CN101948794A - Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof - Google Patents
Engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof Download PDFInfo
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Abstract
The invention discloses engineering lactobacilli for producing plant flavonoid to synthesize related enzymes, construction and application thereof, belonging to the field of lactobacillus reformation of genetic engineering. The engineering lactobacilli is produced by the steps of: synthesizing flavonoid completely from Chinese locally planted soybeans (Glycine max(L.)Merr.) into related enzyme genes PAL, 4CL, CHS and CHI which are connected into a polycistron structure PLA-4CL-CHS-CHI; then constructing the polycistron structure onto a reformed lactobacillus expression vector pMG26e to form a recombinant vector pMG26e-4GS; and then electrically shocking and converting the pMG26e-4GS into lactobacilli to obtain the engineering lactobacilli for producing plant flavonoid to synthesize related enzymes. The preservation number of the recombinant lactobacilli is CGMCC (China General Microbiological Culture Collection Center) No.4086, and the engineering lactobacilli has more abundant physiological functions compared with original lactobacilli and can be applied to the field of food. Through fermentation, the flavonoid output of the obtained recombinant strain is in the level of mg/l, which is higher than the fermentation levels of like recombinant escherichia coli and recombinant yeast.
Description
Invention field
The present invention relates to produce engineering milk-acid bacteria and the structure and the application of the synthetic relevant enzyme of plant flavonoids.Resulting functional engineering lactic bacterium strains can be used for producing the flavonoid material, and this bacterial strain has enormous and latent market, economic benefit and social benefit at grocery trade.
Background technology
Flavonoid (Flavonoids) is one group of polyphenols that extensively is present in the plant, belongs to secondary metabolite, is the natural pigment in the leaf that is present in plant, flower, the fruit, is called as flavonoid because of being yellow more.The flavonoid material is especially removed effects such as free radical and anti-cancer and cancer-preventing, and is had broad application prospects in fields such as medicine, food owing to its important physical function.Yet the content of flavonoid in plant is very low, its extraction cost height, and yield is low; The flavonoid that utilizes the chemical process synthetic is two kinds of mixture of isomers, yet has only just biologically active of (2S)-flavonoid; Also yielded poorly, the cost height, be difficult for restriction such as large scale culturing by the synthetic flavonoid of culture plant cell or plant genetic engineering.In recent years, utilize the synthetic flavonoids of method of microbial fermentation to rise rapidly.Microorganism growth is fast, easily cultivate, and the convenient of genetic engineering technique all is impayable advantage effectively or the like.In microorganism, make up the flavonoid biosynthetic pathway and ferment, be expected to realize extensive, the suitability for industrialized production of flavonoid.The main host that makes up the flavonoid biosynthetic pathway at present is intestinal bacteria and yeast saccharomyces cerevisiae, and milk-acid bacteria (Lactic Acid Bacteria) is as a kind of biological fermentation bacterium, it also is a kind of important probiotic bacterium (Probiotics), it is one of emphasis of future studies that its biotechnology is modified, and can give milk-acid bacteria more physiological function by genetic engineering modified.
Plant flavonoids route of synthesis key gene is recombinated in the milk-acid bacteria, make up flavonoid synthetic genetically engineered milk-acid bacteria, the benefit of the physiological function characteristic of flavonoid and milk-acid bacteria is given birth to effect combine, promote the application of milk-acid bacteria in the functional foodstuff research and development.Plant flavonoids route of synthesis key enzyme mainly comprises phenylalanine ammonia lyase (Phenylalanine aminolyase, PAL), 4-coumaric acid-CoA ligase (p-coumarinic acid CoA ligase, 4CL), chalcone synthetase (Chalcone synthetase, CHS), chalcone isomerase (Chalcone isomerase, CHI) etc., the foreign scholar generally will derive from PAL and the 4CL gene of microorganism, derive from plant CHS and the CHI gene connects into the polycistron structure by proper restriction site, be assembled into intestinal bacteria by vector construction, in the microorganism such as yeast saccharomyces cerevisiae and streptomyces venezuelae.The flavonoid route of synthesis key gene that Shang Weijian will the derive from plant fully report in the milk-acid bacteria of recombinating.Milk-acid bacteria is as a kind of important probiotic bacterium, and it is genetic engineering modified can give milk-acid bacteria more physiological function.The resulting genetically engineered milk-acid bacteria of the present invention can be carried the flavonoid functional factor, and the benefit of its goods are compound flavonoid functional factor and this life of milk-acid bacteria is given birth to effect, strengthens the utility value of milk-acid bacteria.Milk-acid bacteria of the present invention provides brand-new function stem for food industry and dairy industry, widens the picked-up channel of the functional food factor.The pricipal beneficiary group of this product is that old man, cancer are easily sent out crowd, hypertension, hyperlipemia crowd etc.Product form is reinforcement active bacterial strain, reinforcement yogurt and cultured milk prod, and the pure product of biosynthesizing soybean isoflavones of purifying.Have enormous and latent market, economic benefit and social benefit at grocery trade.
