Lactose-free genetically engineered bacteria for efficient production of lactosyl-n-neotetraose and application thereof

Abstract

The application discloses a gene engineering bacterium for efficiently producing lactosyl-N-neotetraose without lactose addition and application thereof, and belongs to the field of metabolic engineering and food fermentation. The CRISPR / Cas9 gene editing technology is adopted to transform a chassis microorganism, the synthesis pathway of lactosyl-N-neotetraose is restructured in a lactose synthesis strain, the key enzyme of a de novo synthesis pathway is up-regulated, the byproduct pathway is weakened, the repression of a repressor protein is released, the extracellular output of a product is enhanced, and the construction of the gene engineering bacterium without antibiotics and the efficient synthesis of lactosyl-N-neotetraose are realized. The recombinant Escherichia coli obtained in the application can efficiently synthesize lactosyl-N-neotetraose by using glycerol and lactose, no antibiotic is added in the fermentation process, the product is safe and harmless, the production cost is low, the product production level is high, the social and economic benefits are strong, and the market development prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to a lactose-free genetically engineered bacterium for efficiently producing lacto-N-neotetraose and its application, and belongs to the field of metabolic engineering and food fermentation. BACKGROUND

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in human milk after lactose and fat, and the content of oligosaccharides in breast milk is 5-15 g / L, which is 100-300 times that of cow's milk. In recent years, with the rapid development of the food industry, the development of functional oligosaccharides has become an important issue in the international biotechnology field, and the oligosaccharide industry has become an important emerging industry applied in food, feed, medicine, chemical industry and other industries.

[0003] Lacto-N-neotetraose (LNnT) is one of the important components of human milk oligosaccharides, which belongs to non-fucosylated neutral breast milk oligosaccharides, and its content in breast milk is about 0.5 g / L. It has important biological functions such as enhancing human immunity, regulating intestinal flora and promoting cell maturation. It has been approved by the European Food Safety Authority (EFSA), the European Union (EU) and the U.S. Food and Drug Administration (FDA) as a nutritional fortifier to be added to infant formula.

[0004] At present, the commercial production of LNnT mainly includes chemical synthesis and biological synthesis. The biological synthesis method only needs cheap carbon source and intracellular renewable donor as raw materials, and obtains high economic output with low environmental cost, so it has more broad application prospects. LNnT (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) is a linear tetrasaccharide composed of D-galactose, N-acetylglucosamine and D-lactose in turn, and its synthesis pathway is relatively complex. At present, there are still some deficiencies in the biological synthesis method, which limit the efficient synthesis of products, such as insufficient supply of intracellular precursors, imbalance of metabolic flow between synthesis pathway and competitive pathway, and excessive metabolic pressure which may affect the genetic stability of the strain.

[0005] Through green biological process, high-value-added products are produced by using cheap biomass, which is an important way to realize carbon neutralization and environment-friendly economy. The development of green, efficient and safe chassis microorganisms is the key to solve the large-scale production and application of HMOs. With the development of metabolic engineering and synthetic biology, the current lacto-N-neotetraose production strain is commonly used in Escherichia coli, and there are few reports and low yield. The production of lacto-N-neotetraose requires the supply of exogenous lactose, and the cost of lactose raw material is high. Developing new technology for lactose synthesis in microorganisms can solve the problem of high cost of lactose in the process of efficient biosynthesis of lacto-N-neotetraose.

[0006] The present application aims to utilize synthetic biology means to construct an antibiotic-free or antibiotic-added lactosyl-N-neotetraose production strain by eliminating byproduct pathways and competitive inhibition pathways, to realize the construction of an engineering strain without lactose addition and the efficient synthesis of lactosyl-N-neotetraose through the construction and combination of key genes in the LNnT pathway, the up-regulation of key enzymes in the de novo synthesis pathway, and the enhancement of the extracellular output of the product. SUMMARY

[0007] [PROBLEMS]

[0008] The prior art has low efficiency in synthesizing lactosyl-N-neotetraose, and the expensive acceptor lactose cannot provide a strain for safe and efficient production of lactosyl-N-neotetraose, nor can it provide a low-cost and environmentally friendly preparation method for lactosyl-N-neotetraose.

[0009] [TECHNICAL SCHEME]

[0010] The present application provides a genetically engineered bacterium for efficiently producing lactosyl-N-neotetraose and a construction method thereof to solve the problems of expensive acceptor lactose and low product yield in the synthesis of lactosyl-N-neotetraose.

