CN105821071A - Unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination - Google Patents
Unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination Download PDFInfo
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Abstract
The invention relates to an unmarked gene knock-out method of pediococcus acidilactici DQ2 based on homologous recombination. The method comprises the following steps: temperature sensitive-type shuttle plasmid pSET4E and knock-out plasmid containing homologous fragments at upstream and downstream parts of target genes to be knocked out are constructed, the knock-out plasmid is subjected to electrotransformation into pediococcus acidilactici, and single commutators generating homologous recombination for the first time and double-exchange mutant strains generating homologous recombination for the second time are screened and identified. The method disclosed by the invention realizes the unmarked gene knock-out of pediococcus acidilactici for the first time, the obtained knock-out bacterial strain does not carry any resistant gene, can be taken as a original strain for subsequent and reconstruction, and also can be used for large-scale industrial production in a safe mode. The method is used for respective knock-out of L-lactate dehydrogenase gene and d-lactate dehydrogenase gene of the pediococcus acidilactici DQ2 (a preservation number is CGMCC NO.7471), the obtained knock-out bacterial strains are respectively named as pediococcus acidilactici ZP26 and TY112, the preservation numbers are CGMCC NO.8665 and CGMCC NO.8664 respectively, and optically pure D-lactic acid and L-lactic acid are respectively generated.
Description
Technical field
The present invention relates to a kind of gene knockout method based on homologous recombination principle, prepared by the marker-free knockout technique particularly relating to a kind of pediococcus acidilactici DQ2 based on homologous recombination and ldh, ldhD gene knocking out pediococcus acidilactici DQ2 produces optical voidness D-ALPHA-Hydroxypropionic acid, the bacterial strain of Pfansteihl.
Background technology
Pediococcus acidilactici (Pediococcusacidilactici, it is called for short P.acidilactici) it is an amphimicrobian gram-positive bacterium of class, belong to a kind of lactic acid bacteria, it carries out homofermentative lactic, the lowest pH value can be tolerated, can also grow under higher temperature and osmotic pressure.The potential of such bacterium profitable probliotics, the lactic acid of generation and pediocin can suppress the growth of other microorganisms, and do not find its toxic effect in the article delivered at present.
Pediococcus acidilactici P.acidilacticiDQ2 (preserving number is CGMCCNO.7471) is that the strain of isolated during cellulose alcoholic fermentation of this laboratory is high temperature resistant, has the Lactic Acid High-yield Strains of higher tolerance to the mortifier produced in lignocellulose preprocessing process simultaneously.This bacterium can be fermented under the high temperature of 48 DEG C normally, and can enough produce the lactic acid of more than 100g/L, does not substantially have the generation of other by-products, has the potentiality building cellulose lactic acid industrial production mode bacterium greatly.But the lactic acid that this bacterial strain is produced is D, L-mixed type lactic acid, limits the application of product.
Lactic acid is a kind of chiral molecule, can be divided into D-ALPHA-Hydroxypropionic acid, Pfansteihl and D, Pfansteihl according to its optical activity.The most important purposes of lactic acid is the degradable poly lactic product (Polymericlacticacid, PLA) producing and can replacing the polymeric material such as polyethylene and polypropylene at present, this important channel being counted as solving the most serious white pollution.The polylactic acid that synthesis performance is good needs optically pure lactic acid monomer, it is necessary to P.acidilacticiDQ2 is carried out strain improvement so that it is produce optically pure lactic acid.What this bacterial strain produced the main dependence of Pfansteihl is LDH (by ldh gene code), produce the main dependence of D-ALPHA-Hydroxypropionic acid is D-lactic acid dehydrogenase (by ldhD gene code), it is considered to take the means transformation bacterial strain of gene knockout to make it produce optically pure lactic acid.
Gene Knockout is the important Protocols in Molecular Biology grown up the eighties in 20th century, is to make specific gene inactivation or the technology of disappearance in cell by certain method.It has, and polarization is strong, insert gene with advantages such as the stable heredity of chromosomal DNA.The conventionally used ultimate principle that knocks out of gene is homologous recombination, if i.e. exogenous genetic fragment has the highest both homology degree with certain fragment on the genome of Host Strains and will exchange.
Generally using suicide plasmid is that vector construction knocks out system, is connected on suicide plasmid and proceeds in Host Strains, target gene upstream and segments downstream or incomplete target gene to knock out or to destroy target gene.The most to colibacillary research, so its gene knockout system also comparative maturity, as the most efficient in Red recombination system.Gene knockout system as the lactic acid bacteria of gram positive bacteria is less than the most perfect, most knock out or uses suicide plasmid.And occur in that the efficient thermal sensitivity of comparison knocks out system in recent years, this system is to be based in knocking out host carrying out the replicon of condition duplication, i.e. at less than a certain temperature, this plasmid can replicate, and the duplication of this plasmid will be closed under higher than a certain temperature conditions.
