CN113897405B - Method for synthesizing 3' -sialyllactose by three-strain coupling fermentation at low cost - Google Patents
Method for synthesizing 3' -sialyllactose by three-strain coupling fermentation at low cost Download PDFInfo
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- CN113897405B CN113897405B CN202110623050.5A CN202110623050A CN113897405B CN 113897405 B CN113897405 B CN 113897405B CN 202110623050 A CN202110623050 A CN 202110623050A CN 113897405 B CN113897405 B CN 113897405B
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Abstract
The invention discloses a method for synthesizing 3' -sialyllactose by three-strain coupling fermentation at low cost, belonging to the field of biological fermentation. The invention constructs and obtains recombinant bacteria by heterologously expressing alpha-2, 3-sialyltransferase and CMP-sialyltransferase in microorganism, adds two recombinant bacteria and Saccharomyces cerevisiae into a fermentation system which takes CMP, sialic acid and lactose as substrates, and the coupled bacteria colony system can directly convert the cheap substrates into 3' -sialyllactose with the conversion rate up to 70%. The fermentation method can obviously reduce the production cost and can be applied to industrialized mass production.
Description
Technical Field
The invention relates to a method for synthesizing 3' -sialyllactose by three-strain coupling fermentation at low cost, belonging to the field of biological fermentation.
Background
The 3' -sialyllactose exists in breast milk, especially in early breast milk in high abundance, plays an important role in the growth of infants, and has a potential and wide market prospect in the food industry, especially in the infant formula milk powder industry. Since sialyllactose is contained in a very low amount in mammalian breast milk, sialyllactose obtained by a natural extraction method is low in yield, high in cost and complicated to operate. Chemical synthesis requires cumbersome protection and deprotection procedures, and enzyme purification is expensive and difficult, so that whole cell catalysis has certain advantages in the production of 3' -sialyllactose.
Breast milk is the best nutrient for neonates after birth, and contains all essential nutrients required for the growth and development of infants. It contains more healthy and beneficial components than milk and milk powder, and part of the functional components are human milk oligosaccharides (human milk oligosaccharides, HMOs). The high content and structural diversity of HMOs is unique to humans, with only trace amounts being present in mature cow milk as well as in infant formulas. The human milk oligosaccharide is used as the third largest solid component with the content next to lactose and lipid in human milk, the concentration changes at the level of 10-15 g/L in mature breast milk, and even reaches 20-30 g/L in colostrum, thus having important effect on the healthy development of infants.
HMOs play an important role in preventing pathogens and toxins from adhering to epithelial surfaces. Due to their similarity to the structure of cytoglycans, viruses and bacteria are prevented from adhering to their carbohydrate mucosal receptors by acting as soluble baits, thereby helping to combat enteric pathogens. As soluble receptors for a variety of bacteria and viruses, HMOs can inhibit adhesion of a variety of bacteria such as Campylobacter jejuni and Streptococcus pneumoniae to intestinal epithelium. In addition, bacterial enterotoxins, such as cholera toxin, can also be protected by competition with cell-bound glycan receptors. HMOs play a promoting role in the intestinal immune system, with prebiotic effects. This is because beneficial bacteria metabolize HMOs and utilize metabolites (e.g., short chain fatty acids) produced by their degradation to promote growth of beneficial bacteria (e.g., bifidobacteria) in the colon, while harmful bacteria fail to metabolize HMOs, thereby improving the microbial intestinal flora of infants and infants to maintain intestinal ecological balance; or inhibit pathogen proliferation by lowering intestinal pH. HMOs have the function of promoting the development of the nervous system and brain. Studies have shown that sialic acid is useful for the synthesis of gangliosides and glycoproteins in infant brain tissue, and sialic acid residues at the ends of HMOs can provide sialic acid to increase brain gangliosides and glycoproteins concentration and promote infant brain maturation.
Currently, more than 200 HMOs have been identified. HMOs consist of five monosaccharide building blocks of glucose (Glc), galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (N-acetylneuraminic acid, neu5Ac, also known as sialic acid), which form linear or branched oligosaccharides under glycosyltransferases. The reducing end of human milk oligosaccharides is typically lactose structures, whereas the non-reducing end typically contains fucose and/or sialic acid moieties, so HMOs can be divided into three types: (1) Neutral oligosaccharides-fucosylated oligosaccharides and nonfucosylated oligosaccharides; (2) an acidic oligosaccharide, sialylated oligosaccharide; (3) Oligosaccharides with both fucosylation and sialylation modifications.
Wherein, the sialyloligosaccharide inhibits the proliferation of intestinal epithelial cells and induces cell differentiation through the interaction of oligosaccharide and carbohydrate on an Epidermal Growth Factor (EGF) receptor, and promotes intestinal maturation. Sialyllactose has anti-infective and immunoregulatory effects. It was found that sialic acid in sialyllactose exists as a glycoconjugate on bacterial surfaces as well as on host cell membranes, has multiple substituted binding sites, and by competing for these binding sites, sialyllactose can prevent or reduce pathogen adhesion in vitro. In addition, sialyllactose has antibacterial and anti-inflammatory activities, and has the effects of promoting the proliferation of probiotics, improving intestinal flora, helping the development and maturation of the infant immune system, promoting the maturation of the infant brain, improving learning ability and the like, and has been attracting more attention in recent years.
