Recombinant yeast for producing cordycepin, construction method and application thereof

Abstract

The invention discloses a recombinant yeast for producing cordycepin, a construction method and an application thereof. The construction method of the recombinant yeast for producing cordycepin comprises the following steps: codon optimization of EA1 and EA2 genes derived from Atlantic locust trees using yeast as a host to obtain optimized EA1 genes and optimized EA2 genes, transforming host cells to obtain recombinant yeast BEA; knocking out adenosine kinase ADO1 gene in the recombinant yeast BEA to obtain recombinant yeast BEA1; and introducing expression cassettes of aminophosphoribosyltransferase ADE4 gene and cpdBN gene into the HO site of the recombinant yeast BEA1 to obtain recombinant yeast BEA2. The recombinant yeasts BEA, BEA1 and BEA2 for producing cordycepin constructed by the invention have cordycepin yields of 420.73 mg / L, 524.23 mg / L and 562.04 mg / L respectively after 96 hours of shake flask fermentation.

Description

Technical Field

[0001] The invention relates to a recombinant yeast for producing cordycepin and a construction method and application thereof, belonging to the field of genetic engineering and application and microbial technology. Background Art

[0002] Cordycepin is a nucleoside antibiotic, also known as 3'-deoxyadenosine, with a molecular formula of C 10 H 13 N5O3, with a relative molecular mass of 251.24. Cordycepin was first isolated from the culture medium of Cordyceps militaris in 1950. As the main active ingredient of Cordyceps militaris, the research on cordycepin in the field of medicinal chemistry has been very active. Cordycepin has anti-cancer, anti-viral and potential anti-leukemia effects. Cordycepin is mainly produced by chemical synthesis and biological fermentation. The research on the chemical synthesis of cordycepin generally has the disadvantages of low yield, large pollution and high cost. Therefore, the current large-scale commercial production of cordycepin is mainly achieved by fermentation of Cordyceps militaris. At present, the research on the biological production of cordycepin mainly focuses on the screening, mutation, hybridization, and gene modification of Cordyceps militaris strains, as well as the optimization of the composition, temperature, pH and other conditions of the fermentation medium. Although the biological fermentation method is the main method for producing cordycepin at present, there are still problems such as long fermentation cycle, difficult to control fermentation conditions, and complex product extraction process, which hinder the large-scale production of cordycepin and cause the price of cordycepin to remain high.

[0003] Due to its outstanding biological activity and medicinal value, cordycepin has attracted more and more scholars to study its biosynthesis pathway. Related gene mining and molecular biology research have also achieved certain results. In 2017, Xia et al. compared the genome sequences of Cordyceps militaris and Aspergillus nidulans, a fungus that can also produce cordycepin, and found a highly conserved (homology>80%) gene cluster in the genomes of the two, which contained four genes, of which the genes from Cordyceps militaris were named CM1, CM2, CM3 and CM4 (the corresponding genes in A. nidulans were named AN1, AN2, AN3 and AN4). The researchers also proved that the CM1 and CM2 genes were involved in the synthesis of cordycepin through a series of gene knockout, complementation and heterologous expression experiments, and predicted the synthesis pathway of cordycepin. Xia et al. predicted that the 3'-OH of adenosine was phosphorylated by an unknown nucleotide kinase to produce 3'-AMP, and then the phosphate group was removed by CM2 to produce an enol structure intermediate. The unstable intermediate spontaneously generates 2'-carbonyl-3'-deoxyadenosine (2'-C-3'-dA), which then generates cordycepin under the reduction of CM1. The researchers also pointed out that 3'-AMP may also be obtained by the mRNA degradation pathway. After Xia et al. discovered the CM1 and CM2 genes, several homologous genes of cordycepin-producing bacteria were discovered. Liu et al. conducted transcriptome analysis on the mycelium, fruiting bodies and sclerotia of cicada fungus (Cordyceps cicadae) and identified differentially expressed genes, and found the cordycepin synthesis pathway in cicada fungus. Zhao et al. used transcriptome and proteomics techniques to analyze Cordyceps skyushuensis and also found a similar conserved gene cluster CK1-CK4.

