Recombinant escherichia coli for producing monophosphoryl lipid A
By constructing recombinant E. coli, replacing the enzyme gene of its lipid A synthesis pathway and introducing specific genes, the pathogenic risk, product complexity and high cost of monophosphoryl lipid A production in the prior art is solved, and safe, green, and large-scale production is achieved.
Patent Information
- Application Number
- CN202311410506.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-27
- Publication Date
- 2025-04-29
AI Technical Summary
The method for producing monophosphoryl lipid A in the prior art has the problem of pathogenic risk, complex product components and difficult to isolate the target product, and high cost.
A recombinant E. coli was constructed to produce monophosphoryl lipid A by replacing the gene of the enzyme encoding the lipid A synthesis pathway with an exogenous gene encoding the enzyme encoding the lipid A synthesis pathway and introducing the exogenous gene encoding the pagL and lpxE.
It has achieved monophosphoryl lipid A production without pathogenic risk, single product and low cost, suitable for large-scale industrial production.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant Escherichia coli for producing monophosphoryl lipid A. Background Art
[0002] Lipid A can be recognized by Toll-like receptor 4 (TLR4) on the surface of host cells, which then triggers a series of physiological and biochemical reactions in the cells, producing a variety of inflammatory factors such as IL-6, IL-8, and TNF-α. The types and quantities of cytokines mainly depend on the structure of lipid A. Among them, monophosphoryl lipid A, a derivative of lipid A, has been developed as a vaccine adjuvant, which can non-specifically enhance the body's immune response to antigens and improve vaccine efficiency. At present, 3-O-deacyl-4'-monophosphoryl lipid A (MPL) is commercially produced by GlaxoSmithKline, mainly extracted from Salmonella by fermentation. However, Salmonella is a conditionally pathogenic bacterium and synthesizes a variety of lipid A with different structures, which is difficult to operate and costly.
[0003] Escherichia coli W3110 is a non-pathogenic bacterium. The Kdo2-lipid A part of its lipopolysaccharide (LPS) has a simple structure, including two 2-keto-3-deoxyoctanoic acids, six fatty acid chains and two phosphate groups at the C1 and C4' positions. The synthesis pathway is very conservative. Therefore, Escherichia coli W3110 is a starting strain for industrial production of lipid A that is superior to Salmonella. Lipid A is synthesized from the inner side of the cell membrane, and the synthesis pathway is relatively conservative. However, it can undergo a variety of modifications during the process of transport to the outer side of the outer membrane to adapt to the external environment. At present, genes related to lipid structure modification have been reported. Using these genes, according to the synthesis and modification mechanism of lipid A molecules, the structure of lipids in Escherichia coli can be modified through genetic engineering technology, which is expected to become an ideal strain for producing monophosphoryl lipid A. Summary of the invention
[0004] The technical problem to be solved by the present invention is to solve the problems in the prior art of producing monophosphoryl lipid A that the method has pathogenic risk, complex product composition, difficulty in separating the target product, and high cost. A recombinant Escherichia coli for producing monophosphoryl lipid A is provided. When the recombinant Escherichia coli is used to produce monophosphoryl lipid A, no pathogenic bacteria are involved, the product is single, and the production cost can be further reduced to adapt to large-scale production.
[0005] The present invention solves the above technical problems through the following technical solutions:
[0006] The first aspect of the present invention provides a recombinant Escherichia coli (Escherichiacoli) for producing monophosphoryl lipid A, wherein the genes encoding enzymes in the lipid A synthesis pathway of the chassis bacteria of the recombinant Escherichia coli are replaced with exogenous genes encoding enzymes in the lipid A synthesis pathway, and the recombinant Escherichia coli contains exogenous genes encoding pagL and exogenous genes encoding lpxE.
[0007] In some embodiments of the present invention, the base bacteria is Escherichia coli W3110 or MG1655.
[0008] In some embodiments of the present invention, the gene encoding pagL is derived from Salmonella sp.
