CN102382788B - Lactic acid bacterium containing isoflavone synthetase (IFS)-cytochromes P450 reductase (CPR) fusion gene and application thereof - Google Patents
Lactic acid bacterium containing isoflavone synthetase (IFS)-cytochromes P450 reductase (CPR) fusion gene and application thereof Download PDFInfo
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Abstract
The invention discloses lactic acid bacterium containing an IFS-CPR fusion gene and application thereof. The recombinant bacterium provided in the invention is recombinant bacterium obtained by introducing coding genes of IFS-CPR into host bacterium, and IFS-CPR is a protein as described in 1) or 2): 1) a protein as represented by sequence 1 in a sequence table; 2) a protein derived by substituting and/or deleting and/or adding one or more amino acid residue in the amino acid residue sequence of sequence 1 in the sequence table. A recombinant lactic acid bacterial strain obtained in the invention can be used for production of isoflavone and has a great potential market, economic benefits and social benefits in the food industry.
Description
Technical field
The present invention relates to recombinant bacterium and application thereof, particularly contain milk-acid bacteria and the application thereof of isoflavone synthase-cytochrome P450 reductase fusion gene.
Background technology
Isoflavones is the important plant estrogen active substance of a class being mainly present in the leguminous plantss such as soybean, comprise daidzin (daidzin) class, Genistoside (genistin) class and C22H22O10 (glycitin) class etc., its functional component is aglycone part, is mainly daidzein (daidzein) and genistein (genistein).The chemical name of genistein is genistein, and molecular formula is C
15h
10o
5, molecular weight is to be for 270.24, No. CAS 446-72-0, structural formula is formula 1; The chemical name of daidzein is 7-hydroxyl-3-(4-hydroxyphenyl)-4-benzopyrone, molecular formula: C
15h
10o
4, molecular weight: 254.24, No. CAS: 486-66-8, structural formula is formula 2.
Formula 1 formula 2
Medical science and Nutritional studies confirm, isoflavones has the climacteric syndrome of improvement symptom, improves osteoporosis, the cancers such as Breast Cancer Prevention, prostate cancer and colorectal carcinoma, reduce blood fat, suppress vascular smooth muscle cell proliferation and atherosclerosis, radioprotective free radical reaction and antioxygenation, the functions such as anti-nervous system disease, raising memory and alleviating physical fatigue.The resource of natural isoflavone is very limited, and soya products is the main food source of isoflavones.The content of isoflavone from soybean very low (< 0.05%), and easily run off in the course of processing.The industrial soybean isoflavones that separates from dregs of beans is main acquiring way, and cost high yield is low.In order to widen and develop the new source of isoflavones, people, by modern genetic engineering and metabolic engineering technology, try hard to build the synthetic genetically engineered of Isoflavone and produce bacterial strain.Milk-acid bacteria is the important zymophyte of foodstuffs industry, diary industry, is also that the probiotic bacterium of human body has many important physiological functions: as alleviated lactose intolerance; Regulate human intestinal microflora balance, form antibacterial biological barrier, safeguard HUMAN HEALTH; Suppress the breeding of spoilage organism, clear up the toxin that spoilage organism produces, remove enteron aisle rubbish; Suppress cholesterol absorption, the effects such as reducing blood-fat, hypotensive and immunomodulatory; The effects such as antitumor, preventing cancer.
Summary of the invention
An object of the present invention is to provide a kind of recombinant bacterium.
Recombinant bacterium provided by the present invention is that the encoding gene of IFS-CPR is imported to the recombinant bacterium that Host Strains obtains, and described IFS-CPR is following 1) or 2) protein:
1) protein shown in the sequence 1 in sequence table;
2) amino acid residue sequence of the sequence in sequence table 1 is passed through to replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and has identical function by 1) derivative protein.
The encoding gene of described IFS-CPR is following 1) or 2) or 3) in arbitrary described DNA molecular:
1) DNA molecular shown in the sequence 2 in sequence table;
2) under stringent condition with 1) DNA molecule hybridize and there is the DNA molecular of identical function;
3) with 1) or 2) DNA molecular there is 90% above homology and there is the DNA molecular of identical function.
