Application of editing cmpks1 gene in creating cordyceps militaris white strain with increased content of beneficial compounds
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-10
- Publication Date
- 2026-08-11
AI Technical Summary
然而，蛹虫草产业发展过程中存在菌种单一、优质菌种缺乏、菌种稳定性难以维持等问题
Smart Images
Figure HDA0005266181290000011 
Figure HDA0005266181290000012 
Figure HDA0005266181290000013
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic breeding technology for edible and medicinal fungi, specifically to the application of editing the Cmpks1 gene in creating white strains of Cordyceps militaris with increased content of beneficial compounds. Background Technology
[0002] Cordyceps militaris (L.) Fr., belonging to the phylum Ascomycota, order Hypocreales, family Cordycipitaceae, and genus Cordyceps, is an edible and medicinal fungus with high nutritional value and tonic activity. According to the *Xinhua Materia Medica*, Cordyceps militaris is "sweet in taste and neutral in nature," and has the effects of "benefiting the lungs and kidneys, replenishing essence, stopping bleeding, and resolving phlegm."
[0003] Cordyceps militaris, containing bioactive components such as cordycepin, cordyceps polysaccharides, γ-aminobutyric acid (GABA), and pentostatin, possesses anti-inflammatory, antioxidant, antitumor, antiviral, and immunomodulatory pharmacological activities, making it a hot topic in scientific research and the development of the edible fungi industry. Because its fruiting bodies can be cultivated artificially on a large scale, Cordyceps militaris has become an extremely important edible and medicinal fungus in my country and even Southeast Asia. my country officially approved it as a new resource food on March 16, 2009 (renamed a new food raw material in 2014), and it has now formed a huge industry. However, the development of the Cordyceps militaris industry faces problems such as single strain, lack of high-quality strains, and difficulty in maintaining strain stability. Due to the limitations of traditional breeding methods, the use of molecular techniques to select new Cordyceps militaris strains is particularly important. Summary of the Invention
[0004] The purpose of this invention is to provide the application of editing the Cmpks1 gene in creating white strains of Cordyceps militaris with increased levels of beneficial compounds.
[0005] In a first aspect, the present invention claims protection for the use of inhibiting the expression and / or activity of CmPKS1 protein in any of the following:
[0006] (A1) Increase the content of beneficial compounds in Cordyceps militaris;
[0007] (A2) Create a white strain of Cordyceps militaris.
[0008] Inhibition of CmPKS1 protein expression and / or activity can be achieved through at least one of the following six regulatory levels: 1) inhibiting the expression of the gene encoding CmPKS1 protein (i.e., the Cmpks1 gene) at the transcriptional level; 2) inhibiting the expression of the Cmpks1 gene at the post-transcriptional level (i.e., inhibiting the expression of CmPKS1 protein through splicing or processing of the primary transcript of the relevant gene); 3) inhibiting the expression of the protein encoded by the Cmpks1 gene at the RNA transport level (i.e., inhibiting the expression of the protein encoded by the Cmpks1 gene by regulating the transport of the mRNA of the relevant gene from the nucleus to the cytoplasm); 4) inhibiting the expression of the protein encoded by the Cmpks1 gene at the translational level; 5) inhibiting the expression of the protein encoded by the Cmpks1 gene at the mRNA degradation level; 6) inhibiting the activity of the protein encoded by the Cmpks1 gene at the post-translational level. The same applies below.
[0009] Secondly, the present invention claims protection for the use of substances capable of inhibiting the expression level and / or activity of CmPKS1 protein in any of the following:
[0010] (A1) Increase the content of beneficial compounds in Cordyceps militaris;
[0011] (A2) Create a white strain of Cordyceps militaris.
[0012] In the first and second aspects above, in (A2), the white strain of Cordyceps militaris may be a white strain of Cordyceps militaris with increased content of beneficial compounds.
[0013] In the first and second aspects mentioned above, the content of the beneficial compound may be the content of the beneficial compound in the fruiting body of Cordyceps militaris.