[0009] In some embodiments of the present invention, the gene encoding lpxE is derived from Francisella sp.
[0010] In some embodiments of the present invention, the genes encoding enzymes in the lipid A synthesis pathway of the recombinant Escherichia coli are selected from one or more of the following genes: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM.
[0011] In some embodiments of the present invention, the exogenous genes encoding enzymes in the lipid A synthesis pathway are one or more of the following: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM; preferably, they are genes encoding enzymes in the lipid A synthesis pathway derived from Salmonella.
[0012] In some embodiments of the present invention, the lacI gene of the chassis bacteria is inactivated.
[0013] In some embodiments of the present invention, the recombinant Escherichia coli does not have kanamycin resistance.
[0014] The second aspect of the present invention provides a method for preparing the recombinant Escherichia coli as described in the first aspect, the method comprising the following steps: replacing the gene encoding the enzyme of the lipid A synthesis pathway of the chassis bacteria of the recombinant Escherichia coli with an exogenous gene encoding the enzyme of the lipid A synthesis pathway; and introducing an exogenous gene encoding pagL and an exogenous gene encoding lpxE.
[0015] In some embodiments of the present invention, the genes encoding enzymes of the lipid A synthesis pathway of the chassis bacteria of the recombinant Escherichia coli are one or more selected from the following: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM.
[0016] In some embodiments of the present invention, the exogenous genes encoding enzymes in the lipid A synthesis pathway are one or more of the following: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM; preferably, they are genes encoding enzymes in the lipid A synthesis pathway derived from Salmonella sp.
[0017] In some embodiments of the present invention, the lacI gene of the chassis bacteria is inactivated.
[0018] In some embodiments of the present invention, the base bacteria is Escherichia coli W3110 or MG1655.
[0019] In some embodiments of the present invention, the gene encoding pagL is derived from Salmonella.
[0020] In some embodiments of the present invention, the gene encoding lpxE is derived from Francisella sp.
[0021] The third aspect of the present invention provides a method for preparing monophosphoryl lipid A, the method comprising:
[0022] (1) culturing the recombinant Escherichia coli described in the first aspect in a culture medium;
[0023] (2) Collect the bacterial cells obtained in (1) and perform lipopolysaccharide hydrolysis and purification.
[0024] In some embodiments of the present invention, in (1), the culture temperature is 25-37° C., and / or the inoculation amount of the recombinant Escherichia coli is 0.2%, and / or the culture medium is LB medium.
[0025] In some embodiments of the present invention, the LPS hydrolysis in (2) comprises the step of separating the LPS using an organic solvent, wherein the organic solvent is selected from one or more of the following: ethanol, acetone, ether, phenol, chloroform and petroleum ether;
[0026] and / or, the hydrolysis of lipopolysaccharide comprises one or more of the following steps: ultrasonic bath, addition of lysis solution and boiling water bath;
[0027] And / or, the purification step comprises extraction and / or centrifugation.
[0028] The fourth aspect of the present invention provides a use of the recombinant Escherichia coli as described in the first aspect in the preparation of a vaccine adjuvant.
[0029] In some embodiments of the present invention, the vaccine adjuvant comprises monophosphoryl lipid A.
[0030] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0031] The reagents and raw materials used in the present invention are commercially available.
