CN113801211A

CN113801211A - Corynebacterium glutamicum protein Ncgl0717 and surface display system and construction method thereof

Info

Publication number: CN113801211A
Application number: CN202110945748.9A
Authority: CN
Inventors: 郑穗平; 林珂瑞; 韩双艳; 林影
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-12-17
Anticipated expiration: 2041-08-17
Also published as: CN113801211B

Abstract

The invention discloses a corynebacterium glutamicum protein Ncgl0717, a surface display system and a construction method thereof. The amino acid sequence of the corynebacterium glutamicum wall protein Ncgl0717 is shown in SEQ ID NO 1, the expression level of the corynebacterium glutamicum wall protein Ncgl0717 in corynebacterium glutamicum is high, and the protein can be used for constructing a surface display system of corynebacterium glutamicum with high display efficiency. The surface display system of the corynebacterium glutamicum is formed by fixing target protein on the cell surface of the corynebacterium glutamicum by taking the corynebacterium glutamicum wall protein Ncgl0717 as an anchor protein. The invention enriches the expression types of endogenous anchoring proteins of a surface display system of corynebacterium glutamicum.

Description

Corynebacterium glutamicum protein Ncgl0717 and surface display system and construction method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a corynebacterium glutamicum protein Ncgl0717, a surface display system thereof and a construction method thereof.

Background

The microbial cell surface display technology is a novel genetic engineering technology for displaying a target fragment on the surface of a microorganism in the form of fusion protein by utilizing a genetic engineering means through a polypeptide fragment (or a protein structural domain), in particular to a technology for fixing protein or polypeptide on the surface of a cell through anchoring protein, wherein the displayed polypeptide or protein can keep a relatively independent spatial structure and biological activity. The microbial cell surface display system comprises a host bacterium, an anchor protein and a target protein, and a connecting (linker) sequence is added between the anchor protein and the target protein. The microbial cell surface display has wide application prospects in the aspects of polypeptide separation, whole-cell catalysts, whole-cell adsorbents, vaccine and antibody production, protein library screening, biosensors, bioremediation and the like.

Currently, phage (Bacteriophages), saccharomyces cerevisiae (saccharomyces cerevisiae), gram-negative bacteria (escherichia coli), gram-positive bacteria (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis), and the like are the main host bacteria applied to a microorganism surface display system. The dockerin used for surface display generally has the following characteristics: (1) is anchored on the cell surface more firmly; (2) can be effectively fused with a target protein sequence without influencing the structure and the function of the target protein. The dockerin proteins of gram-positive bacterial display systems are mainly: (1) membrane associated proteins (with transmembrane domains or lipoproteins); (2) a cell wall-associated protein (having a C-terminal Leu-Pro-X-Thr-Gly (LPXTG) motif or Cell Wall Binding Domain (CWBD)).

The corynebacterium glutamicum has the advantages of strong robustness, low extracellular protease activity, wide natural carbon source substrate and the like, and has very wide application prospect in the aspect of microbial cell surface display. Currently, there are few available anchoring proteins for the display system of C.glutamicum, of only three types: foreign proteins (PgsA), mycoylated proteins (Ncgl1337, porin series proteins PorB, PorC) and membrane proteins (Ncgl1221, also known as the mechanical tunnel protein mscg). These ankyrins have now successfully displayed a variety of enzyme proteins, such as amylases, glucanases, glucosidases, cellulase complexes, etc., on the surface of C.glutamicum. To increase the versatility of the cell surface display technology of C.glutamicum, it is necessary to develop new and efficient anchoring motifs.

Choi et al (Choi J W, Yim S, Jeong K J. development of a potential protein display form in Corynebacterium glutamicum using a mycolic acid layer protein, NCgl1337, as an anchoring motif [ J ]. Biotechnology Journal,2017:1700509.) reported the use of Corynebacterium glutamicum mycolic acid layer protein, Ncgl1337, as an anchor protein for cell surface display of foreign proteins. Yao et al (Display of alpha-amylase on the surface of Corynebacterium glutamicum cells by using NCgl1221 as the anchoring protein, and production of glutamate from stage. electrodes of microbiology.2009) reported the cell surface Display of foreign proteins using Corynebacterium glutamicum membrane protein NCgl1221 as the anchor protein. However, the existing Corynebacterium glutamicum has fewer types and numbers of anchoring proteins, and the display system is single.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a corynebacterium glutamicum protein Ncgl 0717.

Another object of the present invention is to provide a gene encoding said Corynebacterium glutamicum protein Ncgl 0717.

Still another object of the present invention is to provide a C.glutamicum cell surface display system.

The invention also aims to provide a construction method of the corynebacterium glutamicum cell surface display system.

The purpose of the invention is realized by the following technical scheme:

a Corynebacterium glutamicum protein Ncgl0717 has the amino acid sequence shown in SEQ ID NO. 1.

The wall protein of Corynebacterium glutamicum consists of 261 amino acids and has a size of about 26.91 KDa.

The nucleotide sequence of the gene for coding the corynebacterium glutamicum protein Ncgl0717 is shown in SEQ ID NO. 2.

The corynebacterium glutamicum protein Ncgl0717 is used as an anchor protein in a surface display system.

The surface display system is a Corynebacterium glutamicum cell surface display system.

The corynebacterium glutamicum protein Ncgl0717 is used as an anchor protein in a surface display system, wherein the corynebacterium glutamicum cell surface display system is formed by fixing a target protein on the surface of a corynebacterium glutamicum cell by using the corynebacterium glutamicum wall protein Ncgl0717 as an anchor protein.

A Corynebacterium glutamicum cell surface display system is composed of the Corynebacterium glutamicum wall protein Ncgl0717 as anchor protein and target protein fixed on the cell surface of Corynebacterium glutamicum.

The target protein is any one of fluorescent protein or amylase; preferably Enhanced Green Fluorescent Protein (EGFP), red fluorescent protein (mCherry) or alpha-amylase (alpha-amylase).

The Corynebacterium glutamicum is preferably Corynebacterium glutamicum ATCC 13032.

