An escherichia coli engineering bacterium without growth defects, low endotoxin and with high extracellular protein secretion capacity and application thereof

Abstract

The application provides an Escherichia coli engineering bacterium without growth defects, low endotoxin and with high extracellular protein secretion capacity and application, which is classified and named as Escherichia coli CZT3 Escherichia coli (CZT3), which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M20261049 and a preservation time of May 22, 2026. The application is obtained from a metabolic network of ClearColi BL21(DE3) for the first time, does not need to introduce an exogenous resistance marker or metabolic burden, fundamentally repairs the metabolic network of the strain with growth fragility, greatly saves an endotoxin removal step, realizes improvement of product purity and increase of yield, significantly reduces production cost, can synthesize low-endotoxin recombinant proteins, and the cell membrane of the CZT3 strain has higher permeability and protein secretion capacity.

Description

Technical Field

[0001] This invention belongs to the field of protein expression technology and relates to an engineered Escherichia coli strain with no growth defects, low endotoxin, and high extracellular protein secretion capacity, and its applications. Background Technology

[0002] Endotoxins are a common and serious source of contamination in the production of biopharmaceuticals. Endotoxin contamination mainly originates from two aspects: firstly, contamination of the raw material solutions, production environment, and operational processes used in the production process; and secondly, the nature of the producing bacterial strain itself. Endotoxins are inherent components of the cell membranes of Gram-negative bacteria, and lipid A in lipopolysaccharide (LPS) has been proven to be the core structure for endotoxin activity. Therefore, when using Gram-negative bacteria as hosts for recombinant protein expression, endotoxin contamination is unavoidable. Endotoxin contamination occurs when endotoxins from the outer membrane of Gram-negative bacteria are released into the lysate after bacterial death. These endotoxins can interact and bind to biomolecules (including targeted therapeutic compounds). This contamination not only affects the purity of biopharmaceuticals but can also trigger strong immune responses, potentially leading to adverse reactions such as fever and shock in severe cases. Notably, LPS is recognized by the Toll-like receptor 4 / myeloid differentiation factor 2 (TLR4 / MD-2) complex in a structure-dependent manner through its lipid A. LPS molecules must first interact with LPS-binding protein (LBP). LBP presents LPS to the membrane-bound cluster differentiation 14 (CD14) receptor, which in turn delivers LPS to the ectopic domain of the TLR4 / MD-2 complex. LPS recognition leads to homodimerization of the TLR4 / MD-2 complex with another TLR4 / MD-2. This activates a cytoplasmic signaling cascade, ultimately resulting in the transcription of nuclear factor κB (NF-κB), releasing pro-inflammatory cytokines, and also transcribing interferon (IFN) and IFN-induced genes.

[0003] To address the endotoxin contamination issue associated with recombinant protein expression in *E. coli*, Ronald Wesley Woodard's team knocked out the lpxL, lpxM, lpxP, pagP, emtA, kdsD, and gutQ genes involved in LPS synthesis in the BL21(DE3) strain. They also discovered a mutation at amino acid position 148 of the transmembrane flipper protein MsbA, resulting in an endotoxin-free *E. coli* strain, ClearColi BL21(DE3). The deletion of LPS synthesis pathway genes resulted in the modified strain synthesizing only tetraacylated lipid A with four fatty acid chains, i.e., lipid IVA. This structure is almost entirely unrecognizable by immune cells, thus preventing excessive activation of inflammatory factors and inflammation. However, compared to the unmodified BL21(DE3) strain, ClearColi BL21(DE3) exhibited a significantly longer lag phase in the early stages of growth and a significantly smaller final cell mass. This slow growth problem leads to issues such as excessive consumption of human resources, long equipment usage cycles, and low equipment utilization in the recombinant protein production process.

