CN114716519B

CN114716519B - L-serine transport protein and application thereof in improving homoserine yield

Info

Publication number: CN114716519B
Application number: CN202210515655.7A
Authority: CN
Inventors: 张晓梅; 黄易凡; 高宇洁; 叶锦方; 史劲松; 许正宏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2023-02-10
Anticipated expiration: 2042-05-12
Also published as: CN114716519A

Abstract

The invention discloses an L-serine transport protein and application thereof, belonging to the technical field of biological engineering. On the basis of researching amino acid transporters, the invention provides that NCgl0255 sequence in corynebacterium glutamicum has the function of transporting L-serine. The invention further applies NCgl0255 to a production strain of L-serine, which is used for improving the yield of the L-serine, so that the accumulation amount of the L-serine reaches 29.58g/L.

Description

L-serine transport protein and application thereof in improving homoserine yield

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an L-serine transport protein and application thereof in improving the yield of serine.

Background

L-serine is a non-essential amino acid and has wide application in the fields of medicines, foods, cosmetics and the like. The corynebacterium glutamicum is a food safety strain and is widely applied to production of amino acids such as L-glutamic acid, L-lysine, L-valine and the like. However, the general Corynebacterium glutamicum cannot produce L-serine by fermentation using a sugar feedstock.

At present, research on L-serine production by Corynebacterium glutamicum at home and abroad focuses on molecular modification of synthesis and degradation pathways, stolz and the like use Corynebacterium glutamicum ATCC13032 which does not produce L-serine as an original strain, and the L-serine yield is 36.2g/L when a recombinant strain constructed by using glucose and fructose as a mixed carbon source (cited in the documents: stolz M, peters-Wendisch P, etterich H, et al. Reduced folate supply as a key to enhanced L-ine production by Corynebacterium glutamicum Glutaminum. J. Applied & Environmental Microbiology,2007,73 (3): 750.). The enhanced expression of 3-phosphoglycerate kinase (PGK) in Corynebacterium glutamicum ATCC13032 by Neisseria schrencki et al increases the synthesis of L-serine precursor 3-phosphoglycerate and improves the yield of L-serine (from the literature: neisseria schrencki, zhang Yun, liu Shu, et al. Metabolic engineering and Metabolic flux analysis of Corynebacterium glutamicum producing L-serine [ J ]. China science: life sciences, 2012,42 (4): 295-303.).

The inventor screens a wild corynebacterium glutamicum SYPS-062 which can produce L-serine by using sugar raw materials through fermentation in the early stage, and obtains a mutant strain C.glutamicum SYPS-062-33a by taking the wild corynebacterium glutamicum SYPS-062 as an original strain through multiple rounds of chemical mutagenesis. The mutant strain has the advantages that the yield of L-serine is increased by 65 percent and reaches 11.0g/L, and the accumulation of L-alanine and L-valine serving as byproducts is also obviously increased. And further releasing feedback inhibition of key enzyme in an L-serine synthesis pathway on the mutant strain, knocking out a degradation pathway, and blocking and weakening a byproduct accumulation pathway, wherein the shake flask yield of the L-serine of the obtained recombinant bacterium C.glutamicum SYPS-06233a delta SSA (abbreviated as delta SSA) (CGMCC NO.8668) can reach 26.25g/L, which is 3.9 times that of a wild strain.

Although metabolic engineering of genes for L-serine synthesis and degradation pathways has been very effective, the production of L-serine is currently required to be improved in order to be suitable for industrial scale production. The transport of amino acids out of cells is an important prerequisite for their accumulation in culture media, so transport systems are receiving increasing attention in order to achieve large quantities of amino acids. In recent decades, only 7 proteins have been identified as amino acid secretion transporters in Corynebacterium glutamicum, involving secretion of 13 amino acids. BrnFE participates in the secretion of L-methionine, L-isoleucine, L-valine and L-leucine; cgmA is involved in the secretion of L-arginine; lysE is involved in the secretion of L-lysine, L-arginine, L-citrulline and L-ornithine, mscG is involved in the secretion of L-glutamic acid and L-aspartic acid; mscCG2 is involved in the secretion of L-glutamic acid; only the serE and ThrE proteins were identified as having the function of secretory transport of L-threonine and L-serine.

Disclosure of Invention

In order to solve the technical problems, the invention provides an L-serine transport protein and an application thereof in improving the yield of homoserine.

The first purpose of the invention is to provide an L-serine transporter NCgl0255, wherein the amino acid sequence of the L-serine transporter NCgl0255 is shown in SEQ ID NO. 2.

SEQ ID NO.2：MTTDFSYILLVVAVCAVITFALRAVPFLILKPLRESQFVGKMAMWMPAGILAILTASTFRSNAIDLKTLTFGLIAVAITVAAHLLGSRRTLLSVGAGTIVFVGLVNLF*

In one embodiment of the invention, the nucleotide sequence encoding the L-serine transporter NCgl0255 is represented by SEQ ID No. 1.

SEQ ID NO.1：ATGACAACTGATTTCTCCTATATTCTCCTTGTTGTCGCAGTATGTGCAGTCATTACTTTTGCGCTCCGGGCGGTTCCGTTCTTAATCCTCAAGCCCCTGCGTGAATCACAATTTGTGGGCAAAATGGCGATGTGGATGCCAGCAGGAATCCTTGCCATTTTGACCGCATCAACGTTTCGCAGCAATGCGATAGATCTGAAGACTCTAACCTTTGGTCTCATTGCCGTTGCGATTACAGTGGCGGCGCATCTTCTTGGCAGTCGACGCACCTTGTTGAGCGTTGGCGCTGGCACCATCGTTTTTGTTGGACTGGTGAATCTTTTCTAA。

The second object of the present invention is to provide a gene encoding the L-serine transporter NCgl0255.

The third purpose of the invention is to provide a plasmid or a vector containing the gene, wherein the vector is selected from a pXMJ19 vector, a pDXW series vector, a pET series vector or a pPICZ series vector.

The fourth object of the present invention is to provide a cell expressing said L-serine transporter NCgl0255.

In one embodiment of the invention, the host of the cell is Escherichia coli, a coryneform bacterium, a Bacillus, a yeast or a filamentous fungus.

In one embodiment of the invention, the host of the cell is corynebacterium glutamicum SSAAI.

