Recombinant escherichia coli and method for synthesizing sialic acid lactose-N-tetrasaccharide c

Abstract

The invention relates to recombinant escherichia coli and a method for synthesizing sialic acid lactose-N-tetrasaccharide c, and belongs to the technical field of microbial metabolism engineering. According to the research, engineering escherichia coli BAZ for producing LNTri II is taken as an initial strain, two key glycosyl transferases, namely beta 1, 4-galactosyl transferase and alpha 2, 6-sialyltransferase, and three CMP-Neu5Ac pathway enzymes, namely NeuC, NeuB and NeuA, are introduced, and related competitive genes are knocked out; under the conditions of shake-flask culture and fed batch culture, the LST c yield of 1.718 g / L and the LST c yield of 9.745 g / L are achieved through the engineering bacterium respectively, and the huge potential of a microbial cell factory in the aspect of efficient production of complex breast milk oligosaccharides is shown.

Description

Technical Field

[0001] The invention relates to a recombinant Escherichia coli and a method for synthesizing sialyllactose-N-tetrasaccharide C, belonging to the technical field of microbial metabolic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs), as the third largest solid component in breast milk, have been widely recognized as an important standard for high-quality formula milk. With the in-depth study of the nutritional composition of breast milk, the unique health benefits of HMOs have gradually been recognized, promoting their application in infant formula milk. However, due to the complexity and cost of natural extraction, microbial synthesis of metabolically engineered strains has become the main way to industrially produce HMOs.

[0003] HMOs include more than 200 oligosaccharides with different structures, of which the first 15 oligosaccharides account for more than 75% of the total HMOs. However, less than 10 HMOs can be produced on a large scale, mainly simple trisaccharides and tetrasaccharides, such as 2′-fucosyllactose (2′-FL), 3-FL, 3′-sialyllactose (3′-SL), 6′-SL, lactose-N-triose (LNT) and lactose-N-neotetraose (LNnT). Lactose, as the most basic receptor substrate, is glycosylated by various specific glycosyltransferases to extend the sugar chain, such as β-N-acetylglucosyltransferase), β-galactosyltransferase, α-fucosyltransferase and α-galactosyltransferase. These glycosyltransferases usually require nucleotide sugars as donors, such as uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), UDP-galactose, guanosine diphosphate-fucose (GDP-Fucose) and cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac). Therefore, the introduction of specific glycosyltransferases and the increase of the corresponding sugar nucleotide supply are the basic methods for metabolic engineering strains to efficiently biosynthesize HMOs.

[0004] Compared with trisaccharides and tetrasaccharides, the biosynthesis of HMOs with complex structures and long sugar chains is less studied. For example, based on the biosynthesis of two neutral core tetrasaccharides, LNT and LNnT, metabolically engineered strains successfully synthesized several fucosylated pentasaccharides or hexasaccharides.

[0005] However, little research has been done on sialyllactose-N-tetrasaccharide c (LST c), a sialyllactose pentasaccharide, which is a monoaminoacylated complex HMO with a high concentration in breast milk and the only monoaminoacylated complex HMO among the top 15 HMOs. Although existing studies (DOI: 10.1021 / acs.jafc.4c08711) have achieved microbial synthesis of LST c through multivariate modular metabolic engineering, the yield in shake flasks and fed batch cultures is still low and cannot meet existing needs. Summary of the invention

[0006] Purpose of the Invention

[0007] The present invention provides a novel method for synthesizing sialyllactose-N-tetraose C. The method has a high yield of sialyllactose-N-tetraose C, achieves g level, and meets production requirements.

[0008] Technical Solution

[0009] A recombinant Escherichia coli for synthesizing sialyllactose-N-tetrasaccharide C, characterized in that the engineered Escherichia coli E.coli BL21 (DE3) ΔlacZ ΔwecB ΔnagB ΔugD, ΔrecA::P tac -galEΔIS186-1::P tac -lgtA,ΔIS186-2::P tac -lgtA,ΔIS186-4::P tac -lgtA,ΔIS186-5::P tac -lgtA is used as the starting strain, and exogenous CMP-Neu5Ac pathway enzymes are overexpressed in the form of plasmids, wherein the CMP-Neu5Ac pathway enzymes include:

[0010] UDP-N-acetylmannosamine epimerase NeuC, N-acetylneuraminic acid synthetase NeuB, cytosine-5'-monophosphate-N-acetylneuraminic acid synthetase NeuA, β1,4-galactosyltransferase GalT and α2,6-sialyltransferase 6ST.

[0011] The recombinant Escherichia coli is characterized in that the genes corresponding to the UDP-N-acetylmannosamine epimerase NeuC, N-acetylneuraminic acid synthetase NeuB, and cytosine-5'-monophosphate-N-acetylneuraminic acid synthetase NeuA are neuC, neuB, and neuA, respectively, and neuC, neuB, and neuA are connected in series to obtain a gene sequence neuBCA, and the nucleotide sequence is shown in SEQ ID NO.1.

