Method for increasing yield of lactoyl-N-neotetraose based on genome integration and transporter screening

Abstract

The invention discloses a method for increasing the yield of lactoyl-N-neotetraose based on genome integration and transporter screening, and belongs to the field of microbial genetic engineering. According to the invention, a lactoyl-N-trisaccharide II synthesis module and a lactoyl-N-neotetraose synthesis module are rapidly integrated to different sites of an escherichia coli genome in a multi-copy manner by virtue of a CRISPR (clustered regularly interspaced short palindromic repeats)-associated transsposases gene editing technology, so that the use of plasmids is reduced, and the metabolic burden of cells is reduced. And in a 3L bioreactor, the yield of the lactyl-N-neotetraose reaches 30.56 g / L. Through over-expression of the transporter, the transporter which is most obvious in improving the yield of the lactoyl-N-neotetraose is screened to be YbSET, the yield of the lactoyl-N-neotetraose reaches 54.12 g / L, and excellent industrial application prospects are shown.

Description

Technical Field

[0001] The invention relates to a method for increasing the yield of lactoyl-N-neotetraose based on genome integration and transporter protein screening, and belongs to the technical field of genetic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) are complex carbohydrates unique to breast milk, second only to lactose and fat in abundance. Composed primarily of three types of oligosaccharides: fucosylated, non-fucosylated, and sialylated, HMOs possess significant prebiotic, immunomodulatory, and antipathogen properties. Studies have shown that HMOs can selectively promote the proliferation of probiotics such as Bifidobacterium, maintain intestinal microbiome balance, and reduce the risk of infection and allergies in infants by blocking pathogen adhesion and modulating immune responses.

[0003] Over 200 human milk oligosaccharides have been identified, of which lacto-N-neotetraose accounts for approximately 6% of all HMOs. Currently, lacto-N-neotetraose has been approved for production and application, and is used in infant formula and functional foods, demonstrating broad market prospects.

[0004] Approved methods for producing lacto-N-neotetraose (LNT) using chemical methods suffer from harsh reaction conditions, complex processes, low product purity, difficulty in separation, environmental hazards, and difficulty meeting food and pharmaceutical grade standards. Microbial synthesis can utilize inexpensive and readily available carbon sources such as glycerol or glucose to produce LNT, making it environmentally friendly, sustainable, and the current mainstream method for industrial production of LNT. However, most current LNT production strains still rely on multiple plasmid co-expression systems to achieve LNT synthesis. The expression of these multiple plasmids places a significant metabolic burden on the host strain, thus impairing LNT synthesis. Furthermore, studies have found that in these production strains, a large amount of synthesized LNT remains intracellularly, failing to be efficiently and effectively excreted into the extracellular culture medium. This intracellular accumulation may limit the ability of LNT to sustainably synthesize LNT and increase the difficulty and cost of subsequent extraction and purification. Therefore, in order to solve the above problems, it is urgent to develop a lactoyl-N-neotetraose producing strain with low metabolic pressure and high transport efficiency. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem of excessive metabolic pressure caused by the presence of excessive plasmids in cells and excessive intracellular residues of lactoyl-N-neotetraose during the production of lactoyl-N-neotetraose by microorganisms, and to provide an Escherichia coli with greatly improved lactoyl-N-neotetraose production capacity.

[0006] To address the above technical problems, the present invention uses CRISPR-associated transposases gene editing technology to rapidly integrate multiple copies of the lacto-N-triose II synthesis module and the lacto-N-neotetraose synthesis module into different sites of the Escherichia coli genome to reduce the use of plasmids, thereby reducing the metabolic burden of E. coli. Furthermore, by exploiting transport proteins, the efflux of lacto-N-neotetraose is accelerated, the intracellular residue of lacto-N-neotetraose is reduced, and the metabolic pressure of the cell is further reduced. Thus, a lacto-N-neotetraose-producing E. coli strain with greatly improved metabolic pressure and a method for its construction are provided.

[0007] The first technical solution provided by the present invention is to change the target gene from plasmid expression to genomic expression to provide a genetically engineered Escherichia coli strain that reduces the metabolic burden of cellular production of lactoyl-N-neotetraose. The genetically engineered strain uses Escherichia coli as a chassis strain, and multiple copies of the genes of the following modules a)-b) are integrated into the genome of the chassis strain using CRISPR-associated transposases gene editing technology:

[0008] a) Lactoyl-N-triose II synthesis module: β-1,3-N-acetylglucosamine transferase gene lgtA, phosphoglucosamine mutase gene glmM, fusion N-acetylglucosamine-1-phosphate ureyltransferase / glucosamine-1-phosphate acetyltransferase gene glmU, L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS;

[0009] b) Lactoyl-N-neotetraose synthesis module: β-1,4-galactosyltransferase gene lgtB, phosphoglucomutase gene pgm, UTP-glucose-1-phosphate uridyltransferase gene galU, UDP-glucose 4-epimerase gene galE.

[0010] In certain embodiments, the copy number of the multi-copy genome-integrated lacto-N-triose II synthesis module of Escherichia coli is 1-4 copies, preferably 3 copies, and the copy number of the multi-copy genome-integrated lacto-N-neotetraose synthesis module is 3-8 copies, preferably 4 copies.

