CN116179455A

CN116179455A - Genetically engineered bacterium for effectively synthesizing lipopolysaccharide

Info

Publication number: CN116179455A
Application number: CN202211537066.5A
Authority: CN
Inventors: 王建莉; 马文渐; 王小元; 陈姜铧; 叶梓琪; 奚能
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-12-01
Filing date: 2022-12-01
Publication date: 2023-05-30

Abstract

The invention discloses a genetically engineered bacterium for effectively synthesizing lipopolysaccharide, in particular relates to a non-resistant lipopolysaccharide escherichia coli production bacterium, and belongs to the technical fields of genetic engineering and biosynthesis. The genetically engineered bacterium of the present invention is inactivated by at least one deletion mutation of npr, yhbJ and fabF. The invention has the advantages that the synthesis amount of lipopolysaccharide of the genetically engineered bacterium WOZF with the three-gene deletion mutation is increased by 40.6 percent compared with that of a wild type bacterium, the growth condition is good, no exogenous resistance gene sequence is introduced, and the genetically engineered bacterium WOZF is more beneficial to large-scale industrial production.

Description

Genetically engineered bacterium for effectively synthesizing lipopolysaccharide

Technical Field

The invention relates to a genetically engineered bacterium for effectively synthesizing lipopolysaccharide, in particular to a non-resistant lipopolysaccharide escherichia coli production bacterium, and belongs to the technical fields of genetic engineering and biosynthesis.

Technical Field

Lipopolysaccharide is an active ingredient that embodies the virulence of gram-negative bacteria, which can be recognized by TLR4/MD2 on the surface of host immune cells, and is a virulence factor that infects the host. Upon receiving extracellular signals, TLR4 stimulation induces the formation of intracellular protein complexes, thereby activating intracellular signaling cascades. These responses trigger the biosynthesis and secretion of various stimulatory cytokines such as

interleukins

1, 8, 12, alpha tumor necrosis factor (IL-1, IL-8, IL-12, TNF alpha) and produce co-stimulatory molecules that ultimately activate both humoral and cellular responses. The active structure playing an immune role in the lipopolysaccharide structure is lipoid A, wherein the lipoid A contains hexaacylated acyl chains and two phosphoric acid residues, each acyl chain contains 12-14 carbons, and the lipoid A is the most effective stimulator of human TLR4 and can furthest excite immune response. The vaccine can enhance the immunity of the vaccine when the vaccine which naturally contains bacterial components such as LPS or derivatives thereof is inoculated. Such molecules that provide "help" to the antigen are defined as adjuvants. The immune response caused by the vaccine added with the proper adjuvant is enhanced, and the immune range is enlarged.

With intensive studies on the pathogenic mechanism thereof, lipopolysaccharide (LPS) is widely used in animal immunization experiments. But the yield of the E.coli strain naturally synthesizing lipopolysaccharide is low. Therefore, it is necessary to solve the above problems by constructing a strain of Escherichia coli which can efficiently synthesize lipopolysaccharide by genetic engineering. Meanwhile, it is very necessary to obtain genetically engineered bacteria without resistance markers and/or without affecting the growth of the bacteria.

Disclosure of Invention

In order to solve at least one of the problems, the invention provides a novel genetic engineering bacterium which does not have any resistance mark and produces LPS with good growth condition, so as to adapt to mass production. The escherichia coli genetically engineered bacterium disclosed by the invention lacks at least one gene of npr, yhbJ and fabF, does not have any resistance mark, is clear in genetic background and has a good growth condition; e.coli W3110 delta npr delta yhbJ delta fabF (named strain WOZF) with three genes deleted, and the yield of lipopolysaccharide can reach 9.56 mg.g ^-1 DCW produced 40.6% higher lipopolysaccharide than wild E.coli W3110.

The first object of the present invention is to provide a genetically engineered bacterium which can efficiently synthesize lipopolysaccharide, wherein at least one gene in npr, yhbJ, fabF of the genetically engineered bacterium is subjected to deletion mutation.

In one embodiment, the genetically engineered bacterium is deletion mutated on the basis of E.coli W3110.

