CN112852696A - 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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CN112852696A
CN112852696A CN202011466096.2A CN202011466096A CN112852696A CN 112852696 A CN112852696 A CN 112852696A CN 202011466096 A CN202011466096 A CN 202011466096A CN 112852696 A CN112852696 A CN 112852696A
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mannitol
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leuconostoc mesenteroides
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金红星
隋燕妮
成文玉
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Hebei University of Technology
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Abstract

The invention discloses a Leuconostoc mesenteroides mutant strain for high yield of mannitol and an application method thereof, and relates to bacteria, wherein the strain is Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386, ldhA-ee delta adh, mep delta ldh (0503), mep delta ldh (0373), mep strain, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2020, 11 and 19 months and the preservation number of CCTCC No: m2020762. 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 71.58 g/L, and the conversion rate of sucrose to mannitol is 59.65 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 concentration for multiple times, impurity removal, ion exchange, evaporation 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 a 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. After entering leuconostoc cells, cheap sucrose is decomposed into glucose-1-phosphate and fructose, 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.
CN2018115455031 discloses a leuconostoc mesenteroides mutant strain for high-yield mannitol and an application method thereof, wherein the leuconostoc mesenteroides mutant strain is a leuconostoc mesenteroides mutant strain of glucansucrase and D-lactate dehydrogenase gene knockout, acetyl phosphotransferase gene knockout and knock-in of a mannitol dehydrogenase gene, serine/threonine protein kinase gene knockout and knock-in of a mannitol dehydrogenase gene, fructokinase gene knockout and knock-in of a mannitol dehydrogenase gene, acetaldehyde dehydrogenase gene knockout and knock-in of a 1-phosphate mannitol dehydrogenase encoding gene and a mannitol-1-phosphatase encoding gene concatemer, and although the yield is improved compared with that of an initial strain, the leuconostoc mesenteroides 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 (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat with the preservation number of CCTCC M2018815 as a starting strain, and adopts the molecular biology technology to insert an expression element of a D-lactate dehydrogenase gene (ldhA) between 2 coding frames of a coding gene of a mannitol efflux pump (0385/0386), inactivate an ethanol dehydrogenase coding gene and knock in the mannitol efflux pump gene expression cassette, inactivate a lactate dehydrogenase coding gene (0503) and knock in the mannitol efflux pump gene expression cassette, inactivate a lactate dehydrogenase coding gene (0373) and knock in the mannitol pump gene expression cassette, and construct a glucan sucrose removal saccharose gene which is a glucan removal saccharose cassette, D-lactate dehydrogenase gene (1756) knockout, acetylphosphotransferase gene knockout with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knock-in, fructokinase gene knock-in with mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knock-out with mannitol 1-phosphate dehydrogenase encoding gene and mannitol 1-phosphatase encoding gene concatemer knock-in, insertion of a D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of the mannitol efflux pump (0385/0386) encoding gene, ethanol dehydrogenase gene knock-out with mannitol efflux pump gene expression cassette knock-in, lactate dehydrogenase gene (0503) knock-out with mannitol efflux pump gene expression cassette knock-in, and lactate dehydrogenase gene (0373) knock-out with mannitol efflux pump gene expression cassette knock-in, namely, the Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386 with the preservation number of CCTCC M2020762, ldhA-ee delta adh, mep delta ldh (0503), mep delta ldh (0373), mep strain, overcomes the defect that the yield of mannitol produced by taking sucrose as a substrate in the existing Leuconostoc fermentation technology is still not high enough.
The technical scheme adopted by the invention for solving the technical problem is as follows: a Leuconostoc mesenteroides mutant strain for high yield of mannitol is obtained by knocking out a dextransucrase gene, knocking out a D-lactate dehydrogenase gene (1756), knocking out an acetyl phosphate transferase gene and knocking in a mannitol dehydrogenase gene, knocking out a serine/threonine protein kinase gene and knocking in a mannitol dehydrogenase gene, knocking out a fructose kinase gene and knocking in a mannitol dehydrogenase gene, knocking out an acetaldehyde dehydrogenase gene and knocking in a 1-phosphate mannitol dehydrogenase coding gene and a mannitol-1-phosphatase coding gene concatemer, inserting a D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of a mannitol efflux pump (0385/0386) coding gene, knocking in an ethanol dehydrogenase gene and a mannitol efflux pump gene expression cassette, knocking in a lactate dehydrogenase gene (3) and a mannitol efflux pump gene expression cassette, and knocking in a lactate dehydrogenase gene (0373) The Leuconostoc mesenteroides mutant strain knocked into the alcohol efflux pump gene expression cassette is Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386, ldhA-ee delta adh, mep delta ldh (0503), mep delta ldh (0373), mep strain, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2020, 11 and 19 days, the preservation number is CCTCC M2020762, and the preservation unit address is university of Wuhan, Wuhan.
An application method of a Leuconostoc mesenteroides mutant strain for high yield of mannitol comprises the steps of storing the Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386 in a 250 ml triangular flask in a Chinese typical culture collection center (CCTCC) with the preservation date of 2020, 11 and 19 months and the preservation number of CCTCC M2020762, wherein ldhA-ee delta adh comprises mep delta ldh (0503) and mep delta ldh (0373) wherein the mep strain is transferred into an MRS culture medium by 1 percent by weight and cultured for 20 hours at 30 ℃ by a shaking table with the rotating speed of 120 revolutions per minute, the concentration of the mannitol can reach 71.58 g/l, and the conversion rate of sucrose to the mannitol is 59.65 percent.
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, 120 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 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 insert a D-lactate dehydrogenase gene (ldhA) expression element, inactivate and knock in an ethanol dehydrogenase coding gene, inactivate and knock in a mannitol efflux pump gene expression box, inactivate and knock in a lactate dehydrogenase coding gene (0503) and knock in a mannitol efflux pump gene expression box, inactivate and knock in a mannitol efflux pump gene expression box and inactivate a lactate dehydrogenase coding gene (0503) and knock in a mannitol efflux pump gene expression box and inactivate a lactate dehydrogenase coding gene (0373) and knock in a mannitol efflux pump gene expression box in the existing Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat with the preservation number of CCTCC M2018815 to construct a glucan sucrose knock-out gene, knock-out a D-lactate dehydrogenase gene (1756), knock-out and knock in a mannitol dehydrogenase gene expression box, Serine/threonine protein kinase gene knock-out and mannitol dehydrogenase gene knock-in, fructokinase gene knock-out and mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knock-out and 1-phosphomannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding gene concatemer knock-in, D-lactate dehydrogenase gene (hA) expression element is inserted between 2 coding frames of mannitol efflux pump (0385/0386) encoding gene, alcohol dehydrogenase gene knock-out and mannitol efflux pump gene expression cassette knock-in, lactate dehydrogenase gene (0503) knock-out and mannitol efflux pump gene expression cassette knock-in and lactate dehydrogenase gene (0373) knock-out and mannitol efflux pump gene expression cassette knock-in, Leuconostoc mesenteroides mutant strain, i.e., Leuconostoc mesenteroides (Leuconostoc mesenteroides) with preservation number CCTCC M2020762, CCTCC M20150385/0386: (hA-ee:. DELTA.: mep 0500503:. delta. medium: (delta. 0503): Δ mep:. DELTA. medium: (Δ 050881::::: (D) ldh (0373):mep [ delta dts1 delta D-ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp)0385/0386:: ldhA-ee delta adh:: mep delta ldh (0503): mep delta ldh (0373): mep ] strain, overcoming the defect that the yield of the glycerol produced by using sucrose as a substrate in the existing leuconostoc fermentation technology is still not high enough.
(2) The strain is transferred to a delta-MRS medium at the temperature of 30 ℃ and the initial yield of the strain is measured by a shaking table at the speed of 120 hours/min, the initial yield of the strain is measured by a fermentation test of 20-year fermentation of 20-year- Δ adh: (20-year- Δ adh:: mep: (mep- Δ ldh) (0373): mdh:: mep- Δ dh: (mtld-mlp)0385/0386: (mtld-26:: mtld-ha-ee:: mdh: (mlp) of 1 wt% of 1- Δ dhidh strain is transferred to Δ MRS medium by weight percent, and the initial yield of 20-year- Δ ldh Δ pat:: mdh Δ stpk:: mdh Δ fk:: mdh Δ aldh:: mtld-mlp) ] increased by 51.5% and the conversion of sucrose to mannitol increased by 20.27%.
(3) In the application method, the sucrose substrate quality of the MRS culture medium can reach 120 g, the high yield of mannitol can be still ensured, the utilization rate of sucrose is improved, and the industrial popularization and application are facilitated.
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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: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0385/0386 agarose gel electrophoresis of left and right homologous arms in gene homologous recombination vector.
FIG. 2 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0385/0386 gene homologous recombination vector homology arm restriction enzyme digestion verification agarose gel electrophoresis chart.
FIG. 3 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of 0385/0386 gene homologous recombination vector with alpha-amylase marker in the middle.
FIG. 4 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0386 gene expression cassette part sequence, and agarose gel electrophoresis of the initial part sequence of D-lactate dehydrogenase gene (ldhA) expression element and 0386 gene.
FIG. 5 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0386 gene expression cassette partial sequence restriction enzyme digestion verification agarose gel electrophoresis chart.
FIG. 6 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain agarose gel electrophoresis of left and right homologous arms in 0385/0386 gene homologous recombinant vector with D-lactate dehydrogenase gene (ldhA) expression element in the middle.
FIG. 7 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of 0385/0386 gene homologous recombination vector with D-lactate dehydrogenase gene (ldhA) expression element in the middle.
FIG. 8 shows that the preservation number of the invention is CCTCC No: construction of Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386 of M2020762 (ldhA-ee. DELTA. adh): (mep. DELTA. ldh (0503): mep. DELTA. ldh (0373): agarose gel electrophoresis pattern of left and right homologous arms in the gene homologous recombination vector of the mep strain).
FIG. 9 shows that the preservation number of the invention is CCTCC No: construction of an alcohol dehydrogenase gene homologous recombination vector homology arm of Leuconostoc mesenteroides of M2020762 CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): restriction enzyme digestion verification agarose gel electrophoresis image of the mep strain.
FIG. 10 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of an ethanol dehydrogenase gene homologous recombination vector with alpha-amylase marker in the middle.
FIG. 11 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of an alcohol dehydrogenase gene homologous recombination vector with a mannitol efflux pump gene expression cassette in the middle.
FIG. 12 shows that the preservation number of the invention is CCTCC No: construction of Leuconostoc mesenteroides (Leuconostoc mesenteroides) M2020762 CCTCC M20188150385/0386: ldhA-ee. DELTA. adh: mep. DELTA. ldh (0503): mep. DELTA. ldh (0373): construction of mep Strain lactate dehydrogenase gene (0503) agarose gel electrophoresis of the left and right homologous arms in the homologous recombination vector.
FIG. 13 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain lactate dehydrogenase gene (0503) restriction enzyme digestion verification agarose gel electrophoresis picture of homologous recombination vector homology arm.
FIG. 14 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of homologous recombination vector with alpha-starch enzyme labeled lactate dehydrogenase gene (0503) in the middle.
FIG. 15 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of a lactate dehydrogenase gene (0503) homologous recombination vector with a mannitol efflux pump gene expression cassette in the middle.
FIG. 16 shows that the preservation number of the invention is CCTCC No: construction of Leuconostoc mesenteroides (Leuconostoc mesenteroides) M2020762 CCTCC M20188150385/0386: ldhA-ee. DELTA. adh: mep. DELTA. ldh (0503): mep. DELTA. ldh (0373): construction of mep Strain lactate dehydrogenase gene (0373) agarose gel electrophoresis of left and right homologous arms in the homologous recombination vector.
FIG. 17 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain lactate dehydrogenase gene (0373) restriction enzyme digestion verification agarose gel electrophoresis picture of homologous recombination vector homology arm.
FIG. 18 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of the homologous recombination vector with alpha-amylase labeled lactate dehydrogenase gene (0373) in the middle.
FIG. 19 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of a lactate dehydrogenase gene (0373) homologous recombination vector with a mannitol efflux pump gene expression cassette in the middle.
Detailed Description
FIG. 1 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0385/0386 agarose gel electrophoresis of left and right homologous arms in gene homologous recombination 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: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0385/0386 agarose gel electrophoresis of homologous recombination vector homology arm. Two bands generated by cleavage of the recombinant vector are shown: 1. and (3) cutting a band by using a recombinant vector double enzyme, and 2. Marker.
FIG. 3 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and agarose gel electrophoresis image of 0385/0386 gene homologous recombination vector with alpha-amylase marker 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. 4 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0386 gene expression cassette part sequence, and agarose gel electrophoresis of the initial part sequence of D-lactate dehydrogenase gene (ldhA) expression element and 0386 gene. Left and right homology arms are shown: left of the expression element of the D-lactate dehydrogenase gene (ldhA), 2.Marker, 3.0386 gene start part sequence.
