Streptomyces strain and use thereof in synthesis of phosphatidylserine

Abstract

A Streptomyces strain and the use thereof in the synthesis of phosphatidylserine. A fermentation broth produced by means of fermentation of the Streptomyces strain uses soy lecithin and L-serine as raw materials, and can simultaneously convert phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), etc. in lecithin to efficiently produce phosphatidylserine (PS). The Streptomyces strain is named Streptomyces sp. LN2, and was deposited in the China General Microbiological Culture Collection Center on 01 April 2024 with a deposit number of CGMCC NO. 30230.

Description

A Streptomyces and its application in synthesizing phosphatidylserine Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Streptomyces and application thereof in synthesizing phosphatidylserine. Background Art

[0002] Phospholipids, commonly known as egg cithin, narrowly refer to phosphatidylcholine (PC). Broadly speaking, it is a general term for a mixture of phosphatidylglycerides. Phospholipids can be categorized based on the specific phosphatidyl groups they contain: phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidic acid (PA). Phosphatidylserine is the only phospholipid capable of regulating the state of key cell membrane proteins, playing a vital role in maintaining cell membrane structure and function and activating the metabolic synthesis of various enzymes. Due to its strong lipophilicity, PS can rapidly cross the blood-brain barrier after absorption and enter the brain, soothing vascular smooth muscle cells and increasing blood flow to the brain. It has been hailed as an emerging "smart nutrient" after choline, the "brain gold" DHA. Research has shown that PS can prevent Alzheimer's disease, improve memory, alleviate depression, and enhance children's attention. It has been listed as a new resource food and a functional food, and is therefore widely used in health foods, cosmetics, and the pharmaceutical industry. However, the scarcity of natural PS and the complex extraction process have hindered market demand. Therefore, the production of high-purity, high-quality PS products is of great significance.

[0003] Subsequent research revealed that PS, PC, and PE share similar structures and can be converted into each other, leading to the development of enzymatic methods for the preparation of functional phospholipids. Enzymatic methods for the preparation of phosphatidylserine offer high product quality, ease of operation, and mild process conditions. Therefore, the bio-enzymatic synthesis of functional phospholipids is highly promising and can more easily yield products that are difficult to obtain with chemical catalysis.

[0004] Currently, the bioenzymatic production of PS primarily utilizes phospholipase D (PLD) to catalyze the transphosphatidylserine reaction between lecithin (derived from soybeans, sunflower seeds, eggs, etc.) and L-serine to produce phosphatidylserine. PLD is ubiquitous in plants, animals, and microorganisms, with microbial-derived PLD being the most widely used due to its high catalytic performance and simple purification process. Reported phospholipase D-producing microorganisms include Streptomyces, Corynebacterium, Escherichia coli, Salmonella, Pseudomonas, Vibrio harveyi, Bacillus cereus, Achromobacter, and Aspergillus oryzae. Most reported phospholipase D-producing microorganisms use lecithin as a substrate. However, soybean lecithin, in addition to its main component PC, contains several other important components: PE (content approximately 15%) and PI (content approximately 15%). Despite this, these microorganisms can only catalyze the translipidation reaction of PC to produce PS, making it difficult to obtain high-content PS from lecithin with a lower content. This results in a significant decrease in the utilization rate of the raw material lecithin, thereby increasing production costs. Even if PS is synthesized directly from PC, the reaction costs will still increase due to its high price. Therefore, it is necessary to screen for bacterial strains that can use lecithin as a raw material and simultaneously convert PC and PE into PS, and to provide a safe, environmentally friendly, low-cost, and high-raw material utilization method for producing phosphatidylserine, which is of great significance for the industrial production of phosphatidylserine.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to address the deficiencies of the prior art. The present invention first provides a PS-producing strain screened from a soil sample, a Streptomyces sp. LN2, which was deposited on April 1, 2024 at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO. 30230.

[0007] The present invention also provides application of the Streptomyces in synthesizing phosphatidylserine.

[0008] The application of the present invention uses the fermentation broth of the Streptomyces as a catalyst, soybean lecithin and L-serine as raw materials, and homogeneously mixes them for reaction, thereby simultaneously catalyzing the synthesis of phosphatidylserine from phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the raw materials.

