A recombinant Escherichia coli capable of efficiently producing monophospholipid A vaccine adjuvant

Abstract

The present invention discloses a recombinant Escherichia coli capable of efficiently producing monophospholipid A vaccine adjuvant, belonging to the fields of genetic engineering and synthetic biology. The present invention knocks out 132 genes related to membrane wall surface molecules, phospholipid and glucose transport, phosphoethanolamine modification and enoyl-ACP reductase in Escherichia coli MG1655, expresses lipid A modification genes FnlpxE, SepagP and SepagL, and constructs strain MW021 / pTEPL. The growth performance of MW021 / pTEPL is greatly improved and antibiotics and inducers are no longer required during the fermentation process; in a 2-L fermentation tank system, the highest OD of MW021 / pTEPL growth is 600 It can reach 38.54, and can produce 13.84g / L of dry cells and about 88.99mg / L of total lipid A; total lipid A contains only two structures, namely MPL and D-MPLA.

Description

Technical Field

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

[0002] Lipopolysaccharide (LPS) is a crucial component of the outer membrane of Gram-negative bacteria, covering approximately 75% of the cell surface and stabilizing the cell structure and acting as a membrane barrier. LPS is composed of an O-antigen, a core polysaccharide, and lipid A. With the successful delineation of the lipid A biosynthesis pathway in Escherichia coli, precise editing of the molecular structure of E. coli LPS through synthetic biology has become a research hotspot.

[0003] In Escherichia coli, lipid A is the hydrophobic component and active center of the LPS structure, responsible for anchoring the lipopolysaccharide structure to the cell's outer membrane. When pathogens invade the host, LPS is released from the bacterial surface and recognized by TLR4 (Toll-Like Receptor 4) on the surface of host immune cells, leading to the production of various cytokines, such as tumor necrosis factor-α, interleukin-1β, and interferon-γ. This increases the concentration of these cytokines in the host, triggering severe inflammatory responses and even symptoms such as organ failure. However, moderate amounts of these cytokines can actually activate the host's immune system. Research has shown that the structural differences of lipid A determine the quantity and type of cytokines it induces, making certain specialized lipid A structures potentially useful as vaccine adjuvants. MPL (3-deacyl-4'-monophosphoryl lipid A), a lipid A derivative that enhances immune responses, is currently the only commercially available vaccine adjuvant.

[0004] MPL is obtained by chemically treating LPS from Salmonella minnesota R595. Compared to chemically producing MPL, editing the existing lipid A of E. coli through molecular biology and genetic engineering is more in line with industrial standards. Summary of the Invention

[0005] To address the aforementioned technical issues, the present invention first knocks out the gene clusters required for the biosynthesis of pili, flagella, and common intestinal antigens from the Escherichia coli chromosome to obtain strain WQM027 with an optimized membrane wall structure. Subsequently, three genes of the phospholipid transport system (mlaA, mlaC, and pldA), a gene related to the glucose transport system (ptsG), and a phosphoethanolamine modification gene (eptA) are knocked out to obtain strain MW020. Plasmid pTEPL, carrying the lpxE gene from the Francisella chromosome and the pagP and pagL genes from the Salmonella chromosome, is then transformed into strain MW020 to obtain strain MW020 / pTEPL. Finally, the fabI gene in the FabI / triclosan system of strain MW020 / pTEPL is further knocked out to obtain a recombinant strain MW021 / pTEPL. The recombinant strain MW021 / pTEPL can efficiently produce MPL during fermentation without the addition of any additional antibiotics or inducers. The lipid A isolated from MW021 / pWEPL was analyzed by thin layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS). The results showed that MW021 / pWEPL mainly produced two monophosphoryl lipid A, one hexaacylated (MPL) and the other pentaacylated (D-MPLA). The E. coli strain MW021 / pWEPL constructed by this fermentation provides a good alternative for the production of lipid A vaccine adjuvant MPL. By fed-batch fermentation, the highest OD of MW021 / pWEPL growth in a 2-L fermenter system was 600 It can reach 38.54, and can produce 13.84g / L of dry cells and about 88.99mg / L of total lipid A; the total lipid A contains only two structures, which were verified by LC-MS to be MPL and D-MPLA.

[0006] The first object of the present invention is to provide a recombinant Escherichia coli that efficiently produces MPL. The recombinant Escherichia coli knocks out the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, mlaA, mlaC and pldA genes in the phospholipid transport system, ptsG gene in the glucose transport pathway, phosphoethanolamine modification gene eptA and fabI gene in the FabI / triclosan system in the Escherichia coli genome, and overexpresses the dephosphatase (LpxE) from the Francisella genome and the hexadecanoyltransferase (PagP) and deacylase (PagL) from the Salmonella genome.

[0007] In one embodiment, the pilus gene cluster comprises a total of 12 operons.

[0008] In one embodiment, the fimbriae gene cluster comprises 12 operons with a total of 64 genes, namely yagV, yagW, yagX, yagY, yagZ, gltF, yhcA, yhcD, yhcE, yhcF, fimA, fimI, fimC, fimD, fimF, fimG, fimH, sfmA, sfmC, sfmD, sfmH, sfmF, ycbQ, ycbR, ycbS, ycbT, ycbU, ycbV, ycbF, ydeQ, yde R, ydeS, ydeT, yraH, yraI, yraJ, yraK, yadC, yadK, yadL, yadM, htrE, yadV, yadN, yehA, yehB, yehC, yehD, ybgO, ybgP, ybgQ, ybgD, yfcO, yfcP, yfcQ, yfcR, yfcS, yfcT, yfcU, yfcV, ygiL, yqiG, yqiH, yqiI, and the NCBI accession numbers of their sequences are b0289, b0289, and b0289, respectively. 0290, b0291, b0292, b0293, b3214, b3215, b3216, b4569, b3219, b4314, b4315, b4316, b4317, b4318, b4319, b4320 , b0530, b0531, b0532, b0533, b0534, b0938, b0939, b0940, b0941, b0942, b0943, b0944, b1502, b1503, b1504, b15 05, b3142, b3143, b3144, b3145, b0135, b0136, b0137, b0138, b0139, b0140, b0141, b2108, b2109, b2110, b2111, b 0716, b0717, b0718, b0719, b2332, b2333, b2334, b2335, b2336, b2337, b2338, b2339, b3043, b3046, b3047, b3048

[0009] In one embodiment, the flagellar gene cluster comprises a total of 4 operons.

