A recombinant Escherichia coli capable of producing monophospholipid A vaccine adjuvant

Abstract

The present invention discloses a recombinant Escherichia coli capable of producing a monophosphate lipid A vaccine adjuvant, belonging to the fields of genetic engineering and synthetic biology. The present invention knocks out the mlaE and pldA genes of the phospholipid transport system on the Escherichia coli MG1655 genome, the hns gene that regulates PagP expression, and sequentially integrates the FnlpxE gene from Francisella and the SepagL gene from Salmonella by deletion and replacement of other phospholipid transport-related gene clusters ybgC-cpoB and letAB, thereby obtaining the recombinant bacterium WZM012. Through simple fermentation, the WZM012 strain can efficiently synthesize hexaacylated monophosphate lipid A (MPL), producing 32.76 mg / L of MPL in 24 hours of fermentation, with the proportion of MPL in the total lipid A reaching 70%.

Description

Technical Field

[0001] The present invention relates to a recombinant Escherichia coli capable of producing a monophosphate lipid A vaccine adjuvant, and belongs to the fields of genetic engineering and synthetic biology. Background Art

[0002] LPS is a major component of the outer membrane of Escherichia coli, providing protection against bacteria. It consists of three parts: lipid A, core polysaccharide, and O-antigen. The lipid A structure is the biologically active center of LPS and a recognition site for host immune cells. Different lipid A structures can produce different types of cytokines in the body, eliciting distinct immune responses. When pathogens invade host cells, they are recognized by the pathogen recognition receptor TLR4 (Toll-like receptor 4). Some lipid A structures can induce excessive cytokine production, leading to overimmunity and endotoxic shock, while others can induce moderate cytokine production, enhancing the immune response of host cells. Therefore, lipid A derivatives with specific structures can be developed as immune system activators into vaccines or vaccine adjuvants. Aluminum adjuvants are currently the most widely used vaccine adjuvants, but they suffer from the drawback of not inducing strong T cell immunity. There is an urgent need to develop vaccine adjuvants that can activate T cell immune responses to address diseases that rely on T cell-based prevention and treatment, such as tuberculosis and malaria.

[0003] Monophosphoryl lipid A (MPLA) is a lipid A derivative clinically approved for use as a vaccine adjuvant. MPLA exists in various structures, including hepta-acylated monophosphoryl lipid A (P-MPLA), hexa-acylated monophosphoryl lipid A (MPL), and penta-acylated monophosphoryl lipid A (D-MPLA). MPL is the most important component of vaccine adjuvants, and vaccines containing MPL as an adjuvant have been successfully marketed. Currently, commercial MPL can only be obtained through chemical manipulation of the lipid A structure of the Salmonella enterica R595 mutant strain, which is costly and complex. Compared to chemical methods, the rational use of genetic engineering and synthetic biology to modify the existing lipid A of Escherichia coli is more compliant with industrial standards. Existing research, such as the paper "Metabolic engineering of Escherichia coli to produce a monophosphoryl lipid A adjuvant," reports a recombinant Escherichia coli strain that produces MPL at levels of 8-12 mg / L. However, its output is still relatively low and cannot meet the needs of industrial production. Summary of the Invention

[0004] To address these technical issues, the present invention first knocks out two genes related to phospholipid transport (mlaE and pldA) in the E. coli genome, regulates the PagP expression-related gene (hns). The recombinant strain WZM012 is obtained by integrating the Francisella-derived lpxE gene into the genome to replace the other phospholipid transport system-related gene clusters (ybgC-cpoB) and the Salmonella-derived pagL gene into the genome to replace the other phospholipid transport system-related gene clusters (letAB). The recombinant strain MW012 can efficiently produce MPL during fermentation without the addition of any additional antibiotics or inducers.