Described Host Strains is milk-acid bacteria, and described milk-acid bacteria is preferably Lactococcus lactis (Lactococcus lactis).
The encoding gene of described IFS-CPR imports Host Strains by recombinant expression vector.
Described recombinant expression vector is between the XbaI of the encoding gene insertion expression vector pMG36e of IFS-CPR and XhoI restriction enzyme site, to obtain.
Described recombinant bacterium is lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) MG1363-IC, and its preserving number is CGMCC No.4016.
Another object of the present invention is to provide the application of described recombinant bacterium in synthetic isoflavones.
Described isoflavones is genistein and/or daidzein; The chemical name of genistein is genistein, and structural formula is shown in formula 1; The chemical name of daidzein is 7-hydroxyl-3-(4-hydroxyphenyl)-4-benzopyrone, and structural formula is shown in formula 2.
Formula 1 formula 2
Another object of the present invention is to provide a kind of fusion rotein and encoding gene thereof.
Fusion rotein provided by the present invention is following 1) or 2) protein:
1) protein shown in the sequence 1 in sequence table;
2) amino acid residue sequence of the sequence in sequence table 1 is passed through to replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and has identical function by 1) derivative protein.
The encoding gene of described fusion rotein is following 1) or 2) or 3) in arbitrary described DNA molecular:
1) DNA molecular shown in the sequence 2 in sequence table;
2) under stringent condition with 1) DNA molecule hybridize and there is the DNA molecular of identical function;
3) with 1) or 2) DNA molecular there is 90% above homology and there is the DNA molecular of identical function.
Above-mentioned stringent condition is available 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS is hybridized at 65 DEG C and washes film in DNA or RNA hybrid experiment.
The present invention is built into fusion gene IFS-CPR by genetic engineering technique by the cytochrome P450 reductase gene (CPR) that comes from the isoflavone synthase gene (IFS) of soybean and come from bacillus megaterium, the fusion rotein that fusion gene IFS-CPR coding produces has comprised a complete P450 system, can produce and have bioactive isoflavone synthase-redox electron donor complex body at milk-acid bacteria expression in vivo.The recombinant strains of lactic acid bacteria that the present invention obtains can be used for production isoflavones, has huge potential market, economic benefit and social benefit at grocery trade.
Brief description of the drawings
Fig. 1 is the structure iron of lactic acid bacteria expression vectors pMG36e.
Fig. 2 is the structure iron of recombinant vectors pMG36e-IC.
Fig. 3 is the strain morphology figure of Host Strains Lactococcus lactis (Lactococcus lactis ssp.cremoris MG1363).
Fig. 4 is the strain morphology figure of recombinant bacterial strain MG1363-IC.
The PCR qualification result of the positive conversion bacterial strain of Fig. 5.
Fig. 6 is the HPLC result of tunning.
The second order ms collection of illustrative plates of Fig. 7 fermented sample.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Embodiment 1, structure and the application thereof of genetically engineered milk-acid bacteria that contains isoflavone synthase-cytochrome P450 reductase fusion gene
One, the structure of fusion gene IFS-CPR
1, the acquisition of soybean isoflavones synthase gene (IFS)
(1) (public can obtain from China Agricultural University uv induction northern China soybean (Glycine max L.Merr.) Cultivar early soybean series (north rich 12), the non-patent literature of recording this material is: Hao Jia, Ma Huiqin, Chen Shangwu. utilize the polygenic research of SMART method quick clone isoflavonoid biosynthetic pathway. Chinese biological engineering magazine, 2007,27 (6): 66-70) expression of soybean isoflavones synthase gene (IFS) in young shoot, extracts the total RNA of soybean young shoot.
(2) reverse transcription becomes cDNA library.
(3) design special primer, pcr amplification obtains IFS gene
Upstream primer is IFS-F:5 '-GtctagaATGGCTCTGTTATTAGCAGTTTTTCTTGGTTTGTTTGTGTTAGC-3 ', and lowercase is restriction enzyme site,
Downstream primer is IFS-R:5 '-CctcgagAAGCTTggatccAGAAAGGAGTTTAGATGCAAC-3 ', and lowercase is restriction enzyme site,
Wherein the special upstream primer of IFS adds restriction enzyme site XbaI, downstream special primer adds respectively restriction enzyme site BamHI and XhoI, taking the cDNA library of step (2) as template, RT-PCR amplification IFS gene, and obtained PCR product is connected on cloning vector and is checked order.Sequencing result shows, the nucleotides sequence of the fragment that PCR reaction obtains is classified soybean IFS gene order as.
