CN109593696B - Leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof - Google Patents

Leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof Download PDF

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CN109593696B
CN109593696B CN201811545503.1A CN201811545503A CN109593696B CN 109593696 B CN109593696 B CN 109593696B CN 201811545503 A CN201811545503 A CN 201811545503A CN 109593696 B CN109593696 B CN 109593696B
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leuconostoc mesenteroides
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金红星
闫博
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Tianjin Brave Biopharma Technology Co ltd
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Abstract

The invention discloses a mutant strain of Leuconostoc mesenteroides for high yield of mannitol and an application method thereof, and relates to bacteria, wherein the strain is a mutant strain of Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh delta stpk, mdh delta fk, and mtld delta fk, mdh delta aldh, and mtld-mlp, and is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 11-23 days in 2018, and the preservation number is CCTCC No: m2018815. The strain is transferred into an MRS culture medium by 1 percent of weight percentage, and is cultured for 20 hours at 30 ℃ by a shaking table with the rotating speed of 120 r/min, the concentration of mannitol can reach 47.3 g/L, and the conversion rate of sucrose to mannitol is 52.6 percent.

Description

Leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof
Technical Field
The technical scheme of the invention relates to bacteria, in particular to a leuconostoc mesenteroides mutant strain for high yield of mannitol and an application method thereof.
Background
Mannitol (Mannitol) is a hexitol and is widely used in the fields of medicine, food and plastics.
At present, there are two main processes for the industrial production of mannitol in the world. The first method is a seaweed extraction method: 13-15 tons of dried kelp is needed for extracting 1 ton of mannitol, and when the alginate is produced, the kelp soak solution after iodine extraction is subjected to extraction and concentration for multiple times, impurity removal, ion exchange, evaporation and concentration, cooling and crystallization to obtain the kelp extract; the production process produces a large amount of waste water, and has high energy consumption, serious pollution and low yield. The second is a catalytic hydrogenation process: the preparation method comprises the following steps of (1) taking cane sugar or glucose as a raw material, performing hydrolysis, epimerization and enzyme isomerization, and then hydrogenating to obtain the product; the raw material source is stable, the production period is not limited, the cost is low, but the yield is low, and the sorbitol is associated.
There are two more methods for the laboratory production of mannitol. One is an enzymatic conversion method, and enzymatic hydrogenation needs to add expensive coenzyme into the system, so that the method is not economical. The other is a microbial fermentation method, the types of microorganisms capable of synthesizing mannitol in nature are more, and some strains in bacteria, yeast and mould have the capacity of producing mannitol. In the process of converting mannitol by lactic acid bacteria, mannitol is a main product, simultaneously produces lactic acid, acetic acid, ethanol and carbon dioxide, does not produce other by-products such as polyol and the like, and is easy to purify, separate and refine, mild in condition and high in conversion rate.
Many strains produce mannitol by fermentation with fructose as a substrate, while leuconostoc can produce mannitol by taking fructose and sucrose as substrates. Cheap sucrose is decomposed into glucose-1-phosphate and fructose after entering leuconostoc cells, and the fructose is converted into mannitol, so that the reaction steps are relatively few; glucose in the homolactic fermentation lactobacillus is finally converted into mannitol through intermediate products such as glucose-6-phosphate, fructose-6-phosphate, mannitol-1-phosphate and the like, and the reaction steps are relatively more; the chromosome genome of the leuconostoc is only about 2M, so the fermentation period is only about 20 hours; the leuconostoc is oxygen-resistant, so oxygen does not need to be supplied in the fermentation process; therefore, the potential of the leuconostoc to realize large-scale industrial production of mannitol is relatively large.
CN201711169481.9 discloses a leuconostoc mesenteroides mutant strain for high yield of mannitol and an application method thereof, wherein the leuconostoc mesenteroides mutant strain is a leuconostoc mesenteroides mutant strain with genes of dextransucrase and D-lactate dehydrogenase knocked out, genes of acetyl phosphate transferase knocked out and knocked in mannitol dehydrogenase, genes of serine/threonine protein kinase knocked out and knocked in mannitol dehydrogenase, and genes of fructokinase knocked out and knocked in mannitol dehydrogenase, although the yield is improved compared with that of the original strain, the mutant strain is still lower and is not enough to be applied to production.
In summary, in the existing leuconostoc fermentation technology, the yield of mannitol produced by using sucrose as a substrate is not high enough, and needs to be further improved.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the Leuconostoc mesenteroides mutant strain with high mannitol yield and an application method thereof are provided, the Leuconostoc mesenteroides mutant strain takes the existing Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh (Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh) strain (CN201711169481.9 a Leuconostoc mesenteroides mutant strain with high mannitol yield and an application method thereof) with the preservation number of CCTCC M2017578 as a starting strain, and acetaldehyde dehydrogenase coding genes are knocked out by adopting a molecular biology technology and 1-phosphomannitol dehydrogenase coding genes and mannitol-1-phosphatase coding genes are knocked in to construct a glucan sucrase gene, a D-lactate gene, an acetyl phosphate transferase gene and mannitol dehydrogenase gene knock-in, The mutant strain of Leuconostoc mesenteroides with serine/threonine protein kinase gene knockout and mannitol dehydrogenase gene knockout and acetaldehyde dehydrogenase gene knockout and 1-phosphomannitol dehydrogenase coding gene knockout and mannitol-1-phosphatase coding gene, namely the Leuconostoc mesenteroides delta dts1 delta ldh delta pat with the preservation number of CCTCC M2018815, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh stpk, mdh delta fk, mdh delta aldh, mtld-mlp, overcomes the defect that the yield of mannitol produced by taking cane sugar as a substrate in the existing Leuconostoc mesenteroides fermentation technology is not high enough.
