CN105567578B - A kind of ganoderic acid high-yielding engineering bacterial strain kmust-SE - Google Patents

Abstract

The present invention discloses a kind of ganoderic acid high-yielding engineering bacterial strain kmust-SE and its construction method, it is CGMCC NO.11401 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center, ganoderma lucidum engineering bacteria provided by the invention is by selecting ganoderma lucidum strong promoter P-gpdIt is overexpressed encoding squalene monooxygenase SE gene, obtains high yield ganoderic acid ganoderma lucidum engineering bacteria kmust-SE；Show that SE transformant bacterial strain produces monomer Lanost-8-en-26-oic acid,3,7,12-trihydroxy-11,15,23-trioxo-,(3BETA,7BETA,12BETA)- A-Me, GA-T, GA-Mk by shake flask fermentation experiment, the content of GA-S is the 1.2 of WT bacterial strain respectively, 2.1,2.5,1.7 times, therefore, this superior strain can be used as the engineered strain of production ganoderic acid, be with a wide range of applications.

Description

A kind of ganoderic acid high-yielding engineering bacterial strain kmust-SE

Technical field

The invention belongs to genetic engineerings and metabolic engineering field, by genetic engineering to the squalene in ganoderic acid metabolic pathway The method of monooxygenase (SE) gene overexpression, constructs the engineered strain of a ganoderic acid high yield.

Background technique

Ganoderma lucidum (Ganoderma lucidum) it is Basidiomycetes, Polyporaceae, Ganoderma fungi.Lucid ganoderma fungus exists There are more than 20 kinds, including red sesame, Huang Zhi, purple sesame, black sesame, Ganoderma capuse, artist's conk etc. in China.China is existing as drug using ganoderma lucidum More than 2000 years history.Ganoderma lucidum adjusts blood glucose, controls blood pressure, adjuvant therapy chemicotherapy, liver protection shield for enhancing human immunity Liver promotes sleep etc. to all have significant curative effect.Since its special using is worth, the analysis of effective component and pharmacology of ganoderma lucidum It learns research and has caused international extensive concern, especially in Japan, the U.S., the countries such as South Korea.Currently, the research of ganoderma lucidum is Be deep into molecular level, it is some specially publish in succession, describe biological characteristics, the cultivation skill of ganoderma lucidum from different angles Situations such as art, pharmacotoxicological effect and clinical application.

Ganoderic acid is the triterpene substance in molecule structure containing carboxyl, it belongs to highly oxidized from the point of view of structure Lanostane derivative.Since nineteen eighty-two Kubota T et al. for the first time isolated ganoderic acid, have more than 130 kinds of ganoderma lucidums at present Acid is separated.They are mostly tetracyclic triterpenoid, contain 30 carbon atoms, generally have hydroxyl in structure, red There is stronger hydroxyl absorption peak in external spectrum.The characteristic absorption of multiple wavelength is also presented in ultraviolet spectra, majority is 250 Nm, 237 nm, there is absorption peak at 365 nm.Ganoderic acid has many important pharmacological activity such as: anticancer inhibits mouse hepatosarcoma (HTC) proliferation of cell；Protect liver, the ganoderic acid extract in ganoderma lucidum can reinforce its detoxication；AntiHIV1 RT activity；Blood pressure lowering；It is anti- Oxidation；Inhibit platelet aggregation, inhibit histamine release, analgesia inhibits eukaryotic cell dna poly enzymatic activity, inhibits farnesyl egg The effects of white transferase active and promotion humoral immune function.

Squalene monooxygenase (SE) gene is an important gene in ganoderic acid route of synthesis.Ganoderic acid is logical Mevalonate pathway synthesis is crossed, this approach is prevalent in animal, plant, fungi.Mevalonic acid is through mevalonic acid first Three step enzymatic of kinases (MVK), mevalonic acid 5- phosphokinase (PMK) and pyrophosphoric acid mevalonic acid decarboxylase (MDD) Reaction is converted into Isoprenoid (IPP).IPP is further converted into farnesyl pyrophosphate (FPP).FPP is closed in squalene Squalene is synthesized under catalysis at enzyme (SQS), then passes through squalene monooxygenase squalene monooxygenase(SE) Effect generate squalene 2,3- oxide, using lactosterol synthase (LS) effect generate lanosterol.Finally lead to The modification for crossing different enzymes generates the ganoderic acid of different structure.The route of synthesis of ganoderic acid is as follows:

