Genetically engineered bacterium for producing 6 '-sialyllactose as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing 6 '-sialic acid lactose as well as a construction method and application of the genetically engineered bacterium. Escherichia coli K12MG1655 is modified to produce endogenous sialic acid, and the endogenous sialic acid and exogenously added lactose generate sialic acid lactose under the condition of heterologous expression pathway enzyme; further, the de novo synthesis and yield improvement of 6 '-sialyllactose are realized through technologies such as expression element optimization, key enzyme modification, cofactor circulation system construction and high-density fermentation.

Description

Technical Field

[0001] The present invention relates to a genetically engineered bacterium producing 6'-sialyllactose, a construction method thereof and an application thereof, belonging to the fields of synthetic biology and metabolic engineering. Background Art

[0002] 6'-SL is one of the important components of human milk oligosaccharides (HMOs), belonging to the category of sialylated oligosaccharides in acidic HMOs. Its structure is composed of a lactose core and a sialic acid residue linked by an α2,6-glycosidic bond. As one of the HMOs with a relatively high content, 6'-SL has multiple biological functions in infant nutrition and health. Similar to sialic acid, 6'-SL can mimic the sugar chains on the surface of host cells through its unique sialylated structure, competitively bind to pathogens such as rotavirus, Streptococcus pneumoniae and Escherichia coli, thereby blocking their attachment to intestinal epithelial cells and reducing the risk of infection. Secondly, 6'-SL is also a prebiotic, which can promote the proliferation of beneficial bacteria in the intestine such as Bifidobacterium and Lactobacillus, inhibit the growth of harmful bacteria, maintain the intestinal microecological balance, enhance the intestinal barrier function, and reduce the occurrence of intestinal inflammation and infection. In addition, 6'-SL also plays an important role in infant neurodevelopment. Sialic acid is an important component of brain gangliosides and glycoproteins. It participates in synaptic formation, nerve signal transmission and the development of cognitive functions. Therefore, 6'-SL with sialic acid components may play a key role in the neurodevelopment and maturation process of infants. Research also shows that 6'-SL can affect the expression of inflammatory factors by regulating the activities of immune cells (such as macrophages, dendritic cells and T cells), playing an anti-inflammatory and immune-enhancing role, providing support for the development and maturation of the infant immune system. In addition, 6'-SL also has antiviral, antioxidant and anti-apoptotic effects, which can protect intestinal epithelial cells from oxidative stress and inflammatory damage, and promote the repair and regeneration of intestinal tissues. Due to these functional characteristics of 6'-SL, its application in infant formula milk powder has gradually attracted attention. Since its biosynthesis and large-scale production face many technical challenges, how to use synthetic biology technology to achieve large-scale preparation is challenging and of great significance, and it is one of the important directions for future infant nutrition research.

[0003] 6'-SL is one of the core components of HMOs and has important physiological functions such as regulating the infant immune system, promoting the growth of intestinal probiotics and inhibiting pathogen infection. At present, the commercial production of 6'-SL mainly relies on chemical synthesis or enzymatic conversion, but these methods have problems such as high cost, cumbersome steps and low product purity, and it is difficult to meet the large-scale industrial demand. In recent years, the biosynthesis technology based on microbial metabolic engineering has become an important research direction for 6'-SL production due to its advantages such as green, efficient and sustainable.

[0004] Currently, the biosynthesis of 6'-SL mainly relies on model microorganisms such as Escherichia coli, and is achieved by introducing exogenous enzyme catalytic pathways. The core principle is to genetically engineer microorganisms to express a complete sialic acid synthesis pathway and α2,6-sialyltransferase, so as to directly synthesize 6'-SL from inexpensive substrates such as glucose, glycerol and lactose. The synthesis methods mainly include steps such as the construction of host bacteria, the optimization of fermentation conditions, and the separation and purification of products. Generally, key enzymes (such as NeuB, NeuC and NeuA) of the sialic acid synthesis pathway and the α2,6-sialyltransferase gene are introduced into the host bacteria by genetic engineering technology. Since there are three metabolic pathways for synthesizing sialic acid in microorganisms, there will be some differences in the construction of chassis cells. After constructing an engineered strain capable of de novo synthesis of 6'-SL, large-scale fermentation production of 6'-SL by the engineered strain is achieved by optimizing fermentation conditions such as temperature, pH and dissolved oxygen level. During the fermentation process, the yield of 6'-SL can be further increased by adding precursor substances or regulating the metabolic flux. Finally, 6'-SL is separated and purified from the fermentation broth through steps such as centrifugation, filtration, chromatography and crystallization. However, the existing technology still faces some challenges, such as the insufficient natural activity of some key enzymes (such as CMP-sialic acid synthase, sialyltransferase), which affects the overall synthesis efficiency, insufficient cofactor supply, inaccurate expression regulation and other problems. In response to the above problems, existing research mostly adopts single optimization strategies (such as modular pathway construction or fermentation optimization, etc.), but it is often difficult to achieve the efficient synthesis of 6'-SL. Therefore, there is an urgent need to develop a systematic optimization method that integrates multi-dimensional strategies such as enzyme engineering, metabolic regulation and cofactor regeneration to break through the bottleneck of existing technology. Summary of the Invention

