Escherichia coli with high yield of sialyloyl-N-tetrasaccharide as well as construction method and application of escherichia coli

Abstract

The invention belongs to the field of synthetic biology and metabolic engineering, and particularly relates to high-yield sialyl acyl-N-tetrasaccharide a escherichia coli as well as a construction method and application thereof. According to the method, recombinant escherichia coli is taken as an initial strain, and a beta1, 3-galactosyl transferase gene wbgO from escherichia coli O55: H7 is integrated into a chromosome in a multi-copy form, so that a lactyl-N-tetrasaccharide efficient synthesis chassis strain without plasmid dependence is constructed. And overexpression neuC, neuB, neuA and Nm3ST are introduced through plasmids, so that the supply of glycosyl donors is improved. The expression intensity of uridine diphosphate-N-acetylglucosamine synthesis pathway genes glmM and glmUS is regulated through ribosome binding site engineering, and a cytidine triphosphate regeneration module is introduced, so that the yield of sialyloyl-N-tetrasaccharide a is increased. And finally, the maximum titers of the sialyloyl-N-tetrasaccharide a respectively reach 2.238 g / L and 8.178 g / L by shaking a flask and culturing in batches. The invention provides an efficient technical scheme for industrial production of complex breast milk oligosaccharide.

Description

Technical Field

[0001] The present invention belongs to the fields of synthetic biology and metabolic engineering, and particularly relates to a high-yield Escherichia coli producing sialyllacto-N-tetraose a, a construction method thereof, and an application thereof. Background Art

[0002] Human milk oligosaccharides (HMOs) are a collective term for unbound oligosaccharides in human milk. Currently, more than 200 HMOs structures have been identified, which are mainly composed of five basic monosaccharides, namely glucose, galactose, L-fucose, N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (Neu5Ac), connected by different glycosidic bonds. They play a key role in the growth and development of infants. They not only provide prebiotics for intestinal health but also have functions such as antibacterial, antiviral, immunomodulatory, and promoting brain development. It is worth noting that several important single HMOs have been approved as nutritional ingredients and added to infant milk powder, showing great international market potential.

[0003] The large-scale production of HMOs is usually achieved through microbial synthesis of metabolic engineering strains. Currently, most studies focus on the synthesis of simple HMOs, which usually use lactose as a receptor and are synthesized through 1-2 glycosylation reactions. In contrast, the microbial synthesis of complex HMOs such as pentasaccharides and hexasaccharides has been less studied. Based on strains producing lacto-N-tetraose (LNT) or lacto-N-neotetraose (LNnT), the introduction of specific fucosylation or sialylation modules can further synthesize more complex oligosaccharides. Our research group first reported in 2025 that sialyllacto-N-tetraose a (LST-a) was successfully synthesized (CN2025101801289), and its shake flask and fed-batch yields were 1.235 g / L and 4.85 g / L respectively, but the yields were not ideal and there is room for further improvement. Summary of the Invention

[0004] Object of the Invention

[0005] The present invention provides a method for high production of sialyllacto-N-tetraose a.

[0006] Technical Solution

[0007] The first object of the present invention is to provide a recombinant Escherichia coli for efficiently synthesizing sialyllacto-N-tetraose a, which is characterized in that it is recombinant Escherichia coli BL21(DE3)ΔlacZΔwecBΔnagBΔugDΔnanAΔnanTΔnanK,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, β-1,3-galactosyltransferase wbgO integrated in multiple copies under the control of a strong promoter (P J23119 ); free expression of UDP-N-acetylmannosamine epimerase neuC, N-acetylneuraminic acid synthase neuB, cytidine-5'-monophosphate-N-acetylneuraminic acid synthase neuA, and α2,3-sialyltransferase gene; regulation of the expression intensity of UDP-GlcNAc synthesis pathway genes glmM and glmUS through ribosome binding site engineering; introduction of CTP synthase pyrG of the cytidine triphosphate (CTP) regeneration module to enhance CMP-Neu5Ac supply.

[0008] The β1,3-galactosyltransferase wbgO described above is derived from Escherichia coli O55:H7 and is integrated in multiple copies at non-essential gene loci (poxB, pheA, ldhA, arsB) of the host bacterium chromosome, and the nucleotide sequence is as shown in SEQ ID NO.1. Preferably, four copies of wbgO are integrated at the poxB, pheA, ldhA, and arsB gene loci.

[0009] The neuB, neuC, and neuA genes are expressed in tandem to obtain the gene sequence neuBCA, and the sources include Campylobacter jejuni and N. meningitidis. Among them, the nucleotide sequence of neuBCA from Campylobacter jejuni is as shown in SEQ ID NO.2, and the nucleotide sequence of neuBCA from N. meningitidis is as shown in SEQ ID NO.4.