。

It is big by such environmental effects and Wild ganoderma resource is limited, Ganoderma lucidum mycelium training since Wild ganoderma growth cycle is long It forms to produce the important method of active material.With being continuously increased to ganoderma active material requisite, how ganoderic acid is improved Yield and increase ganoderma lucidum effective component have attracted more and more attention from people.Liquid deep layer fermenting technology is to carry out quickly industry The important means that metaplasia produces.Currently marketed ganoderic acid is mainly the mycelium obtained from ganoderma lucidum fruitbody and liquid deep layer fermenting Middle acquisition.Content of the ganoderic acid in glossy ganoderma cell is lower when due to Wild ganoderma liquid deep layer fermenting, and it is also more difficult to isolate and purify, Limit the research and its extensive use to the activity, the mechanism of action of ganoderic acid.In recent years, genetic engineering and metabolic engineering are rapid Development, becomes the important means of modern molecular breeding.Changed by molecular biology methods such as molecular cloning and gene splicings The genome of bacterial strain is made, to improve the content of active constituent increasingly by the favor of scholars.How above to be mentioned from molecular level The yield of high ganoderic acid is current technical problem urgently to be solved.

Summary of the invention

Present invention aim to address wild type ganoderma strains itself to produce the lower problem of ganoderic acid, provides a kind of ganoderic acid Superior strain, the bacterial strain be high yield ganoderic acid ganoderma lucidum (Ganoderma lucidum) engineering bacteria kmust-SE, in On October 23rd, 2015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: court of Beijing The institute 3 of positive area's North Star West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC NO.11401.

The construction method of ganoderic acid high-yielding engineering bacterial strain of the present invention is as follows:

As initial vector and the strong promoter P- of ganoderma lucidum itself is used using PMD19-Tgpd, terminator T-sdhB、cbxBase Cause,cbxGene (having carboxin resistance) is the resistant gene from ganoderma lucidum itself, its main feature is that in mushroom class load The transformation efficiency of homologous marker gene is higher in the transformation system of bacterium, can stablize heredity, resistance.

1, by ganoderma lucidum promoter P-gpdWith terminator T-sdhBIt is connect with PMD19-T, by ganoderma lucidumcbxResistant gene insertion It arrivesPstI restriction enzyme site, to be built into pJW-EXP carrier；Its strong promoter and terminator with ganoderma lucidum, and there is ganoderma lucidum The resistance of itself.

Wherein expand ganoderma lucidum strong promoter P-gpdPrimer sequence are as follows:

P-gpd- F:5 '-TCCAAAGCCGCTCTCATGGCATGGCAC- 3 ',

P-gpd- R:5 '-GCTAGCGTTGAGAGGGGATGAAGAGTGAGTAAGAAG- 3 '；

Expand ganoderma lucidumsdhBThe primer sequence of gene terminator are as follows:

T-sdhB- F:5 '-ATGAGCGGGTCAGAGAGT- 3 ',

T-sdhB-R : 5'-TGCTCTATGTCTTGCCTTGT- 3'；

Expand ganoderma lucidumcbxThe primer of resistant gene are as follows:

cbx- F:5 '-TCTGCTCTTCCCGATTGCTGCATTTGT-3 ',

cbx- R:5 '-CTATGTCTTGCCTTGTCTCGCGTCAACC-3 '；

2, in ganoderic acid route of synthesis key enzyme squalene monooxygenase (SE) cloned from ganoderma lucidum genome it is (used Primer be SE-Nhe-F and SE-Sma-R), and be inserted into pJW-EXP carrier, obtain pJW-EXP-tSE carrier；Primer Sequence are as follows:

SE-Nhe-F:5 '-GCTAGCATGTGGTCCGTCTCGTACGACATCA- 3 '

SE-Sma-R:5 '-CCCGGGTCACGGGCGGATCTCCGTCC- 3 '；

3, the method for mediating protoplast fusion by PEG, pJW-EXP-tSE is transformed into wild type glossy ganoderma cell, Ganoderma lucidum transformant is screened using carboxin as resistance, filters out transformant on the CYM plate containing carboxin resistance.

4, transformant is subjected to secondary culture in the CYM plate of carboxin resistance.

5, the Liquid Culture of glossy ganoderma cell:

PDA culture medium (g/L): glucose 10, agar 20, magnesium sulfate 1.5, potassium dihydrogen phosphate 3, vitamin B1 0.05 and the murphy juice for preparing.

