CN108314713A - L-threonine transport protein thrg and its encoding gene and application - Google Patents

Abstract

The invention discloses an L-threonine transporter ThrG and its coding gene and application. The protein provided by the present invention, named as ThrG protein, is the following (a) or (b): (a) a protein composed of the amino acid sequence shown in sequence 1 in the sequence listing; (b) the amino acid sequence of sequence 1 is processed through a or a substitution and/or deletion and/or addition of several amino acid residues and a protein derived from sequence 1 associated with L-threonine transport. The gene encoding ThrG protein also belongs to the protection scope of the present invention, and it is named as thrG gene. The invention also protects the application of the ThrG protein: as L-threonine transporter; promoting L-threonine transport; promoting L-threonine transport to microorganisms to produce L-threonine in vitro. The invention has great application value.

Description

L-threonine transporter ThrG and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an L-threonine transporter ThrG and its coding gene and application.

Background technique

L-threonine is one of the essential amino acids and is widely used in the fields of medicine, food and feed. In the field of food, it is an important nutritional enhancer, which can strengthen grains, cakes, and dairy products, and has the effect of relieving human fatigue and promoting growth and development. In the field of medicine, it has a water-holding effect on human skin, plays an important role in protecting cell membranes, and can promote phospholipid synthesis and fatty acid oxidation in the body. In the field of feed, it is widely used as an additive for piglet feed, breeding pig feed, broiler feed, prawn feed and eel feed, etc. It can adjust the balance of amino acids in feed to promote growth, improve meat quality, and reduce nitrogen in livestock manure and urine. amount, ammonia concentration and release rate in livestock and poultry houses.

Amino acid transport is one of the important links in cell life activities. Amino acid transport systems exist in both prokaryotic and eukaryotic cells. Cells absorb amino acids from the external environment through the internal transport system for the synthesis of intracellular substances, providing carbon and nitrogen sources and energy for life activities. Metabolites are secreted outside the cell through the export system. Most amino acids can be produced by fermentation. Based on the understanding of the amino acid transport system, it can be modified in the rational transformation of amino acid production strains, so as to achieve the purpose of increasing production.

Contents of the invention

The object of the present invention is to provide an L-threonine transporter ThrG and its coding gene and application.

The protein provided by the present invention is obtained from Corynebacterium glutamicum (C.glutamicum), named as ThrG protein, and is as follows (a) or (b):

(a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing;

(b) A protein derived from Sequence 1 in which the amino acid sequence of Sequence 1 has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to L-threonine transport.

In the above protein, the substitution and/or deletion and/or addition of one or several amino acid residues refers to the substitution and/or deletion and/or addition of no more than ten amino acid residues.

In order to facilitate the purification and detection of the protein in (b), the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in Sequence 1 in the sequence listing can be linked with the tags shown in Table 1.

Table 1 Sequence of tags

The protein in (b) above can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression. The protein-encoding gene in (b) above can be deleted by deleting one or several amino acid residue codons in the DNA sequence shown in Sequence 2 in the sequence listing, and/or carrying out one or several base pairs of missense mutation, and/or link the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.

The gene encoding ThrG protein also belongs to the protection scope of the present invention, and it is named as thrG gene.

The thrG gene is the DNA molecule described in any one of the following 1) to 6):

1) A DNA molecule whose coding region is as shown in nucleotides 1 to 678 of Sequence 2 in the sequence listing;

2) a DNA molecule whose coding region is shown in sequence 2 in the sequence listing;

3) a DNA molecule whose coding region is as shown in nucleotides 14-694 of Sequence 6 in the Sequence Listing;

4) the DNA molecule shown in sequence 6 in the sequence listing;

5) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) or 3) or 4) under stringent conditions and encodes an L-threonine transport-related protein;

6) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) or 2) or 3) or 4) and encoding a protein related to L-threonine transport.

The above-mentioned stringent conditions can be 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution, hybridization at 65° C. in DNA or RNA hybridization experiments and membrane washing.

Recombinant expression vectors, expression cassettes, transgenic cell lines, transgenic plant tissues or recombinant bacteria containing thrG gene all belong to the protection scope of the present invention.

An existing expression vector can be used to construct a recombinant expression vector containing the thrG gene. When using the thrG gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, and they can be used alone or combined with other promoters Use; In addition, when using the thrG gene to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be start codons or adjacent region start codons, etc., but must be compatible with The reading frame of the coding sequences is identical to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of genetically modified microorganisms, the expression vectors used can be processed, such as adding genes that express enzymes or luminescent compounds that can produce color changes in microorganisms, antibiotic markers with resistance, or anti-chemical reagent marker genes Wait. Considering the safety of the transgene, any selectable marker gene may not be added.

The recombinant expression vector can specifically be the following recombinant plasmid: a recombinant plasmid obtained by inserting the thrG gene into the multiple cloning site of the pXMJ19 vector (for example, between XbaI and KpnI restriction sites).

The present invention also protects the application of ThrG protein, which is at least one of the following (c1) to (c7):

(c1) as an L-threonine transporter;

(c2) promoting L-threonine transport;

(c3) promoting the transport of L-threonine to the outside of the microorganism;

(c4) promote the transport of L-threonine to the outside of the bacteria;

(c5) promoting L-threonine transport to Corynebacterium glutamicum in vitro;

(c6) promoting L-threonine to be transported outside Corynebacterium glutamicum m-85 bacterium;

(c7) Production of L-threonine.

The invention also protects a recombinant microorganism obtained by introducing the thrG gene into the target microorganism. The target microorganism is a microorganism capable of producing L-threonine. The target microorganism can be bacteria, specifically Corynebacterium glutamicum, more specifically Corynebacterium glutamicum m-85. Specifically, the thrG gene can be introduced into the target microorganism through the recombinant expression vector. The target microorganism can also be the recombinant bacteria A or recombinant bacteria B described later.

The present invention also protects the use of the recombinant microorganism in the production of L-threonine.