[0012] The recombinant Escherichia coli for synthesizing sialyllactose-N-tetraose c is characterized in that the genes corresponding to the β-1,4-galactosyltransferase GalT include CpsIaJ, NmLgtB, HpGalT, AaGalT or HsGalT, and their nucleotide sequences are shown in SEQ ID NO.2-6 in sequence.

[0013] The recombinant Escherichia coli for synthesizing sialyllactose-N-tetraose c is characterized in that the genes corresponding to the α2,6-sialyltransferase 6ST include HAC1268, Plst6-119, Pst6-224, PdST1P7H / M117A, RO6ST, DES31, ED6ST, SA6ST, PMPD1, TW77 or NS6ST, and their nucleotide sequences are shown in SEQ ID NO.7-17 respectively.

[0014] The recombinant Escherichia coli is characterized in that the recombinant Escherichia coli knocks out the N-acetylneuraminic acid aldolase gene nanA, the N-acetylmannosamine kinase gene nanK, the N-acetylneuraminic acid permease gene nanT, and the N-acyl glucosamine-6-phosphate 2-dimerase gene nanE.

[0015] The recombinant Escherichia coli is characterized in that the nucleotide sequence of nanA is shown in SEQ ID NO.18, the nucleotide sequence of nanK is shown in SEQ ID NO.19, the nucleotide sequence of nanT is shown in SEQ ID NO.20, and the nucleotide sequence of nanE is shown in SEQ ID NO.21.

[0016] The recombinant Escherichia coli is characterized in that the recombinant Escherichia coli knocks out the β-galactosidase gene lacZ, the glucosamine-6-phosphate deaminase gene nagB, the UDP-N-acetylglucosamine 2-acylimase gene wecB, and the UDP-glucose 6-dehydrogenase gene ugd; and integrates the β-1,3-N-acetylglucosamine transferase gene lgtA and the UDP glucose differential enzyme gene galE.

[0017] The recombinant Escherichia coli is characterized in that the nucleotide sequences of lacZ, nagB, wecB, ugd, galE and lgtA are shown in SEQ ID NO.22-27 respectively.

[0018] The recombinant Escherichia coli is characterized in that the engineered Escherichia coli E.coli BL21 (DE3) ΔlacZ ΔwecB ΔnagB ΔugD, ΔrecA::P tac -galEΔIS186-1::P tac-lgtA,ΔIS186-2::P tac -lgtA,ΔIS186-4::P tac -lgtA,ΔIS186-5::P tac -lgtA is obtained by the steps:

[0019] The gene lacZ encoding β-galactosidase, the gene wecB encoding uridine diphosphate-N-acetylglucosamine-2-isomerase, the gene nagB encoding glucosamine-6-deaminase and the gene ugD encoding uridine-glucose-6-dehydrogenase on the Escherichia coli genome were knocked out; the gene lgtA encoding β-1,3-acetylglucosamine transferase was integrated and expressed in multiple copies at the chromosome IS186 locus of the Escherichia coli genome, and the gene galE encoding uridine diphosphate galactose-4-isomerase was integrated and expressed at the recA site of the Escherichia coli genome. A method for producing sialyllactose-N-tetraose c, characterized in that glycerol is used as a carbon source, lactose is used as a substrate, IPTG is used as an inducer, and recombinant Escherichia coli is used as a fermentation strain to ferment and produce sialyllactose-N-tetraose c.

[0020] The method is characterized in that the recombinant Escherichia coli is added to a culture medium containing 25 g / L glycerol and cultured at 37°C and 200 rpm until the OD 600 After the pH value was 0.6 to 0.8, IPTG and lactose were added at final concentrations of 0.5 mM and 5 g / L, respectively, and the culture was induced at 25°C and 200 rpm for at least 72 h.

[0021] Specifically:

[0022] The recombinant Escherichia coli constructed by the present invention is a recombinant Escherichia coli that can fully synthesize LNTriII in patent CN 116536232 A as a starting strain. By introducing two key glycosyltransferases, β1,4-galactosyltransferase and α2,6-sialyltransferase, and three CMP-Neu5Ac pathway enzymes (UDP-N-acetylmannosamine epimerase NeuC, N-acetylneuraminic acid synthetase NeuB, cytosine-5'-monophosphate-N-acetylneuraminic acid synthetase NeuA), the LST c synthesis pathway is successfully constructed. In order to increase the supply of the glycosyl donor CMP-Neu5Ac, the relevant competitive genes are also knocked out. After screening, it was found that the β1,4-galactosyltransferase (HpGalT) from Helicobacter pylori and the α2,6-sialyltransferase (ED6ST) from Vibrio parahaemolyticus have excellent catalytic efficiency and substrate specificity, and are therefore selected as key enzymes for constructing the LST c synthesis pathway. Through these optimization measures, the engineered bacteria achieved LST c production of 1.718 g / L and 9.745 g / L under shake flask culture and fed-batch culture conditions, respectively, which are the highest values ​​reported to date, demonstrating the great potential of microbial cell factories in the efficient production of complex HMOs.

