CN114717250B - Method for improving cordycepin yield by modifying cordycepin based on cofactor metabolic engineering strategy and application - Google Patents

Method for improving cordycepin yield by modifying cordycepin based on cofactor metabolic engineering strategy and application Download PDF

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CN114717250B
CN114717250B CN202210150422.1A CN202210150422A CN114717250B CN 114717250 B CN114717250 B CN 114717250B CN 202210150422 A CN202210150422 A CN 202210150422A CN 114717250 B CN114717250 B CN 114717250B
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cordyceps militaris
cordycepin
gsda
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王亮
康亚然
李倩
黄翠婷
程婧妍
刘冰南
李成
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Dalian Polytechnic University
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Abstract

The invention discloses a method for improving cordycepin yield by modifying cordycepin based on cofactor metabolic engineering strategies and application thereof. The invention utilizes agrobacterium tumefaciens mediated genetic transformation technology to introduce the recombinant vector of the overexpression glucose-6-phosphate dehydrogenase encoding gene gsdA and/or 6-phosphogluconate dehydrogenase encoding gene gndA into cordyceps militaris, so as to construct a cordyceps militaris recombinant strain, strengthen the supply of intracellular NADPH and remarkably improve the fermentation yield of cordycepin through liquid submerged fermentation. The yield of 20d cordycepin obtained by liquid fermentation of the recombinant strain over-expressing gsdA reaches 191.95mg/L, which is 150.86% higher than that of wild Cordyceps militaris; the recombinant strain of the overexpressed gndA has the yield of 20d cordycepin of 764.48mg/L by liquid fermentation, which is 1034.02 percent higher than that of wild cordyceps militaris. The method for reconstructing the cordyceps militaris strain to construct the recombinant engineering strain based on the cofactor metabolic engineering strategy provides a new direction for breeding the cordyceps militaris strain with high cordycepin yield.

Description

Method for improving cordycepin yield by modifying cordycepin based on cofactor metabolic engineering strategy and application
Technical Field
The invention relates to a method for improving cordycepin yield of cordyceps militaris by utilizing cofactor metabolic engineering strategies, and belongs to the technical field of bioengineering.
Background
Cordyceps militaris (Cordyceps militaris) is a traditional fungus for both medicine and food in China, is Cordyceps of Clavipitaceae of Ascomycetes, and has high medicinal value and nutritive value. Researches show that the chemical components of the cordyceps militaris comprise various active components such as Cordycepin, adenosine, polysaccharide, fatty acid, amino acid, ergosterol and the like, and the Cordycepin (Cordycepin) is one of the main active components and is also an important index for evaluating the quality of the cordyceps militaris. The pharmacological activity of cordycepin includes anti-tumor, anti-mutation, antifungal, immunoregulation and other aspects, and the cordycepin has been used as a new anticancer and anti-leukemia medicine in the United states for clinical trial. The traditional cordycepin is extracted from natural cordyceps militaris (but with lower content), and the natural cordyceps militaris grows slowly and is excessively developed, so that market demands cannot be met, and the cordycepin price is increased, and the current selling price exceeds 500000 dollars/kg. Cordycepin can be obtained by chemical synthesis and biological methods. The chemical synthesis method has the advantages of high raw material cost, complex process, low yield and more byproducts, and limits the industrial application of the chemical synthesis method; the biological method for synthesizing cordycepin mainly adopts a liquid fermentation mode, and the liquid fermentation has the advantages of easy control of fermentation process, short period and the like compared with other methods. At present, the measures for improving the cordycepin yield mainly accumulate a great deal of work in the aspects of strain improvement, process technology optimization and the like, such as mutation breeding, carbon source utilization efficiency improvement, precursor supply improvement and the like.
Cofactor metabolic engineering strategies are based on the regulation of cofactors (NADPH/NADP) for intracellular key enzymes + 、NADH/NAD + ATP/ADP, etc.), increases the metabolic pathway flow of the target product, and increases the yield of the target metaboliteHas been successfully applied to various microorganisms such as Saccharomyces cerevisiae, aspergillus niger, etc. Cofactors are basic regulators in organisms, involved in various biological processes (e.g., energy metabolism, anti-redox pressure, cell senescence/death, etc.), and are cooperatively involved in regulating the same biological response. NADP (NADP) + NADPH is one of the important cofactors in organisms, and NADPH acts as a reducing power affecting the synthesis reaction of amino acids, lipids and nucleotides. Thus, adequate NADPH supply is necessary to maintain intracellular redox balance and amino acid biosynthesis.
