A method for the whole-cell catalytic synthesis of sialylated lacto-n-tetraose a

Abstract

A whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a includes the following steps: S1, constructing a recombinant engineered *E. coli* strain: using *E. coli* JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or the α-2,3-sialic transferase gene are expressed in free form to obtain a recombinant engineered *E. coli* strain; S2, whole-cell catalytic synthesis of LSTa: using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates, and IPTG as an inducer, the above-mentioned recombinant engineered *E. coli* strain and *Saccharomyces cerevisiae* as coupled fermentation strains are used for whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a. This whole-cell catalytic synthesis method achieves efficient synthesis of LSTa through the construction of an *E. coli* glycosyltransferase module and a *Saccharomyces cerevisiae* CTP energy regeneration module, with a yield as high as 38.03 g / L.

Description

Technical Field

[0001] This invention relates to the field of sialylated oligosaccharide production technology, and in particular to a whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third largest category of solid nutrients in breast milk, after lactose and fat. As complex natural functional carbohydrates, they play an irreplaceable core role in the healthy development of infants and young children. HMOs not only regulate the structure of the gut microbiota, inhibit the adhesion of pathogenic bacteria to the intestinal mucosa, and enhance the intestinal barrier function to maintain the balance of the infant's gut microbiota, but they also participate in the development and regulation of the immune system and reduce the risk of infectious diseases. Their unique physiological functions are a key reason why formula milk powder cannot accurately replicate them. With the increasing demand for human milk-like components in the infant formula industry, the development of efficient and green HMO synthesis technologies has become a research hotspot in the fields of bioengineering and food science.

[0003] Human milk oligosaccharides (HMOs) can be classified into three main categories based on their core structure: neutral-core HMOs (such as lactose-N-tetrasaccharides (LNT) and lactose-N-neotetrasaccharides (LNnT)), neutrally fucoidylated HMOs (such as 2'-FL and 3'-FL), and sialylated HMOs (such as 3'-SL and 6'-SL). Sialylated human milk oligosaccharides (HMOs) are an important subclass of HMOs, possessing unique physiological activities due to the presence of sialic acid (N-acetylneuraminic acid, Neu5Ac) residues in their structure. Common sialylated derivatives of LNT and LNnT include: sialylated lactose-N-tetrasaccharide a (LSTa), sialylated lactose-N-tetrasaccharide b (LSTb), sialylated lactose-N-tetrasaccharide c (LSTc), and sialylated lactose-N-tetrasaccharide d (LSTd) (e.g., LSTb). Figure 1 (As shown in the image). Among them, sialylated lactose-N-tetrasaccharide a (LSTa), as a typical sialylated HMO, has sialic acid residues linked to the lactose-N-tetrasaccharide (LNT) backbone via α-2,3 glycosidic bonds. It can mimic the functions of key active ingredients in breast milk, showing significant potential in inhibiting viral invasion, regulating nerve cell development, and improving intestinal immune responses. These excellent properties make LSTA a promising candidate for applications in high-end infant formula and functional dietary supplements.

[0004] Currently, the main methods for synthesizing HMOs include chemical synthesis, enzymatic catalysis, and whole-cell catalysis. Chemical synthesis requires complex group protection and deprotection steps, suffers from harsh reaction conditions and poor regioselectivity, and easily generates environmental pollutants, making it difficult to meet the requirements of green production. While pure enzymatic catalysis offers advantages such as high catalytic efficiency and specificity, the enzyme isolation and purification process is cumbersome and costly, and the free enzyme exhibits poor stability in the reaction system. Furthermore, it is limited by natural cofactors, and the problem of cofactor recycling and regeneration has not been fully resolved, further restricting its reusability. Whole-cell catalysis uses intact cells as biocatalysts, leveraging the intracellular natural metabolic network and enzyme system to synergistically synthesize the target product. It eliminates the need for complex enzyme purification steps, the intracellular environment effectively maintains enzyme activity and stability, and the reaction conditions are mild and environmentally friendly, making it the preferred strategy for large-scale HMO synthesis.

[0005] However, the whole-cell catalytic synthesis of LSTA still faces many challenges: First, the synthesis of LSTA requires two steps: the generation of CMP-Neu5Ac (sialic acid donor) and glycosyltransferase, involving the synergistic effect of multiple enzymes, and a single strain is difficult to efficiently coordinate the reaction efficiency of each step; Second, the consumption of CTP (energy donor) during the reaction leads to increased reaction costs, and the regeneration efficiency of CTP directly affects the substrate conversion rate; Third, the reaction conditions of the catalytic system (such as temperature, pH, substrate ratio, etc.) have high requirements for the synergistic adaptation of enzyme activity and cell metabolism, and systematic optimization is required to improve product yield.

[0006] In the field of microbial synthesis research in LSTA, existing reports focus on the construction of multi-enzyme co-expression vectors. For example, CN120249160A discloses a high-yield sialyl-N-tetrasaccharide a *Escherichia coli* strain and its construction method and application. Using recombinant *E. coli* as the starting strain, a plasmid-free, high-efficiency lactyl-N-tetrasaccharide synthesis chassis strain was constructed by integrating the β-1,3-galactosyltransferase gene wbgO from *E. coli* O55:H7 into the chromosome in multiple copies. Overexpression of neuC, neuB, neuA, and Nm3ST was introduced via plasmids to improve glycosyl donor supply. The expression intensity of uridine diphosphate-N-acetylglucosamine synthesis pathway genes glmM and glmUS was regulated through ribosome binding site engineering, and the CTP synthase pyrG was introduced to enhance CMP-Neu5Ac supply, thereby increasing the yield of sialyl-N-tetrasaccharide a. Finally, by shaking flask and batch culture, the maximum titers of sialyl-N-tetrasaccharide a reached 2.238 and 8.178 g / L, respectively, but the product yield of this method still falls short of industrial requirements.

[0007] To address the metabolic incoordination problem in the synthesis of complex products, a modular cell coupling strategy, through the construction of functionally specific modular strains, achieves efficient division of labor and synergistic coupling of each reaction step, providing an effective approach to solving the aforementioned problems. The core of this strategy lies in the deep reconstruction of the biosynthetic network of the cell factory through modularization of metabolic pathways and selective coupling of growth, providing a technical reference for the modular synthesis of complex oligosaccharides. This application proposes a highly efficient modular cell coupling catalytic strategy for the whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a (LSTa), providing technical support for the industrial production of LSTa and offering a new method for the biosynthesis of other complex sialylated HMOs. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a whole-cell catalytic synthesis method for high-yield sialylated lactose-N-tetrasaccharide a.