The technical scheme adopted by the invention for solving the technical problem is as follows: a mutant strain of Leuconostoc mesenteroides with high mannitol yield is a mutant strain of Leuconostoc mesenteroides with the gene of dextransucrase, the gene of D-lactate dehydrogenase, the gene of acetyl phosphotransferase and the gene of mannitol dehydrogenase knocking in, the gene of serine/threonine protein kinase and the gene of mannitol dehydrogenase knocking in, the gene of fructose kinase and the gene of acetaldehyde dehydrogenase knocking in and the gene of 1-phosphomannitol dehydrogenase and the gene of mannitol-1-phosphatase, and is a mutant strain of Leuconostoc mesenteroides with the gene of 1-phosphomannitol dehydrogenase and the gene of mannitol-1-phosphatase coding, wherein the mutant strain of Leuconostoc mesenteroides is DtmdDeltas 1 delta pat, dhmdk, mdh Deltafk, mdhmdh, and the mutant strain is preserved in China center (CCTmdk, preservation No. 56), the preservation date is 11 months and 23 days in 2018, and the preservation number is CCTCC No: m2018815, the address of the preservation unit is China, Wuhan university.
An application method of a leuconostoc mesenteroides mutant strain for high yield of mannitol is to preserve in a 250 ml triangular flask in China Center for Type Culture Collection (CCTCC) with the preservation date of 2018, 11 and 23 days, and the preservation number is CCTCC No: m2018815, Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh delta ldh: (mtld-mlp) ] strain was transferred to MRS medium at 1% by weight, and cultured in a shaker at 120 rpm for 20 hours at 30 ℃, the mannitol concentration could reach 47.3 g/l, and the sucrose to mannitol conversion rate was 52.6%.
The application method of the leuconostoc mesenteroides mutant strain capable of producing mannitol at high yield comprises the following steps: 2 g of yeast extract powder, 90 g of cane sugar, 2 g of ammonium citrate, 5 g of sodium acetate and K2HPO42 g of MnSO4·H2MRS medium was prepared by adjusting the pH of O0.039 g and 1000 ml of water to 6.2 with acetic acid and sterilizing at 121 ℃ for 20 minutes.
The application method of the leuconostoc mesenteroides mutant strain capable of producing mannitol at high yield comprises the steps of obtaining related raw materials, reagents and instruments from commercial sources, wherein related operation processes can be mastered by those skilled in the art.
The invention has the beneficial effects that: compared with the prior art, the invention has the following prominent substantive characteristics and remarkable progress:
(1) the invention adopts molecular biology technology to knock out acetaldehyde dehydrogenase encoding genes in the existing Leuconostoc mesenteroides strain delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh (Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh) with the preservation number of CCTCC M2017578 and knock in 1-phosphomannitol dehydrogenase encoding genes and mannitol-1-phosphatase encoding genes, and constructs the strains as glucan sucrase gene knock-out, D-lactate dehydrogenase gene knock-out, acetyl phosphate transferase gene knock-out and mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knock-out and mannitol dehydrogenase gene knock-in, fructose kinase gene knock-out and mannitol dehydrogenase gene knock-in and acetaldehyde dehydrogenase gene knock-out and 1-phosphomannitol dehydrogenase gene knock-in The leuconostoc mutant strain has a preservation number of CCTCC No: m2018815, namely Leuconostoc mesenteroides delta dts1 delta ldh delta pat: (mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) ] strain, overcomes the defect that the yield of mannitol produced by using sucrose as a substrate in the existing Leuconostoc fermentation technology is still not high enough.
(2) Transferring Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh delta hk:: mdh delta aldh:: mdh delta aldh delta hk: (mtld-mlp) [ Leuconostoc mesenteroides delta aldh Δ s1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh:: mdc delta aldh: (mtld-mlp) ] strain into MRS culture medium at a weight percentage of 1%, culturing the strain in a shaker at a rotation speed of 120 rpm at 30 ℃ for 20 hours, detecting the metabolite, and comparing the result shows that the yield of the Leuconostoc mesenteroides is delta dhmdh delta aldh delta dk-delta aldh delta 90-54-delta dhmdh delta aldh delta dk in the China center for preservation of typical culture (CCTCC), the conversion of sucrose to mannitol increased by 7.0%.
Drawings
FIG. 1 shows that the preservation number of the invention is CCTCC No: m2018815, Leuconostoc mesenteroides delta dts1 delta ldh delta pat: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh delta ldh: (mtld-mlp) ] strain to construct agarose gel electrophoresis images of left and right homologous arms in the acetaldehyde dehydrogenase gene homologous recombination vector;
FIG. 2 shows that the preservation number of the invention is CCTCC No: m2018815, namely Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, Leuconostoc mesenteroides delta dts1 delta ldh, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, constructing an enzyme digestion verification agarose gel electrophoresis image of the homologous recombinant vector homologous arm of the acetaldehyde dehydrogenase gene of the strain;
FIG. 3 shows that the preservation number of the invention is CCTCC No: m2018815, a restriction enzyme digestion verification agarose gel electrophoresis picture of an acetaldehyde dehydrogenase gene homologous recombination vector with an alpha-amylase gene mark in the middle is constructed by the Leuconostoc mesenteroides delta dts1 delta ldh delta pat: (mdh delta stpk:: mdh delta fk mdh delta aldh: (mtld-mlp) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) ];
FIG. 4 shows that the preservation number of the invention is CCTCC No: m2018815, namely Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, Leuconostoc mesenteroides delta dts1 delta ldh, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, constructing an aldehyde dehydrogenase gene homologous recombination vector with a 1-phosphate mannitol dehydrogenase gene and a mannitol-1-phosphate enzyme gene in the middle;
FIG. 5 shows that the preservation number of the invention is CCTCC No: m2018815, an agarose gel electrophoresis image of the mutant strain with acetaldehyde dehydrogenase gene knockout and 1-phosphomannitol dehydrogenase gene knockout and mannitol-1-phosphatase gene knockout verified by PCR for Leuconostoc mesenteroides Deltadts 1 Deltaldh: (mdh Deltastpk:: mdh Deltaldh: (mtld-mlp) [ Leuconostoc mesenteroides Deltadts 1 Deltaldh:: mdh Deltafk:: mdh Deltaldh: (mtld-mlp) ].
