CN105441517A

CN105441517A - Identification and application of synthesis gene cluster of cordycepin

Info

Publication number: CN105441517A
Application number: CN201410425608.9A
Authority: CN
Inventors: 王成树; 夏永亮; 商艳芳
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Center for excellence and innovation in molecular plant science, Chinese Academy of Sciences
Priority date: 2014-08-26
Filing date: 2014-08-26
Publication date: 2016-03-30
Anticipated expiration: 2034-08-26
Also published as: WO2016029802A1; CN105441517B

Abstract

The invention relates to identification and application of a synthesis gene cluster of cordycepin that is a metabolite of cordyceps militaris (L.) link. The gene cluster of cordycepin biosynthesis is found for the first time. The gene cluster comprises Cm1 and Cm2 and/or Cm3, and corresponding homologous genes in aspergillus nidulans are An1, An2 and/or An3 respectively, and therefore a method of achieving efficient cordycepin biosynthesis is disclosed.

Description

The qualification of the synthetic gene of cordycepin bunch and application

Technical field

The present invention relates to gene engineering field, more specifically, the present invention relates to Cordyceps militaris (L.) Link. meta-bolites---the qualification of the synthetic gene of cordycepin bunch and application.

Background technology

Cordycepin (cordycepin), also known as making cordycepin, cordycepin, chemical name cordycepin (3 '-deoxyadenosine) is first nucleoside antibiotics separated from fungi.The fungi that can produce cordycepin only comprises in the Cordyceps few in number such as Cordyceps militaris (L.) Link., kyushu aweto and Aspergillus nidulans, cordycepin has good fungistatic effect, the aging improvement caused at oxidative stress (oxidativestress), the enhancing of body's immunity regulate, suppress Adipocyte Differentiation prevention of obesity occurs, inflammation suppresses, human peripheral blood monocyte produces interleukin 10 effect, leukemia etc. in all play an important role, be one of important evaluation index of Cordyceps quality.The chemical structure of cordycepin is perhaps just resolved as follows many years ago:

Cordycepin:

Current acquisition cordycepin is generally by three approach: extract from Natural C.militaris, synthetic and being extracted from Cordyceps militaris nutrient solution by bionic method.

Have report cordycepin can artificial chemistry synthesis, but process complicated, drop into huge and output is extremely low.Natural cs scarcity of resources, expensive, content is very micro-, and being difficult to becomes the main raw material extracting cordycepin.Therefore how improving the content of cordycepin in Cordyceps militaris (L.) Link. and realize the suitability for industrialized production of cordycepin, is the Main way of research at present.

Although have more enterprise to produce Cordyccps-militaris-(L.)-link. Sporophore domestic at present, because the cordycepin content in natural sporophore is low, the efficiency of extensive extraction purification is very low, is difficult to the new drug development for carrying out according to cordycepin and realizes industrialization to supply raw materials the guarantee of medicine.

Therefore, by bionic method, the efficient synthesis realizing cordycepin has Important Economic and using value.But the synthesis mechanism of cordycepin in fungi is always unclear, bottleneck prepared by this genetic engineering industry becoming cordycepin.

Summary of the invention

The object of the present invention is to provide Cordyceps militaris (L.) Link. meta-bolites---the qualification of the synthetic gene of cordycepin bunch and application.

In a first aspect of the present invention, provide a kind of method of producing cordycepin, described method comprises:

(1) by cordycepin biosynthesis related genes transformed host cell; Wherein, described cordycepin biosynthesis related genes comprises Cm1 and the Cm2 gene of Cordyceps militaris (L.) Link., or comprises An1 and the An2 gene of Aspergillus nidulans;

(2) cultivate the host cell of (1), make it to produce cordycepin.

In a preference, in (1) of described method, described cordycepin biosynthesis related genes comprises Cm1, Cm2 gene and the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1, An2 gene and the An3 gene of Aspergillus nidulans.

In another preference, described host cell to comprise or can the host cell of autologous generation 3 '-AMP.

In another preference, described host cell is fungi; Preferably described fungi includes, but is not limited to: Cordyceps sinensis fungus (as Cordyceps militaris (L.) Link. (Cordycepsmilitaris)), aspergillus fungi (as Aspergillus nidulans (Aspergillusnidulans)), Metarhizium fungi (as Luo Baici green muscardine fungus (Metarhiziumrobertsii)), yeast belong fungi (as yeast saccharomyces cerevisiae, pichia spp).

In another aspect of this invention, provide a kind of cordycepin biological synthesis gene cluster of qualification, described cordycepin biological synthesis gene cluster comprises Cm1 and the Cm2 gene of Cordyceps militaris (L.) Link., or comprises An1 and the An2 gene of Aspergillus nidulans.

In a preference, described cordycepin biological synthesis gene cluster comprises Cm1, Cm2 gene and the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1, An2 gene and the An3 gene of Aspergillus nidulans.

In another preference, described cordycepin biological synthesis gene cluster does not comprise the gene cluster be made up of full-length genome, do not comprise the whole chromosome comprising Cm1 and Cm2 and/or Cm3 gene, do not comprise the whole chromosome comprising An1 and An2 and/or An3 gene yet yet.

In another aspect of this invention, provide recombinant vectors (comprising carrier combinations), described recombinant vectors comprises cordycepin biosynthesis related genes; Described cordycepin biosynthesis related genes comprises Cm1 and Cm2 and/or the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1 and An2 and/or the An3 gene of Aspergillus nidulans; Supplementary condition are: described Cm1 and Cm2 and/or Cm3 gene are positioned at same recombinant vectors or are positioned on different recombinant vectorss; Or described An1 and An2 and/or An3 gene are positioned at same recombinant vectors or are positioned on different recombinant vectorss.

In another aspect of this invention, provide a kind of host cell, described host cell comprises described cordycepin biosynthesis related genes; Described cordycepin biosynthesis related genes comprises Cm1 and Cm2 and/or the Cm3 gene of Cordyceps militaris (L.) Link.; Or described cordycepin biosynthesis related genes comprises An1 and An2 and/or the An3 gene of Aspergillus nidulans; Or comprise described cordycepin biological synthesis gene cluster.

In a preference, described host cell to comprise or can the host cell of autologous generation 3 '-AMP; Preferably, described host cell is fungi; More preferably, described fungi includes, but is not limited to: Cordyceps sinensis fungus (as Cordyceps militaris (L.) Link. (Cordycepsmilitaris)), Metarhizium fungi (as Luo Baici green muscardine fungus (Metarhiziumrobertsii)), aspergillus fungi (as Aspergillus nidulans (Aspergillusnidulans)), yeast belong fungi (as yeast saccharomyces cerevisiae, pichia spp).

In another aspect of this invention, described cordycepin biological synthesis gene cluster or the purposes of described expression vector or described host cell is provided, for the production of cordycepin.

In another aspect of this invention, provide a kind of test kit for the production of cordycepin, described test kit comprises: described expression vector or described host cell.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

The comparison of potential cordycepin biosynthesis gene in Fig. 1, Cordyceps militaris (L.) Link. and Aspergillus nidulans.

(A) the tissue line corresponding relation of cordycepin biological synthesis gene cluster on Cordyceps militaris (L.) Link. and Aspergillus nidulans genome.

(B) Collinearity Diagnosis Analysis of cordycepin biological synthesis gene cluster in Cordyceps militaris (L.) Link. and Aspergillus nidulans.

