Functional signal peptide and fusion protein and application thereof

Abstract

The invention discloses a functional signal peptide and a fusion protein and application thereof. The invention provides a novel enzyme aggregation strategy, and the strategy depends on functional signal peptide as an aggregation induction tag, guides co-localization of enzyme, forms a functional inclusion body, and aims to promote the efficiency of cascade reaction in which the enzyme participates. In the embodiment, two fluorescent proteins are used for characterization, and fluorescent confocal imaging proves that the foreign protein which is subjected to fusion expression with the functional signal peptide realizes co-localization in escherichia coli. Then, the strategy is expanded to a two-step enzyme cascade reaction to realize production of ergothioneine and a three-step enzyme cascade reaction to realize production of 6-hydroxyhexanoic acid.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically, it relates to a functional signal peptide, its fusion protein, and its applications. Background Technology

[0002] In synthetic biology and metabolic engineering, researchers are increasingly focusing on enzyme aggregation, such as the self-assembly of multiple enzymes or the formation of intracellular supramolecular scaffolds. Assembling multiple enzymes to aggregate can reduce substrate loss and improve pathway efficiency. In addition to the widely studied enzyme assembly patterns, the recent proposal of functional inclusion bodies has further enriched enzyme aggregation strategies. Inclusion bodies are generally considered waste reservoirs of non-functional unfolded or misfolded proteins. However, some studies have shown that inclusion bodies generated by fusing target enzymes with coiled-coil domains or easily aggregated proteins in *E. coli* can retain some enzyme function. However, due to the decreased activity of the fused proteins in functional inclusion bodies, the application of functional inclusion bodies, especially those fused with proteins containing coiled-coil domains or easily aggregated signal peptides, remains very limited. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a novel enzyme aggregation strategy that relies on a functional signal peptide as an aggregation-inducing tag to guide enzyme co-localization and form functional inclusion bodies, aiming to improve the efficiency of enzyme-involved cascade reactions. In the examples, two fluorescent proteins were used for characterization, and fluorescence confocal imaging demonstrated that the exogenous protein fused with the functional signal peptide was co-localized in *E. coli*. Subsequently, this strategy was extended to a two-step enzyme cascade for the production of ergothioneine and a three-step enzyme cascade for the production of 6-hydroxyhexanoic acid.

[0004] In one aspect, the present invention provides a functional signal peptide having the amino acid sequence shown in SEQ ID NO: 2.

[0005] In another aspect, the present invention provides a fusion protein comprising a functional signal peptide, a linker peptide, and a target protein; the amino acid sequence of the functional signal peptide is shown in SEQ ID NO: 2, and the amino acid sequence of the linker peptide is shown in SEQ ID NO: 6.

[0006] Preferably, the functional signal peptide, the linker peptide, and the target protein are sequentially linked in the fusion protein.

[0007] In one or more embodiments, the target protein is an enzyme or a fluorescent protein.

[0008] Preferably, the enzyme is selected from one or both of Egt1 and Egt2. Preferably, the enzyme is selected from one or more of ADH, BVMO, and Lactonnase.

[0009] Preferably, the fluorescent protein is selected from one or both of GFP and mCherry.

[0010] In another aspect, the present invention provides a polynucleotide that encodes a functional signal peptide as described in any embodiment herein or one or more fusion proteins as described in any embodiment herein.

[0011] In another aspect, the present invention provides an expression vector comprising a polynucleotide as described in any embodiment herein.

[0012] Preferably, the expression vector is pET28a, pRSFDuet, pMA5, or pMV261.

[0013] In another aspect, the present invention provides a host cell comprising a functional signaling peptide, fusion protein, or polynucleotide as described in any embodiment herein.

[0014] Preferably, the host cell is Escherichia coli, Bacillus subtilis, or Mycobacterium.

[0015] In another aspect, the present invention provides the use of fusion proteins as described in any embodiment herein in cascade reactions catalyzed by two or more enzymes, the fusion proteins serving as catalytic components, the reactions comprising the participation of two or more fusion proteins.

[0016] In one or more embodiments, the reaction involves the participation of fspL4Egt1 and fspL4Egt2, and the product of the reaction is selected from one or more of histidine betaine, histidine betaine cysteine ​​sulfoxide, and ergothioneine; or, the reaction involves the participation of two or more of fspL4ADH1, fspL4BVMO, and fspL4Lactonnase, and the product of the reaction is selected from one or more of cyclohexanone, caprolactone, and 6-hydroxyhexanoic acid.

