KR101185596B1 - Schizochytrium sp. JBF-06, strain for improvement of DHA production and the method of producing DHA using it - Google Patents

Abstract

The present invention relates to marine microorganisms with improved DHA productivity and the production of DHA using the same. In the present invention there is provided a JBF-06 strain of the genus Szozoquitrium, which is a mutant strain that improves DHA productivity. In addition, the present invention provides a method for producing DHA using this strain. In the present invention, through the optimum growth conditions of mutations and strains to increase the productivity in DHA production using marine microorganisms.

Description

DH-06 strain of S jozoquitrium improved DHA productivity and DHA production method using the same {Schizochytrium sp. JBF-06, strain for improvement of DHA production and the method of producing DHA using it}

The present invention relates to the production of DHA using microorganisms, and in particular to the production of marine microorganisms and improved DHA using DHA productivity.

Unsaturated fatty acid

ω-3 unsaturated fatty acids are fatty acids where the first double bond starts from the methyl group of the alkyl chain to the third atom. EPA (Eicosapentaenoic acid, 20: 5n-3) and DHA (Docosahexaenoic acid, 22: 6n-3) Is a representative ω-3 polyunsaturated fatty acid (L. Sijtsma et al. 2004, Anita Nordeng Jakobsen 2008). Their structural formula is shown in the following formula (1). DHA is a polyunsaturated fatty acid consisting of 22 carbon atoms and six double bonds, and EPA is a polyunsaturated fatty acid consisting of 20 carbon atoms and five double bonds. These are essential fatty acids that must be consumed from natural products because they are not easily biosynthetic in the human body. Animals are synthesized from ω-3 fatty acid α-linoleic acid (α-LAN), and the synthesis of PUFAs in microorganisms is synthesized from oleic acid by the same enzyme mechanism found in higher organisms. It consists of two reactions, chain elongation and chain desaturation (Suzette L et al. 2004). Nutritional and pharmacological studies of α-LAN, EPA, and DHA have been extensively conducted since the pioneering work of Needleman et al. was published in the 1970s (P. A. Needleman et al., 1979). In particular, DHA is a major component of human membrane tissue and accounts for more than 60% of the total fatty acids of the rod outer segment of the retina. In addition, since DHA acts as a lipid component of tissues, it is essential for infants' vision and nerve development. However, premature babies and infants cannot synthesize DHA at a sufficient rate, so this ingredient must be supplemented from food to provide the DHA the brain needs quickly (Giusto NM et al., 2000).

Production of Unsaturated Fatty Acids by Microorganisms

DHA is difficult to chemically synthesize and must be obtained from natural products. Currently, these fatty acids are extracted from fish oil and used after separation and purification. The main sources of DHA are herring, mackerel, and sardine, which are rich in n-3 fatty acids and contain more than 30% of DHA (Galli and Simopoulos, 1989). However, the quality of fish oil varies depending on species, season, fishing position, etc., it has an unpleasant taste and smell, it is easy to oxidize, and the composition of fatty acids is very complicated, so it takes a lot of time and money to extract and refine. DHA production from has many problems. Therefore, researches to find a raw material for DHA to replace fish oil are actively underway (Belarbi et al., 2000), and many researchers have proposed many microorganisms as a commercial substitute for DHA (A. P. Bimbo et al. 1987).

The production of DHA by microorganisms is known to produce prokaryotic microorganisms such as bacteria as well as eukaryotic microorganisms such as fungi and algae. However, bacteria are not suitable as production strains because they cannot accumulate high concentrations of triacylglycerols and contain special fatty acids and lipids not found in other systems. Fish fatty acids are known to originate from their prey marine microbes, algae, and phytoplankton. Most microalgae have the ability to produce DHA and have the advantage that the fatty acid composition is very simple compared to fish oil, making it easy to extract and purify (Yongmanitchai et al., 1989). In order to commercially produce DHA using microalgae, it is important to produce high content of DHA and high growth rate according to environmental conditions of microalgae.

