CN109593696A - One plant height produces Leuconostoc mesenteroides mutant strain and its application method of mannitol - Google Patents

One plant height produces Leuconostoc mesenteroides mutant strain and its application method of mannitol Download PDF

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CN109593696A
CN109593696A CN201811545503.1A CN201811545503A CN109593696A CN 109593696 A CN109593696 A CN 109593696A CN 201811545503 A CN201811545503 A CN 201811545503A CN 109593696 A CN109593696 A CN 109593696A
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mdh
mannitol
leuconostoc mesenteroides
gene
dts1
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CN109593696B (en
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金红星
闫博
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Tianjin Brave Biopharma Technology Co ltd
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Abstract

A plant height of the invention produces alcohol Leuconostoc mesenteroides mutant strain and its application method of sweet dew, it is related to bacterium, the bacterial strain is Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp) [Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterial strain, it is in China typical culture collection center (CCTCC) preservation, preservation date is on November 23rd, 2018, and deposit number is CCTCC No:M2018815.The bacterial strain is transferred in MRS culture medium with weight percent 1%, at 30 DEG C, with shaking table culture 20 hours that revolving speed is 120 revs/min, mannitol concentration can achieve 47.3 grams per liters, the conversion ratio 52.6% of sucrose to mannitol.

Description

One plant height produces Leuconostoc mesenteroides mutant strain and its application method of mannitol
Technical field
Technical solution of the present invention is related to bacterium, and a specifically plant height produces the Leuconostoc mesenteroides mutant bacteria of mannitol Strain and its application method.
Background technique
Mannitol (Mannitol) is a kind of hexitol, is obtained extensively in field of medicaments, field of food and plastic applications Application.
Currently, industrial production mannitol mainly has two kinds of techniques in the world.The first is seaweed extraction method: 1 ton of extraction is sweet Dew alcohol about needs 13~15 tons of dry kelps, while producing alginate, will mention the kelp-soaking liquid after iodine, is repeatedly extracted dense Contracting, remove impurity, ion exchange, evaporation and concentration, crystallisation by cooling and obtain；Production process generates a large amount of waste water, and energy consumption is high, and pollution is tight Weight, yield are low.Second is catalytic hydrogenation method: using sucrose or glucose as raw material, by hydrolysis, epimerism and enzyme isomery, Right back end hydrogenation and obtain；Raw material sources are stablized, and product term is unrestricted, at low cost, but its yield is lower, and has sorbierite companion It is raw.
There are also two kinds for the method for Laboratory Production mannitol.First is that enzyme transforming process, enzyme process hydrogenation need be added in system Expensive coenzyme, it is uneconomical.Second is that microbe fermentation method, the microbe species that mannitol can be synthesized in nature are more, There are some bacterial strains that there is the ability for producing mannitol in bacterium, yeast and mould.During lactic acid bacteria converts mannitol, Mannitol is primary product, while lactic acid producing, acetic acid, ethyl alcohol and carbon dioxide, without generating the by-products such as other polyalcohols, because And it is easy to purifies and separates and purification, and mild condition, conversion ratio are higher.
Many bacterial strains generate mannitol by fermenting substrate of fructose, and fructose and sucrose all can serve as bottom by leukonid Object generates mannitol.Cheap sucrose enter leukonid it is intracellular after, resolve into Cori's eater Cori and fructose, fructose converts again For mannitol, reaction step is relatively smaller；And glucose is through glucose 6-phosphate, 6- phosphoric acid fruit in the lactobacillus of homofermentative lactic The intermediate products such as sugar and 1- phosphomamlose alcohol are eventually converted into mannitol, and reaction step is relatively more；The chromosome base of leukonid Because group only has 2M or so, therefore fermentation period only has 20 hours or so；Leukonid is oxytolerant, therefore does not need to mention in fermentation process Oxygen；Therefore leukonid realizes that the potentiality of large-scale industrial production mannitol are bigger.
CN201711169481.9 discloses Leuconostoc mesenteroides mutant strain and its application side that a plant height produces mannitol Method, the leukonid mutant strain are dextransucrase and D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene It knocks out and knocks in mannitol dehydrogenase gene, serine/threonine protein kitase gene knockout and knock in mannitol dehydrogenase base Cause, fructokinase gene knockout and the Leuconostoc mesenteroides mutant strain for knocking in mannitol dehydrogenase gene, although comparing original bacteria Strain improves yield, but relatively low, is not enough to be applied in production.
In short, in existing leukonid fermentation technique, it is still not high enough as the yield of substrate production mannitol using sucrose, also need It further increases.
Summary of the invention
The technical problems to be solved by the present invention are: provide a plant height produce mannitol Leuconostoc mesenteroides mutant strain and Its application method, the Leuconostoc mesenteroides mutant strain are the bright strings of goldbeater's skin for being CCTCC M 2017578 with existing deposit number Pearl bacterium Δ dts1 Δ D-ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh (Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh) bacterial strain (goldbeater's skin of mono- plant height of CN201711169481.9 production mannitol Leukonid mutant strain and its application method) it is bacterium germination, acetaldehyde dehydrogenase is knocked out using Protocols in Molecular Biology and encodes base Cause simultaneously knocks in 1- phosphomamlose alcohol dehydrogenase encoding gene and Mannitol-1-phosphatase encoding gene, is configured to glucansucrase Enzyme gene knockout, D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene knockout and mannitol dehydrogenase gene knock-in, Serine/threonine protein kitase gene knockout and mannitol dehydrogenase gene knock-in, fructokinase gene knockout and mannitol Dehydrogenase gene is knocked in and is knocked out acetaldehyde-dehydrogenase enzyme coding gene and knocks in 1- phosphomamlose alcohol dehydrogenase encoding gene and sweet dew The Leuconostoc mesenteroides mutant strain of alcohol -1- phosphoric acid enzyme coding gene, i.e. deposit number are the bright strings of goldbeater's skin of CCTCC M 2018815 Pearl bacterium △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp) [Leuconostoc Mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterium Strain, overcome in existing leukonid fermentation technique be using sucrose substrate production mannitol the still not high enough defect of yield.
