Cordyceps militaris strain capable of metabolizing xylose and culture method for high yield of cordycepin

Abstract

The present application belongs to the field of biotechnology. Specifically relates to a kind of high-efficiency metabolism xylose Cordyceps militaris (Fr. Cordyceps militaris ​ (L.) Fr.) 20200930147, which is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC No: M 2021415. The present application discloses a method for obtaining high-yield cordycepin by liquid standing culture using Cordyceps militaris strain 20200930147 as carbon source, combined with the optimized combination of amino acids, other nitrogen sources and betaine as culture medium. Under the preferred conditions of 25℃ culture for 25 days, the strain can synthesize 5.30 g / L cordycepin. The present application realizes the low-cost and high-efficiency production of cordycepin by Cordyceps militaris, and the produced cordycepin has important application value and broad market prospect in the fields of medicine and health care, functional food and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a Cordyceps militaris strain capable of efficiently metabolizing xylose and a method for cultivating it to synthesize cordycepin at high levels. Background Technology

[0002] Cordyceps militaris ( Cordyceps militaris (L.)Fr.), also known as North Cordyceps, is taxonomically classified under the subphylum Asomycotina, family Claviticpitaceae, and genus Cordyceps. Cordyceps Cordyceps militaris, a type of fungus, is one of my country's famous traditional Chinese medicinal materials. Through artificial cultivation or mycelial fermentation, Cordycepin, a fungus, can produce various physiologically active substances such as cordycepin, cordycepic acid, polysaccharides, ergosterol, γ-aminobutyric acid, and vitamins. The synthesis level of cordycepin is significantly higher than that of other Cordyceps species or filamentous fungi. Cordycepin, also known as 3'-deoxyadenosine, is composed of adenosine and deoxypentose, with the molecular formula C1. 10 H 13 N5O3 was the first nucleoside antibiotic obtained from fungi. Besides its antibacterial and antiviral activities, cordycepin has been shown to inhibit mRNA polyadenylation, regulate various cellular processes, and exhibit anticancer, anti-inflammatory, and skin-aging-improving pharmacological effects, making it valuable for applications in biomedicine, functional foods, health products, and cosmetics. The chemical synthesis of cordycepin suffers from drawbacks such as complex processes, low efficiency, and high cost. Biosynthesis of cordycepin using superior strains of Cordyceps militaris is an important pathway to achieve the large-scale preparation of this drug.

[0003] The level of cordycepin synthesis in Cordyceps militaris fermentation is influenced by various factors, including strain type, culture medium nutrients, and culture method. Xylose exists in nature as a macromolecular xylan, and is the second most abundant sugar after glucose (a component of cellulose). Converting xylose into high-value-added products through biotechnology is of great significance. Studies have shown that xylose is an ideal carbon source for cordycepin synthesis in Cordyceps militaris, but reported Cordyceps militaris strains have low xylose utilization levels, resulting in low total cordycepin synthesis. Wongsa et al. (2020) reported that Cordyceps militaris strain TBRC7358 could utilize 6 g / L of xylose, achieving a biomass of 41.7% of the biomass obtained from glucose fermentation, with a cordycepin synthesis level per unit biomass twice that of glucose fermentation. Other nutrients in the culture medium, such as nitrogen source type and amino acid addition, also significantly affect cordycepin synthesis in Cordyceps militaris. Furthermore, compared to solid-state fermentation and liquid shaking fermentation, liquid static fermentation is more conducive to high-level cordycepin synthesis in Cordyceps militaris. Taking into account various factors such as strain, nutrient source, and fermentation method, it is possible to further improve the level of cordycepin fermentation. Currently, the reported resources of Cordyceps militaris strains capable of synthesizing cordycepin at high levels are still insufficient. Screening for superior Cordyceps militaris strains and establishing efficient cordycepin fermentation cultivation methods are in line with the growing market demand for cordycepin. Summary of the Invention

[0004] Objective of the Invention: Addressing the current shortage of high-yield cordycepin-producing Cordyceps militaris strains in the market, this invention aims to provide a Cordyceps militaris strain 20200930147 capable of efficiently metabolizing xylose. Another objective of this invention is to provide a cultivation method for high-level cordycepin synthesis using the aforementioned Cordyceps militaris strain 20200930147, thereby achieving low-cost and high-efficiency cordycepin preparation.