[0009] The technical solution adopted by this invention to solve its technical problem is:

[0010] A whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a, comprising the following steps:

[0011] S1. Construction of recombinant engineered Escherichia coli: Using E. coli JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or the α-2,3-sialotransferase gene were expressed in free form to obtain recombinant engineered Escherichia coli; the α-2,3-sialotransferase gene was selected from PmST1M144D from Pasteurella multocida, nst from Neisseria meningitidis, or CstII from Campylobacter jejuni;

[0012] S2. Whole-cell catalytic synthesis of LSTA: Using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates, IPTG (isopropyl-β-D-thiogalactoside) as an inducer, and the above-mentioned recombinant Escherichia coli engineered bacteria and Saccharomyces cerevisiae as coupled fermentation strains, whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a was carried out.

[0013] The first step in the LSTa synthesis route requires the use of high-energy phosphate bonds in CTP to provide energy for the synthesis of CMP-Neu5Ac, which is then used for the next glycosyltransfer module. Considering the high cost of directly using CTP, the applicant chose to introduce Saccharomyces cerevisiae as an energy regeneration module, utilizing the powerful endogenous enzyme system within the yeast to achieve the cyclic regeneration of CTP using CMP as a substrate.

[0014] The applicant constructed an *E. coli* glycosyltransferase module by screening highly active α-2,3-sialyl transferases (nst, CstII, PmST1 M144D). To further reduce the number of strains required while increasing the production rate, a co-expression strategy was adopted to integrate two genes into a single engineered bacterial strain for expression. This invention establishes a glycosyltransferase module and a CTP energy regeneration module in engineered *E. coli* and *Saccharomyces cerevisiae*, respectively, using Neu5Ac and LNT as substrates to achieve LSTA synthesis via whole-cell catalysis. Compared to de novo synthesis, this invention employs whole-cell catalysis, which does not involve gene editing of metabolic pathways and is simpler than other similar studies. However, whole-cell catalysis is more suitable for industrial applications, and this whole-cell catalytic synthesis method achieves an LSTA yield as high as 38.03 g / L.

[0015] The nucleotide sequences of genes neuA, nst, CstII, and PmST1 M144D are shown in SEQ NO.1, SEQ NO.2, SEQ NO.3, and SEQ NO.4, respectively.

[0016] Preferably, the method for constructing recombinant Escherichia coli engineered bacteria includes: inserting the neuA gene of cytosine-5'-monophosphate-N-acetylneuraminic acid synthase with optimized synthetic codons into the vector pET28a(+); inserting the nst, CstII, and PmST1M144D genes of α-2,3-sialotransferase with optimized synthetic codons into the vector pET28a(+); screening for α-2,3-sialotransferase genes that produce high levels of LSTa by testing LSTa synthesis capacity; and then transforming the vector into E. coli JM109(DE3)-neuA and JM109(DE3)-PmST1, recombinant Escherichia coli engineered bacteria expressing the neuA and PmST1M144D genes respectively.

[0017] The procedure for testing LSTA synthesis ability was as follows: 20 mM CMP-Neu5Ac, 20 mM LNT, 20 mM MgCl2, 2% (v / v) surfactant [octadecylamine:ethanol = 1:1 (m / m)], and 50 g / L *E. coli* cells expressing α-2,3-sialic acid transferase were added to a 5 mL test tube containing 1 mL Tris-HCl (100 mM, pH 8.0). The mixture was reacted at 37°C and 200 rpm for 24 h. This LSTA synthesis ability test can rapidly screen for genes that efficiently express α-2,3-sialic acid transferase, laying the foundation for the subsequent construction of engineered *E. coli* strains co-expressing the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or the α-2,3-sialic acid transferase gene neuA and / or the α-2,3-sialic acid transferase gene.

[0018] Alternatively, the method for constructing the recombinant Escherichia coli engineered strain includes: cloning the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA (with optimized synthetic codons) and the α-2,3-sialotransferase gene PmST1M144D (with optimized synthetic codons) into two vectors with different copy numbers, pRSF-Duet1 and pET-Duet1, and then transforming them into E. coli JM109(DE3) to construct four recombinant E. coli engineered strains carrying co-expression plasmids: JM109(DE3)-E01, JM109(DE3)-E02, JM109(DE3)-E03, and JM109(DE3)-E04.

[0019] The catalytic components for the whole-cell catalytic synthesis of LSTA include: 80 mM CMP, 80 mM Neu5Ac, 80 mM MLT, 250 mM KH2PO4, 20 mM MgCl2, 120 mM Tris, 2 mM DTT, 220 mM glycerol, 4 g / L N-polyoxyethyl-N-octadecylamine, 300 mM glucose, and 50 g / L (ww) recombinant Escherichia coli engineered bacteria and Saccharomyces cerevisiae. The recombinant Escherichia coli engineered bacteria are JM109(DE3)-neuA and JM109(DE3)-PmST1, or one of JM109(DE3)-E01, JM109(DE3)-E02, JM109(DE3)-E03 and JM109(DE3)-E04.

[0020] Another technical solution adopted by the present invention to solve its technical problem is:

[0021] In one exemplary embodiment, the whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a employs a shake-flask fermentation catalytic method, comprising the following steps:

[0022] S1. Construction of recombinant engineered Escherichia coli: Using E. coli JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene PmST1M144D were expressed in free to obtain recombinant engineered Escherichia coli JM109(DE3)-E02;

[0023] S2. Whole-cell catalytic synthesis of LSTA: Using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates and IPTG as an inducer, the above-mentioned recombinant Escherichia coli engineered strain JM109(DE3)-E02 and Saccharomyces cerevisiae as coupled fermentation strains were used to synthesize sialylated lactose-N-tetrasaccharide a through whole-cell catalysis.

[0024] The shake-flask fermentation conditions for the whole-cell catalytic synthesis of LSTA are as follows: fermentation temperature of 37-42℃, pH of 8.0-8.5, induction with 0.3-1mM IPTG at 20-25℃, the weight ratio of sialic acid to lactose-N-tetrasaccharide in the substrate is 1:1 to 2:1, and the sialic acid content is ≥160mM; the ratio of the recombinant Escherichia coli engineered strain JM109(DE3)-E02 to Saccharomyces cerevisiae is 1:2.