[0005] To solve the deficiencies of the existing technology, the present invention constructs an engineered Escherichia coli strain for de novo synthesis of 6'-SL through multi-level metabolic engineering transformation. Through optimized metabolic pathways, efficient enzyme combinations, precise expression regulation, and cofactor regeneration system strategies, finally in a 5L bioreactor, through fed-batch fermentation optimization, it is increased by 13 times compared with the initial strain.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a genetically engineered bacterium capable of producing 6'-sialyllactose. The genetically engineered bacterium is obtained by knocking out the nanATEK, nagAB, and lacZ genes of an Escherichia coli host, and expressing sialic acid synthase and UDP-N-acetylglucosamine 2-epimerase using a first plasmid system, and expressing CMP-sialic acid synthase NeuA and α2,6-sialyltransferase using a second plasmid system; the amino acid sequence of the CMP-sialic acid synthase NeuA is as shown in SEQ ID NO.2, or the serine at position 185 of the amino acid sequence shown in SEQ ID NO.2 is mutated to threonine, and the isoleucine at position 194 is mutated to valine; the amino acid sequence of the α2,6-sialyltransferase is as shown in SEQ ID NO.1, or the serine at position 160 of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine.

[0008] In one embodiment of the present invention, the gene number of the sialic acid synthase is WP_002215299.1, and the gene number of the UDP-N-acetylglucosamine 2-epimerase is HGF8732514.1.

[0009] In one embodiment of the present invention, the Escherichia coli host is Escherichia coli K12 MG1655.

[0010] In one embodiment of the present invention, the vector of the first plasmid system is pET28a, and the vector of the second plasmid system is pCDF Deut1.

[0011] In one embodiment of the present invention, the second plasmid system adopts a polycistronic expression structure, which comprises a combination of a high-strength promoter PJ23100, a medium-strength RBS of NeuA, and a low-strength RBS of Pd2,6ST. The nucleotide sequence of the high-strength promoter PJ23100 is as shown in SEQ ID NO.24, the nucleotide sequence of the medium-strength RBS of NeuA is as shown in SEQ ID NO.27, and the nucleotide sequence of the low-strength RBS of Pd2,6ST is as shown in SEQ ID NO.32.

[0012] In one embodiment of the present invention, the genetically engineered bacterium also overexpresses cytidine kinase and / or polyphosphate kinase.

[0013] In one embodiment of the present invention, the number of the cytidine kinase is EC:2.7.4.25, and the number of the polyphosphate kinase is EC:2.7.4.1.

[0014] The second object of the present invention is to provide a method for constructing the genetically engineered bacterium, the method comprising the following steps: using Escherichia coli K12 MG1655 as the chassis bacterium, knocking out the nanATEK, nagAB and lacZ genes therein; expressing sialic acid synthase and UDP-N-acetylglucosamine 2-epimerase with pET28a as the vector, and expressing CMP-sialic acid synthase NeuA and α2,6-sialyltransferase with pCDF Deut1 as the vector.

[0015] The third object of the present invention is to provide a method for fermentatively producing 6'-sialyllactose, the method being to fermentatively produce 6'-sialyllactose with the genetically engineered bacterium.

[0016] In one embodiment of the present invention, during the fermentation process, glycerol and lactose are added as carbon sources and substrates; the initial glycerol concentration is 20-30 g / L and maintained at 5-10 g / L; the initial lactose concentration is 10-30 g / L and maintained at 20-30 g / L.