[0010] In one embodiment, the flagellar gene cluster comprises 4 operons with a total of 50 genes, namely fliE, fliF, fliG, fliH, fliI, fliJ, fliK, fliL, fliM, fliN, fliO, fliP, fliQ, fliR, fliY, fliZ, fliA, fliC, fliD, fliS, fliT, flgN, flgM, flgA, flgB, flg C, flgD, flgE, flgF, flgG, flgH, flgI, flgJ, flgK, flgL, flhE, flhA, flhB, cheZ, cheY, cheB, cheR, tap, tar, cheW, cheA, motB, motA, flhC and flhD. The NCBI accession numbers of their sequences are 946446, 946448, 946451 and 946456, respectively. ,946457,946454,946449,946443,946442,946423,946458,946462,946463,946464,948833,946833,948824,949101,946428,946429,946433,945634,946684,946300,945678,946687,945813 ,945636,945639,945647,946996,947534,947456,945648,945646,946094,946390,946391,946392,946393,946394,946396,946397,946399,946400,946401,946402,947564,947280 and 945442.

[0011] In one embodiment, the intestinal common antigen gene cluster is rfe-rffM.

[0012] In one embodiment, the intestinal common antigen gene cluster contains one operon, and the rfe-rffM operon contains 12 genes, including rfe, wzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wecF, wzyE, and rffM. The NCBI accession numbers of their sequences are "NP_418231.1", "NP_418232.1", "YP_026253.1", "YP_026254.1", "YP_026255.1", "NP_418236.1", "YP_026256.1", "NP_418238.1", "NP_418239.1", "YP_026257.1", "NP_418241.1", and "NP_418242.1";

[0013] In one embodiment, the phospholipid transport system comprises mlaA, mlaC and pldA genes; the accession numbers of their sequences on NCBI are "NP_416848.1", "NP_417659.1" and "NP_418265.1".

[0014] In one embodiment, the glucose transport-related gene, phosphoethanolamine modification-related gene and essential gene in the FabI / triclosan system are ptsG, eptA and fabI respectively; the NCBI accession numbers of their sequences are "NP_415619.1", "NP_418538.2" and "NP_415804.1".

[0015] In one embodiment, the lpxE gene is from the Francisella genome; and the pagP and pagL genes are from Salmonella.

[0016] In one embodiment, the NCBI accession number of the sequence from the Francisella lpxE gene is "WP_159184080.1"; the NCBI accession numbers of the sequences from the Salmonella pagP and pagL genes are "NP_459620.1" and "NP_416848.1", respectively.

[0017] In one embodiment, the E. coli comprises E. coli MG1655.

[0018] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant Escherichia coli, which comprises knocking out the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, related genes in the phospholipid transport system (mlaA, mlaC, pldA), related genes in the glucose transport pathway (ptsG), phosphoethanolamine modification gene (eptA) and essential gene fabI in the FabI / triclosan system in the Escherichia coli genome, and overexpressing the dephosphatase (LpxE) from the Francisella genome and the hexadecanoyltransferase (PagP) and deacylase (PagL) from the Salmonella genome.

[0019] In one embodiment, lpxE from Francisella and pagP and pagL from Salmonella are ligated to the expression plasmid pFT24.

[0020] The third object of the present invention is to provide a method for efficiently producing MPL, wherein the method comprises inoculating the above-mentioned recombinant Escherichia coli into culture medium A to prepare a seed solution; and separating the prepared seed solution according to the initial OD 600 =0.02 was inoculated into culture medium B for fermentation production.

[0021] In one embodiment, the culture medium A contains 4-6 g / L yeast powder, 8-12 g / L peptone, and 8-12 g / L NaCl;

[0022] Medium B contained 10-60 g / L glucose, 7.46 g / L KH2PO4, 15 g / L (NH4)2SO4, 3 g / L yeast extract, 2 g / L citric acid, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4·4H2O, 2 g / L MgSO4·7H2O and was adjusted to pH 7.0.

[0023] In one embodiment, the fermentation reaction conditions are: temperature 37° C., 180-220 rpm.

[0024] The fourth object of the present invention is to provide the application of the recombinant Escherichia coli in the field of biomedicine.

[0025] The fifth object of the present invention is to provide the use of the recombinant Escherichia coli in the production of lipid A vaccine adjuvant.

[0026] Beneficial effects

[0027] (1) The present invention knocks out the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, related genes (mlaA, mlaC, pldA) in the Escherichia coli genome, related genes in the glucose transport pathway (ptsG), phosphoethanolamine modification gene (eptA), and the essential gene fabI in the FabI / triclosan system in Escherichia coli, and overexpresses the dephosphatase (LpxE) from the Francisella genome and the hexadecanoyltransferase (PagP) and deacylase (PagL) from the Salmonella genome to construct the recombinant strain MW021 / pTEPL. In medium A, the growth of the recombinant strain MW021 / pTEPL is similar to that of the starting strain MG1655; in medium B, the growth of the recombinant strain MW021 / pTEPL is better than that of the starting strain.

