Novel cylindrotheca fusiformis strain

Abstract

To provide a strain that maintains both high valuable substance content and high algal productivity, and a culturing method for efficiently proliferating the strain.SOLUTION: The present invention provides a novel strain of Cylindrotheca fusiformis with high productivity of valuable substances such as polyunsaturated fatty acid and carotenoid, and a method for culturing the strain under high glucose conditions.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention provides a novel strain of Cylindrotheca fusiformis, a method for culturing the same, and a method for producing EPA, arachidonic acid, and fucoxanthin. [Background technology]

[0002] Diatoms are single-celled protists found in all aquatic ecosystems, from volcanic seeps to sea ice. They evolved from endosymbiosis between heterotrophic eukaryotes and red algae, and therefore have a unique and diverse set of genes derived from these hosts. Due to this chimeric genetic makeup, there are a great variety of diatom species, estimated to be more than 100,000 in number. Among them, there are diatoms that produce high-value-added products such as polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and ω-6 polyunsaturated fatty acids, such as arachidonic acid (ARA), and carotenoids, such as fucoxanthin.

[0003] It is necessary to search for a strain that can simultaneously have a high content of such valuable products and high algal productivity, and that can easily increase the amount of the product produced, as well as to search for a culture method that can maintain high productivity while maintaining such a desirable content. However, the search for algae that produce valuable substances has not progressed very far.

[0004] For example, the demand for ARA continues to increase in the fields of medicine, nutritional supplements, and the like, but currently, many technologies for industrial-scale production use fungi such as Mortierella alpina (Patent Document 1). In general, heterotrophic microalgae grow quickly and have high algal productivity, but the ARA content is not high. Microalgae with high ARA content have been reported to include the green alga Parietochloris incisa, red algae (Non-Patent Document 1), and Porphyridium purpureum (Non-Patent Document 2), but these algae grow slowly and have low algal productivity. In addition, ARA production is induced under nitrogen depletion conditions, making it difficult to achieve both high ARA content and high algal productivity.

[0005] In addition, while fish oil is the traditional source of EPA, concerns over the depletion of fish resources and potential environmental impacts mean that sustainable alternatives need to be explored. Various microalgae have been proposed as substitutes for fish oil. Microalgae are a promising solution because they can be cultured on a large scale, have a simpler fatty acid composition, and can produce high-purity polyunsaturated fatty acids (Non-Patent Document 3). In addition, EPA production from microalgae has the advantage of being cost-effective and having a high recovery rate. Among microalgae, Phaeodactylum tricornutum, Isochrysis galbana, and Porphyridium crucentum have been considered as one of the best options for producing EPA (Non-Patent Document 4). It is desirable to explore new microalgae as further sources.

[0006] Fucoxanthin is a carotenoid pigment found in brown algae, diatoms, and other seaweeds. Fucoxanthin has been recognized to have anti-obesity, antioxidant, anti-inflammatory, and anti-cancer effects, and may be useful in preventing cardiovascular disease and diabetes. Currently, the main source of fucoxanthin is seaweed such as kelp. However, seaweed has a slow growth rate, a complicated extraction process, and the possibility of heavy metal contamination, making it difficult to meet market demand. Furthermore, the amount of fucoxanthin contained in seaweed is smaller than that of microalgae, and the fucoxanthin content of seaweed is 0.2 to 1.5% by weight of its dry weight (Non-Patent Document 5). On the other hand, some microalgae such as diatoms and haptophytes accumulate fucoxanthin in an amount of 10 to 15% by weight of their dry weight. However, the productivity of fucoxanthin in diatoms is affected by various physiological and environmental factors, such as the species and strain of the diatom, the availability of nutrients, and the types and concentrations of nutrients (Non-Patent Document 6). Therefore, in order to obtain high yields of fucoxanthin, it is necessary to optimize the culture conditions and identify new diatom strains with high fucoxanthin productivity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Pat. No. 5,583,019 [Non-patent literature]

[0008] [Non-Patent Document 1] Bigogno C, Khozin-Goldberg I, Adlerstein D, Cohen Z. Biosynthesis of arachidonic acid in the oleaginous microalga Parietochloris incisa (Chlorophyceae): radiolabeling studies. Lipids. 2002;37(2):209-16. https: / / doi.org / 10.1007 / s11745-002-0882-6

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

[0009] In view of the above problems, the present invention aims to develop a strain that can maintain both a high content of a high-value product and high algal productivity, and to provide a culture method for efficiently growing such a strain and a method for efficiently producing a high-value product from such a strain.