[0025] In one exemplary embodiment, the shake-flask fermentation conditions for the whole-cell catalytic synthesis method are as follows: fermentation temperature is 37°C, pH is 8.0, 0.5 mM IPTG is added at 25°C for induction, the weight ratio of sialic acid to lactose-N-tetrasaccharide in the substrate is 2:1, and the sialic acid content is ≥160 mM; the amount of Escherichia coli and Saccharomyces cerevisiae added is 25 g / L and 50 g / L, respectively.

[0026] The technical solution further adopted by the present invention to solve its technical problem is as follows:

[0027] The whole-cell catalytic synthesis method employs a two-step fermentation process in a bioreactor, including the following steps:

[0028] S1. Induction Culture Strains: The recombinant *E. coli* engineered strain was cultured in LB (Luria-Bertani) medium with an inoculum of 10% (v / v). A dissolved oxygen cascade stirring method was used for control, with a stirring range of 50-800 rpm and an aeration rate of 3 vvm. DO was maintained at 30%, and 25% ammonia was added to maintain the pH at 7.0. The initial culture temperature was 37 ℃. When the glucose residue in the medium decreased to below 1 g / L, feed was promptly added to maintain the glucose concentration at approximately 0.1 g / L. The recombinant *E. coli* engineered strain was cultured until the OD of the fermentation broth reached a certain level. 600 After ≥10, the temperature was gradually reduced to 25℃ and 0.5mM IPTG was added for induction culture for more than 20 hours until the glucose concentration in the fermentation broth was less than 0.1g / L;

[0029] S2. Whole-cell catalytic synthesis of LSTA: 25 g / L yeast cells, 160 mM Neu5Ac, 80 mM LNT, 100 mM Tris, and 20 mM MgCl2 were added to the bioreactor. The initial pH of the reaction solution was adjusted to 8 with NaOH, and the temperature was adjusted to 37°C. The pH was then adjusted to 8.0 with NaOH, and the catalytic reaction was continued for more than 16 hours, preferably 16 hours or 30-35 hours.

[0030] The beneficial effects of the whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a of the present invention are as follows:

[0031] This invention uses *E. coli* JM109(DE3) as the starting strain and constructs a *E. coli* glycosyltransferase module and a *Saccharomyces cerevisiae* CTP energy regeneration module by screening for highly active α-2,3-sialyl transferases (nst, CstII, PmST1 M144D). By screening for the optimal co-expression system and optimizing whole-cell catalytic conditions, the two modules synergistically and efficiently synthesize LSTA. The scalability of this strategy was verified in a 5L bioreactor through optimization of the synergistic catalytic synthesis of LSTA, achieving a final LSTA yield of 38.03 g / L. This provides an effective route for the industrial production of LSTA and offers new insights for the biosynthesis of other complex sialylated HMOs. Attached Figure Description

[0032] Figure 1 —A typical sialylation derivatization pathway diagram for LNT and LNnT, where LNT is (lactose-N-tetrasaccharide); LNnT is (lactose-N-neotetrasaccharide).

[0033] Figure 2—This diagram illustrates the biosynthetic pathway of recombinant Escherichia coli engineered strain and Saccharomyces cerevisiae LSTa, used in the whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a according to the present invention.

[0034] Abbreviations include: Neu5Ac (N-acetylneuraminic acid); CMP-Neu5Ac (cytidine-5′-monophosphate-N-acetylneuraminic acid); CTP (cytidine-5′-triphosphate); CMP (cytidine-5′-monophosphate); LNT (lactose-N-tetrasaccharide); LSTA (sialyllactose-N-tetrasaccharide).

[0035] Figure 3 —A comparative analysis of the effects of α-2,3-sialylate transferase from different species on LSTA production;

[0036] Among them, (A) SDS-PAGE of nst, M: protein marker, 1: uninduced cells, 2: induced cells; (B) SDS-PAGE of CstII, 1: uninduced cells, 2: induced cells; (C) SDS-PAGE of PmST1 M144D, 1: 1: uninduced cells, 2: induced cells; (D) Comparative analysis of LSTA synthesis capacity of different groups, transformation rate: evaluated based on LNT.

[0037] Figure 4 —This is a TLC analysis of the whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a in Example 1, using two-strain coupling and three-strain coupling whole-cell catalytic reactions;

[0038] Among them, (A) is the two-strain coupling experimental group, 1. Neu5Ac standard sample, 2. LNT standard sample, 3. standard sample, 4. catalytic reaction product of two-strain coupling;

[0039] (B) Three-strain coupling experimental group: 1. Control group (without added Saccharomyces cerevisiae catalytic reaction products), 2. LSTA standard sample, 3. Catalytic reaction products of three-strain coupling;

[0040] Figure 5 —This is a comparative analysis diagram of the gene combinations carried by each strain in Example 2 of the present invention and their ability to synthesize LSTA.

[0041] In the figure: (A) Genes carried by four co-expressing recombinant Escherichia coli engineered strains and their expression levels. The black arrows in front of the genes indicate that these genes are regulated by the T7 promoter. Among them, pRSFDuet-1 is defined as high expression level, and pETDuet-1 is defined as medium expression level.

[0042] (B): Comparative analysis of the synthetic capacity of LSTa by four co-expressed recombinant Escherichia coli engineered strains coupled with whole-cell catalytic reaction of Saccharomyces cerevisiae. The conversion rate was calculated relative to LNT.

[0043] Figure 6 —This is a comparative analysis of single-factor (temperature, pH, fermentation time) experiments in a whole-cell catalytic synthesis method (shake flask fermentation) of sialylated lactose-N-tetrasaccharide a in Example 3 of the present invention.

[0044] (A) Effect of temperature on LSTA yield; (B) Effect of reaction pH on LSTA yield; (C) Effect of reaction time on LSTA yield, with conversion calculated relative to LNT.

[0045] Figure 7 —This is a comparative analysis of single-factor experiments (induction conditions, amount of Escherichia coli and Saccharomyces cerevisiae added, and amount of reaction substrate added) in the whole-cell catalytic synthesis method (shake flask fermentation) of sialylated lactose-N-tetrasaccharide a in Example 3 of the present invention.