The construction method of the corynebacterium glutamicum cell surface display system comprises the following steps:

(1) cloning the gene coding the protein Ncgl0717 of the corynebacterium glutamicum into an expression cassette of an expression vector to obtain a surface display expression vector taking the protein Ncgl0717 of the corynebacterium glutamicum wall as an anchor protein;

(2) cloning a gene sequence of a target protein to the upstream of a gene sequence of a corynebacterium glutamicum wall protein Ncgl0717 of the surface display expression vector obtained in the step (1), and forming a fusion gene with the gene of the corynebacterium glutamicum wall protein Ncgl 0717;

(3) transforming corynebacterium glutamicum, and then screening positive transformants according to the screening markers on the expression vectors to obtain the corynebacterium glutamicum cell surface display system.

The construction method of the corynebacterium glutamicum cell surface display system specifically comprises the following steps:

(i) constructing a recombinant plasmid by a homologous recombination mode of a gene for coding the corynebacterium glutamicum protein Ncgl0717, a gene for a target protein and an expression vector;

(ii) and (e) transforming the recombinant plasmid obtained in the step (i) into corynebacterium glutamicum, and selecting a positive transformant to obtain the corynebacterium glutamicum cell surface display system.

The gene sequence encoding the Corynebacterium glutamicum protein Ncgl0717 described in step (i) is shown in SEQ ID NO: 2.

(ii) the target protein in step (i) is any one of fluorescent protein or amylase; preferably Enhanced Green Fluorescent Protein (EGFP), red fluorescent protein (mCherry) or alpha-amylase (alpha-amylase); more preferably an alpha-amylase.

The nucleotide sequence of the Enhanced Green Fluorescent Protein (EGFP) is shown in SEQ ID NO: 5.

The nucleotide sequence of the red fluorescent protein (mCherry) is shown as SEQ ID NO: 17.

The nucleotide sequence of the alpha-amylase (amyE gene) is shown as SEQ ID NO: 21.

The expression vector in step (i) is a Corynebacterium glutamicum expression vector which is conventional in the art and has kanamycin resistance; preferably, the expression vector pEC-XK99e (when pEC-XK99e is used as an expression vector, after the surface display expression vector is constructed, the gene sequence of the target protein is cloned by homologous recombination and connected to the surface display expression vector downstream of the gene sequence of the wall protein of Corynebacterium glutamicum).

The Corynebacterium glutamicum described in step (ii) is preferably Corynebacterium glutamicum ATCC 13032.

The corynebacterium glutamicum protein Ncgl0717, the gene encoding the corynebacterium glutamicum protein Ncgl0717 or the application of a corynebacterium glutamicum cell surface display system in preparing amylase.

The amylase is preferably an alpha-amylase.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a corynebacterium glutamicum wall protein Ncgl0717 which is an endogenous wall protein of corynebacterium glutamicum, has a high expression level in corynebacterium glutamicum, has higher display efficiency compared with an endogenous anchor protein Ncgl1221 which is used for a surface display system of corynebacterium glutamicum, and can be used for constructing a surface display system of corynebacterium glutamicum with high display efficiency.

(2) The surface display system of the corynebacterium glutamicum is formed by taking a corynebacterium glutamicum wall protein Ncgl0717 as an anchor protein and fixing a target protein (such as fluorescent protein or amylase) on the cell surface of the corynebacterium glutamicum, so that the expression types of endogenous anchor proteins of the surface display system of the corynebacterium glutamicum are enriched.

Drawings

FIG. 1 is a diagram showing the results of flow cytometry detection of recombinant bacteria CG/Ncgl0717-EGFP, negative control bacteria CG/EGFP, and positive control bacteria CG/Ncgl1221-EGFP and CG/Ncgl 1337-EGFP.

FIG. 2 is a diagram showing the results of confocal laser microscopy of CG/Ncgl 0717-EGFP.

FIG. 3 is a diagram showing the results of flow cytometry detection of recombinant strain CG/Ncgl0717-mCherry, negative control strain ATCC13032, and positive control strains CG/Ncgl1221-mCherry, CG/Ncgl 1337-mCherry.

FIG. 4 is a graph showing the results of confocal laser microscopy of CG/Ncgl 0717-mCherry.

FIG. 5 is a diagram showing the amylase activity of the recombinant strain (in the drawing, WT: Corynebacterium glutamicum ATCC 13032; NC: negative control strain CG/pEC-XK99 e; PC 1: positive control strain CG/Ncgl 1221-Amy; PC 2: positive control strain CG/Ncgl 1337-Amy; Ncgl 0717: recombinant strain CG/Ncgl 0717-Amy).

FIG. 6 is a graph showing the growth of recombinant strain CG/Ncgl0717-Amy and control strains ATCC13032, CG/pEC-XK99 e; wherein A is a growth curve of the strain when glucose is used as a unique carbon source; b is the growth curve of the strain when starch is used as a unique carbon source.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The following examples are given without reference to specific experimental conditions, and are generally in accordance with conventional experimental conditions. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

Interpretation of terms

LB plate: 10g/L of tryptone, 5g/L of yeast extract, 5g/L of NaCl and 20g/L of agar.

LBH plate: 2.5g/L of yeast powder, 5g/L of peptone, 5g/L of NaCl, 18.5g/L of Brain Heart Infusion (Bacto Brain Heart Infusion), 91g/L of sorbitol and 20g/L of agar.

BHISG medium: 37g/L of brain-heart infusion, 9.1g/L of sorbitol and 10g/L of glucose.

10 × PBS (phosphate buffered saline): 40g NaCl, 1g KCl, 7.1g Na₂HPO₄、1.2g KH₂PO₄The above reagents were dissolved in 400ml of water, the pH was adjusted to 7.4, and the volume was adjusted to 500 ml).

1 × PBS: 10 × PBS dilution 10 times.

MOPS buffer solution: 10ml of 0.5M 3- (N-morpholinyl) propanesulfonic acid (MOPS) (pH 6.9) and diluted 10-fold.

