CN116179581B - Cracking module and system for self-cracking of escherichia coli and application of cracking module and system - Google Patents

Abstract

The invention relates to a cracking module, a system and application thereof for self-cracking of escherichia coli, and relates to the field of self-cracking of escherichia coli, wherein the cracking module is SP-lysozyme-SsrA, the SP is a nucleotide sequence for encoding signal peptide, the amino acid sequence of an SsrA degradation tag is SEQ ID NO 8, the nucleotide sequence of lysozyme is SEQ ID NO 1, the SP is fused at the N-end of lysozyme, and the SsrA is fused at the C-end of lysozyme. The method helps lysozyme (lysozyme) in cytoplasm enter periplasmic space by adopting a simpler and more efficient mode, reduces the number of the lysis modules to reduce metabolic burden on host cells, solves the problem of leakage expression of the lysis modules, reduces cytotoxicity, and improves biomass and target protein yield of the host cells.

Description

Cracking module and system for self-cracking of escherichia coli and application of cracking module and system

Technical Field

The invention relates to the field of self-cracking of escherichia coli, in particular to a cracking module and system for self-cracking of escherichia coli and application thereof.

Background

The production of high value-added recombinant products using expression systems such as bacteria, yeast, animal cells, etc. has become a routine strategy in the biotechnology field, where E.coli systems have been the most popular expression system for their simplicity, economy and high productivity. The first step in the recovery of intracellular proteins of interest in E.coli expression systems is cell lysis. Conventional mechanical methods, such as ultrasonic treatment and high-pressure homogenization, generally achieve the desired cleavage effect, but require additional equipment to increase the cost, and the strong shearing force and high temperature and high pressure generated during the cleavage process easily cause problems such as denaturation and activity loss of the target protein. On the other hand, mechanical disruption methods do not meet the requirement of sample lysis in microplates in high throughput screening techniques.

The escherichia coli self-cracking system completely makes up the defects of a mechanical cracking method, can realize in-situ, mild and economical release of intracellular proteins, avoids the use of an expensive cell breaker, and can maintain the high-activity state of target proteins in the cracking process. Therefore, the system is a promising tool, can be used for industrial large-scale recovery of target products, and can also be applied to directed evolution and high-throughput screening of proteases.

The implementation of the E.coli self-lysis process relies mainly on lysozyme (lysozyme) to cleave the peptidoglycan layer of the periplasmic space, which results in collapse of the E.coli cytoskeleton resulting in cell lysis. Since lysozyme lysozyme is present in the cytoplasm after expression, it cannot reach the peptidoglycan layer of the periplasmic space by itself, phage perforins and some proteins with membrane disruption effects such as phospholipase, D-amino acid oxidase are often co-expressed with lysozyme to assist its entry into the periplasmic space. However, such multi-enzyme coordinated leaky expression of the cleavage module may bring more cytotoxicity to the host cell, while significant metabolic load and limited metabolic resources may result in lower yields of target protein. Therefore, the invention needs to focus on two problems to establish a more rigorous and efficient new method for the self-lysis system of the escherichia coli.

Disclosure of Invention

The invention aims to provide a cracking module and a system for self-cracking of escherichia coli and application thereof. The invention aims to assist lysozyme (lysozyme) in cytoplasm to enter periplasmic space in a simpler and more efficient way, reduce the number of the lysis modules to reduce the metabolic burden on host cells, solve the problem of leakage expression of the lysis modules, reduce cytotoxicity and improve the biomass of the host cells and the yield of target proteins.

The invention solves the technical problems, and the first object is to provide a lysis module for self-lysis of escherichia coli, wherein the lysis module is SP-lysozyme-SsrA, the SP is a nucleotide sequence for coding a signal peptide, the SsrA is a nucleotide sequence for coding an SsrA degradation tag, the amino acid sequence of the SsrA degradation tag is SEQ ID NO:8, lysozyme is a nucleotide sequence for coding lysozyme, the nucleotide sequence of lysozyme is SEQ ID NO:1, the SP is fused at the N-end of lysozyme, and the SsrA is fused at the C-end of lysozyme.