The pentose phosphate pathway is critical for maintaining intracellular NADPH production, and regulation of pentose phosphate pathway flux is an important strategy for cofactor metabolic engineering, and intracellular NADPH/NADP is regulated by increasing the enzymatic activities of glucose-6-phosphate dehydrogenase (G6 PDH), 6-phosphogluconate dehydrogenase (6 PGD), and other key enzymes involved in NADPH synthesis + Balance, and further regulate product synthesis. G6PDH is a key regulator enzyme of the first reaction of the pentose phosphate pathway, catalyzing the conversion of glucose-6-phosphate to 6-phosphogluconolactone and NADPH; the 6PGD catalyzes the third step of the pentose phosphate pathway, catalyzing the 6-phosphogluconate reaction to ribulose-5-phosphate and NADPH. Based on cofactor metabolic engineering strategies, the encoding gene of the over-expressed G6PDH or 6PGD strengthens intracellular NADPH supply, and the method has feasibility of improving cordycepin yield in cordyceps militaris.
Disclosure of Invention
The invention aims to reform Cordyceps militaris strain by over-expressing key enzyme coding genes participating in NADPH synthesis based on cofactor metabolic engineering strategy and applying genetic engineering technology, strengthen intracellular NADPH supply and obtain Cordyceps militaris recombinant strain with high cordycepin yield.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the primary purpose of the invention is to request the application of over-expressed key enzyme coding genes involved in NADPH synthesis in improving cordycepin yield, wherein the gene gsdA for the key enzyme (G6 PDH or 6 PGD) for NADPH synthesis has a sequence shown as SEQ ID NO.3, and the gene gndA has a sequence shown as SEQ ID NO. 4.
The invention provides a method for improving cordycepin yield based on cofactor metabolic engineering strategies, which comprises the steps of constructing a cordycepin mutant strain with target gene over-expression through a genetic engineering means; the target gene is glucose-6-phosphate dehydrogenase (G6 PDH) encoding gene gsdA or glucose-6-phosphate dehydrogenase (6 PGD) encoding gene gndA.
Specifically, the recombinant strain of Cordyceps militaris over-expressing gsdA or gndA gene is constructed by the following method:
(1) Extracting mRNA of Cordyceps militaris C.militaris, reversely recording and preparing cDNA, and cloning gsdA or gndA genes by taking the cDNA as a template;
(2) Connecting a linearization vector with a target gene gsdA or gndA, transferring into an escherichia coli DH5 alpha competent cell, and screening to obtain escherichia coli of a gene overexpression plasmid pDHt-SK-BenA-gsdA/gndA after PCR amplification, enzyme digestion and sequencing verification;
(3) Transferring the plasmid into agrobacterium tumefaciens Agrobacterium tumefaciensAGL, culturing by using a YEB solid screening culture medium (containing 50-100 mug/mL carbenicillin and 50-100 mug/mL kanamycin), and screening after PCR verification and enzyme digestion verification to obtain the agrobacterium tumefaciens containing the plasmid pDHt-SK-BenA-gsdA/gndA;
(4) After agrobacterium tumefaciens (containing plasmid pDHt-SK-BenA-gsdA/gndA) is subjected to induction culture by an IM liquid culture medium, co-culturing with a wild cordyceps militaris C.militaris spore suspension, and screening by using an M-100 solid culture medium (containing cefotaxime and benomyl) resistance to obtain a cordyceps militaris recombinant strain;
(5) Extracting genome from the Cordyceps militaris recombinant strain obtained by screening, performing shake flask liquid fermentation after PCR verification is correct, detecting cordycepin yield in fermentation broth by HPLC, and screening to obtain Cordyceps militaris recombinant strain with improved cordycepin yield;
(6) And carrying out subculture for multiple times and carrying out liquid fermentation verification to finally obtain the Cordyceps militaris recombinant strain with high cordycepin yield and stable inheritance.
Further, the gene symbol of the glucose-6-phosphate dehydrogenase gene in the step (1) is gsdA, genebank nucleotide sequence number CCM_06983; the gene symbol of the gluconic acid-6-phosphate dehydrogenase gene is gndA, genebank nucleotide sequence number CCM_06873. The target gene gsdA (or gndA) is obtained by amplifying cordyceps militaris cDNA by utilizing a PCR technology, wherein the cordyceps militaris is C.militaris CGMCC3.14242, and the gsdA/gndA gene PCR amplification primer pairs are respectively as follows:
gsdA-F：5’-ATGATGCACAAGACCATTAAGAACA-3’；
gsdA-R：5’-TTACAGCTTGTTGCTAGAGTTGTTGT-3’；
gndA-F：5’-ATGTCTGGTCCTGTTGCTCGA-3’；
gndA-R：5’-TTAAGCCTGGTAGGTAGAGGCAG-3’。
further, the linearization vector in the step (2) is pDHt-SK-BenA, and the gene sequence of the BenA is shown in SEQ ID NO. 5.
Further, the components (g/L) of the YEB solid medium in the step (3) are as follows: peptone 10, sucrose 5, yeast extract 1, mgSO 4 ·7H 2 O0.5, agar powder 20.
