Mutant MntP protein and high-yield L-threonine strain expressing same

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to a mutant MntP protein and a high-yield L-threonine strain for expressing the mutant MntP protein. The invention provides a recombinant strain which expresses a mutant MntP manganese ion transport protein, the 25th amino acid of the wild MntP manganese ion transport protein shown as SEQ ID NO: 1 is mutated from glycine to aspartic acid, and the L-threonine yield of the recombinant strain can be improved. The recombinant strain disclosed by the invention is preferably obtained by modifying an escherichia coli THRS strain, an escherichia coli THRS-6 strain, an escherichia coli THRS-YbiI Q86L mutant strain or an escherichia coli THRS-6-YbiI Q86L mutant strain, and the combination of the mutant proteins MntP G25D and YbiI Q86L has a synergistic technical effect, so that the yield of the L-threonine can be further increased.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and in particular relates to a mutant MntP protein and a strain expressing the mutant MntP protein and producing high L-threonine. Background Art

[0002] Threonine is one of the eight amino acids for human and animal growth. It is an important nutritional enhancer and is widely used in medicine, chemical reagents, food enhancers, feed additives, etc. At present, threonine is mainly produced industrially by microbial fermentation. Microorganisms are usually modified to increase the production of L-threonine. A variety of bacteria can be used in the production of L-threonine, such as wild-type induced mutants of Escherichia, Corynebacterium, and Brevibacterium as production strains. With the increasing global demand for threonine, the construction and modification of high-yield threonine strains is particularly important. Specific examples include: U.S. Patent US8293505B2 applied for by Ajinomoto Co., Ltd. in 2006, which used the Escherichia coli MG1655 strain to increase the expression of the pyrE gene encoding orotate phosphoribosyltransferase by increasing the copy number of the pyrE gene encoding orotate phosphoribosyltransferase; Chinese patent CN115572717A applied for by Meihua Group in 2021, by modifying the genome of Escherichia coli MHZ-0215-2 and introducing point mutations to produce the CreC protein variant R77P, thereby improving the production capacity of threonine. The average conversion rate of the modified R77P mutant strain MHZ-0221-4 in the shake flask was 20.6%, an increase of 4.39 percentage points over the starting bacteria.

[0003] The protein encoded by the mntP gene is a manganese ion transporter that regulates the transport of manganese ions (Mn 2+ ) is a key protein in the transport and metabolism of manganese, and plays an important role in maintaining the level of manganese in cells. For organisms, manganese ions play an important role in a variety of biological metabolic processes and are an essential nutrient for intracellular activities. It can serve as a cofactor for a variety of enzymes, including arginase, glutamine synthetase (GS), pyruvate carboxylase and Mn superoxide dismutase (Mn-SOD), among which these enzymes are important components of the amino acid metabolic pathway in organisms.

[0004] However, there is no literature report on increasing threonine production by mutating manganese ion transporters. Summary of the invention

[0005] In order to solve the above technical problems, the present invention performs whole genome sequencing on a mutagenic bacterium FFTHR-1 with high threonine production in the laboratory, and finds that the 25th amino acid sequence of the MntP manganese ion transporter encoded by the mntP gene mutates from glycine to aspartic acid, which is recorded as MntP G25D, and is one of the reasons for the high threonine production of the mutagenic bacterium; introducing its encoding gene into other production strains can also increase the yield of threonine.

[0006] The present invention provides a mutant MntP manganese ion transporter. Relative to the wild-type MntP manganese ion transporter whose sequence is shown in SEQ ID NO: 1, the mutant MntP manganese ion transporter comprises at least the following mutation site: the 25th amino acid of the wild-type MntP manganese ion transporter is mutated from glycine to aspartic acid.

[0007] In some embodiments, the mutant MntP manganese ion transporter may also contain other amino acid substitutions, deletions, insertions, additions or inversions, as long as the mutation does not affect the function of the MntP manganese ion transporter. In a specific embodiment, the amino acid sequence of the mutant MntP manganese ion transporter is as shown in SEQ ID NO: 2.