Summary of the invention
The object of the present invention is to provide a kind of engineering milk-acid bacteria that produces the synthetic relevant enzyme of plant flavonoids.Resulting functional engineering lactic bacterium strains can be used for producing the flavonoid material, in addition, milk-acid bacteria (Lactic Acid Bacteria) also is a kind of important probiotic bacterium (Probiotics), has more physiological function by genetic engineering modified engineering milk-acid bacteria.
Second purpose of the present invention is to provide above-mentioned product plant flavonoids to synthesize the construction process of the engineering milk-acid bacteria of relevant enzyme.
The 3rd purpose of the present invention is to provide above-mentioned product plant flavonoids to synthesize the application of the engineering milk-acid bacteria of relevant enzyme.
The engineering milk-acid bacteria of the synthetic relevant enzyme of product plant flavonoids of the present invention, be that the encoding gene that will derive from the synthetic flavonoid relevant enzyme of plant imports in the milk-acid bacteria and the reorganization bacterium that obtains, described synthetic flavonoid relevant enzyme is phenylalanine transaminase (Phenylalanine aminolyase, PAL), 4-coumaric acid CoA ligase (p-coumarinic acid CoA ligase, 4CL), chalcone synthetase (Chalcone synthetase, CHS) and chalcone isomerase (Chalcone isomerase, CHI).
The original lactobacillus strain of described engineering milk-acid bacteria is lactococcus lactis subsp (Lactococcus lactis subsp.Cremoris) MG1363.
The encoding gene of described synthetic flavonoid relevant enzyme is formed polycistron, and the order of four kinds of structure genes is PAL-4CL-CHS-CHI.
The expression vector that carries synthetic flavonoid relevant enzyme encoding gene is pMG26e.
The encoding gene of described phenylalanine transaminase is shown in SEQ ID No.1; The encoding gene of described 4-coumaric acid CoA ligase is shown in SEQ ID No.2; The encoding gene of described chalcone synthetase is shown in SEQ ID No.3; The encoding gene of described chalcone isomerase is shown in SEQ ID No.4.
Described engineering milk-acid bacteria is preferably lactococcus lactis subsp (Lactococcus lactis subsp.Cremoris) MG1363-4GS, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 17th, 2010, its culture presevation is numbered CGMCC No.4086.Its grown form is spherical, and bacterium colony is creamy white, surface wettability.This bacterial strain can utilize MRS or M17 culture medium culturing, and pH is 6.8-7.0, and growth temperature is 30 ℃-37 ℃.
