Genetic engineering strain for improving settleability of N-acetyl blue product as well as construction method and application of genetic engineering strain

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a gene engineering strain for improving the settleability of an N-acetyl blue product as well as a construction method and application of the gene engineering strain. The engineering bacterium is constructed by over-expressing at least one specific transport protein coding gene of YdeD, rhtB, AexA, YwfM, pecM and ydhE in a host capable of synthesizing N-acetylblue. Compared with a parent strain, the engineering strain can efficiently promote transport and excretion of an N-acetylblue product, breaks the limitation of intracellular accumulation of the product, greatly accelerates the enrichment speed of the product, and also can increase the particle size of the product, so that the sedimentation effect of the product is remarkably improved, the subsequent product recovery process is simpler, more convenient and more efficient, and the production cost is reduced. The method not only simplifies the recovery process, but also effectively reduces the recovery cost, and provides powerful support for the industrial production of N-acetyl blue in the aspects of yield improvement and cost reduction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered strain for improving the sedimentation property of N-acetyl-indigoidine product and a construction method and application thereof. BACKGROUND

[0002] Acetyl-indigoidine (N-Acetyl-indigoidine) is a new type of natural blue pigment synthesized by microorganisms, which belongs to the derivative of indigoidine. Its molecular formula is C 12 H 10 N4O5, which is similar in structure to indigoidine, but acetylation modification occurs at the amino group, making it have unique physicochemical properties and application advantages. At present, natural blue pigments have been widely used in medicine, cosmetics, printing and dyeing, food and other fields. Common natural blue pigments include iridoid derivative blue pigments, phycobilins, indigo pigments, indigoidine, oyster green pigment and anthocyanins. N-acetyl-indigoidine has become an ideal substitute for synthetic indigo and a new generation of natural blue pigment due to its environmental protection, high-efficiency dyeing performance and low-cost production advantage, and has a very broad application prospect in the fields of textiles, food and others.

[0003] However, the synthesis mechanism of N-acetyl-indigoidine in the microbial body has not been completely analyzed. The theoretical gap of product formation, transport and efflux brings great difficulty to yield improvement and product recovery. The size of biological N-acetyl-indigoidine product particles is similar to that of bacterial cells, and the separation of product and bacterial cells is more difficult than that of indigoidine. The product and bacterial cells cannot be directly separated by conventional sedimentation method, and the disc centrifuge equipment, plate centrifuge and filtration equipment used in other biological industries are difficult to separate the product and bacterial cells, and it is difficult to obtain high-purity N-acetyl-indigoidine product or a high process cost is required to obtain high-purity product. There are problems of high industrial production cost and environmental unfriendliness.

[0004] Studies have shown that bacteria release various organic compounds, such as enzymes, toxins, virulence factors, antibiotics, organic acids, amino acids, purine derivatives, vitamins, signal substances, etc. into the surrounding medium during growth. Only some of these compounds can freely diffuse through the cytoplasmic membrane, and most of the compounds are discharged from the cells through transport systems, which are usually composed of membrane transport proteins. In recent years, a variety of genes encoding transport proteins have been identified, which are 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 the most diverse and widely distributed membrane proteins. MATE family membrane proteins are involved in the discharge of quinolone derivatives from cells, which are helpful to improve the resistance of cells to many purine base analogs.

[0005] At present, there are many achievements in promoting product synthesis by using efflux system, for example, overexpression of Snq2p in Saccharomyces cerevisiae can significantly enhance the efflux of beta-carotene, which is 4.04 times higher than that of the control strain, overexpression of cg1298-cg1299 in Saccharomyces cerevisiae can increase the yield of L-cysteine by 17.1%, etc. Therefore, it is an important direction to solve the sedimentation and recovery of N-acetyl kolanin product by excavating suitable product transport and efflux scheme, adjusting the expression level and activity of efflux protein, optimizing the metabolic process of cells, thereby affecting the particle size and morphology of the product, so as to make it more meet the needs of industrial application.

