A cleavage module, system for escherichia coli self-cleavage and application thereof

Abstract

The application relates to a lysis module for E. coli autolysis, a system and application thereof, relates to the field of E. coli autolysis, and the lysis module is SP-lysozyme-SsrA, the SP is a nucleotide sequence for coding a signal peptide, the amino acid sequence of the SsrA degradation label is SEQ ID NO:8, the nucleotide sequence of the lysozyme is SEQ ID NO:1, the SP is fused at the N-terminal of the lysozyme, and the SsrA is fused at the C-terminal of the lysozyme. By adopting a simpler and more efficient mode to assist lysozyme in the cytoplasm to enter the periplasmic space, the number of lysis modules is reduced to reduce the metabolic burden caused to host cells; meanwhile, the problem of lysis module leakage expression is solved, cell toxicity is reduced, and the biomass of host cells and the yield of target proteins are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of E. coli auto-lysis, in particular to a lysis module for E. coli auto-lysis, a system and application thereof. BACKGROUND

[0002] It has become a routine strategy in the field of biotechnology to produce high-value recombinant products using expression systems such as bacteria, yeast, and animal cells. Among them, the E. coli expression system is the most popular due to its simplicity, economy, and high productivity. The first step in recovering intracellular target proteins in the E. coli expression system is cell lysis. Traditional mechanical methods such as ultrasonication and high-pressure homogenization can usually achieve ideal lysis results, but they require additional equipment, increasing costs. In addition, the strong shear force and high temperature and pressure generated during the breaking process can easily cause denaturation and loss of activity of the target protein. On the other hand, mechanical breaking methods cannot meet the needs of sample lysis in micro-well plates in high-throughput screening technology.

[0003] The E. coli auto-lysis system completely compensates for the shortcomings of mechanical breaking methods, enabling the in situ, mild, and economical release of intracellular proteins. Not only does it avoid the use of expensive cell disruptors, but it also maintains the high activity state of the target protein during the lysis process. Therefore, this system is a promising tool that can be used for large-scale recovery of target products in industry and applied to the directed evolution of proteases and high-throughput screening.

[0004] The realization of the E. coli auto-lysis process mainly relies on lysozyme to cut the peptidoglycan layer in the periplasmic space, which leads to the collapse of the E. coli cell skeleton and causes cell lysis. Since lysozyme is expressed in the cytoplasm, it cannot reach the peptidoglycan layer in the periplasmic space on its own, so bacteriophage perforin and some proteins with membrane-destroying effects such as phospholipase and D-amino acid oxidase are often co-expressed with lysozyme to assist its entry into the periplasmic space. However, the leaky expression of this multi-enzyme coordinated lysis module can cause more cytotoxicity to the host cells, and the significant metabolic burden and limited metabolic resources can result in low yield of target proteins. Therefore, the present application needs to focus on two problems to establish a new method for a more rigorous and efficient E. coli auto-lysis system. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a lysis module for E. coli auto-lysis, a system and application thereof. The purpose of the present application is to use a simpler and more efficient way to help lysozyme in the cytoplasm enter the periplasmic space, reduce the number of lysis modules to reduce the metabolic burden on host cells; and to solve the problem of leaky expression of the lysis module, reduce cytotoxicity, and improve the biomass of host cells and the yield of target proteins.

[0006] The present application solves the above technical problems, and the first object is to provide a cleavage module for E. coli auto-cleavage, the cleavage module is SP-lysozyme-SsrA, the SP is a nucleotide sequence encoding a signal peptide, the SsrA is a nucleotide sequence encoding an SsrA degradation tag, the amino acid sequence of the SsrA degradation tag is SEQ ID NO: 8, the lysozyme is a nucleotide sequence encoding lysozyme, the nucleotide sequence of the lysozyme is SEQ ID NO: 1, the SP is fused to the N-terminus of the lysozyme, and the SsrA is fused to the C-terminus of the lysozyme.

[0007] The SP can also be fused to the N-terminus of the lysozyme through a hydrophilic linker, for example, fused to the N-terminus of the lysozyme through a GS linker (Gly-Ser), and the hydrophilic linker sequence is added to prevent the SP signal peptide from being embedded in the lysozyme structure and affecting the secretion effect. The length of the hydrophilic linker should be <10 AA to avoid affecting the cleavage activity of the lysozyme.