The present invention solves technical solution used by the technical problem: the Leuconostoc mesenteroides that a plant height produces mannitol is prominent Become bacterial strain, be glucansucrase gene knockouts, D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene knockout and it is sweet Dew alcohol dehydrogenase gene is knocked in, serine/threonine protein kitase gene knockout and mannitol dehydrogenase gene knock-in, fructose swash Enzyme gene, which knocks out simultaneously mannitol dehydrogenase gene knock-in and knocks out acetaldehyde-dehydrogenase enzyme coding gene and knock in 1- phosphomamlose alcohol, to be taken off The Leuconostoc mesenteroides mutant strain of hydrogen enzyme coding gene and Mannitol-1-phosphatase encoding gene is Leuconostoc mesenteroides △ dts1△ ldh△pat::mdh△stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc Mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterium Strain, in China typical culture collection center (CCTCC) preservation, preservation date is on November 23rd, 2018, and deposit number is CCTCC No:M2018815, depositary institution address are the Wuhan Wuhan University, China.
One plant height produces the application method of the Leuconostoc mesenteroides mutant strain of mannitol, will be in 250 milliliters of triangular flasks China typical culture collection center (CCTCC) preservation, preservation date are on November 23rd, 2018, and deposit number is CCTCC No: The Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh: of M2018815: (mtld-mlp)[Leuconostoc mesenteroides△dts1△ldh△pat::mdh△stpk::mdh△ fk:: Mdh △ aldh::(mtld-mlp)] bacterial strain is transferred in MRS culture medium with weight percent 1%, at 30 DEG C, be with revolving speed 120 revs/min of shaking table culture 20 hours, mannitol concentration can achieve 47.3 grams per liters, and the conversion ratio of sucrose to mannitol is 52.6%.
An above-mentioned plant height produces the application method of the Leuconostoc mesenteroides mutant strain of mannitol, and the MRS culture medium is matched Method processed is: by 2 grams of yeast extract, 90 grams of sucrose, 2 grams of ammonium citrate, 5 grams of sodium acetate, K2HPO42 grams, MnSO4·H2O 0.039 gram and 1000 milliliters of water is sterilized 20 minutes at a temperature of 121 DEG C with acetic acid tune pH to 6.2 and prepares to obtain MRS culture medium.
An above-mentioned plant height produces the application method of the Leuconostoc mesenteroides mutant strain of mannitol, related raw material, reagent With instrument by commercially available, related operating procedure is that those skilled in the art will appreciate that.
The beneficial effects of the present invention are: compared with prior art, the present invention has following substantive distinguishing features outstanding and shows Write progress:
(1) present invention using Protocols in Molecular Biology knock out existing deposit number be CCTCC M 2017578 goldbeater's skin it is bright Beading bacterium Δ dts1 Δ D-ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh (Leuconostoc mesenteroides Δ Dts1 Δ D-ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh) acetaldehyde-dehydrogenase enzyme coding gene in bacterial strain and knock in 1- phosphorus Sour mannitol dehydrogenase encoding gene and Mannitol-1-phosphatase encoding gene are configured to glucansucrase gene knockout, D- Lactic acid dehydrogenase gene knocks out, acetyl phosphate transferase gene knocks out and mannitol dehydrogenase gene knock-in, serine/threonine Protein kinase gene knocks out and mannitol dehydrogenase gene knock-in, fructokinase gene knockout and mannitol dehydrogenase gene knock-in With knock out acetaldehyde dehydrogenase gene and knock in 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase gene goldbeater's skin it is bright Beading bacterium mutant strain, i.e. deposit number are the Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh of CCTCC No:M2018815 △stpk::mdh△fk::mdh △aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterial strain, overcome existing leukonid hair The still not high enough defect of yield of mannitol is produced in ferment technology using sucrose as substrate.
(2) it will be on November 23rd, 2018 in China typical culture collection center (CCTCC) preservation, preservation date, protect Hiding number is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ of CCTCC M2018815 aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ldh△pat::mdh△stpk::mdh△ Fk::mdh △ aldh::(mtld-mlp)] bacterial strain is transferred in MRS culture medium with weight percent 1%, at 30 DEG C, to turn Speed is shaking table culture 20 hours of 120 revs/min, detects metabolite, is proved by comparative test, leukonid mutation The mannitol yield of bacterial strain is the Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ of CCTCCM2017578 than initial deposit number stpk-mdhΔfk-mdh Δpat-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔstpk-mdhΔ Fk-mdh Δ pat-mdh) 15.4% is improved, the conversion ratio of sucrose to mannitol improves 7.0%.
Detailed description of the invention
Fig. 1 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] the building acetaldehyde dehydrogenase gene of bacterial strain The agarose gel electrophoresis figure of left and right homology arm in homologous recombination vector；
Fig. 2 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] the building acetaldehyde dehydrogenase gene of bacterial strain The digestion verification agarose gel electrophoresis figure of homologous recombination vector homology arm；
Fig. 3 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] alphalise starch is had among the building of bacterial strain The digestion verification agarose gel electrophoresis figure of the acetaldehyde dehydrogenase gene homologous recombination vector of enzyme gene label；
Fig. 4 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] 1- phosphoric acid is had among the building of bacterial strain The digestion verification fine jade of the acetaldehyde dehydrogenase gene homologous recombination vector of mannitol dehydrogenase gene and Mannitol-1-phosphatase gene Sepharose electrophoretogram；
Fig. 5 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterial strain to verify acetaldehyde by PCR de- Hydrogenase gene knocks out and the agarose of 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase gene knock-in mutant strain Gel electrophoresis figure.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] the building acetaldehyde dehydrogenase gene of bacterial strain The agarose gel electrophoresis figure of left and right homology arm in homologous recombination vector.Left and right homology arm: 1. left homology arms is shown in figure, 2.Marker, 3. right homology arms.
Fig. 2 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] the building acetaldehyde dehydrogenase gene of bacterial strain The digestion verification agarose gel electrophoresis figure of homologous recombination vector homology arm.Two rules that recombinant vector digestion generates are shown in figure Band: 1.Marker, 2. recombinant vector double digestion bands.
Fig. 3 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] alphalise starch is had among the building of bacterial strain The digestion verification agarose gel electrophoresis figure of the acetaldehyde dehydrogenase gene homologous recombination vector of enzyme gene label.Recombination is shown in figure Two band that carrier digestion generates: 1.Marker, 2. recombinant vector double digestion bands.