[0011] The first object of the present application is to provide a recombinant Escherichia coli, wherein the recombinant Escherichia coli is genetically edited with Escherichia coli BL21 (DE3) as a host, and the recombinant Escherichia coli is (a) or (b):

[0012] (a) knocking out the beta-galactosidase gene lacZ and the UDP-N-acetylglucosamine-2-epimerase gene wecB on the genome; knocking out the glucose-specific transport proteinase EIIABC Glc assembly coding gene crr and ptsG, and integrating setA and glf at the sites of crr and ptsG, respectively; knocking out the pantothenate-dependent pyruvate dehydrogenase gene poxB on the genome, and integrating the beta-1,3-N-acetylglucosamine amino transferase gene lgtA at the site; knocking out the phosphotransacetylase and acetate kinase gene cluster pta-ackA on the genome, and integrating the glutamine-fructose-6-phosphate aminotransferase gene glmS at the site; knocking out the formate cleavage enzyme gene pflB on the genome, and integrating the N-acetylglucosamine-1-phosphate uracil transferase gene glmU at the site; knocking out the UDP-lactose-6-dehydrogenase gene ugd on the genome, and integrating the uridine diphosphate glucose-4-epimerase gene galE at the site; knocking out the lactose kinase gene glk on the genome, and integrating the beta-1,4-galactosyltransferase gene NmlgtB derived from Neisseria meningitidis at the site;

[0013] (b) On the basis of (a), the expression genes of glmS, galE, lgtA and NmlgtB are free.

[0014] In an embodiment, the recombinant Escherichia coli expresses the genes of glmS and galE with pETDuet-1 plasmid, and expresses the genes of lgtA and NmlgtB with pRSFDuet-1 plasmid.

[0015] In an embodiment, the NCBI accession number of the β-galactosidase gene lacZ is NP_414878.1, and the NCBI accession number of the UDP-N-acetylglucosamine-2-epimerase gene wecB is YP_026253.1.

[0016] In an embodiment, the glucose-specific transport protein enzyme EIIABC Glc The component encoding genes include crr and ptsG, the Gene ID of crr is 946880, and the Gene ID of ptsG is 945651.

[0017] In an embodiment, the glucose transport protein gene glf is derived from Zymomonas mobilis, and the sugar efflux transport protein gene setA is derived from Yersinia bercovieri ATCC 43970, and the nucleotide sequences thereof are SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0018] In an embodiment, the β-1, 3-N-acetylglucosamine amino transferase gene lgtA and the β-1, 4-galactosyltransferase NmlgtB gene are derived from Neisseriaceae meningitidis, and the nucleotide sequences thereof are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0019] In an embodiment, the Gene ID of the ubiquinone-dependent pyruvate dehydrogenase gene poxB is 946132, the Gene ID of the phosphate acetyltransferase gene pta is 946778, the Gene ID of the acetate kinase gene ackA is 946775, the Gene ID of the formate cleavage enzyme gene pflB is 945514, the Gene ID of the UDP-glucose-6-dehydrogenase gene ugd is 946571, the Gene ID of the glucose kinase gene glk is 946858, the Gene ID of the uridine diphosphate glucose-4-epimerase gene galE is 945354, the Gene ID of the glutamine-fructose-6-phosphate aminotransferase gene glmS is 948241, and the Gene ID of the N-acetylglucosamine-1-phosphate uracil transferase gene glmU is 948246.

[0020] In one embodiment, the β-1, 3-N-acetylglucosamine aminotransferase gene lgtA, glutamine-fructose-6-phosphate aminotransferase gene glmS, N-acetylglucosamine-1-phosphate uracil transferase gene glmU, uridine diphosphate glucose-4-epimerase gene galE, β-1, 4-galactosyltransferase gene NmlgtB, setA and glf are all expressed using the promoter T7.

[0021] A second object of the present application is to provide a method for producing lacto-N-neotetraose, which is producing lacto-N-neotetraose in a fermentation system using glycerol and glucose as carbon sources with the recombinant E. coli as fermentation strain.

[0022] In one embodiment, the lacto-N-neotetraose is produced by fermentation in a shake flask or a fermenter.

[0023] In one embodiment, the recombinant E. coli is inoculated into a fermentation medium, and glycerol with a final concentration of 20-30 g / L and glucose with a final concentration of 5-10 g / L are added at the beginning of fermentation, and the shake flask culture is carried out at 30-40 °C and 150-250 rpm for 40-60 h.

[0024] In one embodiment, the recombinant E. coli is inoculated into a fermentation medium containing a fermentation medium in a fermenter, and initial fermentation is carried out, and after the initial carbon source is consumed, the carbon source is fed.

[0025] In one embodiment, the recombinant E. coli is cultured at 20-40 °C, and the dissolved oxygen in the fermentation system is maintained at 30±5%, and the pH is maintained at 6.5-7.0.

[0026] In one embodiment, glycerol is fed after the initial glycerol is consumed, so that the concentration of glycerol is 2-3 g / L.

