A genetically engineered bacterium for increasing the production of lactosylsialic acid and its production method

Abstract

The present invention discloses a genetically engineered bacterium for improving the production of lactosylsialic acid and its production method, belonging to the fields of biotechnology and food fermentation engineering. By expressing genes neuB, neuC, neuA, lacY, lst and / or pst6-224, a de novo synthesis pathway of lactosylsialic acid is constructed. Through modular metabolic engineering, the metabolic pathway is balanced, the expression of genes related to the synthesis of the precursor UDP-N-acetylglucosamine is increased, a glutamine cycle system is constructed, the expression of lacZ, nanA, pfkA and nagB in the side branch pathway of the lactosylsialic acid synthesis pathway in the Escherichia coli genome is knocked out, and α-2,3(6) sialyltransferases from different sources are screened, and a production strain for efficiently synthesizing lactosylsialic acid is successfully obtained. In the fermentation culture experiment, the yields of 3'-SL and 6'-SL reach 6.46 g / L and 4.07 g / L respectively.

Description

Technical Field

[0001] The present invention relates to a genetically engineered bacterium for improving the production of sialyllactose and a production method thereof, belonging to the fields of biotechnology and food fermentation engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) have the effects of regulating the activity of intestinal microorganisms, enhancing immune responses, and preventing necrotizing enterocolitis. They are the third largest solid nutrient component in breast milk after lactose and lipids, and play an important role in the homeostasis and development of the infant digestive system, as well as the improvement and establishment of the immune system after birth. Adding HMOs to infant formula milk powder can narrow the nutritional gap between breast milk and formula milk powder. Sialyllactose (3(6)'-sialyllactose, 3(6)'-SL) is an important component of HMOs and is formed by the transgalactosylation reaction of CMP-sialic acid by α-2,3(6) sialyltransferase. In vitro studies have shown that 3'-SL and 6'-SL in HMOs support normal microbial communities and behavioral responses during stress by regulating the gut-brain axis. Therefore, infants fed with breast milk have more perfect brain development, richer neural synapses, and more developed nervous systems. Given the important biological effects of sialyllactose, to further clarify its mechanism of action, a large number of structurally homogeneous compounds of this type need to be prepared. However, the amount obtained by separation and extraction from natural products is very small, far from meeting the research needs. Therefore, obtaining such compounds by artificial synthesis has become the best choice.

[0003] The synthesis methods of sialyllactose mainly include three types: chemical synthesis, enzymatic synthesis, and bioreactor synthesis. The chemical synthesis of sialyllactose requires numerous cumbersome protection and deprotection steps. Enzymatic synthesis makes the cost of synthesized sialyllactose relatively high and is not suitable for industrial application because the donor substrate nucleotide sugar is relatively expensive and the yield is low. Biosynthesis of sialyllactose using genetically engineered strains can produce sialyllactose from inexpensive carbon sources (glucose, glycerol, lactose), and has increasingly attracted wide attention.

[0004] Currently, regarding the preparation of lactosylsialic acid by biological fermentation, in 2008, Eric Samain et al. knocked out nanT, nanA, nanK, nanE, overexpressed neuB, neuA, neuC, and α-2,3 sialyltransferase, and produced 25.5 g / L of 3'-SL; in 2010, the research group constructed the 6'-SL pathway by the same method, only changing the α-2,3 sialyltransferase in the de novo synthesis pathway of 3'-SL to α-2,6 sialyltransferase, and the highest yield of 6'-SL obtained by fermentation was 34 g / L. The currently reported methods for microbial production and the yields of lactosylsialic acid still cannot meet the requirements of industrial production. Therefore, seeking a cheap and high-yield de novo synthesis pathway of lactosylsialic acid to solve the current bottleneck of microbial production and creating a more efficient production strain are urgent problems to be solved currently. Summary of the Invention

[0005] [Technical Problem]

[0006] The cost of producing lactosylsialic acid by existing technologies is relatively high, and the yield is low, which is insufficient to achieve industrial production, unable to provide a strain for efficiently producing lactosylsialic acid, nor can it provide a method for producing lactosylsialic acid that is cheap, green, and efficient.

[0007] [Technical Solution]

[0008] In order to solve the problem of the low yield of lactosylsialic acid synthesized by existing biological methods, the present invention provides a genetically engineered bacterium capable of producing lactosylsialic acid and a method for constructing the same.

[0009] The first object of the present invention is to provide a genetically engineered bacterium for producing lactosylsialic acid, wherein the genetically engineered bacterium knocks out the β-galactosidase-encoding gene lacZ, and heterologously expresses the neuB gene encoding N-acetylneuraminic acid synthase, the neuC gene encoding N-acetylglucosamine isomerase, the neuA gene encoding CMP-sialic acid synthase, the lactose transferase-encoding gene lacY, the glutamine synthetase-encoding gene glnA, and the UDP-N-acetylglucosamine synthesis pathway; overexpresses the gene encoding α-2,3 sialyltransferase or the gene encoding α-2,6 sialyltransferase.

[0010] In one embodiment, the UDP-N-acetylglucosamine synthesis pathway is the glucosamine-6-phosphate synthase-encoding gene glmS, the glucosamine synthase-encoding gene glmM, and / or the UDP-N-acetylglucosamine pyrophosphorylase-encoding gene glmU.

[0011] In one embodiment, the genetically engineered bacterium also knocks out the sialic acid aldolase-encoding gene nanA, the 6-phosphofructokinase-encoding gene pfkA, and / or the glucosamine-6-phosphate deaminase-encoding gene nagB.

[0012] In one embodiment, the N-acetylneuraminic acid synthase gene neuB, N-acetylglucosamine isomerase gene neuC, and CMP-sialic acid synthase gene neuA are all derived from Campylobacter jejuni.

[0013] In one embodiment, the nucleotide sequences of the N-acetylneuraminic acid synthase gene neuB, N-acetylglucosamine isomerase gene neuC, and CMP-sialic acid synthase gene neuA are as shown in SEQ ID NO.1 to SEQ ID NO.3.

[0014] In one embodiment, the lactose permease gene lacY, glucosamine-6-phosphate synthase gene glmS, glucosamine synthase gene glmM, UDP-N-acetylglucosamine pyrophosphorylase gene glmU, and glutamine synthetase gene glnA are all derived from Escherichia coli K-12, and their nucleotide sequences are as shown in SEQ ID NO.6 to SEQ ID NO.10.

[0015] In one embodiment, the genetically engineered bacterium uses Escherichia coli as the host.

[0016] In one embodiment, the Escherichia coli includes MG1655, DH5α, BL21(DE3), JM109 or HB101.

[0017] In one embodiment, the Escherichia coli is Escherichia coli BL21.