The preparation method of murphy juice: removing the peel fresh potato for 200 g and be cut into small pieces, and 1.0 L of deionized water is added and boils 30 Min takes filtrate with eight layers of filtered through gauze, the preparation for PDA culture medium.

Seed culture medium (g/L): glucose 35, peptone 5, yeast extract 2.5, potassium dihydrogen phosphate 1,0.5 and of magnesium sulfate Vitamin B1 0.05.

Fermentation medium (g/L): peptone 5, yeast extract 5, potassium dihydrogen phosphate 1.0, magnesium sulfate 0.5, vitamin B1 0.05, lactose 35 originates pH 5.5.

CYM culture medium (g/L): glucose 20, maltose 10, yeast powder 2, peptone 2, MgSO₄ 0.5、KH₂PO₄ 4.6, agar 10.

Inclined-plane culture: inoculation mycelia is cultivated 5-7 days for 28 DEG C in murphy juice-inclined-plane glucose-agar (PDA).

First order seed culture: 40 mL culture mediums and 10 mL mycelium suspensions are added in 250 mL shaking flasks (from one Obtained in inclined-plane), and cultivated 5 days under 30 DEG C, 120 rpm.

Secondary seed culture: 45 mL culture mediums and 5 mL first order seed culture solutions are added in 250 mL shaking flasks (about 500 mg DW/L, level-one culture are inoculated with after being smashed with bead), it is cultivated 2 days under 30 DEG C, 120 rpm.

Fermented and cultured: 45 mL fermentation mediums and 5 mL secondary seed fermentation liquids are added in 250 mL shaking flasks (about 500 ~ 600 mg DW/L, second level culture are inoculated with after being smashed with bead), it is cultivated under 30 DEG C, 120 rpm.

6, the separation and extraction of monomer ganoderic acid

100 mg of stem cell powder is accurately weighed, 3 mL 70%(v/v are added) ethyl alcohol soaked overnight, ultrasonic treatment 3 times, often Secondary 30 min.10000 rpm obtain supernatant after being centrifuged 5 min.It is dried in 50 DEG C of baking ovens.With 200 μ L hplc grade methanols Thoroughly dissolution, with 0.22 μm of membrane filtration.Ganodenic acid monomer is detected with HPLC.HPLC testing conditions are as follows: HPLC chromatogram column is C18 column (Agilent 1200 series, 5 μm of Agilent Zorbax SB-C18 column, 250 × 4.6 mm)；Sample introduction Measure 20 μ L；1 mL/min of flow velocity；Mobile phase A is methanol/acetic acid (100:0.5(v/v)), Mobile phase B is ultrapure water, 0-20 Min:A phase is 80 %-100 % Gradient elutions；20 min-30 min:A phases are 100 % elution；30 min-35 min:A It is mutually that 80 % are eluted；Ultraviolet detection wavelength is 245 nm；35 min of elution time.Record corresponding peak area and appearance time. The concentration and content of each ganodenic acid monomer are calculated according to standard curve.

Advantages of the present invention and technical effect: show that SE transformant bacterial strain produces monomer ganoderic acid by shake flask fermentation experiment The content of GA-Me, GA-T, GA-Mk, GA-S are the 1.2 of WT bacterial strain, 2.1,2.5,1.7 times respectively.Under square one, use The engineered strain that the present invention constructs can save labour, shorten the production cycle, reduce production cost.Therefore, this superior strain It can be used as the engineered strain of production ganoderic acid, be suitable for industrialized production, be with a wide range of applications.

Detailed description of the invention

Fig. 1 is ganoderma lucidum genome in the present invention；In figure: G is ganoderma lucidum genome, and M is λ-Hind III digest DNA Marker；

Fig. 2 is pJW-EXP carrier structure schematic diagram in the present invention；

Fig. 3 is the electrophoretogram that SE gene is expanded in the present invention；In figure: M is 500bp DNA Ladder (Dye Plus) Nucleic acid standards (Takara)；S is SE gene；

Fig. 4 is pJW-EXP-tSE carrier structure schematic diagram in the present invention；

Fig. 5 is the electrophoretogram that SE positive transformant is verified in the present invention；In figure: M is 500bp DNA Ladder (Dye Plus nucleic acid standards (Takara)), P are positive control, and SE is the positive transformant for turning SE gene, and WT is wild type Bacterial strain, N are negative control.

Specific embodiment

Below by embodiment, invention is further described in detail, but the contents of the present invention are not limited thereto, this Method operating according to a conventional method unless otherwise specified in embodiment, agents useful for same unless otherwise specified use conventional reagent Or the reagent configured according to a conventional method.