[0006] The present application aims to construct a series of efficient and stable engineering strains, which can effectively improve the product efflux capacity of N-acetyl kolanin, reduce the production and recovery cost, and provide more economical and environmentally friendly raw materials for the printing and dyeing industry. SUMMARY

[0007] The present application aims to overcome the shortcomings of the prior art and provide a genetically engineered strain for improving the sedimentation of N-acetyl kolanin product and a construction method and application thereof. The present application verifies and finds a suitable N-acetyl efflux transport system by introducing a variety of sources of main efflux systems, including DMT (drug / metabolite transporter) superfamily, RhtB / LysE family, MATE family, etc., to effectively promote the transport and efflux of N-acetyl kolanin product and improve the extracellular product enrichment speed.

[0008] To achieve the above object, the technical scheme adopted by the present application comprises:

[0009] In a first aspect, the present application provides a genetically engineered strain for improving the sedimentation of N-acetyl kolanin product, which overexpresses a transport protein coding gene in a host strain to construct a genetically engineered strain; the transport protein is at least one of YdeD, rhtB, AexA, YwfM and pecM, and ydhE; the host strain is a strain capable of synthesizing N-acetyl kolanin.

[0010] The present application overexpresses at least one of YdeD, rhtB, AexA, YwfM and pecM and ydhE specific transport protein coding genes in a host capable of synthesizing N-acetyl kolanin, so that the constructed engineering strain can more efficiently transport the intracellularly synthesized N-acetyl kolanin to the extracellular, break the limitation of product accumulation in the intracellular, and provide favorable conditions for continuous synthesis. Compared with the parent strain, the engineering strain can efficiently promote the transport and efflux of N-acetyl kolanin product, greatly accelerate the enrichment speed of the product, and at the same time, it can also increase the particle size of the product, which significantly improves the sedimentation effect of the product, making the subsequent product recovery process more simple and efficient, not only simplifying the recovery process, but also effectively reducing the recovery cost, providing strong support for the industrial production of N-acetyl kolanin in improving yield and reducing cost.

[0011] Preferably, the transport protein is at least one of YdeD, AexA, YwfM and ydhE.

[0012] It is found through experiments that overexpression of the above-mentioned transport protein coding genes can better improve the enrichment speed of N-acetylcoelenterazine product, increase the particle size, thereby significantly improving the sedimentation performance of N-acetylcoelenterazine product, and the introduction of the above-mentioned efflux protein coding genes does not have obvious negative effects on the yield of N-acetylcoelenterazine synthesized by the host bacteria, and can effectively ensure the stability of the product synthesis efficiency.

[0013] Preferably, the amino acid sequences of YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO. 1-6 in sequence.

[0014] Preferably, the nucleotide sequences of the genes coding YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO. 7-12 in sequence.

[0015] Preferably, the host bacteria is Escherichia coli, and the Escherichia coli overexpresses coelenterazine synthase coding gene bpsA, 4'-phosphopantetheinyl transferase coding gene EntD, glutamine synthetase coding gene glnA and N-acetylglutamate synthetase coding gene argA.

[0016] The present application selects Escherichia coli overexpressing bpsA, EntD, glnA and argA genes as host bacteria, which can effectively convert and produce N-acetylcoelenterazine, but the efflux effect is not good. On the basis of the host bacteria, the present application effectively improves the efflux capacity of N-acetylcoelenterazine by introducing specific transport proteins, solves the problem of poor efflux effect of the original host bacteria, reduces the accumulation of the product in the cell, and the particle size of the efflux N-acetylcoelenterazine is increased, which significantly improves its sedimentation performance, so that the subsequent separation and purification process is more simple and efficient.

[0017] Preferably, the YdeD, rhtB and ydhE are derived from Escherichia coli, the AexA and YwfM are derived from Bacillus subtilis, and the pecM is derived from Erwinia.