[0008] The present application has the beneficial effects that: the present application first applies the SsrA degradation tag to the E. coli auto-cleavage system to solve the problem of leaky expression of the cleavage module. Compared with the existing system, the system applying the SsrA degradation tag has higher stringency and lower toxicity than other systems, so that faster growth speed and more target proteins can be accumulated in the same time.

[0009] On the basis of the above technical solution, the present application can also be improved as follows.

[0010] Further, the signal peptide is a Sec pathway signal peptide, a Tat pathway signal peptide or a Sec-Tat dual pathway signal peptide.

[0011] Further, the Sec pathway signal peptide is PelB or PhoA, the amino acid sequence of the PelB is SEQ ID NO: 2, and the amino acid sequence of the PhoA is SEQ ID NO: 3; the Tat pathway signal peptide is TorA or FdoG, the amino acid sequence of the TorA is SEQ ID NO: 4, and the amino acid sequence of the FdoG is SEQ ID NO: 5; the Sec-Tat dual pathway signal peptide is FhuD or MdoG, the amino acid sequence of the MdoG is SEQ ID NO: 6, and the amino acid sequence of the FhuD is SEQ ID NO: 7.

[0012] The beneficial effect of adopting the further scheme is that: through comparative experiments, it is found that the signal peptide is effective for secreting lysozyme into the periplasmic space and establishing a self-cleavage system, and it is first found that the Sec-Tat dual-pathway signal peptide is more suitable for secreting lysozyme into the periplasmic space and establishing a self-cleavage system than the signal peptide of the Sec pathway or the Tat pathway.

[0013] The present application solves the above technical problems, and the second object is to provide: a recombinant vector for E. coli self-cleavage, the recombinant vector comprising the above-mentioned cleavage module.

[0014] The beneficial effect of the present application is to provide a recombinant vector facilitating the expression of the cleavage module.

[0015] Further, the vector is the E. coli expression vector pBAD / His. However, it is not limited to this vector, and the self-cleavage module can be placed after any inducible promoter.

[0016] The beneficial effect of adopting the further scheme is that: the use of the E. coli expression vector pBAD / His enables the cleavage module to achieve better expression in E. coli.

[0017] The present application solves the above technical problems, and the third object is to provide: a system for E. coli self-cleavage, the system for E. coli self-cleavage comprising the above-mentioned recombinant vector.

[0018] The beneficial effect of the present application is that: compared with the existing system, the expression recombinant vector constructed by the cleavage module of the present system is only composed of a single enzyme lysozyme, which not only greatly reduces the metabolic burden on host cells caused by traditional multi-enzyme cleavage modules, but also is more convenient to be applied to other fields.

[0019] Further, the system for E. coli self-cleavage includes a PelB-lysozyme-SsrA system, a PhoA-lysozyme-SsrA system, a TorA-lysozyme-SsrA system, a FdoG-lysozyme-SsrA system, a FhuD-lysozyme-SsrA system, or a MdoG-lysozyme-SsrA system.

[0020] The beneficial effect of the above further scheme is that: the PelB-lysozyme-SsrA system, the PhoA-lysozyme-SsrA system, the TorA-lysozyme-SsrA system, the FdoG-lysozyme-SsrA system, the FhuD-lysozyme-SsrA system or the MdoG-lysozyme-SsrA system established by the signal peptide and the SsrA degradation tag are all effective E. coli self-lysis systems, and the FhuD-lysozyme-SsrA system is the most stringent and efficient E. coli self-lysis system.

[0021] A construction method of the E. coli self-lysis system, the recombinant vector is transformed into the E. coli expression strain BL21 (DE3), and the E. coli self-lysis system is obtained.

[0022] Further, the expression conditions of the E. coli self-lysis system are as follows:

[0023] During the expression of the E. coli self-lysis system in the culture solution, 0.20-0.30% arabinose and 0.4-0.6% Triton X-100, preferably 0.25% arabinose and 0.5% Triton X-100, of the weight of the final culture solution are added, the expression of the lysis module in the recombinant vector is induced for 8-12 h; then according to the final pH of the culture solution, the alkali (for example, 2M NaOH) is used to adjust the culture solution after induction to be slightly alkaline (pH=7.5-9); finally, the cell membrane of E. coli is destroyed at 25°C for 20-40 min to promote the release efficiency of intracellular proteins.