Further, the culture medium in the step (4) comprises the following components:
IM liquid medium (g/L): 400mL of 2.5 XMM salt, 1.8g of glucose, 5mL of glycerol (40 mL of 1M MES and 20mL of 10mMAS were added after sterilization and cooling to 50 ℃). Wherein the MM salt (g/L) comprises: KH (KH) 2 PO 4 3.625、K 2 HPO 4 6.72、MgSO 4 ·7H 2 O 1.250、NaCl 0.375、CaCl 2 0.125、FeSO 4 ·7H 2 O 0.0062、(NH 4 ) 2 SO 4 1.250。
IM solid medium (g/L): 400mL of 2.5 XMM salt, 0.9g of glucose, 5mL of glycerol, and 14g of agar (40 mL of 1M MES and 20mL of 10mMAS were added after sterilization and cooling to 50 ℃).
Further, the cordyceps militaris recombinant strain liquid fermentation medium in the step (5) comprises the following components:
a synthetic medium (ZL 2020111857983) is used, which comprises glucose, inorganic salts, amino acids and vitamin B 1 ；
The method specifically comprises the following steps: glucose 20-50g/L, ammonium sulfate 5-10g/L, dipotassium hydrogen phosphate 0.5-1g/L, potassium dihydrogen phosphate 0.5-1g/L, magnesium sulfate 0.5-10g/L, zinc sulfate 0.5-15g/L, glycine 5-10g/L, 0.5-1g/L aspartic acid, 0.5-2g/L glutamine, 0.1-0.5g/L tyrosine, 0.05-0.2g/L cysteine, 0.5-1g/L leucine, 0.5-2g/L lysine, 0.05-0.5g/L phenylalanine, vitamin B 1 0.1-1g/L。
Compared with the prior art, the invention has the beneficial effects that: the strain with the gene overexpression of the gsdA (or gndA) is constructed by a genetic engineering technical means, compared with a wild strain, the yield of cordycepin is obviously improved by the recombinant strain gsdA-OE and the recombinant strain gndA-OE, the yield of the cordycepin obtained by liquid submerged fermentation of 20d reaches 191.95 +/-7.67 mg/L and 764.48 +/-12.80 mg/L respectively, and the fermentation yield of the strain is respectively improved by 150.86% and 1034.02% compared with that of the wild strain, so that a new direction is provided for breeding the strain with the cordycepin produced in high yield.
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FIG. 1 shows the PCR amplification results of the promoter, terminator and target gene on the over-expression vector constructed according to the present invention. Wherein, (a) M: DL2000 DNA Marker,1-2: gpd master; (b) M: DL2000 DNA Marker,1-2: NOS terminator; (c) M DL5000 DNA Marker,1-2: a gsdA fragment; (d) M: DL2000 DNA Marker,1-2: a gndA fragment; (e) M: DL10000 DNA Marker,1-2: pDHt-SK-BenA double cleavage (SpeI-HF & EcoRI-HF).
FIG. 2 shows the PCR positive identification result of the gsdA overexpression vector constructed according to the invention. Wherein, (a) the M is DL5000 DNA Marker,1-2 is pDHt-SK-BenA-gsdA (E.coli DH5 alpha) bacterial liquid PCR verification; (b) M: DL5000 DNA Marker,1: pDHt-SK-BenA-gsdA (E.coli DH 5. Alpha.) plasmid PCR verification; (c) M: DL15000 DNA Marker,1-3: pDHt-SK-BenA-gsdA (E.coli DH 5. Alpha.) enzyme digestion verification; (d) M: DL5000 DNA Marker,1: pDHt-SK-BenA-gsdA (Agrobacterium tumefaciens AGL 1) bacterial liquid PCR verification; (e) M: DL2000 DNA Marker,1: pDHt-SK-BenA-gsdA (Agrobacterium tumefaciens AGL 1) plasmid PCR verification; (f) M: DL15000 DNA Marker,1-3: pDHt-SK-BenA-gsdA (Agrobacterium tumefaciens AGL 1) enzyme digestion verification.
FIG. 3 shows the PCR positive identification result of the gndA overexpression vector constructed in the invention. Wherein, (a) M is DL5000 DNA Marker,1 is pDHt-SK-BenA-gndA (E.coli DH5 alpha) bacterial liquid PCR verification; (b) M: DL15000 DNA Marker,1-2: pDHt-SK-BenA-gndA (E.coli DH 5. Alpha.) plasmid PCR verification; (c) M: DL15000 DNA Marker,1-3: pDHt-SK-BenA-gndA (E.coli DH 5. Alpha.) enzyme digestion verification; (d) M: DL5000 DNA Marker,1: pDHt-SK-BenA-gndA (Agrobacterium tumefaciens AGL 1) bacterial liquid PCR verification; (e) M: DL10000 DNA Marker,1: pDH t-SK-BenA-gndA (Agrobacterium tumefaciens AGL 1) plasmid PCR verification; (f) M: DL15000 DNA Marker,1-3: pDHt-SK-BenA-gndA (Agrobacterium tumefaciens AGL 1) enzyme digestion verification.