The invention also protects a method for producing L-threonine, comprising the following steps: fermenting the recombinant microorganism, and obtaining L-threonine from the fermentation supernatant. The specific fermentation conditions may be: 30° C., 300 rpm shaking culture. The specific fermentation time can be 72 hours. The culture medium used in the fermentation is the culture medium with glucose as the carbon source. The medium used for the fermentation can specifically be (just an example) (pH7.0): glucose 80g, (NH ₄ ) ₂ SO ₄ 20g, KH ₂ PO ₄ 1.5g, MgSO ₄ ·7H ₂ O 0.4g, MnCl ₂ ·4H ₂ O 10mg, FeSO ₄ ·7H ₂ O 10mg, biotin 50μg, thiamine 200μg, L-methionine 37.5mg, CaCO ₃ 20g, dilute to 1L with water.

The invention also protects a kit for producing L-threonine, including the recombinant microorganism. The kit can also include a conventional or self-optimized fermentation medium. The kit can also include conventional or self-optimized seed culture medium. The seed culture medium can specifically be (just an example): 10 g of peptone, 5 g of yeast powder, 10 g of sodium chloride, 10 g of glucose, and 2 g of urea, and the volume is adjusted to 1 L with water. The specific fermentation medium can be (only for example) (pH7.0): glucose 80g, (NH ₄ ) ₂ SO ₄ 20g, KH ₂ PO ₄ 1.5g, MgSO ₄ ·7H ₂ O 0.4g, MnCl ₂ · 4H ₂ O 10mg, FeSO ₄ ·7H ₂ O 10mg, biotin 50μg, thiamine 200μg, L-methionine 37.5mg, CaCO ₃ 20g, dilute to 1L with water.

The invention also protects a recombinant bacterium, which is recombinant bacterium A or recombinant bacterium B.

The recombinant bacterium A is a recombinant bacterium obtained by silencing the thrG gene in the genome of the target bacterium.

The realization method of silencing the thrG gene in the genome of the target bacterium can specifically be homologous recombination. The realization of the silencing of the thrG gene in the genome of the target bacterium specifically includes the following steps: introducing a specific DNA molecule A or a recombinant plasmid A containing the specific DNA molecule A into the target bacterium, and then screening to obtain all or part of the thrG gene in the genome Knockout recombinant bacteria. The specific DNA molecule A knocks out all or part of the thrG gene through homologous recombination. The specific DNA molecule A may specifically be the DNA molecule shown in sequence 3 of the sequence listing. The recombinant plasmid A can specifically be a recombinant plasmid obtained by substituting the small fragment between the Sal I and EcoR I restriction sites of the pK18mobsacB vector with the double-stranded DNA molecule shown in sequence 3 of the sequence listing.

The recombinant bacterium B is a recombinant bacterium obtained by silencing the thrG gene and thrE gene in the genome of the target bacterium.

The thrE gene is the DNA molecule described in any one of the following 1) to 3):

1) a DNA molecule whose coding region is shown in sequence 4 in the sequence listing;

2) a DNA molecule that hybridizes to the DNA sequence defined in 1) under stringent conditions and encodes an L-threonine transport-related protein;

3) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) and encoding a protein related to L-threonine transport.

The above-mentioned stringent conditions can be 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution, hybridization at 65° C. in DNA or RNA hybridization experiments and membrane washing.

The realization method of silencing the thrG gene and thrE gene in the genome of the target bacterium can specifically be homologous recombination. The implementation of silencing the thrG gene and thrE gene in the genome of the target bacterium specifically includes the following steps: introducing element A and element B into the target bacterium (either at the same time or in steps), and then screening to obtain the thrG gene in the genome Recombinant bacteria with total or partial knockout and thrE gene knockout. The element A is a specific DNA molecule A or a recombinant plasmid A containing the specific DNA molecule A. The specific DNA molecule A knocks out all or part of the thrG gene through homologous recombination. The specific DNA molecule A may specifically be the DNA molecule shown in sequence 3 of the sequence listing. The recombinant plasmid A can specifically be a recombinant plasmid obtained by substituting the small fragment between the Sal I and EcoR I restriction sites of the pK18mobsacB vector with the double-stranded DNA molecule shown in sequence 3 of the sequence listing. The element B is a specific DNA molecule B or a recombinant plasmid B containing the specific DNA molecule B. The specific DNA molecule B knocks out all or part of the thrE gene through homologous recombination. The specific DNA molecule B may specifically be the DNA molecule shown in sequence 5 of the sequence listing. The recombinant plasmid B can specifically be a recombinant plasmid obtained by substituting the small fragment between the Pst I and SmaI restriction sites of the pK18mobsacB vector with the double-stranded DNA molecule shown in sequence 5 of the sequence listing.

The target bacteria can be bacteria, specifically Corynebacterium glutamicum, more specifically Corynebacterium glutamicum RES167.

The present invention discovers a new protein named ThrG protein from Corynebacterium glutamicum RES167, which has the function of transporting L-threonine. The ability of the recombinant strain with thrG gene deletion to transport L-threonine is reduced, and the ability of the recombinant strain expressing thrG gene to transport L-threonine is improved. Introducing the thrG gene into the starting strain producing L-threonine can significantly increase the production of L-threonine in the fermentation supernatant.

The invention has great application value.

Description of drawings

Fig. 1 is the electropherogram of step 2 of embodiment 2.

Fig. 2 is the electropherogram of step 4 of embodiment 2.

Fig. 3 is the electropherogram of step five of embodiment 2.

Fig. 4 is the result graph of embodiment 3.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

pMD19-T simple: Takara Bioengineering (Dalian) Co., Ltd., TAKARA Code: D104.

pK18mobsacB vector: Schafer A et al., Gene, 145(1):69-73, 1994.

pXMJ19 vector: Jakoby M et al., Biotechnology Techniques, 13(6):437-441, 1999.

Corynebacterium glutamicum m-85 (also known as Corynebacterium obtatus m-85): Acta Microbiology 22: 276-283, 1982.

Embodiment 1, the discovery of ThrG protein and its coding gene

After a large number of pre-experiments such as sequence analysis and functional verification, a new protein was found from Corynebacterium glutamicum RES167, named ThrG protein. The ThrG protein is shown in sequence 1 of the sequence listing.