[0005] Technical solution: To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] A strain of Cordyceps militaris, 20200930147, is deposited at the China Center for Type Culture Collection (CCTCC) on April 19, 2021. The deposit address is: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province (opposite to the First Affiliated Primary School of Wuhan University). The accession number is CCTCC No: M 2021415.

[0007] The internal transcribed spacer (ITS) sequence of the Cordyceps militaris strain 20200930147 is shown in SEQ ID NO.1.

[0008] The growth temperature range of the above-mentioned Cordyceps militaris strain is 4℃~32℃.

[0009] The optimal growth temperature for the above-mentioned Cordyceps militaris strain is 23℃~25℃.

[0010] Among them, the carbon sources that the above-mentioned Cordyceps militaris strains can utilize include glucose, xylose, arabinose, fructose, sucrose, maltose, galactose and lactose.

[0011] Among them, the amount of xylose utilized by the above-mentioned Cordyceps militaris strain reached 60 g / L under liquid static culture.

[0012] The above-mentioned method for cultivating high-yield cordycepin from Cordyceps militaris strains includes the following steps:

[0013] 1) Take Cordyceps militaris strain 20200930147 and transfer it to potato dextrose agar (PDA) slant medium. Culture it at 25°C in the dark for 5 days to obtain the slant mother culture.

[0014] 2) Take 5-6 pieces of Cordyceps militaris mycelial culture and inoculate them into Sabouraud II medium, with a medium volume of 20%-50%, and add 8-12 glass beads with a diameter of 4 mm. Incubate at 28℃ and 200 r / min for 4 days to obtain seed culture.

[0015] 3) Take 5 mL of seed culture and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of liquid fermentation medium. Place it in a 25℃ incubator and incubate statically for 25 days. Avoid disturbing the mycelium during the fermentation process.

[0016] 4) After fermentation, the culture medium and mycelium were separated by filtration through nylon cloth. The culture medium was transferred to a new centrifuge tube, and the cordycepin content was determined by high performance liquid chromatography (HPLC). The mycelium was washed to remove residual culture medium from its surface, dried, and its dry weight was determined. The fermentation supernatant was stored at 4 ℃ in the dark, and the mycelium was stored in a desiccator.

[0017] The PDA culture medium, by weight, comprises: 20% peeled potato, 2% glucose, 2% agar, and natural pH.

[0018] The Sabouraud II culture medium, by weight, comprises: 0.5% maltose, 2% glucose, 1% peptone, 0.5% yeast extract, 0.1% K2HPO4, 0.1% KCl, 0.1% MgSO4·7H2O, and pH 6.0.

[0019] The liquid fermentation medium, by weight, comprises: 4% xylose, 1% peptone, 0.5% yeast extract, 0.2% adenosine, 0.2% arginine, 0.25% urea, 0.1% K₂HPO₄, 0.1% KCl, 0.1% MgSO₄·7H₂O, and pH 6. The medium is sealed with gauze, then wrapped in aluminum foil, and sterilized at 115°C for 30 min. A 20% xylose stock solution is prepared, sterilized separately at 115°C for 30 min, and added to the medium according to the specified ratio before use.

[0020] The HPLC method for determining the cordycepin content in the fermentation broth is as follows: 1 mL of fermentation broth is centrifuged at 10,000 r / min for 20 min, the supernatant is collected, filtered through a 0.22 μm filter membrane, and the cordycepin content in the sample is determined by high performance liquid chromatography (HPLC). The analytical column is a C18 reversed-phase column (100 mm × 4.6 mm), the mobile phase is 15% methanol and 85% water, the column temperature is 30℃, the flow rate is 1 mL / min, and the detection wavelength is 260 nm.

[0021] The cordycepin content in the fermentation broth can reach 5.30 g / L.