At present, the preparation of sialyllactose mainly comprises methods of natural extraction, chemical synthesis, enzymatic synthesis, biosynthesis and the like. The natural extraction is to extract from goat milk with high content of galactooligosaccharide (the content of galactooligosaccharide is 4-5 times of that of cow milk and 10 times of that of goat milk), and the 3 '-sialyllactose content in the oligosaccharide mixture obtained by separating and purifying the goat milk by ultrafiltration and nanofiltration is only 0.03-0.05 g/L by utilizing the principle of membrane interception, and the separation and purification are further needed to obtain pure substances only containing 3' -sialyllactose. The chemical synthesis method uses automatic solid phase synthesis, but because of the stereoselectivity control and harsh reaction conditions for constructing the glycosidic bond and a plurality of active groups on the sialyloligosaccharide ring, repeated protection and deprotection processes are needed, the operation is quite complicated, the defects of high energy consumption, serious environmental pollution and the like exist, and the large-scale production of the sialylolactose synthesized by the chemical method is limited to a certain extent. The enzymatic synthesis was performed using enzymes involved in the synthesis of 3 '-sialyllactose, michel Gilbert et al (MICHEL GILBERT R B A E. The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac sylhecase/sialyltransferase fusion [ J ]. Nature biotechnology, 1998, 16 (4)) by constructing a fusion protein expressing the genes of CMP-NeuAc synthase and α -2, 3-sialyltransferase in recombinant E.coli, adding the purified fusion protein to a system starting with lactose, N-acetylneuraminic acid, phosphoenolpyruvate, ATP and CMP, to obtain 3' -sialyllactose. However, the enzyme method needs to be utilized after being purified, which increases the cost and difficulty of the enzyme method synthesis and severely limits the production scale. The biological synthesis components are single-cell synthesis and multicellular coupling, but the synthesis components are easy to cause accumulation of products in cells, produce feedback inhibition, limit the yield of 3' -sialyllactose, or require continuous substrate addition in the fermentation process to ensure the normal operation of the reaction, so that the production cost is high.
Therefore, in the current technology, the operation of sialyllactose is complex, the productivity is low, and the cost of production is high, which hinders the large-scale production and application of 3' -sialyllactose.
Disclosure of Invention
The invention adopts a multi-fungus coupling fermentation method, and the alpha-2, 3-sialyltransferase and the CMP-sialyltransferase are heterologously expressed by microorganisms to construct recombinant bacteria, and the recombinant bacteria are added into a reaction system containing the CMP, and the combined action of saccharomycetes is added, so that 3' -sialyllactose can be synthesized by low-cost substrate reaction. The invention provides a method for producing 3 '-sialyllactose, which adopts a mixed bacteria coupling fermentation mode to produce 3' -sialyllactose by taking CMP, lactose and Neu5Ac as substrates; the mixed bacteria coupling fermentation is a genetic engineering strain which utilizes Saccharomyces cerevisiae, a genetic engineering strain expressing alpha-2, 3-sialyltransferase and a genetic engineering strain expressing CMP-Neu5Ac synthetase.
In one embodiment, the expression of the genetically engineered strain expressing an alpha-2, 3-sialyltransferase is derived fromN. meningitidisIs a. Alpha. -2, 3-sialyltransferase of (A).
In one embodiment, the nucleotide sequence of the gene encoding the α -2, 3-sialyltransferase is shown in SEQ ID NO. 2.
In one embodiment, the expression of the genetically engineered strain expressing CMP-Neu5Ac synthase is derived fromN. meningitidisThe nucleotide sequence of the coding gene of the CMP-Neu5Ac synthetase is shown as SEQ ID NO. 1.
In one embodiment, the genetically engineered bacterium is a host of escherichia coli, yeast or bacillus subtilis.
In one embodiment, the host of the genetically engineered bacterium is preferably escherichia coli, and more preferably, escherichia coli from which beta-galactosidase is knocked out is selected as the host.
In one embodiment, the CMP-Neu5Ac is produced by fermentation using a genetically engineered bacterium expressing the CMP-Neu5Ac synthase with CTP and sialic acid as substrates, or by fermentation using a genetically engineered bacterium expressing the CMP-Neu5Ac synthase and Saccharomyces cerevisiae with CMP and sialic acid.
In one embodiment, the saccharomyces cerevisiae and the genetically engineered bacteria are added into a reaction system according to the mass ratio of (1-2): 1-2, the reaction time is 2-6 hours, the sialic acid addition amount is 50-60 mmol/L, and the Mg 2+ The concentration is 10-20 Mm/L.
In one embodiment, the Saccharomyces cerevisiae has been deposited at the Cantonese province microorganism strain collection, 5.12, 2021 under accession number GDMCC No. 61663.
In one embodiment, the saccharomyces cerevisiae and the genetically engineered bacteria are added into a reaction system in a mass ratio of 2:1, and the reaction time is 2-6 hours, and the saccharomyces cerevisiae and the genetically engineered bacteria are salivaThe addition amount of the liquid acid is 60mmol/L, mg 2+ The concentration was 20Mm/L.
In one embodiment, the Saccharomyces cerevisiae is added in an amount of 100g/L.
In one embodiment, recombinant bacteria expressing CMP-Neu5Ac synthetase and genetically engineered bacteria expressing alpha-2, 3-sialyltransferase are added when CTP, sialic acid and lactose are used as substrates.
In one embodiment, the CTP, sialic acid, and lactose are present in an amount of 60-90mM, 50-90mM, and 70-90mM, respectively.
In one embodiment, when CMP, sialic acid and lactose are used as substrates, saccharomyces cerevisiae, genetically engineered bacteria expressing CMP-Neu5Ac synthetase and genetically engineered bacteria expressing alpha-2, 3-sialyltransferase are added, wherein the ratio of the three bacteria is (1-2): 1-2.
In one embodiment, the three bacteria are added into the reaction system in a mass ratio of 2:1:1, and the added amount of the saccharomyces cerevisiae is 100g/L.
In one embodiment, the content of CMP, sialic acid and lactose is 60-90mM, 50-90mM and 70-90mM, respectively.
