Recombinant cordyceps militaris bacteria, construction method thereof and application of recombinant cordyceps militaris bacteria in production of cordycepin

Abstract

The invention discloses a recombinant cordyceps militaris strain, a construction method thereof and application of the recombinant cordyceps militaris strain in production of cordycepin. A veA gene is overexpressed in the cordyceps militaris strain. The overexpression of the veA gene can promote the generation of secondary metabolin cordycepin and the growth and development of plate hyphae of cordyceps militaris under the condition of liquid standing culture, and compared with a wild strain, the yield of cordycepin under the condition of liquid culture is increased by 29.9%, and the colony diameter and the conidium number under the condition of plate culture are increased by 2.77% and 161.16% respectively. The invention provides a recombinant cordyceps militaris strain OEveA with high yield of cordycepin, provides a new method and direction for high efficiency and high yield of cordycepin, and has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant cordyceps militaris fungus, a construction method thereof, and an application of producing cordycepin through fermentation. Background Art

[0002] Cordyceps militaris, also known as northern cordyceps, is a well-known edible medicinal filamentous fungus, widely praised by Southeast Asia and other countries. At the 1727 Paris Academy of Sciences, Cordyceps militaris was published under the genus and species name Cordyceps militarys. Many bioactive ingredients have been found in Cordyceps militaris, including cordycepin, pentostatin, cordycepic acid, cordyceps polysaccharides and ergosterol. Among them, cordycepin, as one of the most important bioactive ingredients in Cordyceps militaris, has important functions such as anti-tumor, antioxidant, anti-inflammatory, immunomodulatory and for the treatment of new coronavirus. At present, cordycepin is mainly obtained through fruiting body culture and liquid fermentation. Among them, liquid fermentation has the advantages of high efficiency, easy large-scale production and short production cycle, so it is used as an efficient way to obtain cordycepin.

[0003] VeA in the velvet protein family is a well-known global regulatory factor in filamentous fungi and a light-responsive factor. It interacts with other velvet protein members (VelB, VelC, VosA) or other regulatory factors to produce different functions. The veA gene not only regulates secondary metabolism, but also regulates the growth and development of filamentous fungi, including sexual or asexual reproduction. At present, the functional research of the veA gene is mainly in molds, and there are few reports on the study of the function of the veA gene in Cordyceps militaris. One of the strategies to increase the yield of cordycepin is to construct a recombinant cordycepin strain that overexpresses or knocks out the veA gene. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a recombinant Cordyceps militaris with overexpression or knockout of the veA gene, a construction method thereof and an application of producing cordycepin in view of the fact that there are few methods in the prior art to increase the yield of cordycepin by overexpressing or knocking out the veA gene and the problem of low cordycepin yield in the prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A recombinant Cordyceps militaris fungus overexpresses a veA gene in the Cordyceps militaris fungus, wherein the nucleotide sequence of the veA gene is shown in SEQ ID NO.1.

[0007] The Cordyceps militaris is a wild type Cordyceps militaris CGMCC No. 18581, which has been deposited in the China General Microbiological Culture Collection Center, and its detailed information has been disclosed in patent CN 111117896 A.

[0008] The invention also discloses a construction method of the recombinant Cordyceps militaris, and the recombinant Cordyceps militaris is obtained by introducing a veA gene overexpression recombinant plasmid into the Cordyceps militaris through the transformation technology mediated by Agrobacterium tumefaciens.

[0009] The Agrobacterium tumefaciens-mediated transformation technology specifically comprises: introducing the veA gene overexpression recombinant plasmid into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens, and co-culturing the recombinant Agrobacterium tumefaciens with the Cordyceps militaris to obtain the recombinant Cordyceps militaris.

[0010] The construction method of the veA gene overexpression recombinant plasmid is as follows: the veA gene is connected to the plasmid pCAMBIA1303-gpdA-TrpC-Hygro by homologous recombination to obtain the veA gene overexpression recombinant plasmid in which the veA gene is driven by the strong promoter gpdA.

[0011] The plasmid pCAMBIA1303-gpdA-TrpC-Hygro is obtained by removing the EGFP gene from the plasmid pCAMBIA1303-gpdA-EGFP-TrpC-Hygro.

