CN116478894A

CN116478894A - Genetically engineered bacterium for improving sialyllactose yield and production method thereof

Info

Publication number: CN116478894A
Application number: CN202310277210.4A
Authority: CN
Inventors: 张涛; 李晨晨; 李梦丽
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2023-03-21
Filing date: 2023-03-21
Publication date: 2023-07-25

Abstract

The invention discloses a genetic engineering bacterium for improving sialyllactose yield and a production method thereof, belonging to the fields of biotechnology and food fermentation engineering. The invention constructs the secondary synthesis path of sialyllactose by expressing genes neuB, neuC, neuA, lacY, lst and/or pst6-224, balances the metabolic pathway by modularized metabolic engineering, increases the expression of the precursor UDP-acetylglucosamine related synthetic gene, constructs a glutamine circulation system, knocks out lacZ, nanA, pfkA and nagB expression of a bypass path in the escherichia coli genome sialyllactose synthesis path, and screens alpha-2, 3 (6) sialyltransferase from different sources to successfully obtain the production strain for efficiently synthesizing sialyllactose, wherein the 3'-SL and 6' -SL yields respectively reach 6.46g/L and 4.07g/L in fermentation culture experiments.

Description

Genetically engineered bacterium for improving sialyllactose yield and production method thereof

Technical Field

The invention relates to a genetic engineering bacterium for improving sialyllactose yield and a production method thereof, belonging to the fields of biotechnology and food fermentation engineering.

Background

The human milk oligosaccharides (human milk oligosaccharides, HMOs) have the effects of regulating the microbial activity of intestinal tracts, improving immune response, preventing necrotizing enterocolitis and the like, are the third largest solid nutritional ingredient next to lactose and lipid in breast milk, and play an important role in the steady state and development of the digestive system of infants and the perfection and establishment of the postnatal immune system. The addition of HMOs to infant formula can reduce the nutritional gap between breast milk and formula. Sialyllactose (3 (6) '-sialylactose, 3 (6)' -SL) is an important component in HMOs, formed by a-2, 3 (6) sialyltransferase performing a transgalactosylation reaction on CMP-sialic acid, and in vitro studies indicate that: 3'-SL and 6' -SL in HMOs support the normal microflora and behavioral response during stress by modulating the gut-brain axis. Therefore, the infant fed by breast milk has more perfect brain development, richer nerve synapses and more developed nervous system. In view of the important biological efficacy of sialyllactose, to further elucidate its mechanism of action, a large number of compounds of this type with uniform structure need to be prepared, however, the amount of compounds obtained by separation and extraction from natural products is very small and far from the needs of research, so that the obtaining of these compounds by artificial synthesis is the best choice.

The synthesis method of sialyllactose mainly comprises three steps: respectively chemical synthesis, enzymatic synthesis and biological fermentation synthesis. The chemical synthesis of sialyllactose requires numerous cumbersome protection and deprotection steps. Enzymatic synthesis is unsuitable for industrial application because of the relatively high cost and low yield of the donor substrate nucleotide sugar, which makes the cost of the synthesized sialyllactose high. The biosynthesis of sialyllactose using engineered strains, which can produce sialyllactose from inexpensive carbon sources (glucose, glycerol, lactose), is receiving increasing attention.

At present, regarding the preparation of sialyllactose by biological fermentation, 25.5g/L of 3' -SL is generated by knocking out nanT, nanA, nanK, nanE, over-expressing neuB, neuA, neuC and alpha-2, 3 sialyltransferase by Eric Samain et al in 2008; in 2010, the subject group constructed a 6' -SL pathway in the same manner, and only 3' -SL was modified from the α -2,3 sialyltransferase in the de novo synthesis pathway to α -2,6 sialyltransferase, and 34g/L of 6' -SL was obtained by fermentation as the highest yield. The currently reported methods of microbial production and the yields of sialyllactose are still not satisfactory for industrial production. Thus, the search for inexpensive and high-yielding sialyllactose de pathway from the head to solve the bottleneck of current microbial production, and the creation of more efficient production strains is a current urgent need to solve.

Disclosure of Invention

[ technical problem ]

The prior art for producing sialyllactose has relatively high cost and low yield, is not enough to realize industrial production, cannot provide a strain for efficiently producing sialyllactose, and cannot provide a method for producing sialyllactose with low cost and green efficiency.

Technical scheme

The invention provides a genetic engineering bacterium for producing sialyllactose and a construction method thereof, which aim to solve the problem of low yield of sialyllactose synthesized by the existing biological method.

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

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

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

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

In one embodiment, the nucleotide sequences of the acetylneuraminic acid synthetase genes neuB, the N-acetylglucosamine isomerase gene neuC and the CMP-sialic acid synthetase gene neuA are shown as SEQ ID NO. 1-SEQ ID NO. 3.