FIG. 4 shows the PCR positive identification result of the cordyceps militaris transformed by the over-expression vector constructed by the invention. Wherein, (a) M is DL2000 DNA Marker,1-4 is that simple genomic DNA of gsdA over-expression transformant is extracted as template PCRBenA fragment (901 bp); (b) Extracting genomic DNA of the gsdA over-expression transformant as a template for PCR, wherein M is DL2000 DNA Marker,1-5: gsdA-BenA (1980 bp); (c) DL2000 DNA Marker,1-3, extracting simple genomic DNA of the gndA over-expression transformant as a template PCR BenA fragment (901 bp); (d) Extracting genomic DNA of the gndA over-expression transformant as a template for PCR, wherein M is DL2000 DNA Marker,1-2: gndA-BenA (1880 bp).
FIG. 5 shows the liquid fermentation phenotype parameters of the Cordyceps militaris mutant strain over-expressing the gsdA/gndA gene constructed in the invention. Wherein, (a-c): biomass (a), residual sugar concentration (b) and cordycepin concentration (c) profiles of gsdA over-expression and control groups; (d-f): biomass (d), residual sugar concentration (e) and cordycepin concentration (f) profiles for the gndA overexpressed and control groups.
FIG. 6 is a flow chart of a recombinant plasmid over-expressing glucose-6-phosphate dehydrogenase/gluconic acid-6-phosphate dehydrogenase constructed according to the present invention. (a) pDHt-SK-BenA-gsdA; (b) pDHt-SK-BenA-gndA.
Detailed Description
The invention is further illustrated below in connection with specific examples, but the scope of the invention is not limited thereto, as set forth in the following claims. The Cordyceps militaris strain C.militaris and the agrobacterium tumefaciens A.tumefaciens AGL1 are all known strains in the art and can be obtained from commercial sources. The Cordyceps militaris used in the embodiment is C.militaris CGMCC3.14242 and is preserved by China general microbiological culture Collection center.
Example 1: construction of recombinant expression vector pDHt-SK-BenA-gsdA/gndA
The gsdA/gndA gene, gpd promoter and nos terminator fragment (FIG. 1) were PCR amplified using the primers shown in Table 1 with the cDNA of C.militaris CGMCC3.14242 as a template, and the target fragment was recovered using a DNA gel recovery kit (Nannunofizan Biotechnology Co., ltd.). And (3) carrying out gene sequencing on the gel-collected products by the large gene technology Co-Ltd, and selecting PCR gel-collected products with correct sequencing for subsequent experiments.
The pDHt-SK-BenA expression vector stored in this laboratory was subjected to double digestion (SpeI-HF, ecoRI-HF) (FIG. 1), and the gpd promoter fragment, the gsdA/gndA gene fragment, and the nos terminator fragment were ligated to the linearized expression vector pDHt-SK-BenA using ClonExpress Ultra One Step Cloning Kit (Nanjinouzan Biotechnology Co., ltd.), to finally construct the gsdA/gndA overexpression plasmid pDHt-SK-BenA-gsdA/gndA.
TABLE 1 primers used in this experiment
Example 2: agrobacterium tumefaciens AGL 1-mediated pDHt-SK-BenA-gsdA/gndA transfer to Cordyceps militaris
The recombinant plasmid pDHt-SK-BenA-gsdA/gndA is transferred into escherichia coli DH5 alpha by a chemical conversion method to obtain a large number of recombinant expression plasmids, and sequencing verification is carried out by Hua big gene technology Co. Further, the recombinant plasmid pDHt-SK-BenA-gsdA/gndA is transferred into the agrobacterium tumefaciens AGL1 by a chemical conversion method, and the agrobacterium tumefaciens AGL1 containing the pDHt-SK-BenA-gsdA/gndA plasmid is obtained through PCR verification and enzyme digestion verification. YEB medium (g/L) used for Agrobacterium tumefaciens culture: peptone 10, sucrose 5, yeast extract 1, mgSO 4 ·7H 2 O0.5; the solid culture medium is added with 20g of agar powder. LB liquid medium (g/L): tryptone 10, yeast extract 5, naCl 10, pH 7.0; the solid culture medium is added with 20g of agar powder.
Recombinant plasmid using agrobacterium-mediated transformationpDHt-SK-BenA-gsdA/gndA is transferred into Cordyceps militaris Cordyceps militaris CGMCC3.14242 to obtain Cordyceps militaris transformant. The transformation process is as follows: agrobacterium tumefaciens AGL1 containing recombinant expression plasmid was added to liquid YEB resistant medium (carbenicillin and kanamycin), cultured overnight at 28℃at 220rpm, and then the cells were collected, and then an appropriate amount of IM liquid medium was added to resuspend the cells (OD 600 About 0.15), further induced culture at 28℃at 150rpm to OD 600 =0.5 to 0.8. Mixing the induced Agrobacterium tumefaciens AGL1 bacterial liquid and freshly prepared Cordyceps militaris spore suspension in a 1.5mL sterile EP tube, uniformly coating 100 mu L of the mixed liquid on an IM solid culture medium, and culturing for 2-3d at 28 ℃ in an inversion way. Then, a proper amount of M-100 solid resistant medium (containing cefotaxime and benomyl) is added to the plate, and the culture is continued at 25 ℃ until single colonies grow. The colonies were picked up on a new M-100 resistant medium plate for secondary screening, colonies on the secondary screening plate were subjected to shaking culture with SDB liquid medium, mycelium genome was extracted for PCR verification, and primers used are shown in Table 1.