The gene encoding the ThrG protein is named thrG gene, and its coding frame is shown in sequence 2 of the sequence listing.

Embodiment 2, the construction of recombinant bacteria

1. Construction of recombinant plasmid pK18ΔthrG

1. Genomic DNA of Corynebacterium glutamicum RES167 was extracted.

2. Using the genomic DNA obtained in step 1 as a template, a primer pair composed of 0143ZuFk and 0143ZuRk is used for PCR amplification to obtain a PCR amplification product.

0143ZuFk: 5'- CCTGTCGACGATCTTGACGTTGGTAGT -3';

0143ZuRk: 5'-AGTGAGGGTGAGGAAGACAGGAATGAGAGACCAAGGAGGCTGGCTATGAT-3'.

3. Using the genomic DNA obtained in step 1 as a template, a primer pair composed of 0143dFk and 0143dRk is used for PCR amplification to obtain a PCR amplification product.

0143dFk: 5'-ATCATAGCCAGCCTCCTTGGTCTCTCATTCCTGTCTTCCTCACCCTCACT-3';

0143dRk: 5'- GTCGAAGAATTCGAGGTGGACAGGTCGAT -3'.

4. Mix the PCR amplification product obtained in step 2 and the PCR amplification product obtained in step 3 as a template, and perform PCR amplification with a primer pair composed of 0143ZuFk and 0143dRk to obtain a PCR amplification product.

5. Sequencing the PCR amplification product obtained in step 4. Sequencing results show that in the PCR amplification product, the DNA molecule shown in sequence 3 in the sequence table is between the Sal I restriction recognition sequence and the EcoR I restriction recognition sequence. In sequence 3 of the sequence listing, the 1st-567th nucleotide is the upstream arm (the 1st-391st nucleotide is the 391 nucleotides upstream of the thrG gene coding frame, and the 392-567th is the thrG gene coding 176 nucleotides at the 5' end of the frame), 581-1148 nucleotides are downstream arms (581-765 are 185 nucleotides at the 3' end of the thrG gene coding frame, 766-1148 Nucleotides are 383 nucleotides downstream of the coding frame of thrG gene).

6. Ligate the PCR amplification product obtained in step 4 to the cloning vector pMD19-T simple to obtain the recombinant plasmid T-ΔthrGk. After sequencing, in the recombinant plasmid T-ΔthrGk, between the Sal I restriction recognition sequence and the EcoR I restriction recognition sequence is a DNA molecule shown in sequence 3 in the sequence table.

For practical operation, the double-stranded DNA molecule shown in Sequence 3 of the Sequence Listing can also be directly artificially synthesized, and then connected to the cloning vector pMD19-T simple to obtain the recombinant plasmid T-ΔthrGk.

7. Take the recombinant plasmid T-ΔthrGk, perform double digestion with restriction endonucleases Sal I and EcoR I, and recover a fragment of about 1.1 kb.

8. Take the pK18mobsacB vector, perform double digestion with restriction endonucleases Sal I and EcoR I, and recover a vector skeleton of about 5.6 kb.

9. Ligate the fragment obtained in step 7 with the vector backbone obtained in step 8 to obtain the recombinant plasmid pK18ΔthrG. According to the sequencing results, the structure of the recombinant plasmid pK18ΔthrG is described as follows: the small fragment between the Sal I and EcoR I restriction sites of the pK18mobsacB vector was replaced with a double-stranded DNA molecule shown in sequence 3 in the sequence table.

2. Construction of recombinant bacteria RES167ΔthrG (thrG gene knockout bacteria)

The DNA molecule shown in sequence 3 of the sequence listing can undergo homologous recombination with the 5' end segment and the 3' end segment of the thrG gene in the genomic DNA of Corynebacterium glutamicum RES167, thereby obtaining the partial deletion of the thrG gene ( The deletion segment is the recombinant bacterium of nucleotides 177-496 of Sequence 2 in the sequence listing).

1. The recombinant plasmid pK18ΔthrG was electroporated to transform Corynebacterium glutamicum RES167, the transformation solution was spread on the LB solid medium plate containing 10 μg/mL kanamycin, and the transformant with kanamycin resistance was screened and spread on the plate containing 20g/100mL sucrose LB solid medium plate, pick a single colony to obtain a purely cultured strain.

2. Take the bacterial strains obtained in step 1, plant them on LB solid medium plates and LB solid medium plates containing 50 μg/mL kanamycin respectively, and select bacterial strains that meet the following conditions: grow on LB solid medium plates, But it does not grow on the LB solid medium plate containing 50μg/mL kanamycin.

3. Take the bacterial strain obtained in step 2, use the primer pair composed of 0143ZuFk and 0143dRk to carry out PCR amplification, and then perform electrophoresis on the PCR amplification product, and the bacterial strain that meets the following criteria is the target recombinant bacterium with thrG gene deletion: the PCR amplification product is about It is 1.1kb.

The results of electrophoresis are shown in Figure 1 (from top to bottom, the band marked by the first arrow is about 1.45 kb, and the band marked by the second arrow is about 1.1 kb). In FIG. 1 , lane 1 is the PCR amplification product of Corynebacterium glutamicum RES167 in step 3, and lane 2 is the PCR amplification product of the strain obtained in step 2. The results showed that the target recombinant strain with thrG gene deletion was obtained, and it was named recombinant strain RES167ΔthrG.

3. Construction of recombinant plasmid pK18ΔthrE

A protein-ThrE protein related to threonine export has been reported in Corynebacterium glutamicum. The gene encoding the ThrE protein is the thrE gene, and its coding frame is shown in sequence 4 of the sequence listing. In order to avoid interference with the function identification of ThrG protein due to the presence of thrE gene, it is necessary to silence the thrE gene in Corynebacterium glutamicum RES167.

1. Genomic DNA of Corynebacterium glutamicum RES167 was extracted.

2. Using the genomic DNA obtained in step 1 as a template, a primer pair consisting of 2533uFk and 2533uRk is used for PCR amplification to obtain a PCR amplification product.