In one embodiment, the amino acid sequence of the npr, yhbJ, fabF knockout fragment is as shown in NCBI as "BAE77250.1", "BAE77249.1", "BAA35903.1", respectively. The yield of the lipopolysaccharide of the genetically engineered bacterium obtained by single knockout of the npr, yhbJ, fabF gene is respectively improved by 18.6%, 29.6% and 29.0% compared with that of the lipopolysaccharide of the escherichia coli W3110.

In one embodiment, the genetically engineered bacterium is obtained by triple deletion mutation of npr, yhbJ, fabF gene based on Escherichia coli W3110, and the obtained genetically engineered bacterium E.coli W3110 delta npr delta yhbJ delta fabF has a lipopolysaccharide yield increased by 40.6% as compared with that of Escherichia coli W3110 to 9.56 mg.g ^-1 DCW。

In one embodiment, the deletion mutation is a gene knockout in E.coli W3110 using the CRISPR-Cas9 knockout system.

In one embodiment, the knockout is a Cas enzyme-mediated fragment recombination using a CRISPR-Cas9 knockout system, and finally 42oC culture is performed to remove pCas plasmid, thereby obtaining a genetically engineered bacterium for effectively synthesizing lipopolysaccharide without resistance.

The second object of the present invention is to provide a method for producing lipopolysaccharide using the genetically engineered bacterium of the present invention.

In one embodiment, the method is to ferment and culture genetically engineered bacteria; extracting lipopolysaccharide after culturing; wherein lipopolysaccharide is extracted by extracting LPS of Escherichia coli by hot phenol hydrolysis method.

The extraction of the lipopolysaccharide comprises the following steps: centrifuging the fermented genetically engineered bacteria liquid, removing supernatant, adding ddH ₂ O, adding hot phenol with the volume fraction of 90%, screwing the cover, and then placing the cover in a water bath shaking table for shaking to ensure that thalli are fully cracked; cooling, centrifuging, standing, transferring the upper phase to a dialysis bag, and dialyzing in deionized water; after the dialysis is completed, collecting liquid in a dialysis bag, and freeze-drying to obtain white fluffy bulk-like crude LPS; then, crude purification was performed.

The crude sample purification is specifically as follows: re-suspending the crude LPS in Tris-HCl buffer, adding Dnase I and RNase A to remove nucleic acid pollution, and then carrying out water bath reaction for a period of time; adding proteinase K to remove residual protein; and adding water saturated phenol to terminate the reaction, precipitating all proteins, mixing to form a turbid liquid, centrifuging to obtain an upper phase, dialyzing, freeze-drying, re-dissolving in a chloroform-methanol mixed solution, centrifuging to obtain a supernatant, washing, re-lyophilizing, re-dissolving in water, and lyophilizing to obtain a pure LPS product.

In one embodiment, the extraction of lipopolysaccharide is specifically:

extracting LPS of Escherichia coli by hot phenol hydrolysis, culturing overnight, and obtaining initial OD ₆₀₀ =0.02 was transferred to 200mL of LB medium, incubated at 37 ℃ for 16h at 200rpm, and cell concentration was measured and recorded. By empirical value OD ₆₀₀ ＝0.323gDCW·L ^-1 As a standard, 258.4g of dry matter was defined, and the volume of the bacterial liquid (designated as V ₁ ). Bacterial solutions with different volumes and same dry weight are transferred to a large centrifugal bottle, and are centrifuged for 20min at 8000g, and the supernatant is discarded. With ddH ₂ O15 mL is blown through the pellet and transferred entirely into a clean 50mL centrifuge tube. Adding 15mL of hot phenol with volume fraction of 90%, screwing the cover, putting the cover in a water bath shaking table at 68 ℃ for shaking, shaking at 150rpm for 1h,the centrifuge tube was turned upside down every 20 minutes during this period to ensure sufficient lysis of the cells. After the water bath oscillation is finished, cooling to room temperature by ventilation, rotating at 4000g, centrifuging at 4 ℃ for 20min for phase separation, and continuously standing for 4-8 hours. Carefully transfer 5mL of the upper phase (noted as V ₂ ) And (3) putting the solution into a dialysis bag, putting the dialysis bag into deionized water for dialysis for 24 hours, and changing water once every 4 hours. After the dialysis is completed, the liquid in the dialysis bag is completely poured into another clean 50mL centrifuge tube, frozen for 2-3 hours at the temperature of minus 80 ℃, and put into a vacuum freeze dryer after being frozen to be opaque, and freeze-dried for 2 days, so that white fluffy bulk crude LPS is obtained. Purifying a crude sample: crude LPS containing impurities was resuspended in 10mL Tris-HCl buffer (100 mM pH 7.5Tris-HCl,25mM MgCl ₂ ，1mM CaCl ₂ ). Dnase I and RNaseA were added to remove nucleic acid contamination at a working concentration of 1. Mu.g.mg-1, and reacted for 2 hours in a water bath shaker at 37 ℃. 1. Mu.g mg-1 proteinase K was added to remove residual protein, and the mixture was placed in a water bath shaker at 37℃to react for 2 hours. At this time, the reaction was terminated by adding 5mL of water-saturated phenol, and all proteins were precipitated (water-saturated phenol was prepared one day in advance, 80mL of deionized water was added to 300g of phenol, heated in a water bath and stirred until dissolved, then transferred to a 1L separating funnel containing 200mL of deionized water, and mixed by gentle shaking to form an emulsion, and then allowed to stand for 6 hours to separate, the lower colorless transparent liquid was water-saturated phenol, and the separating funnel was allowed to discharge the lower layer into a brown reagent bottle and stored. Mixing to obtain a turbid liquid, centrifuging at 4000rpm for 30 min, collecting the upper phase, and dialyzing for 24 hr. Subsequently, the lyophilization operation was repeated, and the resulting fluffy solid was redissolved in chloroform: methanol=2: 1, the mixture was centrifuged at 12000rpm for 20 minutes, and the supernatant was removed and washed repeatedly. Freeze-drying again, re-dissolving in water, and freeze-drying again to obtain the LPS pure product.

In one embodiment, the fermentation culture is a conventional fermentation process.

The third object of the present invention is to provide a method for producing the genetically engineered bacterium.

In one embodiment, the use is to produce lipopolysaccharide using genetically engineered bacteria, and then to use the lipopolysaccharide for cellular immunization or to prepare a vaccine containing a lipopolysaccharide component.

In one embodiment, the vaccine comprising a lipopolysaccharide component includes, but is not limited to, an attenuated vaccine, an inactivated vaccine.

The invention has the beneficial effects that:

in the invention, the Npr coding gene Npr is singly knocked out, thus solving the inhibition of the Npr on the activity of LpxD enzyme; knockout of yhbJ partially releases negative feedback inhibition of intracellular UDP-GlcNAc, and increases the substrate of the first step of LPS synthesis; the fabF gene is knocked out, so that the activity inhibition of saturated fatty acid on LpxK is reduced; the three genes are knocked out in a combined way, and the strain WOZF is constructed.

The gene engineering bacteria WOZF of the invention, wherein the genes npr, yhbJ and fabF are subjected to deletion mutation and inactivation. Compared with the wild E.coli W3110, the lipopolysaccharide synthesis amount of the strain constructed by the invention is increased by 40.6%, the growth condition is good, no exogenous resistance gene sequence is introduced, and the strain is more beneficial to large-scale industrial production.

Drawings

Fig. 1: genetically engineered bacteria E.coli W3110 delta npr, E.coli W3110 delta yhbJ, E.coli W3110 delta fabF, E.coli W3110

SDS-PAGE silver-stained analysis of Δnpr ΔyhbJ Δfabf (WOZF) lipopolysaccharide structure;

fig. 2: genetically engineered bacteria E.coli W3110 delta npr, E.coli W3110 delta yhbJ, E.coli W3110 delta fabF, E.coli W3110

Δnpr Δyhbj Δfabf (WOZF) lipopolysaccharide yield comparison plot;

fig. 3: strain E.coli W3110 Gene engineering bacteria E.coli W3110 Deltanpr, E.coli W3110 DeltayhbJ, E.coli W3110

Δfabf, e.colliw3110 Δnpr Δyhbj Δfabf (WOZF) growth profile.