[0028] (2) The recombinant strain MW021 / pTEPL 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 two types of monophosphoryl lipid A, one is hexaacylated (MPL) and the other is pentaacylated (D-MPLA), among which MPL is the most important component in vaccine adjuvants.

[0029] (3) The recombinant strain MW021 / pTEPL of the present invention has a maximum OD in a 2-L fermentation tank system. 600 It can reach 38.54, and can produce 13.84g / L of dry cells and about 88.99mg / L of total lipid A. In recent reports, other research groups studied and constructed a recombinant E. coli that can produce purified EcML (MPLA from E. coli W3110), and its total lipid A production is only 8-12mg / L.

[0030] Compared with other methods for preparing MPL, MW021 / pTEPL has more advantages. Some bacteria can naturally express LPS with lower toxicity. For example, Bacteroides thetaiotaomicron and Rhodopseudomonas palustris can express monophosphorylated and pentaacylated lipid A structures, similar to MPL. However, B. thetaiotaomicron and R. palustris are anaerobic bacteria, and the cultivation process is complex and time-consuming. In fact, no strains that directly produce MPL have been found in nature, which means that MPL can only be obtained through chemical synthesis of S. minnesota R595 LPS. However, S. minnesota R595 is a pathogenic bacterium, and the lipid A structure it produces is quite complex, which means that chemically synthesized MPL is composed of different homologs and is expensive. Compared with the above methods, MW021 / pTEPL is a type of Escherichia coli that is easy to culture, grows rapidly, does not require additional antibiotics and inducers, and reduces production costs. MW021 / pTEPL can not only be used safely as a host bacteria in food and pharmaceutical fermentation industrial production, but also as a safer gene expression vector Escherichia coli in laboratory research, which is more in line with the requirements of high-density growth in industrial standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The process of knocking out genes from the Escherichia coli chromosome.

[0032] Figure 2 The construction process of different E. coli mutants and the growth conditions of these strains.

[0033] Figure 3 This is the growth curve of Escherichia coli MW019 / pTEPL and the lipid A structure analysis.

[0034] Figure 4 This paper describes the construction process of Escherichia coli MW021 / pTEPL and the analysis of its lipid A structure.

[0035] Figure 5 These are the results of fed-batch fermentation of Escherichia coli MW021 / pTEPL. 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 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. The pTargetF plasmid involved was disclosed in 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. The WQM021 involved in the paper is disclosed in Qiao, J., Tan, X., Ren, H., Wu, Z., 2021b. Construction of an Escherichiacoli strain lacking fimbriae by deleting 64 genes and its application for efficient production of poly(3-hydroxybutyrate)and L-threonine. AEM.87.e00381-21. The pFT24 involved in the paper is recorded in the Chinese invention patent with publication number CN 111363757 B.

[0039] The strain WQM026 involved in the following examples is described in the Chinese invention patent publication number CN112680393A, and the pWEPL plasmid involved in the following examples is described in the Chinese invention patent publication number CN115181715A.

[0040] The culture medium involved in the following examples is as follows:

[0041] All culture media were prepared using ddH2O and sterilized at 121°C for 15-20 min.

[0042] Medium A (g / L): yeast powder 5, peptone 10, NaCl 10.

[0043] Medium B (g / L): 10-60 g / L glucose, 5 g / L (NH4)2SO4, 2 g / L yeast extract, 2 g / L citric acid, 7.46 g / L KH2PO4, 2 g / L MgSO4·7H2O, 5 mg / L FeSO4·7H2O, 5 mg / L MnSO4·4H2O, (NaOH was used to adjust the medium pH to 7.00).

[0044] The primer sequences involved in the following examples are shown in Table 1:

[0045] Table 1: Primer sequences

[0046]

[0047]

[0048] Example 1: Construction of knockout plasmid

[0049] The CRISPR / Cas9 knockout system was used to knock out the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, genes related to the phospholipid transport system, genes related to glucose transport, genes related to phosphoethanolamine modification, and essential genes in the FabI / triclosan system in Escherichia coli. Eleven knockout plasmids were constructed: pT-fliE-R, pT-fliY-T, pT-flgN-L, pT-flhE-D, pT-wecA-rffM, pT-pldA, pT-mlaA, pT-mlaC, pT-ptsG, pT-eptA, and pT-fabI. The construction process of these plasmids was as follows:

[0050] (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 fliE-R-sgRNA-F, fliY-T-sgRNA-F, flgN-L-sgRNA-F, flhE-D-sgRNA-F, wecA-rffM-sgRNA-F, pldA-sgRNA-F, mlaA-sgRNA-F, mlaC-sgRNA-F, ptsG-sgRNA-F, eptA-sgRNA-F and fabI-sgRNA-F, respectively.

[0051] Using plasmid pTargetF as template, forward primers fliE-R-sgRNA-F, fliY-T-sgRNA-F, flgN-L-sgRNA-F, fl hE-D-sgRNA-F, wecA-rffM-sgRNA-F, pldA-sgRNA-F, mlaA-sgRNA-F, mlaC-sgRNA-F, ptsG-sgRNA-F, eptA-sgRNA-F and fabI-sgRNA-F were used together with reverse primer pTargetF-R to amplify the N-inducible promoter region. 20 The PCR amplification product was verified by electrophoresis and purified and recovered.

[0052] (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.

[0053] (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.