In one embodiment, the reaction is 25 to 40 hours.
In one embodiment, the reaction is carried out at 25-35℃and 150-250 r/min.
The invention has the beneficial effects that:
according to the invention, the alpha-2, 3-sialyltransferase and the CMP-sialyltransferase are heterologously expressed in the microorganism, so that the recombinant bacterium is obtained by constructing, and the recombinant bacterium and the saccharomycete are added into a reaction system containing low-concentration CMP, so that the 3' -sialyllactose can be directly synthesized by taking cheap lactose, sialic acid and CMP as substrates, the production cost is obviously reduced, and the method can be applied to industrialized mass production.
Preservation of biological materials
The saccharomyces cerevisiae provided by the invention has the classification name ofSaccharomyces cerevisiaeThe microorganism strain is preserved in the collection of microorganism strains of Guangdong province at 5.12 of 2021, the preservation number is GDMCC No. 61663, and the preservation address is building 5 of Guangzhou Mitsui 100 institute of Hirship.
Drawings
FIG. 1 is a diagram showing the cleavage verification of recombinant plasmid pET28 a-neuA; and (3) injection: m:10000 A DNA Marker;1: recombinant plasmid after Nde I/Sal I double enzyme cutting.
FIG. 2 is an HPLC plot of the different standards; (A) CMP; (B) CDP; (3) CTP; (4) CMP-Neu5Ac.
FIG. 3 is a schematic view ofE. coliBL21 (DE 3)/pET 28a-neuA synthesized CMP-neu5Ac analysis chart; (A) is a crushed supernatant; (B) adding a substrate to the thalli; (C) crushing supernatant without adding substrate; and (D) the substrate is not added to the thallus.
FIG. 4 is a diagram ofE. coliBL21 (DE 3)/pET 28a-K235NneuA expression synthesis CMP-neu5Ac analysis chart; (A) adding substrate to the crushed supernatant; (B) crushing the supernatant without adding a substrate.
FIG. 5 is a diagram showing the cleavage verification of recombinant plasmid pET28a-nst.
FIG. 6 is a restriction enzyme verification of recombinant plasmid pET22 b-nst; and (3) injection: m:10000 A DNA Marker; warp yarnNcoI/BamH I double digested recombinant plasmid.
FIG. 7 is a SDS-PAGE map of JM109 (DE 3)/pET 28a-neuA recombinant proteins; m: a Marker; lane 1 is before induction; lane 2 is after induction.
FIG. 8 is a SDS-PAGE of JM109 (DE 3)/pET 28a-nst recombinant protein nst; m is Marker, 1 is before induction; 2: after induction.
FIG. 9 is a SDS-PAGE diagram of two genetically engineered expressed proteases of interest; m: protein markers; 1: BL21 (DE 3)/pET 22b-nst pre-induction whole cells; 2: BL21 (DE 3)/pET 22b-nst induced 20 h whole cells; 3: BL21 (DE 3)/pET 28a-nst pre-induction whole cells; 4: BL21 (DE 3)/pET 28a-nst induced 20 h whole cells.
FIG. 10 is an HPLC detection chart of single-cell synthetic CMP-Neu5Ac; a: a CMP-Neu5Ac standard liquid phase diagram; b: liquid phase diagram of single-cell catalytic product (CMP-Neu 5 Ac).
FIG. 11 shows the effect of different cell concentrations on the synthesis of CMP-Neu5Ac by JM109 (DE 3)/pET 28 a-neuA.
FIG. 12 shows the reuse of recombinant JM109 (DE 3)/pET 28 a-neuA.
FIG. 13 is a graph of CMP-Neu5Ac detection; and (3) injection: a: TLC detection: 1: CMP;2: CDP;3: CTP;4: CMP-Neu5Ac;5: double bacteria coupling catalysis; b: standard CMP; c: standard CMP-Neu5Ac; d: double bacteria coupling catalytic liquid phase diagram.
FIG. 14 shows TLC detection of 3' -sialyllactose; and (3) injection: 1: lactose; 2:3' -SL;3.: lactose + CMP-Neu5Ac;4: BL21 (DE 3)/pET 28a-nst cells + lactose; 5: BL21 (DE 3)/pET 28a-nst cell + lactose + CMP-Neu5Ac;6: BL21 (DE 3)/pET 28a-nst disruption supernatant+lactose+CMP-Neu 5Ac;7: BL21 (DE 3)/pET 28a-nst disruption pellet + lactose + CMP-Neu5Ac;8: BL21 (DE 3)/pET 22b-nst cell + lactose; 9: BL21 (DE 3)/pET 22b-nst cell + lactose + CMP-Neu5Ac;10: BL21 (DE 3)/pET 22b-nst disruption supernatant+lactose+CMP-Neu 5Ac.
FIG. 15 shows TLC detection of 3' -sialyllactose; and (3) injection: 1: lactose; 2: sialyllactose; 3: before purification; 4: flow through; 5: washing with water; 6: eluting with 80% acetonitrile.
FIG. 16 shows TLC detection of 3' -sialyllactose; and (3) injection: 1: lactose; 2:3' -SL;3: JM109 (DE 3)/pET 28a-nst cells; 4: JM109 (DE 3)/pET 22b-nst cells.
FIG. 17 shows TLC detection of 3' -sialyllactose; and (3) injection: 1: lactose; 2:3' -SL;3: before purification; 4: flow through; 5: washing with water; 6: eluting with 80% acetonitrile; 7: lactose + sialic acid.
FIG. 18 shows TLC detection of 3' -sialyllactose; and (3) injection: 1: lactose; 2: glucose; 3:3' -SL;4: three bacteria couple the catalytic product.