[0018] In the second aspect, the present application provides a method for constructing a genetically engineered strain for improving the sedimentation performance of N-acetylcoelenterazine product, comprising the following steps: amplifying the transport protein coding gene by PCR, connecting it to a plasmid to obtain a recombinant plasmid, and then transforming the recombinant plasmid into a host bacteria to obtain the genetically engineered strain.

[0019] Preferably, the transport protein is at least one of YdeD, rhtB, AexA, YwfM, pecM and ydhE; and the host bacteria is a strain capable of synthesizing N-acetyl-kanamycin.

[0020] Preferably, the plasmid comprises pACYCduet-1.

[0021] In a third aspect, the application provides the use of the genetically engineered strain in improving the sedimentation of N-acetyl-kanamycin.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] The application overexpresses specific transport protein coding genes in an E. coli host for synthesizing N-acetyl-kanamycin, so that the constructed engineered bacteria can efficiently promote the transport and efflux of intracellularly synthesized N-acetyl-kanamycin to the outside of the cell, effectively breaking the limitation of intracellular accumulation of the product, creating favorable conditions for continuous synthesis, and greatly accelerating the enrichment speed of the product. Moreover, the engineered bacteria can also increase the particle size of the product, which significantly improves the sedimentation effect of the product and is more conducive to subsequent separation and purification. Compared with the parent strain, the engineered bacteria of the application exhibit outstanding advantages in the convenience of product processing and the control of recovery cost of N-acetyl-kanamycin, and have important practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 pACYC-ydhE plasmid map;

[0025] Figure 2 pACYC-ydhE-YdeD plasmid map;

[0026] Figure 3 pACYC-ydhE-rhtB plasmid map;

[0027] Figure 4 pACYC-ydhE-AexA plasmid map;

[0028] Figure 5 pACYC-ydhE-YwfM plasmid map;

[0029] Figure 6 pACYC-ydhE-pecM plasmid map;

[0030] Figure 7 N-acetyl-kanamycin transport protein schematic diagram for improving the particle size of the production product;

[0031] Figure 8 N-acetyl-kanamycin transport protein schematic diagram for improving the sedimentation capacity of the product. DETAILED DESCRIPTION

[0032] In order to make the objects, technical solutions and effects of the present application clearer and more explicit, the present application will be further described in detail below with reference to the examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0033] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0034] The E. coli DH5a (hereinafter referred to as DH5a) used for vector construction can be obtained from commercial channels. The plasmid pACYCDuet-1 is purchased from Biofroge.

[0035] The culture media involved in the following examples are as follows:

[0036] The formula of LB medium is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract.

[0037] The formula of ZYM fermentation medium is: 96 mL ZY medium, 2 mL 50x M salts, 2 mL 50x 5052, 200 μL 1 mol / L magnesium sulfate, 100 μL 1000x trace elements, 0.5 mmol / L IPTG, 20 g / L glucose.

[0038] Among them, ZY medium: 10 g / L peptone, 5 g / L yeast extract, sterilized at 121°C for 20 min and ready for use.

[0039] Among them, 50x M salts: 1.25 mol / L Na2HPO4, 1.25 mol / L KH2PO4, 2.5 mol / L NH4Cl and 0.25 mol / L Na2SO4;

[0040] Among them, 50x 5052: 250 g / L glycerol, 25 g / L glucose; 1 mol / L MgSO4;

[0041] 1000x trace elements: 50 mmol / L FeCl3, 20 mmol / L CaCl2, 10 mmol / L MnCl2, 10 mmol / L ZnSO4, CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H3BO3 each 2 mmol / L.

[0042] In the following examples and comparative examples, the plasmid was transformed into E. coli DH5a by chemical transformation, unless otherwise specified. The chemical transformation included the following steps: the transformation system was added to the thawed DH5a transformation competent cells on ice, and then incubated in an ice bath for 30 min, heated at 42°C for 1 min, incubated in an ice bath for 2 min again, then added with 900 uL of pre-cooled LB, and incubated at 37°C, 220 rpm for 60 min; 100 uL of the incubated bacterial solution was added to the LB medium containing the corresponding antibiotic resistance, and incubated at 30°C / 37°C overnight.