[0018] In one embodiment, the genetically engineered bacterium uses plasmids pETDuet-1, pRSFDuet-1, pCDFDuet-1, pACYCDuet-1 or pCOLADuet-1 to express the genes neuB, neuC, neuA, lacY, glmM, glmS, glmU, glnA, and sialyltransferase-encoding gene.

[0019] In one embodiment, the genetically engineered bacterium uses plasmid pETDuet-1 to express the genes neuB, neuC, neuA, uses plasmid pRSFDuet-1 to express the genes lacY and sialyltransferase-encoding gene.

[0020] In one embodiment, the genetically engineered bacterium uses plasmid pETDuet-1 to express the genes neuB, neuC, neuA, uses plasmid pCDFDuet-1 to express the genes lacY and sialyltransferase-encoding gene.

[0021] In one embodiment, the genetically engineered bacterium uses the pETDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pACYCDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0022] In one embodiment, the genetically engineered bacterium uses the pETDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pCOLADuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0023] In one embodiment, the genetically engineered bacterium uses the pRSFDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pETDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0024] In one embodiment, the genetically engineered bacterium uses the pRSFDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pCDFDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0025] In one embodiment, the genetically engineered bacterium uses the pRSFDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pACYCDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0026] In one embodiment, the genetically engineered bacterium uses the pCDFDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pETDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0027] In one embodiment, the genetically engineered bacterium uses the pCDFDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pRSFDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0028] In one embodiment, the genetically engineered bacterium uses the pCDFDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pACYCDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0029] In one embodiment, the genetically engineered bacterium uses the pCDFDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pCOLADuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0030] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pETDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0031] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pRSFDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0032] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pCDFDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0033] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pCOLADuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0034] In one embodiment, the genetically engineered bacterium uses the pCOLADuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pETDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0035] In one embodiment, the genetically engineered bacterium uses the pCOLADuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pCDFDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0036] In one embodiment, the genetically engineered bacterium uses the pCOLADuet-1 plasmid to express the genes neuB, neuC, and neuA, and uses the pACYCDuet-1 plasmid to express the gene lacY and the sialic acid transferase-encoding gene.

[0037] In one embodiment, the sialic acid transferase gene is an α-2,3 sialic acid transferase gene or an α-2,6 sialic acid transferase gene.

[0038] In one embodiment, the genetically engineered bacterium uses the pETDuet-1 plasmid to express the genes neuB, neuC, and neuA, uses the pRSFDuet-1 plasmid to express the gene lacY and the α-2,3 sialic acid transferase gene, and uses the pCDFDuet-1 plasmid to express the gene glmM.

[0039] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, and neuA using the pETDuet-1 plasmid, expresses genes lacY and the α-2,3 sialyltransferase gene using the pRSFDuet-1 plasmid, and expresses gene glmS using the pCDFDuet-1 plasmid.

[0040] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, and neuA using the pETDuet-1 plasmid, expresses genes lacY and the α-2,3 sialyltransferase gene using the pRSFDuet-1 plasmid, and expresses gene glmU using the pCDFDuet-1 plasmid.

[0041] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, and neuA using the pETDuet-1 plasmid, expresses genes lacY and the α-2,3 sialyltransferase gene using the pRSFDuet-1 plasmid, and expresses genes glmM and glmU using the pCDFDuet-1 plasmid.

[0042] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, and neuA using the pETDuet-1 plasmid, expresses genes lacY and the α-2,3 sialyltransferase gene using the pRSFDuet-1 plasmid, and expresses genes glmM and glmS using the pCDFDuet-1 plasmid.

[0043] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, and neuA using the pETDuet-1 plasmid, expresses genes lacY and the α-2,3 sialyltransferase gene using the pRSFDuet-1 plasmid, and expresses genes glmU and glmS using the pCDFDuet-1 plasmid.

[0044] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, and neuA using the pETDuet-1 plasmid, expresses genes lacY and the α-2,3 sialyltransferase gene using the pRSFDuet-1 plasmid, and expresses genes glmM, glmS, and glmU using the pCDFDuet-1 plasmid.

[0045] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, and neuA using the pACYCDuet-1 plasmid, expresses genes lacY and the α-2,6 sialyltransferase gene using the pCOLADuet-1 plasmid, and expresses gene glmM using the pCDFDuet-1 plasmid.

[0046] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, uses the pCOLADuet-1 plasmid to express the genes lacY and the α-2,6 sialyltransferase gene, and uses the pCDFDuet-1 plasmid to express the gene glmS.

[0047] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, uses the pCOLADuet-1 plasmid to express the genes lacY and the α-2,6 sialyltransferase gene, and uses the pCDFDuet-1 plasmid to express the gene glmU.

[0048] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, uses the pCOLADuet-1 plasmid to express the genes lacY and the α-2,6 sialyltransferase gene, and uses the pCDFDuet-1 plasmid to express the genes glmM and glmU.

[0049] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, uses the pCOLADuet-1 plasmid to express the genes lacY and the α-2,6 sialyltransferase gene, and uses the pCDFDuet-1 plasmid to express the genes glmM and glmS.

[0050] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, uses the pCOLADuet-1 plasmid to express the genes lacY and the α-2,6 sialyltransferase gene, and uses the pCDFDuet-1 plasmid to express the genes glmU and glmS.

[0051] In one embodiment, the genetically engineered bacterium uses the pACYCDuet-1 plasmid to express the genes neuB, neuC, and neuA, uses the pCOLADuet-1 plasmid to express the genes lacY and the α-2,6 sialyltransferase gene, and uses the pCDFDuet-1 plasmid to express the genes glmM, glmS, and glmU.

[0052] In one embodiment, the genetically engineered bacterium uses the pETDuet-1 plasmid to express the genes neuB, neuC, neuA, and glnA, uses the pRSFDuet-1 plasmid to express the genes lacY and the α-2,3 sialyltransferase gene, and uses the pCDFDuet-1 plasmid to express the genes glmM, glmS, and glmU.

[0053] In one embodiment, the genetically engineered bacterium expresses genes neuB, neuC, neuA, and glnA using the pACYCDuet-1 plasmid, expresses the gene lacY and the α-2,6 sialyltransferase gene using the pCOLADuet-1 plasmid, and expresses the genes glmM and glmU using the pCDFDuet-1 plasmid.

[0054] In one embodiment, the gene encoding the α-2,3 sialyltransferase is selected from the gene lst derived from Neisseria meningitidis, the gene Pm0188 derived from Pasteurella multocida, the mutant gene Pm0188*, the gene WQG derived from Bibersteinia trehalosi, the gene Nst derived from Neisseria gonorrhoeae, or the mutant gene Nst*.

[0055] In one embodiment, the gene encoding the α-2,6 sialyltransferase is selected from the gene pst6-224 derived from Photobacterium sp., the gene plst6 derived from Photobacterium leiognathi, the mutant gene plst6*, the gene bst derived from Photobacterium damselae, or the mutant gene bst*.