The fifth object of the present invention is to provide a method for increasing the production of L-serine, comprising the steps of: the L-serine transfer protein NCgl0255 of claim 1 is over-expressed in Corynebacterium glutamicum to obtain genetically engineered bacteria over-expressing the L-serine transfer protein NCgl0255, and the genetically engineered bacteria are used for fermentation.

In one embodiment of the invention, the conditions of the fermentation are: fermenting at 28-32 deg.C under 100-140rpm for 60-150h.

In one embodiment of the invention, the fermentation medium comprises 90-110g/L of sucrose, 20-40g/L of ammonium sulfate, 50-70g/L of calcium carbonate and MgSO ₄ ·7H ₂ O 0.5-1g/L，FeSO ₄ ·7H ₂ O 0.01-0.05g/L，MnSO ₄ ·H ₂ 0.01-0.05g/L of O, 20-40mg/L of protocatechuic acid, 40-60 mu g/L of biotin and 400-500 mu g/L of thiamine.

In one embodiment of the invention, the fermentation medium comprises sucrose 100g/L, ammonium sulfate 30g/L, calcium carbonate 60g/L, mgSO ₄ ·7H ₂ O 0.5g/L，FeSO ₄ ·7H ₂ O 0.02g/L，MnSO ₄ ·H ₂ O0.02 g/L, protocatechuic acid 30mg/L, biotin 50. Mu.g/L, thiamine 450. Mu.g/L, and the initial pH was adjusted to 7.0.

In one embodiment of the invention, the seed medium comprises 37g/L brain heart infusion; 20g/L of glucose; (NH) ₄ ) ₂ SO ₄ 10g/L；MgSO ₄ ·7H ₂ O 0.5g/L；K ₂ HPO ₄ 0.2g/L；NaH ₂ PO ₄ 0.3g/L。

In one embodiment of the invention, the method for gene knockout in corynebacterium glutamicum Δ SSA comprises the following steps:

the method comprises the following steps: extracting the genome of Corynebacterium glutamicum delta SSA (CGMCC NO. 8668) by using a bacterial genome extraction kit of Shanghai Czeri;

step two: using a genome of delta SSA as a template, designing a gene knockout specific primer by using high-fidelity enzyme of Takara company, respectively amplifying an upstream sequence and a downstream segment of a target gene, and obtaining a homologous arm segment of the target gene deletion by a cross PCR method;

step three: connecting the homologous arm fragment to a corynebacterium glutamicum knock-out plasmid pk18mobsacB to construct a recombinant knock-out plasmid, electrically transferring the recombinant knock-out plasmid into a delta SSA competence, screening by using kanamycin and a 10% sucrose plate, and then verifying by PCR to obtain a recombinant bacterium with a knocked-out gene.

In one embodiment of the invention, the method for overexpression of a gene in corynebacterium glutamicum Δ SSA comprises the following steps:

the method comprises the following steps: extracting the genome of Corynebacterium glutamicum delta SSA (CGMCC NO. 8668) by using a bacterial genome extraction kit of the Shanghai strap-down company;

step two: using the genome of delta SSA as a template, and using high-fidelity enzyme of Takara company and gene specific primers to amplify to obtain a gene segment;

step three: connecting the target gene fragment with an expression plasmid PDXW-10, constructing an over-expression recombinant plasmid, electrically transferring the over-expression recombinant plasmid into a delta SSA competence, screening recombinant bacteria by using kanamycin, and extracting the plasmid for verification to obtain correct recombinant bacteria.

In one embodiment of the present invention, the recombinant corynebacterium glutamicum fermentation culture method comprises the following steps:

inoculating recombinant corynebacterium glutamicum on a seed plate, carrying out three-region streaking, culturing for 3d, selecting a single colony, carrying out intensive streaking on the seed plate, culturing for 3d, inoculating bacteria on the plate into 20mL of seed solution, and carrying out overnight culture at 30 ℃ and 120rpm for about 12-16h until OD is reached ₅₆₂ 25, add to 25mL fermentation medium to OD ₅₆₂ Culturing at 120rpm for 5d at 1, 30 deg.C, and measuring OD every 12h ₅₆₂ And amino acid concentration.

The sixth purpose of the present invention is to provide the application of said L-serine transporter NCgl0255 in the fields of medicine, food or cosmetics.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the transport plays an important role in the process of high yield of amino acid by the metabolic engineering strain. To date, a variety of amino acid transporters have been found in Corynebacterium glutamicum, but only the transporters ThrE and SerE have been reported to transport L-serine. The invention provides a novel L-serine transporter NCgl0255, which has a nucleotide sequence shown in SEQ ID NO.1, has the total length of 327 nucleotides, and codes 109 amino acids. By means of genetic engineering, NCgl0255 is overexpressed in Corynebacterium glutamicum delta SSA with high L-serine yield, and the L-serine yield can be increased to 29.58g/L. The method provides a new idea for improving the L-serine yield by metabolic engineering strain.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the embodiments of the present disclosure taken in conjunction with the accompanying drawings, in which

Fig. 1 is an evaluation of the fermentation characteristics of thrE knockout and overexpression recombinant bacteria, wherein a: recombinant strain delta SSA delta thrE; b: the recombinant strain delta SSA-thrE.

FIG. 2 shows the evaluation of fermentation characteristics of NCgl0255 gene knockout recombinant bacteria Δ SSA Δ NCgl0255.

FIG. 3 is the evaluation of fermentation characteristics of recombinant bacteria Δ SSA-NCgl0255 overexpressing NCgl0255 gene.

FIG. 4 shows the transport function verification of NCgl0255 protein.

Detailed Description

The present invention is further described below in conjunction with the drawings and the embodiments so that those skilled in the art can better understand the present invention and can carry out the present invention, but the embodiments are not to be construed as limiting the present invention.

(I) culture Medium

Seed culture medium: 37g/L of brain-heart infusion; 20g/L of glucose; (NH) ₄ ) ₂ SO ₄ 10g/L；MgSO ₄ ·7H ₂ O 0.5g/L；K ₂ HPO ₄ 0.2g/L；NaH ₂ PO ₄ 0.3g/L。

Cgxii minimal medium: glucose 40g/L, urea 5g/L, (NH) ₄ ) ₂ SO ₄ 20g/L，KH ₂ PO ₄ 1g/L，MgSO ₄ 0.25g/L，MOPS 42g/L，CaCl ₂ 10mg/L，FeSO ₄ ·7H ₂ O 10mg/L，MnSO ₄ ·H ₂ O 10mg/L，ZnSO ₄ 1mg/L，CuSO ₄ 0.2mg/L，NiCl ₂ ·6H ₂ 0.02mg/L of O, 0.2mg/L of biotin and 0.03g/L of protocatechuic acid.