The SP may also be fused to the N-terminus of lysozyme by a hydrophilic linker, for example, to the N-terminus of lysozyme by a GS linker (Gly-Ser), which is added to prevent the SP signal peptide from being embedded within the lysozyme structure and affecting secretion. The length of the hydrophilic linker should be <10AA so as not to affect the cleavage activity of lysozyme.

The invention has the beneficial effects that the SsrA degradation tag is firstly applied to the escherichia coli self-lysis system to solve the problem of leakage expression of the lysis module. Compared with the existing system, the system using the SsrA degradation tag has higher stringency and lower toxicity than other systems, so that the system can obtain a faster growth speed and accumulate more target proteins in the same time.

On the basis of the technical scheme, the invention can be improved as follows.

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

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

The beneficial effects of the further scheme are that the signal peptide is found to be effective for secreting lysozyme to the periplasmic space and establishing a self-cleavage system through a comparison experiment, and the Sec-Tat dual-pathway signal peptide is found to be more suitable for secreting lysozyme to the periplasmic space and establishing the self-cleavage system than the signal peptide of the Sec pathway or the Tat pathway for the first time.

The invention solves the technical problem, and a second object is to provide a recombinant vector for self-lysis of escherichia coli, wherein the recombinant vector comprises the lysis module.

The invention has the beneficial effect of providing a recombinant vector which is convenient for the expression of the cleavage module.

Further, the vector is an E.coli expression vector pBAD/His. But is not limited to this vector, the self-cleaving module may be placed after any inducible promoter.

The adoption of the further scheme has the beneficial effect that the adoption of the escherichia coli expression vector pBAD/His enables the lysis module to realize better expression in escherichia coli.

The invention solves the technical problem, and a third object is to provide a system for self-lysing of Escherichia coli, which comprises the recombinant vector.

Compared with the existing system, the expression recombinant vector constructed by the system cracking module is only composed of lysozyme single enzymes, so that the metabolic burden of the traditional multienzyme cracking module on host cells is greatly reduced, and the system is more convenient to apply to other fields.

Further, the E.coli self-lysis system comprises a PelB-lysozyme-SsrA system, a PhoA-lysozyme-SsrA system, a TorrA-lysozyme-SsrA system, a FdoG-lysozyme-SsrA system, a FhuD-lysozyme-SsrA system or a MdoG-lysozyme-SsrA system.

The beneficial effects of the adoption of the further scheme are that the PelB-lysozyme-SsrA system, the PhoA-lysozyme-SsrA system, the TorrA-lysozyme-SsrA system, the FdoG-lysozyme-SsrA system, the FhuD-lysozyme-SsrA system or the MdoG-lysozyme-SsrA system which are obtained by establishing the signal peptide and SsrA degradation tag are all effective escherichia coli self-lysis systems, wherein the FhuD-lysozyme-SsrA system is the most strict and efficient escherichia coli self-lysis system.

The construction method of the system for self-cracking of the escherichia coli comprises the step of transforming the recombinant vector into an escherichia coli expression strain BL21 (DE 3) to obtain the system for self-cracking of the escherichia coli.

Further, the expression conditions of the system for self-lysis of E.coli are:

During the expression of the escherichia coli self-lysing system in the culture medium, arabinose and Triton X-100 which are added in an amount of 0.20-0.30% and preferably 0.25% and Triton X-100 which are added in an amount of 0.4-0.6% by weight of the final culture medium are used for inducing the expression of the lysing modules in the recombinant vector for 8-12 hours, then the induced culture medium is adjusted to be slightly alkaline (pH=7.5-9) by selecting alkali (2M NaOH) according to the final pH of the culture medium, and finally the cell membrane disruption of the escherichia coli is promoted by shaking culture for 20-40min at 25 ℃ to accelerate the release efficiency of intracellular proteins.

The further scheme has the beneficial effect that the release rate of the target protein is higher under the condition.