[0004] The development of synthetic biology and the discovery of key genes for cordycepin synthesis have provided prerequisites for the production of cordycepin using Saccharomyces cerevisiae. Taking full advantage of the rapid reproduction, simple operation, and short cycle of Saccharomyces cerevisiae to synthesize cordycepin can provide a research direction for its production. Huo et al. constructed expression plasmids of CM1 and CM2 genes from Cordyceps militaris and overexpressed them in Saccharomyces cerevisiae S288C, achieving heterologous production of cordycepin in Saccharomyces cerevisiae. After fermentation optimization, the yield reached 137.27 mg / L in a 5L fermenter after 144 hours of fermentation.

[0005] Although cordycepin has been successfully synthesized heterologously in Saccharomyces cerevisiae, the final yield of cordycepin synthesized only by expressing the CM1 and CM2 genes from Cordyceps militaris is still very low and cannot meet the needs of industrial-scale production applications.

[0006] Therefore, there is an urgent need for cordycepin synthases with higher activity to heterologously synthesize cordycepin and to further optimize and construct engineered strains for synthesizing cordycepin, so as to achieve high-efficiency and low-cost synthesis of cordycepin. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant yeast for producing cordycepin.

[0008] The second object of the present invention is to provide a method for constructing a recombinant yeast for producing cordycepin.

[0009] The third object of the present invention is to provide an application of recombinant yeast for producing cordycepin by fermentation.

[0010] The fourth object of the present invention is to provide a second recombinant yeast for producing cordycepin.

[0011] The fifth object of the present invention is to provide a method for constructing a second recombinant yeast for producing cordycepin.

[0012] The sixth object of the present invention is to provide the use of the second recombinant yeast for producing cordycepin by fermentation.

[0013] The seventh object of the present invention is to provide a third recombinant yeast for producing cordycepin.

[0014] The eighth object of the present invention is to provide a method for constructing a third recombinant yeast for producing cordycepin.

[0015] The ninth objective of the present invention is to provide the use of the third recombinant yeast for producing cordycepin by fermentation.

[0016] The technical solution of the present invention is summarized as follows:

[0017] The method for constructing a recombinant yeast for producing cordycepin comprises the following steps: codon optimization of EA1 and EA2 genes from Robinia pseudoacacia using yeast as a host to obtain optimized EA1 gene and optimized EA2 gene, transforming host cells to obtain recombinant yeast BEA for producing cordycepin, wherein the host cell is yeast;

[0018] The nucleotide sequence of the optimized EA1 gene is as shown in SEQ ID No.1;

[0019] The nucleotide sequence of the optimized EA2 gene is shown as SEQ ID No.2.

[0020] Preferably, the yeast is Saccharomyces cerevisiae, Yarrowia lipolytica or Pichia pastoris.

[0021] The recombinant yeast BEA for producing cordycepin constructed by the above construction method.

[0022] Application of recombinant yeast BEA in the production of cordycepin.

[0023] The second method for constructing a recombinant yeast for producing cordycepin comprises the following steps:

[0024] The adenosine kinase ADO1 gene of the recombinant yeast BEA producing cordycepin was knocked out to obtain the recombinant yeast BEA1 producing cordycepin;

[0025] The nucleotide sequence of the adenosine kinase ADO1 gene is shown in SEQ ID NO.3.

[0026] The recombinant yeast BEA1 producing cordycepin constructed by the second construction method mentioned above.

[0027] Application of recombinant yeast BEA1 in the fermentation production of cordycepin.

[0028] The third method for constructing a recombinant yeast for producing cordycepin comprises the following steps:

[0029] The HO site of the recombinant yeast BEA1 producing cordycepin is introduced with the expression cassettes of the aminophosphoribosyltransferase ADE4 gene and the cpdBN gene to obtain the recombinant yeast BEA2 producing cordycepin; the cpdBN gene is a domain of 354 amino acid residues at the N-terminus of the 2',3'-cyclic nucleotide 2'-phosphodiesterase gene;

[0030] The nucleotide sequence of the ADE4 gene is shown in SEQ ID NO.4;

[0031] The nucleotide sequence of the cpdBN gene is shown in SEQ ID NO.5.

[0032] The recombinant yeast BEA2 for producing cordycepin constructed by the third construction method mentioned above.

[0033] Application of recombinant yeast BEA2 in the fermentation production of cordycepin.

[0034] Advantages of the present invention:

[0035] The cordycepin production of the recombinant yeasts BEA, BEA1 and BEA2 constructed by the present invention is 420.73 mg / L, 524.23 mg / L and 562.04 mg / L, respectively, after 96 hours of shake flask fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 .The yield of cordycepin synthesized by the recombinant strains BEA, BEA1 and BEA2. The recombinant strains were all fermented in YPD liquid medium. DETAILED DESCRIPTION

[0037] The present invention will be further described below by means of specific examples.