Fermentation medium: 100g/L of sucrose, 30g/L of ammonium sulfate, 60g/L of calcium carbonate and MgSO ₄ ·7H ₂ O0.5g/L，FeSO ₄ ·7H ₂ O 0.02g/L，MnSO ₄ ·H ₂ O0.02 g/L, protocatechuic acid 30mg/L, biotin 50. Mu.g/L, thiamine 450. Mu.g/L, and the initial pH was adjusted to 7.0.

Method for gene knockout in Corynebacterium glutamicum delta SSA

step two: using a genome of delta SSA as a template, using high-fidelity enzyme of Takara company to design a gene knockout specific primer to respectively amplify an upstream sequence and a downstream segment of a target gene, and obtaining a homologous arm segment of target gene deletion by a cross PCR method;

Method for overexpression of genes in delta SSA of corynebacterium glutamicum

Fermentation culture method of (IV) recombinant corynebacterium glutamicum

Activating the strain: inoculating recombinant Corynebacterium glutamicum on a seed plate, carrying out three-region streaking, culturing for 3d, picking out single colony, carrying out dense streaking on the seed plate, culturing for 3d, inoculating bacteria on the plate into 20mL of seed solution, and culturing at 30 ℃ and 120rpm overnight for about 12-16h until OD is achieved ₅₆₂ 25, add to 25mL fermentation Medium to OD ₅₆₂ Culturing at 120rpm for 5d at 1, 30 deg.C, and measuring OD every 12h ₅₆₂ And amino acid concentration.

(V) verification of L-serine Transporter function

The addition of amino acid dipeptide is a common experimental method for functional verification of amino acid transporters. To verify the L-serine transporter, L-serine dipeptide (L-ser-ser) was synthesized in Nanjing peptide industry for L-serine transport experiments. Activating corynebacterium glutamicum on a solid seed plate, inoculating the corynebacterium glutamicum into a liquid seed culture medium for overnight culture, and growing to a logarithmic growth phase. Collecting thallus in logarithmic growth phase, washing with CGXII minimal medium for 2 times, inoculating to CGXII minimal medium, adding 3mM L-ser-ser dipeptide, and pre-culturing at 30 deg.C for 2 hr. The pre-cultured cells were collected and washed 2 times with pre-cooled CGXII minimal medium. According to initial OD ₅₆₂ =8-10 cells were inoculated into 50mL CGXII minimal medium and L-ser-ser dipeptide was added to start the transport assay to a final concentration of 3mM. Sampling 1mL every 15min, immediately centrifuging the sample, taking out the supernatant, storing the supernatant in a refrigerator at-80 ℃, terminating the reaction after 120h, and then measuring the extracellular L-serine concentration by high performance liquid chromatography.

(VI) determination of L-serine concentration by high performance liquid chromatography

1. Solution preparation

Triethylamine acetonitrile: 0.7mL of triethylamine was added to 4.3mL of acetonitrile.

Phenyl isothiocyanate acetonitrile (PITC) solution: 25. Mu.L of phenylisothiocyanate was added to 2mL of acetonitrile.

Mobile phase A: 15.2g of anhydrous sodium acetate and 1850mL of water were weighed, dissolved, adjusted to pH 6.5 with glacial acetic acid, added with 140mL of acetonitrile, mixed well and filtered through a 0.45 μm organic filter.

And (3) mobile phase B:80% acetonitrile.

2. Amino acid sample derivatization

Taking 200 mu L of amino acid standard sample and diluted fermentation liquor sample, and adding 20 mu L of norleucine internal standard solution into each tube. Then, 100 mu L of triethylamine acetonitrile and 100 mu L of phenyl isothiocyanate acetonitrile solution are respectively added, the mixture is evenly mixed and then is kept stand for 1h at room temperature, 400 mu L of normal hexane is added, the mixture is violently shaken and evenly mixed and then is kept stand for 10min, 200 mu L of lower layer solution is taken out, 800 mu L of ultrapure water is added for dilution, and then the sample is filtered by a 0.45 mu m organic filter membrane and then is loaded.

HPLC reaction conditions

And (3) chromatographic column: venusil AA, 4.6X 250nm,5 μm; violet absorption wavelength: 254nm; column temperature: at 40 ℃; flow rate: 1mL/min; sample injection volume: 10 μ L. Gradient elution of mobile phase, wherein the elution procedure is that 0-4min,100% of mobile phase A is that 4-1semin, 97% of mobile phase A:16-17min,89% mobile phase A:17-32min,79% mobile phase A;32-34min,66% mobile phase A;34-38min,0% mobile phase A;38.01min,100% mobile phase A.

Corynebacterium glutamicum Δ SSA is disclosed in the literature: l-serine Corynebacterium glutamicum SYPS-062 production of Royu, university of south of the Yangtze river 2012.

Example 1 Effect of L-threonine and L-serine Transporter ThrE on L-serine production by Δ SSA

ThrE is a transporter of L-threonine in C.glutamicum. Since L-threonine and L-serine have chemical structural similarity, thrE is reported in the literature to also have the effect of transporting serine, and this example illustrates the effect of ThrE on L-serine production by Corynebacterium glutamicum Δ SSA.

1. The thrE knockout was achieved according to the gene knockout method in Corynebacterium glutamicum. DELTA.SSA.

(1) Extracting the genome of Corynebacterium glutamicum delta SSA (CGMCC NO. 8668) by using a bacterial genome extraction kit of the Shanghai strap-down company;

(2) Amplifying the left homologous arm of the target gene by using specific primers of thrE-1, 5' -GCTCTAGACATCATCATCTGTCAACGAA and thrE-2; amplifying the right homologous arm of the target gene by using specific primers thrE-3;

(3) Connecting the homologous arm fragment to a corynebacterium glutamicum knock-out plasmid pk18mobsacB to construct a recombinant knock-out plasmid, electrically transferring the recombinant knock-out plasmid into a delta SSA competence, screening by using kanamycin and a 10% sucrose plate, and then verifying by PCR to obtain a gene-knocked-out recombinant bacterium delta SSA delta thrE.