FIG. 5 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain 0386 gene expression cassette partial sequence restriction enzyme digestion verification agarose gel electrophoresis chart. Two bands generated by cleavage of the recombinant vector are shown: 1. and (3) cutting a band by using a recombinant vector double enzyme, and 2. Marker.
FIG. 6 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain agarose gel electrophoresis of left and right homologous arms in 0385/0386 gene homologous recombinant vector with D-lactate dehydrogenase gene (ldhA) expression element in the middle. Left and right homology arms are shown: the device comprises a Marker 1, a right homology arm 2 and a left homology arm 3.
FIG. 7 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of 0385/0386 gene homologous recombination vector with D-lactate dehydrogenase gene (ldhA) expression element in the middle. Two bands generated by cleavage of the recombinant vector are shown: 1. double enzyme digestion bands of the recombinant vector, 2. Marker.
FIG. 8 shows that the preservation number of the invention is CCTCC No: construction of Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386 of M2020762 (ldhA-ee. DELTA. adh): (mep. DELTA. ldh (0503): mep. DELTA. ldh (0373): agarose gel electrophoresis pattern of left and right homologous arms in the gene homologous recombination vector of the mep strain). Left and right homology arms are shown: 1. left arm of homology, 2.Marker, 3. right arm of homology.
FIG. 9 shows that the preservation number of the invention is CCTCC No: construction of an alcohol dehydrogenase gene homologous recombination vector homology arm of Leuconostoc mesenteroides of M2020762 CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): restriction enzyme digestion verification agarose gel electrophoresis image of the mep strain. Two bands generated by cleavage of the recombinant vector are shown: 1. recombinant vector double enzyme digestion strip, 2. Marker.
FIG. 10 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of an ethanol dehydrogenase gene homologous recombination vector with alpha-amylase marker in the middle. Two bands generated by cleavage of the recombinant vector are shown: 1. double enzyme digestion bands of the recombinant vector, 2. Marker.
FIG. 11 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of an alcohol dehydrogenase gene homologous recombination vector with a mannitol efflux pump gene expression cassette in the middle. The figure shows two bands generated by enzymatic cleavage of the recombinant vector: 1. double enzyme digestion bands of the recombinant vector, 2. Marker.
FIG. 12 shows that the preservation number of the invention is CCTCC No: construction of Leuconostoc mesenteroides (Leuconostoc mesenteroides) M2020762 CCTCC M20188150385/0386: ldhA-ee. DELTA. adh: mep. DELTA. ldh (0503): mep. DELTA. ldh (0373): construction of mep Strain lactate dehydrogenase gene (0503) agarose gel electrophoresis of the left and right homologous arms in the homologous recombination vector. Left and right homology arms are shown: 1. left arm of homology, 2.Marker, 3. right arm of homology.
FIG. 13 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain lactate dehydrogenase gene (0503) restriction enzyme digestion verification agarose gel electrophoresis picture of homologous recombination vector homology arm. Two bands generated by cleavage of the recombinant vector are shown: 1, Marker, 2, double enzyme digestion of the recombinant vector.
FIG. 14 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of homologous recombination vector with alpha-starch enzyme labeled lactate dehydrogenase gene (0503) in the middle. Two bands generated by cleavage of the recombinant vector are shown: 1. double enzyme digestion bands of the recombinant vector, 2. Marker.
FIG. 15 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of a lactate dehydrogenase gene (0503) homologous recombination vector with a mannitol efflux pump gene expression cassette in the middle. Two bands generated by cleavage of the recombinant vector are shown: 1. double enzyme digestion bands of the recombinant vector, 2. Marker.
FIG. 16 shows that the preservation number of the invention is CCTCC No: construction of Leuconostoc mesenteroides (Leuconostoc mesenteroides) M2020762 CCTCC M20188150385/0386: ldhA-ee. DELTA. adh: mep. DELTA. ldh (0503): mep. DELTA. ldh (0373): construction of mep Strain lactate dehydrogenase gene (0373) agarose gel electrophoresis of left and right homologous arms in the homologous recombination vector. Left and right homology arms are shown: 1. left homology arm, 2. right homology arm, 3. Marker.
FIG. 17 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain lactate dehydrogenase gene (0373) restriction enzyme digestion verification agarose gel electrophoresis picture of homologous recombination vector homology arm. Two bands generated by cleavage of the recombinant vector are shown: 1, Marker, 2, double enzyme digestion of the recombinant vector.
FIG. 18 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of the homologous recombination vector with alpha-amylase labeled lactate dehydrogenase gene (0373) in the middle. Two bands generated by cleavage of the recombinant vector are shown: 1. double enzyme digestion bands of the recombinant vector, 2. Marker.
FIG. 19 shows that the preservation number of the invention is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): construction of mep strain and enzyme digestion verification of a lactate dehydrogenase gene (0373) homologous recombination vector with a mannitol efflux pump gene expression cassette in the middle. Two bands generated by cleavage of the recombinant vector are shown: 1. double enzyme digestion bands of the recombinant vector, 2. Marker.
Example 1
Constructing a variant strain of Leuconostoc mesenteroides with the gene knockout of dextransucrase, the gene knockout of D-lactate dehydrogenase, the gene knockout of acetyl phosphotransferase and the knock-in of mannitol dehydrogenase, the gene knockout of serine/threonine protein kinase and the knock-in of mannitol dehydrogenase, the gene knockout of fructose kinase and the knock-in of mannitol dehydrogenase, the gene knockout of acetaldehyde dehydrogenase and the knock-in of 1-phosphomannitol dehydrogenase coding gene and the concatemer of mannitol-1-phosphatase coding gene, the insertion of an expression element of D-lactate dehydrogenase gene (ldhA) between 2 coding frames of the coding gene of a mannitol efflux pump (0385/0386), the gene knockout of ethanol dehydrogenase and the knock-in of the mannitol efflux pump gene, the gene knockout of lactate dehydrogenase (0503) and the knock-in of the expression cassette of the mannitol efflux pump and the knock-in of the expression cassette of the lactate dehydrogenase (0373) The method comprises the following specific steps:
in the first step, with the existing Leuconostoc mesenteroides (leuconosteroids) delta dts1 delta D-ldh delta pat with the preservation number of CCTCC M2018815: mdh Δ stpk: mdh Δ fk: mdh Δ aldh: (mtld-mlp) is a starting bacterium, and a Leuconostoc mesenteroides mutant strain with a glucan sucrase gene knockout, a D-lactate dehydrogenase gene knockout, an acetyl phosphotransferase gene knockout and mannitol dehydrogenase gene knock-in, a serine/threonine protein kinase gene knockout and mannitol dehydrogenase gene knock-in, a fructose kinase gene knockout and mannitol dehydrogenase gene knock-in, an acetaldehyde dehydrogenase gene knockout and 1-phosphomannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding gene concatemer knock-in, and a D-lactate dehydrogenase gene (ldhA) expression element inserted between 2 coding frames of a mannitol efflux pump (0385/0386) encoding gene is constructed:
(1.1) cloning of the full-length sequence of the encoding gene of the leuconostoc mesenteroides mannitol efflux pump (0385/0386):
the method is characterized in that chromosomal DNA is taken as a template, and the coding gene of a mannitol efflux pump (0385/0386) coding gene with the preservation number of CCTCC M2018815 is cloned into a whole-length continuous sequence of Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat:: mdh delta stpk:: mdh delta fk: (mtld-mlp) [ preservation date is 12 and 8 days in 2018, which is preserved in China Center for Type Culture Collection (CCTCC) and has the preservation number of CCTCC M2018815], wherein the specific operation steps are as follows:
(1.1.1) extraction of Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat with a preservation number of CCTCC M2018815:: mdh delta fk:: mdh delta aldh:: mtld-mlp) total DNA of Leuconostoc mesenteroides:
leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat with the preservation number of CCTCC M2018815 frozen at-80 ℃ is streaked on MRS solid plates and cultured at 30 ℃ overnight, wherein the strain is mdh delta stpk: (mdh delta fk:: mdh delta aldh: (mtld-mlp); selecting a single colony from a solid plate, inoculating the single colony into 5 ml of MRS liquid culture medium, and culturing the single colony 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 mycelia 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.
(1.1.2) PCR amplification of the full-length sequence of the mannitol efflux pump (0385/0386) coding gene:
design a pair of primers 0385/0386 l: 5'-CGGGTACCTAAATATATGAATGAT-3' and 0385/0386 r: 5'-ATGGTACCGTTGATTTTTACTATG-3', taking the total DNA of the Leuconostoc mesenteroides with the preservation number of CCTCC M2018815 (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh:: mtld-mlp) as a template, carrying out PCR amplification to obtain a 3708bp fragment, and connecting the PCR product to a KpnI site of a pUC19 vector by using T4DNA ligase, wherein the recombinant plasmid is named as pUC 19-0385/0386.
(1.1.3) preparation and DNA transformation of competent E.coli DH5 α:
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 to 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 were suspended in solution, i.e., as 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 culture 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 sterilization at 121 ℃ for 20 minutes. Solid medium plus 1.5% agar.
(1.2) construction of 0385/0386 gene homologous recombination vector with alpha-amylase mark in the middle:
(1.2.1) design a pair of primers 0385/0386l 1: 5'-GCGTCTAGAAGAGGTGTTGTTG-3' and 0385/0386l 2: 5'-TATTGGGTACCACTCGTGATGTGC-3' (complementary pair to 0385/0386r 1), and 217bp fragment was obtained by PCR using pUC19-0385/0386 as a template.
(1.2.2) design a pair of primers 0385/0386r 1: 5'-CGAGTGGTACCCAATAGCCTCAAC-3' and 0385/0386r 2: 5'-CCCGAATTCCTGAACTAATACG-3', a 219bp fragment was obtained by PCR using pUC19-0385/0386 as a template.
(1.2.3) the 2 PCR products obtained in (1.2.1) and (1.2.2) above were purified and mixed, and 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 PCR using a pair of primers 0385/0386l 1: 5'-GCGTCTAGAAGAGGTGTTGTTG-3' and 0385/0386r 2: 5'-CCCGAATTCCTGAACTAATACG-3' PCR was performed to obtain a 420bp fragment.
(1.2.4) carrying out double digestion on the overlap extension PCR product obtained in the step (1.2.3) and pUC19 by EcoRI and XbaI, then connecting the two products under the action of T4-DNA ligase, transforming the connected product into escherichia coli DH5 alpha competent cells, and screening a recombinant plasmid pUC19-0385/0386qh to construct a homologous recombinant vector.
(1.2.5) design a pair of primers amyl: 5'-CTTGGTACCTTTGGCGTGATTATCAG-3' and amyr: 5'-TCAGGTACCCGAAGGTGAAGTTATAG-3', using total DNA of the Lactobacillus amylovorus with the preservation number of CGMCC1.3395 as a template, carrying out PCR amplification to obtain a 2131bp fragment, connecting a PCR product to a KpnI site in the middle of a homologous recombination vector pUC19-adhqh homologous arm by using T4-DNA ligase, and naming the recombinant plasmid as pUC19-0385/0386qh-amy, namely constructing the 0385/0386 gene homologous recombination vector with an alpha-amylase mark in the middle.
(1.3) construction of Leuconostoc mesenteroides mutant strain with Glucosansucrase gene knockout, D-lactate dehydrogenase gene knockout, acetylphosphotransferase gene knockout with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knockout with mannitol dehydrogenase gene knock-in, fructokinase gene knockout with mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knockout with 1-phosphomannitol dehydrogenase coding gene and mannitol-1-phosphatase coding gene concatemer knock-in, and alpha-amylase gene expression cassette inserted between 2 coding cassettes of mannitol efflux pump (0385/0386) coding gene:
leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat with the preservation number of CCTCC M2018815 frozen at-80 ℃ is streaked on MRS solid plates and cultured at 30 ℃ overnight, wherein the strain is mdh delta stpk: (mdh delta fk:: mdh delta aldh: (mtld-mlp); selecting a single colony from the 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 for overnight; the culture was continued by 1% transfer to MRS medium containing 0.48. mu.g/ml ampicillin, starting OD6000.048, preservation number of CCTCC M2018815, namely the OD of the Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat:: mdh delta fk:: mdh delta aldh: (mtld-mlp) strain600Collecting thallus when the concentration reaches 0.5, resuspending thallus with LiAc-DTT solution containing 100U/ml lysozyme, 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-adhqh-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 milli-seconds, and then 1 ml of the electric shock cup is addedAn MRS culture medium is applied to a solid plate containing MRS after being recovered for 3h, a single colony is selected after being cultured for 120h for verification, and the plate is screened to obtain a mutant strain of Leuconostoc mesenteroides, i.e. Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat, mdh delta stpk:: mdh delta fhm (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat: mdh delta stpk:: mdh delta dhaldr: (Leuconostoc mesenteroides) delta (Deltadts) delta) and mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knock-in, fructose kinase gene knock-out and fructose dehydrogenase gene knock-in, knock-in and mannitol dehydrogenase gene 2 coding frames of the coding gene of the Leuconosthol (0385/0386) are inserted between 2 mlp)0385/0386, amy strain.