FIG. 19 is a MALDI-TOF-MS analysis of 3' -sialyllactose.
Detailed Description
1. Analytical detection of CTP
(1) TLC thin layer chromatography identification
Deployment of the deployment agent: ethanol: 1M ph6.5 ammonium acetate = 6:4, reference is made to HERMAN H.HIGA J C P. Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glyco-l-, and N-O-diacetylneuraminic acids [ J ]. The Journal of biological chemistry, 1985, 260 (8838-8849).
Spotting: the CMP, CTP and sample were spotted onto TLC plates 2. Mu.L each time, a small number of times, so that the spot diameter of the sample did not exceed 2 mm. And the dried product can be put into a chromatographic cylinder after being dried.
Spreading: the TLC plate was placed obliquely in a chromatography cylinder with the developing agent, with the sample end down, immersed in the developing agent (liquid level below the spot). The developer was taken out and dried when it extended to a distance of 1 to cm from the upper end of the TLC plate.
Color development: color development was performed by ultraviolet irradiation.
(2) HPLC analysis and detection
The conditions for detecting CMP and CTP by HPLC are shown in Table 1.
TABLE 1 HPLC detection of relevant parameters for CTP content
The detection results of the CMP and CTP standard products are shown in figures 2-3, the peak time of the CMP is 5.8 min, and the peak time of the CTP is 7.0 min.
2. Analytical detection of CMP-Neu5Ac
(1) TLC thin layer chromatography identification
The same analysis and detection method as CTP.
(2) HPLC analysis and detection
The conditions for HPLC detection of CMP-Neu5Ac are shown in Table 2.
TABLE 2 HPLC conditions for detecting CMP-Neu5Ac
3. Detection, separation and purification of sialyllactose
(1) Separation and purification
The reacted solution is centrifuged for 10 min by 10000 r/min, and the obtained supernatant is separated and purified by HyperSep Hypercarb solid phase extraction column (SPE column). The purification steps are as follows:
(1) activating: the SPE cartridge was activated with 3 mL methanol.
(2) Balance: the SPE cartridge was equilibrated by adding 3 mL deionized water.
(3) Loading: 500. Mu.L of sample was added and the running-through liquid was noted.
(4) Washing: the SPE cartridge was rinsed with 3 mL deionized water and the resulting liquid was noted as water wash.
(5) Eluting: the SPE cartridge was eluted with 3 mL of 80% acetonitrile and the resulting solution was recorded as purified.
(2) TLC thin layer chromatography identification
Deployment of the deployment agent: n-propanol: water: 25% ammonia = 7.5:3:2 (volume ratio).
Configuration of aniline-diphenylamine-phosphoric acid developer: 4 g diphenylamine, 4 mL aniline and 20 mL of 85% phosphoric acid were co-dissolved in 200 mL acetone.
Color development: the dried TLC sheet was immersed uniformly in the color developing agent, dried and heated at 105℃for 5 min.
Example 1: heterologous expressionneuAConstruction of recombinant bacteria
To be used forN. meningitidisIn (a)neuThe A gene sequence is used as a template, and a primer neuAF (5' -TTC) is designedCATATGGAAAAACAAAATATTGCGGTTATAC-3', underlinedNdeI cleavage site) and neuAR (5' -GACGTCGACTTAGCTTTCCTTGTGATTAAGAATGTTT-3', underlinedSalI cleavage site) to amplifyneuThe gene A, the CMP-Neu5Ac synthetase gene (nucleotide sequence shown as SEQ ID NO. 1) with 687bp size is obtained by PCR, and the PCR product is purified and recovered and then usedNdeI andSali double enzyme cutting, purifying and recovering enzyme cutting fragments. Then the pET28a empty plasmid is digested with the same enzymes, purified and recovered, and then is connected with the fragment obtained before, and the connection product is convertedE. coliJM109 was plated on LB medium plates containing kan with an appropriate amount of the transformation solution, cultured overnight at 37℃and after colony PCR was correct, single colony shake flask culture was performed to extract plasmids and the two fragments were confirmed to be about 5310, bp and 687, bp (the results are shown in FIG. 1). The obtained recombinant expression plasmid pET28a-neuA is subjected to further sequencing verification to obtain the recombinant plasmid pET28a-neuA, and the pET28a-neuA is converted intoE. coliBL21 (DE 3) and knockout of beta-galactosidase-encodinglacZStrains of genesE. coliJM109（DE3）△LacZScreening on LB medium plate containing kan, and screening to obtain the final product containing heavy substancesThe transformant of the group plasmid pET28a-neuA is recombinant strain JM109 (DE 3)/pET 28a-neuA andE. coliBL21（DE3）/pET28a-neuA。
example 2: heterologous expressionnstConstruction of engineering strains of (2)