[0027] In one embodiment, glucose is supplemented after the initial glucose is consumed, so that the concentration of glucose is maintained at 10±0.5 g / L.

[0028] In one embodiment, the fermentation medium contains glycerol 20-30 g / L, glucose 5-10 g / L, potassium dihydrogen phosphate 10-15 g / L, citric acid 1-2 g / L, dihydrogen phosphate 3-5 g / L, magnesium sulfate heptahydrate 1-2 g / L, yeast extract 8-10 g / L, trace metal solution 8-10 mL / L.

[0029] In an embodiment, the trace metal solution contains 8-10 g / L ferric citrate, 2-3 g / L magnesium sulfate heptahydrate, 0.5-1.0 g / L copper sulfate pentahydrate, 0.2-0.5 g / L manganese sulfate monohydrate, 0.2-0.5 g / L borax, 0.1-0.2 g / L ammonium molybdate, and 1-2 g / L calcium chloride dihydrate.

[0030] The application also provides the use of the recombinant E. coli in the production of a product containing lactosyl-N-neotetraose.

[0031] The application has the following beneficial effects:

[0032] The application introduces a biosynthetic pathway of lactosyl-N-neotetraose into E. coli, and realizes the construction of an antibiotic-free strain and the efficient synthesis of lactosyl-N-neotetraose without the addition of exogenous lactose by weakening the byproduct pathway, regulating the central carbon metabolism, up-regulating the key enzymes of the de novo synthesis pathway, relieving the repression inhibition of the repressor protein, and enhancing the extracellular output of the product. The antibiotic-free genetically engineered strain EL6 constructed in the application has the ability to produce lactosyl-N-neotetraose of 1.168 g / L under the condition of shake flask fermentation without the addition of lactose, and the yield of lactosyl-N-neotetraose reaches 15.42 g / L in a 3L fermenter. Meanwhile, the antibiotic-containing genetically engineered strain EL7 constructed in the application has the ability to produce lactosyl-N-neotetraose of 3.41 g / L under the condition of shake flask fermentation without the addition of lactose, and the yield of lactosyl-N-neotetraose reaches 30.05 g / L in a 3L fermenter, which lays a foundation for the industrial production of lactosyl-N-neotetraose. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 A schematic diagram of the metabolic process for producing lactosyl-N-neotetraose by using glucose and glycerol as substrates.

[0034] Figure 2 A yield comparison diagram of the engineering strain for weakening the byproduct pathway and introducing the lactosyl-N-neotetraose pathway.

[0035] Figure 3 A yield comparison diagram of the engineering strain for combining and optimizing the LNnT pathway lactosyl-N-neotetraose pathway.

[0036] Figure 4 A shake flask fermentation of the strain EL7.

[0037] Figure 5 Fed-batch fermentation of the antibiotic-free strain EL6 in a 3L fermenter.

[0038] Figure 6 Fed-batch fermentation of the antibiotic-containing strain EL7 in a 3L fermenter. DETAILED DESCRIPTION

[0039] The following further describes the specific implementation of the present application in combination with examples and drawings. The plasmids, PCR reagents, restriction enzymes, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products, and the specific operations are performed according to the kit instructions.

[0040] The embodiments of the present application are not limited thereto, and other experimental operations and process parameters not mentioned are performed according to conventional techniques.

[0041] The sequencing of DNA products and plasmids is completed by Tianlin Biotechnology (Wuxi) Co., Ltd.

[0042] Preparation of E. coli competence: kit from Shanghai Shenguo Bioengineering Co., Ltd.

[0043] Plasmids involved in the following examples:

[0044] pCOLADuet-1, pACYCDuet-1, pCDFDuet-1, pETDuet-1 are from laboratory preservation and purchased from Shanghai Bai Feng Biotechnology Co., Ltd.; vectors pCas9 and pTargetF are purchased from Addgene.

[0045] Culture media involved in the following examples:

[0046] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0047] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar powder.

[0048] Fermentation medium: glycerol 20 g / L, glucose 10 g / L, potassium dihydrogen phosphate 13.5 g / L, citric acid 1.7 g / L, dihydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 1.4 g / L, yeast extract 10 g / L, trace metal solution 10 mL / L (ferric citrate 10 g / L, magnesium sulfate heptahydrate 2.25 g / L, copper sulfate pentahydrate 1.0 g / L, manganese sulfate monohydrate 0.35 g / L, borax 0.23 g / L, ammonium molybdate 0.11 g / L, calcium chloride dihydrate 2.0 g / L), pH 6.8.

[0049] Methods in the following examples:

[0050] (1) Determination of lactyl-N-neotetraose using HPLC: 1 mL of fermentation broth is boiled at 100°C for 10 min, centrifuged at 12000 r / min for 5 min, and the supernatant is filtered through a 0.22 μm membrane. The generation amount of lactyl-N-neotetraose and the consumption amount of lactose and glycerol are detected by HPLC.