Currently, marine DHA-producing strains are reported as heterotrophic strains ( Thraustochytrium aureum, Scizochytrium sp., Crypthecodinium cohnii, Amphidinium carterae ) and photosynthetic strains ( Isochrysis galbana, Skeletonema costatum , Amphidinium sp., Pavlova lutheri ). Among them, Scizochytrium sp. And Crypthecodinium cohnii are used in the heterotrophic DHA production process. The algae-like Scizochytrium sp. Contains 70% of the total weight of lipids and 35% of the total fatty acids of DHA, and more than 90% of the fat is triglycerides. This strain is currently used for industrial production by Martek Biosciences, and has been used by Scizochytrium sp. The process using SR21 was developed by Nagase Biochemical Industries of Japan. And by Schizochytrium sp. And the DHA productivity using SR21 reports of 13.3 g / L, Swaaf such increased to 19 g / L of acetic acid was used during the glucose using the C. cohnii (L. Sijtsma et al. , 2004).

Lee Hyung-seok (2005). Changes of EPA and DHA Contents according to Environmental Conditions of Marine Microalgae, Gyeongsang National University Anita Nordeng Jakobsen. Compatible solutes and docosahexaenoic acid accumulation of thraustochytrids of the Aurantiochytrium group. NTNU, 2008: 169 A. P. Bimbo, J. Am Oil. 1987. Chem. Soc, 64, 64, 706 A. Singh, S. Wilson and O. P. Ward. 1996. Docosahexaenoic acid (DHA) production by Thraustochytrium sp. ATCC 20892. World J Microbiol Biotechnol. 12: 76-81 Belarbi, E. H., E. Molina and Y. Christi. 2000. A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil. Enzyme Microb. Technol., 26: 516-29. Galli, C. and A. P. Simopoulos. 1989. Dietary ω-3 and ω-6 fatty acids: Biological effects and nutritional essentially. Plenum Press, New York. Giusto NM, Pasquare SJ, Salvador PI, Roque MG (2000) Lipid metabolism in vertebrate retinal rod outer segments. Prog Lipid Res 39: 315 ?? 391 Goldstein S. 1963. Development and nutrition of new species of Thraustochytrium. Am J Bot 50: 271-279 K.V.V.S.N. Bapiraju, P. Sujatha, P. Ellaiah and T. Ramana. 2004. Mutation induced enhanced biosynthesis of lipase. Afr J Biotechnol. 3 618-621 LUC SIMON, MAURICE LALONDE, AND THOMAS D. BRUNS Specific Amplification of 18S Fungal Ribosomal Genes from Vesicular-Arbuscular Endomycorrhizal Fungi Colonizing Roots APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 291-295 Luis A. Meireles, A. Catarina Guedes, F. Xavier Malcata. 2003. Increase of yields of eicosapentaenoic and docosahexaenoic acids by the microalga Pavlova lutheri following random mutagenesis. Biotechnol Bioeng. 81: 50-55 L. Sijtsma. M. E. de Swaaf. Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid. Appl Microbiol Biotechnol (2004) 64: 146-153 P. A. Needleman, M. S. Raz, M. S. Minkes, J. A. Ferrendelli and H. Sprecher, Proc. Natl. Acad. Sci, 76 (2), 944 (1979) Ratledge C, Kanagachandran K, Anderson AJ, Grantham DJ, Stephenson JM (2001) Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source. Lipids 36: 1241 ?? 1246 Simpoulous, R. R. Kifer and R. E. Martin. Health effects of polyunsatuated fatty acids in seafoods, 319, Academic Press (1986) Rinka Yokoyama, Daiske Honda. Taxonomic rearrangement of the genus Schizochitrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov .. Mycoscience 48: 199-211 Suzette L. PEREIRA 1, Amanda E. LEONARD, Yung-Sheng HUANG, Lu-Te CHUANG and Pradip MUKERJI. Identification of two novel microalgal enzymes involved in the conversion of the ω3-fatty acid, eicosapentaenoic acid, into docosahexaenoic acid Biochem. J. (2004) 384, 357 ?? 366 Tom Lewis, Peter D. Nichols, Tom A. McMeekin. 2000. Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J Microbiol Meth 43 107-116 Yongmanitchai, W. and O. P. Ward. 1989. Omega-3 fatty acids: Alternative sources of production. Press Biochem., 24: 117-125

In order to commercially produce DHA using microalgae, it is necessary to increase the DHA productivity of microalgae, and to this end, it is necessary to increase the biomass and intracellular fatty acid content of the microalgae.

In the present invention, to increase the DHA productivity through mutagenesis of the Schizochytrium sp. Strain having DHA productivity, and also to find the optimal growth conditions. The present invention aims to provide a strain with increased biomass and DHA content through mutagenesis, and to provide a high productivity DHA production method using the strain.