The LiAc-DTT solution is a solution prepared from 100 mmol/L LiAc, 10 mmol/L DTT, 0.6 mol/L sucrose, and 10 mmol/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.
(1.4) construction of Leuconostoc mesenteroides mutant strain having expression element of D-lactate dehydrogenase gene (ldhA) inserted between 2 coding frames of gene encoding mannitol efflux pump (0385/0386):
(1.4.1) construction of a part sequence of 0386 gene expression cassette:
(1.4.1.1) designing a pair of primers ldhA-eel: 5'-CCGTCTAGATAGAAAGTGCTTTAAT-3' and ldhA-eer: 5'-TCTGTCATATCCTCCAAAATTTTA-3' (complementary and matched with 0386 l), and obtaining a 179bp fragment by PCR amplification with the leuconostoc mesenteroides chromosome DNA with the preservation number of CGMCC 1.2138 as a template.
(1.4.1.2) design of a pair of primers 0386 l: 5'-TGGAGGATATGACAGATATTAAAC-3' and 0386 r: 5'-CAAGAATTCCTAAGTTCAACAATGA-3', a 811bp fragment was obtained by PCR using pUC19-0385/0386 as a template.
(1.4.1.3) the 2 PCR products obtained in (1.4.1.1) (1.4.1.2) 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 DNA fragments were amplified using a pair of primers ldhA-eel: 5'-CCGTCTAGATAGAAAGTGCTTTAAT-3' and 0386 r: 5'-CAAGAATTCCTAAGTTCAACAATGA-3' PCR was performed to obtain a 974bp fragment.
(1.4.1.4) the double-digested PCR product obtained in (1.4.1.3) and pUC19 were ligated by T4-DNA ligase with XbaI and EcoRI, and the ligated product was transformed into E.coli DH 5. alpha. competent cells to select a recombinant plasmid pUC19-ldhA-ee/0386, that is, a 0386 gene expression cassette was constructed.
(1.4.2) construction of 0385/0386 Gene homologous recombination vector having expression element of D-lactate dehydrogenase Gene (ldhA) in the middle:
(1.4.2.1) design a pair of primers 0385 l: 5'-ATATCTAGAGTCAAAAACAACGTCT-3' and 0385 r: 5'-ACTTTCTATTATTCTGCCCCTTTC-3' (complementary pair to ldhA-ee/0386 l) and 308bp fragment was obtained by PCR using pUC19-0385/0386 as a template.
(1.4.2.2) design a pair of primers ldhA-ee/0386 l: 5'-CAGAATAATAGAAAGTGCTTTAAT-3' and ldhA-ee/0386 r: 5'-ATAGAATTCCCTTGCGTAATAATAA-3', and 862bp of fragment was obtained by PCR using pUC19-ldhA-ee/0386 as a template.
(1.4.2.3) the 2 PCR products obtained in (1.4.2.1) (1.4.2.2) 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 amplified using a pair of primers 0385 l: 5'-ATATCTAGAGTCAAAAACAACGTCT-3' and ldhA-ee/0386 r: 5'-ATAGAATTCCCTTGCGTAATAATAA-3' PCR was performed to amplify a 1154bp fragment.
(1.4.2.4) the double-digested PCR product obtained in (1.4.2.3) and pUC19 were ligated by T4-DNA ligase with XbaI and EcoRI, and the ligated product was transformed into E.coli DH 5. alpha. competent cells to select recombinant plasmid pUC19-0385/ldhA-ee/0386, i.e., 0385/0386 gene homologous recombination vector having an expression element of D-lactate dehydrogenase gene (ldhA) in the middle.
(1.4.3) construction of a Leuconostoc mesenteroides mutant strain in which a D-lactate dehydrogenase gene (ldhA) expression element was inserted between 2 coding frames of a glucan sucrase gene knock-out, a D-lactate dehydrogenase gene knock-out, an acetyl phosphotransferase gene knock-in and a mannitol dehydrogenase gene knock-in, a serine/threonine protein kinase gene knock-out and a mannitol dehydrogenase gene knock-in, a fructokinase gene knock-out and a mannitol dehydrogenase gene knock-in, an acetaldehyde dehydrogenase gene knock-out and a mannitol dehydrogenase 1-phosphate dehydrogenase coding gene concatemer knock-in, and a mannitol efflux pump (0385/0386): in the strain of Leuconostoc mesenteroides obtained in the above (1.3), pUC19-0385/ldhA-ee/0386 was introduced by electrotransformation into Δ dts1 Δ D-ldh Δ pat of mdh Δ stpk: mdh Δ fk: (mdh Δ aldh:) (mtld-mlp)0385/0386, and screening was performed to obtain a gene knockout of dextransucrase, a gene knockout of D-lactate dehydrogenase, a gene knockout of acetyl phosphate transferase and mannitol dehydrogenase, a gene knockout of serine/threonine protein kinase and mannitol dehydrogenase, a gene knockout of fructose kinase and mannitol dehydrogenase, a gene knockout of acetaldehyde dehydrogenase and 1-phosphate mannitol dehydrogenase, a gene knockout of mannitol dehydrogenase and a gene concatemer of mannitol phosphate, a knockout of mannitol and a concatemer of mannitol phosphate, a knockout of mannitol and 2 coding gene of mannitol coding gene (D-lactate dehydrogenase) (insert of D-lactate dehydrogenase) between 2 coding gene concatemers of mannitol coding gene (0385/0386) ) A mutant strain of Leuconostoc mesenteroides expressing the elements, namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat:: mdh delta stpk:: mdh delta fk: (mtld-mlp)0385/0386:: ldhA-ee strain.
Secondly, constructing glucan sucrase gene knockout, D-lactate dehydrogenase gene knockout, acetyl phosphate transferase gene knockout and mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knockout and mannitol dehydrogenase gene knock-in, fructose kinase gene knockout and mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knockout and 1-phosphomannitol dehydrogenase gene concatemer knock-in, D-lactate dehydrogenase (hA) expression element insertion between 2 coding frames of mannitol pump (0385/0386) coding gene efflux, and the like by taking the existing Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat:: mdh delta fk: (mtld-mlp) with the preservation number of CCTCC M2018815 as a starting bacterium, Leuconostoc mesenteroides mutant strain with alcohol dehydrogenase gene knockout and mannitol efflux pump coding gene expression cassette knock-in:
(2.1) cloning of partial sequence of Leuconostoc mesenteroides alcohol dehydrogenase gene:
designing a pair of primers adhl: 5'-AGAGGTGCTGTTGTTCGT-3' and adhr: 5'-TGGGTGTGGTGTAAAATC-3', PCR was carried out using Leuconostoc mesenteroides (Leuconostoc mesenteroides) Δ dts1 Δ D-ldh Δ pat:: mdh Δ stpk:: mdh Δ fk:: mdh Δ aldh: (mtld-mlp)0385/0386:: Leuconostoc mesenteroides chromosomal DNA of hA-ee as a template to obtain a 1033bp fragment, and the PCR product was ligated to T vector pTA2 using T4DNA ligase, and the recombinant plasmid was named pTA 2-adh.
(2.2) construction of an ethanol dehydrogenase gene homologous recombination vector with an alpha-amylase mark in the middle:
(2.2.1) design a pair of primers adhl 1: 5'-AGGTCGACTGATGGTTATG-3' and adhl 2: 5'-ACACCGGTACCACAGGGTCTA-3' (complementary pair to adhr 1), and a 442bp fragment was obtained by PCR using pTA2-adh as a template.
(2.2.2) design a pair of primers adhr 1: 5'-CCTGTGGTACCGGTGTTACAA-3' and adhr 2: 5'-GCGGAATTCACTGTCTCAA-3', using pTA2-adh as a template, a fragment of 508bp was obtained by PCR amplification.
(2.2.3) the 2 PCR products obtained in (2.2.1) and (2.2.2) above were purified and mixed, and 2 gene fragments were extended by 8 cycles of PCR using the PCR product mixture as a template, and then the PCR products were amplified using a pair of primers adhl 1: 5'-AGGTCGACTGATGGTTATG-3' and adhr 2: 5'-GCGGAATTCACTGTCTCAA-3' PCR was performed to amplify a 923bp fragment.
(2.2.4) carrying out double digestion on the overlap extension PCR product obtained in the step (2.2.3) and pUC19 by EcoRI and SalI, then connecting the two products under the action of T4-DNA ligase, transforming the connected product into escherichia coli DH5 alpha competent cells, and screening a recombinant plasmid pUC19-adhqh to construct a homologous recombinant vector.
(2.2.5) design a pair of primers amyl: 5'-CTTGGTACCTTTGGCGTGATTATCAG-3' and amyr: 5'-TCAGGTACCCGAAGGTGAAGTTATAG-3', using total DNA of the Lactobacillus amylovorus with the preservation number of CGMCC1.3395 as a template, carrying out PCR amplification to obtain a 2131bp fragment, connecting a PCR product to a KpnI site in the middle of a homologous recombination vector pUC19-adhqh homologous arm by using T4DNA ligase, and naming a recombinant plasmid as pUC19-adhqh-amy, namely constructing the ethanol dehydrogenase gene homologous recombination vector with alpha-amylase mark in the middle.
(2.3) construction of dextransucrase gene knockout, D-lactate dehydrogenase gene knockout, acetylphosphotransferase gene knockout with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knockout with mannitol dehydrogenase gene knock-in, fructokinase gene knockout with mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knockout with 1-phosphomannitol dehydrogenase coding gene and mannitol-1-phosphatase coding gene concatemer knock-in, insertion of a D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of a mannitol efflux pump (0385/0386) coding gene, ethanol dehydrogenase gene-inactivated Leuconostoc mesenteroides mutant strain:
the homologous recombination vector of the alcohol dehydrogenase gene with the alpha-amylase mark in the middle obtained in (2.2.5) is introduced into Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat mdh delta stpk < mdh delta f fk < mdh delta > aldh < mtld-mlp > 0385/0386 < ldhA-ee strain by an electric shock transformation method, and glucan 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, acetaldehyde dehydrogenase gene knockout and 1-phosphate mannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding concatemer knock-in, and, D-lactate dehydrogenase gene (ldhA) expression elements are inserted among 2 coding frames of the coding gene of the mannitol efflux pump (0385/0386), and the mutant strain of the Leuconostoc mesenteroides with inactivated alcohol dehydrogenase gene, namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp, 0385/0386, ldhA-ee delta adh, amy strain.
(2.4) construction of a Leuconostoc mesenteroides mutant strain in which a glucanosucrase gene is knocked out, a D-lactate dehydrogenase gene is knocked out, an acetylphosphotransferase gene is knocked out and a mannitol dehydrogenase gene is knocked in, a serine/threonine protein kinase gene is knocked out and a mannitol dehydrogenase gene is knocked in, a fructokinase gene is knocked out and a mannitol dehydrogenase gene is knocked in, an acetaldehyde dehydrogenase gene is knocked out and a 1-phosphate mannitol dehydrogenase coding gene concatemer is knocked in, a D-lactate dehydrogenase gene (ldhA) expression element is inserted between 2 coding frames of a mannitol efflux pump (0385/0386) coding gene, and an ethanol dehydrogenase gene is knocked out and a mannitol efflux pump coding gene expression cassette is knocked in:
(2.4.1) construction of an alcohol dehydrogenase gene homologous recombination vector with a mannitol efflux pump coding gene expression cassette in the middle: designing a pair of primers ee/0385-ee/0386 l: 5'-GCGGTACCTAATTTTAAATATATG-3' and ee/0385-ee/0386 r: 5'-CGGGTACCTATGTTAATCAAATAC-3', taking Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat as the specification, mdh delta stpk as the specification, mdh delta fk as the specification, mdh delta aldh as the specification, (mtld-mlp)0385/0386 as the specification, and ldhA-ee chromosomal DNA as a template, carrying out PCR amplification to obtain a 3883bp fragment, connecting a PCR product to a KpnI site in the middle of a homologous recombination vector pUC19-adhqh homologous arm by using T4-DNA ligase, and naming the recombinant plasmid as pUC19-adhqh-ee/0385-ee/0386, namely constructing the alcohol dehydrogenase gene homologous recombination vector with a mannitol efflux pump coding gene expression cassette in the middle.
