Engineering strain for improving yield of cordycepin of cordyceps militaris based on polygene combination expression and precursor engineering and construction method of engineering strain

Abstract

The invention discloses an engineering strain for improving the yield of cordycepin of cordyceps militaris based on polygene combination expression and precursor engineering and a construction method of the engineering strain, a strong promoter gpdA is adopted for driving, a P2A peptide connection strategy is utilized, and two polygene expression modules are constructed: firstly, cordycepin biosynthesis key genes cns1 and cns2 are overexpressed at the same time, and then the cordycepin biosynthesis key genes cns1 and cns2 are overexpressed at the same time; the terminal catalytic efficiency of the synthetic route is enhanced; and 2, the cns1 and a precursor supply gene nt5e are subjected to combined expression, so that the synergistic enhancement of precursor supply and terminal catalysis is realized. And introducing the expression module into cordyceps militaris through an agrobacterium-mediated method to obtain an engineering strain with genetic stability. A fermentation experiment shows that the cordycepin yield of the cns1-P2A-cns2 engineering strain reaches 186.5 mg / L, which is 2.3 times that of a wild type; and the yield of the cns1-P2A-nt5e engineering strain is further increased to 252.6 mg / L, which is 3.1 times that of a wild type. According to the invention, the induction cost is avoided, the construction is simple, the stability is strong, and a new strategy is provided for industrial efficient production of cordycepin.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an engineered strain and its construction method for improving cordycepin production in Cordyceps militaris based on multi-gene combination expression and precursor engineering. Background Technology

[0002] Cordycepin (3'-deoxyadenosine) is a nucleoside-based bioactive substance derived from Cordyceps militaris. It possesses a wide range of pharmacological effects, including antitumor, immunomodulatory, antiviral, and anti-inflammatory properties, and is widely used in the pharmaceutical, health food, and functional food industries. The biosynthetic pathway of cordycepin has been elucidated, among which... cns1 , cns2 and cns3 Participating in the stepwise conversion of adenosine; while the precursor supply gene nt5e (5'-nucleotidase) also plays a key role in regulating adenosine production. However, the natural yield of cordycepin in Cordyceps militaris is low, mainly due to: (1) cns1 and c ns2 (1) Insufficient expression level; (2) Blocked supply of adenosine precursor; (3) Inability to effectively concentrate metabolic flux to the final product cordycepin. Existing methods to increase cordycepin production include: culture medium optimization, random mutagenesis, heterologous expression, dynamic regulation, etc. However, existing methods have drawbacks such as high induction cost, genetic instability, complex construction, or burden on host metabolism. Therefore, it is urgent to develop a genetic engineering method that is simple to construct, highly stable, and can significantly increase cordycepin production. Currently, there are no literature reports on high expression systems based on the strong promoter gpdA, which simultaneously enhance the cordycepin substrate generation gene. cns1 / cns2 and its precursor pathway genes n t5e Combination expression strategies. Summary of the Invention

[0003] To address the aforementioned problems, the present invention aims to provide an engineered strain and its construction method for improving cordycepin production in Cordyceps militaris based on multi-gene combination expression and precursor engineering. By constructing a gpdA-driven multi-gene expression cassette, the key steps of cordycepin biosynthesis and the precursor supply step are simultaneously enhanced, thereby significantly increasing cordycepin production.

[0004] The technical solution adopted in this invention is: Using this method to construct a strong starter driver cns1 -P2A- cns2 A dual-gene expression module, comprising: a promoter: gpdA (derived from...) Aspergillus nidulans ), multi-gene linkage fragments: cns1 -P2A- cns2Transcription terminator: Tcbh1. This module is used to simultaneously enhance the efficiency of the two-step terminal reaction in cordycepin biosynthesis.

[0005] Furthermore, the cordycepin synthesis gene in Cordyceps militaris was analyzed by RT-qPCR. cns1 and cns2 The expression status of P2A peptides was verified to demonstrate that the combined expression strategy of P2A peptides can be applied to Cordyceps militaris. P2A is a 2A peptide with high cleavage efficiency and has been successfully applied to gene editing of various fungi to increase the yield of secondary metabolites.

[0006] This method is used to construct a strong starter driver. cns1 -P2A- nt5e The precursor enhancement module, the expression module includes: a promoter: gpdA, and a multi-gene linker fragment: cns1 -P2A- nt5e Transcription terminator: Tcbh1. By increasing... nt5e The expression of [agent] can enhance the supply of adenosine precursors and simultaneously enhance [the expression of adenosine precursors]. cns1 Catalytic efficiency is improved, achieving "dual-channel enhancement" in both precursor and synthesis processes.