[0022] The specific primers used to amplify the ITS gene sequence of the Cordyceps militaris strain include the following two sequences:

[0023] Upstream primer: 5′-tccgtaggtgaacctgcgg-3′;

[0024] Downstream primer: 5′-tcctccgcttattgatatgc-3′.

[0025] Beneficial Effects: This invention has the following advantages: A novel Cordyceps militaris strain was isolated, which can normally metabolize xylose to accumulate biomass. This strain can synthesize cordycepin at high levels in an optimized xylose-containing medium via liquid surface culture. This invention achieves low-cost and high-efficiency preparation of cordycepin, which is of great value in promoting the large-scale production of cordycepin. Attached Figure Description

[0026] Figure 1 This is a morphological image of the wild fruiting body of Cordyceps militaris strain 20200930147.

[0027] Figure 2 This is a phylogenetic tree of Cordyceps militaris 20200930147 and related fungi constructed based on ITS sequences.

[0028] Figure 3 This is a graph showing the results of Cordyceps militaris strain 20200930147's utilization of different carbon sources.

[0029] Figure 4 This is a graph showing the results of glucose, xylose, and arabinose utilization by Cordyceps militaris strain 20200930147.

[0030] Figure 5 The figure shows the effects of different carbon sources on cordycepin synthesis and biomass of Cordyceps militaris strain 20200930147.

[0031] Figure 6 This figure shows the effects of adding different nitrogen sources on the synthesis of cordycepin and biomass of Cordyceps militaris strain 20200930147.

[0032] Figure 7 The figure shows the effects of adding different amino acids on the synthesis of cordycepin and biomass of Cordyceps militaris strain 20200930147.

[0033] Figure 8 The figure shows the effects of adding different concentrations of betaine on the synthesis of cordycepin and biomass of Cordyceps militaris strain 20200930147.

[0034] Figure 9 The graph shows the results of cordycepin synthesis by Cordyceps militaris strain 20200930147 in an optimized culture medium. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention will be further described below with reference to examples, but these examples are not intended to limit the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] The materials and reagents used in the following examples are as follows:

[0039] PCR reagents and plant genome extraction kits were purchased from TransGenBiotech. D-xylose, D-glucose, L-arabinose, sucrose, etc., were all domestically produced analytical grade reagents (available from general biochemical reagent companies).

[0040] PDA medium: 200 g potato (boiled juice), glucose, 15 g agar powder, add distilled water to 1 L.

[0041] Sabouraud 2 medium: 20 g glucose, 5 g maltose, 10 g peptone, 5 g yeast extract, 1 g K2HPO4, 1 g KCl, 1 g MgSO4·7H2O, pH 6.0, add distilled water to 1 L.

[0042] Basic fermentation medium: 40 g glucose, 10 g peptone, 5 g yeast extract, 1 g K2HPO4, 1 g KCl, 1 g MgSO4·7H2O, 2 g adenosine, pH 6.0, add distilled water to 1 L.

[0043] Example 1: Isolation, Identification, and Biological Characteristics of Cordyceps militaris 20200930147

[0044] On October 3, 2020, a wild Cordyceps militaris fruiting body was collected in a bamboo forest in Leigongshan Nature Reserve, Rongjiang County, Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province (longitude 108.22, latitude 26.38, altitude 1750.76). (See...) Figure 1 The sample was brought back to the laboratory for Cordyceps militaris fungal isolation on PDA medium, yielding a pure isolate numbered 20200930147. The strain 20200930147 was expanded into cells on PDA plates, and total DNA was extracted using a plant genome extraction kit (TransGen, Beijing). The ITS sequence of the strain was obtained by conventional PCR using primer pair ITS1 / ITS4.

[0045] ITS1: 5′-tccgtaggtgaacctgcgg-3′

[0046] ITS4: 5′-tcctccgcttattgatatgc-3′

[0047] The amplified target DNA was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the obtained sequence was compared with the NCBI online BLAST (http: / / www.ncbi.nlm.nih.gov / blast) sequence. The ITS sequence of strain 20200930147 (see SEQ ID NO.1 in the sequence listing) was compared with that in the NCBI gene database. C. militaris (L.)Fr.) exhibits 99.25% sequence identity. A phylogenetic tree was constructed using MEGA 7.0 software with the neighbor-to-neighbor method, and analysis confirmed its similarity to Cordyceps militaris. C. militaris (L.)Fr.) belongs to the same evolutionary branch (see Figure 2 The taxonomic classification of strain 20200930147 was determined to be Cordyceps militaris. C. militaris (L.)Fr.).

