Genetic engineering strain for producing lactyl-N-neotetraose without exogenous addition of lactose and application of genetic engineering strain

Abstract

The invention discloses a genetic engineering strain for producing lactyl-N-neotetraose without exogenous addition of lactose and application of the genetic engineering strain, and belongs to the technical field of genetic engineering. According to the invention, an engineering strain with high LNnT yield and no need of exogenously adding lactose is obtained by gradually optimizing an LNnT biosynthetic pathway. According to the invention, the LNnT synthesis pathway is divided into four modules by applying modular pathway engineering, and the metabolic balance among the four pathways is systematically optimized by simply changing the plasmid copy number of each module. Besides, by optimizing a glucose transport pathway and maintaining the balance of a metabolic pathway, the waste of cell resources and the excessive accumulation of intermediates are avoided, and the metabolic flux of an LNnT biosynthetic pathway is further balanced. Finally, 26.9 g / L of LNnT is produced by the strain.

Description

Technical Field

[0001] The invention relates to a genetic engineering strain for producing lactoyl-N-neotetraose without exogenous lactose addition and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) are a complex mixture of oligosaccharides naturally present in breast milk. They are the third largest component after lactose and fat, accounting for about 8% of human milk. Studies have shown that adding a certain amount of human milk oligosaccharides (HMOs) to infant formula can promote infant growth. Lactoyl-N-neotetraose (LNnT) is the main non-fucosylated neutral HMO in breast milk, with the structure of Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc. It plays a key role in enhancing the immune function of human intestinal epithelial cells and preventing necrotizing enterocolitis. Currently, multiple regulatory agencies such as the National Health Commission of China, the US Food and Drug Administration, the European Commission, and the Australian and New Zealand Food Standards Agency have implemented regulations allowing the addition of LNnT as a nutritional fortifier to infant formula. Therefore, the pursuit of efficient LNnT production has become a prominent research focus.

[0003] There are three main methods for the synthesis of LNnT: chemical synthesis, enzymatic method and microbial synthesis. Although chemical synthesis is effective, it is prone to chemical residue problems and does not meet the strict quality standards required by the food industry. Enzymatic synthesis of LNnT requires acetylglucosamine transferase and galactosyl transferase, and relies on expensive precursors, namely uridine 5′-diphospho-n-acetylglucosamine (UDP-GlcNAc) and uridine 5′-diphospho-galactose (UDP-Gal), which makes them unsuitable for large-scale production. Compared with chemical synthesis and enzymatic catalysis, microbial fermentation has unique advantages such as short production cycle, low cost, simple operation and convenient raw material acquisition, and is the preferred method for the synthesis of LNnT.

[0004] In microbial fermentation, glucose or glycerol is usually used as a carbon source, and sufficient UDP-Gal and UDP-GlcNAc are synthesized by enhancing precursor pathway genes. Then, exogenous lactose is added to assist the synthesis of LNT II. Under the catalysis of β-1,4-galactosyltransferase, UDP-Gal undergoes transglycosylation and combines with LNT II to form LNnT. A lot of research has been done to efficiently produce LNnT in engineered bacteria. For example, in order to optimize the utilization of lactose, β-galactosidase (lacZ) that can hydrolyze lactose in the strain was inactivated. In addition, β-1,3-N-acetylglucosamine transferase (lgtA) and β-1,4-galactosyltransferase (lgtB) were introduced from Neisseria meningitidis to successfully synthesize LNnT. Previous studies have shown that the synthesis efficiency of LNnT can be improved by increasing the content of UDP-Gal and UDP-GlcNAc in cells, optimizing transporter expression, and regulating key rate-limiting enzymes. For example, Aa-β-1,4-GalT (from actinomycete Aggregatibacter NUM4039) and Hp-β-1,4-GalT (from Helicobacter pylori) have high catalytic activity and can increase the synthesis rate of LNnT. However, most of the current reported studies mainly use glycerol as the main carbon source and externally supplement lactose as a metabolic precursor. This method not only complicates the fermentation process, but also increases the production cost. Summary of the invention

[0005] The technical problem to be solved by the present invention is that in the prior art, when synthesizing lactoyl-N-neotetraose by microbial method, exogenous lactose is generally added, resulting in the need for multiple substrates to feed the fermentation, resulting in the problem of complex fermentation process operation.

[0006] In order to solve the above technical problems and realize the efficient production of lactoyl-N-neotetraose, the present invention knocks out the pathway genes that compete with the lactoyl-N-neotetraose synthesis precursor in the host bacteria. In addition, the lactoyl-N-neotetraose synthesis pathway is divided into 5 modules, and the expression level of each module is optimized by changing the plasmid copy number, the metabolic flux is balanced, and the synthesis of lactoyl-N-neotetraose is promoted. A genetically engineered strain with high yield of lactoyl-N-neotetraose and low residual lactoyl-N-triose II and a construction method thereof are provided.