In one embodiment, the lactose transferase gene lacY, the glucosamine-6-phosphate synthase gene glmS, the glucosamine synthase gene glmM, the UDP-acetylglucosamine pyrophosphorylase gene glmU and the glutamine synthase gene glnA are derived from E.coli K-12, and the nucleotide sequences thereof are shown in SEQ ID NO.6 to SEQ ID NO. 10.

In one embodiment, the genetically engineered bacterium hosts escherichia coli.

In one embodiment, the E.coli includes MG1655, DH 5. Alpha., BL21 (DE 3), JM109, or HB101.

In one embodiment, the E.coli is E.coli BL21.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA, lacY, glmM, glmS, glmU, glnA, a sialyltransferase encoding gene using pETDuet-1, pRSFDuet-1, pCDFDuet-1, pACYCDuet-1 or pCOLADuet-1 plasmids.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pETDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pRSFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pETDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pCDFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pETDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pACYCDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pETDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pCOLADuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pRSFDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pETDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pRSFDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pCDFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pRSFDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pACYCDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using the pCDFDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using the pETDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using the pCDFDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using the pRSFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using the pCDFDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using the pACYCDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using the pCDFDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using the pCOLADuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pACYCDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pETDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pACYCDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pRSFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pACYCDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pCDFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pACYCDuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pCOLADuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pcoladat-1 plasmid and gene lacY, sialyltransferase encoding gene using petdouet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pCOLADuet-1 plasmid and gene lacY, sialyltransferase encoding gene using pCDFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using a pcoladat-1 plasmid and gene lacY, a sialyltransferase encoding gene using a pacycdat-1 plasmid.

In one embodiment, the sialyltransferase gene is an alpha-2, 3 sialyltransferase gene or an alpha-2, 6 sialyltransferase gene.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pETDuet-1 plasmid, gene lacY, alpha-2, 3 sialyltransferase gene using pRSFDuet-1 plasmid, and gene glmM using pCDFDuet-1 plasmid.

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

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

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

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

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

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

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

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pACYCDuet-1 plasmid, gene lacY, alpha-2, 6 sialyltransferase gene using pCOLADuet-1 plasmid, and gene glmS using pCDFDuet-1 plasmid.

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pACYCDuet-1 plasmid, gene lacY, alpha-2, 6 sialyltransferase gene using pCOLADuet-1 plasmid, and gene glmU using pCDFDuet-1 plasmid.

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

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

In one embodiment, the genetically engineered bacterium expresses gene neuB, neuC, neuA using pACYCDuet-1 plasmid, gene lacY, alpha-2, 6 sialyltransferase gene using pCOLADuet-1 plasmid, and genes glmU and glmS using pCDFDuet-1 plasmid.

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

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

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

In one embodiment, the gene encoding α -2,3 sialyltransferase is selected from the group consisting of gene lst derived from neisseria meningitidis Neisseria meningitidis, gene Pm0188 derived from pasteurella multocida Pasteurella multocida, mutant gene Pm0188, gene WQG derived from baiptam seaweed Bibersteinia trehalosi, gene Nst derived from neisseria gonorrhoeae, and mutant gene Nst.

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

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

In one embodiment, the nucleotide sequence of gene Pm0188 is shown in SEQ ID No.11, the nucleotide sequence of gene WQG is shown in SEQ ID No.12, the nucleotide sequence of gene Nst is shown in SEQ ID No.13, the nucleotide sequence of gene Pm0188 is shown in SEQ ID No.14, and the nucleotide sequence of gene Nst is shown in SEQ ID No. 15.

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

The invention provides a method for improving sialyllactose yield, which comprises the steps of knocking out beta-galactosidase encoding genes lacZ, sialylaldehyde aldolase encoding genes nanA, 6-phosphofructokinase encoding genes pfkA and glucosamine-6 phosphodeaminase encoding genes nagB in escherichia coli genome, and utilizing an expression vector to heterologously express acetylneuraminic acid synthase genes neuB, N-acetylglucosamine isomerase genes neuC, CMP-sialic acid synthase genes neuA, sialyltransferase genes, lactose transferase genes lacY, glucosamine-6-phosphate synthase genes glmS, glucosamine synthase genes glmM, UDP-acetylglucosamine pyrophosphorylase genes glmU and glutamine synthase genes glnA.

In one embodiment, the α -2,3 sialyltransferase gene is selected from lst, pm0188, WQG, nst, pm0188, or Nst; the alpha-2, 6 sialyltransferase gene is selected from pst6-224, plst6, bst, plst6 or bst.

In one embodiment, the overexpression is performed using pETDuet-1, pRSFDuet-1, pCDFDuet-1, pACYCDuet-1 or pCOLADuet-1 plasmids.

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

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

In one embodiment, the neuB gene, the neuC gene, the neuA gene, and the 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, the glucosamine synthase encoding gene glmM, and/or the UDP-acetylglucosamine pyrophosphorylase encoding gene glmU are overexpressed using pCDFDuet-1.

In one embodiment, the sialyllactose comprises 3'-SL and 6' -SL.

The invention also provides a method for producing sialyllactose, which is characterized in that glycerol is used as a carbon source, lactose is used as a substrate, and the genetic engineering bacteria are used as fermentation strains for fermentation to produce the sialyllactose.