The construction process of the engineering milk-acid bacteria of the synthetic relevant enzyme of above-mentioned product plant flavonoids, with pretreated soybean spire is material, obtain target gene PAL, 4CL, CHS and CHI by the RT-PCR method, by proper restriction site target gene is connected into the polycistron structure in an orderly manner again, be incorporated on the lactic acid bacteria expression vectors, by electric shock transformation method recombinant plasmid vector is imported to the milk-acid bacteria recipient cell, obtain engineering milk-acid bacteria of the present invention.Specifically comprise following operation steps:
(1) the soybean spire with ultraviolet induction is that raw material extracts total RNA, utilizes ThermoScript II that its reverse transcription is become the cDNA library then;
(2) coding gene sequence of soybean phenylalanine transaminase, 4-coumaric acid CoA ligase, chalcone synthetase and the chalcone isomerase of announcing according to GeneBank designs special primer respectively, cDNA library with soybean is a template again, adopts increase the respectively encoding gene of phenylalanine transaminase, 4-coumaric acid CoA ligase, chalcone synthetase and chalcone isomerase of soybean of PCR method;
(3) with SacI and HindIII double digestion expression vector pMG36e, then enzyme to be cut product and nucleotides sequence shown in SEQID No.5 and be listed under the effect of T4 ligase enzyme and be connected, the connection product is called expression vector pMG26e; SEQ ID No.5 is as follows, and 5 ' and 3 ' end is sticky end:
ca
atcgatactgcag
gcggccgctctagagtcgacgcatgcggatc
ctcgagacgcgt
a
tcgagt
tagctatgacgtc
cgccggcgagatctcagctgcgtacgcctag
gagctctgcgcat
tcga
Sac?Ⅰ?Cla?Ⅰ Not?Ⅰ Xba?Ⅰ Xho?Ⅰ HindⅢ
(4) adopt the encoding gene of phenylalanine transaminase, 4-coumaric acid CoA ligase, chalcone synthetase and chalcone isomerase that restriction enzyme cutting expression vector pMG26e and step (2) amplification obtain, then four kinds of goal gene are inserted among the expression vector pMG26e, thereby obtain recombinant vectors pMG26e-4GS;
(5) utilize electric shock transformation method that recombinant vectors pMG26e-4GS is imported among recipient cell lactococcus lactis subsp (the Lactococcus lactis subsp.Cremoris) MG1363, placing height to ooze the reorganization bacterium evenly coats on the MRS flat board of erythromycin resistance after 2h is cultivated in the substratum recovery, cultivated 2-3 days for 37 ℃, pass through the method screening of bacterium colony PCR and extraction plasmid then and identify recombinant bacterial strain, thereby obtain producing the engineering milk-acid bacteria that plant flavonoids synthesizes relevant enzyme; Described height oozes substratum for containing 0.3M sucrose and 0.1MMgCl
2The MRS substratum.
The pre-treatment of described soybean spire is for intermittently growing a week under uv irradiating.
The nucleotide sequence of upstream and downstream primer that is used for amplification coding phenylalanine aminotransferase gene in the step (2) is respectively shown in SEQ ID No.6 and SEQ ID No.7; The nucleotide sequence of upstream and downstream primer that is used for amplification coding 4-coumaric acid CoA ligase gene is respectively shown in SEQ ID No.8 and SEQ ID No.9; The nucleotide sequence of upstream and downstream primer that is used for the amplification coding chalcone synthase gene (chs) is respectively shown in SEQ IDNo.10 and SEQ ID No.11; The nucleotide sequence of upstream and downstream primer that is used for amplification coding chalcone isomerase gene is respectively shown in SEQ ID No.12 and SEQ ID No.13.
The concrete grammar that four kinds of goal gene of the synthetic flavonoid relevant enzyme of coding are inserted on the expression vector pMG26e in the step (4) is as follows:
(a), cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMD 19-CHS-CHI with BamHI and XhoI double digestion recombinant vectors pMD19-CHS and pMD19-CHI; With BamHI and NotI double digestion recombinant vectors pMD19-PAL and pMD19-4CL, cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMD19-PAL-4CL;
(b), cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMG26e-CHS-CHI with XbaI and XhoI double digestion recombinant vectors pMD19-CHS-CHI and expression vector pMG26e;
(c), cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMG26e-4GS with ClaI and NotI double digestion recombinant vectors pMD19-PAL-4CL and recombinant vectors pMG26e-CHS-CHI.
The engineering milk-acid bacteria of the synthetic relevant enzyme of above-mentioned product plant flavonoids is in the application of producing on the flavonoid.This reorganization bacterium can be flavonoid materials such as the synthetic naringenin of fermenting substrate, Liquiritigenin, pinocembrin with phenylalanine, tyrosine etc.Concrete fermentation process is: the inoculum size of engineering milk-acid bacteria by 1wt% is inoculated in the 50mlMRS liquid nutrient medium (containing 5 μ g/ml erythromycin), in substratum, add phenylalanine and/or tyrosine then, making its final concentration is 500mg/L, 37 ℃ of growth 48h, centrifuging and taking substratum supernatant is with the equal-volume ethyl acetate extraction, get organic phase, with the Rotary Evaporators drying, residue is dissolved in the 2.0ml anhydrous methanol, the resulting flavonoid crude product solution that is.
