A s-layer protein-based corynebacterium glutamicum surface display system, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biology and specifically relates to a Corynebacterium glutamicum surface display system based on S-layer protein as well as a preparation method and application thereof. The system uses 134 amino acids at the C-terminal of PS2 protein as an anchor sequence, and displays a target protein on the cell surface in an N-terminal fusion manner through a (GGGGS)3 connecting peptide. The construction method comprises the following steps: cloning the S-layer protein gene into a pH36mob vector, replacing the coding region but retaining the 5'UTR, signal peptide and anchor sequence, and then transforming Corynebacterium glutamicum to obtain a recombinant strain. Experiments show that the display efficiency of the system is much higher than that of the traditional PorB anchor protein, and the whole-cell catalytic lipase activity is 2.47 times that of the PorB system. The system has the advantages of high expression, small host interference and flexible display mode, enriches the Corynebacterium glutamicum surface display toolbox, and is suitable for fields such as whole-cell catalysis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Corynebacterium glutamicum surface display system based on S-layer protein, its preparation method, and its application. Background Technology

[0002] Microbial cell surface display is an important biotechnology that utilizes cell surface anchoring proteins to express and anchor exogenous target proteins (such as enzymes, antibodies, fluorescent proteins, etc.) on the surface of microbial cells. By fusing the target protein with the anchoring protein through genetic engineering, the target protein can be expressed on the cell surface along with the anchoring protein. This technology has broad application prospects in whole-cell catalysis, vaccine development, biosensors, and environmental remediation. In particular, in the field of biocatalysis, displaying enzymes on the cell surface for whole-cell catalysis effectively avoids the limitations of transmembrane transport of substrates and products, simplifies enzyme isolation and purification processes, and improves enzyme stability and reusability, making it an ideal whole-cell catalysis method.

[0003] Corynebacterium glutamicum ( Corynebacterium glutamicum Corynebacterium glutamicum is a key strain in the industrial production of important chemicals such as amino acids and organic acids. It possesses advantages such as robustness, high-density fermentation capability, simple culture conditions, and low extracellular protease content, making it an ideal host for cell surface display. However, compared to model microorganisms like Escherichia coli and yeast, research on cell surface display in Corynebacterium glutamicum is relatively limited. This is manifested in two aspects: First, the types and number of anchoring proteins available are limited. Currently reported anchoring proteins used for cell surface display mainly include exogenous proteins (such as PgsA), mycobacterial laminin (including PorB, PorH, and other proteins such as Ncgl1337), and membrane proteins such as Ncgl1221 (glutamate secretion channel protein). Among these, PgsA, Ncgl1337 and its truncated fragment (Ncgl1337S), and PorB are the more commonly used anchoring proteins. In addition to these traditional anchoring proteins, three Chinese patents with application numbers CN202110941689.8, CN202110941639.X, and CN202110945748.9 disclose three anchoring proteins discovered in Corynebacterium glutamicum: Ncgl2775, Ncgl1307, and Ncgl0717, further enriching the types of anchoring proteins.

[0004] However, existing methods for displaying anchored proteins on the cell surface still have some limitations: most anchored proteins are located in the cell membrane or cell coat, with limited expression levels, thus restricting the amount of target protein displayed; overexpression of these anchored proteins located in the cell membrane or cell coat can crowd out space for normal cell membrane / wall components, affecting the physiological functions of the host cell; currently reported display methods all involve fusing the target protein with the C-terminus of the anchored protein, which lacks flexibility. In particular, when the active site of the target protein is close to the fusion terminus, it may lead to steric hindrance and reduce enzyme activity. Therefore, developing novel, efficient anchoring elements and display systems with minimal impact on the host is a pressing technical problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a Corynebacterium glutamicum surface display system based on S-layer protein, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a Corynebacterium glutamicum cell surface display system using the S-layer protein as an anchor sequence. The system uses the 134 amino acids at the C-terminus of the Corynebacterium glutamicum S-layer protein as an anchor sequence, and fuses the target protein with the anchor sequence through N-terminal fusion to display it on the surface of Corynebacterium glutamicum cells. The amino acid sequence of the anchor sequence is shown in SEQ ID NO. 13. The N-terminal fusion refers to linking the anchor sequence to the target protein using a flexible linker peptide.

[0007] This invention utilizes the high abundance (10%–15% of total cellular protein) and self-assembly characteristics of the endogenous S-layer protein PS2. The C-terminus of PS2 is truncated to a 134-amino acid anchoring sequence, and an N-terminal fusion strategy is employed—i.e., the target protein is attached upstream of the anchoring sequence. This preserves the flexibility of the target protein's active site while avoiding the steric hindrance effects that can occur with traditional C-terminal fusion. Simultaneously, the system retains PS2's 5'UTR and signal peptide, ensuring efficient secretion and localization of the target protein. By constructing a recombinant plasmid, the target gene replaces the coding region of the S-layer protein, retaining only key regulatory elements and the anchoring region, achieving chimeric display of the target protein on the S-layer backbone. Experiments have confirmed that this system can successfully display fluorescent proteins and lipases, with the lipase catalytic efficiency significantly superior to the PorB control, demonstrating not only successful display but also good preservation of protein activity. This concept provides Corynebacterium glutamicum with a new, high-density, low-host-burden, and flexible N-terminal display strategy, expanding its application potential in industrial catalysis and biosensing.