[0011] In certain embodiments, the NCBI sequence number of the amino acid sequence of the β-1,3-acetylglucosamine transferase LgtA is WP_002257440.1, the NCBI sequence number of the amino acid sequence of the β-1,4-galactosyltransferase LgtB is WP_002225824.1, the NCBI accession number of the nucleotide sequence of the phosphoglucomutase gene pgm is ECK0676; the NCBI accession number of the nucleotide sequence of the UTP-glucose-1-phosphate uridyltransferase gene galU is ECK1231; The NCBI accession number of the nucleotide sequence of the UDP-glucose 4-epimerase gene galE is ECK0748; the NCBI accession number of the nucleotide sequence of the phosphoglucosamine mutase gene glmM is ECK3165; the NCBI accession number of the nucleotide sequence of the L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS is ECK3722; and the NCBI accession number of the nucleotide sequence of the fusion N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase gene glmU is ECK3723.

[0012] In certain embodiments, the L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS includes glmS*, which encodes a GlmS mutant GlmS*. The mutation site of the mutant GlmS* is calculated starting from the second amino acid of GlmS (NP_418185), and the following mutations are performed: E14K, D386V, S449P, E524G, wherein E14K indicates that the 14th amino acid E is mutated to K, D386V indicates that the 386th amino acid D is mutated to V, S449P indicates that the 449th amino acid S is mutated to P, and E524G indicates that the 524th amino acid E is mutated to G.

[0013] In certain embodiments, the phosphoglucomutase gene pgm, UTP-glucose-1-phosphate uridyltransferase gene galU, UDP-glucose 4-epimerase gene galE, phosphoglucomutase gene glmM, L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS, and fused N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase gene glmU are all derived from Escherichia coli K-12MG1655.

[0014] In certain embodiments, the genomic integration site of the lacto-N-triose II synthesis module is one or more of wcaJ, ushA, nudK, and nagA.

[0015] In certain embodiments, the genomic integration site of the lacto-N-neotetraose synthesis module is one or more of yadD-panC, aroG-gpmA, ompW-yciE, purT-eda, tktB-ypfG, yghA-exbD, cspA-hokA, and rluF-yjbD.

[0016] In certain embodiments, the plasmids used for multi-copy genomic integration include pQCascade, pDonor-cargo, pTnsABC, and pCutamp-mediated transposition and plasmid elimination.

[0017] In certain embodiments, the chassis strain used is an engineered Escherichia coli strain with lacZ, lacA, nagB, wecB, ugd, gcd, and setA knocked out as reported in patent CN117343889A, herein designated EC. These gene knockouts were achieved by using CRISPR / Cas9 gene editing technology to knock out the β-galactosidase gene lacZ, the galactoside O-acetyltransferase gene lacA, the glucosamine-6-phosphate deaminase gene nagB, the UDP-N-acetylglucosamine 2-imidase gene wecB, the UDP-glucose 6-dehydrogenase gene ugd, the quinone protein glucose dehydrogenase gene gcd, and the sugar export protein gene setA in Escherichia coli.

[0018] In certain embodiments, endogenous transport proteins or exogenous transport proteins are also overexpressed in the genetically engineered bacteria. The endogenous transport protein genes include YajR, mdtM, ​​mdfA, ompC, ompF, and lamB, and the exogenous transport protein genes include PcSET, PsSET, BbSET, sotA, YwbF, LmrC, YuxJ, YbSET, scrY, and cmSET.

[0019] In certain embodiments, the endogenous transporter is a putative transporter gene YajR, a drug MFS transporter gene MdtM, a multidrug efflux pump gene MdfA, an outer membrane porin C gene ompC, an outer membrane porin F gene ompF, and a maltose outer membrane channel protein gene lamB.

[0020] In certain embodiments, the NCBI accession number of the nucleotide sequence of the putative transporter gene YajR is NC_000913.3 (445302-446666); the NCBI accession number of the nucleotide sequence of the drug MFS transporter gene MdtM is NC_000913.3 (4567287-4568519); the NCBI accession number of the nucleotide sequence of the multidrug efflux pump gene MdfA is NC_000913.3 (883673-88490 5); the NCBI accession number of the nucleotide sequence of the outer membrane porin C gene ompC is NC_000913.3 (2311646-2312749); the NCBI accession number of the nucleotide sequence of the outer membrane porin F gene ompF is NC_000913.3 (985894-986982); the NCBI accession number of the nucleotide sequence of the maltose outer membrane channel protein gene lamB is NC_000913.3 (4247971-4249311).

[0021] In certain embodiments, the endogenous transporter is a putative transporter gene YajR, a drug MFS transporter gene MdtM, a multidrug efflux pump gene MdfA, an outer membrane porin C gene ompC, an outer membrane porin F gene ompF, and a maltose outer membrane channel protein gene lamB, all of which are derived from Escherichia coli K12 MG1655.

[0022] In certain embodiments, the exogenous transporter is a sugar efflux transporter gene PcSET, a sugar efflux transporter gene PsSET, a sugar efflux transporter gene BbSET, a sugar efflux transporter A gene sotA, a putative sugar permease gene ywbF, a putative ABC transporter gene LmrC, an acidic stress-induced putative efflux protein gene YuxJ, a major facilitator superfamily protein MFS_1 gene YbSET, an outer membrane porin gene ScrY, or a sugar efflux transporter gene CmSET.