(2.4.2) construction of a mutant strain of Leuconostoc mesenteroides having a glucanosucrase gene knockout, a D-lactate dehydrogenase gene knockout, an acetylphosphotransferase gene knockout with a mannitol dehydrogenase gene knock-in, a serine/threonine protein kinase gene knockout with a mannitol dehydrogenase gene knock-in, a fructokinase gene knockout with a mannitol dehydrogenase gene knock-in, an acetaldehyde dehydrogenase gene knockout with a 1-phosphomannitol dehydrogenase encoding gene and a mannitol-1-phosphatase encoding gene concatemer knock-in, insertion of a D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of a mannitol efflux pump (0385/0386) encoding gene, an ethanol dehydrogenase gene knockout with a mannitol efflux pump encoding gene expression cassette knock-in: by electrotransformation, pUC19-adhqh-ee/0385-ee/0386 was introduced into Leuconostoc mesenteroides (Leuconostoc mesenteroides) Δ dts1 Δ D-ldh Δ pat obtained in (2.3) above, mdh Δ stpk: (mdh Δ fk: (mdh Δ aldh:) (mtld-mlp)0385/0386: (ldhA-ee Δ adh:: amy strain), and dextransucrase 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, acetaldehyde dehydrogenase gene knock-out and 1-phosphate mannitol dehydrogenase gene and mannitol-1-phosphatase gene concatemer knock-in, A Leuconostoc mesenteroides mutant strain with an ethanol dehydrogenase gene knocked out and a mannitol efflux pump coding gene expression cassette knocked in is Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp)0385/0386: ldhA-ee delta adh:: mep strain.
Third, a step of constructing a glucansucrase gene knock-out, a D-lactate dehydrogenase gene knock-out, an acetyl phosphate transferase gene knock-out and mannitol dehydrogenase gene knock-in, a serine/threonine protein kinase gene knock-out and mannitol dehydrogenase gene knock-in, a fructose kinase gene knock-out and mannitol dehydrogenase gene knock-in, an acetaldehyde dehydrogenase gene knock-out and 1-phosphate mannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding gene knock-in, a mannitol efflux pump (0385/0386) an intervening D-lactate dehydrogenase gene (hA) expression element between 2 coding frames of a mannitol efflux pump encoding gene, a D-lactate dehydrogenase gene expression element (0385/0386), a method of expressing a protein, a method of producing a protein-encoded protein-kinase gene-expressed protein-dehydrogenase-protein, The Leuconostoc mesenteroides mutant strain with the ethanol dehydrogenase gene knocked out and the mannitol efflux pump coding gene expression cassette knocked in and the lactic acid dehydrogenase gene (0503) knocked out and the mannitol efflux pump coding gene expression cassette knocked in comprises the following specific steps:
(3.1) cloning of partial sequence of Leuconostoc mesenteroides lactate dehydrogenase gene (0503):
design a pair of primers 0503 l: 5'-CCTCTAGAATGACAAAACAAGTAT-3' and 0503 r: 5'-CCTCTAGACAATTTTATTCACAGC-3', a 953bp fragment was obtained by PCR amplification using a chromosome of Leuconostoc mesenteroides Δ dts1 Δ D-ldh Δ pat:: mdh Δ stpk:: mdh Δ fk:: mdh Δ aldh: (mtld-mlp)0385/0386:: ldhA-ee Δ adh:: mep as a template, and the PCR product was ligated to the XbaI site of pUC19 with T4DNA ligase, and the recombinant plasmid was named pUC 19-0503.
(3.2) construction of a homologous recombination vector of the lactate dehydrogenase gene (0503) with an alpha-amylase mark in the middle:
(3.2.1) cloning of the left homology arm: design a pair of primers 0503 lq: 5'-CAGTCTAGATCCAGCAGTT-3' and 0503 lh: 5'-CAAACGGTACCCAATATCACG-3' (complementary to 0503 rq) and 406bp of the fragment was obtained by PCR using pUC19-0503 as the template.
(3.2.2) cloning of the right homology arm: design a pair of primers 0503 rq: 5'-TATTGGGTACCGTTTGATGTT-3' and 0503 rh: 5'-TATGAATTCGGGTTCTTGC-3', a 449bp fragment was obtained by PCR using pUC19-0503 as a template.
(3.2.3) the 2 PCR products obtained in (3.2.1) and (3.2.2) above were purified and mixed, and 2 gene fragments were extended by 8 cycles of PCR using the PCR product mixture as a template, and then the PCR products were amplified using a pair of primers 0503 lq: 5'-CAGTCTAGATCCAGCAGTT-3' and 0503 rh: 5'-TATGAATTCGGGTTCTTGC-3' PCR was performed to obtain a 827bp fragment.
(3.2.4) carrying out double digestion on the overlap extension PCR product obtained in the step (3.2.3) and pUC19 by EcoRI and XbaI, then connecting the two products under the action of T4-DNA ligase, transforming the connected product into an escherichia coli DH5 alpha competent cell, and screening a recombinant plasmid pUC19-0503qh to construct a homologous recombinant vector.
(3.2.5) design of a pair of primers amyl: 5'-CTTGGTACCTTTGGCGTGATTATCAG-3' and amyr: 5'-TCAGGTACCCGAAGGTGAAGTTATAG-3', using the total DNA of the Lactobacillus amylovorus with the preservation number of CGMCC1.3395 as a template, carrying out PCR amplification to obtain a 2131bp fragment, connecting the PCR product to a KpnI site in the middle of a homologous recombination vector pUC19-0503qh homologous arm by using T4DNA ligase, and naming the recombinant plasmid as pUC19-0503qh-amy, namely constructing the homologous recombination vector of the lactate dehydrogenase gene (0503) with the alpha-amylase mark in the middle.
(3.3) construction of a mutant strain of Leuconostoc mesenteroides with gene expression cassette knock-out of glucansucrase gene, gene knock-out of D-lactate dehydrogenase, gene knock-out of acetylphosphotransferase and mannitol dehydrogenase knock-in, gene knock-out of serine/threonine protein kinase and mannitol dehydrogenase, gene knock-in of fructokinase and mannitol dehydrogenase, gene knock-out of acetaldehyde dehydrogenase and knock-in of 1-phosphomannitol dehydrogenase coding gene and mannitol-1-phosphatase coding gene concatemer knock-in, insertion of D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of mannitol efflux pump (0385/0386) coding gene, gene knock-in of ethanol dehydrogenase and mannitol efflux pump, inactivation of lactate dehydrogenase gene (0503):
the homologous recombinant vector of the lactate dehydrogenase gene (0503) with the alpha-amylase mark in the middle obtained in (3.2.5) is introduced into Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat and mdh delta stpk and mdh delta fk and mdh delta aldh and mtld-mlp and 0385/0386 and ldhA-ee delta adh strains by an electric shock transformation method, and glucan sucrase gene knockout, D-lactate dehydrogenase gene knockout, acetyl phosphate transferase gene knockout and mannitol dehydrogenase gene knockout, serine/threonine protein kinase gene knockout and mannitol dehydrogenase gene knockout, fructose kinase gene knockout and mannitol dehydrogenase gene knockout, acetaldehyde dehydrogenase gene knockout and 1-phosphate mannitol dehydrogenase encoding gene concatemer-in-series are obtained by screening, A mutant strain of Leuconostoc mesenteroides with D-lactate dehydrogenase gene (ldhA) expression elements inserted between 2 coding frames of a coding gene of a mannitol efflux pump (0385/0386), knock-in of an alcohol dehydrogenase gene knock-out and a mannitol efflux pump coding gene expression cassette, and inactivation of a lactate dehydrogenase gene (0503), namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat: mdh delta stpk: mdh delta fk: mdh delta aldh: mtld-mlp)0385/0386: ldhA-ee delta adh: mep delta ldh (0503) and amy strain.
(3.4) Glucosaccharase gene knockout, D-lactate dehydrogenase gene knockout, acetylphosphotransferase gene knockout with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knockout with mannitol dehydrogenase gene knock-in, fructokinase gene knockout with mannitol dehydrogenase gene knock-in, construction of a Leuconostoc mesenteroides mutant strain in which a gene encoding acetaldehyde dehydrogenase gene is knocked out and a gene encoding mannitol phosphate dehydrogenase and a gene encoding mannitol-1-phosphatase are knocked in, a D-lactate dehydrogenase gene (ldhA) expression element is inserted between 2 coding frames of a gene encoding mannitol efflux pump (0385/0386), a gene expression cassette for ethanol dehydrogenase gene knockout and mannitol efflux pump is knocked in, and a gene expression cassette for lactate dehydrogenase gene (0503) knockout and mannitol efflux pump is knocked in:
(3.4.1) construction of a lactic acid dehydrogenase gene (0503) homologous recombination vector with a mannitol efflux pump coding gene expression cassette in the middle: designing a pair of primers ee/0385-ee/0386 l: 5'-GCGGTACCTAATTTTAAATATATG-3' and ee/0385-ee/0386 r: 5'-CGGGTACCTATGTTAATCAAATAC-3', taking Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat as well as mdh delta stpk as well as mdh delta fk as well as mdh delta aldh as well as mtld-mlp as well as 0385/0386 as ldhA-ee delta adh as well as mep delta ldh (0503) as well as amy chromosome DNA as a template, carrying out PCR amplification to obtain a 3883bp fragment, connecting a PCR product to a KpnI site in the middle of a homologous recombination vector pUC19-0503qh homologous arm by using T4DNA ligase, and constructing a homologous recombination vector of the lactate dehydrogenase gene (0503) with a mannitol efflux pump coding gene expression box in the middle, wherein the recombinant vector is named as pUC19-0503 qh-ee/0385-ee/0386.
(3.4.2) knocking out dextransucrase gene, knocking out D-lactate dehydrogenase gene, knocking out acetyl phosphotransferase gene and knocking in mannitol dehydrogenase gene, knocking out serine/threonine protein kinase gene and knocking in mannitol dehydrogenase gene, knocking out fructose kinase gene and knocking in mannitol dehydrogenase gene, construction of a mutant strain of Leuconostoc mesenteroides having an acetaldehyde dehydrogenase gene knockout and 1-phosphomannitol dehydrogenase encoding gene and a mannitol-1-phosphatase encoding gene concatemer knock-in, a D-lactate dehydrogenase gene (ldhA) expression element inserted between 2 coding frames of a mannitol efflux pump (0385/0386) encoding gene, an ethanol dehydrogenase gene knockout and mannitol efflux pump encoding gene expression cassette knock-in, a lactate dehydrogenase gene (0503) knockout and a mannitol efflux pump encoding gene expression cassette knock-in: in the electrocatalytic Leuconostoc mesenteroides obtained in the above (3.3), pUC19-0503qh-ee/0385-ee/0386 was introduced into Leuconostoc mesenteroides Deltadts 1 DeltaD-ldh Deltapat by electrotransformation, mdh Deltastpk:: mdh Deltafk:: mdh Deltaaldh: (mtld-mlp)0385/0386: hadd-ee Deltaadh:: meptaadh (0503): ldamy strain, glucose sucrose kinase 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, acetaldehyde dehydrogenase gene knock-out and 1-phosphate mannitol dehydrogenase gene coding phosphatase-1-knock-in, A Leuconostoc mesenteroides mutant strain with an expression element of D-lactate dehydrogenase gene (ldhA), knock-out of the ethanol dehydrogenase gene and knock-in of a gene expression cassette encoding the mannitol efflux pump, knock-out of the lactate dehydrogenase gene (0503) and knock-in of a gene expression cassette encoding the mannitol efflux pump is inserted between 2 coding frames of a gene encoding the mannitol efflux pump (0385/0386), namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat: mdh delta stpk: mdh delta fk: mdh delta aldh: (mtld-mlp)0385/0386: ldhA-ee delta adh:: mep delta ldh (0503): mep strain.