[0054] (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;

[0055] The knockout plasmids were prepared respectively: pT-fliE-R, pT-fliY-T, pT-flgN-L, pT-flhE-D, pT-wecA-rffM, pT-pldA, pT-mlaA, pT-mlaC, pT-ptsG, pT-eptA and pT-fabI.

[0056] Example 2: Construction of strain MW019

[0057] The specific knockout process of genes using the CRISPR / Cas9 knockout system is as follows ( Figure 1):

[0058] (1) Preparation of E. coli electroporation knockout competent cells MG1655 / pCas

[0059] 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 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.

[0060] (2) Construction of homology arm knockout fragments

[0061] The genome of Escherichia coli WQM026 was extracted and used as a template to amplify the upstream and downstream homology arms using the homology arm primers U-fliE-RF / U-fliE-RR and D-fliE-RF / D-fliE-RR of the target gene cluster fliE-R. The fragments were recovered by gel chromatography, and overlapping PCR was performed using primers U-fliE-F and D-fliE-RR to obtain homology arm knockout fragment 1.

[0062] The upstream and downstream homology arms were amplified using primers U-fliY-TF / U-fliY-TR and D-fliY-TF / D-fliY-TR for knocking out the target gene cluster fliY-T, respectively. The fragments were recovered by gel amplification, and overlapping PCR was performed using primers U-fliY-TF / D-fliY-TR to obtain homology arm knockout fragment 2.

[0063] The upstream and downstream homology arms were amplified using primers U-flgN-LF / U-flgN-LR and D-flgN-LF / D-flgN-LR for knocking out the target gene cluster flgN-L, respectively. The fragments were recovered by gel amplification, and overlapping PCR was performed using primer pair U-flgN-LF / D-flgN-LR to obtain homology arm knockout fragment 3.

[0064] The upstream homology arm and downstream homology arm were amplified using the homology arm primers U-flhE-DF / U-flhE-DR and D-flhE-DF / D-flhE-DR of the target gene cluster flhE-D, respectively. The fragments were recovered by gel and overlap PCR was performed using the primers U-flhE-DF / D-flhE-DR to obtain homology arm knockout fragment 4.

[0065] The upstream and downstream homology arms were amplified using the homology arm primers U-wecA-rffM-F / U-wecA-rffM-R and D-wecA-rffM-F / D-wecA-rffM-R of the target gene cluster wecA-rffM, respectively. The fragments were recovered by gel amplification, and overlapping PCR was performed using the primer pair U-wecA-rffM-F / D-wecA-rffM-R to obtain the homology arm knockout fragment 5.

[0066] 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 for knocking out the target gene pldA, respectively. After gel recovery, overlapping PCR was performed using the primers U-pldA-F / D-pldA-R to obtain homology arm knockout fragment 6;

[0067] The upstream and downstream homology arms were amplified using the homology arm primers U-mlaA-F / U-mlaA-R and D-mlaA-F / D-mlaA-R for knocking out the target gene mlaA, respectively. The fragments were recovered from the gel, and overlapping PCR was performed using the primers U-mlaA-F / D-mlaA-R to obtain homology arm knockout fragment 7.

[0068] The upstream and downstream homology arms were amplified using the homology arm primers U-mlaC-F / U-mlaC-R and D-mlaC-F / D-mlaC-R for knocking out the target gene mlaC, respectively. The fragments were recovered from the gel, and overlapping PCR was performed using the primers U-mlaC-F / D-mlaC-R to obtain homology arm knockout fragment 8.

[0069] The upstream homology arm and downstream homology arm were amplified using the homology arm primers U-ptsG-F / U-ptsG-R and D-ptsG-F / D-ptsG-R of the target gene cluster ptsG, respectively. The fragments were recovered by gel and overlap PCR was performed using the primer pair U-ptsG-F / D-ptsG-R to obtain the homology arm knockout fragment 9.

[0070] (3) Gene knockout by electroporation

[0071] Wash the electroporation cup three times with anhydrous ethanol and blow dry, pre-cool for 20 minutes. Place the E. coli MG1655 / pCas competent cells in step (1) on ice to melt, add 100 ng of the knockout plasmid pTargetF-gene (pT-fliE-R, pT-fliY-T, pT-flgN-L, pT-flhE-D, pT-wecA-rffM, pT-pldA, pT-mlaA, pT-mlaC, pT-ptsG) in Example 1 and 500 ng of the corresponding homology arm knockout fragment in step (2), gently pipette to mix, and aspirate into the groove of the electroporation cup. Ice bath the electroporation cup for 10 minutes, wipe dry and then electroporate. Then, quickly add 1 mL of LB medium to the cuvette, aspirate the entire bacterial suspension into a 1.5 mL EP tube, and resuscitate at 30°C, 100 rpm for 1.5 hours. Plate the cells onto LB plates containing 30 mg / L Kan and 50 mg / L Spe, and incubate them upside down in a 30°C incubator. Using MG1655 as a negative control, screen the transformants using the forward primer for the upstream homology arm and the reverse primer for the downstream homology arm to identify the correct transformants.

[0072] (4) Removal of knockout plasmid pTargetF-gene and temperature-sensitive plasmid pCas

[0073] The correct knockout transformant in step (3) was inoculated into an LB tube supplemented with 30 mg / L Kan and 1 mM IPTG. The enzyme expression of the knockout plasmid pTargetF-gene was induced by IPTG. The cells were cultured at 30°C with shaking for 12 h, and single colonies were isolated by streaking on an LB (Kan+) plate. Single colonies sensitive to spectinomycin were screened, i.e., mutant strains with the pTargetF-gene knockout plasmid removed were obtained. The mutants were inoculated into LB (Kan+) tubes for seed storage, and competent cells could be directly prepared for continuous knockout.