[0046] (A) Effect of induction conditions on LSTA yield; (B) Effect of Escherichia coli and Saccharomyces cerevisiae addition on LSTA yield; (C) Effect of substrate sialic acid and LNT addition on LSTA yield, with conversion rate calculated relative to LNT.

[0047] Figure 8 —This is a graph showing the changes in biomass, GLU concentration, LNT and LSTA content in a whole-cell catalytic synthesis method (bioreactor) for sialylated lactose-N-tetrasaccharide a in Example 4 of the present invention.

[0048] (A) OD in coupled fermentation culture of strain E02 and Saccharomyces cerevisiae 600 The curve showing the change in glucose level versus residual glucose, with the blue arrow indicating the start time of the induction process;

[0049] (B) Curves showing the changes in LNT and LSTA content during whole-cell catalytic synthesis. Detailed Implementation

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0051] 1. Construction of strains and plasmids

[0052] Table 1 lists the strains and plasmids used for gene cloning and construction in this experiment. Plasmids were amplified and preserved using E. coli DH5α, and gene expression and production were performed using E. coli JM109(DE3).

[0053] Table 1. Strains and plasmids used for gene cloning and construction in this invention.

[0054]

[0055] Neu5Ac is first synthesized into CMP-Neu5Ac by the CMP-Neu5Ac synthase, and then LSTa is synthesized by the reaction of CMP-Neu5Ac with LNT via α-2,3-sialyltransferase. Therefore, we selected and screened enzymes related to catalysis. Based on previous research, we selected the CMP-Neu5Ac synthase neuA from Neisseria meningitidis. We synthesized codon-optimized neuA, nst, CstII, and PmST1 M144D genes using GENEWIZ (Suzhou, China). These genes were then inserted into the vector pET28a(+), and the vector was transformed into E. coli JM109(DE3)-neuA to construct a recombinant E. coli engineered bacterium expressing the neuA gene alone.

[0056] Three genes encoding α-2,3-sialic acid transferase from different sources were selected: nst from Neisseria meningitidis, PmST1 M144D from Pasteurella multocida, and CstII from Campylobacter jejuni. The nucleotide sequences of the genes neuA, nst, CstII, and PmST1 M144D are shown in SEQ NO.1, SEQ NO.2, SEQ NO.3, SEQ NO.4, and Table 2, respectively. Through homologous recombination, the codon-optimized gene fragment was successfully integrated into the pET28a(+) vector, and then the vector was transformed into E. coli JM109(DE3) to construct recombinant E. coli engineered strains JM109(DE3)-nst, JM109(DE3)-PmST1, and JM109(DE3)-CstII expressing the neuA gene.

[0057] Table 2. Sequence information of genes neuA, nst, CstII, and PmST1 M144D

[0058]

[0059] Secondly, using the ClonExpress one-step cloning kit from Vazyme Biotech (Nanjing, China), the neuA and PmST1 genes were cloned into two vectors with different copy numbers, pRSFDuet-1 and pET-Duet1, respectively. All successfully constructed expression vectors were transformed into E. coli JM109(DE3) for protein expression, and then transformed into E. coli JM109(DE3) to construct recombinant E. coli engineered strains JM109(DE3)-E01 / E02 / E03 / E04 carrying co-expression plasmids.

[0060] The Saccharomyces cerevisiae cells used in this invention were provided by Shengli Biotechnology Co., Ltd. (Xinjiang, China). The freeze-dried yeast cells were resuspended in 100 mM Tris-HCl (pH 7.0) buffer, centrifuged at 8000 rpm at 4°C for 10 min, and stored at -20°C for the next biotransformation process. The wet weight (w / w) of the yeast cells was determined.

[0061] Alternatively, the brewing yeast may be brewing yeast (GDMCC 61663), brewing yeast (Saccharomyces cerevisiae) xpli, which was deposited on May 12, 2021 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:61663, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province.

[0062] 2. Culture medium, culture conditions and cell culture

[0063] This study used LB (Luria-Bertani) medium for Escherichia coli culture, with 10.0 g / L tryptone, 10.0 g / L sodium chloride, and 5.0 g / L yeast extract.

[0064] The fermentation medium contained: tryptone 10.0 g / L, glucose 10.0 g / L, yeast extract 5.0 g / L, glycerol 5.0 g / L, NH4Cl 2.764 g / L, Na2HPO4 3.549 g / L, KH2PO4 3.402 g / L, Na2SO4 0.7102 g / L, and MgSO4 0.3244 g / L. Based on the plasmid pET28a(+)(Kan) carried by the strain... R ), pRSFDuet-1(Kan R pET-Duet1(Amp) R The properties of the antibiotics were determined by adding them to the culture medium, including 30 μg / mL kanamycin and 100 μg / mL ampicillin.

[0065] The culture process for this strain was as follows: First, the recombinant *E. coli* engineered bacteria were inoculated into 10 mL of LB medium containing antibiotics and cultured at 37°C and 200 rpm for 12-16 h. Then, the bacterial culture was inoculated at a 4% inoculum into 100 mL of LB liquid medium containing the corresponding antibiotics and cultured at 37°C and 200 rpm for 2-3 h until the bacterial culture reached OD500. 600 Once the bacterial count reaches 0.6-0.8, 0.1 mM IMPTG (isopropyl-β-D-thiogalactoside) is added, and the cells are cultured at 15°C and 200 rpm for 24 h. After induction, the cells are collected by centrifugation (8000 rpm, 4°C, 10 min), and the bacterial cells in the centrifuge tubes are stored at -20°C for the next step of biotransformation.

[0066] The collected cells can be used for SDS-PAGE analysis, which consists of a 5% acrylamide concentrate gel and a 12% acrylamide separation gel.

[0067] 3. TLC analysis method for product LSTa

[0068] Silicone 60 F 254 Aluminum plates (Merck Supelco, Darmstadt, Grman) were used as the stationary phase for thin-layer chromatography (TLC) analysis of the products. Products and standards (2 μL) were spotted onto the plate, and a mixture of n-propanol, purified water, and ammonia [15:9:1 (v / v / v)] was used as the developing solvent. After TLC separation and drying, the plates were sprayed with an acetone solution containing 10% phosphoric acid (v / v), 2% aniline (v / v), and 2% diphenylamine (v / v) for color development. After complete drying, the plates were activated at 105 °C for 5 min.

