography. The samples were then analyzed using an Agilent Technologies 7890A GC system.

[0106] The fatty acids extracted from PB31 cells are mainly composed of palmitic acid and DHA. Palmitic acid accounts for nearly 51% of the total fatty acids (TFA), while DHA accounts for the remaining 49% of TFA. Palmitic acid and DHA are also the predominant fatty acids produced by S. limacinum (Yokochi et al., 1998), S. mangrovei (Fan et al., 2001), T. aureum (Bajpaj et al., 1991; Iida et al., 1996), and Thraustochytrium species (Singh et al., 1996).

[0107] For a more concentrated, higher volume fermentation product (5000L), PB31 was grown under PSP conditions to produce the maximum amount of algal bioproduct. PB31 can be cultured on a large scale using cultivation methods such as conventional heterotrophic fermentation, closed system photobioreactors, or open pond systems. The algal culture can be harvested using centrifugation, flocculation, flotation, or filtration. After harvesting the algal product, several drying methods such as spray dryers and drum dryers can be utilized to obtain algal powder. The algal culture can be used directly without harvesting the algal sediment prior to the drying step, which depends on which drying method will be utilized for the particular application. While supercritical fluid extraction is used for the 5000L fermenter product, organic chemical solvents such as Soxhlet or hexane extraction, or mechanical methods such as continuous squeezing, or ultrasound-assisted extraction can be used to extract the entire oil product from the algal powder.

[0108] PB31 cells are composed of 40-80% fat, 5-40% carbohydrate and 10-50% protein. Algal products such as hydrocarbons, carotenoids and extracted oils containing omega 3 and / or 6 fatty acids that are further refined can be used as processed materials for food inclusions (especially for chocolate) and additives, functional foods, pharmaceuticals and cosmetic inclusions. Algal powder can be used for animal feed (especially added or mixed into feed for cattle, pigs, chickens, goats, sheep and pet food) and aquatic animals without further processing, refining or treatment steps. Besides animal feed, algae-derived carbohydrates and proteins (such as dietary fiber and polysaccharides, oligopeptides, protein extracts and essential fatty acids) can be used for cosmetics, bioplastic materials and functional inclusions such as algal flour, vitamins and minerals.

[0109] IV.Metabolic manipulation 1. Transformation and recovery of PB31 recombinant strains Establishing a versatile and reproducible transformation system is an important first step in modulating the production of various biological products in PB31. EF1α is one of the most highly expressed genes in Thraustochytrids. To take advantage of its high expression, the inventors of the present invention attempted to use this locus as a landing site for transformation into the PB31 nuclear genome and select the EF1α promoter and 3' untranslated region to drive the expression of various marker genes. This was achieved by disrupting or knocking out the endogenous EF1α gene and replacing it with neomycin, sucrose invertase or bleomycin marker genes.

[0110] This example describes recombinant strains recovered after transformation of PB31 with plasmids pPB0128, pPB0136 and pPB0240, which contain the S. cerevisiae neomycin resistance gene, the S. cerevisiae sucrose invertase gene and the S. hindustanus bleomycin gene, respectively, under the control of the elongation factor 1 alpha promoter and terminator regions amplified from PB31 genomic DNA.

[0111] Approximately 1500 kb of the elongation factor 1 alpha (EF1α) gene region was amplified from PB31 using specific primers based on the genome sequences of closely related strains in the GenBank database. The purified PCR products were cloned using the pJET1.2 / blunt vector (Fisher Scientific, USA). Plasmid DNA extracted from 3-5 clones was sequenced to verify the amplified region. The sequence of each clone of the PB31 EF1α region showed several polymorphisms in the upstream 5' flanking region and downstream 3' flanking region of the gene coding sequence from the reference strain. Based on the PB31 EF1α region sequence, we decided to use approximately 1000 bp of the EF1α promoter and terminator region as potential recombination flanks in our PB31 transformation experiments.