Other objects and advantages of the present invention will be described below and will be better understood by practice of the present invention.

In the present invention,

Schizochytrium sp. JBF-06 strain (accession number: KACC93083P), which is a mutant strain that induces mutations in the strain of Schizochytrium sp. To increase biomass and DHA content Is provided.

In the present invention,

Provided is a method for producing DHA comprising extracting DHA from JBF-06 strain (Accession No .: KACC93083P) or a culture thereof of Schizochytrium. Preferably, the JBF-06 strain of the genus Sjozukitririum, fructose as a carbon source; Galactose; Incubated in a culture medium containing at least one monosaccharide selected from glucose and containing malt extract or tryptone as a nitrogen source, more preferably at pH 5-8, 15-30 ℃ Let's do it.

In the present invention, the genus JBF-06, a mutant strain with increased biomass and DHA content, was obtained through mutagenesis of the genus Schizochytrium sp. In addition, the present invention can provide a highly productive DHA production method using this strain by finding the optimum growth conditions of the strain. The present invention can be used as a commercial production method of DHA using marine microorganisms.

Figure 1 shows the results of observing the cells of DHA-producing strain JB511 with an optical microscope, Figure 1a shows a cluster of cells having an ovate size of 7 ~ 12㎛, Figure 1b is a cell cultured in a salt-free condition The cells were incubated at 25 ° C. for 3 days and cells were lysed.
2 is a schematic based on the nucleotide sequence of 18S rDNA. Strain JB511 and Schizochytrium sp. FJU-512 showed 99.8% similarity.
Figure 3 is the result of experiments on the effect of the carbon source of the medium on the biomass (biomass) of the JBF-06 strain. As a carbon source, fructose (◆), galactose (■), glucose (▲), lactose (X), and sucrose (○) were added to the medium at 30 g / L, respectively, and cultured as a result of higher galactose and glucose than other carbon sources. Showed growth.
4 is a result of experiments on the effect of the nitrogen source of the medium on the biomass (biomass) of the JBF-06 strain. Yeast extract (◆), peptone (□), tryptone (▲), malt extract (X), and casamino acid (●) were added to 10 g / L of organic nitrogen, and the biomass of the strain was measured. The result is.
5 shows the results of experiments on the effect of pH on the biomass (biomass) of JBF-06 strain. The pH of the medium was adjusted with acetic acid and 10 N NaOH to maintain pH 4, 5, 6, 7, 8 (pH 4: ○, 5: ●, 6: ▲, 7: X, 8: ■), 25 The result of incubation for 72 hours at ℃.
Figure 6 is the result of experimenting the effect of temperature on the biomass (biomass) of JBF-06 strain. Incubation temperature was varied in the range of 15-35 ° C. and incubated at each temperature (15 ° C .: ◆, 20 ° C .: ■, 25 ° C .: ▲, 30 ° C .: X, 35 ° C .: *).
7 is a result of measuring the survival rate of the JB511 strain according to the irradiation time of ultraviolet rays. Irradiation at a distance of 15 cm from the center of the UV lamp, 0, 30, 40, 50 seconds were irradiated. The survival rate was 21% at 50 seconds.

In the present invention, first, the strain JB511 of the genus Szozoquitrium known to have the ability to produce DHA was distributed, and the colonies and cell morphology were observed by sequencing the 18S ribosomal DNA. As a result, it was confirmed to have 99.8% identity with the strain FJU-512 belonging to the genus Schizochytrium .

JB511 has a cell size of about 7 to 12 μm and forms clusters between cells. And since the strain was derived from seawater, it was observed that the cells were difficult to withstand at low osmolarity. As a result of diluting the strain through the liquid culture in sterile distilled water and diluting the cells with an optical microscope, the cells were greatly swollen up and the size was large and the cells burst easily. In addition, the growth of the strain was slow in the culture medium without NaCl compared to the culture medium in the liquid medium containing about 1.8 ~ 2.7% added NaCl. Therefore, the strain was cultured by the addition of sea salt (sea salt) during the culture.