[0023] In certain embodiments, the NCBI sequence number of the amino acid sequence of the sugar efflux transporter PcSET is ZP_03829909.1, or the sugar efflux transporter gene PcSET is derived from Pectobacterium carotovorumsubsp.carotovorum WPP14; the NCBI sequence number of the amino acid sequence of the sugar efflux transporter PsSET is BAP78849.1, or the sugar efflux transporter gene PsSET is derived from Pseudomonas sp.MT-1; the NCBI sequence number of the amino acid sequence of the sugar efflux transporter BbSET is KGQ13398.1, or the sugar efflux transporter gene BbSET is derived from Beauveria bassiana D1-5; the NCBI sequence number of the amino acid sequence of the sugar efflux transporter A is EGC72107.1, or the sugar efflux transporter A gene sotA is derived from Haemophilus parainfluenzae ATCC 33392; the NCBI sequence number of the amino acid sequence of the putative sugar permease is NP_391713.1, or the putative sugar permease gene ywbF is derived from Bacillus subtilis subsp.subtilis str.168; the NCBI sequence number of the amino acid sequence of the putative sugar ABC transporter is ABX00624.1, or the putative ABC transporter gene lmrC is derived from Streptomyces lincolnensis; the NCBI sequence number of the amino acid sequence of the putative acidic stress-induced efflux protein is NP_391026.2, or the putative acidic stress-induced efflux protein gene yuxJ is derived from Bacillus subtilis subsp.subtilis str.168; the NCBI sequence number of the amino acid sequence of the major facilitator superfamily protein MFS_1 is EEQ08298.1, or the major facilitator superfamily protein MFS_1 gene YbSET is derived from Yersinia bercovieri ATCC. 43970; the NCBI sequence number of the amino acid sequence of the outer membrane porin ScrY is AHM80897.1, or the outer membrane porin gene scrY is derived from Klebsiella pneumoniae 30684 / NJST258_2; the NCBI sequence number of the amino acid sequence of the sugar efflux transporter CmSET is WP_024910347.1, or the sugar efflux transporter gene CmSET is derived from Chania multitudinisentens.

[0024] In certain embodiments, the endogenous and exogenous transporters are overexpressed using the pACYCDuet-1 plasmid.

[0025] The second technical solution provided by the present invention is a method for improving the synthesis capacity of lactoyl-N-neotetraose by reducing the metabolic burden of Escherichia coli. The method is to overexpress endogenous transport proteins or exogenous transport proteins in Escherichia coli, wherein the endogenous transport protein genes include yajR, mdtM, ​​mdfA, ompC, ompF, and lamB, and the exogenous transport protein genes include PcSET, PsSET, BbSET, sotA, ywbF, lmrC, yuxJ, YbSET, scrY, and CmSET.

[0026] The third technical solution provided by the present invention is a method for synthesizing lactoyl-N-neotetraose, wherein the method uses glycerol as a carbon source and utilizes the genetically engineered bacteria described in the first technical solution to ferment to obtain lactoyl-N-neotetraose.

[0027] The fourth technical solution provided by the present invention is the use of the genetically engineered bacteria described in the first technical solution, or the method described in the second technical solution, or the method described in the third technical solution in the preparation of lactoyl-N-neotetraose or a product containing lactoyl-N-neotetraose.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention uses CRISPR-associated transposases gene editing technology to rapidly integrate multiple copies of large expression cassettes of the lacto-N-triose II synthesis module and the lacto-N-neotetraose synthesis module into different sites of the Escherichia coli genome, reducing the use of plasmids to reduce the metabolic burden of the cells. The optimal genomic integration copy numbers of lacto-N-triose II and lacto-N-neotetraose were determined to be 3 and 4, respectively. In a 3L bioreactor, the yield of lacto-N-neotetraose reached 30.56g / L.

[0030] (2) Through further screening of overexpressed transporter proteins, the transporter protein that most significantly increased the production of lactoyl-N-neotetraose was YbSET. In a 3L bioreactor, the production of lactoyl-N-neotetraose reached about 54.12 g / L, showing excellent prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the metabolic pathway for the synthesis of lactoyl-N-neotetraose.

[0032] Figure 2(a) shows the results of genome integration of the lactoyl-N-triose II synthesis module; (b) shows the results of shake flasks of the strain with genome integration of the lactoyl-N-triose II synthesis module.

[0033] Figure 3 (a) shows the results of genome integration of the lacto-N-neotetraose synthesis module; (b) shows the results of shake flasks of the strain with genome integration of the lacto-N-neotetraose synthesis module.

[0034] Figure 4 This is a graph showing the fermentation yield of lactoyl-N-neotetraose by the genome-integrated strain in a 3L bioreactor.

[0035] Figure 5 Figure 2 is a graph showing the total yield and extracellular yield of lactoyl-N-neotetraose produced by shake flask fermentation under the condition of overexpression of the transporter.

[0036] Figure 6 This is a graph showing the fermentation yield of lactoyl-N-neotetraose in a 3L bioreactor of a strain overexpressing the transporter protein. DETAILED DESCRIPTION

[0037] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0038] The preparation of fermentation broth samples and the detection conditions of lactoyl-N-neotetraose are as follows:

[0039] Fermentation conditions: The seed solution was transferred to a 250 mL conical flask containing 50 mL fermentation medium at a 5% inoculum volume. The corresponding antibiotics were added to maintain the presence of the plasmid. The culture was carried out at 37°C and 200 rpm for 2.5-3 h. When the OD600 reached 2-4, 0.1 mM IPTG was added to induce enzyme expression. The culture was transferred to an air shaker at 25°C and 200 rpm for 2 h. A final concentration of 5 g·L -1 Lactose, 25 ° C, 200 rpm continued to culture. Fermentation ended after 72 hours.