[0074] The mutant strain with the knockout plasmid removed was transferred to an LB tube and cultured with shaking at 42°C. Single colonies were streaked onto LB plates to isolate the strain. Single colonies sensitive to kanamycin were selected, indicating the pCas-free mutant strain. These colonies were then transferred to LB tubes for seed storage.

[0075] The flagellar gene cluster, intestinal common antigen gene cluster, three phospholipid transport-related genes, mlaA, mlaC and pldA, and glucose transport pathway-related gene ptsG were successfully knocked out from the chromosome of strain WQM026 using the CRISP / Cas9 knockout method, and the LPS-reduced strain MW019 and several process strains ( Figure 2 and shown in Table 2).

[0076] Table 2 Strains and genotypes involved in the above examples

[0077]

[0078] In LB medium, MG1655, MW015, MW016 (the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, and glucose transport-related gene ptsG were knocked out in the MG1655 genome), MW017, MW018 (the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, and phospholipid transport system: pldA, mlaA and mlaC were knocked out in the MG1655 genome), WQM026 (the fimbriae gene cluster was knocked out in the MG1655 genome), WQM027 (the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster were knocked out in the MG1655 genome) and MW019 (the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, glucose transport-related gene ptsG, and phospholipid transport system: pldA, mlaA and mlaC were knocked out in the MG1655 genome) showed similar growth patterns and reached the stationary phase after 12 hours.

[0079] However, these E. coli mutants showed different growth patterns in the fermentation medium: the maximum OD600 values ​​of MG1655, MW015, MW016, MW017, MW018, WQM027 and MW019 were 17.15, 33.66, 28.87, 15.67, 16.93, 21.07 and 32.11, respectively. 600 The values ​​were 1.96, 1.68, and 1.87 times that of MG1655, respectively, indicating that the deletion of ptsG could improve the growth of E. coli in fermentation medium.

[0080] Example 3: Construction of expression plasmid pTEPL

[0081] The specific steps are as follows:

[0082] (1) Using plasmid pWEPL as a template, primers EPL-F and EPL-R were used to amplify the entire fragments of FnlpxE (NCBI accession number "WP_159184080.1"), SepagP (NCBI accession number "NP_459620.1"), and SepagL (NCBI accession number "NP_416848.1") by PCR, and then the fragments were verified by electrophoresis and purified;

[0083] (2) The vector pFT24 was digested with EcoRI endonuclease at 37°C for 30 min. The digested products were then recovered and verified by electrophoresis.

[0084] (3) Mix the recovered enzyme-digested products and gene fragments and react at 37°C for 30 minutes according to the instructions of the one-step cloning kit to obtain a reaction solution; after the reaction, take out the E. coli JM109 competent cells and melt them on ice, add the reaction solution to the competent cells and gently pipette to mix, and place them on ice for 30 minutes; then heat shock the competent cells in a 42°C water bath for 90 seconds, place them on ice for 2 minutes, and quickly add 1 mL of LB culture medium; resuscitate at 37°C, 100 rpm for 1 hour, and then spread on an LB plate supplemented with 0.9 mg / L triclosan, and invert and culture at 37°C to screen knockout transformants; using pFT24 as a negative control, perform colony PCR on the transformants to verify whether the plasmid was successfully constructed; the correct transformants were transferred to a LB liquid test tube supplemented with triclosan, and the plasmid was extracted to obtain pFT24-FnlpxE-SepagP-SepagL, which was named plasmid pTEPL. The plasmid map is shown in the figure below. Figure 3 As shown in A.

[0085] Example 4: Construction of recombinant strain MW019 / pTEPL

[0086] (1) Preparation of Escherichia coli MW019 competent cells

[0087] The E. coli MW019 in Example 2 was inoculated into LB liquid medium and cultured overnight at 37°C, 200 rpm. The seed solution was transferred to 50 mL LB liquid medium at a 2% (v / v) inoculum volume and cultured at 37°C, 200 rpm until the OD 600 =0.4-0.6, ice-bath the culture medium for half an hour and transfer it to a pre-cooled 50 mL centrifuge tube. Centrifuge at 8000 rpm for 10 min at 4°C to collect the bacteria. Wash the precipitate three times with pre-cooled 0.01 M CaCl2, and finally resuspend it with 1 mL of 0.01 M CaCl2. Add 1 mL of 30% glycerol and mix well. Aliquot 200 μL per tube into pre-cooled sterile EP tubes.

[0088] (2) Conversion

[0089] 100-200 ng of the plasmid pTEPL in Example 4 was added to the E. coli MW019 competent cells prepared in step (1), mixed, ice-bathed for 30 min, heat-shocked at 42°C for 90 s, ice-bathed for 2-3 min, and revived by adding 1 mL of LB medium. The cells were incubated at 37°C for 2 h, coated on LB solid plates containing 0.9 mg / L triclosan, and cultured at 37°C. Transformants were selected and cultured in LB liquid medium containing 0.9 mg / L triclosan. The recombinant strain MW019 / pTEPL was obtained.

[0090] The plasmid pTEPL was transformed into MG1655 according to the above method to construct the strain MG1655 / pTEPL.

[0091] (3) Culture of strains

[0092] The introduction of plasmid pTEPL had no effect on the growth of the strain.

[0093] 1) The recombinant strain MG1655 / pTEPL was inoculated into LB medium containing 0.9 mg / L triclosan and cultured at 37°C and 200 rpm for 18 h; samples were taken every 2 h to measure the OD 600 The absorbance value should be recorded; after the strain grows to the stagnant period, two samples should be taken and the OD should be detected. 600 The absorbance value was recorded; the results showed that the highest OD of strain MG1655 / pTEPL 600 It is 4.49.