[0017] The signal peptide of the fusion protein fspL4Egt1 is fsp, the linker peptide is L4, and the target protein is the Egt1 enzyme; the signal peptide of the fusion protein fspL4Egt2 is fsp, the linker peptide is L4, and the target protein is the Egt2 enzyme; the signal peptide of the fusion protein fspL4ADH is fsp, the linker peptide is L4, and the target protein is ADH; the signal peptide of the fusion protein fspL4BVMO is fsp, the linker peptide is L4, and the target protein is BVMO; the signal peptide of the fusion protein fspL4Lactonnase is fsp, the linker peptide is L4, and the target protein is a lactone enzyme.

[0018] Preferably, the Egt1 enzyme is derived from *Nematocephalosporium*; preferably, the Egt2 enzyme is derived from *Nematocephalosporium*; preferably, the ADH is derived from *Lactobacillus brevis*; preferably, the BVMO is derived from *Acinetobacter*; preferably, the lactonease is derived from *Rhodococcus*.

[0019] In another aspect, the present invention provides an application of a fusion protein in cell localization, the fusion protein comprising a functional signal peptide and a target protein; the amino acid sequence of the functional signal peptide is shown in SEQ ID NO: 2, and the target protein is an enzyme or a fluorescent protein; the fusion protein is used to present aggregated fluorescent blocks or chromogenic blocks in cells.

[0020] Preferably, the fluorescent protein is selected from one or both of GFP and mCherry.

[0021] Preferably, the enzyme is used to catalyze a colorimetric reaction of the substrate.

[0022] In another aspect, the present invention provides a method for producing a fusion protein as described in any embodiment herein, the method comprising expression using a polynucleotide or host cell as described in any embodiment herein.

[0023] In another aspect, the present invention provides a method for producing ergothionein, the method comprising expressing fspL4Egt1 and fspL4Egt2 using polynucleotides or host cells as described in any embodiment herein.

[0024] In another aspect, the present invention provides a method for producing 6-hydroxyhexanoic acid, the method comprising expressing fspL4ADH, fspL4BVMO and fspL4Lactonnase using polynucleotides or host cells as described in any embodiment herein.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The functional signal peptide provided by this invention can be fused with GFP as a reporter protein to produce a corresponding fusion protein. The expression location of the protein in the cell can be located by confocal microscopy. The fusion protein using GFP and mCherry as reporter proteins can also achieve dual fluorescence co-localization.

[0027] 2. Based on the aggregation effect guided by functional signal peptides, the functional signal peptides, linker peptides and enzymes of this invention can be fused and expressed to produce corresponding fusion proteins, thereby shortening the distance between enzymes in a two-enzyme or multi-enzyme cascade reaction, significantly promoting the cascade reaction, and increasing the yield of reaction products (ergothioneine or 6-hydroxyhexanoic acid), which is expected to serve as a new strategy for constructing efficient microbial cell factories.

[0028] 3. The aggregation effect of the fusion protein guided by the functional signal peptide provided by the present invention can be achieved in Escherichia coli, Bacillus subtilis and Mycobacterium, suggesting the feasibility of promoting two-enzyme or multi-enzyme cascade reactions in a variety of host cells. Attached Figure Description

[0029] Figure 1 These are laser confocal microscope images of cells expressing GFP, spGFP, and fspGFP.

[0030] Figure 2 These are 3D structural models of sp and fsp; Figure A is the 3D structural model of sp, and Figure B is the 3D structural model of fsp.

[0031] Figure 3 This represents the fluorescence intensity of GFP expressed by fusing different signal peptides and linker peptides. In the figure, whole-cell, supernatant, and precipitation correspond to the fluorescence intensity of cell culture medium, cell culture supernatant, and cell pellet, respectively.

[0032] Figure 4 These are laser confocal microscopy images of Escherichia coli expressing fspL1GFP to fspL5GFP.

[0033] Figure 5 These are laser confocal microscopy images of Escherichia coli co-expressing fspL4GFP and fspL4mCherry.

[0034] Figure 6 This is the pathway for the synthesis of ergothioneine (EGT) catalyzed by a two-enzyme cascade. In the diagram, histidine represents histidine, hercynine represents histidine betaine, hercynylcysteine ​​sulfoxide represents histidine betaine cysteine ​​sulfoxide, and EGT represents ergothioneine.

[0035] Figure 7 It refers to the EGT production in strains Egt1-2, fspEgt1-2, fspL4Egt1-2, and fspL5Egt1-2.

[0036] Figure 8 It is a pathway for the synthesis of 6-hydroxyhexanoic acid (6-HHA) catalyzed by a three-enzyme cascade.

