Mutant mntp protein and strain expressing same for high-yield l-threonine production

Abstract

Provided in the present invention are a manganese ion transporter (MntP) protein mutant and a recombinant strain expressing the mutant for high-yield L-threonine production. The mutant has an amino acid sequence as shown in SEQ ID NO: 2, which is obtained by means of mutating glycine (G) at position 25 to aspartic acid (D) in a wild-type MntP protein sequence as shown in SEQ ID NO: 1. By means of homologous recombination, the mutant is introduced into an Escherichia coli strain, providing the constructed recombinant strain with an increased yield of L-threonine.

Description

A mutant MntP protein and a strain expressing it that produces high levels of L-threonine. Technical Field

[0001] This application belongs to the field of microbial fermentation, specifically relating to a mutant MntP protein and a strain that expresses it and produces high levels of L-threonine. Background Technology

[0002] Threonine is one of the eight essential amino acids for human and animal growth and is an important nutritional fortifier widely used in medicine, chemical reagents, food fortifiers, and feed additives. Currently, threonine is mainly produced industrially through microbial fermentation. Typically, microorganisms are modified to increase L-threonine yield. Various bacteria can be used in L-threonine production, such as wild-type mutant strains induced from *Escherichia coli*, *Corynebacterium*, and *Bryophyceae*. With the increasing global demand for threonine, the construction and modification of high-yield threonine-producing strains are particularly important. Specific examples include: US Patent 8293505B2, filed by Ajinomoto Co., Ltd. in 2006, which utilizes 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; and Chinese Patent CN115572717A, filed by Meihua Group in 2021, which modifies the genome of Escherichia coli MHZ-0215-2, introducing point mutations to generate the CreC protein variant R77P, thereby improving the production capacity of threonine. The modified R77P mutant strain MHZ-0221-4 achieved an average conversion rate of 20.6% in shake flasks, which is 4.39 percentage points higher than the original strain.

[0003] The mntP gene encodes a manganese ion transporter that regulates the transport of manganese ions (Mn) in organisms. 2+ Manganese ions are key proteins involved in transport and metabolism, playing a crucial role in maintaining intracellular manganese levels. For organisms, manganese ions play a vital role in various metabolic processes and are an essential nutrient for intracellular activities. They can act as cofactors for many enzymes, including arginase, glutamine synthase (GS), pyruvate carboxylase, and manganese superoxide dismutase (Mn-SOD), which are important components of amino acid metabolism pathways in organisms.

[0004] However, there are currently no literature reports on increasing threonine production by mutating manganese ion transporters. Summary of the Invention

[0005] This application performed whole-genome sequencing on a high-threonine-producing mutant bacterium, FFTHR-1, in the laboratory. It was found that a mutation occurred at position 25 of the amino acid sequence of the MntP manganese ion transporter protein encoded by the mntP gene, changing from glycine to aspartic acid, denoted as MntP G25D. This mutation is one of the reasons for the high threonine production of this mutant bacterium. Introducing its encoding gene into other producing strains can also increase the yield of threonine.

[0006] In a first aspect, this application provides a mutant MntP manganese ion transporter protein, which, relative to the wild-type MntP manganese ion transporter protein with the sequence shown in SEQ ID NO:1, contains at least the following mutation site: the 25th amino acid of the wild-type MntP manganese ion transporter protein is mutated from glycine to aspartic acid.

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

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

[0009] Secondly, this application provides the application of the mutant MntP manganese ion transporter protein in increasing threonine production in strains that produce threonine.

[0010] Thirdly, this application provides a recombinant strain that produces high levels of L-threonine, wherein the recombinant strain expresses the mutant MntP manganese ion transporter protein.

[0011] In some embodiments, the recombinant strain is Escherichia coli, preferably derived from Escherichia coli THRS strain, THRS-6 strain, THRS-YbiI Q86L mutant strain, or THRS-6-YbiI Q86L mutant strain.

[0012] The Escherichia coli THRS strain mentioned is the Escherichia coli THRS strain in patent CN202411834458.7, which was deposited at the China General Microbiological Culture Collection Center on August 20, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.31694.

[0013] The Escherichia coli THRS-6 strain mentioned is the Escherichia coli THRS-6 strain in patent CN202411834455.3, which was deposited at the China General Microbiological Culture Collection Center on August 20, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.31695.

[0014] The THRS-YbiI Q86L mutant strain is an *E. coli* THRS strain expressing the mutant YbiI protein. The amino acid sequence of the mutant YbiI protein is shown in SEQ ID NO:13, where the wild-type YbiI protein has a glutamine-leucine mutation at position 86. The ybiI gene in the *E. coli* THRS strain was replaced with the mutant ybiI gene to express the mutant YbiI protein, thus obtaining the THRS-YbiI Q86L mutant strain.