Soybean IFS gene also can obtain by synthetic, and method is: the 1st to 1575 Nucleotide of sequence 2 in artificial synthesized sequence table, carry out pcr amplification with primer I FS-F and IFS-R and obtain soybean IFS gene; Obtained PCR product is connected on cloning vector and is checked order.Sequencing result shows, the nucleotides sequence of the fragment that PCR reaction obtains is classified soybean IFS gene order as.
2, the acquisition of cytochrome P450 reductase gene (CPR)
(1) extract bacillus megaterium (Bacillus megaterium) genomic dna.
(2) design special primer, pcr amplification obtains CPR gene
Upstream primer is CPR-F:5 '-AggatccAAAAAGGCAGAAAACGCTC-3 ', lowercase is restriction enzyme site, downstream primer is CPR-B:5 '-TTctcgagTTACCCAGCCCACACGTC-3 ', lowercase is restriction enzyme site, wherein, upstream special primer adds restriction enzyme site BamHI, downstream special primer adds restriction enzyme site XhoI, the genomic dna obtaining taking step (1) is as template, pcr amplification bacillus megaterium CPR gene, and obtained PCR product is connected on cloning vector and is checked order.Sequencing result shows, the nucleotides sequence of the fragment that PCR reaction obtains is classified bacillus megaterium CPR gene order as, i.e. 1576-3309 position Nucleotide in sequence 2.
Bacillus megaterium CPR gene also can obtain by synthetic, and method is: sequence 2 1576-3309 position Nucleotide in artificial synthesized sequence table, carry out pcr amplification with primer CPR-F and CPR-B and obtain bacillus megaterium CPR gene; Obtained PCR product is connected on cloning vector and is checked order.Sequencing result shows, the nucleotides sequence of the fragment that PCR reaction obtains is classified bacillus megaterium CPR gene order as.
3, the structure of fusion gene IFS-CPR
IFS gene and CPR gene are connected into fusion gene IFS-CPR by BamHI and XhoI site.Concrete steps are: after the PCR product purification of the CPR gene that the IFS gene respectively step 1 being obtained and step 2 obtain, TA is cloned into pMD19-T simple carrier (Amp resistance) (purchased from precious biotechnology (Dalian) company limited, catalog number is D104A) upper, obtain plasmid pMD19-IFS and pMD19-CPR; Heat shock transforms bacillus coli DH 5 alpha, blue hickie screening positive clone, and positive single bacterium colony connects LB liquid nutrient medium, and 37 DEG C, 250rpm shake bacterium and extract plasmid; With BamHI and XhoI double digestion plasmid pMD19-IFS and pMD19-CPR, reclaim enzyme and cut product pMD19-IFS (4263bp) and CPR (1734bp); Enzyme is cut to product and connect, transform bacillus coli DH 5 alpha, taking CPR as reporter gene, colony polymerase chain reaction (PCR) method screening positive clone, positive single bacterium colony connects LB liquid nutrient medium, and 37 DEG C, 250rpm shake bacterium and extract plasmid, obtain pMD19-IFS-CPR.
PMD19-IFS-CPR plasmid is checked order, sequencing result shows, the nucleotide sequence of the fusion gene IFS-CPR inserting at the T site place of pMD19-T simple carrier is as shown in the sequence 2 in sequence table, albumen shown in sequence 1 in this gene coded sequence table, by this albumen called after IFS-CPR.In fusion gene, 1-1575 position Nucleotide is IFS gene, and 1576-3309 position Nucleotide is CPR gene.