Fourthly, inserting a D-lactate dehydrogenase gene (hA) expression element (hA) between 2 coding frames of a gene coding for mannitol efflux pump (0385/0386), inserting a D-lactate dehydrogenase gene (hA) expression element between 2 coding frames of a gene coding for mannitol efflux pump (0385/0386), and inserting a D-lactate dehydrogenase gene (hA) expression element between mdt 1 delta D-ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta aldh, mtld-mlp 0385/0386, hal-ee delta adh, mep delta ldh (0503) and mep strain constructing a glucan sucrase gene knockout, a D-lactate dehydrogenase gene knockout, an acetyl phosphate transferase gene knockout and a mannitol dehydrogenase gene knock-in, a serine/threonine protein kinase gene knockout and a mannitol dehydrogenase gene knock-in, a fructose kinase gene knockout and a mannitol dehydrogenase gene knock-in, an acetaldehyde dehydrogenase gene knockout and a mannitol dehydrogenase gene concatemer knock-in, a mannitol dehydrogenase gene coding gene and a mannitol-1 phosphatase coding gene concatemer knock-in, and a mannitol dehydrogenase gene expression element (hA) expression element between 2 coding frames of a gene of a mannitol efflux pump (0385/0386), The leuconostoc mesenteroides mutant strain obtained by knocking out the ethanol dehydrogenase gene and knocking in the mannitol efflux pump coding gene expression cassette, knocking out the lactate dehydrogenase gene (0503) and knocking in the mannitol efflux pump coding gene expression cassette, and knocking out the lactate dehydrogenase gene (0373) and knocking in the mannitol efflux pump coding gene expression cassette comprises the following specific steps:
(4.1) cloning of a partial sequence of a Leuconostoc mesenteroides lactate dehydrogenase gene (0373): design a pair of primers 0373 l: 5'-CGGGTACCATACTATTTTTTAACG-3' and 0373 r: 5'-TAGGTACCGTACAACAGTATTCTC-3', a 977bp fragment was obtained by PCR amplification using a chromosome of Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat:: mdh delta stpk:: mdh delta fk:: mdh delta aldh: (mtld-mlp)0385/0386:: ldhA-ee delta adh:: mep delta ldh (0503): mep as a template, and the PCR product was ligated to the KpnI site of pUC19 using T4DNA ligase to obtain the recombinant plasmid named pUC 19-0373.
(4.2) construction of a lactic acid dehydrogenase gene (0373) homologous recombination vector with an alpha-amylase mark in the middle:
(4.2.1) cloning of the left homology arm: design a pair of primers 0373 lq: 5'-GCGTCTAGAGATGAGTGGGCTG-3' and 0373 lh: 5'-GACTAGGTACCGTGTCGTTTCTGG-3' (complementary pair to 0373 rq) and a 369bp fragment was PCR amplified using pUC19-0373 as a template.
(4.2.2) cloning of the right homology arm: a pair of primers 0373rq was designed: 5'-GACACGGTACCTAGTCTGAAGCCA-3' and 0373 rh: 5'-CGGGAATTCGCTGCCTTCGTAA-3', and a 383bp fragment was obtained by PCR using pUC19-0373 as a template.
(4.2.3) the 2 PCR products obtained in (4.2.1) and (4.2.2) above were purified and mixed, and the PCR product mixture was used as a template to overlap and extend 2 gene fragments by 8 cycles of PCR cycles, and then the PCR products were amplified using a pair of primers 0373 lq: 5'-GCGTCTAGAGATGAGTGGGCTG-3' and 0373 rh: 5'-CGGGAATTCGCTGCCTTCGTAA-3' PCR was performed to obtain a 724bp fragment.
(4.2.4) carrying out double digestion on the overlap extension PCR product obtained in the step (4.2.3) and pUC19 by EcoRI and XbaI, then connecting the two products under the action of T4-DNA ligase, transforming the connected product into escherichia coli DH5 alpha competent cells, and screening a recombinant plasmid pUC19-0373qh to construct a homologous recombination vector.
(4.2.5) design a pair of primers amyl: 5'-CTTGGTACCTTTGGCGTGATTATCAG-3' and amyr: 5'-TCAGGTACCCGAAGGTGAAGTTATAG-3', using total DNA of the Lactobacillus amylovorus with the preservation number of CGMCC1.3395 as a template, carrying out PCR amplification to obtain a 2131bp fragment, connecting a PCR product to a KpnI site in the middle of a homologous recombination vector pUC19-adhqh homologous arm by using T4DNA ligase, and naming a recombinant plasmid as pUC19-0373qh-amy, namely constructing the homologous recombination vector of the lactate dehydrogenase gene (0373) with alpha-amylase mark in the middle.
(4.3) Glucosaccharase gene knockout, D-lactate dehydrogenase gene knockout, acetylphosphotransferase gene knockout with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knockout with mannitol dehydrogenase gene knock-in, fructokinase gene knockout with mannitol dehydrogenase gene knock-in, construction of Leuconostoc mesenteroides mutant strain with acetaldehyde dehydrogenase gene knockout and 1-phosphomannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding gene concatemer knock-in, insertion of D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of mannitol efflux pump (0385/0386) encoding gene, ethanol dehydrogenase gene knockout and mannitol efflux pump encoding gene expression cassette knock-in, lactate dehydrogenase gene (0503) knock-out and mannitol efflux pump gene expression cassette knock-in, lactate dehydrogenase gene (0373) inactivation:
the homologous recombinant vector of the lactate dehydrogenase gene (0373) with the alpha-amylase mark in the middle obtained in (4.2.5) is introduced into Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat as mdh delta stpk as mdh delta fk as mdh delta aldh as mtld-mlp as 0385/0386 as ldhA-ee delta adh as mep delta ldh as 0503 as mep strain, and the genes of glucan sucrase, D-lactate dehydrogenase, acetyl phosphatase and mannitol dehydrogenase, serine/threonine kinase, mannitol dehydrogenase, fructose kinase, mannitol dehydrogenase, acetaldehyde dehydrogenase, 1-phosphate mannitol dehydrogenase and mannitol phosphatase coding gene are knocked in, fructose kinase, mannitol dehydrogenase and mannitol dehydrogenase are knocked in, and the coding gene of 1-phosphate mannitol-1-concatemer-1-phosphate phosphatase is knocked in, A mutant strain of Leuconostoc mesenteroides with an inactivated lactate dehydrogenase gene (0373), namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat, mdh delta stpk, mdh delta fk, mdh delta al: (mtld-mlp)0385/0386: mdhA-ee delta ah: (mep delta ldh) (0503) and mep delta ldh (05073) is inserted between 2 coding frames of a coding gene of the mannitol efflux pump (0385/0386), an expression element of D-lactate dehydrogenase gene (ldhA), a knock-out gene of the mannitol efflux pump coding gene, a knock-in gene expression cassette of the lactate dehydrogenase gene (0503), and a knock-in gene of the lactate dehydrogenase gene (0373).
(4.4) dextransucrase gene knockout, D-lactate dehydrogenase gene knockout, acetylphosphotransferase gene knockout with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knockout with mannitol dehydrogenase gene knock-in, fructokinase gene knockout with mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knockout with 1-phosphomannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding gene concatemer knock-in, insertion of D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of mannitol efflux pump (0385/0386) encoding gene, ethanol dehydrogenase gene knockout with mannitol efflux pump encoding gene expression cassette knock-in, lactate dehydrogenase gene (0503) knockout with mannitol efflux pump encoding gene expression cassette knock-in, lactate dehydrogenase gene (0373) knockout with mannitol efflux pump encoding gene expression cassette knock-in Construction of the mutant strains of the Phyllanthus:
(4.4.1) construction of a lactic acid dehydrogenase gene (0373) homologous recombination vector with a mannitol efflux pump coding gene expression cassette in the middle: designing a pair of primers ee/0385-ee/0386 l: 5'-GCGGTACCTAATTTTAAATATATG-3' and ee/0385-ee/0386 r: 5'-CGGGTACCTATGTTAATCAAATAC-3', taking Leuconostoc mesenteroides delta dts1 delta D-ldh delta pat as the specification, mdh delta stpk as the specification, mdh delta fk as the specification, mdh delta aldh as the specification, mtld-mlp as the specification, 0385/0386 as ldhA-ee delta adh as the specification, mep delta ldh (0503) as the specification, mep delta ldh (0373) as the specification, amy chromosome DNA as a template, PCR is amplified to obtain a 3883bp segment, and a T4DNA ligase is used for connecting a PCR product to a KpnI site in the middle of a homologous recombination vector pUC19-0373qh homologous arm, and the recombination plasmid is named as pUC19-0373qh-ee/0385-ee/0386, namely the lactate dehydrogenase gene (0373) homologous vector with a mannitol efflux pump coding gene expression box in the middle is constructed.
(4.4.2) dextransucrase gene knockout, D-lactate dehydrogenase gene knockout, acetylphosphotransferase gene knockout with mannitol dehydrogenase gene knock-in, serine/threonine protein kinase gene knockout with mannitol dehydrogenase gene knock-in, fructokinase gene knockout with mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knockout with 1-phosphomannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding gene concatemer knock-in, insertion of D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of mannitol efflux pump (0385/0386) encoding gene, ethanol dehydrogenase gene knockout with mannitol efflux pump encoding gene expression cassette knock-in, lactate dehydrogenase gene (0503) knockout with mannitol efflux pump encoding gene expression cassette knock-in, lactate dehydrogenase gene (0373) knockout with mannitol efflux pump encoding gene expression cassette knock-in Construction of a mutant strain of the bead fungus: pUC19-0373qh-ee/0385-ee/0386 was introduced into Leuconostoc mesenteroides obtained in (4.3) by electrotransformation (Leuconostoc mesenteroides) Δ dts1 Δ D-ldh Δ pat: mdh Δ stpk: mdh Δ fk: mdh Δ ldh: (mtld-mlp)0385/0386: ldhA-ee Δ adh: mep Δ ldh (0503) mep Δ ldh (0373) in amy strain, and Glucosansucrase gene knock-out, D-lactate dehydrogenase gene knock-out, acetyl phosphate transferase gene knock-out and mannitol dehydrogenase gene knock-in, serine/threonine kinase gene knock-out and mannitol dehydrogenase gene knock-in, fructose kinase gene knock-out and mannitol dehydrogenase gene knock-in, acetaldehyde dehydrogenase gene knock-out and mannitol dehydrogenase gene knock-in, and mannitol phosphatase gene coding for mannitol-1-knock-in, were obtained by screening, A mutant strain of Leuconostoc mesenteroides, namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) Delta dts1 Delta D-ldh Delta pat, is characterized in that D-lactate dehydrogenase gene (ldhA) expression elements, an alcohol dehydrogenase gene knockout and a knock-in of a gene expression cassette encoding the mannitol efflux pump, a lactate dehydrogenase gene (0503) knockout and a knock-in of a gene expression cassette encoding the mannitol efflux pump are inserted among 2 coding frames of a gene encoding the mannitol efflux pump (0385/0386), and the mutant strain of Leuconostoc mesenteroides, namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) Delta dts1 Delta D-ldh Delta pat, mdhastpk:: mdhafk Delta fhk:: mdh Delta dh: (mtld-mlp)0385/0386:: andea-Delta adhh:: mep Delta ldh:: 0503: mep Delta ldh: (0373): Meuch strain [ CCT is a CCT exosome No. 20262:: 0399). DELTA.05085).
Example 2
The fermentation application of the Leuconostoc mesenteroides mutant strain for high yield of mannitol comprises the following specific steps:
in a 250 ml triangular flask, Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20150385/0386 with the preservation date of 11-19 days in 2020 and the preservation number of CCTCC M2020762 is preserved in a China Center for Type Culture Collection (CCTCC) CCTCC M20188150386, ldhA-ee delta adh, mep delta ldh (0503), mep delta ldh (0373), mep strain is transferred into an MRS culture medium by 1 percent by weight, and is cultured for 20 hours at 30 ℃ by a shaking table with the rotation speed of 120 r/min, and the concentration of mannitol can reach 71.58 g/l.
The preparation method of the MRS culture medium comprises the following steps: 2 g of yeast extract powder, 120 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 shows the yield of mannitol produced by fermentation of Leuconostoc mesenteroides, and the preservation date of China Center for Type Culture Collection (CCTCC) is 11/19/2020, and the preservation number of the Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188185/0386 of the invention is CCHA-ee delta adh, mep delta ldh (0503), mep delta ldh (0373), the mannitol yield of the mep strain is higher than that of the Leuconostoc mesenteroides (Leuconostoc mesenteroides) delta dts1 delta D-ldh delta pat, mdh delta stk, mdh delta ldh, mtdh delta aldh, mtld-mlp and the mannitol conversion rate is improved by 20.27 percent when the sucrose is fermented to be 20.32 g.