In one embodiment, the genetically engineered bacterium is inoculated into a fermentation medium and cultured until the OD ₆₀₀ 10 to 15, addLactose with final concentration of 15-25 g/L and IPTG with 0.2-1.0 mM.

In one embodiment, 600 to 850g/L glycerol and 15 to 25g/LMgSO are fed after the initial carbon source is consumed ₄ ·7H ₂ O; after the initial lactose is consumed, lactose is fed in to maintain the concentration of lactose at 5-10 g/L.

In one embodiment, the fermentation conditions are that the culture temperature is 20-37 ℃, the stirring rotation speed is 200-850 r/min, the ventilation rate is 0.8-2.0 vvm, the pH is 6.5-7.0, and the fermentation time is 15-55 h.

In one embodiment, the fermentation medium has a composition of: 10-20 g/L glycerin, 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.

In one embodiment, the trace metal element comprises the following composition: 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 heptamolybdate and 1.5-2.5 g/L calcium chloride dihydrate.

The invention also provides application of the genetically engineered bacterium or the method for improving the yield of sialyllactose in producing sialyllactose and products containing sialyllactose.

The beneficial effects are that:

according to the invention, the output of sialyllactose is regulated and controlled by using CMP-sialic acid as a node through exogenous expression of acetylneuraminic acid synthase gene neuB, N-acetylglucosamine isomerase gene neuC, CMP-sialic acid synthase gene neuA, alpha-2, 3 sialyltransferase gene lst and/or alpha-2, 6 sialyltransferase gene pst6-224 and overexpression of lactose transferase gene lacY in a modular combination manner. And further regulates the generation of the precursor substance UDP-acetylglucosamine in a combined manner by expressing the genes glmS (glucosamine-6-phosphate synthase), glmM (encoding glucosamine synthase) and glmU (encoding UDP-acetylglucosamine pyrophosphorylase) of the precursor substance UDP-acetylglucosamine in the sialyllactose metabolic pathway, thereby targeting constant flow sialyllactose. Meanwhile, the balance of the glutamine circulation regulating precursor substance glucosamine-6-phosphate is established by expressing the gene glnA (encoding glutamine synthetase). In addition, the β -galactosidase gene lacZ, the sialylaldehyde aldolase gene nanA, the 6-phosphofructokinase gene pfkA and the glucosamine-6-phosphate deaminase gene nagB were knocked out to block the shunt metabolism of the bypass pathway. Finally, the speed limiting enzyme in the synthesis path of alpha-2, 3 (6) sialyltransferase-sialyllactose from different sources is screened, and the aim of improving sialyllactose is achieved through a series of operations.

Through fermentation culture, the capacity of the engineering bacteria constructed by the invention for producing sialyllactose, namely 3'-SL, is improved to 6.46g/L from initial 2.68g/L, and 6' -SL is improved to 4.07g/L from initial 0.51g/L, so that the production capacity of the engineering bacteria lays a foundation for industrial production of sialyllactose.

Drawings

FIG. 1 is a diagram of sialyllactose metabolic pathway;

FIG. 2 is a schematic diagram of modular metabolism.

Detailed Description

The present invention will be further described with reference to examples and drawings, wherein plasmids, PCR reagents, restriction enzymes, plasmid extraction kits, DNA gel recovery kits, etc. used in the following examples are commercially available, and the specific operations are carried out according to the kit instructions.

Embodiments of the invention are not limited thereto, and other unspecified experimental operations and process parameters are conducted in accordance with conventional techniques.

Sequencing of plasmid and DNA products was done by Souzhou Jin Weizhi Biotechnology Inc.

Preparation of E.coli competence: kit for biological engineering (Shanghai) Co., ltd.

LB liquid medium (g/L): yeast extract 5, tryptone 10, sodium chloride 10.

LB solid medium (g/L): yeast extract 5, tryptone 10, sodium chloride 10, agar powder 20.

Fermentation medium (g/L): glycerin 20, citric acid 1.7, monopotassium phosphate 13.5, diammonium phosphate 4, magnesium sulfate heptahydrate 1.4, trace metal solution 10mL/L (ferric citrate 10g/L, zinc sulfate heptahydrate 2.25g/L, cupric sulfate pentahydrate 1.0g/L, manganese sulfate monohydrate 0.35g/L, borax 0.23g/L, ammonium heptamolybdate 0.11g/L, calcium chloride dihydrate 2.0 g/L), and pH 6.80.

The sialyllactose described in the embodiment of the present invention is determined by using High Performance Liquid Chromatography (HPLC), specifically:

the fermentation broth was boiled at 100deg.C for 10min to break the cells, centrifuged at 12000r/min for 10min, and the supernatant was filtered through 0.22 μm membrane and detected by HPLC.