[0005] The first object of the present invention is to provide a recombinant bacterium, which uses Escherichia coli as a starting strain, knocks out the mlaE and pldA genes related to the main phospholipid transport system in the genome of the starting strain, the hns gene that regulates PagP expression, and other phospholipid transport system ybgC-cpoB and letAB gene clusters; and integrates and expresses the dephosphatase LpxE and the deacylase PagL.

[0006] In one embodiment, the NCBI accession numbers of the mlaE and pldA genes are Gene ID: 947732 and Gene ID: 948307, respectively.

[0007] In one embodiment, the NCBI accession number of the hns gene related to the regulation of PagP expression is Gene ID: 945829.

[0008] In one embodiment, the other phospholipid transport system-related ybgC-cpoB gene cluster contains 7 genes, namely ybgC, tolQ, tolR, tolA, tolB, pal and cpoB, and the NCBI accession numbers are Gene ID: 948907, GeneID: 948900, Gene ID: 945328, Gene ID: 946625, Gene ID: 945429, Gene ID: 945004, and GeneID: 947227, respectively.

[0009] In one embodiment, the other phospholipid transport system-related letAB gene cluster comprises two genes, namely letA and letB, whose NCBI accession numbers are Gene ID: 946353 and Gene ID: 946352, respectively.

[0010] In one embodiment, the lpxE gene encoding the dephosphatase LpxE is from Francisella; and the pagL gene encoding the deacylase PagL is from Salmonella.

[0011] In one embodiment, the NCBI accession number of the lpxE gene from Francisella is Gene ID: 63960747; the NCBI accession number of the pagL gene from Salmonella is Gene ID: 945582.

[0012] In one embodiment, the lpxE gene from Francisella and the pagL gene from Salmonella are integrated into the genome by replacing the ybgC-cpoB and letAB gene clusters in the Escherichia coli genome, respectively.

[0013] In one embodiment, the Escherichia coli includes but is not limited to Escherichia coli MG1655.

[0014] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant Escherichia coli, which includes knocking out the mlaE and pldA genes related to the main phospholipid transport system on the Escherichia coli genome, the hns gene that regulates PagP expression, and other phospholipid transport system ybgC-cpoB and letAB gene clusters, and integrating the expression of dephosphatase LpxE and deacylase PagL.

[0015] In one embodiment, the gene encoding the dephosphatase LpxE, lpxE, and the gene encoding the deacylase PagL, pagL, are integrated into the genome by replacing the ybgC-cpoB and letAB gene clusters in the Escherichia coli genome, respectively.

[0016] The third object of the present invention is to provide a method for producing monophosphate lipid A, which comprises inoculating the above-mentioned recombinant bacteria into a fermentation medium and fermenting to produce monophosphate lipid A.

[0017] In one embodiment, the monophospholipid A includes heptaacylated monophospholipid A (P-MPLA), hexaacylated monophospholipid A (MPL) and / or pentaacylated monophospholipid A (D-MPLA). Optionally, the monophospholipid A is hexaacylated monophospholipid A (MPL).

[0018] In one embodiment, the method comprises the following steps:

[0019] (1) inoculating the above-mentioned recombinant bacteria into a seed culture medium, culturing, and obtaining a seed solution;

[0020] (2) The seed solution obtained in step (1) is transferred to a fermentation medium for fermentation to produce monophospholipid A.

[0021] In one embodiment, the seed culture medium contains 4-6 g / L yeast extract, 8-12 g / L peptone, and 8-12 g / L NaCl. Alternatively, the seed culture medium contains 5-15 g / L sucrose, 1-10 g / L yeast extract, 15-30 g / L peptone, 10-20 g / L ammonium sulfate, and 0.5-2 g / L magnesium sulfate.