[0015] The THRS-6-YbiI Q86L mutant strain is an *E. coli* strain THRS-6 expressing the mutant YbiI protein. The amino acid sequence of the mutant YbiI protein is shown in SEQ ID NO:13, where the wild-type YbiI protein has a glutamine-leucine mutation at position 86. The ybiI gene in the *E. coli* THRS-6 strain was replaced with the mutant ybiI gene to express the mutant YbiI protein, thus obtaining the THRS-YbiI Q86L mutant strain.

[0016] In some embodiments, the nucleotide sequence of the mutant ybiI gene is shown in SEQ ID NO:14 (named ybiI Q86L gene).

[0017] Fourthly, this application provides a method for preparing the recombinant strain as described in the third aspect, wherein the method includes the following steps: designing homologous arm primers using the mutant mntP gene as a template, constructing a PCR amplification product, transforming the PCR amplification product into a threonine-producing strain, and replacing the target region through homologous recombination to obtain the recombinant strain. The threonine-producing strain is *Escherichia coli*, preferably *Escherichia coli* THRS strain, THRS-6 strain, THRS-YbiI Q86L mutant strain, or THRS-6-YbiI Q86L mutant strain.

[0018] Fifthly, this application provides a method for producing L-threonine, wherein the method includes the following steps: inoculating a recombinant strain 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.

[0019] In some implementations, the seed culture medium consists of: 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 culture medium consists of: 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 this application include at least the following:

[0021] (1) This application provides a mutant MntP manganese ion transporter protein, which can increase the threonine production of the strain when expressed in a bacterial strain. For example, after expressing the mutant MntP manganese ion transporter protein in the THRS strain, the threonine production of the recombinant strain increased by 5.38%, and the threonine production reached 105.7 g / L after 48 h of fermentation; after expressing the mutant MntP manganese ion transporter protein in the THRS-6 strain, the threonine production of the recombinant strain increased by 3.91%, and the threonine production reached 130.2 g / L after 48 h of fermentation.

[0022] (2) The mutant MntP manganese ion transporter can also be combined with other mutants to further increase threonine production, exhibiting a synergistic effect. For example, the THRS strain expressing both MntP G25D and YbiI Q86L increased threonine production by 15.15%, reaching 115.5 g / L after 48 h of fermentation; the THRS-6 strain expressing both MntP G25D and YbiI Q86L increased threonine production by 20.67%, reaching 151.2 g / L after 48 h of fermentation. Detailed Implementation

[0023] To make the objectives, technical solutions, and beneficial effects of this application clearer, the following detailed description of specific embodiments is provided. It should be understood that the specific embodiments described in the following description are merely illustrative examples of specific implementations of this application and are intended to explain this application, but do not constitute a limitation thereof.

[0024] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them.

[0025] Example 1: Construction of THRS recombinant strain

[0026] 1. Construction of mntP gene knockout strains

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

[0028] Competent E. coli cells were prepared, and the pKD46 plasmid was induced into competent E. coli cells with arabinose stock solution. The cells were then cultured and screened in ampicillin-containing resistant medium. Homologous arms were selected at both ends of the target gene. Primers Pkan-F / Pkan-R, using plasmid pKD13 as a template, were designed for the homologous arms of the kanamycin resistance gene kan. PCR amplification was performed, and the purified PCR product was electroporated into a strain containing the pKD46 plasmid to obtain the pKD46-kan strain. Homologous recombination occurred during culture at 30°C, yielding a homologous recombination strain. The pKD46 temperature-sensitive plasmid was removed by culture at 37°C. Resistance screening was then performed on kanamycin-containing medium to obtain strains with the target gene removed. PCR was performed using primer pair PmntP-F' / PmntP-R' to verify successful removal of the target gene. Subsequently, the pCP20 plasmid was transformed to express the invertase recombinase gene, promoting homologous recombination at the FRT site, ultimately achieving the knockout of the mntP gene. The nmtP gene knockout strain was cultured simultaneously in LB medium and kanamycin-resistant medium. Strains that grew normally in LB medium but not in kanamycin-resistant medium were identified as nmtP gene knockout strains. Finally, the pCP20 temperature-sensitive plasmid was removed by incubation at 42°C. PCR amplification using the PpCP20-F / PpCP20-R primer pair confirmed the mntP knockout strain (named THRS-△mntP strain).

