A genetically engineered strain for improving the sedimentation property of n-acetyl-obscurin product and a construction method and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a genetic engineering strain for improving the sedimentation of N-acetylcoelenterazine product and a construction method and application thereof. The present application is constructed by overexpressing at least one specific transporter protein coding gene in YdeD, rhtB, AexA, YwfM, pecM and ydhE in a host capable of synthesizing N-acetylcoelenterazine. Compared with the parent strain, the engineering strain can efficiently promote the transport and efflux of N-acetylcoelenterazine product, break the limitation of intracellular accumulation of the product, greatly accelerate the enrichment speed of the product, and increase the particle size of the product. This feature significantly improves the sedimentation effect of the product, making the subsequent product recovery process more simple and efficient, simplifying the recovery process, effectively reducing the recovery cost, and providing strong support for the industrial production of N-acetylcoelenterazine in improving yield and reducing cost.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products, its construction method, and its application. Background Technology

[0002] N-Acetyl-indigoidine is a novel natural blue pigment synthesized by microorganisms, belonging to the derivatives of indigoidine. Its molecular formula is C1. 12 H 10 N4O5, structurally similar to indigo, undergoes acetylation modification at the amino group, giving it unique physicochemical properties and application advantages. Currently, natural blue pigments are widely used in medicine, cosmetics, printing and dyeing, and food, among other fields. Common natural blue pigments include iridoid derivatives such as blue pigments, phycocyanin, indigo, indigo, oysterwort, and anthocyanins. N-acetylated indigo, with its environmental friendliness, high dyeing efficiency, and low-cost production advantages, has become an ideal substitute for synthetic indigo and a new generation of natural blue pigments, showing extremely broad application prospects in textiles, food, and other fields.

[0003] However, the synthesis mechanism of N-acetylglucosamine, a novel natural pigment, within microorganisms is not yet fully understood. The theoretical gaps regarding product formation, transport, and efflux pose significant challenges to yield improvement and product recovery. The particle size of biological N-acetylglucosamine is similar to that of bacterial cells, making product separation from bacterial cells more difficult than with traditional speckled blue. It cannot be directly separated using conventional sedimentation methods. Other equipment used in the biotechnology industry, such as disc centrifuges, plate centrifuges, and filtration systems, struggle to separate the product from bacterial cells, making it difficult to obtain high-purity N-acetylglucosamine, or requiring high processing costs to achieve this level of purity. This results in high industrial production costs and environmental concerns.

[0004] Studies have shown that bacteria release various organic compounds into the surrounding environment during their growth: enzymes, toxins, virulence factors, antibiotics, organic acids, amino acids, purine derivatives, vitamins, signaling molecules, etc. Only some of these compounds can freely diffuse across the cell membrane; most are expelled from the cell via transport systems, typically composed of membrane transport proteins. In recent years, numerous genes encoding transport proteins have been identified, involved in the transport of amino acids and purines from bacterial cells. These proteins belong to the DMT and RhtB / LysE protein families, which are among the most diverse and widely distributed membrane proteins. MATE family membrane proteins participate in the expulsion of quinolone derivatives from cells, contributing significantly to cellular resistance to many purine base analogs.

[0005] Currently, numerous studies have been conducted on utilizing efflux systems to promote product synthesis. For example, overexpression of Snq2p in *Saccharomyces cerevisiae* significantly enhances β-carotene efflux, increasing it by 4.04 times compared to the control strain. Overexpression of cg1298-cg1299 in *Glucosamine spp.* increases L-cysteine ​​production by 17.1%. Therefore, exploring suitable product transport and efflux strategies, and optimizing cellular metabolic processes by regulating the expression levels and activity of efflux proteins, thereby influencing product particle size and morphology, is a crucial direction for solving the sedimentation and recovery of N-acetylglucosamine products, making them more suitable for industrial applications.