Example 1: construction of surface display vector pEC-EGFP of Corynebacterium glutamicum wall protein Ncgl0717

(1) Cloning of Corynebacterium glutamicum wall protein Ncgl0717 Gene

Based on the gene sequence (SEQ ID NO:2) of Corynebacterium glutamicum wall protein Ncgl0717 (amino acid sequence shown in SEQ ID NO: 1), the target protein EGFP gene and the sequence characteristics on the Corynebacterium glutamicum plasmid pEC-XK99e (purchased from Novagen), amplification primers were designed:

P1：5'-atgAAAGGAGGCCCTTCAGATGAAAACAGAAACTCGACGAGCCCTC-3'(SEQ ID NO:3)；

P2：5'-CTTATCGTCATCATCCTTGTAATCGCTCGTGGCAGTTGGTTCCAC-3'(SEQ ID NO:4)。

the genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and P1 and P2 were used as primers to amplify the gene sequence of the wall protein Ncgl0717 by PCR method under the following amplification conditions: pre-denaturation at 94 ℃ for 5 min; another 30 cycles of: denaturation at 94 deg.C for 30 seconds, Tm-5 deg.C (Tm is melting temperature; the same applies below), annealing for 30 seconds, and extension at 68 deg.C for 1 minute; and final extension at 68 ℃ for 10 min.

(2) Cloning of target protein EGFP Gene

According to the sequence characteristics of target protein EGFP gene (SEQ ID NO:5) and Corynebacterium glutamicum plasmid pEC-XK99e, an amplification primer is designed:

P3：5'-GATTACAAGGATGATGACGATAAGATGGTGAGCAAGGGCGAGGA-3'(SEQ ID NO:6)；

P4：5'-TCGTCATCATCCTTGTAATCTTACTTGTACAGCTCGTCCATG-3'(SEQ ID NO:7)。

the target protein EGFP gene is amplified by a PCR method by taking the genome DNA of Corynebacterium glutamicum ATCC13032 as a template and P3 and P4 as primers, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 5 min; another 30 cycles of: denaturation at 94 ℃ for 30 seconds, annealing at Tm-5 ℃ for 30 seconds, and extension at 68 ℃ for 1 minute; and final extension at 68 ℃ for 10 min.

(3) Cloning of vector pEC-XK99e

Based on the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 (SEQ ID NO:2), the target protein EGFP gene and the sequence characteristics on the Corynebacterium glutamicum plasmid pEC-XK99e, amplification primers were designed:

P5：5'-GATTACAAGGATGATGACGATAAGggctgttttggcg-3'(SEQ ID NO:8)；

P6：5'-CTGAAGGGCCTCCTTTcatggtctgtttcctgtgtg-3'(SEQ ID NO:9)。

the gene sequence of the vector pEC-XK99e is amplified by a PCR method by taking pEC-XK99e as a template and P5 and P6 as primers, and the amplification conditions are as follows: pre-denaturation at 94 ℃ for 5 min; another 30 cycles of: denaturation at 94 ℃ for 30 seconds, annealing at Tm-5 ℃ for 30 seconds, and extension at 68 ℃ for 2 minutes; and final extension at 68 ℃ for 10 min.

(4) Construction of vector pEC/Ncgl0717-FLAG-EGFP

The PCR product of the gene Ncgl0717 of the wall protein of Corynebacterium glutamicum obtained in step (1), the PCR product of the gene EGFP green fluorescent protein obtained in step (2) and the PCR product of the expression plasmid pEC-XK99e of Corynebacterium glutamicum obtained in step (3) were ligated by Gibson homologous recombination (this step uses the Seamless Assembly cloning kit), and the ligation system was transformed into the E.coli host Top10 (purchased from Novagen). Transformants were selected on LB plates containing 50mg/L kanamycin, positive transformants were picked using identifying primers P7 and P8, plasmids were extracted, identified and sequenced, which indicated that the wall protein Ncgl0717 gene sequence was correctly transformed and that the FLAG tag was upstream of the wall protein Ncgl0717 gene.

The primer sequences involved are as follows:

P7：5'-CACTGCATAATTCGTGTCGCTCAAGGCGCACTCC-3'(SEQ ID NO:10)；

P8：5'-GTCGCCGTCCAGCTCGACCAGGATG-3'(SEQ ID NO:11)。

(5) construction and identification of recombinant Corynebacterium glutamicum surface display system CG/Ncgl0717-EGFP

The plasmid pEC/Ncgl0717-FLAG-EGFP obtained in step (4) was transformed into Corynebacterium glutamicum ATCC13032 by the electrotransformation method, positive transformants were picked on LBH plates and PCR amplification was performed using P7 and P8 as primers, which confirmed that the gene sequence of plasmid pEC/Ncgl0717-FLAG-EGFP was transformed into Corynebacterium glutamicum ATCC13032, and the resulting recombinant strain was named CG/Ncgl 0717-EGFP.

(6) Construction and identification of negative control Strain CG/EGFP and Positive control Strain CG/Ncgl1221-EGFP, CG/Ncgl1337-EGFP of recombinant Corynebacterium glutamicum surface display System CG/Ncgl0717-EGFP

Construction of negative control Strain CG/EGFP: EGFP (SEQ ID NO:5) is taken as a template, and P9 and P4 are taken as primers, and the gene sequence of the EGFP is amplified by a PCR method; the gene sequence of pEC-XK99e is amplified by a PCR method by taking a corynebacterium glutamicum plasmid pEC-XK99e as a template and P5 and P6 as primers; then, the PCR product of the EGFP gene and the PCR product of the expression plasmid pEC-XK99e of corynebacterium glutamicum are subjected to Gibson homologous recombination and connected, and finally a negative control strain CG/EGFP is constructed, wherein the specific preparation and identification methods are shown in the steps (1) to (5);

② construction of a positive control strain CG/Ncgl 1221-EGFP: the gene sequence of Ncgl1221 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P10 and P11 as primers; then, the PCR product of the Ncgl1221 gene, the PCR product of the EGFP gene obtained in the step (2) and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in the step (3) are subjected to Gibson homologous recombination and connected, and finally, a positive control strain CG/Ncgl1221-EGFP is constructed, wherein the identification method is shown in the steps (1) to (5);

construction of Positive control Strain CG/Ncgl 1337-EGFP: the gene sequence of Ncgl1337 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P12 and P13 as primers; then, the PCR product of the Ncgl1337 gene, the PCR product of the EGFP gene obtained in step (2) and the PCR product of the C.glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1337-EGFP was constructed, the identification method being as in steps (1) to (5) above;

the primer sequences involved are as follows:

P9：5'-catgAAAGGAGGCCCTTCAGATGGATTACAAGGATGATGACGATAAGATGGTGAGCAAGGGCGAG-3'(SEQ ID NO:12)；

P10：5'-catgAAAGGAGGCCCTTCAGATGATTTTAGGCGTACCCATTCAATATTTG-3'(SEQ ID NO:13)；

P11：5'-CTTATCGTCATCATCCTTGTAATCAGGGGTGGACGTCGGCGCAACTGTC-3'(SEQ ID NO:14)；

P12：5'-catgAAAGGAGGCCCTTCAGATGGCTCAGCGAAAACTGGCCTC-3'(SEQ ID NO:15)；

P13：5'-CTTATCGTCATCATCCTTGTAATCGGCGTTTACTCGATCTCGCAGGATC-3'(SEQ ID NO:16)。

(7) flow cytometry analysis and confocal microscope analysis of recombinant corynebacterium glutamicum surface display system CG/Ncgl0717-EGFP

The recombinant strain CG/Ncgl0717-EGFP was inoculated into 5ml of BHISG medium containing 0.5mM IPTG (isopropyl. beta. -D-1-thiogalactoside), induced and cultured at 30 ℃ and 220rpm for 24 hours, resuspended after centrifugation, and resuspended after incubation with anti-FLAG monoclonal antibody and goat anti-mouse IgG antibody labeled with Alexa Fluor647 (both antibodies were purchased from Muyunyun). Finally, fluorescence intensity was measured using a C6 Plus flow cytometer. Meanwhile, CG/EGFP was used as a negative control, and CG/Ncgl1221-EGFP and CG/Ncgl1337-EGFP were used as positive controls.

The flow cytometry results are shown in FIG. 1 (M in FIG. 1 represents Comp-FL-A:: average value of APC-A _ Area): the results show that the fluorescence of the recombinant strain CG/Ncgl0717-EGFP is greatly shifted compared with the negative control strains CG/EGFP, positive control strains CG/Ncgl1221-EGFP and CG/Ncgl1337-EGFP, and the wall protein Ncgl0717-EGFP fusion protein is successfully expressed on the cell surface of the recombinant strain CG/Ncgl 0717-EGFP.

Subsequently, the sample was centrifuged and resuspended, then it was coated on a microscope slide, and finally observed by confocal microscopy, which revealed that red fluorescence of the secondary antibody was displayed on the surface of the recombinant bacterium CG/Ncgl0717-EGFP (FIG. 2) (in FIG. 2: EGFP indicates fluorescence excited by EGFP fluorescent protein; Alexa Fluor647 indicates fluorescence excited by Alexa Fluor647 antibody), and it was confirmed again that the cell surface of the recombinant bacterium CG/Ncgl0717-EGFP successfully expressed the fusion protein of Corynebacterium glutamicum wall protein Ncgl 0717-EGFP.

Example 2: construction of Corynebacterium glutamicum wall protein Ncgl0717 surface display vector pEC-Ncgl0717-mCherry

(1) Cloning of Corynebacterium glutamicum wall protein Ncgl0717 Gene

Amplification primers P1 and P2 were designed based on the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 (SEQ ID NO:2), the target protein mCherry gene and the sequence characteristics of Corynebacterium glutamicum plasmid pEC-XK99e, and the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 was obtained by PCR amplification, as described in step (1) of example 1.

(2) Cloning of target protein mCherry Gene

Amplification primers P14 and P15 were designed based on the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 (SEQ ID NO:2), the target protein mCherry gene (SEQ ID NO:17) and the sequence characteristics of Corynebacterium glutamicum plasmid pEC-XK99e, and the target protein mCherry gene was obtained by PCR amplification, as described in example 1.

P14：5'-GATTACAAGGATGATGACGATAAGATGGTTTCCAAGGGCGAGGAGGAC-3'

(SEQ ID NO:18)；

P15：5'-cagccCTTATCGTCATCATCCTTGTAATCTTACTTGTAGAGTTCGTCCATG-3'(SEQ ID NO:19)。

(3) Cloning of vector pEC-XK99e

Amplification primers P5 and P6 were designed based on the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 (SEQ ID NO:2), the target protein mCherry gene and the sequence characteristics of Corynebacterium glutamicum plasmid pEC-XK99e, and the gene sequence of vector pEC-XK99e was amplified by PCR, as described in step (3) of example 1.

(4) Construction of vector pEC/Ncgl0717-mCherry

The PCR product of the wall protein Ncgl0717 gene of Corynebacterium glutamicum obtained in step (1), the PCR product of the mCheerry gene obtained in step (2) and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination (same method as in example 1), and the ligated system was transformed into the E.coli host Top 10. Transformants were selected on LB plates containing 50mg/L kanamycin, positive transformants were picked using identifying primers P7 and P16, plasmids were extracted, identified and sequenced, resulting in the recombinant C.glutamicum surface display expression plasmid pEC/Ncgl0717-mCherry with a FLAG tag upstream of the muramyl Ncgl0717 gene. The primer sequences involved are as follows:

P16：5'-CTTGTAGGTGGTCTTAACCTCAGCGTCGTAGTGACCG-3'(SEQ ID NO:20)。

(5) construction and identification of recombinant Corynebacterium glutamicum surface display system pEC/Ncgl0717-mCherry

Transforming the plasmid pEC/Ncgl0717-mCherry obtained in the step (4) into Corynebacterium glutamicum ATCC13032 by an electrotransfer method, picking up positive transformants on LBH plates, performing PCR amplification by using P7 and P11 as primers, and as a result, confirming that the gene sequence of the plasmid pEC/Ncgl0717-mCherry is transformed into Corynebacterium glutamicum ATCC13032, and the obtained recombinant bacterium is named CG/Ncgl 0717-mCherry.