HPLC detection conditions: a differential refractive detector; the chromatographic column is Rezex ROA-organic acid (Phenomenex, USA) with the column temperature of 50 ℃; h with mobile phase of 0.005mmol/L ₂ SO ₄ The flow rate of the aqueous solution is 0.6mL/min; the sample loading was 10. Mu.L.

The shake flask fermentation culture method comprises the following steps:

engineering bacteria colonies cultured overnight on LB solid medium are selected and inoculated into 5mLLB liquid medium, and cultured for 12 hours at 37 ℃ and 200r/min to be used as seed liquid. Transferring seed solution according to 1% (v/v) inoculum size into 50mL fermentation medium, and culturing at 37deg.C and 200r/min to obtain thallus OD ₆₀₀ The value is 0.6-0.8, IPTG is added to make the final concentration of the mixture be 0.2mmol/L, and lactose is added to make the final concentration of lactose be 10g/L, and the mixture is induced and cultured for 50h under the conditions of 25 ℃ and 200 r/min.

The E.coli expression vector in the embodiment of the invention specifically comprises:

TABLE 1 expression vectors involved in the examples below

Example 1: knockout of E.coli BL21 (DE 3) genomic gene lacZ, nanA, pfkA and nagB

The lacZ, nanA, pfkA and nagB genes in the escherichia coli BL21 (DE 3) genome are knocked out by using a CRISPR-Cas9 gene knockout system, and the specific steps are as follows (the related primer sequences are shown in Table 2):

(1) Homologous upstream and downstream fragments of lacZ, nanA, pfkA and nagB were amplified by PCR using E.coli BL21 (DE 3) genomic DNA as template and 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, respectively. After the product is purified and recovered, homologous upstream and downstream fragments of lacZ, nanA, pfkA and nagB are respectively used as templates, and the primers of 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 are used for amplifying and connecting the upstream and downstream fragments by SOE-PCR technology to obtain homologous repair arms donor-lacZ, donor-nanA, donor-pfkA and donor-nagB, and the required DNA fragments are purified and recovered.

(2) The N20 sequence complementary to the lacZ, nanA, pfkA and nagB sequences was introduced into pTargetF plasmid by PCR amplification using pTargetF plasmid (Addgene: # 62226) as template and lacZ-sg-F/R, nanA-sg-F/R, pfkA-sg-F/R and nagB-sg-F/R as primers, respectively, to obtain pTargetF plasmid with targeting lacZ, nanA, pfkA and nagB (i.e. targeting plasmids pTargetF-lacZ, pTargetF-nanA, pTargetF-pfkA and pTargetF-nagB with lacZ, nanA, pfkA and nagB specific N20 sequences) respectively, E.coli DH 5. Alpha. Competent cells were transformed with PCR amplification products, LB plates (containing spectinomycin) were coated, extracted by overnight culture at 37℃and sequenced.

(3) E.coli BL21 (DE 3) competent cells were thawed on ice for 10min, 5. Mu.L of pCas plasmid (Addgene: # 60847) was added to 100. Mu.L of competent cells and gently mixed. Ice bath for 30min, heat shock at 42 ℃ for 90s, and immediately placing on ice for 2-3 min. 1mL of fresh LB liquid medium is added, after 1h of culture at 30 ℃ and 200r/min, centrifugation is carried out at 3500r/min for 5min, the supernatant is discarded, the thalli are coated on an LB plate containing kanamycin, and the thalli are placed in a constant temperature incubator at 30 ℃ for overnight culture until single colonies of E.coli BL21 (DE 3)/pCas are grown.

(4) Picking up E.coli BL21 (DE 3)/pCas single colony overnight culture seed liquid, transferring LB liquid culture medium according to 1% volume ratio, and culturing OD at 30deg.C ₆₀₀ To 0.2, D-arabinose with a final concentration of 30mmol/L was added to induce pCas-lambda-red system expression, and the culture was continued until the logarithmic growth phase was reached to prepare E.coli BL21 (DE 3)/pCas competence.

(5) E.coli BL21 (DE 3)/pCas competence was electrotransformed with pTargetF-lacZ plasmid (total 500 ng) and gene homology repair arm donor-lacZ (total 1 μg), spread on LB plates (kanamycin and spectinomycin), incubated overnight at 30℃and single colonies on plates were picked for PCR validation, positive transformants were screened and sent to Suzhou Jin Weizhi Biotechnology Co.Ltd.

(6) Culturing the positive clone colony which is successfully knocked out by sequencing and verifying in the step (5) in LB liquid medium containing kanamycin until OD ₆₀₀ The value reaches 0.2, IPTG with the final concentration of 0.5mmol/L is added, the mixture is cultured for 12 to 16 hours at 30 ℃ to remove pTargetF plasmid, then the mixture is cultured for 12 hours at 42 ℃, pCas plasmid is removed, and E.coll BL21 (DE 3) delta lacZ of the genome knockout lacZ gene is obtained.

(7) Taking E.coli BL21 (DE 3) DeltalacZ as host bacteria, sequentially knocking out the genes nanA, pfkA and nagB, and performing knocking-out operation by referring to the knocking-out of the genes lacZ, thereby obtaining corresponding BL21 (DE 3) DeltalacZ DeltananA, deltapfkA and BL21 (DE 3) DeltalacZ DeltapnapkA DeltanagB strains.