[0006] The present invention aims to construct a series of efficient and stable engineered strains that can effectively improve the product efflux capacity of N-acetylglucosamine, reduce production and recycling costs, and provide more economical and environmentally friendly raw materials for the printing and dyeing industry. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a genetically engineered strain that improves the sedimentation of N-acetylglucosamine products, along with its construction method and applications. This invention seeks suitable N-acetylglucosamine efflux transport systems by introducing and validating major efflux systems from various sources, including the DMT (drug / metabolite transporter) superfamily, the RhtB / LysE family, and the MATE family, to effectively promote the transport and efflux of N-acetylglucosamine products and increase the rate of extracellular product accumulation.

[0008] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a genetically engineered strain for improving the sedimentation properties of N-acetylglucosamine products by overexpressing a transporter protein encoding gene in a host bacterium to construct the genetically engineered strain; wherein the transporter protein is at least one of YdeD, rhtB, AexA, YwfM, pecM and ydhE; and wherein the host bacterium is a strain capable of synthesizing N-acetylglucosamine.

[0009] This invention, through overexpression of at least one of the following genes encoding specific transport proteins—YdeD, rhtB, AexA, YwfM, pecM, and ydhE—in a host capable of synthesizing N-acetylglucosamine, enables the constructed engineered bacteria to more efficiently transport intracellularly synthesized N-acetylglucosamine to the extracellular space, overcoming the limitation of product accumulation within the cell and providing favorable conditions for continuous synthesis. Compared to the parent strain, the engineered bacteria can efficiently promote the transport and efflux of N-acetylglucosamine products, significantly accelerating the product enrichment rate. Simultaneously, it can increase the product particle size, a characteristic that significantly improves the product sedimentation effect, making the subsequent product recovery process simpler and more efficient. This not only simplifies the recovery process but also effectively reduces recovery costs, providing strong support for the industrial production of N-acetylglucosamine in terms of increasing yield and reducing costs.

[0010] Preferably, the transporter protein is at least one of YdeD, AexA, YwfM and ydhE.

[0011] Experimental studies have shown that overexpression of the above-mentioned transport protein encoding genes can better improve the enrichment rate of N-acetylglucosamine products and increase their particle size, thereby significantly improving the sedimentation performance of N-acetylglucosamine products. Furthermore, the introduction of the above-mentioned efflux protein encoding genes did not have a significant negative impact on the yield of N-acetylglucosamine synthesized by the host bacteria, and can effectively ensure the stability of product synthesis efficiency.

[0012] Preferably, the amino acid sequences of YdeD, rhtB, ydhE, AexA, YwfM and pecM are as shown in SEQ ID NO. 1-6.

[0013] Preferably, the host bacterium is Escherichia coli, and the Escherichia coli overexpresses the gene encoding bpsA for blue cyanide synthase, the gene encoding EntD for 4'-phosphopantoylthioethylamine transferase, the gene encoding glnA for glutamine synthase, and the gene encoding argA for N-acetylglutamate synthase.

[0014] This invention uses *E. coli* overexpressing the genes bpsA, EntD, glnA, and argA as the host bacterium, which can effectively transform and produce N-acetylglucosamine, but its efflux is poor. Based on this host bacterium, this invention introduces a specific transport protein to effectively enhance the efflux capacity of N-acetylglucosamine, solving the problem of poor efflux in the original host bacterium, reducing product accumulation within the cell, and increasing the particle size of the effluxed N-acetylglucosamine, significantly improving its sedimentation performance and making subsequent separation and purification processes simpler and more efficient.

[0015] Preferably, YdeD, rhtB, and ydhE are derived from Escherichia coli, AexA and YwfM are derived from Bacillus subtilis, and pecM is derived from Erwinia.

[0016] Secondly, the present invention provides a method for constructing a genetically engineered strain that improves the sedimentation property of N-acetylglucosamine products, comprising the following steps: amplifying the transport protein encoding gene by PCR, ligating it into a plasmid to obtain a recombinant plasmid, and then transferring the recombinant plasmid into a host bacterium to obtain the genetically engineered strain. Preferably, the transporter protein is at least one of YdeD, rhtB, AexA, YwfM, pecM and ydhE; the host bacterium is a strain capable of synthesizing N-acetylglucosamine.