[0032] The positive and progressive effects of the present invention are:
[0033] The recombinant E. coli provided by the present invention can be used to produce monophosphoryl lipid A without pathogenicity risk, and the strain itself has no antibiotic screening marker, which is conducive to safe, green, and large-scale industrial production. In addition, the recombinant E. coli of the present invention can also be used as a chassis strain for further modification of lipid A structure. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the flowchart of W3110Mut gene editing. DETAILED DESCRIPTION
[0035] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0036] Example 1 pTarget plasmid construction
[0037] The CRISPR / Cas9 knockout system was used to knock out the lacI gene encoding the DNA-binding transcriptional repressor in the Escherichia coli genome, the Escherichia coli lipid A synthesis pathway enzyme genes (named: ElpxA, Elp xC, ElpxD, ElpxB, ELpxH, ElpxK, ElpxL, EpagP and ELpxM), and at the same time, the Salmonella lipid A synthesis pathway enzyme genes (named: SlpxA, SLpxC, SLpxD, SLpxB, SLpxH, SLpxK, SLpxL, SLpxM, SpagP and SpagL) and the gene encoding lipid A1-phosphatase from Francisella (named: LpxE) were knocked in. The enzyme genes of the Escherichia coli lipid A synthesis pathway come from the paper "Metabolic engineering of Escherichia coli to produce monophosphoryl lipid A adjuvant" (Ji Y, An J, Hwang D, et al. Metabolic engineering of Escherichia coli to produce a monophosphoryl lipid A adjuvant. Metab Eng. 2020; 57: 193-202. doi: 10.1016 / j.ymben.2019.11.009).
[0038] The construction process of these plasmids is as follows:
[0039] (1) Select a 20nt N20 sequence complementary to the target sequence of the target gene, design the upstream and downstream homology arms of the target gene, and introduce them into the pTarget plasmid by PCR amplification and restriction ligation;
[0040] (2) Thaw the competent E. coli DH5α cells on ice, add the ligation product to the competent cells and mix by gently pipetting, and place on ice for 30 minutes. Then, heat shock in a 42°C water bath for 90 seconds, place on ice for 2 minutes, and quickly add 900 μL LB medium. Resuscitate at 37°C, 200 rpm for 45 minutes, then spread on an LB plate supplemented with 50 mg / L spectinomycin, and culture inverted at 37°C to screen for knockout transformants.
[0041] (3) Using blank pTarget as a negative control, perform colony PCR on the transformants and send them for sequencing to verify whether the knockout plasmid is successfully constructed.
[0042] Example 2 Construction of genetically engineered strain W3110Mut
[0043] The CRISPR / Cas9 knockout system was used to modify the lipid A synthesis-related genes of Escherichia coli W3110. The specific knockout process is as follows ( Figure 1 shown):
[0044] (1) Preparation of E. coli electroporated competent cells W3110 / pCas
[0045] Prepare competent E. coli W3110, introduce plasmid pCas into E. coli W3110 by chemical transformation, spread on LB solid plate supplemented with 50 mg / L kanamycin, pick single clone and inoculate into LB liquid test tube, and culture at 30°C overnight; transfer seed liquid to 50 mL LB (containing 50 mg / L kanamycin) medium at 1% (v / v), culture at 30°C, 200 rpm to OD600 = 0.4, add 10 mM arabinose to induce the expression of RED homologous recombinase, continue to culture to OD600 = 0.6, collect bacteria by centrifugation at 4°C, 4000 rpm for 10 min, wash bacteria 3 times with pre-cooled 10% glycerol solution; add 300 μL 10% glycerol solution to resuspend the bacteria, and dispense 80 μL / tube.
[0046] (2) Gene replacement or knockout by electroporation
[0047] First, precool the sterile electroporation cup for 10 minutes. Then, add 100 ng of the knockout plasmid in Example 1 to the Escherichia coli W3110 / pCas competent cells in step (1), gently blow and pipette to mix, and suck it into the groove of the electroporation cup. After electric shock, quickly add 900 μL LB medium to the electroporation cup, aspirate all the bacterial solution into a 1.5 mL sterile EP tube, and resuscitate at 30°C and 200 rpm for 45 minutes. Spread on an LB plate containing 50 mg / L kanamycin and 50 mg / L spectinomycin, and invert and culture overnight in a 30°C incubator. Pick a single clone, verify it by PCR, and screen it by sequencing to obtain the correct mutant strain.
