CN109735556A - The purposes of Priming Glycosyltransferase Gene Involved - Google Patents
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Abstract
The present invention discloses a kind of purposes of Priming Glycosyltransferase Gene Involved, i.e. Priming Glycosyltransferase Gene Involvedcps2E、cps4E is improving the application in lactobacillus plantarum transposon mutagenesis；It willcps2E、cps4E gene and temperature-sensitive plasmid pFED760 recombination to construct knockout carrier, and import in food-borne lactobacillus plantarum competent cell, and then pass through homologous recombination constructioncps2E、cps4E gene knock-out bacterial strain YM 4-3- Δcps2E、YM 4‑3‑Δcps4E and YM 4-3- Δcps2E-4E；The extracellular polysaccharide ability of bacterial strain is measured by phend-sulphuric acid, the discovery extracellular polysaccharide ability of knock-out bacterial strain is below wild-type strain, and double knock-out bacterial strain yield of extracellular polysaccharide are extremely low, thereforecps2E、cps4E gene plays a crucial role in transposon mutagenesis, and the present invention has great potential in exocellular polysaccharide biosynthesis research and application field.
Description
Technical field
The invention belongs to the function of gene and application fields, and in particular to Priming Glycosyltransferase Gene Involvedcps2E、cps4E
Improving the application in lactobacillus plantarum YM 4-3 exocellular polysaccharide biosynthesis.
Background technique
Microbial exopolysaccharides (exopolysaccharide, EPS) are to secrete during its biosynthetic metabolism to thin
It is extracellular and it is normal seep in a kind of saccharide compound of culture medium, compared to plant source and animal sources polysaccharide its with it is with short production cycle,
It is not limited by conditions such as season, pest and disease damage, diseases, so that it is with the stronger market competitiveness and being widely applied property.
Lactic acid bacteria is because of food peculiar flavour corresponding to the correlation of traditional fermented food and its imparting, quality, secure context
Characteristic and it is well-known, growth characteristics make in terms of the preservation of food have original charm.The acid crossed by lactobacillus-fermented
Dish and fermented soya bean become the regular guest of life dining table gradually with the development of the times, and evolution over time makes plus the wisdom of compatriots
Obtaining them gives food richer taste.
The combination of lactic acid bacteria and soybean is the performance of Chinese's wisdom, this is dexterously sent out different from the another of bean curd
It is bright.It allows soybean no longer to make one ventosity under the continuous effect of lactic acid bacteria, people is made to be easier to absorb the nutrition such as sugar therein
Substance.Belong to main kind in lactic acid bacteria lactobacillus plantarum ( Lactobacillus plantarum) it is in Yunnan tradition
The most common strain in fermented food fermented soya bean,Lb. plantarumYM 4-3 is exactly one plant and is isolated from Yunnan traditional fermented food
The lactobacillus lactic acid bacteria of fermented soya bean, produced EPS are one kind by glucose, mannose, fructose, lactose, galactolipin, sandlwood
Sugar, arabinose composition heteroglycan, according to we have discovered that,Lb. plantarumThe produced EPS of bacterial strain YM 4-3 has good
Good anti-oxidant and anti-tumor activity.
With the progress and development of modern society, the health of food becomes the level that people are more concerned about.Lactic acid bacteria is generally acknowledged
The food-grade microorganisms of safety (GRAS, Generally Recognized as Safe), generated EPS have it is natural,
The small characteristic of safety, toxic side effect, the research and development of lactic acid bacteria EPS meet people's health demand just and are concerned.
Can most of lactic acid bacteria EPS yield with excellent physiological activity be not able to satisfy industrial demand, although from culture item
It optimizes also available raising in terms of part, but the uncertainty of its condition of culture and is related to experiment condition and industry is sent out
Research is very few in terms of the changing effect of ferment mode, therefore goes forward side by side from the function that gene level sets about studying EPS synthesis related gene
One step understands its Regulation Mechanism to understand influence of the related gene to lactic acid bacteria EPS biosynthesis, then with improve fermentation condition
This mode is combined with stronger application value.
Summary of the invention
For the deficiency of present Research, the present invention provides a kind of purposes of Priming Glycosyltransferase Gene Involved, i.e. guidance glycosyl
Transferase genecps2E、cps4E improve lactobacillus plantarum (Lactobacillus plantarum) in transposon mutagenesis
Application, the genecpsThe nucleotide sequence of 2E is as shown in SEQ ID NO:1, genecpsThe nucleotide sequence of 4E such as SEQ
Shown in ID NO:2；The present invention willcps2E、cps4E gene and temperature-sensitive plasmid pFED760 recombination to construct knockout carrier
pFED760-Δcps2E、pFED760-Δcps4E、pFED760-Δcps2E-4E, be conducted into lactobacillus plantarum competence into
And pass through homologous recombination constructioncps2E、cps4E gene list knock-out bacterial strain Δcps2E、Δcps4E and double knock-out bacterial strain Δscps2E-4E；By comparing the different proofs of wild-type strain and the Physiology and biochemistry difference of knock-out bacterial strain and EPS yieldcps2E
Withcps4E gene plays a crucial role in EPS synthesis, and the present invention has very big in the research of EPS biosynthesis and application field
Potentiality.