The invention solves the technical problems, and a fourth object is to provide an application of a system for self-cracking of escherichia coli, which is used for releasing protein or screening mutants.

The invention has the beneficial effects that the system for self-cracking of the escherichia coli developed in the invention is a comprehensive system, and can be used for high-throughput screening of mutants in a laboratory and industrial mass production of recombinant proteins.

Drawings

FIG. 1 is a schematic diagram of the system for self-lysing of E.coli according to the present invention;

FIG. 2 is a graph showing the release efficiency of sfGFP of the invention in a self-cleaving system mediated by different signal peptides;

FIG. 3 is a diagram showing the effect of the optimized E.coli FLSA system of the present invention, wherein A is the optimized E.coli FLSA system by perturbing the cell outer membrane in several ways, and B is the distribution of sfGFP in the optimized FLSA system by SDS-PAGE analysis;

FIG. 4 shows the screening of mutants in 96-well plates by E.coli FLSA system according to the present invention, A is the measurement of amylase activity by DNS method, and B is the activity distribution of mutants in two independent experiments.

Detailed Description

The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The methods used in the following examples are conventional experimental methods unless otherwise specified. The reagents and biological materials are commercially available unless otherwise indicated.

Experimental materials and reagents

(1) Strains and vectors E.coli DH 5. Alpha. Strains for constructing recombinant vectors and BL21 (DE 3) pLysS strains for constructing self-lysing systems were purchased from Beijing-family Biotech Co., ltd, and vector pBAD/His was purchased from Wuhan Jin Kairui bioengineering Co., ltd.

(2) The restriction enzymes used in this experiment were purchased from NEB, PRIMESTAR MAX PREMIX (2X) used in the polymerase chain reaction was purchased from Takara, and the kits were purchased from Nanjinotazan Biotechnology Co.Ltd.

(3) Reagents the remaining reagents were purchased from each of the regular dealerships.

Example 1 construction of a cleavage Module recombinant plasmid Using SsrA degradation tag

SsrA degradation tag (SEQ ID NO: 8) is utilized to assist in constructing a cleavage module recombinant plasmid, which comprises the following steps:

The first step is that the T7lysozyme gene is amplified from the escherichia coli (ESCHERICHIA COLI) expression strain BL21 (DE 3) pLysS, the size of the gene is 650bp, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1;

The PCR reaction solution ：25μL PrimeSTAR Max Premix(2X),1.5μL Primer 1(SEQ ID NO：9),1.5μL Primer 2(SEQ ID NO：10),1μL BL21(DE3)pLysS was added in the following ratio to 21. Mu.L of ddH ₂ O, and the mixed reaction solution was subjected to PCR reaction in accordance with the following system (see Table 3 for primer sequences shown in Table 1):

TABLE 1 PCR reaction procedure for amplifying T7lysozyme Gene

Secondly, selecting 2 high-efficiency signal peptides from a bacterial classical secretory pathway (Sec), a double arginine secretory pathway (Tat) and a Sec-Tat double pathway, wherein PelB (SEQ ID NO: 2) and PhoA (SEQ ID NO: 3) are selected from the Sec pathway signal peptides, torr (SEQ ID NO: 4) and FdoG (SEQ ID NO: 5) are selected from the Tat pathway signal peptides, mdoG (SEQ ID NO: 6) and FhuD (SEQ ID NO: 7) are selected from the Sec pathway signal peptides, and the amino acid sequences of the signal peptides are shown in Table 4;

Thirdly, respectively fusing nucleic acid sequences encoding 6 signal peptides at the N-end of a T7 lysozyme gene through a GS linker (Gly-Ser) sequence by utilizing a fusion PCR technology to obtain a group of nucleic acid sequences of a cleavage module SP-lysozyme;

