阅读说明：本技术 一种l-丝氨酸转运蛋白及其应用 (L-serine transport protein and application thereof ) 是由 许正宏 高宇洁 史劲松 张晓梅 颜文斌 杨玲 于 2019-09-30 设计创作，主要内容包括：本发明公开了一种L-丝氨酸转运蛋白及其应用,属于生物工程技术领域。本发明在研究氨基酸转运蛋白基础上,发现了一种来源于谷氨酸棒杆菌的NCgl2054蛋白,其具有转运L-丝氨酸功能。通过基因工程手段,将NCgl2054蛋白在产L-丝氨酸的谷氨酸棒杆菌ΔSSA实现过表达,能够将L-丝氨酸的产量提高至29.9g/L。这为代谢工程改造菌株提高L-丝氨酸产量提供了新思路。(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 transport protein, the invention discovers NCgl2054 protein derived from corynebacterium glutamicum, which has the function of transporting L-serine. The NCgl2054 protein is over-expressed in the L-serine-producing Corynebacterium glutamicum delta SSA by a genetic engineering means, so that the yield of the L-serine can be improved to 29.9 g/L. The method provides a new idea for improving the L-serine yield by metabolic engineering strain.)

1. An L-serine transporter NCgl2054 is characterized by comprising an amino acid sequence shown in SEQ ID NO. 2.

2. A gene encoding the L-serine transporter NCgl2054 of claim 1.

3. Plasmid or vector containing the gene of claim 2, characterized in that the vector includes, but is not limited to, pXMJ19 vector, pDXW series vector, pET series vector or pPICZ series vector.

4. A cell expressing the L-serine transporter NCgl2054 of claim 1.

5. The cell of claim 4, wherein the host of the cell includes, but is not limited to, E.coli, a coryneform bacterium, a Bacillus, a yeast, or a filamentous fungus.

6. The cell of claim 4, wherein the host of the cell is Corynebacterium glutamicum Δ SSA.

7. A method for increasing the production of homoserine, characterized in that serine transporter NCgl2054 of claim 1 is overexpressed in Corynebacterium glutamicum delta SSA to obtain genetically engineered bacteria overexpressing serine transporter NCgl2054, and the genetically engineered bacteria are used for fermentation.

8. The method as claimed in claim 7, wherein the fermentation is carried out at 28-32 ℃ and 100-140rpm for 60-150 h.

9. The method of claim 7 or 8, wherein the fermentation medium comprises sucrose 90-110g/L, ammonium sulfate 20-40g/L, calcium carbonate 50-70g/L, MgSO4 & 7H₂O 0.5-1g/L，FeSO₄·7H₂O 0.01-0.05g/L，MnSO4·H₂0.01-0.05g/L of O, 20-40mg/L of protocatechuic acid, 40-60 mu g/L of biotin and 500 mu g/L of thiamine 400-ketone.

10. Use of the L-serine transporter NCgl2054 as defined in claim 1 in the pharmaceutical, food or cosmetic field.

Technical Field

The invention relates to an L-serine transport protein and application thereof, belonging to the technical field of biological engineering.

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 utilize sugar raw materials to produce L-serine by fermentation.

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: Peters-Wendisch P, Stolz M, Etterich H, et al. metabolic Engineering of Corynebacterium glutamine for L-Serine Production [ J ] Applied and Environmental Microbiology,2005,71(11): 7139-. The enhanced expression of 3-phosphoglycerate kinase (PGK) in Corynebacterium glutamicum ATCC13032 by Neisseria scholaris et al increases the synthesis of L-serine precursor 3-phosphoglycerate and improves the yield of L-serine (from the literature: Neisseria scholaris, Zhang Yun, Liu Tree Wen, etc. the 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.glutamicumYPS-062-33 a 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. Further releasing the feedback inhibition of key enzyme in the L-serine synthesis pathway on the mutant strain, knocking out the degradation pathway, blocking and weakening the byproduct accumulation pathway, and obtaining the recombinant bacterium C.glutamicum SYPS-06233 a delta SSA (abbreviated as delta SSA) (CGMCC NO.8668) with the yield of the L-serine shake flask reaching 26.25g/L which is 3.9 times of that of the 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, in Corynebacterium glutamicum and Escherichia coli, many amino acids have been identified as export proteins and used to improve the production of amino acids using metabolic engineering, such as lysine transporter LysE, threonine transporter ThrE, cysteine transporter EamA, methionine transporter BrnFE, etc., but only threonine transporter ThrE has been reported to transport serine.

Disclosure of Invention

The first purpose of the invention is to provide an L-serine transporter NCgl2054 which comprises an amino acid sequence shown in SEQ ID NO. 2.

The second object of the present invention is to provide a gene encoding the above-mentioned L-serine transporter NCgl 2054.

In one embodiment of the present invention, the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

The third object of the present invention is to provide a plasmid or vector containing the above gene.

In one embodiment of the present invention, the vector includes, but is not limited to, pXMJ19 plasmid vector, pDXW series vector, pET series vector, or pPICZ series vector.