[0023] As a preferred embodiment, the present invention provides a recombinant Escherichia coli that can stably synthesize sialyllactose-N-tetraose c, characterized in that NeuC encoding UDP-N-acetylmannosamine epimerase, NeuB encoding N-acetylneuraminic acid synthetase, NeuA encoding cytosine-5'-monophosphate-N-acetylneuraminic acid synthetase, CpsIaJ encoding β1,4-galactosyltransferase and NS6ST encoding α2,6-sialyltransferase are introduced into the above-mentioned recombinant Escherichia coli chassis in the form of plasmids, thereby realizing the construction of a de novo synthesis pathway for LST c.

[0024] In one embodiment of the present invention, the neuC, neuB and neuA genes are all derived from Campylobacter jejuni, and are expressed in series to obtain the gene sequence neuBCA, the nucleotide sequence of which is shown in SEQ ID NO.1; the CpsIaJ gene is derived from Streptococcus agalactiae, and the nucleotide sequence of which is shown in SEQ ID NO.2; the NS6ST gene is derived from Nicoletella semolina, and the nucleotide sequence of which is shown in SEQ ID NO.17.

[0025] In one embodiment of the present invention, the above genes are introduced into site 1 of vectors pETDuet-1, pRSFDuet-1 and pCDFDuet-1, respectively, to obtain pET-BCA, pRSF-CpsIaJ and pCD-NS6ST.

[0026] The third object of the present invention is to provide a recombinant Escherichia coli with high production of sialyllactose-N-tetraose c by enhancing the supply of donor CMP-Neu5Ac, characterized in that the competitive genes (nanA, nanK, nanT, nanE) for CMP-Neu5Ac synthesis are knocked out in the above-mentioned recombinant Escherichia coli chassis genes to promote the supply of sialylation donors.

[0027] In one embodiment of the present invention, in order to prevent the conversion of neuraminic acid (Neu5Ac) to N-acetylmannosamine (ManNAc), the Neu5Ac aldolase gene (nanA) is knocked out, and the nucleotide sequence is shown in SEQ ID NO.18.

[0028] In one embodiment of the present invention, in order to further block ManNAc kinase from phosphorylating ManNAc to 6-phosphoManNAc, N-acetylmannosamine kinase gene (nanK) is used, and the nucleotide sequence thereof is shown in SEQ ID NO.19.

[0029] In one embodiment of the present invention, the gene encoding Neu5Ac permease (nanT) is knocked out, and the nucleotide sequence is shown in SEQ ID NO.20.

[0030] In one embodiment of the present invention, in order to prevent the conversion of ManNAc6-phosphate into GlcNAc6-1 phosphate, the N-acyl glucosamine-6-phosphate 2-dimerizing iminase gene (nanE) is knocked out, and the nucleotide sequence is shown in SEQ ID NO.21.

[0031] In one embodiment of the present invention, the present invention provides a recombinant Escherichia coli that can stably synthesize sialyllactose-N-tetraose c, characterized in that the Escherichia coli BL21 (DE3) has a β-galactosidase gene (lacZ) knocked out to prevent lactose hydrolysis. In addition, two competing enzymes in the UDP-GlcNAc pathway, glucosamine-6-phosphate deaminase gene (nagB) and UDP-N-acetylglucosamine 2-acylimase gene (wecB) and a competing enzyme gene in the UDP-galactose pathway, UDP-glucose 6-dehydrogenase gene (ugd), are also knocked out to increase the synthesis of LNnT by strengthening the supply of two glycosylation donors. Four copies of the β-1,3-N-acetylglucosaminyltransferase gene (lgtA) from Neisseria meningitidis and one copy of the UDP-glucose differentially expressed enzyme gene (galE) from Escherichia coli K-12MG1655 were integrated to achieve the construction of a pathway for the full gene synthesis of LNTri II and enhance the supply of UDP-galactose.

[0032] In one embodiment of the present invention, the lacZ, nagB, wecB, ugd and galE genes are all derived from Escherichia coli K-12MG1655, and their nucleotide sequences are shown in SEQ ID NO.22-26; the lgtA gene is derived from Neisseria meningitidis, and its nucleotide sequence is shown in SEQ ID NO27.

[0033] As a preferred method, by screening efficient β1,4-galactosyltransferases, a recombinant Escherichia coli with high production of sialyllactose-N-tetraose c is provided, characterized in that the four β1,4-galactosyltransferases obtained by screening replace the above-mentioned CpsIaJ enzyme, and are introduced into the recombinant Escherichia coli chassis together with pET-BCA and pCD-NS6ST plasmids.