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 shows that the preservation number of the invention is CCTCC No: m2018815 Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh:: (mtld-mlp) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh:: mtld-mlp) ] strain, construction of agarose gel electrophoresis pattern of left and right homologous arms in acetaldehyde dehydrogenase gene homologous recombinant vector. Left and right homology arms are shown: 1. left arm of homology, 2.Marker, 3. right arm of homology.
FIG. 2 shows that the preservation number of the invention is CCTCC No: m2018815, namely Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, Leuconostoc mesenteroides delta dts1 delta ldh, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp. Two bands generated by cleavage of the recombinant vector are shown: 1, Marker, 2, double enzyme digestion of the recombinant vector.
FIG. 3 shows that the preservation number of the invention is CCTCC No: m2018815, a restriction enzyme digestion verification agarose gel electrophoresis picture of acetaldehyde dehydrogenase gene homologous recombination vector with alpha-amylase gene mark in the middle is constructed by the Leuconostoc mesenteroides delta dts1 delta ldh delta pat: (mdh delta stpk:: mdh delta aldh: (mtld-mlp) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) ]. Two bands generated by cleavage of the recombinant vector are shown: 1, Marker, 2, double enzyme digestion of the recombinant vector.
FIG. 4 shows that the preservation number of the invention is CCTCC No: m2018815, namely Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, Leuconostoc mesenteroides delta dts1 delta ldh, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, constructing an aldehyde dehydrogenase gene homologous recombination vector with a 1-phosphate mannitol dehydrogenase gene and a mannitol-1-phosphate enzyme gene in the middle. Two bands generated by cleavage of the recombinant vector are shown: 1, Marker, 2, double enzyme digestion of the recombinant vector.
FIG. 5 shows that the preservation number of the invention is CCTCC No: m2018815, agarose gel electrophoresis image of the mutant strain with acetaldehyde dehydrogenase gene knock-out and mannitol-1-phosphate dehydrogenase gene knock-in of Leuconostoc mesenteroides Deltadts 1 Deltaldh:: mdh Deltastpk:: mdh Deltaldh: (mtld-mlp) [ Leuconostoc mesenteroides Deltadts 1 Deltaldh Deltapat:: mdh Deltastpk:: mdh Deltafk:: mdh Deltaaldh: (mtld-mlp) ] and with 1-phosphate mannitol dehydrogenase gene and mannitol-1-phosphate gene. The figure shows that: 1. the leuconostoc mesenteroides delta dts1 delta ldh delta pat-mdh delta stpk-mdh delta fk-mdh strain is used as a template, and 2 the leuconostoc mesenteroides delta dts1 delta ldh delta pat is:: mdh delta stpk:: mdh delta fk:: mdh delta aldh:: amy is used as a template, 3.Marker, 4 the leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) strain is used as a template.
Example 1
The method comprises the following steps of constructing a Leuconostoc mesenteroides mutant strain with an acetaldehyde dehydrogenase gene knocked out and a 1-phosphomannitol dehydrogenase gene and a mannitol-1-phosphatase gene knocked in, and specifically comprising the following steps:
constructing Leuconostoc mesenteroides mutant strains with knocked-in acetaldehyde dehydrogenase gene and knocked-in 1-phosphomanol dehydrogenase gene and mannitol-1-phosphatase gene by taking existing Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta fk-mdh (Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh) with the preservation number of CCTCC M2017578 as starting bacteria:
firstly, cloning a partial sequence of leuconostoc mesenteroides acetaldehyde dehydrogenase gene:
cloning a continuous sequence of an acetaldehyde dehydrogenase gene part of Leuconostoc mesenteroides with a coding sequence length of 1524bp by taking chromosome DNA as a template, wherein the Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta fk-mdh [ the preservation date is 2017, 10 and 25 days, and the preservation number is CCTCC M2017578] (Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh) is preserved in China Center for Type Culture Collection (CCTCC), and the specific operation steps are as follows:
(1.1) extraction of chromosomal DNA of Leuconostoc mesenteroides with a collection number of CCTCCM 2017578. delta. dts 1. delta. D-ldh. delta. pat-mdh. delta. stpk-mdh. delta. fk-mdh (Leuconostoc mesenteroides. delta. dts 1. delta. D-ldh. delta. pat-mdh. delta. stpk-mdh. delta. fk-mdh):
leuconostoc mesenteroides strain delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh (Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh) with the preservation number of CCTCCM2017578 frozen at-80 ℃ is streaked on an MRS solid plate and cultured overnight at 30 ℃; selecting a single colony from a solid plate, inoculating the single colony into 5 ml of MRS liquid culture medium, and culturing overnight by a shaking table at the temperature of 30 ℃ and the rotating speed of 120 r/min; centrifuging 2 ml of the above cultured bacterial liquid at the rotation speed of 10000 rpm for 2 minutes, and collecting thalli; the cells were washed twice with 1 ml of double distilled water; dissolving the thalli in 100 microliters of double distilled water, and uniformly blowing and beating; adding 100 microliter of lysozyme with the concentration of 100 mg/ml, and carrying out water bath at 37 ℃ for 1 h; adding 500 microliters of the extracting solution, and gently mixing uniformly; after incubation at 80 ℃ for 10 minutes, centrifugation was carried out at 14000 rpm for 10 minutes and the supernatant was discarded; add 100 microliter of the suspension to dissolve the DNA; adding equal volume of phenol-chloroform (100 microliters), shaking up lightly, placing in a refrigerator at 4 ℃ for 15 minutes, centrifuging at 4 ℃ at 12500 rpm for 15 minutes, and pumping the supernatant into a new centrifugal tube; repeating the phenol-chloroform extraction operation again; adding 200 microliter of precooled absolute ethyl alcohol with 2 times of volume, and standing for 2 hours in a refrigerator at 4 ℃; centrifuging at 12000 r/min for 20min, and pouring out the supernatant; washing with 70% ethanol for 1 time, centrifuging at 12000 rpm for 10 min, removing supernatant, and air drying; the pellet was dissolved in 20. mu.l of TE (Tris-HCl 100 mmol/l, EDTA 10 mmol/l, pH 8.0).