[0112] Protein sequences of neomycin resistance and sucrose invertase genes from S. cerevisiae and bleomycin resistance gene from Streptoalloteichs hindustanus were downloaded from the public GenBank database. Protein sequences were codon-optimized for optimal expression in PB31 using several publicly available codon tables from closely related Thraustochytrid species. Optimized gene fragments were synthesized using an external supplier (Genewiz, San Francisco, CA, USA). Plasmid DNA extracted from E. coli transformed with synthetic neomycin, sucrose invertase or bleomycin genes cloned into pJET1.2 / blunt vector was sequenced to confirm the original sequence.

[0113] The PB31 EF1α promoter and terminator elements driving the neomycin resistance gene were assembled in the BamHI-cut PUC19 vector using the NEB assembly master mix (New England Biolabs, USA) and named pPB0128. The construct pPB0128 for transformation into PB31 was pPB0128-PB31EF1a::neoR(s)::PB31EF1a It can be written as:

[0114] The sequence of the transforming DNA contained in pPB0128 is shown in FIG. 9. The two BamHI restriction sites used to generate linear DNA for transforming the cassette into PB31 are shown in bold lower case at the beginning and end of the sequence. The underlined sequences in the 5' and 3' flanks of the construct represent the genomic DNA of PB31 that allows for targeted integration of the transforming DNA by homologous recombination at the EF1α region. The 5' flank also acts as a promoter region to drive expression of a downstream neomycin resistance gene that has been codon-optimized for expression in PB31, allowing the PB31 strain to grow in the presence of neomycin selection. ScNeo RThe start ATG and end TGA of the gene are shown in capital italics, and the coding region is shown in lowercase italics. R Immediately following is the underlined 3'-proximal of EF1α, which also contains the terminator region of the gene.

[0115] The inventors of the present invention also generated plasmids pPB136 and pPB240, which contain either sucrose invertase or bleomycin as marker genes. These constructs are as follows: pPB0136-PB31EF1a::ScSUC2::PB31EF1a pPB0240-PB31EF1a::ble::PB31EF1a It can be written as:

[0116] pPB0136 and pPB0240 have the same 5' and 3' EF1a flanking sequences as pPB0128 except that they contain either a sucrose invertase (pPB0136) or bleomycin gene (pPB0240) marker gene in place of the neomycin flanked by the PB31 EF1α flanks. The sequences of the sucrose invertase and bleomycin genes contained in plasmids pPB0136 and pPB0240 are shown in Figures 10 and 11, respectively.

[0117] Plasmids pPB0128, pPB0136 and pPB0240 were introduced into PB31 by a modified lithium acetate (LiAc) transformation method. Briefly, 50 ml of PB31 cell culture grown overnight in growth medium was harvested by centrifugation and washed with 5 ml of wash solution (0.1 M LiAc and 1× Tris EDTA, (pH 8.0)). The cell culture was centrifuged at 3750 rpm and the supernatant was discarded. The cell pellet was resuspended in 500-1000 ml of wash solution and incubated for 1 h at room temperature on an orbital shaker. 150 ml of the cell suspension was transferred to a sterile Eppendorf tube containing 12-15 μg of sterile, digested DNA and incubated for 30 min at room temperature on an orbital shaker. A mock transformation without DNA was also prepared as a control. 750 μl of PEG solution (0.1M LiAc+1×TE+40% PEG-4000) was added to the Eppendorf tube and incubation at room temperature on an orbital shaker was continued overnight. After overnight incubation, the cells were harvested by centrifugation. Optionally, the overnight incubated cells could be further incubated in a water bath at 42° C. for 10-15 minutes before harvesting the cells. After discarding the supernatant, 100-180 μl of growth medium was added to the cells. Primary transformants were selected for heterotrophic growth on media containing the appropriate selection depending on the introduced construct. Cells transformed with pPB0128 (containing the neomycin resistance gene) and pPB0240 (containing the bleomycin gene) were spread onto agar plates containing glucose as a carbon source and selected with neomycin or zeocin using glass beads. Cells transformed with pPB0136 (containing the sucrose invertase gene) were spread on agar plates containing sucrose as the sole carbon source.

[0118] Colonies began to appear after 2-3 days on plates containing cells transformed with pPB0128 and pPB0240, while colonies on plates containing pPB0136 took longer than 10-14 days. No colonies formed on plates containing mock transformed cells. The following table summarizes the results of various transformations of PB31 with pPB0128, pPB0136, and pPB0240 (Table 8).