[0040] Extracellular: Take 1 mL of fermentation broth sample, centrifuge at 12000 rpm for 3-10 min, take the supernatant, boil at 100℃ for 10-15 min, centrifuge at 12000 rpm for 10-15 min, filter the supernatant through a 0.22 μm aqueous needle filter into a liquid phase vial as the sample to be tested.

[0041] Intracellular + extracellular: Take 1 mL of fermentation broth sample, boil at 100℃ for 10-15 min, centrifuge at 12000 rpm for 10-15 min, filter the supernatant after centrifugation through a 0.22 μm aqueous needle filter into a liquid phase vial as the sample to be tested.

[0042] Lactoyl-N-neotetraose was detected by high performance liquid chromatography (HPLC): differential refractive index detector; chromatographic column was Aminex HPX-87H (300×7.8 mm), column temperature was 60°C; mobile phase was 5 mM H2SO4 aqueous solution, flow rate was 0.5 mL / min; injection volume was 10 μL.

[0043] Culture medium formula:

[0044] LB medium: 5.00 g / L yeast extract, 10.00 g / L peptone, and 10.00 g / L sodium chloride.

[0045] LB solid medium: 5.00 g / L yeast extract, 10.00 g / L peptone, 10.00 g / L sodium chloride, and 20 g / L agar powder.

[0046] Shake flask fermentation medium: 30.00 g / L glycerol, 6.70 g / L yeast extract, 3.30 g / L corn steep liquor, 13.50 g / L potassium dihydrogen phosphate, 4.00 g / L diammonium hydrogen phosphate, 1.70 g / L citric acid, 1.40 g / L magnesium sulfate heptahydrate, and 10 mL / L trace element solution. The trace element solution included 10.00 g / L iron (III) citrate, 2.25 g / L zinc sulfate heptahydrate, 1.00 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium tetraborate decahydrate, 0.11 g / L ammonium heptamolybdate, and 2.00 g / L calcium chloride.

[0047] The fermentation medium for the upper tank included 10.00 g / L glycerol, 2.00 g / L yeast extract, 6.00 g / L porcine bone peptone, 8.2 g / L potassium dihydrogen phosphate, 9.20 g / L potassium hydrogen phosphate, 4.00 g / L ammonium sulfate, 2.00 g / L magnesium sulfate heptahydrate, 0.30 g / L citric acid, 0.02 g / L calcium chloride, and 10 mL / L trace element solution. The trace element solution included 10.00 g / L iron (III) citrate, 2.25 g / L zinc sulfate heptahydrate, 1.00 g / L copper sulfate pentahydrate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium tetraborate decahydrate, 0.11 g / L ammonium heptamolybdate, and 2.00 g / L calcium chloride.

[0048] Feed medium: 600 g / L glycerol and 5.00 g / L magnesium sulfate heptahydrate.

[0049] The plasmids and strains involved in the following examples are:

[0050] 1. The CRISPR-associated transposase system involves four plasmids, including the plasmid pDonor (ampicillin resistance Amp r, containing crRNA array for genome targeting, transposase recognition sequences LE and RE, and donor DNA), plasmid pTnsABC (kanamycin resistance Kan r , used to express transposases TnsAB and TnsC), plasmid pQCascade (streptomycin-resistant Sm r , used to express the fusion of Cas protein and transposase TniQ-Cas678) and a plasmid pCutamp (ampramycin resistance Apr r The plasmids were constructed using a sucrose-lethal gene (sacB) and an N20 sequence targeting the AmpR promoter for targeted cleavage of pTnsABC, pQCascade, and pDonor for plasmid elimination. The plasmid sequences are disclosed in the following reference: Yiwen, Zhang, Xiaoman, Sun, Qingzhuo, Wang et al. Correction to Multicopy Chromosomal Integration Using CRISPR-Associated Transposases. [J]. ACS Synth Biol, 2020, 9:0.

[0051] 2. Chassis strain EC: The chassis strain is derived from Escherichia coli BL21 (DE3) ΔlacZΔlacAΔnagBΔwecBΔugdΔgcdΔsetA in patent CN117343889A, and is named EC here.

[0052] 3. Plasmid pCDF-lgtA-glmU-S*-glmM and plasmid pRSF-lgtB-pgm-galE-galU have been disclosed in patent document CN 117343889A.

[0053] Example 1 Construction of Genome-Integrated Strains (Taking Genome Integration of Lactoyl-N-Trisaccharide II Synthesis Module as an Example)

[0054] refer to Figure 1 Metabolic pathways and the construction of genome-integrated strains are as follows:

[0055] 1. Construction of integration plasmid

[0056] 1) Design of genomic integration sites

[0057] After selecting the target integration site, the sgRNAcas9 software was used to predict and select the N32 sequence. Once designed, the N32-4 sequence was synthesized by Anshengda and ligated into the pETDuet-1 vector to construct pET-N32-4.

[0058] 2) Construction of pDonor-N32-4 plasmid

[0059] Using the plasmid pET-N32-4 containing the N32-4 fragment as a template, the N32-4-F / R primer pair was used to amplify the N32-4 fragment, which was then purified and recovered. The recovered N32-4 fragment was inserted into the pDonor plasmid using the Megawhop method. After PCR was completed, 8 μL of the PCR amplification product was taken and the template plasmid in the system was eliminated using Dpn I rapid digestion enzyme. The PCR product was then transformed into Escherichia coli JM109, and the plasmid was extracted and sent for sequencing verification. Successfully verified pDonor-N32-4 was stored at -20°C until use.