[0037] Figure 9 It refers to the production of 6-HHA in strains ABL, fspABL, fspL4ABL, and fspL5ABL.

[0038] Figure 10 This is a laser confocal microscope image of Bacillus subtilis expressing fspGFP.

[0039] Figure 11 This is a laser confocal microscope image of a mycobacterium expressing fspGFP. Detailed Implementation

[0040] In the following embodiments, the information of the relevant plasmids is shown in Table 1 below.

[0041] Table 1: Plasmids and their information

[0042] name Main phenotype or characteristics pET28a-GFP GFP pET28a-spGFP spGFP pET28a-fspGFP fspGFP pET28a-fspL1GFP fspL1GFP pET28a-fspL2GFP fspL2GFP pET28a-fspL3GFP fspL3GFP pET28a-fspL4GFP fspL4GFP pET28a-fspL5GFP fspL5GFP pET28a-fspmCherry fspmCherry pET28a-fspL4mCherry fspL4mCherry pET28a-fspL4GFP-fspL4mCherry fspL4GFP, fspL4mCherry pET28a-Egt1-Egt2 Egt1, Egt2 pET28a-fspEgt1-fspEgt2 fspEgt1, fspEgt2 pET28a-fspL4Egt1-fspL4Egt2 fspL4Egt1, fspL4Egt2 pET28a-fspL5Egt1-fspL5Egt2 fspL5Egt1, fspL5Egt2 pRSFDuet-ADH-BVMO-Lac ADH, BVMO, Lactonnase pRSFDuet-fspADH-fspBVMO-fspLac fspADH, fspBVMO, fspLactonnase pRSFDuet-fspL4ADH-fspL4BVMO-fspL4Lac fspL4ADH, fspL4BVMO, fspL4Lactonnase pRSFDuet-fspL5ADH-fspL5BVMO-fspL5Lac fspL5ADH, fspL5BVMO, fspL5Lactonnase pMA5-fspGFP fspGFP pMV261-fspGFP fspGFP

[0043] The plasmids pET28a, pRSFDuet, pMA5, and pMV261 used in this invention are all commercially available. Unless otherwise specified, the remaining recombinant plasmids were synthesized from the whole genome or obtained by inserting the target gene using conventional molecular biology methods with the above plasmids as a backbone.

[0044] In this invention, the Egt1 gene (Genbank ID: 3872471) expressing the Egt1 enzyme and the Egt2 gene (Genbank ID: 5847244) expressing the Egt2 enzyme are derived from Neurospora crassa; the ADH gene (Genbank ID: MW808993.1) expressing the alcohol dehydrogenase ADH is derived from Lactobacillus brevis ATCC 14869; the BVMO gene (Genbank ID: AB006902.2) expressing the Baeyer-Villiger monooxygenase BVMO is derived from Acinetobacter; and the Lactonnase gene (Genbank ID: MK910756.1) expressing the lactonease is derived from Rhodococcus.

[0045] In this invention, unless otherwise specified, all experimental strains are commercially available.

[0046] In the following examples, the preparation of competent E. coli cells and their plasmid transformation were performed according to conventional methods.

[0047] In the following embodiments, the preparation of Bacillus subtilis competent cells and their plasmid transformation were carried out according to the following methods:

[0048] (1) Pick a single colony of the streaked host bacterium WB600 and inoculate it into a test tube containing 2 mL of SPI medium. Incubate overnight at 37°C and 220 rpm on a shaker.

[0049] (2) Take 40 μL of the overnight culture and inoculate it into 2 mL of SPI medium. After culturing at 37°C and 220 rpm for 5-6 h, take 200 μL and inoculate it into 2 mL of SPII medium. Culturing at 37°C and 220 rpm for 1.5-2 h, and controlling OD600 at 0.6-1.

[0050] (3) Add 20 μL of 100×EGTA solution and incubate at 37℃ and 220 rpm for 10 min. Control OD600 between 0.6 and 1. Aliquot into 500 μL per 1.5 mL centrifuge tube.

[0051] (4) Add an appropriate amount of plasmid (5 μg) to the tube, mix gently, and incubate at 37℃ and 220 rpm for 1.5–2 h.

[0052] (5) Collect the bacterial cells by centrifugation at 4000 rpm for 2 min, discard part of the supernatant, keep 100-200 μL to resuspend the bacterial cells, spread them on the corresponding resistance plates, and incubate overnight at 37°C.

[0053] In the following examples, the SPⅠ culture medium (2 mL) was prepared by uniformly mixing 980 μL of SPI-A salt solution, 980 μL of SPI-B salt solution, 20 μL of 50% glucose, and 20 μL of 100×CAYE solution.

