Engineering escherichia coli for synthesizing sialyloyl-N-tetrasaccharide as well as construction method and application of engineering escherichia coli

Abstract

The invention discloses engineering escherichia coli for synthesizing sialyloyl-N-tetrasaccharide a as well as a construction method and application of the engineering escherichia coli, and belongs to the technical field of microbial metabolism engineering. Recombinant escherichia coli E.coli BL21 (DE3) delta lacZ delta wecB delta nagB delta ugD, delta recA:: Ptac-galE delta ISl86-1:: Ptac-lgtA, delta IS186-2:: Ptac-lgtA, delta IS186-4:: Ptac-lgtA and delta IS186-5:: Ptac-lgtA are taken as initial strains, 5 '-cytidine monophosphate-N-acetyl nerve amino acid pathway genes nenC, nenB and nenA and beta-1, 3-galactosyl transferase genes wbgO and alpha2, 3-galactosyl transferase genes wbgO are over-expressed by knocking out 5'-cytidine monophosphate-N-acetyl nerve amino acid The recombinant escherichia coli capable of stably synthesizing sialic acid lactyl-N-tetrasaccharide a is obtained by adding a 1, 3-sialyltransferase specific gene Pm0188 into the recombinant escherichia coli. By screening more efficient alpha 2, 3-sialyltransferase and optimizing a pathway gene expression combination, the yield of sialyloyl-N-tetrasaccharide a is increased, and the sialyloyl-N-tetrasaccharide a with the yields of 1.235 g / L and 4.85 g / L respectively is finally obtained under shake-flask culture and fed batch culture.

Description

Technical Field

[0001] The invention relates to an engineering Escherichia coli for synthesizing sialyllactyl-N-tetrasaccharide a and a construction method and application thereof, belonging to the technical field of microbial metabolic engineering. Background Art

[0002] Human milk oligosaccharides (HMOs) are a class of oligosaccharides present in breast milk. It is the third most abundant solid component in breast milk, after lipids and lactose, with a content of 5-15g / L. It has many important health effects on breastfed infants, such as shaping the intestinal microbiota, promoting immune regulation and brain development, preventing necrotizing enterocolitis, and having anti-adhesion, antibacterial and antiviral activities.

[0003] At present, the titer of the neutral core human milk oligosaccharide lactoyl-N-tetraose has been greatly improved through various metabolic engineering strategies. Based on these solid research foundations, researchers began to study the metabolic engineering production of complex human milk oligosaccharides derived from lactoyl-N-tetraose. Typical sialylated lactoyl-N-tetraose derivatives include sialyllactoyl-N-tetraose a, which is α2,3-sialylated lactoyl-N-tetraose. et al. estimated the average HMO concentrations of various HMOs in healthy mothers throughout lactation worldwide based on 57 peer-reviewed publications on the concentrations of individual HMOs in mixed milk. Among them, the molar concentration of sialyl lactoyl-N-tetraose a ranked in the top 30 of more than 200 HMOs, accounting for 0.26% of the total HMOs. In vitro, multienzyme cascades have been developed to produce complex sialylated HMOs. Specific sialyltransferases catalyze the sialylation reaction using 5′-cytidylic acid-N-acetylneuraminic acid (CMP-Neu5Ac) as a donor. Yao et al. reported that LNT and CMP-Neu5Ac generated substrates to generate sialyl lactoyl-N-tetraose a through a three-enzyme cascade reaction involving Pasteurella multocida α2,3-sialyltransferase 1 (PmST1). However, the microbial biosynthesis of sialyl lactoyl-N-tetraose a has not been reported.

[0004] Summary of the invention

[0005] Purpose of the Invention

[0006] The invention provides a method for synthesizing sialyllactyl-N-tetrasaccharide a, a recombinant Escherichia coli and applications of the recombinant Escherichia coli.

