Schizochytrium sp. And uses thereof

Abstract

The present disclosure describes an isolated Schizochytrium sp. Strain and uses thereof. Also disclosed are methods of fermentation, methods of producing lipid compositions comprising docosahexaenoic acid (DHA), and methods of producing lipid compositions comprising odd chain fatty acids thereof using the isolated Schizochytrium sp. Strains.

Description

Technical Field

[0001] The present invention relates generally to the field of biotechnology. In particular, the present invention relates to isolated strains of Schizochytrium sp. and their use in fatty acid production.

[0002] Deposit of Microorganisms under the Budapest Treaty

[0003] As disclosed herein, Schizochytrium strain PLU-D was deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures on June 16, 2023 with accession number DSM34665. Background Art

[0004] Traditionally, the components of dietary supplements are obtained either by harvesting or by chemical synthesis. In the current situation, people are seeking alternative sources of such dietary supplements to replace traditional chemical synthesis. This is evident in the application of alternative methods to replace synthetic compounds such as medium-chain triglycerides, dicarboxylic acids and structured lipids. There is a growing demand for omega-3 long-chain fatty acids, especially docosahexaenoic acid (DHA, C22:6n) and eicosapentaenoic acid (EPA, C20:5n). Due to their recognized health benefits, more and more people are consuming them as dietary supplements. In 2019, the global omega-3 fatty acids market was valued at US$2.49 billion and the substance is currently expected to grow at an annual rate of 7% from 2020 to 2027.

[0005] Odd-chain fatty acids, including pentadecanoic acid and heptadecanoic acid, have recently attracted much attention as emerging bioactive lipids. These fatty acids have shown the potential to alleviate a variety of metabolic disorders and have been shown to have positive effects on diabetes, enhancing insulin sensitivity, and glucose regulation. In addition, these odd-chain fatty acids have been shown to have a protective effect against fatty liver and help prevent and control fatty liver. They are considered to have cardiovascular benefits as they have been shown to reduce the risk of cardiovascular disease.

[0006] Traditionally, the main source of these fatty acids, especially DHA, has been marine fisheries, specifically fish oils enriched with EPA and DHA. However, there are concerns that the steady supply of fish oils may be disrupted by unsustainable fish farming, varying lipid compositions and oil quality, and potential contamination by harmful environmental pollutants.

[0007] Therefore, there is an unmet need to provide oil-containing microbial strains and develop methods for producing lipid compositions. Summary of the invention

[0008] In one aspect, the present disclosure relates to a fermentation method for producing a lipid composition using a Schizochytrium sp. strain deposited under accession number DSM34665, wherein the method comprises culturing the Schizochytrium sp. strain to produce the lipid composition.

[0009] In another aspect, the present disclosure relates to a lipid composition obtained by the fermentation method disclosed herein.

[0010] In another aspect, the disclosure relates to an isolated strain of Schizochytrium deposited under accession number DSM34665.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting embodiments and the accompanying drawings, in which:

[0013] Figure 1 Information related to the mangrove samples disclosed herein is shown. Figure 1 A is a geographical map showing the location of Pulau Ubin (arrow); Figure 1 B shows pictures of the collected mangrove and seawater samples.

[0014] Figure 2 is a graph of exemplary growth of Schizochytrium PLU-a, Schizochytrium PLU-B, Schizochytrium PLU-C and Schizochytrium PLU-D strains isolated from mangrove habitats in Singapore grown in glucose and yeast extract (GY) medium in shake flasks.

[0015] Figure 3 This is an unrooted phylogenetic tree of isolated Schizochytrium PLU strains and other publicly available microalgae strains based on their 18S rRNA sequences. Unlike rooted phylogenetic trees that show the ancestors of organisms, unrooted phylogenetic trees only show the relationships between organisms. The tree was constructed using the neighbor-joining method in MEGA 11 software. A bootstrap value of 500 was used for analysis.

[0016] Figure 4 Included are micrographs showing the morphology of Schizochytrium PLU-D strain grown in glucose yeast extract (GY) medium for 3 days. Figure 4 A shows Schizochytrium PLU-D as a vegetative cell; Figure 4B shows Schizochytrium PLU-D as vegetative cells and amebocytes (dark arrows); Figure 4 C shows the release of zoospores from the zoosporangium; Figure 4 D shows cell clusters released from binary cell divisions of octad (white arrows) and decadal (black arrows); Figure 4 E shows an amebocyte (black arrow) and a dyad from binary cell division (white arrow). Figure 4 A. Figure 4 B and Figure 4 The scale bar in C is 20 μm, Figure 4 D and Figure 4 The scale bar in E is 10 μm.

[0017] Figure 5 The biomass change and lipid yield of Schizochytrium PLU-D are shown over a period of about 93 hours when the Schizochytrium PLU-D strain is cultured in a 2-liter fermentor. The pH of the culture medium was controlled at pH 7.0 with 2M NaOH and 0.5M citric acid, and the temperature was maintained at 28°C. In this example, it is contemplated that the glucose batch phase of the claimed method occurs from 0 hours to about 45 hours on the timeline shown in the figure. In this example, the glucose fed-batch phase begins at about 45 hours, as indicated by the "initiate feeding" arrow. DETAILED DESCRIPTION

[0018] Docosahexaenoic acid (DHA, C22:6n) and odd-chain fatty acids (OCFA), such as but not limited to pentadecanoic acid and heptadecanoic acid, have received attention for their potential in alleviating a range of metabolic disorders.

[0019] DHA is an essential omega-3 fatty acid found in the human brain and eyes. It is known to play an important role in the development of the fetal brain and retina during pregnancy and infancy up to two years of age. In addition to infant development, DHA has been reported to have a protective effect on cardiovascular disease, reduce the risk of Alzheimer's disease, improve cognitive abilities in Alzheimer's patients, and reduce cancer cell viability and inhibit cell proliferation.

[0020] Fatty acids, especially docosahexaenoic acid (DHA, C22:6n), are usually obtained from fish oil. However, fish cannot synthesize fatty acids de novo and can only obtain fatty acids from the food they consume, which may include microorganisms (such as but not limited to marine microalgae). Examples of such oil-containing microbial strains can be, but are not limited to, Yarrowia lipolytica, Rhodotorula, Crypthecodinium cohnii, and Schizochytrium.

[0021] Marine microalgae belonging to the genus Schizochytrium are able to produce high levels of docosahexaenoic acid (DHA), accounting for about 26-55% of total fatty acids. There are two pathways for synthesizing DHA in marine microalgae strains, namely, the aerobic elongase-desaturase pathway and the anaerobic polyketide-like synthase (PKS) pathway.

[0022] Saturated fatty acids, such as C16:0 or C18:0, are first synthesized stepwise by the classical fatty acid synthase (FAS) pathway, which is initiated by the condensation of acetyl-CoA molecules and malonyl-CoA molecules. In the aerobic elongase-desaturase pathway, DHA is synthesized by modifying C16:0 or C18:0 fatty acids through a series of alternating elongation and desaturation reactions. These reactions are catalyzed by different elongases and desaturases, wherein the desaturase requires molecular oxygen to introduce double bonds into the fatty acid.