[0060] 3) Construction of pDonor-LNT II plasmid

[0061] Using pCDF-lgtA-glmU-S*-glmM as a template, the LNT II-F / R primer pair was used to amplify the lactoyl-N-triose II synthesis module, and the DNA fragment was purified and recovered. The recovered lactoyl-N-triose II synthesis module fragment was inserted into the pDonor-N32-4 plasmid using the Megawhop method. After the PCR was completed, 8 μL of the PCR amplification product was taken and the template plasmid in the system was eliminated with Dpn I fast-cutting enzyme. The PCR product was then transformed into Escherichia coli JM109, and the plasmid was extracted and sent for sequencing for verification. The successfully verified pDonor-LNT II was stored at -20°C for future use.

[0062] The construction method of the lacto-N-neotetraose synthesis module integration plasmid pDonor-LNnT was the same as above (primers involved in the construction of the genomic integration plasmid are shown in Table 1), wherein the LNnT synthesis module was amplified using the plasmid pRSF-lgtB-pgm-galE-galU as a template.

[0063] Table 1 Primer sequences of genomic integration plasmids

[0064]

[0065]

[0066] 2. CRISPR-associated transposases gene editing tool-mediated large fragment multi-copy Escherichia coli genome integration

[0067] (1) Transformation of pTnsABC plasmid into the base strain

[0068] Electroporate the pTnsABC plasmid into the EC carrier strain, resuscitate at 37°C, 200 rpm for 1 hour, plate onto LB agar medium containing kanamycin resistance, and incubate overnight at 37°C. Pick a single colony from the plate into LB liquid medium containing kanamycin resistance and incubate at 37°C, 200 rpm to preserve the EC-pTnsABC glycerol stock.

[0069] (2) Preparation of EC-pTnsABC competent strain and integration of lacto-N-triose II synthesis module into the E. coli genome

[0070] (1) Pipette an appropriate amount of bacterial solution from a tube stored in a -80℃ refrigerator into LB liquid medium containing kanamycin resistance (30μg / mL), culture at 37℃, 200rpm for about 10-12h, then transfer to a 250mL conical flask containing 50mL LB medium at a 1% inoculum volume, culture at 37℃, 200rpm, and when the OD 600 When the pH value is between 0.4 and 0.6, collect the cells and begin the competent phase. Place the cells on ice for 30 minutes (to stop cell growth). Meanwhile, place the sterilized 50mL centrifuge cup, sterile water, and 10% glycerol in a 4°C refrigerator. Pre-cool the centrifuge used for the competent phase. Transfer the bacterial suspension to a 50mL centrifuge cup in a sterile laminar flow hood. Place the centrifuge cup in a pre-cooled centrifuge and centrifuge at 4000 rpm for 10 minutes at 4°C. Discard the supernatant, add an appropriate amount of pre-cooled aqueous solution, pipette repeatedly to mix the cells, centrifuge at 4000 rpm for 10 minutes at 4°C, and wash twice. Discard the supernatant, add an appropriate amount of pre-cooled 10% glycerol solution, pipette repeatedly to mix the cells, centrifuge at 4000 rpm for 10 minutes at 4°C, and wash twice. Add 500 μL of pre-cooled 10% glycerol, pipette and resuspend with a 1 mL pipette to mix thoroughly, dispense into 1.5 mL centrifuge tubes, 100 μL per tube, and store in a -80°C refrigerator for later use.

[0071] (2) Add the pQcascade plasmid and the pDonor-LNT II plasmid to competent cells of the EC-pTnsABC strain and perform electroporation (1 / 2 mm cuvette, 2.5 kV). Immediately after electroporation, add 1 mL of pre-chilled LB medium and resuscitate at 37°C for about 1 hour. Then, spread the cells onto LB agar medium containing kanamycin resistance, ampicillin resistance, and streptomycin resistance and incubate at 37°C overnight. Once a single colony grows, use it for subsequent transposition experiments.

[0072] (3) Continuous transposition to screen for strains with different copy numbers of the genomic integration module of lactoyl-N-triose II

[0073] Scrape the single colony grown from the plate and resuspend it in fresh LB medium. Dilute the cells 10 5 -10 7 The bacterial solution at different dilutions (each dilution is 10) is plated onto LB agar plates containing kanamycin resistance, ampicillin resistance, and streptomycin resistance for transposition expression. The plate is then incubated at 37°C for approximately 16 hours. Once a single colony has grown, a portion of the colony is scraped and the above process is repeated. The remaining colonies are retained for subsequent screening of strains with different copy numbers. The transposition experiment is completed after approximately five transposition cycles. Single colonies that have undergone different transposition cycles are selected for PCR to screen for strains with genomic integration of different copy numbers of the lacto-N-trisaccharide synthesis module.

[0074] Using the primers in Table 2, colony PCR was performed to verify the genomic integration copy number and integration site of the lacto-N-trisaccharide II synthesis module. Figure 2 (a) shows the genome-integrated strains containing 1-4 copies of the lactoyl-N-triose II synthesis module.