[0043] The Gibson assembly technology (hereinafter referred to as Gibson) was operated according to the 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.).

[0044] The related primer sequences for constructing the recombinant plasmid in the examples are shown in Table 1.

[0045] Table 1 Related primer sequences for constructing the recombinant plasmid

[0046]

[0047]

[0048] The YdeD, rhtB and ydhE in the examples were derived from E. coli, the AexA and YwfM were derived from B. subtilis, and the pecM was derived from Erwinia. The amino acid sequences of the YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO. 1-6, respectively, and the nucleotide sequences of the genes encoding the YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO. 7-12, respectively.

[0049] The genotype of the host strain HG-N-Idg06 in the examples was BL21 (DE3)

[0050] / pRSFDuet-EcglnA-EcargA+pCDFDuet-bpsA-entD, which has overexpressed the biosynthetic genes of cyanophyll synthase bpsA, 4'-phosphopantetheinyl transferase EntD, glutamine synthetase glnA and N-acetylglutamate synthetase argA in E. coli, can effectively convert and produce N-acetyl cyanophyll, which is derived from the patent No. CN2024107899337 HG-N-Idg06.

[0051] Example 1

[0052] The present embodiment provides a genetically engineered strain for improving the sedimentation of N-acetyl cyanophyll product, which is constructed by overexpressing the transport protein coding gene ydhE. The specific construction method is as follows:

[0053] (1) First, the genomic DNA of E. coli BL21(DE3) was used as a template, and the ydhE gene fragment was amplified with the ydhE-F and ydhE-R primer pairs; at the same time, in order to optimize the expression ability of the gene, the original start codon GTG of ydhE was replaced with ATG by primer design;

[0054] (2) The pACYCduet-1 plasmid was used as a template, and the linear carrier fragment was amplified with the pACYC-F and pACYC-R primers; then, the plasmid backbone fragment and the target gene fragment were connected by Gibson assembly, and 10 μL of the connection system was transferred into DH5α competent cells and cultured overnight at 37°C. Positive clones were selected and verified by PCR, and the verification primers were YZ-F and YZ-R. The strains with correct size were sent for sequencing verification, and the successfully constructed plasmids were named pACYC-ydhE (the plasmid map is shown in Figure 1 );

[0055] (3) The constructed plasmid pACYC-ydhE was transformed into the host strain HG-N-Idg06, which was uniformly coated 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 strains were detected by PCR, and the constructed strain was named Ex-ydhE.

[0056] Example 2

[0057] The present embodiment provides a genetically engineered strain for improving the sedimentation of N-acetyl cyanophyll product, which is constructed by overexpressing the transport protein coding gene ydhE, YdeD. The specific construction method is as follows:

[0058] (1) Use the genomic DNA of E. coli BL21(DE3) as a template, and use YdeD-F and YdeD-R primers to amplify the YdeD gene fragment;

[0059] (2) Use the pACYC-ydhE plasmid constructed in Example 1 as a template, and use p-ydhE-F and p-ydhE-R primers to amplify the linear vector fragment; then, connect the plasmid backbone fragment and the target gene fragment by Gibson assembly, and then transfer 10 μL of the connection system into DH5α competent cells for overnight culture at 37°C. Select positive clones for PCR verification, and use YZ-F and YZ-R primers for verification. Select strains with correct sizes for sequencing verification. The successfully constructed plasmids are named pACYC-ydhE-YdeD (see the plasmid map in Figure 2 );

[0060] (3) Transform the constructed plasmid pACYC-ydhE-YdeD into the host strain HG-N-Idg06, and uniformly coat it on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L), and streptomycin (50 mg / L), and then incubate it at 37°C overnight. Then, perform PCR detection on the grown strain, and name the constructed strain as Ex-ydhE-YdeD.