[0012] Specifically, the construction method of the recombinant Cordyceps militaris is as follows:

[0013] (1) The EGFP gene in the binary plasmid pCAMBIA1303-gpdA-EGFP-TrpC-Hygro was removed to obtain the plasmid pCAMBIA1303-gpdA-TrpC-Hygro;

[0014] (2) connecting the veA gene to the plasmid pCAMBIA1303-gpdA-TrpC-Hygro by homologous recombination to obtain a veA gene overexpression recombinant plasmid;

[0015] (3) introducing the veA gene overexpression recombinant plasmid into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

[0016] (4) Cultivate the recombinant Agrobacterium tumefaciens obtained in step (1) to an OD of 600 After the concentration is 0.6 to 1.0 and the concentration is 10 5 ~10 7 The wild-type Cordyceps militaris CGMCC No.18581 conidia of 100 g / mL are mixed in equal volumes and co-cultured for 2 to 5 days to obtain infected Cordyceps militaris fungi; preferably, the recombinant Agrobacterium tumefaciens obtained in step (1) is cultured to an OD of 600 After the concentration is 0.8 and 10 6The conidia of wild-type Cordyceps militaris CGMCC No.18581 with a concentration of 100 / mL were mixed in equal volumes and cultured for 3 days to obtain infected Cordyceps militaris fungi;

[0017] (5) The infected Cordyceps militaris obtained in step (4) is transferred to a resistance plate containing 400-800 μg / mL hygromycin B for culture, and the Cordyceps militaris transformants grown on the resistance plate are picked. After successful PCR verification and sequencing, the recombinant Cordyceps militaris is obtained; preferably, the concentration of hygromycin B in the resistance plate is 600 μg / mL.

[0018] The binary plasmid pCAMBIA1303-gpdA-EGFP-TrpC-Hygro is a public plasmid and can be obtained from commercial channels.

[0019] The Agrobacterium tumefaciens is Agrobacterium tumefaciens AGL1, which is a published strain and can be obtained from commercial channels.

[0020] The invention also discloses the application of the recombinant Cordyceps militaris in producing cordycepin through fermentation.

[0021] The seed liquid of the recombinant Cordyceps militaris is inoculated into a fermentation medium for culturing and fermenting to produce cordycepin; the wet weight of mycelium of the seed liquid is 15-25 g / L; and the inoculation amount of the seed liquid is 5-15% of the volume of the fermentation medium, preferably 10%.

[0022] The culture conditions of the fermentation medium are as follows: temperature of 22-30°C; pH of 5-6.5; culture time of 18-26 days; static culture under dark and / or light conditions, preferably static culture under dark conditions; there are no special requirements for the dark and light conditions.

[0023] The components of the fermentation medium used in the fermentation include glucose, peptone, yeast powder, magnesium ions and precursor substances; the precursor substances are adenosine or adenine.

[0024] Beneficial effects:

[0025] 1. Overexpression of gene veA promotes the production of secondary metabolite cordycepin and the growth and development of plate mycelium of Cordyceps militaris under liquid static culture conditions. The cordycepin yield of recombinant Cordyceps militaris overexpressing veA gene is as high as 6.52g / L under dark liquid culture for 20 days, which is 29.9% higher than that of wild-type Cordyceps militaris; the colony diameter and conidia number of recombinant Cordyceps militaris overexpressing veA gene are 2.77% and 161.16% higher than those of wild-type Cordyceps militaris respectively after plate culture for 20 days under dark conditions. The present invention provides a recombinant Cordyceps militaris OEveA with high cordycepin yield, and at the same time provides a new method and direction for high efficiency and high yield of cordycepin, which has good prospects for industrial application.

[0026] 2. The method for constructing the recombinant Cordyceps militaris provided by the present invention is simple, and only the veA gene needs to be introduced into the Cordyceps militaris, without the need to introduce other genes or perform other metabolic engineering transformation methods on the Cordyceps militaris. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0028] Figure 1 The gel electrophoresis diagram of the PCR amplification products of the upstream and downstream homologous sequences of the veA gene constructed in Example 1, the Hyg gene expression cassette fragment, the linearized knockout recombinant plasmid skeleton, and the colony PCR of the veA gene knockout recombinant plasmid; wherein, Figure a is the PCR amplification result of the upstream homologous sequence of the veA gene, wherein lane M is DL2000 DNA Marker, and lane 1 is the upstream homologous sequence of the veA gene; Figure b is the PCR amplification result of the downstream homologous sequence of the veA gene, wherein lane M is DL2000 DNAMarker, and lane 1 is the downstream homologous sequence of the veA gene; Figure c is the PCR amplification result of the Hyg gene expression cassette, wherein lane M is DL2000 DNAMarker, and lane 1 is the Hyg gene expression cassette; Figure d is the PCR amplification result of the linearized knockout recombinant plasmid skeleton, wherein lane M is DL10000 DNA Marker, lane 1 is the linearized knockout recombinant plasmid skeleton; Figure e is the PCR amplification result of veA gene knockout recombinant plasmid colony PCR, where lane M is DL5000 DNA Marker, and lane 1 is veA gene knockout recombinant plasmid colony PCR.