(6) Construction and identification of negative control Strain CG/pEC-XK99e and Positive control Strain CG/Ncgl1221-mCherry, CG/Ncgl1337-mCherry of recombinant Corynebacterium glutamicum surface display System CG/Ncgl0717-mCherry

Construction of a positive control strain CG/Ncgl 1221-mCherry: the gene sequence of Ncgl1221 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P10 and P11 as primers; then, the PCR product of the Ncgl1221 gene, the PCR product of the mCherry gene obtained in step (2) and the PCR product of the C.glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1221-mCherry was constructed, the identification method being as described in example 1.

② construction of a positive control strain CG/Ncgl 1337-mCherry: the gene sequence of Ncgl1337 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P12 and P13 as primers; then, the PCR product of Ncgl1337 gene, the PCR product of mCherry gene obtained in step (2) and the PCR product of Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally, a positive control strain CG/Ncgl1337-mCherry was constructed, the identification method of which is described in example 1.

(7) Flow cytometry analysis and confocal microscopy analysis of recombinant Corynebacterium glutamicum CG/Ncgl0717-mCherry and control strains

The recombinant strain CG/Ncgl0717-mCherry and the control strain were inoculated into 5ml BHISG medium containing 0.5mM IPTG, induced and cultured at 30 ℃ and 220rpm for 24 hours, resuspended after centrifugation, and resuspended after incubation with anti-FLAG monoclonal antibody and Alexa Fluor 488-labeled goat anti-mouse IgG antibody (both antibodies were purchased from Muyunyun). Finally, fluorescence intensity was measured using a C6 Plus flow cytometer. Meanwhile, the strain ATCC13032 was used as a negative control, and CG/Ncgl1221-mCherry and CG/Ncgl1337-mCherry were used as positive controls.

The results are shown in FIG. 3 (M in FIG. 3 denotes Comp-FL-A:: average value of FITC-A _ Area): the results show that the fluorescence of recombinant bacteria CG/Ncgl0717-mCherry is greatly shifted compared with negative control strains ATCC13032, positive control strains CG/Ncgl1221-mCherry and CG/Ncgl1337-mCherry, and indicate that the fusion protein of the corynebacterium glutamicum wall protein Ncgl0717-mCherry is successfully expressed on the cell surface of the recombinant bacteria CG/Ncgl 0717-mCherry.

Subsequently, the sample was centrifuged and resuspended, then the sample was coated on a microscope slide, and finally observed by confocal microscopy, which revealed that the green fluorescence of the secondary antibody was displayed on the surface of recombinant bacterium CG/Ncgl0717-mCherry (FIG. 4) (in FIG. 4: mCherry represents the fluorescence excited by mCherry fluorescent protein; Alexa Fluor647 represents the fluorescence excited by Alexa Fluor647 antibody), and it was confirmed again that the fusion protein of Corynebacterium glutamicum parin Ncgl0717-mCherry was successfully expressed on the cell surface of recombinant bacterium CG/Ncgl 0717-mCherry.

Example 3: construction of surface display vector pEC-Amy of Corynebacterium glutamicum wall protein Ncgl0717

(1) Cloning of Corynebacterium glutamicum wall protein Ncgl0717 Gene

Amplification primers were designed based on the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 (SEQ ID NO:2), and the gene sequence of Corynebacterium glutamicum wall protein Ncgl0717 was obtained by PCR amplification, as described in step (1) of example 1.

(2) Cloning of the target protein alpha-amylase Gene

Amplification primers P17 and P18 were designed based on the amyE gene of the target protein alpha-amylase (alpha-amylase EC 3.2.1.1) (amyE gene is derived from Bacillus subtilis 168; the sequence is shown in SEQ ID NO: 21), and the gene sequence of the target protein alpha-amylase was amplified by PCR using the genome of Bacillus subtilis 168 as a template, and the preparation method was as shown in example 1:

P17：5'-GATTACAAGGATGATGACGATAAGATGTTTGCAAAACGATTCAAAACCTCTTTACTGCC-3'(SEQ ID NO:22)；

P18：5'-CTTATCGTCATCATCCTTGTAATCTCAATGGGGAAGAGAACCGCTTAAGCCCG-3'(SEQ ID NO:23)。

(3) cloning of vector pEC-XK99e

The gene sequence of the vector pEC-XK99e was amplified by PCR method using amplification primers P5 and P6 designed based on the sequence characteristics of plasmid pEC-XK99e of Corynebacterium glutamicum, see step (3) of example 1.

(4) Construction of vector pEC/Ncgl0717-Amy

The PCR product of the gene Ncgl0717 of the wall protein of Corynebacterium glutamicum obtained in step (1), the PCR product of the gene α -amylase obtained in step (2) and the PCR product of the expression plasmid pEC-XK99e of Corynebacterium glutamicum obtained in step (3) were ligated by Gibson homologous recombination (same procedure as in example 1) to obtain the recombinant surface display expression plasmid pEC/Ncgl0717-Amy, and the obtained pEC/Ncgl0717-Amy plasmid was transformed into the E.coli host Top 10. Transformants were selected on LB plates containing 50mg/L kanamycin, positive transformants were picked, plasmids were extracted, identified and sequenced using the identifying primers P7 and P19, and the result showed that the wall protein Ncgl0717 gene sequence was correctly transformed and the FLAG tag was upstream of the wall protein Ncgl0717 gene. The primer sequences involved are as follows:

P19：5'-GAACGACCAATTCCATGCATGAAGAATGGTTCCGC-3'(SEQ ID NO:24)。

(5) construction of recombinant Corynebacterium glutamicum surface display system CG/Ncgl0717-Amy

The plasmid pEC/Ncgl0717-Amy obtained in step (4) was transformed into Corynebacterium glutamicum ATCC13032 by the electrotransformation method, positive transformants were picked up on LBH plates, PCR amplification was performed using P7 and P14 as primers, and as a result, it was confirmed that the gene sequence of the plasmid pEC/Ncgl0717-Amy was transformed into Corynebacterium glutamicum ATCC13032, and the resulting recombinant strain was named CG/Ncgl 0717-Amy.