In the present invention, the mutation was induced to increase the DHA productivity of the strain. Mutants used are UV and NTG. First, a mutation was induced by using UV, and a total of two strains were obtained through various screening processes. The biomass was measured by dry weight for the mutant strain, and the DHA content was analyzed by gas chromatography, and the maximum biomass and DHA were 26.3% and 77.6% in comparison with the parental strain, respectively. It was confirmed to increase. In order to increase productivity for the mutant strains obtained through UV mutagenesis, mutations were induced by varying the mutagen. NTG was used as a mutagen, and a strain showing high DHA productivity when UV mutagenesis was used as a parental strain. After screening two steps after NTG mutagenesis, one strain was obtained. For one strain obtained, the biomass was measured by dry weight, and the DHA content was analyzed by gas chromatography. The strain was measured by collecting the cells after 7 days incubation in the basic medium. As a result, biomass and DHA were measured at 14.65 and 3.414 g / L, respectively. Induction of NTG mutations in the UV mutants revealed only increased biomass. The mutant strain thus obtained was named JBF-06 in Schizochytrium, and was deposited with the Agricultural Genetic Resource Center of the National Academy of Agricultural Sciences on November 09, 2009 and was assigned accession number KACC93083P.

In the present invention, the biomass was measured by varying the culture conditions to find the optimal culture conditions for the growth of the mutant strain obtained through the above process. In the culture conditions measured, the yield of biomass was measured through OD ₆₀₀ by varying the carbon source, nitrogen source, culture medium pH, and culture temperature. Cultured in a carbon source of fructose, galactose, glucose, lactose and sucrose using 3% carbon source showed the highest biomass yield when glucose was used. Using yeast extract 1%, yeast extract, peptone, tryptone, malt extract, malt extract and casamino acid were cultured in a nitrogen source to measure the biomass. When used, it showed the highest biomass. The pH of the culture medium was found to show the highest growth at pH 6 by measuring the biomass in the range of 4 to 8. After adjusting the initial pH and culturing for 3 days without maintaining the pH, the cell dry weight was measured, and the yield of the highest biomass was obtained at pH 5. It was found that growth occurs well at relatively low pH. At pH 4, growth occurred very slowly. The strains were cultured at different culture temperatures, and the biomass was measured, indicating that they were high at a relatively low temperature of 25 ° C. or less.

In the present invention, the increase in productivity through mutagenesis and the optimum growth conditions were found to increase the biomass and DHA of the strain.

[Example]

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, these examples are only for illustrating the present invention in more detail, the scope of the present invention is not limited by these examples.

Materials and methods

1. Strain

Schizochytrium sp. JB511, sold by Zenith Co., Ltd., was used as a DHA producing strain. This strain is a marine heterotrophic organism isolated from the Pacific Ocean.

 2. Medium and basic culture conditions

The basic medium to which nutrients necessary for the growth of Shizochytrium sp. Were added was used, and the optimum conditions for the growth of the strain were measured by varying the conditions in the basic medium. Cultivation of the strain was carried out in 250 ml Erlenmeyer flask (50 ml) of the basal medium and inoculated with 7x10 ⁷ cells / ㎖ incubated at 250 rpm at 25 ℃. E. coli strains were cultured in LB medium containing ampicillin at a concentration of 20-50 μg / ml depending on the antibiotic resistance of the gene carrier.

1) Composition of basic medium (M medium)

30 g of glucose (Sigma), 10 g of yeast extract (Yeast extract, BD), and 17.5 g of sea salt (Sigma) were dissolved in distilled water to make 1 L, and then sterilized.

2) Composition of LB Medium

10 g of tryptophan, 5 g of yeast extract, and 5 g of sodium chloride were dissolved in distilled water to make 1 L and then sterilized.

3. Restriction Enzymes and Reagents

Restriction enzymes were purchased from Promega (WI, USA), medium was Sigma (MO, USA) and Difco (MI, USA), and other reagents were Sigma or Japanese ER grade reagents.

4. Strain Identification through 18s rRNA Analysis

1) DNA isolation from strain

Plasmid DNA was isolated by alkaline lysis (Sambrook and Russell, 2001), and purified by CsCl-ethidium bromide gradient as needed.

Isolation of plasmid DNA from E. coli was performed using the Wizard Plus SV Miniprep kit (promega, WI).