[0007] A recombinant Escherichia coli for synthesizing sialyllactyl-N-tetrasaccharide a, characterized in that the recombinant Escherichia coli BL21 (DE3) ΔlacZ ΔwecB ΔnagB ΔugD, ΔrecA::Ptac -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 competing pathway genes N-acetylneuraminic acid aldolase nanA, sialic acid transporter nanT, N-acetylmannosamine kinase nanK and N-acylglucosamine-6-phosphate-dimerase nanE were knocked out, and exogenous UDP-N-acetylmannosamine isomerase neuC, N-acetylneuraminic acid synthetase neuB, cytosine-5′-monophosphate-N-acetylneuraminic acid synthetase neuA and β-1,3-galactosyltransferase wbgO and a2,3-sialyltransferase genes were freely expressed.

[0008] The recombinant Escherichia coli for synthesizing sialyl lactyl-N-tetraose a is characterized in that the neuB, neuC and neuA genes are expressed in series to obtain the gene sequence neuBCA, and the nucleotide sequence is shown in SEQ ID NO.1.

[0009] The recombinant Escherichia coli for synthesizing sialyllactyl-N-tetrasaccharide a is characterized in that the nucleotide sequence of the β-1,3-galactosyltransferase wbgO is shown in SEQ ID NO.2.

[0010] The recombinant Escherichia coli for synthesizing sialyl lactyl-N-tetraose a is characterized in that the α2,3-sialyltransferase gene includes Pm0188, BtST1, Hd0053, Vs16 or nst; wherein:

[0011] The nucleotide sequence of Pm0188 is shown in SEQ ID NO. 3;

[0012] The nucleotide sequence of BtST1 is shown in SEQ ID NO.4;

[0013] The nucleotide sequence of Hd0053 is shown in SEQ ID NO.5;

[0014] The nucleotide sequence of Vs16 is shown in SEQ ID NO.6;

[0015] The nucleotide sequence of Nst is shown in SEQ ID NO.7.

[0016] The method for constructing a recombinant Escherichia coli for synthesizing sialyllactyl-N-tetraose a is characterized in that pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 plasmids are respectively used to simultaneously overexpress UDP-N-acetylmannosamine epimerase gene neuC, N-acetylneuraminic acid synthetase gene neuB, cytosine-5′-monophosphate-N-acetylneuraminic acid synthetase gene neuA and β-1,3-galactosyltransferase gene wbgO;

[0017] The pCDFDuet 1 plasmid was used to express the α2,3-sialyltransferase genes Pm0188, BtST1, Hd0053, Vs1 6 and nst to construct the pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 plasmids, respectively.

[0018] A method for producing sialyllactyl-N-tetraose a is characterized in that 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 as a fermentation strain to ferment and produce sialyllactyl-N-tetraose a.

[0019] The method is characterized in that the recombinant Escherichia coli is added to a culture medium containing 25 g / L glycerol, cultured at 37°C and 200 rpm until OD6000 is 0.6-0.8, and then 0.5 mM IPTG and 5 g / L lactose are added at final concentrations, respectively, and induced culture is carried out at 25°C and 200 rpm for at least 72 hours.

[0020] The recombinant Escherichia coli is used in the preparation of sialyl lactoyl-NN sugar a.

[0021] Specifically, the recombinant Escherichia coli constructed in the present invention is an engineering bacterium (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 by knocking out the competing pathway genes N-acetylneuraminic acid aldolase nanA, sialic acid transporter nanT, N-acetylmannosamine kinase nanK and N-acylglucosamine-6-phosphate 2-dimerase nanE in the genome, and freely expressing exogenous UDP-N-acetylmannosamine isomerase neuC, N-acetylneuraminic acid synthetase neuB, cytosine-5′-monophosphate-N-acetylneuraminic acid synthetase neuA and β-1,3-galactosyltransferase wbgO from Escherichia coli K-12 and α2,3-sialyltransferase specific gene Pm0188, a recombinant Escherichia coli that stably synthesizes sialicyl lactoyl-N-tetraose a was obtained, which has the potential for industrial application.

[0022] Further: A recombinant Escherichia coli that can stably synthesize sialyllactyl-N-tetraose a, characterized in that the N-acetylneuraminic acid aldolase gene nanA (Gene ID: 947742), the sialic acid transporter gene nanT (GeneID: 947740), the N-acetylmannosamine kinase gene nanK (Gene ID: 947757) and the N-acyl glucosamine-6-phosphate 2-diiminase gene nanE (Gene ID: 947745) overexpress the UDP-N-acetylmannosamine isomerase gene neuC, the N-acetylneuraminic acid synthetase gene neuB, the cytosine-5'-monophosphate-N-acetylneuraminic acid synthetase gene neuA and the β-1,3-galactosyltransferase gene wbgO and the a2,3-sialyltransferase specific gene Pm0188.

