A FLS spore surface display system based on T7 expression system and application thereof

Abstract

The present application relates to a kind of FLS spore surface display system based on T7 expression system and its application, belong to the field of biotechnology.The present application constructs a kind of spore surface display system based on T7 RNA polymerase, realizes the efficient anchoring and stable expression of target protein on spore surface by screening anchor protein and copy number.In addition, in order to improve the preparation efficiency of spore catalyst, the present application also uses dehydrated tetracycline inducible promoter Ptet to express KinA gene and its variant KinA-q-pas-A, under the premise that sporulation time is shortened by half, it promotes spore production to increase by 3.69 times.The system is used for ligustrazine and its precursor pinacol production, realizes the further improvement of pinacol and ligustrazine yield.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an FLS spore surface display system based on the T7 expression system and its application. Background Technology

[0002] Microbial surface display technology, as a targeted anchoring technique, enables the localization and exposure of functional proteins on the surface of microorganisms through the specific interaction between functional proteins and natural surface proteins, thereby effectively increasing the accessibility of target proteins. Among them, the Bacterial Spore Surface Display System (BSSDS) based on Bacillus subtilis utilizes spore capsid proteins and other elements as anchoring components. During the induction of spore formation, the fused target protein is assembled onto the spore shell; as the mother cell lyses and mature spores are released, the fused target protein is stably presented on the spore surface.

[0003] Despite the unique application potential of spore-based surface display systems, several technical bottlenecks remain in practical applications, primarily concerning poor anchoring protein stability and low functional protein expression efficiency. Regarding anchoring protein stability, the inherent instability of the anchoring proteins directly impacts effective anchoring of functional proteins. While the spore coat's tolerance to extreme environments can enhance the functional stability of target proteins, rapid environmental changes during spore formation can lead to misfolding of anchoring proteins, preventing proper assembly of functional proteins onto the spore coat. Furthermore, some anchoring proteins may be specifically hydrolyzed by proteases secreted by the spore, resulting in the hydrolysis of the anchored protein or failure to achieve the anchoring process. To address this, it is necessary to select suitable anchoring proteins based on the protective properties of the spore coat, develop protease-deficient production strains, and explore anchoring elements resistant to protease hydrolysis to improve the stability of surface-displayed proteins. Regarding the limited expression levels of functional proteins, a key challenge is achieving efficient expression while maintaining spore integrity. The core of this challenge lies in developing promoters with suitable transcriptional strength that can initiate exogenous gene expression at specific stages of spore formation. Although IPTG-inducible promoters have been used to optimize the surface display of α-amylase, their effectiveness is limited by cytotoxicity caused by promoter leakage expression. Therefore, it is necessary to develop more robust inducible expression systems to balance sporulation integrity and efficient functional protein expression. Furthermore, the current main method for producing catalysts for sporulation display systems is through starvation in DSM medium, which suffers from long sporulation cycles, low sporulation efficiency, and large fluctuations in sporulation performance. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems of poor stability of anchored proteins and low expression efficiency of functional proteins in the existing microbial surface display technology.

[0005] To address the aforementioned technical problems, this invention provides an FLS spore surface display system based on the T7 expression system and its applications. This invention constructs a spore surface display system based on T7 RNA polymerase, integrating T7 RANA polymerase into the genome of Bacillus subtilis G600 to obtain the tool strain BSG600-T7L. This strain can effectively reduce the degradation of exogenous proteins by proteases. Subsequently, six capsid proteins—CotB, CotC, CotE, CotG, CotX, and CotY—were selected as anchoring elements to construct fusion expression cassettes containing reporter proteins. These fusion cassettes were then inserted into high-copy vectors (pSTOP-T7LS), medium-copy vectors (pADK-T7LS), low-copy vectors (pHT-T7LS), and single-copy genome integration expression systems. Spore surface display systems that significantly improve the expression of the target gene were then screened. In addition, to improve the efficiency of spore catalyst preparation, this invention also utilizes the dehydrated tetracycline-induced promoter Ptet to express the KinA gene and its variant KinA-q-pas-A, thereby inducing sporulation in Bacillus and constructing an artificially induced sporulation system. Applying this system to the production of tetramethylpyrazine and its precursor acetoin significantly increases the yield of both acetoin and tetramethylpyrazine.