[0017] In one embodiment of the present invention, the fermentation conditions are pH 6-7, temperature 25°C to 37°C, and dissolved oxygen 10% to 30%.

[0018] In one embodiment of the present invention, during fermentation, when OD 600 reaches 10-30, IPTG induction is carried out, and the IPTG induction concentration is 0.05 M to 0.2 M.

[0019] The fourth object of the present invention is to provide the use of the genetically engineered bacterium or the method in the preparation of infant formula foods or nutritional supplements.

[0020] The beneficial effects of the present invention:

[0021] The present invention provides an engineered strain for highly efficient synthesis of 6'-SL, its construction method and application, which have significant technical advantages and industrialization value. First of all, through systematic metabolic engineering modification, the present invention has achieved a breakthrough improvement in the synthesis efficiency of 6'-SL. Specifically, by knocking out competitive pathway genes such as nanATEK, nagAB and lacZ, the loss of metabolic flux is effectively reduced; secondly, the present invention innovatively adopts a multi-level expression regulation strategy, and through the precise combination of promoters and decreasing intensities of RBS, a reasonable distribution of metabolic flux is achieved, and the 6'-SL yield is increased by 91% compared with the initial strain. At the same time, the optimized enzyme combination NeuA-S185T&I194V and Pd2,6ST-S160T is introduced, significantly improving the overall synthesis efficiency. In terms of cofactor supply, by constructing a CTP-ATP coupling cycle system, the problem of insufficient CTP supply is effectively solved, and CTP provides a guarantee for the efficient synthesis of 6'-SL. From the perspective of industrial application, the present invention has outstanding cost advantages and large-scale production potential. By adopting the endogenous sialic acid synthesis pathway, the addition of expensive exogenous Neu5Ac is completely avoided, and the raw material cost is reduced by more than 50%. Using the optimized fed-batch fermentation process in a 5L fermenter, the 6'-SL yield can reach 12.82 g / L, which is 13 times higher than that of the initial strain, and the fermentation process is stable and controllable, and can be directly scaled up to the industrial production scale. In addition, the engineered strain of the present invention has good genetic stability and can still maintain more than 90% of the production capacity after continuous passage for 10 times, laying a foundation for long-term industrial application. Compared with the existing technology, the present invention integrates multi-disciplinary technical means such as metabolic engineering, enzyme engineering and fermentation engineering, not only solves the key bottleneck problems in the biosynthesis of 6'-SL, but also greatly reduces the production cost, provides a reliable technical solution for the industrial production of milk oligosaccharides, and has important economic value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 It is the 6'-SL synthesis pathway and key enzymes in Escherichia coli K12 MG1655;

[0024] Figure 2 It is the plasmid structure and fermentation flow chart;

[0025] Figure 3 It is the schematic diagram of the enzyme plasmid combination for heterologous enzyme screening and its fermentation results;

[0026] Figure 4 Schematic diagram of promoter-RBS combination optimization and its fermentation results;

[0027] Figure 5 Enzyme mutation design and its fermentation results;

[0028] Figure 6 CTP cycle regeneration pathway and its fermentation results;

[0029] Figure 7 Yield curve of the optimal strain in a 5L fermenter. Detailed implementation manners

[0030] The following further elaborates on this invention patent in conjunction with specific examples. These implementation cases are only used to illustrate the invention and not to limit the scope of the invention. In addition, after reading the content taught by this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0031] Detection methods for relevant parameters during the fermentation process:

[0032] Detection of 6'-SL content: Take 1 mL of the fermentation broth into a 2 mL EP tube, boil it at 100 °C for 10 min, then centrifuge at 12000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane, and collect it into a liquid phase vial. Analyze the 6'-SL production using an XBT amide column (Hungpu, Guangzhou, China) and an ultraviolet (VWD) detector through HPLC (Agilent 1260 system). Analysis method: Column oven: 60 °C; VWD at 210 nm. The mobile phase is solvent A (10 mM ammonium formate, pH 4.0) and solvent B (acetonitrile) with an isocratic elution program in a ratio of 30:70; flow rate 1 mL / min. Calculate the 6'-SL content in combination with the standard curve of the standard product.