[0010] The above gene neuBCA is expressed at the MCS1 site of the pETDuet-1 vector. Preferably, the gene is neuBCA from Campylobacter jejuni.

[0011] In one implementation method, the nucleotide sequence of the α2,3-sialyltransferase gene Nm3ST from Neisseria meningitidis (N. meningitidis) is as shown in SEQ ID NO.6.

[0012] In one implementation method, the gene Nm3ST is expressed at the MCS1 site of the pCDFDuet-1 vector.

[0013] In one implementation method, the UDP-GlcNAc synthesis pathway genes include at least one of glmM, glmU, and glmS, wherein the nucleotide sequence of glmM is as shown in SEQ ID NO.7, the nucleotide sequence of glmU is as shown in SEQ ID NO.8, and the nucleotide sequence of glmS is as shown in SEQ ID NO.9.

[0014] In one implementation method, at least one of the genes glmM, glmU, and glmS is expressed at the MCS2 site of the pET-CjBCA vector. Preferably, glmM, glmU, and glmS are tandemly expressed at the MCS2 site of the pET-CjBCA vector.

[0015] In one implementation method, the expression intensities of glmM and glmUS are regulated by ribosome binding site engineering. The ribosome binding sites include RBS29, RBS31, and RBS32. Among them, the nucleotide sequence of RBS29 is as shown in SEQ ID NO.10, the nucleotide sequence of RBS31 is as shown in SEQ ID NO.11, and the nucleotide sequence of RBS32 is as shown in SEQ ID NO.12. Preferably, RBS31 is used to regulate glmM, and RBS32 is used to regulate glmUS.

[0016] In one implementation method, the CTP regeneration module contains at least one of the genes ndk, cmk, and pyrG, wherein the nucleotide sequence of ndk is as shown in SEQ ID NO.13, the nucleotide sequence of cmk is as shown in SEQ ID NO.14, and the nucleotide sequence of pyrG is as shown in SEQ ID NO.15.

[0017] In one implementation method, at least one of the genes ndk, cmk, and pyrG is expressed at the MCS1 site of the pCOLADuet-1 vector. Preferably, pyrG is tandemly expressed at the MCS1 site of the pCOLADuet-1 vector.

[0018] The first object of the present invention is to provide a method for producing sialyllactosyl-N-tetraose a, which uses glycerol as a carbon source, lactose as a substrate, and IPTG as an inducer, and uses the recombinant Escherichia coli to ferment and produce sialyllactosyl-N-tetraose a.

[0019] In one implementation manner, the seed liquid of the recombinant Escherichia coli is added to a medium containing 25 g / L glycerol, and cultured at 37 °C and 200 rpm until OD 600 reaches 0.6 - 0.8, then IPTG with a final concentration of 0.5 mM is added, and at the same time lactose with a final concentration of 5 g / L is added, and induced culture is carried out at 25 °C, 30 °C, and 37 °C respectively at 200 rpm for at least 72 h.

[0020] In one embodiment, the culture medium further contains 4.0 g / L of diammonium hydrogen phosphate, 13.5 g / L of potassium dihydrogen phosphate, 1.4 g / L of magnesium sulfate heptahydrate, 1.7 g / L of citric acid and 10 ml / L of trace metal elements; the trace metal elements include: 0.35 g / L of manganese sulfate monohydrate, 10 g / L of ferrous sulfate, 1.0 g / L of anhydrous copper sulfate, 2.25 g / L of zinc sulfate heptahydrate, 2.0 g / L of calcium chloride dihydrate, 0.23 g / L of sodium borate decahydrate and 0.11 g / L of ammonium molybdate.

[0021] In one embodiment, the seed liquid of the recombinant Escherichia coli is added to a culture medium containing 25 g / L of glycerol and 5 g / L of yeast extract, and cultured at 37 °C and 200 rpm until the OD 600 reaches 0.6 - 0.8, then IPTG with a final concentration of 0.5 mM is added, and at the same time lactose with a final concentration of 5 g / L is added, and the culture is induced at 25 °C, 30 °C and 37 °C respectively at 200 rpm for at least 72 h.

[0022] Preferably, the seed liquid of the recombinant Escherichia coli is added to a culture medium containing 25 g / L of glycerol and 5 g / L of yeast extract, and cultured at 37 °C and 200 rpm until the OD 600 reaches 0.6 - 0.8, then IPTG with a final concentration of 0.5 mM is added, and at the same time lactose with a final concentration of 5 g / L is added, and the culture is induced at 25 °C and 200 rpm for at least 72 h.

[0023] The third object of the present invention is to provide the application of the recombinant Escherichia coli in the production of products containing sialyllactosyl-N-tetraose a.

[0024] 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.