[0048] (3) Removal of gene editing plasmid pTargetF-gene and temperature-sensitive plasmid pCas
[0049] The positive knockout clones in step (2) were inoculated into LB tubes (containing kanamycin resistance), 0.5 mM IPTG was added and cultured overnight at 30°C (inducing the sgRNA in pCAS to cut off the pTarget plasmid), single colonies were isolated by streaking (kanamycin monoclonal antibody plate), 8 single colonies were picked and inoculated into kanamycin LB tubes and spectinomycin LB tubes respectively, and cultured overnight at 30°C. If the kanamycin LB tubes become turbid and the corresponding spectinomycin LB tubes become clear, the plasmid was successfully removed, and the preserved bacteria were named W3110Δgene(kana) bacteria (still containing pCAS plasmid, which can be directly used for the next round of construction). W3110Δgene(kana) bacteria were inoculated into non-resistant LB tubes, subcultured at 37°C, coated with non-resistant LB solid plates, single colonies were picked, and plating was performed on non-resistant and kanamycin resistant LB solid plates by copy printing. The absence of colonies on the kanamycin plate indicated that the pCAS plasmid was removed, and the preserved bacteria were named W3110mut bacteria.
[0050] Example 3
[0051] The glycerol tube of the genetically engineered bacteria W3110mut was inoculated with 0.2% of the LB seed culture medium, and the shaking temperature was set to 37°C and the speed was 220rpm for 8h. Inoculated with 5% of the inoculation volume, it was inoculated into a 50L fermenter (LB culture medium, 30L liquid volume), and the temperature was set to 37°C, the speed was set to 200rpm, and the air flow rate was 25L / min. The pH was automatically controlled to 6.5 with 200ml of 30% ammonia water until it was completed. At this time, the fermentation entered the static culture stage and the pH began to drop. When the pH dropped to about 5, the pH curve showed an inflection point. After 7h after the inflection point appeared, the temperature was lowered to below 30°C for harvesting. The fermentation broth was centrifuged to collect the bacteria, and then the culture medium was washed with deionized water until the washing liquid was clear and transparent and almost colorless.
[0052] Example 4 Lipid A extraction from genetically engineered strain W3110Mut
[0053] The dried cells were first stirred at room temperature for 1 hour in 90% ethanol (v / v) at a concentration of 20 mg / mL and then recovered by vacuum filtration. The cells were subjected to a second ethanol extraction, followed by extraction with acetone and ether (15 minutes each, all based on the initial weight of 40 mg / mL), and the resulting ether powder was air-dried overnight. At the same time, a solution of phenol (89%): chloroform: petroleum ether = 19:45:72 (v / v / v; abbreviated as PCP) was prepared and placed overnight. The ether powder was suspended in PCP and excess water was decanted to a concentration of 70 mg / mL. The solution was stirred for 30 minutes and then centrifuged (3000 x g, 15 minutes, 0-5°C). The supernatant portion was poured into a round-bottom flask and the cell pellet was extracted for the second time with PCP. The supernatant fractions were combined and rotary evaporated at 40°C until most of the volatile solvents were removed. The residual volume was then measured. Water is added dropwise until persistent turbidity is evident, then 5 volumes of acetone and 1 volume of ether (both cooled in an ice bath) are added to the phenol solution and mixed rapidly. The solution is placed in an ice bath for 30 minutes, then the precipitated LPS is recovered by centrifugation (5000×g, 15 minutes, 0-5°C). It is usually necessary to gravity filter the supernatant fraction to recover any LPS that is not retained in the pellet. The LPS is washed once in a minimal volume of cold acetone, recovered by centrifugation / filtration, and then dried under vacuum.
[0054] LPS is suspended in water at a concentration of 10 mg / mL and an ultrasonic bath is used at 45-55°C to help disperse the solid material. The resulting solution should be slightly turbid and no solids can be seen with the naked eye. 1 volume of 0.2N HCl is added to the solution and then placed in a boiling water bath for 15 minutes. The reaction is quenched in an ice bath and then extracted with 5 volumes (relative to the initial LPS solution) of chloroform: methanol = 2: 1 (v / v). The biphasic solution is vortexed and the phases are separated by low speed centrifugation (500-1000×g). The lower phase is recovered and evaporated under nitrogen to obtain a sample powder.