In order to realize above-mentioned purpose of the invention, technical scheme is as follows:
1, Priming Glycosyltransferase Gene Involvedcps2E、cpsThe preparation of 4E knockout carrier, the knockout carrier obtain by the following method
It arrives: using food-borne lactobacillus plantarum genome as template, utilizing primer pair (up-2EF+up-2ER；up-4EF + up-4ER；
down-2EF + down-2ER；Down-4EF+down-4ER) upstream and downstream homology arm is expanded respectively, PCR product is used as template,
Utilize primer pair (up-2EF+down-2ER；Up-4EF+down-4ER) amplification obtain knock out segment, the fragment purification
Afterwards, restriction enzyme is used respectively with temperature-sensitive plasmid pFED760xhoI HeEcoR I andHinIII He of dEcoRI is synchronous
Digestion, digestion products are attached experiment after gel extraction, and connection product imported into bacillus coli DH 5 alpha competent cell
In, it extracts plasmid and obtainscps2E、cps4E gene knockout carrier pFED760- Δcps2E、pFED760-Δcps4E, wherein drawing
Object sequence is as follows:
up-2EF: 5’-CCGGAATTCTGAACAGAT CGATACTGGTG-3 ',
up-2ER: 5'-ACATTTCTCATCTGGCGCGTTTGTGGTTGTACATGAC- 3'；
Down-2EF:5 '-GTCATGTACAACCACAAACGCGCCAGATGAGAAA TGT-3 ',
down-2ER: 5’-CCCTCGAGCATTTTTGCGACTCTCAT-3'；
up-4EF: 5’-CCGGAATTCGGGTTCATTGGC TCGCACTTGGT-3 ',
Up-4ER:5 '-CGTAGCCGTT TCGGAAAATCTCACAGTGTTGTTCGTC AGC-3 ',
Down-4EF:5 '-GCTGACGAACAACACTGTGAGATTTTCCGAAACGGCTACG- 3 ',
down-4ER: 5’-CCCAAG CTTGGGCACATAACTACTGCTCCAA-3 ', underscore are restriction enzyme site.
In the present inventioncps2E、cps4E gene and its upstream and downstream homology arm sequence such as SEQ ID NO:3 and SEQ ID NO:4
It is shown, from food-borne lactobacillus plantarum (Lactobacillus plantarum)；Lactobacillus plantarum is safe and harmless etc. because of its
Feature, is widely used in the fields such as food, drug, thus its gene be also it is safe to the human body harmless, this will be for the invention in the future
Theoretical safety guarantee is provided in the application of exocellular polysaccharide production field.
2, Priming Glycosyltransferase Gene Involvedcps2E、cps4E knock-out bacterial strain construction and screening, including above-mentioned guidance glycosyl turn
Move enzyme genecps2E、cpsThe building of 4E knockout carrier and the conversion of later period knockout carrier and knock-out bacterial strain screening, steps are as follows:
(1) knockout carrier pFED760- Δcps2E and pFED760- ΔcpsThe conversion of 4E: in 90 ~ 100 μ L lactobacillus plantarum senses
By 10 μ L knockout carrier pFED760- Δs are added in statecps2E and pFED760- Δcps4E is mixed gently, and is transferred to after ice bath 5min
In pre-cooling electric shock cup, according to 1.25 kv/cm, the parameter of 200 Ω shocks by electricity, and is added into electric revolving cup rapidly after the completion of electric shock
Mixed liquor is transferred in sterile 1.5mL centrifuge tube, 28 DEG C by the fresh MRS culture solution of 900 μ L after gently blowing and beating mixing with pipette tips
2.5 ~ 3 h of static gas wave refrigerator makes cell recovery.8000 ~ 10000 rpm of bacterium solution is centrifuged 3 min after culture, abandons 900 μ L supernatants, uses
Thallus is resuspended in remaining supernatant, is coated on containing on 5 μ g/mL erythromycin MRS solid plates, 28 DEG C of stationary cultures.
(2)cps2E、cpsThe screening of 4E gene knock-out bacterial strain: the single colonie switching grown in step (1) is in containing 5 μ g/mL
In the MRS fluid nutrient medium of erythromycin, bacterium solution is transferred to 37 DEG C until when bacterium solution OD600 is 0.2 ~ 0.3 by 28 DEG C of stationary cultures
Continue stationary culture to stay overnight, culture bacterium solution dilution 103~105The MRS solid plate containing 5 μ g/mL erythromycin is coated on after times
On, for 24 hours, picking monoclonal is seeded to 1mL and contains in the MRS fluid nutrient medium of 5 μ g/mL erythromycin 37 DEG C 37 DEG C of stationary cultures
Stationary culture is stayed overnight, which is seeded in not antibiotic MRS fluid nutrient medium by 1%, 28 DEG C of stationary culture mistakes
Night, then bacterium solution dilution 103~105It is coated on the MRS solid plate of antibiotic-free after times, 37 DEG C of stationary cultures are to growing Dan Ke
Grand, picking smaller single colonie one-to-one correspondence is lined containing 5 μ g/mL erythromycin and not antibiotic MRS solid plate, and 37 DEG C
For 24 hours, picking can not be grown stationary culture on antibiotic MRS agar plate, and can be on not antibiotic plate
The bacterium colony of growth carries out bacterium solution PCR verifying, to obtain lactobacillus plantarum YM 4-3- Δcps2E and lactobacillus plantarum YM 4-3-
ΔcpsThe mono- knock-out bacterial strain of 4E；What the screening technique improved knock-out bacterial strain sifts out efficiency.