Fig. 2, Cordyceps militaris (L.) Link. wild strain produce comparing of cordycepin with cpABC knockout mutant strain (Δ cpABC).

(A) in SDB fermented liquid, the HPLC detection of cordycepin is compared.

(B) HPLC compares the cordycepin detected in sporophore water extraction sample.Red peak (arrows) is cordycepin peak.

Fig. 3, HPLC detect the cordycepin in the fermented liquid of yeast saccharomyces cerevisiae and all knock-in bacterial strains.KI represents knock-in.

Fig. 4, HPLC detect the synthesis of cordycepin in heterogenous expression cpABC mutant strain.

(A) yeast saccharomyces cerevisiae expresses the detection of cordycepin in cpABC mutant strain and control strain SC fermented liquid.

(B) Luo Baici green muscardine fungus expresses the detection of cordycepin in cpABC mutant strain and wild strain SDB fermented liquid.Red peak (arrows) is cordycepin peak.

Fig. 5, HPLC detect the cordycepin in Cordyceps militaris (L.) Link. wild strain and mutant strain.

(A) detection of cordycepin in fermented liquid is compared.Red peak (arrows) is cordycepin peak.

(B) detection of cordycepin in sporophore water extraction sample is compared.Ko represents knock-out.

Fig. 6, HPLC detect the cordycepin in Aspergillus nidulans wild strain and mutant strain fermented liquid.Red peak is cordycepin peak.

The impact singly knocked out cordycepin output of cpC gene in Fig. 7, Cordyceps militaris (L.) Link..

Fig. 8, cordycepin biosynthetic metabolism approach.

Embodiment

The present inventor, through deep research, have found the biosynthetic genes involved of cordycepin first, comprises Cm1 and Cm2 and/or Cm3, and the homologous gene in corresponding Aspergillus nidulans is An1, An2 and/or An3 respectively.Based on above-mentioned new discovery, the present invention discloses one can realize the biosynthetic method of efficient cordycepin.

As used herein, described " allos " refers to from the relation between the two or more pieces nucleic acid of different sources or protein sequence, knows from the relation between the albumen (or nucleic acid) of different sources and host cell.Such as, if the combination of nucleic acid and host cell is not naturally occurring usually, then nucleic acid is allos for this host cell.Particular sequence is " allos " for its cell inserted or organism.

As used herein, described " cordycepin biosynthesis related genes " refers to the combination for the gene of biosynthesizing cordycepin in fungi, comprises " cordycepin biological synthesis gene cluster ".Described cordycepin biosynthesis related genes comprises Cm1 and the Cm2 gene of Cordyceps militaris (L.) Link., or comprises An1 and the An2 gene of Aspergillus nidulans; More preferably, described cordycepin biosynthesis related genes comprises Cm1, Cm2 gene and the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1, An2 gene and the An3 gene of Aspergillus nidulans.

As used herein, after described " host cell " is included in and is transformed into cordycepin biosynthesis related genes of the present invention, can the summation of cell of biosynthesizing cordycepin; Prokaryotic cell prokaryocyte and eukaryotic cell can be comprised.Preferably, described host cell is fungal cell, prokaryotic cell prokaryocyte or vegetable cell.More preferably, described host cell is fungi.More preferably, described fungi includes, but is not limited to: Cordyceps sinensis fungus (as Cordyceps militaris (L.) Link. (Cordycepsmilitaris)), aspergillus fungi (as Aspergillus nidulans (Aspergillusnidulans)), Metarhizium fungi (as green muscardine fungus), yeast belong fungi (as yeast saccharomyces cerevisiae, pichia spp).

As used herein, described cpA refers to Cm1 or An1, and cpB refers to Cm2 or An2, and cpC refers to Cm3 or An3, and cpD refers to Cm4 or An4.

Cordycepin biosynthesis related genes

Described cordycepin biosynthesis related genes comprises Cm1 and the Cm2 gene of Cordyceps militaris (L.) Link.; Preferably, the Cm3 gene of Cordyceps militaris (L.) Link. is also comprised.The metabolic enzymes that three above-mentioned genes encodings are relevant, the phosphotransferase (ATPphosphoribosyltransferase) that the phosphohydrolase (Metaldependentphosphohydrolase) of wherein Cm1 coding oxydo-reductase (oxidordeuctase), Cm2 coding metal ion dependence and Cm3 coding ATP rely on.Cm1 and Cm2 gene is the key gene that host cell generates cordycepin, and Cm3 gene then more effectively can provide the output of cordycepin.Described Cm1 has the identical sequence of sequence shown in GenBank accession number CCM_04436 or its degenerate sequence or the sequence of the albumen with the same function of albumen coded by CCM_04436 of also encoding, described Cm2 have the identical sequence of sequence shown in GenBank accession number CCM_04437 or its degenerate sequence or the sequence of the albumen with the same function of albumen coded by CCM_04437 of also encoding, described Cm3 have the identical sequence of sequence shown in GenBank accession number CCM_04438 or its degenerate sequence or the sequence of the albumen with the same function of albumen coded by CCM_04438 of also encoding.

Described cordycepin biosynthesis related genes comprises An1 and the An2 gene of Aspergillus nidulans; Preferably, the An3 gene of Aspergillus nidulans is also comprised.The wherein phosphohydrolase (Metaldependentphosphohydrolase) of An1 coding oxydo-reductase (oxidordeuctase), An2 coding metal ion dependence and the phosphotransferase (ATPphosphoribosyltransferase) of An3 coding ATP dependence.An1 and An2 gene is the key gene that host cell generates cordycepin, and An3 gene then more effectively can provide the output of cordycepin.Described An1 has the identical sequence of sequence shown in GenBank accession number AN3333 or its degenerate sequence or the sequence of the albumen with the same function of albumen coded by AN3333 of also encoding, described An2 has the identical sequence of sequence shown in GenBank accession number AN3331 or its degenerate sequence or the sequence of the albumen with the same function of albumen coded by AN3331 of also encoding, described An3 has the identical sequence of sequence shown in GenBank accession number AN3330 or its degenerate sequence or the sequence of the albumen with the same function of albumen coded by AN3330 of also encoding.

In other detected different fungies, not containing Cm1, Cm2 gene and Cm3 gene in any genome, or comprise An1, An2 gene of Aspergillus nidulans and the similar gene of An3 gene, all can not produce cordycepin.

Each gene that cordycepin biosynthesis related genes of the present invention comprises can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.

The invention still further relates to the varient of said gene, comprise and replace varient, Deletion variants and insertion varient.But described varient from the function changing in fact the polypeptide that it is encoded, still can not can realize technique effect of the present invention.

Cordycepin biosynthesis related genes in the present invention preferably provides with the form be separated, and is more preferably purified to homogeneous.

The cordycepin biosynthesis related genes disclosed in the present invention can be naturally occurring, such as its can separated or purifying from microorganism.In addition, described cordycepin biosynthesis related genes also can be artificial preparation, the cordycepin biosynthesis related genes that such as can increase described according to the genetic engineering technique of routine, or prepare described cordycepin biosynthesis related genes by the means of synthetic.Preferably, described cordycepin biosynthesis related genes can be obtained by using gene engineering technique.

Cordycepin biosynthesis related genes of the present invention or its each gene comprised can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.

Once obtain relevant sequence, just relevant sequence can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.In addition, also relevant sequence can be synthesized, when especially fragment length is shorter by the method for synthetic.Usually, by first synthesizing multiple small segment, and then carry out connect can obtain the very long fragment of sequence.