Fig. 4 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] 1- phosphoric acid is had among the building of bacterial strain The digestion verification fine jade of the acetaldehyde dehydrogenase gene homologous recombination vector of mannitol dehydrogenase gene and Mannitol-1-phosphatase gene Sepharose electrophoretogram.Two band that recombinant vector digestion generates: 1.Marker, 2. recombinant vector double digestion items are shown in figure Band.
Fig. 5 is the Leuconostoc mesenteroides △ dts1 △ ldh △ pat: that deposit number of the present invention is CCTCC No:M2018815: mdh△ stpk::mdh△fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ Ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterial strain to verify acetaldehyde by PCR de- Hydrogenase gene knocks out and the agarose of 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase gene knock-in mutant strain Gel electrophoresis figure.Show in figure: 1. with Leuconostoc mesenteroides △ dts1 △ ldh △ pat-mdh △ stpk-mdh △ fk-mdh bacterium Strain is template, 2. Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::amy For template, 3.Marker, 4. Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ Aldh::(mtld-mlp) bacterial strain is template.
Embodiment 1
It constructs acetaldehyde dehydrogenase gene and knocks out simultaneously 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase clpp gene The Leuconostoc mesenteroides mutant strain entered, the specific steps are as follows:
It is the Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat-mdh Δ of CCTCC M 2017578 with existing deposit number stpk-mdh Δfk-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔpat-mdhΔstpk-mdhΔ Fk-mdh) to go out bacterium germination, building acetaldehyde dehydrogenase gene knocks out and 1- phosphomamlose alcohol dehydrogenase gene and mannitol -1- phosphoric acid The Leuconostoc mesenteroides mutant strain that enzyme gene is knocked in:
The first step, the clone of Leuconostoc mesenteroides acetaldehyde dehydrogenase gene partial sequence:
Using chromosomal DNA as template, clone coding sequences length is the Leuconostoc mesenteroides Δ dts1 Δ D-ldh of 1524bp [preservation date is on October 25th, 2017 to Δ pat-mdh Δ stpk-mdh Δ fk-mdh, in China typical culture collection The heart (CCTCC) preservation, deposit number are CCTCC M2017578] (Leuconostoc mesenteroides Δ dts1 Δ D- Ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh) acetaldehyde dehydrogenase gene part continuous sequence, concrete operation step is:
(1.1) deposit number is the Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat-mdh Δ stpk- of CCTCCM2017578 mdh Δfk-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔpat-mdhΔstpk-mdhΔfk- Mdh the extraction of Leuconostoc mesenteroides chromosomal DNA):
The Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat- for being CCTCCM2017578 by the deposit number frozen at -80 DEG C mdh Δstpk-mdhΔfk-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔpat-mdhΔstpk- Mdh Δ fk-mdh) bacterial strain lines on MRS solid plate, is incubated overnight in 30 DEG C；One single bacterium of picking from solid plate It falls and is inoculated into 5 milliliters of MRS fluid nutrient mediums, in 30 DEG C, the shaking table culture that revolving speed is 120 revs/min is stayed overnight；Take 2 milliliters The bacterium solution of above-mentioned culture is 10000 revs/min with revolving speed and is centrifuged 2 minutes, collects thallus；With 1 milliliter of distilled water washing thalline two It is secondary；Thallus is dissolved in 100 microlitres of distilled water, piping and druming mixes；100 microlitres of concentration is added as the bacteriolyze of 100 mg/mls Enzyme, 37 DEG C of water-bath 1h；500 microlitres of extracting solutions are added, mix gently；In 80 DEG C be incubated for after ten minutes, with 14000 revs/min from The heart 10 minutes, abandon supernatant；Add 100 microlitres of suspension, dissolves DNA；Isometric i.e. 100 microlitres of phenol-chloroform is added, gently It shakes up, is put into 4 DEG C of refrigerators and stands 15 minutes, then 4 DEG C, 12500 revs/min are centrifuged 15 minutes, and supernatant is extracted to new Centrifuge tube in；Repeat a phenol-chloroform extraction procedure；The pre-cooling dehydrated alcohol of i.e. 200 microlitres of 2 times of volumes is added, in 4 2h is stood in DEG C refrigerator；12000 revs/min are centrifuged 20 minutes, outwell supernatant；It is cleaned with the ethyl alcohol that percent by volume is 70% 1 time, 12000 revs/min are centrifuged 10 minutes, outwell supernatant, dry；Precipitating is dissolved in 20 microlitres of TE (Tris-HCl 100 MM/l, 10 mM/ls of EDTA, pH 8.0) in.
The composition of above-mentioned MRS culture medium: 3 grams of yeast extract, 10 grams of peptone, 8 grams of beef extract powder, 20 grams of glucose, lemon 2 grams of lemon acid ammonium, 5 grams of sodium acetate, K2HPO42 grams, MgSO4·7H22 grams of O, MnSO4·H20.039 gram of O, 1.6 milli of Tween 80 Rise and 1000 milliliters of water, with acetic acid tune pH to 6.2；121 DEG C of sterilizing 20min.Solid medium adds 1.5% agar.
The composition of said extracted liquid: 240 mM/ls of NaOH, 2.7 mM/ls of EDTA, 74% ethyl alcohol.
The composition of above-mentioned suspension: 0.1 mM/l of EDTA, 50 mM/ls of Tris-HCl, 1% TritonX-100 (pH8.0), 0.5% polysorbas20.
Above-mentioned phenol-chloroform solution is with phenol: chloroform: isoamyl alcohol volume ratio is the solution that 25:24:1 is configured to.
Above-mentioned TE solution is with 100 mM/ls and 10 mM/ls of EDTA preparations of Tris-HCl, pH 8.0.
(1.2) PCR amplification acetaldehyde dehydrogenase gene:
Design pair of primers aldhl:5'-ACTTTGCGAATGAATAATG-3' and aldhr:5'- TCGTGTAACCAATGATAAC -3' is the Leuconostoc mesenteroides Δ dts1 Δ D-ldh of CCTCCM2017578 with deposit number Δpat-mdhΔstpk-mdh Δfk-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔpat-mdh Δ stpk-mdh Δ fk-mdh) Leuconostoc mesenteroides chromosomal DNA be template, PCR amplification obtains the segment for 1524bp, And PCR product is connected on pTA2T carrier with T4 ligase, recombinant plasmid is named as pTA2-aldh.