[0094] Approximately 1 mL of the MG1655 / pTEPL fermentation broth was used for LC-MS analysis of lipid A. The lipid A extracted from MG1655 / pTEPL produced four main peaks in the LC spectrum. Based on previous analysis, these peaks were detected at retention times of 7.8, 8.9, 9.7, and 10.3 min, corresponding to characteristic MS values ​​of m / z = 1490.1, 1716.2, 1728.3, and 1954.5, respectively. These results confirm that the four lipid A structures in MG1655 / pTEPL are D-MPLA, MPLA, MPL, and P-MPLA.

[0095] 2) The recombinant strain MW019 / pTEPL was inoculated into LB medium containing 0.9 mg / L triclosan and cultured at 37°C and 200 rpm for 18 h; samples were taken every 2 h to detect OD 600 The absorbance value should be recorded; after the strain grows to the stagnant period, two samples should be taken and the OD should be detected. 600 The absorbance value was recorded; the results showed that the highest OD of strain MW019 / pTEPL 600 25 mM EDTA was added to the strain MW019 / pTEPL 2 h before the end of fermentation, and the fermented cells were collected and their lipid A was extracted and analyzed by LC-MS.

[0096] The results showed that lipid A extracted from MW019 / pTEPL produced two main peaks in the LC spectrum. According to previous analysis, the two peaks produced at retention times of 7.7 and 9.6 min corresponded to characteristic values ​​of m / z = 1490.1 and 1728.3 in the MS spectrum, respectively. These results confirmed that when 25 mM EDTA was added before fermentation, the two lipid A structures in strain MW019 / pTEPL were D-MPLA and MPL ( Figure 3 ).

[0097] It can be seen that compared with the original strain MG1655 / pTEPL, the lipid A produced by the modified strain MW019 / pTEPL has only two structures, MPL and D-MPLA, that is, the MPL synthesized by the modified strain MW019 / pTEPL has a higher purity.

[0098] Example 5: Construction of Escherichia coli MW021 / pTEPL

[0099] The construction method is the same as in Example 2, and the specific knockout process is as follows:

[0100] (1) Preparation of E. coli electroporation knockout competent cells MW019 / pTEPL-pCas

[0101] The plasmid pCas was transformed into Escherichia coli MW019 / pTEPL to obtain recombinant Escherichia coli MW019 / pTEPL-pCas containing the pCas plasmid. The Escherichia coli MW019 / pTEPL-pCas was activated on LB solid plates supplemented with 30 mg / L kanamycin (Kan) and 0.9 mg / L triclosan (Tri) and inoculated onto LB (Kan). + 、Tri + ) overnight in vitro culture to obtain seed solution; the seed solution was transferred to 25 mL LB (Kan + 、Tri + ) medium and cultured at 30°C and 200 rpm until OD 600 =0.2, add 500 μL 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.

[0102] (2) Construction of homology arm knockout fragments

[0103] 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 eptA knockout using primers U-eptA-F / U-eptA-R and D-eptA-F / D-eptA-R, respectively. The fragments were recovered from the gel and overlap PCR was performed using primers U-eptA-F / D-eptA-R to obtain homology arm knockout fragment 10.

[0104] The upstream homology arm and downstream homology arm were amplified using the homology arm primers U-fabI-F / U-fabI-R and D-fabI-F / D-fabI-R of the knockout target gene cluster fabI, respectively. The fragments were recovered by gel and overlap PCR was performed using the primer pair U-fabI-F / D-fabI-R to obtain the homology arm knockout fragment 11.

[0105] (3) Gene knockout by electroporation

[0106] Wash the electroporation cup three times with anhydrous ethanol and blow dry, and pre-cool for 20 minutes. Place the Escherichia coli MW019 / pTEPL-pCas competent cells in step (1) on ice to melt, add 100ng of the knockout plasmids pTargetF-eptA and pTargetF-fabI in Example 1, and 500ng of the corresponding homology arm knockout fragments in step (2), gently blow and mix, and aspirate into the groove of the electroporation cup. Ice bath the electroporation cup for 10 minutes, wipe dry and then electroporate. Then quickly add 1mL LB culture medium to the electroporation cup, aspirate all the bacterial liquid into a 1.5mL EP tube, 30℃, 100rpm recovery for 1.5h, spread on an LB plate containing 0.9mg / L Tri, 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.

[0107] (4) Removal of knockout plasmid pTargetF-gene and temperature-sensitive plasmid pCas

[0108] The correct knockout transformant in step (3) was inoculated into an LB tube supplemented with 0.9 mg / L triclosan Tri, 30 mg / L Kan, and 1 mM IPTG. The enzyme expression of the knockout plasmid pTargetF-gene was induced by IPTG and cultured at 30°C for 12 h. + Tri + ) plate and streak single colonies. Screen out single colonies that are sensitive to spectinomycin and triclosan, that is, obtain mutant strains with pTargetF-gene knockout plasmid removed, and inoculate them into LB (Kan + Tri + ) tube seed preservation, can directly prepare competent cells for continuous knockout. Remove the knockout plasmid mutant strain and inoculate it into LB (Tri + ) tube, shake culture at 42℃, and streak single colonies on LB plates. Screen out single colonies that are sensitive to kanamycin, that is, obtain a strain without resistance mutants that have removed pCas, and inoculate them onto LB (Tri + )Seed preservation in vitro.

[0109] The eptA gene and fa bI gene were successfully knocked out in the chromosome of strain MW019 / pTEPL using the CRISP / Cas9 knockout method ( Figure 4 The resulting strain was named MW021 / pTEPL.