TABLE 1 yield (g/L) of mannitol produced by fermentation of Leuconostoc mesenteroides
Figure BDA0002834223220000151
In Table 1, CCTCC M2018815 is Leuconostoc mesenteroides with a gene of glucansucrase knockout, a gene of D-lactate dehydrogenase (1756) knockout, a gene of acetylphosphotransferase knockout with a gene of mannitol dehydrogenase knock-in, a gene of serine/threonine protein kinase with a gene of glycerol dehydrogenase knock-in, a gene of fructokinase with a gene of mannitol dehydrogenase knock-in, a gene of acetaldehyde dehydrogenase with a gene of mannitol 1-phosphate dehydrogenase encoding and a gene of mannitol 1-phosphatase concatemer knock-in, CCTCC M20188150385/0386: hald-ee Δ adh: mep Δ ldh (0503): mep Δ ldh (0373): mep Δ ldh (0373): a gene knockout, a gene of D-lactate dehydrogenase (1756) knockout, a gene of acetylphosphotransferase with a gene of mannitol dehydrogenase 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, acetaldehyde dehydrogenase gene knock-out and 1-phosphomannitol dehydrogenase encoding gene and mannitol-1-phosphatase encoding gene concatemer knock-in, insertion of D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of mannitol efflux pump (0385/0386) encoding gene, ethanol dehydrogenase gene knock-out and mannitol efflux pump encoding gene expression cassette knock-in, lactate dehydrogenase gene (0503) knock-out and mannitol efflux pump encoding gene expression cassette knock-in, and Leuconostoc mesenteroides gene (0373) knock-out and mannitol efflux pump encoding gene expression cassette knock-in, namely Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188185/0386: (ldhA-delta) mutant strain of Leuconostoc mesenteroides (MEP. DELTA.0503): Deltaldh: (mep. DELTA. 0373) The mep strain is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 11-19 days in 2020 and the preservation number of CCTCC M2020762, namely the Leuconostoc mesenteroides mutant strain for high yield of mannitol.
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: the procedures described in the laboratory Manual or protocols supplied by the manufacturer. Nothing in this specification is said to apply to the prior art.
Sequence listing
<110> Hebei university of industry
<120> leuconostoc mesenteroides mutant strain capable of producing mannitol in high yield and application method thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3883
<212> DNA
<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)
<400> 1
taattttaaa tatatgaatg attaaaaaat cagtacgaag agtactggtt ttttatgtac 60
ttaaaaatag tgaataattg gtatatttta cattgacagg caaagtaatc tttcctatac 120
tagttgcgtg ttaaatagtt gaaaaatagt attcgatagc gtaatattag tttttaatgt 180
cttgaaagag gaaatgaaat gcaaatttta aaagaacatg caaagcctta tgctcgtgat 240
attatgtttt caattattgc ggtggtgata atggctgttg catcattgtg gcaaccacga 300
ttgcttcagc aagttatgaa agcaattatc gctgatgatc aaaataaggt tgttgcttac 360
ggatttcaac tgttattttt agctttaatc ggattggttg ctggtgtaat taatacaatt 420
tttgcctcca aagtttcaca ggcgattgct gcagatattc gtgcacacga gtataaaaaa 480
atacaatcat tttcatttgg taatattgaa aaattttcag cgggtaactt ggttgtccgt 540
atgacaaatg atgtcaatca agtgcagcaa ttaatcatga tgattttaca gtctttgaca 600
cgcattccaa tattgtttgt tggagccttt attttggcat tgattacatt gccacgttta 660
tggtggattg taattttaat ggttgtactg atttttgtat cttcgcaagt gatttttaaa 720
caaatgggga agtttttctc caaaattcaa acactaattg ataagacgaa tacgttggcc 780
aaggaaaatc ttcagggtgt tcgtgttgtc aagtctttta accaagaaaa aaatgaaaca 840
aaccgtttta cggataattc taatgagttg acgcgtgtca acacgactgt tggctactta 900
ttttcgacta tgatgccgat gttcatgtta atatcttatg ttgccatcgg tacagctatt 960
tggttcgtag gtcgcgatgt tgtcaaacat cccaatgatt tagttgcgat tgcttctttt 1020
acttcttact taatgcaaat attctttgcc attatgattg gtggtatgat ggctaccttt 1080
gcatctcgtg gttttgtctc gctaagacgt attaaagaaa ttttagatac gactccagac 1140
ttaacgtacg ctcaagatgc gcctgatcgt gatttgaatg gaaatatttc attcaaaaat 1200
gtttctttca cctatcctgg agatactaaa ccagctttga aaaacgtatc gtttgatatc 1260
gaagcaggtg agatgatcgg tatcgttggt gcaactggat caggtaaaac aacacttgcc 1320
caattaatcc caagattgta tgatcctact gaaggtgtgg tagctgttgg tggtgtggat 1380
ctcaaagcag taaacgaaaa gtcgttgcga gctgcggttt cgtttgttct acaaaaggcc 1440
atattgttta gtggtacgat tgcagcaaat ttgcgtcaag gtaaaaaaga cgctaatgaa 1500
aaggatttcc aaaaagctgt tgaaattgca caggctgcgg agtttgttaa tcgttatgaa 1560
gataattttg accacattgt tgaagagcgt tctgccaatt tttccggtgg tcaaaaacaa 1620
cgtctttcta tagcaagagg tgttgttggt agtcctaaaa tattgatttt agatgattca 1680
acatcggctt tagatgctcg atccgaaaag ctggttcaag aagcacttga ccaggaatta 1740
aaaggaacga caacaattgt aattgctgaa aaaatcagtt cagttattca tgccaaccgc 1800
attttagtaa tggatgaggg taaggtagta ggcatcggtg cacatcacga gttgcttgaa 1860
acttcaccgg tctatgctga aatttatcaa acacagaaag cgaaaggggc agaataatag 1920
aaagtgcttt aattagtgat taaagcaaag aaaatggaat gggttacatt tgcttaacga 1980
ctgtcatttg taaggggtga aattttttct gaaatctatg cattatatgg gcttaatcgc 2040
gtgcgttagc tcgtgaaata gggtacaatt atagatgaaa taaaattttg gaggatatga 2100
cagatattaa acacgcaatt aaatattttg caaagtatct taagcgttat tggttagcat 2160
tgtctttcgt agttgttgtg acaatagcct caacttattt ccaagttaaa gcaccagtgt 2220
atatgggaaa ggcaattaca gaattatcga cttatttagg tcaatacttg aatcctcaaa 2280
cgcatgctgt tgctagcaaa acacctttct atcatgcgct ggaagcaatg atgattttct 2340
ttactttaac tgcagtaaca atgttcattt ctagttttat atctagtcgt attagttcag 2400
aggcatcagg ccggatgaga attggtctat ttgctaaatt gcaacggatg acgattaaat 2460
attttgacac gcatcaagat ggcgaaattt tatctttgtt tacgtctgat ttagataata 2520
tttttaacgc gatgaaccag gccattttcc aacttttatc acaattcgct ttgtttgttg 2580
gtatcattat tatgatgttt cagcagaacg tcaaattagc atgggtaact atggcttcaa 2640
caccagttgc tattattgtc gcctggtttg tgatcagtcg tgctagaaaa tatattgaca 2700
cgcagcaaga tgaaactggc cgcatgaatg gttatattaa tgagcaaatc aatggtgaga 2760
agattattat tacgcaagga ttgcaacaag aatcaattgc taattttggt ccgtataatg 2820
aacgagtgaa gcaagctact tttaaaggac agatgtattc tggtatgctg tttcctctaa 2880
tgcaaggctt gtcattgttg aacttagcta ttgtgatttt ctttgggtca tggttaattg 2940
ttcatgacgg tatggataag tcggttggtt tgggattaat tgtggtgttt gttaactatt 3000
cacaacagta ttaccaacca atcacacaaa ttacatctat ttataatatg ttgcagttag 3060
ctgttactgg tgcaagacgc ttgagcgatg tccatgaaca agctgaagaa gttaaccccg 3120
aaagtggtca gaaattaact aatttaaagt cgggtgttac cctagaaaat attcactttg 3180
gatacaatga tgacaaagaa attcttcacg gtgtttcaat tgacgttaaa aaaggggaaa 3240
tgatagccct tgttggtccg accggctcag gaaagacaac ggtcatgaac ttattgaatc 3300
gattttatga tgtgactgaa ggatccgtaa agtttgatgg caccgacgtt cgtaagttag 3360
acttgaagtc tttacgtgat catgttggca ttgtgttaca ggaatcagta ttatttagtg 3420
gtactgtcgc agataatatt aaatttggtg agccagatgc taccgaagaa gaaatgattg 3480
atgctgccaa gcaggcgaac atccatgact ttattatgac acttccagaa ggttgatcag 3540
accaaagtag atgacgaaaa ttcggtcttt tcaacaggtc aaaaacaatt attatcaatc 3600
gcccgcacaa ttttgactaa tccggatttc ttaatattag atgaagcaac atctaatgta 3660
gatacggtga cagaggcaaa aatccaagcg gcgatggata atgttattgc tggacgtacg 3720
agttttgtta ttgcgcaccg attgaaaaca atactgggtg ctgataaaat tgttgttctt 3780
aaagatggtc gagttattga aaagggctcg catcaagaat tagttgctga aggcggattt 3840
tattcagaat tgtaccataa tcaaatggta tttgattaac ata 3883
<210> 1
<211> 4894
<212> DNA
<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)
<400> 1
atgaaagcag cagttgttcg taacgtaaat gatggttatg ttgatttgat cgaggattgg 60
cagccacgtg cgttagcatt tggagatgca ttggttgatg ttgaatacgt tggcctttgt 120
cacactgatt tgcactgtgc taacggcgat ttcggtgacc ctaacaagat cggtgaacgt 180
aacggtaatt ttggtcgagt tattggacat gaaggtgttg gacgcgtgtc taaattaggt 240
gaaggtgcta gcgattacct taaagtcggt gaccgtgttt caattgcctg gttctatgac 300
gcttgtggcg tttgtgatta ctgtgtctca ggaaacgaaa cgttctgccg taaggtgcgt 360
aattctggtt acacaattga tggcgcaatg tcacaacaag ttgttgtaaa tgctaagtat 420
gcagttaaag taccagaagg cttagaccct gttaatttta aatatatgaa tgattaaaaa 480
atcagtacga agagtactgg ttttttatgt acttaaaaat agtgaataat tggtatattt 540
tacattgaca ggcaaagtaa tctttcctat actagttgcg tgttaaatag ttgaaaaata 600
gtattcgata gcgtaatatt agtttttaat gtcttgaaag aggaaatgaa atgcaaattt 660
taaaagaaca tgcaaagcct tatgctcgtg atattatgtt ttcaattatt gcggtggtga 720
taatggctgt tgcatcattg tggcaaccac gattgcttca gcaagttatg aaagcaatta 780
tcgctgatga tcaaaataag gttgttgctt acggatttca actgttattt ttagctttaa 840
tcggattggt tgctggtgta attaatacaa tttttgcctc caaagtttca caggcgattg 900
ctgcagatat tcgtgcacac gagtataaaa aaatacaatc attttcattt ggtaatattg 960
aaaaattttc agcgggtaac ttggttgtcc gtatgacaaa tgatgtcaat caagtgcagc 1020
aattaatcat gatgatttta cagtctttga cacgcattcc aatattgttt gttggagcct 1080
ttattttggc attgattaca ttgccacgtt tatggtggat tgtaatttta atggttgtac 1140
tgatttttgt atcttcgcaa gtgattttta aacaaatggg gaagtttttc tccaaaattc 1200
aaacactaat tgataagacg aatacgttgg ccaaggaaaa tcttcagggt gttcgtgttg 1260
tcaagtcttt taaccaagaa aaaaatgaaa caaaccgttt tacggataat tctaatgagt 1320
tgacgcgtgt caacacgact gttggctact tattttcgac tatgatgccg atgttcatgt 1380
taatatctta tgttgccatc ggtacagcta tttggttcgt aggtcgcgat gttgtcaaac 1440
atcccaatga tttagttgcg attgcttctt ttacttctta cttaatgcaa atattctttg 1500
ccattatgat tggtggtatg atggctacct ttgcatctcg tggttttgtc tcgctaagac 1560
gtattaaaga aattttagat acgactccag acttaacgta cgctcaagat gcgcctgatc 1620
gtgatttgaa tggaaatatt tcattcaaaa atgtttcttt cacctatcct ggagatacta 1680
aaccagcttt gaaaaacgta tcgtttgata tcgaagcagg tgagatgatc ggtatcgttg 1740