2533uFk: 5'-GAG CTGCAG AAGGAATATCCCGGAGAACCG-3';

2533uRk: 5'-AGACGCCTAAGCAGGTTGATTTCCGCTGGAAAACATTTTGCAAG-3'.

3. Using the genomic DNA obtained in step 1 as a template, a primer pair consisting of 2533dFk and 2533dRk is used for PCR amplification to obtain a PCR amplification product.

2533dFk: 5'-CTTGCAAAATGTTTTTCCAGCGGAAATCAACCTGCTTAGGCGTCT-3';

2533dRk: 5'-TGT CCCGGG CGTTGCGTTTGAAAAGCCCT-3'.

4. Mix the PCR amplification product obtained in step 2 and the PCR amplification product obtained in step 3 as a template, and perform PCR amplification with a primer pair composed of 2533uFk and 2533dRk to obtain a PCR amplification product.

5. Sequencing the PCR amplification product obtained in step 4. Sequencing results show that in the PCR amplification product, the DNA molecule shown in sequence 5 in the sequence table is between the Pst I restriction recognition sequence and the Sma I restriction recognition sequence. In sequence 5 of the sequence listing, the 1st-700th nucleotide is the upstream arm (the 700 nucleotides upstream of the thrE gene coding frame), and the 701-1400th nucleotide is the downstream arm (the 700 nucleotides upstream of the thrE gene coding frame downstream). 700 nucleotides).

6. Ligate the PCR amplification product obtained in step 4 to the cloning vector pMD19-T simple to obtain the recombinant plasmid T-ΔthrE. After sequencing, in the recombinant plasmid T-ΔthrE, the DNA molecule shown in sequence 5 in the sequence table is between the Pst I restriction recognition sequence and the Sma I restriction recognition sequence.

For practical operation, the double-stranded DNA molecule shown in Sequence 5 of the Sequence Listing can also be directly artificially synthesized, and then connected to the cloning vector pMD19-T simple to obtain the recombinant plasmid T-ΔthrE.

7. Take the recombinant plasmid T-ΔthrE, perform double digestion with restriction endonucleases Pst I and Sma I, and recover a fragment of about 1.4 kb.

8. Take the pK18mobsacB vector, perform double digestion with restriction endonucleases Pst I and Sma I, and recover the vector skeleton of about 5.6 kb.

9. Ligate the fragment obtained in step 7 with the vector backbone obtained in step 8 to obtain the recombinant plasmid pK18ΔthrE. According to the sequencing results, the structure of the recombinant plasmid pK18ΔthrE is described as follows: the small fragment between the Pst I and SmaI restriction sites of the pK18mobsacB vector was replaced by the double-stranded DNA molecule shown in sequence 5 of the sequence table.

4. Construction of recombinant bacteria RES167ΔthrE (thrE gene knockout bacteria)

The DNA molecule shown in sequence 5 of the sequence listing can be homologously recombined with the upstream and downstream fragments of the thrE gene in the genomic DNA of Corynebacterium glutamicum RES167, thereby obtaining the deletion of the thrE gene (the missing segment is the sequence of the sequence listing Nucleotide shown in 4) recombinant bacteria.

1. The recombinant plasmid pK18ΔthrE was electroporated to transform Corynebacterium glutamicum RES167, the transformation liquid was spread on the LB solid medium plate containing 10 μg/mL kanamycin, and the transformant with kanamycin resistance was screened and spread on the plate containing 20g/100mL sucrose LB solid medium plate, pick a single colony to obtain a purely cultured strain.

2. Take the bacterial strains obtained in step 1, plant them on LB solid medium plates and LB solid medium plates containing 50 μg/mL kanamycin respectively, and select bacterial strains that meet the following conditions: grow on LB solid medium plates, But it does not grow on the LB solid medium plate containing 50μg/mL kanamycin.

3. Take the bacterial strain obtained in step 2, use the primer pair composed of 2533uFk and 2533dRk to carry out PCR amplification, and then perform electrophoresis on the PCR amplification product, and the bacterial strain that meets the following criteria is the target recombinant bacterium with thrE gene deletion: the PCR amplification product is about 1.4kb.

The electrophoresis results are shown in Figure 2 (from top to bottom, the band marked by the first arrow is about 2.9 kb, and the band marked by the second arrow is about 1.4 kb). In FIG. 2 , lane 1 is the PCR amplification product of Corynebacterium glutamicum RES167 in step 3, and lane 2 is the PCR amplification product of the strain obtained in step 2. The results showed that the target recombinant strain with deletion of thrE gene was obtained, which was named recombinant strain RES167ΔthrE.

5. Construction of recombinant bacteria RES167ΔthrGΔthrE (thrG gene and thrE gene double knockout bacteria)

1. The recombinant plasmid pK18ΔthrE was electroporated to transform the recombinant strain RES167ΔthrG, the transformation solution was spread on the LB solid medium plate containing 10 μg/mL kanamycin, and the transformants with kanamycin resistance were screened and spread on the plate containing 20 g/100mL kanamycin. Sucrose LB solid medium plate, pick a single colony to obtain a purely cultured strain.

2. Take the bacterial strains obtained in step 1, plant them on LB solid medium plates and LB solid medium plates containing 50 μg/mL kanamycin respectively, and select bacterial strains that meet the following conditions: grow on LB solid medium plates, But it does not grow on the LB solid medium plate containing 50μg/mL kanamycin.

3. Take the strain obtained in step 2, use the primer pair composed of 0143ZuFk and 0143dRk or the primer pair composed of 2533uFk and 2533dRk to carry out PCR amplification, and then perform electrophoresis on the PCR amplification product. The strains that meet the following criteria are thrG gene and thrE gene Target recombinant bacteria with double deletion: the PCR amplification product when using the primer pair consisting of 0143ZuFk and 0143dRk is about 1.1kb, and the PCR amplification product when using the primer pair consisting of 2533uFk and 2533dRk is about 1.4kb.