[0119] [Table 8]

[0120] Single colonies were clonally purified and the insertion of the various marker genes at the EF1α locus was verified by PCR amplification and sequencing of the regions flanking the integration site.

[0121] Disruption of the EF1α gene had no significant effect on the growth pattern in engineered PB31, suggesting that this locus could be used as a neutral site for integrating heterologous gene cassettes to modulate fatty acid composition in subsequent experiments.

[0122] The above examples establish several facts, which are summarized below: 1. PB31 can be efficiently and reproducibly transformed using the modified LiAc transformation method described in our Examples.

[0123] 2. PB31 undergoes homologous recombination and can be used as a tool to modulate various enzyme activities by gene knockdown, knockout, and knockin.

[0124] 3. Homologous recombination in PB31 can be used to enhance or suppress target gene activity by replacing endogenous regulatory elements with heterologous regulatory elements that are up- or down-regulated.

[0125] 4. Specific point mutations can be introduced into endogenous enzymes to affect the modification of the catalytic activity of the target enzyme, resulting in an enhanced product profile or even novel products; The optimal combination of 5.2-4 results in a novel product profile for PB31.

[0126] 2. Disruption of fatty acid synthase genes in PB31 leads to enhanced PKS-dependent accumulation of DPA and DHA After establishing a robust PB31 transformation method, we next attempted to modulate polyunsaturated fatty acid levels in PB31 and decided to disrupt the fatty acid synthase (FAS) gene in PB31 with a neomycin resistance marker.

[0127] This example describes the construction of recombinant plasmid pPB0152, which contains the fatty acid synthase region amplified from PB31 and the Saccharomyces cerevisiae neomycin resistance gene (neoR) linked to the PB31 elongation factor 1 alpha terminator gene fragment amplified from pPB0128 (Example 1 above).

[0128] The upstream 5' flanking region of the fatty acid synthase (FAS) gene coding region was amplified from strain PB31 using primers based on the genomic sequence of closely related strains in the Genbank database. The purified PCR products were then cloned using pJET1.2 / blunt vector (Fisher Scientific, USA). Plasmid DNA extracted from 3-5 clones was sequenced to verify the amplified region. The sequence of each clone of the PB31 FAS region showed several polymorphisms from the reference strain and from the PB31 clone in the upstream 5' flanking region as well as the downstream 3' flanking region of the gene coding sequence, indicating two copies of the FAS gene with several polymorphic bases in the upstream 5' flanking region. pPB0152 was constructed to disrupt and downregulate the expression of the PB31 FAS gene. Based on the upstream 5' flanking FAS region sequence of PB31, approximately 852 bp of the 5' flanking FAS region was designated as the promoter of allele 1, and approximately 1013 bp was designated as the promoter of allele 2. We also amplified the FAS 3' flanking region 804 bp 500 bp downstream from the translation start of the FAS gene, so that the final construct (pPB0152) contained FAS allele 1 as the 5' flanking and 804 bp downstream FAS flanking as the 3' flanking, thereby deleting 500 bp from the FAS coding region and disrupting the FAS coding region upon transformation and integration into the PB31 genome. Plasmid pPB0152 was constructed by assembling the 852 bp FAS allele 1, neomycin resistance gene, EF1 alpha terminator region, and FAS 3' flanking into a pUC19 cloning vector containing a (BamH1) restriction site using NEB assembly master mix (New England Biolabs, USA). The 852 bp of PB31 FAS 5' flanking acted as a promoter driving expression downstream of the neomycin resistance gene. The final pPB0152 construct was as follows: pPB0152-PB31 FAS 5' flanking::Neo(R)-EF1a terminator::PB31 FAS 3' region It can be written as:

[0129] Figure 12 describes the sequence of plasmid construct pPB0152 used to generate linear DNA for cloning and transformation with the BamH1 restriction site shown in bold lower case. The upper case underlined sequences at the beginning and end of the construct represent the FAS 5' flanking of PB31 allele 1 and the PB31 FAS 3' flanking, which allow targeted integration of transforming DNA by homologous recombination at the FAS locus, disrupting allele 1 of the PB31 FAS gene. Proceeding in the 5' to 3' direction, the PB31 FAS 5' flanking acts as a promoter driving expression of the neomycin resistance gene shown in lower case italics. The start ATG and end TGA of neo(R) are shown in upper case italics. The neomycin coding sequence is immediately followed by the PB31 EF1 alpha derived terminator region in small capital letters. The PB31 FAS 3' flanking region (part of the FAS CDS) is shown in underlined capital letters. The final construct was sequenced to ensure the correct reading frame and targeting sequence.