The MRS culture medium comprises the following components: 3 g of yeast extract powder, 10 g of peptone, 8 g of beef extract powder, 20 g of glucose, 2 g of ammonium citrate, 5 g of sodium acetate and K2HPO42 g, MgSO4·7H2O2 g, MnSO4·H20.039 g of O, 801.6 ml of Tween and 1000 ml of water, and adjusting the pH to 6.2 by using acetic acid; sterilizing at 121 deg.C for 20 min. Solid medium plus 1.5% agar.
The composition of the extracting solution is as follows: 240 mmol/l NaOH, 2.7 mmol/l EDTA, 74% ethanol.
Composition of the above suspension: 0.1 mmol/l EDTA, 50 mmol/l Tris-HCl, 1% TritonX-100(pH8.0), 0.5% Tween 20.
The phenol-chloroform solution is prepared by using phenol, chloroform and isoamylol in a volume ratio of 25:24: 1.
The TE solution was prepared using Tris-HCl 100 mmol/l and EDTA 10 mmol/l, and the pH was 8.0.
(1.2) PCR amplification of acetaldehyde dehydrogenase Gene:
designing a pair of primers aldhl: 5'-ACTTTGCGAATGAATAATG-3' and aldhr: 5'-TCGTGTAACCAATGATAAC-3', taking the Leuconostoc mesenteroides chromosome DNA of the Leuconostoc mesenteroides with the preservation number of CCTCCM2017578 delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh (Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh) as a template, carrying out PCR amplification to obtain a 1524bp fragment, connecting the PCR product to a pTA2T vector by using T4 ligase, and naming the recombinant plasmid as pTA 2-aldh.
(1.3) preparation and DNA transformation of competent E.coli DH 5. alpha.:
the Escherichia coli DH5 alpha strain frozen at-80 ℃ is streaked on an LB solid plate and cultured overnight at 37 ℃; picking a single colony from the solid plate, inoculating the single colony into 5 ml of LB liquid culture medium, and culturing the single colony overnight in a shaking table at 37 ℃ and the rotating speed of 150 r/min; transferring 0.2 ml of the bacterial liquid obtained by the culture into 10 ml of liquid culture medium, and performing shaking culture at 37 ℃ and the rotating speed of 150 rpm for 2-3 h until the OD of the bacterial liquid600Is 0.6; taking the OD600Adding 1.0 ml of 0.6 bacterial liquid into a 1.5 ml centrifuge tube, and carrying out ice bath for 10 minutes; centrifuging at 4 deg.C at 10000 rpm for 30 s, and removing supernatant; 1 ml of ice-cold 0.1 mol/l CaCl was added2Suspending cells in the solution, and carrying out ice bath for 30 minutes; centrifuging at 4 deg.C at 10000 rpm for 30 s, and removing supernatant; 100 microliters of ice-cold 0.1 mol/l CaCl was added2The cells are suspended in solution, i.e., the competent cells, i.e., competent E.coli DH5 α.
Adding 10 microliter of recombinant plasmid into the competent cells, and carrying out ice bath for 30 minutes; accurately heat shocking at 42 ℃ for 90 seconds; immediately placed on ice for 3 minutes; adding 400 microliter LB liquid culture medium, and culturing at 37 deg.C for 45 min with shaking; uniformly coating the transformed competent cells on an LB solid medium plate containing ampicillin; the plate was placed in an incubator at 37 ℃ for 30 minutes until the liquid was absorbed; and (5) inverting the plate, and culturing at 37 ℃ for 12-16 h.
And (3) selecting a single colony, culturing in an LB culture medium containing ampicillin, extracting plasmids, and carrying out agarose gel electrophoresis and sequencing identification.
The LB liquid medium described above: 5 g of yeast extract powder, 10 g of peptone, 10 g of NaCl, 1000 ml of distilled water, pH 7.0, and sterilizing at 121 ℃ for 20 minutes. Solid medium plus 1.5% agar.