The electrophoresis results are shown in Figure 3 (from top to bottom, the band marked by the first arrow is about 2.9kb, the band marked by the second arrow is about 1.45kb, the band marked by the third arrow is about 1.4kb, and the band marked by the fourth arrow is about 1.4kb. The band marked by the arrow is about 1.1 kb). In Fig. 3, swimming lane 1 is the PCR amplification product when Corynebacterium glutamicum RES167 adopts the primer pair consisting of 0143ZuFk and 0143dRk, and swimming lane 2 is the PCR amplification product when the strain obtained in step 2 adopts the primer pair consisting of 0143ZuFk and 0143dRk , Lane 3 is the PCR amplification product when Corynebacterium glutamicum RES167 adopts the primer pair consisting of 2533uFk and 2533dRk, and swimming lane 4 is the PCR amplification product when the strain obtained in step 2 adopts the primer pair consisting of 2533uFk and 2533dRk.

The results showed that the target recombinant strain with double deletion of thrG gene and thrE gene was obtained, which was named recombinant strain RES167ΔthrGΔthrE.

6. Construction of recombinant plasmid pZZT1

1. Genomic DNA of Corynebacterium glutamicum RES167 was extracted.

2. Using the genomic DNA obtained in step 1 as a template, perform PCR amplification with a primer pair composed of 0143F and 0143R to obtain a PCR amplification product.

0143F: 5'-GCC TCTAGA AAGGAGGACAACC ATGACAACAGCACAGTTCT-3';

0143R: 5'-CGT GGTACC TTA TTAACCCACTACCCTGCCA-3'.

3. Ligate the PCR amplification product obtained in step 2 to the cloning vector pMD19-T simple to obtain the recombinant plasmid T-thrG. After sequencing, in the recombinant plasmid T-thrG, the DNA molecule shown in sequence 6 in the sequence table is between the XbaI restriction recognition sequence and the KpnI restriction recognition sequence.

For practical operation, the double-stranded DNA molecule shown in sequence 6 of the sequence listing can also be directly artificially synthesized, and then connected to the cloning vector pMD19-T simple to obtain the recombinant plasmid T-thrG.

4. Take the recombinant plasmid T-thrG, perform double digestion with restriction endonucleases XbaI and KpnI, and recover a fragment of about 0.7kb.

5. Take the pXMJ19 vector, perform double digestion with restriction endonucleases XbaI and KpnI, and recover the vector skeleton of about 6.6kb.

6. Ligate the fragment obtained in step 4 with the vector backbone obtained in step 5 to obtain recombinant plasmid pZZT1. According to the sequencing results, the structure of the recombinant plasmid pZZT1 is described as follows: the small fragment between the XbaI and KpnI restriction sites of the pXMJ19 vector was replaced with a double-stranded DNA molecule shown in sequence 6 of the sequence table. In sequence 6 of the sequence listing, nucleotides 14-694 encode ThrG protein.

7. Construction of recombinant bacteria RES167ΔthrGΔthrE/pZZT1

The recombinant plasmid pZZT1 was introduced into the recombinant strain RES167ΔthrGΔthrE, and the obtained recombinant strain was named recombinant strain RES167ΔthrGΔthrE/pZZT1.

8. Construction of recombinant bacteria m-85/pZZT1

The recombinant plasmid pZZT1 was introduced into Corynebacterium glutamicum m-85, and the obtained recombinant strain was named recombinant strain m-85/pZZT1.

9. Construction of recombinant bacteria m-85/pXMJ19

The pXMJ19 vector was introduced into Corynebacterium glutamicum m-85, and the resulting recombinant strain was named recombinant strain m-85/pXMJ19.

Embodiment 3, HPLC method assays the activity of ThrG protein as L-threonine transporter

The test strains were: Corynebacterium glutamicum RES167, recombinant RES167ΔthrG, recombinant RES167ΔthrE, recombinant RES167ΔthrGΔthrE or recombinant RES167ΔthrGΔthrE/pZZT1.

Medium A: Glucose was added to the MMI medium so that the concentration of glucose was 20 mM.

Medium B: Add threonine tripeptide (threonine tripeptide consists of three consecutive threonine residues) and glucose to MMI medium, so that the concentration of threonine tripeptide is 1 mM, and the concentration of glucose is 20mM.

MMI medium (pH7.5): Na ₂ HPO ₄ 12H ₂ O 2.0g, KH ₂ PO ₄ 0.5g, MgSO ₄ 7H ₂ O 0.03g, trace element solution 2mL, biotin 50μg, thiamine 50μg, Make up to 1L with water.

Trace element solution (pH6.0): EDTA 0.500g, ZnSO ₄ 7H ₂ O 0.220g, CaCl ₂ 0.055g, MnCl ₂ 4H ₂ O 0.051g, FeSO ₄ 7H ₂ O 0.0499g, (NH ₄ ) ₆ MO ₇ O ₂₄ ·4H ₂ O 0.011g, CuSO ₄ ·5H ₂ O 0.0157g, CoCl ₂ ·6H ₂ O 0.0161g, H ₂ O 1000m1.

1. Inoculate the test strain into LB liquid medium, culture until the cells grow to the exponential phase, and collect the cells by centrifugation.

2. Take the cells obtained in step 1, wash them twice with medium A, and then suspend them in medium B to obtain a suspension with an _OD600nm value of 7-8.5. Suspension with OD _600nm value of 7-8.5, equivalent to 1.5mg/mL-1.8mg/mL dry cell weight.

3. Take the suspension in step 2, shake it at 30°C and 150rpm, take samples at different reaction times, and immediately centrifuge (4°C, 12000rpm, 3min) to collect the supernatant.

4. Take the supernatant obtained in step 3, filter it with a 0.45 μm filter membrane, and collect the filtrate.

5. Take the filtrate obtained in step 4 and perform HPLC.

HPLC related parameters are as follows:

Agilent 1200 HPLC instrument;

Agilent Zorbax Eclipse-AAA chromatographic column, 4.6×75mm, 3.5μm, automatic pre-column derivatization by OPA method;

Mobile phase is made up of solution A and solution B, and the flow rate of mobile phase is 2mL/min; Solution A is 40mM sodium dihydrogen phosphate aqueous solution (pH 7.8), and mobile phase B is made up of 45 volume parts acetonitrile, 45 volume parts methanol and 10 volume parts water composition. The elution process was carried out according to the operating instructions of Agilent Corporation.