Example 3: cordyceps militaris eggplant bottle culture and preparation of spore suspension
Filling a proper volume of natural solid culture medium into an eggplant bottle, sterilizing, inoculating proper amount of Cordyceps militaris recombinant strain mycelium after the eggplant bottle is solidified, and standing at 25 ℃ for 20-25d. Adding appropriate amount of physiological saline into the bottle of the eggplant with the vigorous Cordyceps militaris mycelia, stirring with a sterile glass rod, filtering the liquid in the bottle with three layers of gauze to obtain spore suspension, and calculating the spore concentration.
Example 4: determination of cordycepin content in cordyceps militaris fermentation broth
The cordycepin content in the fermentation broth is detected by high performance liquid chromatography, and the conditions of the high performance liquid chromatography are as follows: waters e2695, waters 2998, 260nm ultraviolet detector wavelength, venusil MP C18 (2) (4.6X105 mm,5 μm) column temperature setting room temperature, mobile phase condition methanol: water, flow rate 0.8-1.0mL/min, and sample injection amount 10-20 μl.
Example 5: liquid fermentation of recombinant strain of Cordyceps militaris and cordycepin content determination
Selecting Cordyceps militaris strain C.militaris CGMCC3.14242 as a control group, and performing submerged liquid fermentation of the Cordyceps militaris recombinant strain over-expressing gsdA/gndA. A250 mL conical flask is used for carrying out a liquid submerged fermentation experiment, cordyceps militaris spore suspension is inoculated according to 1% of inoculation amount, and the cordyceps militaris spore suspension is subjected to shaking culture for 20-30d at the temperature of 25-28 ℃ and the speed of 160-180r/min, and the biomass concentration, the residual sugar concentration and the cordycepin yield are sampled and measured (figure 5).
When glucose-6-phosphate dehydrogenase (gsdA) is overexpressed, glucose consumption was slower for the first 5 days than for the control, with the rate of glucose consumption being significantly higher at residues 5-10d than in the wild-type (control) and the substrates glucose was substantially depleted for both groups 10 d. From the growth curve, the gsdA-OE group grows slower than the control group, the biomass concentration of the fermentation 10d is 13.13+/-0.32 g/L, and the biomass concentration of the control group reaches 14.73+/-0.42 g/L; the over-expression of glucose-6-phosphate dehydrogenase (gsdA) enhances the synthesis capacity of cordycepin, and the yield of the cordycepin reaches 191.95 +/-7.67 mg/L at 20d, which is improved by 150.86 percent compared with a control group.
When overexpressing 6-phosphogluconate dehydrogenase (gndA), the glucose consumption curve has the same trend as the gsdA-OE group. From the growth curve, the growth rate of the wild strain is higher than that of the gndA-OE group in the first 5 days, the growth rate of the experimental group of 5-10 days is greatly improved, the biomass concentration of the fermentation 10d is 13.78+/-1.14 g/L, and the biomass concentration of the control group reaches 13.63+/-0.62 g/L; from the cordycepin synthesis curve, the yield of the 20d cordycepin reaches 764.48 +/-12.80 mg/L, and is improved by 1034.02 percent compared with the control group.
The result shows that after the gsdA or gndA (encoding the key enzyme G6PDH or 6PGD synthesized by NADPH) is overexpressed, the cordycepin synthesis capability of the recombinant strain of the Cordyceps militaris is obviously improved compared with that of the wild strain, and the enhancement of the supply of the cofactor NADPH is helpful for the biosynthesis of cordycepin.