[0010] The present inventors conducted intensive research to solve the above-mentioned problems and succeeded in developing a more productive strain by culturing a newly isolated strain of Cylindrotheca fusiformis under high glucose conditions, thereby completing the present invention. [Means for solving the problem]

[0011] Therefore, the present application provides the following invention. (1) Cylindrotheca fusiformis strain CYFG6. (2) A method for culturing the strain according to (1), wherein a medium containing sugar components at a total sugar concentration of 5 to 20 g / L is used. (3) The culture method according to (2), wherein the sugar component is one or more sugars selected from the group consisting of glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and glycerol. (4) The strain according to (1), which is capable of growing in a medium containing sugar components at a total sugar component concentration of 5 g / L or more. (5) The strain according to (1) or (4), wherein the productivity of the strain is at least 0.5 g / L / day in terms of the dry weight of the algae of the strain. (6) The strain according to any one of (1), (4), and (5), which contains 5% by weight to 10% by weight of eicosapentaenoic acid per dry weight of the algae of the strain. (7) The strain according to any one of (1) and (4) to (6), which contains 5% by weight to 15% by weight of arachidonic acid per dry weight of the algae of the strain. (8) The strain according to any one of (1) and (4) to (7), which contains 15 mg / g to 30 mg / g of fucoxanthin per dry weight of the algae of the strain. (9) A method for producing polyunsaturated fatty acids, comprising recovering algae cells of the strain according to any one of (1) and (4) to (8) above, and extracting polyunsaturated fatty acids from the recovered algae cells. (10) A method for producing polyunsaturated fatty acids, comprising recovering algal cells of a strain cultured by the culture method according to (2) or (3) above, and extracting polyunsaturated fatty acids from the recovered algal cells. (11) The method according to (9) or (10), wherein the polyunsaturated fatty acid is an omega-3 polyunsaturated fatty acid or an omega-6 polyunsaturated fatty acid. (12) The method according to (11), wherein the omega-3 polyunsaturated fatty acid is eicosapentaenoic acid. (13) The method according to (11), wherein the omega-6 polyunsaturated fatty acid is arachidonic acid. (14) A method for producing fucoxanthin, comprising recovering algae cells of the strain according to any one of (1) and (4) to (8) above, and extracting fucoxanthin from the recovered algae cells. (15) A method for producing fucoxanthin, comprising recovering algal cells of a strain cultured by the culture method according to (2) or (3) above, and extracting fucoxanthin from the recovered algal cells. (16) Cylindrotheca fusiformis strain CYF6. (17) A method comprising growing the strain according to (16) while gradually increasing the concentration of all sugar components in a medium, and acclimatizing the strain so that it can grow in the increased concentration of sugar components in the medium. Effect of the Invention

[0012] According to the present invention, a novel strain has been obtained that is highly productive of valuable substances, such as polyunsaturated fatty acids, such as omega-3 polyunsaturated fatty acids such as EPA and omega-6 polyunsaturated fatty acids, such as ARA, and carotenoids, such as fucoxanthin, and these valuable substances can be mass-produced by an efficient culture method using the strain. [Brief description of the drawings]

[0013] [Figure 1] Figure 1 shows the growth rate (μ) of each CYF clone strain (CYF1 to CYF24 strains) from the first to seventh day of culture, as well as the proportion (wt%) of EPA and ARA per total fatty acid (TFA) contained in the algal cells of each strain, measured after 15 days of growth. [Diagram 2] FIG. 2 shows the time course of growth of the CYF6 strain when grown at various glycerol concentrations. [Diagram 3] FIG. 3 shows the growth of the CYF6 strain in each cycle when the glycerol concentration in the medium was increased stepwise. [Figure 4] FIG. 4 shows the growth and glycerol consumption of the CYF6 and CYFG6 strains over time. [Diagram 5] FIG. 5 is a micrograph showing the CYF6 and CYFG6 strains. [Figure 6] Figure 6 shows the percentage of total fatty acids (TFA) (wt%) per dry weight (DCW) of algae when the CYF6 and CYFG6 strains were grown in a medium containing 10 g / L glycerol, as well as the percentages of EPA and ARA (wt%) per total fatty acids (TFA). Error bars indicate ±SD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present inventors isolated many diatoms, and among them, they discovered Cylindrotheca fusiformis CYF6 strain (hereinafter, sometimes referred to as CYF6 strain), which has high productivity of valuable substances. It is known that the culture rate of Cylindrotheca fusiformis increases when lactic acid is used in the culture medium. On the other hand, it has been reported that Cylindrotheca fusiformis can utilize organic substrates such as lactic acid, succinic acid, malic acid, tryptone, yeast extract, and casamino acid, but may not utilize sugars such as glucose very much (Non-Patent Document 7), and there was a concern that the growth rate would decrease in the presence of high concentrations of sugars.

[0015] However, the present inventors have surprisingly succeeded in evolving the CYF6 strain using a medium with an increased concentration of sugars such as glycerol to obtain a Cylindrotheca fusiformis CYFG6 strain (hereinafter, sometimes referred to as the CYFG6 strain) with higher growth rate. More specifically, the concentration of sugar components was gradually increased to obtain the CYFG6 strain, which can grow efficiently even in a medium containing high concentrations of sugar components that would not be suitable for growth of a normal Cylindrotheca fusiformis strain. Even more surprisingly, the CYFG6 strain produced valuable materials very efficiently. More specifically, the CYFG6 strain had a very high content of polyunsaturated fatty acids such as ω-3 polyunsaturated fatty acids such as EPA and ω-6 polyunsaturated fatty acids such as ARA, and carotenoids such as fucoxanthin, and their productivity was very high.