L-threonine standard substance (purity>99.0%): Beijing Dingguo Biotechnology Co., Ltd.

The retention time of the standard was 6.63min, and the peak appeared at the same retention time after loading the filtrate, which proved that the supernatant obtained in step 3 contained L-threonine. A standard curve was made according to the standard, and the L-threonine content in the supernatant collected at different sampling times was calculated by the standard curve and peak area.

The activity of extracellular transport of L-threonine is the number of micromoles of L-threonine secreted per mg of dry weight of cells.

The results of the activity of extracellular L-threonine transport are shown in Fig. 4 . In Figure 4, ◆ represents Corynebacterium glutamicum RES167, □ represents recombinant RES167ΔthrG, △ represents recombinant RES167ΔthrE, ▼ represents recombinant RES167ΔthrGΔthrE, ▲ represents recombinant RES167ΔthrGΔthrE/pZZT1.

The results showed that: compared with Corynebacterium glutamicum RES167, the activity of the recombinant strain RES167ΔthrG to secrete L-threonine decreased, suggesting that the ThrG protein may be related to the export of L-threonine; compared with the recombinant strain RES167ΔthrGΔthrE, the recombinant strain RES167ΔthrGΔthrE/pZZT1 significantly increased the activity of secreting L-threonine. The results showed that ThrG protein has the function of exporting L-threonine.

Example 4. Preparation of L-threonine by recombinant bacteria m-85/pZZT1

The test strains were: recombinant strain m-85/pZZT1 or recombinant strain m-85/pXMJ19.

Seed medium: peptone 10g, yeast powder 5g, sodium chloride 10g, glucose 10g, urea 2g, dilute to 1L with water.

Fermentation medium (pH7.0): Glucose 80g, (NH ₄ ) ₂ SO ₄ 20g, KH ₂ PO ₄ 1.5g, MgSO ₄ 7H ₂ O 0.4g, MnCl ₂ 4H ₂ O 10mg, FeSO ₄ 7H ₂ O 10mg, biotin 50μg, thiamine 200μg, L-methionine 37.5mg, CaCO ₃ 20g, dilute to 1L with water.

1. Inoculate the test strain into the seed medium, shake and cultivate at 30°C and 300rpm for 16 hours to obtain the seed liquid.

2. Take 1.5mL of seed liquid, inoculate into 15mL of fermentation medium, and culture with shaking at 30°C and 300rpm for 72 hours.

3. After completing step 2, centrifuge at 4°C, 8000rpm for 3min, and collect the supernatant.

4. Take the supernatant obtained in step 3, dilute it appropriately with sterile water, then filter it with a 0.45 μm filter membrane, and collect the filtrate.

5. Take the filtrate obtained in step 4 and perform HPLC.

HPLC related parameters are the same as step 5 of embodiment 3.

The recombinant bacterium m-85/pZZT1 was used as the test strain, and the concentration of L-threonine in the system after step 2 was 3.54 g/L. The recombinant strain m-85/pXMJ19 was used as the test strain, and the concentration of L-threonine in the system after step 2 was 1.73 g/L.

SEQUENCE LISTING

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> L-threonine transporter ThrG and its coding gene and application

<130> GNCYX170191

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 226

<212> PRT

<213> Corynebacterium glutamicum (C. glutamicum)

<400> 1

Met Thr Thr Ala Gln Phe Leu Ala Leu Phe Leu Val Trp Ile Ala Ala

1 5 10 15

Ile Ala Ser Pro Gly Pro Asp Leu Phe Gln Ile Ile Arg Leu Ser Ala

20 25 30

Lys Asn Arg Arg Asp Gly Val Leu Thr Ala Val Gly Ile Met Val Gly

35 40 45

Asn Ser Ile Trp Ile Ile Ala Ser Leu Leu Gly Leu Ser Ala Leu Ile

50 55 60

Ser Thr Tyr Pro Ala Ile Leu Asn Leu Leu Gln Leu Val Gly Gly Gly

65 70 75 80

Tyr Leu Thr Trp Met Gly Ile Gly Ala Val Arg Ser Trp Trp Thr Lys

85 90 95

Arg Ser Thr Gln Gln Ala Ala Ala Asp Ser Gln Ala Val Glu Asn Thr

100 105 110

Leu Val Thr Ala Thr Ala Ala Ser Val Gly Val Trp Pro Ala Ile Arg

115 120 125

Ser Gly Ile Ala Thr Asn Leu Ser Asn Pro Lys Ala Val Leu Phe Phe

130 135 140

Gly Ser Val Phe Ala Gln Phe Val Arg Pro Asp Met Gly Ile Gly Trp

145 150 155 160

Ser Ile Phe Ile Gly Val Phe Leu Thr Leu Thr Gly Leu Leu Trp Phe

165 170 175

Val Gly Phe Ala Val Leu Val Arg Lys Leu Ala Ala Gly Leu Thr Arg

180 185 190

Asn Gly Ala Ile Ile Asp Leu Leu Thr Gly Val Ile Phe Ile Gly Leu

195 200 205

Gly Met Phe Met Ile Phe Glu Gly Val Val Gly Ile Gly Gly Arg Val

210 215 220

Val Gly

225

<210> 2

<211> 681

<212>DNA

<213> Corynebacterium glutamicum (C. glutamicum)