The building of embodiment 1:pJW-EXP carrier

1, the extraction of ganoderma lucidum genomic DNA

The mycelium for weighing about 0.2 g freeze-drying wild type ganoderma lucidum (CCGMC 5.0616) is pulverized in liquid nitrogen Powder is transferred to the CTAB (cetyl trimethylammonium bromide) that 1.5 mL are preheated through 65 DEG C and extracted in buffer by end, and 65 DEG C heat preservation 30 min, then at 4 DEG C, 10000 g be centrifuged 20 min, take supernatant to add isometric chloroform: isoamyl alcohol The mixture of (24:1), gently shakes up 30 min or more, at 4 DEG C, 10000 g be centrifuged 20 min；Supernatant is moved into 1.5 After mL centrifuge tube, the isopropanol of -20 DEG C of 2/3 volume warp pre-coolings is added, gently shakes 5 min, after pulling DNA out with glass bar, uses 75% ethanol washing 2-3 times after room temperature airing, is dissolved in the appropriate TE, 37 DEG C of digestion RNA 30 for containing 20 μ g/mL RNase Ganoderma lucidum genomic DNA can be arrived after min.Agarose gel electrophoresis results are shown: the genome of ganoderma lucidum is single band, and size At 10000 bp or more (see Fig. 1).

2, the clone of ganoderma lucidum strong promoter

Using ganoderma lucidum genomic DNA as template, P- is usedgpd-F、P-gpd- R carries out PCR amplification as primer, and amplification obtains spirit The promoter P- of sesamegpd, primer sequence is as follows:

P-gpd- F:5 '-TCCAAAGCCGCTCTCATGGCATGGCAC- 3 ',

P-gpd- R:5 '-GCTAGCGTTGAGAGGGGGATGAAGAGTGAGTAAGAAG-3 '；

PCR condition is as follows: 95 DEG C of 10 min, 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 90 s, and 72 DEG C 10 min。

3, promoter P-gpdIt is connect with PMD19-T

By the promoter P- after clonegpdGlue recycling is carried out, by recovery product and PMD19-T with T4 ligase at 16 DEG C It is attached, obtains PMD19-T-P intermediate vector.

4, the clone of ganoderma lucidum terminator

Using ganoderma lucidum genomic DNA as template, primer is used

T-sdhB- F:5 '-ATGAGCGGGTCAGAGAGT- 3 '

T-sdhB- R:5 '-TGCTCTATGTCTTGCCTTGT- 3 '

It is expanded, obtains the sequence of 440 bp；PCR condition are as follows: 95 DEG C of 10 min, 95 DEG C of 30 s, 55 DEG C 30 S, 72 DEG C of 30 s, 72 DEG C of 10 min.

5, terminator T-sdhBIt is connect with PMD19-T-P

By the terminator T- after clonesdhBGlue recycling, PMD19-T-P intermediate vector SacI single endonuclease digestion, PCR recycling Digestion products, then with T4 polymerase filling-in restriction enzyme site, then carry out blunt end cloning at 16 DEG C with T4 ligase again, obtain PMD19-T-P-T intermediate vector.

6、cbxThe clone of gene

Using ganoderma lucidum genomic DNA as template, primer is used

cbx- F:5 '-TCTGCTCTTCCCGATTGCTGCATTTGT-3 '

cbx- R:5 '-CTATGTCTTGCCTTGTCTCGCGTCAACC-3 ' carries out PCR and obtains ganoderma lucidumsdhBGene； PCR condition are as follows: 95 DEG C of 10 min, 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 90 s, 72 DEG C of 10 min；Redesign A pair of of rite-directed mutagenesis primer:

sdhB- MR:5'-GAAGATCGTGAGGCAGCGGTATAGGC-3'(is whereinAFor mutational site)

sdhB- MF:5'-GCCTATACCGCTGCCTCACGATCTTC-3'(is whereinTFor mutational site).