[0056] In one embodiment, the nucleotide sequence of the α-2,3 sialyltransferase gene lst is as shown in SEQ ID NO.4, and the nucleotide sequence of the α-2,6 sialyltransferase gene pst6-224 is as shown in SEQ ID NO.5.

[0057] In one embodiment, the nucleotide sequence of the gene Pm0188 is as shown in SEQ ID NO.11, the nucleotide sequence of the gene WQG is as shown in SEQ ID NO.12, the nucleotide sequence of the gene Nst is as shown in SEQ ID NO.13, the nucleotide sequence of the gene Pm0188* is as described in SEQ ID NO.14, and the nucleotide sequence of the gene Nst* is as shown in SEQ ID NO.15.

[0058] In one embodiment, the nucleotide sequence of the gene plst6 is as shown in SEQ ID NO.16, the nucleotide sequence of the gene bst is as shown in SEQ ID NO.17, the nucleotide sequence of the gene plst6* is as shown in SEQ ID NO.18, and the nucleotide sequence of the gene bst* is as shown in SEQ ID NO.19.

[0059] The present invention provides a method for increasing the production of lactosylsialic acid, which involves knocking out the β-galactosidase-encoding gene lacZ, the sialic acid aldolase-encoding gene nanA, the 6-phosphofructokinase-encoding gene pfkA, and the glucosamine-6-phosphate deaminase-encoding gene nagB in the Escherichia coli genome, and then using an expression vector to heterologously express the N-acetylneuraminic acid synthase gene neuB, the N-acetylglucosamine isomerase gene neuC, the CMP-sialic acid synthase gene neuA, the sialyltransferase gene, the lactose transferase gene lacY, the glucosamine-6-phosphate synthase gene glmS, the glucosamine synthase gene glmM, the UDP-N-acetylglucosamine pyrophosphorylase gene glmU, and the glutamine synthase gene glnA.

[0060] In one embodiment, the sialyltransferase gene is an α-2,3 sialyltransferase gene or an α-2,6 sialyltransferase gene.

[0061] In one embodiment, the α-2,3 sialyltransferase gene is selected from lst, Pm0188, WQG, Nst, Pm0188*, or Nst*; the α-2,6 sialyltransferase gene is selected from pst6-224, plst6, bst, plst6*, or bst*.

[0062] In one embodiment, overexpression is carried out using plasmids such as pETDuet-1, pRSFDuet-1, pCDFDuet-1, pACYCDuet-1, or pCOLADuet-1.

[0063] In one embodiment, the neuB gene, the neuC gene, the neuA gene, and the glnA gene are overexpressed using pETDuet-1, the lacY gene and the α-2,3 sialyltransferase-encoding gene are overexpressed using pRSFDuet-1, and the glucosamine-6-phosphate synthase-encoding gene glmS, the glucosamine synthase-encoding gene glmM, and / or the UDP-N-acetylglucosamine pyrophosphorylase-encoding gene glmU are overexpressed using pCDFDuet-1.

[0064] In one embodiment, the neuB gene, the neuC gene, the neuA gene, and the glnA gene are overexpressed using pACYCDuet-1, the lacY gene and the α-2,6 sialyltransferase-encoding gene are overexpressed using pCOLADuet-1, and the glucosamine-6-phosphate synthase-encoding gene glmS, the glucosamine synthase-encoding gene glmM, and / or the UDP-N-acetylglucosamine pyrophosphorylase-encoding gene glmU are overexpressed using pCDFDuet-1.

[0065] In one embodiment, the neuB gene, neuC gene, neuA gene and gene glnA are overexpressed using pETDuet-1 or pACYCDuet-1, the gene lacY and the gene encoding sialyltransferase are overexpressed using pRSFDuet-1 or pCOLADuet-1, and the glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and / or UDP-N-acetylglucosamine pyrophosphorylase encoding gene glmU are overexpressed using pCDFDuet-1.

[0066] In one embodiment, the sialyllactose includes 3'-SL and 6'-SL.

[0067] The present invention also provides a method for producing sialyllactose, which is to use glycerol as a carbon source, lactose as a substrate, and the above-mentioned genetically engineered bacteria as a fermentation strain to ferment and produce sialyllactose.

[0068] In one embodiment, the above-mentioned genetically engineered bacteria are inoculated into a fermentation medium and cultured until the OD 600 reaches 10-15, and lactose with a final concentration of 15-25 g / L and IPTG with a concentration of 0.2-1.0 mM are added.

[0069] In one embodiment, after the initial carbon source is consumed, 600-850 g / L glycerol and 15-25 g / L MgSO4·7H2O are added dropwise; after the initial lactose is consumed, lactose is added dropwise to maintain its concentration at 5-10 g / L.

[0070] In one embodiment, the fermentation conditions are: the culture temperature is 20-37 °C, the stirring speed is 200-850 r / min, the ventilation volume is 0.8-2.0 vvm, the pH is 6.5-7.0, and the fermentation time is 15-55 h.

[0071] In one embodiment, the composition of the fermentation medium is: 10-20 g / L glycerol, 1-2 g / L citric acid, 10-15 g / L potassium dihydrogen phosphate, 2-6 g / L diammonium hydrogen phosphate, 1-2 g / L magnesium sulfate heptahydrate and 7.5-12.5 mL / L trace metal elements.

[0072] In one embodiment, the composition of the trace metal elements is: 8-12 g / L ferric citrate, 2-2.5 g / L zinc sulfate heptahydrate, 0.5-1.5 g / L copper sulfate pentahydrate, 0.2-0.6 g / L manganese sulfate monohydrate, 0.1-0.5 g / L borax, 0.05-0.4 g / L ammonium molybdate heptahydrate, 1.5-2.5 g / L calcium chloride dihydrate.

[0073] The present invention also provides the use of the above-mentioned genetically engineered bacterium or the above-mentioned method for increasing the production of lactosylsialic acid in the production of lactosylsialic acid and products containing lactosylsialic acid.