(the MaguinE such as MaguinE, DuwatP, HegeT, EhrlichD, GrussA.Newthermosensitiveplasmidforgram-positivebacteria .Journalofbacteriology, 1992,174 (17): 5633-5638.) screening obtains a kind of mutant plasmid pVE6002.It is carried out the thermal sensitivity experiment under the conditions of not having antibiotic, will all lose after finding to cultivate 8h at a temperature of this plasmid is more than 37 DEG C, and go out active hardly during cultivation below 30 DEG C.Then on this plasmid, insert one section of multiple clone site, build the carrier pVE6004 being convenient for clone.(the BiswasI such as BiswasI, GrussA, EhrlichSD, MaguinE.High-efficiencygeneinactivationandreplacementsys temforgram-positivebacteria.Journalofbacteriology, 1993,175 (11): 3628-3635.) this cover system has been carried out actual verification, and constructed escherichia coli-lactic acid bacteria shuttle and knock out plasmid pGhost5 so that carrying out the clone of homologous fragment in escherichia coli.(the GoryL such as GoryL, MontelMC, ZagorecM.UseofgreenfluorescentproteintomonitorLactobacil lussakeiinfermentedmeatproducts.FEMSmicrobiologyletters, 2001,194 (2): 127-133.) knock out plasmid pGhost5 as target gene by thermal sensitivity using galactosidase gene gfp fragment to be incorporated on chromosome and expressed.
null(the OKanoK such as OKanoK，ZhangQ，ShinKawaS，YoshidaS，TanaKaT，FuKudaH，KondoA.EfficientproductionofopticallypureD-lacticacidfromrawcornstarchbyusingageneticallymodifiedL-lactatedehydrogenasegene-deficientandα-amylase-secretingLactobacillusplantarumstrain.Appliedandenvironmentalmicrobiology，2009，75 (2): 462-467.) knock out plasmid by pGhost9 successfully the LDH gene (ldhL) on L.plantarum to be knocked out，Reach to produce the purpose of optical voidness D-ALPHA-Hydroxypropionic acid.
(the TaKamatsuD such as TaKamatsuD, OsaKiM, SeKizaKiT.Thermosensitivesuicidevectorsforgenereplacemen tinStreptococcussuis.Plasmid, 2001,46 (2): 140-148.) pGhost3 is transformed, construct a series of more practical Streptococcus suis and knock out plasmid.On pGhost3, insertion derives from the replicon of pUC19, multiple clone site and Spectinomycin resistance labelling thus obtains knocking out plasmid pSET4S.When in escherichia coli, cultivation temperature is 37 DEG C, this plasmid can replicate, and when 37 DEG C, this plasmid can not replicate in Streptococcus suis.Carry out knocking out (being replaced to chloramphenicol resistance gene) to the hemolysin gene sly of S.equi with this plasmid.
There is presently no the report that pediococcus acidilactici carries out gene knockout, the knockout technique hence setting up a kind of pediococcus acidilactici is necessary.
Summary of the invention
It is an object of the invention to provide a kind of gene knockout method of P.acidilacticiDQ2.
Further object is that ldh, ldhD gene by knocking out P.acidilacticiDQ2, it is thus achieved that the bacterial strain of optical voidness D-ALPHA-Hydroxypropionic acid, Pfansteihl can be produced.
nullThe present invention realizes the gene knockout of P.acidilacticiDQ2 and be the technical scheme is that the structure pSET4E plasmid containing erythromycin resistance gene，The target gene upstream and downstream homologous fragment that will knock out is connected on pSET4E the most in certain direction，Obtain target gene knocks out plasmid，Plasmid will be knocked out converted in entrance P.acidilacticiDQ2 by electricity，Obtain containing the bacterial strain knocking out plasmid，Cultivate at the most reproducible higher temperature of plasmid 42 DEG C afterwards，Occur the single-swap that homologous recombination makes plasmid integration to recipient bacterium chromosome for the first time sub in the acquisition on flat board that selects containing erythromycin，Then by the single-swap lower temperature 28 DEG C cultivation that plasmid can replicate under the conditions of without erythromycin，Switching 1-10 time continuously，Cultivate under similarity condition，Until screening obtain occur second time homologous recombination realize, without Erythromycinresistant, the bacterial strain that target gene knocks out.
Above-mentioned pSET4E plasmid carry can replicate in escherichia coli replication origin, in gram-positive bacterium temperature sensitive type replicate replication origin, multiple clone site MCS, erythromycin resistance gene.
The above-mentioned plasmid that knocks out is by being connected in the multiple clone site of pSET4E by the homologous sequence fragment enzyme action the most in certain direction of target gene upstream and downstream to be knocked out on P.acidilacticiDQ2 chromosome, size about 1kb.
The above-mentioned plasmid that knocks out convert entrance P.acidilacticiDQ2 to obtain the method containing the bacterial strain knocking out plasmid being 1 μ g to knock out plasmid (about 10 μ L) mix with 80 μ L pediococcus acidilactici competent cells by electricity, at 2000V, 200 Ω, shock by electricity under the conditions of 25 μ F (1mm shock by electricity cup), rapidly bacterium solution is transferred to after electric shock ice in the resuscitation fluid of 1mL and puts 5min, after transfer in 28 DEG C of incubators cultivate 2.5h, 5000rpm is centrifuged 4min, draw 800 μ L of supernatant, the coating MRS flat board containing erythromycin after remaining mixing, cultivate about 3d for 28 DEG C and grow transformant, picking positive colony carries out PCR checking.
Above-mentioned generation for the first time homologous recombination makes the verification method of the plasmid integration single-swap to recipient bacterium chromosome be to be seeded to add in the MRS culture medium of erythromycin 28 DEG C containing the P.acidilacticiDQ2 knocking out plasmid, logarithmic (log) phase is cultivated under the conditions of 150rpm, a large amount of containing the bacterial strain knocking out plasmid to obtain, then with the inoculum concentration of 1%, above-mentioned bacterium solution is inoculated in MRS culture medium, 42 DEG C (at such a temperature, plasmid can not replicate, what screening obtained has the bacterial strain of resistance to be the bacterial strain that single-swap occurs to erythromycin), stable phase is cultivated under the conditions of 150rpm, above-mentioned bacterium solution is diluted 106The coating MRS flat board 42 DEG C cultivation containing erythromycin after times, obtain and single-swap that homologous recombination makes plasmid integration to recipient bacterium chromosome for the first time occurs, by single-swap 42 DEG C of cultivations in the MRS culture medium containing erythromycin, extracting genomic DNA, design primer carries out PCR checking.