First round PCR primercbx- F andsdhB- MR is expanded, PCR condition are as follows: 95 DEG C of 10 min, 95 DEG C 30 s, 58 DEG C of 30 s, 72 DEG C of 2 min 20 s, 72 DEG C of 10 min obtain segmentsdhB1；Second wheel PCR primercbx- R andsdhB- MF is expanded, PCR condition are as follows: 95 DEG C of 10min, 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1 min 20 s, 72 DEG C of 10 min, obtain segmentsdhB2；After obtaining respective segments, two segments are connected using the method for over-lap PCR It connects.The primer of third round over-lap PCR iscbx- F andcbx- R, amplification condition are as follows: 94 DEG C of 10 min of denaturation, 66 DEG C of annealing 30 S, 72 DEG C of 4 min of extension, totally 35 recycle, 10 min of last 72 DEG C of extensions.It is finally obtained the ganoderma lucidum of 3171 bp mutationsdhBGene order (generates resistance to carboxin antibiotic after rite-directed mutagenesis, we are after rite-directed mutagenesissdhBGene It is defined ascbx) .Confirm that corresponding site is mutated after sequencing.

7, by mutationcbxGene is inserted into PMD19-T-P-T'sPstI restriction enzyme site

PMD19-T-P-T carrier is usedPstI enzyme is at 37 DEG C, 2 h of single endonuclease digestion, the segment after recycling digestion, poly- with T4 Synthase filling-in restriction enzyme site, then with T4 ligase at 16 DEG C handlecbxGene is attached to arrive with segment after the recovery PJW-EXP carrier (see Fig. 2).

The building of embodiment 2:pJW-EXP-tSE carrier

1, the clone of SE gene

Using ganoderma lucidum genomic DNA as template, primer is used

SE-Nhe-F:5 '-GCTAGCATGTGGTCCGTCTCGTACGACATCA- 3 '

SE-Sma-R:5 '-CCCGGGTCACGGGCGGATCTCCGTCC- 3 '；

It carries out PCR and obtains SE gene；PCR condition are as follows: 95 DEG C of 10 min, 95 DEG C of 30 s, 61 DEG C of 30 s, 72 DEG C 2 min, 72 DEG C of 10 min (see Fig. 3), nucleotide sequence is as shown in SEQ ID NO:1, the amino acid sequence of coding Column are as shown in SEQ ID NO:2.

2, SE gene is inserted into pJW-EXP carrier

By pJW-EXP carrier SmaI and NheI double digestion, segment after recycling digestion, with T4 ligase at 16 DEG C, SE gene is inserted between the NheI and SmaI of pJW-EXP carrier and obtains pJW-EXP-tSE carrier (see Fig. 4).

Embodiment 3: it is thin to be transformed into wild type ganoderma lucidum by the method for mediating protoplast fusion by PEG by pJW-EXP-tSE In born of the same parents

1, the preparation and conversion of ganoderma lucidum protoplast

Wild type Ganoderma lucidum mycelium is first prepared into protoplast with lywallzyme, ganoderma lucidum protoplast is then suspended in 100 STC (sorbierite of 0.55 M, the CaCl of 10 mM of μ L₂, the Tris-HCl buffer of 10 mM, pH 7.5) in, then Be added 1 μ g Plasmid DNA and PTC buffer (60% PEG4000 (W/V), the Tris-HCl buffer of 10 mM, PH is the CaCl of 7.5,50 mM₂) ；10 min are cultivated on ice, and the PTC buffer for adding 1 mL is uniformly mixed and in room 20 min of temperature culture；With 10 mL melt CYM solid medium with convert after protoplast mixing inverted plate and be added Carboxin makes final concentration of 2 mg/L of carboxin；Several single colonies can be grown after cultivating 10 days at 30 DEG C.

2, the secondary culture on the plate containing carboxin resistance

Single colonie is transferred in the CYM plate of the carboxin containing 2 mg/L, secondary culture about 7 days at 30 DEG C； Passing in resistant panel by 3 times can get stable transformant, extract the ganoderma lucidum genome after conversion with CTAB method, specifically Operating procedure is shown in 1 step 1 of embodiment.Respectively with convert after ganoderma lucidum genome, pJW-EXP-tSE plasmid (positive control), Wild type (WT) ganoderma lucidum genome, water (negative control) are template, are used

SE-gpd-F:5 '-CAAGGCGGTCAACAGGTAA -3 '

SE-gpd-R:5 '-CGTCCATAGTAGCGGCAAA -3 '

PCR, PCR condition are carried out as verifying primer are as follows: 95 DEG C of 10 min, 95 DEG C of 30 s, 57 DEG C of 30 s, 72 DEG C 3 min, 72 DEG C of 10 min (see Fig. 5).Since P-gpd and SE gene is in fungus expression vector pJW-EXP-tSE It links together, ganoderma lucidum (is located at SE-gpd-FgpdIn the promoter of gene) and SE-gpd-R (be located at ganoderma lucidum SE On gene) it is primer and genomic DNA is that template carries out PCR, the bacterial strains of about 2365 bp bands can be amplified and be believed that It is the transgenic lucid ganoderma for having imported SE gene.As shown in figure 5, can be amplified about from transgenic strain and positive control The band of 2365bp, and do not occur this band in wild type, only there are some non-specific bands.The result shows that: SE base Because being integrated on ganoderma lucidum genome really.