[0023] In contrast, the synthesis of polyunsaturated fatty acids via the anaerobic polyketide-like synthase (PKS) pathway is a more efficient pathway because it is independent of molecular oxygen and requires less nicotinamide adenine dinucleotide (NAD(P)H; a reducing agent that functions as a coenzyme in metabolic pathways). In the PKS pathway, DHA synthesis begins with acetyl-CoA as a precursor and then undergoes repeated cycles of condensation, ketolysis, dehydration, and enoylation, with chain length controlled by regulatory chain length factor proteins.

[0024] Schizochytrium strains usually synthesize DHA anaerobically via the PKS pathway. This is reflected in their long-chain polyunsaturated fatty acid composition, the main components of which are, but not limited to, DHA and docosapentaenoic acid (DPA). This indicates that they are suitable as high-producing microorganisms for DHA.

[0025] Singapore is an island nation with multiple coastal mangrove habitats. However, there are limited reports on the isolation of novel marine microalgae strains from this location. In the present disclosure, the isolation and characterization of microalgae strains isolated from mangrove samples collected from Pulau Ubin, Singapore are described. As described herein, we obtained four isolates of Schizochytrium with high lipid productivity and high DHA content.

[0026] To characterize the strains obtained as described in the present disclosure, the cell morphology phenotype was observed under an optical microscope. The isolated microalgae strains were grown in glucose yeast extract (GY) medium at 25°C and 200 rpm. The morphological characteristics of the microalgae isolates included vegetative cells, sporangia, cytoplasmic reticulum, zoospores, amebocytes, and binary fission (see Figure 4 ).

[0027] Phylogenetic analysis using 18S rRNA indicated that the isolated Schizochytrium strains appeared to be closely related to other Schizochytrium strains, such as Schizochytrium strain LY-2012 (JX847369.1), Schizochytrium strain SH104 (KX379459.1), and Schizochytrium strain ABC101 (MK265698). Therefore, the four strains identified herein were identified as previously unknown Schizochytrium strains. The 18S rRNA sequences of the Schizochytrium strains disclosed herein are referred to as Schizochytrium PLU-A (OP808218), Schizochytrium PLU-B (OP808219), Schizochytrium PLU-C (OP808220), and Schizochytrium PLU-D (OP763747), respectively.

[0028] In the present disclosure, the use of isolated Schizochytrium in the production of lipid compositions is described. In one example, the isolated Schizochytrium strain is but is not limited to PLU-A, PLU-B, PLU-C and PLU-D. In one example, the Schizochytrium is PLU-D. In another example, the isolated strain is Schizochytrium, which was deposited at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ) on June 16, 2023 with accession number DSM34665.

[0029] As shown in the experimental section, after culturing the isolated PLU-D strain using the fermentation method described herein for 84 hours, a biomass of about 175 g / l cell dry weight was produced, corresponding to a lipid yield of about 93.6 g / l. Thus, in one example, a fermentation method for producing a lipid composition using a Schizochytrium strain as described herein, wherein the biomass of the isolated strain increases from the beginning of culturing the Schizochytrium strain to up to about 84 hours.

[0030] Compared to other Schizochytrium fermentation studies known in the art, it was reported that high cell density fermentation of Schizochytrium G13 / 2S strain can achieve biomass of about 65 g / l cell dry weight and about 16 g / l lipid yield, while Schizochytrium ABC101 strain isolated from Korea can produce about 86 g / l cell dry weight and about 37.2 g / l lipid yield after about 84 hours of cultivation under its optimized conditions. In contrast, the method of fermentation using Schizochytrium PLU-D achieved higher biomass and lipid yields in about 84 hours than other disclosed strains.

[0031] In other words, the four isolated Schizochytrium PLU strains disclosed herein can be cultured under similar culture conditions to obtain higher biomass than other microalgae strains known in the art. For example, the cell dry weight obtained by culturing the Schizochytrium F26-b strain known in the art is only about 3.5 g / l, which is lower than the biomass obtained by culturing the PLU strain under similar conditions. In addition, the biomass obtained by culturing the PLU strain cells is also higher than that of other microalgae strains isolated from Southeast Asian mangroves. For example, as known in the art, when cultured under shake flask conditions, the biomass of the Thraustochytrids sp. strain isolated from Thailand is in the range of about 0.6 to about 3.1 g / l cell dry weight. Similarly, the biomass of the Thraustochytrids strain isolated from the Malaysian mangrove habitat is in the range of about 1 to about 5.8 g / l cell dry weight, while in another study, the isolated Aurantiochytrium sp. AW1 strain from the coastal waters of Malaysia was cultured to obtain a cell dry weight of about 13.17 g / l. Thus, when cultured under similar shake flask conditions, the biomass productivity of the PLU-A, PLU-B, PLU-C and PLU-D strains was higher than that of isolated microalgae strains from Southeast Asia known in the art.

[0032] As shown in the present disclosure, the four isolated strains of Schizochytrium disclosed herein exhibited similar growth characteristics when cultured in glucose yeast extract (GY) medium (see Figure 2 When cultured for about 96 hours, the dry cell weights of all four strains in GY medium were between about 13 and about 14 g / l (see Table 1).

[0033] In one example, the scope of the biomass obtained using the method disclosed herein is less than 1g / l to about 200g / l cell dry weight. In another example, the biomass is 9 to 15g / l, 10 to 14g / l, 12 to 15g / l, 13 to 15g / l cell dry weight, 10 to 20g / l cell dry weight, 15 to 30g / l cell dry weight, 25 to 50g / l cell dry weight, 45 to 80g / l cell dry weight, 75 to 100g / l cell dry weight, 80 to 150g / l cell dry weight, 120 to 200g / l cell dry weight, or 150 to 180g / l cell dry weight. In one example, the biomass obtained by cultivating the PLU bacterial strain disclosed herein in glucose yeast extract (GY) is about 13 to about 14 (g / l) cell dry weight. In one example, the biomass obtained using the methods disclosed herein is about 15 g / l, about 20 g / l, about 25 g / l, about 30 g / l, about 35 g / l, about 40 g / l, about 45 g / l, about 50 g / l, about 55 g / l, about 60 g / l, about 65 g / l, about 70 g / l, about 75 g / l, about 80 g / l, about 85 g / l, about 90 g / l, about 95 g / l, about 100 g / l, about In one embodiment, the biomass is 175 g / l dry cell weight.

[0034] As used herein, the term "biomass" refers to the total amount, mass or weight of organisms in a given volume of liquid. The liquid can be, for example, but not limited to, a cell culture medium or a buffer solution. In the present disclosure, biomass is expressed as "grams per liter (g / l) of dry cell weight". As will be appreciated by those skilled in the art, the amount of biomass generated depends on the amount of time spent in culture and a given glucose concentration.

[0035] Lipid content is another parameter used when evaluating the lipid productivity of microalgae strains in large-scale production. As described herein, the lipid productivity of isolated strains can be evaluated by measuring the lipid content in cells. As used herein, the term "lipid content" refers to the percentage of lipid extracted from cells, where the cells are measured by cell dry weight. In the case of the four isolated PLU strains disclosed herein, their lipid content ranged from about 8.9% to about 32.3%, with Schizochytrium PLU-D showing the highest lipid content of about 32.3% (see Table 1).