[0016] [Culture method] Therefore, the present invention provides novel Cylindrotheca fusiformis strains CYF6 and CYFG6, and a method for culturing the CYF6 or CYFG6 strain in a medium containing sugar components at a total sugar component concentration of 5 to 20 g / L. The CYFG6 strain can grow in a medium containing a high total sugar component concentration of 5 g / L or more, for example, 5 to 20 g / L, 7.5 to 15 g / L, 5 to 10 g / L, or 10 to 15 g / L.

[0017] The concentration of the total sugar component refers to the total concentration of all types of sugar components contained in the medium. Examples of sugar components include glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and glycerol, and preferably glycerol.

[0018] In the present invention, various additives other than sugar components may be added to the medium to adjust the composition of the medium to be suitable for culturing the CYFG6 strain or CYF6 strain. Examples of such additives include mineral components, sulfur compounds, organic acids, inorganic acids, organic bases, inorganic bases, and organic nitrogen components. Furthermore, the pH can be appropriately adjusted by adding an appropriate acid or base, if necessary.

[0019] The mineral components are preferably contained at a concentration of total mineral components of usually 1.0 to 20.0 g / L per medium, more preferably 2.5 to 15.0 g / L, and even more preferably 5.0 to 15.0 g / L.

[0020] The concentration of all mineral components refers to the total concentration of all types of sugar components contained in the medium. Examples of mineral components include sodium sulfate, dihydrogen sodium phosphate, sodium metasilicate, boric acid, thiamine hydrochloride, ferric chloride, disodium ethylenediaminetetraacetate, manganese chloride, zinc sulfate, cobalt chloride, copper sulfate, sodium molybdate, artificial sea salts, and natural sea salts.

[0021] The medium preferably contains a sulfur compound and / or sodium metasilicate, such as one or more sulfur compounds selected from the group consisting of magnesium sulfate, sodium sulfate, and sodium sulfide.

[0022] The medium preferably contains an organic nitrogen component, such as tryptone, yeast extract, peptone, polypeptone, proteose peptone, and corn steep liquor, and preferably corn steep liquor and tryptone yeast extract.

[0023] The CYFG6 strain and the CYF6 strain of the present invention are cultured by inoculating the strain in a medium in which the total sugar component concentration has been adjusted as described above, and culturing according to a standard method. The culture conditions are a temperature of 5 to 40° C., preferably 10 to 35° C., particularly preferably 20 to 25° C., for example, at 25° C.±1° C., and may be aeration agitation culture, shaking culture, or stationary culture. Aeration agitation culture, shaking culture, or stationary culture may be employed.

[0024] The strains used in the present invention may be cultured in a culture device having a suitable cell culture means. The term "cell culture means" refers to any means having any function for culturing cells, such as a culture tank, which may have one or more devices selected from a stirring device, a vibration device, a temperature control device, a pH control device, a turbidity measuring device, a light control device, a device for measuring the concentration of specific gases such as O2 and CO2, and a pressure measuring device. The culture tank may be the same tank as the concentration / separation tank, or may be a tank separate from the concentration / separation tank. If the culture tank is a tank separate from the concentration / separation tank, it may be connected to the concentration / separation tank by a suitable means, such as a flow path.

[0025] [Acclimatization method] The present invention also provides a method for acclimating a Cylindrotheca fusiformis strain, such as the CYF6 strain, to the increased concentration of sugar components in the medium by growing the strain while increasing the concentration of all sugar components stepwise. The present invention also provides a composition comprising a Cylindrotheca fusiformis strain, such as the CYF6 strain, for use in the method for acclimating the strain to the increased concentration of sugar components in the medium by growing the strain while increasing the concentration of all sugar components stepwise. The Cylindrotheca fusiformis strain used in this acclimation method is preferably, but not limited to, a strain selected as a strain rich in lipids such as EPA and ARA. The present invention also provides a strain obtained by the acclimation method of the present invention.

[0026] "Increasing the concentration of the total sugar components stepwise" refers to, but is not limited to, gradually increasing the concentration of the total sugar components in the medium over time, and may be, for example, increasing by 1.0 g to 10.0 g / L, more preferably 2.0 g to 7.5 g / L, and more preferably 2.5 g to 5.0 g / L every 2 to 20 cycles, more preferably every 3 to 10 cycles, and even more preferably every 5 to 7 cycles. In this specification, one cycle refers to a period corresponding to the intermediate value of the exponential growth phase. However, the increase does not have to be performed at a constant increase rate, and may be, for example, a mode in which the concentration is increased gently in the first half while checking the culture state, and then increased significantly in the second half, or vice versa, or any mode in which the concentration of the total sugar components is increased within the range of the increase rate. The culture conditions for the acclimation method of the present invention can be the same as the above culture conditions except that the concentration of the total sugar components is increased stepwise.