[0008] Furthermore, the target protein includes a red fluorescent protein and a lipase, the nucleotide sequence of the gene encoding the red fluorescent protein is shown in SEQ ID NO.9, and the nucleotide sequence of the gene encoding the lipase is shown in SEQ ID NO.30.

[0009] Furthermore, the flexible linker peptide is (GGGGS)3.

[0010] This invention provides a method for constructing the Corynebacterium glutamicum cell surface display system, comprising the following steps: S1. The protein-coding gene of the S layer was ligated with the gene of the vector pH36mob by homologous recombination to obtain plasmid pH36-PS2. S2. The gene encoding the target protein is cloned into plasmid pH36-PS2 using homologous recombination, replacing the sequence of the S-layer protein encoding gene while retaining the 5'UTR, signal peptide, and anchoring sequence of the S-layer protein, thus obtaining the recombinant display plasmid. S3. The recombinant display plasmid is transformed into Corynebacterium glutamicum, positive transformants are screened, and recombinant strains are obtained after culturing. The recombinant strains express the display system on their surface.

[0011] Furthermore, the sequence of the S-layer protein-coding gene is shown in SEQ ID NO.3.

[0012] Furthermore, the nucleotide sequences of the S-layer protein 5'UTR and the signal peptide encoding gene are shown in SEQ ID NO.12 and SEQ ID NO.15, respectively.

[0013] Furthermore, the culture is carried out in LB medium supplemented with 1%–3% sodium lactate at 25–35°C and 100–300 rpm for 30–40 h. Furthermore, the conversion method is electroconversion.

[0014] This invention provides the application of the Corynebacterium glutamicum cell surface display system in the preparation of lipases.

[0015] The present invention has the following beneficial effects: This invention provides a method for displaying the S-layer protein of Corynebacterium glutamicum. The S-layer protein is an endogenous protein in Corynebacterium glutamicum, which not only has a high expression level but also can completely cover the cell surface. With the help of the 5'UTR, signal peptide and C-terminal anchoring sequence of the S-layer protein, the efficient secretion and display of the target protein can be ensured, which has a higher display efficiency than the conventional anchoring protein PorB.

[0016] Compared to existing display methods that all use C-terminal fusion of the target protein, the cell surface display system provided by this invention fuses the target protein with the C-terminal anchoring structure of the S-layer protein through N-terminal fusion to achieve cell surface fixation of the target protein, which can effectively make up for the lack of flexibility of existing display methods. Attached Figure Description

[0017] Figure 1 For PS2 C134 The mCherry expression vector plasmid map is displayed to show the anchored sequence.

[0018] Figure 2 For PS2 C134 This is an electrophoresis result of the S-layer protein expressing the anchored sequence, showing the red fluorescent protein. Lane 1 is the positive control for PorB expressing the anchored sequence, lane 2 is the negative control expressing wild-type S-layer protein, and lane 3 is the negative control expressing PS2. C134 Experimental group displaying red fluorescent protein for anchoring sequences.

[0019] Figure 3 For PS2 C134 Laser focusing microscopy results showing the anchor sequence for displaying red fluorescent protein; PS2 represents the negative control expressing wild-type S-layer protein, and PorB represents the positive control displaying mCherry using PorB as the anchor sequence. C134 Represented by PS2 C134 The experimental group showing the anchored sequence for mCherry.

[0020] Figure 4 For PS2 C134 The LpaA expression vector plasmid map is displayed to show the anchoring sequence.

[0021] Figure 5 For PS2 C134 The image shows the S-layer electrophoresis results of LpaA with the anchor sequence PorB. Lane 1 is the positive control for LpaA with PorB as the anchor sequence, and lane 2 is the positive control with PorB as the anchor sequence. C134 Lane 3 is the experimental group that displays the anchored sequence LpaA, while lane 3 is the negative control group that expresses wild-type S-layer protein.

[0022] Figure 6 For PS2 C134 Laser focusing microscopy results of LpaA as the anchor sequence are shown; PS2 represents expression of wild-type S-layer protein, and PorB represents a positive control displaying LpaA using PorB as the anchor sequence. C134 Represented by PS2 C134 LpaA is displayed for the anchor sequence.

[0023] Figure 7The image shows the whole-cell catalytic effect of recombinant Corynebacterium glutamicum for LpaA; PS2 represents the negative control expressing wild-type S-layer protein, and PorB represents the positive control expressing LpaA using PorB as the anchor sequence. C134 Represented by PS2 C134 LpaA is displayed for the anchor sequence. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0025] The LB medium consisted of 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl.

[0026] The LBHIS medium consisted of 2.5 g / L yeast extract, 5 g / L tryptone, 5 g / L sodium chloride, 18.5 g / L bovine brain and heart extract, 91 g / L sorbitol, and pH 7.0.

[0027] The LB sodium lactate medium consists of 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, and 20 g / L sodium lactate, with a pH of 7.0.

[0028] Unless otherwise specified, all corresponding plate culture media are prepared by adding 17 g / L agar powder to the liquid culture medium.