[0004] LPS is a glycolipid located on the surface of Gram-negative bacterial cells. Lipid A, the hydrophobic group in its structure, is relatively conserved. It consists of an acylated glucosamine disaccharide containing 1,4-bisphosphorylated β(1→6) glycosidic bonds. Lipid A is primarily synthesized on the cytoplasmic side of the inner membrane (IM) via the lipid A biosynthesis pathway (Raetz pathway). This pathway mainly involves nine enzymes: UDP-N-acetylglucosamine acyltransferase (LpxA), lipid A disaccharide synthase (LpxB), UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC), UDP-3-O-acylglucosamine N-acyltransferase (LpxD), UDP-2,3-diacylglucosamine pyrophosphate hydrolase (LpxH), tetraacylbiose 4'-kinase (LpxK), phosphoethanolamine transferase (EptA), lipid A biosynthesis lauroyltransferase (LpxL), and lipid A biosynthesis myristyltransferase (LpxM). First, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is reacted by LpxA to add a β-hydroxymyristate to the 3'-OH position of the acyl carrier protein, generating uridine diphosphate-3-O-acyl-N-acetylglucosamine (UDP-3-O-(acy)-GlcNAc). This reaction is generally thermodynamically unfavorable. Subsequently, LpxC deacetylates the protein, removing the acetyl group from its amino group. This step is the rate-limiting step in lipid A synthesis and is generally considered the first step in LPS synthesis. Then, LpxD adds another β-hydroxymyristate to the 2'-OH position, generating uridine diphosphate-2,3-diacylglucosamine (UDP-2,3-diacylglucosamine). Under the catalysis of LpxH, the pyrophosphate bond is cleaved, generating lipid X (LipidX). Subsequently, under the action of lipid A disaccharide synthase LpxB, the Lipid X molecule undergoes a condensation reaction with the UDP-2,3-diacylglucosamine molecule to form the initial backbone of Lipid A, which contains two glucosamines, four hydroxy fatty acid chains, and a 1' phosphate group. Then, phosphokinase LpxK phosphorylates the glucosamine in the initial Lipid A backbone, adding a new phosphate group at the 4' position. The resulting molecule is usually called tetraacyl-β-1,6-D-glucosinolate-1,4'-bisphosphate (LipidIV). In *E. coli*, the 3-deoxy-D-mannooctulose acid (Kdo) group is synthesized from the D-ribulose 5-phosphate molecule, forming D-arabinose 5-phosphate (A5P) under the catalysis of D-arabinose-5-phosphate isomerase (KdsD or GutQ).Following the action of 2-dehydro-3-deoxy ... It is then flipped into the periplasm by the transmembrane protein MsbA. There, it binds to the synthesized O antigen and ultimately attaches to the extracellular membrane, forming a complete LPS molecule.

[0005] Because endotoxins can trigger pyrogenic and inflammatory responses in the human body, and in severe cases even lead to organ failure and shock, recombinant proteins produced by *E. coli* must undergo rigorous endotoxin removal and protein purification processes until only extremely low levels of endotoxins remain. Although various endotoxin removal methods exist, such as ultrafiltration, extraction, ion exchange chromatography, affinity chromatography, and membrane adsorption, problems such as low removal rates, high costs, and complex operations persist. Therefore, developing cost-effective endotoxin removal methods remains an ongoing challenge. Furthermore, the lack of an efficient protein secretion system in *E. coli* causes recombinant proteins to remain primarily in the cytoplasm or periplasmic space, limiting the production of soluble proteins and resulting in low overall protein yield.

[0006] Therefore, there is an urgent need for an Escherichia coli that combines the characteristics of no growth defects, low endotoxin levels, and high protein secretion capacity. Summary of the Invention

[0007] To address the growth defects and poor extracellular protein secretion capabilities of the ClearColi BL21(DE3) strain, this invention constructs a new Escherichia coli chassis strain with no growth defects, low endotoxin levels, and high extracellular protein secretion capabilities, restoring its growth level to that of the ordinary BL21(DE3) strain. This strain can also serve as a universal host for the expression of various recombinant proteins.

[0008] The technical solution adopted in this invention is:

[0009] The primary objective of this invention is to provide an engineered *Escherichia coli* strain that is free from growth defects, low in endotoxins, and possesses highly efficient extracellular protein secretion capabilities. This low-endotoxin engineered *Escherichia coli* strain is classified as *Escherichia coli* CZT3, and is deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20261049, and on May 22, 2026.

[0010] As a preferred embodiment of this application, the engineered Escherichia coli strain is a mutant strain of the BL21(DE3) series of Escherichia coli strains.

[0011] As a preferred embodiment of this application, the nucleotide sequence of the msbA gene of the engineered Escherichia coli is shown in SEQ ID NO.1.