[0025] Beneficial effects:

[0026] Based on the preparation method of sialyllactosyl-N-tetraose a disclosed in the patent CN2025101801289, the shake flask and fed-batch yields of the present invention are 1.235 g / L and 4.85 g / L respectively, and the yields need to be improved. The present invention aims to improve the productivity of sialyllactosyl-N-tetraose a by using a multi-dimensional metabolic engineering strategy. Using recombinant Escherichia coli BL21(DE3)ΔlacZΔwecBΔnagBΔugDΔnanAΔnanTΔnanK,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 synthetic pathway of sialyllacto-N-tetraose a was constructed by combining genomic integration and plasmid expression. First, through gene integration, multiple copies of β1,3-galactosyltransferase wbgO were integrated into the chassis host, and through medium optimization, the yield of the precursor lacto-N-lactose was successfully increased, providing an adequate supply of precursors. Then, strategies such as plasmid combination optimization, ribosome binding site (RBS) engineering, and enhancing the supply of cytidine triphosphate (CTP) were used to increase the yield of sialyllacto-N-tetraose a. Through shake flask and batch cultivation, the final titers reached 2.238 and 8.178 g / L respectively, showing the potential for industrial application.

[0027] Furthermore: The present invention conducts optimization and screening:

[0028] 1. Optimization of the integration method: CN2025101801289 uses the plasmid expression method, in which the plasmid expression system requires antibiotics and the plasmid is prone to loss, and the quality is unstable during scale-up production; therefore, the present invention uses the method of integrating related genes into the genome, but

[0029] The following problems still exist when integrating into the genome: There is a risk of off-target during gene integration, which may cause accidental gene mutations; the integration efficiency is low; the selection of gene integration sites that have no effect on the metabolic flux and the growth of Escherichia coli; and the balance between the integration gene copy number and the impact on production efficiency and metabolic burden.

[0030] Therefore, the inventor conducts the following optimizations:

[0031] Using strain LSTa-QL00 as the starting strain, the four-copy wbgO gene controlled by a strong promoter (PJ23119) was integrated into the poxB, ldhA, pheA, and arsB sites in the genome of the starting strain using the CRISPR-Cas9 gene editing system. After adding 5 g / L yeast extract to the DM medium, the yield of lacto-N-tetraose almost doubled. Escherichia coli integrating three copies of the β1,3-galactosyltransferase gene wbgO achieved the highest yield of lacto-N-tetraose, and the four-copy wbgO was slightly lower than the three-copy. There was no significant difference in the yield of lacto-N-tetraose of LSTa-QL01-04 in the DM medium without added yeast extract. It shows that the engineered bacteria integrating wbgO not only have an impact on the copy number but also are selective for the medium. However, if plasmid pCD-Nm3ST is present, the engineered strains based on four-copy wbgO (such as LSTa-QL09-12) usually show higher productivity of sialyllacto-N-tetraose a than the engineered strains based on three-copy wbgO (LSTa-QL05-08), and the optimal temperature is also 30 °C.

[0032] 2. Optimization of free expression of neuBCA from different sources at different temperatures:

[0033] Among the neuBCA from four sources, the neuBCA from Campylobacter jejuni achieved the best yield of 1.374 g / L at 25 °C. The neuBCA from N. meningitidis obtained a yield of 0.634 g / L at 25 °C. The yields of lactosyl-N-tetraose a of neuBCA from Streptococcus agalactiae and E. coli K1 were extremely low, being 0.055 g / L and 0.072 g / L respectively. Meanwhile, the yields of 3'-sialyllactose were also low, indicating that the neuBCA from these two sources might provide a very small supply of CMP-Neu5Ac. This shows that the neuBCA from four different sources have a huge impact on the yield of lactosyl-N-tetraose a, and not all sources of neuBCA have the same effect. Also, it is optimal at 25 °C.

[0034] 3. Effect of engineering the ribosome binding site on the expression intensity of genes glmM and glmUS in the UDP-N-acetylglucosamine synthesis pathway on the yield of lactosyl-N-tetraose a

[0035] Among them, strain LSTa-QL19 showed the highest yield of lactosyl-N-tetraose a, with a yield of 1.912 g / L, that is, glmM and glmUS were optimal under the control of low-intensity RBS31 and medium-intensity RBS32 respectively, which indicates that it is not that the stronger the expression of glmM and glmUS, the better, but rather a delicate metabolic balance needs to be sought.