(6) Construction and identification of Positive control strains CG/Ncgl1221-Amy, CG/Ncgl1337-Amy and negative control Strain CG/pEC-XK99e of the recombinant Corynebacterium glutamicum surface display System Ncgl0717-Amy

Construction of a Positive control Strain CG/Ncgl1221-Amy (PC 1): the gene sequence of Ncgl1221 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P10 and P11 as primers; then, the PCR product of the Ncgl1221 gene, the PCR product of the Amy gene obtained in step (2), and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1221-Amy was constructed, and the identification method was described in example 1.

② construction of a positive control strain CG/Ncgl1337-Amy (PC 2): the gene sequence of Ncgl1337 was amplified by the PCR method using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and P12 and P13 as primers; then, the PCR product of the Ncgl1337 gene, the PCR product of the Amy gene obtained in step (2), and the PCR product of the Corynebacterium glutamicum expression plasmid pEC-XK99e obtained in step (3) were ligated by Gibson homologous recombination, and finally a positive control strain CG/Ncgl1337-Amy was constructed, as identified in example 1.

Construction of negative control Strain CG/pEC-XK99 e: the vector pEC-XK99e was directly transformed into Corynebacterium glutamicum ATCC13032, and finally a negative control strain CG/pEC-XK99e was constructed, the identification method being described in example 1.

(7) Fermentation of recombinant Corynebacterium glutamicum CG/Ncgl0717-Amy and determination of amylase activity

CG/Ncgl0717-Amy was inoculated into 5mL of BHISG medium for overnight culture, and then transferred to 30mL of BHISG medium containing 0.5mM IPTG (resistant strain supplemented with 25. mu.g/mL kanamycin) and induced and cultured at 30 ℃ and 220rpm for 24 hours to obtain a cell fermentation broth. The determination method of amylase activity adopts EnzChek^TMAn amylase detection kit (cat # E33651). The reaction substrate comprises cell suspension, supernatant and cell fermentation liquor, wherein the supernatant and the cell suspension are obtained by centrifuging the cell fermentation liquor, the cell suspension needs to be washed by MOPS buffer solution for 3 times and then resuspended, and the centrifugation condition is 6000 rpm. One unit of enzyme activity (U/ml) is defined as the amount of enzyme required to release 1 mg of maltose from starch at 20 ℃ in 3 minutes at pH 6.9. Corynebacterium glutamicum ATCC13032, (WT) and CG/pEC-XK99e (NC) were used as negative controls, and CG/Ncgl1221-Amy (PC1) and CG/Ncgl1337-Amy (PC2) were used as positive controls.

The results of the amylase activity assay are shown in FIG. 5: the results showed that the enzyme activity of CG/Ncgl0717-Amy (Ncgl0717) cell suspension reached 0.16U/ml after 24h fermentation, which was higher than that of the positive control CG/Ncgl1221-Amy (0.13U/ml). It was shown that the alpha-amylase was successfully displayed in active form on the cell surface of C.glutamicum by the Ncgl0717 protein.

(6) Determination of growth Curve for recombinant Corynebacterium glutamicum CG/Ncgl0717-Amy

The recombinant strain CG/Ncgl0717-Amy selected from the plate was inoculated into BHIS (resistant strain supplemented with kanamycin to a final concentration of 25. mu.g/mL) medium and cultured overnight at 30 ℃ and 220 rpm. The medium was inoculated with an appropriate volume of bacteria using glucose or soluble starch ((Tianjin Dacron chemical Co., Ltd.; CAS No:9005-84-9)) (4%, w/w) solution as the sole carbon source, and then cultured in 12-well plates (resistant strains supplemented with kanamycin to a final concentration of 25. mu.g/mL). The initial OD600nm was about 0.3, and the culture conditions were 30 ℃ and 280 rpm. The OD600 optical density was measured every 4 hours for 32 hours.

ATCC13032, CG/pEC-XK99e and CG/Ncgl0717-Amy strains had similar growth curves in glucose medium (FIG. 6). The cells of the negative controls ATCC13032 and CG/pEC-XK99e grew poorly in starch medium because they were unable to utilize starch. In contrast, the recombinant strain CG/Ncgl0717-Amy grew better in starch medium and grew towards similar trends in starch and glucose media, including the time point of entry into stationary phase and the maximum OD600 value. This indicates that the alpha-amylase from CG/Ncgl0717-Amy was successfully displayed in active form on the cell surface of C.glutamicum and that it has good starch utilization.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> university of southern China's science

<120> Corynebacterium glutamicum protein Ncgl0717 and surface display system and construction method thereof

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 261

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Corynebacterium glutamicum protein Ncgl0717

<400> 1

Met Lys Thr Glu Thr Arg Arg Ala Leu Val Phe Ile Val Ala Gly Cys

1 5 10 15

Leu Ala Ala Thr Ala Leu Gly Phe Met Val Trp Gln Met Ser Ser Pro

20 25 30

Ser Arg Pro Thr Ser Asp Ile Ala Thr Ser Thr Thr Thr Ser Thr Thr

35 40 45

Gln Thr Gln Ala Arg Tyr Asp Ser Pro Gly Asn Thr Glu Thr Lys Glu

50 55 60

Ala Glu Pro Asp Leu Glu Asn Gln Thr Leu Ala Pro Ile Asn Thr Glu

65 70 75 80

Asp Pro Tyr Leu Pro Pro Asn Ala Phe Val Arg Pro Asp Asn Gly Arg

85 90 95

Ser Ser Gly Leu Thr Pro Ser Gly Ser Ser Pro Thr Thr Thr Ser Arg

100 105 110

Val Ser Ser Pro Ser Ser Ala Gly Ser Ala Ser Pro Thr Gln Ile Thr

115 120 125

Ser Arg Ser Asn Glu Pro Ser Glu Pro Gly Asp Glu Ser Thr Ala Ala

130 135 140

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

145 150 155 160

Asp Pro Thr Gly Pro Ser Glu Pro Thr Glu Pro Thr Asp Pro Ile Glu

165 170 175

Thr Thr Asp Pro Ile Glu Thr Thr Asp Pro Val Ala Pro Ser Thr Pro

180 185 190

Pro Thr Ser Asp Asp Ser Thr Ser Thr Pro Gln Pro Asp Glu Ser Asp

195 200 205

Thr Pro Pro Thr Asp Phe Val Glu Glu Pro Thr Ala Pro Leu Asn Pro

210 215 220

Asp Gln Pro Ala Gly Ser Thr Thr Asp Ala Thr Pro Asn Ala Thr Pro

225 230 235 240

Ser Ala Pro Ala Asp Thr Thr Ser Asn Ser Val Ala Asn Ser Val Glu

245 250 255

Pro Thr Ala Thr Ser

260

<210> 2

<211> 786

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gene sequence of Corynebacterium glutamicum protein Ncgl0717