[0034] In one embodiment of the present invention, the four β1,4-galactosyltransferase genes screened are CpsIaJ, NmLgtB, HpGalT, AaGalT and HsGalT, and their nucleotide sequences are shown in SEQ ID NO.2-6.

[0035] In one embodiment of the present invention, the pRSFDuet-1 plasmid is used to overexpress the above four β-1,4-galactosyltransferase genes to obtain plasmids pRSF-CpsIaJ, pRSF-NmLgtB, pRSF-HpGalT, pRSF-AaGalT, and pRSF-HsGalT.

[0036] As a preferred method, by screening efficient α2,6-sialyltransferases, a recombinant Escherichia coli with high production of sialyllactose-N-tetraose c is provided, characterized in that the ten α2,6-sialyltransferases obtained by screening are used to replace the above-mentioned NS6ST enzyme, and are introduced into the recombinant Escherichia coli chassis together with pET-BCA and pRSF-HpGalT plasmids.

[0037] In one embodiment of the present invention, the ten α2,6-sialyltransferase genes screened are HAC1268, Plst6-119, Pst6-224, PdST1 P7H / M117A, RO6ST, DES31, ED6ST, SA6ST, PMPD1, and TW77, and their nucleotide sequences are shown in SEQ ID NOs. 7-16.

[0038] In one embodiment of the present invention, the pCDFDuet-1 plasmid is used to overexpress the above ten α2,6-sialyltransferase genes to obtain plasmids pCD-NS6ST, pCD-HAC1268, pCD-Plst6-119, pCD-Plst6-224, pCD-PdST1 P7H / M117A, pCD-RO6ST, pCD-DES31, pCD-ED6ST, pCD-SA6ST, pCD-PMPD1, and pCD-TW77.

[0039] As a preferred embodiment, a method for producing sialyllactose-N-tetraose c is provided, wherein glycerol is used as a carbon source, lactose is used as a substrate, IPTG is used as an inducer, and the recombinant Escherichia coli is used to ferment and produce sialyllactose-N-tetraose c.

[0040] In one embodiment of the present invention, the seed solution of the recombinant Escherichia coli is added to a culture medium containing 25 g / L glycerol and cultured at 37°C and 200 rpm until the OD 600 After the pH value reached 0.6 to 0.8, IPTG was added at a final concentration of 0.5 mM, and lactose was added at a final concentration of 5 g / L. The culture was induced at 25°C and 200 rpm for at least 72 h.

[0041] As a preferred embodiment, the present invention provides the use of the recombinant Escherichia coli in producing a product containing sialyllactose-N-tetrasaccharide c.

[0042] As a preferred embodiment, the present invention provides the use of the recombinant Escherichia coli in the fields of food, chemical industry and medicine.

[0043] Beneficial effects:

[0044] The present invention relates to an engineered Escherichia coli constructed by metabolic engineering means, which is used for efficiently synthesizing sialyllactose-N-tetrasaccharide c, and belongs to the technical field of microbial metabolic engineering. The research is based on the patent CN 116536232A to produce LNTri II engineered Escherichia coli E.coli BL21 (DE3) ΔlacZ ΔwecB ΔnagB ΔugD, ΔrecA::P tac -galEΔIS186-1::P tac -lgtA,ΔIS186-2::P tac -lgtA,ΔIS186-4::P tac -lgtA,ΔIS186-5::P tac -lgtA was used as the starting strain, and the LST c pathway was constructed by introducing two key glycosyltransferases, β1,4-galactosyltransferase and α2,6-sialyltransferase, and three CMP-Neu5Ac pathway enzymes, NeuC, NeuB, and NeuA. The relevant competitive genes were knocked out to increase the supply of the glycosyl donor CMP-Neu5Ac. The β1,4-galactosyltransferase (HpGalT) from Helicobacter pylori J99 and the α2,6-sialyltransferase (ED6ST) from Vespertiliibacter pulmonis showed excellent catalytic efficiency and substrate specificity, and were therefore selected as the key enzymes for constructing the LST c synthesis pathway. Through these optimization measures, the engineered bacteria achieved LST c production of 1.718 g / L and 9.745 g / L under shake flask culture and fed-batch culture conditions, respectively, which are the highest values ​​reported to date. The existing technology (DOI: 10.1021 / acs.jafc.4c08711) reported that the LST c concentration of the recombinant strain in shake flask culture reached 220.9 mg / L, and the LST c concentration in 3L fed-batch fermentation reached 922.2 mg / L, demonstrating the great potential of microbial cell factories in the efficient production of complex human milk oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The metabolic pathway diagram for the biosynthesis of sialyllactose-N-tetraose c by recombinant Escherichia coli;

[0046] Figure 2 UPLC-MS detection diagram of sialyllactose-N-tetrasaccharide c standard (A) and sample (B);

[0047] Figure 3 A comparison of sialyllactose-N-tetraose c production by knocking out genes in different competing pathways;

[0048] Figure 4 This is a comparison chart of sialyl lactose-N-tetraose c production by different β1,4-galactosyltransferases;

[0049] Figure 5 The figure is a comparison of the production of sialyl lactose-N-tetraose c by different α2,6-sialyltransferases;

[0050] Figure 6 This is a graph showing the fermentation yield of sialyllactose-N-tetraose c by the BL21-LSTc16 engineered bacteria in a 5L fermenter. DETAILED DESCRIPTION

[0051] 1. The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are carried out in accordance with the kit instructions.