[0023] In one embodiment of the present invention, the neuB, neuC 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.

[0024] In one embodiment of the present invention, the nucleotide sequence of the gene wbgO encoding β-1,3-galactosyltransferase is shown as SEQ ID NO.2.

[0025] In one embodiment of the present invention, the gene encoding α2,3-sialyltransferase is selected from one of the following genes:

[0026] The gene Pm0188 from P. Multocida, the nucleotide sequence of which is shown in SEQ ID NO. 3;

[0027] The gene BtST1 from Bibersteinia trehalosi USDA-ARS-USMARC-192, whose nucleotide sequence is shown in SEQ ID NO.4;

[0028] Gene Hd0053 from Haemophilus ducreyi, the nucleotide sequence of which is shown in SEQ ID NO.5;

[0029] Gene Vs16 from Vibrio sp. JT-FAJ-16, the nucleotide sequence of which is shown in SEQ ID NO.6;

[0030] The nucleotide sequence of the gene nst derived from Neisseria meningitidis MC58 is shown in SEQ ID NO.7.

[0031] In one embodiment of the present invention, the NCBI accession number of the gene Pm0188 is AAK02272.1; the NCBI accession number of the gene BtST1 is AGH37861.1; the NCBI accession number of the gene Hd0053 is AAP95068.1;

[0032] The NCBI accession number of the gene Vs16 is BAF91160.1; the NCBI accession number of the gene nst is U60660.

[0033] In one embodiment of the present invention, pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 plasmids are used respectively to simultaneously overexpress UDP-N-acetylmannosamine isomerase gene neuC, N-acetylneuraminic acid synthetase gene neuB, cytosine-5′-monophosphate-N-acetylneuraminic acid synthetase gene neuA and β-1,3-galactosyltransferase gene wbgO.

[0034] In one embodiment of the present invention, the pCDFDuet-1 plasmid is used to express the α2,3-sialyltransferase genes Pm0188, BtST1, Hd0053, Vs16 and nst, respectively.

[0035] In one embodiment of the present invention, the α2,3-sialyltransferase gene nst is overexpressed using pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 plasmids, respectively.

[0036] The second object of the present invention is to provide a method for producing sialyllactyl-N-tetraose a, 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 sialyllactyl-N-tetraose a.

[0037] 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 was 0.6-0.8, add final concentrations of 0.05, 0.2, 0.5 and 1.0 mM IPTG, and at the same time add lactose with a final concentration of 5 g / L, and induce culture at 25°C and 200 rpm for at least 72 h.

[0038] 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 0.6-0.8, add IPTG with a final concentration of 0.5 mM and lactose with a final concentration of 5 g / L, and induce culture at 28, 25 and 22°C, 200 rpm for at least 72 h.

[0039] The third object of the present invention is to provide the use of the recombinant Escherichia coli in producing products containing sialyllactyl-N-tetrasaccharide a.

[0040] The fourth object of the present invention is to provide the application of the recombinant Escherichia coli in the fields of food, chemical industry and medicine.

[0041] Beneficial effects:

[0042] The present invention is to recombinant 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 further genetically modified to obtain a recombinant Escherichia coli that efficiently produces sialyllactyl-N-tetraose a. The present invention, for the first time, constructs a sialyllactyl-N-tetraose a synthesis pathway through three consecutive glycosylation steps of β-1,3-N-acetylglucosaminylation, β-1,3-galactosylation and α2,3-sialylation. At the same time, by knocking out the CMP-Neu5Ac synthesis competitive genes nanA, nanT, nanK and nanE, and introducing the CMP-Neu5Ac synthesis pathway genes neuC, neuB and neuA, and screening more efficient α2,3-sialyltransferases and combining optimized pathway gene expression, the yield of sialyllactyl-N-tetraose a was increased. Through shake flask culture and fed-batch culture, sialyllactyl-N-tetraose a with titers of 1.235 and 4.85 g / L were finally obtained, proving the feasibility of efficient production of complex sialylated human milk oligosaccharides by microorganisms, and having good industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The metabolic pathway diagram for the biosynthesis of sialyllactyl-N-tetraose a by recombinant Escherichia coli;