[0051] HPLC detection conditions: differential refractive index detector; the chromatographic column was Rezex ROA-organic acid (Phenomenex, USA), the column temperature was 50 DEG C; the mobile phase was 0.005 mol / L H2SO4 aqueous solution, the flow rate was 0.6 mL / min; the injection volume was 10 μL.

[0052] (2) Shake flask fermentation of antibiotic-free strain: the single colony of the engineering bacteria was inoculated into LB liquid medium, 37 DEG C, 200 rpm, shake flask culture for 12 h to obtain seed liquid; the seed liquid was inoculated into 50 mL fermentation medium at 2% (v / v) inoculation amount, and cultured at 37 DEG C, 200 rpm for 48 h.

[0053] (3) Shake flask fermentation of antibiotic-containing strain: the single colony of the engineering bacteria was inoculated into LB liquid medium, 37 DEG C, 200 rpm, shake flask culture for 12 h to obtain seed liquid; the seed liquid was inoculated into 50 mL fermentation medium at 2% (v / v) inoculation amount, and the corresponding antibiotic was added, 37 DEG C, 200 rpm, shake flask culture until OD 600 0.6, IPTG was added to a final concentration of 0.4 mM, the temperature was changed to 25 DEG C, 200 rpm, and the culture was induced for 48 h.

[0054] Example 1: Elimination of byproduct pathway and competitive pathway inhibition, construction of antibiotic-free lactyl-N-neotetraose production strain

[0055] The synthesis of lactyl-N-neotetraose requires the supply of lactose, in order to prevent metabolic overflow of the glycolytic pathway, the byproduct pathway is weakened and the lactyl-N-neotetraose synthesis pathway is introduced. Using E. coli BL21 (DE3) as the host bacteria, the CRISPR-Cas9 gene editing system was used to knock out the β-galactosidase gene lacZ and the UDP-N-acetylglucosamine-2-epimerase gene wecB of the engineering bacteria, obtaining EL0 E. coli BL21 (DE3) ΔlacZ ΔwecB; on the basis of EL0 E. coli BL21 (DE3) ΔlacZ ΔwecB, the glucose-specific transport protein enzyme EIIABC Glc assembly encoding gene crr and ptsG, and setA and glf were integrated at the crr and ptsG sites respectively, obtaining EL1 E. coli BL21 (DE3) ΔlacZ ΔwecB, ΔptsG::P T7 -glf, Δcrr::P T7 -setA.

[0056] EL1 E. coli BL21 (DE3) ΔlacZ ΔwecB, ΔptsG::P T7 -glf, Δcrr::P T7-setA is a starting strain, the ubiquinone-dependent pyruvate dehydrogenase gene poxB is knocked out by using the CRISPR-Cas9 gene editing system, and the β-1, 3-N-acetylglucosamine amino transferase gene lgtA is integrated at the site; the phosphotransferase and acetate kinase gene cluster pta-ackA is knocked out, and the glutamine-fructose-6-phosphate aminotransferase gene glmS is integrated at the site; the formate cleavage enzyme gene pflB is knocked out, and the N-acetylglucosamine-1-phosphate uracil transferase gene glmU is integrated at the site; the UDP-lactose-6-dehydrogenase gene ugd is knocked out, and the uridine diphosphate glucose-4-epimerase gene galE is integrated at the site; finally, the lactose kinase gene glk is knocked out, and the β-1, 4-galactosyltransferase NmlgtB is integrated at the site.

[0057] The metabolic pathway of lactyl-N-neotetraose with glycerol and glucose as substrates in the lactose-producing strain is as shown in Figure 1 The specific steps of gene knockout and integration are as follows (the primer sequences involved are shown in Table 1):

[0058] (1) Taking the example of knocking out poxB and integrating the β-1, 3-N-acetylglucosamine amino transferase gene lgtA gene in the chromosome, the specific target gRNA (20 bp) of poxB gene is found through http: / / www.regenome.net / cas-offinder, the upstream and downstream primers poxB-gRNA-F / poxB-gRNA-R are used to amplify the product by PCR using pTargetF plasmid (Addgene: #62226) as the template, and the amplification product is digested with restriction endonuclease Dpn I to remove the excess circular plasmid pTargetF. Then the amplification product is transformed into E. coli DH5α competent cells, the plasmid is extracted, and the successful knockout plasmid is named pTargetF-poxB through sequencing identification.

[0059] (2) Using the E. coli EL1 strain genome as the template, three sequence fragments are amplified by using the upstream homologous arm primers poxB-US-F / poxB-US-R, the middle homologous arm primers lgtA-MS-F / lgtA-MS-R and the downstream homologous arm primers poxB-DS-F / poxB-DS-R, respectively, and the product is purified and recovered, and then the SOE-PCR method is used to connect the three fragments to obtain the gene homologous repair template by using the primers poxB-US-F / poxB-DS-R.