(1.3) preparation of competent E.coli DH5 α and DNA conversion:
The e.colistraindh5α frozen at -80 DEG C is lined on LB solid plate, 37 DEG C are incubated overnight；From solid One single colonie of picking is inoculated into 5 milliliters of LB liquid mediums on plate, in 37 DEG C with revolving speed be 150 revs/min of shaking table mistakes Night culture；The bacterium solution for taking 0.2 milliliter of above-mentioned culture to obtain is transferred in 10 milliliters of liquid culture mediums, in 37 DEG C with revolving speed be 150 Rev/min 2~3h of shaken cultivation to bacterium solution OD600It is 0.6；Take above-mentioned OD6001.0 milliliters of bacterium solution for 0.6 are added to 1.5 millis It rises in centrifuge tube, ice bath 10 minutes；It is 10000 revs/min in 4 DEG C with revolving speed to be centrifuged 30 seconds, abandons supernatant；1 milliliter of ice is added 0.1 cold mol/L CaCl2Solution suspension cell, ice bath 30 minutes；It is 10000 revs/min in 4 DEG C with revolving speed and is centrifuged 30 Second, abandon supernatant；100 microlitres of 0.1 ice-cold mol/L CaCl are added2Solution suspension cell, as competent cell, namely Competent E.coli DH5 α.
10 microlitres of recombinant plasmid are added in above-mentioned competent cell, ice bath 30 minutes；In 42 DEG C of accurate heat shocks 90 Second；It places 3 minutes on ice immediately；400 microlitres of LB liquid mediums are added, in 37 DEG C shaken cultivation 45 minutes；By conversion Competent cell is spread evenly across in LB solid medium tablets with ampicillin；Plate is placed in 37 DEG C of incubators 30 Minute, until liquid is absorbed；It is inverted plate, in 37 DEG C of 12~16h of culture.
Picking single colonie is cultivated in LB culture medium with ampicillin, plasmid is extracted, by agarose gel electrophoresis It is identified with sequencing.
Above-mentioned LB liquid medium: 5 grams of yeast extract, 10 grams of peptone, 10 grams of NaCl, 1000 milliliters of distilled water, pH 7.0,121 DEG C sterilize 20 minutes.Solid medium adds 1.5% agar.
Second step, the building of acetaldehyde dehydrogenase gene homologous recombination vector of the centre with alpha-amylase label:
(2.1) pair of primers aldhl1:5'-ACAGAATTCGCAGAGATATTAAACA-3' and aldhl2:5'- are designed ACATACTCTAGATATTCACTTGATCGTA-3'(and aldhr1 complementary pairing), using pTA2-aldh as template, PCR amplification Obtain the segment for 453bp.
(2.2) pair of primers aldhr1:5'-TGAATATCTAGAGTATGTACTTCGTCTA-3' and aldhr2 are designed: 5'-TATAAGCTTCTCAGGTAATGTTCCA-3', using pTA2-aldh as template, PCR amplification obtains the segment for 507bp.
(2.3) by the purified rear mixing of 2 PCR products obtained in above-mentioned (2.1) (2.2), it is with PCR product mixture Template makes 2 genetic fragment overlap-extension PCRs by 8 wheel PCR cycles, then utilizes pair of primers aldhl1:5'- ACAGAATTCGCAGAGATATTAAACA-3' and aldhr2:5'-TATAAGCTTCTCAGGTAATGTTCCA-3' is carried out again PCR, amplification obtain the segment for 948bp.
(2.4) Overlap extension PCR product obtained in above-mentioned (2.3) and pUC19 are subjected to double enzymes with EcoRI and Hind III After cutting, the two connects under the action of T4-DNA ligase, then the product of connection is converted bacillus coli DH 5 alpha competent cell, Recombinant plasmid pUC19-aldhqh is screened, that is, is configured to homologous recombination vector.
(2.5) pair of primers amyl:5'-CTATCTAGATTTGGCGTGATTATCAG-3' and amyr:5'- are designed TACTCTAGACGAAGGTGAAGTTATAG-3' is the food starch lactobacillus chromosomal DNA of CGMCC1.3395 with deposit number For template, PCR amplification obtains the segment for 2131bp, and PCR product is connected to homologous recombination vector with T4 ligase On the site XbaI among pUC19-aldhqh homology arm, recombinant plasmid is named as pUC19-aldhqh-amy, that is, is configured to Between with alpha-amylase label acetaldehyde dehydrogenase gene homologous recombination vector.
Third step, glucansucrase gene knockout, D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene strike Except and mannitol dehydrogenase gene knock-in, serine/threonine protein kitase gene knockout and mannitol dehydrogenase gene knock-in, The Leuconostoc mesenteroides mutation of fructokinase gene knockout and mannitol dehydrogenase gene knock-in and acetaldehyde dehydrogenase gene inactivation The building of bacterial strain:
The Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat- for being CCTCCM2017578 by the deposit number frozen at -80 DEG C mdh Δstpk-mdhΔfk-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔpat-mdhΔstpk- Mdh Δ fk-mdh) bacterial strain lines on MRS solid plate, is incubated overnight in 30 DEG C；One single bacterium of picking from solid plate It falls and is inoculated into 5 milliliters of MRS fluid nutrient mediums, stayed overnight in 30 DEG C with revolving speed for 120 revs/min of shaking table cultures；It is transferred to 1% MRS continues to cultivate in the culture medium containing 0.48 mcg/ml ampicillin, initial OD600It is for 0.048 deposit number The Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh of CCTCCM2017578 The OD of (Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh) bacterium solution600 Thallus is collected when reaching 0.5, the LiAc-DTT solution suspension thalline again for being 100U/ milliliters with lysozyme concentration, in 30 DEG C Be incubated for 20 minutes, washed twice with ice-cold PBS solution, then use 50 microlitres of ice-cold PBS solution suspension thallines, addition 5 microlitres Above-mentioned homologous recombination vector plasmid (pUC19-aldhqh-amy), ice bath carry out electrotransformation, electric converter used after ten minutes For Bio-Rad Gene Pulser XCellTM, shock parameters are electric shock cup spacing 0.1cm, 1400V, 25 μ F, 300 Ω, electric shock Time is 4 milliseconds, then 1 milliliter of MRS culture medium of addition, after recovery 3h, is coated on solid plate containing MRS, chooses after cultivating 120h Take single colonie to verify, with prove to screen from plate glucansucrase gene knocks out, D- lactic acid dehydrogenase gene knocks out, Acetyl phosphate transferase gene knock out and mannitol dehydrogenase gene knock-in, serine/threonine protein kitase gene knockout simultaneously Mannitol dehydrogenase gene knock-in, fructokinase gene knockout and the mistake of mannitol dehydrogenase gene knock-in and acetaldehyde dehydrogenase gene Leuconostoc mesenteroides mutant strain living, i.e. Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh△aldh::amy(Leuconostoc mesenteroides△dts1△ldh△pat::mdh △stpk::mdh △ fk::mdh △ aldh::amy) bacterial strain.