Secondly, constructing a acetaldehyde dehydrogenase gene homologous recombination vector with an alpha-amylase mark in the middle:
(2.1) design a pair of primers aldhl 1: 5'-ACAGAATTCGCAGAGATATTAAACA-3' and aldhl 2: 5'-ACATACTCTAGATATTCACTTGATCGTA-3' (complementary pair with aldhr 1), and a 453bp fragment was obtained by PCR using pTA2-aldh as a template.
(2.2) designing a pair of primers aldhr 1: 5'-TGAATATCTAGAGTATGTACTTCGTCTA-3' and aldhr 2: 5'-TATAAGCTTCTCAGGTAATGTTCCA-3', using pTA2-aldh as template, and PCR amplifying to obtain 507bp fragment.
(2.3) purifying and mixing the 2 PCR products obtained in (2.1) and (2.2), overlapping and extending 2 gene fragments by 8 PCR cycles by using the PCR product mixture as a template, and then performing PCR amplification by using a pair of primers aldhl 1: 5'-ACAGAATTCGCAGAGATATTAAACA-3' and aldhr 2: 5'-TATAAGCTTCTCAGGTAATGTTCCA-3' PCR was performed to obtain a 948bp fragment.
(2.4) after double digestion of the overlap extension PCR product obtained in (2.3) and pUC19 with EcoRI and Hind III, the two were ligated by T4-DNA ligase, the ligated product was transformed into E.coli DH 5. alpha. competent cells, and recombinant plasmid pUC19-aldhqh was selected to construct a homologous recombinant vector.
(2.5) design a pair of primers amyl: 5'-CTATCTAGATTTGGCGTGATTATCAG-3' and amyr: 5'-TACTCTAGACGAAGGTGAAGTTATAG-3', using the chromosome DNA of the Lactobacillus amylovorus with the preservation number of CGMCC1.3395 as a template, obtaining a 2131bp fragment by PCR amplification, connecting the PCR product to an XbaI site in the middle of a homologous recombination vector pUC19-aldhqh homologous arm by using T4 ligase, and naming the recombinant plasmid as pUC19-aldhqh-amy, namely constructing the acetaldehyde dehydrogenase gene homologous recombination vector with alpha-amylase mark in the middle.
Thirdly, constructing the Leuconostoc mesenteroides mutant strain with the gene knockout of dextransucrase, the gene knockout of D-lactate dehydrogenase, the gene knockout of acetyl phosphotransferase and the gene knock-in of mannitol dehydrogenase, the gene knockout of serine/threonine protein kinase and the gene knock-in of mannitol dehydrogenase, the gene knockout of fructose kinase and the gene knock-in of mannitol dehydrogenase and the gene inactivation of acetaldehyde dehydrogenase:
leuconostoc mesenteroides strain delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh (Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta stpk-mdh delta fk-mdh) with the preservation number of CCTCCM2017578 frozen at-80 ℃ is streaked on an MRS solid plate and cultured overnight at 30 ℃; selecting a single colony from a solid plate, inoculating the single colony into 5 ml of MRS liquid culture medium, and performing shake culture at 30 ℃ at a rotating speed of 120 r/min overnight; the culture was continued by 1% transfer to MRS medium containing 0.48. mu.g/ml ampicillin, starting OD6000.048 of the OD of the Leuconostoc mesenteroides Deltadts 1 DeltaD-ldh Deltapat-mdh Deltastpk-mdh Deltafk-mdh (Leuconostoc mesenteroides Deltadts 1 DeltaD-ldh Deltapat-mdh Deltastpk-mdh Deltafk-mdh) bacterial liquid with the preservation number of CCTCCM2017578600Collecting thallus when reaching 0.5, resuspending thallus with LiAc-DTT solution containing 100U/ml lysozyme concentration, incubating at 30 deg.C for 20min, washing twice with ice-cold PBS solution, suspending thallus with 50 μ l of ice-cold PBS solution, adding 5 μ l of the above homologous recombination vector plasmid (pUC19-aldhqh-amy), performing electric transformation after ice bath for 10 min, using an electric transformation apparatus of Bio-Rad Gene Pulser XCellTMThe electric shock parameters are that the distance between electric shock cups is 0.1cm, 1400V, 25 muF and 300 omega, the electric shock time is 4 milliseconds, then 1 ml of MRS culture medium is added, after 3 hours of recovery, the mixture is coated on a solid plate containing MRS, after 120 hours of culture, a single colony is picked for verification, so as to prove that the glucan sucrase gene knockout, the D-lactate dehydrogenase gene knockout, the acetyl phosphate transferase gene knockout and the mannitol dehydrogenase gene knock-in, the serine/threonine protein kinase gene knockout and the mannitol dehydrogenase gene knock-in, the fructose kinase gene knockout and the mannitol dehydrogenase gene knock-in and the acetaldehyde dehydrogenase gene knock-in are obtained by screening on the plateA mutant strain of inactivated Leuconostoc mesenteroides, i.e., Leuconostoc mesenteroides Deltadts 1 Deltaldh Delta0 pat:: mdh Deltafk:: mdh Deltaaldh:: amy (Leuconostoc mesenteroides Deltadts 1 Deltaldh Deltapat:: mdh Deltastpk:: mdh Deltaldh:: amy) strain, was used.