[0110] Example 6: Extraction and structural verification of lipid A structure of recombinant strain MW021 / pTEPL

[0111] The specific steps are as follows:

[0112] The recombinant strain MW021 / pTEPL prepared in Example 5 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 OD 600 =0.02 was transferred to 200 mL of culture medium B and cultured at 37°C and 200 rpm for 18 h. Subsequently, 25 mM EDTA was added and the culture was continued for 2 h. After that, the cells were collected by centrifugation at 8000 rpm for 10 min and their lipid A was extracted (no 25 mM EDTA was added before the end of fermentation as a control).

[0113] The detection method of Escherichia coli lipid A structure is as follows:

[0114] 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, sonicated for 10 minutes, and incubated at 100°C for 30 minutes to cleave sugar chains. 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 blowdown device, and lipid A was stored at -20°C until use.

[0115] 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.

[0116] 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.

[0117] The results show that: Figure 4 As shown, the highest OD of strain MW021 / pTEPL in LB medium 600 It is 4.31.

[0118] LC-MS analysis of lipid A samples from MW021 / pTEPL was performed. Without the addition of 25 mM EDTA 2 hours before the end of fermentation, lipid A extracted from MW021 / pTEPL produced three main peaks in the LC spectrum. Based on previous analysis, the three peaks at retention times of 7.6, 9.5, and 10.2 minutes corresponded to characteristic MS values ​​of m / z = 1490.1, 1728.3, and 1954.5, respectively, confirming the structures of these lipid A compounds as D-MPLA, MPL, and P-MPLA.

[0119] When 25 mM EDTA was added 2 h before the end of fermentation, lipid A extracted from MW021 / pTEPL produced only two main peaks in the LC spectrum. The two peaks produced at retention times of 7.6 and 9.55 min corresponded to characteristic values ​​of m / z = 1490.1 and 1728.3 in the MS spectrum, namely D-MPLA and MPL, respectively.

[0120] These results demonstrate that the knockout of the fabI gene in MW021 / pTEPL strain can efficiently produce MPL without affecting its growth in LB medium. Importantly, E. coli MW021 / pTEPL does not require the addition of any additional antibiotics (such as triclosan) or chemical reagents during its growth.

[0121] Example 7: Fed-batch fermentation of recombinant strain MW021 / pTEPL

[0122] The specific steps are as follows:

[0123] (1) Activation of strains: E. coli MG1655 and MW021 / pTEPL were first cultured in 5 mL of LB medium at 37°C and 200 rpm for 18 h;

[0124] (2) The activated strain in step (1) was transferred to 50 mL of STF medium (10 g / L glucose, 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;

[0125] (3) Transfer 50 mL of the seed solution obtained in step (2) into a fermentation tank containing 1 L of fermentation medium.

[0126] 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)

[0127] 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 fell below 10 g / L, glucose was added. After 26 hours, 25 mM EDTA (ethylenediaminetetraacetic acid) was added and the culture was continued for another 2 hours before fermentation was stopped. The bacterial cells were collected by centrifugation for subsequent experiments.

[0128] The results showed that the fed-batch fermentation of strain MG1655 ( Figure 5 As shown in Figure A, after 10 hours of fermentation, strain MG1655 consumed 30 g / L of glucose to 9 g / L, and then supplemented with glucose to 29 g / L for the first time. In the following 6 hours, MG1655 continued to grow and its sugar consumption continued to increase. After 16 hours of fermentation, the glucose concentration was supplemented to 22 g / L for the second time. No more glucose was supplemented until 26 hours of fermentation. The highest OD value of strain MG1655 after 26 hours of fermentation was 600 It is 22.48, with a total sugar consumption of 55g.

[0129] The strain MW021 / pTEPL was subjected to fed-batch fermentation ( Figure 5As shown in Figure B, after 10 h of fermentation, strain MW021 / pTEPL consumed 30 g / L of glucose to 13 g / L, and then supplemented it to 31 g / L. In the following 6 h, MW021 / pTEPL grew rapidly, and the glucose concentration of 31 g / L was consumed to 10 g / L. After 16 h of fermentation, the glucose concentration was supplemented again to 20 g / L. Glucose was then supplemented every 2 h until 26 h of fermentation. The highest OD value of strain MW021 / pTEPL after 26 h of fermentation was 600 It is 38.54, and the total sugar consumption is 86g.

[0130] The cells of the MW021 / pTEPL high-density fermentation were dried and weighed, revealing that MW021 / pTEPL produced 13.84 g / L of dry cell weight in a 2-L fermenter. Extraction of the lipid A structure of these stem cells yielded approximately 88.99 mg / L of total lipid A. Based on the ratio of MPL to D-MPLA on the TLC plate, MW021 / pTEPL produced approximately 62.30 mg / L of MPL in a 2-L fermenter.

[0131] The lipid A sample of the strain MW021 / pTEPL was then subjected to LC-MS analysis ( Figure 5 The lipid A sample produced two main peaks in the LC spectrum. According to previous analysis, the three peaks produced at retention times of 7.7 and 9.6 min corresponded to characteristic values ​​of m / z = 1490.1 and 1728.3 in the MS spectrum, confirming that the structures of these lipid A samples were D-MPLA and MPL, respectively.

[0132] These results indicate that compared with the previously constructed MW019 / pWEPL and MW019 / pTEPL, strain MW021 / pTEPL can also efficiently produce MPL. Although its growth is slightly affected, strain MW021 / pTEPL does not need to rely on antibiotics to maintain plasmid stability or inducers to induce gene expression in a high-density fermentation system.