[0048] Cordyceps militaris 20200930147 was inoculated onto PDA plates and cultured in the dark at 25 °C for 7 days. Colony growth was observed. On PDA plates, the strain exhibited radial growth of white hyphae, forming loose, nearly circular colonies. Further culture under light resulted in yellow colonies. Microscopic observation revealed that the sporulating cells on the hyphae were solitary or clustered, resembling Penicillium. Conidia were oval or columnar.

[0049] Example 2: Utilization of Cordyceps militaris 20200930147 by different carbon sources

[0050] Fermentation media with different carbon sources were prepared by replacing 40 g / L glucose in the basal fermentation medium with 60 g / L xylose (D-type), glucose (D-type), arabinose (L-type), fructose, galactose, maltose, sucrose, or lactose, and a fermentation medium without added carbon source was also prepared. Cordyceps militaris strain 20200930147 was transferred to PDA slant medium and cultured at 25℃ in the dark for 5 days to obtain the slant mother culture. Cordyceps militaris mycelial blocks (3-5 pieces, 0.5 × 0.5 cm) were inoculated into 250-mL Erlenmeyer flasks containing 100 mL Sabouraud dextrose medium (containing 10 glass beads with a diameter of 4 mm) and cultured at 28℃ and 200 r / min with shaking for 4 days to obtain the bacterial suspension. 2 mL of the bacterial suspension was inoculated into 50 mL of fermentation medium (in 250-mL Erlenmeyer flasks) with different carbon sources and cultured at 25℃ and 150 r / min for 10 days. After fermentation, mycelia were collected by filtration and dried in a 60℃ oven until constant weight. The biomass was then measured using an electronic balance. The results are as follows: Figure 3 As shown, this Cordyceps militaris can utilize all carbon sources for growth. The highest biomass was obtained from glucose, fructose, galactose, and sucrose, reaching 22.2 g / L - 23.8 g / L; the next highest biomass was obtained from maltose and xylose, at 21.1 g / L and 20.3 g / L, respectively; the lowest biomass was obtained from arabinose and lactose, at 16.3 g / L and 13.0 g / L, respectively; and the control group obtained a biomass of 7.4 g / L.

[0051] Meanwhile, the utilization of glucose, xylose, and arabinose by Cordyceps militaris was analyzed. Under the above fermentation conditions, 1 mL of fermentation broth was collected every 2 days, centrifuged at 10,000 r / min for 20 min, filtered (0.22 µm), and the content of the corresponding monosaccharides in the samples was analyzed by high performance liquid chromatography (HPLC) according to the literature (Jiang K., Li L., Long L., Ding S. Comprehensive evaluation of combining hydrothermal pretreatment (autohydrolysis) with enzymatic hydrolysis for efficient release of monosaccharides and ferulic acid from corn bran, Industrial Crops and Products, 2018, 113, 348-357.). The results are as follows. Figure 4 As shown, this *Cordyceps militaris* strain can rapidly utilize glucose and xylose. On day 8, the residual glucose and xylose levels were 2.6 g / L and 11.1 g / L, respectively; by day 10, both sugars (initially 60 g / L) had been fully utilized. The strain's ability to utilize arabinose was relatively weak; after 10 days of culture, it utilized 37.9 g / L of arabinose. Therefore, *Cordyceps militaris* 20200930147 exhibits excellent xylose metabolism capabilities.