[0014] In the first and second aspects above, the beneficial compounds may be all or part of the following: cordycepin, denticulaflavonol, dehydrocorydalin, PC(34:5) (PC(34:5) is a phosphatidylcholine compound), trigonelline, nevirapine, daurisoline, coumestrol, and tenofovir.
[0015] In the first and second aspects mentioned above, inhibiting the expression and / or activity of the CmPKS1 protein may include either completely preventing the expression of the CmPKS1 protein or completely eliminating its activity, or reducing the expression and / or activity of the CmPKS1 protein but not to zero.
[0016] In the first and second aspects described above, inhibiting the expression and / or activity of the CmPKS1 protein can be achieved by knocking out or reducing the expression of the gene encoding the CmPKS1 protein in the Cordyceps militaris genome. Accordingly, in the second aspect described above, the substance can be a substance capable of knocking out or reducing the expression of the gene encoding the CmPKS1 protein in the Cordyceps militaris genome.
[0017] Furthermore, in the first and second aspects above, inhibiting the expression and / or activity of the CmPKS1 protein can be achieved by introducing a CRISPR / Cas9 editing tool, siRNA, or shRNA targeting the gene encoding the CmPKS1 protein in the Cordyceps militaris genome into Cordyceps militaris. Accordingly, in the second aspect above, the substance can be a CRISPR / Cas9 editing tool targeting the gene encoding the CmPKS1 protein in the Cordyceps militaris genome;
[0018] In one embodiment of the present invention, the target sequence of the CRISPR / Cas9 editing tool is shown in SEQ ID No. 1.
[0019] Thirdly, the present invention claims protection for any of the following methods:
[0020] Method I: A method for increasing the content of beneficial compounds in Cordyceps militaris may include the following steps: inhibiting the expression level and / or activity of CmPKS1 protein in the Cordyceps militaris genome, thereby increasing the content of beneficial compounds in Cordyceps militaris.
[0021] Method II: A method for creating a white strain of Cordyceps militaris, comprising the following steps: inhibiting the expression level and / or activity of CmPKS1 protein in the Cordyceps militaris genome, thereby creating a white strain of Cordyceps militaris.
[0022] In Method II, the white Cordyceps militaris strain may be a white Cordyceps militaris strain with increased content of beneficial compounds.
[0023] In the method, the content of the beneficial compound can be the content of the beneficial compound in the fruiting body of Cordyceps militaris.
[0024] The beneficial compounds may be all or part of the following: cordycepin, denticulaflavonol, dehydrocorydalin, PC (34:5), trigonelline, nevirapine, daurisoline, coumestrol, and tenofovir.
[0025] Furthermore, in the method, the expression level and / or activity of the CmPKS1 protein in the Cordyceps militaris genome can be inhibited by knocking out or reducing the expression of the gene encoding the CmPKS1 protein in the Cordyceps militaris genome.
[0026] Furthermore, in the method, the expression level and / or activity of the CmPKS1 protein in the Cordyceps militaris genome can be suppressed by introducing a CRISPR / Cas9 editing tool that targets the gene encoding the CmPKS1 protein in the Cordyceps militaris genome into Cordyceps militaris.
[0027] In one embodiment of the present invention, the target sequence of the CRISPR / Cas9 editing tool is shown in SEQ ID No. 1.
[0028] In one embodiment of the present invention, inhibiting the expression level and / or activity of the CmPKS1 protein in the Cordyceps militaris genome specifically involves inserting a single base A between positions 6407 and 6408 of the gene encoding the CmPKS1 protein shown in SEQ ID No. 3 of the Cordyceps militaris genome (i.e., the Cmpks1 gene) (thus forming a frameshift mutation) (corresponding to the mutant strain ΔCmpks1 in the embodiment) or deleting the segment between positions 6397 and 6413 of SEQ ID No. 3 (thus forming a frameshift mutation) (corresponding to the mutant strain ΔCmpks2 in the embodiment).
[0029] In the aforementioned relevant aspects, the CmPKS1 protein may be any of the following:
[0030] (B1) A protein with the amino acid sequence SEQ ID No. 2;
[0031] (B2) A protein derived from Cordyceps militaris with the same function, having undergone substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2.