Detailed Description

Detection and quantification of LPS:

polyacrylamide gel electrophoresis and silver staining development: polyacrylamide gel electrophoresis (SDS-PAGE) detection: the lyophilized crude LPS was dissolved in 1mL deionized water and sonicated until a pale yellow homogeneous solution was obtained. Commercial loading buffer solution SDS loading buffer is added into LPS solution according to the volume ratio of 4:1, and the water bath kettle is heated at 100 DEG CRemoving spatial structure, boiling for 10min, and completely denaturing LPS. After completion of the water bath, the mixture was cooled slightly to room temperature, spotted, and 10. Mu.L was added to the gel wells. When LPS sample is concentrated in 5% of the upper layer, constant current is set to 12mA, and when the strip runs to the interface of the separation gel, the constant current is switched to 25mA. And stopping electrophoresis when the blue band runs to about 1cm from the bottom. Silver staining and developing: firstly, preparing a fixing solution: 30% ethanol, 10% glacial acetic acid; oxidizing liquid: 30% ethanol, 10% glacial acetic acid, 0.7% periodic acid; silver ammonia solution: 28mL of 0.1 mol.L-1 NaOH,1g AgNO ₃ 125mL of deionized water and 2mL of ammonia water, and the existing preparation is needed; color development liquid: 0.05g.L-1 citric acid, 0.02% formaldehyde. And (3) rinsing in the silver staining step (1). Washing off impurities on the gel surface by deionized water; (2) fixing. Fixing solution for 20min, and increasing LPS dyeing sensitivity; (3) oxidation. Oxidizing the oxidizing solution for 20min; (4) rinsing. The deionized water is gently shaken and washed for 20min, and the washing is repeated for three times, so that the rinsing time can be properly prolonged; (5) silver staining. The prepared silver ammonia solution is treated for 10min, and sometimes the oxidation time can be reduced due to the excessive concentration of the sample; (6) Washing with double distilled water for 20min, repeating the treatment with the color development liquid for three times (7) until the gel shows LPS band, rapidly and carefully pouring out the color development liquid to prevent excessive reaction, and concentrating the band too much.

LPS quantification: (1) standard curve determination: weighing a certain amount of purified LPS on a precise balance, and carrying out vortex vibration and ultrasonic dissolution to prepare a gradient standard solution of 2-20 mg.mL < -1 >. Samples of the solution were taken at different concentration gradients, 50 μl each, in triplicate, placed in clean 1.5mL centrifuge tubes and placed on ice. The freshly prepared cysteine hydrochloride with the mass fraction of 6% was protected from light and placed on ice as well. A blank was set and 50. Mu.L deionized water was added. The volume ratio of sulfuric acid to water in the concentrated sulfuric acid used in the experiment is 6:1, because of the large amount of heat released, it is necessary to prepare and cool in advance, and put into the ice bank as well. 450. Mu.L of concentrated sulfuric acid was added to a centrifuge tube containing 50. Mu.L of LPS solution; 5. Mu.L of cysteine hydrochloride solution was added to the centrifuge tube and, after vigorous shaking, reinserted into ice; after 3 minutes, the sample was discarded in boiling water and boiled for 20 minutes; after 1 hour, transferring 100 mu L of the reaction mixture into a 96-well plate, measuring the values of the absorption light wavelength at 505 nm and 545nm by using an enzyme-labeled instrument, and performing difference making to obtain a required difference value; a standard curve was prepared based on LPS concentration (X) and the difference (Y). Various mutants with the yield required to be measured were tested by quantifying the crude LPS sample extracted, diluting the sample to 1mL with deionized water, and detecting the concentration of LPS solution. The procedure was as for the standard curve determination above.

Example 1: construction of npr, yhbJ, fabF Single Gene, tri-Gene deletion mutant

1. Construction of knockout fragment and pTargetF-gene plasmid

The CRISPR-Cas9 knockout system is adopted, a genome fragment takes a W3110 genome as a template, a fragment derived from a plasmid takes a corresponding plasmid as a template, a corresponding primer is added, and 2 x pfx polymerase is used for PCR to obtain the genome fragment. And (3) recovering and purifying the fragments, and after confirming the correct strip by nucleic acid gel electrophoresis, obtaining the fragments by using a gel recovery kit.