[0022] In one embodiment, the fermentation medium contains: 10-60 g / L glucose, 5-8 g / L KH2PO4, 10-20 g / L ammonium sulfate, 1-5 g / L yeast extract, 1-5 g / L citric acid, 1-10 mg / L FeSO4·7H2O, 1-10 mg / L MnSO4·4H2O, and 1-5 g / L MgSO4·7H2O. Alternatively, the fermentation medium contains: 10-60 g / L glucose, 7.46 g / L KH2PO4, 15 g / L ammonium sulfate, 3 g / L yeast extract, 2 g / L citric acid, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4·4H2O, and 2 g / L MgSO4·7H2O. Alternatively, the fermentation medium contains: 1-10 g / L ammonium sulfate, 10-60 g / L glucose, 5-10 g / L KH2PO4, 1-5 g / L yeast extract, 1-5 g / L citric acid, 1-5 g / L MgSO4·7H2O, 1-5 mg / LFeSO4·7H2O. Optionally, the fermentation medium contains: 5 g / L ammonium sulfate, 30 g / L glucose, 7.46 g / L KH2PO4, 2 g / L yeast extract, 2 g / L citric acid, 2 g / L MgSO4·7H2O, 5 mg / L FeSO4·7H2O.

[0023] In one embodiment, the fermentation temperature is 25-40°C, optionally, the fermentation temperature is 37°C.

[0024] In one embodiment, the fermentation pH is 5-10, optionally, the pH is 6-9, optionally, the pH is 7.

[0025] In one embodiment, the fermentation dissolved oxygen is 10-50%, optionally, the dissolved oxygen is 20-30%.

[0026] In one embodiment, when the glucose concentration is lower than 5-10 g / L during the fermentation process, glucose is fed to maintain the glucose concentration at no less than 5-10 g / L.

[0027] The fourth object of the present invention is to provide the above-mentioned recombinant bacteria, or the application of the above-mentioned method in the field of biomedicine.

[0028] In one embodiment, the use includes using the recombinant bacteria or the method for producing lipid A vaccine adjuvant.

[0029] Beneficial effects:

[0030] (1) The recombinant strain WZM012 described in the present invention has simple fermentation conditions and does not require the addition of any antibiotics or inducers. Whether it is shake flask fermentation or fed-batch fermentation, it mainly produces three types of monophosphoryl lipid A, one is heptaacylated monophosphoryl lipid A (P-MPLA), another is hexaacylated monophosphoryl lipid A (MPL), and the last is pentaacylated monophosphoryl lipid A (D-MPLA). Among them, MPL is the most important component in vaccine adjuvants. Among the monophosphoryl lipid A produced by the strain described in the present invention, MPL accounts for 70% of the total lipid A content.

[0031] (2) The recombinant strain WZM012 of the present invention was fermented in a 2-L fermentation tank system for 24 h, and the highest OD 600 It can reach 19.40, the cell dry weight can reach 2.86g / L, the total lipid A content can reach 46.80mg / L, of which the hexaacylated monophosphate lipid A (MPL) content can reach 32.76mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The process of gene knockout in the Escherichia coli genome.

[0033] Figure 2 The genotypes of different E. coli mutants (A) and the growth conditions of these strains (B).

[0034] Figure 3 LC-MS analysis of lipid A structure of different E. coli mutants, A is the liquid chromatogram, B is the mass spectrum.

[0035] Figure 4 These are the results of fed-batch fermentation of Escherichia coli WZM012. A is the OD and glucose content-time curves. B is the strain OD, biomass, lipid A production, and MPL production test results. C is the TLC chromatogram. D is the LC-MS analysis diagram. DETAILED DESCRIPTION

[0036] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available or can be prepared by known methods.

[0038] The Escherichia coli JM109 strain involved in the following examples was purchased from NEB, and the Escherichia coli MG1655 strain involved was purchased from ATCC with the deposit number CGSC 6300; the pCas plasmid involved was disclosed in the paper Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S., 2015. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 81(7), 2506-2514., and the pTargetF plasmid involved was disclosed in the paper Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S., 2015. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 81(7), 2506-2514.

[0039] The culture medium involved in the following examples is as follows:

[0040] All culture media were prepared using ddH2O and sterilized at 121°C for 15-20 min.

[0041] Medium A (g / L): yeast powder 5, peptone 10, NaCl 10.