[0074] Beneficial effects:

[0075] In the present invention, through the exogenous expression of N-acetylneuraminic acid synthase gene neuB, N-acetylglucosamine isomerase gene neuC, CMP-sialic acid synthase gene neuA, α-2,3 sialyltransferase gene lst and / or α-2,6 sialyltransferase gene pst6-224, and the overexpression of lactose transferase gene lacY, the production of lactosylsialic acid is regulated by modular combination with CMP-sialic acid as the node. And by expressing the genes glmS (glucosamine-6-phosphate synthase), glmM (encoding glucosamine synthase), and glmU (encoding UDP-N-acetylglucosamine pyrophosphorylase) for the precursor UDP-N-acetylglucosamine in the lactosylsialic acid metabolic pathway, the production of the precursor UDP-N-acetylglucosamine is further regulated by combined regulation to target the constant flow of lactosylsialic acid. At the same time, by expressing the gene glnA (encoding glutamine synthase), a glutamine cycle is established to regulate the balance of the precursor glucosamine-6-phosphate. In addition, the β-galactosidase gene lacZ, sialic acid aldolase gene nanA, 6-phosphofructokinase gene pfkA, and glucosamine-6-phosphate deaminase gene nagB are knocked out to block the shunt metabolism of the side branch pathway. Finally, α-2,3(6) sialyltransferases from different sources, the rate-limiting enzyme in the lactosylsialic acid synthesis pathway, are screened, and the purpose of increasing the production of lactosylsialic acid is achieved through the above series of operations.

[0076] Through fermentation culture, the ability of the engineered bacterium constructed in the present invention to produce lactosylsialic acid - 3'-SL is increased from the initial 2.68 g / L to 6.46 g / L, and 6'-SL is increased from the initial 0.51 g / L to 4.07 g / L, laying a foundation for the industrial production of lactosylsialic acid. Description of the drawings

[0077] Figure 1 is the lactosylsialic acid metabolic pathway diagram;

[0078] Figure 2 is the modular metabolism schematic diagram. Detailed implementation manners

[0079] The following further describes the specific implementation of the present invention in combination with examples and drawings. The plasmids, PCR reagents, restriction endonucleases, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercial products, and the specific operations are carried out according to the kit instructions.

[0080] Embodiments of the present invention are not limited thereto, and other unmentioned experimental operations and process parameters are carried out according to conventional techniques.

[0081] The sequencing of plasmids and DNA products was completed by Genewiz (Suzhou) Co., Ltd.

[0082] Preparation of Escherichia coli competent cells: Kit from Sangon Biotech (Shanghai) Co., Ltd.

[0083] LB liquid medium (g / L): Yeast extract 5, Tryptone 10, Sodium chloride 10.

[0084] LB solid medium (g / L): Yeast extract 5, Tryptone 10, Sodium chloride 10, Agar powder 20.

[0085] Fermentation medium (g / L): Glycerol 20, Citric acid 1.7, Potassium dihydrogen phosphate 13.5, Diammonium hydrogen phosphate 4, Magnesium sulfate heptahydrate 1.4, Trace metal solution 10 mL / L (Ferric citrate 10 g / L, Zinc 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 tetrahydrate 0.11 g / L, Calcium chloride dihydrate 2.0 g / L), pH 6.80.

[0086] The sialyllactose described in the embodiments of the present invention was determined by high performance liquid chromatography (HPLC), specifically as follows:

[0087] Take the fermentation broth, boil it at 100 °C for 10 min to break the cells, centrifuge at 12000 r / min for 10 min, take the supernatant, filter it through a 0.22 μm membrane, and detect it by HPLC.

[0088] HPLC detection conditions: Refractive index detector; Chromatographic column is Rezex ROA-organic acid (Phenomenex, USA), column temperature is 50 °C; Mobile phase is 0.005 mmol / L H2SO4 aqueous solution, flow rate is 0.6 mL / min; Injection volume is 10 μL.

[0089] The shake flask fermentation culture method is as follows:

[0090] Pick the colonies of the engineered bacteria cultured overnight on the LB solid medium and inoculate them into 5 mL of LB liquid medium, culture at 37 °C and 200 r / min for 12 h as the seed liquid. Transfer the seed liquid to the fermentation medium with a loading volume of 50 mL according to an inoculation amount of 1% (v / v), and culture at 37 °C and 200 r / min until the cell OD 600The value is 0.6 - 0.8. IPTG is added to make its final concentration 0.2 mmol / L, and at the same time lactose is added to make the final lactose concentration 10 g / L. Cultivate under the conditions of 25 °C and 200 r / min for 50 h.

[0091] The E. coli expression vector described in the embodiment of the present invention is specifically:

[0092] Table 1 Expression vectors involved in the following examples

[0093]

[0094] Example 1: Knockout of genes lacZ, nanA, pfkA, and nagB in the chromosome group of Escherichia coli BL21(DE3)

[0095] Use the CRISPR-Cas9 gene knockout system to knockout genes lacZ, nanA, pfkA, and nagB in the genome of Escherichia coli BL21(DE3). The specific steps are as follows (the primer sequences involved are shown in Table 2):

[0096] (1) Using the genomic DNA of E. coli BL21(DE3) as a template, use primer pairs lacZ-up-F / R and lacZ-down-F / R, nanA-up-F / R and nanA-down-F / R, pfkA-up-F / R and pfkA-down-F / R, nagB-up-F / R and nagB-down-F / R to amplify the homologous upstream and downstream fragments of lacZ, nanA, pfkA, and nagB by PCR respectively. After the products are purified and recovered, then using the homologous upstream and downstream fragments of lacZ, nanA, pfkA, and nagB as templates respectively, use primers lacZ-up-F / lacZ-down-R, nanA-up-F / nanA-down-R, pfkA-up-F / pfkA-down-R, and nagB-up-F / nagB-down-R to amplify and connect the upstream and downstream fragments by SOE-PCR technology to obtain gene homologous repair arms donor-lacZ, donor-nanA, donor-pfkA, donor-nagB, and purify and recover to obtain the required DNA fragments.

[0097] (2) Using the pTargetF plasmid (Addgene:#62226) as a template, and lacZ-sg-F / R, nanA-sg-F / R, pfkA-sg-F / R, and nagB-sg-F / R as primers, PCR amplification was used to introduce N20 sequences complementary to the lacZ, nanA, pfkA, and nagB sequences into the pTargetF plasmid, respectively obtaining pTargetF plasmids targeting lacZ, nanA, pfkA, and nagB (i.e., the targeting plasmids pTargetF-lacZ, pTargetF-nanA, pTargetF-pfkA, and pTargetF-nagB with lacZ, nanA, pfkA, and nagB specific N20 sequences). The PCR amplification products were respectively transformed into E.coli DH5α competent cells, spread on LB plates (containing spectinomycin), and cultured overnight at 37°C. The plasmids were extracted and sequenced.

[0098] (3) Place the E.coli BL21(DE3) competent cells on ice to thaw for 10 min. Take 5 μL of the pCas plasmid (Addgene:#60847) and add it to 100 μL of the competent cells, and gently mix. Incubate on ice for 30 min, heat shock at 42°C for 90 s, and immediately place on ice for 2 - 3 min. Add 1 mL of fresh LB liquid medium, culture at 30°C and 200 r / min for 1 h, then centrifuge at 3500 r / min for 5 min, discard the supernatant, spread the bacteria on an LB plate containing kanamycin, and place it in a 30°C constant temperature incubator to culture overnight until single colonies of E.coli BL21(DE3) / pCas grow.