As optimal way of the present invention, can carry out codon optimized to each gene that cordycepin biosynthesis related genes or it comprise, to improve expression efficiency.Such as, in order to promote its recombinant expressed in yeast host, the codon optimized of yeast cell preference can be carried out to it.

After obtaining careless plain biosynthesis related genes, be connected in suitable expression constructs (as expression vector), then proceeded to suitable host cell.Finally by cultivating the host cell after transforming, obtain the meta-bolites cordycepin of host cell.

Described expression constructs can comprise cordycepin biosynthesis related genes or its each gene comprised, and also can comprise the expression regulation sequence be connected with the sequence being operational of described gene, so that the expression of albumen.The design of described expression regulation sequence is well known in the art.In expression regulation sequence, according to different needs, can apply the promotor of induction type or composing type, the promotor of induction type can realize more controlled expression and meta-bolites is produced, and is conducive to industrial applications.For prokaryotic cell prokaryocyte or eukaryotic cell, which applicable expression vector known in the art is, therefore people can choose the skeleton carrier of suitable expression vector as clones coding gene.

Produce the method for cordycepin

The present invention discloses a kind of method utilizing host cell heterologous production cordycepin.Utilize biotechnology, express the gene relevant to synthesis cordycepin, generate cordycepin.

The biosynthetic pathway of cordycepin comprises: adenosine is phosphorylated generation 3 '-AMP comprising under the special phosphotransferases such as cpC or protein kinase effect, certainly, 3 '-AMP also can by 2 ', 3 '-phosphodiesterase in 3 '-cAMP-adenosine metabolic pathway produces 2 ', 3 '-cAMP effect.Gained 3 '-AMP is dephosphorylation under the effect of cpB, obtains a unstable enolization compound, and the spontaneous isomery of this compound forms carbonyl, is responsible for carbonyl reduction to become hydroxyl, generates cordycepin under the effect of cpA reductase enzyme.

Therefore, the invention provides a kind of method of producing cordycepin, described method comprises: (1) is by cordycepin biosynthesis related genes transformed host cell; Wherein, described cordycepin biosynthesis related genes comprises Cm1 and Cm2 and/or the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1 and An2 and/or the An3 gene of Aspergillus nidulans; (2) cultivate the host cell of (1), make it to produce cordycepin.Described host cell comprises the host cell that maybe can generate 3 '-AMP, and wherein 3 '-AMP is as the substrate specificity of cpB.

According to the difference of adopted host cell, the mode of cultivating host cell can change, and this is that those skilled in the art can carry out based on existing knowledge.

The method being separated cordycepin from the cultured products of host cell is also that those skilled in the art can carry out based on existing knowledge.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, per-cent is calculated by weight percentage.

Materials and methods

1, Cordyceps militaris (L.) Link. test relating operation

1.1 asexually nourish and generate and the induction of sporophore

The growth of the asexual vegetative hyphae of Cordyceps militaris (L.) Link. can be induced and be carried out in solid rich medium PDA and liquid rich medium SDB, 25 DEG C of cultivations.Except asexual mycelial growth, Cordyceps militaris (L.) Link. can form hydrophobic circular conidium on PDA flat board.Different from the conidium that solid plate is formed, the asexual spore produced in SDB liquid culture is corynebacterium, and tool wetting ability, is called blastospore.

Under experiment condition, the induction of Cordyccps-militaris-(L.)-link. Sporophore is carried out on rice medium or in body silkworm chrysalis, generally chooses fresh conidium spore suspension and inoculates.The induction of sporophore on rice medium, after inoculation 25 DEG C of dark culturing of 1 week advanced behavior phase, after asexual mycelia fully grows, then carry out the light cycle induction (12h illumination/12h is dark) of sporophore under going to 22 DEG C of conditions.Due to insect body wall to external world illumination there is shielding effect, silkworm chrysalis sub-entities induction without the need to carry out early stage dark processing, can directly cultivate under light cycle condition.In fruiting-body inducement process, strictly controlled environment humidity, ensures that humidity is not less than 80%.From being seeded to sporophore maturation, about 50 ~ 60 days.

1.2 Cordyceps militaris (L.) Link. genomes extract

(1) Cordyceps militaris (L.) Link. CM01 inoculation is on PDA flat board, is just putting cultivation one week for 25 DEG C.

(2) from the appropriate mycelia of scraping the cultivation flat board of a week, be inoculated in SDB liquid nutrient medium (SabouraudDextroseBroth, Difco), lower 25 DEG C of dark condition, 200rpm, cultivate 48h.

(3) after 50mlBD pipe collected by centrifugation thalline, then with vacuum pump collected by suction mycelium, and wash once with clear water.

(4) the dry mycelia after suction filtration is collected, after liquid nitrogen grinding to fine and smooth powder, add DNA extraction liquid (0.1MTris-HCl (pH8.0), 1.0%SDS, 2%TritonX-100,10mMEDTA, 0.1MNaCl) and quartz sand continues to be crushed to microscopy time almost can't see complete spore.

(5) add beta-mercaptoethanol (DES: beta-mercaptoethanol=110:1), mix rear 65 DEG C of temperature bath 30min, every 10min puts upside down mixing once.

(6) add the saturated phenol of equal-volume Tris, put upside down mixing, 5,000rpm, 5min are centrifugal.

(7) get supernatant to new centrifuge tube, add equal-volume CIA (chloroform/primary isoamyl alcohol 24:1) mixing, 5,000rpm, 5min are centrifugal.Repeat once.

(8), in careful absorption supernatant to new centrifuge tube, add equal-volume Virahol, after mixing ,-20 DEG C staticly settle, and the time is not shorter than 30min.

(9) 12,000rpm, 15min, centrifugally remove supernatant.

(10) ice-cold 70-75% (v/v) ethanol purge twice.

(11), after drying, appropriate ddH is added according to precipitation capacity size ₂o dissolves.

(12) get (2-4 μ l) solution 1% agarose gel electrophoresis and detect DNA quality and concentration.Remaining genome solution is put-20 DEG C of preservations.

The conversion that 1.3 agrobacterium tumefaciens AGL-1 mediate

1.3.1. agrobacterium tumefaciens AGL-1 competence preparation

(1) picking original strain AGL-1 (HellenR,, in YEB flat board (containing 50 μ g/mlCarbs) above streak culture activate MullineauxP (2000) AguidetoAgrobacteriumbinaryTivectorsanupdate.TrendsPlant Sci5 (10): 446-451).(2) single bacterium colony on picking flat board, is seeded in 4mlYEB liquid nutrient medium (containing 50 μ g/mlCarb), 28 DEG C, 200rpm, overnight incubation.(3) getting the above-mentioned bacterium liquid of 2ml is forwarded in 50mlYEB liquid nutrient medium (containing 50 μ g/mlCarb), and 28 DEG C, 200rpm, is cultured to bacterium liquid OD ₆₀₀be 0.5 (generally about 8h).(4) bacterium liquid is taken out, ice bath 30min, 4 DEG C, 8000rpm, 5min collected by centrifugation thalline.(5) abandon supernatant, add 10ml20mMCaCl ₂resuspended bacterial sediment.(6) 4 DEG C, 8,000rpm, 5min, recentrifuge collects thalline.(7) supernatant, with 2ml20mMCaCl ₂resuspended thalline again, adding sterile glycerol to final concentration is 15 ~ 20%.Divide and be filled to 1.5mlEP pipe, 100 μ l/ manage, and are stored in-80 DEG C after liquid nitrogen flash freezer.