[0042] Culture medium B (g / L): 10-60 glucose, 5 (NH4)2SO4, 2 yeast powder, 2 citric acid, 7.46KH2PO4, 2g / LMgSO4·7H2O, 0.005FeSO4·7H2O, 0.005MnSO4·4H2O, (NaOH was used to adjust the pH of the culture medium to 7.00).

[0043] The primer sequences involved in the following examples are shown in Table 1:

[0044] Table 1: Primer sequences

[0045]

[0046]

[0047] Table 2: Strains and plasmids involved in the following examples

[0048]

[0049]

[0050] Example 1: Construction of knockout plasmid

[0051] The CRISPR / Cas9 knockout system was used to knock out the major phospholipid transport system-related mlaE and pldA genes in E. coli, the hns gene that regulates PagP expression, and the ybgC-cpoB and letAB gene clusters in other phospholipid transport systems. Five knockout plasmids were constructed: pT-mlaE, pT-pldA, pT-hns, pT-ybgC-cpoB, and pT-letAB. The construction process of these plasmids was as follows:

[0052] (1) Select 20 nt N complementary to the target sequence of the target gene 20 sequences, and these sequences were modified to the 5' end of the forward primer of plasmid pTargetF to obtain forward primers mlaE-sgRNA-F, pldA-sgRNA-F, hns-sgRNA-F, ybgC-cpoB-sgRNA-F and letAB-sgRNA-F, respectively.

[0053] Using plasmid pTargetF as a template, forward and reverse primers mlaE-sgRNA-F / R, pldA-sgRNA-F / R, hns-sgRNA-F / R, ybgC-cpoB-sgRNA-F / R and letAB-sgRNA-F / R were used to amplify the N 20 The PCR amplified product was verified by electrophoresis and purified and recovered.

[0054] (2) Since the recovered product may contain the template plasmid pTargetF, which will affect subsequent experiments, DpnⅠ was added to the recovered product and reacted at 37°C for 2 h to digest the template plasmid.

[0055] (3) Phosphorylate the recovered product after digestion of the template plasmid using T4 polynucleotide kinase (T4 PNK), react at 37°C for 30 min, and then heat inactivate at 65°C for 10 min.

[0056] (4) Add 1 μL T4 DNA ligase to the phosphorylated reaction system and react at 22°C for 4 hours to obtain a ligation solution. After the reaction is completed, take out the Escherichia coli JM109 competent cells and melt them on ice. Add the ligation solution to the competent cells and gently pipette to mix, and place on ice for 30 minutes. Then heat shock the competent cells in a 42°C water bath for 90 seconds, place on ice for 2 minutes, and quickly add 1 mL LB culture medium. Resuscitate at 37°C, 100 rpm for 1 hour, then spread on an LB plate supplemented with 50 mg / L of spectinomycin (Spe), and culture inverted at 37°C to screen knockout transformants. Using pTargetF as a negative control, perform colony PCR on the transformants to verify whether the knockout plasmid is successfully constructed. The correct transformant is inoculated into an LB liquid test tube supplemented with spectinomycin (Spe), and the plasmid is extracted to obtain the knockout plasmid pTargetF-gene;

[0057] Knockout plasmids were prepared respectively: pT-mlaE, pT-pldA, pT-hns, pT-ybgC-cpoB and pT-letAB.

[0058] Example 2: Construction of strain WZM012

[0059] The CRISPR / Cas9 knockout system was used to construct strain MW012. The specific knockout process is as follows ( Figure 1 ):

[0060] (1) Preparation of E. coli electroporation knockout competent cells MG1655 / pCas