[0052] 2. The routine operation methods such as colony PCR, nucleic acid agarose gel electrophoresis, protein SDS-PAGE gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells and extraction and storage of bacterial genome were carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0053] 3. The sequencing of plasmids and DNA products was completed by Shanghai Shenggong Bioengineering Co., Ltd.

[0054] 4. Preparation of competent E. coli: TAKARA kit.

[0055] 5. Sialyl lactose-N-tetraose c fermentation process and detection:

[0056] (1) LB liquid medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0057] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0058] (3) Fermentation medium: 20 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate and 10 ml / L trace metal elements; the trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate.

[0059] (4) Sialyl lactose-N-tetraose c fermentation process: The constructed strain was inoculated into LB liquid medium, cultured overnight at 37°C, 200 rpm for 12 h to obtain seed solution, 500 μL of seed solution was inoculated into 25 mL of fermentation culture, and cultured at 37°C, 200 rpm until OD 600 The final concentration of IPTG was 0.5 mM, and 5 g / L lactose was added at 25°C and 200 rpm for 72 h. 1 mL of fermentation broth was taken and centrifuged at 10,000 rpm for 10 min. The supernatant was taken for HPLC determination.

[0060] (5) High performance liquid chromatography (HPLC) analysis conditions: Agilent 1260 Infinity II system was used for detection; XBT Amide was used as the chromatographic column; the detector was Agilent 1260 Infinity II VWD detector; the mobile phase consisted of 10 mM ammonium formate solution (adjusted to pH 4.0 with formic acid) from pump A and pure acetonitrile from pump B, with a volume ratio of A to B of 30:70; the flow rate was set at 1.0 mL / min; the UV detection wavelength was set at 210 nm; the column temperature was maintained at 35 °C; and the injection volume was 10 μL.

[0061] Example 1: Construction of recombinant expression vector

[0062] The specific steps for constructing the recombinant expression vector are as follows (the primer sequences involved are shown in Table 1):

[0063] The gene neuBCA (nucleotide sequence shown in SEQ ID NO.1) derived from Campylobacter jejuni was constructed into site 1 of pETDuet-1 to obtain the recombinant plasmid pET-1BCA.

[0064] The gene CpsIaJ (nucleotide sequence shown in SEQ ID NO.2) derived from Streptococcus agalactiae was constructed into site 1 of pRSFuet-1 to obtain the recombinant plasmid pRSF-CpsIaJ.

[0065] The gene NmLgtB (nucleotide sequence shown in SEQ ID NO.3) from Neisseria meningitidis MC58 was constructed and inserted into site 1 of pRSFuet-1 to obtain the recombinant plasmid pRSF-NmLgtB.

[0066] The gene HpGalT (nucleotide sequence shown in SEQ ID NO.4) from Helicobacter pylori J99 was constructed into the pRSFuet-1 site to obtain the recombinant plasmid pRSF-HpGalT.

[0067] The gene AaGalT (nucleotide sequence shown in SEQ ID NO.5) derived from Aggregatibacter actinomycetemcomitans NUM4039 was constructed into site 1 of pRSFuet-1 to obtain the recombinant plasmid pRSF-AaGalT.

[0068] The gene HsGalT (nucleotide sequence shown in SEQ ID NO.6) from Histophilus somni was constructed and inserted into site 1 of pRSFuet-1 to obtain the recombinant plasmid pRSF-HsGalT.

[0069] The gene HAC1268 (nucleotide sequence shown in SEQ ID NO.7) from Helicobacter acinonychis ATCC 51104 was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-HAC1268.

[0070] The gene Plst6-119 (nucleotide sequence as shown in SEQ ID NO.8) derived from Photobacterium leiognathi strain JTSHIZ-119 was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-Plst6-119.

[0071] The gene Plst6-224 (nucleotide sequence shown in SEQ ID NO.9) derived from Photobacterium sp. JT-ISH-224 was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-Plst6-224.

[0072] The gene PdST1 P7H / M117A (nucleotide sequence shown in SEQ ID NO.10) from Pasteurella dagmatis was constructed and inserted into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-PdST1P7H / M117A.