Design pair of primers aldhyq:5'-GCAGAGATATTAAACAAAA-3' and aldhyh:5'- TGGTGGAACATTACCTGAG-3' extracts chromosomal DNA, carries out PCR, the above-mentioned bright beading of goldbeater's skin by template of chromosomal DNA Bacterium mutant strain, i.e. Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::amy (Leuconostoc mesenteroides△dts1△ldh△pat::mdh△stpk::mdh△fk::mdh△aldh:: Amy) bacterial strain obtains length as the amplified production of 3079bp, and Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ stpk-mdh Δ fk- mdhΔpat-mdh (Leuconostoc mesenteroidesΔdts1ΔD-ldhΔstpk-mdhΔfk-mdhΔpat- Mdh, deposit number CCTCCM2017578) obtain the amplified production that length is 1125bp.
Above-mentioned LiAc-DTT solution be with 100 mM/ls of LiAc, 10 mM/ls of DTT, 0.6 mol/L sucrose, The solution of 10 mM/ls of Tris-HCl (pH7.5)；
Above-mentioned PBS solution is K2HPO4-KH2PO41 mM/l, MgCl21 mM/l and 0.5 mol/L of sucrose The solution of preparation, pH 6.9.
4th step, glucansucrase gene knockout, D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene strike Except and mannitol dehydrogenase gene knock-in, serine/threonine protein kitase gene knockout and mannitol dehydrogenase gene knock-in, Fructokinase gene knockout and mannitol dehydrogenase gene knock-in and acetaldehyde dehydrogenase gene knock out simultaneously 1- phosphomamlose alcohol dehydrogenase The building of the Leuconostoc mesenteroides mutant strain of enzyme gene and Mannitol-1-phosphatase gene knock-in:
(4.1) clone of 1- phosphomamlose alcohol dehydrogenase gene expression cassette:
(4.1.1) designs pair of primers ldh1:5'-ATAGAATTCAGTGCTTTAATTAGTG-3' and ldh2:5'- TCTAACATAAGATCCTCCAAAATT-3'(and mt1d1 complementary pairing), the bright string of goldbeater's skin for being CGMCC 1.2138 with deposit number Pearl bacterium chromosomal DNA is template, and PCR amplification obtains the segment for 177bp.
(4.1.2) designs pair of primers mt1d1:5'-AGGATCTTATGTTAGACGTACATT-3' and mt1d2:5'- ATGAATTCATCGAACTACTTTGCT-3', using the lactobacillus plantarum chromosomal DNA that deposit number is CGMCC 1.2437 as mould Plate, PCR amplification obtain the segment for 1164bp.
(4.1.3) mixes the purified rear mixing of 2 PCR products obtained in above-mentioned (4.1.1) (4.1.2) with PCR product Conjunction object is template, makes 2 genetic fragment overlap-extension PCRs by 8 wheel PCR cycles, then utilizes pair of primers ldh1:5'- ATAGAATTCAGTGCTTTAATTAGTG-3' and mt1d2:5'-ATGAATTCATCGAACTACTTTGCT-3' carries out PCR again, Amplification obtains the segment for 1341bp.
After Overlap extension PCR product obtained in above-mentioned (4.1.3) and pUC19 are carried out digestion with EcoRI by (4.1.4), The two connects under the action of T4-DNA ligase, then the product of connection is converted bacillus coli DH 5 alpha competent cell, screening Recombinant plasmid pUC19-mt1de, that is, the 1- phosphomamlose alcohol dehydrogenase gene expression cassette cloned.
(4.2) clone of Mannitol-1-phosphatase expression casette:
(4.2.1) designs pair of primers ldh3:5'-AGTCTAGAAGTGCTTTAATTAGTG-3' and ldh4:5'- TCTGCCATAAGATCCTCCAAAATT-3'(and m1p1 complementary pairing), the bright string of goldbeater's skin for being CGMCC 1.2138 with deposit number Pearl bacterium chromosomal DNA is template, and PCR amplification obtains the segment for 177bp.
(4.2.2) designs pair of primers m1p1:5'-AGGATCTTATGGCAGAGACTGAGTG-3' and m1p2:5'- TCTCTAGATTAGGGTTTAGCGTTTG-3', using the chemically synthesized m1p coded sequence of Sheng Gong bio-engineering corporation as template, PCR amplification obtains the segment for 946bp.
(4.2.3) mixes the purified rear mixing of 2 PCR products obtained in above-mentioned (4.2.1) (4.2.2) with PCR product Conjunction object is template, makes 2 genetic fragment overlap-extension PCRs by 8 wheel PCR cycles, then utilizes pair of primers ldh3:5'- AGTCTAGAAGTGCTTTAATTAGTG-3' and m1p2:5'-TCTCTAGATTAGGGTTTAGCGTTTG-3' carries out PCR again, expands Increasing obtains the segment for 1107bp.
After Overlap extension PCR product obtained in above-mentioned (4.2.3) and pUC19 are carried out digestion with XbaI by (4.2.4), two Person connects under the action of T4-DNA ligase, then the product of connection is converted bacillus coli DH 5 alpha competent cell, screening weight Group plasmid pUC19-m1pe, that is, the Mannitol-1-phosphatase expression casette cloned.
(4.3) clone of 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase gene multiple repeats:
(4.3.1) designs pair of primers mt1de1:5'-TACGAATTCAGTGCTTTAATTAGTG-3' and mt1de2: 5'-CTTCTAGATAGAGGTACCTACTACTTTGCTG-3', using pUC19-mt1de as template, PCR amplification obtains being 1371 The segment of bp.