All primers used in the present invention are listed in Table 1.

Taking the construction of W31105 Δnpr as an example, the gene knockout operation in the present invention will be briefly described. The site CHOPCHOP (uib.no) was used to predict the NGG sequence that could be recognized by pTargetF-npr, which should be in the middle of the npr gene sequence. The primer pair sg-npr-F/sg-npr-R is designed by introducing a 20bp sequence before NGG in the npr sequence at the 5' end of the primer, and pTargetF-npr linear plasmid is obtained by PCR by taking pTargetF as a template. The transformants were introduced into JM109 chemocompetence, spread on spectinomycin selection plates, cultured overnight and selected. Taking N20-npr-F/N20-R as a primer, picking a single colony as a template, and carrying out colony PCR verification. The correct transformant obtained by screening is inoculated into a liquid medium containing spectinomycin, and plasmid pTargetF-npr is extracted. Using the W3110 genome as template, the upstream fragment was amplified using the primer pair npr-U-F/npr-U-R, and the downstream fragment was amplified using npr-D-F/npr-D-R. The upstream and downstream fragments were overlapped by fusion PCR through the reverse complement on the primer to give the knockout fragment npr-UD.

Construction of W3110 ΔyhbJ: the pTargetF-yhbJ plasmid, N20-yhbJ-F/N20-R validation plasmid was constructed using the primer pair sg-yhbJ-F/sg-yhbJ-R. The upstream fragment was knocked out using yhbJ-U-F/yhbJ-U-R and the downstream fragment was amplified using yhbJ-D-F/yhbJ-D-R. When overlapping the upstream and downstream, the primer yhbJ-U-F/yhbJ-D-R is selected to obtain a knockout fragment yhbJ-UD.

Construction of W3110 ΔfabZ: the pTargetF-fabF plasmid, N20-fabF-F/N20-R verification plasmid was constructed using the primer pair sg-fabF-F/sg-fabF-R. The upstream fragment was knocked out using fabF-U-F/fabF-U-R and the downstream fragment was knocked out using fabF-D-F/fabF-D-R. When overlapping the upstream and downstream, the primer fabF-U-F/fabF-D-R is selected to obtain the knocked-out fragment fabF-UD.

Constructing WOZF: npr, yhbJ, fabF was knocked out separately, and the corresponding primers were as described above. PCR was verified to be the correct band size.

2. Obtaining of the knockout Strain

Taking the npr knockout as an example, 200ng of knockout plasmid and 300ng of knockout fragment are subjected to electric shock transfer into W3110/pCas9 electric transfer competence, the mixture is cultured on a spectinomycin and kanamycin double antibiotic plate for 20 hours at 30 ℃, the transformant is verified by using a primer pair npr-U-F/npr-D-R, and the W3110 is used as negative control, so that the transformant after correct knockout is obtained. The same is true for the remaining two genes, either knocked out separately or in tandem.

3. Removing resistance to obtain aseptic strain

Kanamycin and IPTG were added to remove pTargetF-npr, and the primer pair was verified to be N20-npr-F/N20-R. And transferring at 42 ℃ for two generations to remove pCas9, and carrying out non-resistance verification of transformants and colony PCR verification (verification primer pair is npr-U-F/npr-D-R). Finally, W3110 delta npr which does not contain other plasmids and is knocked out successfully is obtained. The same is true for the remaining two genes, either knocked out separately or in tandem. And performing PCR verification of the corresponding gene knockout. Finally, the genetically engineered bacteria E.coli W3110 delta npr, E.coli W3110 delta yhbJ, E.coli W3110 delta fabF and E.coli W3110 delta npr delta yhbJ delta fabF (named WOZF) are obtained.