[0061] The plasmid pCas was transformed into Escherichia coli MG1655 to obtain recombinant Escherichia coli MG1655 / pCas containing the pCas plasmid. The Escherichia coli MG1655 / pCas was activated on an LB solid plate supplemented with 30 mg / L kanamycin (Kan) and inoculated onto an LB solid plate (Kan). + ) overnight in vitro culture to obtain seed solution; the seed solution was transferred to 25 mL LB (Kan+) medium at 1% (v / v) and cultured at 30°C and 200 rpm until OD 600 =0.2, add 500 μL of 0.5 mM L-arabinose solution for induction, and continue to culture until OD 600 = 0.5, ice bath for 30 min; centrifuge at 4000 rpm for 10 min at 4°C to collect the cells, wash the cells three times with pre-cooled 10% glycerol solution; add 300 μL 10% glycerol solution to resuspend the cells, and distribute them into sterile 1.5 mL EP tubes, 80 μL / tube.

[0062] (2) Construction of homology arm knockout fragments

[0063] The genome of Escherichia coli MG1655 was extracted and used as a template to amplify the upstream and downstream homology arms of the target gene mlaE knockout using primers U-mlaE-F / U-mlaE-R and D-mlaE-F / D-mlaE-R, respectively. The fragments were recovered by gel chromatography, and overlapping PCR was performed using primers U-mlaE-F and D-mlaE-R to obtain homology arm knockout fragment 1.

[0064] The upstream and downstream homology arms were amplified using the homology arm primers U-pldA-F / U-pldA-R and D-pldA-F / D-pldA-R of the target gene pldA knockout, respectively. The fragments were recovered from the gel, and overlapping PCR was performed using the primers U-pldA-F / D-pldA-R to obtain homology arm knockout fragment 2.

[0065] The upstream and downstream homology arms of the target gene hns knockout were amplified using primers U-hns-F / U-hns-R and D-hns-F / D-hns-R, respectively. The fragments were recovered by gel amplification, and overlapping PCR was performed using primers U-hns-F / D-hns-R to obtain homology arm knockout fragment 3.

[0066] The homology arm primer ybgC-cpoB::P FnlpxE -FnlpxE-F1 / ybgC-cpoB::P FnlpxE -FnlpxE-R1,ybgC-cpoB::P FnlpxE -FnlpxE-F2 / ybgC-cpoB::P FnlpxE -FnlpxE-R2,ybgC-cpoB::P FnlpxE -FnlpxE-F3 / ybgC-cpoB::P FnlpxE -FnlpxE-R3, using Francisella genomic DNA as a template, the upstream homology arm, the target inserted gene fragment and the downstream homology arm were amplified, gel recovery was performed, and the primer pair ybgC-cpoB::P FnlpxE -FnlpxE-F1 / ybgC-cpoB::P FnlpxE - Overlap PCR was performed on FnlpxE-R3 to obtain homology arm insertion knockout fragment 4;

[0067] The homology arm primer letAB::P SepagL -SepagL-F1 / letAB::P SepagL -SepagL-R1, letAB::P SepagL -SepagL-F2 / letAB::P SepagL -SepagL-R2, letAB::PSepagL -SepagL-F3 / letAB::P SepagL -SepagL-R3, using Salmonella genomic DNA as a template, the upstream homology arm, the target inserted gene fragment and the downstream homology arm were amplified, recovered by gel, and the primer pair letAB::P SepagL -SepagL-F1 / letAB::P SepagL -SepagL-R3 was subjected to overlapping PCR to obtain homology arm insertion knockout fragment 5.

[0068] (3) Gene knockout by electroporation

[0069] Wash the electroporation cup three times with anhydrous ethanol and blow dry, and pre-cool for 20 minutes. Place the Escherichia coli MG1655 / pCas competent cells in step (1) on ice to melt, add 300ng of the knockout plasmid pTargetF-gene (pT-mlaE, pT-pldA, pT-hns, pT-ybgC-cpoB or pT-letAB) in Example 1 and 500ng of the corresponding homology arm knockout fragment in step (2), gently blow and mix, and aspirate into the groove of the electroporation cup. The electroporation cup is then ice-bathed for 10 minutes, wiped dry and then electroporated. Then quickly add 1mL LB culture medium to the electroporation cup, aspirate all the bacterial liquid into a 1.5mL EP tube, resuscitate at 30℃, 100rpm for 1h, spread on an LB plate containing 30mg / L Kan and 50mg / L Spe, and culture upside down in a 30℃ incubator. Using MG1655 as a negative control, the transformants were screened using the forward primer of the upstream homology arm and the reverse primer of the downstream homology arm to select the correct transformants.