The structure of naringenin, Liquiritigenin and pinocembrin is as follows:
The chemical name of naringenin (Naringenin) is 4 ', 5, the 7-trihydroxyflavone; English name: 4 ', 5,7-Trihydroxyflavanone, another name: 4,5,7-Trihydroxyflavone; Naringenin; Molecular formula: C
15H
12O
5, molecular weight: 272.25; CAS number: 480-41-1, its structural formula is:
The chemical name of Liquiritigenin (Liquiritigenin) is 4 ', the 7-dihydroxyflavone; English name: Liquiritigenin; Another name: (2S)-7-Hydroxy-2-(4-hydroxyphenyl)-2,3-dihydro-4H-1-benzopyran-4-one; 4 ', 7-Dihydroxyflavanone; 4H-1-Benzopyran-4-one, 2,3-dihydro-7-hydroxy-2-(4-hydroxyphenyl); Molecular formula: C
15H
12O
4Molecular weight: 256.25; CAS number: 578-86-9, its structural formula is:
Pinocembrin (Pinocembrin) pinocembrin; 7-dihydroxyflavanone; Molecular formula: C
15H
12O
4Molecular weight: 256.25; CAS number: 68745-38-0.
Beneficial effect of the present invention: its individual encoded gene source is in prokaryotic organism in the existing engineering bacteria that contains the synthetic relevant enzyme encoding gene of flavonoid, as 4CL from streptomyces coelicolor (Streptomyces coelicolor), and gene used in the present invention all comes from soybean, eukaryotic gene is expressed the codon preference that relates in prokaryotic organism, problems such as expression efficiency, and the difficulty of milk-acid bacteria genetic engineering technique itself is bigger, moreover, intestinal bacteria, streptomyces venezuelaes etc. belong to the non-food product fermenting bacteria, can not directly apply to food, milk-acid bacteria is as important food fermentation bacterial strain, the resulting recombinant bacterial strain of the present invention is through fermentation, flavonoid output is higher than the level of recombination bacillus coli and recombination microzyme fermentation in the mg/L level.
Description of drawings
Fig. 1 is the structure schema of recombinant lactic acid bacteria pMG26e-4GS;
Fig. 2 is the structure iron of expression vector pMG36e;
Fig. 3 is the structure iron of expression vector pMG26e;
Fig. 4 is the structure iron of expression vector pMG26e-4GS;
Fig. 5 is the HPLC figure of recombinant bacterial strain tunning of the present invention;
A: standard substance Liquiritigenin; B: the experimental strain tunning, wherein, and 1 HPLC color atlas for the tunning of the contrast bacterium MG1363/pMG26e that changes empty carrier pMG26e, 2 is the HPLC color atlas of the tunning of recombinant lactic acid bacteria pMG26e-4GS;
Fig. 6 is the electrophorogram of pcr amplification target gene;
Wherein, A is PAL electrophorogram (M:DNA ladder DL2000,1 is the PAL goal gene), B is 4CL electrophorogram (M:DNA ladder DL2000,1 is the 4CL goal gene), C is CHS electrophorogram (M:DNA ladder DL2000,1 is the CHS goal gene), D is CHI electrophorogram (M:DNA ladderDL2000,1 is the CHI goal gene);
Fig. 7 is ClaI and the NotI double digestion checking electrophorogram of recombinant vectors pMG26e-4GS.
1、2：pMG26e-4GS；M：DNA?ladder?DL15000
Fig. 8 is the bacterium colony PCR checking electrophorogram that has transformed the Lactococcus lactis MG1363 of recombinant vectors pMG26e-4GS.
A: with CHS is the bacterium colony PCR electrophorogram of reporter gene; Wherein, 1-4: reorganization bacterium; +: positive control; CK: blank; M:DNA ladder DL2000
B: with 4CL is the bacterium colony PCR electrophorogram of reporter gene.Wherein, 1-4: reorganization bacterium; +: positive control; CK: blank; M:DNA ladder DL2000
Embodiment
Following examples do not limit the present invention, unspecified operation steps please refer to " molecular cloning experiment guide " second edition corresponding section (J. Sa nurse Brooker E.F. is Ritchie etc. not, Science Press) or consults the specification sheets of used kit or reagent among the embodiment.
Below among two embodiment the prescription of used substratum as follows:
MRS culture medium preparation: contain potassium primary phosphate 2g, Triammonium citrate 2g, anhydrous sodium acetate 5g, Tryptones 10g, yeast powder 5g, glucose 20g, sal epsom 0.5g, manganous sulfate 0.25g, extractum carnis 10g, tween 80 1mL in every 1000mL substratum, surplus is a distilled water, transfers pH to be: 6.8-7.0.