[0044] Figure 2 A comparison of sialyl lactyl-N-tetraose a production by knocking out genes in different competitive pathways;

[0045] Figure 3 The figure is a comparison of the production of sialyl lactyl-N-tetraose a by different α2,3-sialyltransferases;

[0046] Figure 4 The figure is a comparison of the yield of sialyllactyl-N-tetraose a under different shake flask fermentation conditions;

[0047] Figure 5 This is a graph showing the fermentation yield of sialyllactyl-N-tetraose a by the engineered bacteria BL21-LSTa9 in a 5L fermenter. DETAILED DESCRIPTION

[0048] The present invention is further described below through specific implementation examples.

[0049] All commercial products such as PCR amplification enzymes, plasmids, DNA gel recovery kits and column plasmid extraction kits used in the present invention are specifically operated in accordance with the kit instructions. Escherichia coli competent preparation: TAKARA kit; nucleic acid agarose gel electrophoresis, water bath heat shock transformation, electroporation transformation, competent cell preparation, colony PCR and bacterial genome extraction and other conventional molecular biology experimental operations are performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition). The construction of plasmids and sequencing of PCR amplification products were completed by Suzhou Anshengda Company.

[0050] The culture medium involved in the following examples is as follows:

[0051] LB solid medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar powder.

[0052] LB liquid medium: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L peptone.

[0053] Fermentation medium: 25 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, and then adjust the pH to 6.8 with sodium hydroxide.

[0054] Fermentation tank culture medium: 30 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, and then adjust the pH to 6.8 with sodium hydroxide.

[0055] Trace metal elements: 2.25 g / L zinc sulfate heptahydrate, 10 g / L ferrous sulfate, 0.35 g / L manganese sulfate monohydrate, 1.0 g / L anhydrous copper sulfate, 0.23 g / L sodium borate decahydrate, 2.0 g / L calcium chloride dihydrate, 0.11 g / L ammonium molybdate, dissolved in 5 M hydrochloric acid.

[0056] Antibiotic concentration: ampicillin 100 mg / L (liquid culture medium), ampicillin 200 mg / L (solid culture medium), kanamycin 50 mg / L, streptomycin 50 mg / L, chloramphenicol 34 mg / L.

[0057] Inducer concentration: During shake flask fermentation, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mM.

[0058] Feed solution for batch fed fermentation: 600 g / L glycerol, 20 g / L magnesium sulfate heptahydrate, 0.2 g / L thiamine, 100 mg / L ampicillin, 50 mg / L streptomycin. pH control: 14% ammonia water (w / v).

[0059] The strain culture and fermentation involved in the following examples are as follows:

[0060] Sialyl lactoyl-N-tetraose a shake flask fermentation process: On the corresponding LB solid plate, pick a single colony with normal colony morphology and put it into a 4mL test tube LB culture liquid (containing the corresponding antibiotics). After culturing for 10-12 hours, transfer the seed liquid in the test tube to a 25mL shake flask DM medium at a 2% (v / v) inoculation volume. The culture conditions are 37°C and 200rpm. When OD 600 When the concentration reached 0.6-0.8, IPTG with a final concentration of 0.5 mM and lactose of 5 g / L were added, and the induction culture was continued at 25°C and 200 rpm for 72 h.