[0073] The gene RO6ST (nucleotide sequence as shown in SEQ ID NO.11) derived from Muribacter muris was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-RO6ST.

[0074] The gene DES31 (nucleotide sequence shown in SEQ ID NO.12) derived from Otariodibacter oris was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-DES31.

[0075] The gene ED6ST (nucleotide sequence shown in SEQ ID NO.13) from Vespertiliibacter pulmonis was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-ED6ST.

[0076] The gene SA6ST (nucleotide sequence shown in SEQ ID NO. 14) from Alysiella filiformis DSM 16848 was constructed and inserted into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-SA6ST.

[0077] The gene PMPD1 (nucleotide sequence shown in SEQ ID NO.15) derived from Erwiniaceae bacterium PD-1 was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-PMPD1.

[0078] The gene TW77 (nucleotide sequence shown in SEQ ID NO.16) from Pseudoalteromonas rubra was constructed and inserted into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-TW77.

[0079] The gene NS6ST (nucleotide sequence shown in SEQ ID NO.17) from Nicoletella semolina was constructed into site 1 of pCDFuet-1 to obtain the recombinant plasmid pCD-NS6ST.

[0080] The primer sequences involved in the following examples are shown in Table 1.

[0081] Table 1: Primer sequence information

[0082]

[0083]

[0084] Example 2: Knockout of genes in the CMP-Neu5Ac synthesis competition pathway (nanA, nanK, nanT and nanE) in the recombinant strain

[0085] The recombinant strain BAZ (E. coli BL21 (DE3) ΔlacZ ΔwecB ΔnagB ΔugD, ΔrecA::P tac -galEΔIS186-1::P tac -lgtA,ΔIS186-2::P tac-lgtA,ΔIS186-4::P tac -lgtA,ΔIS186-5::P tac -lgtA) as the starting strain. CRISPR-Cas9 gene editing system was used to further knock out nanA, nanK, nanT and nanE genes in the genome of the starting strain. The specific steps are as follows (the primer sequences used are shown in Table 2):

[0086] (1) Using the original pTargetF plasmid as a template and the nanA-sg-F / R primer pair, the N20 sequence on the original pTargetF plasmid was replaced with an N20 sequence complementary to the nanA sequence by PCR amplification to obtain the pTargetF plasmid pTargetF-nanA targeting the nanA gene. The PCR product was digested with DpnI to remove the template DNA, and then transformed into Escherichia coli JM109 competent cells, spread on LB plates containing spectinomycin, and cultured at 37°C. The plasmid was extracted and sequenced.

[0087] (2) Using the E. coli BL21 genome as a template, primers nanA-UP-F / R and nanA-DH-F / R were used to amplify the upstream and downstream fragments of nanA by PCR, respectively, and the fragments were recovered by gel. The two fragments were connected by PCR to obtain the donor DNA fragment.

[0088] (3) The pCas plasmid was transformed into the competent cells of the recombinant bacterium BAZ, spread on LB plates containing kanamycin, and cultured in a 37°C incubator overnight to obtain the BAZ-pCas strain.

[0089] (4) A single BAZ-pCas colony was selected and inoculated into LB medium, cultured at 30°C for 12 h, and then L-arabinose was added at a final concentration of 10 mM to induce the expression of the pCas-λ-red system. 600 When the value reaches 0.5-0.6, prepare BAZ-pCas electroporation competent cells.

[0090] (5) 200-500 ng of pTargetF-nanA plasmid and 400-800 ng of donor DNA fragment were electroporated into BAZ-pCas competent cells, spread on LB plates containing kanamycin and spectinomycin, and cultured at 30°C for 24 h. The knockout effect of the nanA gene was verified by PCR. The colonies with knockout of the nanA gene were positive clones.

[0091] (6) The positive clone colony obtained in step (5) was inoculated into 4 mL of LB liquid culture medium, IPTG and 50 mg / L kanamycin were added at a final concentration of 1 mM, and cultured at 30°C for 8-16 h to remove the pTargetT-nanA plasmid, and then cultured at 42°C for 12 h to remove the pCas plasmid, and finally the BAZA strain with the nanA gene knocked out was obtained.

[0092] (7) Using the same method, the genes nanK, nanT, and nanE in the strain were knocked out in turn to obtain the corresponding four recombinant Escherichia coli BAZA, BAZAK, BAZAKT, and BAZAKTE.

[0093] (8) Plasmids pET-BCA, pRSF-CpsIaJ and pCD-NS6ST were introduced into five recombinant Escherichia coli (BAZ

[0094] , BAZA, BAZAK, BAZAKT, BAZAKTE) were used to obtain the recombinant bacteria BL21-LSTc1, BL21-LSTc2, BL21-LSTc3, BL21-LSTc4 and BL21-LSTc5, thus realizing the construction of the recombinant strains.