[0024] The beneficial effect of the above further scheme is that: under this condition, the release rate of the target protein is higher.

[0025] The present application solves the above technical problems, and the fourth object is to provide: an application of the E. coli self-lysis system, the E. coli self-lysis system is used for protein release or screening of mutants.

[0026] The beneficial effect of the present application is that: the E. coli self-lysis system developed in the present application is a comprehensive system, which can be used for high-throughput screening of mutants in the laboratory, and can also be applied to large-scale production of recombinant proteins in industry. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A schematic diagram of the E. coli self-lysis system of the present application;

[0028] Figure 2Figure for release efficiency of sfGFP in different signal peptide-mediated self-cleavage systems of the application;

[0029] Figure 3 Figure for optimized E. coli FLSA system of the application; wherein A is the optimization of E. coli FLSA system by disturbing outer membrane of cells in several ways; B is the distribution of sfGFP in the optimized FLSA system analyzed by SDS-PAGE;

[0030] Figure 4 Figure for screening of mutants in 96-well plate by E. coli FLSA system of the application; A is the measurement of amylase activity by DNS method; B is the activity distribution of mutants in two independent experiments. DETAILED DESCRIPTION

[0031] The principles and features of the application are described below, and the examples are only used to explain the application, and are not used to limit the scope of the application. The methods used in the following examples are conventional experimental methods unless otherwise specified. The reagents and biological materials, unless otherwise specified, can be obtained commercially.

[0032] Experimental materials and reagents

[0033] (1) Strains and vectors: E. coli DH5a strain for constructing recombinant vectors and BL21(DE3) and BL21(DE3)pLysS strains for establishing self-cleavage system were purchased from Beijing Genesee Biotechnology Co., Ltd.; vector pBAD / His was purchased from Wuhan Jin Kai Rui Biological Engineering Co., Ltd.

[0034] (2) Enzymes and kits: The restriction enzymes used in this experiment were purchased from NEB company, PrimeSTAR Max Premix(2X) used in polymerase chain reaction was purchased from Takara company, and the kits were purchased from Nanjing Novozyme Biological Technology Co., Ltd.

[0035] (3) Reagents: The remaining reagents were purchased from regular distributors.

[0036] Example 1: Use of SsrA degradation tag to assist construction of cleavage module recombinant plasmid

[0037] Use of SsrA degradation tag (SEQ ID NO: 8) to assist construction of cleavage module recombinant plasmid, including the following steps:

[0038] First step: T7 lysozyme gene with a size of 650 bp was amplified from E. coli expression strain BL21(DE3)pLysS, and the sequence of its nucleotides is shown in SEQ ID NO: 1;

[0039] PCR reaction solution was added in the following proportion: 25 μL PrimeSTAR Max Premix (2X), 1.5 μL Primer 1 (SEQ ID NO: 9), 1.5 μL Primer 2 (SEQ ID NO: 10), 1 μL BL21(DE3)pLysS bacterial solution, 21 μL ddH2O, and the mixed reaction solution was subjected to PCR reaction in the following system (as shown in Table 1, the sequence of the primer is shown in Table 3):

[0040] Table 1 PCR reaction process for amplifying T7 lysozyme gene

[0041]

[0042] Second step: two high-efficiency signal peptides were selected from the bacterial classical secretion pathway (Sec), the double arginine secretion pathway (Tat), and the Sec-Tat double pathway, respectively, wherein the Sec pathway signal peptide selected PelB (SEQ ID NO: 2) and PhoA (SEQ ID NO: 3), the Tat pathway signal peptide selected TorA (SEQ ID NO: 4) and FdoG (SEQ ID NO: 5), and the Sec-Tat double pathway signal peptide selected MdoG (SEQ ID NO: 6) and FhuD (SEQ ID NO: 7), the amino acid sequence of the signal peptide is shown in Table 4;

[0043] Third step: using fusion PCR technology, the nucleic acid sequences encoding the six signal peptides were fused to the N-terminus of the T7 lysozyme gene through a GS linker (Gly-Ser) sequence, respectively, to obtain a group of nucleic acid sequences of the cleavage module SP-lysozyme;