[0054] The preparation method of SPI-A salt solution is as follows: weigh 0.4% (NH4)2SO4, 2.8% K2HPO4·3H2O, 1.2% KH2PO4, and 0.2% sodium citrate dihydrate according to the final volume based on w / v, prepare the solution with deionized water, and sterilize at 121℃ for 20 min.

[0055] The preparation method of SPI-B salt solution is as follows: Weigh 0.04% MgSO4·7H2O according to the final volume based on w / v, prepare the solution with deionized water, and sterilize at 121℃ for 20 min;

[0056] The method for preparing 100×CAYE solution is as follows: weigh 2% casein amino acids and 10% yeast extract according to the final volume of preparation, based on w / v, and sterilize at 121℃ for 20 min.

[0057] In the following examples, the SPⅡ medium was prepared by uniformly mixing 2 mL of SPI medium, 20 μL of 50 mM calcium chloride, and 20 μL of 250 mM magnesium chloride. In the following examples, the 100×EGTA solution was prepared by preparing a 10 mM / L EGTA solution, adding a small amount of sodium hydroxide to adjust the pH to 8.0 during dissolution.

[0058] In the following examples, the Super-Rich culture medium was prepared as follows: 25g of peptone, 20g of yeast extract, 3g of dipotassium hydrogen phosphate, and 30g of glucose were weighed per liter of culture medium, and autoclaved at 115°C for 20 minutes. In the following examples, the preparation of competent mycobacterial cells was carried out according to the following method:

[0059] (1) Dip the glycerol bacteria of Mycobacterium oxysporum into solid LB medium, streak it, and place it in an incubator at 30°C for 3 days.

[0060] (2) In a clean bench, use a sterile pipette tip to pick up a single colony and place it in a 5 mL LB liquid test tube. Incubate at 30°C with shaking for 3 days.

[0061] (3) Transfer 3 mL to 30 mL of seed culture medium and culture at 30 °C and 220 rpm with shaking until the OD600 is about 1.5 to 2. Then add glycine solution to the final concentration of 2% and continue to culture for 12 to 24 h until the cells turn orange-yellow.

[0062] (4) Incubate the bacterial culture in an ice water bath for 30 min, transfer it to a 50 mL sterile centrifuge tube, centrifuge at 4000 rpm for 10 min at 4 °C, and discard the supernatant.

[0063] (5) Take 3 mL of pre-cooled 10% glycerol solution into a centrifuge tube, then fully resuspend the precipitated bacterial cells, centrifuge at 4000 rpm for 10 min at 4℃, and discard the supernatant.

[0064] (6) After repeating step (5) 3 times, the bacterial precipitate is suspended in 2 mL of 10% glycerol solution, dispensed and stored at -80℃ for later use.

[0065] In the following examples, plasmid transformation of mycobacterial competent cells was performed according to the following method:

[0066] Before electroporation, remove the electrode cup stored in ethanol and air dry it in a clean bench. Place competent cells of mycobacteria in an ice-water bath for 5 minutes to thaw them.

[0067] (1) Add an appropriate amount of recombinant plasmid to the thawed competent cells, mix thoroughly, and place in ice water for 20 min.

[0068] (2) Transfer the mixture of plasmid and competent state to a pre-cooled electrode cup and electroporate it twice with a voltage of 2.5kV, with a 5s interval between the two electroporations.

[0069] (3) Place the electrode cup after electric shock on ice for 3 minutes, add 500 μL of antibiotic-free LB liquid medium to fully resuspend the cells, transfer to a new sterile 1.5 mL centrifuge tube, seal with sealing film, and incubate at 30°C and 220 rpm for 3-4 hours.

[0070] (4) Dilute with sterile water at an appropriate ratio, take 100 μL and spread it evenly on a solid culture medium containing the corresponding antibiotic, and incubate upside down in a 30℃ incubator for about 3 days.

[0071] In the following examples, the fermentation system for Escherichia coli to produce ergothioneine was 10 mL of phosphate buffer (0.2 M) containing 10 mM His, 30 mM SAM, 10 mM Cys and 10 mM PLP, pH 7.4.

[0072] In the following examples, the fermentation system for Escherichia coli to produce 6-hydroxyhexanoic acid contains 10 mM cyclohexanol and 0.1 mM NADP. + 10 mL of phosphate buffer (0.2 M), pH 7.4.