[0029] Figure 2The PCR amplification product gel electrophoresis diagrams of the veA gene fragment, linearized overexpression recombinant plasmid skeleton, and veA gene overexpression recombinant plasmid colony PCR constructed in Example 2; wherein, Figure a is the PCR amplification result of the veA gene fragment, wherein lane M is DL2000 DNA Marker, and lane 1 is the veA gene fragment; Figure b is the PCR amplification result of the linearized overexpression recombinant plasmid skeleton, wherein lane M is DL15000 DNA Marker, and lane 1 is the linearized overexpression recombinant plasmid skeleton; Figure c is the PCR amplification result of the veA gene overexpression recombinant plasmid colony, wherein lane M is DL2000 DNA Marker, and lane 1 is the veA gene overexpression recombinant plasmid colony PCR.

[0030] Figure 3 The colony PCR of AGL1 containing the recombinant plasmid, the colony PCR of the recombinant Cordyceps militaris containing the recombinant plasmid, the gel electrophoresis of the PCR amplification products, the growth of the recombinant Cordyceps militaris, and the statistical graph of the relative expression of the veA gene in the recombinant Cordyceps militaris constructed in Example 3. Wherein, Figure a is the colony PCR amplification result of AGL1 containing the recombinant plasmid, wherein lane M is DL2000 DNA Marker, lane 1 is the colony PCR of AGL1 containing the veA gene knockout recombinant plasmid, and lane 2 is the colony PCR of AGL1 containing the veA gene overexpression recombinant plasmid; Figure b is the growth situation of ΔveA; Figure c is the growth situation of OEveA; Figure d is the colony PCR amplification result of the recombinant Cordyceps militaris, wherein lane M is DL2000 DNA Marker, lane 1 is the colony PCR of ΔveA, and lane 2 is the colony PCR of OEveA; Figure e is the statistical graph of the relative expression of the veA gene in the recombinant Cordyceps militaris and the wild-type Cordyceps militaris.

[0031] Figure 4 The figures are actual pictures of the plate colony growth of the recombinant Cordyceps militaris and the wild-type Cordyceps militaris in Example 4 and the corresponding statistical graphs of colony diameter data, microscopic morphology of hyphae, and statistical graphs of conidia yield; wherein, Figure a is a physical picture of the plate colony growth of OEveA, ΔveA and WT cultured on a plate for 20 days; Figure b is a statistical graph of the plate colony diameter data of OEveA, ΔveA and WT cultured on a dark plate; Figure c is a statistical graph of the plate colony diameter data of OEveA, ΔveA and WT cultured on a light plate; Figure d is a microscopic morphology of mycelium of OEveA, ΔveA and WT; Figure e is a statistical graph of the conidia yield of OEveA, ΔveA and WT cultured on a dark plate; Figure f is a statistical graph of the conidia yield of OEveA, ΔveA and WT cultured on a light plate.

[0032] Figure 5Figure 5 is a colony growth diagram and colony diameter data statistical diagram of the recombinant Cordyceps militaris and wild-type Cordyceps militaris on different single carbon source plates in Example 5; wherein, Figure a is a colony growth diagram of OEveA, ΔveA and WT on different single carbon source plates; Figure b is a colony diameter data statistical diagram of OEveA, ΔveA and WT on different single carbon source plates under dark culture; Figure c is a colony diameter data statistical diagram of OEveA, ΔveA and WT on different single carbon source plates under light culture.

[0033] Figure 6 These are statistical graphs of the liquid static fermentation culture data of the recombinant Cordyceps militaris and the wild-type Cordyceps militaris under dark conditions in Example 8; wherein, Figure a is a statistical graph of the residual sugar data in the liquid static culture fermentation broth of OEveA, ΔveA and WT; Figure b is a statistical graph of the pH data of the liquid static culture fermentation broth of OEveA, ΔveA and WT; Figure c is a statistical graph of the biomass data in the liquid static culture fermentation broth of OEveA, ΔveA and WT; Figure d is a statistical graph of the cordycepin yield data of the liquid static culture of OEveA, ΔveA and WT.

[0034] Figure 7 The figures are statistical graphs of the liquid static fermentation culture data of the recombinant Cordyceps militaris and the wild-type Cordyceps militaris under light conditions in Example 8; wherein, Figure a is a statistical graph of the residual sugar data in the liquid static culture fermentation broth of OEveA, ΔveA and WT; Figure b is a statistical graph of the pH data of the liquid static culture fermentation broth of OEveA, ΔveA and WT; Figure c is a statistical graph of the biomass data in the liquid static culture fermentation broth of OEveA, ΔveA and WT; Figure d is a statistical graph of the cordycepin yield data of the liquid static culture of OEveA, ΔveA and WT. DETAILED DESCRIPTION

[0035] The present invention is further described below based on the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0036] If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased through regular channels.

[0037] The Cordyceps militaris and Agrobacterium tumefaciens AGL1 used in the following examples are both known strains and can be obtained from commercial channels.

[0038] The wild type Cordyceps militaris used in the examples was provided by the Institute of Biotechnology of Nanjing High-Tech University. The strain has been deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 18581. Its detailed information is disclosed in patent CN 111117896 A.