In heretofore described bacterium solution PCR verifying using positioned at upstream homology arm upstream gene group preceding primer (cps2E-
Uu-F:5 '-GGGTCTTGGCACAGGTTACGG- 3 ',cps4E-uu-F:5 '-TATGATTTAGTGCAGCAGGG- 3 '), position
In downstream homology arm downstream gene group rear primer (cps2E-uu-R:5 '-CAACAGCACGAAACCAATAC- 3 ',cps4E-
Dd-R:5 '-CTTACGAAGAACTTCTAGCC-3 ')；Wild-type strain PCR fragment ratiocpsThe big 502bp of 2E knock-out bacterial strain, ratiocpsThe big 421bp of 4E knock-out bacterial strain.
(3)cps2E、cpsThe construction method of the bis- knock-out bacterial strains of 4E is identical as single knock-out bacterial strain, will as shown in step (1)
pFED760-Δcps2E knockout carrier electricity is transferred to lactobacillus plantarum YM 4-3- ΔcpsIn 4E competence, plate is carried out after recovery
Coating, is screened according still further to method shown in step (2), the primer and lactobacillus plantarum YM 4-3- ΔcpsThe mono- knockout bacterium of 2E
Strain is identical.
3, food-borne lactobacillus plantarum is demonstrated using the successful knock-out bacterial strain of buildingcps2E、cps4E gene and plant
It plays a crucial role in lactobacillus EPS synthesis.
One of features of the present invention is to study gene from food-borne food lactobacillus, has safety, can be used for
Later period field of food fermentation.
The two of the features of the present invention are that the method for gene knock-out bacterial strain screening improves the screening efficiency of knock-out bacterial strain.
The three of feature of the present invention are, it was demonstrated that research gene of the present inventioncps2E、cps4E gene is in transposon mutagenesis
In key effect, provide certain theoretical basis for the EPS research and development for synthesizing functional food, and it has further been discovered that this gene exists
Effect in cellular morphology and strain growth.
Compared with prior art present invention has the advantage that 1) research gene source has peace in food-grade microorganisms
Quan Xing；2) present invention research genecps2E、cps4E gene plays key effect in EPS synthesis.
Detailed description of the invention
Fig. 1 is knock-out bacterial strain bacterium solution PCR of the present invention verifying, wherein figure A:YM 4-3- Δcps4E bacterial strain screening, M are
2000bp DNA molecular Marker, swimming lane 1-6 are YM 4-3- Δcps4E bacterial strain, swimming lane 7 are YM 4-3 strain control, swimming lane 8
For blank control (sterile ddH2O) PCR product compares；Scheme B:YM 4-3- Δ cps 2E bacterial strain screening, M is 2000bp DNA points
Sub- Marker, swimming lane 1-3 are YM 4-3- Δcps2E bacterial strain, swimming lane 4 are YM 4-3 strain control；Scheme C:YM 4-3- Δcps2E-4E bacterial strain screening, M are 2000bp DNA molecular Marker, and swimming lane 4 is YM 4-3- Δcps2E-4E bacterial strain, swimming lane 1-
3 be YM 4-3 strain control, and swimming lane 5-7 is failed control；
Fig. 2 is that wild type of the present invention and knockout type bacterial strain yield of extracellular polysaccharide compare, and wherein WT indicates wild type YM 4-3 bacterial strain；
Qc2E is indicatedcps2E gene knock-out bacterial strain YM 4-3- Δcps2E；Qc4E is indicatedcps4E gene knock-out bacterial strain YM 4-3- Δcps4E；Qc2E-4E is indicatedcps2E andcpsDouble knock-out bacterial strain YM 4-3- Δs of 4E genecps2E-4E。
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples, but the scope of the present invention is not limited to
The content, reagent and method used in embodiment are all made of conventional reagent and use conventional method unless otherwise specified；
Temperature-sensitive plasmid pPED760 is given by University of Illinois Michael J doctor Federle；In following embodiments
As a result unless otherwise instructed, it is duplicate average value three times.