Designing a pair of primers aldhyq: 5'-GCAGAGATATTAAACAAAA-3' and aldhey: 5'-TGGTGGAACATTACCTGAG-3', extracting chromosome DNA, carrying out PCR by taking the chromosome DNA as a template, wherein the Leuconostoc mesenteroides mutant strain, namely Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh:: amy (Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta afk, delta dhΔ aldh:: amy) strain obtains an amplification product with the length of 3079bp, while the Leuconostoc mesenteroides delta dts1 delta D-ldh delta stpk-mdh delta fk-mdh (Leuconostoc mesenteroides delta dts1 delta ddm Δ ddh Δ stfk-1125-delta dhm < 78 is obtained by using the number of the Leuconostoc mesenteroides delta dts1 delta ldh delta Δ dts as a and delta ldh as a template.
The LiAc-DTT solution is prepared by using 100 millimole/L LiAc, 10 millimole/L DTT, 0.6 molar sucrose and 10 millimole/L Tris-HCl (pH7.5);
the PBS solution is K2HPO4-KH2PO41 mmol/l MgCl 21 mmol/l and 0.5 mol/l sucrose, pH 6.9.
Fourthly, constructing the Leuconostoc mesenteroides mutant strain with the gene knockout of dextransucrase, the gene knockout of D-lactate dehydrogenase, the gene knockout of acetyl phosphotransferase and the gene knock-in of mannitol dehydrogenase, the gene knockout of serine/threonine protein kinase and the gene knock-in of mannitol dehydrogenase, the gene knockout of fructose kinase and the gene knock-in of mannitol dehydrogenase and the gene knock-in of acetaldehyde dehydrogenase, and the gene knock-in of 1-phosphomannitol dehydrogenase and mannitol-1-phosphatase:
(4.1) cloning of 1-phosphomannitol dehydrogenase Gene expression cassette:
(4.1.1) design a pair of primers ldh 1: 5'-ATAGAATTCAGTGCTTTAATTAGTG-3' and ldh 2: 5'-TCTAACATAAGATCCTCCAAAATT-3' (complementary pairing with mt1d 1), and taking leuconostoc mesenteroides chromosome DNA with the preservation number of CGMCC 1.2138 as a template, and obtaining a 177bp fragment by PCR amplification.
(4.1.2) design a pair of primers mt1d 1: 5'-AGGATCTTATGTTAGACGTACATT-3' and mt1d 2: 5'-ATGAATTCATCGAACTACTTTGCT-3', using plant lactobacillus chromosome DNA with preservation number CGMCC 1.2437 as template, PCR amplifying to obtain 1164bp segment.
(4.1.3) the 2 PCR products obtained in (4.1.1) (4.1.2) above were purified and mixed, 2 gene fragments were extended by 8 cycles of PCR using the PCR product mixture as a template, and then the PCR products were separated by a pair of primers ldh 1: 5'-ATAGAATTCAGTGCTTTAATTAGTG-3' and mt1d 2: 5'-ATGAATTCATCGAACTACTTTGCT-3' PCR was performed to obtain a 1341bp fragment.
(4.1.4) the PCR product of the overlap extension obtained in (4.1.3) above and pUC19 were digested with EcoRI, and the two were ligated by T4-DNA ligase, and the ligated product was transformed into E.coli DH 5. alpha. competent cells, and recombinant plasmid pUC19-mt1de, which is the cloned expression cassette of the 1-phosphomannitol dehydrogenase gene, was selected.
(4.2) cloning of mannitol-1-phosphatase Gene expression cassette:
(4.2.1) design a pair of primers ldh 3: 5'-AGTCTAGAAGTGCTTTAATTAGTG-3' and ldh 4: 5'-TCTGCCATAAGATCCTCCAAAATT-3' (complementary pairing with m1p 1), and taking leuconostoc mesenteroides chromosome DNA with the preservation number of CGMCC 1.2138 as a template, and obtaining a 177bp fragment by PCR amplification.
(4.2.2) design a pair of primers m1p 1: 5'-AGGATCTTATGGCAGAGACTGAGTG-3' and m1p 2: 5'-TCTCTAGATTAGGGTTTAGCGTTTG-3', using m1p coding sequence chemically synthesized by Biochemical engineering as template, PCR amplifying to obtain 946bp fragment.
(4.2.3) the 2 PCR products obtained in (4.2.1) and (4.2.2) above were purified and mixed, 2 gene fragments were extended by 8 cycles of PCR using the PCR product mixture as a template, and then the PCR products were separated by a pair of primers ldh 3: 5'-AGTCTAGAAGTGCTTTAATTAGTG-3' and m1p 2: 5'-TCTCTAGATTAGGGTTTAGCGTTTG-3' PCR was performed to obtain a 1107bp fragment.
(4.2.4) the overlap extension PCR product obtained in (4.2.3) above and pUC19 were digested with XbaI, and the two were ligated by T4-DNA ligase, and the ligated product was transformed into E.coli DH 5. alpha. competent cells, and recombinant plasmid pUC19-m1pe, which is a cloned mannitol-1-phosphatase gene expression cassette, was selected.
(4.3) cloning of concatemers of the mannitol-1-phosphate dehydrogenase gene and the mannitol-1-phosphatase gene:
(4.3.1) design a pair of primers mt1de 1: 5'-TACGAATTCAGTGCTTTAATTAGTG-3' and mt1de 2: 5'-CTTCTAGATAGAGGTACCTACTACTTTGCTG-3', and a 1371bp fragment was obtained by PCR using pUC19-mt1de as a template.
(4.3.2) after the PCR product obtained in (4.3.1) above and pUC19 were digested with EcoRI and XbaI, they were ligated by T4-DNA ligase, and the ligated product was transformed into E.coli DH 5. alpha. competent cells to select recombinant plasmid pUC19-mt1 d.