[0069] 4. High-performance liquid chromatography (HPLC) detection of product LSTA

[0070] The preparation methods for all samples are as follows: The catalytically reacted solution was centrifuged at 10,000 rpm for 15 min. The supernatant was diluted and filtered through a 0.22 μm aqueous membrane. The products were then detected using high-performance liquid chromatography (HPLC; Shimadzu LC2010, Japan). The chromatographic column was an Aminex HPX-87H ion exchange column (7.8 mm × 300 mm), the mobile phase was pure water containing 5 mM H₂SO₄, the flow rate was 0.6 mL / min, and the column temperature was maintained at 60 ℃. Diluted samples (10 μL) were analyzed at a detection wavelength of 210 nm for 30 min.

[0071] Comparative Example

[0072] Reference Figures 2-4A whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a, comprising the following steps:

[0073] S1. Construction of recombinant engineered Escherichia coli: Using E. coli JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene were expressed in free form to obtain recombinant engineered Escherichia coli; the α-2,3-sialotransferase gene was selected from PmST1M144D from Pasteurella multocida, nst from Neisseria meningitidis, or CstII from Campylobacter jejuni; the recombinant engineered Escherichia coli included JM109(DE3)-neuA, JM109(DE3)-nst, JM109(DE3)-PmST1, and JM109(DE3)-CstII;

[0074] S2. Whole-cell catalytic synthesis of LSTA: Using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates, IPTG (isopropyl-β-D-thiogalactoside) as an inducer, and recombinant Escherichia coli engineered strain JM109(DE3)-neuA+JM109(DE3)-PmST1 as the coupled fermentation strain, whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a was carried out.

[0075] The components of sialylated lactose-N-tetrasaccharide a synthesized by whole-cell catalysis without the addition of Saccharomyces cerevisiae are as follows: 80 mM MCPTP, 80 mM Neu5Ac, 80 mM LNT, 250 mM KH2PO4, 20 mM MgCl2, 120 mM Tris, 2 mM DTT, 220 mM glycerol, 4 g / L N-polyoxyethyl-N-octadecylamine, and 50 g / L (ww) recombinant Escherichia coli engineered strain JM109(DE3).

[0076] Example 1

[0077] Reference Figures 2-4 A whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a, comprising the following steps:

[0078] S1. Construction of recombinant engineered Escherichia coli: Using E. coli JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene were expressed in free form to obtain recombinant engineered Escherichia coli; the α-2,3-sialotransferase gene was selected from PmST1M144D from Pasteurella multocida, nst from Neisseria meningitidis, or CstII from Campylobacter jejuni; the recombinant engineered Escherichia coli included JM109(DE3)-neuA, JM109(DE3)-nst, JM109(DE3)-PmST1, and JM109(DE3)-CstII;

[0079] S2. By testing the ability to synthesize LSTA, we screened for α-2,3-sialotransferase genes that produce high levels of LSTA.

[0080] S3. Whole-cell catalytic synthesis of LSTA by three strains coupled together: using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates, IPTG (isopropyl-β-D-thiogalactoside) as inducer, and using recombinant Escherichia coli engineered strain JM109(DE3)-neuA+JM109(DE3)-PmST1 and Saccharomyces cerevisiae as coupled fermentation strains, the whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a was carried out.

[0081] In this embodiment, the components for whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a by adding Saccharomyces cerevisiae are as follows:

[0082] 80 mM CMP, 80 mM Neu5Ac, 80 mM LNT, 250 mM KH2PO4, 20 mM MgCl2, 120 mM MTris, 2 mM DTT, 220 mM glycerol, 4 g / L N-polyoxyethyl-N-octadecylamine, 300 mM glucose, 50 g / L (ww) recombinant Escherichia coli engineered strain JM109(DE3)-neuA + JM109(DE3)-PmST1 and Saccharomyces cerevisiae.

[0083] In S2, the verification experiment of the ability to synthesize LSTA in the test tube:

[0084] LSTa synthesis capacity experiment: 20 mM CMP-Neu5Ac, 20 mM LNT, 20 mM MgCl2, 2% (v / v) surfactant [octadecylamine:ethanol = 1:1 (m / m)] and 50 g / L *E. coli* cells expressing α-2,3-sialyl transferase were added to a 5 mL test tube containing 1 mL Tris-HCl (100 mM, pH 8.0). The mixture was reacted at 37 ℃ and 200 rpm for 24 h. The test results are as follows: Figure 3 As shown.

[0085] Depend on Figure 3 It was found that the PmST1 gene clone from Pasteurella multocida exhibited the highest activity for LSTA synthesis, with a conversion rate of 22.77%. The lower activities of the other two enzymes may be related to heterologous expression, indicating that these genes lack suitable expression conditions in Escherichia coli; or the temperature and pH conditions of the catalytic system may not be within the optimal range required by nst and CstII enzymes.

[0086] Based on the high α-2,3-sialic acid transferase activity of the engineered Escherichia coli strain JM109(DE3)-PmST1, LST1 was synthesized by two-strain coupling whole-cell catalysis using engineered Escherichia coli strains JM109(DE3)-neuA + JM109(DE3)-PmST1.

[0087] The catalytic products obtained by the whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a in the comparative examples and this embodiment were analyzed by TLC, and the TLC analysis chromatograms are shown below. Figure 4 As shown.

[0088] Depend on Figure 4 -B indicates that LSTA can be synthesized through whole-cell catalysis using a three-strain coupling method by adding Saccharomyces cerevisiae, proving that the energy regeneration strategy can effectively synthesize LSTA. Moreover, compared with the comparative example, the whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a in this embodiment, by adding CMP to replace CTP in the catalytic reaction component of the two-strain coupling fermentation, achieves the recycling and regeneration of CTP using CMP as a substrate through the powerful endogenous enzyme system in Saccharomyces cerevisiae, which significantly reduces the raw material cost of LSTA and provides an effective reference for cost reduction in industrial production.

[0089] The applicant obtained a highly active α-2,3-sialic acid transferase from the PmST1M144D gene of Pasteurella multocida through screening in this embodiment, constructed an Escherichia coli glycosyltransferase module, and clarified the feasibility of the whole-cell catalytic synthesis method of LSTA.