[0006] The first objective of this invention is to provide an FLS spore surface display system based on the T7 expression system, the FLS spore surface display system comprising a driving module and an expression module:

[0007] The driving module includes a first expression box containing LacI repressor protein and a second expression box containing LacO operon and T7 RNA polymerase.

[0008] The target gene expression module includes a fusion protein expression frame containing an anchor protein and a target protein connected by a linker, and the expression of the fusion protein expression frame is driven by the T7 promoter.

[0009] The second expression cassette is located in the pADK plasmid, pHT plasmid, or integrated into the host bacterial genome.

[0010] Furthermore, the anchoring protein and the target protein are connected by a linker.

[0011] Furthermore, the T7 promoter and the fusion protein expression cassette contain a LacO operon.

[0012] Furthermore, the anchoring protein is selected from one or more of CotB, CotC, CotE, CotG, CotX, and CotY.

[0013] Furthermore, the amino acid sequence of the LacI repressor protein is shown in SEQ ID NO.12;

[0014] The amino acid sequence of the LacO operon is shown in SEQ ID NO.13;

[0015] The amino acid sequence of the T7 RNA polymerase is shown in SEQ ID NO.14;

[0016] The amino acid sequence of the T7 promoter is shown in SEQ ID NO.15.

[0017] Furthermore, the FLS spore surface display system also includes an induction module comprising a histidine kinase with an amino acid sequence as shown in SEQ ID NO. 8 or 9, the induction module being expressed by the promoter Ptet.

[0018] Furthermore, the amino acid sequence of the promoter Ptet is shown in SEQ ID NO.16.

[0019] A second objective of this invention is to provide a recombinant bacterium containing the aforementioned FLS spore surface display system.

[0020] The third objective of this invention is to provide an application of the above-mentioned recombinant bacteria in the preparation of acetoin and tetracycline, wherein the target protein is a formaldehyde lyase encoding gene, the anchoring protein is located upstream of formaldehyde lyase (i.e., anchoring protein-linker-formaldehyde lyase), and the expression module is a single copy.

[0021] The fourth objective of this invention is to provide a method for producing tetramethylpyrazine and acetoin, wherein the recombinant bacteria are inoculated into a culture medium for fermentation and culture, and an inducer containing isopropyl-β-D-thiogalactoside is added to induce expression.

[0022] In the recombinant bacteria, the target protein is formaldehyde lyase, the anchoring protein is located upstream of formaldehyde lyase, and the expression module is a single copy.

[0023] Furthermore, the inducing agent also includes thiamine pyrophosphate.

[0024] Furthermore, the inducing agent also includes dehydrated tetracycline.

[0025] Furthermore, the culture medium contains 0.5-2.5 mol / L of acetaldehyde, and the fermentation culture temperature is 20-60℃.

[0026] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0027] This invention provides a technical solution combining a T7 expression system with a spore surface display system. By combining the T7 expression system with the spore surface display system, the problem of low functional protein expression efficiency is solved while avoiding the toxic side effects of leaked expression on spore formation. This achieves efficient anchoring and stable expression of functional proteins on the spore surface, providing an innovative technical solution for the industrial application of spore surface display systems. Addressing the problems of poor enzyme catalyst stability and inability to reuse in the bio-chemical coupling method for producing tetramethylpyrazine, this invention utilizes the aforementioned spore surface display system to display FLS on the surface of Bacillus subtilis spores, realizing the bio-chemical synthesis of tetramethylpyrazine and further verifying the feasibility of the system. Furthermore, addressing the problems of long time consumption and low sporulation efficiency in DSM culture medium using starvation induction, this invention also constructs an artificial sporulation induction system induced by dehydrated tetracycline expression to improve the sporulation efficiency of Bacillus subtilis, further optimizing the sporulation time and inducer concentration to achieve efficient preparation of the spore catalyst. Attached Figure Description