[0038] The present invention uses Saccharomyces cerevisiae BY4741 (ATCC4040002) as the starting strain; other Saccharomyces cerevisiae and Yarrowia lipolytica with ATCC number 20460 or Pichia pastoris with ATCC number 14356 can also be used in the present invention.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] Saccharomyces cerevisiae ATCC4040002, purchased in June 2016, website: (https: / / www.atcc.org / )

[0042] The EA1 gene derived from Robinia pseudoacacia has the sequence shown in GenBank Accession No. KAG9252265.1 or its degenerate sequence.

[0043] The EA2 gene derived from Robinia pseudoacacia has the sequence shown in GenBank Accession No. KAG9252268.1 or its degenerate sequence.

[0044] Example 1

[0045] The method for constructing a recombinant yeast for producing cordycepin comprises the following steps:

[0046] The gene expression modules of cordycepin synthesis-related genes EA1 and EA2 from Robinia pseudoacacia were introduced into the Ura3 site of the Saccharomyces cerevisiae BY4741 genome to obtain recombinant yeast BEA.

[0047] EA1 and EA2 genes are derived from Emericellopsis atlantica. They were codon-optimized by General Biotech Co., Ltd. using Saccharomyces cerevisiae as the host and then integrated into the PUC57 plasmid to obtain PUC57-EA1 and PUC57-EA2. The nucleic acid sequences of EA1 and EA2 after codon optimization are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0048] (1) Expression cassette construction

[0049] As shown in Table 1, the Uura fragment was PCR amplified using Uura-F (SEQ ID NO. 6) and Uura-R-PGK1p (SEQ ID NO. 7) using the genome of Saccharomyces cerevisiae ATCC4040002 as a template;

[0050] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify P using Uura-PGK1p-F (SEQ ID NO.8) and PGK1t-R-EA1 (SEQ ID NO.9). PGK1 Fragment;

[0051] Using plasmid PUC57-EA1 as template, EA1 fragment was amplified by PCR using PGK1p-EA1-F (SEQ ID NO. 10) and EA1-R-ADH2t (SEQ ID NO. 11);

[0052] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify T using EA1-ADH2t-F (SEQ ID NO.12) and ADH2t-R-TEF1p (SEQ ID NO.13). ADH2 Fragment;

[0053] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify P using PCR using ADH2t-TEF1p-F (SEQ ID NO.14) and TEF1p-R-EA2 (SEQ ID NO.15). TEF1 Fragment;

[0054] Using PUC57-EA2 as a template, PCR amplified the EA2 fragment using TEF1p-EA2-F (SEQ ID NO. 16) and EA2-R-CYC1t (SEQ ID NO. 17);

[0055] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify T using EA2-CYC1t-F (SEQ ID NO.18) and CYC1t-R-LEU2 (SEQ ID NO.19). CYC1 Fragment;

[0056] Using prs405 plasmid (ATCC, USA) as template, PCR amplified LEU2 fragment using CYC1t-LEU2-F (SEQ ID NO. 20) and LEU2-R-Dura (SEQ ID NO. 21);

[0057] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify the Dura fragment using LEU2-Dura-F (SEQ ID NO.22) and Dura-R (SEQ ID NO.23). PGK1 , EA1 and T ADH2 Fragment fusion to obtain Uura-P PGK1-EA1-T ADH2 expression cassette, the amplified P TEF1 ,EA2,T CYC1 , LEU2 and Dura fragments were fused to obtain P TEF1 -EA2-T CYC1 -LEU2-Dura expression cassette.

[0058] (2) Saccharomyces cerevisiae transformation

[0059] A single colony of Saccharomyces cerevisiae BY4741 was inoculated into 3ml YPD liquid medium and cultured in a shaker at 30℃ and 220rpm for 12h; the cultured yeast seed liquid was transferred to a new 3ml YPD liquid medium and cultured at 30℃ and 220rpm for 5h; 1ml of bacterial liquid was taken into a 1.5ml centrifuge tube that had been sterilized in advance, centrifuged at 4000rpm for 3min, the supernatant was discarded, and the bacteria were collected; 1ml of sterile water was washed once, centrifuged at 4000rpm for 3min, the supernatant was discarded, and the bacteria were collected. Then 1ml of 100mM LiAc aqueous solution was added to the bacteria and mixed, and allowed to stand at room temperature for 5min. After standing, the cells were centrifuged at 4000rpm for 3min, and the LiAc liquid was removed with a pipette. Salmon sperm DNA (Solarbio, 10mg / ml) was boiled in boiling water for 5min, and then quickly placed on the prepared ice for cooling.