<400> 2

atgaaaacag aaactcgacg agccctcgtc ttcatcgtcg ccggctgttt agccgccacc 60

gccctgggtt ttatggtctg gcagatgtcc agcccaagcc gacccacctc tgatattgcc 120

acgtctacta ctacgtctac cacccaaacc caggctaggt acgattcccc aggtaataca 180

gagaccaaag aggcggaacc tgacctagaa aaccaaactt tggcgcccat caacaccgaa 240

gatccatatc ttccaccgaa tgcttttgtg cgtccagaca atggccgaag ctccggttta 300

accccttctg gcagttctcc aactaccacc tctcgggtga gttctccctc ctcagcagga 360

tcggcaagcc cgactcaaat cacctccagg tcaaacgagc ctagtgaacc tggtgatgag 420

tcaactgctg ctacacaacc gtcgagccca gacaggccaa ccgaacctac aaatccggta 480

gacccaactg gaccttctga acctacggaa cccaccgatc cgattgagac aaccgatccg 540

attgagacaa ccgatccggt agctccgtcc accccgccaa cgagcgatga ttcaacaagc 600

actccccaac cagatgagtc tgatacgcca cctaccgatt tcgtagagga acctactgct 660

cctctcaatc cggatcagcc agccggttca actactgatg cgacgccaaa cgcaacacca 720

agcgcaccag ctgacacaac atccaattct gtagctaact ctgtggaacc aactgccacg 780

agctaa 786

<210> 3

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P1

<400> 3

atgaaaggag gcccttcaga tgaaaacaga aactcgacga gccctc 46

<210> 4

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P2

<400> 4

cttatcgtca tcatccttgt aatcgctcgt ggcagttggt tccac 45

<210> 5

<211> 720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> EGFP Gene

<400> 5

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 742

<210> 6

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P3

<400> 6

gattacaagg atgatgacga taagatggtg agcaagggcg agga 44

<210> 7

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P4

<400> 7

tcgtcatcat ccttgtaatc ttacttgtac agctcgtcca tg 42

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P5

<400> 8

gattacaagg atgatgacga taagggctgt tttggcg 37

<210> 9

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P6

<400> 9

ctgaagggcc tcctttcatg gtctgtttcc tgtgtg 36

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P7

<400> 10

cactgcataa ttcgtgtcgc tcaaggcgca ctcc 34

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P8

<400> 11

gtcgccgtcc agctcgacca ggatg 25

<210> 12

<211> 65

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P9

<400> 12

catgaaagga ggcccttcag atggattaca aggatgatga cgataagatg gtgagcaagg 60

gcgag 67

<210> 13

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P10

<400> 13

catgaaagga ggcccttcag atgattttag gcgtacccat tcaatatttg 50

<210> 14

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P11

<400> 14

cttatcgtca tcatccttgt aatcaggggt ggacgtcggc gcaactgtc 49

<210> 15

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P12

<400> 15

catgaaagga ggcccttcag atggctcagc gaaaactggc ctc 43

<210> 16

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P13

<400> 16

cttatcgtca tcatccttgt aatcggcgtt tactcgatct cgcaggatc 49

<210> 17

<211> 711

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mCherry gene

<400> 17

atggtttcca agggcgagga ggacaacatg gcaatcatca aggaattcat gcgcttcaag 60

gttcacatgg agggctccgt caacggtcac gagttcgaaa tcgagggcga gggcgaaggt 120

cgtccatacg agggcaccca gaccgctaag ctcaaggtta ctaagggcgg tccactgcct 180

ttcgcatggg acatcctctc cccacagttc atgtacggct ctaaggctta cgttaagcac 240

ccagcagata tccctgacta cctgaagctt tccttcccag agggcttcaa gtgggaacgc 300

gtcatgaact tcgaggacgg tggcgttgtt accgtcaccc aggattcctc cctccaggac 360

ggcgagttca tctacaaggt gaagctgcgt ggtaccaact tcccatctga cggccctgtt 420

atgcagaaga agactatggg ctgggaagct tcctccgagc gcatgtaccc agaggatggt 480

gcactcaagg gcgaaatcaa gcagcgtctg aagcttaagg acggcggtca ctacgacgct 540

gaggttaaga ccacctacaa ggcaaagaag ccagtccagc tccctggcgc ttacaacgtt 600

aacatcaagc tggatatcac ctcccacaac gaggactaca ctatcgttga acagtacgag 660

cgcgcagagg gccgtcactc taccggtggc atggacgaac tctacaagta a 711

<210> 18

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P14

<400> 18

gattacaagg atgatgacga taagatggtt tccaagggcg aggaggac 48

<210> 19

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P15

<400> 19

cagcccttat cgtcatcatc cttgtaatct tacttgtaga gttcgtccat g 51

<210> 20

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P16

<400> 20

cttgtaggtg gtcttaacct cagcgtcgta gtgaccg 37

<210> 21

<211> 1980

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amyE Gene

<400> 21

atgtttgcaa aacgattcaa aacctcttta ctgccgttat tcgctggatt tttattgctg 60

tttcatttgg ttctggcagg accggcggct gcgagtgctg aaacggcgaa caaatcgaat 120

gagcttacag caccgtcgat caaaagcgga accattcttc atgcatggaa ttggtcgttc 180

aatacgttaa aacacaatat gaaggatatt catgatgcag gatatacagc cattcagaca 240

tctccgatta accaagtaaa ggaagggaat caaggagata aaagcatgtc gaactggtac 300

tggctgtatc agccgacatc gtatcaaatt ggcaaccgtt acttaggtac tgaacaagaa 360

tttaaagaaa tgtgtgcagc cgctgaagaa tatggcataa aggtcattgt tgacgcggtc 420

atcaatcata ccaccagtga ttatgccgcg atttccaatg aggttaagag tattccaaac 480

tggacacatg gaaacacaca aattaaaaac tggtctgatc gatgggatgt cacgcagaat 540

tcattgctcg ggctgtatga ctggaataca caaaatacac aagtacagtc ctatctgaaa 600

cggttcttag acagggcatt gaatgacggg gcagacggtt ttcgatttga tgccgccaaa 660

catatagagc ttccagatga tggcagttac ggcagtcaat tttggccgaa tatcacaaat 720