[0060] (3) Take pCas9 plasmid (Addgene: #62225) and E. coli EL1 electrotransformation competent cells, place on ice for 5 min, then thaw the competent cells. Take 10 μL plasmid and add to 100 μL competent cells, mix gently. Transfer the plasmid and electrotransformation competent cells into a pre-cooled electrotransformation cup, 2.5 kV shock for 5 ms. After shocking, quickly add pre-cooled liquid LB, mix gently, then transfer the mixed medium containing plasmid and competent cells to a new centrifuge tube for expansion culture for 1.5 h. Centrifuge at 6000 r / min for 2 min, discard the supernatant, and spread the bacteria on LB plates containing kanamycin, and place in a 30°C incubator for overnight culture.

[0061] (4) Pick a single colony of E. coli EL1 / pCas9 in LB medium, 30°C for 1.0 h, add L-arabinose to a final concentration of 30 mM to induce the expression of the λ-red system. When the OD 600 reaches 0.6-0.8, prepare E. coli EL1 / pCas9 competent cells.

[0062] (5) Electrotransform 500 ng of the targeting plasmid pTargetF with poxB-specific target gRNA (20 bp) constructed in step (1) and 1000 ng of the homologous repair template constructed in step (2) into the E. coli EL1 / pCas9 competent cells prepared in step (4), and spread on LB plates (kanamycin and spectinomycin), 30°C for 16-24 h. Perform colony PCR on the single colonies grown on the plates to verify positive transformants and perform gene sequencing.

[0063] (6) Eliminate pTargetF-poxB and pCas9 plasmids from the verified single colonies. Inoculate the single colonies in LB liquid medium (kanamycin resistance) and culture at 30°C, 200 r / min to the logarithmic growth phase. Add IPTG to a final concentration of 0.5 mmol / L and incubate overnight to induce the inactivation of pTargetF-poxB plasmid. Streak the bacterial solution on LB plates containing kanamycin and incubate at 30°C, 200 r / min for 12 h. Spot the single colonies on plates with double resistance to kanamycin and spectinomycin. If no colonies grow, it indicates that the pTargetF-poxB plasmid has been successfully eliminated.

[0064] (7) The pCas9 plasmid is a temperature-sensitive plasmid. The single colony of the pTargetF-poxB plasmid successfully eliminated is transferred to LB liquid medium without resistance, and the pCas9 plasmid is eliminated by subculture at 42°C. After streaking the bacterial liquid on the LB plate without resistance and incubating at 37°C, the single colony is spotted on the LB medium containing kanamycin. If the single colony does not grow, it indicates that the pCas9 plasmid is successfully eliminated. The constructed gene deletion strain without the pTargetF-poxB plasmid and the pCas9 plasmid is stored at -80°C for standby.

[0065] (8) The knockout of the genes lacZ and wecB is performed according to the above steps. The knockout of the genes poxB, pta-ackA, pflB, ugd and glk and the integration of the corresponding lgtA, glmS, glmU, galE and NmlgtB are performed according to the above steps. The construction steps of the gene editing strains involved in other embodiments are all referred to Example 1.

[0066] Table 1. Gene knockout and integration primers

[0067]

[0068]

[0069]

[0070] In the recombinant engineering bacteria, the accumulation of glycolysis pathway byproducts is not only toxic to cell growth, but also competes with carbon source materials for the synthesis of lactyl-N-trisaccharide. In order to strengthen the synthesis of the precursor lactyl-N-trisaccharide, the present application blocks the generation of byproducts formic acid and acetic acid through gene knockout. By regulating five enzymes that may be involved in the competing pathways, the efficient production of lactyl-N-neotetraose in the engineering Escherichia coli is further realized. The engineering strains constructed in this embodiment are shown in Table 2. The synthesis ability of lactyl-N-neotetraose, the biomass of the bacterial cells and the accumulation of byproducts of the engineering strains are detected. The results show that after the deletion of poxB and the introduction of lgtA gene, the lactyl-N-neotetraose production strain is preliminarily constructed. The lactyl-N-neotetraose titer of the EL2 strain is 0.23 g / L, and there is 3.15 g / L of residual lactose in the fermentation supernatant. After removing all the glycolysis pathway byproducts, no accumulation of acetic acid and formic acid is detected during the fermentation of the recombinant strain. The strain EL6 shows higher biomass, and the lactose synthesis yield reaches 2.40 g / L. In addition, the extracellular output content of lactyl-N-neotetraose reaches 0.73 g / L. Figure 2 The above indicates that weakening the glycolysis pathway makes more carbon flow to the lactyl-N-neotetraose and lactose synthesis pathways, which is beneficial to the efficient biosynthesis of lactyl-N-neotetraose.