[0060] 120 μl PEG3350 (50 g PEG3350 / 100 ml water), 18 μl 1.0 M LiAc aqueous solution, 5 μl salmon sperm DNA after boiling water for 5 min and cooling, 400 ng Uura-P were added to the centrifuge tube of bacterial precipitation in sequence. PGK1 -EA1-T ADH2 fragment, 400ng P TEF1 -EA2-T CYC1 -LEU-Dura fragment, the final volume is supplemented to 180μl with sterile water. Then gently blow with a pipette for 1min to mix it; place the mixed centrifuge tube in a 42℃ water bath for 30min, centrifuge at 4000rpm for 3min, remove the supernatant; continue to add clean 1ml YPD liquid culture medium, 30℃, 220rpm shaker for recovery culture for 2h; centrifuge the recovered centrifuge tube at 4000rpm for 3min, discard the upper culture medium, and wash twice with 1ml sterile water; finally, add 200μl of sterile water to the centrifuge tube containing the bacteria and mix it, spread it on the leucine-deficient SC culture medium plate, place it in a 30℃ constant temperature incubator for 2 days, wait for a single colony to grow, and screen to obtain the recombinant bacteria BEA.

[0061] Table 1 Primers and templates for constructing recombinant bacteria BEA

[0062]

[0063]

[0064] (3) Culture medium used in the examples

[0065] YPD liquid medium: final concentration of glucose is 25g / L, final concentration of yeast extract powder is 10g / L, final concentration of peptone is 20g / L, prepared with distilled water. YPD solid medium is prepared by adding 20g / L agar powder to YPD liquid medium.

[0066] LB liquid medium: the final concentration of peptone is 10 g / L, the final concentration of yeast extract is 5 g / L, the final concentration of sodium chloride is 10 g / L, and it is prepared with distilled water.

[0067] SC medium: final concentration of glucose is 20 g / L, final concentration of amino-free yeast nitrogen source (YNB) is 6.7 g / L, final concentration of amino acid mixture is 0.2 g / L, 20 g / L agar powder, prepared with distilled water, missing amino acid mixture means removing the corresponding components in the amino acid mixture.

[0068] Amino acid mixture: glycine, 2.0g; alanine, 2.0g; methionine, 2.0g; lysine, 2.0g; arginine, 2.0g; serine, 2.0g; asparagine, 2.0g; aspartic acid, 2.0g; phenylalanine, 2.0g; cysteine, 2.0g; proline, 2.0g; tyrosine, 2.0g; glutamic acid, 2.0g; valine, 2.0g; threonine, 2.0g; isoleucine, 2.0g; inositol, 2.0g; glutamine, 2.0g; p-aminobenzoic acid 0.2g; adenine, 0.5g; leucine 10g; methionine, 2g; tryptophan, 2g; histidine, 2g; uracil, 2g.

[0069] The corresponding recombinant strain can be obtained by replacing the Saccharomyces cerevisiae BY4741 of this example with Yarrowia lipolytica with ATCC number 20460 or Pichia pastoris with ATCC number 14356, while remaining the same as in this example.

[0070] Example 2 Fermentation process of recombinant bacteria BEA and detection of cordycepin content

[0071] (1) Fermentation process of recombinant bacteria

[0072] The recombinant strain BEA was inoculated into a 3 mL YPD liquid medium test tube and cultured overnight at 30°C in a shaking incubator for about 16 h. When the strain grew to the logarithmic phase, it was transferred to a 250 mL shake flask containing 50 mL YPD liquid medium. The inoculation volume was controlled so that the initial OD600 The fermentation temperature was 0.05 and the culture was carried out on a shaker at 30°C and 220 rpm for 96 h.

[0073] (2) Cordycepin content detection

[0074] Use a pipette to draw 1 mL of fermentation liquid into a centrifuge tube and centrifuge at 12000 rpm for 2 min. Use a pipette to draw the supernatant, use a syringe to draw the supernatant through a 0.22 μm microporous membrane into a liquid phase vial for the detection of cordycepin. Use ultraviolet high performance liquid chromatography for detection.