[0075] Table 2 Primer sequences for genomic integration site verification

[0076]

[0077]

[0078] 3. Elimination of pTnsABC, pQcascade, pDonor-LNT II, ​​and pCutamp plasmids

[0079] Elimination of pTnsABC, pQcascade, and pDonor-LNT II plasmids:

[0080] Plasmid elimination was performed on the obtained genome-integrated strains with 1-4 copies of the lactoyl-N-triose II synthesis module. First, competent cells of the genome-integrated strains containing different numbers of lactoyl-N-triose II copies were prepared, and the pcutamp plasmid was electroporated into the competent cells. After incubation at 37°C, 200 rpm for about 1 hour, the electroporated bacterial liquid was spread on LB agar medium containing 20 mM rhamnose + apramycin resistance, and placed in a 37°C constant temperature incubator for about 12 hours to obtain single colonies. Subsequently, single colonies were picked with a sterile toothpick and inoculated onto LB agar containing kanamycin resistance, ampicillin resistance, and streptomycin resistance, as well as apramycin resistance plus 20 mM rhamnose. Transformants were incubated upside down at 37°C for approximately 12 hours to observe their sensitivity to kanamycin resistance, ampicillin resistance, and streptomycin resistance. If transformants grew only on LB agar containing apramycin resistance plus 20 mM rhamnose but not on the corresponding positions on LB agar containing kanamycin resistance, ampicillin resistance, or streptomycin resistance, this indicated that the pTnsABC, pQcascade, and pDonor-LNT II / LNnT plasmids had been eliminated. Once these three plasmids were successfully eliminated, the copy number was again determined by colony PCR, which was the final genomic integration copy number.

[0081] pCutamp plasmid elimination:

[0082] The positive transformants that successfully eliminated the pTnsABC, pQcascade, and pDonor-LNT II / LNnT plasmids were transferred to the culture medium containing 15 g·L -1 Incubate the cells in LB liquid medium containing sucrose at 37°C, 200 rpm, for approximately 6 hours. Then, streak a small amount of the bacterial suspension onto LB agar medium containing the same concentration of sucrose. Incubate the cells in an incubator at 37°C, inverted, for 10-12 hours. Once a single colony has grown, use a sterile toothpick to pick a single colony and spot it onto LB agar medium containing apramycin and LB agar without the antibiotic. Observe the transformant's sensitivity to apramycin resistance. If the transformant does not grow on LB agar containing apramycin resistance but does not grow at the corresponding position on LB solid medium without the antibiotic, this indicates that pCutamp has been eliminated.

[0083] Finally, a genome-integrated strain with 1 to 4 copies of the lactoyl-N-triose II synthesis module was obtained, named EC1-4. The shake flask fermentation results were as follows: Figure 2 As shown in (b), the strain EC3 with 3 copies of the lacto-N-triose II synthesis module integrated into the genome had the highest LNT II production, so EC3 was selected for the subsequent genomic integration of the lacto-N-neotetraose synthesis module.

[0084] 4. Genomic integration of the lacto-N-neotetraose synthesis module was performed using the same method as described above. Strain EC3, which had the highest LNT II production, was selected for subsequent genomic integration of the lacto-N-neotetraose synthesis module. Ultimately, strain EC5-10 was obtained, which had both the lacto-N-triose II synthesis module and the lacto-N-neotetraose synthesis module integrated into their genomes. Information on the strains used in this example is shown in Table 3.

[0085] Table 3 Strain information

[0086]

[0087] The results of the integration of the lactoyl-N-neotetraose synthesis module are shown in Figure 3 (a). Shake flask fermentation results are as follows Figure 3 As shown in (b), the strain EC6 with 4 copies of the genomic integration module for lacto-N-neotetraose synthesis had the highest lacto-N-neotetraose production, so this strain was selected for subsequent 3L bioreactor validation.

[0088] Example 2: 3L bioreactor validation of genome-integrated strains

[0089] The strain EC6 with the highest lactoyl-N-neotetraose production was selected for fermentation verification in a 3L bioreactor. The prepared seed solution was inoculated at a 10% inoculum into a 3L bioreactor containing 0.9L fermentation medium. The bioreactor parameters were set as follows: at the initial inoculation stage, culture was performed at 37°C and 300 rpm. When the dissolved oxygen dropped to about 30%, the dissolved oxygen and rotation speed (300-900 rpm) were coupled to control the dissolved oxygen at about 30%. 50% ammonia (v·v) was automatically added to the reactor. -1 ) Control the pH at around 6.8. 600 When the temperature reaches about 30, the culture temperature is set to 25°C, and IPTG is added at a final concentration of 0.1mM. After 2 hours of induction, 5.00g / L lactose is added. The pH value of the culture medium is adjusted with 50% ammonia water to maintain it at about 6.8. Feed medium and 200g / L lactose solution are added to the 3L bioreactor to maintain the growth of the strain and the continuous production of lacto-N-neotetraose. When the fermentation is completed, the maximum yield of lacto-N-neotetraose reaches 30.56g / L ( Figure 4 ).

[0090] Example 3: Construction of overexpression transporter plasmid and shake flask verification

[0091] 1. Plasmid construction

[0092] The steps for constructing the overexpression plasmid using pACYC-mdfA as an example are as follows. The primer sequences involved are shown in Table 2.