3, inclined-plane culture

Positive transformant is transferred in solid PDA medium (carboxin containing 2 mg/L), at 28 DEG C Culture 5-7 days.

4, first order seed culture

40 mL seed culture mediums and 10 mL mycelium suspensions (obtaining from an inclined-plane) are added in 250 mL shaking flasks , and cultivated 5 days under 28 DEG C, 120 rpm.

5, secondary seed culture

45 mL seed culture mediums and 5 mL first order seed culture solution (about 500 mg are added in 250 mL shaking flasks DW/L, level-one culture are inoculated with after being smashed with bead), it is cultivated 2 days under 28 DEG C, 120 rpm.

6, fermented and cultured

45 mL fermentation mediums and 5 mL secondary seed fermentation liquids (about 500 ~ 600 are added in 250 mL shaking flasks Mg DW/L, second level culture are inoculated with after being smashed with bead), it is cultivated under 28 DEG C, 120 rpm.

7, the separation and extraction of monomer ganoderic acid

100 mg of stem cell powder is accurately weighed, 3 mL 70%(v/v are added) ethyl alcohol soaked overnight, ultrasonic treatment 3 times, often Secondary 30 min.10000 rpm obtain supernatant after being centrifuged 5 min.It is dried in 50 DEG C of baking ovens.With 200 μ L hplc grade methanols Thoroughly dissolution, with 0.22 μm of membrane filtration.Ganodenic acid monomer is detected with HPLC.HPLC testing conditions are as follows: HPLC chromatogram column is C18 column (Agilent 1200 series, 5 μm of Agilent Zorbax SB-C18 column, 250 × 4.6 mm)；Sample introduction Measure 20 μ L；1 mL/min of flow velocity；Mobile phase A is methanol/acetic acid (100:0.5(v/v)), Mobile phase B is ultrapure water, 0-20 Min:A phase is 80 %-100 % Gradient elutions；20 min-30 min:A phases are 100 % elution；30 min-35 min:A It is mutually that 80 % are eluted；Ultraviolet detection wavelength is 245 nm；35 min of elution time.Record corresponding peak area and appearance time. The concentration and content of each ganodenic acid monomer are calculated according to standard curve.

Pass through the measurement of wild type and the content of SE bacterial strain ganoderic acid, the results showed that show by shake flask fermentation experiment The content that SE transformant bacterial strain produces monomer Lanost-8-en-26-oic acid,3,7,12-trihydroxy-11,15,23-trioxo-,(3BETA,7BETA,12BETA)- A-Me, GA-T, GA-Mk, GA-S is the 1.2,2.1,2. of WT bacterial strain respectively 5,1.7 times, therefore, this superior strain can be used as the engineered strain of production ganoderic acid, be with a wide range of applications.

Sequence table

<110>Kunming University of Science and Technology

<120>a kind of ganoderic acid high-yielding engineering bacterial strain kmust-SE