[0007] Furthermore, the nucleotide sequence of the strong promoter gpdA is shown in SEQ ID NO: 1; the key gene for cordycepin synthesis in Cordyceps militaris. cns1 The nucleotide sequence is shown in SEQ ID NO: 2; the key gene for cordycepin synthesis in Cordyceps militaris. cns2 The nucleotide sequence is shown in SEQ ID NO: 3; the key gene for the synthesis of the cordycepin precursor from Cordyceps militaris. nt5e The nucleotide sequence is shown in SEQ ID NO: 4, and the nucleotide sequence of the P2A peptide is shown in SEQ ID NO: 5.

[0008] Furthermore, the recombinant expression plasmid was constructed using the following method: S1: The plasmid containing pCAMBIA-PgpdA-EGFP-Tcbh1- hyg -PtrpC E. coli were cultured overnight at 37°C in LB, and then the plasmid pCAMBIA-PgpdA-EGFP-Tcbh1-hyg-PtrpC was extracted using a plasmid extraction kit. S2: Amplify the plasmid pCAMBIA-PgpdA-EGFP-Tcbh1- obtained in step S1 using primers Vector-F / Vector-R. hyg -PtrpC, to obtain linearized vector fragments; S3: Using primer P2A- cns1 -F / P2A- cns2 -R、P2A- cns2 -F / P2A- cns2 -R、P2A- nt5e -F / P2A- nt5e -R amplification of Cordyceps militaris strain genomic DNA, obtaining cns1 , cns2 , nt5e Target gene fragment. During primer design, the P2A sequence was used as a homologous arm, resulting in a fragment of the target gene containing the P2A peptide. S4: Combine the linearized carrier from step S2 with the carrier from step S3. cns1 , cns2 , nt5e Gene fragments were ligated using a seamless cloning kit to construct the recombinant plasmid pCAMBIA-PgpdA- cns1 -P2A- cns2 -EGFP-Tcbh1- hyg -PtrpC, pCAMBIA-PgpdA- cns1 -P2A- nt5e -EGFP-Tcbh1- hyg -PtrpC.

[0009] Furthermore, the Cordyceps militaris genetically engineered bacteria were constructed according to the following steps: the recombinant plasmid pCAMBIA-PgpdA- cns1 -P2A- cns2 -EGFP-Tcbh1- hyg -PtrpC, pCAMBIA-PgpdA- cns1 -P2A-- nt5e -EGFP-Tcbh1- hyg -PtrpC is transferred into Agrobacterium to obtain recombinant Agrobacterium; the recombinant Agrobacterium is used to infect the Cordyceps militaris host bacteria to obtain the Cordyceps militaris genetically engineered bacteria.

[0010] Furthermore, the Agrobacterium is Agrobacterium tumefaciens (Gastrointestinal rust) Agrobacteriumtumefaciens ) AGL1 The host fungus is Cordyceps militaris ( Cordycepsmilitaris ).

[0011] Furthermore, the fermentation culture method of Cordyceps militaris genetically engineered bacteria is as follows: Cordyceps militaris genetically engineered bacteria are inoculated into Sabouraud dextrose agar containing 200 μg / mL hygromycin B and cultured at 25℃ in the dark for 7 days. Colonies are then picked and inoculated into fermentation medium and cultured on a shaker at 22–25℃ and 120–180 rpm (preferably 25℃ and 150 rpm). Sabouraud dextrose agar consists of 10 g / L peptone, 5 g / L glucose, and distilled water as the solvent, with a natural pH. If a solid medium is to be prepared, 20 g / L agar powder is added. The fermentation medium consists of 20 g / L glucose, 10 g / L peptone, 0.5 g / L K₂HPO₄·3H₂O, 0.5 g / L KH₂PO₄, 0.5 g / L MgSO₄·7H₂O, and 1 g / L L-glycine, with distilled water as the solvent and a natural pH.

[0012] Compared with existing technologies, the advantages of this invention include: avoiding the induction costs and possible growth inhibition of dynamic regulation systems; gpdA is a stable and strong promoter in fungi, with high expression levels that are unaffected by conditions; it can achieve synergistic enhancement of precursor supply and terminal catalysis; it is flexible in combination and can form a variety of expression modules; it is simple to construct, easy to scale up for production, and suitable for industrialization. Attached Figure Description

[0013] Figure 1 This is a plasmid map after the recombinant plasmid construction in Example 1 was completed.

[0014] Figure 2 The target gene for Example 1 cns1- P2A -cns2 PCR results of Agrobacterium colonies.