[0061] Example 3

[0062] This example provides a genetically engineered strain for improving the sedimentation property of N-acetyl-obscurine product. The genetically engineered strain is constructed by overexpressing the transporter protein coding gene ydhE and rhtB. The specific construction method is as follows:

[0063] (1) Use the genomic DNA of E. coli BL21(DE3) as a template, and use rhtB-F and rhtB-R primers to amplify the rhtB gene fragment;

[0064] (2) Use the pACYC-ydhE plasmid constructed in Example 1 as a template, and use p-ydhE-F and p-ydhE-R primers to amplify the linear vector fragment; then, connect the plasmid backbone fragment and the target gene fragment by Gibson assembly, and then transfer 10 μL of the connection system into DH5α competent cells for overnight culture at 37°C. Select positive clones for PCR verification, and use YZ-F and YZ-R primers for verification. Select strains with correct sizes for sequencing verification. The successfully constructed plasmids are named pACYC-ydhE-rhtB (see the plasmid map in Figure 3 );

[0065] (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.

[0066] Example 4

[0067] 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:

[0068] (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;

[0069] (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 using Gibson assembly, and then 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 using YZ-F and YZ-R primers. Strains of the correct size were selected for sequencing verification. The successfully constructed plasmids were named pACYC-ydhE-AexA (see plasmid map). Figure 4 );

[0070] (3) The constructed plasmid pACYC-ydhE-AexA was transformed into the host bacterium HG-N-Idg06. The plasmid 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.

[0071] Example 5

[0072] 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:

[0073] (1) With YwfM amino acid sequence in Bacillus subtilis as template, codon optimization aiming at E. coli host was carried out to synthesize target gene; then primers YwfM-F / YwfM-R were used to amplify YwfM gene fragment respectively;

[0074] (2) The pACYC-ydhE plasmid constructed in embodiment 1 was used as template, and the p-ydhE-F and p-ydhE-R primer pair was used to amplify the linear vector fragment; then, the plasmid backbone fragment and the target gene fragment were connected by Gibson assembly, 10 μL of the connection system was transferred into DH5α competent cells and cultured at 37℃ overnight, positive clones were selected and verified by PCR, the verification primers were YZ-F and YZ-R, the strain with correct size was selected for sequencing verification, and the successfully constructed plasmids were named as pACYC-ydhE-YwfM (the plasmid map is shown in Figure 5 );

[0075] (3) The constructed plasmid pACYC-ydhE-YwfM was transformed into the host strain HG-N-Idg06, which was uniformly coated on solid LB medium containing chloramphenicol (50 mg / L), kanamycin (50 mg / L) and streptomycin (50 mg / L) and cultured at 37℃ overnight, then the grown strain was detected by PCR, and the constructed strain was named as Ex-ydhE-YwfM.

[0076] Embodiment 6

[0077] The embodiment provides a genetically engineered strain for improving the sedimentation property of N-acetylglucosamine product, and the genetically engineered strain is constructed by overexpressing the transporter protein coding gene ydhE and pecM. The specific construction method is as follows:

[0078] (1) With pecM amino acid sequence in Erwinia as template, codon optimization aiming at E. coli host was carried out to synthesize target gene, and then primers pecM-F and pecM-R were used to amplify pecM gene fragment respectively;

[0079] (2) The pACYC-ydhE plasmid constructed in embodiment 1 was used as template, and the p-ydhE-F and p-ydhE-R primer pair was used to amplify the linear vector fragment; then, the plasmid backbone fragment and the target gene fragment were connected by Gibson assembly, 10 μL of the connection system was transferred into DH5α competent cells and cultured at 37℃ overnight, positive clones were selected and verified by PCR, the verification primers were YZ-F and YZ-R, the strain with correct size was selected for sequencing verification, and the successfully constructed plasmids were named as pACYC-ydhE-pecM (the plasmid map is shown in Figure 6 );

[0080] (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.

[0081] Example 1

[0082] 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:

[0083] 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.