Embodiment 1: Priming Glycosyltransferase Gene Involved (cps2E、cps4E) knock out homology arm clone
1, PCR amplification upstream and downstream homology arm
Food-borne lactobacillus plantarum is extracted using TAKARA bacterial genomes DNA extraction kit (precious bioengineering Co., Ltd)
YM 4-3 genome, concrete operations are carried out according to kit specification.For genecps2E, to extract genome as template, divide
Not with up-2EF (CCGGAATTCTGAACAGATCG ATACTGGTG, underscore are restriction enzyme siteEcoRⅠ)、up-2ER(ACA
) and down-2EF (GTCATGTACAACCACAAACGCGCCAGATG TTTCTCATCTGGCGCGTTTGTGGTTGTACATGAC
AGAAATGT)、down-2ER(CCCTCGAGCATT TTTGCGACTCTCAT, underscore are restriction enzyme sitexhoI) it is expanded
Obtain upstream and downstream homology arm cps2E-up(1066bp), cps2E-down(1048bp).For genecps4E, to extract gene
Group is template, respectively with primer pair up-4EF (CCGGAATTCGGGTTCATTGGCTCGCACTTGGT, underscore are digestion position
PointEcoR I), up-4ER (CGTAGCCGTTTCGGAAAATCTCACAGTGTTGTTCGTCAGC) and down-4EF (GCTG ACG
AACAACACTGTGAGATTTTCCGAAACGGCTACG)、down-4ER (CCCAAGCTTGGGC
ACATAACTACTGCTCCAA, underscore are restriction enzyme siteHinD III) amplification upstream and downstream homology arm cps4E-up(922bp),
Cps4E-down(1174b), PCR reaction system and amplification condition are as follows:
(1) PCR reaction system
；
(2) PCR amplification condition
95 DEG C of 3 min of initial denaturation；95 DEG C of 15 s of denaturation；55 DEG C of 15 s of annealing；72 DEG C of 50 s of extension；Circulation 30 times；72 DEG C extend 5
Min, 12 DEG C of preservations.5 μ L are taken after the reaction was completed, and electrophoretic analysis is carried out in 1% Ago-Gel.
2, gene knockout segment clone building and sequencing
Using each 1 μ L of upstream and downstream homology arm PCR product as template, up-2EF and down-2ER and up-4EF and down-4ER are to draw
Object carries out over-lap PCR according to above-mentioned PCR reaction system and amplification condition (extension of time is changed to 2 min)；Gel extraction is expected big
Small PCR product (i.e. gene knockout segment), according to Dalian treasured bioengineering Co., Ltd TA Cloning Kit specification, by PCR
Product is connected in pMD19-T carrier；Connection product is imported in bacillus coli DH 5 alpha competent cell by heat-shock transformed method,
It is coated on Amp-LB plate；After 37 DEG C are incubated overnight, 10~15 single colonies are randomly selected, extract plasmid in its cell, and divide
It does not useEcoRⅠ、xhoI HeEcoRⅠ、HinD III carries out digestion verification, and positive plasmid send sequencing company to be sequenced.
Embodiment 2:cps2E、cpsThe building of 4E gene list knockout carrier
Use restriction enzymeEcoRⅠ、xhoI HeEcoRⅠ、HinD III is respectively to the correct gene knockout segment of sequencing and temperature
Degree responsive type plasmid pFED760 synchronizes digestion.cps2E digestion system are as follows:EcoR I, 2 μ L；xhoI, 2 μ L；1×H
Buffer, 4 μ L；Gene knockout segment or pFED760,10 ~ 16 μ L；Add sterile deionized water to 20 μ L.cps4E digestion body
System are as follows:EcoR I, 1 μ L；HinD III, 1 μ L；1 × M buffer, 4 μ L；Gene knockout segment or pFED760,30 μ L；Add
Sterile deionized water is to 40 μ L.Digestion products are recycled after 37 DEG C of 4 h of digestion, according to target gene: carrier=4:1~2:1(rubs
Your ratio) after sample-adding, T4 DNA ligase is added in 16 DEG C of 12~16 h of connection；Connection product is imported greatly using heat-shock transformed method
In enterobacteria DH5 α competent cell, then it is coated on erythromycin-LB solid plate；After 28 DEG C are incubated overnight, extraction 10~
Plasmid in 15 single colonie cells, and double digestion verifying is carried out with corresponding restriction endonuclease to obtain positive plasmid, it is named as pFED760-
Δ cps2E and pFED760- Δ cps4E.
Embodiment 3:cps2E、cpsThe building of 4E gene list knock-out bacterial strain
1、cps2E、cps4E gene knockout carrier imports lactobacillus plantarum competent cell
It is thin that lactobacillus plantarum competence is prepared referring to Fei Yongtao (2015, South China Science & Engineering University's master thesis) report method
Born of the same parents；10 μ L gene knockout carrier pFED760- Δ cps2E and pFED760- are added in 90 ~ 100 μ L lactobacillus plantarum competence
Δ cps4E, mixes gently, and is transferred in pre-cooling electric shock cup after ice bath 5min, and according to 12.5kv/cm, the parameter of 200 Ω carries out electricity
It hits；The fresh MRS culture solution of 900 μ L is added after the completion of electric shock into electric revolving cup rapidly, after gently blowing and beating mixing with pipette tips, will mix
It closes liquid to be transferred in sterile 1.5mL centrifuge tube, 28 DEG C of 2.5 ~ 3 h of static gas wave refrigerator make cell recovery.8000 rpm of bacterium solution after culture
3 min are centrifuged, 900 μ L supernatants are abandoned, thallus is resuspended with remaining supernatant, are coated on flat containing 5 μ g/mL erythromycin MRS solids
On plate, 28 DEG C of stationary cultures.