[0039] Example 1 Construction of veA gene knockout recombinant plasmid

[0040] Using Cordyceps militaris genomic DNA as a template, the upstream homologous sequence and downstream homologous sequence of the veA gene were amplified by primers veAUF / veAUR and veADF / veADR, respectively. Using pCAMBIA1303-gpdA-EGFP-TrpC-Hygro binary plasmid (Wuhan Miaoling Biotechnology Co., Ltd., product number: P29287) as a template, the Hyg gene expression cassette fragment was amplified by primers HygF / HygR. Using pCAMBIA1303-gpdA-EGFP-TrpC-Hygro binary plasmid as a template, the linearized knockout recombinant plasmid skeleton was amplified by primers 1303F / 1303R. The purified linearized knockout recombinant plasmid skeleton was combined with the upstream homologous sequence and downstream homologous sequence of the veA gene and the Hyg gene expression cassette using a seamless cloning kit -UniSeamless Cloning and Assembly Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number: CU101-01) was used for homologous recombination ligation, and the ligation product was transformed into Escherichia coli DH5α competent cells (purchased from Nanjing Novogene Biotechnology Co., Ltd., product number: C502-02), spread on LB agar plates containing kanamycin (50 μg / mL), and cultured at 37°C until a single colony grew. A single colony was picked and colony PCR verification and sequencing were performed using JPHygF / JPHygR as primers. If the sequencing was correct, the veA gene knockout recombinant plasmid (pCAMBIA1303-veAU-Hyg-veAD) was successfully obtained.

[0041] The primer sequences used in the above steps are shown in Table 1.

[0042] Figure 1It is the PCR amplification result of colony PCR of the upstream and downstream homologous sequences of the veA gene constructed in this example, the Hyg gene expression cassette fragment, the linearized knockout recombinant plasmid backbone, and the veA gene knockout recombinant plasmid. Among them, Figure a is the PCR amplification result of the upstream homologous sequence of the veA gene, wherein lane M is DL2000 DNA Marker, and lane 1 is the upstream homologous sequence of the veA gene; Figure b is the PCR amplification result of the downstream homologous sequence of the veA gene, wherein lane M is DL2000 DNA Marker, and lane 1 is the downstream homologous sequence of the veA gene; Figure c is the PCR amplification result of the Hyg gene expression cassette, wherein lane M is DL2000 DNA Marker, and lane 1 is the Hyg gene expression cassette; Figure d is the PCR amplification result of the linearized knockout recombinant plasmid backbone, wherein lane M is DL10000 DNA Marker, and lane 1 is the linearized knockout recombinant plasmid backbone; Figure e is the PCR amplification result of the veA gene knockout recombinant plasmid colony PCR, wherein lane M is DL5000 DNA Marker, and lane 1 is the veA gene knockout recombinant plasmid colony PCR.

[0043] Table 1 Primers used to construct veA gene knockout recombinant plasmid

[0044]

[0045]

[0046] Example 2 Construction of veA gene overexpression recombinant plasmid

[0047] The veA gene was amplified using primers veAF / veAR with Cordyceps militaris genomic DNA as a template, and the linearized overexpression recombinant plasmid skeleton without EGFP gene was amplified using primers 1303f / 1303r with pCAMBIA1303-gpdA-EGFP-TrpC-Hygro binary plasmid as a template. The linearized overexpression recombinant plasmid skeleton was connected to the veA gene using homologous recombination technology, and the connection product was transformed into Escherichia coli DH5α competent cells. The cells (purchased from Nanjing Novozyme Biotechnology Co., Ltd., product number: C502-02) were spread on LB agar plates containing kanamycin (50 μg / mL) and cultured at 37°C until a single colony grew. A single colony was picked for colony PCR verification and sequencing using JPHygF / JPHygR as primers. If the sequencing was correct, the veA gene overexpression recombinant plasmid (pCAMBIA1303-gpdA-veA) in which the veA gene was driven by the strong promoter gpdA was obtained.

[0048] The primer sequences used in the above steps are shown in Table 2.

[0049] Figure 2 The PCR amplification product gel electrophoresis diagram of the veA gene fragment, linearized overexpression recombinant plasmid skeleton, and veA gene overexpression recombinant plasmid colony PCR constructed in this embodiment. Among them, Figure a is the PCR amplification result of the veA gene fragment, wherein lane M is DL2000 DNA Marker, and lane 1 is the veA gene fragment; Figure b is the PCR amplification result of the linearized overexpression recombinant plasmid skeleton, wherein lane M is DL15000 DNA Marker, and lane 1 is the linearized overexpression recombinant plasmid skeleton; Figure c is the PCR amplification result of the veA gene overexpression recombinant plasmid colony, wherein lane M is DL2000 DNA Marker, and lane 1 is the veA gene overexpression recombinant plasmid colony PCR.