[0017] Preferably, the plasmid includes pACYCduet-1.

[0018] Thirdly, the present invention provides the application of the genetically engineered strain in improving the sedimentation properties of N-acetylglucosamine products.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through overexpression of a specific transporter protein encoding gene in the *E. coli* host that synthesizes N-acetylascan, enables the constructed engineered bacteria to efficiently promote the extracellular transport and efflux of intracellularly synthesized N-acetylascan, effectively overcoming the limitations of intracellular product accumulation and creating favorable conditions for continuous synthesis, thus significantly accelerating product enrichment. Furthermore, this engineered bacteria can increase product particle size, a characteristic that significantly improves product sedimentation and facilitates subsequent separation and purification. Compared to the parent strain, the engineered bacteria described in this invention exhibit outstanding advantages in the ease of N-acetylascan product processing and cost control, possessing significant practical application value. Attached Figure Description

[0020] Figure 1 The pACYC-ydhE plasmid map; Figure 2 The pACYC-ydhE-YdeD plasmid map; Figure 3 The pACYC-ydhE-rhtB plasmid map; Figure 4 The pACYC-ydhE-AexA plasmid map; Figure 5 The pACYC-ydhE-YwfM plasmid map; Figure 6 The pACYC-ydhE-pecM plasmid map; Figure 7 This is a schematic diagram illustrating how the N-acetylglucosamine transporter of the present invention improves the particle size of the production product. Figure 8 This is a schematic diagram illustrating how the N-acetylglucosamine transporter enhances the sedimentation ability of products according to the present invention. Detailed Implementation

[0021] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0023] Escherichia coli DH5α (hereinafter referred to as DH5α) was used for vector construction, and all of them were commercially available. The plasmid pACYCDuet-1 was purchased from BioWind.

[0024] The culture media involved in the following examples are as follows: The LB medium formula is: 10 g / L sodium chloride, 10 g / L peptone, and 5 g / L yeast extract.

[0025] The ZYM fermentation medium formula is as follows: 96 mL ZY medium, 2 mL 50×M salts, 2 mL 50×5052, 200 μL 1 mol / L magnesium sulfate, 100 μL 1000× trace elements, 0.5 mmol / L IPTG, and 20 g / L glucose.

[0026] ZY medium consisted of 10 g / L peptone and 5 g / L yeast extract, sterilized at 121°C for 20 min before use.

[0027] Among them, 50×M salts: 1.25mol / L Na2HPO4, 1.25mol / L KH2PO4, 2.5mol / L NH4Cl and 0.25mol / L Na2SO4; Among them, 50×5052: 250g / L glycerol, 25g / L glucose; 1mol / L MgSO4; 1000× Trace elements: 50 mmol / L FeCl3, 20 mmol / L CaCl2, 10 mmol / L MnCl2, 10 mmol / L ZnSO4, 2 mmol / L each of CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H3BO3.

[0028] Unless otherwise specified, the plasmid transformation into Escherichia coli DH5α in the following examples and comparative examples are all chemical transformations. The chemical transformation includes the following steps: the transformation system is added to DH5α competent cells that have been thawed on ice, incubated on ice for 30 min, then heat-shocked at 42°C for 1 min, incubated on ice again for 2 min, then 900 μL of pre-cooled LB medium is added, and incubated at 37°C and 220 rpm for 60 min; 100 μL of the incubated bacterial solution is added to LB medium containing the corresponding antibiotic resistance, and cultured overnight at 30°C / 37°C.

[0029] Gibson assembly technology (hereinafter referred to as Gibson) is performed with reference to existing literature ([1] Gibson, D.G., et al. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343-345. [2] Gibson, D.G., et al. (2010). Creation of a bacterial cell controlled by a chemically synthesized genome. Science, 329(5987), 52-56.).

[0030] The primer sequences used to construct the recombinant plasmids in the examples are shown in Table 1.