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 1722

<212> DNA

<213>ganoderma strain CCGMC 5.0616

<400> 1

atgtggtccg tctcgtacga catcatcatc gtcggtgccg gcatcgccgg ctgcgccctc 60

gctcacgccc tctccgccgc cgccatcaaa cgcaaccgtc ctcttcgcat atgtctgctc 120

gagcggtccc tcgcggagcc cgaccgcatc gtcggcgaac tcctccagcc aggaggcatc 180

atcgcactcc agaagctcgg cctcgaggac tgcgtcgacg gcattgacgc gatccccacc 240

tacggctact gcgtcgtcct cgacggcgcg cccgtccaca tcccctaccc cgacggccac 300

gagggacgcg cgttccacca cggccgcttc atccagcagc ttcgcaagaa ggcgaaggcg 360

gccgagcgcg tcgaagtcgt cgaggcgacc gtgtccgagc tcatcgagtg ccccctgaca 420

ggccgcatcc tgggcgtgcg cgcaacccgc aaagaggagg gcgcggtcga gaaggaggcg 480

ttcttcgcgg acctcaccgt cgtcgcggac gggtgcttct ccaacttccg gagcaaggtc 540

ctcggcaagg cccaacatcc gggcgtcacg aagggccact tctgcgggat catattggag 600

gacgcgacgc tgccgatacc gaagcacggg acggtcatgc tcgtgaaggg gtacgggccc 660

gtgctgtcat accagatcgg gacccacgac acgagggtgc tcttcgacgt caagcatcca 720

cccccatcgg acctgaaggt gcgtcgtcgc ccatattttc ttttcttttc gcatctctta 780

gcgttatccc ccgtccccca ggagcaaatc ctacacaaca tcatccccca actcccagcc 840

gccctccacc ttcccgcgca aaaagccctc gagaaagacc gcatccgacg catgccgaac 900

acattcatcc cgcccgcgca gcagggcaag cagacgacag agggcgcatt cctcctcggg 960

gactcgtgga acatgcgcca tccgctcacg ggcgggggca tgacgtgtgc gttcaacgac 1020

gtcgtcgtcc tccgcgacct cctgctcgac gtcaaggacc tcgcggactg ggagcaggtc 1080

gcacctgtgc taaatcgatg gttctgggtg cgcaagccgc tcgcgtcgac ggtgaacatt 1140

ctgagtgtcg cgttatacga tcttttcggc gcagatggtg cgtgtggcgc ggtttccctt 1200

actccactcg aacgtttgct tatccacggc gtccttgcag atccgcatct ggaggtgctc 1260

cggaccggct gcttcaaata cttcgagctt ggcggtgcgt gcgtccgcga acccgtcagc 1320

cttctcgctg cgtatgtgcc ctttccctcg ctattcccgt ccctatgtat gtattccccc 1380

ctttcgcccc tcttacttac gctgcctgcg tttccaggat cgcgcaagcc ccagtcctgc 1440

tcttccgaca cttcttcact gtcgcgctct actcgatctg gatcttgttc acgcatccgc 1500

gaaagatggg tacgtcgctg gacgggaagc cgctcctccg gcggccgcgg cccgatgaat 1560

ggccgctctt ggcactcaag agcgtgcgag tggtacgtcg tgcttcgtcc cgtggtccca 1620

tcggtgttcg cgctggctga cgactctctg tttctttgtg cacagttcta cacggcatgc 1680

gtggtatttt tgccgctact atggacggag atccgcccgt ga 1722

<210> 2

<211> 573

<212> PRT

<213>ganoderma strain CCGMC 5.0616

<400> 2

MET Trp Ser Val Ser Tyr Asp Ile Ile Ile Val Gly Ala Gly Ile Ala Gly Cys Ala Leu

1 10 20

Ala His Ala Leu Ser Ala Ala Ala Ile Lys Arg Asn Arg Pro Leu Arg Ile Cys Leu Leu

30 40

Glu Arg Ser Leu Ala Glu Pro Asp Arg Ile Val Gly Glu Leu Leu Gln Pro Gly Gly Ile

50 60

Ile Ala Leu Gln Lys Leu Gly Leu Glu Asp Cys Val Asp Gly Ile Asp Ala Ile Pro Thr

70 80

Tyr Gly Tyr Cys Val Val Leu Asp Gly Ala Pro Val His Ile Pro Tyr Pro Asp Gly His

90 100

Glu Gly Arg Ala Phe His His Gly Arg Phe Ile Gln Gln Leu Arg Lys Lys Ala Lys Ala

110 120

Ala Glu Arg Val Glu Val Val Glu Ala Thr Val Ser Glu Leu Ile Glu Cys Pro Leu Thr

130 140

Gly Arg Ile Leu Gly Val Arg Ala Thr Arg Lys Glu Glu Gly Ala Val Glu Lys Glu Ala

150 160

Phe Phe Ala Asp Leu Thr Val Val Ala Asp Gly Cys Phe Ser Asn Phe Arg Ser Lys Val

170 180

Leu Gly Lys Ala Gln His Pro Gly Val Thr Lys Gly His Phe Cys Gly Ile Ile Leu Glu