[0073] In the following examples, the pretreatment method of the fermentation broth before use for detecting ergothioneine content is as follows:

[0074] Take 2 mL of fermentation broth into a plastic screw-cap centrifuge tube for EGT extraction and quantification. Centrifuge at 12000 rpm for 2 min at room temperature to collect the cells. Wash the cells twice with an equal volume of PBS buffer and resuspend them in 70% acetonitrile-water solution. Add an appropriate amount of 0.1 mm silica beads. Place the cell in a shaker, pre-cool to -4℃, set to 55 Hz, and break the cells for 5 min. Then, let it stand at -20℃ for 2 min (repeat twice), centrifuge at 12000 rpm for 30 min, collect the supernatant, and filter the extract through a 0.22 μm filter membrane. Use the filtrate as the sample to be tested.

[0075] In the following embodiments, the parameters for detecting the ergothioneine content in the fermentation broth sample using high-performance liquid chromatography are as follows:

[0076] An Agilent 1100 liquid chromatograph (purchased from Agilent Technologies) was used. The parameters were set as follows: ZORBAX Eclipse×DB C18 column (250 mm × 4.6 mm, 5 μm); column temperature 30 °C; mobile phase acetonitrile:water = 3:97 (v / v); flow rate 0.6 mL / min; detection wavelength 254 nm; injection volume 10 μL.

[0077] In the following examples, the pretreatment method for the fermentation broth before use in detecting the 6-hydroxyhexanoic acid content is as follows:

[0078] Take 1 mL of the reaction solution and add 25 μL of hydrochloric acid (10 mM) to terminate the reaction; add an equal volume of ethyl acetate, vortex for 10 min, and centrifuge at 12000 rpm for 15 min; take the supernatant and transfer it to a new EP tube for centrifugation, add an appropriate amount of anhydrous sodium sulfate to remove water, and finally filter the extract using a 0.22 μm polypropylene centrifuge tube. The filtrate is used as the sample to be tested.

[0079] In the following examples, the parameters for detecting the 6-hydroxyhexanoic acid content in the fermentation broth sample using gas chromatography are as follows:

[0080] An Agilent 7820A gas chromatograph (purchased from Agilent Technologies) was used, with a flame ionization detector (FID); the column was an HP-5 (30m × 0.25mm, 0.25μm); the column oven temperature was initially 40℃ and held for 1 min, then increased to 140℃ at a rate of 12℃ / min, and then increased to 280℃ at a rate of 50℃ / min.

[0081] Example 1: Expression of target protein containing signal peptide

[0082] This embodiment investigated the effect of signal peptides on the expression of target proteins in *E. coli*, using GFP as an example. Information on the signal peptides is shown in Table 2.

[0083] Table 2: Signal peptide sequence listing

[0084] name amino acid sequence sp MGRRVVSFLIVVAALSGALWLGGLALA(SEQ ID NO: 1) fsp MGRRVVSFLIVVAAPSGALWLGGRALA (SEQ ID NO: 2)

[0085] pET28a-GFP, pET28a-spGFP, and pET28a-fspGFP were transformed into competent E. coli cells using standard methods. After culturing for 24 hours, the recombinant E. coli cells expressing fluorescent proteins were collected by centrifugation. The cells were washed three times with PBS, resuspended in physiological saline, and approximately 10 μL of the bacterial culture was transferred onto a glass slide. The cells were observed and collected under a laser confocal microscope. Figure 1The confocal image shown is an overlay of the GFP and Bright field images.

[0086] Depend on Figure 1 It is evident that the fusion expression of the original signal peptide sp or the functional signal peptide fsp leads to a decrease in GFP protein expression. However, the fluorescent particles in the fspGFP group are spatially localized near the polar regions of the cell, indicating that fspGFP exhibits polar localization in *E. coli*, while both the GFP and spGFP groups produce fluorescence dispersed in the cytoplasm. Analysis of subcellular localization characteristics revealed one or more large GFP fluorescent foci in the cytoplasm of *E. coli* in the fspGFP group.

[0087] By performing homology modeling on sp and fsp, we obtain, as follows Figure 2 The results show that the 3D structure of sp is an α-helical polypeptide chain. Figure 2 A), while the 3D structure of fsp consists of two antiparallel α-helical polypeptide chains ( Figure 2 B), forming a The antiparallel β-sheet units of fsp. Based on subcellular localization features and modeling results, it is speculated that in cells expressing fspGFP, the antiparallel β-sheet units of fsp may interact and self-assemble to form supramolecular structures, which leads to the polar localization of the target protein (GFP) and the generation of fluorescent foci in the fspGFP group.