[0015] Figure 3 The results of PCR identification of the hygromycin B resistance gene in the recombinant engineered bacteria in Example 1 are shown.

[0016] Figure 4 In Example 2 cns1 Genes and cns2 Gene RT-qPCR results.

[0017] Figure 5 This is a plasmid map after the recombinant plasmid was constructed in Example 3.

[0018] Figure 6 The target gene in Example 3 cns1- P2A -nt5e PCR results of Agrobacterium colonies.

[0019] Figure 7 The results of PCR identification of the hygromycin B resistance gene in the recombinant engineered bacteria in Example 3 are shown.

[0020] Figure 8This is the standard curve of cordycepin in Example 4.

[0021] Figure 9 To implement the plasmid map after the recombinant plasmid construction in Comparative Example 1 was completed.

[0022] Figure 10 To implement the target gene in Comparative Example 1 cns1 PCR results of Agrobacterium colonies.

[0023] Figure 11 To conduct PCR identification of the hygromycin B resistance gene in the recombinant engineered bacteria in Comparative Example 1.

[0024] Figure 12 Wild-type Cordyceps militaris strain, PgpdA- cns1- P2A -cns2 Recombinant strains and PgpdA- cns1- P2A - nt5e Recombinant strains and overexpressed single genes cns1 A bar graph showing the extracellular cordycepin content of recombinant strains. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0026] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0027] (1) Culture medium used for Cordyceps militaris strain Sabouraud medley medium: 10 g / L peptone, 5 g / L glucose, distilled water as solvent, natural pH; if you want to prepare a solid medium, add 20 g / L agar powder.

[0028] Fermentation medium: glucose 20 g / L, peptone 10 g / L, K2HPO4·3H2O 0.5 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.5 g / L, L-glycine 1 g / L, solvent: distilled water, pH: natural.

[0029] (2) Culture medium used for bacterial strains LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, distilled water as solvent, pH 7.0; if you want to prepare a solid medium, add 20 g / L agar powder.

[0030] IM medium: KH₂PO₄ 1.45 g / L, K₂HPO₄ 2.05 g / L, MgSO₄·7H₂O 0.6 g / L, NaCl 0.1 g / L, CaCl₂ 2 0 The following ingredients were added: 0.001 g / L FeSO4, 0.5 g / L NH4NO3, 5 mL / L glycerol, 40 mL / L MES buffer (1 mol / L, pH 5.5), 5 mL / L trace element stock solution, and 2 g / L glucose. The solvent was distilled water. The trace element stock solution consisted of: 0.1 g / L ZnSO4·7H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L H3BO3, and 0.1 g / L Na2MoO4·7H2O. If a solid culture medium was to be prepared, the final glucose concentration was reduced to 1 g / L, and 20 g / L agar powder was added.

[0031] AIM medium: IM medium containing a final concentration of 200 μmol / L acetosyringone.

[0032] Selective medium: IM solid medium containing 200 μg / mL hygromycin B, 100 μg / mL ceftriaxone sodium and 50 μg / mL kanamycin.

[0033] Binary vector pCAMBIA-PgpdA-Tcbh1- hyg -The construction of PtrpC is based on the following reference: Cai Linian. Study on the directed expression of cellulase in marine Aspergillus niger and its salt and heat tolerance mechanism [D]. Zhejiang University, 2021.

[0034] Example 1 Construction cns1 and cns2 Combination expression strains 1. Construction of recombinant expression vectors Genomic DNA extraction from Cordyceps militaris: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] Cordycepsmilitaris (Purchased from Shanghai Preservation Microbiology Co., Ltd.) Inoculated onto solid Sabouraud broth and activated by incubation at 25°C in the dark for 7 days. Then, a 0.5 cm sample was taken. 2Mycelial blocks of various sizes were inoculated into fermentation medium and cultured at 25℃ and 150 rpm for 7 days to obtain a large amount of Cordyceps militaris mycelium. The mycelium was filtered and the surface moisture was blotted dry with filter paper before use. Genomic DNA was extracted from the Cordyceps militaris mycelium using a rapid fungal genomic DNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: B518229-0050). Specifically: ① 200 mg of fresh mycelium was ground into a fine powder using liquid nitrogen and added to a 1.5 mL centrifuge tube. 400 μL of Buffer Digestion and 4 μL of β-mercaptoethanol were added, the mixture was shaken well, and incubated at 65℃ for 1 h until complete cell lysis. ② 200 μL of Buffer PF was added, the mixture was thoroughly mixed by inverting, and the mixture was placed at -20℃ for 5 min. ③ The mixture was centrifuged at 10000 rpm for 5 min at room temperature, and the supernatant (500~550 μL) was transferred to a new 1.5 mL centrifuge tube. ④ Add an equal volume of isopropanol, invert 8 times to mix thoroughly, and let stand at room temperature for 3 min. Centrifuge at 10,000 rpm for 5 min at room temperature and discard the supernatant; ⑤ Add 1 ml of 75% ethanol, invert and rinse for 3 min, centrifuge at 10,000 rpm for 2 min, and discard the supernatant; ⑥ Repeat step ⑤ once; ⑦ Open the lid and invert at room temperature for 10 min until the residual ethanol has completely evaporated; ⑧ Dissolve the obtained DNA in 100 μL TE buffer. The extracted DNA can be used immediately for the next experiment or stored at -20℃.