[0036] Therefore, in one example, the lipid content of the PLU strain of separation described herein can be about 5% to about 40% of dry cell weight. In another example, lipid content is about 10% to about 20% of dry cell weight, about 20% to about 30% of dry cell weight, or about 30% to about 40% of dry cell weight. In another example, lipid content is about 9% to about 35% of dry cell weight, about 8% to about 33% of dry cell weight, or about 8.9% to about 32.3% of dry cell weight. In another example, lipid content is about 5%, about 8%, about 10%, about 12%, about 15%, about 18%, about 20%, about 25%, about 30%, about 35%, or about 40%. In one example, the lipid content in the isolated strain is about 8.9% of dry cell weight. In another example, the lipid content in the isolated strain is about 12.7% of dry cell weight. In another example, the lipid content of the isolated strain is about 19.2%. In yet another example, the lipid content in the isolated strain is about 32.3% of the dry cell weight. In one example, the Schizochytrium PLU-D strain comprises a lipid content of about 32.3% of the dry cell weight.

[0037] Compared to other isolated microalgae strains from Southeast Asia known in the art, the lipid content of the isolated PLU strain is also higher than the Thraustochytrium strain isolated in Malaysia (about 3.29% to about 15.76% of dry cell weight) and the Thraustochytrium strain isolated in Thailand (about 3.5% to about 19.4% of dry cell weight).

[0038] As described herein, lipid productivity can be represented by lipid output. As used herein, term " lipid output " refers to the total amount of lipid gathered in the crops from the cultured cells of microalgae. Microalgae is usually defined in volume units. This output is usually represented by grams of lipid (g / l) per liter of culture, but can also be represented by other equivalent units, such as but not limited to mg / ml or mg / l. Lipid output disclosed herein can be in the scope of about 0.5g / l to 10g / l, about 1g / l to 5g / l, about 1.5g / l to 5g / l, about 2g / l to 4g / l, about 3g / l to 5g / l, about 4g / l to 5g / l or about 4.5g / l to 5g / l. In another example, the lipid yield is about 1 g / l, about 1.5 g / l, about 2 g / l, about 2.5 g / l, about 3 g / l, about 3.5 g / l, about 4 g / l, about 4.5 g / l, about 5 g / l, about 6 g / l, about 6.5 g / l, about 7 g / l, about 7.5 g / l, about 8 g / l, about 8.5 g / l, about 9 g / l, about 9.5 g / l, or about 10 g / l. In one example, culturing the isolated Schizochytrium strain as described herein can yield a lipid yield of about 1.29 to about 4.43 g / l. In another example, the lipid yield is about 1.29 g / l. In another example, the lipid yield is about 1.70 g / l. In another example, the lipid yield is about 2.62 g / l. In yet another example, the lipid yield is about 4.43 g / l.

[0039] The isolated strains described herein can be used to produce or obtain the lipid compositions disclosed herein. In one example, a method for producing a lipid composition using a Schizochytrium strain is described. In another example, the method disclosed herein is a fermentation method. As used herein, the Schizochytrium strain is cultured under conditions known in the art to produce a lipid composition. As described herein, the Schizochytrium strain can be a strain deposited with accession number DSM34665. Therefore, the present disclosure also describes a fermentation method for producing a lipid composition using a Schizochytrium strain deposited with accession number DSM34665, wherein the method includes culturing a Schizochytrium strain to produce a lipid composition.

[0040] As used herein, the term "fermentation" refers to the process of using microorganisms to anaerobicly decompose high-energy compounds (such as, but not limited to, carbohydrates) into carbon dioxide and alcohols or organic acids by enzyme catalysis. Such microorganisms can be, for example, bacteria, yeast or microalgae.

[0041] In one example, the fermentation method includes a glucose batch stage and a glucose batch feed stage for accumulating biomass. In an exemplary method, the first stage of the method is a batch fermentation, which is carried out until glucose and a nitrogen source are exhausted. Once these nutrient sources are exhausted, glucose and a nitrogen source are additionally supplemented to the cells in batches. Therefore, in one example, glucose is present in two stages of the disclosed method. In one example, when the glucose in the culture medium is completely consumed, it is considered that the glucose batch stage is completed. For example, it can be indicated by a rise or surge in dissolved oxygen levels, or any other method that can be used by those skilled in the art. It is believed that at this time, changing the process from the glucose batch stage to the glucose batch feed stage can promote further increase in lipid accumulation and biomass. In one example, the accumulation of biomass is obtained using a glucose batch stage, wherein the cells of the Schizochytrium strain are cultured in the culture medium for about 36 to about 48 hours. In another example, the cells of the Schizochytrium strain are cultured in the culture medium for about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or about 48 hours. In one example, the cells are cultured for about 36 to about 46 hours, about 36 to about 44 hours, about 36 to about 42 hours, about 36 to about 40 hours, about 38 to about 48 hours, about 38 to about 46 hours, about 38 to about 44 hours, about 38 to about 42 hours, about 38 to about 40 hours, about 40 to about 48 hours, about 40 to about 46 hours, about 40 to about 44 hours, about 40 to about 42 hours, about 42 to about 48 hours, about 42 to about 46 hours, about 42 to about 44 hours, about 44 to about 48 hours, about 44 to about 46 hours, or about 46 to about 48 hours. In another example, the feed solution is provided for about 45 hours. In another example, cells of the Schizochytrium strain are cultured for about 45 hours. In another example, the glucose batch phase is performed at about 28° C. and pH 7.0. In another example, the pH is maintained at pH 7.0 with 2M NaOH and 0.5M citric acid.

[0042] As shown in the experimental section below, the initial glucose concentration supplied to the fermentor was about 200 g / l and was almost completely consumed at about 45 hours, as shown by the dissolved oxygen spike. This glucose concentration resulted in a biomass of about 128 g / l dry cell weight (DCW) and a lipid yield of about 67.4 g / l lipid.

[0043] As described herein, the purpose of the glucose batch feed stage is to promote lipid accumulation in the cell and further increase biomass. In an example, the glucose batch feed stage starts after the glucose batch stage is completed. Those skilled in the art will be able to use methods known in the art to determine when the glucose batch stage is completed. In an example, the glucose batch feed stage can start 24 hours after the glucose batch stage starts. In an example, the glucose batch feed stage can start 24 hours to about 45 hours after the glucose batch stage starts. In another example, the glucose batch feed stage can start about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, or about 45 hours after the glucose batch stage starts. In another example, the glucose batch feeding stage can start at about 25 to about 44 hours, about 25 to about 42 hours, about 25 to about 40 hours, about 25 to about 38 hours, about 25 to about 36 hours, about 25 to about 34 hours, about 25 to about 32 hours, about 25 to about 30 hours, about 25 to about 28 hours, about 28 to about 40 hours, about 28 to about 36 hours, about 28 to about 32 hours, about 28 to about 30 hours, about 30 to about 42 hours, about 30 to about 38 hours, about 30 to about 34 hours, about 32 to about 42 hours, about 32 to about 36 hours, about 32 to about 34 hours. In an example, the glucose batch feeding stage starts about 45 hours after the glucose batch feeding stage starts. In another example, the glucose batch feeding stage is started by introducing a feeding solution. In an example, a feeding solution is provided at a single time point. In another example, a feeding solution is provided continuously, i.e., provides over time. In one example, the feed solution comprises glucose and yeast extract. In another example, the feed solution is provided to the cells of the Schizochytrium strain for about 36 to about 48 hours. In one example, the feed solution is provided for about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, or about 48 hours.In one example, the feed solution is provided for about 36 to about 46 hours, about 36 to about 44 hours, about 36 to about 42 hours, about 36 to about 40 hours, about 38 to about 48 hours, about 38 to about 46 hours, about 38 to about 44 hours, about 38 to about 42 hours, about 38 to about 40 hours, about 40 to about 48 hours, about 40 to about 46 hours, about 40 to about 44 hours, about 40 to about 42 hours, about 42 to about 48 hours, about 42 to about 46 hours, about 42 to about 44 hours, about 44 to about 48 hours, about 44 to about 46 hours, or about 46 to about 48 hours. In another example, the feed solution is provided for about 45 hours.