The fusion PCR was performed by mixing a PCR reaction solution of 25. Mu. L PRIMESTAR Max Premix (2X), 1.5. Mu.L of Primer 3-8 (forward primers of 6 signal peptides, SEQ ID NO:11 to SEQ ID NO: 16), 1.5. Mu.L of Primer 9 (SEQ ID NO: 17), 20ng of the T7 lysozyme fragment amplified in the first step, 10ng of the signal peptide fragment, and adding ddH ₂ O to a final volume of 50. Mu.L according to the following system, and performing a PCR reaction of the mixed reaction solution according to the following system (as shown in Table 2, primer sequences are shown in Table 3):

TABLE 2 fusion PCR reaction procedure

The fourth step is to clone the nucleic acid sequences of the 6 cleavage modules into the E.coli expression vector pBAD/His (NcoI/Hi III) respectively by using a one-step cloning kit (product number C112) developed by Nanjinouzan Biotechnology Co., ltd. However, in the process of constructing the vector, it was found that the recombinant plasmid strain cannot grow by participating in construction of the Tat pathway signal peptides TorA, fdoG and Sec-Tat dual pathway signal peptides MdoG and FhuD, and the cleavage of the cloned strain is caused by the leaky expression of the cleavage module. Therefore, the nucleic acid sequences encoding SsrA degradation tags are fused to the C-terminal of the 6 cleavage modules (SP-T7 lysozyme) respectively, to obtain a group of nucleic acid sequences of SP-lysozyme-SsrA cleavage modules.

The fusion PCR was performed by mixing the PCR reaction solution (25. Mu. L PRIMESTAR Max Premix (2X), 1.5. Mu.L of Primer 3-8 (forward primers of 6 signal peptides, SEQ ID NO:11 to SEQ ID NO: 16), 1.5. Mu.L of Primer 10/11 (SEQ ID NO:18 and SEQ ID NO: 19), 30ng of SP-T7 lysozyme template, and adding ddH ₂ O to a final volume of 50. Mu.L according to the system used in the third step, and performing the PCR reaction according to the mixed reaction solution (Primer sequences are shown in Table 3)

And fifthly, cloning the nucleic acid sequences of 6 SP-lysozyme-SsrA into an escherichia coli expression vector pBAD/His by using a one-step cloning kit (product number C112) developed by Nanjinouzan biotechnology Co., ltd. Sequencing and verification obtain 6 correct recombinant plasmids.

TABLE 3 primer sequences used in PCR reactions

TABLE 4 amino acid sequence of Signal peptides

Example 2 results of cleavage Module verification of the efficiency of cleavage of recombinant plasmids

The result verification of the efficiency of the recombinant plasmid cleavage by the cleavage module comprises the following steps:

In the first step, the recombinant plasmid of the cleavage module which is verified to be correct is co-transformed into E.coli expression strain BL21 (DE 3) with reporter gene expression plasmid pET28a-sfGFP, respectively, according to the following system and method. 50. Mu.L BL21 (DE 3) competent cells were added with 2. Mu.L pBAD/His-SP-lysozyme-SsrA cleavage module recombinant plasmid and 2. Mu.L reporter plasmid, mixed well, left on ice for 5min, heat-shocked in a 42℃water bath for 35s, then 500. Mu.L fresh LB medium was added to the system, incubated for 1h in a 37℃shaker, and then coated with ampicillin/kanamycin double resistance plates, and placed in a 37℃incubator overnight until colonies grew.

The LB medium consisted of 1% tryptone (tryptone), 0.5% yeast extract (yeast extract) and 1% sodium chloride (NaCl).

Then, the successfully transformed monoclonal strains are respectively picked and inoculated in 2mL of LB culture medium for overnight culture at 37 ℃, transferred into 10mL of fresh LB culture medium in the next day, cultured at 37 ℃ until OD ₆₀₀ = 0.6-0.8, added with 0.5mM IPTG (isopropyl thiogalactoside) to the sample for inducing sfGFP to express for 20h at 18 ℃, and added with 0.25% arabinose to induce a lysis module to express for 10h in the culture solution in the expression process.