To be used forN. meningitidisAlpha-2, 3-sialyltransferase genesnstThe gene sequence was used as template, and primer nstF (5'-ATGCCATGGGGTCTGAAAAAGGTCTGTCT-3', cleavage site:Ncoi) And nstR (5'-CGGGATCCTTATTTGGATTTACCATGAGATTGGTTTTTGT-3', cleavage site:BamHI) to amplifynstThe sialyllactose transferase gene (nucleotide sequence shown as SEQ ID NO. 2) with 1137bp is obtained by PCR, and the PCR product is purified and recovered and then usedNcoI andBamh I double enzyme cutting and purifying and recovering the enzyme cutting segment fragments. Then the pET28a empty plasmid is digested with the same enzymes, purified and recovered, and then connected with the fragment obtained before, and the connection product is converted intoE. coliJM109 (DE 3). Coating proper amount of the conversion solution on LB culture medium plate containing kan, culturing overnight at 37 ℃ until single colony is grown, picking single colony, shake flask culturing, extracting plasmid, enzyme cutting and verifying, and performing recombinant pET28a-nstNcoI andBamh I double enzyme digestion and electrophoresis results are shown in FIG. 5. The obtained recombinant expression plasmid pET28a-nst is consistent with the target gene in the sequencing result, which shows that the recombinant plasmid pET28a-nst is successfully constructed, and the recombinant bacterium containing pET28a-nst is JM109 (DE 3)/pET 28a-nst. Transformation of pET28a-nst toE. coliBL21 (DE 3) and strainsE. coliJM109 (DE 3) was selected on a kan-containing LB medium plate, and the selected transformant containing the recombinant plasmid pET28a-neuA was recombinant JM109 (DE 3)/pET 28a-nst andE. coliBL21（DE3）/pET28a-nst。
in the above waynstThe gene is constructed to a pET22b vector to obtain a recombinant plasmid pET22b-nst. The plasmid was subjected toNcoI andBamh I double enzyme digestion and electrophoresis results are shown in FIG. 6. As can be seen from FIG. 6, the two fragments obtained by electrophoresis after cleavage of the recombinant plasmid pET28a-nst were about 5400 bp (5471 bp after cleavage of pET28 a) and about 1100 bp, respectively (nst gene is encoded by 1137bp bases). The recombinant plasmid is sent to sequencing, and the sequencing resultThe recombinant plasmid pET22b-nst is consistent with the target gene, which shows that the construction is successful. Transformation of pET22b-nst toE. coliBL21 (DE 3) and strainsE. coliJM109 (DE 3) was selected on a kan-containing LB medium plate, and the selected transformants containing the recombinant plasmid pET22b-nst were recombinant JM109 (DE 3)/pET 22b-nst andE. coliBL21（DE3）/pET22b-nst。
example 3: inducible expression and detection of recombinant mycoprotein
(1) Heterologous expressionneuAProtein induction and detection of recombinant bacteria
Recombinant JM109 (DE 3) constructed in example 1 was transformed△LacZinoculating/pET 28a-neuA into 10 mL LB liquid medium containing 20 μg/mL Kan, shake-flask culturing at 37deg.C and 200 r/min for 12 h, transferring into 100mL LB liquid medium containing 20 μg/mL Kan according to 2% (2 mL/100 mL), shake-flask culturing at 37deg.C and 200 r/min for OD 600 After about 0.6, induction was performed by adding IPTG at a final concentration of 0.1 mmol/L, and shaking culture was performed at 200 r/min for 20: 20 h. The cells were collected by centrifugation at 8 r/min at 4℃for 10 min. And respectively mixing bacterial solutions before induction and 20 h with SDS-PAGE Loading buffer solution, and heating at 100 ℃ for 10 min to obtain samples before and after induction, wherein the SDS-PAGE detects the expression of the target protein.
As can be seen from FIG. 7, the inducible group had a distinct expression band between 25 kD and 35 kD compared to the control group (uninduced recombinant), consistent with the band size reported previously (30.4 kDa), indicating successful expression of the NeuA enzyme in engineering strain JM109 (DE 3)/pET 28 a-neuA.
(2) Heterologous expressionnstProtein induction and detection of recombinant bacteria
Recombinant bacteria JM109 (DE 3)/pET 28a-nst, BL21 (DE 3)/pET 28a-nst and obtained in example 2 were obtained in the same manner as in step 1E. coliBL21 (DE 3)/pET 22b-nst induced expression of the target protein, respectively.
FIG. 8 shows the protein expression patterns of strain JM109 (DE 3)/pET 28a-nst before and after induction, and it can be seen from FIG. 8 that a distinct protein band was added between 35 kD and 45 kD, which is consistent with the target protein (43.3 kDa), compared to the control group (non-induced recombinant). This indicates that the Nst enzyme in engineering strain JM109 (DE 3)/pET 28a-Nst successfully achieves expression in E.coli.
FIG. 9 shows protein expression patterns of strains BL21 (DE 3)/pET 28a-nst and E.coli BL21 (DE 3)/pET 22b-nst before induction, and it can be seen from the patterns that the protein band (lane 4) of BL21 (DE 3)/pET 28a-nst induced 20 h is newly increased by a distinct protein band between 35 kD and 45 kD compared with the control group (lane 3), and the value is consistent with that of the target protein (43.3 kD). BL21 (DE 3)/pET 22b-nst (lane 2) also enables expression of the protein of interest.
Example 4: synthesis of CMP-Neu5Ac
1. Synthesis of CMP-Neu5Ac by single bacterial transformation
Conversion conditions: 20mM MgCl 2 1 mM DTT, 150 mM Tris,60 mM CTP, 60mM Naeu5 Ac and 50g/L JM109 (DE 3)/pET 28a-neuA cells (wet weight) were reacted at 30℃and 200 r/min for 4 h. Thus, CMP-Neu5Ac (FIG. 10) was obtained, indicating that the production of CMP-Neu5Ac was achieved by recombinant JM109 (DE 3)/pET 28 a-neuA.