After PCR product obtained in above-mentioned (4.3.1) and pUC19 are carried out double digestion with EcoRI and XbaI by (4.3.2), The two connects under the action of T4-DNA ligase, then the product of connection is converted bacillus coli DH 5 alpha competent cell, screening Recombinant plasmid pUC19-mt1d.
(4.3.3) designs pair of primers m1pe1:5'-CTGGTACCTTAGTAGAAAGTGCTT-3' and m1pe2:5'- CTGGTACCTTAGGGTTTAGCGTTTG-3', using pUC19-m1pe as template, PCR amplification obtains the segment for 1123bp.
After PCR product obtained in above-mentioned (4.3.3) and pUC19 are carried out digestion with KpnI by (4.3.4), the two is in T4- It is connected under the action of DNA ligase, then the product of connection is converted into bacillus coli DH 5 alpha competent cell, screen recombinant plasmid PUC19-mt1d-m1p, that is, the 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase gene multiple repeats cloned.
(4.4) the intermediate acetaldehyde for having 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase gene multiple repeats The building of dehydrogenase gene homologous recombination vector: design pair of primers mpl:5'-CGTCTAGAAGTGCTTTAATTAGTG-3' and Mpr:5'-TATCTAGATAGAGGTACCTTAGGGT-3', using pUC19-mt1d-m1p as template, PCR amplification obtain for The segment of 2469bp, and PCR product is connected among homologous recombination vector pUC19-aldhqh homology arm with T4 ligase On the site XbaI, recombinant plasmid is named as pUC19-aldhqh-mt1d-m1p, that is, is configured to intermediate with 1- phosphomamlose alcohol The acetaldehyde dehydrogenase gene homologous recombination vector of dehydrogenase gene and Mannitol-1-phosphatase gene multiple repeats.
(4.5) glucansucrase gene knockout, D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene knock out And mannitol dehydrogenase gene knock-in, serine/threonine protein kitase gene knockout and mannitol dehydrogenase gene knock-in, fruit Sugar kinase genes knock out and mannitol dehydrogenase gene knock-in and acetaldehyde dehydrogenase gene knock out simultaneously 1- phosphomamlose alcohol dehydrogenase The building of the Leuconostoc mesenteroides mutant strain of gene and Mannitol-1-phosphatase gene knock-in: with electrotransformation by pUC19- Aldhqh-mt1d-m1p imported into Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ obtained in above-mentioned third step stpk::mdh△fk::mdh△aldh::amy(Leuconostoc mesenteroides△dts1△ldh△pat::mdh △ stpk::mdh △ fk::mdh △ aldh::amy) in bacterial strain, screening obtain glucansucrase gene knock out, D-ALPHA-Hydroxypropionic acid it is de- Hydrogenase gene knocks out, acetyl phosphate transferase gene knocks out and mannitol dehydrogenase gene knock-in, serine/threonine protein kinase Enzyme gene knocks out and mannitol dehydrogenase gene knock-in, fructokinase gene knockout and mannitol dehydrogenase gene knock-in and acetaldehyde Dehydrogenase gene knocks out and the Leuconostoc mesenteroides of 1- phosphomamlose alcohol dehydrogenase gene and Mannitol-1-phosphatase gene knock-in Mutant strain, i.e. Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh:: (mt1d-m1p)[Leuconostoc mesenteroides△dts1△ldh△pat::mdh△stpk::mdh△fk::mdh △ aldh::(mt1d-m1p)] bacterial strain.
Design pair of primers aldhyq:5'-GCAGAGATATTAAACAAAA-3' and aldhyh:5'- TGGTGGAACATTACCTGAG-3' extracts chromosomal DNA, carries out PCR, the above-mentioned bright beading of goldbeater's skin by template of chromosomal DNA Bacterium mutant strain, i.e. Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh:: (mt1d-m1p)[Leuconostoc mesenteroides△dts1△ldh△pat::mdh△stpk::mdh△fk::mdh △ aldh::(mt1d-m1p)] bacterial strain obtains length as the amplified production of 3417bp, and Leuconostoc mesenteroides Δ dts1 Δ D- ldhΔpat-mdh Δstpk-mdhΔfk-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔpat- Mdh Δ stpk-mdh Δ fk-mdh, deposit number CCTCCM2017578) obtain the amplified production that length is 1125bp.
Embodiment 2
Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mt1d- m1p) [Leuconostoc mesenteroides△dts1△ldh△pat::mdh△stpk::mdh△fk::mdh△ Aldh::(mt1d-m1p)] bacterial strain, in China typical culture collection center (CCTCC) preservation, preservation date is 2018 years November 23, deposit number are CCTCC M2018815), i.e., a plant height of the invention produces the Leuconostoc mesenteroides mutant bacteria of mannitol The fermentation application of strain, the specific steps are as follows:
In 250 milliliters of triangular flasks, will be in China typical culture collection center (CCTCC) preservation, preservation date On November 23rd, 2018, deposit number are the Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ of CCTCC M2018815 stpk::mdh△fk::mdh△aldh::(mt1d-m1p)[Leuconostoc mesenteroides△dts1△ldh△ Pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mt1d-m1p)] bacterial strain with weight percent 1% be transferred to MRS training Support in base, at 30 DEG C, with revolving speed for shaking table culture 20 hours of 120 revs/min, mannitol concentration can achieve 9.73 grams/ It rises, the conversion ratio 97.3% of fructose moiety in sucrose.
The preparation method of above-mentioned MRS culture medium is: by 2 grams of yeast extract, 90 grams of sucrose, 2 grams of ammonium citrate, sodium acetate 5 Gram, K2HPO42 grams, MnSO4·H20.039 gram of O is sterilized at a temperature of 121 DEG C with 1000 milliliters of water with acetic acid tune pH to 6.2 Preparation in 20 minutes obtains MRS culture medium.