[0036] 4. Effect on the yield of lactosyl-N-tetraose a after strengthening the supply of cytidine triphosphate

[0037] Based on strain LSTa-QL19, three genes related to CTP regeneration were overexpressed through pCOLADuet-1, including ndk (uridine / cytidine kinase), pyrG (CTP synthase), and cmk (cytidylate kinase). The yield of lactosyl-N-tetraose a of pyrG overexpression (LSTa-QL28) was the best (2.238 grams per liter), far higher than the control strain, while the LST-a yields of strains LSTa-QL27 and LSTa-QL29 into which ndk and cmk were introduced respectively were 1.694 grams per liter and 1.522 grams per liter. The LST-a yield of strain LSTa-QL30 into which these three genes were introduced simultaneously was only 1.420 grams per liter.

[0038] Note: Only the single overexpression of pyrG (LSTa-QL28) is the best. However, the introduction of ndk or cmk separately, or the simultaneous introduction of pyrG, ndk, and cmk is less effective than the control strain. Description of the Drawings

[0039] Figure 1 It is a metabolic pathway diagram of recombinant Escherichia coli for biosynthesis of lactosyl-N-tetraose a

[0040] Figure 2 It is a comparison chart of lactosyl-N-tetraose production with different copies of wbgO

[0041] Figure 3 It is a comparison chart of lactosyl-N-tetraose a production with different sources of neuBCA and different culture temperatures

[0042] Figure 4 It is a comparison chart of lactosyl-N-tetraose a production with different RBS-regulated glmMUS expression intensities

[0043] Figure 5 It is a comparison chart of lactosyl-N-tetraose a production with different CTP regeneration modules

[0044] Figure 6 It is a fermentation production result chart of lactosyl-N-tetraose a by the best engineered strain in a 5L fermenter. Detailed Implementation Modes

[0045] For all commercial products such as PCR amplification enzymes, plasmids, DNA gel recovery kits, and column plasmid extraction kits used in the following examples, the specific operations are carried out according to the kit instructions. Preparation of Escherichia coli competent cells: TAKARA kit; Conventional molecular biology experimental operations such as nucleic acid agarose gel electrophoresis, water bath heat shock transformation, electroporation transformation, competent cell preparation, colony PCR, and bacterial genome extraction are carried out according to Molecular Cloing: A Laboratory Manua (Fourth Edition). The sequencing work of the constructed plasmids and PCR amplification products was completed by Suzhou Anshengda Company.

[0046] The media involved in the following examples are as follows:

[0047] (1) LB solid medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, 15 g / L agar powder.

[0048] (2) LB liquid medium: 10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L peptone.

[0049] (3) Fermentation medium: 25 g / L glycerol, 5 g / L yeast extract, 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.

[0050] (4) Fermenter 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.

[0051] (5) 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 copper sulfate anhydrous, 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.

[0052] (6) Antibiotic concentration: Ampicillin 100 mg / L (liquid medium), Ampicillin 200 mg / L (solid medium), Kanamycin 50 mg / L, Streptomycin 50 mg / L

[0053] (7) Inducer concentration: During the shake flask fermentation process, the final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) added is 0.5 mM.

[0054] (8) Fed-batch fermentation feeding solution: Glycerol 600 g / L, Magnesium sulfate heptahydrate 20 g / L, Thiamine 0.2 g / L, Ampicillin 100 mg / L, Streptomycin 50 mg / L. pH regulation: 14% ammonia water (w / v).

[0055] The strain culture and fermentation involved in the following examples are as follows:

[0056] The shake flask fermentation process of sialyllactosyl-N-tetraose a: On the corresponding LB solid plate, pick a single colony with normal colony morphology into 4 mL test tube LB culture liquid (containing the corresponding antibiotics). After culturing for 10 - 12 h, transfer the seed liquid in the test tube to 25 mL shake flask DM medium at an inoculation amount of 2% (v / v). The culture conditions are 37 °C and 200 rpm. When OD 600 reaches 0.6 - 0.8, add IPTG with a final concentration of 0.5 mM and 5 g / L lactose, and continue the induction culture at 25 °C and 200 rpm for 72 h.

[0057] Fed-batch fermentation process of sialyllactosyl-N-tetraose a: Pick a single colony of recombinant Escherichia coli from the plate and inoculate it into 4 mL of LB medium containing ampicillin and streptomycin for overnight culture as the primary seed solution; inoculate 2.5 mL of the primary seed solution into 250 mL of fermentation medium and culture it at 37 °C and 200 rpm for scale-up culture. Transfer it to a 5 L fermenter for culture when the OD 600 reaches approximately 1.6. Fed-batch fermentation is carried out in a 5 L fermenter containing 2.5 L of fermenter medium. The initial temperature is maintained at 37 °C. When the OD 600 reaches approximately 25, 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. Adjust the pH to 6.8 with 14% (w / v) NH3·H2O and control the foam by adding antifoaming agent. Control the dissolved oxygen by adjusting the stirring speed (200 - 800 rpm) and the aeration rate (2 - 8 L / min). Further add carbon sources (including 600 g / L glycerol, 20 g / L MgSO4·7H2O, 0.2 g / L thiamine, and 200 g / L lactose solution) to the fermentation medium.