[0027] [CYFG6 strain] The novel CYFG6 strain obtained by the above-mentioned acclimation method can grow in a medium containing sugar components at a total sugar component concentration of 5 g / L or more. When cultured in such a medium, the algal cell productivity of the CYFG6 strain is at least 0.5 g / L / day, preferably at least 0.6 g / L / day, more preferably at least 0.7 g / L / day, and even more preferably at least 0.8 g / L / day, calculated as the dry weight of the algal cells. The algal cell productivity is the value calculated by converting the amount of algal cells (g) in the medium (L) after culture per day.

[0028] The total fatty acid (TFA) concentration per algal dry weight (DCW) of the CYFG6 strain is usually 20% by weight to 60% by weight, preferably 30% by weight to 50% by weight, and more preferably 35% by weight to 45% by weight (TFA / DCW).

[0029] The polyunsaturated fatty acid is preferably an omega-3 polyunsaturated fatty acid or an omega-6 polyunsaturated fatty acid. A preferred omega-3 polyunsaturated fatty acid is EPA and a preferred omega-6 polyunsaturated fatty acid is ARA.

[0030] The CYFG6 strain generally contains 1 to 30% by weight, preferably 2.5 to 15% by weight, more preferably 5 to 10% by weight, and even more preferably 7 to 10% by weight of EPA per dry weight of algal cells (DCW) (EPA / DCW).

[0031] The concentration of EPA per total fatty acids (TFA) contained in the CYFG6 strain is usually 5% by weight to 40% by weight, preferably 10% by weight to 30% by weight, and more preferably 15% by weight to 25% by weight (EPA / TFA).

[0032] The CYFG6 strain generally contains 1 to 30% by weight, preferably 2.5 to 20% by weight, more preferably 5 to 15% by weight, and even more preferably 7 to 15% by weight of ARA per dry weight of algal cells (DCW) (ARA / DCW).

[0033] The concentration of ARA per total fatty acids (TFA) contained in the CYFG6 strain is usually 5% by weight to 40% by weight, preferably 15% by weight to 30% by weight, and more preferably 20% by weight to 25% by weight (ARA / TFA).

[0034] The CYFG6 strain generally contains 10 mg / g to 40 mg / g, preferably 15 mg / g to 30 mg / g, and more preferably 15 mg / g to 25 mg / g of fucoxanthin per algal dry weight (DCW).

[0035] The fucoxanthin productivity of the CYFG6 strain is usually 5 mg / g / day to 50 mg / g / day, preferably 8 mg / g / day to 40 mg / L / day, and more preferably 10 mg / L / day to 30 mg / L / day per dry weight (DCW) of algae. The productivity is a value calculated by converting the amount of fucoxanthin (g) in the medium (L) per day.

[0036] [Manufacturing method] The present invention further provides a method for producing polyunsaturated fatty acids such as EPA and ARA, which comprises recovering algal cells of the CYFG6 strain and extracting polyunsaturated fatty acids from the recovered algal cells.The present invention further provides a method for producing fucoxanthin, which comprises recovering algal cells of the CYFG6 strain and extracting fucoxanthin from the recovered algal cells.The method for producing polyunsaturated fatty acids or fucoxanthin of the present invention is characterized in that the CYFG6 strain produces polyunsaturated fatty acids or fucoxanthin.

[0037] The CYFG6 strain used in the production method of the present invention is preferably one cultured by the above-mentioned culture method. In particular, a culture method using a medium containing sugar components at a total sugar component concentration of 5 to 20 g / L is preferable. The culture period is usually 1 to 20 days, preferably 3 to 15 days, for example, 6 to 10 days, etc.

[0038] The polyunsaturated fatty acids such as EPA and ARA and fucoxanthin produced by the CYFG6 strain of the present invention can be analyzed by methods known to those skilled in the art. For example, fatty acids can be measured using gravimetric analysis as described in the Examples. Polyunsaturated fats such as EPA and ARA can be measured using gas chromatography as described in the Examples. For example, fucoxanthin can be measured using high performance liquid chromatography as described in the Examples.

[0039] The algal cells can be obtained by, for example, culturing and growing the CYFG6 strain by the above-mentioned method, and recovering the algal cells from the obtained culture solution by a conventional method such as centrifugation, filtration, etc. The recovered algal cells of the CYFG6 strain may be dried, for example, by freeze-drying or drying by heating, or may be concentrated and then used in a wet, non-dried state for extraction of polyunsaturated fatty acids or fucoxanthin.