[0075] Chromatographic detection conditions:

[0076] Fluidity: 20% methanol in water; Flow rate: 1 mL / min; Detection wavelength: 260 nm; Column temperature: 30°C;

[0077] Standard solutions of cordycepin at 10 mg / L, 25 mg / L, 50 mg / L, 100 mg / L and 250 mg / L were prepared respectively to draw standard curves.

[0078] (3) Measurement results

[0079] The cordycepin production of the recombinant bacteria BEA was 420.73 mg / L.

[0080] Example 3 Construction method of recombinant bacteria BEA1

[0081] The ADO1 (adenosine kinase) gene knockout expression cassette was introduced into the recombinant strain BEA to obtain the recombinant strain BEA1.

[0082] The nucleotide sequence of the adenosine kinase ADO1 gene is shown in SEQ ID NO.3;

[0083] (1) Construction of gene expression cassette

[0084] As shown in Table 2, the UADO1 fragment was PCR amplified using UADO1-F (SEQ ID NO. 24) and UADO1-R (SEQ ID NO. 25) using the genome of Saccharomyces cerevisiae ATCC4040002 as a template;

[0085] Using PXP218 plasmid (purchased from Addgene, Inc. www.addgene.org) as a template, PCR amplified the URA3 fragment using URA3-F (SEQ ID NO. 26) and URA3-R (SEQ ID NO. 27);

[0086] The DADO1 fragment was amplified by PCR using the genome of Saccharomyces cerevisiae ATCC4040002 as a template using DADO1-F (SEQ ID NO.28) and DADO1-R (SEQ ID NO.29).

[0087] The amplified UADO1, URA3 and DADO1 fragments were fused to obtain the UADO1-URA3-DADO1 gene expression cassette.

[0088] According to the transformation method of step (2) of Example 1, 800 ng of the UADO1-URA3-DADO1 gene expression cassette obtained in step (1) was transformed into the recombinant bacteria BEA, and the recombinant bacteria were spread on SC medium plates lacking uracil for screening to obtain the recombinant bacteria BEA1.

[0089] (2) Cordycepin content test results of recombinant bacteria BEA1

[0090] The fermentation of the recombinant bacteria BEA1 and the detection of the cordycepin content were carried out according to the method of Example 2. The detection results showed that the cordycepin content of the recombinant bacteria BEA1 was 524.23 mg / L.

[0091] Table 2 Primers and templates for constructing recombinant bacteria BEA1

[0092]

[0093] Example 4 Construction method of recombinant bacteria BEA2

[0094] The expression cassettes of ADE4 (aminophosphoribosyltransferase) gene and cpdBN gene (cpdBN gene is the domain of 354 amino acid residues at the N-terminus of 2',3'-cyclic nucleotide 2'-phosphodiesterase gene) were simultaneously introduced into the HO site of recombinant strain BEA1 to obtain recombinant strain BEA2. The cpdBN gene was derived from Escherichia coli BL21, and the cpdBN nucleic acid sequence was synthesized by General Biotech Co., Ltd.

[0095] The nucleotide sequence of the ADE4 gene is shown in SEQ ID NO.4;

[0096] The nucleotide sequence of the cpdBN gene is shown in SEQ ID NO.5.

[0097] (1) Expression cassette construction

[0098] As shown in Table 3, the Uho fragment was amplified by PCR using Uho-F (SEQ ID NO. 30) Uho-R-TDH3p (SEQ ID NO. 31) with the genome of Saccharomyces cerevisiae ATCC4040002 as a template;

[0099] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify P using Uho-TDH3p-F (SEQ ID NO.32) and TDH3p-R-ADE4 (SEQ ID NO.33). TDH3 Fragment;

[0100] Using the genome of Saccharomyces cerevisiae ATCC4040002 as a template, the ADE4 fragment was PCR amplified using TDH3p-ADE4-F (SEQ ID NO.34) and ADE4-R-ADH1t (SEQ ID NO.35);

[0101] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify T using PCR using ADE4-ADH1t-F (SEQ ID NO.36) and ADH1t-R-PGK1p (SEQ ID NO.37). ADH1 Fragment;

[0102] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify P using PCR using ADH1t-PGK1p-F (SEQ ID NO.38) and PGK1p-R-cpdBN (SEQ ID NO.39). PGK1 Fragment;