[0028] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0029] Figure 1 This invention relates to a spore surface display technology based on the T7 expression system;

[0030] Figure 2 It is an anchoring protein gene PCR amplification;

[0031] Figure 3 This is an observation of sporulation of recombinant Bacillus, where (a)-(f) represent the recombinant strains corresponding to the anchoring proteins CotB, CotC, CotE, CotG, CotX, and CotY, respectively.

[0032] Figure 4 These are images of recombinant spores observed under a confocal microscope; where (a)-(f) represent recombinant spores displaying GFP by anchoring proteins CotB, CotC, CotE, CotG, CotX, and CotY, respectively.

[0033] Figure 5 It is an evaluation of the anchoring effect;

[0034] Figure 6These are the plasmid maps of pHT-ptet-KinA and pHT-ptet-KinA-q-pas-A; where a is the plasmid map of pHT-ptet-KinA; and b is the plasmid map of pHT-ptet-KinA-q-pas-A.

[0035] Figure 7 These are PCR results of Bacillus subtilis transformed colonies;

[0036] Figure 8 The sporulation results of KinA and KinA-q-pas-A are characterized, where a is the sporulation efficiency analysis; b is the bright-field observation image under fluorescence microscopy; and c is the observation image under fluorescence microscopy at 488 nm excitation wavelength.

[0037] Figure 9 The results show the sporulation results of a single-copy CotC-FLS; where a is the sporulation flowchart of the nutrient depletion method and the induction method; b is the sporulation efficiency of different combinations.

[0038] Figure 10 These are plasmid maps showing two different protein element expression sequences; where a is the pADK-cotE-fls plasmid map; and b is the pADK-fls-cotE plasmid map.

[0039] Figure 11 These are agarose gel electrophoresis images of PCR amplification products. In Figure a, lane M: 2000 bp DNA marker; lanes 1 and 2: cotB; lanes 3 and 4: cotC; lanes 5 and 6: cotE; lanes 7 and 8: cotY. In Figure b, lane M: 2000 bp DNA marker; lanes 1 and 2: fls. In Figure c, lane M: 10000 bp DNA marker; lanes 1 and 2: pHT; lanes 3 and 4: pADK; lanes 5 and 6: pcrF19.

[0040] Figure 12 It is a characterization and screening method for strains displayed on the spore surface;

[0041] Figure 13 This is the result of optimizing the production conditions for acetoin;

[0042] Figure 14 It is the result of the enzyme-catalyzed chemical coupling method. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0044] Bacillus subtilis G600 was published in the paper “A genetic toolkit for efficient production of secretory protein in Bacillus subtilis”, DOI number “https: / / doi.org / 10.1016 / j.biortech.2022.127885”.

[0045] DNA polymerase was purchased from Novizan, the seamless cloning kit was purchased from Ibotek Biotechnology Co., Ltd., the plasmid extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the PCR product nucleic acid purification kit was purchased from ThermoScientific.

[0046] LB medium (for plasmid construction and cell culture) mainly contains NaCl (10 g / L), tryptone (10 g / L), and yeast extract (5 g / L). DSM medium (for sporulation) mainly contains peptone (5 g / L), beef extract (3 g / L), KCl (1 g / L), MgSO4·7H2O (0.25 g / L), and MnCl2·H2O (0.002 g / L). Antibiotics used during culture mainly include kanamycin (50 mg / L), ampicillin (100 mg / L), tetracycline (25 mg / L), and chloramphenicol (5 mg / L). TB medium (for sporulation) mainly contains yeast extract (24 g / L), peptone (12 g / L), glycerol (4 mL / L), and phosphate buffer (100 mM).