Genomic DNA of Schizochytrium sp. Was isolated as follows. Inoculate 7X10 ⁷ cells / mL of the strain into 50 ml of the basal liquid medium, and incubate for 24 hours to harvest the strain. Add 400 μl SET buffer (75 mM NaCl, 25 mM EDTA, 20 mM Tris (pH 7.5)), mix well, add 12 μl of lysozyme (50 mg / ml), add beads, and add bead beater. After 30 seconds of reaction, the supernatant is transferred to a new microcentrifuge tube. 10% SDS of 1/10 volume of the supernatant and 13 μl of proteinase K (20 mg / ml) were added and mixed, followed by reaction at 55 ° C. for 2 hours. Mix well by adding 1/3 volume of 5M NaCl and 1 volume of chloroform and leave for 30 minutes at room temperature. The supernatant was taken by centrifugation at 12,000 rpm for 10 minutes. Add 1 volume of isopropanol, mix well, and centrifuge for 7 minutes at 12,000 rpm. Remove the supernatant, mix well with 70% ethanol and centrifuge for 5 min at 12,000 rpm to remove supernatant. After drying the DNA band for 15 minutes using a centrifugal vaporizer (EYELA, CVE-100), 30μl of sterilized distilled water was put in at 4 ° C overnight, and the DNA was confirmed by electrophoresis.

2) PCR method for 18S rDNA amplification

PCR was used as a method for obtaining the 18S rDNA gene portion. Primers used in this experiment were used referring to Simon et al. (1992). The primers used are summarized in Table 1 below. DNA polymerase for PCR was reacted using Ex Tag DNA polymerase (Takara, Ohtsu, Japan), denaturated at 94 ° C for 30 seconds, annealed at 55 ° C, and extended at 72 ° C. The cycle was reacted.

3) E. coli transformation

E. coli was inoculated into 10 ml LB medium and incubated for 16 hours, after which 1 ml was inoculated into 100 ml LB medium and shaken at 37 ° C. to OD ₆₀₀ to 0.3. Left for 30 minutes. After centrifugation at 4 ° C for 10 minutes at 3,000 rpm, the precipitated cells were washed with 20 ml of cold sterile distilled water and centrifuged at 3,000 rpm for 10 minutes. The supernatant was discarded, and 5 ml of cold 0.1 M CaCl ₂ solution was added to the precipitate and allowed to stand in an ice bath for 30 minutes. Centrifuge at 3,000 rpm for 10 minutes and 1 ml of 0.1 M CaCl ₂ The solution was added, the deposits were thoroughly mixed, and then 100 μl of the mixed solution and DNA were added to a new tube and left in an ice bath for 30 minutes. This was given a heat shock at 42 ° C. for 90 seconds, and 2 ml of LB medium was added thereto, followed by shaking culture at 60 ° C. for 60 minutes at 37 ° C. The culture solution was centrifuged at 200 rpm for 5 minutes and 200-300 μl of the precipitated cells were plated in LB medium containing ampicillin or kanamycin.

4) DNA sequencing

PCR products were purified using a QIAquick PCR purification kit (Qiagen). Primers used to analyze the overall sequence of 18s rDNA are NS1, NS3, NS5 (Table 1). A DNA sequencer was used to analyze the nucleotide sequence using the ABI PRISM 3730XL Analyzer. The analyzed sequences were analyzed using the nucleotide-nucleotide Basic Local Alignment Search Tool (BLAST) algorithm (blastn).

5. Mutation induced by UV

The parent strain is incubated for 18 hours at 250 rpm at a temperature of 25 ° C. in the base medium. The parent strain used in this experiment was JB511 strain. 1 ml of 1 × 10 ⁵ cells / ml culture solution is placed in an empty Petri dish, shaken evenly, and exposed to UV-light. In order to measure the survival rate for the mutagenesis experiment by UV, strains were cultured at different exposure times. The exposure time to ultraviolet rays is 0, 30, 40 and 50 seconds, respectively. The UV light used in this experiment was UVP Model R-52 at 254 nm, and the distance between the lamp and the sample was 15 cm. 100 μl of the culture medium exposed to UV light was inoculated into the base medium to which agar was added, followed by incubation at 25 ° C. for 48 hours.

6. Mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (NTG)

The parent strain is incubated for 18 hours at 250 rpm at a temperature of 25 ° C. in the base medium. The parent strain used in the NTG mutagenesis experiment used mutant strain CNU402 obtained by UV mutagenesis. Treat 10 g of the culture solution diluted to 1X10 ⁶ cells / ml with different concentrations of NTG and react for 30 minutes. Treated NTG concentrations are 0, 0.03, 0.05, 0.07 mg / ml. After the supernatant is removed by centrifugation, 100 μl of the supernatant is washed, inoculated into agar medium containing agar, and then incubated for 48 hours at 25 ° C. to measure the number of colonies formed. . NTG used was TCI (Tokyo Chemical Industry) product, which used more than 95.0% purity.