[0033] Detection methods for glycerol and lactose: Analyze extracellular compounds (lactose and glycerol) using a RezexTM ROA-H+ (8%) organic acid column (Phenomenex, USA) and a refractive index (RI) detector through HPLC (Agilent 1260 system). Analysis method: Column oven: 55 °C; mobile phase is 5 mM H2SO4; flow rate: 0.6 mL / min.

[0034] Biomass is detected using an ultraviolet spectrophotometer at OD 600 to evaluate cell growth.

[0035] The technical solution of the present invention will be described in detail below in combination with specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be obtained from commercial sources, or prepared by conventional methods, or commonly used in this industry.

[0036] Example 1: Construction of the 6'-SL synthesis pathway

[0037] (1) Using Escherichia coli K12 MG1655 as the host strain, optimize the metabolic network by targeting and knocking out key genes in the competing pathways, including the sialic acid metabolism-related genes nanATEK, N-acetylglucosamine metabolism gene nagAB, and lactose metabolism gene lacZ( Figure 1 ), and the sgRNA sequences and primers used for gene knockout are shown in Tables 1-3. A dual-plasmid co-expression system was used: NeuB (sialic acid synthase; WP_002215299.1) and NeuC (UDP-N-acetylglucosamine 2-epimerase; HGF8732514.1) were constructed on the pET28a vector (named BC), and NmCSS (CMP-sialic acid synthase; WP_002225824.1) and Pd2,6ST (α2,6-sialyltransferase, amino acid sequence as shown in SEQ ID NO.1) were constructed on the pCDFDuet-1 vector (named CP). After 72 h of shake-flask fermentation, the 6'-SL yield reached 0.98 g / L. The engineered strain E. coli K12 MG1655△nanATEK&△nagAB&△lacZ / pET28a-BC / pCDFDuet 1-CP( Figure 2 ) was obtained.

[0038] Table 1 sgRNA sequence list for knockout genes in the competing pathway during gene editing

[0039]

[0040] Table 2 Primer names and their sequence lists for constructing the targeting recognition plasmid during knockout in the competing pathway of gene editing

[0041]

[0042] Table 3 Primer names and their sequence lists for constructing the target gene repair template during knockout in the competing pathway of gene editing

[0043]

[0044]

[0045] (2) Based on the above NmCSS and Pd2,6ST enzyme sequences, sequence homology analysis was performed using the National Center for Biotechnology Information (NCBI) database. The screening criteria were set as microbial-derived enzymes with sequence similarity of 20%-100% and an amino acid total difference within about 20%. One CMP-sialic acid synthase (NeuA; EC 2.7.7.43, amino acid sequence shown in SEQ ID NO.2) and one sialyltransferase (Nm2,6ST; WP_118948975.1) from Neisseria meningitidis were randomly selected within the range, and in addition, one sialyltransferase from Campylobacter jejuni (Cj2,6ST; WP_224397596.1). After codon optimization of these genes, using pCDFDuet-1 as the vector, 6 different plasmid combination strains were constructed: BC-CP (control), BC-CN, BC-CC, BC-AP, BC-AN, and BC-AC, where BC represents pET28a carrying NeuB and NeuC; CP represents pCDFDuet 1 carrying NmCSS and Pd2,6ST; CN represents pCDFDuet 1 carrying NmCSS and the sialyltransferase Nm2,6ST from Neisseria meningitidis; CC represents pCDFDuet 1 carrying NmCSS and the sialyltransferase Cj2,6ST from Campylobacter jejuni; AP represents pCDFDuet 1 carrying the CMP-sialic acid synthase NeuA from Neisseria meningitidis and Pd2,6ST; AN represents pCDFDuet1 carrying the CMP-sialic acid synthase NeuA from Neisseria meningitidis and the sialyltransferase Nm2,6ST from Neisseria meningitidis; AC represents pCDFDuet 1 carrying the CMP-sialic acid synthase NeuA from Neisseria meningitidis and the sialyltransferase Cj2,6ST from Campylobacter jejuni. Flask fermentation experiments showed that the combination of NeuA and Pd2,6ST (BC-AP) exhibited the optimal 6'-SL synthesis ability, with a yield reaching 1.26 g / L( Figure 3 ).