[0070] (4) Removal of knockout plasmid pTargetF-gene and temperature-sensitive plasmid pCas

[0071] The correct knockout transformant in step (3) was inoculated into an LB test tube supplemented with 30 mg / L Kan and 1 mM IPTG. The enzyme expression of the knockout plasmid pTargetF-gene was removed by IPTG induction. The strain was cultured at 30°C with shaking for 24 hours, and single colonies were isolated by streaking on an LB (Kan+) plate. Single colonies sensitive to spectinomycin were screened out, i.e., mutant strains with the pTargetF-gene knockout plasmid removed were obtained. These strains were inoculated into an LB (Kan+) test tube for seed preservation, and competent strains could be directly prepared for continuous knockout. The mutant strains with the knockout plasmid removed were inoculated into an LB test tube, cultured at 42°C with shaking, and single colonies were isolated by streaking on an LB plate. Single colonies sensitive to kanamycin were screened out, i.e., mutant strains without resistance to pCas were obtained. These strains were inoculated into an LB test tube for seed preservation.

[0072] The CRISP / Cas9 knockout method was used to successfully knock out the major phospholipid transport-related mlaE and pldA genes, and the gene hns that regulates PagP expression in the MG1655 genome. The other phospholipid transport-related gene clusters ybgC-cpoB and letAB were replaced by deletion. The lpxE gene from Francisella and the pagL gene from Salmonella were sequentially integrated to obtain strain WZM012 and several process strains (such as Figure 2 and shown in Table 3).

[0073] Table 3: Strains and their genotypes

[0074]

[0075] Example 3: Extraction and structural verification of lipid A from strain WZM012 and other mutants

[0076] The specific steps are as follows:

[0077] The recombinant strain prepared in Example 2 was streaked on a medium A (LB solid medium) plate and cultured in a 37°C incubator for 18 h until a single colony appeared; a single colony was picked and transferred to medium A (LB liquid medium, generally 5 mL) for pre-culture until OD 600 ≈1.0; the pre-cultured bacterial solution was adjusted to the initial OD 600 =0.02 was transferred to 200 mL of culture medium B, cultured at 37°C and 200 rpm for 18 h, and then centrifuged at 4000 r / min for 20 min to collect the bacteria and extract their lipid A.

[0078] The specific method for detecting the structure of Escherichia coli lipid A is as follows:

[0079] After washing the cells once with ddH2O, they were suspended in a Bligh-Dyer monophasic system (chloroform / methanol / water, 1:2:0.8, v / v / v) and magnetically stirred for 1 hour. The phases were separated by centrifugation at 2000 rpm for 20 minutes. Cell debris was washed two to three times using the monophasic system. 27 mL of 12.5 mmol / L sodium acetate (pH 4.5) was added, and the mixture was sonicated for 10 minutes. The sugar chains were cleaved by incubation at 100°C for 30 minutes. After cooling to room temperature, 30 mL of chloroform and 30 mL of methanol were added to create a Bligh-Dyer two-phase system (chloroform / methanol / water, 2:2:1.8, v / v / v). The mixture was centrifuged at 2000 rpm for 10 minutes, and the phase was removed and transferred to a rotary evaporator for rotary evaporation. Finally, chloroform / methanol solution (4:1, v / v) was added to elute lipid A. The organic solvent was dried using a nitrogen blower, and lipid A was stored at -20°C until use.