[0035] Target gene amplification: The wild-type Cordyceps militaris DNA extracted in step (1) was amplified using primers P2A- from Table 1. cns1 -F / P2A- cns1 -R、P2A- cns2 -F / P2A- cns2 -R was amplified by PCR using the PCR reaction system shown in Table 2 to obtain the target gene fragment, namely the genomic DNA of Cordyceps militaris. cns1 Gene (nucleotide sequence shown in SEQ ID NO: 2) and cns2 Gene (nucleotide sequence shown in SEQ ID NO: 3). PCR amplification program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 66℃ annealing for 30 s, 72℃ extension for 1 min 50 s, 3 cycles; 94℃ denaturation for 30 s, 70℃ annealing for 30 s, 72℃ extension for 1 min 50 s, 27 cycles; 72℃ fill-in for 10 min; stored at 4℃.

[0036] Table 1 Primers for cns1 and cns2 gene amplification

[0037] Table 2 PCR amplification system

[0038] pCAMBIA-PgpdA-EGFP-Tcbh1- hyg - PtrpC vector linearization: The plasmid extracted from *E. coli* was amplified by PCR using primers Vector-F and Vector-R (Table 3) according to the PCR reaction system in Table 4. PCR amplification program: 94℃ pre-denaturation for 5 min, cycle 1; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 6 min, cycle 25; 72℃ final extension for 10 min, cycle 1. 1 μL of DpnI enzyme was added to the amplified product, and the mixture was digested in a 37℃ water bath for 2 h to remove the remaining template DNA. The DpnI enzyme was then inactivated by heating in an 80℃ water bath for 15 min. The product was identified by 1% agarose gel electrophoresis, and the length of the bands was used to preliminarily determine whether the sequence amplification was successful. The PCR amplification product was recovered from the gel using a SanPrep column DNA gel extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: B518131). The linearized vector DNA solution was obtained by following the specific operating steps of the gel extraction kit and stored at -20℃.

[0039] Table 3. Vector linearization primers

[0040] Table 4 Linearized PCR Reaction System

[0041] Recombinant expression vectors: Using the pEASY-UniSeamless Cloning and Assembly Kit (Beijing TransGen Biotech Co., Ltd., product catalog number: CU101-02), according to Table 5, vectors containing the P2A homologous arm were... cns1 Genes and cns2 Gene inserts, linearized vectors, and 2xAssembly Mix were gently mixed and incubated at 50°C for 15 minutes. After the reaction, the centrifuge tubes were placed on ice to cool for a few seconds to obtain the recombinant expression vector pCAMBIA-PgpdA- cns1 -P2A- cns2 -EGFP-Tcbh1- hyg -PtrpC, its spectrum, as follows Figure 1 As shown.

[0042] Table 5 Seamless Cloning Reaction System

[0043] 2. Transformation of Escherichia coli DH5α 7 µL of the constructed recombinant expression vector was added to 100 µL of *E. coli* DH5α competent cells and incubated on ice for 30 min. The cells were then heat-shocked in a 42 °C water bath for 30 s, followed by cooling on ice for 2 min. 800 µL of LB broth was added to the competent cells, and the cells were incubated at 37 °C with shaking at 180 rpm for 1 h. The cells were centrifuged at 3500 rpm for 10 min, and the supernatant was discarded on a clean bench, retaining 100 µL of the bacterial suspension. The resuspended suspension was spread onto LB broth (kanamycin concentration 100 μg / mL) and incubated at 37 °C for 24 h. Single colonies were picked and PCR was performed using primers Ide-F / Ide-R (Table 6) for verification. The transformation success was checked according to the reaction system (Table 7).