[0007] The first object of the present invention is to provide a genetically engineered bacterium, wherein the genetically engineered strain has knocked out the genes of the pathway competing for the precursor of lactoyl-N-neotetraose synthesis, and overexpresses the genes of the following modules (a) to (e):

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

[0009] (b) Downstream 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] (c) Lactose synthesis module: β-1,4-galactosyltransferase gene GalTpm1141;

[0011] (d) Coordinated supply module: ribose bisphosphate kinase gene prsA, glucose 6-phosphate isomerase gene pgi,

[0012] (e) Glucose supply module: glucose transporter glf.

[0013] In one embodiment, the genetically engineered bacteria uses Escherichia coli as a starting strain.

[0014] In one embodiment, the genes for the competitive pathway of the lacto-N-neotetraose synthesis precursor include β-galactosidase gene lacZ, galactoside O-acetyltransferase gene lacA, glucosamine-6-phosphate deaminase gene nagB, UDP-N-acetylglucosamine 2-acylimase gene wecB, UDP-glucose 6-dehydrogenase gene ugd, quinone protein glucose dehydrogenase gene gcd, sugar export protein gene setA, EIICB Glc Protein gene ptsG, phosphate acetyltransferase gene ptA.

[0015] In one embodiment, the deletion is knocked out using the CRISPR / Cas9 system.

[0016] In one embodiment, the phosphoglucomutase gene pgm, UTP-glucose-1-phosphate uridyltransferase gene galU, UDP-glucose 4-epimerase gene galE, phosphoglucoamine mutase gene glmM, L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS, fusion N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase gene glmU, ribose phosphate diphosphate kinase gene prs and glucose 6-phosphate isomerase gene pgi are all derived from Escherichia coli K-12.

[0017] In one embodiment, the genes of modules d) and e) are expressed on a low copy plasmid; the genes of modules b) and c) are expressed on a high copy plasmid; and the genes of module a) are expressed on a medium copy plasmid.

[0018] In one embodiment, the high copy plasmid includes but is not limited to pRSFDuet-1; the medium copy plasmid includes but is not limited to pETDuet-1; the low copy plasmid includes but is not limited to pCDFDuet-1.

[0019] In one embodiment, the genetically engineered bacteria express the coding genes of the downstream lactoyl-N-neotetraose synthesis module and the lactose synthesis module in a high copy plasmid.

[0020] In one embodiment, the genetically engineered bacteria express the gene encoding the upstream lactoyl-N-trisaccharide II synthesis module in a medium copy plasmid.

[0021] In one embodiment, the genetically engineered bacteria express the coding gene of the coordinated supply module in the form of a low copy plasmid.

[0022] In one embodiment, the genetically engineered bacteria express the gene encoding the glucose supply module in the form of a low copy plasmid.

[0023] In one embodiment, the genetically engineered bacteria express the β-1,4-galactosyltransferase gene lgtB, the phosphoglucomutase gene pgm, the UTP-glucose-1-phosphate uridyltransferase gene galU, and the UDP-glucose 4-epimerase gene galE using a high copy plasmid.

[0024] In one embodiment, the genetically engineered bacteria express the β-1,3-acetylglucosamine transferase gene lgtA, the phosphoglucosamine mutase gene glmM, the fusion N-acetylglucosamine-1-phosphate urea transferase / glucosamine-1-phosphate acetyl transferase gene glmU, and the L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS or glmS* using a medium copy plasmid.

[0025] In one embodiment, the genetically engineered bacteria express the ribose bisphosphate kinase gene prsA and the glucose 6-phosphate isomerase gene pgi in a low copy plasmid.

[0026] In one embodiment, the genetically engineered bacteria is based on the strain BL21 (DE3) ΔlacZΔlacAΔnagBΔwecBΔugdΔgcdΔsetAΔptsGΔptA in which the lacZ, lacA, nagB, wecB, ugd, gcd, setA and ptsG genes are knocked out, and the genes lgtB, pgm, galU, galE, Galtpm1141 are expressed with the high copy plasmid pRSFDuet-1, lgtA, glmM, glmU, glmS* are expressed with the medium copy plasmid pETDuet-1, and the genes prsA, pgi and glf are expressed with the low copy plasmid pCDFDuet-1.

[0027] In one embodiment, the L-glutamine-D-fructose-6-phosphate aminotransferase has at least one of the following mutations based on the starting sequence of GlmS (NP_418185) (calculated from the second amino acid): E14K, D386V, S449P, E524G; wherein E14K indicates that the 14th glutamic acid (E) is mutated to K, D386V indicates that the 386th aspartic acid (D) is mutated to valine (V), S449P indicates that the 449th serine (S) is mutated to proline (P), and E524G indicates that the 524th glutamic acid (E) is mutated to glycine (G).