[0012] As a preferred embodiment of this application, the amino acid sequence of the MsbA protein encoded by the msbA gene of the engineered Escherichia coli is shown in SEQ ID NO.2.

[0013] As a preferred embodiment of this application, the asparagine (N) at position 529 of the MsbA protein encoded by the msbA gene of the engineered Escherichia coli is mutated to lysine (K).

[0014] As a preferred embodiment of this application, the BL21(DE3) series Escherichia coli strain is ClearColi BL21(DE3), which has had genes related to lipopolysaccharide synthesis in Escherichia coli knocked out using gene editing technology. These related genes include the lpxL gene, lpxM gene, lpxP gene, pagP gene, eptA gene, kdsD gene, and gutQ gene.

[0015] The second objective of this invention is to provide a method for constructing *Escherichia coli* that is free from growth defects, has low endotoxin levels, and possesses highly efficient extracellular protein secretion capabilities. This method includes using a BL21(DE3) series *Escherichia coli* strain as the starting strain, modifying the starting strain through a spontaneous mutation strategy, and mutating the asparagine at position 529 of the MsbA protein encoded by the msbA gene of the modified engineered strain to lysine.

[0016] A third objective of this invention is to provide the use of the aforementioned growth-defect-free, low-endotoxin, and highly efficient extracellular protein secretion strain as a host bacterium expressing low-endotoxin recombinant proteins.

[0017] The fourth objective of this invention is to provide an engineered Escherichia coli strain expressing a low-endotoxin recombinant protein. This engineered strain is obtained by introducing a recombinant vector expressing the recombinant protein into the engineered Escherichia coli strain that is free from growth defects, has low endotoxin levels, and possesses highly efficient extracellular protein secretion capabilities, and then screening the resulting recombinant strain.

[0018] As a preferred embodiment of this application, the recombinant vector expressing the low endotoxin recombinant protein is obtained by cloning the gene encoding the recombinant protein into the pET28a vector.

[0019] As a preferred embodiment of this application, the expression vector is pET28a.

[0020] As a preferred embodiment of this application, the low-endotoxin recombinant protein includes green fluorescent protein (GFP), amylase protein (AmyK), or β-galactosidase protein (LacZ).

[0021] As a preferred embodiment of this application, the gene (sfgfp) encoding the green fluorescent protein has the accession number KF020493 in GenBanK.

[0022] As a preferred embodiment of this application, the gene (amyE) encoding the amylase protein has the accession number KF751392 in GenBanK.

[0023] As a preferred embodiment of this application, the gene (lacZ) encoding the β-galactosidase protein has the accession number CP107553 in GenBanK.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. Overcoming traditional technical bottlenecks to construct low-endotoxin chassis strains without growth defects:

[0026] This invention abandons the traditional modification strategy that relies on exogenous gene compensation. For the first time, it starts from the genome of endotoxin-free Escherichia coli ClearColi BL21(DE3) and successfully obtains the mutant strain CZT3, which combines low endotoxin characteristics with normal growth ability, through spontaneous mutation and phenotypic screening. This strategy eliminates the metabolic burden caused by exogenous elements from the root, without the need to introduce exogenous resistance markers or heterologous genes. At the same time, it repairs the fragile metabolic network of the ClearColi BL21(DE3) strain, providing a robust chassis strain for subsequent cell engineering modification.

[0027] 2. Significantly simplifies the production process, enabling low-cost, high-purity recombinant protein preparation:

[0028] The low-endotoxin mutant strain constructed in this invention reduces the risk of endotoxin contamination at the source, significantly simplifying the endotoxin removal steps in downstream purification processes. Traditional E. coli expression systems require complex processes such as multiple rounds of chromatography and ultrafiltration to remove endotoxins, which is not only time-consuming and labor-intensive but also results in significant loss of the target protein. In contrast, the recombinant protein produced by the strain of this invention has extremely low endotoxin content, and high-purity products can be obtained directly through simple clarification steps such as centrifugation and filtration. This improves product purity and recombinant protein yield, significantly reduces production costs, and enables the efficient synthesis of low-endotoxin recombinant proteins that are both economical and highly active.