[0071] Table 2. Detailed information of the engineered bacteria (without antibiotic resistance) of genomic integrated lactyl-N-neotetraose metabolic pathway

[0072]

[0073] Example 2: Construction of recombinant plasmid of de novo synthesis pathway of lactyl-N-neotetraose

[0074] The specific steps of constructing the recombinant plasmid are as follows (the primer sequences involved are shown in Table 3):

[0075] (1) Obtaining of galE and glmS gene fragments: Taking the genome of E. coli K12 as the template, and taking galE-F / galER and glmS-F / glmS-R as primers, the galE and glmS gene fragments were amplified respectively, and the DNA fragments were recovered by gel. The recovered galE and glmS gene fragments were connected to the BamHI / Sai I and Bgi II / Xho I enzyme digestion sites of the vector pACYDuet-1 by the seamless cloning kit (Nanjing Novozyme Life Science Co., Ltd.) respectively, and finally the plasmid pACY-glmS-galE was obtained. Taking the plasmid pACY-glmS-galE as the template, the glmS-galE gene fragment was amplified by using the primers glmS-galE-F / glmS-galE-R (the primer sequences are shown in Table 3), and the DNA fragment was recovered by gel. The recovered glmS-galE gene fragment was connected to the Bgi II / Xho I enzyme digestion sites of the vectors pETYCDuet-1, pCDFDuet-1 and pRSFDuet-1 by the seamless cloning kit (Nanjing Novozyme Life Science Co., Ltd.) respectively, and the plasmids pET-glmS-galE, pCDF-glmS-galE and pRSF-glmS-galE were obtained.

[0076] (2) Obtaining of lgtA and NmlgtB gene fragments: lgtA gene sequence derived from Neisseria meningitidis was synthesized by Tianlin Biotech (Shanghai) Co., Ltd. The synthesized lgtA gene fragment was ligated into the BamH I / Sai I enzyme cutting sites of the vector pETFDuet-1 by using a seamless cloning kit (Nanjing Novozyme Life Science Co., Ltd.) to obtain plasmid pET-lgtA. Then, the NmlgtB gene fragment was ligated into the Bgi II / Xho I enzyme cutting sites of the vector plasmid pET-lgtA, and the final obtained plasmid was pET-lgtA-NmlgtB. The lgtA-NmlgtB gene fragment was amplified from the plasmid pET-lgtA-NmlgtB by using primers lgtA-NmlgtB-F / lgtA-NmlgtB-R (the primer sequences are shown in Table 3), and the DNA fragment was recovered by using a gel recovery kit. The recovered lgtA-NmlgtB gene fragment was ligated into the Bgi II / Xho I enzyme cutting sites of the vectors pACYCDuet-1, pCDFDuet-1 and pRSFDuet-1 by using a seamless cloning kit (Nanjing Novozyme Life Science Co., Ltd.) to obtain plasmids pAC-lgtA-NmlgtB, pCDF-lgtA-NmlgtB and pRSF-lgtA-NmlgtB.

[0077] Table 3. Primers for plasmid construction

[0078]

[0079] Example 3: Construction, combination and fine-tuning of modular LNnT pathway key genes

[0080] The plasmids expressing the glmS-galE gene fragment obtained in Example 2 (pCDF-glmS-galE, pACY-glmS-galE, pRSF-glmS-galE, pET-glmS-galE) and the plasmids expressing the lgtA-NmlgtB gene fragment (pAC-lgtA-NmlgtB, pCDF-lgtA-NmlgtB, pRSF-lgtA-NmlgtB, pET-glmS-galE) were combined and transformed into the ELO Escherichia coli BL21(DE3)AlacZAwecB constructed in Example 1, to obtain 12 different antibiotic-resistant strains, denoted as SA1 to SA12 (Table 4). The yields of lacto-N-neotetraose after shake flask fermentation of the 12 different antibiotic-resistant strains were 1.168 g / L, 0.423 g / L, 0.693 g / L, 0 g / L, 0.645 g / L and 0.452 g / L, 0.365 g / L, 0.420 g / L, 0.463 g / L, 0.105 g / L, 0.243 g / L and 0 g / L, respectively. The highest yield of 1.168 g / L was obtained by the engineered bacteria containing the recombinant plasmids pET-glmS-galE and pRSF-lgtA-NmlgtB (i.e. strain SA1) (see Table 4). Figure 3 ).