[0052] Example 3: Effect of carbon source on cordycepin production by liquid static fermentation of Cordyceps militaris 20200930147

[0053] The seed culture of Cordyceps militaris was prepared in the same manner as in Example 2. Fermentation media with different carbon sources were prepared by replacing 40 g / L glucose in the basal fermentation medium with 40 g / L xylose (D-type), glucose (D-type), arabinose (L-type), fructose, galactose, maltose, sucrose, or lactose, and a fermentation medium without added carbon source was also prepared. 5 mL of seed culture was inoculated into 100 mL of liquid fermentation medium with different carbon sources (in 250-mL Erlenmeyer flasks, sealed with 8 layers of gauze and covered with a layer of aluminum foil, the same applies below), and placed in a 25℃ incubator for static incubation for 25 days. After fermentation, the mycelia were collected by filtration and dried in a 60℃ oven until constant weight. The weight of the mycelia was measured using an electronic balance to obtain the biomass. Meanwhile, 1 mL of fermentation supernatant was taken and centrifuged at 10,000 r / min for 20 min. The supernatant was filtered through a filter membrane, and the cordycepin content was determined using an Agilent 1260 high performance liquid chromatography (HPLC) system (Agilent Technologies, USA). The analytical column was a C18 reversed-phase column (100 mm × 4.6 mm), the mobile phase was 15% methanol and 85% water, the column temperature was 30℃, the flow rate was 1 mL / min, and the detection wavelength was 260 nm.

[0054] The results are as follows Figure 5 As shown, *Cordyceps militaris* produced the highest biomass (18.8 g / L) using glucose as a carbon source, and the lowest (13.0 g / L) using xylose. Biomass from other carbon sources ranged from 14.9 g / L to 18.8 g / L. This strain produced the highest level of cordycepin (3.08 g / L) using xylose as a carbon source; cordycepin production from other carbon sources ranged from 1.72 g / L to 2.22 g / L, with glucose producing 1.85 g / L. Therefore, xylose is the optimal carbon source for cordycepin synthesis in *Cordyceps militaris* 20200930147.

[0055] Example 4: Effect of nitrogen source on cordycepin production during liquid static fermentation of Cordyceps militaris 20200930147

[0056] The seed culture of *Cordyceps militaris* was prepared according to the method described in Example 2. Simultaneously, fermentation media containing different nitrogen sources were prepared, namely, replacing glucose in the basal fermentation medium with an equal mass concentration of xylose, and adding 0.5% ammonium sulfate, 0.25% urea, 0.5% ammonium citrate, or 0.5% sodium nitrate. A control group without additional nitrogen sources was also set up. Urea was prepared as a 25 g / L solution, filtered and sterilized, and added separately. 5 mL of the seed culture was inoculated into 100 mL of fermentation medium containing different nitrogen sources and cultured statically in the dark at 25°C for 20 days. After fermentation, the mycelia were collected by filtration and dried at 60°C to constant weight, then weighed. As in Example 3, the cordycepin content in each fermentation supernatant was determined by HPLC. The results are as follows: Figure 6 As shown, the addition of ammonium sulfate, urea, or ammonium citrate had no significant effect on the biomass of Cordyceps militaris, but all of them could increase the yield of cordycepin. The cordycepin yield in the urea-treated group reached 4.7 g / L, while that in the control group was 3.2 g / L, an increase of 46.9%. The addition of ammonium nitrate greatly inhibited mycelial growth and cordycepin synthesis.

[0057] Example 5: Effect of amino acids on cordycepin production from liquid static fermentation of Cordyceps militaris 20200930147

[0058] The seed culture of *Cordyceps militaris* was prepared according to the method described in Example 2. Simultaneously, fermentation media containing different amino acids were prepared by replacing glucose in the basal fermentation medium with an equal mass concentration of xylose, and adding 0.2% of either methionine, tyrosine, aspartic acid, glycine, alanine, arginine, histidine, or threonine. A control group without added amino acids was also included. Five mL of the seed culture was inoculated into 100 mL of fermentation media containing different amino acids and cultured statically in the dark at 25°C for 25 days. After fermentation, the mycelia were collected by filtration and dried at 60°C to constant weight, then weighed. As in Example 3, the cordycepin content in each fermentation supernatant was determined by HPLC. The results are as follows: Figure 7 As shown, except for arginine and histidine, the addition of other amino acids can increase the biomass of Cordyceps militaris to varying degrees; all treatments with added amino acids can increase the yield of cordycepin, with the cordycepin yields of the treatments with added aspartic acid, glycine, arginine and histidine being 4.5 g / L, 4.3 g / L, 4.1 g / L and 4.1 g / L, respectively, compared to 3.3 g / L in the control group.