[0009] When the homogeneous mixing is carried out for reaction, the reaction temperature is 40-45° C. and the reaction time is 6-8 hours.

[0010] After the reaction is completed, water is added to the reaction solution to dissolve L-serine, centrifugation is performed to obtain an aqueous phase and a precipitate, the precipitate is washed with water, and centrifugation is performed again to obtain an aqueous phase and a precipitate, the aqueous phases are combined, concentrated by membrane filtration to recover L-serine, and the precipitates are combined and vacuum freeze-dried to obtain a phosphatidylserine product. Beneficial effects:

[0011] The invention uses the fermentation broth of screened Streptomyces as a catalyst and soybean lecithin and L-serine as raw materials, and can simultaneously convert phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) in lecithin to efficiently generate phosphatidylserine (PS), thereby improving the utilization rate of raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings further illustrate the present invention, but the contents in the accompanying drawings do not constitute any limitation to the present invention.

[0013] Figure 1: Colony morphology of the solid culture of the strain Streptomyces sp. LN2 of the present invention;

[0014] FIG2 is a phylogenetic tree diagram of the strain Streptomyces sp. LN2 of the present invention;

[0015] FIG3 is a liquid phase detection diagram of the strain Streptomyces sp. LN2 of the present invention used as a raw material for phosphatidylserine synthesis reaction;

[0016] FIG4 is a liquid phase detection diagram showing the completion of the phosphatidylserine synthesis reaction using the strain Streptomyces sp. LN2 of the present invention.

[0017] Biomaterial deposit information:

[0018] Streptomyces sp. LN2 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 1, 2024, and its deposit number is CGMCC NO.30230. DETAILED DESCRIPTION

[0019] The technical solutions of the present invention are described in detail below in conjunction with specific examples so that those skilled in the art can better understand and implement the technical solutions of the present invention. The reagents or materials used in the examples, unless otherwise specified, were obtained from commercial sources.

[0020] Example 1: Determination of lipid transfection activity of crude fermentation enzyme solution

[0021] (1) Determination by catalyzing PC and L-serine to produce PS:

[0022] An appropriate amount of soybean-derived phosphatidylcholine (PC) was dissolved in 10 mL of chromatographically pure chloroform (final concentration 5 mg / mL) as the organic phase. Separately, 10 mL of the crude fermentation enzyme solution was added to form the aqueous phase with L-serine (final concentration 50 mg / mL), Triton X-100 (v / v, 2.4%), and CaCl2 (final concentration 80 mM). The organic and aqueous phases were mixed in a 1:1 ratio in a conical flask and placed on a constant temperature shaker at 37°C for 1-8 h.

[0023] (2) Liquid phase detection method:

[0024] After the reaction, 500 μl of the reaction solution was centrifuged at 12,000 rpm for 10 min. The upper organic phase was aspirated and evaporated. The organic phase was then dissolved with mobile phase A consisting of n-hexane-isopropanol-acetic acid-triethylamine (820:170:10:0.8, volume ratio) and passed through a 0.45 μm organic membrane for HPLC analysis.

[0025] Chromatographic conditions: Lichrosphere 100 diol (4 mm x 125 mm) column, ELSD evaporative light scattering detector, mobile phase: mobile phase A: n-hexane-isopropanol-acetic acid-triethylamine (820:170:10:0.8, volume ratio), mobile phase B: isopropanol-water-acetic acid-triethylamine (850:140:10:0.8, volume ratio); flow rate: 1 mL / min; measurement temperature: 30°C; injection volume: 10 μL; evaporation temperature: 80°C; nebulization temperature: 50°C; nitrogen flow rate: 1 L / min; gradient elution: performed according to Table 1.

[0026] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of phosphatidylcholine to 1 μmol of phosphatidylserine per minute at 37°C.