[0031] Table 1. Primer sequences for constructing recombinant plasmids In the embodiments, YdeD, rhtB, and ydhE are derived from *Escherichia coli*, AexA and YwfM are derived from *Bacillus subtilis*, and pecM is derived from *Erwinia*. The amino acid sequences of YdeD, rhtB, ydhE, AexA, YwfM, and pecM are shown in SEQ ID NO. 1-6, respectively.

[0032] The host bacterium HG-N-Idg06 described in the example has the genotype BL21 (DE3) / pRSFDuet-EcglnA-EcargA+pCDFDuet-bpsA-entD, which means that the blue oxaliplatin synthase encoding gene bpsA, the 4'-phosphopantoinyl thioethylamine transferase encoding gene EntD, the glutamine synthase encoding gene glnA, and the N-acetylglucoamyl synthase encoding gene argA have been overexpressed in Escherichia coli, and can be effectively transformed to produce N-acetylglucoamyl, which is derived from HG-N-Idg06 with patent number CN2024107899337.

[0033] Example 1 This embodiment provides a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products. The strain was constructed by overexpressing the transporter protein encoding gene ydhE. The specific construction method is as follows: (1) First, using the genomic DNA of E coli BL21 (DE3) as a template, the ydhE gene fragment was amplified using primer pairs ydhE-F and ydhE-R respectively; at the same time, in order to optimize the gene expression, the original start codon GTG of ydhE was replaced with ATG using primer design. (2) Using pACYCduet-1 plasmid as a template, the linear vector fragment was amplified using pACYC-F and pACYC-R primers; subsequently, the plasmid backbone fragment and the target gene fragment were ligated using Gibson assembly, and 10 μL of the ligation system was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected for PCR verification, and the verification primers were YZ-F and YZ-R. Strains of the correct size were selected for sequencing verification. The successfully constructed plasmids were named pACYC-ydhE (see plasmid map). Figure 1 ); (3) The constructed plasmid pACYC-ydhE was transformed into the host bacterium HG-N-Idg06, and spread evenly on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L). After overnight culture at 37°C, the grown strain was detected by PCR. The constructed strain was named Ex-ydhE.

[0034] Example 2 This embodiment provides a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products. The strain was constructed by overexpressing the transport protein encoding genes ydhE and YdeD. The specific construction method is as follows: (1) Using E coli BL21 (DE3) genomic DNA as a template, the YdeD gene fragment was amplified using YdeD-F and YdeD-R primers; (2) Using the pACYC-ydhE plasmid constructed in Example 1 as a template, the linear vector fragment was amplified using the p-ydhE-F and p-ydhE-R primer pairs; subsequently, the plasmid backbone fragment and the target gene fragment were ligated by Gibson assembly, and 10 μL of the ligation system was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected for PCR verification, and the verification primers were YZ-F and YZ-R. Strains of the correct size were selected for sequencing verification. The successfully constructed plasmids were named pACYC-ydhE-YdeD (see plasmid map). Figure 2 ); (3) The constructed plasmid pACYC-ydhE-YdeD was transformed into the host bacterium HG-N-Idg06. It was evenly spread on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L) and cultured overnight at 37°C. The grown strain was then subjected to PCR detection. The constructed strain was named Ex-ydhE-YdeD.

[0035] Example 3 This embodiment provides a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products. The strain was constructed by overexpressing the transport protein encoding genes ydhE and rhtB. The specific construction method is as follows: (1) Using E coli BL21 (DE3) genomic DNA as a template, the rhtB gene fragment was amplified using rhtB-F and rhtB-R primers; (2) Using the pACYC-ydhE plasmid constructed in Example 1 as a template, the linear vector fragment was amplified using the p-ydhE-F and p-ydhE-R primer pairs; subsequently, the plasmid backbone fragment and the target gene fragment were ligated by Gibson assembly, and 10 μL of the ligation system was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected for PCR verification, and the verification primers were YZ-F and YZ-R. Strains of the correct size were selected for sequencing verification. The successfully constructed plasmids were named pACYC-ydhE-rhtB (see plasmid map). Figure 3 ); (3) The constructed plasmid pACYC-ydhE-rhtB was transformed into the host bacterium HG-N-Idg06, and spread evenly on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L). After overnight culture at 37°C, the grown strain was detected by PCR. The constructed strain was named Ex-ydhE-rhtB.