[0044] In another example, the fermentation method further includes cultivating the isolated strain in the presence of sodium propionate. In another example, cultivating the isolated strain in the presence of sodium propionate results in a higher concentration of odd-chain fatty acids (OCFA) in the lipid composition. Those skilled in the art will be able to use methods known in the art, such as but not limited to high pressure liquid chromatography (HPLC), gas chromatography (GC), gas chromatography-mass spectrometer (GCMS), gas chromatography-mass spectrometry coupled with hydrogen flame ionization detector (GCMS / FID) or any other suitable quantitative or qualitative analysis method to determine the specific concentration, output or composition of the compounds disclosed herein and the lipid composition.

[0045] In another example, sodium propionate is used to increase the output of odd-chain fatty acids in lipid compositions disclosed herein. In one example, sodium propionate concentration is 1 to 20 g / l. In another example, sodium propionate concentration is about 1 g / l, about 2 g / l, about 3 g / l, about 4 g / l, about 5 g / l, about 6 g / l, about 7 g / l, about 8 g / l, about 9 g / l, about 10 g / l, about 11 g / l, about 12 g / l, about 13 g / l, about 14 g / l, about 15 g / l, about 16 g / l, about 17 g / l, about 18 g / l, about 19 g / l or about 20 g / l. In another example, the sodium propionate concentration is about 5 to about 20 g / l, about 5 to about 15 g / l, about 5 to about 10 g / l, about 2.5 to about 7.5 g / l, about 2.5 to about 5 g / l, about 1 to about 5 g / l, or about 1 to about 2.5 g / l. In another example, the sodium propionate concentration is about 5 g / l. In one example, the fermentation method described herein includes the steps of culturing cells of the Schizochytrium strain at about 25° C. in the presence of sodium propionate for about 5 days.

[0046] In summary, the fermentation methods disclosed herein result in an increase in the concentration of the lipid composition compared to the concentration of the lipid composition obtained using the control strain. In one example, culturing the Schizochytrium strain results in an increase in the concentration of (docosahexaenoic acid) DHA compared to the control strain. In one example, culturing the Schizochytrium strain results in an increase in the concentration of odd-chain fatty acids (OCFA) compared to the control strain. In one example, the control strain is Schizochytrium ABC101 or Schizochytrium G13 / 2S.

[0047] The fatty acid profile of the lipid composition obtained by the fermentation method using the Schizochytrium PLU strain was compared with the fatty acid profiles of two commercially available DHA algae oils (i.e., Nordic Naturals and GNC) on the market (see Table 2). The fatty acid profile of the lipid composition obtained by the fermentation method disclosed in the present invention is different from the fatty acid profiles of these two commercially available algae oils.

[0048] In addition, the fermentation method described herein can also produce a lipid composition composed of polyunsaturated fatty acids and polysaturated fatty acids. Therefore, the present invention also describes a lipid composition obtained from the fermentation method described herein.

[0049] As described herein, the fatty acid profiles of lipid compositions obtained from culturing cells of four isolated Schizochytrium PLU strains were also analyzed.The terms "composition of lipids" and lipid composition are used interchangeably in this disclosure.

[0050] In one example, the lipid composition comprises docosahexaenoic acid (DHA). In another example, the lipid composition comprises odd-chain fatty acids (OCFA). In one example, the lipid composition obtained by cultivating the isolated PLU strain disclosed herein is described. In another example, the lipid composition is obtained from the fermentation method disclosed herein.

[0051] In another example, the lipid composition comprises about 45% to about 50% of the docosahexaenoic acid (DHA, C22:6) of the total lipid yield. In another example, the lipid composition comprises about 32% palmitic acid (C16:0). In another example, the lipid composition comprises about 1% to about 3.35% eicosapentaenoic acid (EPA, C20:5). In another example, the lipid composition comprises about 4.5% docosapentaenoic acid (DPA, C22:5).

[0052] It was reported that isolated Thraustochytrids from strains from Japan and Fiji contained five different types of polyunsaturated fatty acid profiles in their lipid components, namely DHA / DPA, DHA / DPA / EPA, DHA / EPA, DHA / DPA / EPA / arachidonic acid (AA), and DHA / DPA / EPA / AA / docosatetraenoic acid. On the other hand, the polyunsaturated fatty acid profiles of the four isolated PLU strains contained DHA / DPA / EPA. As described in the experimental section, the DHA content of the isolated PLU strain ranges from about 44.51% to about 48.72% of the total lipid yield, which is higher than other strains known in the art such as Schizochytrium ATCC20888, which contains about 15.68 to about 38.30% DHA of the total lipid yield, Schizochytrium ATCC20889, which contains about 15.56% to about 26.25% DHA of the total lipid yield, and Schizochytrium mangrovei FB1, which contains about 37.8% to about 39.14% DHA of the total lipid yield.

[0053] As described herein, culturing the isolated PLU strain as described herein also produces odd-chain fatty acids in its lipid composition. In one example, culturing cells of the Schizochytrium strain in the presence of sodium propionate causes a change in the lipid composition ratio compared to culturing cells of the Schizochytrium strain in the absence of sodium propionate. That is, culturing cells or performing the methods disclosed herein in the absence of sodium propionate can still obtain the desired lipid composition. In other words, the presence of sodium propionate has been shown to increase the amount of odd-chain fatty acids in the lipid composition produced thereby. In one example, when cells of the Schizochytrium strain are cultured in the absence of sodium propionate, the C15:0 fat yield of the lipid composition accounts for about 3% to about 25% of its total fat yield. In another example, the percentage of C15:0 in the lipid composition obtained by culturing cells of the Schizochytrium strain in the absence of sodium propionate is about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 25%, about 15% to about 20%, or about 20% to about 25%. In another example, the percentage of C15:0 in the lipid composition obtained by culturing cells of the Schizochytrium strain in the absence of sodium propionate is about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, or about 20%.