[0008] In some embodiments, the mntP gene sequence encoding the wild-type MntP manganese ion transporter is shown as SEQ ID NO: 3. In some embodiments, the mutant mntP gene sequence encoding the mutant MntP manganese ion transporter is shown as SEQ ID NO: 4 (named as mntP G25D gene).

[0009] The present invention also provides the use of the mutant MntP manganese ion transporter in increasing the threonine yield in a threonine-producing strain.

[0010] The present invention also provides a recombinant strain capable of producing high amounts of L-threonine, wherein the recombinant strain expresses the mutant MntP manganese ion transporter.

[0011] In some embodiments, the recombinant strain is Escherichia coli, preferably modified based on Escherichia coli THRS strain, THRS-6 strain, THRS-YbiIQ86L mutant strain or THRS-6-YbiIQ86L mutant strain.

[0012] The Escherichia coli THRS is the Escherichia coli THRS strain in patent CN202411834458.7, which was deposited in the China General Microbiological Culture Collection Center on August 20, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.31694, and the classification name is Escherichia coli.

[0013] The Escherichia coli THRS-6 is the Escherichia coli THRS-6 strain in patent CN202411834455.3, which was deposited in the China General Microbiological Culture Collection Center on August 20, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.31695, and the classification name is Escherichia coli.

[0014] The THRS-YbiIQ86L mutant strain is an Escherichia coli THRS strain expressing a mutant YbiI protein, the amino acid sequence of which is shown in SEQ ID NO: 13, wherein the 86th glutamine of the wild-type YbiI protein is mutated to leucine. The ybiI gene of the Escherichia coli THRS strain is replaced with a mutant ybiI gene to express the mutant YbiI protein, thereby obtaining the THRS-YbiIQ86L mutant strain.

[0015] The THRS-6-YbiIQ86L mutant strain is an Escherichia coli THRS-6 strain expressing a mutant YbiI protein, the amino acid sequence of which is shown in SEQ ID NO: 13, wherein the 86th glutamine of the wild-type YbiI protein is mutated to leucine. The ybiI gene of the Escherichia coli THRS-6 strain is replaced with a mutant ybiI gene to express the mutant YbiI protein, thereby obtaining the THRS-YbiIQ86L mutant strain.

[0016] In some embodiments, the nucleotide sequence of the mutant ybiI gene is shown in SEQ ID NO: 14 (named as ybiIQ86L gene).

[0017] The present invention also provides a method for preparing the recombinant strain, comprising the following steps: using the mutant mntP gene as a template to design homology arm primers, constructing PCR amplification products, transforming the PCR amplification products into a threonine production strain, replacing the target region by homologous recombination, and obtaining a recombinant strain. The threonine production strain is Escherichia coli, preferably Escherichia coli THRS strain, THRS-6 strain, THRS-YbiIQ86L mutant strain or THRS-6-YbiIQ86L mutant strain.

[0018] The present invention also provides a method for producing L-threonine, comprising the following steps: inoculating the recombinant strain on a seed culture medium for cultivation to obtain a seed solution, transferring the seed solution into a fermentation culture medium at an inoculation rate of 20% to produce L-threonine.

[0019] In some embodiments, the seed culture medium is: 5 g / L corn steep liquor powder, 20 g / L glucose, 5 g / L yeast powder, 2 g / L KH2PO4, 1 g / L magnesium sulfate, 20 mg / L FeSO4·7H2O, and 20 mg / L MnSO4·H2O; the fermentation medium is: 20 g / L glucose, 2 g / L potassium dihydrogen phosphate, 3 g / L yeast powder, 1 g / L betaine, 1 g / L magnesium sulfate, 10 mg / L FeSO4·7H2O, 10 mg / L MnSO4·H2O, 8 g / L corn steep liquor powder, and 10 mg / L vitamin B1.