190 200

Asp Ala Thr Leu Pro Ile Pro Lys His Gly Thr Val MET Leu Val Lys Gly Tyr Gly Pro

210 220

Val Leu Ser Tyr Gln Ile Gly Thr His Asp Thr Arg Val Leu Phe Asp Val Lys His Pro

230 240

Pro Pro Ser Asp Leu Lys Val Arg Arg Arg Pro Tyr Phe Leu Phe Phe Ser His Leu Leu

250 260

Ala Leu Ser Pro Val Pro Gln Glu Gln Ile Leu His Asn Ile Ile Pro Gln Leu Pro Ala

270 280

Ala Leu His Leu Pro Ala Gln Lys Ala Leu Glu Lys Asp Arg Ile Arg Arg MET Pro Asn

290 300

Thr Phe Ile Pro Pro Ala Gln Gln Gly Lys Gln Thr Thr Glu Gly Ala Phe Leu Leu Gly

310 320

Asp Ser Trp Asn MET Arg His Pro Leu Thr Gly Gly Gly MET Thr Cys Ala Phe Asn Asp

330 340

Val Val Val Leu Arg Asp Leu Leu Leu Asp Val Lys Asp Leu Ala Asp Trp Glu Gln Val

350 360

Ala Pro Val Leu Asn Arg Trp Phe Trp Val Arg Lys Pro Leu Ala Ser Thr Val Asn Ile

370 380

Leu Ser Val Ala Leu Tyr Asp Leu Phe Gly Ala Asp Gly Ala Cys Gly Ala Val Ser Leu

390 400

Thr Pro Leu Glu Arg Leu Leu Ile His Gly Val Leu Ala Asp Pro His Leu Glu Val Leu

410 420

Arg Thr Gly Cys Phe Lys Tyr Phe Glu Leu Gly Gly Ala Cys Val Arg Glu Pro Val Ser

430 440

Leu Leu Ala Ala Tyr Val Pro Phe Pro Ser Leu Phe Pro Ser Leu Cys MET Tyr Ser Pro

450 460

Leu Ser Pro Leu Leu Leu Thr Leu Pro Ala Phe Pro Gly Ser Arg Lys Pro Gln Ser Cys

470 480

Ser Ser Asp Thr Ser Ser Leu Ser Arg Ser Thr Arg Ser Gly Ser Cys Ser Arg Ile Arg

490 500

Glu Arg Trp Val Arg Arg Trp Thr Gly Ser Arg Ser Ser Gly Gly Arg Gly Pro MET Asn

510 520

Gly Arg Ser Trp His Ser Arg Ala Cys Glu Trp Tyr Val Val Leu Arg Pro Val Val Pro

530 540

Ser Val Phe Ala Leu Ala Asp Asp Ser Leu Phe Leu Cys Ala Gln Phe Tyr Thr Ala Cys

550 560

Val Val Phe Leu Pro Leu Leu Trp Thr Glu Ile Arg Pro ***

570 573

<210> 3

<211> 27

<212> DNA

<213>artificial sequence

<400> 3

tccaaagccg ctctcatggc atggcac 27

<210> 4

<211> 37

<212> DNA

<213>artificial sequence

<400> 4

gctagcgttg agagggggat gaagagtgag taagaag 37

<210> 5

<211> 18

<212> DNA

<213>artificial sequence

<400> 5

atgagcgggt cagagagt 18

<210> 6

<211> 20

<212> DNA

<213>artificial sequence

<400> 6

tgctctatgt cttgccttgt 20

<210> 7

<211> 27

<212> DNA

<213>artificial sequence

<400> 7

tctgctcttc ccgattgctg catttgt 27

<210> 8

<211> 28

<212> DNA

<213>artificial sequence

<400> 8

ctatgtcttg ccttgtctcg cgtcaacc 28

<210> 9

<211> 31

<212> DNA

<213>artificial sequence

<400> 9

gctagcatgt ggtccgtctc gtacgacatc a 31

<210> 10

<211> 26

<212> DNA

<213>artificial sequence

<400> 10

cccgggtcac gggcggatct ccgtcc 26

<210> 11

<211> 26

<212> DNA

<213>artificial sequence

<400> 11

gaagatcgtg aggcagcggt ataggc 26

<210> 12

<211> 26

<212> DNA

<213>artificial sequence

<400> 12

gcctataccg ctgcctcacg atcttc 26

<210> 13

<211> 19

<212> DNA

<213>artificial sequence

<400> 13

caaggcggtc aacaggtaa 19

<210> 14

<211> 19

<212> DNA

<213>artificial sequence

<400> 14

cgtccatagt agcggcaaa 19

Claims (1)

1. a kind of ganoderic acid high-yielding engineering bacterial strain kmust-SE, in China Committee for Culture Collection of Microorganisms's commonly micro- life The deposit number at object center is CGMCC NO.11401.