TABLE 1 primer sequences for the npr, yhbJ, fabF knockdown of the genes of the invention

EXAMPLE 2 lipopolysaccharide semi-quantitative analysis of mutant strains

1. Extraction of lipopolysaccharide from mutant strains

Extracting LPS of Escherichia coli by hot phenol hydrolysis, culturing overnight, and obtaining initial OD ₆₀₀ =0.02 was transferred to 200mL of LB medium, incubated at 37 ℃ for 16h at 200rpm, and cell concentration was measured and recorded. By empirical value OD ₆₀₀ ＝0.323gDCW·L ^-1 As a standard, 258.4g of dry matter was defined, and the volume of the bacterial liquid (designated as V ₁ ). Bacterial solutions with different volumes and same dry weight are transferred to a large centrifugal bottle, and are centrifuged for 20min at 8000g, and the supernatant is discarded. With ddH ₂ O15 mL is blown through the pellet and transferred entirely into a clean 50mL centrifuge tube. 15mL of hot phenol with the volume fraction of 90% is added, the cap is screwed, and then the mixture is placed in a water bath shaking table at 68 ℃ for shaking, the shaking time is 1h at the rotating speed of 150rpm, and the centrifuge tube is turned upside down every 20 minutes during the shaking time, so that the sufficient cracking of thalli is ensured. After the water bath oscillation is finished, cooling to room temperature by ventilation, rotating at 4000g, centrifuging at 4 ℃ for 20min for phase separation, and continuously standing for 4-8 hours. Carefully transfer 5mL of the upper phase (noted as V ₂ ) And (3) putting the solution into a dialysis bag, putting the dialysis bag into deionized water for dialysis for 24 hours, and changing water once every 4 hours. After the dialysis is completed, the liquid in the dialysis bag is completely poured into another clean 50mL centrifuge tube, frozen for 2-3 hours at the temperature of minus 80 ℃, and put into a vacuum freeze dryer after being frozen to be opaque, and freeze-dried for 2 days, so that white fluffy bulk crude LPS is obtained. Purifying a crude sample: crude LPS containing impurities was resuspended in 10mL Tris-HCl buffer (100 mM pH 7.5Tris-HCl,25mM MgCl ₂ ，1mM CaCl ₂ ). Dnase I and RNaseA were added to remove nucleic acid contamination at a working concentration of 1. Mu.g.mg-1, and reacted for 2 hours in a water bath shaker at 37 ℃. 1. Mu.g mg-1 proteinase K was added to remove residual protein, and the mixture was placed in a water bath shaker at 37℃to react for 2 hours. At this time, the reaction was terminated by adding 5mL of water-saturated phenol, and all proteins were precipitated (water-saturated phenol was prepared one day in advance, 80mL of deionized water was added to 300g of phenol, heated in a water bath and stirred until dissolved, then transferred to a 1L separating funnel containing 200mL of deionized water, and mixed by gentle shaking to form an emulsion, and then allowed to stand for 6 hours to separate, the lower colorless transparent liquid was water-saturated phenol, and the separating funnel was allowed to discharge the lower layer into a brown reagent bottle and stored. Mixing to obtain a turbid liquid at 4000rpmCentrifuging for 30 min, collecting the upper phase, and dialyzing for 24 hr. Subsequently, the lyophilization operation was repeated, and the resulting fluffy solid was redissolved in chloroform: methanol=2: 1, the mixture was centrifuged at 12000rpm for 20 minutes, and the supernatant was removed and washed repeatedly. Freeze-drying again, re-dissolving in water, and freeze-drying again to obtain the LPS pure product.