[0036] Example 4 This embodiment provides a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products. The strain was constructed by overexpressing the transport protein encoding genes ydhE and AexA. The specific construction method is as follows: (1) Using the AexA amino acid sequence in Bacillus subtilis as a template, codon optimization was performed for the host Escherichia coli to synthesize the target gene; then the AexA gene fragment was amplified using primers AexA-F / AexA-R respectively; (2) Using the pACYC-ydhE plasmid constructed in Example 1 as a template, the linear vector fragment was amplified using the p-ydhE-F and p-ydhE-R primer pairs; subsequently, the plasmid backbone fragment and the target gene fragment were ligated by Gibson assembly, and 10 μL of the ligation system was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected for PCR verification, and the verification primers were YZ-F and YZ-R. Strains of the correct size were selected for sequencing verification. The successfully constructed plasmids were named pACYC-ydhE-AexA (see plasmid map). Figure 4 ); (3) The constructed plasmid pACYC-ydhE-AexA was transformed into the host bacterium HG-N-Idg06. It was evenly spread on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L) and cultured overnight at 37°C. The grown strain was then subjected to PCR detection. The constructed strain was named Ex-ydhE-AexA.

[0037] Example 5 This embodiment provides a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products. The strain was constructed by overexpressing the transporter protein encoding genes ydhE and YwfM. The specific construction method is as follows: (1) Using the YwfM amino acid sequence in Bacillus subtilis as a template, codon optimization was performed for the host Escherichia coli to synthesize the target gene; then the YwfM gene fragment was amplified using primers YwfM-F / YwfM-R respectively; (2) Using the pACYC-ydhE plasmid constructed in Example 1 as a template, the linear vector fragment was amplified using the p-ydhE-F and p-ydhE-R primer pairs; subsequently, the plasmid backbone fragment and the target gene fragment were ligated by Gibson assembly, and 10 μL of the ligation system was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected for PCR verification, and the verification primers were YZ-F and YZ-R. Strains of the correct size were selected for sequencing verification. The successfully constructed plasmids were named pACYC-ydhE-YwfM (see plasmid map). Figure 5 ); (3) The constructed plasmid pACYC-ydhE-YwfM was transformed into the host bacterium HG-N-Idg06, and spread evenly on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L). After overnight culture at 37°C, the grown strain was detected by PCR. The constructed strain was named Ex-ydhE-YwfM.

[0038] Example 6 This embodiment provides a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products. The strain was constructed by overexpressing the transporter protein encoding genes ydhE and pecM. The specific construction method is as follows: (1) Using the pecM amino acid sequence in Erwinia as a template, codon optimization was performed for the host Escherichia coli to synthesize the target gene, and then the pecM gene fragment was amplified using primers pecM-F and pecM-R respectively. (2) Using the pACYC-ydhE plasmid constructed in Example 1 as a template, the linear vector fragment was amplified using the p-ydhE-F and p-ydhE-R primer pairs; subsequently, the plasmid backbone fragment and the target gene fragment were ligated by Gibson assembly, and 10 μL of the ligation system was transformed into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected for PCR verification, and the verification primers were YZ-F and YZ-R. Strains of the correct size were selected for sequencing verification. The successfully constructed plasmids were named pACYC-ydhE-pecM (see plasmid map). Figure 6 ); (3) The constructed plasmid pACYC-ydhE-pecM was transformed into the host bacterium HG-N-Idg06, and spread evenly on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L). After overnight culture at 37°C, the grown strain was detected by PCR. The constructed strain was named Ex-ydhE-pecM.