1.3.2. Plastid transformation agrobacterium tumefaciens AGL-1 competence

(1) the Agrobacterium competence that taking-up-80 DEG C is frozen, is placed in thawed on ice and is about 10min.(2) in competence, add 0.5 ~ 1 μ g object plasmid, after mixing, leave standstill 30min on ice.(3) process is as follows successively: liquid nitrogen 5min, 37 DEG C of water-bath 5min, are placed in 2min on ice after process immediately.(4) 1ml nonreactive YEB liquid nutrient medium is added, 28 DEG C, 150rpm, recovery 3h.(5) 8,000rpm, 2min collected by centrifugation thalline.(6) abandon supernatant, to be spread evenly across YEB flat board (containing 50 μ g/mlCarb and 50 μ g/mlKan) after the resuspended thalline of 100 μ l nonreactive YEB, be inverted cultivation 2 days for 28 DEG C.(7) PCR verifies transformant, and picking positive transformant spends the night and shakes bacterium, 28 DEG C with 3mlYEB liquid (containing 50 μ g/mlCarb and 50 μ g/mlKan), 220rpm.(8) bacterium liquid is got part and be stored in-80 DEG C, final glycerol concentration is 15%.

1.3.3. Agrobacterium tumefaciens mediated Cordyceps militaris (L.) Link. genetic transformation

(1) preparation of Cordyceps militaris (L.) Link. conidiospore suspension: choose PDA (PotatoDextroseAgar, Difco) the illumination cultivation fresh Cordyceps militaris (L.) Link. of a week on flat board, the appropriate bacterium colony of scraping is to the aseptic 0.05%Tween-20 aqueous solution of 1ml, and vortex oscillation is separated mycelia and conidium; Filter with glass silk flosssilk wadding or three layers of lens wiping paper and remove mycelia, collect filtrate; 12,000rpm, 1min, centrifugal bacterium liquid collects spore; Abandon supernatant, with the resuspended spore of 1ml sterilized water, remove remaining nutrition; 12,000rpm, 1min, recentrifuge collects spore; Can repeating step 4 ~ 5 once, thoroughly to remove the remaining nutrition of spore surface; Abandon supernatant, with the resuspended spore of appropriate amounts of sterilized water, after Hematocyte Counter statistics spore number, spore suspension concentration is adjusted to 5 × 10 ⁵spore/ml, is used for subsequent experimental operation as working fluid.(2) Cordyceps militaris (L.) Link. transforms: the frozen bacterium of AGL-1 of the successful conversion respective carrier of transferring to 3ml liquid YEB (containing 50 μ g/mlCarb and 50 μ g/mlKan), 28 DEG C, 220rpm, overnight incubation; 8,000rpm, 2min, collected by centrifugation thalline; Abandon supernatant, with the appropriate resuspended thalline of IM liquid, be adjusted to OD ₆₅₀be 0.15; 28 DEG C, 150rpm, inducing culture is to OD ₆₅₀be 0.5 ~ 0.8, generally need 6h; Get each 100 μ l of Cordyceps militaris (L.) Link. spore suspension of Agrobacterium bacterium liquid and the fresh preparation of having induced, be mixed evenly in 1.5mlEP pipe; Get above-mentioned mixed solution 100 μ l and coat IM flat board, 28 DEG C of Dual culture 48h; M-100 solid medium temperature after subject to sterilization is down to 60 DEG C, adds corresponding microbiotic (Reflin, glufosinates/Totomycin); Every block Dual culture IM is dull and stereotyped to be covered with the above-mentioned M-100 substratum of 15ml, cultivates for 25 DEG C and occurs to single resistant clones for 7 ~ 10 days; Picking above-mentioned resistant clones picking, on new M-100 resistance culture base flat board, carries out programmed screening; Shake bacterium with SDB liquid nutrient medium to the resistant clones on postsearch screening flat board to cultivate, and carry out mark in the corresponding position at the dull and stereotyped back side of M-100; Suction filtration SDB cultivates mycelia, and extracting genome carries out PCR checking.

1.4 for the substratum of Cordyceps militaris (L.) Link. and the genetic manipulation of Luo Baici green muscardine fungus

For preparing 2.5 × MM salts solution (1L) of IM:

KH ₂PO ₄	3.625g
		K ₂HPO ₄·3H ₂O	6.72g
MgSO ₄·7H ₂O	1.250g
		NaCl	0.375g
CaCl ₂	0.125g
		FeSO ₄·7H ₂O	0.0062g
(NH ₄) ₂SO ₄	1.250g
		ddH ₂O	Add to final volume 1L

M-100 trace element solution (500ml):

H ₃BO ₃	30mg
		MnCl ₂·4H ₂O	70mg
ZnCl ₂	200mg
		Na ₂MoO ₄·2H ₂O	20mg
FeCl ₃·6H ₂O	50mg
		CuSO ₄·5H ₂O	200mg
ddH ₂O	Add to final volume 500mL

For preparing the M-100 salts solution of M-100:

KH ₂PO ₄	16g
		Na ₂SO ₄·10H ₂O	9.064g
KCl	8g
		MgSO ₄·7H ₂O	2g
CaCl ₂	1g
		M-100Trace Element Solution	8ml
ddH ₂O	Add to final volume 1L

IM (InductionMedium) (liquid) (200ml)

1 × MM salt	2.5 × the MM of 80ml
		10mM glucose	0.36g

0.5% glycerine	1ml
		ddH ₂O	Add to final volume 192mL

Add after being cooled to 50 DEG C after sterilizing:

40mM MES	8ml 1M stock
		200μM AS	4ml 10mM AS

IM (inducing culture, InductionMedium) (solid) (400ml)

1 × MM salt	160ml 2.5×MM
		5mM glucose	0.36g
0.5% glycerine	2ml
		ddH ₂O	Add to final volume 376mL
1.5％Agar	6g

Add after being cooled to 50 DEG C after sterilizing:

40mM MES	16ml 1M stock
		200μM AS	8ml 10mM AS

M-100(1L)

M-100 salts solution	62.5 ml
		Glucose	10 g
KNO ₃	3 g
		ddH ₂O	Add to final volume 1L
1.5％Agar	15 g

MES and AS mother liquor:

	Storage liquid concentration	PH (adjusting with 5 M KOH)
			MES	1 M	5.3
AS	10 mM	8

2, Aspergillus nidulans test relating operation

The 2.1 Aspergillus nidulans inductions life history

(1) asexually to nourish and generate: the asexual process induction in solid rich medium PDA and liquid rich medium SDB (auxotrophic strain can add uridine and uridylic) of nourishing and generating of Aspergillus nidulans is carried out, 28 DEG C or 37 DEG C of illumination cultivation; Or cultivate in YGUUTE substratum.On solid plate, main induction produces asexual spore, the mycelium pellet that in liquid nutrient medium, main induced synthesis is made up of mycelium.Bacterium liquid in liquid nutrient medium can be directly used in HPLC and detect the metabolite be secreted in substratum (extracellular) after crossing 0.22 μm of aqueous phase pin type filter; And mycelium pellet 50 DEG C of oven dry after sterile water wash, extract with coordinative solvent after liquid nitrogen grinding powdered, can be used for detecting the metabolite in Aspergillus nidulans cell.