[0059] Example 6: Effect of betaine on cordycepin production from liquid static fermentation of Cordyceps militaris 20200930147

[0060] The seed culture of *Cordyceps militaris* was prepared according to the method described in Example 2. Simultaneously, fermentation media containing different concentrations of betaine were prepared; that is, glucose in the basal fermentation medium was replaced with an equal mass concentration of xylose, and betaine was added to a final concentration of 0.1%, 0.2%, 0.3%, 0.4%, 1.0%, 5.0%, or 10%. A control group without added betaine was also included. 5 mL of the seed culture was inoculated into 100 mL of the above fermentation medium and cultured statically in the dark at 25°C for 25 days. After fermentation, the mycelia were collected by filtration and dried at 60°C to constant weight, then weighed. As in Example 3, the cordycepin content in each fermentation supernatant was determined by HPLC. The results are as follows: Figure 8As shown, the addition of 0.2% to 1.0% betaine can increase the synthesis of cordycepin, with the highest cordycepin yield (3.45 g / L) in the 1.0% betaine treatment group, which is 10% higher than the control group (3.08 g / L). At the same time, the addition of 1.0% betaine can promote mycelial growth, and its biomass is increased by 25.6% compared with the control group.

[0061] Example 7: Cordycepin Production from Liquid Static Fermentation of Cordyceps militaris 20200930147 under Optimized Conditions

[0062] Based on the results of Examples 3 to 6, the cordycepin fermentation medium was optimized. Its formula was: xylose 40 g, peptone 10 g, yeast extract 5 g, K₂HPO₄ 1 g, KCl 1 g, MgSO₄·7H₂O 1 g, urea 2.5 g, arginine 2 g, betaine 10 g, adenosine 2 g, pH 6.0, with distilled water added to 1 L. The urea was prepared as a 25 g / L solution, filtered and sterilized, and then added to the medium in the specified proportion. The seed culture of *Cordyceps militaris* was prepared according to the method in Example 2. 5 mL of the seed culture was inoculated into 100 mL of the above fermentation medium and cultured statically in the dark at 25°C for 25 days. During fermentation, 1 mL of fermentation broth was collected every 5 days, centrifuged to remove mycelia, and then frozen at -20°C. After fermentation, the mycelia were collected by filtration and dried at 60°C to constant weight, then weighed. Same as in Example 3, the cordycepin content in the fermentation supernatant was determined by HPLC. The results are as follows: Figure 9 As shown, cordycepin was detected at 0.09 g / L on day 5 of fermentation. Subsequently, cordycepin accumulated rapidly in the fermentation broth, reaching 3.07 g / L on day 15. The increase then slowed slightly, followed by a rapid increase after day 20, reaching a maximum of 5.30 g / L on day 25. With continued cultivation, the cordycepin yield showed a decreasing trend. After 30 days of fermentation, the mycelial biomass was 14.5 g / L.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Claim 1 claims protection for a strain of Cordyceps militaris ( Cordyceps militaris )20200930147, characterized in that, It is deposited at the China Center for Type Culture Collection on April 26, 2021, with accession number CCTCC No: M2021415.

2. The Cordyceps militaris strain according to claim 1, characterized in that, The growth temperature of the Cordyceps militaris strain is 4℃~32℃.

3. The Cordyceps militaris strain according to claim 1, characterized in that, The optimal growth temperature for the Cordyceps militaris strain is 23℃~25℃.

4. The Cordyceps militaris strain according to claim 1, characterized in that, The Cordyceps militaris strain can grow in a culture medium with one of glucose, xylose, arabinose, fructose, sucrose, galactose, maltose or lactose as a single carbon source.

5. The Cordyceps militaris strain according to claim 1, characterized in that, The Cordyceps militaris strain utilized 60 g / L of xylose under static liquid culture conditions.

6. The application of a Cordyceps militaris strain according to claim 1 in the production of cordycepin.

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