Two, the structure of recombinant expression vector
With XbaI and XhoI digested plasmid pMD19-IFS-CPR, reclaim fusion gene fragment; (public can obtain from China Agricultural University to cut carrier pMG36e with XbaI and XhoI enzyme, the non-patent literature of recording this material is: M.VAN DE GUCHTE, J.M.B.M.VAN DER VOSSEN, J.KOK, G.VENEMA.Construction of a lactococcal expression vector:Expression of hen egg white lysozyme in Lactococcus lactis subsp.lactis[J] .Applied and Environmental Microbiology, 1989,55 (1): 224-228), reclaim carrier large fragment; The fusion gene fragment being recovered to is connected with carrier large fragment, obtains recombinant vectors.
The connection product of recombinant vectors transforms bacillus coli DH 5 alpha, taking CPR as reporter gene, and colony polymerase chain reaction (PCR) method screening positive clone, positive single bacterium colony connects LB liquid nutrient medium, and 37 DEG C, 250rpm shake bacterium and spend the night, and extract plasmid, obtain recombinant vectors pMG36e-IFS-CPR.Plasmid pMG36e-IFS-CPR is checked order, and sequencing result shows, between the XbaI of pMG36e and XhoI restriction enzyme site, inserted the fusion gene shown in the sequence 2 in sequence table, and positive recombinant vectors is denoted as to pMG36e-IC (Fig. 2).
Three, transform lactic bacterium strains
(public can obtain from China Agricultural University to utilize electric shock transformation method that recombinant vectors pMG36e-IC is imported to Lactococcus lactis (Lactoccous lactis ssp.cremoris MG1363), the non-patent literature of recording this material is: PASCALLE G.G.A.DE RUYTER, OSCAR P.KUIPERS, AND WILLEM M.DE VOS.Controlled Gene Expression Systems for Lactococcus lactis with the Food-Grade Inducer Nisin[J] .APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1996, 62 (10): 3662-3667) (the strain morphology figure that Fig. 3 is this bacterial strain) in recipient cell, electric shock Transformation Parameters is 2.0KV, 800 Ω, 25 μ F, after conversion, cell oozes substratum MRSSM (MRS, 0.3M sucrose, 0.1M MgCl in height
2) middle recovery cultivation 2h, evenly coat on the MRS flat board of erythromycin resistance, cultivate 2-3 days for 37 DEG C.
Meanwhile, establish and turn empty carrier pMG36e Lactococcus lactis and wild-type Lactococcus lactis for contrast.
Four, transform the qualification of lactic bacterium strains
(1) the positive screening that transforms lactic bacterium strains
Method by bacterium colony PCR and extraction plasmid is identified.Taking CPR gene as reporter gene, carry out bacterium colony PCR, the positive lactic bacterium strains that transforms of qualification, choose altogether 4 single bacterium colonies, PCR result show to be positive transformant (Fig. 5, swimming lane 1,2,3,4 is recombinant bacterium ,+positive contrast, the negative contrast of ck, M is DL2000 DNA marker); This 4 strain positive transformant is connect to liquid MRS culture medium culturing, adopt traditional method for extracting plasmid, result shows, 4 plasmids that strains conversion bacterial strains all contain and expectation size meets.Determine that this 4 strain bacterium is the positive recombinant bacterial strain that turns pMG36e-IC.
The wherein strain recombinant bacterial strain obtaining is lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) MG1363-IC, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 07 20th, 2010, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4016.
The strain morphology of this recombinant bacterial strain is as Fig. 4.
Five, the application of recombinant bacterial strain
Add the substrates such as naringenin, Liquiritigenin, carry out the fermenting experiment of recombinant bacterium; The extraction preparation of target product and HPLC qualification.
Genistein standard substance are purchased from Xi'an Xu Huang Bioisystech Co., Ltd, and catalog number is 100114; Daidzein standard substance are purchased from Xi'an Xu Huang Bioisystech Co., Ltd, and catalog number is 091212; Naringenin is purchased from Xi'an Xu Huang Bioisystech Co., Ltd, and catalog number is 100113; Liquiritigenin is purchased from Nanjing Zelang Pharmaceutical Technology Inc..