(2) induction of sexual reproduction: under experiment condition, the induction of Aspergillus nidulans sexual reproduction is mainly carried out on solid plate.Different from the asexual process of nourishing and generating of Aspergillus nidulans, the derived need of sexual reproduction is carried out under hypoxemia dark condition.37 DEG C of mycelium pellets cultivating 14h are transferred on solid plate, with sealed membrane (splicing tape is better) by plate seal with reduce or avoid gaseous interchange, with masking foil or other can lucifuge film by whole flat board bag tight, flat board is placed in 37 DEG C and cultivates 20-24h, the formation of induction sexual structure.

2.2 substratum or reagent

Aspergillus is with nutrient enriched medium (YGUUTE) (1L)

0.5% yeast extract, 2% glucose, 5mM uridine (1.221g), 10mM uridylic (1.121g), 1 × vitamine mixture (2ml500 ×), 1 × basic salt ion (1ml1000 ×).The time of falling solid plate add 1.5% agar.

Protoplastis preparation and the agent prescription needed in transforming:

Solution I (100ml)

Solution II (100ml)

Solution III (100ml)

Solution IV (100ml)

Solution V (100ml)

Protoplasma solution (shaking 50ml bacterium liquid 20ml enzymatic hydrolysis system)

2.3 protoplastis preparation and conversions

(1) F-strain A.nidulansTN02A7 (purchased from FungalGeneticsStockCenter, the http://www.fgsc.net/) spore of YGUUTE cultured on solid medium 2.5d is collected in the Tween-80 of 0.02% (v/v).Spore is collected in 50ml centrifuge tube, 5,500rpm, 5min, with the resuspended spore of aseptic washing twice, 1ml sterilized water.

(2) get 10 μ l spore suspensions and dilute 100 times, blood counting chamber counts, finally according to 2 × 10 ⁸-1 × 10 ⁹spore total amount join in 50mlYGUUTE liquid nutrient medium, 37 DEG C, 220rpm, 6-7h.

(3) microscopy, receives bacterium when the spore germination until more than 50% and germ tube are within a spore length, 6,000rpm, 5min.

(4) directly spore is resuspended in 20ml protoplasma solution, 30 DEG C, 150rpm, 2.5-3h.

(5) microscopy, when most spore all forms protoplastis, when there is cavity in cell, 3,000rpm, 10min collect.Can not rotating speed be improved, blot surplus liquid with thick rifle head, do not outwell the protoplastis of suspension.

(6) wash twice by the solution III of precooling, precipitation be resuspended in 1ml solution V, ice bath spends the night.

(7) pre-sniper's shot head, gets protoplast suspension (competence) prepared by 100 μ l and is sub-packed in the centrifuge tube of 15ml for subsequent use.

(8) in 100 μ l protoplastiss, add a certain amount of fusion built when transforming and knock out fragment (1-20 μ l, 1-40 μ g), light dialling mixes.

(9) add in 50 μ l solution IV, ice bath 20min.

(10) 1ml solution IV is added, 25 DEG C or room temperature 20min.

(11) in the suspension after conversion, add the YAGK (0.8%Agar of 15ml liquefaction, melt before heat shock), put upside down several times, pour into previously prepared containing in 15mlYAGK (1.5%Agar) bottom culture medium flat plate, just putting a night for 30 DEG C, after 37 DEG C of inversion 2-3d, choose transformant by mycelia and screen.

YAGK fills a prescription: 0.5% yeast extract, 20mM glucose (4g), 1M sucrose (342.29g), 0.6M Repone K, adds the agar (Agar) of various dose as required.

The qualification of 2.4 homologous recombination transformants

Transformant general edge irregularity, selects this kind of bacterium colony, and the sticky spore that takes a morsel carries out spore PCR and verifies.Spore is put into 1.5mlEP pipe, by EP pipe high fire heating more than 5min in microwave oven, the triangular flask putting a water in stove is explosion-proof.Then by EP pipe in-80 DEG C of freezing more than 30min.Add 30 μ l sterilized waters afterwards, concussion, leave standstill, 14,000rpm, 5min are centrifugal, get 1 μ l supernatant as template.Use checking primer An12-F/An12-R to carry out PCR checking, judge whether transformant is homologous recombination transformant according to electrophoresis result.Select the transformant that homologous recombination occurs.

3, Yeast assay relating operation

3.1 substratum and reagent

YPD substratum (YeastExtractPeptoneDextroseMedium) (1L):

121 DEG C of moist heat sterilizations 20 minutes, solid medium adds 20g agar powder.

SC minimum medium:

Component	Content (w/v)
		Yeast nitrogen	0.67％
Carbon source (i.e. glucose or raffinose)	2％
		Aminoacid mixture 1	0.01％
Aminoacid mixture 2	0.005％
		Agar (for flat board)	2％

Mentioned component is dissolved in except carbon source in 900ml deionized water that (carbon source is glucose.If carbon source is raffinose, be then dissolved in 800ml deionized water), solid medium adds the agar powder of 2%, 121 DEG C of moist heat sterilizations 20 minutes.Be cooled to about 50 DEG C, add the raffinose that glucose that 100ml concentration degerming is after filtration 20% (w/v) or the degerming after filtration concentration of 200ml are 10% (w/v).Liquid or good plate are inverted in 4 DEG C of storages.

Amino acid/11 00 × powder, is fully ground (1L)

Leucine (Leucine), tryptophane (Tryptophan) and uridylic (Uracil) be made into 100 respectively × mother liquor put 4 DEG C for subsequent use.

Inducing culture:

Preparation composition and step and SC substratum basically identical, only when sterilizing is cooled to about 50 DEG C, the carbon source added changes the semi-lactosi that 100ml concentration degerming is after filtration 20% (w/v) into.

10 × TE: dissolve 1.21gTrisbase and 0.37gEDTA (sodium-salt form) in 90ml deionized water; With concentrated hydrochloric acid pH value be transferred to 7.5 and make cumulative volume reach 100ml; Filtration sterilization is also stored in room temperature.Diluted 10 times and 1 × TE.

10 × LiAc: dissolve 10.2glithiumacetate in 90ml deionized water; With Glacial acetic acid pH value is transferred to 7.5 and overall solution volume is transferred to 100ml; Filtration sterilization is also stored in room temperature.Diluted 10 times and 1 × LiAc.

1 × LiAc/0.5 × TE: mixing 5ml10 × TE and 10ml10 × LiAc; 100ml is adjusted to deionized water; Filtration sterilization is also stored into room temperature.

1 × LiAc/40%PEG-3350/1 × TE: mixing 10ml10 × LiAc, 10ml10 × TE and 40gPEG-3350; Add deionized water and fully dissolve PEG (can suitably heated solution fully to dissolve PEG) to 100ml; 121 DEG C of moist heat sterilization 20min, room temperature preservation.Note: this solution preferably uses front now with the current.

The conversion of 3.2 yeast

(1) from 30 DEG C cultivate YPD flat boards picking yeast list colony inoculation in 10mlYPD substratum, 30 DEG C, 220rpm shaken overnight cultivate.

(2) OD of the bacterium liquid that spends the night is measured ₆₀₀value, with 50mlYPD substratum by its OD ₆₀₀value is diluted to 0.4,30 DEG C, and 220rpm continues shaking culture 2-4h.

(3) 3000-8000rpm collects yeast cell in centrifugal 5 minutes, removes supernatant.40ml1 × TE re-suspended cell is used to precipitate again.