[0029] In this embodiment of the invention, *Corynebacterium glutamicum* S9114 was purchased from the Shanghai Institute of Industrial Microbiology; *Corynebacterium glutamicum* ATCC 13032 is a wild-type model bacterium, disclosed in the paper "Taxonomical Study of Glutamic Acid Accumulating Bacteria, *Micrococcus glutamicus* nov. sp."; *Escherichia coli* JM109 is disclosed in the paper "Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors". The pH36mob expression vector is disclosed in the paper "Engineering *Corynebacterium glutamicum* triggers glutamic acid accumulation in biotin-rich corn stover hydrolysate". The mCherry expression plasmid pBBR1MCS2-Tac-mCherry was purchased from Beina Chuanglian Biotechnology Co., Ltd., with the product number BNCC356416.

[0030] Example 1: Construction of S-layer protein expression plasmid pH36-PS2.

[0031] I. Cloning of the PS2 encoding gene of Corynebacterium glutamicum S layer protein.

[0032] Primers P1 and P2 were designed to amplify the complete S-layer protein gene sequence, including the 5' untranslated region, based on the sequence information of the S-layer protein encoding gene in Corynebacterium glutamicum S9114 and the characteristic sequence on the expression plasmid pH36mob. The nucleotide sequence of P1 is shown in SEQ ID NO.1, and the nucleotide sequence of P2 is shown in SEQ ID NO.2.

[0033] SEQ ID NO. 1: gtaggagtagcatgggatccaccatgaacgacaggctgctaagctgc.

[0034] SEQ ID NO. 2: ttctgatttaatctgtatcattagaacttaacgataccggagagg.

[0035] Using genomic DNA from Corynebacterium glutamicum S9114 as a template and primers P1 and P2, the complete S-layer protein gene sequence, including the 5' untranslated region, was amplified by PCR. The amplification conditions were: pre-denaturation at 98°C for 30 seconds; followed by up to 30 cycles of denaturation at 98°C for 10 seconds, annealing at 58°C for 5 seconds, extension at 72°C for 10 seconds, and a final extension at 72°C for 2 minutes. The PCR product was purified using a product purification kit. The S-layer protein gene sequence is shown in SEQ ID NO. 3.

[0036]

[0037] II. Cloning of plasmid vector pH36mob.

[0038] Based on the complete S-layer protein gene sequence of Corynebacterium glutamicum and the sequence characteristics on the Corynebacterium glutamicum expression plasmid vector pH36mob, amplification primers P3 and P4 were designed. The nucleotide sequence of P3 is shown in SEQ ID NO.4, and the nucleotide sequence of P4 is shown in SEQ ID NO.5.

[0039] SEQ ID NO.4: catggtggatcccatgctact.

[0040] SEQ ID NO. 5: tgatacagattaaatcagaacgcag.

[0041] Using pH36mob as a template and P3 and P4 as primers, the gene sequence of the vector pH36mob was amplified by PCR. The amplification conditions were: 98℃ pre-denaturation for 30 seconds; followed by up to 30 cycles: 98℃ denaturation for 10 seconds, 58℃ annealing for 5 seconds, 72℃ extension for 35 seconds; and a final extension at 72℃ for 2 minutes. The amplified nucleotide sequence of pH36mob is shown in SEQ ID NO. 6.

[0042]

[0043] III. Construction of plasmid pH36-PS2.

[0044] The obtained Corynebacterium glutamicum S-layer protein gene was ligated into the PCR product of the Corynebacterium glutamicum expression plasmid pH36mob using homologous recombination. Ligation was performed using the Seamless Assembly cloning kit. The ligation product was transformed into Escherichia coli JM109 and then plated on LB agar plates containing 50 mg / L kanamycin. Transformants were validated by colony PCR. Plasmids from positive transformants were extracted and sequenced for verification, indicating successful construction of plasmid pH36-PS2.

[0045] Example 2: Construction of an mCherry cell surface display system for C-terminal anchoring sequences of S-layer proteins.

[0046] I. Cloning of the target gene mCherry.

[0047] Based on the encoding gene of the target protein mCherry and the sequence of the expression plasmid pH36-PS2, amplification primers P5 and P6 were designed to amplify the mCherry gene containing a Flag tag at the 5' end and a (GGGGS)3 linker peptide at the 3' end. The nucleotide sequence of P5 is shown in SEQ ID NO.7, and the nucleotide sequence of P6 is shown in SEQ ID NO.8.

[0048] SEQ ID NO. 7: tagctatcccagcattcgctgattataaagatgacgatgacaaagtgagcaagggcgaggagg.

[0049] SEQ ID NO. 8: tgatccgccacctccagagccacctccgcctgaaccgcctccacctgacttgtacagctcgtccatgcc.

[0050] The amplification conditions were as follows: pre-denaturation at 98°C for 30 seconds; followed by up to 30 cycles: denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 10 seconds; and a final extension at 72°C for 2 minutes. The nucleotide sequence of the mCherry gene is shown in SEQ ID NO. 9.

[0051] SEQ ID NO.9:

[0052] II. Cloning of plasmid vectors displayed on cell surface.

[0053] Based on the sequence characteristics of the expression plasmid pH36-PS2 and the encoding gene of the target protein mCherry, amplification primers P7 and P8 were designed. The nucleotide sequence of P7 is shown in SEQ ID NO.10, and the nucleotide sequence of P8 is shown in SEQ ID NO.11.

[0054] SEQ ID NO. 10: ctctggaggtggcggatcagaggacgcagcttacgctg.

[0055] SEQ ID NO. 11: agcgaatgctgggatagctacg.