[0040] Polyunsaturated fatty acids or fucoxanthin can be extracted from the obtained dry algae or wet algae. The extraction method is not particularly limited, and can be performed by known methods such as organic solvent extraction, squeezing, and supercritical extraction. Examples of organic solvents used in organic solvent extraction include hexane, acetone, chloroform, methanol, ethanol, and diethyl ether, and can be used alone or in a mixture of two or more liquids such as a polar solvent and a nonpolar solvent. The algae may be disrupted before extraction, and may be disrupted by known methods such as, but not limited to, chemical disruption using alkali or acid, mechanical disruption using a homogenizer, ultrasonic waves, or a bead mill, or biological disruption using enzymes. After the above extraction, the obtained extract may be concentrated and purified by methods known to those skilled in the art, for example, column chromatography using silica gel or acid clay, high performance liquid chromatography, liquid-liquid distribution, and urea addition method. EXAMPLES

[0041] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these. Experiment 1: Isolation and cultivation of CYFG6 strain A surface biofilm sample collected from the intertidal zone of Nabetahama Beach near Shimoda Marine Research Institute, Shimoda City, Kanagawa Prefecture, Japan, was used. A mixed phytoplankton culture was obtained from this sample, and after gradient filtration, single cells were picked up and isolated from Cylindrotheca fusiformis Reimann and Lewin. Further pure cultures were established by sonication of these isolates and subsequent cell washing with an antibiotic mixture, as described by Buckner et al., 2008. Taxonomic identification was performed by comparing the morphology of the frustule with the taxonomic guide described in Reimann et al., 1965. This resulted in the isolated Cylindrotheca fusiformis strains CYF1-24.

[0042] An f / 2+Si medium with the following composition was prepared (Guillard and Ryther, 1962). In Table 1, the artificial seawater salt mixture refers to a Coral Pro Instant sea salt mixture (Red Sea Inc.). Using this f / 2+Si medium, CYF1 to 24 strains were grown at 25°C under a combination of red light and blue light in a ratio of 80:20, with a concentration of 100 μmol / m 2 ·s 1 The plants were cultured for 15 days under a 12:12 hour light:dark photoperiod with a light intensity of 1000 Hz.

[0043] In order to further optimize the medium for culturing these CYF1-24 strain cultures at high cell density, 5f+Si medium (composition below) with a higher concentration than the f / 2+Si medium was used and cultured for an additional 15 days at the same temperature, light intensity and dark photoperiod as above. [Table 1]

[0044] The CYF1-24 strains thus cultured were isolated into single cells using a micropipette under an inverted microscope. Individual cells were placed into each well of a 96-well culture plate containing 200 μl of f / 2+Si medium, and the culture plate was incubated for 7 days under the same conditions as above to allow sufficient growth. A clone library (n=96) was established using each of the single cell isolates. From these 96 clonal cultures, 26 clones were selected that were evaluated as having high growth potential based on cell density estimated from the number of cells counted using a hemocytometer. These 26 clones were transferred from the culture plate into a 15 ml test tube containing 10 ml of f / 2+Si medium, scaled up three-fold, grown under the same conditions as above, and used for measuring fatty acids.

[0045] Fatty acid quantification: Fatty acids were extracted from 1 g of freeze-dried biomass using a modified Bligh and Dyer method for algal lipids according to the method described in Non-Patent Document 8. The amount of fatty acids was measured using gravimetric analysis as the amount in dry cells according to the method described in Non-Patent Document 9. More specifically, an aliquot (10 mg) of the fatty acid extract was transesterified. FAME profiles were generated using a Shimadzu Nexis GC2030 gas chromatograph (Shimadzu Corporation, Tokyo, Japan) equipped with a BPX-70 column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness), nitrogen was used as the carrier gas to achieve FAME separation, and the column temperature was increased from 120 ° C to 250 ° C at 3 ° C / min. Fatty acid peaks were identified using a FAME standard mixture (Supelco, Bellefont, PA) and quantified by comparing with the peak area of ​​the internal standard (tricosylic acid, C23:0) run under the same conditions. The amount of fatty acids measured in this way was taken as the amount of total fatty acids (TFA). Similarly, EPA and ARA were quantified by comparing the peak areas with those of the internal standards of EPA and ARA, respectively, according to the method described in Non-Patent Document 9.

[0046] Statistical analysis: All data were collected from triplicate samples and expressed as mean ± standard deviation as final values. All statistical analyses were performed using SPSS version 21.0 software (SPSS Inc., Chicago, IL, USA).

[0047] Lipid analysis showed that the CYF6 strain had the highest EPA and ARA contents, 8.92% and 8.99%, respectively. The average growth rate of the 26 clones was 0.64 (μ). The strain with the highest growth rate was CYF8, with a growth rate of 0.76 (μ), and the strain with the lowest growth rate was CYF3, with a growth rate of 0.58 (μ) (Figure 1). Although the growth rate of CYF6 was 0.71 (μ), which was slightly lower than that of CYF8, the CYF6 strain was selected due to its high EPA and ARA contents, and was used in the application method described below.