[0088] Example 2: Expression of target protein containing signal peptide and linker peptide

[0089] To control the distance between the functional signal peptide and the target protein and reduce interference, this embodiment investigated the effects of signal peptides and linkers on the expression of the target protein in *E. coli*, using GFP as an example. The signal peptides are shown in Table 1, and the linker information is shown in Table 3. L1 and L2 are flexible linkers, while L3, L4, and L5 are rigid linkers; L1–L3 are known linkers, and L4 and L5 are linkers designed by the applicant.

[0090] Table 3: Information on Connecting Peptides

[0091]

[0092]

[0093] Plasmids pET28a-GFP, pET28a-spGFP, pET28a-fspGFP, pET28a-fspL1GFP, pET28a-fspL2GFP, pET28a-fspL3GFP, pET28a-fspL4GFP, and pET28a-fspL5GFP were transformed into competent *E. coli* cells using standard methods. After culturing for 24 h, the recombinant *E. coli* cells expressing fluorescent proteins were collected by centrifugation. The cells were washed three times with PBS and resuspended in physiological saline. 200 μL of the resuspended cells were added to a 96-well cell culture plate, and the fluorescence value was measured using a microplate reader. Figure 3 The quantitative results of fluorescence in each group of cells are shown. Additionally, approximately 10 μL of the bacterial suspension was transferred onto a glass slide, and the cells were observed and collected using a laser confocal microscope. Figure 4 The confocal image shown is an overlay of the GFP and Bright field images.

[0094] Depend on Figure 3 As can be seen, the fluorescence intensity of all groups of cells expressing the signal peptide was lower than that of the GFP group without the signal peptide (fluorescence intensity 47618). The fluorescence intensity of green fluorescent protein fused with the original signal peptide sp (spGFP) was the lowest (only 124), indicating that the original signal peptide sp significantly affected the expression of the target protein. The fluorescence intensity of green fluorescent protein fused with the functional signal peptide fsp (fspGFP) was 596, which was higher than that of spGFP, indicating that the functional signal peptide improved the expression of the target protein compared to the original signal peptide. Among the proteins expressed by fused signal peptides and linker peptides, the fluorescence intensity increased with the increase of the linker peptide length, with fspL5GFP exhibiting the highest fluorescence intensity.

[0095] Depend on Figure 4 It can be seen that the polar localization of GFP can still be observed when fsp and L1-L5 expression are fused. However, in the fspL5GFP group, fluorescence diffusion occurs, which is speculated to be due to the high protein expression level affecting the fsp-mediated protein aggregation.

[0096] In summary, among proteins that express both signal peptides and linker peptides, fspL4GFP showed the best expression performance and did not exhibit intracellular diffusion.

[0097] Example 3: Co-expression of a dual-target protein containing a functional signal peptide and a linker peptide

[0098] To investigate the co-localization of the two proteins, this embodiment fused the genes encoding fspL4GFP and fspL4mCherry into pET28a to construct the plasmid pET28a-fspL4GFP-fspL4mCherry. This plasmid was then transformed into *E. coli* using conventional methods, achieving co-expression of fspL4GFP and fspL4mCherry in *E. coli*. To visualize the formation of intracellular aggregates, specific excitation and emission filters were used for GFP and mCherry. *E. coli* cells co-expressing fspL4GFP and fspL4mCherry proteins were observed using fluorescence microscopy. The results are as follows: Figure 5 As shown, the Merge column image is a superimposed image of the GFP, mCherry, and Bright field columns. It is evident that the confocal fluorescence image displays GFP and mCherry fluorescence, indicating co-expression of the two fluorescent proteins in *E. coli*. Furthermore, fluorescent particles at the cell poles of both proteins can be observed, suggesting co-localization of the two fluorescent proteins at both ends of *E. coli*.

[0099] Example 4: Enzymes containing functional signal peptides and linker peptides are used to catalyze two-enzyme cascade reactions.

[0100] Ergothioneine is a sulfur-containing small metabolite (229 Da) synthesized by various bacteria and fungi, which can accumulate to millimolar levels in the tissues or cells of higher eukaryotes (such as erythrocytes). Due to its protective and antioxidant functions, it is commonly marketed as a dietary supplement. The cascade reaction for ergothioneine synthesis can be achieved using two enzymes: Egt1, a bifunctional enzyme from *Streptococcus*, and Egt2, a PLP (pyridoxal phosphate)-binding C–S lyase that catalyzes the synthesis of ergothioneine from histidine betaine cysteine ​​sulfoxide.