gtgcaactgg atcaggtaaa acaacacttg cccaattaat cccaagattg tatgatccta 1800
ctgaaggtgt ggtagctgtt ggtggtgtgg atctcaaagc agtaaacgaa aagtcgttgc 1860
gagctgcggt ttcgtttgtt ctacaaaagg ccatattgtt tagtggtacg attgcagcaa 1920
atttgcgtca aggtaaaaaa gacgctaatg aaaaggattt ccaaaaagct gttgaaattg 1980
cacaggctgc ggagtttgtt aatcgttatg aagataattt tgaccacatt gttgaagagc 2040
gttctgccaa tttttccggt ggtcaaaaac aacgtctttc tatagcaaga ggtgttgttg 2100
gtagtcctaa aatattgatt ttagatgatt caacatcggc tttagatgct cgatccgaaa 2160
agctggttca agaagcactt gaccaggaat taaaaggaac gacaacaatt gtaattgctg 2220
aaaaaatcag ttcagttatt catgccaacc gcattttagt aatggatgag ggtaaggtag 2280
taggcatcgg tgcacatcac gagttgcttg aaacttcacc ggtctatgct gaaatttatc 2340
aaacacagaa agcgaaaggg gcagaataat agaaagtgct ttaattagtg attaaagcaa 2400
agaaaatgga atgggttaca tttgcttaac gactgtcatt tgtaaggggt gaaatttttt 2460
ctgaaatcta tgcattatat gggcttaatc gcgtgcgtta gctcgtgaaa tagggtacaa 2520
ttatagatga aataaaattt tggaggatat gacagatatt aaacacgcaa ttaaatattt 2580
tgcaaagtat cttaagcgtt attggttagc attgtctttc gtagttgttg tgacaatagc 2640
ctcaacttat ttccaagtta aagcaccagt gtatatggga aaggcaatta cagaattatc 2700
gacttattta ggtcaatact tgaatcctca aacgcatgct gttgctagca aaacaccttt 2760
ctatcatgcg ctggaagcaa tgatgatttt ctttacttta actgcagtaa caatgttcat 2820
ttctagtttt atatctagtc gtattagttc agaggcatca ggccggatga gaattggtct 2880
atttgctaaa ttgcaacgga tgacgattaa atattttgac acgcatcaag atggcgaaat 2940
tttatctttg tttacgtctg atttagataa tatttttaac gcgatgaacc aggccatttt 3000
ccaactttta tcacaattcg ctttgtttgt tggtatcatt attatgatgt ttcagcagaa 3060
cgtcaaatta gcatgggtaa ctatggcttc aacaccagtt gctattattg tcgcctggtt 3120
tgtgatcagt cgtgctagaa aatatattga cacgcagcaa gatgaaactg gccgcatgaa 3180
tggttatatt aatgagcaaa tcaatggtga gaagattatt attacgcaag gattgcaaca 3240
agaatcaatt gctaattttg gtccgtataa tgaacgagtg aagcaagcta cttttaaagg 3300
acagatgtat tctggtatgc tgtttcctct aatgcaaggc ttgtcattgt tgaacttagc 3360
tattgtgatt ttctttgggt catggttaat tgttcatgac ggtatggata agtcggttgg 3420
tttgggatta attgtggtgt ttgttaacta ttcacaacag tattaccaac caatcacaca 3480
aattacatct atttataata tgttgcagtt agctgttact ggtgcaagac gcttgagcga 3540
tgtccatgaa caagctgaag aagttaaccc cgaaagtggt cagaaattaa ctaatttaaa 3600
gtcgggtgtt accctagaaa atattcactt tggatacaat gatgacaaag aaattcttca 3660
cggtgtttca attgacgtta aaaaagggga aatgatagcc cttgttggtc cgaccggctc 3720
aggaaagaca acggtcatga acttattgaa tcgattttat gatgtgactg aaggatccgt 3780
aaagtttgat ggcaccgacg ttcgtaagtt agacttgaag tctttacgtg atcatgttgg 3840
cattgtgtta caggaatcag tattatttag tggtactgtc gcagataata ttaaatttgg 3900
tgagccagat gctaccgaag aagaaatgat tgatgctgcc aagcaggcga acatccatga 3960
ctttattatg acacttccag aaggttgatc agaccaaagt agatgacgaa aattcggtct 4020
tttcaacagg tcaaaaacaa ttattatcaa tcgcccgcac aattttgact aatccggatt 4080
tcttaatatt agatgaagca acatctaatg tagatacggt gacagaggca aaaatccaag 4140
cggcgatgga taatgttatt gctggacgta cgagttttgt tattgcgcac cgattgaaaa 4200
caatactggg tgctgataaa attgttgttc ttaaagatgg tcgagttatt gaaaagggct 4260
cgcatcaaga attagttgct gaaggcggat tttattcaga attgtaccat aatcaaatgg 4320
tatttgatta acataggtgt tacaatgtac aaagccttga aagtcggtga aactaagcct 4380
ggtgagtggg tgtcagttca cggtgctggt ggtctaggta atttggctgt tcaatatgca 4440
cacagtgttt tcggtgctca cgttgcagtt attgatggaa acgatgataa attagcagcc 4500
gcaaaagcta atggagcgga agtactagtt aatcgtaaga aggaagatgt tgtagctaag 4560
gtgaatgaat taactggcgg cgttcacaat gctcaagtga cagctgttaa cgatgctgca 4620
ttctcccagg ctgtgaatgt gcttcgtcca atgggcaagc ttgtagccgt tgctttgcca 4680
caaggtgata tggctttgaa tattgctaaa acagtccttg atggtattga agttcgcggt 4740
tcattggttg gaacacgtgc agacctaaag gaagctttcc aatttggtgc tgaaggaaaa 4800
gttaagccaa ttgttgagac agttgccttc caagatatga atgaaattat tgatgaaatg 4860
aaagggtcag gtcattgatt ttacaacact ttaa 4894
<210> 1
<211> 4761
<212> DNA
<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)
<400> 1
atgacaaaac aagtattctt accagatgat attccagcag ttgggaaaaa gattcttgaa 60
gaagcaggat tagaagtagt tgttggttct ggtcgtgatc atgaaaagat gaaggctgaa 120
ggggtagagg caagtgctgt attgattggt acacaaaagt ttgatgcaga tataatggac 180
gctatgccaa atttaaaggt tattgcacgt aacggcgttg gttatgatgc tgttgacgtg 240
gatgctgcaa cccagcgcgg tatctatgtt gtgaacacac ctaaagcttt atcaggatca 300
gttgctgaaa cggctgtctc tgagttatta gctatcagca agaacttata tcaagattca 360
aaagcaattc atgatgataa ttggaattat cgaaaggcac acccaggccg tgatattgta 420
attttaaata tatgaatgat taaaaaatca gtacgaagag tactggtttt ttatgtactt 480
aaaaatagtg aataattggt atattttaca ttgacaggca aagtaatctt tcctatacta 540
gttgcgtgtt aaatagttga aaaatagtat tcgatagcgt aatattagtt tttaatgtct 600
tgaaagagga aatgaaatgc aaattttaaa agaacatgca aagccttatg ctcgtgatat 660
tatgttttca attattgcgg tggtgataat ggctgttgca tcattgtggc aaccacgatt 720
gcttcagcaa gttatgaaag caattatcgc tgatgatcaa aataaggttg ttgcttacgg 780
atttcaactg ttatttttag ctttaatcgg attggttgct ggtgtaatta atacaatttt 840
tgcctccaaa gtttcacagg cgattgctgc agatattcgt gcacacgagt ataaaaaaat 900
acaatcattt tcatttggta atattgaaaa attttcagcg ggtaacttgg ttgtccgtat 960
gacaaatgat gtcaatcaag tgcagcaatt aatcatgatg attttacagt ctttgacacg 1020
cattccaata ttgtttgttg gagcctttat tttggcattg attacattgc cacgtttatg 1080
gtggattgta attttaatgg ttgtactgat ttttgtatct tcgcaagtga tttttaaaca 1140
aatggggaag tttttctcca aaattcaaac actaattgat aagacgaata cgttggccaa 1200
ggaaaatctt cagggtgttc gtgttgtcaa gtcttttaac caagaaaaaa atgaaacaaa 1260
ccgttttacg gataattcta atgagttgac gcgtgtcaac acgactgttg gctacttatt 1320
ttcgactatg atgccgatgt tcatgttaat atcttatgtt gccatcggta cagctatttg 1380
gttcgtaggt cgcgatgttg tcaaacatcc caatgattta gttgcgattg cttcttttac 1440
ttcttactta atgcaaatat tctttgccat tatgattggt ggtatgatgg ctacctttgc 1500
atctcgtggt tttgtctcgc taagacgtat taaagaaatt ttagatacga ctccagactt 1560
aacgtacgct caagatgcgc ctgatcgtga tttgaatgga aatatttcat tcaaaaatgt 1620
ttctttcacc tatcctggag atactaaacc agctttgaaa aacgtatcgt ttgatatcga 1680
agcaggtgag atgatcggta tcgttggtgc aactggatca ggtaaaacaa cacttgccca 1740
attaatccca agattgtatg atcctactga aggtgtggta gctgttggtg gtgtggatct 1800
caaagcagta aacgaaaagt cgttgcgagc tgcggtttcg tttgttctac aaaaggccat 1860
attgtttagt ggtacgattg cagcaaattt gcgtcaaggt aaaaaagacg ctaatgaaaa 1920
ggatttccaa aaagctgttg aaattgcaca ggctgcggag tttgttaatc gttatgaaga 1980
taattttgac cacattgttg aagagcgttc tgccaatttt tccggtggtc aaaaacaacg 2040
tctttctata gcaagaggtg ttgttggtag tcctaaaata ttgattttag atgattcaac 2100
atcggcttta gatgctcgat ccgaaaagct ggttcaagaa gcacttgacc aggaattaaa 2160
aggaacgaca acaattgtaa ttgctgaaaa aatcagttca gttattcatg ccaaccgcat 2220
tttagtaatg gatgagggta aggtagtagg catcggtgca catcacgagt tgcttgaaac 2280
ttcaccggtc tatgctgaaa tttatcaaac acagaaagcg aaaggggcag aataatagaa 2340
agtgctttaa ttagtgatta aagcaaagaa aatggaatgg gttacatttg cttaacgact 2400
gtcatttgta aggggtgaaa ttttttctga aatctatgca ttatatgggc ttaatcgcgt 2460
gcgttagctc gtgaaatagg gtacaattat agatgaaata aaattttgga ggatatgaca 2520
gatattaaac acgcaattaa atattttgca aagtatctta agcgttattg gttagcattg 2580
tctttcgtag ttgttgtgac aatagcctca acttatttcc aagttaaagc accagtgtat 2640
atgggaaagg caattacaga attatcgact tatttaggtc aatacttgaa tcctcaaacg 2700
catgctgttg ctagcaaaac acctttctat catgcgctgg aagcaatgat gattttcttt 2760
actttaactg cagtaacaat gttcatttct agttttatat ctagtcgtat tagttcagag 2820
gcatcaggcc ggatgagaat tggtctattt gctaaattgc aacggatgac gattaaatat 2880
tttgacacgc atcaagatgg cgaaatttta tctttgttta cgtctgattt agataatatt 2940
tttaacgcga tgaaccaggc cattttccaa cttttatcac aattcgcttt gtttgttggt 3000
atcattatta tgatgtttca gcagaacgtc aaattagcat gggtaactat ggcttcaaca 3060
ccagttgcta ttattgtcgc ctggtttgtg atcagtcgtg ctagaaaata tattgacacg 3120
cagcaagatg aaactggccg catgaatggt tatattaatg agcaaatcaa tggtgagaag 3180
attattatta cgcaaggatt gcaacaagaa tcaattgcta attttggtcc gtataatgaa 3240
cgagtgaagc aagctacttt taaaggacag atgtattctg gtatgctgtt tcctctaatg 3300
caaggcttgt cattgttgaa cttagctatt gtgattttct ttgggtcatg gttaattgtt 3360
catgacggta tggataagtc ggttggtttg ggattaattg tggtgtttgt taactattca 3420
caacagtatt accaaccaat cacacaaatt acatctattt ataatatgtt gcagttagct 3480
gttactggtg caagacgctt gagcgatgtc catgaacaag ctgaagaagt taaccccgaa 3540
agtggtcaga aattaactaa tttaaagtcg ggtgttaccc tagaaaatat tcactttgga 3600
tacaatgatg acaaagaaat tcttcacggt gtttcaattg acgttaaaaa aggggaaatg 3660
atagcccttg ttggtccgac cggctcagga aagacaacgg tcatgaactt attgaatcga 3720
ttttatgatg tgactgaagg atccgtaaag tttgatggca ccgacgttcg taagttagac 3780
ttgaagtctt tacgtgatca tgttggcatt gtgttacagg aatcagtatt atttagtggt 3840
actgtcgcag ataatattaa atttggtgag ccagatgcta ccgaagaaga aatgattgat 3900
gctgccaagc aggcgaacat ccatgacttt attatgacac ttccagaagg ttgatcagac 3960
caaagtagat gacgaaaatt cggtcttttc aacaggtcaa aaacaattat tatcaatcgc 4020
ccgcacaatt ttgactaatc cggatttctt aatattagat gaagcaacat ctaatgtaga 4080
tacggtgaca gaggcaaaaa tccaagcggc gatggataat gttattgctg gacgtacgag 4140
ttttgttatt gcgcaccgat tgaaaacaat actgggtgct gataaaattg ttgttcttaa 4200
agatggtcga gttattgaaa agggctcgca tcaagaatta gttgctgaag gcggatttta 4260
ttcagaattg taccataatc aaatggtatt tgattaacat agtttgatgt taaggtcatt 4320
gcctttgatc cctttgccaa ggatgtgcct ggtgttgaat tggttgatcg tgaaacgatt 4380
tttaaaactg ccgactatgt gatggtccat ttgccggcat tacctgagac tcagcattca 4440
attggcgcag atgagtttaa gttaatgaaa aatgacgcat ttctaattaa tatggctcgt 4500
ggttctattc tggttgaatc tgacttggtc ttggctctga aatcgggtga gatagctggt 4560