2、cps2E、cpsThe screening and verifying of 4E gene knock-out bacterial strain
2~3 single colonies are randomly selected, are transferred in the MRS fluid nutrient medium containing 5 μ g/mL erythromycin, 28 DEG C of stationary cultures
Until bacterium solution OD600When being 0.2 ~ 0.3, bacterium solution is transferred to 37 DEG C of continuation stationary cultures and is stayed overnight, culture bacterium solution dilution 103~105
It is coated on the MRS solid plate containing 5 μ g/mL erythromycin after times, for 24 hours, picking monoclonal is seeded to 37 DEG C of stationary cultures
1mL contains 37 DEG C of stationary cultures in the MRS fluid nutrient medium of 5 μ g/mL erythromycin and stays overnight, which is seeded to not by 1%
In antibiotic MRS fluid nutrient medium, 28 DEG C of stationary cultures are stayed overnight, then bacterium solution dilution 103~105No antibiosis is coated on after times
The MRS solid plate of element, 37 DEG C of stationary cultures are lined to monoclonal, the smaller single colonie one-to-one correspondence of picking is grown containing 5 μ g/
ML erythromycin and not antibiotic MRS solid plate, 37 DEG C of 24 h of stationary culture, picking are flat in antibiotic MRS agar
The bacterium colony that can not be grown on plate, and can grow on not antibiotic plate carries out bacterium solution PCR verifying.cps2E knocks out strain
Bacterium solution PCR verifying: preceding primer (cps2E-uu-F:5 '-GGGTCTTGGCACAGGTTACGG -3 ') it is located on the homology arm of upstream
It swims on genome, rear primer (cps2E-dd-R:5 '-CAACAGCACGAAACCAATAC-3 ') it is located at downstream homology arm downstream base
Because in group；Wild-type strain PCR fragment is 502bp bigger than knock-out bacterial strain, obtains knock-out bacterial strain YM 4-3- Δcps2E。cps4E strikes
Except strain bacterium solution PCR is verified: preceding primer (cps4E-uu-F:5 '-TATGATTTAGTGCAGCAGGG -3 ') it is located at upstream homology arm
In upstream gene group, rear primer (cps4E-dd-R:5 '-CTTACGAAGAACTTCTAGCC -3 ') it is located under the homology arm of downstream
It swims on genome；Wild-type strain PCR fragment is 421bp bigger than knock-out bacterial strain, obtains single knock-out bacterial strain YM 4-3- Δcps4E；It should
Screening technique improves the efficiency of sifting out of knock-out bacterial strain, the result is shown in Figure 1.
Embodiment 4: double knock-out bacterial strain buildings
Referring to the method for step 1 in embodiment 3, preparationcps4E gene list knock-out bacterial strain YM 4-3 Δcps4E competence, and will
Knockout carrier pFED760- Δ cps2E point is transferred to YM 4-3- ΔcpsIn 4E competent cell, it is coated with after 28 DEG C of cultures, centrifugation
In containing on 5 μ g/mL erythromycin MRS solid plates, then at 28 DEG C of stationary cultures；Referring to step 2 in embodiment 3 forcps2E
The screening technique that gene list knocks out filters out knockout, to obtaincps2E、cpsThe bis- knock-out bacterial strain YM 4-3- Δs of 4Ecps2E-
4E。
Embodiment 5: knock-out bacterial strain yield of extracellular polysaccharide detection
1, the extraction of exocellular polysaccharide
Activate wild-type plant lactobacillus strain YM 4-3 and knockout type lactobacillus plantarum strain YM 4-3 Δcps2E、YM 4-
3Δcps4E and YM 4-3- Δcps2E-4E is inoculated in MRS fluid nutrient medium respectively according to 2% inoculum concentration, 37 DEG C of cultures
24h；12000 rpm/min are centrifuged 10min, take supernatant, and 4% trichloroacetic acid (TCA) is added and removes removing protein, and rotor is added in magnetic
30min is stirred on power blender；12000rpm/min is centrifuged 10min, takes supernatant, 3 times of volume dehydrated alcohols, -20 DEG C of items are added
Precipitates overnight under part；12000rpm/min is centrifuged 15min, removes supernatant, and precipitating is dissolved with appropriate distilled water, is packed into pre-treatment
It dialyses in bag filter, it is primary that every 12h changes water, dialyses 2 days；Gained dialyzate in bag filter is dispensed, -80 DEG C of sufficiently freezings
Afterwards, vacuum freeze drying obtains EPS.
2, yield of extracellular polysaccharide detects
Phend-sulphuric acid measurement referring to Kanmani Paulraj et al. (Bioresour Technol, 2011) is extracellular more
Sugared content, wild-type plant lactobacillus strain YM 4-3 exocellular polysaccharide content is 122.642 ± 0.781 to Fig. 2 as the result is shown
Mg/L, and single knockout type bacterial strain YM 4-3- Δ cps2E exocellular polysaccharide content is 61.49 ± 1.41 mg/L, single knock-out bacterial strain
YM 4-3- Δ cps4E exocellular polysaccharide content is 110.22 ± 2.42 mg/L, double knock-out bacterial strain YM 4-3- Δ cps 2E-4E
Exocellular polysaccharide polyoses content only has 17.72 ± 1.35 mg/L；It is lower than wild-type strain, and imitated under the conditions of double knockouts
Fruit becomes apparent from, and thus proves Priming Glycosyltransferase Gene Involvedcps2E、cps4E is extracellular more in lactobacillus plantarum YM 4-3 bacterial strain
It plays a key effect in sugared synthesis process, and the synthesis of the possible coordinated regulation exocellular polysaccharide of the two.Therefore, subsequent to pass through
The mode of amount expression guidance glycosyl transferase improves its yield of extracellular polysaccharide, is plant cream bar to achieve the purpose that expanding production
The application of bacterium YM 4-3 bacterial strain industrially is provided fundamental basis.