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

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

[0031] 3. Construction of the ybiI Q86L gene mutant strain

[0032] Using the mutant ybiI gene sequence (SEQ ID NO:14, named ybiI Q86L) as a template, homologous arm primers PybiI-F / PybiI-R (sequences shown in SEQ ID NO:15 and SEQ ID NO:16, respectively) were designed to construct the PCR amplification product of the exogenous ybiI Q86L gene. The PCR amplification product was transformed into E. coli THRS, and the target region was replaced by homologous recombination. The specific implementation method was the same as in 1, and the ybiI Q86L mutant gene was finally knocked in. The above recombinant strain was verified by PCR and sequenced. The sequencing results are shown in SEQ ID NO:14, that is, the ybiI Q86L mutant strain (named THRS-YbiI Q86L mutant strain) was successfully obtained.

[0033] 4. Construction of strains containing both mntP G25D and ybiI Q86L gene mutants

[0034] Competent cells were prepared from another high-threonine-producing bacterium (ybiI Q86L gene mutant strain). Using the aforementioned homologous arm primers PmntP-F / PmntP-R, a PCR amplification product of the exogenous mntP G25D gene was constructed. This PCR amplification product was then transformed into competent cells of the ybiI Q86L gene mutant strain. Homologous recombination was used to replace the target region, following the same implementation method as above, ultimately achieving the knock-in of the mntP G25D mutant gene. The recombinant strain was validated by PCR and sequenced. The sequencing results are shown in SEQ ID NO:4, indicating that a mutant strain containing both the mntP G25D and ybiI Q86L genes was successfully obtained (named THRS-MntP G25D-YbiI Q86L mutant strain).

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

[0036] The THRS-YbiI Q86L mutant strain, THRS-ΔmntP strain, THRS-MntP G25D mutant strain, THRS-MntP G25D-YbiI Q86L mutant strain, and the original THRS strain obtained in Example 1 were inoculated onto seed culture medium for cultivation to obtain seed solutions. The seed culture medium contained the following components at the following concentrations: corn steep liquor 4.2 g / L, glucose 10 g / L, yeast extract 2.5 g / L, KH2PO4 2 g / L, magnesium sulfate 1.2 g / L, FeSO4·7H2O 20 mg / L, MnSO4·H2O 20 mg / L, and biotin 30 mg / L. Each experiment was conducted in triplicate.

[0037] The seed culture obtained using the above cultivation method was transferred into fermentation medium at an inoculum size of 20%.

[0038] 1.5L Seed Tank Process Control

[0039] a. Set the temperature to 37℃, pH to 7.0, fan speed to 500 rpm, and airflow to 0.3 m³ / h. 3 / h, with the temperature controlled at 37℃ throughout the process, the tank pressure at 0.05~0.08MPa, and the culture cycle at 10h;

[0040] b. Transplanting standard: OD600: 12-15;

[0041] c. The seed culture medium consisted of 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.

[0042] Fermentation process control in a 2.5L fermenter

[0043] a. Set the temperature to 37℃, pH to 7.0, initial rotation speed to 300 rpm, and airflow to 0.3 m³ / h. 3 / h, with the temperature controlled at 37℃ throughout the process and the tank pressure at 0.05~0.08MPa;

[0044] c.DO control: At 0h, the air volume is 0.3m³. 3 / h, 300rpm, tank pressure 0.05MPa;

[0045] d. When DO drops below 30%, adjust the aeration rate and stirring speed to control the dissolved oxygen level at 30% until fermentation ends;

[0046] e. The fermentation medium consists of 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.

[0047] 3. Method for determining threonine:

[0048] (1) Sample preparation: Take 1 mL of fermentation broth after 48 h of fermentation, centrifuge at 12000 rpm for 10 min to remove the bacterial cells and collect 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) Analysis method: OPA pre-column derivation;

[0050] (3) Chromatographic conditions:

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

[0052] ② Column temperature: 40℃;

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

[0054] Mobile phase B: Weigh 3.01 g of anhydrous sodium acetate into a beaker; dissolve in ultrapure water and bring the volume 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 this solution, mix and filter, then place in an ultrasonic cleaning pot to purge for 20 min, and set aside.

[0055] ④ Flow rate: 1.0 ml / min;

[0056] ⑤ Ultraviolet detector: 338nm;

[0057] ⑥ Column temperature: 40℃.

[0058] Validation of the production performance of the strain in a 4.5L fermenter

[0059] Table 1: Performance of strains in L-threonine production in a 5L fermenter

[0060] The results showed that replacing glycine at position 25 of the amino acid sequence encoded by the mntP gene with aspartic acid significantly increased L-threonine production compared to the original strain. MntP G25D and YbiI Q86L acted synergistically to further increase L-threonine production. The THRS-MntP G25D-YbiI Q86L mutant strain was designated FFTHR-38.