[0054] In another example, the percentage of C15:0 in the lipid composition obtained by culturing cells of the Schizochytrium strain in the presence of sodium propionate is about 20% to about 55%. In another example, the percentage of C15:0 in the lipid composition obtained by culturing cells of the Schizochytrium strain in the presence of sodium propionate is about 20% to about 30%, about 25% to about 35%, about 34% to about 45%, or about 42% to about 54%. In another example, the percentage of C15:0 in the lipid composition resulting from culturing cells of a Schizochytrium strain in the presence of sodium propionate is at least about 30% to at least about 50%, at least about 30% to at least about 45%, at least about 30% to at least about 40%, at least about 30% to at least about 35%, at least about 35% to at least about 50%, at least about 35% to at least about 50%, at least about 35% to at least about 45%, at least about 35% to at least about 40%, at least about 40% to at least about 55%, at least about 40% to at least about 50%, at least about 40% to at least about 45%, at least about 45% to at least about 55%, at least about 45% to at least about 50%, or at least about 50% to at least about 55%. In other examples, the percentage of C15:0 in the lipid composition of cells of the Schizochytrium strain cultured in the presence of sodium propionate is at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, or at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, or at least about 55%. In another example, the percentage of C15:0 is about 47%. In view of the presence of these OCFAs in the isolated PLU strain, the lipid composition obtained from the isolated PLU strain is different from other producers of high DHA microalgae oils.

[0055] So far, countries and regions including but not limited to the United States, the European Union, Australia and China have approved the use of Schizochytrium strains for the production of DHA algae oil. For example, recently the multinational giant DSM commercialized its DHA products derived from Schizochytrium, such as Life's and Life's TMOMEGA. In addition, new strains and new processes related to the production of DHA algae oil from Schizochytrium have also been developed. For example, the Schizochytrium ABC101 strain was isolated from a seawater sample near Jeju Island, South Korea, and obtained about 86 grams per liter (g / l) of cell dry weight, about 37.2 g / l of lipids, and about 16.7 g / l of DHA under optimized fermentation process conditions. In another study, the Schizochytrium HX-308 strain isolated from a Chinese seawater sample produced about 19 g / l of lipids containing 60% DHA during the lipid accumulation stage by adding about 4 g / l of malic acid. Through adaptive laboratory evolution, the Schizochytrium HX-308 strain was continuously optimized, and a DHA yield of about 38.1 g / l was further obtained under fermentation conditions. In addition, the Schizochytrium PKU#Mn4 strain isolated from a coastal mangrove in China increased its DHA content in lipids from about 42.6% to about 60.7% by atmospheric pressure room temperature plasma (ARTP) mutagenesis, producing about 0.98 g / l DHA in a shake flask. Therefore, it is necessary to discover and isolate new Schizochytrium strains with high biomass, high lipids and high DHA productivity.

[0056] In one example, fermentation method as described herein produces the lipid composition comprising docosahexaenoic acid (DHA, C22:6).In one example, the percentage of DHA in the lipid composition obtained from fermentation method disclosed herein is at least about 30% to at least about 55% of total lipid production.Therefore, the percentage of DHA content in the lipid composition obtained from the fermentation method using fission yeast strain is at least about 30% of total lipid production, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49% or at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54% or at least about 55%. In one example, the percentage of DHA is at least about 35% to at least about 50%, at least about 35% to at least about 45%, at least about 35 to at least about 40%, at least about 40 to at least about 55%, at least about 40% to at least about 50%, at least about 45 to at least about 50% or at least about 45% to at least about 50% of the total lipid yield. In one example, a lipid composition as described herein is described, which includes docosahexaenoic acid (DHA). In one example, compositions are as described herein, wherein the percentage of docosahexaenoic acid (DHA) is in the range of about 45% to about 50%. In another example, compositions are as described herein, wherein the percentage of docosahexaenoic acid (DHA) is about 44.5%. In another example, lipid compositions as described herein include about 44.95% docosahexaenoic acid (DHA). In another example, the docosahexaenoic acid (DHA) percentage in lipid compositions as described herein is about 45.43%. In yet another example, the lipid composition described herein comprises about 48.72% docosahexaenoic acid (DHA).

[0057] The amount of DHA produced using the Schizochytrium PLU-D strain in the fermentation method disclosed herein was about 25.6 g / l. In contrast, the amount of DHA produced using Schizochytrium G13 / 2S and Schizochytrium ABC101 was about 6 g / l and about 16.7 g / l, respectively. Thus, the fermentation method described herein results in an increase in the concentration of docosahexaenoic acid (DHA) compared to the control strain.

[0058] In another example, fermentation methods as disclosed herein have been shown to produce lipid compositions comprising odd-chain fatty acids. The percentage of C15:0 odd-chain fatty acids in the obtained lipid composition is at least about 25% to at least about 55% of the total lipid yield. Thus, in the lipid composition of a cultured Schizochytrium strain, the level of C15:0 fatty acids is at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, In one example, the percentage of C15:0 fatty acids is at least about 25% to at least about 55%, at least about 25% to at least about 50%, at least about 25% to at least about 45%, at least about 25% to at least about 40%, at least about 25% to at least about 35%, at least about 25% to at least about 30%, at least about 30% to at least about 55%, at least about 30% to at least about 50%, at least about 30% to at least about 45%, at least about 30% to at least about 55%, at least about 30% to at least about 50%, at least about 30% to at least about 45%, at least about 30% to at least about 50% of the total lipid production. In one example, lipid compositions as described herein are described, and it comprises odd-chain fatty acids (OCFA). In one example, compositions as described herein, wherein the percentage of odd-chain fatty acids (OCFA) is in the range of about 30% to about 55%. In another example, the percentage of OCFA in lipid compositions is about 47%. In another example, the percentage of OCFA is about 51%.

[0059] In another example, the lipid composition obtained from the fermentation method disclosed herein further includes, but is not limited to, the following fatty acids: C11:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0 fatty acids, eicosapentaenoic acid (EPA, C20:5), docosapentaenoic acid (DPA, C22:5), docosahexaenoic acid (DHA, C22:6), and combinations thereof.

[0060] Also described herein are lipid compositions comprising C11:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:5, C22:5 or C22:6 fatty acids. In one example, the percentage of C11:0 fatty acids is at least about 0.01% to at least about 0.03% of the total lipid production, the percentage of C12:0 fatty acids is at least about 0.23% to at least about 0.33% of the total lipid production; the percentage of C13:0 fatty acids is at least about 0.08% to at least about 0.12% of the total lipid production; the percentage of C14:0 fatty acids is at least about 3.25% to at least about 4.34% of the total lipid production; the percentage of C15:0 fatty acids is at least about 4.43% to at least about 4.97% of the total lipid production; the percentage of C16:0 fatty acids is at least about 32.24% to at least about 33.97% of the total lipid production. about 33.7%; the percentage of C17:0 fatty acids is at least about 0.82% to at least about 1.03% of the total lipid production; the percentage of C18:0 fatty acids is at least about 0.71% to at least about 1.09% of the total lipid production; the percentage of eicosapentaenoic acid (EPA, C20:5) is at least about 1.07% to at least about 3.35% of the total lipid production; the percentage of docosapentaenoic acid (DPA, C22:5) is at least about 4.43% to at least about 4.9% of the total lipid production; and the percentage of docosahexaenoic acid (DHA, C22:6) is at least about 44.95% to at least about 48.72% of the total lipid production.