[0020] Compared with the prior art, the beneficial effects of the present invention include at least:

[0021] 1. The present invention provides a mutant MntP manganese ion transporter, which can increase the threonine production of the strain when expressed in the strain. For example, after the THRS strain expresses the mutant MntP manganese ion transporter, the threonine production of the recombinant strain increases by 5.38%, and the threonine production after 48h fermentation reaches 105.7g / L; after the THRS-6 strain expresses the mutant MntP manganese ion transporter, the threonine production of the recombinant strain increases by 3.91%, and the threonine production after 48h fermentation reaches 130.2g / L.

[0022] 2. The mutant MntP manganese ion transporter can also be combined with other mutations to further increase threonine production, which has a synergistic technical effect. For example, the THRS strain contains both the expression of MntP G25D and YbiIQ86L, and the threonine production is increased by 15.15%, and the 48h fermentation threonine production reaches 115.5g / L; the THRS-6 strain contains both the expression of MntP G25D and YbiIQ86L, and the threonine production is increased by 20.67%, and the 48h fermentation threonine production reaches 151.2g / L. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0024] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0025] Example 1: Construction of THRS recombinant strain

[0026] 1. Construction of mntP gene knockout strain

[0027] The mntP gene in the THRS strain (deposit number: CGMCC NO.31694) was knocked out by homologous recombination. The specific steps are as follows:

[0028] Prepare competent E. coli cells, induce pKD46 plasmid into competent E. coli cells with arabinose stock solution, and culture and screen in a resistance medium containing ampicillin. Select homologous arms at both ends of the target gene, design the homologous arm primers Pkan-F / Pkan-R of the kanamycin resistance gene kan using plasmid PKD13 as a template for PCR amplification, and transfer the purified PCR product into a strain containing pKD46 plasmid by electrotransformation to obtain a pKD46-kan strain. Homologous recombination occurs during cultivation at 30°C to obtain a homologous recombination strain, and the pKD46 temperature-sensitive plasmid is removed by cultivation at 37°C. Resistance screening is performed on a medium containing kanamycin to obtain a strain with the target gene removed, and PCR is performed with primers PmntP-F' / PmntP-R' to verify whether the target gene is successfully removed. Subsequently, the pCP20 plasmid is introduced and transformed to express the flippase recombinase gene, promote homologous recombination of the FRT site itself, and finally achieve the knockout of the mntP gene. The nmtP gene knockout strain was cultured in LB medium and kanamycin resistance medium at the same time. The strain that grew normally in LB medium but did not grow in resistance medium was the nmtP gene knockout strain. Finally, it was cultured at 42°C to remove the pCP20 thermosensitive plasmid and verified by PCR amplification with PpCP20-F / PpCP20-R primer pair to obtain the mntP knockout strain (named THRS-△mntP strain).

[0029] 2. Construction of mntP G25D gene mutant strain

[0030] Using the mutant sequence of mntP G25D as a template, homology arm primers PmntP-F / PmntP-R were designed, and PCR amplification products of the exogenous mntPG25D gene were constructed. The PCR amplification products were transformed into Escherichia coli THRS, and the target region was replaced by homologous recombination to finally achieve the knock-in of the mntP G25D mutant gene. The recombinant strain was verified by PCR and sequenced. The sequencing results are shown in SEQ ID NO: 4, and the mntP G25D gene mutant strain (named THRS-MntP G25D mutant strain) was successfully obtained.

[0031] 3. Construction of ybiIQ86L mutant strain

[0032] Using the mutant ybiI gene sequence (SEQ ID NO: 14, named ybiIQ86L) as a template, homology arm primers PybiI-F / PybiI-R (sequences are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively) were designed to construct a PCR amplification product of the exogenous ybiIQ86L gene. The PCR amplification product was transformed into Escherichia coli THRS, and the target region was replaced by homologous recombination. The specific implementation method was the same as 1, and finally the knock-in of the ybiIQ86L mutant gene was achieved. The above-mentioned recombinant strain was verified by PCR and sequenced. The sequencing results are shown in SEQ ID NO: 14, that is, the ybiIQ86L mutant strain (named THRS-YbiIQ86L mutant strain) was successfully obtained.