[0027] Table 1 Gradient elution program

[0028] Example 2: Screening and identification of Streptomyces sp. LN2

[0029] According to Bergey's Handbook of Bacterial Identification, the strain was first identified as Streptomyces through morphological observation and physiological and biochemical tests. The Streptomyces sp. LN2 strain exhibits typical Streptomyces characteristics: when cultured on solid matter, its hyphae are branched, lack septa, are Gram-positive, and intertwine to form small, dense colonies that are dry, opaque, and difficult to pick. The colonies of this strain reach a diameter of 3-4 mm after 5-7 days. The hyphae are initially white, then become light gray and fuzzy, as shown in Figure 1.

[0030] Subsequently, the 16S rDNA cluster analysis method was used to commission General Biotechnology (Anhui) Co., Ltd. to determine the 16S rDNA sequence of the isolated strain. The reported 16S rDNA sequence of Streptomyces was downloaded from NCBI. After analysis and comparison using the software MEGA10.0, a phylogenetic tree of the strain's 16S rDNA was obtained. The results are shown in Figure 2, showing that the physiological and biochemical results of strain LN2 are consistent with those of Streptomyces and belong to the genus Streptomyces.

[0031] 16SrDNA sequence determination results:

[0032] Specific screening steps of the strain of the present invention are:

[0033] (1) Plate screening: The collected soil samples were processed and cultured at 28°C using lecithin as substrate and inducer and bromocresol purple as color developer. The strains that can produce white transparent circles around the colonies are the strains that can transform lecithin. Then, single colonies with transparent circles were picked and cultured on solid plates for 72 hours before the next step of rescreening.

[0034] The solid culture medium used was: yeast extract 4 g / L, maltodextrin 10 g / L, glucose 4 g / L, agar 15 g / L, and sterilized at 115°C for 20 min.

[0035] (2) Shake flask rescreening: The strains obtained from the initial plate screening were inoculated onto seed culture medium and cultured in shake flasks at 28°C for 5 days to obtain seed culture fluid. The seed culture medium used was: 20 g / L soy peptone, 20 g / L maltodextrin, 4 g / L glucose, 1 g / L K2HP04, 1 g / L MgSO4·7H2O, 0.1 g / L CaCl2, and 6 g / L Na2HPO4·12H2O, sterilized at 115°C for 20 min.

[0036] (3) Fermentation detection of enzyme activity: The obtained seed culture solution was then inoculated into a basic fermentation medium with an inoculation amount of 2% of the volume of the basic fermentation medium. The culture was cultured at 28°C for 48 hours to obtain a fermentation culture solution. The basic fermentation medium used was: soy peptone 30g / L, maltodextrin 20g / L, glucose 7g / L, K2HP041g / L, MgS04·7H2O 1g / L, CaCl20.1g / L, Na2HPO4·12H2O 6g / L, and sterilized at 115°C for 20min. The fermentation culture solution was centrifuged at 6000rpm for 30min to remove the Streptomyces cells, and the supernatant was then ultrafiltered through an ultrafiltration membrane to remove salts and impurities to obtain a crude enzyme solution. The enzyme activity was determined according to the above-mentioned enzyme activity determination method, and strains with high translipidase activity were screened for the preparation of phosphatidylserine. The results screened out a strain with the highest translipidase activity of 58.4 U / ml, and named it Streptomyces sp. LN2.

[0037] Example 3: Bacteria fermentation

[0038] The Streptomyces sp. LN2 strain screened in Example 2 was fermented and expanded under the following conditions:

[0039] (1) Plate culture: Prepare a plate culture medium containing 20 g / L soy peptone, 20 g / L maltodextrin, 4 g / L glucose, 1 g / L K2HP04, 1 g / L MgSO4·7H2O, 0.1 g / L CaCl2, 6 g / L Na2HPO4·12H2O, and 15 g / L agar. Sterilize at 115°C for 20 min. Inoculate Streptomyces sp. LN2 onto the plate culture medium and culture at 28°C for 72 h.

[0040] (2) Primary seed culture: Take a 250 mL conical flask, add 6 glass beads, and prepare 60 mL of culture medium: 20 g / L soy peptone, 20 g / L maltodextrin, 4 g / L glucose, 1 g / L K2HP04, 1 g / L MgSO4·7H2O, 0.1 g / L CaCl2, and 6 g / L Na2HPO4·12H2O. Sterilize at 115°C for 20 min. Several large colonies of Streptomyces sp. LN2 obtained in step (1) were selected and inoculated into the primary seed culture medium. Culture was performed at 200 rpm and 28°C for 48 h.