Recombinant bacterium, the fresh bacterium liquid that turns empty carrier contrast bacterium and wild-type Lactococcus lactis are inoculated in to 50mlMRS liquid nutrient medium (containing 5ug/ml erythromycin) by 1%, add 0.05mM naringenin and/or Liquiritigenin, 37 DEG C of growth 48h, centrifuging and taking supernatant, with the extraction of equal-volume ethyl acetate, organic phase is dry with Rotary Evaporators, residue was with 5ml methyl alcohol: HCl (9: 1, vol/vol) solution dissolves, and mixes after 1h, in 55 DEG C of constant temperature water bath 15min.Solution evaporation falls, and residue is dissolved in 2ml anhydrous methanol to be HPLC.
HPLC analyzes tunning, and condition is that liquid is composed post: C-18 150X4.6; Column temperature: 30 DEG C; Mobile phase A is the phosphoric acid buffer of 0.01M pH2.8, and Mobile phase B is 100% methyl alcohol; Sample size: 10ul; Flow velocity: 1ml/min; It is 260nm that genistein and daidzein detect wavelength.
3 repetitions are established in experiment, and result is taken the mean.
Analyze through HPLC, recombinant bacterium sample contrast bacterium and wild mushroom and has compared the obvious peaks increase of two places with turning empty carrier, and retention time is (Fig. 6 consistent with genistein and daidzein respectively, wherein, the HPLC collection of illustrative plates that in Fig. 6, A is standard substance, D is daidzein, retention time 36m283s; G is genistein, retention time 46m456s; In Fig. 6, B is the HPLC analytical results of restructuring strain fermentation sample, and filled arrows represents daidzein, retention time 36.283 '; Hollow arrow represents genistein, retention time 46.206 ').By analysis, the productive rate of genistein is 0.41mg/L supernatant liquor, and the productive rate of daidzein is 0.31mg/L supernatant liquor.Turn in empty carrier control strain and wild strain fermented sample and genistein and daidzein do not detected.
Tunning is carried out to the analysis of electron spray(ES) ion trap tandem mass spectrometry, condition: instrument, Esquire 6000 (Brooker dalton company, the U.S.); ESI source, negative ions detects; 250 DEG C of capillary temperatures; Dry gas (N2) flow velocity 4L/min.; Assisted gas (N2), pressure 15psi; Collision gas is He, collision energy 40V; Adopt negative ions scan mode, sweep limit m/z 50-800.
(Fig. 7) by analysis, in fermented sample, the sample of retention time 36m668s and 46m206s position is distinguished A and B in corresponding mass spectrum 7, corresponding molecular weight is 255 and 271, is defined as daidzein (A in Fig. 7) and genistein (B in Fig. 7).
Claims (2)
1. lactococcus lactis subsp (Lactococcus lactis subsp.cremoris) MG1363-IC, its preserving number is CGMCC No.4016.
2. the application of bacterium in synthetic isoflavones described in claim 1;
Described isoflavones is genistein and/or daidzein; The chemical name of genistein is genistein, and structural formula is shown in formula 1; The chemical name of daidzein is 7-hydroxyl-3-(4-hydroxyphenyl)-4-benzopyrone, and structural formula is shown in formula 2
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| TWI580784B (en) * | 2016-01-13 | 2017-05-01 | 國立臺南大學 | Method of manufacturing methoxy-isoflavones by biotransformation and use thereof |
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| CN1944634A (en) * | 2006-03-08 | 2007-04-11 | 沈阳农业大学 | Hydrolyzed soy bean isoflavone glycosidase engineering strain, its construction method and its use |
| CN1948460A (en) * | 2005-10-14 | 2007-04-18 | 中国科学院大连化学物理研究所 | Yeast system capable of coexpressing CYP and CPR |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1948460A (en) * | 2005-10-14 | 2007-04-18 | 中国科学院大连化学物理研究所 | Yeast system capable of coexpressing CYP and CPR |
| CN1944634A (en) * | 2006-03-08 | 2007-04-11 | 沈阳农业大学 | Hydrolyzed soy bean isoflavone glycosidase engineering strain, its construction method and its use |
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| Title |
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| Dae Hwan Kim et al.Enhancement of isoflavone synthase activity by co-expression of P450 reductase from rice.《Biotechnology Letters 》.2005,第27卷1291-1294. |
| Enhancement of isoflavone synthase activity by co-expression of P450 reductase from rice;Dae Hwan Kim et al;《Biotechnology Letters 》;20051231;第27卷;1291-1294 * |
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