[0050] Table 2 Primers required for constructing veA gene overexpression recombinant plasmid

[0051]

[0052] Example 3 Agrobacterium tumefaciens-mediated veA gene knockout and overexpression recombinant plasmids into Cordyceps militaris

[0053] The veA gene knockout and overexpression recombinant plasmids constructed in Examples 1 and 2 were introduced into Agrobacterium tumefaciens AGL1 by electroporation. Colony PCR verification was performed using JPHygF / JPHygR as primers and AGL1 containing veA gene knockout and overexpression recombinant plasmids as templates. After successful verification, AGL1 containing veA gene knockout and overexpression recombinant plasmids were cultured in LB liquid culture medium at 28°C and 200rpm for about 16 hours. The LB liquid culture medium contained 50 μg / mL of kanamycin. Then, the bacteria in the LB liquid medium were centrifuged at 5000 rpm for 3 min, and the sludge was resuspended twice with 5 mL of induction medium IM (0.0025% v / v glycerol, 1.8 g / L glucose, 40% v / v MM salt (MM salt formula: 3.625 g / L potassium dihydrogen phosphate, 6.72 g / L dimethyl phosphate, 1.25 g / L magnesium sulfate heptahydrate, 0.375 g / L sodium chloride, 0.125 g / L calcium chloride, 0.0062 g / L ferrous sulfate heptahydrate, 1.25 g / L ammonium sulfate)), IM containing 200 μmol / L acetosyringone as an inducer. The OD of the bacteria was increased by IM. 600 The value was adjusted to 0.2, and the cells were cultured again at 28°C and 200 rpm until the OD 600 When the temperature was 0.8, the mixture was temporarily stored in a 4°C refrigerator. The wild-type Cordyceps militaris CGMCC No. 18581 (WT) mycelium on the PDA plate was scraped with a sterile scalpel and filtered to obtain 10 6200 μL of the cultured OD 600 0.8 AGL1 and 200 μL 10 6 The conidia suspension of 100 μL / mL was mixed evenly, 200 μL of the mixed bacterial solution was evenly spread on a sterile filter paper, and the sterile filter paper was placed in a co-culture medium (CO-IM) (20 g / L agar was added to the IM medium). After co-culture at 26°C for 72 hours, the filter paper was transferred to a PDA plate containing 600 μg / mL hygromycin B (as a selection agent for Cordyceps militaris transformants) and 100 μg / mL timentin (to inhibit the growth of Agrobacterium tumefaciens), and cultured at 26°C until Cordyceps militaris transformants grew out. The Cordyceps militaris transformants were verified by colony PCR using primers JPHygF / JPHygR, and the translation elongation factor tef1 was used as an internal reference gene, and its nucleotide sequence is shown in SEQ ID As shown in NO.2, real-time fluorescence quantitative PCR was performed with tef1F / tef1R and qPveAF / qPveAR as primers to detect the relative expression levels of the veA gene in the two transformants. If the relative expression levels were verified to be correct, the veA gene mutant strains (veA gene overexpressing recombinant Cordyceps militaris OEveA and veA gene knockout recombinant Cordyceps militaris ΔveA) were successfully obtained.

[0054] The primer sequences used in the above steps are shown in Table 3.

[0055] Figure 3 The colony PCR of AGL1 containing the recombinant plasmid, the colony PCR of the recombinant Cordyceps militaris containing the recombinant plasmid, the gel electrophoresis of the PCR amplification product, the growth of the recombinant Cordyceps militaris, and the statistical graph of the relative expression of the veA gene in the recombinant Cordyceps militaris constructed for this embodiment. Wherein, Figure a is the colony PCR amplification result of AGL1 containing the recombinant plasmid, wherein lane M is DL2000 DNA Marker, lane 1 is the colony PCR of AGL1 containing the veA gene knockout recombinant plasmid, and lane 2 is the colony PCR of AGL1 containing the veA gene overexpression recombinant plasmid; Figure b is the growth situation of ΔveA; Figure c is the growth situation of OEveA; Figure d is the colony PCR amplification result of the recombinant Cordyceps militaris, wherein lane M is DL2000 DNA Marker, lane 1 is the colony PCR of ΔveA, and lane 2 is the colony PCR of OEveA; Figure e is the statistical graph of the relative expression of the veA gene in the recombinant Cordyceps militaris and the wild-type Cordyceps militaris.

[0056] Depend on Figure 3 As shown in Figure e, the relative expression level of gene veA in ΔveA is 0, proving that the recombinant Cordyceps militaris with gene veA knocked out was successfully obtained; the relative expression level of gene veA in OEveA is 1.49 times that of WT, proving that the recombinant Cordyceps militaris with gene veA overexpressed was successfully obtained.