[0028] In one embodiment, the L-glutamine-D-fructose-6-phosphate aminotransferase is a mutant E14K / D386V / S449P / E524G obtained by performing quadruple mutations on the starting sequence of GlmS (NP_418185).

[0029] In one embodiment, the NCBI accession number of the nucleotide sequence of the β-galactosidase gene lacZ is CP053602.1 (334064-337138); the NCBI accession number of the nucleotide sequence of the galactoside O-acetyltransferase gene lacA is CP053602.1 (332082-332693); the NCBI accession number of the nucleotide sequence of the glucosamine-6-phosphate deaminase gene nagB is CP053602.1 (663983-664783); the nucleotide sequence of the UDP-N-acetylglucosamine 2-acylimase gene wec ... The NCBI accession number of the nucleotide sequence of the UDP-glucose 6-dehydrogenase gene ugd is CP053602.1 (3861671-3862801); the NCBI accession number of the nucleotide sequence of the UDP-glucose 6-dehydrogenase gene ugd is CP053602.1 (1995548-1996714); the NCBI accession number of the nucleotide sequence of the quinone protein glucose dehydrogenase gene gcd is CP053602.1 (141640-144030); the NCBI accession number of the nucleotide sequence of the sugar export protein setA is CP053602.1 (80426-81604); the EIICB Glc The NCBI accession number of the nucleotide sequence of the protein gene ptsG is CP053602.1 (1161734-1163167).

[0030] In one embodiment, the NCBI sequence number of the amino acid sequence of the β-1,3-acetylglucosamine transferase LgtA is WP_002257440.1, or the β-1,3-acetylglucosamine transferase is derived from the Neisseria meningitidis gene lgtA, and the nucleotide sequence is shown in SEQ ID NO.1; the NCBI sequence number of the amino acid sequence of the β-1,4-galactosyltransferase LgtB is WP_002225824.1, or the β-1,4-galactosyltransferase gene lgtB is derived from Neisseria meningitidis, and the nucleotide sequence is shown in SEQ ID NO.2; 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 phosphoglucomutase 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 fusion N-acetylglucosamine-1-phosphate uridyltransferase / glucose The NCBI accession number of the nucleotide sequence of the glucosamine-1-phosphate acetyltransferase gene glmU is ECK3723; the NCBI accession number of the nucleotide sequence of the ribose phosphate diphosphate kinase gene prsA is ECK1195; the NCBI accession number of the nucleotide sequence of the glucose 6-phosphate isomerase gene pgi is ECK4017; the NCBI accession number of the nucleotide sequence of the glucose transporter gene glf is AEC04686; the NCBI sequence number of the amino acid sequence of the β-1,4 galactosyltransferase gene GalTpm1141 is AEC04686, or the β-1,4 galactosyltransferase is derived from the pestis gene GalTpm1141, and the nucleotide sequence is shown in SEQ ID NO.3.

[0031] The present invention also provides a method for producing lactoyl-N-neotetraose by fermentation culture using the above genetically engineered bacteria, the method comprising the following steps:

[0032] (1) inoculating the genetically engineered bacteria into a culture medium to obtain a bacterial liquid;

[0033] (2) transferring the bacterial solution to a fermentation medium to ferment and produce lactoyl-N-neotetraose.

[0034] In one embodiment, the culture medium in step (1) comprises LB medium.

[0035] In one embodiment, the culture conditions in step (1) are culturing for 8 h to 12 h, 30 to 40° C., and 200 to 220 rpm.

[0036] Preferably, the culture condition is culturing at 37°C for 12 hours.

[0037] In one embodiment, the bacterial OD during the transfer in step (2) is 600 It is 1 to 3.

[0038] In one embodiment, the fermentation medium in step (2) is: 30-50 g / L carbon source, 3-4 g / L yeast extract, 0.25-1 g / L peptone, 10-15 g / L potassium dihydrogen phosphate, 2-6 g / L diammonium hydrogen phosphate, 1-2 g / L citric acid, 1-2 g / L magnesium sulfate heptahydrate and 8-12 mL / L trace element solution. The trace element solution includes 8-12 g / L iron (III) citrate, 1-3 g / L zinc sulfate heptahydrate, 0.5-1.5 g / L copper sulfate pentahydrate, 0.1-0.5 g / L manganese sulfate monohydrate, 0.1-0.3 g / L sodium tetraborate decahydrate, 0 / 05-0.2 g / L ammonium heptamolybdate and 1-3 g / L calcium chloride.

[0039] In one embodiment, the fermentation medium is: 30.00 g / L carbon source, 5.00 g / L yeast extract, 0.50 g / L peptone, 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 includes 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.

[0040] In one embodiment, the fermentation culture temperature in step (2) is 20-30°C, preferably 25°C.