7. Screening of Mutant Strains

As a result of preliminary experiments, we found the most suitable concentration of Doxorubicin (doxorubicin) for screening strains. As a result, the cells were not grown when grown for 4 hours after plating about 4X10 ⁶ cells / ml in solid culture medium containing 1% concentration. Appeared. In addition, when inoculation of about 3X10 ⁴ cells / ml was performed in the basic medium containing 1% doxorubicin and agar, it was found that the bacteria did not grow. Doxorubicin has the characteristic of inhibiting growth against strains with low production capacity of DHA. When cultured strains with low DHA production capacity in the doxorubicin-added medium it was observed that they do not grow. However, it could be confirmed that the growth of a single cell in a certain concentration of doxorubicin did not grow, but when the cells existed in clusters and grew between cells, they were grown in solid medium. After colonization of the strain, when colonies are formed in the agar-added base solid medium, the strain is transferred to the base solid medium to which 1% doxorubicin is added by replica plating. After incubation at 25 ° C. for 120 hours, viable strains were isolated (primary screening). The strains screened here were incubated in primary medium, diluted to 1X10 ⁵ cells / ml, inoculated with 3 μl of solid primary medium containing 1% doxorubicin, and cultured for 72 hours, followed by screening for colony formation (secondary). screening).

8. DHA Analysis by Gas Chromatography

1) Pretreatment of Sample

Pretreatment of the sample for gas chromatography of fatty acids produced by the strain was performed by the following method with reference to Tom Lewis et al. Strains incubated in 50 ml basal media for 48 hours were inoculated with 5% (v / v) in 50 ml basal media and incubated for 72 hours. 20 ml of the culture solution is placed in a conical tube, followed by centrifugation at 3200 rpm for 10 minutes to remove the supernatant, and the precipitate is recovered, lyophilized, and weighed. 3 ml of 4% H ₂ SO ₄ methanol solution was added to the precipitate, followed by heat treatment at 100 ° C. for 1 hour. At this time, vortex treatment is performed three times during the heat treatment. After the heat treatment, leave at room temperature to lower the temperature of the tube, add 2 ml sterile distilled water and 2 ml hexane (hexane), vortex for 30 seconds, and leave for about 30 seconds to separate the layers, and then the supernatant is placed in a GC vial Contain and analyze. The internal standard was used by adding 100 ug / ml of heptadecaenoic acid (Sigma) to hexane, and the standard was 1 mg / ml of DHA methyl ester (Sigma).

2) Operating conditions of GC

Fatty acid separated during pretreatment was analyzed using a column for gas chromatography (GC-2010, SHIMADZU) and a hydrogen flame ionization detector (FID). The column used for omega 3 fatty acid analysis was Omegawax 320 (Capillary column; polyethyleneglycol 100%; 30 mx 0.32 mm x 0.52 μm), inlet temperature is 250 ° C., column temperature is 200 ° C., detector temperature is 260 ° C., The flow rate is 1.0 ml / min, the split ratio is 10: 1, and the sample injection amount is 1-2 mu l.

9. Determination of dry weight of cells

Biomass of the strain was measured by the dry weight of the cell. The cultured strain is placed in a conical tube 10ml of the culture solution and centrifuged for 10 minutes at 3200 rpm. The supernatant was removed and placed in a 75 ° C. dry oven for 24 hours and weighed. At this time, the empty conical tube is placed in a drying oven in advance and weighed.

10. Growth as Carbon Sources Change

In culturing the strains, the growth of the strains was compared against five kinds of carbon sources. The OD ₆₀₀ value was measured using a spectrophotometer (spectrophotometer, Eppendorf, Biophotometer) as a method for measuring growth. Yeast extract and sea salt were fixed to the basic medium, and 50 kinds of carbon sources were added to each of 30 g / L to make 50 ml, and then sterilized. The types of carbon source used were fructose (Fructose, C ₆ H ₁₂ O ₆ ), galactose (galactose, C ₆ H ₁₂ O ₆ ), glucose (C ₆ H ₁₂ O ₆ ), lactose (C ₁₂ H ₂₂ O ₁₁₎ + H ₂ O), sucrose (sucrose, C ₁₂ H ₂₂ O ₁₁ + H ₂ O) and the like. First, the strain was incubated for 48 hours in 50 ml of the basic medium, and then inoculated with 5% (v / v) of each basic medium containing 5 ml of 5 different carbon sources and incubated for 144 hours. Growth was measured. To determine the OD ₆₀₀ value, the value measured by diluting the culture 10-fold was used.