<400> 2

atgacaacag cacagtttct agcgcttttt ctggtgtgga tcgcagcaat tgcatcccct 60

gggccagacc ttttccagat catcaggcta agtgccaaaa accgccgtga tggcgtactg 120

actgccgtag gcatcatggt gggaaactcc atctggatca tagccagcct ccttgggctc 180

tcggcactga tctccacgta tccagcaatt ttgaacctgt tgcagctcgt cggtggcggt 240

tatttgacct ggatgggcat cggggcggtg aggtcatggt ggacgaaacg ctccacacag 300

caagctgcag cggattctca agctgtagag aatacgttgg tgacagccac ggctgcatct 360

gtcggagtgt ggccagctat tcgatctggc attgctacca acttgtccaa ccccaaagct 420

gtgctgtttt ttggttccgt tttcgcccaa tttgttagac ctgacatggg aatcgggtgg 480

agtattttca ttggagtctt cctcaccctc actggcctgc tgtggtttgt ggggttcgcc 540

gtcttggtcc gcaaactagc cgctggcctc acccgaaatg gagccatcat cgacctgcta 600

acgggggtga ttttcatcgg gctgggaatg ttcatgatct tcgaggggggt tgtaggaatc 660

ggtggcaggg tagtggggtta g 681

<210> 3

<211> 1148

<212>DNA

<213> Artificial sequence

<400> 3

gatcttgacg ttggtagttt tccaagtggg tgtcacctgg catgctgtgc tgtccaaacg 60

ggaagggttc cgtcaagcat tcgcccaatt cgatgttgca aaagtagccg ccttcaatga 120

ggacgacgtg gaacgcctac ttgatgatct acagattttt agaaaccgaa gaaaaatcaa 180

cgctgccatc accaatgcca aagcgttgct ggagttaaac gatgaaacag gcacctttga 240

ctcaattatt gccgaccact caactgacgc cacagccatg gtgaagcatc tcaaagcctt 300

gggctttacc catatcggac tgacctcctt gagcatcctc cagcaggcca ttggggtcac 360

agagctgaag gctgcctaag atataactcc gatgacaaca gcacagtttc tagcgctttt 420

tctggtgtgg atcgcagcaa ttgcatcccc tgggccagac cttttccaga tcatcaggct 480

aagtgccaaa aaccgccgtg atggcgtact gactgccgta ggcatcatgg tgggaaactc 540

catctggatc atagccagcc tccttggtct ctcattcctg tcttcctcac cctcactggc 600

ctgctgtggt ttgtggggtt cgccgtcttg gtccgcaaac tagccgctgg cctcacccga 660

aatggagcca tcatcgacct gctaacgggg gtgattttca tcgggctggg aatgttcatg 720

atcttcgagg gggttgtagg aatcggtggc agggtagtgg gttagccccg cccccaggac 780

gtcactagac ttaggcacta tgcaacctga agaagtgcac atcaaggacg agaccatcaa 840

gttaggtcag ttcatcaaat tggccaacct tgtcgaatca ggcggagcgg ccaaagatgc 900

catcgctaac ggtgatgtca ccgtcaatgg tgaagtggat acccgaaggg gtaagacact 960

tcgcgatggc gatgtggtgt gcatcggcga ggtatgcgcg caggtgtcta ctggtgacgc 1020

agccgacgac gattattttg acgaagccac cgcaaacgat gacttcgatc ccgaaaagtg 1080

gaggaacatg taatgccagc ctttgaggca atgccaggaa tgccgtattg gatcgacctg 1140

tccacctc 1148

<210> 4

<211> 1470

<212>DNA

<213> Corynebacterium glutamicum (C. glutamicum)