[0133] 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 Escherichia coli, characterized in that The recombinant Escherichia coli knocks out the fimbriae gene cluster, flagellar gene cluster, intestinal common antigen gene cluster, phospholipid transport system related genes: mlaA, mlaC and pldA genes, glucose transport related gene: ptsG gene, phosphoethanolamine modification related gene: eptA gene and FabI / triclosan system related essential gene: fabI gene on the Escherichia coli genome, and overexpresses the dephosphatase from the Francisella genome and the hexadecanoyltransferase and deacylase from the Salmonella genome; The fimbriae gene cluster consists of 64 genes in 12 operons, namely yagV, yagW, yagX, yagY, yagZ, gltF, yhcA, yhcD, yhcE, yhcF, fimA, fimI, fimC, fimD, fimF, fimG, fimH, sfmA, sfmC, sfmD, sfmH, sfmF, ycbQ, ycbR, ycbS, ycbT, ycbU, ycbV, ycbF, ydeQ, ydeR, ydeS, ydeT, yraH, yraI, yraJ, yraK, yadC, yadK, yadL, yadM, htrE, yadV, yadN, yehA, yehB, yehC, yehD, ybgO, ybgP, ybgQ, ybgD, yfcO, yfcP, yfcQ, yfcR, yfcS, yfcT, yfcU, yfcV, ygiL, yqiG, yqiH, yqiI, and the NCBI accession numbers of their sequences are b0289, b0290, b0291, b0292, b0293, b3214, b3215, b3216, b4569, b3219, b4314, b4315, b4316, b4317, b4318, b4319, b4320, b05 30, b0531, b0532, b0533, b0534, b0938, b0939, b0940, b0941, b0942, b0943, b0944, b1502, b1503, b1504, b1505, b3142, b3143, b3144, b3145, b0135, b0136, b0137, b0138, b0139, b0140, b0141, b2108, b2109, b2110, b2111, b07 16, b0717, b0718, b0719, b2332, b2333, b2334, b2335, b2336, b2337, b2338, b2339, b3043, b3046, b3047, b3048; The flagellar gene cluster consists of 50 genes in 4 operons, namely fliE, fliF, fliG, fliH, fliI, fliJ, fliK, fliL, fliM, fliN, fliO, fliP, fliQ, fliR, fliY, fliZ, fliA, fliC, fliD, fliS, fliT, flgN, flgM, flgA, flgB, flgC, and flgD , flgE, flgF, flgG, flgH, flgI, flgJ, flgK, flgL, flhE, flhA, flhB, cheZ, cheY, cheB, cheR, tap, tar, cheW, cheA, motB, motA, flhC and flhD, and the NCBI accession numbers of their sequences are 946446, 946448, 946451, 946456, 946464, respectively. 457,946454,946449,946443,946442,946423,946458,946462,946463,946464,948833,946833,948824,949101,946428,946429,946433,945634,946684,946300,945678,946687,945813,9 45636,945639,945647,946996,947534,947456,945648,945646,946094,946390,946391,946392,946393,946394,946396,946397,946399,946400,946401,946402,947564,947280 and 945442; The intestinal common antigen gene cluster consists of one operon, and the rfe-rffM operon consists of 12 genes, namely rfe, wzzE, wecB, wecC, rffG, rffH, rffC, wecE, wzxE, wecF, wzyE, and rffM. The NCBI accession numbers of the sequences are NP_418231.1, NP_418232.1, YP_026253.1, YP_026254.1, YP_026255.1, NP_418236.1, YP_026256.1, NP_418238.1, NP_418239.1, YP_026257.1, NP_418241.1, and NP_418242.1, respectively; The phosphate transport system-related genes: mlaA, mlaC and pldA, whose sequences have the NCBI accession numbers NP_416848.1, NP_417659.1 and NP_418265.1; The glucose transport-related gene: ptsG gene, the phosphoethanolamine modification-related gene: eptA gene, and the essential gene related to the FabI / triclosan system: fabI gene, the NCBI accession numbers of their sequences are NP_415619.1, NP_418538.2, and NP_415804.1, respectively; The NCBI accession number of the sequence from the Francisella lpxE gene is WP_159184080.1; the NCBI accession numbers of the sequences from the Salmonella pagP and pagL genes are NP_459620.1 and NP_416848.1, respectively.

2. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli overexpresses the dephosphatase from the Francisella genome and the hexadecanoyl transferase and deacylase from the Salmonella genome using the pFT24 vector.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that The Escherichia coli is Escherichia coli MG1655.

4. A method for producing monophospholipid A, characterized in that: The method comprises fermenting and producing monophospholipid A using the recombinant Escherichia coli according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The recombinant Escherichia coli is inoculated into an antibiotic-free culture medium for culture.

6. The method according to claim 5, characterized in that The method comprises the following steps: inoculating the above-mentioned recombinant Escherichia coli into a seed culture medium to prepare a seed solution; and isolating the prepared seed solution according to the initial OD 600 The inoculum size is 0.02 and inoculated into the fermentation medium for fermentation. MPL is extracted from the recombinant Escherichia coli after 2 to 18 hours of fermentation.

7. The method according to claim 6, characterized in that The seed culture medium contains 4-6 g / L yeast powder, 8-12 g / L peptone, and 8-12 g / L NaCl; the fermentation culture medium contains 10-60 g / L glucose, 7.46 g / L KH2PO4, 15 g / L (NH4)2SO4, 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, and the pH is adjusted to 7.

0.

8. Use of the recombinant Escherichia coli according to any one of claims 1 to 3 in the production of lipid A vaccine adjuvant.

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