[0039] Example 1 This example uses different genetically engineered strains constructed in Examples 1-6 as experimental subjects to explore the effects of different engineered strains on the yield of N-acetylglucosamine. The specific methods are as follows: Single colonies of the engineered bacteria described in Examples 1-6 were selected and cultured for 16 h each in LB liquid medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L), and streptomycin (50 mg / L). The HG-N-Idg06 strain, which did not overexpress the transporter protein, was used as a control group, cultured in LB liquid medium containing kanamycin (50 mg / L) and streptomycin (50 mg / L). The inoculum was then transferred at a 1% inoculum to ZYM medium containing the corresponding antibiotics and cultured at 25°C with shaking at 220 rpm for 72 h. At 24 h and 48 h, 10 g / L sugar, 10 g / L glycerol, and 2.5 g / L glutamate were added, and the yield was measured after 72 h. Specific results are shown in Table 2.

[0040] Table 2 Table 2 shows that, compared with the control group, among the engineered strains constructed by overexpressing the specific transporter protein in Examples 1-6, only the N-acetylglucosamine yield of the engineered strain described in Example 6 decreased slightly, while the engineered strains in Examples 1-5 showed almost no decrease. This indicates that the introduction of the specific transporter protein described in this invention will not have a significant impact on the N-acetylglucosamine yield and can effectively ensure the stability of the product synthesis efficiency.

[0041] Example 2 This effect example uses different genetically engineered strains constructed in Examples 1-6 as experimental subjects to compare the particle size of N-acetylglucosamine products in the efflux effect of different engineered strains. The specific method is as follows: (1) Single colonies of the engineered bacteria described in Examples 1-6 were picked and cultured for 16 h each in LB liquid medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L), and streptomycin (50 mg / L). The HG-N-Idg06 strain, which did not overexpress the transporter protein, was used as the control group. The control group was cultured in LB liquid medium containing kanamycin (50 mg / L) and streptomycin (50 mg / L). The inoculum was then transferred to ZYM medium containing the corresponding antibiotics at a 1% inoculum and cultured at 25°C and 220 rpm for 72 h. At 24 h and 48 h, 10 g / L sugar, 10 g / L glycerol, and 7.5 g / L glutamate were added respectively. Samples were taken at 48 h for microscopic examination. (2) Take bacterial suspensions from different groups for 48 hours and measure their OD. Dilute the OD600 to 0.5-0.8 with physiological saline. Take a clean glass slide, sterilize it by flame burning with an alcohol lamp, cool it, add the diluted bacterial suspension, spread it evenly into a thin layer with a diameter of about 1 cm, let it air dry naturally or dry it slightly above an alcohol lamp flame, add cedar oil to the area to be observed, and observe and record the results using an eyepiece (10×) and an oil immersion lens (100× objective lens). See the results below. Figure 7 .

[0042] Figure 7 The results showed that, compared with the control group, the particle size of the N-acetylglucosamine product in Example 1 was increased, and the visible particles were more obvious; the particle size of the product in Example 2 was significantly larger than that of the control group, and the product morphology was more rounded; in Example 3, there were very few large particles, and the individual size was slightly larger than that of the control group and Example 1; the product in Example 4 showed irregular particle size, but there were many visible product aggregates with relatively large individuals, and these were the largest of all examples; the product particles in Examples 5-6 were slightly larger than those of the control group and Example 1. The results indicate that using efflux proteins from different sources for product transport can yield significant results, and the particle size of the products in Examples 1-6 was significantly larger than that of the original strain.