Sequence listing
<110> university of Dalian industry
<120> method for improving cordycepin yield by modifying cordycepin based on cofactor metabolic engineering strategy and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1035
<212> DNA/RNA
<213> gpd promoter sequence (Artificial sequence)
<400> 1
ggccaatttg tctattctgg tagttccgcc gtggccaact tgacggttca ctggttttgt 60
gtcttgatga ctgacaaaga gggccagaga tgagacgacg agctgccggt tgcaccatca 120
gccaaccgtt cgcagaaatc atcagttgcg ctacagtcaa catagcgggt ggtcaaagtt 180
tgcgtgggca cgggttgccg cacgagttgg aacgtcagta aagtggggga ggggaagcaa 240
ccagaacgag gccaattgaa tgggatgaag gcgtctggga gggaagcacg agcgtgaatg 300
aatggacggt tgcgtgactc tggtctcgcg atttcgatcg agtcaaggaa cccgtatggt 360
tgcctcttgt cttggagagc tcgtcgaggc ccaagctgcg agtatatggt tggtgatgcg 420
ctgggcattt gcccctctct gcccctccat ggacggacct gcagctcttg ggctggcaag 480
gtgttgcgct acgtcgagat gcggttaggt aagctggcat aagctggcat aaggttggct 540
gttcttgaga aatcaggcca gcagtcttgc acctctctgg ctacaggctc tccagcggtc 600
agccaccgcc actccagcgc ctgtttttca ctgacgatgg ccttgttttt ttgttgctct 660
ggcagctggc aagctgggtc ccggcagctg ccacggccaa gccacccagg actacaaaac 720
catttgccca cccctcctct tcgccgtgcg tctcttcttt ttctcccctc cattacacct 780
cattcttcag ttcaaaggaa caaaagtgag ttttccatcc gtcccacacc tggctccatc 840
atctcgagct tcattgcttc ttgaccatct accacgctct accctccaac agcttcgctc 900
acccaagagc tgcctgcttt tcccctcctc tccacgaccc cgccatccac cacttaacct 960
aaagttaccc ctcgctaacc gccaaacgtc ccaacaggtt tttcttctct cacctcactt 1020
ttctaatcaa gaaca 1035
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gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atc 253
<210> 3
<211> 1830
<212> DNA/RNA
<213> gsdA Gene sequence (Artificial sequence)
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atgatgcaca agaccattaa gaacatccag aacacctact acaagaccga caccgccgcc 60
ggccagaccc ccggcgccgg ccccggcgtt acctacctgg aatctgaccg tgctttcggc 120
taccagcgaa ttattcccag cagcgctatt gctctggtta gcaagtcgcg tcccaagctc 180
gacctccagc agctgaacgg cctgtcgtct tctagcttcc ccctgtctcc cacccgccac 240
tcgtcgtctt ggaccgcttc tagcctccga ttcaacttcc gaccctcgcg tccccccccc 300
tggtctaagc ccctgccaat ggagctgaag gagaacacca ccatcgttgt cctgggcgcc 360
tccggcgacc tcgctaagaa aaaaacctac cccgctctgt tcggcctgta ccgtaaccag 420
ttcctgccca aggacgtccg cattgttggc tacgcccgaa ccaagatgga ccacgaggag 480
tacctgcgtc gtattaagtc gtacatgaaa acccccacca aggagattga gcagcagctg 540
gaggagttct gcaagatttg ctcttacgtt tcgggccagt acgacaagga cgagtctttc 600
gaggctctca ccaagcacat ggaggagtta gaaaagggcc gtcccgagca caaccgactg 660
ttctacatgg ctctgccccc ctcggttttc accattgttt ctcagcacct caagaagtgc 720
tgctacccct ctaagggcat tgctcgtgtt atcgttgaga agcccttcgg caaggacctg 780
gccagcagcc gtgagttaca gaagtctctc gaacccgact ggaaggagga ggagctgtac 840
cgaattgacc actacctggg caaggagatg gttaagaaca tcctgattat gcgattcggc 900
aactctttcc tgggcgccac ctggaaccgt caccacattg acaacgtcca gatcaccttc 960
aaggagccct tcggcaccga gggccgtggc ggctacttcg acgagttcgg catcatccgt 1020
gacgtcatgc agaaccacct cctccaggtt ctcaccctcc tcgctatgga gcgtcccgtt 1080
tctttcgact ctgaggacat ccgtgacgag aaggtccgtg ttctgcgtgc tattagcgct 1140
ttggaaccca agaacgttat cattggccag tacggccgat cgctcgacgg ctctaagccc 1200
gcttacaagg aggacgacac cgttccccag gactcgcgat gccccacctt ctgcgctctg 1260
gttgcttaca ttaagaacga gcgatgggac ggcgttccct tcatcatgaa ggccggcaag 1320
gctctcaacg agcaaaagac cgagatcagg atacagttca aggacgttac ctcgggcatt 1380
ttcaaggaca ttccccgtaa cgagctggtt atgcgtatac agcccaacga gtcggtttac 1440
attaagatga actctaagct gcccggcctg tctatgcaga ccgttgttac cgagctggac 1500
ctcacctacc gccgtcgatt cagcgacctg aagattcccg aggcttacga gtcgctggtc 1560
ctggactgcc tgaagggcga ccactccaac ttcgttcgtg acgacgagct ggacgcttct 1620
tggcgtattt tcacccccct cctgcactac ctcgacgaca acaaggagat cattcctatg 1680
gagtacccct acggctcgcg tggccccgct gtcctggacg acttcaccgc ttcttacggc 1740
tacaagttct ctgacgccgc cggctaccag tggcccacca ccagcgctgt tccctctaac 1800
aacaacaaca actctagcaa caagctgtaa 1830
<210> 4
<211> 1593
<212> DNA/RNA
<213> gndA Gene sequence (Artificial sequence)