[0041] In one embodiment, in step (2), the OD of the bacterial cells is controlled before fermentation production. 600 10 to 20, preferably, OD 600 is 15.

[0042] In one embodiment, an inducer is also added during the fermentation production in step (2), and preferably, the inducer is IPTG.

[0043] In one embodiment, the final concentration of the inducer is 0.05-0.4 mM, preferably 0.1 mM.

[0044] In one embodiment, the induction time is 2 to 4 hours, preferably 2 hours.

[0045] In one embodiment, after the inducer is added, the fermentation is cultured for no less than 40 hours, preferably 72 hours.

[0046] In one embodiment, in the fermentation production in step (2), feed medium needs to be added to maintain the carbon source concentration not less than 5 g / L.

[0047] In one embodiment, the feed medium comprises: 400-800 g / L carbon source, 15-30 g / L MgSO4·7H2O, 2-8 g / L yeast extract, and 1-5 g / L peptone.

[0048] In one embodiment, the feed medium is: 600 g / L carbon source, 22.02 g / L MgSO4·7H2O, 5.76 g / L yeast extract, and 2.88 g / L peptone.

[0049] In one embodiment, the genetically engineered bacteria are cultured in LB medium for 8 h to 12 h at 30 to 40 ° C and 200 to 220 rpm to obtain a seed solution, and the seed solution is added to a fermentation medium containing a carbon source at a rate of 5 to 10%, and cultured until OD 600 is 1 to 2, IPTG is added so that the concentration of IPTG in the reaction system is 0.05 to 0.4 mM, and the induction culture time is not less than 72 h.

[0050] In one embodiment, the genetically engineered bacteria are inoculated into LB medium and cultured overnight at 37°C for 12 hours to prepare a fermentation seed solution. The seed solution is then inoculated into a fermentation medium containing glucose as a carbon source at an inoculum rate of 1%. 600 When the pH value reached 1, IPTG with a final concentration of 0.05 mM was added as an inducer, and the culture was continued at 25°C for 2 h; the fermentation was continued at 25°C for 72 h.

[0051] In one embodiment, the genetically engineered bacteria are cultured at 30-40°C and 200-220 rpm to obtain a seed solution, and the seed solution is added to a 3L tank fermentation medium at an amount of 5-10%, and cultured until the OD 600 The induction culture time is not less than 50h.

[0052] In one embodiment, the genetically engineered bacteria are cultured at 37°C and 200 rpm for 12 h to obtain a seed solution, and the seed solution is added to a 3 L tank fermentation medium at 10% and cultured until the OD 600The induction culture time was not less than 40 h.

[0053] In one embodiment, the carbon source is glucose.

[0054] The present invention also provides the use of the genetically engineered bacteria in fermenting and producing lactoyl-N-neotetraose or a product containing lactoyl-N-neotetraose.

[0055] In one embodiment, the application includes food, chemical, and pharmaceutical fields.

[0056] Beneficial effects:

[0057] (1) The present invention blocks the competitive pathways of lactose, uridine-5′-diphosphate-galactose (UDP-Gal) and uridine-5′-diphosphate-N-acetyl-glucosamine (UDP-GlcNAc), weakens the extracellular transport pathway of lactoyl-N-trisaccharide II, an intermediate product in the synthesis of lactoyl-N-neotetraose, and knocks out the ptsG gene encoding the EIICBGlc protein, thereby improving the strain's ability to transport glucose and effectively utilize it. A modular engineering strategy was used to divide the lactoyl-N-neotetraose synthesis pathway into trisaccharide synthesis module, tetrasaccharide synthesis module, lactose synthesis module, and auxiliary synthesis module. The expression level of each module was optimized by changing the plasmid copy number, and the optimal combination was determined as: pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-lgtA-glmM-glmU-S*, pCDFDuet-prsA-pgi, which achieved a lactoyl-N-neotetraose production of 2.47 g / L at the shake flask level.

[0058] To enhance the glucose supply capacity, by optimizing the expression level, the best combination was determined as: pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-lgtA-glmM-glmU-S*, pCDFDuet-prsA-pgi-glf, so that the production of lactoyl-N-neotetraose at the shake flask level reached 4.43 g / L.

[0059] (2) The present invention has also been verified by a large-scale fermentation process in a 3L bioreactor. Without the need for exogenous lactose supplementation, the yield of lactoyl-N-neotetraose can reach 26.9 g / L after 61.5 h of fermentation, which has excellent industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Metabolic pathway for the synthesis of lactoyl-N-neotetraose.

[0061] Figure 2This is the yield diagram of lactoyl-N-neotetraose under different module combinations, including tetrasaccharide synthesis pathway (MUEB): lgtB, pgm, galU-E; trisaccharide synthesis pathway (SUMA): lgtA, glmM, glmU-S*; lactose synthesis pathway (GALT): Galtpm1141; auxiliary synthesis pathway (PP): prsA, pgi; glucose supply pathway (GS): glf; L: high copy plasmid; M: medium copy plasmid; S: low copy plasmid.