[0044] The operation process of fusion PCR is as follows: the PCR reaction solution is mixed in the following system: 25 μL PrimeSTAR Max Premix (2X), 1.5 μL Primer 3-8 (the forward primers of the six signal peptides, SEQ ID NO: 11 to SEQ ID NO: 16, respectively), 1.5 μL Primer 9 (SEQ ID NO: 17), 20 ng of the T7 lysozyme fragment amplified in the first step, 10 ng of the signal peptide fragment, and supplemented with ddH2O to a final volume of 50 μL, and the mixed reaction solution was subjected to PCR reaction in the following system (as shown in Table 2, the sequence of the primer is shown in Table 3):

[0045] Table 2 Operation process of fusion PCR reaction

[0046]

[0047] Fourth step: using a one-step cloning kit (C112) developed by Nanjing Novozyme Biotech Co., Ltd. to clone the nucleic acid sequences of the six lysis modules into the E. coli expression vector pBAD / His(NcoI / Hind III), respectively. The specific operation method can be referred to the product manual. However, it was found that the recombinant plasmid strains constructed by the Tat pathway signal peptide TorA, FdoG and the Sec-Tat dual pathway signal peptide MdoG, FhuD could not grow, because the leakage expression of the lysis module caused the lysis of the cloned strain. Therefore, the nucleic acid sequence encoding SsrA degradation tag was fused to the C-terminus of the six lysis modules (SP-T7 lysozyme), respectively, to obtain a group of SP-lysozyme-SsrA lysis module nucleic acid sequences.

[0048] The operation process of fusion PCR is as follows: mix the PCR reaction liquid according to the following system: 25 μL PrimeSTAR Max Premix (2X), 1.5 μL Primer 3-8 (forward primers of the six signal peptides, SEQ ID NO: 11 to SEQ ID NO: 16), 1.5 μL Primer 10 / 11 (SEQ ID NO: 18 and SEQ ID NO: 19), 30 ng SP-T7 lysozyme template, and supplement ddH2O to a final volume of 50 μL. The mixed reaction liquid is subjected to PCR reaction according to the system used in the third step: (the primer sequence is shown in Table 3)

[0049] Fifth step: using a one-step cloning kit (C112) developed by Nanjing Novozyme Biotech Co., Ltd. to clone the nucleic acid sequences of the six SP-lysozyme-SsrA into the E. coli expression vector pBAD / His, respectively. The specific operation method can be referred to the product manual. Sequencing verification obtained six correct recombinant plasmids.

[0050] Table 3 Primer sequence used in PCR reaction

[0051]

[0052]

[0053] Table 4 Amino acid sequence of signal peptide

[0054]

[0055] Case 2: verification of the efficiency of the lysis of the lysis module recombinant plasmid

[0056] Verification of the efficiency of the lysis of the lysis module recombinant plasmid includes the following steps:

[0057] First, the correct lysing module recombinant plasmid and the reporter gene expression plasmid pET28a-sfGFP were co-transformed into the E. coli expression strain BL21 (DE3) according to the following system and method. 2 μL of pBAD / His-SP-lysozyme-SsrA lysing module recombinant plasmid and 2 μL of reporter gene plasmid were added to 50 μL of BL21 (DE3) competent cells, mixed, and then placed on ice for 5 min. Then, the system was heated in a water bath at 42°C for 35 s, and then 500 μL of fresh LB culture solution was added. After incubation at 37°C for 1 h, the sample was plated on an ampicillin / kanaamycin double-antibiotic plate and placed in a 37°C incubator for overnight growth until colonies grew.

[0058] The composition of the LB culture solution was as follows: tryptone 1%, yeast extract 0.5%, and NaCl 1%.

[0059] Then, the successfully transformed single colony strains were inoculated in 2 ml of LB culture medium and incubated at 37°C overnight. The next day, the sample was transferred to 10 mL of fresh LB culture medium and incubated at 37°C until the OD 600 = 0.6-0.8. Then, 0.5 mM IPTG (isopropyl thiogalactoside) was added to the sample to induce the expression of sfGFP at 18°C for 20 h. During the expression process, 0.25% arabinose was added to the culture solution to induce the expression of the lysing module for 10 h.