Above-mentioned screening occur second time homologous recombination realize the method for the bacterial strain that target gene knocks out be single-swap is inoculated in the liquid MRS culture medium containing erythromycin 42 DEG C, 150rpm cultivates to stable phase, above-mentioned bacterium solution is inoculated in MRS culture medium by the inoculum concentration with 1%, stable phase is cultivated to obtain culture fluid for the first time under the conditions of 28 DEG C of (activate the plasmid fragments being incorporated on genome at low temperatures and carry out rolling-circle replication, sheared by homologous recombination mode), 150rpm.Same being inoculated in MRS culture medium with the inoculum concentration of 1% is cultivated afterwards, thus obtains secondary culture fluid, and at most switching 10 times realizes, until screening generation second time homologous recombination, the bacterial strain that target gene knocks out.The bacterium solution dilution 10 that will every time cultivate6Times after coating MRS flat board, 42 DEG C of cultivation, the single bacterium colony grown with toothpick picking, respectively dibbling containing erythromycin with do not contain on the MRS flat board of erythromycin, 42 DEG C of cultivations.For can grow on the flat board do not contain erythromycin and containing the bacterial strain that can not grow on erythromycin flat board, it should be occur double crossing over homologous recombination bacterial strain.By the 42 DEG C of cultivations in MRS culture medium of double crossing over bacterial strain, extracting genomic DNA, design primer carries out PCR checking, thus obtains the mutant that target gene knocks out.
The invention provides a kind of marker-free knockout technique based on homologous recombination, take the lead in that marker-free is knocked out technology in the world and be applied to P.acidilactici, any one gene can be realized in P.acidilactici and polygenic knock out, and not remaining any antibiotics resistance gene.
The present invention can knock out the gene of P.acidilactici fast, stably, efficiently, cannot be only used for studying function and the metabolic mechanism of P.acidilactici gene, realize the hereditary character transformation of P.acidilactici, and the mutant obtained does not carries any antibiotics resistance gene, both genetic modification can be carried out as starting strain, it is also possible to can be safely used for large-scale industrial production.
The ldh gene using this knockout technique to knock out P.acidilacticiDQ2 has obtained producing the bacterial strain ZP26 of optical voidness D-ALPHA-Hydroxypropionic acid, the ldhD gene knocking out P.acidilacticiDQ2 has obtained producing the bacterial strain TY112 of optical pure L-lactic acid, the two bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 31st in December in 2013 and (is called for short CGMCC, address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preservation registration number is respectively CGMCCNO.8665 and CGMCCNO.8664, its Classification And Nomenclature is pediococcus acidilactici (Pediococcusacidilactici).
[accompanying drawing explanation]
Fig. 1. for the structure of the pSET4E plasmid of gene knockout；
Fig. 2. knock out the structure of plasmid pSET4E-Δ ldh；
Fig. 3. knock out the structure of plasmid pSET4E-Δ ldhD；
The structure schematic flow sheet of Fig. 4 .P.acidilacticiDQ2ldh gene knockout；
The structure schematic flow sheet of Fig. 5 .P.acidilacticiDQ2ldhD gene knockout；
The PCR proof diagram of Fig. 6 .P.acidilacticiZP26, P.acidilacticiTY112 gene knockout；
Being labeled as in accompanying drawing 6: M, DL5,000DNAMarker；1, utilize the ldh gene of primer ldh-F and ldh-R amplification P.acidilacticiDQ2 genome；2, utilize ldh gene and upstream and downstream homology arm thereof in primer up-F-ldh and down-R-ldh amplification P.acidilacticiDQ2 genome；3, utilize the subregion of ldhD gene in primer ldhD-F and ldhD-R amplification P.acidilacticiDQ2 genome；4, utilize ldhD gene and upstream and downstream homology arm thereof in primer up-F-ldhD and down-R-ldhD amplification P.acidilacticiDQ2 genome；5, utilize the ldh gene of primer ldh-F and ldh-R amplification P.acidilacticiZP26 genome；6, utilize ldh gene and upstream and downstream homology arm thereof in primer up-F-ldh and down-R-ldh amplification P.acidilacticiZP26 genome；7, utilize the subregion of ldhD gene in primer ldhD-F and ldhD-R amplification P.acidilacticiTY112 genome；8, utilize ldhD gene and upstream and downstream homology arm thereof in primer up-F-ldhD and down-R-ldhD amplification P.acidilacticiTY112 genome；
Fig. 7 .P.acidilacticiDQ2, P.acidilacticiZP26, P.acidilacticiTY112 growth curve in simplifying MRS culture medium；
Fig. 8 .P.acidilacticiDQ2, P.acidilacticiZP26, P.acidilacticiTY112 add calcium carbonate simplification MRS culture medium in glucose consumption and lactic acid formation curve.
[detailed description of the invention]
The detailed description of the invention of the present invention presented below " the marker-free knockout technique of a kind of pediococcus acidilactici DQ2 based on homologous recombination ".