[0048] The CYF6 strain (Accession No.: FERM P-22479) was deposited at the depository institution as follows: The deposit was made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. (i) The name and address of the depositary institution; Name: National Institute of Technology and Evaluation, Patent Organism Depositary Center (NITE-IPOD) Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (ii) Entrustment date: July 11, 2023 (iii) Accession number: FERM P-22479

[0049] Experiment 2: Effect of glycerol concentration on mixotrophic growth of strain CYF6 To identify the minimum concentration of glycerol that caused substrate inhibition, 5f+Si medium containing 2 g / L, 5 g / L, and 10 g / L glycerol was used. To examine the effect of glycerol on growth, CYF6 strain was cultured at various glycerol concentrations (0, 2, 5, and 10 g / L). At glycerol concentrations of 5 g / L and 10 g / L, substrate inhibition occurred and the lag phase was prolonged to about 6 days, but such a long lag phase was not observed at a glycerol concentration of 2 g / L per medium (Figure 2). At glycerol concentrations of 5 g / L and 10 g / L, the final cell number was 1.5-fold (5 g / L) and 2.9-fold (10 g / L) lower than that at 2 g / L, respectively. This is likely due to the longer lag phase at 5 g / L and 10 g / L. However, even at a glycerol concentration of 2 g / L, the growth was insufficient, and the dry cell weight (DCW) of the CYF6 strain per L of medium was low at 1.74 g / L. It is very difficult to obtain a sufficient amount of lipids with such a low growth rate. Therefore, in order to develop a strain that can utilize large amounts of resources such as glycerol and shorten the lag phase of the CYF6 strain, which is prolonged due to substrate inhibition, the CYF6 strain was subjected to adaptive evolution using the following method.

[0050] Experiment 3: Obtaining the CYFG6 strain by adaptive laboratory evolution (ALE) The CYF6 strain was used as a seed culture medium, which was obtained by culturing in a 500-ml Erlenmeyer flask containing 200 ml of f / 2+Si medium with reciprocal shaking at 100 revolutions per minute. The seed culture thus obtained had an initial cell density of 4 × 10 in the medium, depending on the density of the seed culture medium. 4 The cells were inoculated into 5f+Si medium at a concentration of 1000 cells / ml and cultured under the same conditions as in experiment 1, except that the glycerol concentration was increased stepwise as described below. The glycerol concentration in the medium was increased according to the increase or decrease in the growth ability by measuring the cell density every other day for each cycle by growing each in triplicate under the same conditions. One ALE cycle lasted for 5 days, which corresponds to the midpoint of the exponential growth phase. The glycerol concentration in the medium was gradually increased from 5 g / L, the concentration at which CYF6 showed substrate inhibition, to 5, 7.5, 10, 15, and 20 g / L. Seven ALE cycles were performed with an initial glycerol concentration of 5 g / L, and then the substrate (glycerol) concentration was gradually increased while decreasing from 7 to 5 ALE cycles. For comparison, the CYF6 strain was cultured under the same conditions, except that the glycerol concentration in the medium was always adjusted to 2 g / L. A total of 28 ALE cycles were performed for a total of 140 days.

[0051] The results are shown in Figure 3. In Figure 3, the system in which the glycerol concentration was increased from 2 g / L to 20 g / L is indicated as CYFG6 and marked with a black circle, and the system with a glycerol concentration of 2 g / L is indicated as CYF6 and marked with a white circle. As shown in Figure 3, the cell density of the strain with the increased glycerol concentration was 480 × 10 in the first ALE cycle (glycerol concentration 5 g / L). 4 cells / mL, which gradually increased, and the cell density stabilized and showed favorable adaptation from ALE cycles 19 to 23 (glycerol concentration 15 g / L), and a slight increase in cell density was observed from ALE cycles 24 to 28 (corresponding to a glycerol concentration of 20 g / L), with the final cell density at ALE cycle 28 (glycerol concentration 20 g / L) being 2163 × 104 The strain obtained at the end of the 28th ALE cycle was designated as CYFG6 strain.

[0052] The CYFG6 strain (Accession No.: FERM P-22466) was deposited at the depository institution as follows: The deposit was made in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. (i) The name and address of the depositary institution; Name: National Institute of Technology and Evaluation, Patent Organism Depositary Center (NITE-IPOD) Address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (ii) Entrustment date: February 7, 2023 (iii) Accession number: FERM P-22466

[0053] Experiment 4: Growth and glycerol concentration of CYF6 and CYFG6 strains The CYF6 and CYFG6 strains obtained from experiments 1 to 3 were cultured for 10 days under the same conditions as in experiment 3, except that a medium with an initial glycerol concentration of 10 g / L was used and the glycerol concentration was not adjusted after the start of culture, and growth and glycerol consumption were observed.

[0054] The results are shown in Figure 4. Even under high glycerol concentrations with an initial glycerol concentration of 10 g / L, the CYFG6 strain showed vigorous growth and achieved a final algal mass of 1.74 g / L after 10 days of culture. On the other hand, the growth of the CYF6 strain only reached 0.78 g / L. In addition, during the 10 days of culture, the CYFG6 strain consumed 5.79 g of glycerol, but the CYF6 strain consumed only 2.07 g, indicating a two-fold increase in glycerol consumption by the CYFG6 strain. Furthermore, although glycerol consumption was only observed after 144 hours in the CYF6 strain, no such substrate inhibition was observed in the CYFG6 strain.