[0099] (4) Pick a single colony of E.coli BL21(DE3) / pCas for overnight culture of the seed solution, then transfer it to LB liquid medium at a volume ratio of 1%, and culture at 30°C until the OD 600 reaches 0.2. Add D - arabinose with a final concentration of 30 mmol / L to induce the expression of the pCas-λ-red system, and continue to culture until the logarithmic growth phase to prepare E.coli BL21(DE3) / pCas competent cells.

[0100] (5) Electroporate the pTargetF-lacZ plasmid (total amount 500 ng) and the gene homologous repair arm donor-lacZ (total amount 1 μg) into E.coli BL21(DE3) / pCas competent cells, spread on an LB plate (kanamycin and spectinomycin), culture overnight at 30°C, pick single colonies on the plate for PCR verification, screen positive transformants and send them to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing.

[0101] (6) Culture the positive clone colonies with successful knockout verified by sequencing in LB liquid medium containing kanamycin. When the OD 600 value reaches 0.2, add IPTG with a final concentration of 0.5 mmol / L, and culture at 30 °C for 12 - 16 h to remove the pTargetF plasmid, then culture at 42 °C for 12 h to remove the pCas plasmid, obtaining E. coli BL21(DE3)ΔlacZ with the lacZ gene knocked out in the genome.

[0102] (7) Using E. coli BL21(DE3)ΔlacZ as the host bacterium, sequentially knock out the genes nanA, pfkA, and nagB. The knockout operation refers to the knockout of the gene lacZ above, thereby obtaining the corresponding strains BL21(DE3)ΔlacZΔnanA, BL21(DE3)ΔlacZΔnanAΔpfkA, and BL21(DE3)ΔlacZΔnanAΔpfkAΔnagB.

[0103] Table 2 sgRNA and knockout primers

[0104]

[0105]

[0106] Example 2: Construction of recombinant bacteria for de novo synthesis pathway of sialyllactose

[0107] The specific steps for constructing the recombinant bacteria are as follows (the primer sequences involved are shown in Table 3):

[0108] (1) Obtaining of neuB, neuC, neuA, lst, and pst6 - 224 gene fragments: Entrust Sangon Biotech (Shanghai) Co., Ltd. to synthesize the gene sequences of neuB, neuC, and neuA derived from Campylobacter jejuni, the lst gene sequence derived from Neisseria meningitidis, and the pst6 - 224 gene sequence derived from Photobacterium sp.

[0109] Using the synthesized neuB gene fragment as a template, amplify the neuB gene fragment with neuB - F / neuB - R as primers, purify and recover the DNA fragment, and ligate the recovered gene fragment neuB between the NcoI / BamHI digestion sites of the vector pETDuet - 1 through a seamless cloning kit (Nanjing Novoprotein Science & Technology Co., Ltd.) to obtain the plasmid pET - neuB;

[0110] Using the synthesized neuC gene fragment as a template, the neuC gene fragment was amplified with neuC-F / neuC-R as primers. The DNA fragment was purified and recovered, and the recovered neuC gene fragment was ligated between the PstI / HindⅢ restriction enzyme sites of the vector plasmid pET-neuB to obtain the plasmid pET-BC;

[0111] Using the synthesized neuA gene fragment as a template, the neuA gene fragment was amplified with neuA-F / neuA-R as primers. The DNA fragment was purified and recovered, and the recovered neuA gene fragment was ligated between the NdeI / XhoI restriction enzyme sites of the vector pET-BC to obtain the plasmid pET-BCA.

[0112] Using the synthesized lst gene fragment as a template, the lst gene fragment was amplified with lst-F / lst-R as primers. The DNA fragment was purified and recovered, and the recovered lst gene fragment was ligated between the NcoI / BamHI restriction enzyme sites of the vector pRSFDuet-1 to obtain the plasmid pRS-lst.

[0113] Using the synthesized pst6-224 gene fragment as a template, the pst6-224 gene fragment was amplified with pst6-224-F / pst6-224-R as primers. The DNA fragment was purified and recovered, and the recovered pst6-224 gene fragment was ligated between the NcoI / BamHI restriction enzyme sites of the vector pRSFDuet-1 to obtain the plasmid pRS-pst6.

[0114] (2) Obtaining the lacY gene fragment: Using the genome of Escherichia coli K-12 as a template, the lacY gene fragment was amplified with lacY-F / lacY-R as primers. The DNA fragment was purified and recovered, and the recovered lacY gene fragment was ligated between the NdeI / XhoI restriction enzyme sites of the plasmids pRS-lst and pRS-pst6 through a seamless cloning kit (Nanjing Novoprotein Science & Technology Co., Ltd.) to finally obtain the plasmids pRS-LY and pRS-PY.

[0115] Table 3 Primers for plasmid construction

[0116]

[0117]

[0118] (3) According to the key genes in the sialyllactose metabolic synthesis pathway, the plasmids pET-BCA and pRS-LY obtained in step (1) were transferred into E. coli BL21(DE3)ΔlacZ obtained in Example 1 to obtain the engineered strain BC1; the plasmids pET-BCA and pRS-PY were transferred into E. coli BL21(DE3)ΔlacZ to obtain the engineered strain BK1. After fermentation and culture of these two strains, the products were identified as sialyllactose by HPLC and LC-MS, and their yields were 2.68 g / L of 3'-SL and 0.51 g / L of 6'-SL respectively (see Table 4). The sialyllactose metabolic pathway diagrams of strains BC1 and BK1 are as Figure 1 shown.

[0119] Example 3: Influence of modular metabolic engineering theory on the de novo synthesis pathway of sialyllactose

[0120] In order to optimize the sialyllactose synthesis pathway, the theory of modular metabolic engineering was introduced. The de novo synthesis pathway was divided into upstream and downstream modules, and the metabolic flux of the modules was changed by plasmids with 5 different copy numbers to balance the synthesis pathway.

[0121] The expression level value of the module is jointly determined by the strength of the promoter and the plasmid copy number. The plasmid copy numbers of the 5 plasmids pCOLADuet-1 (CoIA ori), pACYCDuet-1 (p15A ori), pCDFDuet-1 (CDF ori), pETDuet-1 (pBR322 ori), pRSFDuet-1 (RSF ori) used in this example were defined as 10, 20, 40, 60, 100 respectively, and the strength of the T7 promoter was defined as 5.