[0101] This embodiment describes a two-enzyme one-pot reaction catalyzing the synthesis of ergothioneine (EGT) from histidine, cysteine, and S-adenosyl-L-methionine using Egt1 and Egt2, which express functional signal peptides and linker peptides. The aim is to explore the application of enzymes containing functional signal peptides and linker peptides in two-enzyme cascade reactions. The reaction route is as follows: Figure 6 As shown.

[0102] Plasmids pET28a-Egt1-Egt2, pET28a-fspEgt1-fspEgt2, pET28a-fspL4Egt1-fspL4Egt2, and pET28a-fspL5Egt1-fspL5Egt2 were transformed into competent *E. coli* cells using standard methods. The cells were then incubated on a shaker at 30°C and 220 rpm for 12 h. The fermentation broth was pretreated, and the ergothioneine content was measured. The results for each group are shown below. Figure 7 As shown. Figure 7 The strains Egt1-2, fspEgt1-2, fspL4Egt1-2, and fspL5Egt1-2 correspond to plasmids pET28a-Egt1-Egt2, pET28a-fspEgt1-fspEgt2, pET28a-fspL4Egt1-fspL4Egt2, and pET28a-fspL5Egt1-fspL5Egt2, respectively.

[0103] Depend on Figure 7 As can be seen, the EGT yield of the dual-enzyme co-expression strains fused with fspL4 was 73% higher than that of the free dual-enzyme co-expression strains, while the EGT yield of the dual-enzyme co-expression strains fused with fspL5 was only slightly higher than that of the free dual-enzyme co-expression strains. It is speculated that although the combination of fsp-L5 may have increased the expression of the target protein compared to fsp-L4, the diffusion of the target protein significantly reduced the overall catalytic effect of fsp-L5, thus the increase in EGT yield was not significant. The yield of the dual-enzyme co-expression strains fused with fsp was lower than that of the dual-enzyme co-expression strains without fsp, indicating that the addition of fsp affected the enzyme expression level, which is consistent with the results of Example 2.

[0104] Example 5: Enzymes containing functional signal peptides and linker peptides are used to catalyze three-enzyme cascade reactions.

[0105] 6-Hydroxyhexanoic acid can be used both as a platform chemical and as a building block for an important polymer (polycaprolactone). The three-enzyme cascade reaction for the production of 6-hydroxyhexanoic acid involves the conversion of cyclohexanol to cyclohexanone via an alcohol dehydrogenase (ADH) from *Lactobacillus brevis* ATCC 14869, where NADP... + Cyclohexanone is reduced to NADPH. Then, Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter converts cyclohexanone to ε-caprolactone, consuming NADPH in the process. Finally, lactonase from Rhodococcus converts ε-caprolactone to 6-hydroxyhexanoic acid. Since cofactor regeneration is crucial for the economic performance of the cascade, the two enzymes, ADH and CHMO, form a redox equilibrium system in which NADPH is recycled through a hydrogen-borrowing process.

[0106] This embodiment explores the application of enzymes containing functional signal peptides and linker peptides in a three-enzyme cascade reaction by expressing ADH, BVMO, and Lactonnase containing functional signal peptides and linker peptides to catalyze the conversion of cyclohexanol to 6-hydroxyhexanoic acid. The reaction route is as follows: Figure 8 As shown.

[0107] Plasmids pRSFDuet-ADH-BVMO-Lac, pRSFDuet-fspADH-fspBVMO-fspLac, pRSFDuet-fspL4ADH-fspL4BVMO-fspL4Lac, and pRSFDuet-fspL5ADH-fspL5BVMO-fspL5Lac were transformed into competent *E. coli* cells using standard methods. The cells were then incubated on a shaker at 30°C and 220 rpm for 12 h. The fermentation broth was pretreated, and the content of 6-hydroxyhexanoic acid was measured. The results for each group are shown below. Figure 9 As shown. Figure 9 The strains ABL, fspABL, fspL4ABL, and fspL5ABL were transformed into plasmids pRSFDuet-ADH-BVMO-Lac, pRSFDuet-fspADH-fspBVMO-fspLac, pRSFDuet-fspL4ADH-fspL4BVMO-fspL4Lac, and pRSFDuet-fspL5ADH-fspL5BVMO-fspL5Lac, respectively.

[0108] Depend on Figure 9 It is evident that the 6-HHA yield of the three-enzyme co-expression strains fused with fspL4 was 11% higher than that of the strains co-expressing the three enzymes without fsp, while the 6-HHA yield of the three-enzyme co-expression strains fused with fspL5 was slightly higher than that of the strains co-expressing the three enzymes without fsp. This is presumably because fsp-L5 causes target protein diffusion, thus the increase in 6-HHA yield is not significant. The 6-HHA yield of the three-enzyme co-expression strains fused with fsp was lower than that of the three-enzyme co-expression strains without fsp, indicating that the addition of fsp affects the enzyme expression level, which is consistent with the results of Example 2.