[0061] Fed-batch fermentation process of sialic acid lactyl-N-tetraose a: a single colony of recombinant Escherichia coli was picked from the plate and inoculated into 4 mL LB medium containing ampicillin and streptomycin for overnight culture as the primary seed liquid; 2.5 mL of the primary seed liquid was inoculated into 250 mL of fermentation medium and cultured at 37 ° C and 200 rpm until OD 600 The OD was about 1.6 and then transferred to a 5L fermenter for culture. Fed-batch fermentation was carried out in a 5L fermenter containing 2.5L fermenter medium. The initial temperature was maintained at 37°C. 600 When the temperature reached about 22°C, the mixture was cooled to 25°C and IPTG was added to a final concentration of 0.2 mM and lactose was added to a final concentration of 5 g / L. 3 ·H 2 O was used to adjust the pH to maintain at 6.8, and the foam was controlled by adding a defoamer. The dissolved oxygen was controlled by adjusting the stirring speed (100-900 rpm) and the ventilation volume (2-8 vvm). Carbon sources (including 600 g / L glycerol, 20 g / L MgSO 4 7H 2 O and 0.2 g / L thiamine, and 200 g / L lactose solution).

[0062] The detection methods involved in the following embodiments are as follows:

[0063] Production Detection of Sialyl Lactoyl-N-tetraose a

[0064] Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, and take the supernatant for HPLC determination.

[0065] HPLC detection conditions: by high performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II); chromatographic column: XBT Amide; detector: Agilent 1260 Infinity II VWD detector; mobile phase: A pump is 10 mM ammonium formate solution (pH value adjusted to 4.0 with formic acid), B pump is acetonitrile, A and B solvent ratio is 30:70; flow rate: 1.0 mL / min; detection wavelength: 210 nm; column temperature: 35°C; injection volume: 10 μL.

[0066] Example 1: Knockout of genes in the CMP-Neu5Ac synthesis competition pathway in the recombinant strain

[0067] 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. The CRISPR-Cas9 gene editing system was used to further knock out the nanA, nanK, nanT and nanE genes in the genome of the starting strain. The specific steps are as follows (the primer sequences involved are shown in Table 1):

[0068] (1) Using the original pTargetF plasmid as template and nanA-sg-F / R as primers, PCR amplification was performed to remove the N 20 The sequences were replaced with N complementary to the nanA sequence. 20 The pTargetF plasmid pTargetF-nanA with the target gene nanA was obtained by sequencing. The PCR product was transformed into competent E. coli JM109 after removing the template DNA with DpnI enzyme, coated on LB plates (containing spectinomycin), expanded at 37°C, and the plasmid was extracted and sequenced.

[0069] (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.

[0070] (3) The pCas plasmid was transferred into the competent cell of the recombinant bacterium BAZ, and the transformed bacterial solution was spread on an LB plate containing kanamycin and cultured overnight in a 37°C incubator to obtain BAZ-pCas.

[0071] (4) Pick a single BAZ-pCas colony in LB medium, culture at 30°C for 1.0 h, and add L-arabinose with a final concentration of 10 mM to induce the expression of the pCas-λ-red system. 600 When the concentration reaches 0.5-0.6, prepare the BAZ-pCas electroporation competent state.

[0072] (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 (kanamycin and spectinomycin), cultured at 30°C for 24 h, and PCR was used to verify the knockout effect of the nanA gene. The nanA knockout colonies were positive clones.

[0073] (6) The positive clone colonies obtained in step (5) were picked into a 4 mL LB liquid test tube, 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. The BAZA strain with the nanA gene knocked out was obtained.

[0074] (7) Using the same method, the genes nanK, nanT, and nanE in the strain were knocked out in turn to obtain the corresponding three recombinant Escherichia coli BAZAK, BAZAKT, and BAZAKTE.

[0075] Table 1: Primer sequences and targeting plasmid information

[0076]

[0077]

[0078] Example 2: Construction of recombinant expression vector

[0079] The specific steps for constructing the recombinant expression vector are as follows (the primer sequences involved are shown in Table 1):

[0080] The gene neuBCA from Campylobacter jejuni (the nucleotide sequence is shown in SEQ ID NO.1) and the gene wbgO from Escherichia coli K-12 (the nucleotide sequence is shown in SEQ ID NO.2) were constructed into MCS1 and MCS2 of five expression vectors, namely pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pCOLADuet-1, respectively, to form recombinant plasmids pET-1BCA-2wbgO, pAC-1BCA-2wbgO, pCD-1BCA-2wbgO, pRSF-1BCA-2wbgO, and pCO-1BCA-2wbgO.