(4) 3000-8000rpm collects yeast cell in centrifugal 5 minutes, removes supernatant.Use 2ml1 × LiAc/0.5 × TE re-suspended cell precipitation again.

(5) incubated at room cell 10 minutes.

(6) plasmid DNA that need be transformed by 1 μ g and 100 μ g salmon sperm DNAs (being placed in immediately on ice after boiling water boiling 5min in advance) mix with the sterilised yeast suspension of 100 μ l step 5 gained.

(7) 700 μ l1 × LiAc/40%PEG-3350/1 × TE are added, and mixing of fully vibrating rapidly.

Hatch 30 minutes for (8) 30 DEG C.

(9) add 88 μ lDMSO, fully mix, 42 DEG C of heat shocks 7 minutes.

The centrifugal 2-3 minute of (10) 5,000-8,000rpm, removes supernatant.

(11) by 1 × TE re-suspended cell precipitation, and with 1ml1 × TE re-suspended cell.

(12) re-suspended cell is deposited in 50-100 μ l1 × TE, and is uniformly coated on corresponding screening flat board.

3.3 induction expression of recombinant proteins

(1) picking is grown on 30 DEG C and selects the dull and stereotyped upper mono-clonal containing expression plasmid, and being transferred to 15ml carbon source is in the SC Selective agar medium of raffinose or glucose, 30 DEG C, 220rpm incubated overnight.

(2) OD of the bacterium liquid that spends the night is measured ₆₀₀value, calculates sampling amount and makes it add OD in 50ml inducing culture ₆₀₀be 0.4.

(3) corresponding bacterium liquid is drawn, 4 DEG C, centrifugal 5 minutes of 3,000g by calculating in step 2.

(4) by 1-2ml inducing culture re-suspended cell precipitation, and be forwarded in 50ml inducing culture, 30 DEG C, 220rpm shakes cultivation.

(5) respectively 0,2,4,6,8 and 10h (according to test requirements document, also can time expand or at point At All Other Times) sample from inducing culture.Can 5ml bacterium liquid be got at each time point respectively and measure OD ₆₀₀value.

(6) 4 DEG C, the centrifugal bacterium liquid of 3,000g 5 minutes (as detected the metabolite be secreted in substratum, the supernatant reservation in this can being walked, in order to detecting use later).

(7) outwell supernatant, and precipitate with 500 μ l sterilized water re-suspended cells.

(8) cell suspension is transferred in the aseptic EP pipe of 1.5ml, 4 DEG C, centrifugal 30 seconds of 13,000rpm.

(9) remove supernatant, cell precipitation is stored into-80 DEG C of refrigerators for subsequent use.Also the expression of recombinant protein can be detected by lysing cell immediately.

4, high performance liquid chromatography (HPLC) relating operation

High performance liquid chromatograph is Shimadzu LC-20AD type high performance liquid chromatograph.Detection chromatographic column is the InertsilODS-SP liquid-phase chromatographic column (4.6 × 250mm, 5 μm) of Shimadzu Corporation, and concrete wash-out conditional parameter is:

Determined wavelength	260nm
		Column temperature	36℃
Flow velocity	1ml/min
		Moving phase	Water (A): methyl alcohol (B)=85:15 constant ratio wash-out
Sample size	20μl

When there being multiple sample will carry out batch processing, allow the elution time of each sample a little long, to avoid the residual peak of a upper sample on the impact of next sample overall peak type as far as possible.

5, primer

The primer related in subsequent embodiment is listed in table 2.

Table 2

Embodiment

The acquisition of the biosynthetic latent gene of embodiment 1, cordycepin bunch

By by Cordyceps militaris (L.) Link. (CM01 bacterial strain) and pattern filamentous fungi A. nidulans (Aspergillusnidulans, A4 bacterial strain) icp gene group analysis, the present inventor have found the biosynthetic latent gene of cordycepin bunch (genecluster), they are Cm1 (GenBank accession number: CCM_04436) respectively, Cm2 (CCM_04437), Cm3 (CCM_04438) and Cm4 (CCM_04439) (on Figure 1A), homologous gene in corresponding Aspergillus nidulans is An1 (AN3333) respectively, An2 (AN3331), An3 (AN3330) and An4 (AN3329) (under Figure 1A).

By dot matrix (dotplot), sequence alignment analysis is carried out to the cordycepin synthetic gene of these two species bunch, find that the coding region of each gene shows good collinearity (Figure 1B).Structure function domain analysis shows, the metabolic enzymes that three genes encodings in this gene cluster are relevant, be that Cm1 encode phosphohydrolase (Metaldependentphosphohydrolase) that metal ion relies on and the Cm3 of oxydo-reductase (oxidordeuctase), Cm2 that encode encodes the phosphotransferase (ATPphosphoribosyltransferase) that ATP relies on respectively, remaining gene C m4 encodes the translocator (ABC-typetransporter) (table 1) of an ABC type.

Table 1

For ease of describing, below Cm1/An1 is referred to as cpA, Cm2/An2 is called cpB, and Cm3/An3 is called cpC, and Cm4/An4 is called cpD.Experiment proves that cpD gene is not the translocator of cordycepin.

Embodiment 2, Cordyceps militaris (L.) Link. wild strain CM01 produce comparing of cordycepin with cpABC knockout mutant strain

For the latent gene bunch that above-mentioned analysis obtains, first the present inventor has knocked out three enzyme genes cpA, cpB and cpC in Cordyceps militaris (L.) Link. wild strain CM01 strain gene group simultaneously.

1, the structure of gene knockout carrier

Cordyceps militaris (L.) Link. deletion mutant body builds the gene replacement method depended on based on homologous recombination, and the fungal transformation technology that agrobacterium tumefaciens AGL-1 mediates.With binary vector pDHt-Bar (DuanZ, ChenY, HuangW, ShangY, ChenP, WangC. (2013) Linkageofautophagytofungaldevelopment, lipidstorageandvirulenceinMetarhiziumrobertsii.Autophagy 9 (4): 538-549) (comprise Glufosinate ammonium resistant gene (Bar), for forming homology arm-Bar-downstream, upstream homology arm structure, respective section gene in genome is knocked out by homologous recombination) set out, carry out the structure of gene knockout plasmid.Primer cpABC-Uf/cpABC-Ur is used to amplify target gene bunch ORF upstream homology arm fragment by PCR method, by being linked in sequence after EcoRI single endonuclease digestion in the EcoRI site, upstream of pDHt-Bar carrier, be connected with the pDHt-Bar carrier of upstream homology arm; Amplify target gene bunch ORF downstream homology arm fragment with cpABC-Df/cpABC-Dr by PCR method, be inserted into the pDHt-Bar carrier downstream SpeI site that is connected with upstream homology arm to construct finally for the knockout carrier plasmid of Agrobacterium-mediated Transformation by order after SpeI single endonuclease digestion.

2, Plastid transformation

Knock out plasmid and carry out Cordyceps militaris (L.) Link. conversion (see above and state 1.3) for the fungal transformation technology that agrobacterium tumefaciens AGL-1 mediates.Obtaining positive transformant is the bacterial strain that producer knocks out.

Cordyceps militaris (L.) Link. wild type strain CM01 and three enzyme gene is knocked to the comparative analysis of the HPLC detected result of the SDB fermented liquid of mutant strain, find to go out peak position at cordycepin standard substance, and compared to wild strain (on Fig. 2 A), the cordycepin peak of mutant strain disappears (under Fig. 2 A).The water extraction sample detection result of wild-type and mutant strain sporophore also shows, and relative to wild strain (on Fig. 2 B), the cordycepin product peak of mutant strain can not be detected (under Fig. 2 B).