[0032] (B3) is a protein that has 99%, 95%, 90%, 85% or 80% identity with any of the amino acid sequences defined in (B1)-(B2) and is derived from Cordyceps militaris and has the same function.
[0033] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.
[0034] The 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or higher level of identity can be at least 95%, 96%, 97%, 98%, or 99% identity.
[0035] In all the aforementioned relevant aspects, the gene encoding the CmPKS1 protein (i.e., the Cmpks1 gene) may be any of the following:
[0036] (C1) The DNA molecule shown in SEQ ID No. 3;
[0037] DNA molecules that have 99%, 95%, 90%, 85%, or 80% identity with the DNA sequences defined by (C2) and (C1) and encode the CmPKS1 protein.
[0038] For the genes mentioned above, nucleotide sequence identity can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastn as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of nucleotide sequences to calculate the identity value (%), then the identity value can be obtained.
[0039] In the aforementioned genes, the 95% or higher identity can be at least 96%, 97%, or 98% identity. The 90% or higher identity can be at least 91%, 92%, 93%, or 94% identity. The 85% or higher identity can be at least 86%, 87%, 88%, or 89% identity. The 80% or higher identity can be at least 81%, 82%, 83%, or 84% identity.
[0040] This invention utilizes CRISPR / Cas9 gene editing technology to edit and knock out the Cmpks1 gene, which produces pigments in Cordyceps militaris, creating a Cordyceps militaris strain with a different fruiting body color trait than the parent strain (the parent strain has yellow fruiting bodies, while the Cmpks1 gene-edited strain has white fruiting bodies). Furthermore, the single fruiting body of the Cmpks1 gene-edited Cordyceps militaris strain created in this invention is relatively heavy, and its biotransformation rate is not significantly different from that of the wild type (parent strain). More importantly, the content of beneficial compounds such as cordycepin and denticulaflavonol in the fruiting bodies of the Cmpks1 gene-edited Cordyceps militaris strain is increased. Attached Figure Description
[0041] Figure 1 pAMA1-Cas9-sgRNA Cmpks1 A schematic diagram of a gene-editing vector.
[0042] Figure 2 Sequencing results for the edited mutant strains ΔCmpks1 and ΔCmpks2 of Cmpks1.
[0043] Figure 3 To identify plasmid loss in ΔCmpks1 and ΔCmpks2 mutant strains. The positive control was a Cordyceps militaris strain containing hygromycin resistance.
[0044] Figure 4 The difference in growth rate between ΔCmpks1, ΔCmpks2 and wild-type strains.
[0045] Figure 5 The primordium differentiation times of ΔCmpks1, ΔCmpks2, and wild-type strains are consistent. The scale bar in the figure is 1 cm.
[0046] Figure 6 The images show the fruiting bodies of strains ΔCmpks1, ΔCmpks2, and the wild-type strain. The scale bar in the image is 2 cm.
[0047] Figure 7 Comparison of fruiting body lengths between ΔCmpks1, ΔCmpks2, and wild-type strains. Different lowercase letters indicate significant differences (P<0.05).
[0048] Figure 8 Biotransformation comparisons of ΔCmpks1, ΔCmpks2, and wild-type strains. n=3, p<0.1 (no significant difference).
[0049] Figure 9 Cordycepin content in the fruiting bodies of ΔCmpks1, ΔCmpks2, and wild-type strains. Different lowercase letters indicate significant differences (P<0.05).
[0050] Figure 10 Differential metabolites for yellow and white fruiting bodies. WT: wild type, yellow fruiting body; ΔCmpks: mutant ΔCmpks1, white fruiting body. Detailed Implementation
[0051] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0053] In the quantitative detections involved in the following examples, at least three replicates were performed, and the average of the results was taken.
[0054] The amino acid sequence of the Cordyceps militaris CmPKS1 protein involved in the following examples is shown in SEQ ID No. 2, and the nucleotide sequence of its encoding gene (i.e., Cmpks1 gene) in the Cordyceps militaris genome is shown in SEQ ID No. 3.