<400> 4

atgttgagtt ttgcgaccct tcgtggccgc atttcaacag ttgacgctgc aaaagccgca 60

cctccgccat cgccactagc cccgattgat ctcactgacc atagtcaagt ggccggtgtg 120

atgaatttgg ctgcgagaat tggcgatatt ttgctttctt caggtacgtc aaatagtgac 180

accaaggtac aagttcgagc agtgacctct gcgtacggtt tgtactacac gcacgtggat 240

atcacgttga atacgatcac catcttcacc aacatcggtg tggagaggaa gatgccggtc 300

aacgtgtttc atgttgtagg caagttggac accaacttct ccaaactgtc tgaggttgac 360

cgtttgatcc gttccattca ggctggtgcg accccgcctg aggttgccga gaaaatcctg 420

gacgagttgg agcaatcccc tgcgtcttat ggtttccctg ttgcgttgct tggctgggca 480

atgatgggtg gtgctgttgc tgtgctgttg ggtggtggat ggcaggtttc cctaattgct 540

tttattaccg cgttcacgat cattgccacg acgtcatttt tgggaaagaa gggtttgcct 600

actttcttcc aaaatgttgt tggtggtttt attgccacgc tgcctgcatc gattgcttat 660

tctttggcgt tgcaatttgg tcttgagatc aaaccgagcc agatcatcgc atctggaatt 720

gttgtgctgttggcaggttt gacactcgtg caatctctgc aggacggcat cacgggcgct 780

ccggtgacag caagtgcacg atttttcgaa acactcctgt ttaccggcgg cattgttgct 840

ggcgtgggtt tgggcattca gctttctgaa atcttgcatg tcatgttgcc tgccatggag 900

tccgctgcag cacctaatta ttcgtctaca ttcgcccgca ttatcgctgg tggcgtcacc 960

gcagcggcct tcgcagtggg ttgttacgcg gagtggtcct cggtgattat tgcggggctt 1020

actgcgctga tgggttctgc gtttattac ctcttcgttg tttattagg ccccgtctct 1080

gccgctgcga ttgctgcaac agcagttggt ttcactggtg gtttgcttgc ccgtcgattc 1140

ttgattccac cgttgattgt ggcgattgcc ggcatcacac caatgcttcc aggtctagca 1200

atttaccgcg gaatgtacgc caccctgaat gatcaaacac tcatgggttt caccaacatt 1260

gcggttgctt tagccactgc ttcatcactt gccgctggcg tggttttggg tgagtggatt 1320

gcccgcaggc tacgtcgtcc accacgcttc aacccatacc gtgcatttac caaggcgaat 1380

gagttctcct tccaggagga agctgagcag aatcagcgcc ggcagagaaa acgtccaaag 1440

actaatcaga gattcggtaa taaaaggtaa 1470

<210> 5

<211> 1400

<212>DNA

<213> Artificial sequence

<400> 5

aaggaatatc ccggagaacc ggtgaaccat tcaccgtcga tccaatgatg tgtatgaagc 60

ttgcagaggc tctcgacctt gatccagtag aggttttaag tgcagcgaaa gttcccgaat 120

ctgaatggcc aaatttttcg aagatcatct cccaaagtga ctatgtgaag catgtagaca 180

taacaagact gaccgtccgc cagcaagatc tagtcatcaa tctggtcaac gaatttaagg 240

agcttaattt gaacacgcca tatgaaaagt gactaatttc aacccaaacg ggagcctaag 300

tgaaatgaaa taatcccctc accaactggc gacattcaaa caccgtttca tttccaaaca 360

tcgagccaag ggaaaagaaa gcccctaagc cccgtgttat taaatggaga ctttttggag 420

acctcaagcc aaaaaggggc attttcatta agaaaatacc cctttgacct ggtgttattg 480

agctggagaa gagacttgaa ctctcaacct acgcattaca agtgcgttgc gctgccaatt 540

gcgccactcc agcaccgcag atgctgatga tcaacaacta cgaatacgta tcttagcgta 600

tgtgtacatc acaatggaat tcggggctag agtatctggt gaaccgtgca taaacgacct 660

gtgattggac tctttttcct tgcaaaatgt tttccagcgg aaatcaacct gcttaggcgt 720

ctttcgctta aatagcgtag aatatcgggt cgatcgcttt taaacactca ggaggatcct 780

tgccggccaa aatcacggac actcgtccca ccccagaatc ccttcacgct gttgaagagg 840

aaaccgcagc cggtgcccgc aggattgttg ccacctattc taaggacttc ttcgacggcg 900

tcactttgat gtgcatgctc ggcgttgaac ctcagggcct gcgttacacc aaggtcgctt 960

ctgaacacga ggaagctcag ccaaagaagg ctacaaagcg gactcgtaag gcaccagcta 1020

agaaggctgc tgctaagaaa acgaccaaga agaccactaa gaaaactact aaaaagacca 1080

ccgcaaagaa gaccacaaag aagtcttaag ccggatctta tatggatgat tccaatagct 1140

ttgtag ttgt tgctaaccgt ctgccagtgg atatgactgt ccaccccagat ggtagctata 1200

gcatctcccc cagccccggt ggccttgtca cggggctttc ccccgttctg gaacaacatc 1260

gtggatgttg ggtcggatgg cctggaactg tagatgttgc acccgaacca tttcgaacag 1320

atacgggtgttttgctgcac cctgttgtcc tcactgcaag tgactatgaa ggcttctacg 1380

agggcttttc aaacgcaacg 1400

<210> 6

<211> 697

<212>DNA

<213> Artificial sequence

<400> 6

aaggaggaca accatgacaa cagcacagtt tctagcgctt tttctggtgt ggatcgcagc 60

aattgcatcc cctgggccag accttttcca gatcatcagg ctaagtgcca aaaaccgccg 120

tgatggcgta ctgactgccg taggcatcat ggtgggaaac tccatctgga tcatagccag 180

cctccttggg ctctcggcac tgatctccac gtatccagca attttgaacc tgttgcagct 240

cgtcggtggc ggttattga cctggatggg catcggggcg gtgaggtcat ggtggacgaa 300

acgctccaca cagcaagctg cagcggattc tcaagctgta gagaatacgt tggtgacagc 360

cacggctgca tctgtcggag tgtggccagc tattcgatct ggcattgcta ccaacttgtc 420

caaccccaaa gctgtgctgt tttttggttc cgttttcgcc caatttgtta gacctgacat 480

gggaatcggg tggagtattt tcattggagt cttcctcacc ctcactggcc tgctgtggtt 540

tgtggggttc gccgtcttgg tccgcaaact agccgctggc ctcacccgaa atggagccat 600

catcgacctg ctaacggggg tgattttcat cgggctggga atgttcatga tcttcgaggg 660

ggttgtagga atcggtggca gggtagtggg ttaataa 697

Claims (10)

1. A protein that is (a) or (b): (a) a protein consisting of the amino acid sequence shown in Sequence 1 in the Sequence Listing; (b) A protein derived from Sequence 1 in which the amino acid sequence of Sequence 1 has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to L-threonine transport. 2. A gene encoding the protein of claim 1. 3. The gene according to claim 2, characterized in that: the gene is the DNA molecule described in any one of the following 1) to 6): 1) A DNA molecule whose coding region is as shown in nucleotides 1 to 678 of Sequence 2 in the sequence listing; 2) a DNA molecule whose coding region is shown in sequence 2 in the sequence listing; 3) a DNA molecule whose coding region is as shown in nucleotides 14-694 of Sequence 6 in the Sequence Listing; 4) the DNA molecule shown in sequence 6 in the sequence listing; 5) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) or 3) or 4) under stringent conditions and encodes an L-threonine transport-related protein; 6) A DNA molecule having more than 90% homology with the DNA sequence defined in 1) or 2) or 3) or 4) and encoding a protein related to L-threonine transport. 4. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium containing the gene of claim 2 or 3. 5. The application of the protein according to claim 1, which is at least one of the following (c1) to (c7): (c1) as an L-threonine transporter; (c2) promoting L-threonine transport; (c3) promoting the transport of L-threonine to the outside of the microorganism; (c4) promote the transport of L-threonine to the outside of the bacteria; (c5) promoting L-threonine transport to Corynebacterium glutamicum in vitro; (c6) promoting L-threonine to be transported outside Corynebacterium glutamicum m-85 bacterium; (c7) Production of L-threonine. 6. A recombinant microorganism obtained by introducing the gene of claim 2 or 3 into the target microorganism. 7. the application of the recombinant microorganism described in claim 6 in the production of L-threonine. 8. A method for producing L-threonine, comprising the steps of: fermenting the recombinant microorganism according to claim 6, and obtaining L-threonine from the fermentation supernatant. 9. A test kit for producing L-threonine, comprising the recombinant microorganism according to claim 6. 10. A recombinant bacterium, which is obtained by silencing the gene encoding the protein according to claim 1 in the genome of the target bacterium.