[0062] Figure 3 This is the fermentation yield of lactoyl-N-neotetraose by strain S8 in a 3L bioreactor.

[0063] Figure 4 This is the fermentation yield of lactoyl-N-neotetraose by strain S9 in a 3L bioreactor. DETAILED DESCRIPTION

[0064] Culture medium formula:

[0065] LB medium: 5.00 g / L yeast extract, 10.00 g / L peptone and 10.00 g / L sodium chloride.

[0066] LB solid medium: 5.00 g / L yeast extract, 10.00 g / L peptone and 10.00 g / L sodium chloride, 20 g / L agar powder.

[0067] Fermentation medium: 30.00 g / L glucose, 5.00 g / L yeast extract, 0.50 g / L peptone, 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 includes 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.

[0068] Feed medium: 600 g / L glucose, 22.02 g / L MgSO4·7H2O, 5.76 g / L yeast extract, and 2.88 g / L peptone.

[0069] The fermentation broth treatment method and the detection conditions of lactoyl-N-neotetraose described in the embodiment of the present invention are as follows: the sample preparation and analysis method are as follows: the fermentation broth sample is boiled at 100°C for 15 minutes, and then centrifuged at 12000rpm for 15 minutes using a high-speed centrifuge. The supernatant after centrifugation is filtered through a 0.22μm filter membrane as a sample to be detected. Lactoyl-N-neotetraose is detected by high-performance liquid chromatography (HPLC): differential refractive index detector; chromatographic column is Aminex HPX-87H (300×7.8mm), column temperature is 50°C; mobile phase is 5mM H2SO4 aqueous solution, flow rate is 0.5mL / min; injection volume is 10μL.

[0070] Example 1: Construction of gene knockout chassis cells

[0071] 1. Construction of knockout plasmid (using knockout of lacZ as an example)

[0072] Replacement of N20: Use sgRNAcas9 software to select the predicted N20 sequence on the lacZ gene, design primers, use the original pTargetF as a template, and replace the N20 sequence on the template by PCR replacement. Transform the PCR product into E. coli JM109, extract the plasmid and send it for sequencing verification. The successfully verified pTarget-lacZ is stored at -20℃ for future use.

[0073] Preparation of lacZ homology arms: Using the genome of the strain to be knocked out as a template, use the lacZ-up-F / R and lacZ-down-F / R primer pairs to amplify the upstream and downstream homology arms required for knocking out lacZ, respectively, and purify and recover the two DNA fragments. Further, use the lacZ-up-F / lacZ-down-R primer pair to fuse the two fragments of the lacZ upstream and downstream homology arms into one fragment by overlapping PCR, and purify and recover the DNA fragments.

[0074] The lacZ homology arms were integrated into pTarget-lacZ to construct a complete knockout plasmid pTarget-ΔlacZ: the lacZ homology arms were connected to the EcoR I and HiNd III restriction sites of the vector pTarget-lacZ using a seamless cloning kit (Nanjing Novogene Biotech Co., Ltd.), the ligation products were transformed into Escherichia coli JM109, the plasmids were extracted and sent for sequencing for verification, and the successfully verified pTarget-ΔlacZ was stored at -20°C for future use.

[0075] The methods for knocking out plasmids of other genes lacA, nagB, wecB, nagB, ugd, gcd, setA, ptsG, and ptA are the same as above (primers involved in the construction of knockout plasmids are shown in Table 1).

[0076] Table 1 Primer sequences for gene knockout

[0077]

[0078]

[0079]

[0080] 2. Knockout of Escherichia coli BL21 (DE3) genomic genes (using lacZ knockout as an example)

[0081] The pcas9 plasmid was transformed into the host bacteria BL21 (DE3).

[0082] The pcas9 plasmid was transferred into BL21 (DE3), cultured at 30°C for 16-24h, single clones were picked, transferred to 10mL LB (containing kanamycin) medium, and cultured at 30°C, 200rpm for about 12h-16h.

[0083] Prepare competent cells containing pcas9 plasmid and knock out lacZ gene. The specific steps are as follows:

[0084] (1) Transfer the cells from the sterile tube or streaked plate stored in a -80°C refrigerator (the strain used contains the cas9 plasmid) to 10 mL LB medium (containing kanamycin) and culture at 30°C, 200 rpm for about 12 h-16 h. Transfer to sterile LB medium (containing kanamycin) with an inoculum size of 1%. When the cells grow to OD 600 When OD was 0.1, arabinose was added at a final concentration of 10 mmol / L to induce λ-Red-mediated homologous recombination. 600 When the concentration reaches 0.4-0.6, the bacteria are collected and competent cells are prepared.