[0033] 4. Construction of strains containing both mntP G25D and ybiIQ86L gene mutations

[0034] Another strain of threonine high-producing bacteria (ybiIQ86L gene mutant strain) was used to prepare competent cells. The PCR amplification product of the exogenous mntP G25D gene was constructed using the above homology arm primers PmntP-F / PmntP-R, and the PCR amplification product was transformed into the competent cells of the ybiIQ86L gene mutant strain, and the target region was replaced by homologous recombination. The specific implementation method was the same as above, and finally the knock-in of the mntP G25D mutant gene was achieved. The above recombinant strain was subjected to PCR verification and sequencing analysis. The sequencing results are shown in SEQ ID NO: 4, that is, a mutant strain containing both mntP G25D and ybiIQ86L genes (named THRS-MntP G25D-YbiIQ86L mutant strain) was successfully obtained.

[0035] Example 2: Fermentation production of L-threonine by recombinant strains

[0036] The THRS-YbiIQ86L mutant strain, THRS-△mntP strain, THRS-MntP G25D mutant strain, THRS-MntP G25D-YbiIQ86L mutant strain and the original THRS strain obtained in Example 1 were inoculated on a seed culture medium for culture to obtain a seed solution; the seed culture medium included the following components in concentrations: corn steep liquor 4.2 g / L, glucose 10 g / L, yeast powder 2.5 g / L, KH2PO4 2 g / L, magnesium sulfate 1.2 g / L, FeSO4·7H2O 20 mg / L and MnSO4·H2O 20 mg / L, and biotin 30 mg / L. Three parallel experiments were set for each group of experiments.

[0037] The seed culture obtained by the above-mentioned culture method is transferred into the fermentation medium at a 20% inoculation rate for culture:

[0038] 1. 5L seed tank process control

[0039] a. Adjust the temperature to 37℃, pH 7.0, speed to 500rpm, air volume to 0.3m 3 / h, the whole process temperature is controlled at 37℃, the tank pressure is 0.05~0.08MPa, and the culture period is 10h;

[0040] b. Transplantation standard: OD600: 12-15.

[0041] c. The seed culture medium is corn steep liquor powder 5g / L, glucose 20g / L, yeast powder 5g / L, KH2PO4 2g / L, magnesium sulfate 1g / L, FeSO4·7H2O 20mg / L, MnSO4·H2O 20mg / L;

[0042] 2. 5L fermentation tank fermentation process control

[0043] a. Adjust the temperature to 37℃, pH 7.0, initial speed to 300rpm, air volume to 0.3m 3 / h, the whole process temperature is controlled at 37℃, and the tank pressure is 0.05~0.08MPa;

[0044] c.DO control: 0h, air volume 0.3m 3 / h, 300rpm, tank pressure 0.05MPa;

[0045] d. When DO drops below 30%, the dissolved oxygen level is controlled at 30% by adjusting the ventilation volume and stirring speed until the fermentation ends;

[0046] e. The fermentation medium is glucose 20 g / L, potassium dihydrogen phosphate 2 g / L, yeast powder 3 g / L, betaine 1 g / L, magnesium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, corn steep liquor powder 8 g / L, vitamin B1 10 mg / L.

[0047] 3. Determination method of threonine:

[0048] (1) Sample treatment: Take 1 mL of the fermentation liquid after 48 hours of fermentation, centrifuge at 12000 rpm for 10 min to remove the bacteria and take the supernatant. Dilute the supernatant appropriately with deionized water and then filter it through a filter membrane with a pore size of 0.22 μm.

[0049] (2) Analytical method: OPA pre-column derivatization

[0050] (3) Chromatographic conditions:

[0051] ①Chromatographic column: Chromatographic column C18 (250×4.6) mm

[0052] ② Column temperature: 40℃

[0053] ③ Mobile phase A: Weigh 3.01g of anhydrous sodium acetate into a beaker, add ultrapure water to dissolve and dilute to 1L, then add 200μL of triethylamine, and adjust the pH to 7.20±0.05 with 5% acetic acid; add 5mL of tetrahydrofuran after filtration, mix and filter with a 0.22μm inorganic filter membrane, then put into an ultrasonic cleaning pot to exhaust for 20min, and set aside.