gcggctttag atgtttttga agaagagcca ttgcctgtta caaacccatt agttgcattg 4620
gaaaatgttt tgctcacacc acatattgct tctaatactg ttgaaactaa ggcgcgtatg 4680
gcggttgatg caaccaatga cattgttcgg gtattatctg gcaagaaccc agaatcagct 4740
gtgaataaaa ttggtaaata a 4761
<210> 1
<211> 4728
<212> DNA
<213> Leuconostoc mesenteroides (Leuconostoc mesenteroides)
<400> 1
atgaaaatac tattttttaa cgtctcagct gtagagaaaa atattatcga tgagtgggct 60
gacacacaac tgatttctgt taaaacgtta cctgtttcga ttgactacga taacattgac 120
ttaactcgca attttgatgc tgttattttt tatccaggaa aatcttttca aactgatgaa 180
aagttatatg ctcgattggc agagaatggt atgaaacaaa tttctgtcaa atcaacaggt 240
tatgataata ttaatttcga atttgctgaa aaatatcacc tgacaataac taatgttcct 300
gattattcgc cagaatctgt atcgcatttt acagtgatga gcgtgctcat gttgttacgt 360
aatttaccgc gatatcttga tagcccagaa acgacactaa ttttaaatat atgaatgatt 420
aaaaaatcag tacgaagagt actggttttt tatgtactta aaaatagtga ataattggta 480
tattttacat tgacaggcaa agtaatcttt cctatactag ttgcgtgtta aatagttgaa 540
aaatagtatt cgatagcgta atattagttt ttaatgtctt gaaagaggaa atgaaatgca 600
aattttaaaa gaacatgcaa agccttatgc tcgtgatatt atgttttcaa ttattgcggt 660
ggtgataatg gctgttgcat cattgtggca accacgattg cttcagcaag ttatgaaagc 720
aattatcgct gatgatcaaa ataaggttgt tgcttacgga tttcaactgt tatttttagc 780
tttaatcgga ttggttgctg gtgtaattaa tacaattttt gcctccaaag tttcacaggc 840
gattgctgca gatattcgtg cacacgagta taaaaaaata caatcatttt catttggtaa 900
tattgaaaaa ttttcagcgg gtaacttggt tgtccgtatg acaaatgatg tcaatcaagt 960
gcagcaatta atcatgatga ttttacagtc tttgacacgc attccaatat tgtttgttgg 1020
agcctttatt ttggcattga ttacattgcc acgtttatgg tggattgtaa ttttaatggt 1080
tgtactgatt tttgtatctt cgcaagtgat ttttaaacaa atggggaagt ttttctccaa 1140
aattcaaaca ctaattgata agacgaatac gttggccaag gaaaatcttc agggtgttcg 1200
tgttgtcaag tcttttaacc aagaaaaaaa tgaaacaaac cgttttacgg ataattctaa 1260
tgagttgacg cgtgtcaaca cgactgttgg ctacttattt tcgactatga tgccgatgtt 1320
catgttaata tcttatgttg ccatcggtac agctatttgg ttcgtaggtc gcgatgttgt 1380
caaacatccc aatgatttag ttgcgattgc ttcttttact tcttacttaa tgcaaatatt 1440
ctttgccatt atgattggtg gtatgatggc tacctttgca tctcgtggtt ttgtctcgct 1500
aagacgtatt aaagaaattt tagatacgac tccagactta acgtacgctc aagatgcgcc 1560
tgatcgtgat ttgaatggaa atatttcatt caaaaatgtt tctttcacct atcctggaga 1620
tactaaacca gctttgaaaa acgtatcgtt tgatatcgaa gcaggtgaga tgatcggtat 1680
cgttggtgca actggatcag gtaaaacaac acttgcccaa ttaatcccaa gattgtatga 1740
tcctactgaa ggtgtggtag ctgttggtgg tgtggatctc aaagcagtaa acgaaaagtc 1800
gttgcgagct gcggtttcgt ttgttctaca aaaggccata ttgtttagtg gtacgattgc 1860
agcaaatttg cgtcaaggta aaaaagacgc taatgaaaag gatttccaaa aagctgttga 1920
aattgcacag gctgcggagt ttgttaatcg ttatgaagat aattttgacc acattgttga 1980
agagcgttct gccaattttt ccggtggtca aaaacaacgt ctttctatag caagaggtgt 2040
tgttggtagt cctaaaatat tgattttaga tgattcaaca tcggctttag atgctcgatc 2100
cgaaaagctg gttcaagaag cacttgacca ggaattaaaa ggaacgacaa caattgtaat 2160
tgctgaaaaa atcagttcag ttattcatgc caaccgcatt ttagtaatgg atgagggtaa 2220
ggtagtaggc atcggtgcac atcacgagtt gcttgaaact tcaccggtct atgctgaaat 2280
ttatcaaaca cagaaagcga aaggggcaga ataatagaaa gtgctttaat tagtgattaa 2340
agcaaagaaa atggaatggg ttacatttgc ttaacgactg tcatttgtaa ggggtgaaat 2400
tttttctgaa atctatgcat tatatgggct taatcgcgtg cgttagctcg tgaaataggg 2460
tacaattata gatgaaataa aattttggag gatatgacag atattaaaca cgcaattaaa 2520
tattttgcaa agtatcttaa gcgttattgg ttagcattgt ctttcgtagt tgttgtgaca 2580
atagcctcaa cttatttcca agttaaagca ccagtgtata tgggaaaggc aattacagaa 2640
ttatcgactt atttaggtca atacttgaat cctcaaacgc atgctgttgc tagcaaaaca 2700
cctttctatc atgcgctgga agcaatgatg attttcttta ctttaactgc agtaacaatg 2760
ttcatttcta gttttatatc tagtcgtatt agttcagagg catcaggccg gatgagaatt 2820
ggtctatttg ctaaattgca acggatgacg attaaatatt ttgacacgca tcaagatggc 2880
gaaattttat ctttgtttac gtctgattta gataatattt ttaacgcgat gaaccaggcc 2940
attttccaac ttttatcaca attcgctttg tttgttggta tcattattat gatgtttcag 3000
cagaacgtca aattagcatg ggtaactatg gcttcaacac cagttgctat tattgtcgcc 3060
tggtttgtga tcagtcgtgc tagaaaatat attgacacgc agcaagatga aactggccgc 3120
atgaatggtt atattaatga gcaaatcaat ggtgagaaga ttattattac gcaaggattg 3180
caacaagaat caattgctaa ttttggtccg tataatgaac gagtgaagca agctactttt 3240
aaaggacaga tgtattctgg tatgctgttt cctctaatgc aaggcttgtc attgttgaac 3300
ttagctattg tgattttctt tgggtcatgg ttaattgttc atgacggtat ggataagtcg 3360
gttggtttgg gattaattgt ggtgtttgtt aactattcac aacagtatta ccaaccaatc 3420
acacaaatta catctattta taatatgttg cagttagctg ttactggtgc aagacgcttg 3480
agcgatgtcc atgaacaagc tgaagaagtt aaccccgaaa gtggtcagaa attaactaat 3540
ttaaagtcgg gtgttaccct agaaaatatt cactttggat acaatgatga caaagaaatt 3600
cttcacggtg tttcaattga cgttaaaaaa ggggaaatga tagcccttgt tggtccgacc 3660
ggctcaggaa agacaacggt catgaactta ttgaatcgat tttatgatgt gactgaagga 3720
tccgtaaagt ttgatggcac cgacgttcgt aagttagact tgaagtcttt acgtgatcat 3780
gttggcattg tgttacagga atcagtatta tttagtggta ctgtcgcaga taatattaaa 3840
tttggtgagc cagatgctac cgaagaagaa atgattgatg ctgccaagca ggcgaacatc 3900
catgacttta ttatgacact tccagaaggt tgatcagacc aaagtagatg acgaaaattc 3960
ggtcttttca acaggtcaaa aacaattatt atcaatcgcc cgcacaattt tgactaatcc 4020
ggatttctta atattagatg aagcaacatc taatgtagat acggtgacag aggcaaaaat 4080
ccaagcggcg atggataatg ttattgctgg acgtacgagt tttgttattg cgcaccgatt 4140
gaaaacaata ctgggtgctg ataaaattgt tgttcttaaa gatggtcgag ttattgaaaa 4200
gggctcgcat caagaattag ttgctgaagg cggattttat tcagaattgt accataatca 4260
aatggtattt gattaacata tagtctgaag ccagagttaa ttggatcagg catagaattt 4320
gttgatttta aacagttatt agttgaaagc gatgtgttga gcattcacgt gccactaaat 4380
aaacaaactt tccataaatt tgactttgat aatcttagtt taatgaaaca aaatgccata 4440
ttaattaaca cagctcgagg tgagatagtt gatacatcag cactgataac gcacttacag 4500
caaggaaaat ttcaaggttt ggcacttgat gcattggaag atgaggatac gaaaggattt 4560
gcaacaaatc cctattacca gtcacttcat aaatttgaga atgtattgct gacaccgcat 4620
attgcgtatt ttacgaaggc agctgttcgt gatattgcaa taactgcatt agaaaatgca 4680
cgtgatattg ttattgaagg taaatcagag aatactgttg tacaatag 4728

Claims (3)

1. A leuconostoc mesenteroides mutant strain for high yield of mannitol is characterized in that: is a Leuconostoc mesenteroides mutant strain with the gene knockout of dextransucrase, the gene knockout of D-lactate dehydrogenase (1756), 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, the gene knockout of acetaldehyde dehydrogenase and the gene knock-in of 1-phosphomannitol dehydrogenase and the gene concatemer of mannitol-1-phosphatase coding gene, the insertion of a D-lactate dehydrogenase gene (ldhA) expression element between 2 coding frames of the coding gene of a mannitol efflux pump (0385/0386), the gene knockout of ethanol dehydrogenase and the knock-in of the gene expression cassette of the mannitol efflux pump, the knockout of the gene cassette of the lactate dehydrogenase (0503) and the knock-in of the gene expression cassette of the mannitol efflux pump and the knock-in of the gene expression cassette of the lactate dehydrogenase (0373), is Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386, ldhA-ee delta adh, mep delta ldh (0503), mep delta ldh (0373), mep strain, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2020, 11 and 19 days and the preservation number of CCTCC No: m2020762.
2. The application method of the leuconostoc mesenteroides mutant strain with high mannitol yield 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), the preservation date is 11 months and 19 days in 2020, and the preservation number is CCTCC No: m2020762 Leuconostoc mesenteroides (Leuconostoc mesenteroides) CCTCC M20188150385/0386: ldhA-ee delta adh: mep delta ldh (0503): mep delta ldh (0373): mep strain is transferred into MRS culture medium by weight percent of 1%, 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 71.58 g/L, and the conversion rate of sucrose to mannitol is 59.65%.
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, 120 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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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107881140A (en) * 2017-11-22 2018-04-06 河北工业大学 The Leuconostoc mesenteroides mutant strain of one plant height production mannitol and its application process
CN109593696A (en) * 2018-12-17 2019-04-09 天津博瑞威生物医药科技有限公司 One plant height produces Leuconostoc mesenteroides mutant strain and its application method of mannitol
CN109593699A (en) * 2018-12-20 2019-04-09 河北工业大学 One plant height produces Leuconostoc mesenteroides mutant strain and its application method of mannitol

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107881140A (en) * 2017-11-22 2018-04-06 河北工业大学 The Leuconostoc mesenteroides mutant strain of one plant height production mannitol and its application process
CN109593696A (en) * 2018-12-17 2019-04-09 天津博瑞威生物医药科技有限公司 One plant height produces Leuconostoc mesenteroides mutant strain and its application method of mannitol
CN109593699A (en) * 2018-12-20 2019-04-09 河北工业大学 One plant height produces Leuconostoc mesenteroides mutant strain and its application method of mannitol

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
李自强: "明串珠菌产双乙酰的研究", 《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》 *
王小芳等: "利用代谢工程构建D-甘露醇生产菌株", 《生物工程学报》 *

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