2. SDS-PAGE silver staining analysis

Polyacrylamide gel electrophoresis and silver staining development: polyacrylamide gel electrophoresis (SDS-PAGE) detection: the lyophilized crude LPS was dissolved in 1mL deionized water and sonicated until a pale yellow homogeneous solution was obtained. Commercial loading buffer solution SDS loading buffer is added into the LPS solution according to the volume ratio of 4:1, the space structure is removed by heating in a water bath kettle at 100 ℃, and the LPS is completely denatured after boiling for 10 min. After completion of the water bath, the mixture was cooled slightly to room temperature, spotted, and 10. Mu.L was added to the gel wells. When LPS sample is concentrated in 5% of the upper layer, constant current is set to 12mA, and when the strip runs to the interface of the separation gel, the constant current is switched to 25mA. And stopping electrophoresis when the blue band runs to about 1cm from the bottom. Silver staining and developing: firstly, preparing a fixing solution: 30% ethanol, 10% glacial acetic acid; oxidizing liquid: 30% ethanol, 10% glacial acetic acid, 0.7% periodic acid; silver ammonia solution: 28mL of 0.1 mol.L-1 NaOH,1g of AgNO3, 125mL of deionized water and 2mL of ammonia water, and needs to be prepared on site; color development liquid: 0.05g.L-1 citric acid, 0.02% formaldehyde. And (3) rinsing in the silver staining step (1). Washing off impurities on the gel surface by deionized water; (2) fixing. Fixing solution for 20min, and increasing LPS dyeing sensitivity; (3) oxidation. Oxidizing the oxidizing solution for 20min; (4) rinsing. The deionized water is gently shaken and washed for 20min, and the washing is repeated for three times, so that the rinsing time can be properly prolonged; (5) silver staining. The prepared silver ammonia solution is treated for 10min, and sometimes the oxidation time can be reduced due to the excessive concentration of the sample; (6) Washing with double distilled water for 20min, repeating the treatment with the color development liquid for three times (7) until the gel shows LPS band, rapidly and carefully pouring out the color development liquid to prevent excessive reaction, and concentrating the band too much. The LPS bands of each strain were compared in size, depth and thickness, and the relative content was analyzed. As shown in FIG. 1, the SDS-PAGE silver staining results showed that the LPS size of each mutant was consistent with that of W3110, but the LPS band was thicker than that of the wild-type W3110, indicating that the content was increased.

EXAMPLE 3 LPS content analysis of mutant strains

1. LPS standard curve determination

Weighing a certain amount of purified LPS on a precise balance, and carrying out vortex vibration and ultrasonic dissolution to prepare a gradient standard solution of 2-20 mg.mL < -1 >. Samples of the solution were taken at different concentration gradients, 50 μl each, in triplicate, placed in clean 1.5mL centrifuge tubes and placed on ice. The freshly prepared cysteine hydrochloride with the mass fraction of 6% was protected from light and placed on ice as well. A blank was set and 50. Mu.L deionized water was added. The volume ratio of sulfuric acid to water in the concentrated sulfuric acid used in the experiment is 6:1, because of the large amount of heat released, it is necessary to prepare and cool in advance, and put into the ice bank as well. 450. Mu.L of concentrated sulfuric acid was added to a centrifuge tube containing 50. Mu.L of LPS solution; 5. Mu.L of cysteine hydrochloride solution was added to the centrifuge tube and, after vigorous shaking, reinserted into ice; after 3 minutes, the sample was discarded in boiling water and boiled for 20 minutes; after 1 hour, transferring 100 mu L of the reaction mixture into a 96-well plate, measuring the values of the absorption light wavelength at 505 nm and 545nm by using an enzyme-labeled instrument, and performing difference making to obtain a required difference value; a standard curve was prepared based on LPS concentration (X) and the difference (Y).

2. LPS quantification of each mutant Strain

Various mutants with the yield required to be measured were tested by quantifying the crude LPS sample extracted, diluting the sample to 1mL with deionized water, and detecting the concentration of LPS solution. The procedure was as for the standard curve determination above.

The strain culture method comprises the following steps: activating Escherichia coli W3110 and its mutant strain on plate, picking single colony, inoculating into test tube, culturing overnight, and using initial OD ₆₀₀ =0.02 was transferred to a 500mL volume flask containing 200mL of LB medium, and the cells were cultured at 37 ℃ for 16h at 200rpm, the cell concentration was measured, recorded, and fermentation was completed. Wherein, the LB medium contains 50g/L yeast extract, 100g/L peptone and 100g/LNaCl.

The LPS quantification results showed that each mutant had increased LPS compared to wild type E.coli W3110, with the triple knockout mutant WOZF having the greatest LPS yield. The lipopolysaccharide yields of E.coli W3110, E.coli W3110 Δnpr, E.coli W3110 ΔyhbJ, E.coli W3110 ΔfabF, E.coli W3110 Δnpr ΔyhbJ ΔfabF were 6.80mg/g DCW, 8.06mg/g DCW, 8.81mg/g DCW, 8.77mg/g DCW, 9.56mg/g DCW, respectively, and the lipopolysaccharide yields of E.coli W3110 Δnpr, E.coli W3110 ΔyhbJ, E.coli W3110 ΔfabF, E.coli W3110 Δnpr ΔyhbJ ΔΔfabF were increased by 18.6%, 29.6%, 29.0%, and 40.6%, respectively, as compared with wild type E.coli W3110.