[0029] 3. Enhance extracellular protein secretion performance and expand the potential for large-scale production and application:

[0030] The CZT3 mutant strain obtained in this invention achieves a significant enhancement in extracellular protein secretion performance through spontaneous mutation. Experimental results show that after 20 hours of induction, the extracellular GFP fluorescence signal of the CZT3 strain is 1.91 times and 2.24 times that of the BL21(DE3) and ClearColiBL21(DE3) strains, respectively; after 24 hours of induction, the extracellular GFP fluorescence signal of the CZT3 strain is 2.37 times and 2.71 times that of the BL21(DE3) and ClearColiBL21(DE3) strains, respectively. Furthermore, with prolonged induction time, the extracellular protein secretion of the CZT3 strain increases significantly and continuously, while the extracellular protein secretion of the BL21(DE3) and ClearColiBL21(DE3) strains only increases slightly. This characteristic increases the extracellular secretion capacity of the recombinant protein, further reduces endotoxin release and host protein contamination, and lowers the difficulty of downstream purification. Meanwhile, extracellular secreted proteins are more likely to maintain their native conformation and biological activity, reducing complex process steps such as inclusion body refolding, and significantly improving production efficiency and product quality.

[0031] 4. Possesses universal expression host characteristics, suitable for the production of various recombinant proteins:

[0032] The low-endotoxin Escherichia coli mutant strain constructed in this invention not only possesses excellent low-endotoxin protein expression capability but also enhanced extracellular protein secretion performance. It exhibits universal expression host characteristics and is suitable for the production of recombinant proteins compatible with various Escherichia coli strains, including but not limited to therapeutic proteins, vaccine antigens, diagnostic enzymes, and industrial enzyme preparations. The high secretion performance and low endotoxin characteristics of this strain make it a promising candidate for large-scale recombinant protein production, meeting the high-quality, low-cost demands of different fields for recombinant protein products. Attached Figure Description

[0033] Figure 1 This is a map of the expression plasmid for the green fluorescent protein GFP.

[0034] Figure 2This is a plasmid map of the expression of the amylase protein AmyK.

[0035] Figure 3 This is a plasmid map of the expression of the β-galactosidase protein LacZ.

[0036] Figure 4 The growth curves are for BL21(DE3), ClearColi BL21(DE3), and the mutant CZT3.

[0037] Figure 5 This is a gel image used for PCR validation of genes involved in the endotoxin synthesis pathway.

[0038] Figure 6 This is a sequence comparison diagram of the msbA gene of ClearColi BL21(DE3) and CZT3 strains. Detailed Implementation

[0039] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0040] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0041] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0042] The starting strain of this invention, ClearColi BL21(DE3), was purchased from Beijing Bomei Gene Technology Co., Ltd.

[0043] LB liquid medium components: 10 g / L peptone, 5 g / L NaCl and 5 g / L yeast extract.

[0044] LB solid plate composition: Prepare solid culture medium by adding 1.5g of agar powder to every 100mL of liquid culture medium.

[0045] Example 1: Obtaining a low-endotoxin mutant without growth defects

[0046] (1) Specific steps of the spontaneous mutation experiment: ClearColi BL21(DE3) strain was activated on a solid LB plate and then incubated upside down in a 37℃ incubator. Single colonies were picked and inoculated into 3ml liquid LB medium in a test tube and cultured at 37℃ and 180rpm for 12h with shaking, which is the seed culture. The seed culture was continuously subcultured every day (once every 12h). The seed culture cultured for 12h was subcultured into 3mL of LB liquid medium at an inoculation rate of 1% and cultured at 37℃ and 180rpm with shaking. Samples were taken every 12 subcultures and serially diluted 10-fold. The serially diluted solutions were evenly spread on LB solid plates and incubated overnight in a 37℃ incubator. The growth of single colonies was observed. The mutant strains were initially screened by differences in the size of single colonies. Single colonies with significantly larger diameters were selected and preserved. Subsequent screening was conducted, and the band size of the LPS synthesis pathway genes (lpxL, lpxM, lpxP, pagP, emtA, kdsD, and gutQ genes) in the strains was verified by PCR to determine whether the endotoxin synthesis pathway had been reconstructed. The strains that were verified to be correct were the spontaneous mutants without growth defects and were named CZT3.

[0047] (2) Procedure for determining the growth curve:

[0048] The seed culture was transferred to fresh LB liquid medium at an inoculation rate of 1%, and 200 μL was added to the honeycomb plate. Each sample was divided into 3 parallel groups and cultured in a fully automated growth curve analyzer. The absorbance of the culture in each well was measured at 600 nm every 0.5 h.