[0130] To determine its effect on fatty acid profile, pPB0152 was transformed into PB31 and primary transformants were selected on growth medium containing neomycin. Single clone-purified colonies were grown under standard lipid production conditions in shake flasks. Fatty acid profiles of lipids from shake flask assays of representative derivative lines resulting from transformation of wild-type PB31 with pPB0152 are shown in Table 9.

[0131] [Table 9]

[0132] Disruption of FAS allele 1 in PB31 resulted in subtle but significant changes in the fatty acid profile of the derivative transgenic lines. Representative derivative lines PB31;152-1, PB31;152-2, and PB31;152-3 showed increased DPA (C22:5n6; 7.8%, 6.91%, and 8.48%, respectively, versus 5.54% in wild-type PB31) and DHA (C22:6n3; 35.55%, 35.21%, and 40.27%, respectively, versus 33.28% in wild-type PB31). The increase in DPA and DHA came at the expense of C14:0 and C16:0, which showed a consistent decrease in all representative derivative lines. Compared to wild-type PB31, the highest DPA and DHA were found in the derivative lines of the PB31;152-3 set, and thus a significant decrease in C14:0 and C16:0 appeared in this set. This data provides a strong indication that disruption of the FAS gene in PB31 simultaneously results in increased PKS-derived DPA and DHA with decreased C16:0 synthesis in the derivative strains. PB31;152-1 and PB31;152-3 were preserved as Phycoil engineered strains PES-100 and PES-101, respectively.

[0133] 3. Introduction of specific point mutations into the polyunsaturated fatty acid synthase (Pfs) B gene of PB31 results in recombinant strains that produce low or no C22:5n6. Approximately 1500 bases upstream and part of the polyunsaturated fatty acid synthase (Pfs) B coding region were amplified from PB31 genomic DNA. The amplified PCR product was purified, ligated into a blunt vector (pJET1.2), and transformed into competent cells. Plasmid DNA was extracted, sequenced, and verified against the locus of the reference genome sequence from Genbank. Three point mutations were introduced into the coding sequence of Pfs B to modify specific amino acids. The first mutation was introduced at 65 bases from the translation start to convert phenylalanine to leucine (F65L), the second mutation was introduced at 230 bases from the translation start for phenylalanine to leucine (F230L), and the third mutation was introduced at 231 bases from the translation start for isoleucine to threonine (I231T). These mutations, when introduced into the Aurantiochytrium sp. OH4 DHA synthase gene, were recently reported to trigger EPA production in a heterologous E. coli host (Hayashi et al., 2019). However, the functionality and specificity of these mutations to produce EPA in a Thraustochytrid host was not demonstrated. We sought to test the effect of these mutations in the PB31 Thraustochytrid host strain, and constructed plasmid pPB0218 for this purpose. The construct pPB218 introduced for expression in PB31 was: pPB0218-PB31_4-2 pfsBp 5' flanking: ScNeo-Ap bicistronic signal: PB31_4-2 PfsB F65L F230L I231T ORF 3' flanking It can be written as:

[0134] The sequence of plasmid pPB0218 is shown in FIG. 13. The NotI restriction enzyme sites used to generate linear DNA and for cloning are shown in lowercase bold typeface and delimit the 5' and 3' ends of the transforming DNA. The underlined uppercase letters on the 5' and 3' flanks of the construct represent the genomic DNA of Thraustochytrid PB31 that allows targeted integration of the transforming DNA by homologous recombination at the pfs B allele 2 locus. The 5' flank acts as a promoter driving expression of the downstream neomycin phosphotransferase II gene (Neo, codon-optimized for expression in A. protothecoides and encodes neomycin phosphotransferase II, allowing the strain to grow on the aminoglycoside antibiotic G418). The start ATG and end TGA of Neo are shown in uppercase italics, while the remaining sequence is shown in lowercase italics. The neomycin coding sequence is followed by a 101 nucleotide sequence from A. protothecoides in small capital letters, acting as a bicistronic signal to allow production of neomycin and mutated Pfs B allele 2 proteins from a single transcript. Immediately following this bicistronic sequence is the 3' pfs B flanking sequence, which contains the F65L, F230L and I231T mutations. The final construct was sequenced to ensure the correct reading frame and targeting sequence. Once transformed and integrated into the PB31 genome, we envisioned that Pfs B protein containing the intended modifications would be produced in recombinant strains selected by neomycin resistance.

[0135] pPB0218 was transformed into the PB31 Thraustochytrid strain, and primary transformants were selected on growth medium supplemented with G418. Single clone-purified colonies were grown under standard lipid production conditions in shake flasks. The profiles obtained from a representative set of derivative clones resulting from transformation with construct pPB0218 are shown in Table 10.

[0136] [Table 10]

[0137] Introduction of the F65L F230L I231T mutation in Pfs B allele 2 in PB31 did not enhance EPA accumulation in the derivative transgenic strains, in contrast to previously reported results obtained in the heterologous host E. coli (Table 10) (Hayashi et al., 2019). However, there was a significant reduction in DPA in all derivative transgenic lines. All of the derivative transgenic lines produced less than 2% DPA compared to 5.3% DPA in the parent PB31, except for PB31;218-4, which produced 3.3% DPA. PB31;218-5, PB31;218-9, and PB31;218-13 produced minimal amounts of DPA (1.16%, 0.95%, and 0.86%, respectively) compared to the control PB31. With the exception of PB31;218-13, there was a very slight reduction in DHA in the derivative lines, concomitant with a minor increase in C16:0 levels. In the derivative line PB31;218-13, the significant decrease in DPA and DHA levels was strongly associated with the increase in C16:0. These results demonstrate that the F65L F230L and I231T mutations modulate the Pfs B activity of PB31, resulting in DHA oil containing very low amounts of DPA. PB31;218-5, which produces 1.16% DPA, was preserved as Phycoil-engineered strain PES-102 and used as the parent strain in subsequent experiments. Since pPB0218 was targeted to introduce the F65L F230L and I231T mutations in Pfs B allele 2, we hypothesized that the unmutated allele 1 of Pfs B was likely responsible for the remaining DPA found in the derivative transgenic lines. To further reduce DPA levels, another construct (pPB0258) was made that introduced the same F65L F230L and I231T mutations in Pfs B allele 1. For this construct, we used the bleomycin gene from Streptoalloteicus hindustanus as a selectable marker. Construct pPB0258 contains the following: pPB0258-PB31_4-1 pfsBp 5' flanking: ble-Ap biscistronic signal: PB31_4-2 PfsB F65L F230L I231T ORF 3' flanking It can be written as:

[0138] The sequence of plasmid pPB0258 is shown in Figure 14. The NotI restriction enzyme site used to generate linear DNA and for cloning is shown in lowercase bold typeface and delimits the 5' and 3' ends of the transforming DNA. The underlined uppercase letters on the 5' and 3' flanks of the construct represent genomic DNA from Thraustochytrid PB31 that allows targeted integration of the transforming DNA by homologous recombination at the pfsB allele 1 locus. The 5' flank acts as a promoter driving expression of downstream bleomycin (Ble, codon-optimized for expression in PB31, allowing the strain to grow on Zeocin selection). The start ATG and end TGA of Ble are shown in uppercase italics, while the remaining sequence is shown in lowercase italics. The bleomycin coding sequence is followed by a 101 nucleotide bicistronic signal from A. protothecoides in small capital letters, allowing production of both bleomycin and the mutated Pfs B allele 1 protein from a single transcript. Immediately following this bicistronic sequence is the 3' pfs B flanking, which contains the F65L, F230L and I231T mutations. The final construct was sequenced to ensure the correct reading frame and targeting sequence.