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

[0062] Following the method in Example 1, based on the THRS-6 strain (CGMCC NO.31695), mutant strains THRS-6-YbiI Q86L, THRS-6-ΔmntP, THRS-6-MntP G25D, and THRS-6-MntP G25D-YbiI Q86L were constructed. The results of the strains' production performance verification in a 5L fermenter are shown in the table below.

[0063] Table 2: Performance of strains in L-threonine production in a 5L fermenter

[0064] The results showed that after replacing the strain with THRS-6, the substitution of aspartic acid at position 25 of the amino acid sequence encoding the mntP gene protein with glycine significantly increased L-threonine production compared to THRS-6. Similarly, in THRS-6, MntP G25D and YbiI Q86L synergistically increased L-threonine production. Therefore, MntP G25D is applicable to different strains, effectively increasing L-threonine production, and exhibits a synergistic effect with YbiI Q86L. The THRS-6-MntP G25D-YbiI Q86L mutant strain is designated FFTHR-39.

[0065] The nucleotide and amino acid sequences involved in this application are shown below:

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

[0067] SEQ ID NO:2: (Mutant MntP protein)

[0068] SEQ ID NO:3: (mntP gene)

[0069] SEQ ID NO:4: (mntP mutant gene)

[0070] SEQ ID NO:5: (Pkan-F primer sequence 5'→3')

[0071] SEQ ID NO:6: (Pkan-R primer sequence 5'→3')

[0072] SEQ ID NO:7: (PmntP-F primer sequence 5'→3')

[0073] SEQ ID NO:8: (PmntP-R primer sequence 5'→3')

[0074] SEQ ID NO:9: (PmntP-F' primer sequence 5'→3')

[0075] SEQ ID NO:10: (PmntP-R' primer sequence 5'→3')

[0076] SEQ ID NO:11: (PpCP20-F primer sequence 5'→3')

[0077] SEQ ID NO:12: (PpCP20-R primer sequence 5'→3')

[0078] SEQ ID NO:13: (Mutant YbiI protein sequence)

[0079] SEQ ID NO:14: (nucleotide sequence of the mutant ybiI gene)

[0080] SEQ ID NO:15: (PybiI-F primer sequence 5'→3')

[0081] SEQ ID NO:16: (PybiI-R primer sequence 5'→3')

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and do not constitute a limitation on the content of this application. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solutions of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in this application and all fall within the protection scope of this application.

Claims

1. Application of a mutant MntP manganese ion transporter protein in increasing threonine production in threonine-producing strains, wherein, Compared to the wild-type MntP manganese ion transporter with the sequence shown in SEQ ID NO:1, the mutant MntP manganese ion transporter contains 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 application as described in claim 1, wherein, The amino acid sequence of the mutant MntP manganese ion transporter is shown in SEQ ID NO:

2.

3. A recombinant strain that produces high levels of L-threonine, expressing a mutant MntP manganese ion transporter protein, wherein, Compared to the wild-type MntP manganese ion transporter with the sequence shown in SEQ ID NO:1, the mutant MntP manganese ion transporter contains 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 as described in claim 3, wherein, The amino acid sequence of the mutant MntP manganese ion transporter is shown in SEQ ID NO:

2.

5. The recombinant strain as described in claim 3, wherein, The recombinant strain is Escherichia coli.

6. The recombinant strain as described in claim 5, wherein, The recombinant strain was modified based on Escherichia coli THRS strain, THRS-6 strain, THRS-YbiI Q86L mutant strain or THRS-6-YbiI Q86L mutant strain; The THRS-YbiI Q86L mutant strain is an Escherichia coli THRS strain expressing mutant YbiI protein, and the THRS-6-YbiI Q86L mutant strain is an Escherichia coli THRS-6 strain expressing mutant YbiI protein. The amino acid sequence of the mutant YbiI protein is shown in SEQ ID NO:

13.

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

8. The method of claim 7, wherein the threonine-producing strain is Escherichia coli.

9. The method of claim 7, wherein the threonine-producing strain is an Escherichia coli THRS strain, THRS-6 strain, THRS-YbiI Q86L mutant strain, or THRS-6-YbiI Q86L mutant strain; in, The THRS-YbiI Q86L mutant strain is an Escherichia coli THRS strain expressing mutant YbiI protein, and the THRS-6-YbiI Q86L mutant strain is an Escherichia coli THRS-6 strain expressing 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, comprising the following steps: inoculating the recombinant strain according to any one of claims 3-6 onto 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.

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