[0090] Example 2

[0091] To reduce the number of strains while increasing production rate, a co-expression strategy was adopted to integrate two genes into one strain for expression. neuA and PmST1 M144D were cloned into two vectors with different copy numbers, pRSF-Duet1 and pET-Duet1, and then transformed into E. coli JM109(DE3). Four recombinant E. coli engineered strains carrying the co-expression plasmid were constructed: JM109(DE3)-E01, JM109(DE3)-E02, JM109(DE3)-E03, and JM109(DE3)-E04.

[0092] Reference Figure 2 and Figure 5 The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a according to this embodiment includes the following steps:

[0093] S1. Construction of recombinant engineered Escherichia coli: Using E. coli JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene PmST1M144D were expressed in free form to obtain recombinant engineered Escherichia coli.

[0094] S2. Whole-cell catalytic synthesis of LSTA: Using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates at the shake-flask level, and IPTG (isopropyl-β-D-thiogalactoside) as an inducer, and any one of the recombinant Escherichia coli engineered strains (JM109(DE3)-E01, JM109(DE3)-E02, JM109(DE3)-E03, and JM109(DE3)-E04) and Saccharomyces cerevisiae as coupled fermentation strains, whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a was carried out.

[0095] In this embodiment, the components for whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a by adding Saccharomyces cerevisiae are as follows:

[0096] 80 mM CMP, 80 mM Neu5Ac, 80 mM LNT, 250 mM KH2PO4, 20 mM MgCl2, 120 mM MTris, 2 mM DTT, 220 mM glycerol, 4 g / L N-polyoxyethyl-N-octadecylamine, 300 mM glucose, 50 g / L (ww) recombinant Escherichia coli engineered strain JM109(DE3)-E01 / E02 / E03 / E04 and Saccharomyces cerevisiae.

[0097] Recombinant Escherichia coli engineered strains JM109(DE3)-E01 / E02 / E03 / E04, co-expressing plasmids, were coupled with Saccharomyces cerevisiae for whole-cell catalytic synthesis of LST. The composition of different recombinant Escherichia coli engineered strains and their corresponding LST synthesis yields are shown in the figure. Figure 5 As shown.

[0098] Depend on Figure 5 As shown in section -B, the LSTA yield of high-copy plasmid strains was significantly higher than that of medium-copy plasmid strains, and the LSTA yield synthesized by recombinant *E. coli* engineered strain JM109(DE3)-E02 and whole-cell catalysis of *Saccharomyces cerevisiae* was the highest, at 19.95 g / L. This is because the higher expression level of the target protein increased the activity of α-2,3-sialyl transferase, thereby promoting substrate transformation. The applicant will further optimize the reaction conditions of the recombinant *E. coli* engineered strain JM109(DE3)-E02 and whole-cell catalysis of *Saccharomyces cerevisiae* for synthesis.

[0099] Example 3

[0100] To improve enzyme activity and substrate conversion, the applicant systematically optimized various conditions of the whole-cell catalytic reaction system using single-factor optimization. First, they optimized the key factors directly determining enzyme activity: temperature, pH, and cell induction conditions. Second, they optimized several important parameters in the synthesis process: cell biomass, substrate addition amount, and catalytic reaction time.

[0101] Reference Figure 6 and Figure 7 The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a according to this embodiment includes the following steps:

[0102] S1. Construction of recombinant engineered Escherichia coli: Using E. coli JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene PmST1M144D were expressed in free to obtain recombinant engineered Escherichia coli JM109(DE3)-E02;

[0103] S2. Whole-cell catalytic synthesis of LSTA: At the shake-flask level, using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates, IPTG (isopropyl-β-D-thiogalactoside) as an inducer, and recombinant Escherichia coli engineered strain JM109(DE3)-E02 and Saccharomyces cerevisiae as coupled fermentation strains, sialylated lactose-N-tetrasaccharide a was synthesized through whole-cell catalysis.

[0104] In this embodiment, the components for whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a by adding Saccharomyces cerevisiae are as follows:

[0105] 80 mM CMP, 80 mM Neu5Ac, 80 mM LNT, 250 mM KH2PO4, 20 mM MgCl2, 120 mM MTris, 2 mM DTT, 220 mM glycerol, 4 g / L N-polyoxyethyl-N-octadecylamine, 300 mM glucose, 50 g / L (ww) recombinant Escherichia coli engineered strain JM109(DE3)-E01 / E02 / E03 / E04 and Saccharomyces cerevisiae.

[0106] Regarding the temperature of the catalytic reaction, according to Figure 6 As shown in Figure -A, the whole-cell catalytic synthesis of LSTa exhibited similar catalytic effects and substrate conversion rates at 37 °C and 42 °C, indicating that the optimal temperature range for the enzyme required for the whole-cell catalytic reaction coincides with the optimal temperature range of *E. coli*, where it exhibits optimal activity. Considering the cell's temperature tolerance, we will subsequently choose to conduct the catalytic reaction at 37 °C.

[0107] Under certain catalytic conditions, the applicant used reaction solutions with an initial pH of 7.0-9.5 for whole-cell catalytic synthesis of LST. The yield of LST varied significantly depending on the initial pH of the catalytic reaction solution. Figure 6 As shown in Figure B, in a catalytic reaction system with an initial pH of 8.0-8.5, it is evident that neuA and PmST1 M144D target proteins have corresponding optimal pH values, and the reaction is more favorable under alkaline conditions. Referring to previous experiments, the control group without pH adjustment showed almost no product formation (see...). Figure 4 (The control group in -B) Due to the presence of high-energy phosphoric acid compounds and acidic substances such as sialic acid in the catalytic system, the initial pH of the system was low, indicating that the acidic environment severely inhibited enzyme activity. The highest LSTA yield was observed at an initial pH of 8.0. Subsequently, the initial pH of the system was adjusted to 8.0 using NaOH to promote the reaction.

[0108] Since the plasmids used in this study are all based on the T7 promoter, in order to obtain more active target proteins, we need to optimize the induction conditions of the engineered strains, including the induction temperature and IPTG concentration. For strain culture, the engineered bacteria were cultured according to the above method, and when OD... 600 When the concentration reached 0.6-0.8, different induction schemes (20 ℃, 20 h; 25 ℃, 20 h; 30 ℃, 20 h) and inducer concentrations (0.1, 0.3, 0.5 and 1 mM) were used to generate a total of 12 experimental groups. The obtained strains were used to synthesize LSTA, the fermentation broth was collected by centrifugation, and the yield of LSTA was measured.