TABLE 2sgRNA and knockout primers

Example 2: construction of sialyllactose de novo synthesis pathway recombinant bacteria

The construction of recombinant bacteria comprises the following specific steps (the related primer sequences are shown in Table 3):

(1) neuB, neuC, neuA, lst and pst6-224 Gene fragment acquisition: the sequence of neuB, neuC, neuA gene derived from Campylobacter jejuni, lst gene derived from Neisseria meningitidis Neisseria meningitidis, and pst6-224 gene derived from Photobacterium sp.

Amplifying a neuB gene fragment by taking the synthesized neuB gene fragment as a template and a neuB-F/neuB-R as a primer, purifying and recovering a DNA fragment, and connecting the recovered gene fragment neuB to NcoI/BamHI cleavage sites of a vector pETDuet-1 through a seamless cloning kit (Nanjinouzan life technologies Co., ltd.) to obtain a plasmid pET-neuB;

amplifying a neuC gene fragment by taking the synthesized neuC gene fragment as a template and a neuC-F/neuC-R as a primer, purifying and recovering a DNA fragment, and connecting the recovered neuC gene fragment between PstI/HindIII enzyme cutting sites of a vector plasmid pET-neuB to obtain a plasmid pET-BC;

the synthesized neuA gene fragment is used as a template, the neuA-F/neuA-R is used as a primer to amplify the neuA gene fragment, the DNA fragment is purified and recovered, and the recovered neuA gene fragment is connected between NdeI/XhoI cleavage sites of a vector pET-BC to obtain a plasmid pET-BCA.

The synthesized lst gene fragment is used as a template, the lst-F/lst-R is used as a primer to amplify lst gene fragment, the DNA fragment is purified and recovered, and the recovered lst gene fragment is connected between NcoI/BamHI cleavage sites of the vector pRSFDuet-1 to obtain plasmid pRS-lst.

The synthesized pst6-224 gene segment is used as a template, the pst6-224-F/pst6-224-R is used as a primer to amplify the pst6-224 gene segment, the DNA segment is purified and recovered, and the recovered pst6-224 gene segment is connected between NcoI/BamHI enzyme cutting sites of a vector pRSFDuet-1 to obtain a plasmid pRS-pst6.

(2) Obtaining of lacY Gene fragment: the genome of Escherichia coli K-12 is used as a template, lacY-F/lacY-R is used as a primer to amplify a lacY gene fragment, DNA fragments are purified and recovered, the recovered lacY gene fragment is respectively connected between NdeI/XhoI cleavage sites of plasmids pRS-lst and pRS-pst6 through a seamless cloning kit (Nanjinopran life technologies Co., ltd.) to finally obtain plasmids pRS-LY and pRS-PY.

TABLE 3 plasmid construction primers

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(3) Transferring the plasmids pET-BCA and pRS-LY obtained in the step (1) into E.coli BL21 (DE 3) DeltalacZ obtained in the example 1 according to key genes in a sialyllactose metabolic synthesis path to obtain engineering bacteria BC1; the plasmids pET-BCA and pRS-PY were transferred into E.coli BL21 (DE 3) ΔlacZ to obtain engineering bacterium BK1. After fermentation culture of these two strains, the product was confirmed to be sialyllactose by HPLC and LC-MS identification, and the yields were 2.68g/L of 3'-SL and 0.51g/L of 6' -SL, respectively (see Table 4). The sialyllactose metabolic pathway diagrams of strains BC1 and BK1 are shown in fig. 1.

Example 3: influence of modularized metabolic engineering theory on production of sialyllactose from the head synthesis pathway

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

The expression level of the module is determined by the promoter strength and plasmid copy number. The 5 plasmids used in this example, pCOLADuet-1 (CoIA ori), pACYCDuet-1 (p 15A ori), pCDFDuet-1 (CDF ori), pETDuet-1 (pBR 322 ori), pRSFDuet-1 (RSF ori) were defined as 10, 20, 40, 60, 100, respectively, and the degree of T7 promoter was defined as 5.

The gene fragment neuB was ligated between NcoI/BamHI cleavage sites of vectors pRSFDuet-1, pCDFDuet-1, pACYCDuet-1 and pCOLADuet-1, respectively, using the construction method of example 2 to obtain plasmids pRS-neuB, pCD-neuB, pAC-neuB and pCO-neuB; the gene fragment neuC is respectively connected between PstI/HindIII cleavage sites of plasmids pRS-neuB, pCD-neuB, pAC-neuB and pCO-neuB to obtain plasmids pRS-BC, pCD-BC, pAC-BC and pCO-BC; finally, the gene fragment neuA was ligated between NdeI/XhoI cleavage sites of plasmids pRS-BC, pCD-BC, pAC-BC and pCO-BC, respectively, to obtain plasmids pRS-BCA, pCD-BCA, pAC-BCA and pCO-BCA.