[0139] pPB0258 was transformed into strain PES-102, and primary transformants were selected on growth medium supplemented with G418 and Zeocin. Single clone-purified colonies were grown under standard lipid production conditions in shake flasks. The profiles obtained from a representative set of derivative clones resulting from transformation with construct pPB0218 are shown in Table 11.

[0140] [Table 11]

[0141] As hypothesized, the F65L F230L and I231T mutations introduced into both alleles of Pfs B resulted in strains with almost no DPA. The DPA peaks found in PES-102;258-2 and PES-102;258-3 were below the detection level of GC and did not translate into measurable numbers (Table 10). The PES-102;258-2 and PES-102;258-3 derivative lines were processed a second time with similar results. Taken together, our data demonstrate that the F65L F230L and I231T mutations introduced into the Pfs B protein of Thraustochytrid PB31 can modulate the polyketide synthase (PKS) system so that DHA oil with very little or no DPA can be produced on demand. We expect to further fine-tune the ability to produce DHA oil with adjusted DPA content (between 0 and 5%) by controlling the fermentation conditions depending on the scale. PES-102;258-2 and PES-102;258-3 were preserved as phycoil-engineered strains PES-103 and PES-104, respectively.

[0142] The studies presented above demonstrate our ability to produce novel LCPUFAs by manipulating both the FAS and PKS systems.

Claims

1. A method for producing biomass comprising culturing and fermenting the microorganism PB31, also referred to as ATCC Accession No. PTA-123692.

2. 2. The method of claim 1, wherein the biomass contains 70-80% by weight lipids, the lipids containing 30-55% by weight long chain polyunsaturated fatty acids (LCPUFA).

3. 2. The method of claim 1, wherein the polyunsaturated fatty acid is one or more selected from the group consisting of ARA, EPA, DPA and DHA.

4. 2. The method of claim 1, wherein the culturing and fermentation is carried out heterotrophically by conventional heterotrophic fermentation, closed system photobioreactor or open pond system.

5. The culture and fermentation is carried out at a concentration of 5 μmol / m ２ 2. The method of claim 1 , wherein the method is performed under conditions that provide an optical signal having an optical intensity less than s.

6. 2. The method of claim 1, wherein the microorganism is a Schizochytrium species.

7. The method of claim 5 , wherein the optical signal is generated by continuous and / or discontinuous light.

8. The method of claim 5 , wherein the optical signal is generated by full spectrum light or light having a specific wavelength.

9. The method of claim 1 , wherein the culturing and fermentation is carried out using a medium containing a carbon source and a nitrogen source.

10. 9. The method of claim 8, wherein the carbon source is at least one selected from the group consisting of glucose, fructose, galactose, mannose, sucrose, arabinose, xylose, sodium acetate and glycerol.

11. 1. A method for preparing a bio-oil, the method comprising: (1) culturing PTA-123692; and (2) harvesting the cultured cell biomass and extracting and isolating a bio-oil containing omega-3 and / or omega-6 polyunsaturated fatty acids; and

12. 12. The method of claim 11, wherein the bio-oil comprises 30-55 wt. % polyunsaturated fatty acids based on total fatty acids.

13. 12. The method of claim 11, wherein said culturing in step (1) is carried out in a manner selected from the group consisting of batch, semi-batch and continuous culture.

14. The method of claim 11 , wherein step (2) further comprises disrupting the cells.

15. 12. The method of claim 11, further comprising the step (3) of refining the bio-oil containing polyunsaturated fatty acids.

16. Methods of using the microorganism PTA-123692 or mutants thereof as a commercial-scale biotechnology platform for the production of polyunsaturated fatty acids.

17. 17. The method of claim 16, wherein a variant of PTA-123692 is used and the long chain polyunsaturated fatty acid is one or more selected from the group consisting of DHA with DPA in the range of 0-8% by weight.

18. The method of claim 16, wherein the PTA-123692 has the 18s rDNA sequence of SEQ ID NO:

1.

19. A feed, food, pharmaceutical or cosmetic composition comprising a biomass produced by PTA-123692, or a bio-oil, concentrate or dry product thereof.

20. Use of Schizochytrium sp. PB31 (PTA-123692) or a mutant thereof to engineer a commercial-scale biotechnology platform for producing polyunsaturated fatty acids.

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