[0044] Table 6. Colony PCR Primers

[0045] Table 7 Colony PCR Reaction System

[0046] The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; followed by 72℃ fill-in for 10 min. The results of 1% agarose gel electrophoresis can be used to preliminarily determine whether the clone is positive and to verify the success of the transformation.

[0047] Positive E. coli clones were inoculated into LB medium, and plasmid DNA was extracted using the SanPrep column-based plasmid DNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: B518131). The obtained DNA was dissolved in 35 µL ddH2O. The extracted DNA can be used immediately for the next experiment or stored at -20℃.

[0048] 3. Transformation of Agrobacterium tumefaciens Add 5 µL of the extracted plasmid DNA solution to 100 µL of Agrobacterium tumefaciens AGL-1 (purchased from Shanghai Jiqi Biotechnology Co., Ltd.) competent cells, freeze in liquid nitrogen for 8 s, and incubate at 37℃ for 5 min; add 800 µL of LB liquid medium to the competent cells, and incubate at 28℃, 180 rpm, shaking for 1 h; centrifuge at 3500 rpm for 10 min, discard the supernatant on a clean bench, and retain 100 µL of bacterial suspension; resuspend the bacterial suspension and spread it on IM medium containing 25 µg / mL rifampicin and 50 µg / mL kanamycin, incubate at 28℃ for 36 h, pick single colonies, and screen for Agrobacterium containing recombinant plasmids.

[0049] Single colonies were selected and identified by colony PCR using primers Ide-F / Ide-R (Table 6). The PCR reaction system is shown in Table 7. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, cycle 1; 94℃ denaturation for 30 s, 65℃ annealing for 30 s, 72℃ extension for 1 min, cycle 30; 72℃ final extension for 10 min, cycle 1. The success of the recombinant engineered bacteria transformation was determined by the results of 1% agarose gel electrophoresis. (See Table 7 for the results.) Figure 2 As shown, lanes 1, 2, 3, 4, and 5 contain the target gene. cns1 -P2A- cns2 Agrobacterium colony PCR products.

[0050] 4. Agrobacterium tumefaciens-mediated genetic transformation of filamentous fungi Single colonies of Agrobacterium containing the recombinant plasmid were picked and inoculated into 3 mL of LB liquid medium containing 50 µg / mL kanamycin (Kan), and cultured at 28°C with shaking at 180 rpm for 36 h. The culture was then transferred to AIM liquid medium at a volume concentration of 1%, and cultured at 28°C with constant temperature at 180 rpm for 8 h until the bacterial cell concentration reached OD500. 600 =0.80, and Agrobacterium tumefaciens bacterial solution was obtained.

[0051] Wild-type Cordyceps militaris was inoculated into solid Sabouraud dextrose agar and cultured at 25°C in the dark for 7 days (spore suspension preparation generally uses slant or solid culture; liquid culture conditions are not conducive to spore production or result in lower spore concentrations). The solid culture medium was then repeatedly rinsed with sterile water on a clean bench to obtain a Cordyceps militaris mycelial suspension. The mycelium was removed by filtering the suspension through four layers of sterile gauze, yielding a Cordyceps militaris spore suspension. A small amount of the Cordyceps militaris spore suspension was used to determine the spore concentration using a hemocytometer. 200 µL of the 10⁻⁶ spores prepared in step 2 was then... 6 / mL of Cordyceps militaris spore suspension and the prepared OD 600 =0.80 Agrobacterium bacterial suspension and mix them evenly to obtain a mixed bacterial suspension.

[0052] Sterile cellophane was tightly adhered to the surface of Sabouraud dextrose agar, and the mixed bacterial suspension was then evenly spread onto the cellophane surface. After co-culturing at 25°C in the dark for 48 hours, the cellophane was transferred to a new selective medium surface (strain side up, medium thickness approximately 2 mm). Another layer of selective medium was then poured on top to completely cover the cellophane (medium thickness approximately 2 mm). After culturing at 25°C in the dark for 5 days, hyphae that penetrated the upper selective medium were selected as potential positive clones and inoculated onto Sabouraud dextrose agar containing 200 µg / mL hygromycin B. The clones were then incubated at 25°C in the dark for 5 days. Once the hyphae had grown, they were transferred to culture the next generation. A total of five generations were cultured to obtain stably inherited clones containing... cns1 and cns2A recombinant cordyceps militaris hygromycin-resistant strain with the gene (nucleotides shown in SEQ ID NO.2 and SEQ ID NO.3).