[0046] Example 2: Screening of key enzyme expression elements

[0047] This example aims to optimize the expression of NeuA and Pd2,6ST by screening promoters and RBSs with different strengths to determine the combination that maximizes the 6'-SL yield. NeuA and Pd2,6ST are expressed as a polycistronic system, sharing the same promoter, but each protein coding sequence (CDS) uses a different RBS. We selected three J23 series promoters with different expression strengths (Table 4): P J23100 (high strength), PJ23108 (Medium strength) and P J23115 (Low strength). The initial RBS strengths of NeuA and Pd2,6ST were calculated using the RBS calculator (https: / / www.denovodna.com / software / ) (Table 5). For NeuA, the initial RBS strength was 878 (medium strength), and the predicted maximum and minimum strengths were 3057 (high strength) and 503.67 (low strength), respectively. For Pd2,6ST, the initial RBS strength was 100000 (high strength), 5000 (low strength), and 25000 (medium strength) as alternative strengths. After arranging and combining these promoters and RBSs, 27 different strains were constructed. After 72 h of shake-flask fermentation, P J23100 The combined strain with the medium-strength RBS of NeuA and the low-strength RBS of Pd2,6ST achieved the highest 6'-SL yield of 2.41 g / L( Figure 4 ).

[0048] Table 4 Promoter names and sequences used in the present invention

[0049]

[0050] Table 5 RBS sequences used in the present invention

[0051]

[0052] Example 3: Semi-rational design of key enzymes

[0053] (1) The amino acid conservation and mutation frequencies of NeuA and Pd2,6ST in the pathway were analyzed using the DeMaSk software( Figure 5 A and B in). Among the 684 sites of NeuA, 41 were identified as potentially enhancing enzyme activity after residue substitution. Ten predicted highly active mutants were selected for fermentation verification. Among them, the yields of 6 mutants D123E, I147V, L158I, S185T, I194V, and H225Q increased significantly, and the yield of the S185T mutant was the highest, reaching 3.02 g / L( Figure 5 C in). In addition, combinatorial mutations were introduced at the S185T site with five other sites to obtain the S185T&I194V mutation, and the 6'-SL yield reached 3.67 g / L( Figure 5 D in).

[0054] (2) For Pd2,6ST, 10 sites with the highest predicted activity were identified: T151S, S160T, E203D, S208T, T250S, E304D, K307R, K324R, I396V, and S400T. Among them, the yield of the S160T mutant increased significantly, reaching 4.27 g / L in shake-flask fermentation ( Figure 5 in F).

[0055] Example 4: Recycling of CTP cofactor

[0056] In the last two reactions of the 6'-SL metabolic pathway, CTP is the only nucleotide cofactor involved in the reaction. It reacts with CMP-sialic acid under the catalysis of NeuA to generate 6'-SL ( Figure 6 C). Based on the analysis of pyrimidine metabolism in Escherichia coli and the CTP regeneration strategy in the multi-enzyme cascade study of whole-cell catalysis synthesis, we designed two directions to investigate how the increase in intracellular CTP content affects the final yield of 6'-SL.

[0057] (1) First, aiming to promote CTP synthesis, we overexpressed CTP synthase to increase the content of CTP. Three CTP synthases from Escherichia coli, Corynebacterium glutamicum, and Bacillus subtilis were selected and overexpressed based on the combined mutant. The fermentation results showed that the overexpression of all three CTP synthases decreased the shake-flask yield of 6'-SL to varying degrees ( Figure 6 A).

[0058] (2) At the same time, the second scheme was implemented. By coupling the CTP cycle with the ATP cycle, the cycle process from CMP to CDP and then to CTP was promoted, thereby increasing the CTP supply rate. During the entire cycle coupling process, three enzymes needed to be overexpressed, namely cytidine kinase (cmK, EC: 2.7.4.25), nucleoside diphosphate kinase (ndK, EC: 2.7.4.6), and polyphosphate kinase (ppK, EC: 2.7.4.1). The entire cycle reaction pathway is shown in Figure 6 C. Similarly, we constructed and overexpressed them in the combined mutant strains. Finally, the overexpression of cmK and ppK in the CTP regeneration pathway increased the yield of 6'-SL, which were 4.47 g / L and 4.56 g / L respectively. Then cmK and ppK were co-expressed, and the fermentation verification showed that the yield of 6'-SL was 4.92 g / L ( Figure 6 A).