[0041] (3) Secondary seed culture: Take a 2L conical flask, add 8 glass beads, and prepare 300mL of culture medium: 30g / L soy peptone, 20g / L maltodextrin, 4g / L glucose, 1g / L K2HP04, 1g / L MgSO4·7H2O, 0.1g / L CaCl2, and 6g / L Na2HPO4·12H2O. Sterilize at 115°C for 20min. Add the entire primary seed culture obtained in step (2) and culture at 200rpm, 28°C, for 48h.

[0042] (4) Fermentation broth culture: A 5 L fermentation tank was used to prepare 3 L of fermentation medium: 30 g / L soy peptone, 20 g / L maltodextrin, 7 g / L glucose, 1 g / L K2HP04, 1 g / L MgSO4·7H2O, 0.1 g / L CaCl2, 6 g / L Na2HPO4·12H2O, and 1 mL / L defoamer. The mixture was sterilized at 115°C for 20 min. The secondary seed culture obtained in step (3) was inoculated into the fermentation medium. The initial stirring speed was 200 rpm, the ventilation volume was 1 vvm, the temperature was 28°C, and the culture time was 48 h to obtain a fermentation culture medium.

[0043] (5) Centrifugal ultrafiltration to obtain crude enzyme solution: The fermentation culture obtained in step (4) is allowed to stand for a period of time, and then centrifuged at 6000 rpm for 30 min to remove Streptomyces sp. LN2 bacteria, and the supernatant is then ultrafiltered through an ultrafiltration membrane to remove salts and foreign proteins to obtain a crude enzyme solution.

[0044] Example 4: Preparation of phosphatidylserine

[0045] Take an appropriate amount of the crude fermentation enzyme solution from Example 3 above, prepare 270 ml of an enzyme-containing aqueous solution with an enzyme activity of 10 U / mL, and adjust the pH to 7.0; add 50 g of soybean lecithin raw material (phosphatidylcholine content of 44.33%) and 40 g of L-serine to the enzyme-containing aqueous solution, stir homogenously, and react at 40-45°C for 6-8 hours;

[0046] After the reaction, 500 μl of the reaction solution was centrifuged at 12,000 rpm for 10 min. The upper organic phase was aspirated and evaporated. The organic phase was then dissolved with mobile phase A consisting of n-hexane-isopropanol-acetic acid-triethylamine (820:170:10:0.8, volume ratio) and passed through a 0.45 μm organic membrane for HPLC analysis.

[0047] Next, 270 ml of water was added to the reaction solution, and the serine was dissolved by stirring. The aqueous phase was centrifuged to obtain 90 g of a centrifugal precipitate. The centrifugal precipitate was washed with 450 ml of pure water, and the aqueous phase was centrifuged. Approximately 990 ml of the aqueous phase separated by the two centrifugations was filtered and concentrated to 150 ml using a reverse osmosis membrane to obtain an L-serine aqueous solution for the next batch reaction. 90 g of the L-serine aqueous solution was obtained by the second centrifugation. The two centrifugal precipitates were vacuum freeze-dried to obtain 43 g of a product containing high-purity phosphatidylserine.

[0048] The reaction raw material soybean lecithin and the liquid phase detection results at the end of the reaction are shown in Figures 3-4 below: the substances detected in the reaction raw materials in Figure 3 are, according to the peak time, PA retention time 4.553min, PE retention time 8.012min, PC retention time 8.755min, and PI retention time 10.873min; the substances detected at the end of the reaction in Figure 4 are, according to the peak time, PA retention time 4.276min, PE retention time 7.879min, PC retention time 8.599min, the new peak, i.e., the product PS retention time 10.192min, and PI retention time 10.715min; the percentage content of each component in the reactant is shown in Table 2. The results show that the Streptomyces sp. LN2 of the present invention can not only convert the main component PC in the substrate lecithin into PS, but also significantly reduce the content of PE and PI, indicating that the Streptomyces sp. LN2 can convert the main component PC in the substrate lecithin into PS, and the content of PE and PI also decreases significantly, indicating that the Streptomyces sp. sp.LN2 also has the activity of converting PE and PI to PS, making full use of the raw materials, and the PC conversion rate is as high as 109.77%.