[0053] 5. Identification of genes in Cordyceps militaris strains resistant to hygromycin Genomic DNA was extracted from the recombinant Cordyceps militaris hygromycin-resistant strain, and the obtained DNA was dissolved in 100 µL TE buffer. The extracted DNA can be used immediately for the next experiment or stored at -20°C. The extracted genomic DNA of the Cordyceps militaris hygromycin-resistant strain was then used... hyg -F / hyg -R primers were used for PCR verification to check if the transformation was successful. Primers for PCR positive identification are shown in Table 8, and the reaction system is shown in Table 9. The results of 1% agarose gel electrophoresis can be used to determine whether a clone is positive. The results are as follows: Figure 3 As shown, lanes 1-4 are parallel samples.

[0054] Table 8 Primers for Hygromycin Identification

[0055] Table 9 PCR reaction system for hygromycin identification

[0056] PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ final extension for 10 min.

[0057] Example 2 Target Gene cns1 and cns2 RT-qPCR detection of expression levels 1. Cordyceps militaris RNA extraction Wild-type and recombinant strains were inoculated into liquid fermentation medium and cultured at 25°C and 150 rpm for 7 days. Mycelia were collected, and total RNA was extracted using a plant / fungus total RNA extraction kit (Vazyme FastPure UniversalPlant Total RNA Isolation Kit, product number: RC411) according to the manufacturer's instructions. An appropriate amount of RNA was measured for concentration and purity using NanoDrop; the A260 / A280 ratio should be between 1.8 and 2.1.

[0058] 2. cDNA synthesis 500 ng of total RNA was reverse transcribed into cDNA using a reverse transcription kit (such as HiScript IV All-in-One Ultra RT SuperMix, product number: R433). The reaction conditions were performed according to the kit's recommended procedure, and the reaction system is shown in Table 10.

[0059] Table 10 cDNA Synthesis Reaction System

[0060] Reaction procedure: 50℃ for 5 min; 85℃ for 5 s.

[0061] 3. qPCR reaction system and conditions The SYBR Green qPCR system (Vazyme SupRealQ Ultra SYBR qPCR Master Mix, product number: Q713) was used for detection on an ABI QuantStudio 5 instrument. The internal control gene was Cordyceps militaris elongation factor 1-alpha. tef1 (CCM_00809) gene. The primer sequences for the target gene and internal reference gene in the reaction are shown in Table 11, and the reaction system is shown in Table 12.

[0062] Table 11 qPCR primers

[0063] Table 12 qPCR reaction system name Volume (µL) 2x SupRealQ Ultra SYBR qPCR Master Mix 10 -F 0.4 -R 0.4 Recombinant Cordyceps militaris DNA solution 2 <![CDATA[ddH2O]]> 7.2 total 20 qPCR amplification program: 95℃ pre-denaturation for 30s; 95℃ denaturation for 10s, 60℃ annealing for 30s, 40 cycles; melting curve, instrument default settings.

[0064] 4. Data Processing Methods Each experiment was repeated three times, using 2 −ΔΔCt The method was used to calculate the relative expression level of mRNA. The results are as follows: Figure 4 As shown, when expressed in common, cns1 and cns2 Expression levels were significantly increased in all gene types, with the cns1 gene expression level increasing by 4.2-fold. cns2 Gene expression levels increased by 2.1-fold. This demonstrates that P2A can be applied to the multi-gene expression of Cordyceps militaris.

[0065] Example 3: Construction using precursor engineering strategies cns1 and nt5e Combination expression strains Using primer P2A- from Table 13 nt5e -F / P2A- nt5e -R amplification yielded Cordyceps militaris genomic DNA nt5e Gene (SEQ ID NO: 4), linearized binary vector pCAMBIA-PgpdA-Tcbh1- using primers Vector-F / Vector-R (Table 3). hyg -PtrpC, using the pEASY-UniSeamless Cloning and Assembly Kit (Beijing TransGen Biotech Co., Ltd., product catalog number: CU101-02), linked the target gene fragment with the linearized vector to obtain the recombinant vector pCAMBIA-gpdA- cns1 -p2A- nt5e -EGFP, plasmid map can be found here. Figure 5 As shown.

[0066] Table 13 Primers for nt5e gene amplification Primers Primer sequence 5'to3' P2A--F aaagcaagcaggagacgTGGAAGAAAACCCCGGTCCTATGAAGACTGCTGCTACTCTC P2A--R gaagtgggaaagctcgaaCTAAATCGCCAAAGCCATG Other operations are the same as in Example 1, to obtain expression containing the constitutive promoter. cns1 and nt5e The recombinant Cordyceps militaris engineered strain was verified by PCR using primers Ide-F / Ide-R (Table 6) to check the success of the transformation. The results are as follows: Figure 6 As shown in Table 8, the genomic DNA of the extracted Cordyceps militaris hygromycin-resistant strain was taken and processed using the primers listed in Table 8. hyg -F / hyg -R is used for PCR verification to check whether the transformation was successful. The results are as follows: Figure 7 As shown in lanes 1-4, preserve the transformants that have a positive identification result.