[0085] (2) Add the pTarget-ΔlacZ knockout plasmid to about 100 μL of competent cells containing the pCas9 plasmid and perform electroporation (1 / 2 mm cuvette, 2.5 kV). Immediately after electroporation, add 1 mL of pre-cooled LB liquid medium to suspend the cells, resuscitate at 30°C for 1-2 hours, spread on LB agar medium containing kanamycin and spectinomycin, and culture at 30°C overnight. PCR was used to verify the lacZ knockout effect.

[0086] Elimination of pTarget-ΔlacZ: The positive clones containing pCas9 and pTarget-ΔlacZ were inoculated into LB medium containing kanamycin and IPTG (0.5 mmol / L) and cultured for 8-16 h to remove pTarget-ΔlacZ and obtain the gene knockout strain BL21(DE3)ΔlacZ.

[0087] 3. Knockout of lacA, nagB, wecB, ugd, gcd, setA, ptsG, ptA genes

[0088] The genes lacA, nagB, wecB, ugd, gcd, setA, ptsG, and ptA were knocked out using the same strategy as in steps 1-2 above, and the gene knockout strain BL21 (DE3) ΔlacZ ΔlacA ΔnagB ΔwecB Δugd Δgcd ΔsetA ΔptsG ΔptA was finally obtained.

[0089] Example 2: Construction and screening of host engineering bacteria for efficient production of lactoyl-N-neotetraose

[0090] 1. Plasmid construction

[0091] The glmU and glmS genes are located adjacent to each other on the genome, so they are abbreviated as glmU-S (i.e., glmU and glmS genes) below. The steps for constructing an overexpression plasmid using pETDuet-lgtA-glmU-S*-glmM (S* represents the quadruple mutant of glmS* E14K / D386V / S449P / E524G) as an example are as follows, and the primer sequences involved are shown in Table 2.

[0092] (1) Using the lgtA gene sequence (nucleotide sequence as shown in SEQ ID NO. 1) as a template and 1-2lgtA-F / R as primers, PCR was performed to amplify the lgtA gene fragment, and the DNA fragment was recovered by gel electrophoresis;

[0093] (2) Using 2×Phanta Max Master Mix (Nanjing Novogene Biotechnology Co., Ltd.), the lgtA gene fragment and pETDuet-1 were recombined and connected by MEGAWHOP. The ligation product was transferred into Escherichia coli JM109, and the plasmid was extracted and sent for sequencing for verification. It was verified that pETDuet-lgtA was successfully obtained.

[0094] (3) Using the genome of Escherichia coli K-12 as a template and glmU-SF / R as primers, PCR was performed to amplify the glmU-S gene fragment, and the DNA fragment was recovered by gel. Using the glmM gene sequence (nucleotide sequence as shown in SEQ ID NO.5) as a template and 1-1glmM-F / R as primers, PCR was performed to amplify the glmM gene fragment, and the DNA fragment was recovered by gel; the 1-1glmU-SF / 1-1glmM-R primer pair was further used to fuse the glmU-S* and glmM DNA fragments into one fragment by overlapping PCR, and the DNA fragment was purified and recovered.

[0095] (4) 2×Phanta Max Master Mix premixed enzyme (Nanjing Novogene Biotechnology Co., Ltd.) was used, and the glmU-S and glmM fusion fragments were recombined and connected with pETDuet-lgtA by MEGAWHOP method. The ligation product was transferred into Escherichia coli JM109, and the plasmid was extracted and sent for sequencing for verification. It was verified that pETDuet-lgtA-glmU-S-glmM was successfully obtained.

[0096] (5) Using pETDuet-lgtA-glmU-S-glmM as a template, glmS(E14K,S449P)-F / R were added to the reaction system for PCR amplification. After the amplification, 7.5 μL of the amplified product was taken, 1.5 μL of Dpn I enzyme and 1 μL of enzyme buffer were added and mixed. The mixture was treated in a 37°C water bath for 2 h to digest the template in the system. The digested PCR product was then transferred into Escherichia coli JM109, and the plasmid was extracted and sent for sequencing for verification. pETDuet-lgtA-glmU-S(E14K,S449P)-glmM was successfully obtained. Using pETDuet-lgtA-glmU-S(E14K,S449P)-glmM as a template, glmS*-F / R were added to the reaction system for PCR amplification. After the amplification, 7.5 μL of the amplified product was taken, 1.5 μL of Dpn I enzyme was added and mixed. The mixture was treated in a 37°C water bath for 2 h to digest the template in the system. The digested PCR product was then transferred into Escherichia coli JM109, and the plasmid was extracted and sent for sequencing for verification. Mix I fast-digesting enzyme and 1 μL fast-digesting enzyme buffer, treat in a 37°C water bath for 2 h, digest the template in the system, and then transfer the digested PCR product into Escherichia coli JM109. The plasmid is extracted and sent for sequencing for verification. After successful verification, the plasmid pETDuet-lgtA-glmU-S*-glmM is finally obtained.