[0049] Table 2 Content of each component in the reactants

[0050] Note: PC conversion rate = product PS content ÷ raw material PC content x 100%

[0051] Example 5: Verification of the activity of Streptomyces sp. LN2 in converting PE to PS

[0052] Take an appropriate amount of phosphatidylethanolamine (PE) dissolved in 10 mL of chromatographic-grade chloroform (final concentration 5 mg / mL) as the organic phase; take 10 mL of the crude fermentation enzyme solution, add L-serine (final concentration 50 mg / mL), Triton X-100 (v / v, 2.4%), and CaCl2 (final concentration 80 mM) to it as the aqueous phase; mix the organic phase and the aqueous phase in a 1:1 ratio in a conical flask, place it on a constant temperature shaking platform at 37°C and shake for 1-8 hours.

[0053] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of phosphatidylethanolamine to 1 μmol of phosphatidylserine per minute at 37°C.

[0054] The phosphatidylserine content was determined according to the sample preparation method and HPLC-ELSD detection method in Example 1, and the enzyme activity was calculated. The result showed that the activity of Streptomyces sp. LN2 in converting PE to PS was 33.7 U / ml.

[0055] Example 6: Verification of the activity of Streptomyces sp. LN2 in converting PI to PS

[0056] Take an appropriate amount of phosphatidylinositol (PI) dissolved in 10 mL of chromatographic grade chloroform (final concentration 5 mg / mL) as the organic phase; take another 10 mL of the fermentation crude enzyme solution, add L-serine (final concentration 50 mg / mL), Triton X-100 (v / v, 2.4%), and CaCl2 (final concentration 80 mM) to it as the aqueous phase; mix the organic phase and the aqueous phase in a 1:1 ratio in a conical flask, place it on a constant temperature shaking shaker at 37°C and react for 1-8 hours.

[0057] Enzyme activity is defined as the amount of enzyme required to catalyze the conversion of phosphatidylinositol to 1 μmol of phosphatidylserine per minute at 37°C.

[0058] The phosphatidylserine content was determined according to the sample preparation method and HPLC-ELSD detection method in Example 1, and the enzyme activity was calculated. The result showed that the activity of Streptomyces sp. LN2 in converting PI to PS was 12.6 U / ml.

[0059] The technical principles of the present invention have been described above with reference to specific embodiments. These descriptions are intended solely to illustrate the principles of the present invention and are not to be construed in any way as limiting the scope of protection of the present invention. Based on the explanations herein, those skilled in the art will be able to devise other specific embodiments of the present invention without inventive effort, and such equivalent variations or substitutions are intended to be encompassed within the scope of the claims of this application.

Claims

1. A Streptomyces sp., characterized in that The deposit number of the Streptomyces sp. LN2 is CGMCC NO. 30230.

2. Use of the Streptomyces according to claim 1 in synthesizing phosphatidylserine.

3. The use according to claim 2, characterized in that The fermentation liquid of the streptomyces is used as a catalyst, soybean lecithin and L-serine are used as raw materials, and the mixture is homogeneously mixed to react, thereby simultaneously catalyzing the phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the raw materials to synthesize phosphatidylserine.

4. The use according to claim 3, characterized in that: When the mixture is homogeneously mixed and reacted, the reaction temperature is 40° C. to 45° C., and the reaction time is 6 to 8 hours.

5. The use according to claim 3, characterized in that After the reaction is completed, water is added to the reaction solution to dissolve L-serine, and the mixture is centrifuged to obtain an aqueous phase and a precipitate. The precipitate is washed with water, and centrifuged again to obtain an aqueous phase and a precipitate. The aqueous phases are combined and concentrated by membrane filtration to recover L-serine. The combined precipitates are vacuum freeze-dried to obtain a phosphatidylserine product.

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