[0080] Lipid A was dissolved in chloroform / methanol (4:1, v / v) and spotted on a gel 60 TLC plate using a developing solvent consisting of chloroform / methanol / water / ammonia (40:25:4:2, v / v / v / v). After chromatography, the remaining developing solvent on the plate was dried and carbonized with 10% sulfuric acid in ethanol. The plate was then placed on a hot plate and developed at 170°C.

[0081] Lipid A was dissolved in chloroform / methanol (4:1, v / v) and detected by mass spectrometry on a WATERS SYNAPT Q-TOF Mass Spectrometer. Negative ion detection was used, with a detection range below m / z 2500. Data were acquired and analyzed using MassLynx V4.1 software.

[0082] Lipid A was dissolved in chloroform / methanol (4:1, v / v) and spotted on a gel 60 TLC plate using a developing solvent consisting of chloroform / methanol / water / ammonia (40:25:4:2, v / v / v / v). After chromatography, the remaining developing solvent on the plate was dried and carbonized with 10% sulfuric acid in ethanol. The plate was then placed on a hot plate and developed at 180°C.

[0083] Lipid A was dissolved in chloroform / methanol (4:1, v / v) and detected by mass spectrometry on a WATERS SYNAPT Q-TOF Mass Spectrometer. Negative ion detection was used, with a detection range below m / z 2500. Data were acquired and analyzed using MassLynx V4.1 software.

[0084] The shake flask fermentation results showed that: Figure 3 As shown, the lipid A product of wild-type MG1655 is a typical hexaacylated lipid A; the lipid A product of strain WZM004 is hexaacylated lipid A and heptaacylated lipid A, but the proportion of heptaacylated lipid A is insufficient; the proportion of heptaacylated lipid A in strain WZM005 is significantly increased; further, in order to obtain a strain producing monophosphorylated lipid A, the FnlpxE gene of heterologous Francisella was integrated to construct WZM010, which can produce heptaacylated monophosphorylated lipid A and hexaacylated monophosphorylated lipid A; finally, in order to obtain the product MPL, the SepagL gene of Salmonella was additionally integrated to construct WZM012. The lipid A extracted from WZM012 was detected in the LC spectrum ( Figure 3 A) produced three main peaks. Analysis showed that the three peaks produced at retention times of 7.6, 9.5, and 10.1 min corresponded to characteristic values ​​of m / z = 1490.1, 1728.3, and 1954.5 in the MS spectrum, respectively, and were D-MPLA, MPL, and P-MPLA.

[0085] Example 4: Fed-batch fermentation of recombinant strain WZM012

[0086] The specific steps are as follows:

[0087] (1) Strain activation: Escherichia coli WZM012 was first cultured in 5 mL of LB medium at 37°C and 200 rpm for 18 h;

[0088] (2) The activated strain in step (1) was transferred to 50 mL of STF medium (10 g / L sucrose, 5 g / L yeast extract, 20 g / L peptone, 15 g / L ammonium sulfate, and 1 g / L magnesium sulfate), and cultured at 37°C and 200 rpm for 8 h to prepare a seed solution;

[0089] (3) Transfer 50 mL of the seed solution obtained in step (2) into a fermentation tank containing 1 L of fermentation medium.

[0090] The fermentation medium is: 5 g / L ammonium sulfate, 30 g / L glucose (supplemented as needed during the process), 7.46 g / L KH2PO4, 2 g / L yeast extract, 2 g / L citric acid, 2 g / L MgSO4·7H2O, 5 mg / L FeSO4·7H2O, pH adjusted to 7.0)

[0091] During the fed-batch fermentation, the temperature was controlled at 37°C, the aeration rate was set at 0.74 vvm, the pH was maintained at 6.0-7.0 with NH4OH, and the DO (dissolved oxygen) level was controlled at 20-30% by adjusting the stirring speed (250-800 rpm). Before the start of fermentation, glucose was fed at a rate of 50 mL / min until the initial glucose concentration reached 30 g / L. During the fermentation, 1 mL of the fermentation broth was collected every 2 h and the OD was measured. 600 When the glucose concentration was lower than 10 g / L, glucose was added to maintain the glucose concentration at or above 10 g / L. Bacterial cells were collected by centrifugation for subsequent experiments.