Test material:
Used by the present invention, pediococcus acidilactici P.acidilacticiDQ2 is obtained by this laboratory screening, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 12nd, 2013 and (is called for short CGMCC, address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCCNO.7471, and its Classification And Nomenclature is pediococcus acidilactici (Pediococcusacidilactici).E.colistrain XL1 blue used is by Laboratories Accession.MvandeGuchte is seen in the source of described pMG36e plasmid, JMvanderVossen, JKoKandGVenema, ConstructionofaLactococcalExpressionVector:ExpressionofH enEggWhiteLysozymeinLactococcuslactissubsp.Lactis, Appliedandenvironmentalmicrobiology, 1989,55 (1): 224-228.TaKamatsuD is seen in the source of described pSET4S plasmid, OsaKiM, SeKizaKiT, ThermosensitivesuicidevectorsforgenereplacementinStrepto coccussuis, Plasmid, 2001,46 (2): 140-148.
One, reagent and culture medium
PrimeSTARHSDNA polymerase, T4DNA ligase are purchased from TaKara bio-engineering corporation；The restricted enzyme such as PstI, EcoRI, HindIII, BamHI, SpeI are purchased from Fermentas company；Little test kit, the DNA gel recovery test kit taken out of plasmid is purchased from Shanghai Jierui Biology Engineering Co., Ltd；Bacterial genomes extraction agent box is purchased from Omega bio-engineering corporation；PCR primer purification kit is purchased from Sheng Gong biological engineering company limited；MegazymeD-/L-LacticacidKit test kit is purchased from Megazyme company；Erythromycin, spectinomycin are purchased from Bo Zun bio tech ltd；Other medicine, reagent are purchased from Shanghai Ling Feng chemical reagents corporation or Shanghai traditional Chinese medicines chemical reagent group if no special instructions, and are all analytical pure；Primer synthesis completes in Shanghai Jierui Biology Engineering Co., Ltd.
MRS culture medium (g/L):
Liquid: glucose 20, peptone 10, yeast powder 4, Carnis Bovis seu Bubali cream 8, sodium acetate 3, diammonium hydrogen citrate 2, dipotassium hydrogen phosphate 2, Magnesium sulfate heptahydrate 0.2, manganese sulfate monohydrate 0.05, Tween 80 lmL/L；Solid: the extra agar adding 15g/L, 115 DEG C of high pressure steam sterilization 20min in liquid medium within；
MRS culture medium containing erythromycin: add the erythromycin of final concentration 5 μ g/mL on MRS medium base；
Simplification MRS fluid medium (g/L): glucose 20, peptone 10, yeast powder 10, sodium acetate 5, diammonium hydrogen citrate 2, dipotassium hydrogen phosphate 2, Magnesium sulfate heptahydrate 0.58, manganese sulfate monohydrate 0.25,115 DEG C of high pressure steam sterilization 20min；
Resuscitation fluid (g/L): sucrose 171, glucose 20, peptone 10, yeast powder 4, Carnis Bovis seu Bubali cream 8, sodium acetate 3, diammonium hydrogen citrate 2, dipotassium hydrogen phosphate 2, Magnesium sulfate heptahydrate 0.2, manganese sulfate monohydrate 0.05, Tween 80 lmL/L, 115 DEG C of high pressure steam sterilization 20min；
LB culture medium (g/L):
Liquid: yeast leaching powder 5, peptone 10, sodium chloride 10；Solid: the extra agar adding 15g/L, 115 DEG C of high pressure steam sterilization 20min in liquid medium within；
LB culture medium containing erythromycin: add the erythromycin of final concentration 150 μ g/mL on LB medium base；LB culture medium containing spectinomycin: add the spectinomycin of final concentration 200 μ g/mL on LB medium base.
Two, key instrument used:
Mastercycler type PCR instrument (Eppendorf company)；GenePulserXcellTMType electroporation apparatus (Bio-Rad company)；EPS-100 type DNA electrophoresis system (Shanghai Tian Neng company)；The full-automatic ultraviolet of FR-200A type and visible analytical equipment (Fu scientific & technical corporation)；DU-800 type nucleic acid protein analyser (Beckman company)；HZ-2111KB type lands constant temperature oscillation shaking table (granary Hua Lida company limited)；5415R type tabletop refrigerated centrifuge (Eppendorf company)；LC-20AD type high performance liquid chromatography (Shimadzu Corporation)；SDC-6 type low temperature tank (Xin Zhi bio tech ltd, Ningbo)；DK-8D type three hole electric heating constant temperature tank (Shanghai Yiheng Scientific Instruments Co., Ltd)；GHP-9160 type water isolation type constant incubator (Shanghai Yiheng Scientific Instruments Co., Ltd)；Forma-86C type ultra cold storage freezer (Thermo company).
The structure of embodiment 1:pSET4E plasmid
Thermal sensitivity suicide plasmid pSET4S is a kind of escherichia coli-gram-positive bacterium width host range fabric shuttle-type plasmid, DaisuKeTaKamatsu is seen in the source of this plasmid, MaKotoOsaKi, andTsutomuSeKizaKi, ThermosensitiveSuicideVectorsforGeneReplacementinStrepto coccussuis, Plasmid, 2001,46 (2): 140 148.This plasmid can replicate when in escherichia coli, cultivation temperature is 37 DEG C, consequently facilitating the clone of homologous fragment, and can not replicate when 37 DEG C in Streptococcus suis.This plasmid cannot be only used for the gene knockout of Streptococcus suis, it may also be used for the gene knockout of some other gram-positive bacterium, therefore considers that suitably knocking out plasmid with this plasmid as initial plasmid structure carries out the gene knockout of P.acidilactici.