The gene fragments lst and pst6-224 were ligated between NcoI/BamHI cleavage sites of the vectors pETDuet-1, pCDFDuet-1, pACYCDuet-1 and pCOLADuet-1, respectively, using the construction method of example 2, 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 NdeI/XhoI cleavage sites of plasmids pET-lst, pCD-lst, pAC-lst, pCO-lst, pET-pst6, pCD-pst6, pAC-pst6 and pCO-pst6, respectively, to obtain plasmids pET-LY, pCD-LY, pAC-LY, pCO-LY, pET-PY, pCD-PY, pAC-PY and pCO-PY.

The metabolic synthesis pathway was divided into two modules with CMP-sialic acid as the modular node, neuB, neuC, neuA for the upstream gene and lacY, lst and/or pst6-224 downstream (FIG. 2). By combining plasmids pET-BCA, pRS-BCA, pCD-BCA, pAC-BCA and pCO-BCA of gene fragments upstream of the expression module, and plasmids pET-LY, pRS-LY, pCD-LY, pAC-LY, pCO-LY, pET-PY, pRS-PY, pCD-PY, pAC-PY and pCO-PY of gene fragments downstream of the expression module, respectively, 18 engineering bacteria producing 3'-SL and 18 engineering bacteria producing 6' -SL were obtained, which were designated BC1 to BC18 and BK1 to BK18, respectively. The fermentation culture method is the same as in example 2, and the engineering bacteria containing the recombinant plasmids pET-BCA and pRS-LY (namely the strain BC 1) and the engineering bacteria containing the recombinant plasmids pAC-BCA and pCO-PY (namely the strain BK 15) respectively obtain the highest yields of 2.68g/L and 0.98g/L after fermentation of the engineering strain.

TABLE 4 engineering bacteria detailed information

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Example 4: effect of expression of precursor substances of the de novo Synthesis pathway on sialyllactose

(1) Optimized expression of the precursor UDP-acetylglucosamine

Acetylmannosamine is an intermediate in sialic acid synthesis and is easily derived from cells. Also, sialic acid can be reversibly transported by the NanT enzyme. The availability of acetylmannosamine and sialic acid has a certain impact on the efficiency of sialyllactose synthesis. UDP-acetylglucosamine is a precursor of acetylmannosamine and sialic acid in the sialyllactose biosynthetic pathway. Improving intracellular flux to the precursor substance UDP-acetylglucosamine to enhance the production of acetylmannosamine and sialic acid, and then targeting the flux to sialyllactose. Three genes, including glmS (encoding glucosamine-6-phosphate synthase), glmM (encoding glucosamine synthase) and glmU (encoding UDP-acetylglucosamine pyrophosphorylase), are critical in the production of UDP-acetylglucosamine. For this, the gene fragments of glmS, glmM and glmU were amplified with primers glmS-F/R, glmM-F/R and glmU-F/R (primer sequences are shown in Table 3) respectively, and the DNA fragments were recovered by purification, and the recovered glmS, glmM and glmU gene fragments were ligated between NcoI/BamHI cleavage sites of vector pCDFDuet-1 by the construction method of example 2, respectively, using a seamless cloning kit (Nannoopran life technologies Co., ltd.) to obtain plasmids pCD-glmS, pCD-glmM and pCD-glmU; pCD-SM, pCD-SU, pCD-MU, pCD-SMU were obtained by the same construction method.

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 engineering bacteria, which were designated as F1 to F7, respectively. Similarly, 7 plasmids were expressed on the basis of the strain BK15 in example 3, and 7 different engineering bacteria were obtained, which were denoted as G1 to G7. Carrying out shake flask fermentation culture on 14 different engineering bacteria respectively, wherein engineering bacteria F7 obtain the highest 3' -SL yield of 3.94g/L; the engineering bacterium G6 obtains the highest 6' -SL yield of 1.63G/L. The results indicate that the appropriate expression of UDP-acetylglucosamine can increase the production of sialyllactose.

(2) Construction and expression of glutamine circulation System

Fructose-6-phosphate and glucosamine-6-phosphate are important precursors for the de novo synthesis pathway of 3 (6)' -SL. The cyclic utilization of L-glutamine + fructose-6-phosphate, L-glutamic acid + glucosamine-6-phosphate, can be achieved by constructing a glutamine circulating system (figure 1). Two genes, glmS (encoding glucosamine-6-phosphate synthase) and glnA (encoding glutamine synthase), catalyze this cycle. For this, the glnA gene fragment was amplified with the E.coli K-12 genome as a template and with the primers glnA-F/R (primer sequences shown in Table 3), and the DNA fragment was recovered by purification, and the recovered glnA gene fragment was connected between the SlaI/PacI cleavage sites of pET-BCA and pAC-BCA, respectively, by the construction method of example 2, using a seamless cloning kit (Nannuo-Tokida Seiko Co., ltd.) to obtain plasmids pET-BCA and pAC-BCA.