[0081] The gene nst (nucleotide sequence is shown in SEQ ID NO.7) was constructed into the MCS1 of the expression vectors pETDuet-1, pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, and pCOLADuet-1 to form recombinant plasmids pET-NST, pAC-NST, pCD-NST, pRSF-NST, and pCO-NST.

[0082] The gene Pm0188 (nucleotide sequence as shown in SEQ ID NO.3) was constructed into MCS1 of the expression vector pCDFDuet-1 to form the recombinant plasmid pCD-Pm0188.

[0083] The gene BtST1 (nucleotide sequence shown in SEQ ID NO.4) was constructed into MCS1 of the expression vector pCDFDuet-1 to form the recombinant plasmid pCD-BtST1.

[0084] The gene Hd0053 (nucleotide sequence as shown in SEQ ID NO.5) was constructed into MCS1 of the expression vector pCDFDuet-1 to form the recombinant plasmid pCD-Hd0053.

[0085] The gene Vs16 (nucleotide sequence as shown in SEQ ID NO.6) was constructed into MCS1 of the expression vector pCDFDuet-1 to form the recombinant plasmid pCD-Vs16.

[0086] Example 3: Fermentation production of sialyllactyl-N-tetrasaccharide by recombinant strains of different α2,3-sialyltransferases

[0087] The pCD-Pm0188, pCD-BtST1, pCD-Hd0053, pCD-Vs16, and pCD-NST constructed in Example 2 were combined with pET-1BCA-2wbgO and transferred into the host BAZAKT constructed in Example 1 to obtain recombinant strains BL21-LSTa4, BL21-LSTa6, BL21-LSTa7, BL21-LSTa8, and BL21-LSTa9.

[0088] 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 sialic acid lactyl-N-tetraose a is shown in Table 1. The highest yield of sialic acid lactyl-N-tetraose a was 1.235 g / L ( Figure 3 ). Therefore, the nst gene from Neisseria meningitidis may be a good candidate for microbial synthesis of sialyl lactoyl-N-tetraose a, followed by Pm0188, BtST1 and Vs16, and Hd0053 is the worst.

[0089] Example 4: Fermentation production of sialyllactyl-N-tetraose a by recombinant strains expressing different gene pathways

[0090] The pET-1BCA-2wbgO, pAC-1BCA-2wbgO, pCD-1BCA-2wbgO, pRSF-1BCA-2wbgO, pCO-1BCA-2wbgO constructed in Example 2 were combined with pET-NST, pAC-NST, pCD-NST, pRSF-NST, pCO-NST and transferred into the constructed host BAZAKT. The specific information of the obtained recombinant strain is shown in Table 1.

[0091] 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 600The 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 and cultured for 72 h at 25 °C and 200 rpm. 1 mL of fermentation broth was taken, and the supernatant was used for HPLC determination. The yield of sialyl lactoyl-N-tetraose a is shown in Table 2. The engineering bacteria BL21-LSTa9 containing the recombinant plasmids pET-1BCA-2wbgO and pCD-NST obtained the highest yield of 1.235 g / L.

[0092] Table 2: Recombinant strains and corresponding sialyllactyl-N-tetrasaccharide a production information

[0093]

[0094]

[0095]

[0096] BL21-LSTa9 was used as the fermentation strain. Under the same shaking flask conditions, the culture was induced at 28, 25 and 22°C. The results showed that the highest yield of sialic acid lactyl-N-tetraose a was 1.233 g / L ( Figure 4 A).

[0097] Using BL21-LSTa9 as the fermentation strain, the final concentration of IPTG was changed to 0.05, 0.2, 0.5 and 1.0 mM under the condition of the other shake flask conditions unchanged. The results showed that the highest yield of sialic acid lactyl-N-tetraose a was 1.233 g / L when the final concentration of IPTG was 0.5 mM ( Figure 4 B).

[0098] Example 5: Production of sialyllactyl-N-tetraose a by fed-batch culture in a fermenter

[0099] The strain BL21-LSTa9 was selected to carry out a fed-batch fermentation experiment of sialyllactyl-N-tetraose a in a 5L fermenter.