<400> 4
atgtctggtc ctgttgctcg actcgccaac atcaagcttg gctcccacga cagcaacaac 60
ccctcgtcca cgtctcctgc cactggcagt actcatccat tctcaagtgc taacaatgcc 120
cccagcgcgg atctcggcct cattggcctc gctgtcatgg gacagaacct gatcctcaac 180
atggccgatc acggcttcac catctgcgct ttcaaccgaa ccgtctccaa ggtcgaccac 240
ttcttggaaa atgaggccaa aggcaagtcc atcgtcggtg cccactccgt cgaagagttc 300
gtctccaagc tcaagacccc ccgccgcgtc atgctccttg tccaggccgg ccaggctgtc 360
gatgactgga ttgagaagct tctgcccctc ctggagaagg gtgacatcat tattgatggt 420
ggcaactcgc atttccccga ctctaaccgc cgcaccaagt acctcaccag caagggcctt 480
cgcttcgtcg gctccggtgt ttctggtggt gaggaaggtg cccgttacgg cccctccatc 540
atgcctggtg gtgatgagga agcttggcct catatcaagg aaatcttcca gggcatttct 600
gccaagtctg atggtgaggc ctgctgcgag tgggtcggtg acgagggtgc tggccactat 660
gttaagatgg ttcacaacgg tattgagtat ggtgacatgc agctcatctg cgaggcctac 720
gatatcatga agcgtgccct cggcctctcc aataaggaga tcggcgatgt cctcgccaag 780
tggaacaagg gtgttcttga ctctttcctc atcgaaatca cccgcgacat catgtacttc 840
aacgatgatg atggtaccgc cctcgtcgag aagattctcg acaaggccgg tcagaagggt 900
accggcaagt ggactgccgt caacgctctc gacctcggta tgcccgtcac cctcattgct 960
gaggctgtcc ttgccagatg cctgtctggc atcaaggagg agcgtgaggt tgcctcgacc 1020
aagctccagt acgtttcgcg caacagcggc aagttcgagg gcaacaagga gcaattcctc 1080
gatgatcttg agcaggctct ctacgcgtcc aagattatct cgtacgccca aggtttcatg 1140
ctgatgcagg aggctgccaa ggagtacggc tggaaactga acaagccctc gattgccctc 1200
atgtggcgtg gcggctgcat catccgctcc gtcttcctca aggacatcac cgccgcctac 1260
cgcaaggaca atgaccttgc caacctcctc ttcgacgact tcttcaacaa ggccatccac 1320
aaggcccagc ccggctggag agacgtcgtc gcccaggctg ctcagcttgg tatccctacc 1380
cccgccttct ctaccgctct gtcgtggttc gacggctacc gcaccaagga cctgcctgcc 1440
aacctgctcc aggcccagcg tgattacttt ggtgctcaca ccttccacat caagcctgag 1500
gctgccaacg ccaagtacca ggtcggcaag gacattcacg tcaactggac tggccgtggt 1560
ggcaacgtct ctgcctctac ctaccaggct taa 1593
<210> 5
<211> 1344
<212> DNA/RNA
<213> BenA Gene sequence (Artificial sequence)
<400> 5
atgcgtgaga ttgtccactt gcaaaccggc cagtgcggca accagattgg cgccgctttc 60
tggcagacca tctcgggcga gcacggcctc gacggctcgg gcgtttacaa cggcacctct 120
gacctccagc tagagcgtat gaacgtttac ttcaacgagg cgtccggcaa caagtacgtc 180
ccccgtgctg ttctggttga cctggagccc ggcactatgg acgctgttcg tgccggcccc 240
ttcggccagc tgttccgtcc cgacaacttc gttttcggcc agtctggcgc tggcaacaac 300
tgggctaagg gccactacac cgagggcgct gaactggttg accaggtcct ggacgttgtc 360
cgtcgtgagg ctgagggctg cgactgccta caaggcttcc agattaccca cagcctgggc 420
ggcggcaccg gcgccggcat gggcaccctc ctcatctcta agatccgtga ggagttcccc 480
gaccgaatga tggctacctt ctcggttgtc ccctcgccca aggtatcgga caccgttgtt 540
gagccctaca acgctaccct cagcgtccac cagctggtcg agaactctga cgctaccttc 600
tgcattgaca acgaggctct gtacgacatt tgtatgcgaa ccctgaagct gtctaacccc 660
tcctacggcg acctgaacca cctggttagc gctgttatgt cgggcgtcac cacctgcctg 720
cgattccccg gccagctcaa ctcggacctc cgaaagctcg ccgttaacat ggtccccttc 780
ccccgactgc acttcttcat ggttggcttc gctcccctca ccagccgcgg cgcccactct 840
ttccgcgccg ttaccgtccc cgagctgacc cagcagatgt acgaccccaa gaacatgatg 900
gccgcctcag acttccgaaa cggccgatac ctcacctgct ccgccatttt ccgtggcaag 960
gttagcatga aggaggtcga ggaccagatg cgtaacgttc agaacaagaa ctcttcttac 1020
ttcgtcgagt ggattcccaa caacgttcag accgccctgt gctctattcc cccccgtggc 1080
ctcaagatgt cgtctacctt cgttggcaac tctacctcta ttcaggagct gttcaagcgt 1140
gttggcgacc agttcaccgc tatgttccgt cgtaaggctt tcctccactg gtacaccggc 1200
gagggcatgg acgagatgga gttcaccgag gctgagtcta acatgaacga cctggtttcg 1260
gagtaccagc agtaccagga tgcctcgatc tctgagggcg aggaggagta cgaggaggag 1320
gttcccattg agggcgagga gtaa 1344

Claims (4)

1. A method for improving cordycepin yield by modifying cordycepin based on cofactor metabolic engineering strategy is characterized in that:
the construction method of the cordyceps militaris recombinant strain comprises the following steps:
s1, extracting cordyceps militarisCordyceps militarisIs reversely recorded to prepare cDNA, and uses the cDNA as a template to clonegsdAOr (b)gndAA gene; genegsdAHas a sequence shown as SEQ ID NO.3, and a genegndAHas a sequence shown as SEQ ID NO. 4;