Sequence table
<110>Kunming University of Science and Technology
<120>purposes of Priming Glycosyltransferase Gene Involved
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 678
<212> DNA
<213>lactobacillus plantarum YM 4-3 (Lactobacillus plantarum YM 4-3)
<400> 1
gtgaagcaag tcgaaattaa tgaaacagtt aaagttggtc atgtacaacc acaaacggat 60
tatcgttttc caactatgat tggaaagcca gttacaggct ggaaactatt tgtaaaacgg 120
atttttgacc ttactgttgg gttaatcggg acggttttga gttcgccaat tgtcctagtg 180
tttgccattc ttgtaaaact gacttctaaa ggaccagcat tttataagca agaacgggtt 240
ggcctaatgg gaaagagttt caatgtaatt aagctgcgat ctatgtatca ggatgccgaa 300
gcgcgaacag gtgcggtttg ggctcaaaag aatgatccgc gaattacgcc cattggtcgg 360
tttatgcgta aaactcgagt agacgaacta ccgcaatttt ggaatgtgtt aaaaggtgat 420
atgagtttgg taggaccgcg accagaacgg ccagtgctga cagaggagtt tagtcggcaa 480
ttttcagatt tccctaaacg gttacgtatt attcctggta ttacagggta tgcacaaatt 540
aatggtggct acgatattac gccagatgag aaatgtaagc tggataatta ctatattgaa 600
catttttcgg tttggttcga tattaagatg ttattaggaa cagtcaaaat tgtgtttacc 660
ggagatggag cccgatga 678
<210> 2
<211> 672
<212> DNA
<213>lactobacillus plantarum YM 4-3 (Lactobacillus plantarum YM 4-3)
<400> 2
gtgagcatgc acgaagttga gattaatgaa tcaatcgtgg ctgacgaaca acactgtgaa 60
tttattagca aaattggtgt tccggtcacc ggctggaaga tggatctaaa gcggtgtttc 120
gatctagcag tcggcttatt gggcacactg ctcagcgttc ccatcgtaat tgtgtttgcc 180
atacttatca agttgacttc tgacggtccc gttttctaca agcaggagcg agtcggttgg 240
atggggacga cctttgacgt gattaagctg cgatcaatgt atcaggacgc tgaggcgcag 300
acgggtgcga tctgggcaca aaaaaatgat cctcgggtga cgccggttgg acgctggatg 360
cgcaaaactc ggatcgatga gttaccacaa ttttggaatg ttttgaaggg cgatatgagt 420
ttggtcggac ctcgcccgga acggccagaa ctaaccgaac aattcagtga acgctatcct 480
gattttccga aacggctacg aatcattcct ggcattaccg gctatgcgca gatcaatggt 540
ggctatgata ttacaccggg tgccaagtgt cagtacgata actattacat tgagcacttt 600
tcgatttggt ttgatatcaa gatgctgatg ggaacggtta gggtgattat ttccggggat 660
ggtgcgcggt ag 672
<210> 3
<211> 2616
<212> DNA
<213>artificial sequence (Artificial)
<400> 3
tgaacagatc gatactggtg agattgtgat gcctagaaaa cagcgaaagc actggttgtt 60
ttaaaagaag gagcgagtta aatgaaagca ttagtaactg gaggagcggg atttattggt 120
tcccaccttg tggatcactt ggtctcagaa ggtttggacg ttgtcgtagt tgataacttg 180
tccatggggg acttacataa tattaagtat caggatgaag tcactattta tgttgaagat 240
gtccgcaacg aaaaattcat gcaacaattg ctacaggaag aacagcctga ttatatttac 300
tttttagcag ctgttgctag tgttgccgat tcgattgaac gtccagctga aacccattca 360
gtgaatcaaa ctgcagtatt caatatgttg gaatatattc ggaagactaa tttaccaatt 420
aaacagttcc tatttacgtc ttcggcagct gtttacggta atcttccaga attgcctaag 480
aaagaagact cacgagtcga tccattatca ccatatgcga ttgataagta tgcgacggaa 540
cgttttgtac tagcatatgg tgaactttat gatttaccaa ctgtctgtgt gcgcttcttc 600
aacgtttatg gtcccggtca aaaccccagc tcgccatatt caggggtact gtcgattttg 660
accgattgtc tcaataataa gaagccattt acattgtacg gagatgggag tcagacacga 720
gattttgtgt atgttgaaga tgttatccaa gcattatggc tgataactaa gagtaatacg 780
gagcatgaag tctttaatat tgccaatggt aatgaaacca gtttaagtgc cattatcgaa 840
acgtacgaga aagttgcggg gacttcatta caggttaaga aagcgccggg tcgtgaaggg 900
gaagttaagc gttcggtggc taacattggt aaattaatta agttgggata cacgacgagg 960
tggtcgttag aagcgggttt gagcaaatat tgggaagggg cgcaacgacg tgaagcaagt 1020
cgaaattaat gaaacagtta aagttggtca tgtacaacca caaacggatt atcgttttcc 1080
aactatgatt ggaaagccag ttacaggctg gaaactattt gtaaaacgga tttttgacct 1140
tactgttggg ttaatcggga cggttttgag ttcgccaatt gtcctagtgt ttgccattct 1200
tgtaaaactg acttctaaag gaccagcatt ttataagcaa gaacgggttg gcctaatggg 1260