[0047] Colorimetric determination of acetoin: Take 16 μL of the reaction supernatant and add 184 μL of colorimetric solution (5% α-naphthol / 40% KOH / 0.5% creatine = 1∶1∶1). Then incubate at 30℃ for 20 min and measure at 522 nm using spectrophotometry.

[0048] HPLC determination of acetoin and tetramethylpyrazine: HPLC detection conditions for acetoin: Aminex HPX-87H (Bio-Rad) column; detection wavelength 210 nm; mobile phase: 5 mM sulfuric acid; flow rate: 0.6 mL / min; injection volume: 5 μL; column temperature: 30℃; HPLC detection conditions for TMP: XBridge C18 5 μm (4.6 × 250 mm) column; detection wavelength: 278 nm; mobile phase: 0.1% formic acid (80%) and acetonitrile (20%); flow rate: 0.8 mL / min; injection volume: 10 μL.

[0049] To better evaluate the catalytic efficiency of different combinations, this study defined enzyme activity units as the amount of enzyme required to release 1 µg of acetoin per minute under the assay conditions. The enzyme activity conversion formula is as follows:

[0050] Y=

[0051] Where: FLS enzyme activity (U / mL) of the Y-spore surface display system;

[0052] M - Dilution factor of the reaction solution;

[0053] OD 522- The absorbance of a 200 µL acetoin colorimetric system at 522 nm;

[0054] The slope of the K-standard curve;

[0055] T - Reaction time (min);

[0056] V: The system volume of the catalytic reaction (mL);

[0057] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, cell biology, recombinant DNA technology, and related fields.

[0058] The sequences involved in the following embodiments are shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062]

[0063] Example 1: Design and Construction of a Spore Surface Display System Based on the T7 Expression System

[0064] Design framework of spore surface display system based on T7 expression system, as follows: Figure 1As shown: First, module 1 (the driver module) was integrated into the genome of *B. subtilis* G600. This module mainly contains two core expression cassettes: the LacI repressor protein expression cassette and the T7 RNA polymerase expression cassette with the LacO operon. Without an inducer, the LacI protein represses T7 RNA polymerase transcription by binding to LacO. Then, module 2 (the expression module) was transformed into the tool strain BSG600-T7L, which already incorporated module 1. The core element of this module is the Cot-anchored protein-sfGFP fusion expression cassette driven by the T7 promoter. There is also a LacO operon between the T7 promoter and the fusion protein; transcription of the fusion cassette is also repressed without an inducer.

[0065] To investigate the effect of gene copy number on surface display, the T7 expression cassette was linked to plasmid backbones with different copy numbers, resulting in recombinant plasmids of varying copy numbers. pSTOP was a high-copy plasmid in *B. subtilis*, pADK a medium-copy plasmid, and pHT a low-copy plasmid; these recombinant plasmids existed as free circular plasmids in the BSG600-T7L strain. Single-copy T7 expression cassettes were site-directedly integrated into the BSG600-T7L genome using the CRISPR / Cpf1 gene editing system. By adding IPTG inducer to the sporulation medium, transcription of modules 1 and 2 was activated, leading to the expression of the Cot-anchored protein-sfGFP fusion cassette. The sfGFP reporter protein was then anchored to the spore surface during sporulation.

[0066] Six endogenous capsid proteins of *B. subtilis* (CotB, CotC, CotE, CotG, CotX, and CotY) were selected as anchoring proteins. These anchoring proteins and sfGFP were linked via a linker (GSGGGGS). The amino acid sequences corresponding to the different anchoring proteins and sfGFP are shown in Table 1. Specific primers were designed based on the gene sequences corresponding to these anchoring proteins in the NCBI database. Using the *B. subtilis* G600 genome as a template, the corresponding fragments were amplified and then gel-cleaved for recovery. Figure 2 Subsequently, the recovered fragment was fused with sfGFP and ligated into the corresponding plasmid backbone using overlap extension PCR. After sequencing verification, the plasmid was transformed into BSG600 strain to obtain recombinant Bacillus (BSG600-T7L strain) containing the T7 RNA polymerase-driven module and the Cot-sfGFP expression control module.