[0095] Table 2: Primer sequences and targeting plasmid information

[0096]

[0097]

[0098] Example 3: Fermentation production of sialyllactose-N-tetrasaccharide c by recombinant strains of different β1,4-galactosyltransferases

[0099] The pRSF-CpsIaJ, pRSF-NmLgtB, pRSF-HpGalT, pRSF-AaGalT, and pRSF-HsGalT constructed in Example 1 were combined with pET-1BCA and pCD-NS6ST, respectively, and transformed into the host BAZAKT constructed in Example 2 to obtain recombinant strains BL21-LSTc4, BL21-LSTc6, BL21-LSTc7, BL21-LSTc8, and BL21-LSTc9.

[0100] The above recombinant strains were inoculated into LB liquid medium containing the corresponding antibiotics and cultured overnight at 37°C and 200 rpm for 12 h to obtain seed liquid. Then, the seed liquid was inoculated into 25 mL DM fermentation medium and cultured at 37°C and 200 rpm until OD 600When the value reached 0.8, the induction was performed. During the induction, IPTG was added at a final concentration of 0.5 mM, and 5 g / L lactose was added at the same time, and then the culture was continued for 72 h at 25 ° C and 200 rpm. 1 mL of the fermentation broth was taken, centrifuged and the supernatant was taken for HPLC determination. The yield results of sialyllactose-N-tetrasaccharide c are shown in Table 2, among which the highest yield of sialyllactose-N-tetrasaccharide c was 1.457 g / L ( Figure 4 ). Therefore, the HpGal gene from Helicobacter pylori J99 may be a good candidate gene for microbial synthesis of sialyllactose-N-tetraose c.

[0101] Example 4: Fermentation production of sialyllactose-N-tetrasaccharide c by recombinant strains of different α2,6-sialyltransferases

[0102] pCD-HAC1268, pCD-Plst6-119, pCD-Plst6-224, pCD-PdST1P7H / M117A, pCD-RO6ST, pCD-DES31, pCD-ED6ST, pCD-SA6ST, pCD-PMPD1, pCD-TW77, and pCD-NS6ST constructed in Example 1 were respectively mixed with pET-1BCA, pRSF-HpGalT The combination was transferred into the host BAZAKT constructed in Example 2 to obtain recombinant strains BL21-LSTc8, BL21-LSTc10, BL21-LSTc11, BL21-LSTc12, BL21-LSTc13, BL21-LSTc14, BL21-LSTc15, BL21-LSTc16, BL21-LSTc17, BL21-LSTc18 and BL21-LSTc19.

[0103] The recombinant strains were inoculated into LB liquid medium containing the corresponding antibiotics, cultured overnight at 37°C, 200 rpm for 12 h to obtain seed solution, and the seed solution was inoculated into 25 mL DM fermentation medium at 37°C, 200 rpm to OD 600 The mixture was induced at 0.8, and a final concentration of 0.5 mM IPTG was added. At the same time, 5 g / L lactose was added. The mixture was cultured for 72 h at 25 °C and 200 rpm. 1 mL of the fermentation broth was taken, and the supernatant was used for HPLC determination. The yield of sialyllactose-N-tetraose c is shown in Table 3. The highest yield of sialyllactose-N-tetraose c was 1.718 g / L ( Figure 5 ). Therefore, the gene ED6ST from Vespertiliibacter pulmonis may be a good candidate for the microbial synthesis of sialyllactose-N-tetraose c.

[0104] The recombinant strains involved in the following examples and the corresponding production of sialyllactose-N-tetraose c are shown in Table 3.

[0105] Table 3: Recombinant strains and corresponding sialyllactose-N-tetraose c production information

[0106]

[0107]

[0108] Example 5: Production of sialyllactose-N-tetrasaccharide c by fed-batch culture in a fermenter

[0109] In order to further verify the effectiveness of the synthesis method of sialyllactose-N-tetraose c and improve the yield of sialyllactose-N-tetraose c, the seed liquid of the engineering bacteria BL21-LSTc16 was inoculated into a fermentation medium with a working volume of 2.5L at a 10% (v / v) inoculation rate. The fermentation temperature of the fermenter was 37°C, the stirring speed was 800r / min, the ventilation volume was 1vvm, and the pH was 7.0 (automatically controlled by adding ammonia water). Fermentation for 10h (OD 600 About 18), add 5g / L lactose and 0.2mM IPTG, and culture at 25°C. During this period, glycerol and lactose were manually fed: when the glycerol concentration in the reaction system was lower than 3g / L, 30mL of mother liquor (mother liquor glycerol concentration 600g / L) was added, and when the lactose concentration was lower than 3g / L, 30mL of mother liquor (mother liquor lactose concentration 200g / L) was added to maintain the growth of the bacteria and the synthesis of BL21-LSTc16 seed liquid. After the whole process of cultivation reached 60h, the OD600 of the bacteria reached 89.31, and the yield of sialyllactose-N-tetrasaccharide c reached the highest, reaching 9.745g / L ( Figure 6 ).