[0060] Second, the total fluorescence of the induced sample was measured by a fluorescence microplate reader, and part of the sample was centrifuged at 12000 rpm and 4°C for 10 min to collect the supernatant for measuring the supernatant fluorescence.

[0061] The calculation formula of the lysis efficiency was as follows: lysis efficiency = (supernatant fluorescence / total fluorescence) * 100%.

[0062] The schematic diagram of the E. coli self-lysing system is shown in Figure 1 .

[0063] The lysis efficiency of the six lysing module recombinant plasmids was calculated, and the results are shown in the following table. Figure 2 The lysis efficiency of the six lysing module recombinant plasmids was calculated, and the results are shown in the following table. Figure 2The results show that the cleavage efficiency of lysozyme-SsrA module fused with Sec signal peptide PelB and PhoA is 8.4±0.7% and 12.5±0.3% respectively; the cleavage efficiency of lysozyme-SsrA module fused with Tat signal peptide TorA and FdoG is 34.7±2.3% and 47.7±3.1% respectively; the cleavage efficiency of lysozyme-SsrA module fused with Sec-Tat dual pathway signal peptide FhuD and MdoG is 58.3±0.7% and 52.0±1.4% respectively.

[0064] Case 3: Optimization of E. coli FLSA system for expressing sfGFP

[0065] Based on the results of Example 2, the self-cleavage system established by the recombinant plasmid of the highest efficiency FhuD-lysozyme-SsrA cleavage module is named as E. coli FLSA (FhuD-lysozyme-SsrA autolytic) system.

[0066] Optimization of E. coli FLSA system for expressing sfGFP includes the following steps:

[0067] First step, according to the method described in the first step of Example 2, induce expression of reporter gene sfGFP in E. coli FLSA system. During the expression process, add 0.25% arabinose and 0.5% Triton X-100 (polyethylene glycol octylphenyl ether) to induce the expression of cleavage module for 10h; after the expression is completed, according to the final pH of the culture solution, adjust the induced culture solution to slightly alkaline (pH=8.0) with 2M NaOH, and culture at 25℃ for 30min to promote cell membrane damage and accelerate the release efficiency of intracellular protein.

[0068] Second step, measure the total fluorescence of the induced sample with a fluorescence microplate reader, and measure the supernatant fluorescence of the supernatant collected by centrifuging part of the sample at 12000rpm, 4℃ for 10min.

[0069] Third step, calculate the release efficiency of sfGFP in the optimized FLSA system: release efficiency=(supernatant fluorescence / total fluorescence)*100%.

[0070] The release efficiency of sfGFP in the optimized FLSA system is shown in Figure Figure 3 A, the release efficiency of sfGFP in the optimized FLSA system is increased from 58% to 82%. As shown in Figure Figure 3As shown in Figure B, the yield of sfGFP in the optimized FLSA system was the same as the control group without inducing the expression of the cleavage module, indicating that the cleavage module in the FLSA system did not leak expression to be toxic to the host cells and did not occupy the metabolic resources of the target protein sfGFP.

[0071] Case 4: Using the E. coli FLSA system to lyse cells in a 96-well plate for screening an α-amylase mutant library

[0072] Using the E. coli FLSA system to lyse cells in a 96-well plate for screening an α-amylase mutant library includes the following steps:

[0073] First, the laboratory previously isolated and modified a kind of alkaline α-amylase N-Amy (article PMID number: 26926401) from Bacillus, and to further improve its activity, a mutant library of N-Amy was established, and 12 mutants were randomly selected to determine the activity in the E. coli FLSA system. The 12 mutants are Y220S, E199A, G372Q, Y322F, Y372W, G379R, S354Y, D353S, L416V, D32G, G379M, and G372E.

[0074] The mutants were transformed into the E. coli FLSA system according to the following method. 50 μL of BL21(DE3) competent cells were added with 2 μL of pBAD / His-FhuD-lysozyme-SsrA cleavage module recombinant plasmid and 2 μL of mutant plasmid, mixed, and placed on ice for 5 min, then heated in a 42°C water bath for 35 s, then 500 μL of fresh LB culture solution was added, and the system was incubated at 37°C for 1 h, then plated on ampicillin / kanaamycin double-antibiotic plates, and placed in a 37°C incubator overnight to grow colonies.