It is a fourth object of the present invention to provide a cell expressing the above-mentioned L-serine transporter NCgl 2054.

In one embodiment of the invention, the host of the cell includes, but is not limited to, 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 Δ SSA.

The method for constructing the corynebacterium glutamicum delta SSA is disclosed in the literature: metabolic engineering of corynebacterium glutamicum SYPS-062, L-serine, rogowski, njianche, 2012.

The fifth purpose of the invention is to provide a method for improving the yield of serine, which comprises the steps of firstly over-expressing serine transporter NCgl2054 in corynebacterium glutamicum delta SSA to obtain genetically engineered bacteria over-expressing serine transporter NCgl2054, and then fermenting by using the genetically engineered bacteria.

In one embodiment of the invention, the culture medium for fermentation comprises 90-110g/L of sucrose, 20-40g/L of ammonium sulfate, 50-70g/L of calcium carbonate, and MgSO4 & 7H₂O 0.5-1g/L，FeSO₄·7H₂O 0.01-0.05g/L，MnSO4·H₂0.01-0.05g/L of O, 20-40mg/L of protocatechuic acid, 40-60 mu g/L of biotin and 500 mu g/L of thiamine 400-ketone.

In one embodiment of the present invention, the fermentation is performed at 28-32 deg.C and 100-140rpm for 60-150 h.

The sixth object of the present invention is to provide the use of the above-mentioned L-serine transporter NCgl2054 in the fields of pharmaceuticals, foods or cosmetics.

The invention has the beneficial effects that:

the invention provides a novel L-serine transporter NCgl2054, which has a nucleotide sequence shown in SEQ ID NO.1, has a full length of 936 nucleotides and codes 312 amino acids. The NCgl2054 protein is over-expressed in the L-serine-producing Corynebacterium glutamicum delta SSA by a genetic engineering means, so that the yield of the L-serine can be improved to 29.9 g/L. The method provides a new idea for improving the L-serine yield by metabolic engineering strain.

Drawings

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

FIG. 2 shows the evaluation of fermentation characteristics of NCgl2054 knock-out recombinant bacteria Δ SSA Δ 2054.

FIG. 3 is the evaluation of fermentation characteristics of recombinant bacteria Δ SSA-2054 overexpressing NCgl2054 gene.

Detailed Description

The present invention will be further described with reference to the following examples.

(I) culture Medium

Seed culture medium: 37g/L of brain-heart infusion; 20g/L of glucose; (NH)₄)₂SO₄ 10g/L；MgSO₄·7H₂O 0.5g/L；K₂HPO₄ 0.2g/L；NaH₂PO₄ 0.3g/L。

Cgxii minimal medium: glucose 40g/L, Urea 5g/L, (NH)₄)₂SO₄ 20g/L，KH₂PO₄ 1g/L，MgSO₄0.25g/L，MOPS 42g/L，CaCl₂ 10mg/L，FeSO₄·7H₂O 10mg/L，MnSO₄·H₂O 10mg/L，ZnSO₄ 1mg/L，CuSO₄ 0.2mg/L，NiCl₂·6H₂O0.02 mg/L, biotin 0.2mg/L, protocatechuic acid 0.03 g/L.

Fermentation medium: 100g/L of sucrose, 30g/L of ammonium sulfate, 60g/L of calcium carbonate, MgSO4 & 7H₂O 0.5g/L，FeSO₄·7H₂O 0.02g/L，MnSO4·H₂O0.02 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

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, 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;

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.

Method for overexpression of genes in Corynebacterium glutamicum delta SSA

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 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.

Fermentation culture method of (IV) recombinant corynebacterium glutamicum

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 reached₅₆₂25, add to 25mL fermentation Medium to OD₅₆₂Culturing at 120rpm for 5d at 1, 30 deg.C, and measuring OD every 12h₅₆₂And 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. Collecting pre-cultured thallus, and using pre-cooled thallusCgxii minimal medium was washed 2 times. According to initial OD₅₆₂The cells were inoculated into 50mL of CGXII minimal medium (8-10) and the transport experiment was started by adding L-ser-ser dipeptide to a final concentration of 3 mM. 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 μ 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.

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 phenyl isothiocyanate acetonitrile solution are respectively added, the mixture is evenly mixed and then stands for 1h at room temperature, 400 mu L of normal hexane is added, the mixture is violently shaken and evenly mixed and then stands for 10min, 200 mu L of lower layer solution is taken out, 800 mu L of ultrapure water is added for dilution, and then sample loading is carried out after filtration by a 0.45 mu m organic filter membrane.

HPLC reaction conditions

A chromatographic column: venusil AA, 4.6X 250nm, 5 μm; violet absorption wavelength: 254 nm; column temperature: 40 ℃; flow rate: 1 mL/min; sample introduction volume: 10 μ L. Gradient elution with mobile phase, wherein the elution procedure is 0-4min, 100% of mobile phase A is 4-16min, 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.