[0081] Table 4. Detailed information of the engineered bacteria constructed by modular combination of key genes of the lacto-N-neotetraose metabolic pathway

[0082]

[0083]

[0084] Example 4: Fermentation of the combination of plasmids and genome-antibiotic-resistant strains

[0085] The optimal plasmid combination pET-glmS-galE and pRSF-lgtA-NmlgtB selected in Example 2 was transformed into EL6 constructed in Example 3 to produce strain EL7. The constructed engineered bacteria EL7 was inoculated into LB liquid medium, and cultured at 37°C, 200 rpm in a shake flask for 12 h to obtain a seed solution; then the seed solution was inoculated into 50 mL fermentation medium at a seeding amount of 2 mL / 100 mL, and cultured at 37°C, 200 rpm until the OD 600 was 0.6; IPTG was added to a final concentration of 0.4 mM, and glucose was added to a glucose concentration of 10 g / L, and the culture was induced at 25°C, 200 rpm for 48 h.

[0086] Fermentation medium: glycerol 20 g / L, glucose 10 g / L, potassium dihydrogen phosphate 13.5 g / L, citric acid 1.7 g / L, dihydrogen phosphate 4.0 g / L, magnesium sulfate heptahydrate 1.4 g / L, yeast extract 10 g / L, trace metal solution 10 mL / L (ferric citrate 10 g / L, magnesium sulfate heptahydrate 2.25 g / L, copper sulfate pentahydrate 1.0 g / L, manganese sulfate monohydrate 0.35 g / L, borax 0.23 g / L, ammonium molybdate 0.11 g / L, calcium chloride dihydrate 2.0 g / L), pH 6.8.

[0087] Fermentation broth was sampled at regular intervals and the OD of the bacteria was measured 600 1 mL of fermentation broth was boiled for 15 min to completely break the cells, centrifuged at 12000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm membrane. During the fermentation process, the production of lactosyl-N-neotetraose and the consumption of glucose and glycerol were detected by HPLC. Figure 4 The results showed that the concentration of extracellular lactosyl-N-neotetraose reached 3.41 g / L after fermentation (total fermentation time 48 h).

[0088] Table 5. Detailed information of the genome-integrated plasmid lactosyl-N-neotetraose metabolic pathway engineering bacteria

[0089]

[0090] Example 5: Batch feeding production of lactosyl-N-neotetraose in a 3L fermenter

[0091] In order to produce high-yield lactosyl-N-neotetraose, high-density fed-batch fermentation was carried out in a 3L fermenter using antibiotic-free strain EL6 and antibiotic-containing strain EL7, wherein the fermentation medium of antibiotic-free strain EL6 did not contain antibiotics and did not require IPTG induction, and the fermentation medium of antibiotic-containing strain EL7 should contain antibiotics and IPTG with a final concentration of 0.4 mM.

[0092] Fermentation conditions: 50 mL of overnight seed liquid was inoculated into 1 L of fermentation medium, the culture temperature was 37℃, the initial concentrations of glycerol and glucose were 20 and 10 g / L, and NH4OH was used to control the tank pH to be constant at 6.80 during the whole fermentation. In order to maintain the growth of the bacteria and the synthesis of lactosyl-N-neotetraose, after the initial glycerol was consumed, glycerol with a concentration of 800 g / L (containing 20 g / L of MgSO4·7H2O) was added to supplement the carbon source, and the glycerol concentration in the fermentation system was maintained at a low concentration level (glycerol was used for bacterial growth and metabolism, and the concentration was about 2-3 g / L) by pH feedback regulation (the flow rate was set to 20 mL / h) until the end of fermentation. After the initial glucose was consumed, glucose with a concentration of 300 g / L was manually added to maintain the final concentration of glucose in the fermentation system at about 10±0.5 g / L, and if the glucose consumption was low during the fermentation, the glucose was continuously added until the end of fermentation. During the fermentation process, the system was cascadedly controlled by adjusting the rotation speed, the aeration amount and the oxygen to make the dissolved oxygen in the tank to be 30±5%.

[0093] Fermentation whole-time sampling and determination of bacterial OD 600 , 1 mL of fermentation liquid was boiled for 15 min to break the cells completely, centrifuged at 12000 r / min for 10 min, the supernatant was filtered through a 0.22 μm membrane, and the generation amounts of lactose and lactosyl-N-neotetraose and the consumption amounts of glucose and glycerol were detected by HPLC during the fermentation process. Figure 5 and Figure 6 The results showed that the product lactose was maintained at 6-10 g / L during the fermentation process, and after the fermentation (a total of 64 hours), the abilities of the lactose-free genetically engineered bacteria EL6 and the lactose-free genetically engineered bacteria EL7 to produce lactosyl-N-neotetraose without adding lactose were 15.42 g / L and 30.05 g / L, respectively.