[0093] (1) Using the genome of Escherichia coli K-12MG1655 or Bacillus subtilis as a template, the mdfA gene fragment was amplified using the mdfA-F / R primer pair, and the DNA fragment was recovered by gel extraction;

[0094] (2) Using pACYCDuet-1 as a template and the recovered mdfA fragment as a large primer, the recovered mdfA fragment and pACYCDuet-1 were added to the reaction system for PCR. The recovered mdfA fragment was inserted into the pACYCDuet-1 plasmid using the Megawhop method. After the PCR was completed, 8 μL of the PCR amplification product was taken and the template plasmid in the system was eliminated with Dpn I fast-cutting enzyme. The PCR product was then transformed into the competent E. coli JM109. After a single colony grew, a single colony was picked and cultured in liquid LB. The plasmid was extracted and sent for Sanger sequencing for verification. The successfully verified plasmid pACYC-mdfA was stored at -20°C for future use.

[0095] The methods for obtaining the YajR, mdtM, ​​mdfA, ompC, ompF, lamB, YwbF, YuxJ, scrY, PsSET, PcSET, LmrC, BbSET, sotA, YbSET, and CmSET gene fragments and constructing the plasmids pACYC-YajR, pACYC-mdtM, ​​pACYC-mdfA, pACYC-ompC, pACYC-ompF, pACYC-lamB, pACYC-YwbF, pACYC-YuxJ, pACYC-scrY, pACYC-PsSET, pACYC-PcSET, pACYC-LmrC, pACYC-BbSET, pACYC-sotA, pACYC-YbSET, and pACYC-CmSET are the same as above.

[0096] Table 4 Primers for constructing transporter overexpression plasmids

[0097]

[0098]

[0099]

[0100] 2. Shake flask fermentation

[0101] The transporter protein plasmid constructed above and the plasmid sent to the company for gene synthesis were electroporated into the EC6 strain, and 16 strains expressing different transporter proteins were obtained. After shake flask fermentation and HPLC detection, the total production of lactoyl-N-neotetraose and the extracellular production of lactoyl-N-neotetraose were as follows: Figure 5As shown. The results showed that the three transporters with the best secretion effect on lacto-N-neotetraose were BbSET (91.61%), YbSET (85.14%), and scrY (81.33%), which were significantly improved compared to the control secretion rate. However, the production of lacto-N-neotetraose did not improve, and the overexpression of YbSET caused a significant decrease in the production of lacto-N-neotetraose. After co-expression of BbSET and scrY, the production of lacto-N-neotetraose was improved compared to single expression, and the secretion rate was basically the same as that of BbSET. ( Figure 5 ).

[0102] Example 4: 3 L bioreactor validation of transporter overexpression strains

[0103] The strain co-expressing BbSET and scrY was selected for fermentation verification in a 3L tank. The prepared seed solution was inoculated into a 3L bioreactor containing 0.9L fermentation medium at a 10% inoculum volume. The parameters of the bioreactor were set as follows: at the initial inoculation stage, culture was carried out at 37°C and 300 rpm. When the dissolved oxygen dropped to about 30%, the dissolved oxygen and the rotation speed (300-900 rpm) were coupled to control the dissolved oxygen at about 30%. The dissolved oxygen was automatically increased by adding 50% ammonia (v / v -1 ) Control the pH at around 6.8. When OD600 reaches around 30, set the culture temperature to 25°C and add IPTG with a final concentration of 0.1mM. After 2 hours of induction, add 5.00g / L lactose. Use 50% ammonia water to adjust the pH so that the pH value of the culture medium is maintained at around 6.8. Add feed medium and 200g / L lactose solution to the 3L bioreactor to maintain the growth of the strain and the continuous production of lacto-N-neotetraose. At the end of fermentation, the yield of lacto-N-neotetraose is only 24.19g / L ( Figure 6 (a)).

[0104] According to the above fermentation conditions, YbSET with low shake flask yield but high secretion efficiency was selected for tank verification. The highest yield of LNnT was 54.12 g / L, and all the products were transported to the extracellular space in the late fermentation period ( Figure 6 (b)).

[0105] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered Escherichia coli strain that reduces the metabolic burden of cellular production of lactoyl-N-neotetraose, characterized in that: The genetically engineered bacteria uses Escherichia coli as a base strain, and integrates multiple copies of the genes of the following modules a)-b) into the base strain genome through CRISPR-associated transposases gene editing technology: a) Lactoyl-N-triose II synthesis module: β-1,3-N-acetylglucosamine transferase gene lgtA, phosphoglucosamine mutase gene glmM, fusion N-acetylglucosamine-1-phosphate ureyltransferase / glucosamine-1-phosphate acetyltransferase gene glmU, L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS; b) Lactoyl-N-neotetraose synthesis module: β-1,4-galactosyltransferase gene lgtB, phosphoglucomutase gene pgm, UTP-glucose-1-phosphate uridyltransferase gene galU, UDP-glucose 4-epimerase gene galE.

2. The genetically engineered Escherichia coli according to claim 1, characterized in that The copy number of the lacto-N-triose II synthesis module is 1-4 copies, and the copy number of the lacto-N-neotetraose synthesis module is 3-8 copies.

3. The genetically engineered Escherichia coli according to claim 1 or 2, characterized in that The genomic integration site of the lactoyl-N-triose II synthesis module is one or more of wcaJ, ushA, nudK and nagA; The genomic integration site of the lacto-N-neotetraose synthesis module is one or more of yadD-panC, aroG-gpmA, ompW-yciE, purT-eda, tktB-ypfG, yghA-exbD, cspA-hokA and rluF-yjbD.