acatctgcag agttccaata cggagaaatc ctgcaggata gtgcctccag agatgctgca 780

tatgcgaatt atatggatgt gacagcgtct aactatgggc attccataag gtccgcttta 840

aagaatcgta atctgggcgt gtcgaatatc tcccactatg catctgatgt gtctgcggac 900

aagctagtga catgggtaga gtcgcatgat acgtatgcca atgatgatga agagtcgaca 960

tggatgagcg atgatgatat ccgtttaggc tgggcggtga tagcttctcg ttcaggcagt 1020

acgcctcttt tcttttccag acctgaggga ggcggaaatg gtgtgaggtt cccggggaaa 1080

agccaaatag gcgatcgcgg gagtgcttta tttgaagatc aggctatcac tgcggtcaat 1140

agatttcaca atgtgatggc tggacagcct gaggaactct cgaacccgaa tggaaacaac 1200

cagatattta tgaatcagcg cggctcacat ggcgttgtgc tggcaaatgc aggttcatcc 1260

tctgtctcta tcaatacggc aacaaaattg cctgatggca ggtatgacaa taaagctgga 1320

gcgggttcat ttcaagtgaa cgatggtaaa ctgacaggca cgatcaatgc caggtctgta 1380

gctgtgcttt atcctgatga tattgcaaaa gcgcctcatg ttttccttga gaattacaaa 1440

acaggtgtaa cacattcttt caatgatcaa ctgacgatta ccttgcgtgc agatgcgaat 1500

acaacaaaag ccgtttatca aatcaataat ggaccagaga cggcgtttaa ggatggagat 1560

caattcacaa tcggaaaagg agatccattt ggcaaaacat acaccatcat gttaaaagga 1620

acgaacagtg atggtgtaac gaggaccgag aaatacagtt ttgttaaaag agatccagcg 1680

tcggccaaaa ccatcggcta tcaaaatccg aatcattgga gccaggtaaa tgcttatatc 1740

tataaacatg atgggagccg agtaattgaa ttgaccggat cttggcctgg aaaaccaatg 1800

actaaaaatg cagacggaat ttacacgctg acgctgcctg cggacacgga tacaaccaac 1860

gcaaaagtga tttttaataa tggcagcgcc caagtgcccg gtcagaatca gcctggcttt 1920

gattacgtgc taaatggttt atataatgac tcgggcttaa gcggttctct tccccattga 1980

<210> 22

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P17

<400> 22

gattacaagg atgatgacga taagatgttt gcaaaacgat tcaaaacctc tttactgcc 59

<210> 23

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P18

<400> 23

cttatcgtca tcatccttgt aatctcaatg gggaagagaa ccgcttaagc ccg 53

<210> 24

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P19

<400> 24

gaacgaccaa ttccatgcat gaagaatggt tccgc 35

Claims

1. A corynebacterium glutamicum protein Ncgl0717, characterized in that: the amino acid sequence is shown as SEQ ID NO. 1.

2. A gene coding for the protein Ncgl0717 of corynebacterium glutamicum of claim 1 characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.

3. Use of the corynebacterium glutamicum protein Ncgl0717 as described in claim 1 as an anchor protein in a surface display system characterized in that: the surface display system is a Corynebacterium glutamicum cell surface display system.

4. A corynebacterium glutamicum cell surface display system, comprising: a method for producing a protein of Corynebacterium glutamicum comprising immobilizing a target protein on the cell surface of Corynebacterium glutamicum, wherein the protein is Ncgl0717 of the wall protein of Corynebacterium glutamicum as defined in claim 1.

5. The C.glutamicum cell surface display system of claim 4, wherein: the target protein is any one of fluorescent protein or amylase.

6. The C.glutamicum cell surface display system of claim 5, wherein: the target protein is enhanced green fluorescent protein, red fluorescent protein or alpha-amylase.

7. The method for constructing a C.glutamicum cell surface display system of any one of claims 4 to 6, comprising the steps of:

(1) cloning a gene encoding the corynebacterium glutamicum protein Ncgl0717 of claim 1 into an expression cassette of an expression vector to obtain a surface display expression vector with the corynebacterium glutamicum wall protein Ncgl0717 as an anchor protein;

8. The method for constructing a cell surface display system of Corynebacterium glutamicum as set forth in claim 7, comprising the steps of:

(i) constructing a recombinant plasmid by homologous recombination of a gene encoding the Corynebacterium glutamicum protein Ncgl0717 of claim 1, a gene encoding a target protein and an expression vector;

(ii) (ii) transforming the recombinant plasmid obtained in the step (i) into corynebacterium glutamicum, and selecting a positive transformant to obtain a corynebacterium glutamicum cell surface display system;

(ii) the target protein in step (i) is any one of fluorescent protein or amylase; further enhancing green fluorescent protein, red fluorescent protein or alpha-amylase;

the nucleotide sequence of the enhanced green fluorescent protein is shown as SEQ ID NO. 5;

the nucleotide sequence of the red fluorescent protein is shown as SEQ ID NO. 17;

the nucleotide sequence of the alpha-amylase is shown as SEQ ID NO. 21;

(ii) the expression vector in step (i) is a corynebacterium glutamicum expression vector with kanamycin resistance; further is a Corynebacterium glutamicum expression vector pEC-XK99 e.

9. Use of the corynebacterium glutamicum protein Ncgl0717 of claim 1, a gene encoding the corynebacterium glutamicum protein Ncgl0717 of claim 1 or a corynebacterium glutamicum cell surface display system of any of claims 4-6 in the preparation of an amylase.

10. Use according to claim 9, characterized in that: the amylase is alpha-amylase.