[0054] Mobile phase B: Weigh 3.01 g of anhydrous sodium acetate into a beaker; add ultrapure water to dissolve and dilute to 200 mL; adjust the pH to 7.20±0.05 with 5% acetic acid; then add 400 mL of acetonitrile and 400 mL of methanol to the solution, mix and filter, place in an ultrasonic cleaning pot to exhaust for 20 minutes, and set aside.

[0055] ④Flow rate: 1.0ml / min;

[0056] ⑤Ultraviolet detector: 338nm;

[0057] ⑥Column temperature: 40℃.

[0058] 4. Verification of strain production performance in 5L fermenter

[0059] strain Yield (g / L) Conversion rate (%) THRS strains 100.3g / L 30.2％ THRS-YbiIQ86L mutant strain 107.8g / L 29.7％ THRS-ΔmntP strain 91.2g / L 29.3％ THRS-MntPG25D mutant strain 105.7g / L 31.1％ THRS-MntPG25D-YbiIQ86L 115.5g / L 30.7％

[0060] The results showed that after the 25th glycine in the amino acid sequence of the protein encoded by the mntP gene was replaced by aspartic acid, the L-threonine production was significantly improved compared with the original strain. MntP G25D and YbiIQ86L can work synergistically to further increase the L-threonine production. The THRS-MntP G25D-YbiIQ86L mutant strain was recorded as FFTHR-38.

[0061] Example 3: Construction of THRS-6 recombinant strain and production of L-threonine

[0062] According to the method of Example 1, based on the THRS-6 strain (deposit number CGMCC NO.31695), THRS-6-YbiIQ86L mutant strain, THRS-6-△mntP strain, THRS-6-MntP G25D mutant strain, THRS-6-MntP G25D-YbiIQ86L mutant strain were constructed. The verification results of the strain production performance in the 5L fermentation tank are as follows:

[0063] strain Yield (g / L) Conversion rate (%) THRS-6 125.3g / L 42％ THRS-6-YbiIQ86L 142.1g / L 44％ THRS-6-△mntP 105.2g / L 29％ THRS-6-MntPG25D 130.2g / L 43.3％ THRS-6-MntPG25D-YbiIQ86L 151.2g / L 45.1％

[0064] As shown in the results, after the strain was replaced with the THRS-6 strain, the 25th glycine in the amino acid sequence of the protein encoded by the mntP gene was replaced by aspartic acid, and the L-threonine production was also significantly improved relative to the THRS-6 strain. Similarly, in the THRS-6 strain, MntP G25D and YbiIQ86L can work synergistically to further increase the L-threonine production. It can be seen that the MntP G25D of the present invention is suitable for different strains, can increase the L-threonine production, and has a synergistic effect with YbiIQ86L. The THRS-6-MntP G25D-YbiIQ86L mutant strain is recorded as FFTHR-39.

[0065] The nucleotide sequence and amino acid sequence involved in the present invention are as follows:

[0066] SEQ ID NO: 1: (MntP protein)

[0067] MNITATVLLAFGMSMDAFAASIGKGATLHKPKFSEALRTGLIFGAVETLTPLIGWGMGMLASRFVLEWNHWIAFVLLIFLGGRMIIEGFRGADDEDEEPRRRHGFWLLVTTAIATSLDAMAVGVGLAFLQVNIIATALAIGCATLIMSTLGMMVGRFIGSIIGKKAEILGGLVLIGIGVQILWTHFHG

[0068] SEQ ID NO: 2: (Mutant MntP protein)

[0069] MNITATVLLAFGMSMDAFAASIGKDATLHKPKFSEALRTGLIFGAVETLTPLIGWGMGMLASRFVLEWNHWIAFVLLIFLGGRMIIEGFRGADDEDEEPRRRHGFWLLVTTAIATSLDAMAVGVGLAFLQVNIIATALAIGCATLIMSTLGMMVGRFIGSIIGKKAEILGGLVLIGIGVQILWTHFHG