[0049] Table 1. Growth parameters of BL21(DE3), ClearColi BL21(DE3), and mutant CZT3

[0050]

[0051] Depend on Figure 4 , Figure 5 As shown in Table 1, the generation time of the CZT3 mutant strain was 0.572 h, which was 3.38 times shorter than that of the parent strain ClearColiBL21(DE3) (1.362 h); the maximum specific growth rate of the CZT3 mutant strain was 1.211 h. -1The growth rate was 2.38 times that of the parent strain and 82.8% of that of the BL21 (DE3) strain, far exceeding that of the ClearColi BL21 (DE3) strain and close to that of the BL21 (DE3) strain. This indicates that the spontaneous mutant strain CZT3 has a significantly improved growth ability compared to the ClearColi BL21 (DE3) strain, and no reconstruction of the endotoxin synthesis pathway occurred. This shows that the spontaneous mutation experiment successfully obtained a mutant strain with restored growth, and this mutant strain will be used for subsequent experiments.

[0052] Furthermore, this invention identified the newly added mutation site MsbA in the mutant strain using resequencing technology. N529K The sequencing alignment results are as follows Figure 6 As shown.

[0053] Example 2: Expression of low-endotoxin green fluorescent protein GFP, amylase AmyK, and β-galactosidase LacZ using a low-endotoxin mutant without growth defects.

[0054] The induction and expression process of low-endotoxin recombinant green fluorescent protein, amylase, and β-galactosidase proteins specifically includes the following steps:

[0055] Step 1: Construction of recombinant expression vectors and recombinant strains for expressing green fluorescent protein, amylase, and β-galactosidase proteins.

[0056] Primers were designed to amplify linear fragments of the green fluorescent protein gene sfgfp, the β-galactosidase gene lacZ, and the pET28a plasmid. This operation was performed using 2×Phanta®Max Master Mix according to its standard procedure. Subsequently, the purified linearized vectors were ligated to the sfgfp and lacZ gene fragments, respectively, using the pEASY®-Basic Seamless Cloning and Assembly Kit (Trans), and incubated at 50°C for 15 min to obtain the pET28a-sfgfp and pET28a-lacZ plasmids. The amyK gene, synthesized by General Biotech Inc., was directly ligated to the pET28a plasmid. The recombinant plasmids pET28a-sfgfp, pET28a-amyK, and pET28a-lacZ were transformed into the low-endotoxin Escherichia coli mutant strains without growth defects screened in Example 1 using chemical transformation, resulting in strains CZT3 / pET28a-sfgfp, CZT3 / pET28a-amyK, and CZT3 / pET28a-lacZ. Recombinant green fluorescent protein, amylase, and β-galactosidase proteins were prepared using these strains and the control strain.

[0057] The specific plasmid map is attached. Figures 1-3 As shown.

[0058] Step 2: Investigation of the expression capacity of recombinant proteins from low-endotoxin strains without growth defects

[0059] (1) Preparation of seed culture: In a clean bench, pick a single colony from the Km transformation plate in Example 1 and inoculate it into a test tube containing 3 mL of LB liquid medium. Add kanamycin Km (50 mg / mL) and place it in a shaker at 37°C and 180 rpm for 12 h to obtain seed culture.

[0060] (2) Fermentation and induction:

[0061] Transfer the seed culture at a 1% inoculum to a shake flask containing 50 mL of LB liquid medium and incubate at 37°C and 180 rpm on a shaker until OD (Organic Dose) is reached. 600 The concentration was in the range of 0.4-0.6. IPTG was added to induce expression to a final concentration of 0.1 mM, and then the mixture was transferred to a shaker at 16 °C (GFP and LacZ) or 30 °C (AmyK) at 180 rpm for 12 h.

[0062] Purification steps for recombinant proteins:

[0063] (1) Transfer the induced bacterial culture to a 50 mL centrifuge tube, centrifuge at 8000 rpm and 4℃ for 10 min, discard the supernatant, and retain the bacterial precipitate. Wash the bacterial precipitate in the tube three times with 100 mM PBS buffer (pH=7.2). Resuspend the bacterial precipitate in 10 mL PBS buffer and vortex thoroughly. Dissolve the mixed bacterial culture using an ultrasonic disruptor, ensuring an ice-water bath throughout the disruption process. The operating procedure is as follows: Amplitude bar No. 6, power 35 W, 4 s operation, 3 s pause, total time 30 min.