(1) The effect of engineering bacteria cell concentration on the synthesis of CMP-Neu5Ac was analyzed, the cell amounts in the above steps were set to 50g/L, 100g/L, 150g/L, 200g/L, respectively, and fermentation was performed using engineering bacteria JM109 (DE 3)/pET 28a-neuA, and the corresponding yield results are shown in FIG. 11. As can be seen from FIG. 11, when the bacterial concentration is 200g/L, the CMP-Neu5Ac synthesized by JM109 (DE 3)/pET 28a-neuA reaches the maximum value at 3h, and the CMP-Neu5Ac is gradually degraded with the increase of fermentation time; when the bacterial concentration is 100g/L and 150g/L, the synthesis amount of JM109 (DE 3)/pET 28a-neuA reaches the maximum value at 3 and 4 hours, especially the concentration of CMP-Neu5Ac generated by the bacterial concentration of 100g/L is highest and reaches about 29g/L, and then the CMP-Neu5Ac is rapidly degraded with the increase of time; when the bacterial concentration is 50g/L, the synthesis amount of CMP-Neu5Ac of JM109 (DE 3)/pET 28a-NeuA is increased with the increase of time, the maximum value of CMP-Neu5Ac is reached at the reaction time of 4 hours, the conversion rate is about 26g/L, the conversion rate is about 72%, and then the concentration of the synthesized CMP-Neu5Ac is basically kept stable with the increase of time and is kept about 22 g/L. Therefore, 50g/L of engineering strain concentration is selected as the optimal concentration for synthesizing CMP-Neu5Ac by using the JM109 (DE 3)/pET 28 a-neuA.
(2) Number of times of reuse of engineering strain JM109 (DE 3)/pET 28a-neuA
On the basis of determining the condition that the optimal thallus concentration is 50g/L, the influence of the recycling of engineering strain JM109 (DE 3)/pET 28a-neuA on the catalytic synthesis of CMP-Neu5Ac is studied. The results are shown in FIG. 12. The conversion rate of the engineering bacteria is set to be 100% for the first time, and after 4 times of repeated utilization, the conversion rate of the engineering bacteria still can reach 40% for the first time, so that the engineering strain JM109 (DE 3)/pET 28a-neuA is suitable for fed-batch fermentation.
2. Double-bacteria coupled fermentation synthesis of CMP-Neu5Ac
Fermentation conditions: 80mM CMP, 80mM Neu5Ac as substrate, 300 mM glucose, 248.3 mM KH 2 PO 4 1 mM DTT, 150 mM Tris, 10 mL/L glycerol, 6 mL/L acetaldehyde, mgCl of a certain concentration 2 50g/L JM109 (DE 3)/pET 28a-neuA and 100g/L Saccharomyces cerevisiae (Saccharomyces cerevisiae GDMCC No: 61663) were reacted at 30℃and 200 r/min.
The TLC detection results are shown in FIG. 13. As can be seen from the figure, after catalysis by the coupled catalytic system, CMP-Neu5Ac was successfully detected in the fermentation broth. To further determine the product, CMP-Neu5Ac standard and Saccharomyces cerevisiae and JM109 (DE 3)/pET 28a-neuA coupled catalysis products were analyzed by HPLC, and the peak time of the double bacteria coupled catalysis products was identical to that of the standard CMP-Neu5Ac (FIG. 13C and FIG. 13D).
Example 5: enzymatic synthesis of 3' -sialyllactose
The 3' -sialyllactose is synthesized by utilizing the broken liquid enzyme method of the engineering strain.
Enzymatic synthesis conditions: engineering strains BL21 (DE 3)/pET 28a-nst and BL21 (DE 3)/pET 22b-nst are respectively inoculated into LB culture medium (kanamycin), after IPTG induction of 20 h, the thalli are collected by centrifugation, and the thalli are treated with a proper amount of buffer solution (150 mmol.L) -1 Tris-HCl、20 mmol·L -1 MgCl 2 、1 mmol·L -1 DTT, pH 8.0) re-suspended ultrasonication, 4 ℃,8000 r min -1 Centrifuging for 20 min to obtain a crushed supernatant fraction and a crushed precipitate fraction. Equivalent amount of buffer for crushing sedimentAnd (5) liquid resuspension. mu.L of the disrupted supernatant, 250. Mu.L of the disrupted precipitated heavy suspension, and 50. 50g/L final concentration of wet cells (as whole cell-synthesized cells) were taken, 80mM CMP-Neu5Ac and 80 mmol.L of the resulting suspension were added, respectively -1 Lactose, 20 mL.L -1 Glycerol, 10 mL L -1 Xylene, 248.3 mmol.L -1 KH 2 PO 4 、5 g·L -1 MgCl 2 、4 g·L -1 Nymeen S-215, composed of 500. Mu.L system, at 30℃and 200 r. Mu.min -1 Under the condition of 30 h, the conditions of three parts of engineering bacteria BL21 (DE 3)/pET 28a-nst (or BL21 (DE 3)/pET 22 b-nst) thallus, broken supernatant and broken sediment are explored to catalyze and synthesize 3' -sialyllactose. The reacted catalyst liquid is 8000 r min -1 Centrifuging for 5 min, and retaining supernatant.
The production of 3' -sialyllactose was detected by TLC. The TLC detection results are shown in fig. 14. Both the catalytic solution of the disruption supernatants of the two engineering bacteria BL21 (DE 3)/pET 28a-nst and BL21 (DE 3)/pET 22b-nst (lanes 6, 10) and the catalytic solution precipitated after disruption (lanes 7, 11) had an oligosaccharide moiety identical to the Rf value of the standard 3' -sialyllactose (L2), whereas the catalytic solution of the whole cell (lanes 5, 9) did not produce the same substance, and it can be seen that there were almost no substances in lanes 5 and 9 identical to the Rf value of the standard lactose (lane 1).
To further confirm whether lane 6 (engineering bacterium BL21 (DE 3)/pET 28a-nst disruption supernatant) had 3 '-sialyllactose as the standard 3' -sialyllactose (lane 2) in the same Rf value. Crushing the reaction 30 h to obtain a crushed supernatant, and treating the crushed supernatant with a catalyst liquid of 8000 r min -1 Centrifugation was performed for 5 min, and the obtained supernatant was purified by SPE cartridge and analyzed by TLC, and the results are shown in FIG. 15. From the figure, it can be seen that the 80% acetonitrile elution fraction (lane 6) has the same Rf value as the standard 3 '-sialyllactose (lane 2), and thus the product can be determined to be 3' -sialyllactose. This indicates that: the constructed recombinant plasmids pET28a-nst and pET22b-nst have sialyltransferase activity, and can synthesize 3' -sialyllactose by using lactose and CMP-Neu5Ac.