2. Overexpression of thrE was achieved as described for overexpression in C.glutamicum. DELTA.SSA.

(2) Using the genome of Corynebacterium glutamicum. DELTA.SSA as a template, a high fidelity enzyme from Takara, and a gene-specific primer thrE-F:5' -GAAGATCTAGAAGGATATACCATGTTGGAGTTTGCGACCT and thrE-R:5' -CCCAAGCTTTTACTTTATTACCGAAC) to obtain a gene fragment;

(3) Connecting the target gene fragment with an expression plasmid, constructing an over-expression recombinant plasmid, electrically transferring the over-expression recombinant plasmid into a delta SSA competence, screening recombinant bacteria by using kanamycin, and extracting the plasmid for verification to obtain correct recombinant bacteria.

The fermentation characteristics of the recombinant bacteria Δ SSA Δ thrE and Δ SSA-thrE were evaluated in a fermentation medium containing 100g/L sucrose as a substrate to explain the effect on L-serine production by Δ SSA. As can be seen from FIG. 1A, the knockout of the L-threonine transporter, thrE, had no significant effect on strain growth and L-serine production; as can be seen from FIG. 1B, the L-serine production did not increase after over-expression of ThrE, indicating that ThrE, the L-threonine transporter, had no significant effect on increasing L-serine production.

Example 2 Effect of knockout of NCgl0255 Gene on L-serine production by Δ SSA

Comparison of the strain Δ SSA with the transcriptome sequencing of ATCC13032 revealed that the transcription levels of NCgl2463 (see SEQ ID NO.3 and SEQ ID NO. 4), NCgl0074 (see SEQ ID NO.5 and SEQ ID NO. 6) and NCgl0255 (see SEQ ID NO.1 and SEQ ID NO. 2) were averagely up-regulated, which was suspected to be associated with the export of L-serine.

The NCgl0255 gene knockout procedure is as follows:

(2) Amplifying the left homologous arm of the target gene by using specific primers NCgl0255-1, namely CTATGACATGATTACGAATTAGCCAGAAACGTTCACCA and NCgl0255-2, namely GGTACTTCTTCTTCTTGGCATGCCCTCAATTTGAAGG; amplifying the right homologous arm of the target gene by using specific primers NCgl0255-3, namely CAAATTGAGGGCATGCCAAGAAGAGTACCGGATG and NCgl0255-4, namely TGCCTGCAGGGTCGACTCTAGAATGCTGGTCATCGCCTC, and obtaining a homologous arm fragment with target gene deletion by a cross-PCR method;

(3) Connecting the homologous arm fragment to a corynebacterium glutamicum knock-out plasmid pk18mobsacB to construct a recombinant knock-out plasmid, electrically transferring the recombinant knock-out plasmid into a delta SSA competence, screening by using kanamycin and a 10% sucrose plate, and then verifying by PCR to obtain a gene-knocked-out recombinant strain delta SSA delta NCgl0255.

The fermentation characteristics of the recombinant strain Δ SSA Δ NCgl0255 were evaluated in a fermentation medium using 100g/L sucrose as a substrate, and the results are shown in FIG. 2. As can be seen from FIG. 2, the production of L-serine was significantly reduced by knocking out NCgl0255. After fermentation for 120h, the L-serine accumulation of the recombinant strain delta SSA delta NCgl0255 is 21.7g/L, which is reduced by 17.3% compared with the original strain delta SSA, thus indicating that NCgl0255 is an extremely important transport protein for L-serine.

Example 3 functional verification of the L-serine Transporter NCgl0255

This example is intended to verify the L-serine transport function of the L-serine transporter NCgl0255.

The addition of amino acid dipeptide is a common experimental method for functional verification of amino acid transporters. To verify the L-serine transporter, L-serine dipeptide (L-ser-ser) was synthesized in Nanjing peptide industry for L-serine transport experiments. Activating Corynebacterium glutamicum on a solid seed plate, and inoculating toCulturing in liquid seed culture medium overnight until logarithmic phase. Collecting thallus in logarithmic growth phase, washing with CGXII minimal medium for 2 times, inoculating to CGXII minimal medium, adding 3mM L-ser-ser dipeptide, and pre-culturing at 30 deg.C for 2 hr. The pre-cultured cells were collected and washed 2 times with pre-cooled CGXII minimal medium. According to initial OD ₅₆₂ =8-10 cells were inoculated into 50mL CGXII minimal medium and L-ser-ser dipeptide was added to start the transport assay to a final concentration of 3mM. Sampling 1mL every 15min, immediately centrifuging the sample, taking out the supernatant, storing the supernatant in a refrigerator at-80 ℃, terminating the reaction after 120h, and then measuring the extracellular L-serine concentration by high performance liquid chromatography.

As can be seen from FIG. 3, when L-serine dipeptide was not added to CGXII medium, the L-serine concentration in the culture medium of the knockout recombinant bacterium Δ SSA Δ NCgl0255 was reduced by 14.9% as compared with Δ SSA, i.e., the L-serine transport function of the NCgl 0255-knocked protein was rapidly decreased, indicating that NCgl0255 has the function of transporting L-serine.

Example 4 enhanced expression of NCgl0255 improves L-serine production

The L-serine transport protein is applied to the research of improving the production of homoserine, and a recombinant strain delta SSA-NCgl0255 for enhancing the expression of NCgl0255 is constructed in corynebacterium glutamicum delta SSA.

The steps for enhancing expression of NCgl0255 are as follows:

(2) Using the genome of Corynebacterium glutamicum delta SSA as a template, and using high-fidelity enzyme of Takara company and gene specific primers NCgl0255-F, 5 '-TTCACACACAGGAAACAGAATTCAGACAACTGATTTCCTA and NCgl0255-R, 5' -CATCCCGCCAAAACAAGCTTTTAGAAAAGATTCACCAGTC to amplify to obtain a gene fragment;

(3) Connecting the target gene fragment with an expression plasmid, constructing an over-expression recombinant plasmid, electrically transferring the over-expression recombinant plasmid into delta SSA competence, screening recombinant bacteria by using kanamycin, and extracting the plasmid for verification to obtain correct recombinant bacteria delta SSA-0255.

The fermentation characteristics of the recombinant strain Δ SSA-NCgl0255 were evaluated in a fermentation medium using 100g/L sucrose as a substrate, and the results are shown in FIG. 4. As can be seen from FIG. 4, the L-serine accumulation amount after over-expression of NCgl0255 was 29.58g/L, which is 12.7% higher than that of the starting strain Δ SSA, indicating that NCgl0255 was successfully applied to increase the L-serine production.