[0056] Using pH36-PS2 plasmid as a template and P7 and P8 primers, an expression plasmid vector retaining the 5'UTR, signal peptide, and 134 amino acid anchoring sequence at the C-terminus of the S-layer protein was amplified by PCR. The amplification conditions were: 98℃ pre-denaturation for 30 seconds; followed by up to 30 cycles: 98℃ denaturation for 10 seconds, 60℃ annealing for 5 seconds, 72℃ extension for 35 seconds; and a final extension at 72℃ for 2 minutes.

[0057] The nucleotide sequence of the 5'UTR is shown in SEQ ID NO.12.

[0058] SEQ ID NO. 12: AACGACAGGCTGCTAAGCTGCTAGTTCGGTGGCCTAGTGAGTGGCGTTTACTTGAATGAAAAGTAATCCCATGTCGTGATCAGCCAATTTGGGTTGTGTCAAAGCAATTCAAAGGTTTCATCTTTCGATATCCTATTCAAGGAGACCCTCGCCTCT.

[0059] The 134 amino acid coding sequence is shown in SEQ ID NO.13.

[0060] SEQ ID NO. 13: EDAAYAEEKRQFALLTYAKVFINGIWQEEVKYVQNLDEGARAEAARIEAERLADEAYRAEQLRIAQEAADAQKAIAEALAKEAENGNNNGGDNSSDDKETGSSDIGSWGPFAAIAAIIAAIAAIFPFLSGIVKF.

[0061] The nucleotide sequence of the 134 amino acids is shown in SEQ ID NO.14.

[0062] SEQ ID NO.14: GAGGACGCAGCTTACGCTGAGGAGAAGCGCCAGTTCGCTCTCCTGACCTACGCAAAGGTCTTCATCAATGGTATCTGGCAGGAAGAAGTCAAGTACGTCCAGAACCTGGATGAAGGAGCTCGCGCAGAAGCAGCTCGCATCGAGGCAGAGCGTCTTGCAGACGAGGCATACCGCGCTGAGCAGCTCCGCATCGCTCAGGA AGCAGCTGACGCTCAGAAGGCAATCGCTGAGGCTCTTGCTAAGGAAGCAGAGAACGGCAACAACAACGGTGGCGACAACTCCTCCGACGACAAGGAGACCGGTTCTTCTGACATCGGATCCTGGGGACCTTTCGCAGCAATTGCAGCTATCATCGCAGCCATTGCAGCTATCTTCCCATTCCTCTCCGGTATCGTTAAGTTCTAA.

[0063] The nucleotide sequence of the signal peptide is shown in SEQ ID NO.15.

[0064] SEQ ID NO. 15: ATGTTTAACAACCGTATCCGCACTGCAGCTCTCGCTGGTGCAATCGCAATCTCCACCGCAGCTTCTGGCGTAGCTATCCCAGCATTCGCT.

[0065] The nucleotide sequence of the expression plasmid vector is shown in SEQ ID NO.16.

[0066]

[0067] III. Displaying plasmid pH36-PS2sp-Flag-mCherry-PS2 C134 The construction.

[0068] The obtained red fluorescent protein mCherry gene was ligated into the obtained expression plasmid vector via homologous recombination using a Seamless Assembly cloning kit. The ligation product was transformed into *E. coli* JM109 and then plated on LB agar plates containing 50 mg / L kanamycin. Transformants were validated by colony PCR, and plasmids from positive transformants were extracted and sequenced for verification. The results indicated successful construction of the expression plasmid vector. Figure 1 The pH36-PS2sp-Flag-mCherry-PS2 shown is shown. C134 Plasmid.

[0069] IV. Construction and identification of the recombinant Corynebacterium glutamicum surface display system CG-Flag-mCherry-C134.

[0070] The plasmid pH36-PS2sp-Flag-mCherry-PS2 was converted using the Corynebacterium glutamicum electroporation method. C134 Corynebacterium glutamicum ATCC 13032 was transformed. Positive transformants were picked from LBHIS plates supplemented with 50 mg / L kanamycin for PCR identification. Positive transformants with band sizes meeting the requirements were considered to have successfully transformed the plasmid pH36-PS2sp-Flag-mCherry-PS2. C134 The recombinant strain was transformed into Corynebacterium glutamicum ATCC 13032 and named CG-Flag-mCherry-C134.

[0071] V. Construction and identification of negative control strain CG-PS2.

[0072] The S-layer protein expression plasmid pH36-PS2 was transformed into Corynebacterium glutamicum ATCC 13032 by electroporation. Positive transformants were picked from LBHIS plates supplemented with 50 mg / L kanamycin and identified by colony PCR amplification. Positive transformants with matching electrophoretic band sizes were considered recombinant bacteria that had been successfully transformed from pH36-PS2 into ATCC 13032, and the corresponding negative control recombinant bacteria CG-PS2 were obtained.

[0073] VI. Construction and identification of the positive control bacterium CG-PorB-Flag-mCherry.

[0074] The amplification method for the pH36mob expression plasmid vector is the same as in Example 1. The gene sequence of the vector pH36mob (SEQ ID NO.6) was amplified by PCR.