[0084] Table 2

[0085] Group 48h yield (g / L) 72h yield (g / L) Control 3.05 8.33 Example 1 3.02 8.57 Example 2 2.96 8.05 Example 3 3.03 8.24 Example 4 3.29 8.77 Example 5 3.07 8.41 Example 6 2.58 7.86

[0086] 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.

[0087] Example 2

[0088] 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:

[0089] (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, and 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 with shaking. 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.

[0090] (2) OD values ​​of bacterial suspensions from different groups were measured after 48 hours. The OD values ​​were diluted with physiological saline to 0.5-0.8 from 600. A clean glass slide was sterilized by flame burning with an alcohol lamp. After cooling, the diluted bacterial suspension was added and evenly spread into a thin layer approximately 1 cm in diameter. The slide was allowed to air dry naturally or lightly dried over an alcohol lamp flame. Cedarwood oil was then added to the area to be observed. The results were observed and recorded using an eyepiece (10×) and an oil immersion lens (100× objective). See [see attached image]. Figure 7 .

[0091] 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.

[0092] Example 3

[0093] 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:

[0094] (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, and 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 with shaking. 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.

[0095] (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.

[0096] Table 3

[0097]

[0098] 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.

[0099] 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.

[0100] The above results show that by introducing specific efflux systems from different sources into the N-acetyl-obscurin producing strain, the product transport capacity to the extracellular can be obviously promoted, the product particle size is improved, and the obvious settling capacity is obtained. Among them, the strains Ex-ydhE-YdeD, Ex-ydhE-AexA, Ex-ydhE-YwfM, Ex-ydhE-pecM have more obvious product settling capacity compared with the original strain, especially the bacteria and product in Ex-ydhE-AexA have obvious precipitation separation, thereby the product recovery difficulty and recovery cost can be effectively reduced.

[0101] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A genetically engineered bacterial strain for improving the sedimentation property of N-acetylcolinesterase product, characterized by, The gene engineering strain is constructed by overexpressing a transport protein coding gene in a host bacterium, wherein the transport protein is at least one of YdeD, rhtB, AexA, YwfM, pecM and ydhE, and the host bacterium is a strain capable of synthesizing N-acetyl-oxazone.

2. The genetically engineered bacterial strain of claim 1, wherein, The transport protein is at least one of YdeD, AexA, YwfM and ydhE.

3. The genetically engineered bacterial strain of claim 1, wherein, The amino acid sequences of YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO. 1-6, respectively.

4. The genetically engineered bacterial strain of claim 1, wherein, The nucleotide sequences of the genes encoding YdeD, rhtB, ydhE, AexA, YwfM and pecM are shown in SEQ ID NO. 7-12, respectively.

5. The genetically engineered bacterial strain of claim 1, wherein, The host bacterium is Escherichia coli, and the Escherichia coli overexpresses oxazone synthase coding gene bpsA, 4'-phosphopantetheinyl transferase coding gene EntD, glutamine synthetase coding gene glnA and N-acetylglutamate synthetase coding gene argA.

6. The genetically engineered bacterial strain of claim 1, wherein, The YdeD, rhtB and ydhE are derived from Escherichia coli, the AexA and YwfM are derived from Bacillus subtilis, and the pecM is derived from Erwinia.

7. A method for constructing a genetically engineered bacterial strain having improved sedimentation properties of N-acetylcolinesterase product, characterized by, The method comprises the following steps: The transport protein coding gene is amplified by PCR, connected to a plasmid to obtain a recombinant plasmid, and then the recombinant plasmid is transformed into the host bacterium to obtain the gene engineering strain. The transport protein is at least one of YdeD, rhtB, AexA, YwfM, pecM and ydhE, and the host bacterium is a strain capable of synthesizing N-acetyl-oxazone.

8. The construction method of claim 7, wherein, The plasmid comprises pACYCduet-1.

9. Use of the gene engineering strain according to any one of claims 1-6 in the production of N-acetyl-oxazone product.

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