[0122] Using the construction method of Example 2, the gene fragment neuB was respectively ligated between the NcoI / BamHI restriction sites of the vectors pRSFDuet-1, pCDFDuet-1, pACYCDuet-1 and pCOLADuet-1 to obtain the plasmids pRS-neuB, pCD-neuB, pAC-neuB and pCO-neuB; the gene fragment neuC was respectively ligated between the PstI / HindⅢ restriction sites of the plasmids pRS-neuB, pCD-neuB, pAC-neuB and pCO-neuB to obtain the plasmids pRS-BC, pCD-BC, pAC-BC and pCO-BC; finally, the gene fragment neuA was respectively ligated between the NdeI / XhoI restriction sites of the plasmids pRS-BC, pCD-BC, pAC-BC and pCO-BC to obtain the plasmids pRS-BCA, pCD-BCA, pAC-BCA and pCO-BCA.

[0123] Using the construction method of Example 2, the gene fragments lst and pst6-224 were respectively ligated between the NcoI / BamHI restriction sites of vectors pETDuet-1, pCDFDuet-1, pACYCDuet-1, and pCOLADuet-1 to obtain plasmids pET-lst, pCD-lst, pAC-lst, pCO-lst, pET-pst6, pCD-pst6, pAC-pst6, and pCO-pst6; the gene fragment lacY was ligated between the NdeI / XhoI restriction sites of plasmids pET-lst, pCD-lst, pAC-lst, pCO-lst, pET-pst6, pCD-pst6, pAC-pst6, and pCO-pst6 to obtain plasmids pET-LY, pCD-LY, pAC-LY, pCO-LY, pET-PY, pCD-PY, pAC-PY, and pCO-PY.

[0124] Taking CMP-sialic acid as a modular node, the metabolic synthesis pathway was divided into two modules. The upstream genes of the module were neuB, neuC, and neuA, and the downstream of the module was lacY, lst, and / or pst6-224( Figure 2 ). By combining the plasmids pET-BCA, pRS-BCA, pCD-BCA, pAC-BCA, and pCO-BCA expressing the upstream gene fragments of the module and the plasmids pET-LY, pRS-LY, pCD-LY, pAC-LY, pCO-LY, pET-PY, pRS-PY, pCD-PY, pAC-PY, and pCO-PY expressing the downstream gene fragments of the module respectively, 18 engineered bacteria producing 3'-SL and 18 engineered bacteria producing 6'-SL were obtained, which were respectively designated as BC1 to BC18 and BK1 to BK18. The fermentation culture method was the same as that of Example 2. The engineered bacteria containing the recombinant plasmids pET-BCA and pRS-LY (i.e., strain BC1) and the engineered bacteria containing the recombinant plasmids pAC-BCA and pCO-PY (i.e., strain BK15) obtained the highest yields of 2.68 g / L and 0.98 g / L respectively after fermentation.

[0125] Table 4 Details of each engineered bacterium

[0126]

[0127]

[0128] Example 4: Effect of expression of de novo synthesis pathway precursor substances on sialyllactose

[0129] (1) Optimization of expression of the precursor UDP-N-acetylglucosamine

[0130] N - acetylmannosamine is an intermediate in sialic acid synthesis and is easily exported from cells. Similarly, sialic acid can be reversibly transported by the NanT enzyme. The availability of N - acetylmannosamine and sialic acid has a certain impact on the synthesis efficiency of sialyllactose. UDP - N - acetylglucosamine is the precursor of N - acetylmannosamine and sialic acid in the sialyllactose biosynthetic pathway. Improving the intracellular flux to the precursor UDP - N - acetylglucosamine can enhance the production of N - acetylmannosamine and sialic acid, and then target the fixed flow to sialyllactose. Three genes, including glmS (encoding glucosamine - 6 - phosphate synthase), glmM (encoding glucosamine synthase), and glmU (encoding UDP - N - acetylglucosamine pyrophosphorylase), are crucial for the production of UDP - N - acetylglucosamine. For this purpose, using the genome of Escherichia coli K - 12 as a template, gene fragments of glmS, glmM, and glmU were amplified with primers glmS - F / R, glmM - F / R, and glmU - F / R (the primer sequences are shown in Table 3), respectively. The DNA fragments were purified and recovered, and the recovered glmS, glmM, and glmU gene fragments were ligated between the NcoI / BamHI restriction sites of the vector pCDFDuet - 1 through a seamless cloning kit (Nanjing Novozymes Biotechnology Co., Ltd.) using the construction method of Example 2 to obtain plasmids pCD - glmS, pCD - glmM, and pCD - glmU; pCD - SM, pCD - SU, pCD - MU, and pCD - SMU were obtained using the same construction method.

[0131] Based on the strain BC1 in Example 3, pCD - glmS, pCD - glmM, pCD - glmU, pCD - SM, pCD - SU, pCD - MU, and pCD - SMU were expressed respectively to obtain 7 different engineered bacteria, which were designated as F1 - F7 respectively. Similarly, based on the strain BK15 in Example 3, the above 7 plasmids were expressed respectively to obtain 7 different engineered bacteria, which were designated as G1 - G7 respectively. The 14 different engineered bacteria were respectively subjected to shake - flask fermentation culture. Among them, the engineered bacterium F7 obtained the highest 3'-SL yield of 3.94 g / L; the engineered bacterium G6 obtained the highest 6'-SL yield of 1.63 g / L. The results showed that appropriate expression of UDP - N - acetylglucosamine could increase the yield of sialyllactose.

[0132] (2) Construction and expression of the glutamine cycle system

[0133] Fructose - 6 - phosphate and glucosamine - 6 - phosphate are important precursors in the de novo synthesis pathway of 3(6)'-SL. By constructing a glutamine cycle system, the recycling of L - glutamine + fructose - 6 - phosphate —— L - glutamate + glucosamine - 6 - phosphate can be achieved ( Figure 1)。Two genes, glmS (encoding glucosamine-6-phosphate synthase) and glnA (encoding glutamine synthetase), catalyze this cycle. For this purpose, using the Escherichia coli K-12 genome as a template, the glnA gene fragment was amplified with primers glnA-F / R (primer sequences are shown in Table 3), the DNA fragment was purified and recovered, and the recovered glnA gene fragment was ligated between the SlaI / PacI restriction sites of pET-BCA and pAC-BCA respectively through a seamless cloning kit (Nanjing Novoprotein Science & Technology Co., Ltd.) using the construction method of Example 2 to obtain plasmids pET-BCAA and pAC-BCAA.

[0134] Using Escherichia coli BL21(DE3)ΔlacZ as the host, strains F8 and G8 (Table 5) were formed by expressing the glnA gene, and 4.27 g / L and 1.87 g / L of 3'-SL and 6'-SL were obtained by shake-flask fermentation culture. This indicates that overexpression of the glnA gene promotes the production of sialyllactose.