[0064] (2) The lysate is the whole cell solution. The supernatant obtained by centrifuging the lysate at 4℃ and 12000rpm for 5-10min is the soluble crude enzyme solution. Transfer the supernatant to a clean centrifuge tube, resuspend the precipitate in 8mM urea, vortex thoroughly, and let stand for 2-5min to allow the precipitate to dissolve.

[0065] (3) The protein concentration of the sample was determined using the Beyotime BCA protein concentration assay kit.

[0066] (4) Purify the target protein using 1 mL of a nickel-iminodiacetic acid-6% highly crosslinked agarose gel (Ni-IDA 6FF, Pre-Packed Gravity Column). Remove the nickel column from the 4°C freezer and allow the 20% ethanol in the packed column to flow out naturally. Rinse with 30 mL of Buffer A.

[0067] (5) Load the sample slowly and pass all the prepared protein samples through the column. Use 30 mL of high salt solution to remove impurities.

[0068] (6) Slowly add 30 mL of 80 mM imidazole to elute the impurities.

[0069] (7) Finally, use an appropriate volume of Buffer B to elute the target protein.

[0070] (8) Clean the nickel column with 100 mL of ultrapure water. Finally, fill the nickel column with 20% ethanol and store it in a refrigerator at 4°C.

[0071] Determination of the fluorescence value of green fluorescent protein:

[0072] The bacterial culture induced at 16℃ and the supernatant obtained by centrifugation were transferred to 96-well plates, and fluorescence was measured using a microplate reader. Fluorescence detection parameters: excitation wavelength and emission wavelength were 488 nm and 520 nm, respectively.

[0073] Table 2. Expression parameters of GFP, AmyK, and LacZ proteins in BL21(DE3), ClearColi BL21(DE3), and CZT3.

[0074]

[0075] The results in Table 2 show that, compared with the parental ClearColi BL21(DE3) strain, the CZT3 mutant strain obtained in this invention has significantly enhanced protein expression capabilities. The expression levels of GFP, AmyK, and LacZ proteins in CZT3 are 3.0 times, 2.8 times, and 3.2 times that of ClearColi BL21(DE3) strain, respectively. The fluorescence value of the supernatant of CZT3 strain is 3.0 times that of ClearColi BL21(DE3) strain. The protease activities of AmyK and LacZ in CZT3 strain are 1.1 times and 2.9 times that of ClearColi BL21(DE3) strain, respectively.

[0076] Step 3: Investigation of the extracellular GFP protein content and inner and outer membrane permeability of low-endotoxin strains without growth defects.

[0077] Table 3: Comparison of inner and outer membrane permeability and extracellular GFP fluorescence intensity for each strain

[0078]

[0079] Table 3 shows that the cell membrane of the CZT3 mutant strain obtained in this invention has higher permeability than that of ClearColi BL21(DE3) and BL21(DE3) strains, which may be more suitable for the secretion of intracellular substances. The extracellular GFP protein fluorescence intensity clearly shows that after 20 h of induction, the extracellular fluorescence signal of the CZT3 strain was 1.91 times and 2.24 times that of the BL21(DE3) and ClearColi BL21(DE3) strains, respectively; after 24 h of induction, the extracellular fluorescence signal of the CZT3 strain was 2.37 times and 2.71 times that of the BL21(DE3) and ClearColi BL21(DE3) strains, respectively. With prolonged induction time, the extracellular protein secreted by CZT3 increased significantly, while the GFP secreted by BL21(DE3) and ClearColi BL21(DE3) increased slightly. This indicates that the CZT3 mutant strain obtained in this invention has better protein secretion ability.

[0080] Step 4: Determination of endotoxin content in recombinant protein expressed by low-endotoxin strains without growth defects.