[0055] Example 1: Application of Cmpks1 gene knockout in creating white Cordyceps militaris strains with increased content of beneficial compounds
[0056] I. Creation of White Cordyceps militaris strains using CRISPR / Cas9 gene editing technology
[0057] 1. The Cmpks1 gene, which is related to pigment synthesis in the Cordyceps militaris genome, was selected as the target gene.
[0058] 2. Identify the sgRNA targeting the Cmpks1 gene and construct a CRISPR-Cas9 gene editing vector for this sgRNA.
[0059] The target sequence for the Cmpks1 gene is as follows:
[0060] 5'-CCTTTGGACGTTCTGTCGCAGAGT-3' (SEQ ID No. 1).
[0061] The target sequence (SEQ ID No. 1) was cloned into the BstEII restriction site of the vector pAMA1-Cas9-hygR (described in the article "Meng et al. Efficient CRISPR / Cas9 system based on autonomously replicating plasmid with an AMA1 sequence and precisely targeted gene deletion in the ediblefungus, Cordyceps militaris. Microbial Biotechnology. 2022; 00: 1–13", which is publicly available from the applicant and can only be used to repeat the experiments of this invention, and may not be used for other purposes), to obtain the recombinant CRISPR / Cas9 gene editing vector, which was named pAMA1-Cas9-sgRNA after being verified by sequencing. Cmpks1 carrier ( Figure 1 ).
[0062] 3. The pAMA1-Cas9-sgRNA obtained in step 2 Cmpks1 The vector was transformed into protoplasts of Cordyceps militaris (CGMCC3.16323) to obtain mutant strains with the Cmpks1 gene edited.
[0063] 4. Mutant strains with the Cmpks1 gene edited were obtained through phenotypic screening (the color was observed after exposure to light; the wild type was yellow, while the Cmpks1 gene-edited mutant strain of this invention was white).
[0064] 5. At the genomic DNA level, the edited sites in the Cmpks1 gene of the mutant strain were determined using Sanger sequencing.
[0065] 6. The identified positive transformants were transferred into PDA plates without hygromycin resistance for subculture, for a total of three generations.
[0066] 7. The positive transformants, after three generations of subculture, were transferred into PDA plates containing hygromycin resistance and those without hygromycin resistance, respectively. When the transformants could not grow on the PDA plates containing hygromycin resistance but grew normally on the PDA plates without hygromycin resistance, the inactivated strains of Cordyceps militaris Cmpks1 without exogenous gene insertion and without vector loss were obtained. Two of these strains were named ΔCmpks1 and ΔCmpks2, respectively.
[0067] Sequencing results for mutant strains ΔCmpks1 and ΔCmpks2 can be found in [link to documentation]. Figure 2 As can be seen from the figure:
[0068] Compared with the unmutated wild-type Cordyceps militaris Cmpks1 gene, the mutant strain ΔCmpks1 has a single-base insertion mutation near the target sequence in the Cmpks1 gene. Specifically, a single base A is inserted between positions 6407 and 6408 of the Cmpks1 gene sequence shown in SEQ ID No. 3, which forms a frameshift mutation and causes premature termination of translation.
[0069] Compared with the unmutated wild-type Cordyceps militaris Cmpks1 gene, the mutant strain ΔCmpks2 has a 17bp deletion in the Cmpks1 gene near the target sequence, specifically a deletion mutation at positions 6397-6413 of the Cmpks1 gene sequence shown in SEQ ID No. 3, which forms a frameshift mutation and causes premature termination of translation.
[0070] The plasmid loss verification results for mutant strains ΔCmpks1 and ΔCmpks2 are as follows: Figure 3 As shown in the figure. The results showed that the transformants could not grow on plates containing hygromycin resistance, but could grow normally on plates without hygromycin resistance. This indicates that the plasmids in ΔCmpks1 and ΔCmpks2 have been lost.
[0071] II. Phenotypic Identification of Cordyceps militaris Mutants with Cmpks1 Gene Knockout
[0072] 1. Detection of mycelial growth rate and primordium differentiation time of ΔCmpks1, ΔCmpks2 and wild-type strains under dark conditions
[0073] ΔCmpks1, ΔCmpks2 and wild-type strains were inoculated on PDA plates. After the mycelia were cultured in the dark for 14 days, the colony diameter (cm) was measured. The colony diameter / culture days (cm / d) was calculated as the mycelial growth rate.