[0067] Example 2: Preparation of recombinant spores and evaluation of anchoring effect

[0068] The verified recombinant Bacillus strains were inoculated into 10 mL of LB liquid medium containing the corresponding antibiotic and cultured overnight at 37 °C with shaking. Then, they were transferred to 25 mL of DSM sporulation medium containing the corresponding antibiotic at a 1% (v / v) inoculation ratio. After 4 h of culture, 0.5 mM IPTG inducer was added, and the culture was continued for 48 h at 37 °C and 220 rpm. A suitable amount of bacterial culture was observed under an electron microscope to monitor sporulation. Figure 3 As shown, the recombinant strains corresponding to the six anchoring proteins all exhibited typical oval spore morphology.

[0069] like Figure 4 The results showed that further observation of recombinant spores with six anchoring proteins characterized by GFP using laser confocal microscopy (taking recombinant spores expressing CotB, CotC, CotE, CotG, CotX, and CotY via medium-copy plasmids as examples) revealed significant green fluorescence on the surface of all six recombinant spores, with a particularly prominent ring of green light on the spore surface. These results indicate that all six anchoring proteins can effectively anchor GFP on the spore surface.

[0070] The bacterial suspension was transferred to a 50 mL centrifuge tube and centrifuged at 8000 rpm for 20 min. After discarding the supernatant, the suspension was washed 2-3 times with deionized water and resuspended in 2 mL of sterile water. Lysozyme was then added to a final concentration of 2 mg / L, and the suspension was incubated in a 37 ℃ water bath for 2 h to eliminate non-spore-forming proliferating cells. The suspension was then washed 2-3 times with deionized water and resuspended in sterile water to obtain a purified recombinant spore suspension. The spore suspension was transferred to flow cytometry tubes, and the fluorescence intensity of each combination was detected using flow cytometry to evaluate the surface display effect.

[0071] like Figure 5 As shown, the fluorescence intensities of the high-copy CotG and CotE combinations in the library were 63 and 28, respectively. This study concludes that these two combinations lack surface display capability. The fluorescence levels of the remaining combinations were significantly higher than those of the negative control (approximately 50). The fluorescence intensities, ranked from highest to lowest, were: single-copy CotE, low-copy CotE, medium-copy CotE, single-copy CotC, low-copy CotB, and medium-copy CotY. The median fluorescence intensity of these six combinations exceeded 10,000, indicating strong protein expression and display efficiency.

[0072] Example 3: Design and Construction of an Artificial Sporulation Induction System

[0073] KinA is a key kinase in the sporulation initiation program of Bacillus subtilis. It initiates a phosphate transfer cascade through autophosphorylation to activate the master regulator Spo0A, and its internal PAS-A domain is primarily responsible for sensing intracellular and extracellular environmental signals. Based on these gene characteristics, a pHT plasmid containing KinA and KinA-q-pas-A sporulation genes, induced by dehydrotetracycline, was designed in Bacillus subtilis. Figure 6 KinA-q-pas-A refers to KinA with the PAs-A domain removed, and is named pHT-ptet-KinA and pHT-ptet-KinA-q-pas-A respectively.

[0074] Primers were designed based on plasmid maps, and the target gene was amplified by PCR. Subsequently, the corresponding plasmid was constructed via Gibson ligation. After sequencing verification, the plasmid was transformed into the BSG600-T7L strain. Simultaneously, the T7 RNA polymerase-driven module and the Cot-FLS expression control module were transformed into this strain, resulting in a recombinant Bacillus strain (BSG600-T7L-KinA) containing the T7 RNA polymerase-driven module, the Cot-FLS expression control module, and the dehydrated tetracycline-induced module. Colony PCR verified the correct construction. Figure 7 ).