[0057] Table 3 Primer sequences used in this example

[0058]

[0059] Example 4 Effect of veA gene on growth characteristics of Cordyceps militaris

[0060] The wild-type Cordyceps militaris strain (WT) and the veA gene mutant strain (veA gene overexpression recombinant Cordyceps militaris OEveA and veA gene knockout recombinant Cordyceps militaris ΔveA) were inoculated on PDA plates and cultured in the dark at 26°C for 14 days. Mycelium was scraped with a sterile scalpel and dissolved in sterile pure water. After filtering with 4 layers of sterile gauze, the number of cells was counted using a hemocytometer to obtain 10 6 Conidia suspension with a concentration of conidia / mL. 5 μL of the spore suspension of the corresponding strain was inoculated on a fresh PDA plate and cultured at 26°C in the dark or under light (FSL (Foshan Lighting), 8W / 865, 750Lm, the distance between the light source and the plate was 30 cm). The diameter of the colony was measured every 2 days within 20 days to evaluate the growth of the colony. In order to observe the microscopic structure of the hyphae, a sterilized cover glass was inserted into the PDA plate at 45°, and the microscopic growth state of the hyphae of the wild-type Cordyceps militaris and the veA gene mutant was evaluated under an optical microscope on the 20th day. Within 21 days, the initial spore suspension of each strain was obtained every 7 days according to the above method, and the conidia yield of the wild-type strain and the veA gene mutant was counted using a hemocytometer. The results are expressed as "conidia / mm 2 "express.

[0061] Figure 4 It is a physical picture of the plate colony growth of the recombinant Cordyceps militaris and wild-type Cordyceps militaris of this embodiment and the corresponding colony diameter data statistics, hyphae micromorphology, and conidia yield statistics. Among them, Figure a is a physical picture of the plate colony growth of OEveA, ΔveA and WT cultured on a plate for 20 days (D: Dark, L: Light); Figure b is a statistical chart of the plate colony diameter of OEveA, ΔveA and WT cultured on a dark plate; Figure c is a statistical chart of the plate colony diameter of OEveA, ΔveA and WT cultured on a light plate; Figure d is a microscopic morphology of OEveA, ΔveA and WT hyphae; Figure e is a statistical chart of the conidia yield of OEveA, ΔveA and WT cultured on a dark plate; Figure f is a statistical chart of the conidia yield of OEveA, ΔveA and WT cultured on a light plate.

[0062] Figure 4The results showed that, regardless of darkness or light conditions, at 20 days, OEveA had the largest colony diameter and ΔveA had the smallest colony diameter. Many wrinkles formed on the back of ΔveA, which were more obvious under light conditions. OEveA had more yellow pigment accumulation on its back than WT under darkness or light conditions. Optical microscopy observations of OEveA, WT, and ΔveA revealed that under darkness, OEveA had many and long hyphal branches at the end of its hyphae, while WT had shorter branches at the end of its hyphae, and ΔveA had almost no branches formed. Under light conditions, the end of OEveA hyphae grew very densely and had shorter branches, while WT had longer branches at the end of its hyphae, and ΔveA also had very few branches formed. With the extension of the culture time, the spore yields of the three increased under darkness or light conditions, and the light conditions had more spore yields than the dark conditions. On the 21st day of cultivation, the spore production of OEveA, WT and ΔveA under light conditions were 91.74%, 134.64% and 118.9% higher than those under dark conditions, respectively; OEveA had the highest spore production in both dark and light conditions, and ΔveA had the lowest; under dark conditions, the conidia production of OEveA was 161.16% higher than that of WT.

[0063] Example 5 Effect of veA gene on single carbon source utilization of Cordyceps militaris

[0064] Referring to the method of Example 4, 10 of the wild-type strain and the veA gene mutant were respectively 6 The spore suspension of conidia / mL was added dropwise to MM medium (ammonium sulfate 1g / L, dipotassium hydrogen phosphate 7g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 0.1g / L, sodium citrate 0.5g / L, agar powder 20g / L) containing 5g / L of different carbon sources (glucose, lactose, fructose, galactose, sucrose), and cultured at 26°C in the dark or in the light for 20 days. The colony diameter was measured and the colony growth was observed.

[0065] Figure 5 Figure 1 is a colony growth diagram and colony diameter data statistical diagram of the recombinant Cordyceps militaris and wild-type Cordyceps militaris on different single carbon source plates in this embodiment. Among them, Figure a is a colony growth diagram of OEveA, ΔveA and WT on different single carbon source plates (D: Dark, L: Light); Figure b is a colony diameter data statistical diagram of OEveA, ΔveA and WT on different single carbon source plates under dark culture; Figure c is a colony diameter data statistical diagram of OEveA, ΔveA and WT on different single carbon source plates under light culture.