And secondly, measuring the total fluorescence of the culture solution by using a fluorescence enzyme-labeled instrument on the induced sample, and simultaneously taking part of the sample, centrifuging for 10min at 12000rpm and 4 ℃, and collecting the supernatant to measure the fluorescence of the supernatant.

Calculation formula of cleavage efficiency cleavage efficiency= (supernatant fluorescence/total fluorescence) ×100%;

the schematic diagram of the system of the invention for the self-lysis of E.coli is shown in FIG. 1.

The cleavage efficiency obtained by the recombinant plasmids of the 6 cleavage modules was calculated as shown in FIG. 2. As can be seen from the results of FIG. 2, the cleavage efficiencies obtained for the lysozyme-SsrA modules fused to the Sec signal peptide PelB and PhoA were 8.4.+ -. 0.7% and 12.5.+ -. 0.3%, respectively, the cleavage efficiencies obtained for the lysozyme-SsrA modules fused to the Tat signal peptide Torr and FdoG were 34.7.+ -. 2.3% and 47.7.+ -. 3.1%, respectively, and the cleavage efficiencies obtained for the lysozyme-SsrA modules fused to the Sec-Tat dual-pathway signal peptides FhuD and MdoG were 58.3.+ -. 0.7% and 52.0.+ -. 1.4%, respectively.

Embodiment 3 optimization of E.coli FLSA System for expression of sfGFP

Based on the example 2, the self-cleavage system established by the FhuD-lysozyme-SsrA cleavage module recombinant plasmid with highest efficiency is named as an escherichia coli FLSA (FhuD-lysozyme-SsrA autolytic) system.

The E.coli FLSA system was optimized for expression of sfGFP, comprising the steps of:

In the first step, the expression of reporter gene sfGFP was induced in the E.coli FLSA system according to the method described in the first step of example 2. During the expression process, 0.25% of arabinose and 0.5% of Triton X-100 (polyethylene glycol octyl phenyl ether) are added to induce the expression of the cleavage module for 10 hours, after the expression is finished, the induced culture solution is adjusted to be slightly alkaline (pH=8.0) by 2M NaOH according to the final pH of the culture solution, and the culture solution is subjected to shaking culture for 30 minutes at 25 ℃ to promote the cell membrane disruption so as to accelerate the release efficiency of intracellular proteins.

And secondly, measuring the total fluorescence of the culture solution by using a fluorescence enzyme-labeled instrument on the induced sample, and simultaneously taking part of the sample, centrifuging for 10min at 12000rpm and 4 ℃, and collecting the supernatant to measure the fluorescence of the supernatant.

Third, the release efficiency of sfGFP in the optimized FLSA system was calculated as release efficiency= (supernatant fluorescence/total fluorescence) ×100%.

The result of the release efficiency of sfGFP in the optimized FLSA system is shown in FIG. 3A, and the release efficiency of sfGFP in the optimized FLSA system is improved from 58% to 82%. As shown in FIG. 3B, the yield of sfGFP in the optimized FLSA system is the same as that of the control group without inducing expression of the cleavage module, which indicates that the cleavage module in the FLSA system does not leak expression to cause toxicity to host cells and does not occupy the metabolic resources of the target protein sfGFP.

Embodiment 4 lysis of cells in 96 well plates Using E.coli FLSA System for screening of alpha-Amylase mutant pool

Cells were lysed in 96-well plates using the E.coli FLSA system for screening of a library of alpha-amylase mutants comprising the steps of:

In the first step, alkaline alpha-amylase N-Amy (PMID number: 26926401) is isolated and modified from bacillus in the early stage of the laboratory, a mutant library of N-Amy is established for further improving the activity of the alkaline alpha-amylase, and 12 mutants are randomly selected from the mutant library to determine the activity in an escherichia coli FLSA system. The 12 mutants were Y220S, E199A, G372Q, Y322F, Y372W, G379R, S354Y, D353S, L416V, D32G, G379M, G372E, respectively.