[0058] The detection methods involved in the following examples are as follows:

[0059] Detection of the yield of sialyllactosyl-N-tetraose a

[0060] Take 1 mL of the fermentation broth, centrifuge it at 10,000 rpm for 10 min, and take the supernatant for HPLC determination.

[0061] HPLC detection conditions: Through a high-performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II); chromatographic column: XBT Amide; detector: Agilent 1260 Infinity II VWD detector; mobile phase: Pump A is 10 mM ammonium formate solution (adjusted to pH 4.0 with formic acid), pump B is acetonitrile, and the ratio of solvents A and B is 30:70; flow rate: 1.0 mL / min; detection wavelength: 210 nm; column temperature: 35 °C; injection volume: 10 μL.

[0062] Detection of glycerol, lactose, and lactosyl-N-tetraose

[0063] Take 1 mL of the fermentation broth, centrifuge it at 10,000 rpm for 10 min, and take the supernatant for HPLC determination.

[0064] HPLC detection conditions: Detection is carried out through a high-performance liquid chromatography (HPLC) system (Waters Corporation). The detection conditions are Rezex ROA organic acid column and differential refractive index detector. The mobile phase is 5 mM sulfuric acid, the column temperature is 60 °C, and the injection volume is 10 μL.

[0065] Example 1: Multi-copy integration of β1,3-galactosyltransferase gene in recombinant strain

[0066] Using strain LSTa-QL00 (E. coli BL21(DE3)ΔlacZΔwecBΔnagBΔugDΔnanAΔnanTΔnanK, 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. Using the CRISPR-Cas9 gene editing system, four copies of the wbgO gene (nucleotide sequence shown in SEQ ID NO.1) under the control of a strong promoter (P J23119 ) were integrated at the poxB, ldhA, pheA, and arsB sites in the genome of the starting strain. The specific steps are as follows (the primer sequences involved are shown in Table 1):

[0067] (1) Using the original pTargetF plasmid as a template and poxBsg-F / R as primers, the N 20 sequences on the original pTargetF plasmid were replaced with N 20 sequences complementary to the poxB sequence by PCR amplification to obtain the pTargetF plasmid pTargetF-poxB with the targeted gene poxB. After removing the template DNA with DpnI enzyme from the PCR product, it was transformed into competent E. coli JM109, spread on an LB plate (containing spectinomycin), and cultured at 37°C for expansion. The plasmid was extracted and sequenced.

[0068] (2) Using the E. coli BL21 genome as a template and primers poxB-UP-F / R and poxB-DH-F / R, the upstream and downstream fragments of the poxB site were amplified by PCR respectively, recovered by gel electrophoresis, and the two fragments were ligated by PCR to obtain the donor DNA fragment.

[0069] (3) Transfer the pCas plasmid into the chemically competent cells of the recombinant strain LSTa-QL00, spread the transformed bacterial solution on an LB plate containing kanamycin, and culture it overnight in a 37°C incubator to obtain LSTa-QL00-pCas.

[0070] (4) Pick a single colony of LSTa-QL00-pCas into LB medium, culture it 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. When OD 600When it reaches 0.5 - 0.6, prepare electrocompetent cells of LSTa-QL00-pCas.

[0071] (5) Electroporate 200 - 500 ng of pTargetF-poxB plasmid and 400 - 800 ng of donor DNA fragment into LSTa-QL00-pCas electrocompetent cells, spread them on an LB plate (kanamycin and spectinomycin), culture at 30 °C for 24 h, and verify the integration effect of gene wbgO at the poxB locus by PCR. The colonies with gene wbgO integrated at the poxB locus are positive clone colonies.

[0072] (6) Pick the positive clone colonies obtained in step (5) into a 4 mL LB liquid test tube, add IPTG with a final concentration of 1 mM and kanamycin at 50 mg / L, culture at 30 °C for 8 - 16 h to remove the pTargetT-poxB plasmid, and then culture at 42 °C for 12 h to remove the pCas plasmid. Obtain the LSTa-QL01 strain with gene wbgO integrated at the poxB locus.

[0073] (7) Using the same method, sequentially integrate wbgO at the ldhA, pheA, and arsB loci in the strain. That is, integrate 1 wbgO at poxB to obtain the LSTa-QL01 strain, integrate 2 wbgOs at poxB and ldhA to obtain LSTa-QL02, integrate 3 wbgOs at poxB, ldhA, and pheA to obtain LSTa-QL03, and integrate 4 wbgOs at poxB, ldhA, pheA, and arsB to obtain LSTa-QL04. (See Figure 1 、 Figure 2 shown).