[0081] Endotoxin detection method: This study used a micro-kinetic chromogenic tachypleus amebocyte lysate (LTAL) assay to determine the endotoxin content of recombinant proteins. The reagent used was the Micro Kinetic Chromogenic Tachypleus Amebocyte Lysate Assay Kit from Xiamen Limulus Amebocyte Reagent Biotechnology Co., Ltd. The 50 EU / mL endotoxin standard solution was serially diluted to prepare four concentrations: 5 EU / mL, 0.5 EU / mL, 0.05 EU / mL, and 0.005 EU / mL. Each concentration of endotoxin standard solution was vortexed for at least 3 minutes during preparation. Endotoxin-free microstrips were placed on a microplate rack. 25 μL each of endotoxin test water (negative control) and endotoxin standard solution were added to the corresponding wells, followed by an equal volume of LTAL reagent for reaction. The standard curve should satisfy |r| ≥ 0.980, and the T value at the lowest point of the standard curve should be less than the T value of the negative control.

[0082] Dynamic photometric assay procedures and template settings were performed using the Gen5 3.08 software on a Biotec Synergy H1 multi-functional microplate reader: incubation at 37℃ for 10 min; medium speed, plate shaking for 5 s; plate reading time 90-120 min, reading interval 30-150 s; detection wavelength 405 nm; Onset OD was set according to the quality inspection report. Kinetic parameters were set: the extrapolation factor was set to 1 for standard curve fitting.

[0083] Table 4: Determination of GFP protein endotoxin content in each strain

[0084]

[0085] Although the GFP expressed by strain CZT3 carried 11.8 times more endotoxin than that of strain ClearColi BL21(DE3), it was 9 times lower than that of strain BL21(DE3). This may be because the consumables used in the assay were not completely pyrogen-free, which could explain the low endotoxin content in strain ClearColi BL21(DE3).

[0086] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An engineered *Escherichia coli* strain with no growth defects, low endotoxin levels, and highly efficient extracellular protein secretion capacity, characterized in that... The engineered Escherichia coli strain is classified and named Escherichia coli CZT3. It is deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with accession number CCTCCNO: M20261049 and deposit date of May 22, 2026.

2. The engineered *Escherichia coli* strain according to claim 1, characterized by being free of growth defects, low in endotoxins, and possessing highly efficient extracellular protein secretion capabilities, is characterized in that... The engineered Escherichia coli strain is a mutant strain of the BL21(DE3) series of Escherichia coli strains, and the nucleotide sequence of the msbA gene in the strain is shown in SEQ ID NO.

1.

3. The engineered *Escherichia coli* strain according to claim 1 or 2, characterized by being free of growth defects, low in endotoxins, and possessing highly efficient extracellular protein secretion capabilities, is characterized in that... The asparagine at position 529 of the MsbA protein encoded by the msbA gene of the engineered Escherichia coli was mutated to lysine.

4. The use of the engineered Escherichia coli strain according to any one of claims 1-3, which is free from growth defects, has low endotoxin levels, and has a high capacity for extracellular protein secretion, as a host bacterium for expressing low-endotoxin recombinant proteins.

5. An engineered *Escherichia coli* strain capable of expressing a low-endotoxin recombinant protein and achieving extracellular secretion, characterized in that... The engineered bacterium is a recombinant strain obtained by introducing a recombinant vector expressing recombinant protein into the engineered Escherichia coli strain according to any one of claims 1-3 and then screening it.

6. The engineered *Escherichia coli* strain capable of expressing low-endotoxin recombinant proteins and achieving extracellular secretion according to claim 5, characterized in that: The vector for expressing recombinant proteins is obtained by cloning the gene encoding the recombinant protein into the pET28a vector.

7. The engineered *Escherichia coli* strain capable of expressing low-endotoxin recombinant proteins and achieving extracellular secretion according to claim 5, characterized in that... The low-endotoxin recombinant protein includes green fluorescent protein, amylase protein, or β-galactosidase protein.

8. The engineered *Escherichia coli* strain capable of expressing low-endotoxin recombinant proteins and achieving extracellular secretion according to claim 7, characterized in that: The gene encoding the green fluorescent protein has the GenBanK accession number KF020493; the gene encoding the amylase protein has the GenBanK accession number KF751392; and the gene encoding the β-galactosidase protein has the GenBanK accession number CP107553.

9. The use of any one of the engineered Escherichia coli strains capable of expressing low-endotoxin recombinant proteins and achieving extracellular secretion according to claims 5-8 in the preparation of low-endotoxin recombinant proteins.

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