[0055] Example 5 Mass spectrometry analysis of genetically engineered strain W3110Mut MPL
[0056] The sample was detected by Agilent 6545Q-Tof LC / MS. Electrospray ionization (ESI) source was used in negative ion mode, and the detection range was less than m / z 2000. A peak of 1729.28656 existed in the MS spectrum, corresponding to the molecular weight of hexaacylated MPL. This indicated that the genetically engineered bacteria obtained by the present invention can synthesize MPL.
Claims
1. A recombinant Escherichia coli for producing monophosphoryl lipid A, characterized in that: The genes encoding enzymes in the lipid A synthesis pathway of the chassis bacteria of the recombinant Escherichia coli are replaced with exogenous genes encoding enzymes in the lipid A synthesis pathway, and contain exogenous genes encoding pagL and exogenous genes encoding lpxE.
2. The recombinant Escherichia coli according to claim 1, characterized in that The bottom plate bacteria is Escherichia coli W3110 or MG1655.
3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that The gene encoding pagL is derived from Salmonella; and / or, the gene encoding lpxE is derived from Francisella; And / or, the genes encoding enzymes of lipid A synthesis pathway of the chassis bacteria of the recombinant Escherichia coli are selected from one or more of the following genes: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM; And / or, the exogenous gene encoding an enzyme in the lipid A synthesis pathway is one or more of the following: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM; preferably, a gene encoding an enzyme in the lipid A synthesis pathway derived from Salmonella; And / or, the lacI gene of the chassis bacteria is inactivated; And / or, the recombinant Escherichia coli does not have kanamycin resistance.
4. A method for preparing the recombinant Escherichia coli according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: The genes encoding enzymes in the lipid A synthesis pathway of the recombinant Escherichia coli are replaced with exogenous genes encoding enzymes in the lipid A synthesis pathway; and exogenous genes encoding pagL and exogenous genes encoding lpxE are introduced.
5. The method according to claim 4, characterized in that The genes encoding enzymes of the lipid A synthesis pathway of the chassis bacteria of the recombinant Escherichia coli are one or more selected from the following: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM; And / or, the exogenous gene encoding an enzyme in the lipid A synthesis pathway is one or more of the following: lpxA, lpxC, lpxD, lpxB, lpxH, lpxK, lpxL, pagP and lpxM; preferably, a gene encoding an enzyme in the lipid A synthesis pathway derived from Salmonella; And / or, the lacI gene of the chassis bacteria is inactivated; And / or, the bottom plate bacteria is Escherichia coli W3110 or MG1655; and / or, the gene encoding pagL is derived from Salmonella; And / or, the gene encoding lpxE is derived from Francisella.
6. A method for preparing monophosphoryl lipid A, characterized in that: The method comprises: (1) culturing the recombinant Escherichia coli according to any one of claims 1 to 3 in a culture medium; (2) Collect the bacterial cells obtained in (1) and perform lipopolysaccharide hydrolysis and purification.
7. The method according to claim 6, characterized in that In (1), the culture temperature is 25-37° C., and / or the inoculation amount of the recombinant E. coli is 0.2%, and / or the culture medium is LB medium.
8. The method according to claim 6, characterized in that (2) further comprising the step of using an organic solvent to separate lipopolysaccharide, wherein the organic solvent is selected from one or more of the following: ethanol, acetone, ether, phenol, chloroform and petroleum ether; and / or, the hydrolysis of lipopolysaccharide comprises one or more of the following steps: ultrasonic bath, addition of lysis solution and boiling water bath; And / or, the purification step comprises extraction and / or centrifugation.
9. Use of the recombinant Escherichia coli according to any one of claims 1 to 3 in the preparation of a vaccine adjuvant; Preferably, the vaccine adjuvant comprises monophosphoryl lipid A.