[0067] Example 4: Fermentation of engineered strains and detection of cordycepin 1. Strains culture Pick 0.1cm 2 Wild-type Cordyceps militaris mycelium blocks were inoculated into fermentation medium and cultured at 25°C and 150 rpm for 3 days to prepare seed culture. The recombinant Cordyceps militaris hygromycin-resistant strains prepared in Examples 1 and 3 were inoculated into Sabouraud dextrose agar containing 200 μg / mL hygromycin B and cultured at 25°C in the dark for 5 days. 0.1 cm samples were then picked. 2 The bacterial blocks were inoculated into fermentation medium and cultured in a constant temperature shaker at 25℃ and 150 rpm for 3 days to prepare seed culture. The seed culture of wild-type strain and recombinant strain was transferred to a new fermentation medium at 3% (V / V) and cultured in a constant temperature shaker at 25℃ and 150 rpm for 7 days to obtain fermentation broth. The broth was centrifuged at 8000 rpm for 10 min, and the supernatant and precipitate were collected.

[0068] 2. Cordycepin standard curve Accurately pipette 1 μL, 5 μL, 10 μL, 20 μL, 50 μL, and 100 μL of 5 mg / mL cordycepin standard stock solution, respectively, and dilute to 1 mL with purified water. Filter through a 0.22 μm microporous membrane to obtain a series of cordycepin standard solutions with concentrations of 5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 250 mg / L, and 500 mg / L. Determine the cordycepin content using HPLC, injecting 10 μL for each concentration, repeating three times, and plotting a standard curve as shown below. Figure 8 As shown.

[0069] The high-performance liquid chromatography (HPLC) conditions were as follows: an Agilent 1260 HPLC system (Agilent, China), a Welch Ultimate AQ-C18 column (4.6 × 250 mm, 5 µm), a mobile phase of 85% water and 15% methanol, an injection volume of 10 µL, a column temperature of 40 °C, a flow rate of 1 mL / min, and three replicates.

[0070] 3. Cordycepin content detection The supernatant of the fermentation broth obtained in step 2 was filtered through a 0.22 μm microporous membrane, and the cordycepin content was determined by high performance liquid chromatography (HPLC) in step 2. The results are shown in the figure. Figure 12 The results showed that, compared with the wild-type strain, the cordycepin synthesis gene was co-expressed. cns1 and cns2 The recombinant Cordyceps militaris strain achieved an extracellular cordycepin yield of 186.5 mg / L, which is 2.3 times that of the wild-type strain. The cordycepin synthesis gene was co-expressed. cns1 and precursor synthesis genes nt5e The recombinant Cordyceps militaris strain achieved an extracellular cordycepin yield of 252.6 mg / L, which is 3.1 times that of the wild-type strain. This indicates that precursor engineering is also a key strategy for increasing cordycepin yield in Cordyceps militaris.

[0071] Comparative Example 1: Construction of a single gene cns1 overexpression strains Using the primers in Table 14 cns1 -F / cns1 -R amplification yielded Cordyceps militaris genomic DNA cns1 Gene (SEQ ID NO: 2), using the primers Vector-F / Vector-R in Table 3, linearized the binary vector pCAMBIA-PgpdA-Tcbh1-hyg-PtrpC, and then used the pEASY-UniSeamless Cloning and Assembly Kit (Beijing TransGen Biotech Co., Ltd., product catalog number: CU101-02) to ligate the target gene fragment with the linearized vector, obtaining the recombinant vector pCAMBIA-PgpdA- cns1-EGFP-Tcbh1-hyg-PtrpC, such as Figure 9 As shown.

[0072] Table 14 cns1 Gene amplification primers Primers Primer sequence 5'to3' cns1-F tcccatcccttattccttATGGCCATGAACGAGAACGC cns1-R gaagtgggaaagctcgaaTCAGGCTATGCCCACCTTGG The other operations are the same as in Example 1, to obtain gpdA-driven expression containing only the target gene. cns1 The recombinant Cordyceps militaris engineered strain was verified by PCR using primers Ide-F / Ide-R (Table 6) to check the success of the transformation. The results are as follows: Figure 10 As shown, lanes 1-5 are parallel samples. Genomic DNA of the extracted Cordyceps militaris hygromycin-resistant strain was taken and analyzed using the primers listed in Table 8. hyg -F / hyg -R is used for PCR verification to check whether the transformation was successful. The results are as follows: Figure 11 As shown, lanes 1-4 are parallel samples, preserving transformants with positive identification results; co-expressed with wild-type Cordyceps militaris. cns1 and cns2 The transgenic Cordyceps militaris strain was fermented simultaneously under the same conditions, and the extracellular cordycepin content of the transgenic strain was determined. The extracellular cordycepin content was as follows: Figure 12 As shown.