[0103] Using the nucleic acid sequence of the cpdBN gene (SEQ ID NO.5) as a template, PCR amplified the cpdBN fragment using PGK1p-cpdBN-F (SEQ ID NO.40) and cpdBN-R-ADH2t (SEQ ID NO.41);

[0104] The genome of Saccharomyces cerevisiae ATCC4040002 was used as a template to amplify T using cpdBN-ADH2t-F (SEQ ID NO.42) and ADH2t-R-HIS (SEQ ID NO.43). ADH2 Fragment;

[0105] Using PXP320 plasmid (purchased from Addgene, Inc. www.addgene.org) as a template, PCR amplified the HIS fragment using ADH2t-HIS-F (SEQ ID NO. 44) and HIS-R (SEQ ID NO. 45);

[0106] The Dho fragment was amplified by PCR using Dho-F (SEQ ID NO.46) and Dho-R (SEQ ID NO.47) using the genome of Saccharomyces cerevisiae ATCC4040002 as a template. TDH3 Fusion with ADE4 to obtain Uho-P TDH3-ADE4 expression cassette, the amplified T ADH1、 P PGK1 Fusion with cpdBN fragment to obtain T ADH1 -P PGK1 -cpdBN gene expression cassette, the amplified T ADH2 , HIS and Dho fusion to obtain T ADH2 -HIS-Dho gene expression cassette.

[0107] According to the transformation method of step (2) of Example 1, 200 ng Uho-P obtained in step (1) was TDH3 -ADE4 fragment, 200ng T ADH1 -P PGK1 -cpdBN fragment and 200ng T ADH2 -HIS-Dho was transformed into the recombinant bacterium BEA1, and the recombinant bacterium was spread on a SC medium plate lacking histidine for screening to obtain the recombinant bacterium BEA2.

[0108] (2) Cordycepin content test results of recombinant bacteria BEA2

[0109] The fermentation of the recombinant bacteria BEA2 and the detection of the cordycepin content were carried out according to the method of Example 2. The detection results showed that the cordycepin content of the recombinant bacteria BEA2 was 562.04 mg / L.

[0110] Table 3 Primers and templates for constructing recombinant bacteria BEA2

[0111]

Claims

1. A method for constructing a recombinant yeast for producing cordycepin, characterized in that The following steps are involved: The EA1 and EA2 genes from Emericellopsis atlantica were codon optimized using yeast as a host to obtain optimized EA1 and optimized EA2 genes, and the optimized genes were transformed into host cells to obtain recombinant yeast BEA that produces cordycepin, wherein the host cell is Saccharomyces cerevisiae; The nucleotide sequence of the optimized EA1 gene is as shown in SEQ ID No.1; The nucleotide sequence of the optimized EA2 gene is shown as SEQ ID No.

2.

2. The recombinant yeast BEA for producing cordycepin constructed by the construction method of claim 1.

3. Use of the recombinant yeast BEA according to claim 2 in the production of cordycepin by fermentation.

4. A method for constructing a recombinant yeast for producing cordycepin, characterized in that The following steps are involved: Knocking out the adenosine kinase ADO1 gene of the recombinant yeast BEA producing cordycepin according to claim 2 to obtain the recombinant yeast BEA1 producing cordycepin; The nucleotide sequence of the adenosine kinase ADO1 gene is shown in SEQ ID NO.

3.

5. The recombinant yeast BEA1 for producing cordycepin constructed by the construction method of claim 4.

6. Use of the recombinant yeast BEA1 according to claim 5 in fermentation production of cordycepin.

7. A method for constructing a recombinant yeast for producing cordycepin, characterized in that The following steps are involved: The HO site of the recombinant yeast BEA1 producing cordycepin according to claim 5 is introduced into the expression cassette of the aminophosphoribosyltransferase ADE4 gene and the cpdBN gene to obtain the recombinant yeast BEA2 producing cordycepin; the cpdBN gene is a domain of 354 amino acid residues at the N-terminus of the 2',3'-cyclic nucleotide 2'-phosphodiesterase gene; The nucleotide sequence of the ADE4 gene is shown in SEQ ID NO.4; The nucleotide sequence of the cpdBN gene is shown in SEQ ID NO.

5.

8. The recombinant yeast BEA2 for producing cordycepin constructed according to the construction method of claim 7.

9. Use of the recombinant yeast BEA2 according to claim 8 in fermentation production of cordycepin.

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