11. Growth according to the change of nitrogen source

In culturing the strains, the growth of the strains was compared against the five nitrogen sources. The OD ₆₀₀ value was measured as a method for measuring growth. Glucose and sea salt were added to the basic medium at a constant concentration, and 50 ml of each of the five nitrogen sources were added and sterilized. Nitrogen sources used were yeast extract, peptone, tryptone, malt extract and casamino acd. First, the strain was incubated for 48 hours in 50 ml of the basic medium, and then 5% (v / v) was inoculated into each basic medium containing 50 ml of five different nitrogen sources to grow the strain according to the culture for 144 hours. Was measured. To determine the OD ₆₀₀ value, the value measured by diluting the culture 10-fold was used.

12. Growth with temperature

The growth of the strain according to the various culture temperatures in the culture conditions were investigated. The culture temperature was cultured at 15, 20, 25, 30, 35 ℃, the growth was confirmed by measuring the OD ₆₀₀ value. The strains were incubated for 48 hours in 50 ml of the basal medium and inoculated with 5% (v / v) in 50 ml of the basal medium and incubated for 144 hours. To determine the OD ₆₀₀ value, the value measured by diluting the culture 10-fold was used.

13. Growth by changing pH

The growth was examined by varying the pH of the culture medium in the culture conditions. The pH range of the culture medium was incubated at 4, 5, 6, 7, 8, and the pH was measured every 24 hours according to the culture of the strain. The pH of the initial medium was 4, 5, 6, 7, 8, respectively. Calibration was also done every 24 hours to adjust and maintain pH upon incubation. Growth of the strain was determined by measuring the OD ₆₀₀ value. Strains were incubated for 48 hours in 50 ml of the basal medium and inoculated with 5% (v / v) in 50 ml of the basal medium and measured for 144 hours. To determine the OD ₆₀₀ value, the value measured by diluting the culture 10-fold was used.

result

1. Characteristics of strain

As a result of observing JB511, a DHA producing strain with an optical microscope, as shown in FIGS. 1A and 1B, the diameter of the strain was about 7-12 μl. After culturing for 96 hours in a 50 ml basal medium, the cells were observed to grow and form clusters between the cells with vacuole inside the cells.

In addition, it is presumed to be heterotrophic microorganisms that do not photosynthesize because the growth of the strain does not occur when the light is supplied and cultured in a medium containing only a small amount of nitrogen source without adding a carbon source.

The cultured strains were diluted in sterile distilled water without added salt and observed under a microscope. As a result, the cells were compared with those observed after dilution in sterile distilled water added with 3% salt (Fig. 1A). Easily lysed, the vacuoles inside the cells can be seen to escape (Fig. 1b). Strain JB511 is a strain isolated from seawater that appears to be susceptible to osmotic shock.

2. Identification of Strains

JB511, a DHA-producing strain, was designed to identify the species of the strain through analysis of 18S rDNA. First, 18S rDNA portions were isolated from the genomic DNA of the strain using NSS and NS8, 18S rDNA primers. Then, after purification, the entire sequence was determined by plasmid sequencing using primers inserted into the vector to detect the intermediate portion of 18S rDNA through cloning. The results were confirmed by NCBI blast search. Schizochytrium sp. 99.8% identity with FJU-512 (FIG. 2). The genus Schizochytrium sp. Belongs to the family of Thraustochytriidae and is characterized by multiplication by binary cell division in vegetative cells (Goldstein and Belsky 1964, Yokoyama and Honda 2007).