aaagagtttc aatgtaatta agctgcgatc tatgtatcag gatgccgaag cgcgaacagg 1320
tgcggtttgg gctcaaaaga atgatccgcg aattacgccc attggtcggt ttatgcgtaa 1380
aactcgagta gacgaactac cgcaattttg gaatgtgtta aaaggtgata tgagtttggt 1440
aggaccgcga ccagaacggc cagtgctgac agaggagttt agtcggcaat tttcagattt 1500
ccctaaacgg ttacgtatta ttcctggtat tacagggtat gcacaaatta atggtggcta 1560
cgatattacg ccagatgaga aatgtaagct ggataattac tatattgaac atttttcggt 1620
ttggttcgat attaagatgt tattaggaac agtcaaaatt gtgtttaccg gagatggagc 1680
ccgatgaatg gggtgtatta atacatgaac aggatagaag tttcaattat attgcctgta 1740
tataatagcc aaaaattcat tcgtgagaca attaattccg ttttgagtca atcatttaaa 1800
ctatttgaat tgctcattat caacgatgca tcaactgatg atactgaaaa aatcattcaa 1860
tcgtataacg atccacgaat tgtttataag aaattttcaa ggaatcgtgg cgttgcaaat 1920
gctcgtaatt atggtattaa tctggctgag ggtgaatttg ttgcatttat tgatagcgat 1980
gacatctgga atagcgataa gttacaacaa caattgcaag aaatgcaggc aaaatgcata 2040
aattttagtt attcaaatta cgagcttgta aatgaatctg ggagaacaat caagtatatt 2100
gaaaatttac ctaaatggaa cacgtataaa agcttattaa tgaccaatag cattccgttg 2160
ctaactgtaa ttataaaaaa aaatctgatt aagggtaatc agtttataaa tattcgtcat 2220
gaggattatg ctacatggtt acaaatacta agaaagtctg atgaaaaggc gtggcttttt 2280
ccagaaataa ccgctaaata tcgagtaagg gatgattcga taagcagtaa taagttgaaa 2340
tcattaagtt ggacatggaa cgtttatcga aacagtgaac acttatcact cattaagtcg 2400
gcttattatt tgggatgcaa tgcattacat ggacttttaa agcatcaaca gattttcaag 2460
agcgtataaa tgaaagacaa tatccaaagg agtggttgag gttgaaagtt agcttcccaa 2520
ttgattttgt tgtgacttgg gtcaatagta atgatactat ttggcaacaa aagaaagaac 2580
attacgaatt gagcgaaaat gagagtcgca aaaatg 2616
<210> 4
<211> 2347
<212> DNA
<213>artificial sequence (Artificial)
<400> 4
tggctttgag gtggtggtag acgatctgtc aatgggggcc atcagcaata tcaagcattg 60
ggaacagatt acgatatacg tcgcggatgt ctgtgacgat aaattcatgc aacaattgtt 120
agcagacgaa cggccagact atatttattt tctggcagcg attgcgagtg tggcggattc 180
gattgagcgg ccggcagaaa cgcatgctgt caatcatacc gcggtattta acttgctcga 240
acatattcgc caaattcgct tgcctattaa gcaatttcta ttcacttctt cggcggcggt 300
ttatggcaat ctacctgaat taccaaaacg cgaggattcg cgcgtcgcac ccgtgtcacc 360
gtatgctatt gataagtatg cgactgaacg ttttgtattg gcatacggtg agttgtacga 420
tttgccgacc gtttgtgtgc ggttcttcaa cgtttatgga ccgcgtcaga atccaagctc 480
accgtattcg ggggttttgt cgattttgac ggattgtttg aaaacacagc gaccatttac 540
attattcggt gatggaacgc agacccgtga ttttgtatat gtaagtgatg ttatcaaggc 600
attatggttg attacagaac atcaagtgca acatgaagtg atcaatattg ccaatggctt 660
agaaactagt ttaaatggca ttattcagat gtatcaagag attgctggtc aacaacttga 720
aatcaaacgg gccgagcagc ggggtggtga agttgatcat tcagttgcaa gtattggcaa 780
gttggcacgc ttaaattatg aatcagagtg gccgttgaga agaggattaa ctaagtactg 840
ggaaggggaa tgtgagcatg cacgaagttg agattaatga atcaatcgtg gctgacgaac 900
aacactgtga atttattagc aaaattggtg ttccggtcac cggctggaag atggatctaa 960
agcggtgttt cgatctagca gtcggcttat tgggcacact gctcagcgtt cccatcgtaa 1020
ttgtgtttgc catacttatc aagttgactt ctgacggtcc cgttttctac aagcaggagc 1080
gagtcggttg gatggggacg acctttgacg tgattaagct gcgatcaatg tatcaggacg 1140
ctgaggcgca gacgggtgcg atctgggcac aaaaaaatga tcctcgggtg acgccggttg 1200
gacgctggat gcgcaaaact cggatcgatg agttaccaca attttggaat gttttgaagg 1260
gcgatatgag tttggtcgga cctcgcccgg aacggccaga actaaccgaa caattcagtg 1320
aacgctatcc tgattttccg aaacggctac gaatcattcc tggcattacc ggctatgcgc 1380
agatcaatgg tggctatgat attacaccgg gtgccaagtg tcagtacgat aactattaca 1440
ttgagcactt ttcgatttgg tttgatatca agatgctgat gggaacggtt agggtgatta 1500
tttccgggga tgtggcgcgg tagtgaagat tgtttacatc attactcaag cgacttgggg 1560
tggggcccag gcgcatctat atagtttgat caaagcgcaa gtgatgcgtg gcaatgccgt 1620