[0075] Example 4: Optimization and Characterization of Sporulation Efficiency of Artificial Sporulation Induction System

[0076] like Figure 8 As shown in Figure a, to determine the optimal sporulation conditions, we conducted a two-factor optimization experiment on the inducing agent addition time and sporulation time in a 2 mL LB liquid medium system. We found that both gene-mediated Bacillus subtilis strains exhibited the highest sporulation efficiency when the inducing agent addition time was 4 h and the sporulation time was 24 h. We then observed the sporulation suspension under these conditions using a bright-field fluorescence microscope and a 488 nm excitation wavelength, further demonstrating that KinA and KinA-q-pas-A can effectively mediate the production of sporulation spores with surface display activity. Figure 8 (b and c).

[0077] After verifying the feasibility of inducible expression of the genes KinA and KinA-q-pas-A mediating sporulation in Bacillus subtilis, we transformed the plasmids pHT-ptet-KinA and pHT-ptet-KinA-q-pas-A into a single-copy CotC-FLS strain, respectively, and induced sporulation through fermentation in 50 mL LB and TB liquid media, using 50 mL DSM liquid media with nutrient depletion sporulation as a control. Figure 9As shown, the highest sporulation rate was achieved by inducing expression of the KinA-q-pas-A gene in TB liquid medium. Compared with the nutrient depletion method, the sporulation time was shortened by half, and the sporulation rate was increased by 3.69 times under the same culture conditions.

[0078] Example 5: Biochemical Synthesis of Tetramethylpyrazine Based on Spore Surface Display System

[0079] Based on the six combinations selected in Example 2, plasmids were designed in which the FLS-coding gene replaced the GFP-coding gene. The resulting basic plasmids were named pcrf19-cotC-fls, pcrf19-cotE-fls, pHT-cotB-fls, pHT-cotE-fls, pADK-cotE-fls, and pADK-cotY-fls. Figure 10 (Taking pADK-cotE-fls plasmid as an example). Considering that the expression order of protein elements may affect the display efficiency, six basic plasmids with the FLS encoding gene first were further constructed and named pcrf19-fls-cotC, pcrf19-fls-cotE, pHT-fls-cotB, pHT-fls-cotE, pADK-fls-cotE, and pADK-fls-cotY. Figure 10 (For example, pADK-fls-cotE plasmid).

[0080] Primers were designed based on plasmid maps, the target gene was amplified by PCR, and verified by nucleic acid gel electrophoresis. Figure 11 Subsequently, the corresponding plasmid was constructed via Gibson ligation. After sequencing verification, the plasmid was transformed into the BSG600-T7L strain. Then, the dehydrated tetracycline induction module was also transformed into the strain, and the biochemical synthesis of tetramethylpyrazine was achieved using an artificial sporulation induction system.

[0081] Furthermore, based on the successfully constructed and validated surface-displaying FLS recombinant strain, spore catalysts were prepared. Catalytic experiments were conducted in a 200 µL system to quantitatively evaluate the catalytic performance of 12 combined catalysts. Under uniform sporulation induction and reaction conditions, the single-copy CotC-FLS combination exhibited the most outstanding catalytic performance. Figure 12 The acetoin yield was significantly higher than that of the other 11 groups, so this strain was selected as the target strain for subsequent fermentation optimization.