[0074] After inoculating the strain into wheat culture medium and culturing it in the dark for 10 days, the bacteria were scratched and then cultured under light for another 10 days to observe primordium differentiation.
[0075] The results showed that under dark conditions, the mycelial growth rate of the inactivated Cmpks1 strains ΔCmpks1 and ΔCmpks1 was consistent with that of the wild type (WT), with no significant difference. Figure 4 ); Primordial differentiation time is consistent with wild type ( Figure 5 ).
[0076] 2. Identification of fruiting body characteristics of ΔCmpks1, ΔCmpks2 and wild-type strains
[0077] Fruiting bodies were harvested after 60 days of growth, and their height was measured.
[0078] The results showed that under light, the fruiting bodies of the ΔCmpks1 and ΔCmpks2 mutant strains grew slower than the wild type (WT), were white, and after 60 days of growth, the fruiting bodies of the inactivated Cmpks1 strain were thicker and more robust. Figure 6 The fruiting bodies of the inactivated Cmpks1 strain were significantly shorter than those of the wild type. Figure 7 )
[0079] 3. Detection of biotransformation rates of ΔCmpks1, ΔCmpks2 and wild-type strains
[0080] The fresh weight of Cordyceps militaris fruiting bodies was measured after 60 days of growth. The bioconversion rate was calculated as: fresh weight of fruiting bodies / dry weight of material.
[0081] The results showed that one-way ANOVA (p<0.1) indicated no significant difference in biotransformation rates between the ΔCmpks1 and ΔCmpks2 mutants and the wild-type (WT). Figure 8 ).
[0082] 4. Cordycepin content analysis of ΔCmpks1, ΔCmpks2 and wild-type strains
[0083] Three parallel samples each of wild-type and ΔCmpks1 fruiting bodies cultured under the same conditions were dried at 45℃, ground, and 0.1 g of the dried sample powder was added to 80 mL of 20% ethanol. After ultrasonic extraction for 30 min, the volume was adjusted to 100 mL, and 1 mL was filtered through a 0.45 μm filter membrane for high-performance liquid chromatography (HPLC) detection. The HPLC detection conditions were: phase A: H2O, phase B: methanol, ratio 95:5 (V / V), flow rate 1 mL / min, and detection column: C18 column (AQ-C18, 5 μm, 4.6*250 mm, GLsciences).
[0084] The results showed that one-way ANOVA (p<0.1) indicated that the cordycepin content in the fruiting bodies of the ΔCmpks1 and ΔCmpks2 mutant strains was significantly higher than that in the wild type (WT). Figure 9 ).
[0085] 5. Non-target metabolomics analysis of ΔCmpks1, ΔCmpks2 and wild-type strains
[0086] Four parallel samples each of wild-type and ΔCmpks1 fruiting bodies cultured under the same conditions were submitted to Bestway Biotechnology Co., Ltd. for non-target metabolomics data determination. After data acquisition, noise reduction / normalization was performed, followed by univariate and multivariate statistical analysis.
[0087] The results showed that by measuring the non-target metabolome, the metabolite composition of Cordyceps militaris was obtained. Further comparison of the metabolite differences between the wild type and ΔCmpks1 revealed that the white Cordyceps militaris fruiting body ΔCmpks1 contained higher levels of flavonoids such as denticulaflavonol, dehydrocorydalin, PC(34:5), trigonelline, nevirapine, daurisoline, coumestrol, and / or tenofovir. Figure 10 These compounds contain physiological activities such as antioxidant, antitumor, and antiviral activity. The increased content of numerous beneficial compounds in the white Cordyceps militaris fruiting body ΔCmpks1 in this invention provides a solid foundation for exploring the potential compounds and medicinal value of Cordyceps militaris.
[0088] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. Inhibition of CmPKS1 protein expression and / or activity in any of the following: (A1) Increase the content of beneficial compounds in Cordyceps militaris; (A2) Create a white strain of Cordyceps militaris.