tgccttagta tacggcgttg aaggacgcct gagtgcaagc gtcgcgaaag aatttcaaga 1680
cgtgcaagtt gtcagagttg ccagcctggt acatccgatt gcaccgctga gtgatttgaa 1740
agcaatctac acgttaagga aattagtaaa aaattggcag ccagatatta ttcatttgca 1800
ttcttcgaag gctggtatga ttgggattgt cggcgatggt agcttgagcg cacacatcgc 1860
taaacagatt gcagccaccc cgcaaatacg gtggttaggg tttaaaagta acccttacaa 1920
atacatgcgg catgcaaaag ttgtgctgtc gacgtccaaa tcagacgcgt ttggattgac 1980
catggtcgag gcagccttat tgggtgcgat tccgtttgcc ccccgcattg ggggcatctc 2040
tcaaacggcc gcaagggtaa atggcctggt ttatgcgagt gatgctgaat tattggcagt 2100
actgacacgt ctcttttcgg atgggaaatt ttatcaggaa accaaggcta aaatagcagc 2160
ggtcgatttt agtgactatg aacaaaggca attcattcag cggatccagc aagtttataa 2220
aggagtaatg gaacgatgac tgcagtaaat cgtcagattg gtgggccatt cttaagacta 2280
ggactattga ttgccggatt ttacctgatt taccaaccca acttttggag cagtagttat 2340
gtgcccc 2347
<210> 5
<211> 29
<212> DNA
<213>artificial sequence (Artificial)
<400> 5
ccggaattct gaacagatcg atactggtg 29
<210> 6
<211> 37
<212> DNA
<213>artificial sequence (Artificial)
<400> 6
acatttctca tctggcgcgt ttgtggttgt acatgac 37
<210> 7
<211> 37
<212> DNA
<213>artificial sequence (Artificial)
<400> 7
gtcatgtaca accacaaacg cgccagatga gaaatgt 37
<210> 8
<211> 26
<212> DNA
<213>artificial sequence (Artificial)
<400> 8
ccctcgagca tttttgcgac tctcat 26
<210> 9
<211> 32
<212> DNA
<213>artificial sequence (Artificial)
<400> 9
ccggaattcg ggttcattgg ctcgcacttg gt 32
<210> 10
<211> 40
<212> DNA
<213>artificial sequence (Artificial)
<400> 10
cgtagccgtt tcggaaaatc tcacagtgtt gttcgtcagc 40
<210> 11
<211> 40
<212> DNA
<213>artificial sequence (Artificial)
<400> 11
gctgacgaac aacactgtga gattttccga aacggctacg 40
<210> 12
<211> 31
<212> DNA
<213>artificial sequence (Artificial)
<400> 12
cccaagcttg ggcacataac tactgctcca a 31
<210> 13
<211> 21
<212> DNA
<213>artificial sequence (Artificial)
<400> 13
gggtcttggc acaggttacg g 21
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 14
caacagcacg aaaccaatac 20
<210> 15
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 15
tatgatttag tgcagcaggg 20
<210> 16
<211> 20
<212> DNA
<213>artificial sequence (Artificial)
<400> 16
cttacgaaga acttctagcc 20
Claims (1)
1. Priming Glycosyltransferase Gene Involvedcps2E、cps4E improve lactobacillus plantarum (Lactobacillus plantarum)
Application in transposon mutagenesis, the genecpsThe nucleotide sequence of 2E is as shown in SEQ ID NO:1, genecpsThe core of 4E
Nucleotide sequence is as shown in SEQ ID NO:2.
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| CN112961878A (en) * | 2021-03-08 | 2021-06-15 | 昆明理工大学 | Application of gene of lactobacillus plantarum in folic acid biological generation |
| CN113832167A (en) * | 2021-11-01 | 2021-12-24 | 昆明理工大学 | Gene and application thereof in improving yield of phenethyl alcohol and tryptophol |
| CN114634938A (en) * | 2022-03-06 | 2022-06-17 | 昆明理工大学 | Application of lactobacillus plantarum gene fol KE in folic acid biosynthesis |
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| CN112280795A (en) * | 2020-11-17 | 2021-01-29 | 昆明理工大学 | Use of glycosyltransferase genes |
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| CN112852844A (en) * | 2021-03-05 | 2021-05-28 | 昆明理工大学 | Application of hydroxymethyl dihydropterin pyrophosphokinase gene folK |
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| CN114634938A (en) * | 2022-03-06 | 2022-06-17 | 昆明理工大学 | Application of lactobacillus plantarum gene fol KE in folic acid biosynthesis |
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