The mutants were transformed into E.coli FLSA systems, respectively, as follows. 50. Mu.L BL21 (DE 3) competent cells were added with 2. Mu.L pBAD/His-FhuD-lysozyme-SsrA cleavage module recombinant plasmid and 2. Mu.L mutant plasmid, mixed well, left on ice for 5min, heat-shocked in a 42℃water bath for 35s, then 500. Mu.L fresh LB medium was added to the system, incubated for 1h in a 37℃shaker, and then coated with ampicillin/kanamycin double resistant plates, and placed in a 37℃incubator overnight until colonies grew out.

Second, single colonies were inoculated into 96-deep well plates (about 400. Mu.L LB medium per well), and after the samples were cultured at 37℃for about 2-3 hours, 0.5mM IPTG was added to the samples to induce expression of the alpha-amylase mutant for 20 hours;

thirdly, adding 0.25% of arabinose and 0.5% of TritonX-100 to induce the lysis module to express for 10 hours in the expression process;

fourth, after the expression is completed, the activity of the alpha-amylase mutant is directly measured by taking soluble starch as a substrate.

3. Mu.L of the lysate and 100. Mu.L of 1% soluble starch were mixed and reacted at 37℃for 10min, and the reducing sugar content produced by hydrolysis of the soluble starch by the alpha-amylase mutant was determined by the method of 3, 5-dinitrosalicylic acid. As shown in FIG. 4, the E.coli FLSA system was able to stably achieve E.coli cell self-lysis in a 96-well plate format (FIG. 4A), and all of the 12 mutants tested were tested for their corresponding activities (FIG. 4B).

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (8)

1. A cleavage module for self-cleavage of escherichia coli, wherein 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, lysozyme is a nucleotide sequence encoding lysozyme, the nucleotide sequence of lysozyme is SEQ ID No. 1, the SP is fused at the N-terminus of lysozyme by a hydrophilic linker, and the SsrA is fused at the C-terminus of lysozyme;

the signal peptide is Sec pathway signal peptide, tat pathway signal peptide or Sec-Tat double pathway signal peptide;

The Sec pathway signal peptide is PelB or PhoA, the amino acid sequence of the PelB is SEQ ID NO. 2, the amino acid sequence of the PhoA is SEQ ID NO. 3, the Tat pathway signal peptide is TorrA or FdoG, the amino acid sequence of TorrA is SEQ ID NO. 4, the amino acid sequence of FdoG is SEQ ID NO. 5, the Sec-Tat double pathway signal peptide is FhuD or MdoG, the amino acid sequence of MdoG is SEQ ID NO. 6, and the amino acid sequence of FhuD is SEQ ID NO. 7.

2. A recombinant vector for self-lysis of e.coli, comprising the lysis module of claim 1.

3. A recombinant vector for self-cleavage of e.coli according to claim 2, characterized in that said vector is e.coli expression vector pBAD/His.

4. A system for self-lysing of e.coli, characterized in that said system for self-lysing of e.coli comprises the recombinant vector of claim 2 or 3.

5. The system for self-lysing according to claim 4, wherein said system for self-lysing of E.coli comprises a PelB-lysozyme-SsrA system, a PhoA-lysozyme-SsrA system, a TorrA-lysozyme-SsrA system, a FdoG-lysozyme-SsrA system, a FhuD-lysozyme-SsrA system or a MdoG-lysozyme-SsrA system.

6. The method for constructing a system for self-lysis of Escherichia coli according to claim 4 or 5, wherein the system for self-lysis of Escherichia coli is obtained by transforming the recombinant vector into Escherichia coli.

7. The method for constructing a system for self-lysis of Escherichia coli according to claim 6, wherein the expression conditions of the system for self-lysis of Escherichia coli are:

During the systematic expression of the self-lysing of the escherichia coli, arabinose which is 0.20-0.30% of the weight of the final culture solution and Triton X-100 which is 0.4-0.6% of the weight of the final culture solution are added, and the expression of a lysis module in the recombinant vector is induced to be 8-12 h.

8. Use of a system for self-lysing e.coli according to claim 4 or 5 for the release of proteins of interest or for screening mutants.