[0075] Using genomic DNA of Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primers P9 and P10 to obtain the PorB anchoring protein encoding gene containing a (GGGGS)3 linker peptide at the 3' end. The amplification conditions were: 98℃ pre-denaturation for 30 seconds; followed by up to 30 cycles: 98℃ denaturation for 10 seconds, 56℃ annealing for 5 seconds, 72℃ extension for 10 seconds; and a final extension at 72℃ for 2 minutes. The nucleotide sequence of the PorB anchoring protein encoding gene is shown in SEQ ID NO. 17. The nucleotide sequence of P9 is shown in SEQ ID NO. 18, and the nucleotide sequence of P10 is shown in SEQ ID NO. 19.

[0076] SEQ ID NO.17: ATGAAGCTTTCAACCGCATCGCAGCAATGGCAGCAACCGCAGGCATCACAGTGGCAGCATTCGCAGCACCTGCTTCCGCATCCGACTTCGCAAACCTATCCTCCACCAACAAAGAACTGTCCCCTCAGTACAACTGGGTTGCTTGCGGCATCCTTGAGGGTGGCCTCAAAGCAGCTGGCGTCCCTT GAAGAAGGCCAGTACAACCGCGAGCTCGCAGAAGCAATCGCCGCAAAGGGTGAAGGCTTCTGGACCACTCAGTTCCCACAAATCGGTGATTGGAACGAAGATCAGGCAGCAGCACTTGCCGACCGCGCACAAACCTGTGGCCTAGTTAAGGCTGACACCTACCTTTCTGAGCTGTCCTCCAACTTCTCTTCC.

[0077] SEQ ID NO. 18: gtagcatgggatccaccatgatgaagctttcacaccgc.

[0078] SEQ ID NO. 19: tgatccgccacctccagagccacctccgcctgaaccgcctccacctgaggaagagaagttggaggacag.

[0079] Using the mCherry expression plasmid pBBR1MCS2-Tac-mCherry as a template, PCR amplification was performed using primers P11 and P12 to obtain the mCherry gene containing a Flag tag at its 5' end. The amplification conditions were: 98℃ pre-denaturation for 30 seconds; followed by up to 30 cycles: 98℃ denaturation for 10 seconds, 60℃ annealing for 5 seconds, 72℃ extension for 10 seconds; and a final extension at 72℃ for 2 minutes. The nucleotide sequence of the mCherry gene containing the Flag tag at its 5' end is shown in SEQ ID NO. 20. The nucleotide sequence of P11 is shown in SEQ ID NO. 21. The nucleotide sequence of P12 is shown in SEQ ID NO. 22.

[0080] SEQ ID NO.20:GATTATAAAGATGACGATGACAAAGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA。

[0081] SEQ ID NO.21:gctctggaggtggcggatcagattataaagatgacgatgacaaagtgagcaagggcgaggagg。

[0082] SEQ ID NO.22:ttctgatttaatctgtatcattacttgtacagctcgtccatgcc。

[0083] The three PCR products obtained above were ligated using homologous recombination using the Seamless Assembly cloning kit. The ligation products were transformed into *E. coli* JM109 and then plated on LB agar plates containing 50 mg / L kanamycin. Transformants were validated by colony PCR. Plasmids from positive transformants were extracted and sequenced, confirming successful construction of the pH36-PorB-Flag-mCherry plasmid. The obtained pH36-PorB-Flag-mCherry plasmid was transformed into *Corynebacterium glutamicum* ATCC 13032 via electroporation. Positive transformants were picked from LBHIS plates supplemented with 50 mg / L kanamycin for colony PCR amplification. Positive transformants with matching electrophoretic band sizes were considered successful recombinant bacteria transformed with pH36-PorB-Flag-mCherry into ATCC 13032, yielding the corresponding negative control recombinant bacteria CG-PorB-Flag-mCherry.

[0084] VII. SDS-PAGE analysis of S-layer proteins extracted from recombinant bacteria.

[0085] The negative control recombinant strain CG-PS2, the positive control strain CG-PorB-Flag-mCherry, and the experimental group recombinant strain CG-Flag-mCherry-C134 were cultured as follows: A single colony was picked and inoculated into 5 mL of LB liquid medium containing kanamycin (final concentration 50 mg / L), and cultured overnight at 30℃ and 200 rpm for 12 h with shaking, as the seed culture. 600 μL of the seed culture was then inoculated into 30 mL of LB medium containing kanamycin (final concentration 50 mg / L) and supplemented with 2% sodium lactate, and cultured in the same constant-temperature shaker for 36 h with shaking. Every 8 h, the pH of the medium was adjusted to 7.0 with 2 mol / L HCl or NaOH. After the culture was completed, the bacterial culture was placed in an ice bath for 10 min, centrifuged at 4℃ and 12000 rpm for 10 min, and the supernatant was completely discarded. The bacterial suspension was resuspended in an equal volume of pre-chilled 1×PBS solution. The absorbance (OD600) of the bacterial suspension was measured at 600 nm after appropriate dilution. The OD600 value of the bacterial suspension was calculated based on the measured OD600 value. A portion of the bacterial suspension with an OD600 value of 10 was then placed in a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 10 min at 4 °C. After discarding the supernatant, 250 μL of Tris-HCl buffer (pH 6.8, 60 mmol / L) containing 2% SDS was added to the precipitate. The precipitate was thoroughly resuspended by vortexing and then boiled in a 100 °C water bath for 5 min to fully dissolve and denature the S-layer protein. After cooling to room temperature, the sample was centrifuged again at 12000 rpm for 10 min. The supernatant was collected as the extracted S-layer protein sample and used for SDS-PAGE electrophoresis.