s2. The target GenegsdAOr (b)gndAAfter being connected with a linearization vector pDHt-SK-BenA, the gene is transferred intoIn the competent cells of the escherichia coli, the transformant is screened by PCR amplification, enzyme digestion and gene sequencing verification to obtain the recombinant vector containing pDHt-SK-BenA-gsdAOr pDHt-SK-BenA-gndAE.coli of (E.coli);
s3, extracting plasmids from escherichia coli containing the recombinant vector obtained in the step 2 by liquid culture, transferring the recombinant vector into competent cells of agrobacterium tumefaciens AGL-1, culturing and screening transformants by using a YEB solid medium containing 50-100 mug/mL carbenicillin and 50-100 mug/mL kanamycin, and obtaining the recombinant vector containing pDHt-SK-BenA after PCR amplification and enzyme digestion verificationgsdAOr pDHt-SK-BenA-gndAIs a root agrobacterium of (2);
s4, the recombinant vector pDHt-SK-BenA obtained in the step S3 is addedgsdAOr pDHt-SK-BenA-gndAAfter inducing and culturing the agrobacterium tumefaciens in an IM liquid culture medium, co-culturing the agrobacterium tumefaciens and a cordyceps militaris spore suspension on an IM solid culture medium, covering an M-100 solid culture medium containing cefotaxime and benomyl, screening cordyceps militaris transformants, further carrying out secondary screening on the cordyceps militaris transformants on the M-100 solid culture medium containing cefotaxime and benomyl, and obtaining the overexpression glucose-6-phosphate dehydrogenase encoding gene through PCR amplification and gene sequencing verificationgsdAOr 6-phosphogluconate dehydrogenase encoding genegndACordyceps militaris recombinant strain;
target genegsdAOr (b)gndAIn order to obtain the Cordyceps militaris cDNA by using PCR technology as template, the primer pair used for PCR amplification is
gsdA-F：5’-ATGATGCACAAGACCATTAAGAACA-3’；
gsdA-R：5’-TTACAGCTTGTTGCTAGAGTTGTTGT-3’；
gndA-F：5’-ATGTCTGGTCCTGTTGCTCGA-3’；
gndA-R：5’-TTAAGCCTGGTAGGTAGAGGCAG-3’；
The linearization carrier contains benomyl resistance geneBenA expression vector pDHt-SK-BenA,Benthe A gene sequence is shown as SEQ ID NO. 5.
2. The use of the recombinant strain of Cordyceps militaris constructed by the method of claim 1 for accumulating cordycepin in a submerged liquid fermentation system.
3. The use according to claim 2, wherein the recombinant strain of Cordyceps militaris is prepared by subjecting a recombinant strain of Cordyceps militaris to submerged fermentation in a synthetic medium comprising glucose, inorganic salts, amino acids and vitamin B to accumulate cordycepin 1 。
4. Use according to claim 3, characterized in that the synthetic medium comprises in particular: 20-50g/L glucose, 5-10g/L ammonium sulfate, 0.5-1g/L dipotassium phosphate, 0.5-1g/L potassium dihydrogen phosphate, 0.5-10g/L magnesium sulfate, 0.5-15g/L zinc sulfate, 5-10g/L glycine, 0.5-1g/L aspartic acid, 0.5-2g/L glutamine, 0.1-0.5g/L tyrosine, 0.05-0.2g/L cysteine, 0.5-1g/L leucine, 0.5-2g/L lysine, 0.05-0.5g/L phenylalanine, 0.1-1 g/L vitamin B 1 。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112239728A (en) * 2020-10-30 2021-01-19 大连工业大学 Synthetic culture medium containing reductive glutathione and suitable for cordyceps militaris culture, preparation method and application

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CN112239728A (en) * 2020-10-30 2021-01-19 大连工业大学 Synthetic culture medium containing reductive glutathione and suitable for cordyceps militaris culture, preparation method and application

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CCM_06983 glucose-6-phosphate 1-dehydrogenase (G6PD) [Cordyceps militaris CM01];无;NCBI Reference Sequence: XP_006672184.1;1-2 *
Cordyceps militaris CM01 6-phosphogluconate dehydrogenase (CCM_06873), partial mRNA;无;NCBI Reference Sequence: XM_006672011.1;1-2 *
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