[0135] Table 5 Details of each engineered strain

[0136]

[0137] Example 5: Effects of knocking out genes nanA, pfkA, and nagB on the yield of sialyllactose

[0138] To increase the synthesis efficiency of sialyllactose, using Escherichia coli BL21(DE3)ΔlacZ as the starting strain through the steps described in Example 1, the nanA encoding sialic acid aldolase gene, the pfkA encoding 6-phosphofructokinase gene, and the nagB encoding glucosamine-6-phosphate deaminase gene were knocked out using the CRISPR / Cas9 system, thereby blocking the loss of other metabolic pathways of precursor substances, and the knockout strains BL21(DE3)ΔlacZΔnanA, BL21(DE3)ΔlacZΔnanAΔpfkA, and BL21(DE3)ΔlacZΔnanAΔpfkAΔnagB were obtained. The plasmid combinations in Example 4 were respectively transformed into the knockout strains BL21(DE3)ΔlacZΔnanA, BL21(DE3)ΔlacZΔnanAΔpfkA, and BL21(DE3)ΔlacZΔnanAΔpfkAΔnagB to obtain strains F9 to F11 and G9 to G11. The highest 3'-SL yield of 5.94 g / L and 6'-SL yield of 3.69 g / L could be obtained by shake-flask fermentation culture (see Table 6). The yields were increased by 1.39 times and 1.97 times compared with F8 and G8 respectively, indicating that blocking the side branches of sialyllactose helps to improve the conversion of 3'-SL and 6'-SL, and the knockout of the pfkA gene has the most significant effect on the increase in the yield of sialyllactose.

[0139] Table 6 Detailed information of each engineered strain

[0140]

[0141] Example 6: Screening of sialyltransferases from different sources

[0142] Sialyltransferases allow for the structural modification of substrates to achieve the enzymatic conversion of the donor CMP-sialic acid and the acceptor lactose to the product sialyllactose. Currently, although bacterial sialyltransferases have been widely explored and their properties have been deeply characterized, their application in sialyllactose is still insufficient. For this reason, four α-2,3 sialyltransferases from prokaryotes, namely PM0188 (protein ID: AAK02272.1), WQG (protein ID: AGH37861.1), Nst (protein ID: AAC44539.1) and Lst (protein ID: AAF41330.1), and three α-2,6 sialyltransferases from different sources, namely Pst6-224 (protein ID: BAF92026.1), Plst6 (protein ID: BAI49484.1) and Bst (protein ID: BAA25316.1), and their mutants PM0188* (R313N / T265S), Nst* (I411T / L433T), Plst6* (ΔN2-15aa) and Bst* (ΔN2-15 aa) were screened.

[0143] Obtaining of Pm0188, WQG, Nst, plst6, bst, Pm0188*, Nst*, plst6* and bst* gene fragments: The gene sequences of Pm0188 and Pm0188* from Pasteurella multocida, the WQG gene sequence from Bibersteinia trehalosi, the Nst and Nst* gene sequences from Neisseria gonorrhoeae, the plst6 and plst6* gene sequences from Photobacterium leiognathi, and the bst and bst* gene sequences from Photobacterium damselae were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0144] Using the plasmid pRS-LY in Example 3 as a template, referring to the construction method in Example 2, and using Pm0188-F / Pm0188-R, WQG-F / WQG-R, Nst-F / Nst-R, Pm0188*-F / Pm0188*-R, Nst*-F / Nst*-R as primers, the plasmids pRS-Pm0188-lacY, pRS-WQG-lacY, pRS-Nst-lacY, pRS-Pm0188*-lacY, and pRS-Nst*-lacY were obtained by PCR amplification respectively;

[0145] Similarly, using the plasmid pAC-PY in Example 3 as a template, referring to the construction method in Example 2, and using plst6-F / plst6-R, bst-F / bst-R, plst6*-F / plst6*-R, bst*-F / bst*-R as primers, the plasmids pRS-plst6-lacY, pRS-bst-lacY, pRS-plst6*-lacY, and pRS-bst*-lacY were obtained by PCR amplification respectively.

[0146] Using the knockout strain BL21(DE3)ΔlacZΔnanAΔpfkAΔnagB in Example 1 as the host strain, α-2,3 sialyltransferase genes from different sources were transformed to form strains F12 - F16; α-2,6 sialyltransferase genes from different sources were transformed to form strains G12 - G15 (see Table 7 for details).

[0147] Table 7 Details of each engineered strain

[0148]

[0149] Example 7: Fed-batch production of sialyllactose in a 3L fermenter

[0150] To further verify the effectiveness of the sialyllactose synthesis method and improve the yield of sialyllactose.

[0151] The genetically engineered strains F13 and G13 constructed in Example 6 were respectively inoculated into 100 mL of LB liquid medium and cultured in a shaker flask at 37 °C and 200 r / min for 12 h to obtain seed solutions; the seed solutions were inoculated into a fermentation medium with a working volume of 1 L at an inoculation amount of 10% by volume. The fermentation temperature was 37 °C, the stirring speed was 800 r / min, the aeration rate was 1 vvm, and the pH was 6.80 (automatically controlled by adding ammonia water). Fermentation was carried out for 18 h (OD 600Approximately 15), lactose with a final concentration of 20 g / L and IPTG with a concentration of 0.5 mmol / L were added. To maintain the growth of the bacterial cells and the synthesis of sialyllactose, glycerol (containing 20 g / L of MgSO4·7H2O) at a concentration of 750 g / L was fed to supplement the carbon source after the initial carbon source was consumed, and lactose at a concentration of 200 g / L was fed after the initial lactose was consumed to maintain its concentration in the system at approximately 10 g / L, and fermentation was carried out for 55 h.

[0152] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A genetically engineered bacterium for producing lactosylsialic acid, characterized in that, Knock out the β-galactosidase-encoding gene lacZ, and heterologously express the neuB gene encoding N-acetylneuraminate synthase, the neuC gene encoding N-acetylglucosamine isomerase, the neuA gene encoding CMP-sialic acid synthase, the lactose permease-encoding gene lacY, the glutamine synthetase-encoding gene glnA, and the UDP-N-acetylglucosamine synthesis pathway; overexpress the gene encoding sialyltransferase; The UDP-N-acetylglucosamine synthesis pathway is the glucosamine-6-phosphate synthase-encoding gene glmS, the glucosamine synthase-encoding gene glmM, and the UDP-N-acetylglucosamine pyrophosphorylase-encoding gene glmU; The genetically engineered bacterium also knocks out the sialic acid aldolase-encoding gene nanA, the 6-phosphofructokinase-encoding gene pfkA, and the glucosamine-6-phosphate deaminase-encoding gene nagB; The sialyltransferase gene is the α-2,3 sialyltransferase gene; The α-2,3 sialyltransferase gene is selected from the mutant Pm0188* of Pasteurella multocida Pasteurella multocida ; Use pETDuet-1 to overexpress the neuB gene, neuC gene, neuA gene, and gene glnA, use pRSFDuet-1 to overexpress the gene lacY and the gene encoding sialyltransferase, and use pCDFDuet-1 to overexpress the glucosamine-6-phosphate synthase-encoding gene glmS, the glucosamine synthase-encoding gene glmM, and the UDP-N-acetylglucosamine pyrophosphorylase-encoding gene glmU; The nucleotide sequences of the neuB gene, neuC gene, and neuA gene are shown in SEQ ID NO.1 to SEQ ID NO.3 respectively; The nucleotide sequence of the lactose permease-encoding gene lacY is shown in SEQ ID NO:6; The nucleotide sequences of the glucosamine-6-phosphate synthase-encoding gene glmS, the glucosamine synthase-encoding gene glmM, and the UDP-N-acetylglucosamine pyrophosphorylase-encoding gene glmU are shown in SEQ ID NO.7 to SEQ ID NO.9 respectively; the nucleotide sequence of the glutamine synthetase-encoding gene glnA is shown in SEQ ID NO:10; The nucleotide sequence of the gene mutant Pm0188* is shown in SEQ ID NO.14; The genetically engineered bacterium uses Escherichia coli as the host.