[0066] Figure 5The results showed that ΔveA had the least aerial hyphae in all single carbon source media, the colonies grew thin and transparent, and the colony diameter was smaller than that of OEveA and WT regardless of dark or light conditions. Compared with dark conditions, light conditions were conducive to the radial growth of colonies, while the hyphae density of colonies under dark conditions was large and the growth was thicker. OEveA can efficiently utilize glucose and sucrose, followed by fructose and lactose, and finally galactose. Compared with OEveA and WT, ΔveA's ability to utilize various single carbon sources decreased.

[0067] Example 6 Liquid Static Dark Cultivation of Wild-Type Cordyceps Militaris Strain and VeA Gene Mutant

[0068] 1) Preparation of Cordyceps militaris fermentation medium: Accurately weigh 40 g of glucose, 5 g of peptone, 10 g of yeast powder, 0.2 g of potassium dihydrogen phosphate, 0.2 g of dipotassium hydrogen phosphate, 0.2 g of anhydrous magnesium sulfate, and 4 g of adenine, add them into pure water, fully dissolve and mix, adjust the volume to 1 L, and adjust the pH to 5.8.

[0069] 2) The culture medium prepared in step 1) was dispensed into 500 mL Erlenmeyer flasks with a working volume of 100 mL. After complete dispensing, the flasks were sealed, sterilized at 115° C. for 20 min, and cooled to obtain the culture medium.

[0070] 3) Cordyceps militaris seed culture: In a clean bench, an alcohol lamp was used to burn and sterilize the cooled inoculation loop to scrape off all the OEveA, WT, and ΔveA Cordyceps militaris mycelia in the PDA medium, and then the mycelia were transferred to a seed medium (glucose 40 g / L, peptone 5 g / L, yeast powder 10 g / L, potassium dihydrogen phosphate 0.2 g / L, potassium hydrogen phosphate 0.2 g / L, anhydrous magnesium sulfate 0.2 g / L, pH 5.8), respectively, at 26° C., 150 rpm, and cultured in the dark for 5 days, at which time the wet weight of the mycelium in the seed solution was 20 g / L.

[0071] 4) The Cordyceps militaris seed solution cultured for 5 days was transferred to the fermentation medium of step 2) at a transfer volume of 10% (v / v), and cultured in the dark at 26° C. for 20 days.

[0072] Example 7 Liquid static light culture of wild-type Cordyceps militaris strains and veA gene mutants

[0073] 1) Preparation of Cordyceps militaris fermentation medium: Accurately weigh 40 g of glucose, 5 g of peptone, 10 g of yeast powder, 0.2 g of potassium dihydrogen phosphate, 0.2 g of dipotassium hydrogen phosphate, 0.2 g of anhydrous magnesium sulfate, and 4 g of adenine, add them into pure water, fully dissolve and mix, adjust the volume to 1 L, and adjust the pH to 5.8.

[0074] 2) The culture medium prepared in step 1) was dispensed into 500 mL Erlenmeyer flasks with a working volume of 100 mL. After complete dispensing, the flasks were sealed, sterilized at 115° C. for 20 min, and cooled to obtain the culture medium.

[0075] 3) Cordyceps militaris seed culture: In a clean bench, an alcohol lamp was used to burn and sterilize the cooled inoculation loop to scrape off all the OEveA, WT, and ΔveA Cordyceps militaris mycelia in the PDA medium, and then the mycelia were transferred to a seed medium (glucose 40 g / L, peptone 5 g / L, yeast powder 10 g / L, potassium dihydrogen phosphate 0.2 g / L, potassium hydrogen phosphate 0.2 g / L, anhydrous magnesium sulfate 0.2 g / L, pH 5.8), respectively, at 26° C., 150 rpm, and cultured in the dark for 5 days, at which time the wet weight of the mycelium in the seed solution was 20 g / L.

[0076] 4) The Cordyceps militaris seed solution cultured for 5 days was transferred to the fermentation medium of step 2) at a transfer volume of 10% (v / v), and the mixture was allowed to stand at 26° C. and cultured under light (FSL (Foshan Lighting), 8W / 865, 750Lm, with a distance of 30cm between the light source and the fermentation liquid surface) for 20 days.

[0077] Example 8 Cordycepin yields of wild-type Cordyceps militaris strains and veA gene mutants

[0078] During the fermentation and cultivation process of Examples 6 and 7, samples were taken to determine the residual sugar concentration, pH, biomass concentration, and cordycepin content in the fermentation broth.

[0079] The detection conditions of the HPLC method were as follows: ZORBAX 300SB-C18 column (250 mm × 4.6 mm, 5 μm), column temperature 30°C, detection wavelength 254 nm, mobile phase of 25% methanol (chromatographic grade) and 75% 10 mmol / L potassium dihydrogen phosphate aqueous solution, flow rate 0.600 mL / min, injection volume 20 μL.