[0097] Plasmid pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pRSFDuet-pgm-galU-galE-lgtB, pRSFDuet-prsA-pgi-Galtpm1141, pRSFDuet-lgt A-glmM-glmU-S*-Galtpm1141,pETDuet-Galtpm1141,pETDuet-prsA-pgi-Galtpm1141,pRSFDuet-lgtA-glmM-glmU-S*,pETDuet-pg m-galU-galE-lgtB,pCDFDuet-prsA-pgi,pCDFDuet-prsA-pgi-Galtpm1141,pETDuet-prsA-pgi,pRSFDuet-pgm-galU-galE-lgtB-p rsA-pgi,pETDuet-lgtA-glmM-glmU-S*-glf,pCDFDuet-prsA-pgi-glf,pETDuet-prsA-pgi-glf,pCDFDuet-lgtA-glmM-glmU-S*-glf

[0098] The construction method is the same as above.

[0099] Table 2 Primers for overexpression plasmid construction

[0100]

[0101]

[0102]

[0103]

[0104] 2. Construction of host engineering bacteria for efficient production of lactoyl-N-neotetraose and modular combination optimization screening based on different plasmid copy numbers

[0105] The 19 overexpression plasmids constructed in step 1 were combined and matched to obtain 12 combinations:

[0106] ⑴pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-lgtA-glmM-glmU-S*, pCDFDuet-prsA-pgi;

[0107] ⑵pRSFDuet-pgm-galU-galE-lgtB, pETDuet-lgtA-glmM-glmU-S* and

[0108] pCDFDuet-prsA-pgi-Galtpm1141;

[0109] (3) pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-prsA-pgi and

[0110] pCDFDuet-lgtA-glmM-glmU-S*;

[0111] (4) pRSFDuet-pgm-galU-galE-lgtB-prsA-pgi, pETDuet-Galtpm1141 and

[0112] pCDFDuet-lgtA-glmM-glmU-S*;

[0113] (5) pRSFDuet-pgm-galU-galE-lgtB, pETDuet-prsA-pgi and pCDFDuet-lgtA-glmM-glmU-S*-Galtpm1141;

[0114] (6) pRSFDuet-prsA-pgi-Galtpm1141, pETDuet-pgm-galU-galE-lgtB and

[0115] pCDFDuet-lgtA-glmM-glmU-S*;

[0116] (7) pRSFDuet-lgtA-glmM-glmU-S*-Galtpm1141, pETDuet-pgm-galU-galE-lgtB and pCDFDuet-prsA-pgi;

[0117] (8) pRSFDuet-lgtA-glmM-glmU-S*, pETDuet-pgm-galU-galE-lgtB and

[0118] pCDFDuet-prsA-pgi-Galtpm1141;

[0119] (9) pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-lgtA-glmM-glmU-S*, pCDFDuet-prsA-pgi-glf;

[0120] ⑽pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-lgtA-glmM-glmU-S*-glf and pCDFDuet-prsA-pgi;

[0121] ⑾pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-prsA-pgi-glf and

[0122] pCDFDuet-lgtA-glmM-glmU-S*;

[0123] ⑿pRSFDuet-pgm-galU-galE-lgtB-Galtpm1141, pETDuet-prsA-pgi and

[0124] pCDFDuet-lgtA-glmM-glmU-S*-glf.

[0125] The above 12 combined recombinant plasmids were respectively electroporated into the knockout strain BL21 (DE3) ΔlacZ ΔlacA ΔnagB ΔwecB Δugd Δgcd ΔsetA ΔptsG ΔptA of Example 1, and 12 different engineered bacteria were obtained, which were named S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11 and S12 (see Table 3).

[0126] The above 8 strains of engineered bacteria were cultured in fermentation medium at 25°C and 200rpm for 72h. The results showed that the lactoyl-N-neotetraose production of the 12 strains of engineered bacteria were 2.47g / L, 1.76g / L, 1.74g / L, 1.53g / L, 0.62g / L, 0.68g / L, 1.02g / L, 1.54g / L, 4.43g / L, 3.64g / L, 1.145g / L, 0.675g / L; the lactose production were 0.54g / L, 0.67g / L, 1.2g / L, 1.06g / L, 0.44g / L, 0.46g / L, 0.44g / L, 0.33g / L, 1.3g / L, 1.47g / L, 1.025g / L, 0.37g / L. Among them, the engineered bacteria S1 containing plasmids pRSFDuet-lgtB-pgm-galU-E-Galtpm1141, pETDuet-lgtA-glmM-glmU-glmS*, and pCDFD-prsA-pgi-glf obtained the highest lactoyl-N-neotetraose production of 4.43 g / L.