[0074] Table 1: Primer sequences and information of targeting plasmids

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] Example 2: Construction of recombinant expression vector

[0082] The codon-optimized Campylobacter jejuni, Streptococcus agalactiae, N. meningitidis, and E. coli K1, among which the neuBCA genes (nucleotide sequences are shown in SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4) from Campylobacter jejuni were respectively constructed into MCS1 of the pETDuet-1 expression vector to form recombinant plasmids pET-CjBCA, pET-SaBCA, pET-NmBCA, and pET-EkBCA.

[0083] Taking pET-CjBCA as an example, the specific construction steps are as follows (the primer sequences involved are shown in Table 1):

[0084] Using the neuBCA gene sequence from Campylobacter jejuni as a template and CjneuBCA-F / R as primers, the CjneuBCA gene fragment was amplified by PCR, and the DNA fragment was recovered by gel extraction; using pET-V-F / R as primers, the corresponding vector fragment was amplified with pETDuet-1 as a template, and the DNA fragment was recovered by gel extraction.

[0085] The amplified CjneuBCA gene fragment and vector fragment were ligated through the Gibson kit (NEB reagent company, USA) to obtain plasmid pET-CjBCA.

[0086] Construct the following recombinant expression vectors according to the above steps;

[0087] The codon-optimized gene Nm3ST from Neisseria meningitidis (nucleotide sequence is shown in SEQ ID NO.6) was constructed into MCS1 of the expression vector pCDFDuet-1 to form recombinant plasmid pCD-Nm3ST.

[0088] The codon-optimized genes glmM, glmU, and glmS (nucleotide sequences are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9) were constructed into MCS2 of the expression vector pET-CjBCA to form recombinant plasmids pET-CjBCA-glmM, pET-CjBCA-glmU, pET-CjBCA-glmS, pET-CjBCA-glmMU, pET-CjBCA-glmMS, pET-CjBCA-glmUS, pET-CjBCA-glmMUS.

[0089] Insert the RBS29, RBS31, and RBS32 fragments (nucleotide sequences are shown in SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12) before the glmM and glmUS gene fragments of the expression vector pET-CjBCA-glmMUS to construct pET-CjBCA-glmM l -glmUS l 、pET-CjBCA-glmM l -glmUS m 、pET-CjBCA-glmM l -glmUS h 、pET-CjBCA-glmM m -glmUS l 、pET-CjBCA-glmM m -glmUS m 、pET-CjBCA-glmM m -glmUS h 、pET-CjBCA-glmM h -glmUS l 、pET-CjBCA-glmM h -glmUS m 、pET-CjBCA-glmM h -glmUS h 。

[0090] Construct the codon-optimized genes ndk, cmk, and pyrG (nucleotide sequences are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15) into MCS1 of the expression vector pCOLADuet-1 to form recombinant plasmids pCO-ndk, pCO-cmk, pCO-pyrG, and pCO-ndk-cmk-pyrG.

[0091] Example 3: Shake-flask fermentation production of recombinant strains

[0092] (1) Effects of different copy numbers of the β1,3-galactosyltransferase gene wbgO and the culture medium on the yield of lactosyl-N-tetraose

[0093] Inoculate the recombinant strains LSTa-QL01, LSTa-QL02, LSTa-QL03, and LSTa-QL04 constructed in Example 1 into LB liquid medium containing the corresponding antibiotics, and culture them overnight at 37°C and 200 rpm for 12 h to obtain seed solutions. Take the seed solutions and inoculate them into 25 mL of DM fermentation medium, and culture them at 37°C and 200 rpm until OD 600Induce when it is 0.8, add IPTG with a final concentration of 0.5 mM, and at the same time add 5 g / L lactose, and continue to culture at 25 °C and 200 rpm for 72 h. Take 1 mL of the fermentation broth, take the supernatant, and use it for HPLC determination. The highest yield of lactoyl-N-tetraose is 6.070 g / L of LSTa-QL03( Figure 2 ).

[0094] The results showed that Escherichia coli integrating three copies of the β1,3-galactosyltransferase gene wbgO achieved the highest yield of lactoyl-N-tetraose. The yield of four copies of wbgO was slightly lower than that of three copies. This may be because the increase in copy number increased the metabolic burden of Escherichia coli, resulting in a decrease in yield. It is worth noting that after adding 5 g / L yeast extract, the yield of lactoyl-N-tetraose almost doubled. Taking the three-copy as an example, the yield of lactoyl-N-tetraose increased from 3.026 g / L to 6.070 g / L. The high yield of lactoyl-N-tetraose provided an adequate precursor guarantee for sialyllactoyl-N-tetraose.