(4.3.3) design a pair of primers m1pe 1: 5'-CTGGTACCTTAGTAGAAAGTGCTT-3' and m1pe 2: 5'-CTGGTACCTTAGGGTTTAGCGTTTG-3', and 1123bp of fragment was obtained by PCR using pUC19-m1pe as a template.
(4.3.4) the PCR product obtained in (4.3.3) above and pUC19 were digested with KpnI, and then both were ligated by T4-DNA ligase, and the ligated product was transformed into E.coli DH 5. alpha. competent cells, and recombinant plasmid pUC19-mt1d-m1p, which is a cloned 1-phosphomannitol dehydrogenase gene and mannitol-1-phosphatase gene concatemer, was selected.
(4.4) construction of acetaldehyde dehydrogenase gene homologous recombination vector with 1-phosphomannitol dehydrogenase gene and mannitol-1-phosphatase gene concatemer in the middle: designing a pair of primers mpl: 5'-CGTCTAGAAGTGCTTTAATTAGTG-3' and mpr: 5'-TATCTAGATAGAGGTACCTTAGGGT-3', using pUC19-mt1d-m1p as a template, obtaining a 2469bp fragment by PCR amplification, connecting a PCR product to an XbaI site in the middle of a homologous recombination vector pUC19-aldhqh homologous arm by using T4 ligase, and naming a recombinant plasmid as pUC19-aldhqh-mt1d-m1p, namely constructing the acetaldehyde dehydrogenase gene homologous recombination vector with a 1-phosphomannitol dehydrogenase gene and a mannitol-1-phosphatase gene concatemer in the middle.
(4.5) construction of Leuconostoc mesenteroides mutant strains by Glucosucrase gene knock-out, D-lactate dehydrogenase gene knock-out, acetylphosphotransferase gene knock-out with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knock-out with mannitol dehydrogenase gene knock-in, fructokinase gene knock-out with mannitol dehydrogenase gene knock-in, and acetaldehyde dehydrogenase gene knock-out with mannitol 1-phosphate dehydrogenase gene and mannitol 1-phosphate gene knock-in: introducing pUC19-aldhqh-mt1D-m1p into Leuconostoc mesenteroides obtained in the third step by electrotransformation method, mdh delta stpk:: mdh delta fk:: mdh delta aldh:: amy (Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta kinase:: amy), screening to obtain Glucan sucrase gene knock-out, D-lactate dehydrogenase gene knock-out, acetyl phosphate transferase gene knock-out and mannitol dehydrogenase gene knock-in, serine/threonine protein kinase knock-out and mannitol dehydrogenase gene knock-in delta kinase gene and mannitol dehydrogenase knock-out and mannitol dehydrogenase gene knock-in of Leuconostoc mesenteroides found in strain obtained in the third step 1, and mannose phosphate knock-down delta kinase gene (Leuconostoc mesenteroides 1-5 < mu > separately from Leuconostoc mesenteroides obtained in the third step mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mt1d-m1p) ] strain.
Designing a pair of primers aldhyq: 5'-GCAGAGATATTAAACAAAA-3' and aldhey: 5'-TGGTGGAACATTACCTGAG-3', extracting chromosome DNA, and carrying out PCR by using the chromosome DNA as a template, wherein the Leuconostoc mesenteroides mutant strain, namely Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh delta pat: (mt1D-m1p) [ Leuconostoc mesenteroides delta 1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta afk:: mdh delta palatal Δ:: mt1D-m1p) ] strain obtains an amplification product with the length of 3417bp, and the Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat-mdh delta pfk-mdhk-delta fduk-delta leuton-delta leucotact (Lemendotock is accession number is 7545-3578 delta dhmdh delta.
Example 2
Leuconostoc mesenteroides delta dts1 delta ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mt1d-M1p, Leuconostoc mesenteroides delta dts1 delta ldh, mdh delta stpk, mdh delta fk, mdh delta aldh, mt1d-M1p, preserved in China Center for Type Culture Collection (CCTCC) with preservation date of 2018, 11 and 23 months and preservation number of CCTCC M2018815, namely the fermentation application of the high-yield mannitol Leuconostoc mesenteroides mutant strain of the invention comprises the following specific steps:
in a 250 ml triangular flask, Leuconostoc mesenteroides delta dts1 delta ldh delta pat with the preservation date of 11 and 23 days in 2018 and the preservation number of CCTCC M2018815 is mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mt1d-M1p) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk delta aldh:: mdh delta Δ aldh:: mt1d-M1p) ] strain is transferred to MRS culture medium by the weight percentage of 1%, and the concentration of mannitol in a shaking table can reach 20 hours at the rotating speed of 120 r/min under the temperature of 30 ℃, the conversion rate of mannitol in the sucrose can reach 9.73 g, and the conversion rate of fructose in the sucrose part reaches 97.3.97%.
The preparation method of the MRS culture medium comprises the following steps: 2 g of yeast extract powder, 90 g of cane sugar, 2 g of ammonium citrate, 5 g of sodium acetate and K2HPO42 g of MnSO4·H2MRS medium was prepared by adjusting the pH of O0.039 g and 1000 ml of water to 6.2 with acetic acid and sterilizing at 121 ℃ for 20 minutes.