[0086] SDS-PAGE electrophoresis results are as follows: Figure 2 As shown, the positive control PorB (lane 1) did not detect S-layer protein because it is not an S-layer protein. In contrast, the negative control CG-PS2 clearly showed a single band of S-layer protein (lane 2). The experimental group's recombinant bacterium CG-Flag-mCherry-C134 also showed a protein band of the same size as Flag-mCherry-C134 (lane 3), indicating that the C134 anchoring sequence can display Flag-mCherry on the cell surface.

[0087] 8. Laser confocal analysis of recombinant bacteria.

[0088] After culturing recombinant Corynebacterium glutamicum displaying the target protein, a portion of the fermentation broth was collected and centrifuged at 5000 rpm for 10 min, then resuspended in 1×PBS solution. The OD600 was measured, and the cells were appropriately diluted to adjust the OD600 value to 0.2. The adjusted bacterial suspension was centrifuged at 5000 rpm for 10 min, washed twice with an equal volume of 1×PBS solution, and the cells were resuspended in 0.2 mL of 1×PBS solution containing 1% BSA (blocking solution) and incubated at room temperature for 1 h for blocking. Then, an excess of 1 μL of mouse anti-Flag primary antibody (Flag Tag Mouse Monoclonal Antibody, Beyotime, AF2852-50μ) solution was added directly to the 0.2 mL solution, and the cells were incubated at room temperature for 2 h, gently agitated to maintain bacterial suspension. The cells were centrifuged at 2000 rpm for 5 min, and then washed three times with an equal volume of 1×PBS to thoroughly remove unbound primary antibody. Secondary antibody was administered using Alexa Fluor 488-labeled goat anti-mouse IgG antibody (AF488-labeled goat anti-mouse IgG (H+L), Beyotime, A0428). 1 μL of Alexa Fluor 488-labeled goat anti-mouse IgG antibody was added to the bacterial suspension after incubation and washing with primary antibody, and the suspension was incubated at room temperature for 1 h, gently agitated to maintain cell suspension. The cells were centrifuged at 2000 rpm for 5 min, washed three times with an equal volume of 1×PBS, and then resuspended with an equal volume of 1×PBS. The bacterial suspension was diluted to a cell concentration of 0.5 × 10⁻⁶ cells / mL. 6The cell density should be measured in cells / mL for microscopic observation, avoiding overly dense cell concentrations that could affect the observation results. Heat 10 mL of 0.7% agarose in a constant-temperature water bath until completely melted to fix the cells; keep warm for later use. Take 10 μL of the above bacterial suspension and quickly mix it with 20 μL of melted 0.7% agarose, then immediately drop it onto the center of a sterile glass slide. Slowly cover with a confocal coverslip, avoiding air bubbles, and gently wipe away excess liquid around the coverslip with lint-free paper. Let it stand at room temperature in the dark for 10 minutes until the agarose solidifies, completing the slide preparation. The prepared sample can be stored for a short period at 4°C in the dark. Then, laser confocal imaging observation was performed: First, the microscope room environment was confirmed to be 24℃ with moderate humidity. Strong light was turned off, and the environment was kept dark to reduce fluorescence interference. Next, the instrument status was checked: the laser module, stage, and objective lens were confirmed (a 63× oil immersion lens was preferred for its high resolution suitability for bacterial detection). Then, the sample was placed and the objective lens was adjusted: the objective lens was switched to 10× low magnification for quick field of view positioning. Finally, one drop of cedarwood oil was added to the 63× oil immersion lens, ensuring the drop was uniform and free of air bubbles. The prepared coverslip was placed face down on the microscope stage and secured firmly to prevent movement. The field of view was adjusted to a suitable position, and the excitation wavelength was set to 488nm and the emission wavelength to 520nm for sample observation. If red fluorescence of mCherry needs to be detected, a 560-585nm laser was used for excitation, and a 610-645nm bandpass filter was used for detection.

[0089] The results of laser confocal microscopy observation are as follows Figure 3 As shown, no red fluorescence (fluorescence emitted by mCherry) was detected in the negative control, while red fluorescence was detected in both the positive control and experimental groups, indicating that mCherry in both groups was successfully expressed. Since the negative controls did not contain the Flag tag, no green fluorescence (fluorescence emitted by Alexa Fluor488) was detected, ruling out interference from non-specific binding. Green fluorescence was detected in both the positive control and experimental groups, indicating that Flag-tagged mCherry was successfully displayed on the cell surface. The green fluorescence intensity was comparable between the positive control and experimental groups, indicating that C134, as an anchoring protein, can achieve display effects comparable to PorB.

[0090] Example 3: Construction of a cell surface display system for LpaA lipase with C-terminal anchoring sequence of S-layer protein.

[0091] I. Cloning of the target gene LpaA.

[0092] Based on the coding gene sequence of the target protein LpaA and the sequence characteristics of the expression plasmid pH36-PS2, amplification primers P13 and P14 were designed. The nucleotide sequence of P13 is shown in SEQ ID NO.23, and the nucleotide sequence of P14 is shown in SEQ ID NO.24.

[0093] SEQ ID NO. 23: tagctatcccagcattcgctgattataaagatgacgatgacaaagctgaacacaatccagtcgtt.

[0094] SEQ ID NO. 24: tgatccgccacctccagagccacctccgcctgaaccgcctccacctgaattcgtattctggccccc.