[0095] (2) Yield of sialyllactoyl-N-tetraose a from different sources of neuBCA at different temperatures

[0096] Combine the pCD-Nm3ST constructed in Example 2 with pET-CjBCA, pET-SaBCA, pET-NmBCA and pET-EkBCA and transfer them into the constructed hosts LSTa-QL03 and LSTa-QL04. The specific information of the obtained recombinant strains is shown in Table 2.

[0097] Inoculate the recombinant strains into LB liquid medium containing the corresponding antibiotics, culture overnight at 37 °C and 200 rpm for 12 h to obtain seed liquid. Take the seed liquid and inoculate it into 25 mL of DM fermentation medium, culture at 37 °C and 200 rpm until OD 600 Induce when it is 0.8, add IPTG with a final concentration of 0.5 mM, and at the same time add 5 g / L lactose, and continue to culture at 25 °C, 30 °C and 37 °C and 200 rpm for 72 h. Take 1 mL of the fermentation broth, take the supernatant, and use it for HPLC determination. The highest yield of sialyllactoyl-N-tetraose a is 1.374 g / L of LSTa-QL11 at 25 °C( Figure 3 ).

[0098] As Figure 3As shown, all strains showed the highest yield of sialyllacto-N-tetraose a when induced at 25°C, and the titer decreased significantly with the increase of the induction temperature. Interestingly, in the case of containing plasmid pCD-Nm3ST, engineered strains based on the four-copy wbgO strain LSTa-QL04 (such as LSTa-QL09-12) generally showed higher productivity of sialyllacto-N-tetraose a than the engineered strains (LSTa-QL05-08) based on the three-copy wbgO strain LSTa-QL03, although the yield of lacto-N-tetraose of LSTa-QL04 was slightly lower than that of LSTa-QL03. Among the four sources of neuBCA, Campylobacter jejuni-derived neuBCA achieved the best yield of 1.374 g / L at 25°C. The yield of N. meningitidis-derived neuBCA was 0.634 g / L at 25°C. While the yields of sialyllacto-N-tetraose a from Streptococcus agalactiae and E. coli K1-derived neuBCA were extremely low, 0.055 g / L and 0.072 g / L respectively. At the same time, the yield of 3'-sialyllactose was also low, indicating that the supply of CMP-Neu5Ac provided by these two sources of neuBCA might be very small. However, lacto-N-tetraose accumulated in large amounts in these strains. When induced at 30°C, the yields of lacto-N-tetraose of LSTa-QL10 and LSTa-QL12 reached 6.394 g / L and 5.915 g / L respectively. This indicates that the optimal temperatures for the production of lacto-N-tetraose and sialyllacto-N-tetraose a are different, and here the optimal temperature is 30°C.

[0099] (3) Effect of engineering the expression intensity of genes glmM and glmUS in the UDP-N-acetylglucosamine synthesis pathway at the ribosome binding site on the yield of sialyllacto-N-tetraose a

[0100] The pCD-Nm3ST constructed in Example 2 was combined with pET-CjBCA-glmM, pET-CjBCA-glmU, pET-CjBCA-glmS, pET-CjBCA-glmMU, pET-CjBCA-glmMS, pET-CjBCA-glmUS, pET-CjBCA-glmMUS, pET-CjBCA-glmM l -glmUS l 、pET-CjBCA-glmM l -glmUS m 、pET-CjBCA-glmM l -glmUS h 、pET-CjBCA-glmM m-glmUS l 、pET-CjBCA-glmM m -glmUS m 、pET-CjBCA-glmM m -glmUS h 、pET-CjBCA-glmM h -glmUS l 、pET-CjBCA-glmM h -glmUS m 、pET-CjBCA-glmM h -glmUS h were combined and transferred into the constructed host LSTa-QL04, and the specific information of the obtained recombinant strains is shown in Table 2.

[0101] The recombinant strains were respectively inoculated into LB liquid medium containing the corresponding antibiotics, cultured at 37 °C and 200 rpm overnight for 12 h to obtain seed solutions. The seed solutions were inoculated into 25 mL of DM fermentation medium, cultured at 37 °C and 200 rpm until OD 600 reached 0.8 for induction, IPTG with a final concentration of 0.5 mM was added, and at the same time 5 g / L of lactose was added, and the culture was continued at 25 °C and 200 rpm for 72 h. 1 mL of the fermentation broth was taken, and the supernatant was taken for HPLC determination. The highest yield of sialyllactosyl-N-tetraose a was 1.912 g / L of LSTa-QL19 (glmM was controlled by RBS31, and glmUS was controlled by RBS29) ( Figure 4 ).