[0075] Second, single colonies were inoculated into a 96-deep well plate (about 400 μL of LB medium per well), and after the sample was cultured at 37°C for about 2-3 h, 0.5 mM IPTG was added to induce the expression of the α-amylase mutant for 20 h;

[0076] Third, during the expression process, 0.25% arabinose and 0.5% Triton X-100 were added to induce the expression of the cleavage module for 10 h;

[0077] Fourth, after the expression was completed, the activity of the α-amylase mutant was directly determined using soluble starch as the substrate.

[0078] Take 3 μL lysate and 100 μL 1% soluble starch mixed, after 10 min reaction at 37℃, the reducing sugar content produced by α-amylase mutant hydrolysis of soluble starch was determined by 3,5-dinitrosalicylic acid method. As shown in the accompanying Figure 4 Figure 2, the E. coli FLSA system can stably achieve E. coli cell self-lysis in a 96-well plate small system (the accompanying Figure 4 A), the corresponding activity of the 12 mutants tested was detected (the accompanying Figure 4 B).

[0079] Although the embodiments of the present application have been shown and described above, it is to be understood that the above-mentioned embodiments are exemplary and are not to be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above-mentioned embodiments within the scope of the present application.

Claims

1. A lysis module for E. coli auto-lysis, characterized in that, The cleavage module is SP-lysozyme-SsrA, the SP is a nucleotide sequence encoding a signal peptide, the SsrA is a nucleotide sequence encoding an SsrA degradation tag, the amino acid sequence of the SsrA degradation tag is SEQ ID NO: 8, the lysozyme is a nucleotide sequence encoding lysozyme, the nucleotide sequence of the lysozyme is SEQ ID NO: 1, the SP is fused to the N-terminus of the lysozyme through a hydrophilic linker, and the SsrA is fused to the C-terminus of the lysozyme; The signal peptide is a Sec pathway signal peptide, a Tat pathway signal peptide, or a Sec-Tat dual pathway signal peptide; The Sec pathway signal peptide is PelB or PhoA, the amino acid sequence of the PelB is SEQ ID NO: 2, and the amino acid sequence of the PhoA is SEQ ID NO: 3; the Tat pathway signal peptide is TorA or FdoG, the amino acid sequence of the TorA is SEQ ID NO: 4, and the amino acid sequence of the FdoG is SEQ ID NO: 5; the Sec-Tat dual pathway signal peptide is FhuD or MdoG, the amino acid sequence of the MdoG is SEQ ID NO: 6, and the amino acid sequence of the FhuD is SEQ ID NO:

7.

2. A recombinant vector for E. coli auto-lysis, characterized in that, The recombinant vector comprises the cleavage module of claim 1.

3. The recombinant vector for E. coli auto-cleavage according to claim 2, wherein, The vector is an Escherichia coli expression vector pBAD / His.

4. A system for E. coli auto-lysis, characterized by, The system for self-cleavage of Escherichia coli comprises the recombinant vector of claim 2 or 3.

5. The system for E. coli auto-lysis according to claim 4, wherein, The system for self-cleavage of Escherichia coli comprises a PelB-lysozyme-SsrA system, a PhoA-lysozyme-SsrA system, a TorA-lysozyme-SsrA system, a FdoG-lysozyme-SsrA system, a FhuD-lysozyme-SsrA system, or a MdoG-lysozyme-SsrA system.

6. A method for constructing a system for self-cleavage of E. coli according to claim 4 or 5, characterized in that, The recombinant vector is transformed into Escherichia coli, thereby obtaining the system for self-cleavage of Escherichia coli.

7. The method of claim 6, wherein the system is constructed by the steps of: The expression conditions of the system for self-cleavage of Escherichia coli are as follows: During the expression of the system for self-cleavage of Escherichia coli, 0.20-0.30% arabinose and 0.4-0.6% Triton X-100 by weight of the final culture medium are added, and the expression of the cleavage module in the recombinant vector is induced for 8-12 h.

8. Use of a system according to claim 4 or 5 for the self-lysis of E. coli, characterized in that, The system is used for the release of a target protein or the screening of mutants. The system is used for the release of a target protein or the screening of mutants.

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