Table 1 lists the output of mannitol produced by fermentation of various Leuconostoc strains, and the result shows that the yield of mannitol of the Leuconostoc mesenteroides delta dts1 delta ldh delta pat is improved in a region of 5-15% of the original preservation number of the Leuconostoc mesenteroides delta dhm Δ stpk (23 days 11 and 2018) and the preservation number of the Leuconostoc mesenteroides delta dts 2018815, mdh delta stpk:: mdh delta fk:: mdh delta aldh delta hdr:: mdh delta pat 1-M1 p) [ Leuconostoc mesenteroides dts delta 1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh aldh delta pat:: mdh delta [ mt 1-M1 p) ] is higher than the yield of the Leuconostoc mesenteroides delta dhm Δ dts Δ dt-5% of sucrose delta dhmdho Δ dhods Δ dh Δ pat-5% of the original preservation number of the Leuconostoc mesenteroides delta dhm culture collection number of the original preservation number of 2017578, and the yield of sucrose Δ dhmdho Δ dhmsk-5-delta dhmson Δ dh Δ.
TABLE 1 yield (g/L) of mannitol produced by fermentation of Leuconostoc mesenteroides
Figure GDA0001970146820000101
In Table 1, the original strain is original Leuconostoc mesenteroides which is not modified, the delta dts1 delta ldh delta pat-mdh delta stpk-mdh delta fk-mdh is a mutant strain of Leuconostoc mesenteroides with gene knockout of glucan sucrase, gene knockout of D-lactate dehydrogenase, gene knockout of acetyl phosphate transferase and gene knock-in of mannitol dehydrogenase, gene knock-out of serine/threonine protein kinase and gene knock-in of mannitol dehydrogenase, and the mutant strain of Leuconostoc mesenteroides with gene knock-in of fructose kinase is the mutant strain of Leuconostoc mesenteroides delta dts1 delta ldh delta pat-mdh delta stpk-mdh delta fk-mdh (Leuconostoc mesenteroides delta dts delta dt 1 delta ldh delta pat-mdh delta pk-mdh) and the delta ldds 539 delta ldd 3624 delta dhdhdhdhdhdpk-mdh delta dp 1 and gene knockout of glucan 1 delta dhdhdhdhmdh delta dp 54 A Leuconostoc mesenteroides mutant strain with acetyl phosphotransferase gene knock-out and mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knock-out and mannitol dehydrogenase gene knock-in, fructokinase gene knock-out and mannitol dehydrogenase gene knock-in and acetaldehyde dehydrogenase encoding gene knock-out and 1-phosphomannitol dehydrogenase encoding gene knock-in and mannitol-1-phosphatase encoding gene is Leuconostoc mesenteroides delta dts1 delta ldh delta pat: mdh Δ stpk: mdh Δ fk: mdh Δ aldh: (mtld-mlp) [ Leuconostoc mesenteroides Δ dts1 Δ ldh Δ pat: mdh Δ stpk: mdh Δ fk: mdh Δ aldh: (mtld-mlp) ] strain, the mutant strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 11 and 23 months in 2018 and the preservation number of CCTCC M2018815), namely the mutant strain of leuconostoc mesenteroides for high yield of mannitol is provided.
In the above examples, the raw materials, reagents and instruments are commercially available, and the procedures involved are those familiar to those skilled in the art and are not specifically described, and are generally performed according to conventional conditions, such as molecular cloning: methods described in the Experimental handbook "or protocols supplied by the manufacturer.
Sequence listing
Partial sequence deletion of acetaldehyde dehydrogenase gene and insertion of 1-phosphomannitol dehydrogenase gene expression cassette and nucleotide sequence of mannitol-1-phosphatase gene expression cassette
<110> Tianjin Boruiwei biomedical science and technology Co Ltd
<120> leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof
<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 leuconostoc mesenteroides mutant strain for high yield of mannitol is characterized in that: is a mutant strain of Leuconostoc mesenteroides with glucosan sucrase gene knockout, D-lactate dehydrogenase gene knockout, acetyl phosphotransferase gene knockout and mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knock-in and mannitol dehydrogenase gene knock-in, fructose kinase gene knock-out and mannitol dehydrogenase gene knock-in and acetaldehyde dehydrogenase encoding gene knock-out and 1-phosphate mannitol dehydrogenase encoding gene knock-in and mannitol-1-phosphatase encoding gene, and is Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta spk:: mdh delta fk:: mdh delta aldh delta Δ aldh:: mtld-mlp) [ Leuconostoc mesenteroides Delta dts1 delta ldh:: mdh delta stpk:: mdh delta dh delta mdh:: delta mdh: (preservation) (CCT-mlp) and culture date of China center for CCT 23 and 75, the preservation number is CCTCC No: m2018815.
2. The application method of the leuconostoc mesenteroides mutant strain capable of highly producing mannitol as claimed in claim 1 is characterized in that: in a 250 ml triangular flask, the culture is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 20187, 11 and 23 days and the preservation number of CCTCC No: m20187815 Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp) [ Leuconostoc mesenteroides delta dts1 delta ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh delta ldh: (mtld-mlp) ] strain was transferred to MRS medium in a weight percentage of 1%, and cultured in a shaker at a rotation speed of 120 rpm for 20 hours at 30 ℃ to reach a mannitol concentration of 47.3 g/l and a sucrose to mannitol conversion rate of 52.6%.
3. The application method of the leuconostoc mesenteroides mutant strain capable of highly producing mannitol according to claim 2, which is characterized in that: the preparation method of the MRS culture medium comprises the following steps: 2 g of yeast extract powder, 90 g of cane sugar, 2 g of ammonium citrate, 5 g of sodium acetate and K2HPO42 g of MnSO4·H2MRS medium was prepared by adjusting the pH of O0.039 g and 1000 ml of water to 6.2 with acetic acid and sterilizing at 121 ℃ for 20 minutes.
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