Height oozes the preparation of substratum MRSSM: adding sucrose in the MRS substratum, to make its final concentration be 0.3mol/L, adds MgCl
2Making its final concentration is 0.1mol/L.
The acquisition of embodiment 1 lactococcus lactis subsp pMG26e-4GS
The whole flow process of the acquisition of lactococcus lactis subsp pMG26e-4GS is seen Fig. 1.
(1) extraction of the pre-treatment of soybean spire and total RNA thereof
Soybean (Glycine max L.Merr.) Cultivar is a northeast spring soybean series (north rich 12) (this laboratory preservation or market are bought), illumination cultivation is when growing 10 to 15 tender leafs, transfer in the culturing room of intermittent irradiation under the ultraviolet lamp, cultivate a week, getting tender leaf is material, and the Trizol method is extracted the total RNA of blade (Trizol reagent is available from Invitrogen); Uv irradiating condition: wavelength, 254nm; Power: 20W; Apart from about 1.5m;
(2) the RNA reverse transcription becomes the cDNA library: the total RNA that obtains with step (1) is a template, synthetic its first chain cDNA under the effect of M-MLV ThermoScript II (Promega);
(3) pcr amplification goal gene (PAL, 4CL, CHS, CHI)
(the GeneBank accession number is: X13094), (the GeneBank accession number is 4CL: X69955), (the GeneBank accession number is CHS: AY237728), (the GeneBank accession number is CHI: AF276302) gene order design special primer (primer sequence such as table 1) according to soybean PAL that GeneBank announced, the reverse transcription product that obtains with step (2) is a template, under the guiding of corresponding primer, carry out pcr amplification respectively, thereby obtain the corresponding target gene, 50 μ L PCR reaction systems are: 10 * buffer damping fluid (contains Mg
+): 5 μ L, 2.5mM dNTPs:4 μ L, Primers (100ng/ μ L): each 1 μ L, EX-Taq DNA Polymerase:0.5 μ L, cDNA:1 μ L add ddH
2O mends flat to 50 μ L; The PCR response procedures is: pre-94 ℃ of 5min of sex change, and 94 ℃ of 30s of sex change, the 50-60 ℃ of 30s that anneal extends 72 ℃ of 2min, circulates 30 times, replenishes and extends 10min.Pcr amplification product is carried out 1% agarose gel electrophoresis detect (seeing A, B, C, the D of Fig. 6), reclaim fragment length respectively and be the purpose fragment of 2142bp (PAL), 1689bp (4CL), 1170bp (CHS), 657bp (CHI) and it is carried out purifying, four fragments that purifying reclaims are cloned into respectively among the pMD19-Tsimple (available from precious biotechnology (Dalian) company limited), cut evaluation through conversion, screening, enzyme and obtain containing the segmental recombinant plasmid of purpose, and to its evaluation of checking order;
Table 1 PAL, 4CL, CHS, CHI target gene PCR amplimer
Annotate: have underscore partly to be respective limits restriction enzyme site sequence
Wherein, added the ClaI restriction enzyme site in the upstream primer of PAL, downstream primer has added BamHI and NotII restriction enzyme site respectively; The 4CL upstream primer has added the BamHI restriction enzyme site, and downstream primer has added the NotI restriction enzyme site; The upstream primer of CHS has added the XbaI enzyme cutting site, and downstream primer has added BamHI and XhoI restriction enzyme site, and CHI has added the upstream BamHI restriction enzyme site, and downstream primer adds the XhoI restriction enzyme site;
(4) make up PAL-4CL and the dual-gene structure of CHS-CHI.Concrete grammar is: with BamHI and NotI double digestion pMD19-PAL and pMD19-4CL, reclaim enzyme and cut product open chain pMD19-PAL and 4CL fragment, connecting under the effect of T4 ligase enzyme about 16h then, connect product transformed into escherichia coli DH5 α, cut through screening and enzyme and identify and obtain recombinant vectors pMD19-PAL-4CL; With BamHI and XhoI double digestion pMD19-CHS and pMD19-CHI, reclaim enzyme and cut product open chain pMD19-CHS and CHI fragment, connecting under the effect of T4 ligase enzyme about 16h then, connecting product transformed into escherichia coli DH5 α, cutting through screening and enzyme and identify and obtain recombinant vectors pMD19-CHS-CHI;
(5) (structure of pMG36e sees also M.VAN DE GUCHTE to transform lactic acid bacteria expression vectors pMG36e, 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, the pMG36e that this experiment is used is so kind as to give as China Agricultural University's Food science and associate professor Hao Yanling of nutrition engineering college) be pMG26e.Wherein, the structure iron of expression vector pMG36e sees Table 2, and the structure iron of expression vector pMG26e sees Table 3.