The above results illustrates, enzyme gene cpA, cpB and cpC are the key genes that cordyceps militaris link bacterial strain CM01 produces cordycepin, and it is called cordycepin biological synthesis gene cluster (cpABC) by the present inventor.

Embodiment 3, cpA, cpB and cpC heterogenous expression in green muscardine fungus and yeast saccharomyces cerevisiae

1, cordycepin biological synthesis gene cluster (cpABC) proceeds in Luo Baici green muscardine fungus and expresses

Cordyceps militaris (L.) Link. and Aspergillus nidulans and the Luo Baici green muscardine fungus genome that completed order-checking contrast by the present inventor, to find in Luo Baici green muscardine fungus genome not with the sequence of three gene very high homology in cordycepin synthetic gene bunch, and to Luo Baici green muscardine fungus carry out cultivation extract detected result show, Luo Baici green muscardine fungus cannot produce cordycepin.Therefore, cordycepin biological synthesis gene cluster (cpABC) proceeds in Luo Baici green muscardine fungus and expresses by the present inventor.

The present inventor devises pair of primers cpABC-MrF/cpABC-MrR (table 2), from the genome sequence of Cordyceps militaris (L.) Link. (Cordycepsmilitaris) (CM01 bacterial strain), these three enzyme genes is increased out (9321bp) in the lump together with their promoter sequence, the method of equally cutting connection through enzyme is implemented in the EcoRI/EcoRI site of pDHt-Ben plasmid (transforming by the Glufosinate ammonium resistant gene Bar of above-mentioned pDHt-Bar carrier is changed to benomyl resistance gene Ben), Luo Baici Metarhizium Strains Ma23 (USDA-ARSCollectionofEntompathogenicFungi is proceeded to after agriculture bacillus mediated, http://www.ars.usda.gov/News/docs.htm? docid=12125) in, the knock-in expression strain of three gene co-expressings is obtained through resistance screening.Concrete Agrobacterium-mediated Transformation, resistance screening system and whole conversion process all transform the same with Cordyceps militaris (L.) Link..

HPLC comparative analysis Ma23 wild strain and knock-in expression strain are in the fermented liquid of SDB substratum, find the position at cordycepin peak, knock-in expression strain has obviously had more a peak (Fig. 4 B), LC-MS result confirms that the molecular size range at this peak is consistent with the molecular size range of cordycepin standard model, and namely this peak is the peak of cordycepin.

By above-mentioned experiment, prove that three genes cpA, cpB and cpC in gene cluster are responsible for the biosynthesizing of cordycepin.

2, cordycepin biological synthesis gene cluster (cpABC) proceeds to heterogenous expression in yeast saccharomyces cerevisiae

In order to further checking, and be convenient to subsequent biochemical analysis and illustrate the biosynthetic pathway of cordycepin, the present inventor is by cordycepin biological synthesis gene cluster (cpABC) heterogenous expression in yeast saccharomyces cerevisiae.

In order to heterogenous expression cordycepin synthetic gene, the present inventor have selected the INVSc1-pYES2 expression system of Invitrogen company.INVSc1 is Histidine, leucine, tryptophane and uracil auxotrophy bacterial strain.PYES2 grows on the auxotrophic selective medium of uridylic because only can recover INVSc1 bacterial strain, in order to illustrate the concrete effect of three enzyme gene individual genes in gene cluster, the present inventor also needs to build the plasmid that can recover to grow on the substratum of other two seed amino acid defects.For this reason, consider the Polyclonal restriction enzyme site on three enzyme gene orders and pYES2 plasmid, the present inventor selects to have increased respectively Leu2 and Trp1 sequence from pRS315 and pRS314 plasmid (available from Shanghai plant physiological ecology institute), be inserted into HheI and the NcoI site of pYES2 (purchased from Invitrogen) respectively, build pYES2-Leu2, pYES2-Trp1 plasmid.

With the genome of Cordyceps militaris (L.) Link. (Cordycepsmilitaris) (CM01 bacterial strain) for template, primer cpA-ScF/cpA-ScR, cpB-ScF/cpB-ScR, cpC-ScF/cpC-ScR (table 2) is used to go out the cDNA sequence of three enzyme genes through pcr amplification respectively, and connect the KpnI/XbaI site being implemented in the KpnI/EcoRI site of expression plasmid pYES2-Trp1, the HindIII/XbaI site of pYES2-Leu2 and pYES2 respectively, obtain pYES2-Trp1-cpA, pYES2-Leu2-cpB and pYES2-cpC induction expression plasmid respectively.Simultaneously for ease of follow-up expression checking, the present inventor is connected to 6 × His label at the C end of each gene.

Proceed to yeast saccharomyces INVSc1 bacterial strain (Genotype:MATahis3 Δ 1leu2trp1-289ura3-52/MAT α his3 Δ 1leu2trp1-289ura3-52 respectively or simultaneously; Phenotype:His-, Leu-, Trp-, Ura-) (purchased from Invitrogen), after auxotrophy screening, the present inventor obtains the transformant KIcpABC that simultaneously have expressed three enzymes respectively, have expressed the transformant KIcpAB of cpA and cpB simultaneously, have expressed the transformant KIcpBC of cpB and cpC simultaneously and individually expresses cpA, cpB, the transformant KIcpA of cpC, KIcpB, KIcpC.

By transformant bacterial strain and the INVSc1 bacterial strain KIvector proceeding to empty plasmid in SC liquid selective medium after abduction delivering, HPLC detection is carried out to fermented liquid, found that to only have the transformant bacterial strain (KIcpABC and KIcpAB) of simultaneously expressing cpA and cpB relative to the yeast mutant or other transformant bacterial strains that have only proceeded to an empty plasmid, had more a peak (Fig. 3) in the position of standard cordycepin.LC-MS result confirms that the molecular size range at this peak is consistent with the molecular size range of cordycepin standard model, and namely this peak is the peak of cordycepin.

The mutant strain of single expression gene cpA (Fig. 3 KIcpA) or cpB (Fig. 3 KIcpB) all can not produce cordycepin, only have two genes simultaneously abduction delivering could synthesize cordycepin (Fig. 3 KIcpAB).

These results suggest that, cpA and cpB is two very necessary genes for the synthesis of cordycepin, and needs synergy to produce cordycepin.

HPLC detects and compares discovery, and Saccharomyces cerevisiae transformant (under Fig. 4 A) of expressing cordycepin gene cluster has had more a peak with Luo Baici green muscardine fungus transformant (under Fig. 4 B) respectively in the position of standard cordycepin.LC-MS result confirms that the molecular size range at this peak is consistent with the molecular size range of cordycepin standard model, and namely this peak is the peak of cordycepin, further demonstrates the biosynthesizing that three genes are responsible for cordycepin.

Singly knocking out or two knock-out experiment for cpA, cpB and cpC in embodiment 4, Cordyceps militaris (L.) Link.