[0127] Table 3 Detailed information of each genetically engineered strain and yield of fermentation products

[0128]

[0129]

[0130] Example 3: Fermentation of lactoyl-N-neotetraose by efficient production strain in 3L bioreactor

[0131] Lactoyl-N-neotetraose was produced in a 3 L bioreactor (Applikon). The genetically engineered strains S8 and S9 were cultured at 37 °C and 200-220 rpm to prepare seed solution, which was inoculated into a fermentation medium with a working volume of 1 L at a 10% inoculum. The parameters of the bioreactor were set as follows: the culture temperature was 37 °C, the dissolved oxygen was maintained at 30%, and the pH was set to 7.0. When OD 600 When the culture temperature reached 15, the culture temperature was set to 25°C, and IPTG was added at a final concentration of 0.1 mM. The pH of the culture medium was adjusted with 50% ammonia water to maintain the pH value of the culture medium at 7.0. Feed medium was added to the 3L bioreactor to maintain the glucose concentration at 5-15 g / L. The lactoyl-N-neotetraose production during the fermentation process was detected. After 61.5 hours of fermentation, the lactoyl-N-neotetraose production of strain S8 reached a maximum of 18.45 g / L, and the OD 600 Reaching a maximum of 160 ( Figure 3 ); After 79 hours of fermentation, the yield of lactoyl-N-neotetraose of strain S9 reached a maximum of 26.9 g / L, OD 600 Reaching a maximum of 250 ( Figure 4 ).

[0132] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 bacterium, characterized in that: The genes involved in the competition for precursors of lactoyl-N-neotetraose synthesis were knocked out, and the genes of modules (a) to (e) were overexpressed: (a) Upstream lactoyl-N-triose II synthesis module: β-1,3-acetylglucosamine transferase gene lgtA, phosphoglucosamine mutase gene glmM, fusion N-acetylglucosamine-1-phosphate urea transferase / glucosamine-1-phosphate acetyltransferase gene glmU, L-glutamine-D-fructose-6-phosphate aminotransferase gene glmS; (b) Downstream 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; (c) Lactose synthesis module: β-1,4-galactosyltransferase gene GalTpm1141; (d) Coordinated supply module: ribose bisphosphate kinase gene prsA, glucose 6-phosphate isomerase gene pgi; (e) Glucose supply module: glucose transporter glf.

2. The genetically engineered bacterium according to claim 1, characterized in that: The genetically engineered bacteria uses Escherichia coli as a starting strain.

3. The genetically engineered bacterium according to claim 1 or 2, characterized in that: The genes of the lactoyl-N-neotetraose synthesis precursor competition pathway include β-galactosidase gene lacZ, galactoside O-acetyltransferase gene lacA, glucosamine-6-phosphate deaminase gene nagB, UDP-N-acetylglucosamine 2-acylimase gene wecB, UDP-glucose 6-dehydrogenase gene ugd, quinone protein glucose dehydrogenase gene gcd, sugar export protein gene setA, EIICB Glc Protein gene ptsG, phosphate acetyltransferase ptA.

4. The genetically engineered bacterium according to any one of claims 1 to 3, characterized in that: The coding genes of the downstream lactoyl-N-neotetraose synthesis module and the lactose synthesis module are expressed with a high copy plasmid, the coding gene of the upstream lactoyl-N-triose II synthesis module is expressed with a medium copy plasmid, and the coding gene of the coordinated supply module is expressed with a low copy plasmid.

5. The genetically engineered bacterium according to any one of claims 1 to 3, characterized in that: The L-glutamine-D-fructose-6-phosphate aminotransferase has at least one of the following mutations based on the amino acid sequence shown in Genbank accession number NP_418185: E14K, D386V, S449P, E524G.

6. A method for producing lactoyl-N-neotetraose by fermentation, characterized in that: Fermentation is carried out using the genetically engineered bacteria described in any one of claims 1 to 5.

7. The method according to claim 6, characterized in that The method comprises the following steps: (1) inoculating the genetically engineered bacteria into a culture medium to obtain a bacterial liquid; (2) Transferring the bacterial solution obtained in step (1) to a fermentation medium to ferment and produce lactoyl-N-neotetraose.

8. The method according to claim 7, characterized in that An inducer is also added during the fermentation process of step (2), and the inducer is IPTG.

9. The method according to any one of claims 6 to 8, characterized in that: In step (2), feed medium is also added during the fermentation process to maintain the carbon source concentration at not less than 5 g / L.

10. Use of the genetically engineered bacteria according to any one of claims 1 to 5, or the method according to any one of claims 6 to 9 in the fermentation production of lactoyl-N-neotetraose or a product containing lactoyl-N-neotetraose.

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