2. A genetically engineered bacterium for producing lactosylsialic acid, characterized in that, Knock out the β-galactosidase-encoding gene lacZ, and heterologously express the neuB gene encoding N-acetylneuraminate synthase, the neuC gene encoding N-acetylglucosamine isomerase, the neuA gene encoding CMP-sialic acid synthase, the lactose permease-encoding gene lacY, the glutamine synthetase-encoding gene glnA, and the UDP-N-acetylglucosamine synthesis pathway; overexpress the gene encoding sialyltransferase; The UDP-N-acetylglucosamine synthesis pathway is the glucosamine synthase-encoding gene glmM and the UDP-N-acetylglucosamine pyrophosphorylase-encoding gene glmU; The genetically engineered bacterium also knocks out the sialic acid aldolase encoding gene nanA, the 6-phosphofructokinase encoding gene pfkA, and the glucosamine-6-phosphate deaminase encoding gene nagB; The sialyltransferase gene is an α-2,6 sialyltransferase gene; The α-2,6 sialyltransferase gene is selected from the mutant plst6* of Photobacterium leiognathi Photobacterium leiognathi ; Using pETDuet-1 to overexpress the neuB gene, neuC gene, neuA gene, and glnA gene, using pRSFDuet-1 to overexpress the lacY gene and the sialyltransferase encoding gene, and using pCDFDuet-1 to overexpress the glucosamine synthase encoding gene glmM and the UDP-N-acetylglucosamine pyrophosphorylase encoding gene glmU; The nucleotide sequences of the neuB gene, neuC gene, and neuA gene are shown in SEQ ID NO.1 to SEQ ID NO.3 respectively; The nucleotide sequence of the lactose permease encoding gene lacY is shown in SEQ ID NO.6; The nucleotide sequences of the glucosamine synthase encoding gene glmM and the UDP-N-acetylglucosamine pyrophosphorylase encoding gene glmU are shown in SEQ ID NO:8 to SEQ ID NO:9 respectively; The nucleotide sequence of the glutamine synthase encoding gene glnA is shown in SEQ ID NO:10; The nucleotide sequence of the gene mutant plst6* is shown in SEQ ID NO.18; The genetically engineered bacterium uses Escherichia coli as the host.

3. A method for increasing the production of lactosylsialic acid, characterized in that, Knock out the β-galactosidase encoding gene lacZ, the sialic acid aldolase encoding gene nanA, the 6-phosphofructokinase encoding gene pfkA, and the glucosamine-6-phosphate deaminase encoding gene nagB on the Escherichia coli genome, Using pETDuet-1 to overexpress the neuB gene, neuC gene, neuA gene, and glnA gene, using pRSFDuet-1 to overexpress the lacY gene and the sialyltransferase encoding gene, and using pCDFDuet-1 to overexpress the glucosamine-6-phosphate synthase encoding gene glmS, the glucosamine synthase encoding gene glmM, and the UDP-N-acetylglucosamine pyrophosphorylase encoding gene glmU; The sialyltransferase gene is an α-2,3 sialyltransferase gene; The α-2,3 sialyltransferase gene is selected from the mutant Pm0188* of Pasteurella multocida Pasteurella multocida; The nucleotide sequences of the neuB gene, neuC gene, and neuA gene are shown in SEQ ID NO.1 to SEQ ID NO.3 respectively; The nucleotide sequence of the lacY gene is shown in SEQ ID NO:6; The nucleotide sequences of the glucosamine-6-phosphate synthase encoding gene glmS, the glucosamine synthase encoding gene glmM, and the UDP-N-acetylglucosamine pyrophosphorylase encoding gene glmU are shown in SEQ ID NO:7 to 9 respectively; The nucleotide sequence of the gene glnA is shown as SEQ ID NO.10; the nucleotide sequence of the gene mutant Pm0188* is shown as SEQ ID NO.

14.

4. A method for increasing the production of lactosylsialic acid, characterized in that, Knock out the β-galactosidase encoding gene lacZ, the sialic acid aldolase encoding gene nanA, the 6-phosphofructokinase encoding gene pfkA, and the glucosamine-6-phosphate deaminase encoding gene nagB on the Escherichia coli genome. Overexpress the neuB gene, the neuC gene, the neuA gene, and the gene glnA using pETDuet-1, overexpress the gene lacY and the sialyltransferase encoding gene using pRSFDuet-1, and overexpress the glucosamine synthase encoding gene glmM and the UDP-N-acetylglucosamine pyrophosphorylase encoding gene glmU using pCDFDuet-1. The sialyltransferase gene is the α-2,6 sialyltransferase gene. The α-2,6 sialyltransferase gene is selected from the gene mutant plst6* of Photobacterium leiognathi Photobacterium leiognathi ; The nucleotide sequences of the neuB gene, the neuC gene, and the neuA gene are shown as SEQ ID NO.1 to SEQ ID NO.3 respectively. The nucleotide sequence of the gene lacY is shown as SEQ ID NO:

6. The nucleotide sequences of the glucosamine synthase encoding gene glmM and the UDP-N-acetylglucosamine pyrophosphorylase encoding gene glmU are shown as SEQ ID NO:8 to 9 respectively. The nucleotide sequence of the gene glnA is shown as SEQ ID NO.

10. The nucleotide sequence of the gene mutant plst6* is shown as SEQ ID NO.

18.

5. A method for producing lactosylsialic acid, characterized in that, Using glycerol as the carbon source and lactose as the substrate, ferment and produce lactosylsialic acid using the genetically engineered bacterium described in claim 1 or 2 as the fermentation strain.

6. Use of the genetically engineered bacterium described in claim 1 or 2, or the method described in any one of claims 3-5, in the production of lactosylsialic acid and products containing lactosylsialic acid.

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