[0043] Example 3 This effect example uses different genetically engineered strains constructed in Examples 1-6 as test subjects to compare the product sedimentation ability in the N-acetylglucosamine efflux effect of different engineered strains. The specific method is as follows: (1) Single colonies of the engineered bacteria described in Examples 1-6 were picked and cultured for 16 h in LB liquid medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L), and streptomycin (50 mg / L). The HG-N-Idg06 strain, which did not overexpress the transporter protein, was used as the control group. The control group was cultured in LB liquid medium containing kanamycin (50 mg / L) and streptomycin (50 mg / L). The inoculum was then transferred to ZYM medium containing the corresponding antibiotics at a 1% inoculum and cultured at 25°C and 220 rpm for 72 h. At 24 h and 48 h, 10 g / L sugar, 10 g / L glycerol, and 7.5 g / L glutamate were added respectively. Samples were taken at 72 h for static precipitation comparison. (2) Take 8-10 mL of the 72-hour shake-flask fermentation broth from each group, mix well, and let stand at room temperature for 12 hours. Observe the sedimentation ability of the product. The results are shown in […]. Figure 8 The supernatant from different groups was collected for N-acetylglucosamine production determination, and the specific results are shown in Table 3.

[0044] Table 3 Table 3 shows that, compared with the natural sedimentation results of the control group, there was no significant difference in the fermentation broth of Examples 1 and 3, and the product had a weak self-settling ability, but the residual amount of supernatant was significantly lower than that of the control group; in Example 6, the supernatant was basically transparent, light in color, and the precipitate was visible, with only 0.14 g / L of product residue in the supernatant; in Examples 2 and 5, the supernatant was transparent, extremely light in color, and the precipitate was clearly visible, with the product residue in the supernatant being less than 0.1 g / L; in Example 4, the supernatant was transparent, with no residue detected in the supernatant, and the white bacteria and black product were clearly distinguishable in the bottom precipitate.

[0045] The results showed that the products in Examples 6, 2, and 5 settled naturally with very little product remaining in the supernatant; in Example 4, there was almost no product residue in the supernatant, and the product and bacterial cells could be clearly separated. This demonstrates that using the specific efflux protein described in this invention to enhance the extracellular transport capacity of the product can significantly increase the particle size of the product, promote product enrichment, and improve product collection capacity.

[0046] The above results demonstrate that by introducing specific efflux systems from different sources into the N-acetylglucosamine-producing bacteria, this invention can significantly promote the extracellular transport of products, increase product particle size, and achieve significant sedimentation ability. Among these strains, Ex-ydhE-YdeD, Ex-ydhE-AexA, Ex-ydhE-YwfM, and Ex-ydhE-pecM exhibit more pronounced product sedimentation ability compared to the original strain. In particular, Ex-ydhE-AexA shows significant separation of bacterial cells and products through precipitation, effectively reducing the difficulty and cost of product recovery.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A genetically engineered strain that enhances the sedimentation properties of N-acetylglucosamine products, characterized in that, A genetically engineered strain was constructed by overexpressing a transporter protein encoding gene in a host bacterium; the transporter protein is at least one of YdeD, rhtB, AexA, YwfM, pecM and ydhE; the host bacterium is a strain capable of synthesizing N-acetylglucosamine. The amino acid sequences of YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO.1-6, respectively.

2. The genetically engineered strain as described in claim 1, characterized in that, The transporter protein is at least one of YdeD, AexA, YwfM and ydhE.

3. The genetically engineered strain as described in claim 1, characterized in that, The host bacterium is Escherichia coli, which overexpresses the following genes: bpsA (encoding blue cyanide synthase), EntD (encoding 4'-phosphopantoylthioethylamine transferase), glnA (encoding glutamine synthase), and argA (encoding N-acetylglutamate synthase).

4. A method for constructing a genetically engineered strain that improves the sedimentation properties of N-acetylglucosamine products, characterized in that, Includes the following steps: The transport protein encoding gene is amplified by PCR, ligated into a plasmid to obtain a recombinant plasmid, and then the recombinant plasmid is transferred into a host bacterium to obtain the genetically engineered strain. The transporter protein is at least one of YdeD, rhtB, AexA, YwfM, pecM and ydhE; the host bacterium is a strain capable of synthesizing N-acetylglucosamine. The amino acid sequences of YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO.1-6, respectively.

5. The construction method as described in claim 4, characterized in that, The plasmid includes pACYCduet-1.

6. The use of the genetically engineered strain as described in any one of claims 1-3 in the production of N-acetylglucosamine products.

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