Find that P.acidilacticiDQ2 has the highest resistance (> 150 μ g/mL to spectinomycin), and to erythromycin-sensitive, it is contemplated that the spectinomycin resistance gene on pSET4S is replaced with erythromycin resistance gene.
Sequence (SEQIDNO:1) design primer Emr-F (SEQIDNO:2) and Emr-R (SEQIDNO:3) according to plasmid pMG36e expand erythromycin resistance gene Emr sequence (SEQIDNO:4) therein, obtaining erythromycin resistance gene fragment Emr with plasmid pMG36e for masterplate PCR amplification, PCR amplification system is as follows:
PCR reaction condition: 94 DEG C of denaturations 3min, 94 DEG C of degeneration 30s, 55 DEG C of annealing 15s, 72 DEG C extend 1min, 30 rear 72 DEG C of extension 10min of circulation.With SpeI restricted enzyme, Emr genetic fragment after purification and plasmid pSET4S (SEQIDNO:5) are carried out single endonuclease digestion respectively, then cut glue and reclaim purpose fragment.In order to avoid in connection procedure, the plasmid fragments of single endonuclease digestion occurs, from connecting, the plasmid after single endonuclease digestion to be carried out dephosphorylation process.Dephosphorylation system is following (50 μ L):
In PCR instrument, setting program reacts, and 65 DEG C of reaction 10min, reaction is purified with PCR primer purification kit after terminating.Finally carry out the connection of genes of interest fragment and plasmid fragments, convert E.colistrain XL1 blue, select positive bacteria and drop into row bacterium colony PCR, extract plasmid and carry out enzyme action qualification, obtain the pSET4E (SEQIDNO:6) (see Fig. 1) successfully constructed.
1 μ gpSET4E plasmid (about 10 μ L) is mixed with 80 μ LP.acidilacticiDQ2 competent cells, shock by electricity under the conditions of 2000V, 200 Ω, 25 μ F (1mm shock by electricity cup), rapidly bacterium solution is transferred to after electric shock ice in the resuscitation fluid of 1mL and puts 5min, after transfer in 28 DEG C of incubators cultivate 2.5h, then 5000rpm is centrifuged 4min, draw 800 μ L of supernatant, it is coated on after remaining mixing on the MRS culture medium flat plate containing 5 μ g/mL erythromycin, cultivate about 3d for 28 DEG C and grow transformant, carry out PCR checking with primer Emr-F and Emr-R.
The unmarked of embodiment 2:P.acidilacticiDQ2ldh gene knocks out
(1) structure of ldh gene knockout plasmid pSET4E-Δ ldh
The upstream and downstream DNA sequence of ldh gene (NCBI-GI=304328039) in type strain DSM20284 according to Pediococcusacidilactici, designs corresponding primer up-F-ldh, up-R-ldh, down-F-ldh, down-R-ldh (SEQIDNO:7-10) PCR amplification ldh gene upstream and downstream sequence up-ldh (sequence of the about 1kb size of LDH gene start codon upstream) (SEQIDNO:11), down-ldh (sequence of the about 1kb size in LDH gene end codon downstream) (SEQIDNO:12).
With the genomic DNA of P.acidilacticiDQ2 as template, with upstream and downstream homologous sequence up-ldh, down-ldh of primer up-F-ldh, up-R-ldh and down-F-ldh, down-R-ldh PCR clone ldh gene respectively, PCR amplification system is with the most consistent.
The amplification condition of PCR is as follows: 94 DEG C of denaturations 3min, 94 DEG C of degeneration 30s, 58 DEG C of annealing 15s, and 72 DEG C extend 70s, 30 rear 72 DEG C of extension 10min of circulation.The PCR primer of gained detects through the agarose gel electrophoresis of 0.7%, obtains the electrophoretic band that size is about about 1kb, by PCR primer purification.nullUtilize BamHI and PstI double digestion up-ldh and plasmid pSET4E respectively，Afterwards digestion products is attached，Convert E.colistrain XL1 blue，Identify through PCR and enzyme action and obtain positive strain，Obtain being cloned into up-ldh the plasmid pSET4E-up-ldh of pSET4E multiple clone site，The most again with EcoRI and BamHI double digestion down-ldh and pSET4E-up-ldh respectively，E.colistrain XL1 blue is converted after being connected by digestion products，Identify through PCR and enzyme action and obtain positive strain，Successfully obtain being cloned into ldh gene upstream and downstream homologous sequence LDH gene knockout plasmid pSET4E-Δ ldh (SEQIDNO:13) (see Fig. 2) of pSET4E.
(2) there is the screening of the single-swap bacterial strain of homologous recombination for the first time
The gene knockout plasmid pSET4E-Δ ldh electricity built is transformed into the competent cell of P.acidilacticiDQ2, its conversion condition is consistent with holding before, bacterium solution is coated on the MRS flat board containing 5 μ g/mL erythromycin 28 DEG C and cultivates about 3d, select positive bacterium colony, carry out PCR checking with primer Emr-F and Emr-R.By containing knock out the mono-colony inoculation of P.acidilacticiDQ2 of plasmid in the MRS culture medium add 5 μ g/mL erythromycin 28 DEG C, cultivate about 24h to logarithmic (log) phase under the conditions of 150rpm, then with the inoculum concentration of 1%, above-mentioned bacterium solution is inoculated in MRS culture medium, 42 DEG C, cultivate about 12h under the conditions of 150rpm to stable phase, bacterium solution is diluted 106The coating MRS flat board containing 5 μ g/mL erythromycin, 42 DEG C of cultivations again.The resistance list bacterium colony grown is single-swap bacterial strain.Picking resistance list bacterium colony is connected in the MRS culture medium containing 5 μ g/mL erythromycin 42 DEG C, cultivates under the conditions of 150rpm, extract genomic DNA, the form of the first single-swap that design primer single-swap 1 (ldh)-F, single-swap 1 (ldh)-R (SEQIDNO:14-15) detection is recombinated by up-ldh, the form (see Fig. 4) of the second single-swap that primer single-swap 2 (ldh)-F, single-swap 2 (ldh)-R (SEQIDNO:16-17) detection is recombinated by down-ldh.