[0059] (3) By analyzing the effect of intracellular CTP content on the yield, it was found that the increase in the content of cofactor CTP did indeed increase the yield of 6'-SL ( Figure 6 as shown in B).

[0060] Example 5: Verification in a 5L Fermenter

[0061] Fed-batch fermentation was carried out in a 5L bioreactor. The liquid volume of the fermentation medium was 2L, and the temperature was controlled at 30°C. The dissolved oxygen level was maintained at 30%, and the stirring speed was combined with the dissolved oxygen data (stirring speed range: 50 rpm to 900 rpm). The pH was fixed at 6.8, and the fermentation process started with an initial glycerol concentration of 30 g / L. When the glycerol concentration was lower than 5 g / L, continuous feeding was immediately started, and the glycerol concentration was maintained at 5 - 10 g / L to prevent the accumulation of by-products. When OD 600 reached 15 - 20, IPTG was immediately added to induce protein expression. The initial substrate lactose concentration was controlled at 30 g / L. During fermentation, when the lactose level decreased to about 20 g / L, intermittent feeding was started to increase the lactose concentration to 30 g / L. At 76 h of fermentation, OD 600 could reach above 60, and the maximum concentration of 6'-SL reached 12.82 g / L( Figure 7 ).

[0062] The examples provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art in combination with the existing well-known common knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A genetically engineered bacterium producing 6'-sialyllactose, characterized in that: The genetically engineered bacteria are prepared by knocking out the nanATEK, nagAB, and lacZ genes of an Escherichia coli host, using a first plasmid system to express sialic acid synthase and UDP-N-acetylglucosamine 2-epimerase, and using a second plasmid system to express CMP-sialic acid synthase NeuA and α2,6-sialyltransferase; the amino acid sequence of the CMP-sialyl synthase NeuA is as shown in SEQ ID NO.2, or the serine at position 185 of the amino acid sequence shown in SEQ ID NO.2 is mutated to threonine, and the isoleucine at position 194 is mutated to valine; the amino acid sequence of the α2,6-sialyltransferase is as shown in SEQ ID NO.1, or the serine at position 160 of the amino acid sequence shown in SEQ ID NO.1 is mutated to threonine.

2. The genetically engineered bacterium according to claim 1, characterized in that The Escherichia coli host is Escherichia coli K12MG1655.

3. The genetically engineered bacterium according to claim 1 or 2, characterized in that The vector of the first plasmid system is pET28a, and the vector of the second plasmid system is pCDF Deut1.

4. The genetically engineered bacterium according to claim 1 or 3, characterized in that The second plasmid system adopts a polycistronic expression structure, comprising a combination of a high-strength promoter PJ23100, a medium-strength RBS of NeuA, and a low-strength RBS of Pd2,6ST. The nucleotide sequence of the high-strength promoter PJ23100 is shown in SEQ ID NO.24, the nucleotide sequence of the medium-strength RBS of NeuA is shown in SEQ ID NO.27, and the nucleotide sequence of the low-strength RBS of Pd2,6ST is shown in SEQ ID NO.

32.

5. The genetically engineered bacterium according to claim 1, characterized in that The genetically engineered bacteria also overexpress cytidine kinase and / or polyphosphate kinase.

6. A method for producing 6'-sialyllactose by fermentation, characterized in that: The method comprises fermenting and producing 6'-sialyllactose using the genetically engineered bacteria according to any one of claims 1 to 5.

7. The method according to claim 6, characterized in that During the fermentation process, glycerol and lactose are added as carbon sources and substrates; the initial glycerol concentration is 20-30 g / L and maintained at 5-10 g / L; the initial lactose concentration is 10-30 g / L and maintained at 20-30 g / L.

8. The method according to claim 6, characterized in that The fermentation conditions are pH 6-7, temperature 25° C.-37° C., and dissolved oxygen 10%-30%.

9. The method according to claim 6, characterized in that During fermentation, when OD 600 When the cell proliferation rate reaches 10-30, IPTG induction is performed, and the IPTG induction concentration is 0.05M to 0.2M.

10. Use of the genetically engineered bacteria according to any one of claims 1 to 5 or the method according to any one of claims 6 to 9 in the preparation of infant formula or nutritional supplements.

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