Example 6: production of 3' -sialyllactose by multi-bacterium coupling fermentation
(1) Engineering bacterium host and expression vector for single cell fermentation selection
(1) Production of 3' -sialyllactose Using engineering bacterium JM109 (DE 3)/pET 28a-nst or JM109 (DE 3)/pET 22b-nst as fermentation strain
Fermentation conditions: 2mL System contains 1 mL of CMP-Neu5Ac synthesized as described in example 4 and 1 mL 80mM lactose, 20 mL/L glycerol, 10 mL/L xylene, 248.3 mM KH 2 PO 4 、5 g/L MgCl 2 A solution of 4 g/L Nymeen S-215, 50g/L JM109 (DE 3)/pET 28a-nst (or JM109 (DE 3)/pET 22 b-nst) was reacted at 30℃and 200 r/min for 30 h. The reaction solution was purified by SPE cartridge and analyzed by TLC. The results are shown in FIG. 14.
(2) Production of 3' -sialyllactose using engineering bacterium BL21 (DE 3)/pET 22b-nst as fermentation strain
As shown in FIG. 14, it can be seen from FIG. 14 that the supernatant and the pellet of engineering bacterium BL21 (DE 3)/pET 22b-nst cells after disruption have sialyltransferase activity, and 3 '-sialyllactose can be synthesized by fermenting and synthesizing sialyllactose by using the strain BL21 (DE 3)/pET 22b-nst, but the whole cell reaction of the strain BL21 (DE 3)/pET 28a-nst does not generate 3' -sialyllactose, and the exogenously added lactose substrate is consumed. The main reason for this phenomenon should be that the E.coli BL21 (DE 3) strain contains β -galactosidase, and exogenous lactose is hydrolyzed by endogenous β -galactosidase (LacZ) for self-growth or endogenous metabolism, but not for synthesis of 3' -sialyllactose; the disrupted supernatant and pellet are mainly used for the synthesis of 3' -sialyllactose, due in part to the fact that the cells are disrupted and the β -galactosidase is present, but the metabolic activity of the cells is terminated and thus the lactose is degraded to glucose and galactose with a greatly reduced need. Given that E.coli strains (e.g., DH 5. Alpha. And JM series) lacking a portion of the lacZ gene encoding beta-galactosidase are incapable of assimilating lactose or utilizing lactose, these E.coli strains can be used for the production of 3' -sialyllactose. Thus, E.coli JM109 (DE 3) capable of overexpressing proteins under the control of the T7 promoter was selected as an alternative host for 3' -SL production.
Thus, E.coli JM109 (DE 3) capable of overexpressing proteins under the control of the T7 promoter was selected as an alternative host for 3' -sialyllactose production.
Inoculating the obtained recombinant JM109 (DE 3)/pET 28a-nst and JM109 (DE 3)/pET 22b-nst into 10 mL LB liquid medium containing 20 μg/mL Kan, shake-flask culturing at 37deg.C and 200 r/min for 12 h, transferring into 100mL LB liquid medium containing 20 μg/mL Kan at 2%, shake-flask culturing at 37deg.C and 200 r/min to OD 600 After about 0.6, induction was performed by adding IPTG at a final concentration of 0.1 mmol/L, shaking culture was performed at 200 r/min for 20 hours, centrifugation was performed at 4℃at 8000 r/min for 10 minutes to collect cells, and fermentation was performed according to the conditions of example 5. The results are shown in FIG. 16. As can be seen from the figure, 3 '-sialyllactose can be synthesized by JM109 (DE 3)/pET 28a-nst and JM109 (DE 3)/pET 22b-nst whole cell catalysis, and the JM109 (DE 3)/pET 28a-nst whole cell catalysis can obtain more 3' -sialyllactose than JM109 (DE 3)/pET 22 b-nst; l4 (JM 109 (DE 3)/pET 22b-nst cells) had the same Rf value as the standard lactose (lane 1) but a lower concentration than the fermentation product of JM109 (DE 3)/pET 28a-nst cells. Thus, JM109 (DE 3)/pET 28a-nst strain having a higher conversion rate was selected as the fermentation strain.
(2) Double bacteria coupling catalysis condition
Conversion conditions: 2mL of reaction system, 60mM CTP, 60mM Neu5Ac and 80mM lactose are used as substrates, and 20mM MgCl is used as a substrate 2 1 mM DTT, 150 mM Tris, 20 mL/L glycerol, 10 mL/L xylene, 4 g/L Nymen S-215, 248.3 mM KH 2 PO 4 In 50g/L JM109 (DE 3)/pET 28a-neuA, 50g/L JM109 (DE 3)/pET 28a-nst solution, 30 h were reacted at 30℃and 200 r/min. The reaction solution was purified by SPE cartridge and analyzed by TLC.
The results are shown in FIG. 17. In the figure, the standard 3' -sialyllactose (lane 2) and the fermentation broth (lane 3) can be seen: in lane 3, the same oligosaccharide as the standard Rf exists, and the different components of the fermentation broth purified by the SPE column are sequentially lane 4, L5 and lane 6, wherein the Rf value of L6 is completely the same as that of the standard pure 3' -sialyllactose, but lactose exists at the same time, because 80% acetonitrile can not only elute acidic oligosaccharides, but also elute neutral oligosaccharides. This indicates that: under the condition of coupling fermentation of CTP, sialic acid and lactose serving as substrates, JM109 (DE 3)/pET 28a-neuA and JM109 (DE 3)/pET 28a-nst, 3' -sialyllactose is successfully synthesized.