[0061] In another example, the percentage of C11:0 fatty acids is at least about 0.18% to at least about 0.40%; the percentage of C12:0 fatty acids is at least about 0.09% to at least about 0.22%; the percentage of C13:0 fatty acids is at least about 1.4% to at least about 3.0%; the percentage of C14:0 fatty acids is at least about 0.63% to at least about 0.96%; the percentage of C15:0 fatty acids is at least about 27% to at least about 32%; the percentage of C16:0 fatty acids is at least about 9.6% to at least about 13%; the percentage of C17:0 fatty acids is at least about 1.9% to at least about 2.4%; the percentage of C18:0 fatty acids is at least about 2.5% to at least about 5.8%; the percentage of eicosapentaenoic acid (EPA, C20:5) is at least about 2.1% to at least about 3.2%; the percentage of docosapentaenoic acid (DPA, C22:5) is at least about 2.8% to at least about 4.2%; and the percentage of docosahexaenoic acid (DHA, C22:6) is at least about 35% to at least about 43%.

[0062] As disclosed herein, four strains of microalgae were isolated from rotten mangrove samples collected from Pulau Ubin, Singapore, and identified as Schizochytrium by phylogenetic analysis. Compared with other wild-type microalgae strains isolated from Southeast Asia, these PLU isolated strains were cultivated in shake flask experiments to produce higher biomass, higher lipid content, and higher docosahexaenoic acid (DHA) content. In addition, culturing the PLU isolated strains in the presence of sodium propionate will produce OCFA. When the Schizochytrium PLU-D strain is cultivated under conditions of batch feed fermentation, it is possible to produce significantly higher amounts of biomass and higher amounts of lipids, which have comparable or higher concentrations of DHA compared to two commercially available algae oils. This makes the Schizochytrium PLU-D strain a suitable host for commercial production of DHA.

[0063] The invention exemplarily described herein can be appropriately implemented in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Therefore, for example, the terms "include", "comprise", "contain", etc. should be read broadly without limitation. In addition, the terms and expressions used herein are used as illustrative rather than restrictive terms, and the use of these terms and expressions does not exclude any equivalent features or parts of the features or parts thereof shown and described, and it should be recognized that various modifications may fall within the scope of the present invention. It should be understood that although the present invention is specifically disclosed by preferred embodiments and optional features, those skilled in the art can make modifications, improvements and changes by virtue of the invention disclosed herein, and therefore it should be considered that such modifications, improvements and changes fall within the scope of the present invention.

[0064] As used in this application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "genetic marker" may include a plurality of genetic markers, including mixtures and combinations thereof.

[0065] As used herein, the term "about" in the context of formulation component concentrations typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0066] In the present disclosure, certain embodiments are presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity, and should not be interpreted as a hard limit to the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, a range description such as 1 to 6 should be considered to have specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as each number within the range, such as 1, 2, 3, 4, 5 and 6. This is generally applicable, regardless of the breadth of the range.

[0067] Certain embodiments may also be described broadly and generically herein. Each narrower species and subgeneric grouping falling within the general disclosure also forms part of the present disclosure. This includes the general description of the embodiments with a proviso or negative limitation deleting any subject matter from the genus, whether or not the deleted material is specifically cited herein.

[0068] The invention is described broadly and generically herein. Each narrower species and subgeneric grouping falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation deleting any subject matter from the genus, whether or not the deleted material is specifically cited herein.

[0069] Other embodiments are within the appended claims and non-limiting examples. In addition, when features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention can also be described in terms of any individual component or subgroup of the Markush group.

[0070] Experimental Section

[0071] Sample collection and strain isolation

[0072] Decayed mangrove samples, including fallen leaves, stems, flowers, and fruits, were collected from Pulau Ubin (1.4126°N, 103.9577°E) off the northeast coast of Singapore (see Figure 1). The samples were first washed with sterile seawater containing 0.5 grams per liter (g / l) of penicillin G and 0.5 g / l of streptomycin sulfate. The suspension was then filtered through a 40 μm filter to remove debris and then streaked on glucose, peptone, yeast extract (GPY) plates for separation. GPY plates contained 1 g / l peptone, 2 g / l yeast extract, 4 g / l glucose, 0.5 g / l penicillin G, 0.5 g / l streptomycin sulfate, 0.1 g / l fluconazole, and 10 g / l agar in half-strength seawater. The plates were then sealed and incubated at 25°C in the dark for 2 to 5 days. Microalgae-like colonies were aseptically picked and streaked onto new GPY plates. Single colonies were repeatedly isolated until a sterile culture was obtained. The cell morphology was then examined under an optical microscope equipped with a color camera to verify the authenticity of the microalgae-like morphology. Pure isolates were stored in 20% glycerol at -80°C. After several rounds of cultivation and purification, four pure microalgae microbial strains were obtained, named PLU-A, PLU-B, PLU-C and PLU-D. After incubation at 25°C for 4 days, the strains formed opaque white, flat, creamy-looking colonies on agar plates.

[0073] Staining culture and dry cell weight (DCW) measurement

[0074] The isolated microalgae strains were cultured in 250 ml baffled flasks containing 50 ml of glucose yeast extract (GY) medium containing 30 g / l glucose, 10 g / l yeast extract and half-strength seawater, pH 6.0. The cultures were incubated in a rotary shaker at 200 rpm and 25° C., and cell growth was monitored by measuring wet cell mass. At the end of the culture, cells were harvested for lipid extraction to determine lipid yield and fatty acid composition.

[0075] To determine the dry cell weight, 20 ml of culture fluid was collected and the cells were pelleted by centrifugation at 3500 g for 10 minutes at room temperature. The cell pellet was then washed twice with 20 ml of distilled water and re-centrifuged as above and then dried at 60°C until the weight remained constant.

[0076] The dry cell mass, lipid content, and lipid yield of the isolated Schizochytrium strains PLU-A, PLU-B, PLU-C, and PLU-D are shown in Table 1 .

[0077] Table 1

[0078]

[0079] * DCW: dry cell weight; # Lipid content: lipid weight on DCW

[0080] Strain identification by 18S rRNA sequence and morphological analysis

[0081] Genomic DNA was extracted from isolated Schizochytrium species. 18S rRNA sequences were amplified from genomic DNA using primers PF: 5'-CTGGTTGATCCTGCCAGTAGTC-3' and PR: 5'-GTTAAGACTACGATGGTATCTAA-3'. Each PCR reaction mixture contained 100 ng template genomic DNA, 1x PCR buffer, 1.0 mM MgSO4, 0.2 mM dNTPs, 0.3 μM of each primer, and 1.0 U KOD DNA polymerase in a 50 μl reaction volume. PCR amplification was performed using the following program: initial denaturation at 94°C for 2 minutes, [denaturation at 94°C for 15 seconds, annealing at 52°C for 30 seconds, extension at 68°C for 1 minute] for 30 cycles, and final extension at 68°C for 5 minutes. PCR products were analyzed by agarose gel electrophoresis. The desired bands were cut from the gel and purified. The purified products were then sequenced.

[0082] The obtained 18S rRNA sequences were subjected to BLAST analysis with the 18S rRNA sequences available in the GenBank database using nucleotide BLAST. Multiple 18S rRNA sequences were selected for multiple sequence alignment analysis, which was performed using the MEGA 11 software using the MUSCLE algorithm. The neighbor-joining algorithm was then used to generate an unrooted phylogenetic tree, which shows the relationships between species but does not describe their common ancestor. The results are shown in Figure 3 shown.