2. The application of substances capable of inhibiting the expression level and / or activity of CmPKS1 protein in any of the following: (A1) Increase the content of beneficial compounds in Cordyceps militaris; (A2) Create a white strain of Cordyceps militaris.
3. Use according to claim 1 or 2, characterized in that: In (A2), the white strain of Cordyceps militaris is a white strain of Cordyceps militaris with increased content of beneficial compounds; and / or The content of the beneficial compounds is the content of beneficial compounds in the fruiting body of Cordyceps militaris; and / or The beneficial compounds are all or part of the following: cordycepin, denticulaflavonol, dehydrocorydaline, PC (34:5), trigonelline, nevirapine, guarsulinine, estrogens, and tynofovir. and / or Inhibition of the expression and / or activity of the CmPKS1 protein is achieved by knocking out or reducing the expression of the gene encoding the CmPKS1 protein in the Cordyceps militaris genome; The substance is a substance capable of knocking out or reducing the expression of the gene encoding the CmPKS1 protein in the Cordyceps militaris genome.
4. The application according to any one of claims 1-3, characterized in that: Inhibition of the expression and / or activity of the CmPKS1 protein was achieved by introducing a CRISPR / Cas9 editing tool into Cordyceps militaris that targets the gene encoding the CmPKS1 protein in the Cordyceps militaris genome; The substance is a CRISPR / Cas9 editing tool that targets the gene encoding the CmPKS1 protein in the Cordyceps militaris genome; Furthermore, the target sequence of the CRISPR / Cas9 editing tool is shown in SEQ ID No.
1.
5. Any of the following methods: Method I: A method for increasing the content of beneficial compounds in Cordyceps militaris, comprising the following steps: inhibiting the expression level and / or activity of CmPKS1 protein in the Cordyceps militaris genome, thereby increasing the content of beneficial compounds in Cordyceps militaris; Method II: A method for creating a white strain of Cordyceps militaris, comprising the following steps: inhibiting the expression level and / or activity of CmPKS1 protein in the Cordyceps militaris genome, thereby creating a white strain of Cordyceps militaris.
6. The method according to claim 5, characterized in that: In Method II, the white Cordyceps militaris strain is a white Cordyceps militaris strain with increased content of beneficial compounds; and / or The content of the beneficial compounds is the content of beneficial compounds in the fruiting body of Cordyceps militaris; and / or The beneficial compounds are all or part of the following: cordycepin, denticulaflavonol, dehydrocorydaline, PC(34:5), trigonelline, nevirapine, gersoline, estrogens, and tynofovir.
7. The method according to claim 5 or 6, characterized in that: In the method, the expression level and / or activity of the CmPKS1 protein in the Cordyceps militaris genome are inhibited by knocking out or reducing the expression of the gene encoding the CmPKS1 protein in the Cordyceps militaris genome.
8. The method according to any one of claims 5-7, characterized in that: In the method, the expression level and / or activity of the CmPKS1 protein in the Cordyceps militaris genome are suppressed by introducing a CRISPR / Cas9 editing tool that targets the gene encoding the CmPKS1 protein in the Cordyceps militaris genome into Cordyceps militaris. Furthermore, the target sequence of the CRISPR / Cas9 editing tool is shown in SEQ ID No.
1.
9. The application or method according to any one of claims 1-8, characterized in that: The CmPKS1 protein is any one of the following: (B1) A protein with the amino acid sequence SEQ ID No. 2; (B2) The amino acid sequence shown in SEQ ID No. 2 is modified by substitution and / or deletion and / or addition of one or more amino acid residues; (B3) is a protein that has 99%, 95%, 90%, 85% or 80% identity with any of the amino acid sequences defined in (B1)-(B2) and is derived from Cordyceps militaris and has the same function.
10. The application or method according to any one of claims 1-9, characterized in that: The gene encoding the CmPKS1 protein is any one of the following: (C1) The DNA molecule shown in SEQ ID No. 3; DNA molecules that have 99%, 95%, 90%, 85%, or 80% identity with the DNA sequences defined by (C2) and (C1) and encode the CmPKS1 protein.