Comparative example 1

Knocking out other proteins NCgl2463 according to a gene knockout method provided by the application to obtain a recombinant bacterium delta SSA delta NCgl2463.

The fermentation characteristics of the recombinant strain delta SSA delta NCgl2463 are evaluated in a fermentation medium taking 100g/L sucrose as a substrate, and the result shows that after NCgl2463 is knocked out, the strain is knocked out for 120h, the L-serine accumulation amount of the recombinant strain delta SSA delta 2463 is 26.3g/L, compared with the original strain delta SSA, the L-serine is increased by 1.9%, and no significant influence is generated on the growth of the strain and the L-serine yield.

Comparative example 2

Knocking out other proteins NCgl0074 according to the gene knockout method provided by the application to obtain recombinant bacteria delta SSA delta NCgl0074.

The fermentation characteristics of the recombinant strain delta SSA delta NCgl0074 are evaluated in a fermentation medium taking 100g/L sucrose as a substrate, and the result shows that after NCgl0074 is knocked out, the strain is knocked out for 120h, the accumulation amount of L-serine of the recombinant strain delta SSA delta Y is 27.5g/L, compared with the original strain delta SSA, the L-serine is improved by 4.8%, and the growth of the strain and the yield of the L-serine are not influenced.

Protein NCgl2463 sequence case:

ATGGCTAACGCCACCGCACAGAAGGGCCGCTTCGGCCTTCCCGGCTGGATGACTGGCTTTGGTGCCCAGGTTATCGCCGGCCTCATTCTTGGCCTTATTCTCGGCCTTGTCGCCCGAGGCATGGACAGCGGCGCTACAGACGGTGAAGCAAGCTGGCTTACCGGTCTTCTTGGCGGCGTCGGTTCTGCTTATGTTTCCCTACTTAAAGTAATGGTTCCACCACTGGTGTTCGCTGCAGTGGTTACCAGTGTGGCAAAGCTGCGCGAGGTAGCTAACGCTGCTCGTCTGGCTGTTTCCACCTTGGTGTGGTTCGCCATTACTGCATTCTTCTCTGTGCTCGCGGGTATCGCCGTAGCGCTGATTATGCAGCCTGGTGTTGGATCCACTGTTGACGCATCTAATGCTGCAGATCCTTCCCATGTTGGCAGCTGGCTGGGCTTTATCCAGTCCGTTATTCCATCAAACATTCTGGGACTTTCCGGTTCTTACAGTGAGAACTCTGGTGTGAACCTGTCCTTCAACGTGCTGCAGATCCTGGTGATCTCCATTGCGATTGGTGTTGCAGCGCTAAAGGCTGGCAAGTCCGCTGAGCCTTTCTTGAAGTTCACCGAGTCCTTCCTCAAGATCATCCAGATAGTGTTGTGGTGGATTATTCGCCTGGCTCCAATTGGTTCCGCTGCGCTGATCGGTAATGCTGTTGCTACCTACGGTTGGTCTGCACTTGGATCCCTGGGCAAGTTTGTTCTTGCGATCTACGTTGGTCTGGCAATCGTCATGTTCGTTATCTACCCAGTCGTGCTGAAGCTCAATGGAATTCCTGTTCTTGGATTCTTTAAGCGCGTTTGGCCTGTCACAAGCCTTGGCTTTGTTACCCGTTCCTCCATGGGCGTTATGCCAGTTACCCAGCGCGTTACTGAGCAGTCCTTGGGTGTTCCATCTGCGTACGCTTCCTTTGCTATCCCACTGGGTGCGACCAGCAAGATGGACGGCTGCGCTGCTGTCTACCCAGCTGTTGCCGCTATCTTCGTGGCACAGTTCTACGGCATTGACTTGAGCATCATGGATTACGTACTGATCATGATCGTCTCTGTCCTGGGCTCTGCTGCTACTGCAGGCACCACTGGCGCAACCGTCATGCTGACCCTGACCCTATCCACCTTGGGTCTGCCACTTGCTGGTGTTGGTCTGCTGCTGGCTATCGAGCCAATCATCGACATGGGACGTACCGCAACCAACGTCACCGGTCAGGCACTGGTTCCTGCGATCGTTGCTAAGCGCGAGGGCATTCTGGATCAGGATGTGTGGGATGCTGCTGAAAAGGGTGGCGCTGCTATTGATAAGGCAACCGTCTCTGAGAAAGAAACTGAGCCTGCAGAGGTTCGCTCCTAA(SEQ ID NO.3)

MANATAQKGRFGLPGWMTGFGAQVIAGLILGLILGLVARGMDSGATDGEASWLTGLLGGVGSAYVSLLKVMVPPLVFAAVVTSVAKLREVANAARLAVSTLVWFAITAFFSVLAGIAVALIMQPGVGSTVDASNAADPSHVGSWLGFIQSVIPSNILGLSGSYSENSGVNLSFNVLQILVISIAIGVAALKAGKSAEPFLKFTESFLKIIQIVLWWIIRLAPIGSAALIGNAVATYGWSALGSLGKFVLAIYVGLAIVMFVIYPVVLKLNGIPVLGFFKRVWPVTSLGFVTRSSMGVMPVTQRVTEQSLGVPSAYASFAIPLGATSKMDGCAAVYPAVAAIFVAQFYGIDLSIMDYVLIMIVSVLGSAATAGTTGATVMLTLTLSTLGLPLAGVGLLLAIEPIIDMGRTATNVTGQALVPAIVAKREGILDQDVWDAAEKGGAAIDKATVSEKETEPAEVRS*(SEQ ID NO.4)。

protein NCgl0074 sequence condition:

ATGTCTAACGCATCTTTTAAAGGCGACGATAAAGCACTCATTGGCATAGTTTTATCAGTTCTCACATTTTGGCTTTTTGCTCAGTCAACCCTAAATATCGGCCCAGATATGGCAACTGATTTAGGGATGAGCGATGGCACCATGAACATAGCTGTCGTGGCCGCCGCGTTATTCTGTGGAACATTTATCGTCGCAGCCGGCGGCATCGCAGATGTCTTTGGCCGAGTACGAATCATGATGATTGGCAACATCCTTAACATCCTGGGATCTCTCCTCATCGCCACGGCAACGACTTCTTTAGCCACCCAAATGGTGATCACCGGCCGAGTTCTCCAAGGACTGGCAGCAGCGGCCATCATGTCTGCATCCCTAGCATTAGTTAAGACATATTGGTTAGGTACTGACCGCCAACGAGCAGTCTCCATTTGGTCCATTGGTTCATGGGGTGGCACCGGATTCTGCGCACTTTTCGCGGGTCTTGTTGTAGCAAGCCCCTTTGGCTGGAGAGGAATCTTCGCCCTCTGCGCGATCGTCTCCATCGTTGCTATTGCCCTTACCCGCCACATCCCGGAATCCCGTCCGGCTCAACCCATTGGCATGCACTTGGATTGGAGTGGCATCATCGTTCTTGCCCTCAGTGTTCTATCTCTTGAATTGTTTATTACCCAAGGAGAATCACTTGGCTGGACGCACTGGATGGCCTGGACTCTCCTTGCCGTTTCTTTGACATTTCTCGCTGTTTTCGTCTTCATTGAACGCGTCGCCAGCTGGCCAGTTCTCGACTTCAACCTTTTCAAAGACCACGCCTTCAGCGGTGCGACCATCACCAACTTCATTATGAGCGCTACTGGCGGAGTAGTTGCCGTTGTCATGTGGGTTCAGCAAATGGGATGGGGTGTCTCCCCAACAATCTCGGGACTCACCAGCATCGGCTTCGCAGCCTTTGTCATCCTTTTCATTCGAGTTGGAGAAAAGGCCATGCAGAAAGTTGGCGCCCGAGCAGTGATCATCACCGCTGGCATCTTGGTAGCGACTGCGACCGCCCTCCTAATGATCACCGCAGTCAGCGAGTCAACGTACATCGTCATCTCCCTCGCCGGCTTCTCCCTTTATGGCCTTGGCCTCGGACTCTTCGCCACCCCAGTCACAGATACCGCCCTTGGAACACTTCCCAAAGACCGTACCGGCGCTGGTGCAGGTGTATTCAAGATGTCTTCTTCCCTCGGCGCAGCACTCGGCATCGCAATCTCCACTTCAGTGTTCCTCGCACTTCGCGACGGCACCTCCATCAACTCCGACGTCGCACTCGCCGGAACAGTTTCACTTGGCATCAACGTTGTATTCGCAGCAACAGCCACCATCACCGCAGCAGTCCTTATTCCAAAAGCCGCTGGCAAAGTCTCACAAACCAGCATCACCCTTCCTGAGCCAGCTATCGCTGTAAAAATCTAA(SEQ ID NO.5)。

MSNASFKGDDKALIGIVLSVLTFWLFAQSTLNIGPDMATDLGMSDGTMNIAVVAAALFCGTFIVAAGGIADVFGRVRIMMIGNILNILGSLLIATATTSLATQMVITGRVLQGLAAAAIMSASLALVKTYWLGTDRQRAVSIWSIGSWGGTGFCALFAGLVVASPFGWRGIFALCAIVSIVAIALTRHIPESRPAQPIGMHLDWSGIIVLALSVLSLELFITQGESLGWTHWMAWTLLAVSLTFLAVFVFIERVASWPVLDFNLFKDHAFSGATITNFIMSATGGVVAVVMWVQQMGWGVSPTISGLTSIGFAAFVILFIRVGEKAMQKVGARAVIITAGILVATATALLMITAVSESTYIVISLAGFSLYGLGLGLFATPVTDTALGTLPKDRTGAGAGVFKMSSSLGAALGIAISTSVFLALRDGTSINSDVALAGTVSLGINVVFAATATITAAVLIPKAAGKVSQTSITLPEPAIAVKI*(SEQ ID NO.6)。

it should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Various other modifications and alterations will occur to those skilled in the art upon reading the foregoing description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> L-serine transporter and application thereof in improving production of serine

<130> 6

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 327

<212> DNA

<213> (Artificial Synthesis)

<400> 1

atgacaactg atttctccta tattctcctt gttgtcgcag tatgtgcagt cattactttt 60

gcgctccggg cggttccgtt cttaatcctc aagcccctgc gtgaatcaca atttgtgggc 120

aaaatggcga tgtggatgcc agcaggaatc cttgccattt tgaccgcatc aacgtttcgc 180

agcaatgcga tagatctgaa gactctaacc tttggtctca ttgccgttgc gattacagtg 240

gcggcgcatc ttcttggcag tcgacgcacc ttgttgagcg ttggcgctgg caccatcgtt 300

tttgttggac tggtgaatct tttctaa 327

<210> 2

<211> 108

<212> PRT

<213> (Artificial Synthesis)

<400> 2

Met Thr Thr Asp Phe Ser Tyr Ile Leu Leu Val Val Ala Val Cys Ala

1 5 10 15

Val Ile Thr Phe Ala Leu Arg Ala Val Pro Phe Leu Ile Leu Lys Pro

20 25 30

Leu Arg Glu Ser Gln Phe Val Gly Lys Met Ala Met Trp Met Pro Ala

35 40 45

Gly Ile Leu Ala Ile Leu Thr Ala Ser Thr Phe Arg Ser Asn Ala Ile

50 55 60

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

65 70 75 80

Ala Ala His Leu Leu Gly Ser Arg Arg Thr Leu Leu Ser Val Gly Ala

85 90 95

Gly Thr Ile Val Phe Val Gly Leu Val Asn Leu Phe

100 105

<210> 3

<211> 1389

<212> DNA

<213> (Artificial Synthesis)