[0095] Using the Bacillus subtilis B168 genome as a template and primers P13 and P14, the LpaA gene, which contains a Flag tag at the 5' end and a (GGGGS)3 linker peptide coding sequence at the 3' end, was amplified by PCR. The amplification conditions were: 98℃ pre-denaturation for 30 seconds; followed by up to 30 cycles: 98℃ denaturation for 10 seconds, 60℃ annealing for 5 seconds, 72℃ extension for 10 seconds; and a final extension at 72℃ for 2 minutes. The nucleotide sequence of the LpaA gene is shown in SEQ ID NO. 25.

[0096] SEQ ID NO.25:

[0097] II. Demonstration of the construction of plasmid pH36-PS2sp-Flag-mCherry-PS2C134.

[0098] The obtained LpaA PCR product was ligated to the expression plasmid vector amplification product (SEQ ID NO. 12) obtained in Example 2 using homologous recombination ligation. The ligation product was transformed into *E. coli* JM109 and then plated on LB agar plates containing 50 mg / L kanamycin. Transformants were validated by colony PCR, and plasmids were extracted from positive transformants for sequencing verification. The results indicated successful construction of the expression plasmid vector. Figure 4 The pH36-PS2sp-Flag-LpaA-PS2 shown is shown. C134 Plasmid.

[0099] III. Construction and identification of the recombinant Corynebacterium glutamicum surface display system CG-Flag-LpaA-C134.

[0100] The plasmid pH36-PS2sp-Flag-LpaA-PS2 was converted using the Corynebacterium glutamicum electroporation method. C134 Corynebacterium glutamicum ATCC 13032 was transformed. Positive transformants were picked from LBHIS plates supplemented with 50 mg / L kanamycin for PCR identification. Positive transformants with band sizes meeting the requirements were considered to have successfully transformed the plasmid pH36-PS2sp-Flag-LpaA-PS2. C134 The recombinant strain was transformed into Corynebacterium glutamicum ATCC 13032 and named CG-Flag-LpaA-C134.

[0101] IV. Construction and identification of positive control strain CG-PorB-Flag-LpaA.

[0102] Using P4 and P15 as primers and pH36-PorB-Flag-mCherry as a template, PCR amplification was performed to obtain the expression plasmid backbone. The amplification conditions were: 98℃ pre-denaturation for 30 seconds; followed by up to 30 cycles: 98℃ denaturation for 10 seconds, 58℃ annealing for 5 seconds, 72℃ extension for 10 seconds; and a final extension at 72℃ for 2 minutes. The nucleotide sequence of P15 is shown in SEQ ID NO.26.

[0103] SEQ ID NO. 26: tgatccgccacctccagag.

[0104] The nucleotide sequence of the expression plasmid backbone is shown in SEQ ID NO.27.

[0105]

[0106] Using the Bacillus subtilis 168 genome as a template and primers P16 and P17, PCR amplification was performed to obtain the LpaA encoding gene product containing a Flag tag at the 5' end. The amplification conditions were: 98℃ pre-denaturation for 30 seconds; followed by up to 30 cycles: 98℃ denaturation for 10 seconds, 58℃ annealing for 5 seconds, 72℃ extension for 10 seconds; and a final extension at 72℃ for 2 minutes. The nucleotide sequence of P16 is shown in SEQ ID NO.28, and the nucleotide sequence of P17 is shown in SEQ ID NO.29.

[0107] SEQ ID NO. 28: gctctggaggtggcggatcagattataaagatgacgatgacaaagctgaacacaatccagtcgtt.

[0108] SEQ ID NO. 29: ttctgatttaatctgtatcattaattcgtattctggcccccg.

[0109] The nucleotide sequence of the LpaA encoding gene containing a Flag tag at its 5' end is shown in SEQ ID NO.30.

[0110] SEQ ID NO.30:

[0111] The two PCR products obtained above were ligated for homologous recombination using the Seamless Assembly cloning kit. The ligation products were transformed into *E. coli* JM109 and then plated on LB agar plates containing 50 mg / L kanamycin. Transformants were validated by colony PCR. Plasmids from positive transformants were extracted and sequenced, confirming successful construction of the pH36-PorB-Flag-LpaA plasmid. The obtained pH36-PorB-Flag-LpaA plasmid was transformed into *Corynebacterium glutamicum* ATCC 13032 via electroporation. Positive transformants were picked from LBHIS plates supplemented with 50 mg / L kanamycin and identified by colony PCR amplification. Positive transformants with matching electrophoretic band sizes were considered successful recombinant bacteria transformed with pH36-PorB-Flag-LpaA into ATCC 13032, yielding the corresponding positive control recombinant bacteria CG-PorB-Flag-LpaA.

[0112] V. SDS-PAGE analysis of S-layer proteins extracted from recombinant bacteria.

[0113] The negative control CG-PS2, the positive control CG-PorB-Flag-LpaA, and the experimental group CG-Flag-LpaA-C134 recombinant bacteria were cultured according to the method in Example 2, and the S-layer protein was extracted using the same method and then detected by SDS-PAGE electrophoresis.

[0114] SDS-PAGE electrophoresis results are as follows: Figure 5 As shown, the positive control CG-PorB-Flag-LpaA did not express the S-layer protein, so there was no band (lane 1). The negative control CG-PS2 clearly showed the S-layer protein band, and the band was single (lane 3). The experimental group recombinant bacteria CG-Flag-LpaA-C134 also showed a protein band of the same size as Flag-LpaA-C134 (lane 2), indicating that the C134 anchoring sequence can display Flag-LpaA on the cell surface.