(3) there is the screening of the gene knock-out bacterial strain of second time homologous recombination
Single-swap is inoculated in the liquid MRS culture medium containing erythromycin 42 DEG C, 150rpm cultivate about 12h to stable phase, above-mentioned bacterium solution is inoculated in MRS culture medium by the inoculum concentration with 1%, 28 DEG C, 150rpm cultivate about 24h to stable phase, it is forwarded in fresh MRS culture medium cultivate under similarity condition 24h with the inoculum concentration of 1% the most again, at most switching 10 times, until screening the bacterial strain that producer knocks out.Collect a part of bacterium solution every time cultivated, with normal saline dilution 106Being coated on after Bei on the MRS flat board without antibiotic, 42 DEG C of cultivations, with the single bacterium colony grown on toothpick picking flat board, dibbling is not containing on erythromycin and the MRS flat board containing 5 μ g/mL erythromycin respectively, 42 DEG C of cultivations.For growing without on the flat board of antibiotic, and the bacterium colony that can not grow on the flat board containing antibiotic, it should it is the bacterial strain that second time homologous recombination occurs.By 42 DEG C, 150rpm cultivation in inoculation to MRS fluid medium, extract genomic DNA, carry out PCR amplification with primer up-F-ldh and down-R-ldh, additionally carry out PCR amplification with primer ldh-F, ldh-R (SEQIDNO:18-19) of amplification ldh gene.
In double crossing over that second time homologous recombination occurs, one is that back mutation becomes wild type, another kind to be exactly ldh gene knockout mutant strain (principle is shown in Fig. 4).
For can only amplify the PCR primer (containing up-ldh and down-ldh) of about 2kb size with primer up-F-ldh and down-R-ldh, ldh-F and ldh-R can not expand the bacterial strain of correspondingly sized product, is the bacterial strain that second time homologous recombination ldh gene knockout occurs.
The Strain Designation of the strain ldh gene knockout obtained by the method is PediococcusacidilacticiZP26 (Fig. 6 is shown in PCR checking), within 31st, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in December in 2013 and (is called for short CGMCC, address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCCNO.8665, and its Classification And Nomenclature is pediococcus acidilactici (Pediococcusacidilactici).
The unmarked of embodiment 3:P.acidilacticiDQ2ldhD gene knocks out
(1) structure of ldhD gene knockout plasmid pSET4E-Δ ldhD
According to ldhD gene (NCBI-GI=304329050) upstream and downstream DNA sequence in PediococcusacidilacticiDSM20284, design corresponding primer up-F-ldhD, up-R-ldhD, down-F-ldhD, down-R-ldhD (SEQIDNO:20-23) PCR and expand its upstream and downstream sequence up-ldhD (sequence of the about 1kb size of D-lactic acid dehydrogenase gene start codon upstream) (SEQIDNO:24), down-ldhD (sequence of the about 1kb size in D-lactic acid dehydrogenase gene end codon downstream) (SEQIDNO:25).
With the genomic DNA of P.acidilacticiDQ2 as template, with upstream and downstream homologous sequence up-ldhD and down-ldhD of primer up-F-ldhD, up-R-ldhD and down-F-ldhD, down-R-ldhD PCR clone ldhD gene respectively, PCR amplification system is consistent with holding before.
The amplification condition of PCR is as follows: 94 DEG C of denaturations 3min, 94 DEG C of degeneration 30s, 58 DEG C of annealing 15s, and 72 DEG C extend 60s, 30 rear 72 DEG C of extension 10min of circulation.The PCR primer of gained detects through the agarose gel electrophoresis of 0.7%, obtains the electrophoretic band that size is about about 1kb, by PCR primer purification.Utilize HindIII and BamHI double digestion up-ldhD and plasmid pSET4E respectively, E.colistrain XL1 blue is converted after being connected by digestion products, identify through PCR and enzyme action and obtain positive strain, obtain being cloned into up-ldhD the recombiant plasmid pSET4E-up-ldhD of pSET4E multiple clone site, the most again with BamHI and EcoRI double digestion down-ldhD and pSET4E-up-ldhD respectively, digestion products converts E.colistrain XL1 blue after connecting, identify through PCR and enzyme action and obtain positive strain, successfully obtain D-lactic acid dehydrogenase gene knockout plasmid pSET4E-Δ ldhD (SEQIDNO:26) (see Fig. 3).
(2) there is the screening of the single-swap bacterial strain of homologous recombination for the first time
Consistent with the single-swap bacterial strain screening step that ldh gene occurs homologous recombination for the first time, but carry out the primer during the checking of single-swap different, the form of the first single-swap that design primer single-swap 1 (ldhD)-F, single-swap 1 (ldhD)-R (SEQIDNO:27-28) detection is recombinated by up-ldhD, the form (see Fig. 5) of the second single-swap that single-swap 2 (ldhD)-F, single-swap 2 (ldhD)-R (SEQIDNO:29-30) detection is recombinated by down-ldhD.