[0080] Figure 6 and Figure 7 The data of liquid static fermentation culture of the recombinant Cordyceps militaris and wild-type Cordyceps militaris in the present embodiment under dark and light conditions are statistical graphs. Among them, Figure a is a statistical graph of residual sugar data in OEveA, ΔveA and WT liquid static culture fermentation broth; Figure b is a statistical graph of pH data in OEveA, ΔveA and WT liquid static culture fermentation broth; Figure c is a statistical graph of biomass data in OEveA, ΔveA and WT liquid static culture fermentation broth; Figure d is a statistical graph of cordycepin (COR) production data in OEveA, ΔveA and WT liquid static culture.

[0081] Figure 6The results showed that OEveA and WT consumed glucose faster than ΔveA. On the 20th day, all the glucose in the fermentation broth of OEveA and WT was consumed, while ΔveA still had 10 g / L of glucose remaining. In terms of the pH change of the fermentation broth, OEveA and WT recovered in a shorter time, and the pH of ΔveA began to rise after 15 days. WT consumed more sugar, resulting in more biomass accumulation. On the 20th day, the cordycepin production of OEveA (6.52 g / L) was 29.9% higher than that of WT (5.02 g / L). Figure 7 The results showed that under light conditions, the glucose of OEveA and WT was eventually consumed, and ΔveA remained relatively high (about 16 g / L). The pH of OEveA and WT reached the inflection point earlier. The cordycepin production of OEveA exceeded that of WT after 15 days. The final cordycepin production of OEveA (5.19 g / L) was 14.06% higher than that of WT (4.55 g / L), while the cordycepin production of ΔveA was the lowest (1.51 g / L). Compared with dark culture, light culture had no obvious promoting effect on the production of cordycepin by OEveA.

[0082] The invention discloses a recombinant Cordyceps militaris fungus, a construction method thereof, and an application of cordycepin production, and discloses that overexpression of the veA gene promotes the production of secondary metabolites cordycepin and the growth and development of plate mycelium of Cordyceps militaris under liquid static culture conditions. After 20 days of dark culture, compared with the wild-type strain, the cordycepin yield under liquid culture conditions increased by 29.9%, and the colony diameter and conidia number under plate culture conditions increased by 2.77% and 161.16%, respectively. The invention provides a recombinant Cordyceps militaris strain OEveA with high cordycepin production, and at the same time provides a new method and direction for high efficiency and high cordycepin production, with industrial application prospects.

[0083] The present invention provides a recombinant Cordyceps militaris and its construction ideas and methods, and there are many methods and approaches to specifically implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in the technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not specified in this embodiment can be implemented with existing technology.

Claims

1. A recombinant Cordyceps militaris, characterized in that: The veA gene is overexpressed in Cordyceps militaris, and the nucleotide sequence of the veA gene is shown in SEQ ID NO.

1.

2. The recombinant Cordyceps militaris according to claim 1, characterized in that The cordyceps militaris is Cordyceps militaris CGMCC No.18581.

3. The method for constructing the recombinant Cordyceps militaris according to claim 1 or 2, characterized in that: The veA gene overexpression recombinant plasmid is introduced into the Cordyceps militaris through the transformation technology mediated by Agrobacterium tumefaciens, so as to obtain the recombinant Cordyceps militaris.

4. The construction method according to claim 3, characterized in that: The Agrobacterium tumefaciens-mediated transformation technology specifically comprises: introducing the veA gene overexpression recombinant plasmid into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens, and co-culturing the recombinant Agrobacterium tumefaciens with the Cordyceps militaris to obtain the recombinant Cordyceps militaris.

5. The construction method according to claim 3, characterized in that: The method for constructing the veA gene overexpression recombinant plasmid is as follows: the veA gene is connected to the plasmid pCAMBIA1303-gpdA-TrpC-Hygro to obtain the recombinant plasmid.

6. The construction method according to claim 5, characterized in that: The plasmid pCAMBIA1303-gpdA-TrpC-Hygro is obtained by removing the EGFP gene from the plasmid pCAMBIA1303-gpdA-EGFP-TrpC-Hygro.

7. Use of the recombinant Cordyceps militaris according to claim 1 or 2 in fermentation to produce cordycepin.

8. The use according to claim 7, characterized in that: The seed liquid of the recombinant Cordyceps militaris is inoculated into a fermentation medium for culturing and fermenting to produce cordycepin; the mycelium wet weight of the seed liquid is 15-25 g / L; and the inoculation amount of the seed liquid is 5-15% of the volume of the fermentation medium.

9. The use according to claim 7, characterized in that: The culture conditions are: temperature of 22-30° C., pH of 5-6.5, and culture time of 18-26 days.

10. The use according to claim 7, characterized in that: The components of the fermentation medium include glucose, peptone, yeast powder, magnesium ions and precursor substances; the precursor substances are adenosine or adenine.

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