[0083] The 18S rRNA sequences of PLU-A, PLU-B, PLU-C and PLU-D isolates were deposited in the NCBI database and the corresponding GenBank accession numbers were obtained. Phylogram analysis showed three clusters, in which the four PLU strains were closely clustered with Schizochytrium TK6 (OK244290.1), as shown in Figure 2. Figure 3 The cell morphology phenotype was observed under an optical microscope. The selected microalgae isolates were grown in glucose yeast extract (GY) medium at 25°C and 200 rpm. The morphological characteristics of the microalgae isolates included vegetative cells, sporangia, cytoplasmic reticulum, zoospores, amebocytes, and binary fission (see Figure 4 ).

[0084] The Schizochytrium PLU-D strain was further selected for morphological study. Under the microscope, the vegetative cells showed the typical spherical shape of Schizochytrium strains. These vegetative cells were dispersed in the culture in the form of single cells, with a size diameter ranging from 9 to 20 μm ( Figure 4A and 4B). Vegetative cells undergo a series of binary fissions to form dyads, octads, decadas, etc., forming small cell clusters ( Figure 4 D and Figure 4 E). The zoosporangium that develops from the vegetative cell is relatively large, with a diameter of about 10 to 25 μm. The zoospores are released from the sporangium when the zoosporangium wall ruptures ( Figure 4 C). Slender deformed cells with a diameter of about 15-20 μm were also observed ( Figure 4 B and Figure 4 E) The amebocyte becomes spherical and matures into a zoosporangium, releasing zoospores.

[0085] Measurement of total lipid and fatty acid composition

[0086] First, 40 ml of crude cell culture of Schizochytrium from each of the four PLU isolates was precipitated by centrifugation at 3500G for 10 minutes. The cell pellet was then resuspended with 20 ml of 0.1 M TrisHCl at pH 8.0, and 100 μL of calciferol and 100 μL of cellulase (volume / volume) were added to the cell suspension to initiate enzymatic hydrolysis. To promote complete enzymatic hydrolysis, the suspension was incubated at 50°C for 6 hours with constant stirring. The lipids were then extracted with an equal volume of hexane, and the extraction was repeated twice. The hexane organic layers were then combined and the organic solvent was removed by evaporation under reduced pressure using a concentrator. The extracted lipid components were weighed and stored at -20°C until further analysis.

[0087] Before the fatty acid composition analysis, the extracted lipids were derivatized into fatty acid methyl esters (FAME). 50 mg of lipids were added to 1 ml of 3M hydrogen chloride methanol solution, incubated in a 60 ° C water bath for 20 minutes, and then cooled to room temperature. Next, 400 μl of hexane and 200 μl of water were added to the reaction mixture, vortexed for 1 minute, and then the mixture was centrifuged at 3500g for 5 minutes. The upper layer was then aliquoted into new vials for analysis of FAME.

[0088] FAMEs were analyzed by gas chromatography-mass spectrometry coupled with flame ionization detector (GCMS / FID). The gas chromatograph was equipped with a HP-88 column of 100 m x 0.25 mm ID x 0.25 μm film thickness and a mass spectrometer. The samples were injected with a split ratio of 1:10. Helium was used as carrier gas and the column flow rate was set to 1.3 ml per minute (ml / min). The initial oven temperature was 100 °C and the inlet temperature was 250 °C. After 5 minutes, the oven temperature was increased to 200 °C at a rate of 4 °C per minute and maintained at 200 °C for 10 minutes and then further increased to 220 °C at a rate of 1 °C per minute. The temperature of the flame ionization detector (FID) was set to 250 °C, the feed flow was 30 ml / min, the hydrogen flow was 40 ml / min, and the air flow was 400 ml / min. The injection volume was set to 1 μl. The peaks were then identified based on the retention time of the FAME standards and mass spectrometry analysis. The FAME standard is a commercially available, certified reference material and contains the following analytes: 2 weight (wt.) % cis-13,16-docosadienoic acid methyl ester; 2 wt. % cis-4,7,10,13,16,19-docosahexaenoic acid methyl ester; 2 wt. % cis-11,14-eicosadienoic acid methyl ester; 2 wt. % cis-5,8,11,14,17-eicosapentaenoic acid methyl ester; 2 wt. % cis-8,11,14-eicosatrienoic acid methyl ester; 2 wt. % cis-11,14,17-eicosatrienoic acid methyl ester; 4 wt. % arachidic acid methyl ester; 2 wt. % arachidonic acid methyl ester; 4 wt. % methyl behenate; 4 wt. % methyl butyrate; 4 wt. % methyl decanoate; 2 wt. % cis-13-docosaenoic acid methyl ester; 4 wt. % methyl dodecanoate; 4 wt. % cis-11-eicosene 2 wt% of methyl ester of anisole; 2 wt% of methyl ester of anisole; 2 wt% of methyl ester of docosanoate; 2 wt% of methyl ester of heptadecanoate; 2 wt% of methyl 10-heptadecenoate; 4 wt% of methyl caproate; 2 wt% of methyl linoleate; 2 wt% of methyl linoleate; 2 wt% of methyl linolenate; 2 wt% of methyl γ-linolenate; 4 wt% of methyl myristate; 2 wt% of methyl myristoleate; 4 wt% of methyl octanoate; 4 wt% of methyl oleate; 6 wt% of methyl palmitate; 2 wt% of methyl palmitoleate; 2 wt% of methyl pentadecanoate; 2 wt% of methyl 10-pentadecanoate; 4 wt% of methyl stearate; 4 wt% of methyl tetracosanoate; 2 wt% of methyl 15-tetracosenoate; 2 wt% of methyl tridecanoate; 2 wt% of methyl undecanoate. An example of a FAME standard is commercially available from Sigma-Aldrich (FAMEMix, C4-C24, 18919-1AMP).The fatty acid composition of the lipid composition extracted from the isolated Schizochytrium strain is shown in Table 2.

[0089] The two most important fatty acids in the extracted lipid composition are docosahexaenoic acid (DHA, C22:6) and palmitic acid (C16:0), which account for 45% and 32% of the total fatty acids in the extracted lipid fraction, respectively, as shown in Table 2. In addition to DHA, the PLU strain also synthesizes other types of polyunsaturated fatty acids, including eicosapentaenoic acid (EPA, C20:5) and docosapentaenoic acid (DPA, C22:5), accounting for 1.07% to 3.35% and about 4.5%, respectively.

[0090] Table 2

[0091]

[0092]

[0093] * CO-1 refers to algae ω from Nordic Naturals; # CO-2 refers to GoldMinds DHA200 from GNC; EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid. All values ​​are provided as percentages ("%").