(3) there is the screening of the gene knock-out bacterial strain of second time homologous recombination
The screening process that the gene knock-out bacterial strain of second time homologous recombination occurs with ldh gene is consistent, carry out PCR amplification with primer up-F-ldhD and down-R-ldhD when except for the difference that carrying out PCR checking, additionally carry out PCR checking with primer ldhD-F, the ldhD-R (SEQIDNO:31-32) in amplification ldhD Gene Partial region.
For about 2kb size PCR primer (containing up-ldhD and down-ldhD) can only be amplified with up-F-ldhD and down-R-ldhD, ldhD-F and ldhD-R can not expand the bacterial strain of correspondingly sized product, is the bacterial strain that second time homologous recombination ldhD gene knockout occurs.
The Strain Designation being obtained a strain ldhD gene knockout by the method is PediococcusacidilacticiTY112 (Fig. 6 is shown in PCR checking), within 31st, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center in December in 2013 and (is called for short CGMCC, address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCCNO.8664, and its Classification And Nomenclature is pediococcus acidilactici (Pediococcusacidilactici).
Embodiment 4: the application of gene knock-out bacterial strain
After structure obtains two pnca gene knock-out bacterial strains, need to compare it with the key property of original strain.
The mensuration of configuration of lactic acid: the glycerol pipe that P.acidilacticiDQ2, P.acidilacticiZP26, P.acidilacticiTY112-80 DEG C frozen is seeded to respectively 20mL and simplifies in MRS culture medium, 42 DEG C, 150rpm cultivate after 12h, be forwarded to 50mL with 10% inoculum concentration and simplify MRS fluid medium and (add 12g/LCaCO3In), 42 DEG C, 150rpm cultivation, take 12h sample and utilize the optical purity of MegazymeD-/L-LacticacidKit kit measurement lactic acid, result such as table 1, P.acidilacticiZP26, P.acidilacticiTY112 can produce the optical purity lactic acid more than 99%, illustrate D, the knocking out to have played and should have effect of LDH gene.
The comparison of growth curve: the glycerol pipe that P.acidilacticiDQ2, P.acidilacticiZP26, P.acidilacticiTY112-80 DEG C frozen is seeded to respectively 20mL and simplifies in MRS culture medium, 42 DEG C, 150rpm cultivate 12h, it is forwarded to 50mL with 10% inoculum concentration afterwards and simplifies in MRS fluid medium, 42 DEG C, 150rpm cultivation, survey growth curve, such as Fig. 7.As can be seen from the figure P.acidilacticiDQ2, P.acidilacticiZP26 growth difference is little, and P.acidilacticiTY112 is slightly worse, illustrate the two gene to knock out the growth effect to bacterial strain less.
Generate the comparison of lactic acid: the glycerol pipe that P.acidilacticiDQ2, P.acidilacticiZP26, P.acidilacticiTY112-80 DEG C frozen is inoculated in respectively 20mL and simplifies in MRS culture medium, 42 DEG C, 150rpm cultivate 12h, be forwarded to 50mL with 10% inoculum concentration afterwards and simplify MRS fluid medium and (add 12g/LCaCO3In), 42 DEG C, 150rpm cultivation, period sampling measuring lactic acid and concentration of glucose, such as Fig. 8.The sugar consumption of DQ2 is quicker, and other two kinds of bacterium are the slowest.During 10h, DQ2 runs out of sugar, ZP26 and TY112 also has residual sugar to exist, and the lactic acid yield of DQ2, ZP26, TY112 is respectively 85%, 82.3% and 95.4%.These results suggest that the two gene to knock out the Influence of production to lactic acid the least.
The mensuration of table 1 mutant configuration of lactic acid
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, without departing from the inventive concept of the premise; can also make some improvements and modifications, these improvements and modifications also should be regarded as in protection scope of the present invention.
Claims (5)
1. a marker-free knockout technique of pediococcus acidilactici DQ2 based on homologous recombination, step includes:
(1) structure of pSET4E plasmid: pSET4S plasmid is a kind of responsive to temperature type escherichia coli-gram-positive bacterium width host range fabric shuttle-type plasmid, at 37 DEG C, this plasmid can replicate in escherichia coli, but can quickly lose in some streptococcus such as Streptococcus suis, streptococcus equi, streptococcus dysgalactiae；PSET4S plasmid contains spectinomycin resistance gene, and pediococcus acidilactici DQ2 has the highest resistance to spectinomycin, but very sensitive to erythromycin, therefore obtains pSET4E plasmid with the spectinomycin resistance gene of erythromycin resistance gene replacement pSET4S；
(2) each about 1kb of the target gene upstream and downstream homologous fragment that will knock out is cloned on pSET4E respectively, it is thus achieved that target gene knock out plasmid；
(3) plasmid will be knocked out converted in entrance pediococcus acidilactici DQ2 by electricity, obtain containing the bacterial strain knocking out plasmid；
(4) will cultivate at the higher temperature 42 DEG C that plasmid replication is obstructed containing the bacterial strain knocking out plasmid, at single-swap selecting to screen generation homologous recombination i.e. plasmid integration for the first time on flat board to recipient bacterium chromosome containing erythromycin；
(5) single-swap is cultivated at without erythromycin and lower temperature 28 DEG C, continuously switching 1-10 time cultivation under similarity condition, until screening obtains occurring second time homologous recombination to realize, without Erythromycinresistant, the bacterial strain that target gene knocks out；
It is characterized in that:
The construction method of the pSET4E plasmid described in step (1) is: replace the spectinomycin resistance gene of pSET4S with erythromycin resistance gene；Erythromycin resistance gene Emr (SEQIDNO:4) is expanded as template, design primer Emr-F (SEQIDNO:2) and Emr-R (SEQIDNO:3) PCR using pMG36e plasmid (SEQIDNO:1)；With restricted enzyme SpeI, Emr genetic fragment and plasmid pSET4S are carried out single endonuclease digestion respectively, in order to avoid in connection procedure, the plasmid fragments of single endonuclease digestion occurs from connecting, plasmid after single endonuclease digestion is carried out dephosphorylation process, afterwards genes of interest fragment is attached with plasmid, convert E.colistrain XL1 blue, utilizing primer Emr-F and Emr-R to carry out PCR checking, digestion verification obtains correct plasmid pSET4E simultaneously；
The target gene that obtains described in step (2) knocks out the method for plasmid and is: design primer PCR method expands the upstream and downstream of pediococcus acidilactici target gene respectively, size is about the homologous fragment of 1kb, successively being cloned into the multiple clone site of pSET4E in certain direction, obtain this target gene knocks out plasmid；
Step (3) is converted by electricity and enters the bacterial strain method that pediococcus acidilactici DQ2 obtains containing knocking out plasmid by knocking out plasmid and be: mixed with 80 μ L pediococcus acidilactici DQ2 competent cells by 1 μ g plasmid (about 10 μ L), at 2000V, 200 Ω, shock by electricity under the conditions of 25 μ F (1mm shock by electricity cup), rapidly bacterium solution is transferred to after electric shock ice in the resuscitation fluid of 1mL and puts 5min, after transfer in 28 DEG C of incubators cultivate 2.5h, 5000rpm is centrifuged 4min, draw 800 μ L of supernatant, the MRS flat board containing erythromycin is coated after remaining mixing, cultivate about 3d for 28 DEG C and grow transformant, picking positive colony carries out PCR checking；
Step (4) is described occurs the verification method of single-swap that for the first time homologous recombination makes plasmid integration to recipient bacterium chromosome to be: will be seeded to add in the MRS culture medium of erythromycin 28 DEG C containing the pediococcus acidilactici DQ2 knocking out plasmid, logarithmic (log) phase is cultivated under the conditions of 150rpm, a large amount of containing the bacterial strain knocking out plasmid to obtain, then with the inoculum concentration of 1%, above-mentioned bacterium solution is inoculated in the MRS culture medium without erythromycin, 42 DEG C (at such a temperature, plasmid can not replicate, what screening obtained has the bacterial strain of resistance to be the bacterial strain that single-swap occurs to erythromycin), stable phase is cultivated under the conditions of 150rpm, above-mentioned bacterium solution dilution 106Coating 42 DEG C of cultivations on the MRS flat board containing erythromycin after Bei, it is thus achieved that single-swap that homologous recombination makes plasmid integration to recipient bacterium chromosome for the first time occurs, design primer carries out PCR checking；
nullThe described screening of step (5) occurs second time homologous recombination to realize the method for the bacterial strain that target gene knocks out: single-swap is inoculated in the liquid MRS culture medium containing erythromycin 42 DEG C、150rpm cultivates to stable phase，Above-mentioned bacterium solution is inoculated in MRS culture medium by the inoculum concentration with 1%，28 DEG C (are activated the plasmid fragments being incorporated on genome at low temperatures and carry out rolling-circle replication，Sheared by homologous recombination mode)、Stable phase is cultivated under the conditions of 150rpm，Culture fluid is inoculated in MRS culture medium with the inoculum concentration of 1%，The same terms is cultivated to obtain second time culture fluid，It is inoculated into equally in MRS culture medium after growing to stable phase and cultivates，Thus obtain the culture fluid of third time，At most switching 10 times realizes, until screening generation second time homologous recombination, the bacterial strain that target gene knocks out；The bacterium solution dilution 10 that will every time cultivate6Times after coating MRS flat board, 42 DEG C of cultivation, the single bacterium colony grown with toothpick picking, respectively dibbling containing erythromycin with do not contain on the MRS flat board of erythromycin, 42 DEG C of cultivations；For can grow on the flat board do not contain erythromycin and containing the bacterial strain that can not grow on erythromycin flat board, it should be occur double crossing over homologous recombination bacterial strain；Design primer carries out PCR checking, thus obtains the mutant that target gene knocks out.
The marker-free knockout technique of a kind of pediococcus acidilactici DQ2 based on homologous recombination the most according to claim 1, is characterized in that: described pSET4E plasmid is that the Spc gene of pSET4S plasmid is obtained by the replacement of Emr gene.
The marker-free knockout technique of a kind of pediococcus acidilactici DQ2 based on homologous recombination the most according to claim 1, is characterized in that: described in knock out the structure of plasmid be the multiple clone site homologous fragment of the upstream and downstream size about 1kb of gene to be knocked out being cloned into the most in certain direction pSET4E.
4. the L of pediococcus acidilactici DQ2 that prepared by this gene knockout method, D-lactic acid dehydrogenase gene knock-out bacterial strain P.acidilacticiZP26, P.acidilacticiTY112, its preserving number is respectively CGMCCNO.8665, CGMCCNO.8664.
L the most according to claim 4, D-lactic acid dehydrogenase gene knock-out bacterial strain P.acidilacticiZP26 and P.acidilacticiTY112, it is characterized in that fermenting and producing optical voidness D-ALPHA-Hydroxypropionic acid, Pfansteihl, gene knockout is less to the growth effect of this two strains bacterium, and the impact on fermenting property is the least.
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