Concrete grammar is: with SacI and HindIII double digestion carrier pMG36e, cut the big fragment of glue recovery 3561bp behind 1% agarose gel electrophoresis, then with the joint (Adaptor of itself and synthetic, sequence is shown in SEQ ID No.5) link to each other, method of attachment is: 10 * buffer, 1 μ L, enzyme cut carrier segments 2 μ L, joint 6 μ L, ligase enzyme 1 μ L, 16 ℃ of connections are spent the night.Connect product and transform DH5 α, coat screening positive clone on the LB flat board of erythromycin resistance (erythromycin concentration 300ug/ml) then, the several bacterial plaques of picking are shaken bacterium upgrading grain at random, carry out enzyme with the restriction enzyme site that increases newly (ClaI, NotI, XbaI, XhoI) and cut the evaluation of identifying or directly check order, obtaining positive colony is pMG26e, and this expression vector pMG26e has increased restriction enzyme sites such as ClaI, NotI, XbaI, XhoI;
5 ' and 3 ' end of synthetic joint is sticky end, and the sequence of double chain nucleotide is as follows:
ca
atcgatactgcag
gcggccgctctagagtcgacgcatgcggatc
ctcgagacgcgt
a
tcgagt
tagctatgacgtc
cgccggcgagatctcagctgcgtacgcctag
gagctctgcgcat
tcga
Sac?Ⅰ?Cla?Ⅰ Not?Ⅰ Xba?Ⅰ Xho?Ⅰ HindⅢ
(6) with XbaI and XhoI double digestion recombinant vectors pMD19-CHS-CHI and lactic acid bacteria expression vectors pMG26e, reclaim big fragment of pMG26e and CHS-CHI fragment, under the effect of T4 ligase enzyme, product is connected about 16h behind the purifying, to connect product transformed into escherichia coli DH5 α then, transformed bacteria liquid is coated on the LB flat board of erythromycin resistance, is reporter gene with CHS, bacterium colony PCR method screening positive clone, the several positive colonies of picking shake bacterium upgrading grain, cut evaluation with XbaI and XhoI enzyme; Obtain recombinant vectors pMG26e-CHS-CHI.
With ClaI and NotI double digestion recombinant vectors pMD19-PAL-4CL and recombinant vectors pMG26e-CHS-CHI, under the effect of T4 ligase enzyme, product is connected about 16h after reclaiming the big fragment of pMG26e-CHS-CHI and PAL-4CL fragment purification, to connect product transformed into escherichia coli DH5 α then, transformed bacteria liquid is coated on the LB flat board of erythromycin resistance, with 4CL is reporter gene, bacterium colony PCR method screening positive clone, the several positive colonies of picking shake bacterium upgrading grain, cut evaluation (as Fig. 7) with ClaI and NotI enzyme, obtain recombinant vectors pMG26e-4GS, structure iron is seen Fig. 4.
(7) utilize electric shock transformation method that recombinant vectors pMG26e-4GS is imported to lactococcus lactis subsp (Lactoccous lactis subsp.cremoris) MG1363 (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[J] .1996,62 (10): 3662-3667, the lactococcus lactis subsp MG1363 that uses in this experiment is so kind as to give as professor Cao Yusheng of Chinese and Germanic Union Research Inst. of University Of Nanchang) in, it is 2KV/mm that electric shock transforms parameter, 800 Ω, 25 μ F; Transform the back bacterial strain and ooze substratum MRSSM (MRS, 0.3M sucrose, 0.1M MgCl in height
2) middle recovery cultivation 2h, evenly coat then on the MRS flat board of erythromycin resistance (erythromycin concentration is 5 μ g/ml), cultivated 2-3 days for 37 ℃.Simultaneously, establish commentaries on classics empty carrier pMG26e Lactococcus lactis MG1363/pMG26e and wild-type Lactococcus lactis MG1363 and be contrast.