For the effect of further clear and definite cordycepin synthetic gene bunch each gene in cordycepin synthesis, the present inventor has carried out singly knocking out to cpA, cpB, cpC gene respectively and twoly to have knocked out in Cordyceps militaris (L.) Link. (CM01 bacterial strain) genome.In order to realize knocking out, the vector construction related to and method for transformation identical with the method " knocking out three enzyme genes cpA, cpB and cpC in cordyceps militaris link bacterial strain genome " in embodiment 2 simultaneously, the only amplimer difference (table 2) in view of needing the section that knocks out different:

Carry out the amplimer that cpA gene list knocks out upstream and downstream homology arm and be respectively cpA-Uf/cpA-Ur, cpA-Df/cpA-Dr, knock out positive strain with primer cpA-F/cpA-R checking.The marker gene adopted is all Glufosinate ammonium resistant gene.

Carry out the amplimer that cpB gene list knocks out upstream and downstream homology arm and be respectively cpB-Uf/cpB-Ur, cpB-Df/cpB-Dr, knock out positive strain with primer cpB-F/cpB-R checking.The marker gene adopted is all Glufosinate ammonium resistant gene.

Carry out the amplimer that cpC gene list knocks out upstream and downstream homology arm and be respectively cpC-Uf/cpC-Ur, cpC-Df/cpC-Dr, knock out positive strain with primer cpC-F/cpC-R checking.The marker gene adopted is all Glufosinate ammonium resistant gene.

The amplimer carrying out the upstream and downstream homology arm that cpA, cpB two genes knock out altogether is respectively cpAB-Uf/cpAB-Ur, cpAB-Df/cpAB-Dr, knocks out positive strain with primer cpAB-F/cpAB-R checking.The marker gene adopted is all Glufosinate ammonium resistant gene.

The amplimer carrying out the upstream and downstream homology arm that cpC, cpB two genes knock out altogether is respectively cpBC-Uf/cpBC-Ur, cpAB-Df/cpAB-Dr, knocks out positive strain with primer cpBC-F/cpBC-R checking.The marker gene adopted is all Glufosinate ammonium resistant gene.

For wild-type cordyceps militaris link bacterial strain, utilize Agrobacterium to transform, obtain positive transformant.

Respectively HPLC detection is carried out to positive knock-out bacterial strain.Result shows, as long as relate to knocking out muton and all cannot producing cordycepin (Fig. 5) of cpA or cpB gene.

Although cpC gene does not participate in the synthesis of cordycepin directly, along with the disappearance of cpC gene, the output of cordycepin decreases (Fig. 7), and prompting cpC affects the generation of cordycepin by other approach.

For two knock-out experiments of cpA, cpB in embodiment 5, Aspergillus nidulans

The fragment (Gene A n1 and An2) of knocking out of cpA and cpB gene corresponding in Aspergillus nidulans is carried out two knocking out by the present inventor equally.

1, obtain fusion and knock out fragment

Adopt the method for fusion DNA vaccine, its principle and design of primers are see document: SzewczykE etc. (2006), FusionPCRandgenetargetinginAspergillusnidulans.NatProtoc .1 (6): 3111-3120).(1) increase homology arm and marker gene: with wild strain filamentous fungi A. nidulans (A.nidulans) A4 (Glasgow wild-type (veA+); Purchased from FungalGeneticsStockCenter, http://www.fgsc.net/) genomic dna (10-100ng) is template, use primer An12-Uf/An12-Ur to amplify upstream homology arm fragment, use primer An12-Df/An12-Dr to amplify downstream homology arm fragment; With the plasmid pXDRFP4 (1-10ng) containing pyrG gene for template, primer An12-Gf/An12-Gr is used to amplify marker gene fragment (concrete primer sequence is in table 2).(2) upstream and downstream homology arm and middle marker gene fusion: the PCR primer of previous step is cut glue and reclaim target fragment (can directly carry out the recovery of PCR primer purifying when mixing and be with), measure the concentration reclaiming product with Nanodrop.Take a morsel recovery fragment, by left homology arm: marker: right homology arm mol ratio 1:2:1 mixes as template, uses primer An12-Wf/An12-Wr (concrete primer sequence is in table 2) to carry out pcr amplification and go out fusion and knock out fragment.

2, conversion is knocked out

Merge and knock out fragment for protoplast transformation (see above and state 2.3 and 2.4).Obtaining positive transformant is the bacterial strain that producer knocks out.

Show through HPLC detected result, the muton knocking out An1 and An2 cannot produce cordycepin (Fig. 6).

Sum up

Based on above-mentioned heterogeneic biological function explore, the present inventor draws the biosynthetic pathway (Fig. 8) of cordycepin, namely adenosine is phosphorylated generation 3 '-AMP under special protein kinase effect, certain 3 '-AMP also can by 2 ', 3 '-phosphodiesterase in 3 '-cAMP-adenosine metabolic pathway produces 2 ', 3 '-cAMP effect.Gained 3 '-AMP is dephosphorylation under the effect of cpB, obtains a unstable enolization compound, and the spontaneous isomery of this compound forms carbonyl, is responsible for carbonyl reduction to become hydroxyl, generates cordycepin under the effect of cpA reductase enzyme.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Sequence table

Claims

1. produce a method for cordycepin, it is characterized in that, described method comprises:

(2) cultivate the host cell of (1), make it to produce cordycepin.

2. the method for claim 1, is characterized in that, in (1), described cordycepin biosynthesis related genes comprises Cm1, Cm2 gene and the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1, An2 gene and the An3 gene of Aspergillus nidulans.

3. the method for claim 1, is characterized in that, described host cell is the host cell comprising 3 '-AMP or autologous the generation 3 '-AMP of energy.

4. method as claimed in claim 3, it is characterized in that, described host cell is fungi; Preferably described fungi comprises: Cordyceps sinensis fungus, aspergillus fungi, Metarhizium fungi, yeast belong fungi.

5. the cordycepin biological synthesis gene cluster be separated, it is characterized in that, described cordycepin biological synthesis gene cluster comprises Cm1 and the Cm2 gene of Cordyceps militaris (L.) Link., or comprises An1 and the An2 gene of Aspergillus nidulans.

6. the cordycepin biological synthesis gene cluster be separated as claimed in claim 5, it is characterized in that, described cordycepin biological synthesis gene cluster comprises Cm1, Cm2 gene and the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1, An2 gene and the An3 gene of Aspergillus nidulans.

7. recombinant vectors, is characterized in that, described recombinant vectors comprises cordycepin biosynthesis related genes; Described cordycepin biosynthesis related genes comprises Cm1 and Cm2 and/or the Cm3 gene of Cordyceps militaris (L.) Link.; Or described cordycepin biosynthesis related genes comprises An1 and An2 and/or the An3 gene of Aspergillus nidulans.

8. a host cell, is characterized in that, described host cell comprises cordycepin biosynthesis related genes; Described cordycepin biosynthesis related genes comprises Cm1 and Cm2 and/or the Cm3 gene of Cordyceps militaris (L.) Link., or comprises An1 and An2 and/or the An3 gene of Aspergillus nidulans.

9. host cell as claimed in claim 8, is characterized in that, described host cell is the host cell comprising 3 '-AMP or autologous the generation 3 '-AMP of energy; Preferably, described host cell is fungi; More preferably, described fungi comprises: Cordyceps sinensis fungus, Metarhizium fungi, aspergillus fungi, yeast belong fungi.

10. the purposes of the cordycepin biological synthesis gene cluster described in claim 5 or 6 or expression vector according to claim 7 or the arbitrary described host cell of claim 8-9, for the production of cordycepin.

11. 1 kinds, for the production of the test kit of cordycepin, is characterized in that, described test kit comprises:

Expression vector according to claim 7 or the arbitrary described host cell of claim 8-9.