Strains F8 and G8 (Table 5) were formed by expressing the glnA gene using E.coli BL21 (DE 3) DeltalacZ as a host, and 4.27G/L and 1.87G/L of 3'-SL and 6' -SL were obtained by shake flask fermentation culture. This suggests that overexpression of the glnA gene promotes sialyllactose production.

TABLE 5 engineering bacteria detailed information

Example 5: effect of knockout genes nanA, pfkA and nagB on sialyllactose production

In order to increase the synthesis efficiency of sialyllactose, the steps described in example 1 were used to knock out the nanA of the encoding sialylaldehyde aldolase gene, the pfkA of the 6-phosphofructokinase gene and the nagB of the glucosamine-6 phosphodeaminase gene using the CRISPR/Cas9 system with escherichia coli BL21 (DE 3) Δlacz as an initial strain, thereby blocking the loss of other metabolic pathways of the precursor substances, and obtaining knock-out strains BL21 (DE 3) Δlacz Δnana, BL21 (DE 3) Δlacz Δpnak, and BL21 (DE 3) Δlacz Δnana Δpfka Δnagb. The plasmid combinations of example 4 were transformed into knockout strains BL21 (DE 3) ΔlacZ ΔnanA, BL21 (DE 3) ΔlacZ ΔnanA ΔpfkA and BL21 (DE 3) ΔlacZ ΔnanA ΔpfkA ΔnagB, respectively, to give strains F9 to F11 and G9 to G11, and shake flask fermentation culture gave the highest yields of 5.94G/L for 3'-SL and 3.69G/L for 6' -SL (see Table 6), which were increased by 1.39-fold and 1.97-fold, respectively, indicating that blocking the side-branch pathways of sialyllactose contributed to the increased conversion of 3'-SL and 6' -SL, respectively, with the knockout of gene pfkA being most pronounced for the increased yields of sialyllactose.

TABLE 6 engineering bacteria detailed information

Example 6: screening of sialyltransferases of different origins

Sialyltransferases allow for structural modification of the substrate to effect enzymatic conversion of donor CMP-sialic acid and acceptor lactose to product sialyllactose. At present, although bacterial sialyltransferases have been widely explored and their properties have been deeply characterized, their use in sialyllactose is still inadequate. For this purpose 4 α -2,3 sialyltransferases from prokaryotes were selected, namely PM0188 (protein ID: AAK 02272.1), WQG (protein ID: AGH 37861.1), NSt (protein ID: AAC 44539.1) and Lst (protein ID: AAF 41330.1) and 3 α -2,6 sialyltransferases of different origin, namely Pst6-224 (protein ID: BAF 92026.1), plst6 (protein ID: BAI 49484.1) and Bst (protein ID: BAA 25316.1) and their mutants PM0188 (R313N/T265S), NSt (I411T/L433T), plst6 (DeltaN 2-15 aa) and Bst (DeltaN 2-15 aa).

Pm0188, WQG, nst, plst6, bst, pm0188, nst, plst6 and bst gene fragments were obtained: the division of the attorney bioengineering (Shanghai) company synthesizes the Pm0188 and Pm0188 gene sequences from Pasteurella multocida Pasteurella multocida, the WQG gene sequences from Pasteurella alga Bibersteinia trehalosi, the Nst and Nst gene sequences from Neisseria gonorrhoeae Neisseria gonorrhoeae, the plst6 and plst6 gene sequences from P.leiognathi Photobacterium leiognathi, and the bst and bst gene sequences from P.mermairei Photobacterium damselae.

The plasmids pRS-LY in example 3 were used as templates and the plasmids pRS-Pm0188-lacY, pRS-WQG-lacY and pRS-Nst-lacY, pRS-Pm0188-lacY and pRS-Nst-lacY were obtained by PCR amplification with the primers Pm0188-F/Pm0188-R, WQG-F/WQG-R, nst-F/Nst-R, pm-F/Pm 0188-R, nst-F/Nst-R, respectively, and pRS-Pm 0188-Nst-lacY and pRS-Nst-lacY were used as primers in the construction method of example 2;

similarly, plasmids pRS-plst6-lacY, pRS-bst-lacY, pRS-plst6-lacY and pRS-bst-lacY were obtained by PCR amplification using plasmid pAC-PY of example 3 as a template and plst6-F/plst6-R, bst-F/bst-R, plst6-F/plst6-R, bst-F/bst-R as primers, respectively, in accordance with the construction method of example 2.

The knock-out strain BL21 (DE 3) DeltalacZ Deltanana DeltapfkA DeltanagB in example 1 is used as a host strain, and alpha-2, 3 sialyltransferase genes of different sources are transformed to form strains F12-F16; the various sources of the α -2,6 sialyltransferase genes were transformed to form strains G12 to G15 (see Table 7 for details).

TABLE 7 engineering bacteria detailed information

Example 7:3L fermentation tank batch feeding production of sialyllactose

To further verify the effectiveness of the sialyllactose synthesis process, the production of sialyllactose was increased.