Table 1 lists the yield that various leukonid fermentations produce mannitol, it is seen that Chinese Typical Representative culture of the invention is protected Hiding center (CCTCC) preservation, preservation date are on November 23rd, 2018, and deposit number is the bright beading of goldbeater's skin of CCTCC M2018815 Bacterium △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mt1d-m1p) [Leuconostoc Mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mt1d-m1p)] bacterial strain Mannitol yield than initial deposit number be CCTCCM2017578 Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat-mdh Δstpk-mdh Δfk-mdh(Leuconostoc mesenteroidesΔdts1ΔD-ldhΔpat-mdhΔstpk-mdh Δ fk-mdh) 15.4% is improved, the conversion ratio of fructose moiety improves 7.0% in sucrose.
The fermentation of 1. Leuconostoc mesenteroides of table produces the yield (g/L) of mannitol
In table 1, original bacteria is the original Leuconostoc mesenteroides not being modified, Δ dts1 Δ ldh Δ pat-mdh Δ stpk- Mdh Δ fk-mdh is glucansucrase gene knockout, D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene strike Except and mannitol dehydrogenase gene knock-in, serine/threonine protein kitase gene knockout and mannitol dehydrogenase gene knock-in With the Leuconostoc mesenteroides mutant strain of fructokinase gene knockout and mannitol dehydrogenase gene knock-in, the as bright beading of goldbeater's skin Bacterium Δ dts1 Δ ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh (Leuconostoc mesenteroides Δ dts1 Δ Ldh Δ pat-mdh Δ stpk-mdh Δ fk-mdh) bacterial strain, Δ dts1 Δ ldh Δ pat::mdh Δ stpk::mdh Δ fk:: Mdh Δ aldh::(mt1d-m1p) it is glucansucrase gene knockout, the transfer of D-lactic acid dehydrogenase gene knockout, acetyl phosphate Enzyme gene knocks out and mannitol dehydrogenase gene knock-in, serine/threonine protein kitase gene knockout and mannitol dehydrogenase Gene knock-in, fructokinase gene knockout and mannitol dehydrogenase gene knock-in and knockout acetaldehyde-dehydrogenase enzyme coding gene are simultaneously knocked in The Leuconostoc mesenteroides mutant strain of 1- phosphomamlose alcohol dehydrogenase encoding gene and Mannitol-1-phosphatase encoding gene is Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh △ aldh::(mtld-mlp) [Leuconostoc mesenteroides△dts1△ldh△pat::mdh△ stpk::mdh△fk::mdh△aldh:: (mtld-mlp)] bacterial strain, in China typical culture collection center (CCTCC) preservation, preservation date is 2018 year November 23 Day, deposit number is CCTCC M2018815), i.e., a plant height of the invention produces the Leuconostoc mesenteroides mutant strain of mannitol.
In above-described embodiment, by commercially available, related operating procedure is this for related raw material, reagent and instrument Field technical staff will appreciate that the specific experiment method being not specified such as " molecular cloning: is tested usually according to normal condition Handbook " described in method or manufacturer provide scheme carry out.
Sequence table
Partial sequence lacks and is inserted into 1- phosphomamlose alcohol dehydrogenase gene expression cassette and sweet dew herein in acetaldehyde dehydrogenase gene The nucleotide sequence of alcohol -1- phosphatase gene expression cassette
<110>Tianjin Bo Ruiwei biological medicine Science and Technology Ltd.
<120>one plant heights produce Leuconostoc mesenteroides mutant strain and its application method of mannitol
<160> 1
<210> 1
<211> 3682
<212> DNA
<213>Leuconostoc mesenteroides (Leuconostoc mesenteroides)
〈400〉1
atgagctatc aaacaattaa tccctttaac gacgaagtta ttcaaacatt tgacaatcat 60
gatgacgctt atgttgagaa ggccattgcc gaaggtcatg cactgtataa aaagtggcgc 120
aatgacccgg ctagtagtcg cgcagagata ttaaacaaaa ttgctgactt gatggaagaa 180
gatgctgatc atttagctaa ggtacttact attgaaatgg gtaagcgatt tgtcgaggct 240
caaggtgaag tagcattaag tgtttcaatt gctcgttact acgccaaaaa tggtgcagat 300
tttcttaagc cagaaccaat caaatcctcg atgggggatg cgcaagtaat ttcgcgcccc 360
actggggtat tgatgatggt tgaaccatgg aattttcctt actatcaaat tattcgtgta 420
tttgcaccaa attatatagc tggaaaccca atgcttttga agcacgcaag caatacgcca 480
atggctgcat cagaatttga aaaaattgtt gaacgggctg gtgcacctac tggtgcgttt 540
gctaatttat tcattgatta cgatcaagtg aatatctaga agtgctttaa ttagtgatta 600
aagcaaagaa aatggaatgg gttacatttg cttaacgact gtcatttgta aggggtgaaa 660
ttttttctga aatctatgca ttatatgggc ttaatcgcgt gcgttagctc gtgaaatagg 720
gtacaattat agatgaaata aaattttgga ggatcttatg ttagacgtac attttggcgc 780
agggaatatt ggtcggggct tcattggcga aaccttggct gacaacgggt ttaagattac 840
tttcgttgat gttaacgata ctttgattga cgaattaaac aaacggaatg gttataccat 900
tgaattggct gcagaaggtc aaaaacatat tgaagttcac gatgttaagg gtattaacaa 960
cggtaaggat cctaaggcgg ttgctgaaga aattgcccaa gccgatatgg ttacgactgc 1020
gattggacct aagatcttga agttcatcgc gccattaatt gctgacgggt taaagttacg 1080
gcaagctaac aacaatacga caccaattga tatcattgct tgtgaaaaca tgattggcgg 1140
gagtcagtca ttaaagaaat ccgtttatga atcattaaat gaagacgaac aagcttgggc 1200
tgaccaaaat gcaggcttcc ccaatgccgc tgttgaccgg atcgtaccgc ttcaaaagca 1260
tgatgatcca ttgttcgttt cagttgaacc attcaaggaa tgggttatcg acaagtccca 1320
gatgaagaac cctaagattc aattaaaggg tgttgattat gctgacgact tggaaccata 1380