<400> 3

atggctaacg ccaccgcaca gaagggccgc ttcggccttc ccggctggat gactggcttt 60

ggtgcccagg ttatcgccgg cctcattctt ggccttattc tcggccttgt cgcccgaggc 120

atggacagcg gcgctacaga cggtgaagca agctggctta ccggtcttct tggcggcgtc 180

ggttctgctt atgtttccct acttaaagta atggttccac cactggtgtt cgctgcagtg 240

gttaccagtg tggcaaagct gcgcgaggta gctaacgctg ctcgtctggc tgtttccacc 300

ttggtgtggt tcgccattac tgcattcttc tctgtgctcg cgggtatcgc cgtagcgctg 360

attatgcagc ctggtgttgg atccactgtt gacgcatcta atgctgcaga tccttcccat 420

gttggcagct ggctgggctt tatccagtcc gttattccat caaacattct gggactttcc 480

ggttcttaca gtgagaactc tggtgtgaac ctgtccttca acgtgctgca gatcctggtg 540

atctccattg cgattggtgt tgcagcgcta aaggctggca agtccgctga gcctttcttg 600

aagttcaccg agtccttcct caagatcatc cagatagtgt tgtggtggat tattcgcctg 660

gctccaattg gttccgctgc gctgatcggt aatgctgttg ctacctacgg ttggtctgca 720

cttggatccc tgggcaagtt tgttcttgcg atctacgttg gtctggcaat cgtcatgttc 780

gttatctacc cagtcgtgct gaagctcaat ggaattcctg ttcttggatt ctttaagcgc 840

gtttggcctg tcacaagcct tggctttgtt acccgttcct ccatgggcgt tatgccagtt 900

acccagcgcg ttactgagca gtccttgggt gttccatctg cgtacgcttc ctttgctatc 960

ccactgggtg cgaccagcaa gatggacggc tgcgctgctg tctacccagc tgttgccgct 1020

atcttcgtgg cacagttcta cggcattgac ttgagcatca tggattacgt actgatcatg 1080

atcgtctctg tcctgggctc tgctgctact gcaggcacca ctggcgcaac cgtcatgctg 1140

accctgaccc tatccacctt gggtctgcca cttgctggtg ttggtctgct gctggctatc 1200

gagccaatca tcgacatggg acgtaccgca accaacgtca ccggtcaggc actggttcct 1260

gcgatcgttg ctaagcgcga gggcattctg gatcaggatg tgtgggatgc tgctgaaaag 1320

ggtggcgctg ctattgataa ggcaaccgtc tctgagaaag aaactgagcc tgcagaggtt 1380

cgctcctaa 1389

<210> 4

<211> 462

<212> PRT

<213> (Artificial Synthesis)

<400> 4

Met Ala Asn Ala Thr Ala Gln Lys Gly Arg Phe Gly Leu Pro Gly Trp

1 5 10 15

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

20 25 30

Ile Leu Gly Leu Val Ala Arg Gly Met Asp Ser Gly Ala Thr Asp Gly

35 40 45

Glu Ala Ser Trp Leu Thr Gly Leu Leu Gly Gly Val Gly Ser Ala Tyr

50 55 60

Val Ser Leu Leu Lys Val Met Val Pro Pro Leu Val Phe Ala Ala Val

65 70 75 80

Val Thr Ser Val Ala Lys Leu Arg Glu Val Ala Asn Ala Ala Arg Leu

85 90 95

Ala Val Ser Thr Leu Val Trp Phe Ala Ile Thr Ala Phe Phe Ser Val

100 105 110

Leu Ala Gly Ile Ala Val Ala Leu Ile Met Gln Pro Gly Val Gly Ser

115 120 125

Thr Val Asp Ala Ser Asn Ala Ala Asp Pro Ser His Val Gly Ser Trp

130 135 140

Leu Gly Phe Ile Gln Ser Val Ile Pro Ser Asn Ile Leu Gly Leu Ser

145 150 155 160

Gly Ser Tyr Ser Glu Asn Ser Gly Val Asn Leu Ser Phe Asn Val Leu

165 170 175

Gln Ile Leu Val Ile Ser Ile Ala Ile Gly Val Ala Ala Leu Lys Ala

180 185 190

Gly Lys Ser Ala Glu Pro Phe Leu Lys Phe Thr Glu Ser Phe Leu Lys

195 200 205

Ile Ile Gln Ile Val Leu Trp Trp Ile Ile Arg Leu Ala Pro Ile Gly

210 215 220

Ser Ala Ala Leu Ile Gly Asn Ala Val Ala Thr Tyr Gly Trp Ser Ala

225 230 235 240

Leu Gly Ser Leu Gly Lys Phe Val Leu Ala Ile Tyr Val Gly Leu Ala

245 250 255

Ile Val Met Phe Val Ile Tyr Pro Val Val Leu Lys Leu Asn Gly Ile

260 265 270

Pro Val Leu Gly Phe Phe Lys Arg Val Trp Pro Val Thr Ser Leu Gly

275 280 285

Phe Val Thr Arg Ser Ser Met Gly Val Met Pro Val Thr Gln Arg Val

290 295 300

Thr Glu Gln Ser Leu Gly Val Pro Ser Ala Tyr Ala Ser Phe Ala Ile

305 310 315 320

Pro Leu Gly Ala Thr Ser Lys Met Asp Gly Cys Ala Ala Val Tyr Pro

325 330 335

Ala Val Ala Ala Ile Phe Val Ala Gln Phe Tyr Gly Ile Asp Leu Ser

340 345 350

Ile Met Asp Tyr Val Leu Ile Met Ile Val Ser Val Leu Gly Ser Ala

355 360 365

Ala Thr Ala Gly Thr Thr Gly Ala Thr Val Met Leu Thr Leu Thr Leu

370 375 380

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

385 390 395 400

Glu Pro Ile Ile Asp Met Gly Arg Thr Ala Thr Asn Val Thr Gly Gln

405 410 415

Ala Leu Val Pro Ala Ile Val Ala Lys Arg Glu Gly Ile Leu Asp Gln

420 425 430

Asp Val Trp Asp Ala Ala Glu Lys Gly Gly Ala Ala Ile Asp Lys Ala

435 440 445

Thr Val Ser Glu Lys Glu Thr Glu Pro Ala Glu Val Arg Ser

450 455 460

<210> 5

<211> 1452

<212> DNA

<213> (Artificial Synthesis)