[0070] SEQ ID NO: 3: (mntP gene)

[0071] atgaatatcactgctactgttcttcttgcgtttggtatgtcgatggatgcatttgctgcatcaatcggtaaaggtgccaccctccataaaccgaaattttctgaagcattgcgaaccggccttatttttggtgccgtcgaaaccctgacgccgctgatcggctggggaatgggcatgttagccagccggtttgtccttgaatggaaccactggattgcgtttgtgctgctgatattcctcggcgggcgaatgattattgagggttttcgtggcgcagatgatgaagatgaagagccgcgccgtcgacacggtttctggctactggtaaccaccgcgattgccaccagcctggatgccatggctgtgggtgttggtcttgctttcctgcaggtcaacattatcgcgaccgcattggccattggttgtgcaaccttgattatgtcaacattagggatgatggttggtcgctttatcggctcaattattgggaaaaaagcggaaattctcggcgggctggtgctgatcggcatcggcgtccagatcctctggacgcacttccacggttaa

[0072] SEQ ID NO: 4: (mntP mutant gene)

[0073] atgaatatcactgctactgttcttcttgcgtttggtatgtcgatggatgcatttgctgcatcaatcggtaaagatgccaccctccataaaccgaaattttctgaagcattgcgaaccggccttatttttggtgccgtcgaaaccctgacgccgctgatcggctggggaatgggcatgttagccagccggtttgtccttgaatggaaccactggattgcgtttgtgctgctgatattcctcggcgggcgaatgattattgagggttttcgtggcgcagatgatgaagatgaagagccgcgccgtcgacacggtttctggctactggtaaccaccgcgattgccaccagcctggatgccatggctgtgggtgttggtcttgctttcctgcaggtcaacattatcgcgaccgcattggccattggttgtgcaaccttgattatgtcaacattagggatgatggttggtcgctttatcggctcaattattgggaaaaaagcggaaattctcggcgggctggtgctgatcggcatcggcgtccagatcctctggacgcacttccacggttaa

[0074] SEQ ID NO:5: (Pkan-F primer sequence 5'→3')

[0075] CGATGATGTGTTTTATGGATACCCCGGTCAGGACATTGTCAGGCTGGAGCTGCT TC

[0076] SEQ ID NO:6: (Pkan-R primer sequence 5'→3')

[0077] GCCAGACGGACTTTAATATTCACCTCTGGCAGCGTTCTTATCCGGGGATCCGTC GACC

[0078] SEQ ID NO:7: (PmntP-F primer sequence 5'→3')

[0079] CGATGATGTGTTTTATGGATACCCCGGTCAGGACATTGTCATGAATATCACTGCTACTGTTCTTCTTGC

[0080] SEQ ID NO:8: (PmntP-R primer sequence 5'→3')

[0081] GCCAGACGGACTTTAATATTCACCTCTGGCAGCGTTCTTATTAACCGTGGAAGTGCGTCC

[0082] SEQ ID NO:9: (PmntP-F' primer sequence 5'→3')

[0083] TTATGGATACCCCGGTCAGGACATTGTC

[0084] SEQ ID NO:10: (PmntP-R' primer sequence 5'→3')

[0085] GGACTTTAATATTCACCTCTGGCAGCGTTCTTA

[0086] SEQ ID NO:11: (PpCP20-F primer sequence 5'→3')

[0087] ATGTCTGAATTAGTTGTTTTCAAAGAAATGAAC

[0088] SEQ ID NO:12: (PpCP20-R primer sequence 5'→3')

[0089] GATCCTTCCGTATTTAGCCAGTATGTTCT

[0090] SEQ ID NO:13: (mutant YbiI protein sequence)

[0091] MASGWANDDAVNEQINSTIEDAIARARGEIPRGESLDECEECGAPIPQARREAIPG VRLCIHCQQEKDLQKPAYTGYNRRGSKDSLLR

[0092] SEQ ID NO:14: (Nucleotide sequence of mutant ybiI gene)