[0109] Example 6: Validation of the expression of functional signal peptides in Bacillus subtilis and Mycobacterium.

[0110] Bacillus subtilis is widely used in the production of important industrial biochemical products and pharmaceutical compounds, while mycobacteria play a crucial role in disease prevention and control and industrial production. To explore whether fsp can be applied to multiple model strains, this example transforms plasmids containing the fspGFP expression gene into Bacillus subtilis and mycobacteria for expression verification.

[0111] The plasmid pMA5-fspGFP was transformed into Bacillus subtilis competent cells. Verified positive transformants were inoculated into 5 mL of LB liquid medium and cultured overnight at 37°C and 200 rpm. The transformed cells were then inoculated into Super-Rich medium at a 2% inoculum and cultured at 37°C for 24 h. The transformed cells were observed and collected using a laser confocal microscope. Figure 10 The confocal image shown.

[0112] The plasmid pMV261-fspGFP was transformed into competent Mycobacterium cells. Verified positive transformants were inoculated into test tubes containing 5 mL of LB broth and cultured overnight at 37°C and 200 rpm on a shaker. A 2% inoculum was then transferred to a shake flask containing 50 mL of LB broth and cultured at 37°C for 24 h. The transformed cells were observed and collected using a laser confocal microscope. Figure 11 The confocal image shown.

[0113] Depend on Figure 10 Clear fspGFP fluorescent particles can be observed in Bacillus subtilis cells; Figure 11 As can be seen, fspGFP fluorescent particles can be observed at both ends of the mycobacterial cells, indicating the polar localization of fspGFP. This shows that fspGFP fluorescent foci can still be formed in Bacillus subtilis and mycobacteria, thus verifying the feasibility of further extending the functional signal peptide to Bacillus subtilis and mycobacterial hosts to establish a multi-enzyme cascade reaction system.

Claims

1. A functional signal peptide, characterized in that, The amino acid sequence of the functional signal peptide is shown in SEQ ID NO:

2.

2. A fusion protein, characterized in that, The fusion protein comprises a functional signal peptide, a linker peptide, and a target protein; the amino acid sequence of the functional signal peptide is shown in SEQ ID NO: 2, and the amino acid sequence of the linker peptide is shown in SEQ ID NO:

6.

3. The fusion protein as described in claim 2, characterized in that, The target protein is an enzyme or a fluorescent protein.

4. A polynucleotide, characterized in that, The polynucleotide encodes the functional signal peptide as described in claim 1 or one or more fusion proteins as described in claim 2 or 3.

5. A host cell, characterized in that, The host cell contains the functional signaling peptide as described in claim 1, the fusion protein as described in claim 2 or 3, or the polynucleotide as described in claim 4.

6. The application of the fusion protein as described in claim 2 or 3 in a cascade reaction catalyzed by two or more enzymes, characterized in that, The fusion protein is used as a catalytic component, and the reaction involves the participation of two or more fusion proteins.

7. The application as described in claim 6, characterized in that, The reaction involves the participation of fspL4Egt1 and fspL4Egt2, and the product of the reaction is selected from one or more of histidine betaine, histidine betaine cysteine ​​sulfoxide, and ergothioneine; or, the reaction involves the participation of two or more of fspL4ADH1, fspL4BVMO, and fspL4Lactonnase, and the product of the reaction is selected from one or more of cyclohexanone, caprolactone, and 6-hydroxyhexanoic acid.

8. The application of a fusion protein in cellular localization, characterized in that, The fusion protein comprises a functional signal peptide and a target protein; the amino acid sequence of the functional signal peptide is shown in SEQ ID NO: 2, and the target protein is an enzyme or a fluorescent protein; the fusion protein is used to present aggregated fluorescent blocks or chromogenic blocks in cells.

9. A method for producing the fusion protein as described in claim 2 or 3, characterized in that, The method includes expression using the polynucleotide as described in claim 4 or the host cell as described in claim 5.

10. A method for producing ergothioneine, characterized in that, The method includes expressing fspL4Egt1 and fspL4Egt2 using the polynucleotide as described in claim 4 or the host cell as described in claim 5.

11. A method for producing 6-hydroxyhexanoic acid, characterized in that, The method includes expressing fspL4ADH1, fspL4BVMO, and fspL4Lactonnase using the polynucleotides of claim 4 or the host cells of claim 5.

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