[0082] like Figure 13As shown, after determining single-copy CotC-FLS as the optimal strain, we systematically optimized the catalytic conditions of the single-copy CotC-FLS combined surface display system in a 200 μL system. First, addressing the coenzyme shielding problem caused by the dense spore structure, experiments confirmed that exogenous addition of 0.5 mM THDP significantly increased the yield of acetoin. Spore concentration (OD) 600 Gradient testing revealed that acetoin production increased with increasing spore concentration. Considering the limited spore quantity, OD... 600 The concentration of acetoin was set at 20 for subsequent optimization experiments. Reaction time kinetics showed that acetoin accumulation was rapid in the first 8 hours, followed by a significant slowdown in yield growth; therefore, 8 hours was chosen as the subsequent catalytic reaction time. Finally, by examining acetaldehyde concentration gradients (0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M) and reaction temperature gradients (20℃, 30℃, 40℃, 60℃, 80℃), it was found that acetoin yield showed a trend of first increasing and then decreasing. Therefore, 1.5 M acetaldehyde concentration and 40℃ were determined to be the optimal conditions for the FLS spore surface display catalyst.

[0083] like Figure 14 As shown, catalysis was carried out in a 1 mL reaction system, and the spore concentration OD was increased. 600 The concentration was set to 25, and the acetaldehyde concentration was set to 1.5 M. After the reaction was completed, the concentration of acetoin in the fermentation broth reached 16.7 g / L. Finally, 43.7 mg of ammonium dihydrogen phosphate (molar ratio of acetoin to ammonium dihydrogen phosphate = 1:2) was added to the fermentation broth, and the mixture was incubated in a water bath at 90°C for 3 h.

[0084] Standard curves for acetoin and tetramethylpyrazine were established by high performance liquid chromatography (HPLC), and the catalytic end products were detected. The concentration of acetoin in the fermentation broth was calculated to be 16.7 g / L, and the concentration of tetramethylpyrazine reached 9.6 g / L after further reaction. The conversion rates of acetoin and tetramethylpyrazine were 25.3% and 18.9%, respectively. Figure 14 ).

[0085] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A FLS spore surface display system based on the T7 expression system, characterized in that, The FLS spore surface display system includes a driving module and an expression module; The driving module includes a first expression box containing LacI repressor protein and a second expression box containing LacO operon and T7 RNA polymerase. The target gene expression module includes a fusion protein expression frame containing an anchor protein and a target protein connected by a linker, and the expression of the fusion protein expression frame is driven by the T7 promoter. The second expression cassette is located in the pADK plasmid, pHT plasmid, or integrated into the host bacterial genome.

2. The FLS spore surface display system according to claim 1, characterized in that, The T7 promoter and the fusion protein expression cassette contain the LacO operon.

3. The FLS spore surface display system according to claim 1, characterized in that, The anchoring protein is selected from one or more of CotB, CotC, CotE, CotG, CotX, and CotY.

4. The FLS spore surface display system according to claim 1, characterized in that, The spore surface display system further includes an induction module comprising a histidine kinase with an amino acid sequence as shown in SEQ ID NO. 8 or 9, the induction module being expressed by the promoter Ptet.

5. Recombinant bacteria containing the FLS spore surface display system according to any one of claims 1-4.

6. The application of the recombinant bacteria according to claim 5 in the preparation of acetoin and tetramethylpyrazine, characterized in that, The target protein is formaldehyde lyase, the anchoring protein is located upstream of formaldehyde lyase, and the expression module is a single copy.

7. A method for producing tetramethylpyrazine and acetoin, characterized in that, The recombinant bacteria described in claim 5 were inoculated into a culture medium for fermentation and culture, and an inducer containing isopropyl-β-D-thiogalactoside was added to induce expression. In the recombinant bacteria, the target protein is formaldehyde lyase, the anchoring protein is located upstream of formaldehyde lyase, and the expression module is a single copy. When producing tebufenozide, ammonium dihydrogen phosphate is added to the fermentation broth after fermentation culture.

8. The production method according to claim 7, characterized in that, The inducing agent also includes dehydrated tetracycline.

9. The production method according to claim 8, characterized in that, The inducer also includes thiamine pyrophosphate.

10. The production method according to claim 7, characterized in that, The culture medium contains 0.5-2.5 mol / L of acetaldehyde, and the fermentation culture temperature is 20-60℃.

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