[0094] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A recombinant Escherichia coli strain, characterized in that, The recombinant E. coli is genetically edited with E. coli BL21 (DE3) as a host, and the recombinant E. coli is (a) or (b): (a) Knocking out β-galactosidase gene lacZ and UDP-N-acetylglucosamine-2-epimerase gene wecB on the genome; knocking out glucose-specific transporter enzyme EIIABC on the genome Glc Knocking out β-galactosidase gene lacZ and UDP-N-acetylglucosamine-2-epimerase gene wecB on the genome; knocking out glucose-specific transporter enzyme EIIABC on the genome Knocking out β-galactosidase gene lacZ and UDP-N-acetylglucosamine-2-epimerase gene wecB on the genome; knocking out glucose-specific transporter enzyme EIIABC on the genome Knocking out β-galactosidase gene lacZ and UDP-N-acetylglucosamine-2-epimerase gene wecB on the genome; knocking out glucose-specific transporter enzyme EIIABC on the genome Knocking out β-galactosidase gene lacZ and UDP-N-acetylglucosamine-2-epimerase gene wecB on the genome; knocking out glucose-specific transporter enzyme EIIABC on the genome Knocking out β-galactosidase gene lacZ and UDP-N-acetylglucosamine-2-epimerase gene wecB on the genome; knocking out glucose-specific transporter enzyme EIIABC on the genome (b) On the basis of (a), the free expression genes are glmS, galE, lgtA and NmlgtB; The glucose transporter gene glf is derived from Z. mobilis, the sugar efflux transporter gene setA is derived from Yersinia bercovieri ATCC 43970, and the nucleotide sequences thereof are SEQ ID NO. 1 and SEQ ID NO. 2, respectively; the β-1,3-N-acetylglucosamine amino transferase gene lgtA and the β-1,4-galactosyltransferase NmlgtB gene are derived from Neisseriaceae meningitidis, and the nucleotide sequences thereof are shown as SEQ ID NO. 3 and SEQ ID NO. 4, respectively; The Gene ID of the uridine diphosphate glucose-4-epimerase gene galE is 945354, the Gene ID of the glutamine-fructose-6-phosphate aminotransferase gene glmS is 948241, and the Gene ID of the N-acetylglucosamine-1-phosphate uronic acid transferase gene glmU is 948246. The recombinant E. coli expresses the genes glmS and galE with pETDuet-1 plasmid, and expresses the genes lgtA and NmlgtB with pRSFDuet-1 plasmid.

2. The recombinant E. coli of claim 1, wherein, The NCBI accession number of the β-galactosidase gene lacZ is NP_414878.1, the NCBI accession number of the UDP-N-acetylglucosamine-2-epimerase gene wecB is YP_026253.1, and the glucose-specific transporter enzyme EIIABC Glc The component encoding genes include crr and ptsG, the Gene ID of crr is 946880, and the Gene ID of ptsG is 945651.

3. The recombinant E. coli of claim 1, wherein, The Gene ID of the ubiquinone-dependent pyruvate dehydrogenase gene poxB is 946132, the Gene ID of the phosphate acetyltransferase gene pta is 946778, the Gene ID of the acetate kinase gene ackA is 946775, the Gene ID of the formate cleavage enzyme gene pflB is 945514, the Gene ID of the UDP-glucose-6-dehydrogenase gene ugd is 946571, and the Gene ID of the glucose kinase gene glk is 946858.

4. The recombinant E. coli of claim 1, wherein, The β-1,3-N-acetylglucosamine amino transferase gene lgtA, the glutamine-fructose-6-phosphate aminotransferase gene glmS, the N-acetylglucosamine-1-phosphate uronic acid transferase gene glmU, the uridine diphosphate glucose-4-epimerase gene galE, the β-1,4-galactosyltransferase gene NmlgtB, setA and glf are all expressed by using the promoter T7.

5. A method for producing lactosyl-N-neotetraose, characterized by, The method is to use the recombinant E. coli of any one of claims 1-4 as a fermentation strain to produce lactosyl-N-neotetraose in a fermentation system with glycerol or glucose as a carbon source.

6. The method of claim 5, wherein, The recombinant E. coli of any one of claims 1-4 is inoculated into a fermenter containing a fermentation medium to perform initial fermentation, and after the initial carbon source is consumed, the carbon source is added; the carbon source is glycerol or glucose.

7. The method of claim 6, wherein, After the initial glycerol is consumed, glycerol is added to make the concentration of glycerol 2-3 g / L; after the initial glucose is consumed, glucose is added to maintain the concentration of glucose at 10±0.5 g / L. After the initial glycerol is consumed, glycerol is added to make the concentration of glycerol 2-3 g / L; after the initial glucose is consumed, glucose is added to maintain the concentration of glucose at 10±0.5 g / L.

8. Use of the recombinant E. coli of any one of claims 1 to 4 for the production of a product comprising lacto-N-neotetraose.

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