[0093] ttaacgtaacaggctgtctttcgaacctctgcgattatatcctgtataagctggtttttgtaaatctttctcctgctgacaatgaatacataagcgcacgccaggaatggcttcccgacgggcctgcggggatg ggggcaccgcactcttcacattcatccaggctttcgccgcgcggaatttcaccccgagcgcgggcaatcgcatcttcaattgtactgttgatctgttcgttgacggcgtcatcgttagcccaaccggatgccat

[0094] SEQ ID NO:15: (PybiI-F primer sequence 5'→3')

[0095] AAATACAAATTATACTCGCTATGCATCTCGCCAGTTGTAGTTAACGTAACAGGCTGTCTTTCGAACCTC

[0096] SEQ ID NO:16: (PybiI-R primer sequence 5'→3')

[0097] CTACACTTAACTGTACAAGTATTGATATGGGGAGGTCGATATGGCATCCGGTTG GGCTAAC

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. An application of a mutant MntP manganese ion transporter to increase threonine production in a threonine-producing strain, characterized in that: Relative to the wild-type MntP manganese ion transporter whose sequence is shown in SEQ ID NO: 1, the mutant MntP manganese ion transporter comprises at least the following mutation site: the 25th amino acid of the wild-type MntP manganese ion transporter is mutated from glycine to aspartic acid.

2. The use according to claim 1, characterized in that The amino acid sequence of the mutant MntP manganese ion transporter is shown in SEQ ID NO:

2.

3. A recombinant strain for high production of L-threonine, characterized in that: The recombinant strain expresses a mutant MntP manganese ion transporter. Relative to the wild-type MntP manganese ion transporter whose sequence is shown in SEQ ID NO: 1, the mutant MntP manganese ion transporter comprises at least the following mutation site: the 25th amino acid of the wild-type MntP manganese ion transporter is mutated from glycine to aspartic acid.

4. The recombinant strain according to claim 3, characterized in that The amino acid sequence of the mutant MntP manganese ion transporter is shown in SEQ ID NO:

2.

5. The recombinant strain according to claim 3, characterized in that The recombinant strain is Escherichia coli.

6. The recombinant strain according to claim 5, characterized in that The recombinant strain is transformed based on the Escherichia coli THRS strain, THRS-6 strain, THRS-YbiIQ86L mutant strain or THRS-6-YbiIQ86L mutant strain; The THRS-YbiIQ86L mutant strain is an Escherichia coli THRS strain expressing a mutant YbiI protein, and the THRS-6-YbiIQ86L mutant strain is an Escherichia coli THRS-6 strain expressing a mutant YbiI protein. The amino acid sequence of the mutant YbiI protein is shown in SEQ ID NO:

13.

7. The method for preparing the recombinant strain according to any one of claims 3 to 6, characterized in that: include: The homology arm primers were designed using the gene encoding the mutant MntP manganese ion transporter as a template, PCR amplification products were constructed, the PCR amplification products were transformed into a threonine production strain, and the target region was replaced by homologous recombination to obtain a recombinant strain.

8. The method according to claim 7, wherein the threonine producing strain is Escherichia coli.

9. The method according to claim 7, wherein the threonine producing strain is an Escherichia coli THRS strain, a THRS-6 strain, a THRS-YbiIQ86L mutant strain or a THRS-6-YbiIQ86L mutant strain; The THRS-YbiIQ86L mutant strain is an Escherichia coli THRS strain expressing a mutant YbiI protein, and the THRS-6-YbiIQ86L mutant strain is an Escherichia coli THRS-6 strain expressing a mutant YbiI protein. The amino acid sequence of the mutant YbiI protein is shown in SEQ ID NO:

13.

10. A method for producing L-threonine, characterized in that: The method comprises the following steps: inoculating the recombinant strain according to any one of claims 3 to 6 on a seed culture medium to obtain a seed solution, and transferring the seed solution into a fermentation culture medium at an inoculation rate of 20% to produce L-threonine.