[0109] The results are as follows Figure 7 As shown in -A, the strain achieved the highest LSTA yield under the induction conditions of 25 °C and 0.5 mM IPTG, indicating that the appropriate induction temperature and inducer concentration play a key role in the soluble expression of the target gene.

[0110] In whole-cell catalysis, the biomass of *E. coli* and *Saccharomyces cerevisiae* significantly affects mass transfer and energy exchange during the reaction. Therefore, an appropriate biomass is essential for the forward propagation of the catalytic reaction and the accumulation of products. Optimization results can be found in [link to optimization results]. Figure 7 -B, the highest yield was obtained when the addition amounts of E. coli and yeast were 25 g / L and 50 g / L (ratio 1:2, wet weight), respectively. This indicates that the CTP cycle regeneration within the Saccharomyces cerevisiae plays a crucial role in the next reaction step. However, higher biomass in the reaction is not necessarily better; excessive biomass can affect the entry, exit, and transfer of substances during the reaction process, further leading to a decrease in yield.

[0111] like Figure 7 As shown in Figure -C, the highest yield and substrate conversion rate are obtained when the ratio of Neu5Ac to LNT is 2:1. In contrast, the effect is poor when the ratio is 1:1. Similarly, more substrate is not necessarily better; there is a suitable range.

[0112] Based on the optimal catalytic and induction conditions described above, the reaction time was optimized to obtain the best reaction duration. During the first 15 hours of the reaction, the product rapidly accumulated, then gradually decomposed, and began to rise again after 25 hours, reaching a peak yield of 36.99 g / L at 30 hours. Figure 6 -C is shown. This may be due to changes in enzyme activity caused by pH fluctuations during the reaction, as well as the presence of intermediate products in the catalytic solution, leading to variations in the amount of LSTA synthesized. To obtain a higher product accumulation during the reaction and reduce production costs, it is appropriate to control the catalytic reaction time to 30-35 h.

[0113] Example 4

[0114] Reference Figure 8 This embodiment describes a whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a, employing a two-step fermentation process in a bioreactor, comprising the following steps:

[0115] S1. Construction of recombinant engineered Escherichia coli: Using E. coli JM109(DE3) as the starting strain, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene PmST1M144D were expressed in free form to obtain recombinant engineered Escherichia coli.

[0116] S2. Whole-cell catalytic synthesis of LSTA: Using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates, IPTG (isopropyl-β-D-thiogalactoside) as inducer, and recombinant Escherichia coli engineered strain JM109(DE3)-E02 and Saccharomyces cerevisiae as coupled fermentation strains, whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a was carried out.

[0117] The specific operation was carried out in a 5 L bioreactor (Eppendorf, Germany) containing 2.5 L of fermentation medium;

[0118] The recombinant *E. coli* engineered strain was cultured in LB (Luria-Bertani) medium with an inoculum of 10% (v / v). A dissolved oxygen cascade stirring method was used for control, with a stirring range of 50–800 rpm, an aeration rate of 3 vvm, and DO maintained at 30%. 25% ammonia was added to maintain the pH at 7.0, and the initial culture temperature was 37 °C. When the OD600 reached 10, the temperature was lowered to 25 °C, and then IPTG was added to a final concentration of 0.5 mM. Simultaneously, when the glucose concentration dropped below 0.1 g / L, induction was performed for 20 h until the glucose concentration in the fermentation broth was less than 0.1 g / L. Then, 25 g / L yeast cells, along with reaction substrates of 160 mM Neu5Ac, 80 mM LNT, 100 mM Tris, and 20 mM MgCl2, were added to the bioreactor. The temperature was adjusted to 37 °C, and the pH was adjusted to 8.0 with NaOH. The reaction was continuously catalyzed for 30–35 h.

[0119] The changes in biomass and residual glucose concentration, LSTA yield and conversion rate with reaction time during the whole-cell catalytic synthesis reaction in this embodiment are as follows: Figure 8 As shown. During the first 16 hours of the reaction, the biomass of the whole-cell catalytic reaction system increased exponentially, and then stabilized after 16 hours. The glucose concentration in the reaction system decreased significantly in the first 8 hours, being consumed by cell growth, and was subsequently maintained at 0.1 g / L through feeding. Figure 8 -A is shown.

[0120] In the first 16 hours of the reaction, the synthesis of LSTA rapidly accumulated, reaching 33.85 g / L, which was positively correlated with the accumulation of biomass in the whole-cell catalytic reaction system. The yield then began to decline within 2 hours, before gradually increasing again. At 32 hours, the LSTA yield reached its peak at 38.03 g / L, with an LNT conversion rate of 46.57%. The product then degraded within the following 3 hours, such as... Figure 8-B shows the fluctuations in LSTA yield during the reaction process. This may be due to the natural degradation of the product, byproducts generated during microbial metabolism, and pH changes affecting enzyme activity and stability. To shorten the production cycle and the feeding cycle of subsequent catalytic reactions, the catalytic reaction time can be prioritized when the recombinant E. coli engineered bacteria and Saccharomyces cerevisiae fermentation culture enter the stable growth phase, such as 16 hours in this embodiment, thereby reducing the amount of LSTA product naturally degraded in subsequent reactions. To obtain a higher product yield, the catalytic reaction time can be considered to be controlled at the boundary between the stable growth phase and the decay phase, such as 32 hours in this embodiment.

[0121] In the description of this invention, it should be understood that features specified as "first" or "second" may explicitly or implicitly include one or more of those features. In the description of this invention, unless otherwise stated, "a plurality of" means two or more. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

Claims

1. A whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a, characterized in that, Includes the following steps: S1. Constructing recombinant engineered Escherichia coli: using E. coli JM109(DE3) was the starting strain. The cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene were expressed in free form to obtain recombinant Escherichia coli engineered bacteria. The α-2,3-sialotransferase gene was selected from PmST1M144D from Pasteurella multocida, nst from Neisseria meningitidis, or CstII from Campylobacter jejuni. S2. Whole-cell catalytic synthesis of LSTA: Using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates, IPTG as an inducer, and the above-mentioned recombinant Escherichia coli engineered strain and Saccharomyces cerevisiae as coupled fermentation strains, whole-cell catalytic synthesis of sialylated lactose-N-tetrasaccharide a was carried out.

2. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 1, characterized in that, The method for constructing the recombinant Escherichia coli engineered strain includes: inserting the neuA gene of cytosine-5'-monophosphate-N-acetylneuraminic acid synthase with optimized synthetic codons into the vector pET28a(+); inserting the nst, CstII, and PmST1M144D genes of α-2,3-sialotransferase with optimized synthetic codons into the vector pET28a(+); screening for high-yield LSTa-producing α-2,3-sialotransferase genes by testing LSTa synthesis capacity; and then transforming the vector into E. coli. coli Recombinant Escherichia coli engineered strains JM109(DE3)-neuA and JM109(DE3)-PmST1, which express the neuA and PmST1M144D genes respectively, were constructed from JM109(DE3).

3. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 2, characterized in that, The procedure for testing the ability to synthesize LSTA was as follows: 20 mM CMP-Neu5Ac, 20 mM LNT, 20 mM MgCl2, 2% (v / v) surfactant [octadecylamine:ethanol = 1:1 (m / m)] and 50 g / L E. coli cells expressing α-2,3-sialyltransferase were added to a 5 mL test tube containing 1 mL Tris-HCl (100 mM, pH 8.0). The mixture was reacted at 37 °C and 200 rpm for 24 h.

4. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 1, characterized in that, The method for constructing the recombinant Escherichia coli engineered strain includes: cloning the cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA (with optimized synthetic codons) and the α-2,3-sialotransferase gene PmST1M144D (with optimized synthetic codons) into two vectors with different copy numbers, pRSF-Duet1 and pET-Duet1, and then transforming them into E. coli. coli Four recombinant Escherichia coli strains, JM109(DE3)-E01, JM109(DE3)-E02, JM109(DE3)-E03, and JM109(DE3)-E04, carrying co-expression plasmids, were constructed from JM109(DE3).

5. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 4, characterized in that, The recombinant Escherichia coli engineered strain carrying the co-expression plasmid is JM109(DE3)-E02.

6. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 2 or 4, characterized in that, The catalytic components for the whole-cell catalytic synthesis of LSTA include: 80 mM CMP, 80 mM Neu5Ac, 80 mM LNT, 250 mM KH2PO4, 20 mM MgCl2, 120 mM Tris, 2 mM DTT, 220 mM glycerol, 4 g / L N-polyoxyethyl-N-octadecylamine, 300 mM glucose, and 50 g / L (ww) recombinant Escherichia coli engineered bacteria and Saccharomyces cerevisiae. The recombinant Escherichia coli engineered bacteria are JM109(DE3)-neuA and JM109(DE3)-PmST1, or one of JM109(DE3)-E01, JM109(DE3)-E02, JM109(DE3)-E03 and JM109(DE3)-E04.

7. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 6, characterized in that, The whole-cell catalytic synthesis method for sialylated lactose-N-tetrasaccharide a employs a shake-flask fermentation catalytic method, comprising the following steps: S1. Constructing recombinant engineered Escherichia coli: using E. coli JM109(DE3) was the starting strain. The cytosine-5'-monophosphate-N-acetylneuraminic acid synthase gene neuA and / or α-2,3-sialotransferase gene PmST1M144D were expressed in free form to obtain the recombinant Escherichia coli engineered strain JM109(DE3)-E02. S2. Whole-cell catalytic synthesis of LSTA: Using sialic acid Neu5Ac and lactose-N-tetrasaccharide LNT as substrates and IPTG as an inducer, the above-mentioned recombinant Escherichia coli engineered strain JM109(DE3)-E02 and Saccharomyces cerevisiae as coupled fermentation strains were used to synthesize sialylated lactose-N-tetrasaccharide a through whole-cell catalysis. The shake-flask fermentation conditions for the whole-cell catalytic synthesis of LSTA are as follows: fermentation temperature of 37-42℃, pH of 8.0-8.5, induction with 0.3-1mM IPTG at 20-25℃, the weight ratio of sialic acid to lactose-N-tetrasaccharide in the substrate is 1:1 to 2:1, and the sialic acid content is ≥160mM; the ratio of the recombinant Escherichia coli engineered strain JM109(DE3)-E02 to Saccharomyces cerevisiae is 1:

2.

8. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 7, characterized in that, The shake-flask fermentation conditions for the whole-cell catalytic synthesis method are as follows: fermentation temperature is 37℃, pH is 8.0, 0.5mM IPTG is added at 25℃ for induction, the weight ratio of sialic acid to lactose-N-tetrasaccharide in the substrate is 2:1, and the sialic acid content is ≥160mM; the amount of recombinant Escherichia coli engineered strain JM109(DE3)-E02 and Saccharomyces cerevisiae added are 25g / L and 50g / L, respectively.

9. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 1, characterized in that, The whole-cell catalytic synthesis method employs a two-step fermentation process in a bioreactor, including the following steps: S1. Induction Culture Strains: Recombinant Escherichia coli engineered strain JM109(DE3)-E02 was fermented in LB (Luria-Bertani) medium with an inoculum size of 10% (v / v). Dissolved oxygen cascade stirring was used for control, with a stirring range of 50-800 rpm and an aeration rate of 3 vvm. DO was maintained at 30%, and 25% ammonia was added to maintain the pH at 7.

0. The initial culture temperature was 37 ℃. When the glucose residue in the medium decreased to below 1 g / L, feed was promptly added to maintain the glucose concentration at approximately 0.1 g / L. The recombinant Escherichia coli engineered strain was cultured until the OD of the fermentation broth reached a certain level. 600 After ≥10, the temperature was gradually reduced to 25℃ and 0.5mM IPTG was added for induction culture for more than 20 hours until the glucose concentration in the fermentation broth was less than 0.1g / L; S2. Whole-cell catalytic synthesis of LSTA: 25 g / L yeast cells, 160 mM Neu5Ac, 80 mM LNT, 100 mM Tris, and 20 mM MgCl2 were added to the bioreactor. The initial pH of the reaction solution was adjusted to 8 using NaOH, and the temperature was adjusted to 37℃. The catalytic reaction was continued for more than 16 hours.

10. The whole-cell catalytic synthesis method of sialylated lactose-N-tetrasaccharide a as described in claim 9, characterized in that, In S2, the catalytic reaction time is shown to be 16 h or 30-35 h.

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