Recombinant bacterial strain is screened and identified to method by bacterium colony PCR and extraction plasmid.With CHS and/or 4CL gene is reporter gene, carries out bacterium colony PCR, identifies the positive lactic bacterium strains (as Fig. 8) that transforms; Again positive transformant is connect liquid MRS culture medium culturing, adopt the traditional method for extracting plasmid, carry out enzyme and cut evaluation, the 1st reorganization bacterium is that preserving number is the lactococcus lactis subsp Lactoccous lactis subsp.cremoris MG1363-4GS of CGMCC No.4086 among Fig. 8.
(1) be the reorganization bacterium MG1363-4GS overnight growth of CGMCC No.4086 with preserving number, fresh bacterium liquid (grows to logarithmic phase, OD
600=2.0) inoculum size by 1wt% is inoculated in the 50ml MRS liquid nutrient medium (containing 5 μ g/ml erythromycin), adds phenylalanine and tyrosine that final concentration is 500mg/L simultaneously, 37 ℃ of growth 48h;
(2) with the nutrient solution of step (1) under the 6000rmp condition, centrifugal 10min gets supernatant, with the equal-volume ethyl acetate extraction, gets organic phase then, with the Rotary Evaporators drying, residue is dissolved in the 2.0ml anhydrous methanol, promptly obtains the flavonoid crude product solution;
(3) adopt HPLC that the flavonoid crude product that step (2) obtains is carried out proximate analysis, carry out the HPLC chromatogram under the following conditions, Liquiritigenin standard substance (be about to Liquiritigenin be dissolved in preparation concentration is the solution of 0.15mg/mL in the anhydrous methanol) in contrast are set simultaneously, liquid spectrum post: C-18 150X4.6; Column temperature: 30 ℃; 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; The detection wavelength is 280nm.
Present method is provided with the contrast bacterium simultaneously: the contrast bacterium MG1363/pMG26e and the wild-type Lactococcus lactis MG1363 that change empty carrier pMG26e.
The results are shown in Figure 5.Fig. 5-A shows, Liquiritigenin standard substance retention time is 52.252 ', and the HPLC color atlas of the tunning of the contrast bacterium MG1363/pMG26e of Fig. 5-B transfer empty carrier pMG26e is not have new peak near 52.252 ' in retention time, and the HPLC color atlas of the tunning of recombinant lactic acid bacteria pMG26e-4GS of the present invention has been located a significantly new peak 52.598 ', be Liquiritigenin, the productive rate that calculates Liquiritigenin is 5.4mg/L.
Claims (10)
1. produce the engineering milk-acid bacteria of the synthetic relevant enzyme of plant flavonoids, be that the encoding gene that will derive from the synthetic flavonoid relevant enzyme of plant imports in the milk-acid bacteria and the reorganization bacterium that obtains, described synthetic flavonoid relevant enzyme is phenylalanine transaminase, 4-coumaric acid CoA ligase, chalcone synthetase and chalcone isomerase.
2. according to the engineering milk-acid bacteria of the synthetic relevant enzyme of the described product plant flavonoids of claim 1, it is characterized in that the original lactobacillus strain of described engineering milk-acid bacteria is lactococcus lactis subsp (Lactococcus lactis subsp.Cremoris) MG1363.
3. according to the engineering milk-acid bacteria of the described recombinant plant flavonoid of claim 1 synthetase-coding gene, it is characterized in that the encoding gene of described synthetic flavonoid relevant enzyme is formed polycistron.
4. according to the engineering milk-acid bacteria of the synthetic relevant enzyme of the described product plant flavonoids of claim 1, it is characterized in that the expression vector that carries synthetic flavonoid relevant enzyme encoding gene is pMG26e.
5. according to the engineering milk-acid bacteria of the synthetic relevant enzyme of the described product plant flavonoids of claim 1, it is characterized in that the encoding gene of described phenylalanine transaminase is shown in SEQ ID No.1; The encoding gene of described 4-coumaric acid CoA ligase is shown in SEQ ID No.2; The encoding gene of described chalcone synthetase is shown in SEQ IDNo.3; The encoding gene of described chalcone isomerase is shown in SEQ ID No.4.