[0100] A single colony of recombinant E. coli BL21-LSTa9 was picked from the plate and inoculated into 4 mL LB medium containing ampicillin and streptomycin for overnight culture as the primary seed liquid; 2.5 mL of the primary seed liquid was inoculated into 250 mL of fermentation medium and cultured at 37°C and 200 rpm until OD 600 The OD was about 1.6 and then transferred to a 5L fermenter for culture. Fed-batch fermentation was carried out in a 5L fermenter containing 2.5L fermenter medium. The initial temperature was maintained at 37°C. 600When the temperature reaches about 22°C, the temperature is lowered to 25°C, and IPTG with a final concentration of 0.2 mM and lactose with a final concentration of 5 g / L are added to induce gene expression. Subsequently, the feed solution is added by batch feeding to ensure that the glycerol is maintained at a final concentration of 3-10 g / L, the lactose is maintained at a final concentration of 3-5 g / L, the pH is maintained at 6.8±0.1 throughout the process, and the foam is controlled by adding a defoamer. The dissolved oxygen is controlled by adjusting the stirring speed (100-900 rpm) and the ventilation volume (2-8 vvm).

[0101] After 80 h of fermentation, the yield of sialic acid lactyl-NN sugar a reached 4.85 g / L ( Figure 5 B), OD 600 The highest reached 105.

[0102] 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 sialyllactyl-N-tetrasaccharide a, characterized in that: The recombinant Escherichia 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 competing pathway genes N-acetylneuraminic acid aldolase nanA, sialic acid transporter nanT, N-acetylmannosamine kinase nanK and N-acylglucosamine-6-phosphate-dimerase nanE were knocked out, and exogenous UDP-N-acetylmannosamine isomerase neuC, N-acetylneuraminic acid synthetase neuB, cytosine-5′-monophosphate-N-acetylneuraminic acid synthetase neuA and β-1,3-galactosyltransferase wbgO and α2,3-sialyltransferase genes were freely expressed.

2. The recombinant Escherichia coli for synthesizing sialyl lactyl-N-tetrasaccharide a according to claim 1, characterized in that: The neuB, neuC and neuA genes are expressed in series to obtain the gene sequence neuBCA, the nucleotide sequence of which is shown in SEQ ID NO.

1.

3. The recombinant Escherichia coli for synthesizing sialyllactyl-N-tetrasaccharide a according to claim 1, characterized in that: The nucleotide sequence of the β-1,3-galactosyltransferase wbgO is shown in SEQ ID NO.

2.

4. The recombinant Escherichia coli for synthesizing sialyl lactyl-N-tetrasaccharide a according to claim 1, characterized in that: The α2,3-sialyltransferase gene includes Pm0188, BtST1, Hd0053, Vs16 or nst; wherein: The nucleotide sequence of Pm0188 is shown in SEQ ID NO. 3; The nucleotide sequence of BtST1 is shown in SEQ ID NO.4; The nucleotide sequence of Hd0053 is shown in SEQ ID NO.5; The nucleotide sequence of Vs16 is shown in SEQ ID NO.6; The nucleotide sequence of Nst is shown in SEQ ID NO.

7.

5. The method for constructing a recombinant Escherichia coli for synthesizing sialyllactyl-N-tetrasaccharide a according to claim 4, characterized in that: Plasmids pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 were used to simultaneously overexpress UDP-N-acetylmannosamine epimerase gene neuC, N-acetylneuraminic acid synthetase gene neuB, cytosine-5′-monophosphate-N-acetylneuraminic acid synthetase gene neuA and β-1,3-galactosyltransferase gene wbgO; Among them, the pCDFDuet-1 plasmid was used to express the α2,3-sialyltransferase genes Pm0188, BtST1, Hd0053, Vs16 and nst to construct the pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 plasmids respectively.

6. A method for producing sialyllactyl-N-tetraose a, 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 4 as a fermentation strain to produce sialyllactyl-N-tetraose a.

7. The method according to claim 6, characterized in that The recombinant E. coli was added to a culture medium containing 25 g / L glycerol and cultured at 37°C and 200 rpm until 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.

8. Use of the recombinant Escherichia coli according to any one of claims 1 to 4 in the preparation of sialyl lactyl-N-tetrasaccharide a.

Images