[0055] Experiment 5: Morphological characteristics and productivity of CYF6 and CYFG6 strains After cultivation in Experiment 4, the CYF6 and CYFG6 strains were photographed and the morphological characteristics shown in Table 2 were measured. The amounts of DWC, TFA, ARA and EPA were measured using the same methods as in Experiment 1, and the amount of fucoxanthin was also measured using the method described below.

[0056] Fucoxanthin Quantification: Pigments were extracted from freeze-dried algae (1 g) using ethanol. Pigment profiles were generated using a high-performance liquid chromatograph (HPLC) (Agilent, Tokyo, Japan) equipped with a RP C-18 column (5 mm particle size, 250 mm, 4.6 mm ID, Waters, Tokyo, Japan). Identification and estimation of fucoxanthin were performed by comparing the peak timing and calibration curves generated based on peak area with true standards run under the same conditions.

[0057] Regarding the dry weight, the algae were collected and centrifuged. The supernatant was removed and washed with 0.5 M ammonium formate solution. After washing, the algae were collected using a GFC filter, dried at 60°C for 6 hours, and weighed to obtain dried algae. Furthermore, the morphological characteristics listed in Table 2 were also measured.

[0058] The morphological characteristics and productivity of the CYF6 and CYFG6 strains are shown in Table 2, Figure 5, and Figure 6. The amount of algae indicates the dry weight (DCW) (g) of algae per L of medium. The productivity of algae is the value calculated by converting the amount of algae per medium into productivity per day. [Table 2]

[0059] As can be seen from Table 2 and Figure 5, the CYFG6 strain was larger in size and had higher algal productivity, total fatty acids, ARA, EPA, and fucoxanthin contents compared to the CYF6 strain. The cell size of the CYFG6 strain was much larger than that of CYF6 (57.7%), and although there was a slight morphological change in cell shape related to the decrease in proboscis length, there was no abnormal change in cell shape, and only the size increased. This suggests that this may be due to a change in metabolic activity rather than osmotic stress caused by the addition of glycerol.

[0060] Figure 6 shows the percentage (wt%) of total fatty acids (TFA) per dry cell weight (DCW). The TFA, ARA, and EPA contents were all much higher than those of the CYF6 strain (Figure 6). The CYFG6 strain was able to accumulate 40.06% lipids per dry cell weight (DCW), which was higher than that of the CYF6 strain (26.55%) (Table 2). In addition, the EPA and ARA contents (wt%) per TFA in the CYFG6 strain reached a total of 41.24%, which was 2.3 times higher than that of the CYF6 strain (Table 2).

[0061] Experiment 6: Comparison of CYFG6 strain with other algae The CYF6 and CYFG6 strains obtained in Experiments 1 to 3 were cultured for 10 days under the same conditions as in Experiment 3, except that glycerol was added every 2 to 3 days so that the glycerol concentration in the medium was always 10 g / L.

[0062] The results of Experiment 6 are shown in Table 3-5 in comparison with other algae, fungi, etc. Algal productivity, TFA productivity, ARA productivity, and fucoxanthin productivity are values ​​calculated by converting the amount of algal body, TFA, ARA, and fucoxanthin per medium into productivity per day. [Table 3] [Table 4] [Table 5]

[0063] As shown in Table 3-5, the CYFG6 strain has the following advantages over other algae: With reference to Table 3, the ARA productivity may be higher when using fungi such as Morterella aplina, but these fungi are CO2 excreting fungi, which causes a problem of environmental load. In addition, the CYFG6 strain has three times more ARA productivity than ARA-producing algae such as Parietochloris incisa and Lobosphaera bisecta. With reference to Table 4, the EPA productivity is highest in Nitzschia laevis, but not only does it require glucose as a nutrient source, which is costly, but since many organisms use glucose as a nutrient, the CYFG6 strain, which uses other nutrient sources such as glycerol, is preferable from the viewpoint of not competing with other organisms. With reference to Table 5, the fucoxanthin content may be higher when using algae that produce fucoxanthin, such as Tisochrysis lutea, but it has a slow growth rate and is inferior to the CYFG6 strain in terms of fucoxanthin productivity.