<400> 5

atgtctaacg catcttttaa aggcgacgat aaagcactca ttggcatagt tttatcagtt 60

ctcacatttt ggctttttgc tcagtcaacc ctaaatatcg gcccagatat ggcaactgat 120

ttagggatga gcgatggcac catgaacata gctgtcgtgg ccgccgcgtt attctgtgga 180

acatttatcg tcgcagccgg cggcatcgca gatgtctttg gccgagtacg aatcatgatg 240

attggcaaca tccttaacat cctgggatct ctcctcatcg ccacggcaac gacttcttta 300

gccacccaaa tggtgatcac cggccgagtt ctccaaggac tggcagcagc ggccatcatg 360

tctgcatccc tagcattagt taagacatat tggttaggta ctgaccgcca acgagcagtc 420

tccatttggt ccattggttc atggggtggc accggattct gcgcactttt cgcgggtctt 480

gttgtagcaa gcccctttgg ctggagagga atcttcgccc tctgcgcgat cgtctccatc 540

gttgctattg cccttacccg ccacatcccg gaatcccgtc cggctcaacc cattggcatg 600

cacttggatt ggagtggcat catcgttctt gccctcagtg ttctatctct tgaattgttt 660

attacccaag gagaatcact tggctggacg cactggatgg cctggactct ccttgccgtt 720

tctttgacat ttctcgctgt tttcgtcttc attgaacgcg tcgccagctg gccagttctc 780

gacttcaacc ttttcaaaga ccacgccttc agcggtgcga ccatcaccaa cttcattatg 840

agcgctactg gcggagtagt tgccgttgtc atgtgggttc agcaaatggg atggggtgtc 900

tccccaacaa tctcgggact caccagcatc ggcttcgcag cctttgtcat ccttttcatt 960

cgagttggag aaaaggccat gcagaaagtt ggcgcccgag cagtgatcat caccgctggc 1020

atcttggtag cgactgcgac cgccctccta atgatcaccg cagtcagcga gtcaacgtac 1080

atcgtcatct ccctcgccgg cttctccctt tatggccttg gcctcggact cttcgccacc 1140

ccagtcacag ataccgccct tggaacactt cccaaagacc gtaccggcgc tggtgcaggt 1200

gtattcaaga tgtcttcttc cctcggcgca gcactcggca tcgcaatctc cacttcagtg 1260

ttcctcgcac ttcgcgacgg cacctccatc aactccgacg tcgcactcgc cggaacagtt 1320

tcacttggca tcaacgttgt attcgcagca acagccacca tcaccgcagc agtccttatt 1380

ccaaaagccg ctggcaaagt ctcacaaacc agcatcaccc ttcctgagcc agctatcgct 1440

gtaaaaatct aa 1452

<210> 6

<211> 483

<212> PRT

<213> (Artificial Synthesis)

<400> 6

Met Ser Asn Ala Ser Phe Lys Gly Asp Asp Lys Ala Leu Ile Gly Ile

1 5 10 15

Val Leu Ser Val Leu Thr Phe Trp Leu Phe Ala Gln Ser Thr Leu Asn

20 25 30

Ile Gly Pro Asp Met Ala Thr Asp Leu Gly Met Ser Asp Gly Thr Met

35 40 45

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

50 55 60

Ala Ala Gly Gly Ile Ala Asp Val Phe Gly Arg Val Arg Ile Met Met

65 70 75 80

Ile Gly Asn Ile Leu Asn Ile Leu Gly Ser Leu Leu Ile Ala Thr Ala

85 90 95

Thr Thr Ser Leu Ala Thr Gln Met Val Ile Thr Gly Arg Val Leu Gln

100 105 110

Gly Leu Ala Ala Ala Ala Ile Met Ser Ala Ser Leu Ala Leu Val Lys

115 120 125

Thr Tyr Trp Leu Gly Thr Asp Arg Gln Arg Ala Val Ser Ile Trp Ser

130 135 140

Ile Gly Ser Trp Gly Gly Thr Gly Phe Cys Ala Leu Phe Ala Gly Leu

145 150 155 160

Val Val Ala Ser Pro Phe Gly Trp Arg Gly Ile Phe Ala Leu Cys Ala

165 170 175

Ile Val Ser Ile Val Ala Ile Ala Leu Thr Arg His Ile Pro Glu Ser

180 185 190

Arg Pro Ala Gln Pro Ile Gly Met His Leu Asp Trp Ser Gly Ile Ile

195 200 205

Val Leu Ala Leu Ser Val Leu Ser Leu Glu Leu Phe Ile Thr Gln Gly

210 215 220

Glu Ser Leu Gly Trp Thr His Trp Met Ala Trp Thr Leu Leu Ala Val

225 230 235 240

Ser Leu Thr Phe Leu Ala Val Phe Val Phe Ile Glu Arg Val Ala Ser

245 250 255

Trp Pro Val Leu Asp Phe Asn Leu Phe Lys Asp His Ala Phe Ser Gly

260 265 270

Ala Thr Ile Thr Asn Phe Ile Met Ser Ala Thr Gly Gly Val Val Ala

275 280 285

Val Val Met Trp Val Gln Gln Met Gly Trp Gly Val Ser Pro Thr Ile

290 295 300

Ser Gly Leu Thr Ser Ile Gly Phe Ala Ala Phe Val Ile Leu Phe Ile

305 310 315 320

Arg Val Gly Glu Lys Ala Met Gln Lys Val Gly Ala Arg Ala Val Ile

325 330 335

Ile Thr Ala Gly Ile Leu Val Ala Thr Ala Thr Ala Leu Leu Met Ile

340 345 350

Thr Ala Val Ser Glu Ser Thr Tyr Ile Val Ile Ser Leu Ala Gly Phe

355 360 365

Ser Leu Tyr Gly Leu Gly Leu Gly Leu Phe Ala Thr Pro Val Thr Asp

370 375 380

Thr Ala Leu Gly Thr Leu Pro Lys Asp Arg Thr Gly Ala Gly Ala Gly

385 390 395 400

Val Phe Lys Met Ser Ser Ser Leu Gly Ala Ala Leu Gly Ile Ala Ile

405 410 415

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

420 425 430

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

435 440 445

Ala Ala Thr Ala Thr Ile Thr Ala Ala Val Leu Ile Pro Lys Ala Ala

450 455 460

Gly Lys Val Ser Gln Thr Ser Ile Thr Leu Pro Glu Pro Ala Ile Ala

465 470 475 480

Val Lys Ile

Claims

1. A method for increasing the yield of L-serine, comprising the steps of: over-expressing an L-serine transporter NCgl0255 shown as SEQ ID No.2 in corynebacterium glutamicum to obtain a genetic engineering bacterium over-expressing the L-serine transporter NCgl0255, and then fermenting by using the genetic engineering bacterium; the preservation number of the corynebacterium glutamicum is CGMCC NO.8668.

2. The method according to claim 1, characterized in that the conditions of the fermentation: fermenting at 28-32 deg.C for 60-150 hr at 100-140 rpm.

3. The method of claim 1, wherein the fermentation medium comprises sucrose 90-110g/L, ammonium sulfate 20-40g/L, calcium carbonate 50-70g/L, mgSO ₄ ·7H ₂ O 0.5-1 g/L，FeSO ₄ ·7H ₂ O 0.01-0.05 g/L，MnSO ₄ ·H ₂ 0.01-0.05g/L of O, 20-40mg/L of protocatechuic acid, 40-60 mu g/L of biotin and 400-500 mu g/L of thiamine.