3. Mutations by UV Mutation

Schzochytrium sp. 7 shows the results of measuring the survival rate of the strain according to the irradiation time of ultraviolet rays in the process of inducing mutations by irradiating the ultraviolet rays to JB511. When 1 × 10 ⁴ cells / ml of the strain was irradiated with 254 nm UV at a distance of 15 cm, the survival rate was 21% and 79% of the strains were killed. Survival by mutation induction by UV was determined to be most appropriate when the number of colonies for replica plating was 21% during the screening of mutant strains. A total of 1049 strains were obtained through the first screening of colonies obtained by exposure to UV light twice. The obtained strains were selected from two strains (CNU402 and CNU507) identically formed in three basic solid media to which 1% doxorubicin was added through the second screening process. The biomass of CNU402 and CNU507 was 5.92 and 7.50 g / L, which was 99.7% and 126.3% compared with the parent strain. As a result of quantifying DHA through gas chromatography for the two strains, the content of DHA was 1.90 and 1.49 g / L, which was 77.6% and 39.3% higher than that of the parent strain.

4. Mutations caused by NTG mutagenesis

Mutant strains obtained by UV mutagenesis were used to induce mutations using NTG to increase the content of DHA. The strain used for NTG mutagenesis was used as the parent strain, CNU402, which showed lower biomass but higher DHA content than the parent strain. Survival measurement according to NTG treatment concentration showed that the number of colonies formed was the best for the first screening when using 0.03 mg / mL NTG, which showed a survival rate of about 11%. As a result, a total of 433 strains were obtained. Of these, 10 strains were obtained through the primary screening process. A secondary screening procedure was performed on these strains to ensure one strain CNUN106. The biomass was measured by cell dry weight for this strain, and the content of DHA was measured by gas chromatography. The biomass and DHA were 14.65 and 3.414 g / L, respectively, when they were incubated at 25 ° C for 7 days in a 50 ml basal medium, showing only an increase in biomass compared to the parent strain. The strain CNUN106 was named JBF-06 in the Skeezokitrium and was deposited with the Agricultural Genetic Resources Center of the National Academy of Agricultural Sciences on November 09, 2009 and was assigned accession number KACC93083P.

5. Effect of culture conditions

The effect of the culture conditions on the JBF-06 was observed. First, after using fructose, galactose, glucose, lactose and sucrose as carbon sources of the medium, growth curves were measured according to the culture time. As a result, the fructose, galactose, and glucose showed similar growth rates when observed at 72 hours of culture. Among them, galactose and glucose showed similar large growth, but there was no significant difference between the three monosaccharide carbon sources. On the other hand, lactose and sucrose showed slow growth. The monosaccharides fructose, galactose and glucose seem to be relatively easy to use strains, but the disaccharides lactose and sucrose seems to be low growth because the strain is difficult to use for growth (Fig. 3).

Growth differences were observed for five types of organic nitrogen sources of yeast extract, peptone, tryptone, malt extract, and casamino acid, and the results are shown in FIG. 4. The growth of strains showed the largest growth in yeast extracts based on 72 hours of culture, and trypton showed the highest growth after yeast extracts and did not show much difference with yeast extracts. On the other hand, when cultured using malt extract showed the lowest growth graph.

As the pH of the culture medium was changed, the growth of the strain was observed while 144 hours of incubation while the pH was adjusted to the initial pH. As a result, when 72 hours incubation was found to show the highest biomass at pH 6, but with a slight difference, did not show a significant difference in the pH 6 ~ 8 range (Fig. 5). On the other hand, the growth of the strain did not occur in the acidity of pH 4. In addition, the pH of the culture solution was measured every 24 hours, and the change in alkaline (about pH 8.3) was observed.

When the strains were observed by culturing at various culture temperatures, the DHA producing strains were found to show large growth at relatively low temperatures (FIG. 6). It was confirmed that the fastest growth was observed when incubated at 25 ℃, the largest growth was seen at the lowest temperature of 15 ℃ but reached the highest growth after 144 hours. On the other hand, the lowest growth was seen at 35 ℃.

Agricultural Biotechnology Research Institute KACC93083P 20091222

Claims (4)

JBF-06 strain (Accession No. KACC93083P) of the genus Schizochytrium sp., With increased biomass and DHA content. A method of producing DHA comprising extracting DHA from JBF-06 strain (Accession No .: KACC93083P) or its culture in Sjozoquitium with increased biomass and DHA content. 3. The method of claim 2, wherein the carbon source is fructose; Galactose; A method for producing DHA comprising culturing the JBF-06 strain of the genus Sjozkitrium in a culture medium containing at least one monosaccharide selected from glucose and containing malt extract or tryptone as a nitrogen source. . The method for producing DHA according to claim 3, wherein the JBF-06 strain of Schizochytrium is incubated at pH 5-8, 15-30 ° C.

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