[0110] Table 4. Dynamic changes of biomass and synthesis during fermentation

[0111]

[0112] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli for synthesizing sialyllactose-N-tetrasaccharide C, characterized in that: The engineered E. coli BL21 (DE3) ΔlacZ ΔwecB ΔnagB ΔugD, ΔrecA::P tac -galEΔIS186-1::P tac -lgtA,ΔIS186-2::P tac -lgtA,ΔIS186-4::P tac -lgtA,ΔIS186-5::P tac -lgtA is used as the starting strain, and exogenous CMP-Neu5Ac pathway enzymes are overexpressed in the form of plasmids, wherein the CMP-Neu5Ac pathway enzymes include: UDP-N-acetylmannosamine epimerase NeuC, N-acetylneuraminic acid synthetase NeuB, cytosine-5'-monophosphate-N-acetylneuraminic acid synthetase NeuA, β1,4-galactosyltransferase GalT and α2,6-sialyltransferase 6ST.

2. The recombinant Escherichia coli according to claim 1, characterized in that The genes corresponding to the UDP-N-acetylmannosamine epimerase NeuC, N-acetylneuraminic acid synthetase NeuB, and cytosine-5'-monophosphate-N-acetylneuraminic acid synthetase NeuA are neuC, neuB, and neuA, respectively. The gene sequence neuBCA is obtained by connecting neuC, neuB, and neuA in series, and the nucleotide sequence is shown in SEQ ID NO.

1.

3. The recombinant Escherichia coli for synthesizing sialyllactose-N-tetraose c according to claim 1, characterized in that: The genes corresponding to the β-1,4-galactosyltransferase GalT include CpsIaJ, NmLgtB, HpGalT, AaGalT or HsGalT, and their nucleotide sequences are shown in SEQ ID NO.2-6.

4. The recombinant Escherichia coli for synthesizing sialyllactose-N-tetraose c according to claim 1, characterized in that: The genes corresponding to the α2,6-sialyltransferase 6ST include HAC1268, Plst6-119, Pst6-224, PdST1 P7H / M117A, RO6ST, DES31, ED6ST, SA6ST, PMPD1, TW77 or NS6ST, and their nucleotide sequences are shown in SEQ ID NO.7-17 respectively.

5. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli is knocked out of the N-acetylneuraminic acid aldolase gene nanA, the N-acetylmannosamine kinase gene nanK, the N-acetylneuraminic acid permease gene nanT, and the N-acyl glucosamine-6-phosphate 2-dimerase gene nanE.

6. The recombinant Escherichia coli according to claim 5, characterized in that The nucleotide sequence of nanA is shown in SEQ ID NO.18, the nucleotide sequence of nanK is shown in SEQ ID NO.19, the nucleotide sequence of nanT is shown in SEQ ID NO.20, and the nucleotide sequence of nanE is shown in SEQ ID NO.

21.

7. The recombinant Escherichia coli according to claim 1, characterized in that The recombinant Escherichia coli knocks out the β-galactosidase gene lacZ, the glucosamine-6-phosphate deaminase gene nagB, the UDP-N-acetylglucosamine 2-acylimase gene wecB, and the UDP-glucose 6-dehydrogenase gene ugd; and integrates the β-1,3-N-acetylglucosamine transferase gene lgtA and the UDP glucose differential enzyme gene galE.

8. The recombinant Escherichia coli according to claim 1, characterized in that The nucleotide sequences of lacZ, nagB, wecB, ugd, galE and lgtA are shown in SEQ ID NOs. 22-27 respectively.

9. The recombinant Escherichia coli according to claim 1, characterized in that: Engineered E. coli BL21(DE3)ΔlacZΔwecBΔnagBΔugD,ΔrecA::P tac -galEΔIS186-1::P tac -lgtA,ΔIS186-2::P tac -lgtA,ΔIS186-4::P tac -lgtA,ΔIS186-5::P tac -lgtA is obtained by the steps: The genes lacZ encoding β-galactosidase, wecB encoding UDP-N-acetylglucosamine-2-isomerase, nagB encoding glucosamine-6-deaminase and ugD encoding uridine-glucose-6-dehydrogenase in the Escherichia coli genome were knocked out; the gene lgtA encoding β-1,3-acetylglucosamine transferase was integrated and expressed in multiple copies at the chromosomal IS186 locus of the Escherichia coli genome, and the gene galE encoding UDP-galactose-4-isomerase was integrated and expressed at the recA site of the Escherichia coli genome.

10. A method for producing sialyllactose-N-tetraose C, characterized in that: Using glycerol as a carbon source, lactose as a substrate, IPTG as an inducer, and the recombinant Escherichia coli according to any one of claims 1 to 9 as a fermentation strain to produce sialyllactose-N-tetraose c.

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