(3) Three-bacteria coupling catalysis condition
Conversion conditions: 2mL of a reaction system, which uses 70 mM CMP, 60mM Neu5Ac and 80mM lactose as substrates, contains 300 mM glucose and 20mM MgCl 2 、248.3 mM KH 2 PO 4 1 mM DTT, 150 mM Tris, 20 mL/L glycerol, 6 mL/L acetaldehyde, 10 mL/L xylene, 4 g/L Nymeen S-215, 100g/L Saccharomyces cerevisiae (Saccharomyces cerevisiae GDMCC No: 61663), 50g/L JM109 (DE 3)/pET 28a-neuA, 50g/L JM109 (DE 3)/pET 28a-nst solution were reacted at 30℃under 200 r/min for 30 h. The reaction solution was purified by SPE cartridge and analyzed by TLC.
As a result, as shown in FIG. 18, the fermentation broth (L3) contained a component having the same Rf value as that of the standard 3' -sialyllactose, and was further characterized by mass spectrometry (FIG. 19), and the molecular weight wasM/z632([M-H] - ) The molecular weight is correct. This indicates that: the synthesis of 3' -sialyllactose is successfully realized by using a three-strain coupled fermentation system.
Comparative example 1: from different sourcesneuAConstruction, expression and conversion of engineering bacteria of fragments into CMP-Neu5Ac activity
The N-terminal sequence (HindIII and BamH I cleavage site) of NeuA enzyme from Escherichia coli K235 is connected with PET28a vector by the steps of cleavage and connection to obtain expression vector pET28a-K235NeuA, and transferred intoE. coliBL21 (DE 3) competent cells, obtainedE. coliBL21 (DE 3)/pET 28a-K235NneuA transformants. Then according to the system conversion conditions: 20 mmol.L -1 MgCl 2 、1 mmol·L -1 DTT、150 mmol·L -1 Tris，60 mmol·L -1 CTP、60 mmol·L - 1 Neu5Ac and g.L -1 Engineering bacterial cells (wet weight) at 30 ℃ and 200 r min -1 Reaction 2 h under conditions. The results were analyzed by HPLC. Is similar to the standardRatio (figure 2D),E. colisynthesis of CMP-Neu5Ac (FIG. 3) and BL21 (DE 3)/pET 28a-neuAE. coliIn the analysis of BL21 (DE 3)/pET 28a-K235NneuA synthesis of CMP-Neu5Ac (FIG. 4), onlyE. coliBL21 (DE 3)/pET 28a-neuA expressed enzymes have the ability to catalyze the synthesis of CMP-Neu5Ac.
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.
SEQUENCE LISTING
<110> university of Jiangnan
<120> method for synthesizing 3' -sialyllactose by three-strain coupling fermentation at low cost
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<170> PatentIn version 3.3
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Claims (6)
1. A method for producing 3 '-sialyllactose, which is characterized in that the 3' -sialyllactose is produced by adopting a mixed bacteria coupled fermentation mode and taking CMP, lactose and Neu5Ac as substrates; the mixed bacteria coupling fermentation is to utilize Saccharomyces cerevisiae, gene engineering strain expressing alpha-2, 3-sialyltransferase and gene engineering strain expressing CMP-Neu5Genetically engineered strains of Ac synthase; adding Saccharomyces cerevisiae, genetic engineering bacteria expressing CMP-Neu5Ac synthetase and genetic engineering bacteria expressing alpha-2, 3-sialyltransferase, wherein the ratio of the three bacteria is (1-2); the Saccharomyces cerevisiae has been deposited with the microorganism strain collection of Guangdong province at 5.12 of 2021 with the deposit number of GDMCC No. 61663; the gene engineering strain expressing the CMP-Neu5Ac synthetase is expressed fromN. meningitidisThe nucleotide sequence of the coding gene of the CMP-Neu5Ac synthetase is shown as SEQ ID NO.1, and the expression of the genetic engineering strain is derived fromN. meningitidisThe nucleotide sequence of the alpha-2, 3-sialyltransferase encoding gene is shown as SEQ ID NO.2, and the genetically engineered bacterium takes escherichia coli as a host.
2. The method of claim 1, wherein the CMP-Neu5Ac is produced by fermentation using CTP and sialic acid as substrates using genetically engineered bacteria expressing CMP-Neu5Ac synthase, or by fermentation using CMP and sialic acid by coupled fermentation using genetically engineered bacteria and yeast expressing CMP-Neu5Ac synthase.
3. The method according to claim 2, wherein the CMP-Neu5Ac is synthesized by double-bacteria coupled fermentation of Saccharomyces cerevisiae and a genetically engineered strain expressing CMP-Neu5Ac synthase, wherein the Saccharomyces cerevisiae and the genetically engineered strain are added into a reaction system in a mass ratio of (1-2) for 2-6 hours, the sialic acid addition amount is 50-60 mmol/L, and Mg 2+ The concentration is 10-20 Mm/L; the Saccharomyces cerevisiae was deposited with the microorganism strain collection of Guangdong province at 5.12 of 2021 under the accession number GDMCC No. 61663.
4. The method according to claim 1, wherein the contents of CMP, sialic acid and lactose are 60-90mm, 50-90mm and 70-90mm, respectively.
5. The method according to any one of claims 1 to 4, wherein the reaction time is 25 to 40 hours.
6. The method according to claim 5, wherein the fermentation is carried out at a temperature of 30-35℃and a rotational speed of 200 r/min.
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