Respectively inoculating the genetically engineered bacteria F13 and G13 constructed in the example 6 into 100mL LB liquid medium, culturing at 37 ℃ for 12h in a shaking flask at 200r/min to obtain seed liquid; the seed solution was inoculated into a fermentation medium having a working volume of 1L at a fermentation temperature of 37℃and a stirring speed of 800r/min at an inoculum size of 10% by volume, and was aerated at 1vvm at pH 6.80 (automatic control of additional ammonia). Fermentation for 18h (OD) ₆₀₀ About 15), and IPTG was added at a final concentration of 20g/L lactose and 0.5 mmol/L. To maintain the growth of the cells and the synthesis of sialyllactose, 750g/L of glycerol (20 g/L of MgSO) was fed after the initial carbon source was consumed ₄ ·7H ₂ O) to supplement carbon source, 200g/L lactose is added after the initial lactose is consumed, and the concentration of lactose in the system is maintained to be about 10g/L, and fermentation culture is carried out for 55h.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A genetically engineered bacterium for producing sialyllactose, characterized in that the β -galactosidase encoding gene lacZ is knocked out and the neuB gene encoding acetylneuraminic acid synthase, the neuC gene encoding N-acetylglucosamine isomerase, the neuA gene encoding CMP-sialyl synthase, the lactose transferase encoding gene lacY, the glutamine synthetase encoding genes glnA and the UDP-acetylglucosamine synthesis pathway are expressed heterologous; overexpression of a gene encoding sialyltransferase;

the UDP-acetylglucosamine synthesis pathway is glucosamine-6-phosphate synthase encoding gene glmS, glucosamine synthase encoding gene glmM and/or UDP-acetylglucosamine pyrophosphorylase encoding gene glmU.

2. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium further knocks out sialic acid aldolase encoding genes nanA, 6-phosphofructokinase encoding genes pfkA and/or glucosamine-6 phosphate deaminase encoding genes nagB.

3. The genetically engineered bacterium of claim 1, wherein the sialyltransferase gene is an α -2,3 sialyltransferase gene or an α -2,6 sialyltransferase gene;

the gene encoding alpha-2, 3 sialyltransferase is selected from gene lst from neisseria meningitidis neisseria gmenptitis, gene Pm0188 from pasteurella multocida, mutant gene Pm0188, gene WQG from babestini reehalosi of babitentan alga, gene Nst from neisseria gonorrhoeae neisseria gonorhoeae or mutant gene Nst;

the encoding alpha-2, 6 sialyltransferase gene is selected from a gene pst6-224 derived from Photobacterium sp, a gene plst6 derived from Photobacterium leiognathi, a mutant gene plst6 and a gene bst or a mutant gene bst derived from Photobacterium mermairei Photobacterium damselae.

4. The genetically engineered bacterium of any one of claims 1 to 3, wherein the plasmid pETDuet-1, pRSFDuet-1, pCDFDuet-1, pACYCDuet-1 or pCOLADuet-1 is used for overexpression.

5. The genetically engineered bacterium of claim 4, wherein the neuB gene, the neuC gene, the neuA gene, and the gene glnA are overexpressed by pETDuet-1 or pACYCDuet-1, the gene lacY and the gene encoding sialyltransferase are overexpressed by pRSFDuet-1 or pCOLADuet-1, and the gene encoding glucosamine-6-phosphate synthase glmS, the gene encoding glucosamine synthase glmM, and/or the gene encoding UDP-acetylglucosamine pyrophosphorylase glmU are overexpressed by pCDFDuet-1.

6. The genetically engineered bacterium of any one of claims 1 to 5, wherein the genetically engineered bacterium is a host of e.

7. A method for improving sialyllactose yield is characterized in that beta-galactosidase encoding gene lacZ, sialylaldehyde aldolase encoding gene nanA, 6-phosphofructokinase encoding gene pfkA and glucosamine-6 phosphodeaminase encoding gene nagB on the genome of escherichia coli are knocked out, and an expression vector is utilized to heterologously express acetylneuraminic acid synthase gene neuB, N-acetylglucosamine isomerase gene neuC, CMP-sialyltransferase gene neuA, sialyltransferase gene, lactose transferase gene lacY, glucosamine-6-phosphate synthase gene glmS, glucosamine synthase gene glmM, UDP-acetylglucosamine pyrophosphorylase gene glmU and glutamine synthase gene glnA.

8. The method of claim 7, wherein the overexpression is performed using pETDuet-1, pRSFDuet-1, pCDFDuet-1, pACYCDuet-1 or pCOLADuet-1 plasmids.

9. A method for producing sialyllactose, which is characterized in that glycerol is used as a carbon source, lactose is used as a substrate, and the genetic engineering bacteria of any one of claims 1 to 6 are used as fermentation strains for fermentation production of sialyllactose.

10. Use of the genetically engineered bacterium of any one of claims 1 to 6, or the method of claim 7 or 8, for the production of sialyllactose and sialyllactose-containing products.