6. the engineering milk-acid bacteria of synthesizing relevant enzyme according to the described product plant flavonoids of claim 1, it is characterized in that, described engineering milk-acid bacteria is lactococcus lactis subsp (Lactococcus lactis subsp.Cremoris) MG1363-4GS, and its deposit number is CGMCC No.4086.
7. the construction process of the engineering milk-acid bacteria of the synthetic relevant enzyme of the described product plant flavonoids of claim 1 is characterized in that, comprises following operation steps:
(1) the soybean young shoot with ultraviolet induction is that raw material extracts total RNA, utilizes ThermoScript II that its reverse transcription is become the cDNA library then;
(2) coding gene sequence of soybean phenylalanine transaminase, 4-coumaric acid CoA ligase, chalcone synthetase and the chalcone isomerase of announcing according to GeneBank designs special primer respectively, cDNA library with soybean is a template again, adopts increase the respectively encoding gene of phenylalanine transaminase, 4-coumaric acid CoA ligase, chalcone synthetase and chalcone isomerase of soybean of PCR method;
(3) with SacI and HindIII double digestion expression vector pMG36e, then enzyme to be cut product and nucleotides sequence shown in SEQID No.5 and be listed under the effect of T4 ligase enzyme and be connected, the connection product is called expression vector pMG26e;
(4) adopt the encoding gene of phenylalanine transaminase, 4-coumaric acid CoA ligase, chalcone synthetase and chalcone isomerase that restriction enzyme cutting expression vector pMG26e and step (2) amplification obtain, then four kinds of goal gene are inserted among the expression vector pMG26e, thereby obtain recombinant vectors pMG26e-4GS;
(5) utilize electric shock transformation method that recombinant vectors pMG26e-4GS is imported among recipient cell lactococcus lactis subsp (the Lactococcus lactis subsp.Cremoris) MG1363, placing height to ooze the reorganization bacterium evenly coats on the MRS flat board of erythromycin resistance after 2h is cultivated in the substratum recovery, cultivated 2-3 days for 37 ℃, pass through the method screening of bacterium colony PCR and extraction plasmid then and identify recombinant bacterial strain, thereby obtain producing the engineering milk-acid bacteria that plant flavonoids synthesizes relevant enzyme; Described height oozes substratum for containing 0.3M sucrose and 0.1MMgCl
2The MRS substratum.
8. the construction process that synthesizes the engineering milk-acid bacteria of relevant enzyme according to the described product plant flavonoids of claim 7, it is characterized in that the nucleotide sequence of upstream and downstream primer that is used for amplification coding phenylalanine aminotransferase gene in the step (2) is respectively shown in SEQ ID No.6 and SEQ ID No.7; The nucleotide sequence of upstream and downstream primer that is used for amplification coding 4-coumaric acid CoA ligase gene is respectively shown in SEQ ID No.8 and SEQ ID No.9; The nucleotide sequence of upstream and downstream primer that is used for the amplification coding chalcone synthase gene (chs) is respectively shown in SEQID No.10 and SEQ ID No.11; The nucleotide sequence of upstream and downstream primer that is used for amplification coding chalcone isomerase gene is respectively shown in SEQ ID No.12 and SEQ ID No.13.
9. synthesize the construction process of the engineering milk-acid bacteria of relevant enzyme according to the described product plant flavonoids of claim 7, it is characterized in that, the concrete grammar that four kinds of goal gene of the synthetic flavonoid relevant enzyme of coding are inserted on the expression vector pMG26e in the step (4) is as follows:
(a), cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMD19-CHS-CHI with BamHI and XhoI double digestion recombinant vectors pMD19-CHS and pMD19-CHI; With BamHI and NotI double digestion recombinant vectors pMD19-PAL and pMD19-4CL, cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMD19-PAL-4CL;
(b), cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMG26e-CHS-CHI with XbaI and XhoI double digestion recombinant vectors pMD19-CHS-CHI and expression vector pMG26e;
(c), cut evaluation through connection, conversion, screening and enzyme and obtain recombinant vectors pMG26e-4GS with ClaI and NotI double digestion recombinant vectors pMD19-PAL-4CL and recombinant vectors pMG26e-CHS-CHI.
10. the application of engineering milk-acid bacteria in producing flavonoid of the synthetic relevant enzyme of the arbitrary described product plant flavonoids of claim 1-6.
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