4. The genetically engineered Escherichia coli according to claim 1 or 2, characterized in that The NCBI sequence number of the amino acid sequence of the β-1,3-acetylglucosamine transferase LgtA is WP_002257440.1, the NCBI sequence number of the amino acid sequence of the β-1,4-galactosyltransferase LgtB is WP_002225824.1, the NCBI accession number of the nucleotide sequence of the phosphoglucomutase gene pgm is ECK0676; the NCBI accession number of the nucleotide sequence of the UTP-glucose-1-phosphate uridyltransferase gene galU is ECK1231; the UDP-glucose The NCBI accession number of the nucleotide sequence of the glucose 4-epimerase gene galE is ECK0748; the NCBI accession number of the nucleotide sequence of the phosphoglucosamine mutase gene glmM is ECK3165; the NCBI accession number of the nucleotide sequence of the L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS is ECK3722; the NCBI accession number of the nucleotide sequence of the fusion N-acetylglucosamine-1-phosphate uridyltransferase / glucosamine-1-phosphate acetyltransferase gene glmU is ECK3723; Optionally, the L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS includes glmS*, which encodes a GlmS mutant GlmS*. The mutation site of the mutant GlmS* is calculated starting from the second amino acid of the GlmS amino acid, and the following mutations are performed: E14K, D386V, S449P, E524G, wherein E14K indicates that the 14th amino acid E mutates to K, D386V indicates that the 386th amino acid D mutates to V, S449P indicates that the 449th amino acid S mutates to P, and E524G indicates that the 524th amino acid E mutates to G.

5. The genetically engineered Escherichia coli according to claim 1 or 2, characterized in that The chassis strain was Escherichia coli BL21(DE3)ΔlacZΔlacAΔnagBΔwecBΔugdΔgcdΔsetA.

6. The genetically engineered Escherichia coli according to claim 1 or 2, characterized in that At the same time, endogenous transport proteins or exogenous transport proteins are overexpressed in the genetically engineered bacteria, wherein the endogenous transport proteins include putative transport protein YajR, drug MFS transport protein MdtM, multidrug efflux pump protein MdfA, outer membrane porin C OmpC, outer membrane porin F OmpF, and maltose outer membrane channel protein LamB, and the exogenous transport protein genes include sugar efflux transport protein PcSET, sugar efflux transport protein PsSET, sugar efflux transport protein BbSET, sugar efflux transport protein A SotA, putative sugar permease YwbF, putative sugar ABC transport protein LmrC, major facilitator superfamily protein MFS_1YbSET, acidic stress-induced putative efflux protein YuxJ, outer membrane porin ScrY, and sugar efflux transport protein cmSET.

7. The genetically engineered Escherichia coli according to claim 6, characterized in that The NCBI accession number of the amino acid sequence of the putative transporter YajR is NP_414961.4; the NCBI accession number of the amino acid sequence of the drug MFS transporter MdtM is NP_418757.1; the NCBI accession number of the amino acid sequence of the multidrug efflux pump protein MdfA is NP_415363.1; the NCBI accession number of the amino acid sequence of the outer membrane porin C OmpC is NP_416719.1; the NCBI accession number of the amino acid sequence of the outer membrane porin F OmpF is NP_415449.1; the NCBI accession number of the amino acid sequence of the maltose outer membrane channel protein LamB is NP_418460.1; The NCBI sequence number of the amino acid sequence of the sugar efflux transporter PcSET is ZP_03829909.1, the NCBI sequence number of the amino acid sequence of the sugar efflux transporter PsSET is BAP78849.1, the NCBI sequence number of the amino acid sequence of the sugar efflux transporter BbSET is KGQ13398.1, the NCBI sequence number of the amino acid sequence of the sugar efflux transporter A is EGC72107.1, the NCBI sequence number of the amino acid sequence of the putative sugar permease is NP_391713.1, and the putative sugar The NCBI sequence number of the amino acid sequence of the ABC transporter is ABX00624.1, the NCBI sequence number of the amino acid sequence of the putative efflux protein induced by acidic stress is NP_391026.2, the NCBI sequence number of the amino acid sequence of the major facilitator superfamily protein MFS_1 is EEQ08298.1, the NCBI sequence number of the amino acid sequence of the outer membrane porin ScrY is AHM80897.1, and the NCBI sequence number of the amino acid sequence of the sugar efflux transporter CmSET is WP_024910347.

1.

8. A method for improving the ability of Escherichia coli to synthesize lactoyl-N-neotetraose by reducing the metabolic burden of Escherichia coli, characterized in that: The method is to overexpress an endogenous transport protein or an exogenous transport protein in Escherichia coli, wherein the endogenous transport protein genes include YajR, mdtM, ​​mdfA, ompC, ompF, and lamB, and the exogenous transport protein genes include PcSET, PsSET, BbSET, sotA, YwbF, LmrC, YuxJ, YbSET, scrY, and CmSET.

9. A method for synthesizing lactoyl-N-neotetraose, characterized in that: The method comprises the steps of using glycerol as a carbon source and utilizing the genetically engineered bacteria according to any one of claims 1 to 7 to perform fermentation to obtain lactoyl-N-neotetraose.

10. Use of the genetically engineered bacterium according to any one of claims 1 to 7, or the method according to claim 8, or the method according to claim 9 in the preparation of lactoyl-N-neotetraose or a product containing lactoyl-N-neotetraose.

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