[0064] The references listed in Table 3-5 are listed below. Reference 1: Hao, G., Chen, H., Gu, Z. et al. Metabolic engineering of Mortierella alpina for arachidonic acid production with glycerol as carbon source. Microb Cell Fact 14, 205 (2015). https: / / doi.org / 10.1186 / s12934-015-0392-4 Table 2:Tababa, HG, Hirabayashi, S. & Inubushi, K. Media optimization of Parietochloris incisa for arachidonic acid accumulation in an outdoor vertical tubular photobioreactor. J Appl Phycol 24, 887–895. https: / / doi.org / 10.1007 / s10811-011-9709-9 Table 3:Gao, B.; Huang, L.; Lei, X.; Meng, G.; Liu , J. ; Zhang, C. Evaluation and Transcriptome Analysis of the Novel Oleaginous Microalga Lobosphaera bisecta (Trebouxiophyceae, Chlorophyta) for Arachidonic Acid Production. ;. https: / / doi.org / 10.3390 / md1 Table 4: Wen, .ZY., Chen, .F. A perfusion-cell bleeding culture strategy for enhancing the productivity of eicosapentaenoic acid by Nitzschia laevis . Appl Microbiol Biotechnol 57, 316–322. https: / / doi.org / 10.1007 / s002530100786 Reference 5: Tanaka T, et al. Production of eicosapentaenoic acid by high cell density cultivation of the marine oleaginous diatom Fistulifera solaris. Bioresour. Technol. 2017; 245: 567-572. https: / / doi: 10.1016 / j.biortech.2017.09.005 Reference 6: Lu, X.; Sun, H.; Zhao, W.; Cheng, K.-W.; Chen, F.; Liu, B. A Hetero-Photoautotrophic Two-Stage Cultivation Process for Production of Fucoxanthin by the Marine Diatom Nitzschia laevis. Mar. Drugs 2018, 16, 219. https: / / doi.org / 10.3390 / md16070219 Reference 7: Mohamadnia, S., Tavakoli, O., Faramarzi, M.A., 2021. Enhancing production of fucoxanthin by the optimization of culture media of the microalga Tisochrysis lutea. Aquaculture 533, 736074. https: / / doi.org / 10.1016 / j.aquaculture.2020.736074. Reference 8: Xia, S.; Wang, K.; Wan, L.; Li, A.; Hu, Q.; Zhang, C. Production, Characterization, and Antioxidant Activity of Fucoxanthin from the Marine Diatom Odontella aurita. Mar. Drugs 2013, 11, 2667-2681. https: / / doi.org / 10.3390 / md11072667 Table 9:McClure, DD, Luiz, A., Gerber, B., Barton, GW, & Kavanagh, JM (2018). An investigation into the effect of culture conditions on fucoxanthin production using the marine microalgae Phaeodactylum tricornutum. Algal Research, 29, 41-48. Table 10:Petrushkina M, Gusev E, Sorokin B, Zotko N, Mamaeva A, Filimonova A, et al. Fucoxanthin production by heterokont microalgae. Algal Res. Rev. 2017;24:387–9 https: / / doi.org / 10.1016 / j.algal.2017.03.016

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[0065] FERM P-22479 FERM P-22466

Claims

1. Cylindrotheca fusiformis strain CYFG6.

2. The method for culturing the strain according to claim 1, wherein a medium containing sugar components at a total sugar component concentration of 5 to 20 g / L is used.

3. The culture method according to claim 2 , wherein the sugar component is one or more sugars selected from the group consisting of glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and glycerol.

4. The strain according to claim 1 , which is capable of growing in a medium containing sugar components at a total sugar component concentration of 5 g / L or more.

5. The strain according to claim 1 or 4, wherein the productivity of the strain is at least 0.5 g / L / day in terms of dry weight of the algal cells of the strain.

6. The strain according to any one of claims 1, 4 and 5, comprising 5% to 10% by weight of eicosapentaenoic acid per dry weight of the algae of the strain.

7. The strain according to any one of claims 1 and 4 to 6, comprising 5% to 15% by weight of arachidonic acid per dry weight of the algae of the strain.

8. The strain according to any one of claims 1 and 4 to 7, comprising 15 mg / g to 30 mg / g of fucoxanthin per dry weight of algal cells of the strain.

9. 9. A method for producing polyunsaturated fatty acids, comprising: recovering algal cells of the strain according to any one of claims 1 and 4 to 8; and extracting polyunsaturated fatty acids from the recovered algal cells.

10. A method for producing polyunsaturated fatty acids, comprising: recovering algal cells of a strain cultured by the culture method according to claim 2 or 3; and extracting polyunsaturated fatty acids from the recovered algal cells.

11. The method according to claim 9 or 10, wherein the polyunsaturated fatty acid is an ω-3 polyunsaturated fatty acid or an ω-6 polyunsaturated fatty acid.

12. The method according to claim 11, wherein the omega-3 polyunsaturated fatty acid is eicosapentaenoic acid.

13. The method according to claim 11, wherein the omega-6 polyunsaturated fatty acid is arachidonic acid.

14. A method for producing fucoxanthin, comprising: recovering algal cells of the strain according to any one of claims 1 and 4 to 8; and extracting fucoxanthin from the recovered algal cells.

15. A method for producing fucoxanthin, comprising: recovering algal cells of a strain cultured by the culture method according to claim 2 or 3; and extracting fucoxanthin from the recovered algal cells.

16. Cylindrotheca fusiformis strain CYF6.

17. A method for acclimatizing the strain according to claim 16 to the increased concentrations of total sugar components in the medium by growing the strain in the medium while increasing the concentration of the total sugar components in the medium stepwise.

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