[0115] VI. Laser confocal analysis of recombinant bacteria.

[0116] The recombinant bacteria were observed using a laser confocal microscope according to the method described in Example 2. The observation results are as follows: Figure 6 As shown, the negative controls, lacking the Flag tag, did not exhibit any green fluorescence (fluorescence emitted by Alexa Fluor 488), thus ruling out interference from non-specific binding. Both the positive control and experimental groups showed detectable green fluorescence, indicating that the Flag-tagged LpaA was successfully displayed on the cell surface. The comparable green fluorescence intensity between the positive control and experimental groups suggests that C134, as an anchoring protein, achieves display performance comparable to PorB.

[0117] VII. Verification of the whole-cell catalytic effect of LpaA on cell surface.

[0118] First, prepare the substrate solution for the whole-cell catalytic reaction—a 0.1 mol / L Tris-HCl buffer (pH 7.5) containing 2 mmol / L p-nitrophenol palmitate (p-NPP). Weigh 37.75 mg of p-NPP substrate, add 5 mL of isopropanol and 0.5 mL of Triton X-100 to aid dissolution, and sonicate in an ultrasonic cleaner for 10 min until the p-NPP is completely dissolved. Then, add 0.1 mol / L Tris-HCl buffer to a final volume of 50 mL to obtain the substrate solution. Take 1 mL of cell sample with OD600=10, centrifuge at 5000 rpm for 10 min at 4℃, discard the supernatant, resuspend in an equal volume of 0.1 mol / L Tris-HCl buffer (pH 7.5), then add 1 mL of the solution, mix well, and incubate in a 37℃ water bath for 30 min. Immediately after the reaction, add 0.3 mL of 0.5 mol / L NaOH solution to terminate the reaction. Then, centrifuge the sample at 12000 rpm for 10 min, collect the supernatant, and measure the absorbance at 405 nm using a spectrophotometer. Use 0.1 mol / L Tris-HCl buffer (pH 7.5) containing different concentrations of p-nitrophenol as standard solutions to measure the absorbance at 405 nm and plot a standard curve. Then, calculate the amount of product produced based on the standard curve and the absorbance measured from the whole-cell catalytic supernatant. The results obtained after whole-cell catalysis are as follows: Figure 7 As shown.

[0119] As can be seen from the figure, the negative control produced almost no p-nitrophenol, the positive control produced 0.32±0.03 mg / L of p-nitrophenol, while the experimental group produced 0.79±0.01 mg / L of p-nitrophenol, which was 2.47 times that of the positive control group. Therefore, it can be seen that using PS2... C134 Compared to traditional positive controls, anchor sequences can achieve better catalytic effects.

[0120] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0121] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0122] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A Corynebacterium glutamicum cell surface display system using S-layer protein as an anchor sequence, characterized in that, Using the C-terminal 134 amino acids of the S-layer protein of Corynebacterium glutamicum as the anchoring sequence, the target protein was fused with the anchoring sequence via N-terminal fusion and displayed on the cell surface of Corynebacterium glutamicum. The amino acid sequence of the anchoring sequence is shown in SEQ ID NO.

13. The N-terminal fusion refers to linking the anchor sequence to the target protein using a flexible linker peptide.

2. The Corynebacterium glutamicum cell surface display system according to claim 1, characterized in that, The target protein includes a red fluorescent protein and a lipase. The nucleotide sequence of the gene encoding the red fluorescent protein is shown in SEQ ID NO.9, and the nucleotide sequence of the gene encoding the lipase is shown in SEQ ID NO.

30.

3. The Corynebacterium glutamicum cell surface display system according to claim 2, characterized in that, The flexible linker peptide is (GGGGS)3.

4. The method for constructing the Corynebacterium glutamicum cell surface display system according to claim 1, characterized in that, Includes the following steps: S1. The protein-coding gene of the S layer was ligated with the gene of the vector pH36mob by homologous recombination to obtain plasmid pH36-PS2. S2. The gene encoding the target protein is cloned into plasmid pH36-PS2 using homologous recombination, replacing the sequence of the S-layer protein encoding gene while retaining the 5'UTR, signal peptide, and anchoring sequence of the S-layer protein, thus obtaining the recombinant display plasmid. S3. The recombinant display plasmid is transformed into Corynebacterium glutamicum, positive transformants are screened, and recombinant strains are obtained after culturing. The recombinant strains express the display system on their surface.

5. The construction method according to claim 4, characterized in that, The sequence of the S-layer protein encoding gene is shown in SEQ ID NO.

3.

6. The construction method according to claim 4, characterized in that, The nucleotide sequences of the S-layer protein 5'UTR and the signal peptide encoding gene are shown in SEQ ID NO.12 and SEQ ID NO.15, respectively.

7. The construction method according to claim 4, characterized in that, The culture was carried out in LB medium supplemented with 1% to 3% sodium lactate, at 25 to 35°C and 100 to 300 rpm for 30 to 40 hours.

8. The construction method according to claim 4, characterized in that, The conversion method is electroconversion.

9. The application of the Corynebacterium glutamicum cell surface display system of claim 3 in the preparation of lipase.

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