[0094] Fed-batch fermentation for DHA production

[0095] The isolated Schizochytrium PLU-D strain was subjected to fed-batch fermentation in a 2-liter Biostat B bioreactor to determine its lipid and DHA productivity. A seed culture was first prepared in a 500 ml shake flask containing 100 ml of medium containing 90 grams / liter (g / l) glucose, 22.5 g / l yeast extract and 20 g / l sea salt and incubated at 28° C. with shaking at 150 rpm for 3 days. Then 70 ml of the seed culture was inoculated into 700 ml of fermentation medium containing 50 g / l yeast extract, 2.5 g / l NaCl, 4 g / l KH2PO4, 2.5 g / l MgSO4·7H2O, 0.5 g / l KCl, 9.7 g / l Na2SO4, 0.1 g / l CaCl2, 200 g / l glucose, 10 ml / l trace element solution and 2 ml / l vitamin solution. The trace element solution contained 6 g / L of Edta-Na2, 0.174 g / L of FeCl3, 6.84 g / L of H3BO3, 0.55 g / L of MnCl2, 60 mg / L (mg / L) of ZnCl2, 26 mg / L of CoCl2·6H2O, 52 mg / L of NiSO4·6H2O, 2 mg / L of CuSO4·5H2O and 4.26 mg / L of Na2MoO4 and was sterilized by filtration with a 0.22 μm membrane, while the vitamin solution contained 100 mg / L of thiamine and 0.5 mg / L of biotin and was sterilized by filtration with a 0.22 μm membrane. When the dissolved oxygen (DO) level was observed to peak or rise, a feed solution containing 100 g / L of yeast extract and 400 g / L of glucose was added. The feed solution flow rate is first set to provide 0.14ml / minute (ml / min) of feed solution, and is increased every 8 hours to ensure that the total feed solution flow rate remains on 0.185ml / liter of fermentation volume / minute. After 90 hours of fermentation, a total of 500ml of feed solution is added to the fermentor tank. During the fermentation process, the air flow rate is set to 2.5 liters per minute (l / minute) and is reduced to 1.5l / minute when the feed is started, and the O2 flow rate remains on 0.5l / minute during the entire fermentation process. During the entire fermentation process, the pH is maintained at 7.0 with 2M NaOH and 0.5M citric acid, the temperature is maintained at 28°C, and the stirring speed is set to 700rpm.

[0096] The Schizochytrium PLU-D strain was selected for the fermentation production of DHA microalgae oil. The fermentation process started with a glucose batch phase for biomass accumulation, followed by a glucose batch feed phase when glucose was completely consumed (as indicated by a peak in dissolved oxygen levels) to promote lipid accumulation and further increase biomass. Figure 5As shown, in the first 10 hours, biomass slightly increased to 7.3 grams per liter (g / l) cell dry weight from 2.0 grams per liter (g / l), and then in 10 to 38 hours, cell dry weight increased sharply to 109.9g / l from 7.3g / l. The initial glucose concentration provided to the fermentation tank was 200g / l, which was almost exhausted at about 45 hours, and the dissolved oxygen peak showed this point, and produced the biomass of 128g / l cell dry weight and the lipid accumulation of 67.4g / l lipid. After 45 hours, the feed solution was progressively introduced into the fermentation tank, and continued for another 45 hours, causing the biomass to slowly and progressively increase in 45 to 93 hours. In the fermentation process of about 84 hours, the PLU- strain obtained the biomass up to 175g / l cell dry weight, which was equivalent to the lipid yield of 93.6g / l.

[0097] Yield of odd-chain fatty acids in shake flasks

[0098] Cells of Schizochytrium PLU-D were cultured in 50 ml medium containing 30 g / l glucose, 10 g / l yeast extract and 5 g / l sodium propionate in a 250 ml shake flask. After culturing at 25°C for 5 days, the cells were harvested and the lipids in the cells were extracted. The fatty acid composition of the extracted lipids was measured by GC-MS. The concentrations of each fatty acid in the lipid extract are shown in Table 3. All values ​​are expressed as a percentage of the total fatty acid composition. Odd-chain fatty acids C15:0 fatty acids reached 31.5%, and C17:0 fatty acids reached 2.3%.

[0099] Table 3

[0100]

[0101]

[0102] EPA: eicosapentaenoic acid; DPA: docosapentaenoic acid; DHA: docosahexaenoic acid; all values ​​are expressed as percentage ("%") of the total fatty acid composition.

[0103] Fed-batch fermentation for the production of odd-chain fatty acids

[0104] Schizochytrium was cultured in a 2-liter fermenter, and the fermentation medium contained 150 to 200 g / l glucose, 30 to 50 g / l yeast extract, 5 g / l sodium propionate, and a trace element solution. The trace element solution used herein contained 6 g / l EDTA-Na2, 0.174 g / l FeCl3, 6.84 g / l H3BO3, 0.55 g / l MnCl2, 60 mg / l ZnCl2, 26 mg / l CoCl2·6H2O, 52 mg / l NiSO4·6H2O, 2 mg / l CuSO4·5H2O, and 4.26 mg / l Na2MoO4. The dissolved oxygen peak indicated that the glucose was almost completely consumed at around 40 to 45 hours. Subsequently, a feed solution comprising 400 g / l to 500 g / l glucose, 70 to 100 g / l yeast extract and 5 g / l to 15 g / l sodium propionate is gradually introduced into the fermenter for another 40 to 60 hours. The final cell dry weight obtained is 65 g / l, the lipid yield is 40 g / l, the percentage of odd-chain fatty acids in the lipid composition is about 51%, including 47% C15:0 fatty acids and 4% C17:0 fatty acids, and the percentage of DHA in the lipid composition is about 24%.

[0105] Sequence Listing

[0106]

Claims

1. A fermentation method for producing a lipid composition using a Schizochytrium sp. strain deposited under accession number DSM34665, wherein the method comprises culturing the Schizochytrium sp. strain to produce the lipid composition.

2. The fermentation process of claim 1, wherein the lipid composition comprises docosahexaenoic acid (DHA).

3. The fermentation process of claim 1, wherein the lipid composition comprises odd-chain fatty acids (OCFA).

4. The fermentation method according to claim 3, further comprising culturing the isolated strain in the presence of sodium propionate.

5. The fermentation process of claim 4, wherein culturing the isolated strain in the presence of sodium propionate results in a higher concentration of odd-chain fatty acids (OCFA) in the lipid composition.

6. The fermentation method according to any one of claims 1 to 3, wherein the biomass of the isolated strain increases from the start of culturing the Schizochytrium strain up to 84 hours.

7. The fermentation process according to any one of claims 1 to 6, wherein culturing the Schizochytrium strain results in an increase in the concentration of the lipid composition compared to the concentration of the lipid composition obtained using a control strain.

8. The fermentation process according to any one of claims 1 to 7, wherein culturing the Schizochytrium strain results in an increase in docosahexaenoic acid (DHA) concentration compared to a control strain.

9. The fermentation process according to any one of claims 1 to 7, wherein culturing the Schizochytrium strain results in an increase in the concentration of odd-chain fatty acids (OCFA) compared to a control strain.

10. The fermentation process according to any one of claims 7 to 9, wherein the control strain is Schizochytrium ABC101 or Schizochytrium G13 / 2S.

11. A lipid composition obtained by the fermentation method according to any one of claims 1 to 9.

12. The composition of claim 10, comprising C11:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:5, C22:5 or C22:6 fatty acids.

13. The composition of claim 10, comprising docosahexaenoic acid (DHA).

14. The composition of claim 10, comprising odd-chain fatty acids (OCFA).

15. The composition of claim 12, wherein the concentration of docosahexaenoic acid (DHA) ranges from 35% to 55%.

16. The composition of claim 13, wherein the concentration of odd-chain fatty acids (OCFA) ranges from 30% to 55%.

17. An isolated strain of Schizochytrium deposited under accession number DSM 34665.

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