[0092] The results are as follows Figure 4 As shown in the figure, after 16 hours of fermentation, strain WZM012 consumed 30g / L of glucose to 11g / L, and then supplemented sugar to 29g / L for the first time; in the following 8 hours, the growth of WZM012 slowed down and the sugar consumption also continued to slow down until the fermentation reached 24 hours, during which time sugar was no longer supplemented. The highest OD of strain WZM012 after 24 hours of fermentation 600 It is 19.40, with a total sugar consumption of 29g.

[0093] The cells of strain WZM012 fermented at high density were dried and weighed. WZM012 could produce 2.86 g / L of dry cell weight in a 2-L fermenter system. The lipid A structure of these stem cells was extracted to collect about 46.80 mg / L of total lipid A. Based on the ratio of MPL and D-MPLA in the TLC chromatogram, WZM012 could produce about 32.76 mg / L of MPL in a 2-L fermenter system. Figure 4 B) in.

[0094] The lipid A sample of the strain WZM012 was then subjected to LC-MS analysis ( Figure 4 The lipid A sample produced three main peaks in the LC spectrum. The three peaks produced at retention times of 7.8, 9.6, and 10.3 min corresponded to characteristic values ​​of m / z = 1490.1, 1728.3, and 1954.5 in the MS spectrum, respectively, confirming that the structures of these lipid A samples were D-MPLA, MPL, and P-MPLA, respectively.

[0095] These results indicate that strain WZM012 does not need to rely on antibiotics to maintain plasmid stability or inducers to induce gene expression in a high-density fermentation system, and can produce 32.76 mg / L of MPL after 24 hours of fermentation.

[0096] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant bacterium, characterized in that The recombinant bacteria uses Escherichia coli as the starting strain, and knocks out the mlaE Gene 、pldA Gene, hns Gene, ybgC-cpoB Gene clusters and letAB Gene cluster; integrated expression of dephosphatase LpxE and deacylase PagL; mlaE 、 pldA and hns The Gene IDs of the genes are 947732, 948307, and 945829; described ybgC-cpoB The gene cluster contains 7 genes, namely ybgC 、 tolQ 、 tolR 、 tolA 、 tolB 、 pal and cpoB , whose Gene IDs are 948907, 948900, 945328, 946625, 945429, 945004, and 947227; described letAB The gene cluster contains two genes, namely letA and letB , whose Gene IDs are 946353 and 946352 respectively; Encoding the dephosphatase LpxE lpxE The NCBI accession number of the gene is Gene ID: 63960747, encoding the deacylase PagL pagL The NCBI accession number of the gene is Gene ID: 945582.

2. The recombinant bacterium according to claim 1, characterized in that The Escherichia coli includes Escherichia coli MG1655.

3. A method for producing monophospholipid A, characterized in that: The method comprises inoculating the recombinant bacteria according to claim 1 or 2 into a fermentation medium to ferment and produce monophospholipid A.

4. The method according to claim 3, characterized in that The method comprises the following steps: (1) inoculating the recombinant bacteria into a seed culture medium, culturing, and obtaining a seed solution; (2) The seed solution obtained in step (1) is transferred to a fermentation medium for fermentation to produce monophosphate lipid A.

5. The method according to claim 4, characterized in that The fermentation temperature is 25-40°C.

6. The method according to claim 4, characterized in that The pH of the fermentation is 5-10.

7. The method according to claim 4, characterized in that The dissolved oxygen in the fermentation is 10-50%.

8. The method according to claim 7, characterized in that When the glucose concentration was lower than 5-10 g / L during the fermentation process, glucose was fed to maintain the glucose concentration at not lower than 5-10 g / L.

9. Use of the recombinant bacterium according to claim 1 or 2 or the method according to any one of claims 3 to 8 in the production of lipid A vaccine adjuvant.

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