[0073] Figure 12 This demonstrates that the constitutive promoter gpdA drives the expression of a single gene for cordycepin synthesis in Cordyceps militaris. cns1 The recombinant strain achieved a yield of 127.2 mg / L. Although it increased cordycepin content, it did not co-express... cns1 and cns2 The recombinant strains of the gene produced higher yields. This indicates that multi-gene expression methods are more effective in increasing cordycepin production.

Claims

1. An engineered strain for improving cordycepin yield in Cordyceps militaris based on multi-gene combination expression and precursor engineering, characterized in that, The engineered strain integrates a multi-gene expression cassette driven by the strong promoter gpdA. The multi-gene expression cassette contains at least two genes linked by a P2A peptide, and the at least two genes are selected from Cordyceps militaris. cns1 Genes, Cordyceps militaris cns2 Genes and nt5e Genes: The multi-gene expression cassette is operatively linked to the transcription terminator Tcbh1.

2. The engineered strain as described in claim 1, characterized in that, Multi-gene expression cassette contains cns1 Genes and cns2 Genes are formed by linking P2A peptides. cns1 –P2A– cns2 Expression unit.

3. The engineered strain as described in claim 1, characterized in that, Multi-gene expression cassette contains cns1 Genes and nt5e Genes are formed by linking P2A peptides. cns1 –P2A– nt5e Expression unit.

4. The engineered strain as described in claim 1, characterized in that, The nucleotide sequence of the strong promoter gpdA is shown in SEQ ID NO:1; cns1 The nucleotide sequence of the gene is shown in SEQ ID NO:2; cns2 The nucleotide sequence of the gene is shown in SEQ ID NO:3; nt5e The nucleotide sequence of the gene is shown in SEQ ID NO:4; the nucleotide sequence of the P2A peptide is shown in SEQ ID NO:

5.

5. A method for constructing an engineered strain as described in any one of claims 1-4, characterized in that, Includes the following steps (1) Construction of recombinant expression vector: using plasmid pCAMBIA-PgpdA-EGFP-Tcbh1- hyg -Based on PtrpC, the target gene fragment is inserted through seamless cloning to obtain a recombinant plasmid, wherein the target gene fragment is the cns1 and cns2 genes or the cns1 and nt5e genes linked by the P2A peptide. (2) The recombinant plasmid was transformed into Agrobacterium tumefaciens AGL1 to obtain recombinant Agrobacterium; (3) The recombinant Agrobacterium was used to infect the Cordyceps militaris host strain, and a stable genetically engineered strain was obtained through screening.

6. The method as described in claim 5, characterized in that, The specific process of constructing the recombinant expression vector in step (1) is as follows: (1) Using Cordyceps militaris genomic DNA as a template, PCR amplification was performed to obtain the target gene fragment with the P2A homologous arm; (2) Linearize the plasmid pCAMBIA-PgpdA-EGFP-Tcbh1-hyg-PtrpC using primers Vector-F / Vector-R; (3) The target gene fragment was ligated to the linearized vector using a seamless cloning kit to construct the recombinant plasmid pCAMBIA-PgpdA-cns1-P2A-cns2-EGFP-Tcbh1-hyg-PtrpC or pCAMBIA-PgpdA-cns1-P2A-nt5e-EGFP-Tcbh1-hyg-PtrpC.

7. The method as described in claim 5, characterized in that, The screening in step (3) is as follows: resistance screening is performed using Sabouraud solid medium containing hygromycin B, and gene integration is verified by PCR.

8. The application of an engineered strain as described in claims 1-4 in the production of cordycepin, characterized in that, Includes the following steps: (1) The engineered strain was inoculated into Sabouraud broth and cultured in the dark for activation; (2) The activated strain was inoculated into the fermentation medium and cultured. The fermentation broth was collected and cordycepin was extracted. The fermentation medium consisted of: 20 g / L glucose, 10 g / L peptone, 0.5 g / L K2HPO4·3H2O, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1 g / L L-glycine, with distilled water as the solvent and natural pH.

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