cattgaacgg aaactcttct ccgttaatac tggtcatgcc accgtagcct atactggtaa 1440
catgaaaggc tacaagacga ttggcgaagc ggtcaaggat gacagtgtcg ttgaccaagc 1500
taagcacgtt ttaggtgaaa ccggcgactt gttgattcaa aagtggggct ttgatcctga 1560
agtacaccat gcttatcaaa agaagatttt gagtcggttt gaaaacccat atatctctga 1620
tgatattgaa cgggtcggcc ggacaccaat tcggaaatta ggtttcaatg aacgttttat 1680
tcgcccaatt cgggaattga aggaacgtgg tcgtgattac agtgccttag ttgatacagt 1740
tggtgaaatg ttcttcttca actatcctaa tgatagtgaa agtgttaagt tacaacaatt 1800
attgaaggat gaaccaatcg aacaagtcat tcgcgaaaca actgacttga aagatgaaga 1860
tttagttaat gaaatcaaag ctgcgtacga aaagcactta gctgcagcaa agtagtaggt 1920
accttagtag aaagtgcttt aattagtgat taaagcaaag aaaatggaat gggttacatt 1980
tgcttaacga ctgtcatttg taaggggtga aattttttct gaaatctatg cattatatgg 2040
gcttaatcgc gtgcgttagc tcgtgaaata gggtacaatt atagatgaaa taaaattttg 2100
gaggatctta tggcagagac tgagtggact ccggaggcgc tttctgggcg ttacgaggag 2160
ataaaaagct gcattccgca gcagctggag gcttacgcgc ggtttctgcg cgaggccgcg 2220
ccggaagacc tccgccgctg gcaacaaatt gcgcaagatt taaaacttga attgaattta 2280
gaaaacggaa gaataaaata caaaaaagaa ttcaaaccac tggagcttcc cgtggacatt 2340
tgctacatcc gccacggcaa gacgcagggc aacacggagc cccgggtttt tcagggccag 2400
gtggactacg caaacaacca gctgacgcag caggggcagc agcaagcagc agcagcagca 2460
acaaaactag aagcaatggc agcagcaaaa gaattcattc cggatttgct gctgtcttct 2520
ccgctgctgc gagcagtcca cacggcgcag cccttcgtag acgcaaaccc taaacccctt 2580
tttagggttt tgccagaact cgcggaaatg gcgttcgggg aatgggacaa cagaaaggtg 2640
gcagaactcg aaaaagatga ccccgcgcat ttgttttact tgcaacaaaa tgctgttatt 2700
aaggcgaaag ggccccacag gatttgctgc caactttggc aaagtcccga gtggctcgag 2760
gggaaaaaag aactgcccgc tgagaacttt ctcgagtgtc tggacagaca gcgcaaagcc 2820
ctcatcaagg ttggggaaat cgccaaagag ctttgcggcc cttcttgcgg agaaaggaag 2880
cctcgggttg cggtgtacgg acacagcatg gccggagccg ctgtatccgt ccttttaggg 2940
tttgggaaag aagaccaatt agggtttcta gggtttgacg gaaactacat catgcccaac 3000
gccacgccta ccatcctaat cccaaacgct aaaccctaag gtacctctat ctagagtatg 3060
tacttcgtct aaacggttta ttgtaaccga aaaaaattat gatgcggtac ttacaatgtt 3120
aaaagatgcc tttgctgaag caaaactagg cgacccattg ttggaagata cgacattagc 3180
accattaagt accagcaagg ctaagaaaaa cttgaccaaa caagtgaaag cggcagttga 3240
tgccggtgct actcttgaat atggtagtgt tgtccaagat aaaccagctg cactgtttga 3300
tcccgttatt ttaactggta ttacaaaaga caacccagct tattatcaag agttcttcgg 3360
tccagttgga caagtctaca aagtgaaaga tgaagaagag gcaattacac tagctaatga 3420
ttctaattat ggcttatcgg gcgtggtatt tggtggttca cctgagcatg cgacggaagt 3480
tgcttctcgt attgagacgg gagcggttta tgtgaatagt tttggtggaa cattacctga 3540
gttaccattt ggtggtgtta aaaattctgg ctatggacgt gagctaggac gctttggtat 3600
cgaaaccttt gtgaacaagg aacttattgt tactaaaaag gaaccaattg atttagataa 3660
tgcttttggt ggatttgttt aa

Claims (3)

1. a plant height production mannitol Leuconostoc mesenteroides mutant strain, it is characterised in that: be glucansucrase gene knock out, D-lactic acid dehydrogenase gene knockout, acetyl phosphate transferase gene knocks out and mannitol dehydrogenase gene knock-in, serine/Soviet Union's ammonia Pka acid gene knockout and mannitol dehydrogenase gene knock-in, fructokinase gene knockout and mannitol dehydrogenase clpp gene Enter and knocks out acetaldehyde-dehydrogenase enzyme coding gene and knock in 1- phosphomamlose alcohol dehydrogenase encoding gene and Mannitol-1-phosphatase volume The Leuconostoc mesenteroides mutant strain of code gene is Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh △ fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ldh△pat::mdh△ Stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterial strain, it is protected at China typical culture collection center (CCTCC) Hiding, preservation date is on November 23rd, 2018, and deposit number is CCTCC No:M2018815.
2. the application method that a plant height described in claim 1 produces the Leuconostoc mesenteroides mutant strain of mannitol, it is characterised in that: It will be in November, 20187 in China typical culture collection center (CCTCC) preservation, preservation date in 250 milliliters of triangular flasks 23, deposit number was the Leuconostoc mesenteroides △ dts1 △ ldh △ pat::mdh △ stpk::mdh of CCTCC No:M20187815 △fk::mdh△aldh::(mtld-mlp)[Leuconostoc mesenteroides△dts1△ldh△pat::mdh△ Stpk::mdh △ fk::mdh △ aldh::(mtld-mlp)] bacterial strain is transferred in MRS culture medium with weight percent 1%, in At 30 DEG C, with shaking table culture 20 hours that revolving speed is 120 revs/min, mannitol concentration can achieve 47.3 grams per liters, and sucrose arrives The conversion ratio 52.6% of mannitol.
3. a plant height produces the application method of the Leuconostoc mesenteroides mutant strain of mannitol, feature according to claim 2 Be: the preparation method of the MRS culture medium is: by 2 grams of yeast extract, 90 grams of sucrose, 2 grams of ammonium citrate, 5 grams of sodium acetate, K2HPO42 grams, MnSO4·H20.039 gram of O and 1000 milliliters of water acetic acid tune pH to 6.2, at a temperature of 121 DEG C, sterilizing 20 Minute prepares and obtains MRS culture medium.
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