EXAMPLE 4 comparison of growth status of strains

Five strains of E.coli W3110, E.coli W3110 Δnpr, E.coli W3110 ΔyhbJ, E.coli W3110 ΔfabF, E.coli W3110 Δnpr ΔyhbJ ΔfabF (WOZF) were activated on LB solid plates, and five single colonies were picked up and inoculated into 5mL of LB liquid medium, respectively. OD after 6h of test tube culture ₆₀₀ About 2.5 transit, according to the initial OD ₆₀₀ =0.02 was transferred to a 250mL triangular flask containing 50mL of LB liquid medium, incubated at 37 ℃ for 24h, bacterial solutions at different time points were taken, and bacterial solution OD was determined. The process was repeated three times and an average value was obtained. The results are shown in fig. 3, which shows: e.coli W3110 Δnpr, E.coli W3110 ΔyhbJ, E.coli W3110 ΔfabF, E.coli W3110 Δnpr ΔyhbJ ΔfabF were grown substantially identical to the wild type, i.e., the growth of the gene-deleted was good.

In particular, the mutant WOZF has high lipopolysaccharide yield, no resistance mark, no pollution hidden trouble and good growth condition, and is beneficial to large-scale industrial production.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications may be made therein by those skilled in the art without departing from the spirit and scope of the invention, which is therefore to be limited only by the appended claims.

Claims

1. A genetically engineered bacterium for efficiently synthesizing lipopolysaccharide, characterized in that at least one gene in npr, yhbJ, fabF of the genetically engineered bacterium is subjected to deletion mutation.

2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium has a triple deletion mutation of the npr, yhbJ, fabF gene.

3. The genetically engineered bacterium of claim 1, wherein the amino acid sequences of the npr, yhbJ, fabF knockout fragment are the sequences shown in NCBI as "BAE77250.1", "BAE77249.1", "BAA35903.1", respectively.

4. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is deletion mutated on the basis of e.coli W3110.

5. The genetically engineered bacterium of claim 1, wherein the deletion mutation is a gene knockout in e.coli W3110 using a CRISPR-Cas9 knockout system.

6. A method for producing lipopolysaccharide using the genetically engineered bacterium according to any one of claims 1 to 5.

7. The method according to claim 6, wherein the method comprises fermenting and culturing genetically engineered bacteria; extracting lipopolysaccharide after culturing; wherein lipopolysaccharide is extracted by extracting LPS of Escherichia coli by hot phenol hydrolysis method.

8. The method according to claim 7, characterized in that the lipopolysaccharide extraction is in particular: centrifuging the fermented genetically engineered bacteria liquid, removing supernatant, adding ddH ₂ O, adding hot phenol with the volume fraction of 90%, screwing the cover, and then placing the cover in a water bath shaking table for shaking to ensure that thalli are fully cracked; cooling, centrifuging, standing, transferring the upper phase to a dialysis bag, and dialyzing in deionized water; after the dialysis is completed, collecting liquid in a dialysis bag, and freeze-drying to obtain white fluffy bulk-like crude LPS; then purifying the crude sample; the crude sample purification is specifically as follows: the crude LPS was resuspended in Tris-HCl buffer, dnaseI, RNaseA was added to remove nucleic acid contamination, and then reacted in a water bathA period of time; adding proteinase K to remove residual protein; and adding water saturated phenol to terminate the reaction, precipitating all proteins, mixing to form a turbid liquid, centrifuging to obtain an upper phase, dialyzing, freeze-drying, re-dissolving in a chloroform-methanol mixed solution, centrifuging to obtain a supernatant, washing, re-lyophilizing, re-dissolving in water, and lyophilizing to obtain a pure LPS product.

9. Use of the genetically engineered bacterium of any one of claims 1-5.

10. The use according to claim 9, wherein the use is to use genetically engineered bacteria to produce lipopolysaccharide, which is then used for cellular immunization or to prepare a vaccine containing a lipopolysaccharide component.