[0102] In this example, the effect of glmMUS overexpression on LST-a biosynthesis was studied. When glmMUS was inserted into another multiple cloning site of pET-CjneuBCA, the newly generated strain LSTa-QL17 produced 1.559 g / L of sialyllactosyl-N-tetraose a, slightly higher than the control strain LSTa-QL11 without glmMUS overexpression. Interestingly, some of the engineered strains (LSTa-QL13-16) with selected glmMUS overexpression produced slightly lower amounts of sialyllactosyl-N-tetraose a ( Figure 4)。Based on the strain LSTa-QL17, ribosome binding site (RBS) engineering was attempted to fine-tune the glmMUS expression module to enhance the production of LST-a. Here, glmM and glmUS (obtained by tandem of glmU and glmS) were respectively controlled by different RBSs to fine-tune the UDP-GlcNAc synthesis pathway. By controlling glmM and glmUS with different RBS combinations, nine new strains were obtained and named LSTa-QL18-26. Seven of these nine strains had a higher yield of lacto-N-tetraose a sialic acid than the control strain LSTa-QL17. Among them, strain LSTa-QL19, that is, glmM l and glmUSm were respectively under the control of the low-intensity RBS31 and the medium-intensity RBS32, showing the highest yield of lacto-N-tetraose a sialic acid, with a yield of 1.912 g / L. This indicates that it is not the stronger the expression of glmM and glmUS is, the better, but a delicate metabolic balance needs to be sought. Note: RBS29 is high-intensity, RBS31 is low-intensity, and RBS32 is medium-intensity.

[0103] (4) Effect of strengthening the supply of cytidine triphosphate on the yield of lacto-N-tetraose a sialic acid

[0104] The pCD-Nm3ST and pET-CjBCA-glmM constructed in Example 2 l -glmUS m were combined with pCO-ndk, pCO-cmk, pCO-pyrG, and pCO-ndk-cmk-pyrG and transferred into the constructed host LSTa-QL04. The specific information of the resulting recombinant strains is shown in Table 2.

[0105] The recombinant strains were respectively inoculated into LB liquid medium containing the corresponding antibiotics, cultured overnight at 37 °C and 200 rpm for 12 h to obtain seed solutions. The seed solutions were inoculated into 25 mL of DM fermentation medium, cultured at 37 °C and 200 rpm until the OD 600 reached 0.8 for induction, 0.5 mM IPTG was added at a final concentration, and at the same time 5 g / L lactose was added, and the culture was continued at 25 °C and 200 rpm for 72 h. 1 mL of the fermentation broth was taken, the supernatant was taken for HPLC determination, and the highest yield of lacto-N-tetraose a sialic acid was 2.238 g / L of LSTa-QL28( Figure 5 ).

[0106] Based on strain LSTa-QL19, three genes related to CTP regeneration, including ndk (uridine / cytidine kinase), pyrG (CTP synthase), and cmk (cytidylate kinase), were overexpressed via pCOLADuet-1. Only the yield of sialyllacto-N-tetraose a (2.238 g / L) of the strain with overexpressed pyrG (LSTa-QL28) was higher than that of the control strain, while the LST-a yields of strains LSTa-QL27 and LSTa-QL29 into which ndk and cmk were introduced respectively were 1.694 g / L and 1.522 g / L. The LST-a yield of strain LSTa-QL30 into which all three genes were introduced simultaneously was only 1.420 g / L( Figure 5 ).

[0107] Table 2: Yield information of recombinant strains and corresponding sialyllacto-N-tetraose a during fermentation at 25 °C

[0108]

[0109]

[0110]

[0111] Example 5: Fed-batch culture in a fermenter for the production of sialyllacto-N-tetraose a

[0112] Strain LSTa-QL28 was selected for a fed-batch fermentation experiment of sialyllacto-N-tetraose a in a 5 L fermenter.

[0113] A single colony of recombinant Escherichia coli LSTa-QL28 was picked from the plate and inoculated into 4 mL of LB medium containing ampicillin, kanamycin, and streptomycin for overnight culture as the primary seed solution; 2.5 mL of the primary seed solution was inoculated into 250 mL of fermentation medium and cultured at 37 °C and 200 rpm for scale-up culture. When the OD 600 reached approximately 1.6, it was transferred to a 5 L fermenter for culture. Fed-batch fermentation was carried out in a 5 L fermenter containing 2.5 L of fermenter medium. The initial temperature was maintained at 37 °C. When the OD 600 reached approximately 25, the temperature was lowered to 25 °C, and IPTG with a final concentration of 0.2 mM and lactose at 5 g / L were added for gene induction expression. Subsequently, the fed-batch fermentation feed solution was added to ensure that glycerol was maintained at a final concentration of 3 - 5 g / L and lactose was maintained at a final concentration of 3 - 5 g / L. The pH was maintained at 6.8 ± 0.1 throughout the process, and foam was controlled by adding an antifoaming agent. Dissolved oxygen was controlled by adjusting the stirring speed (200 - 800 rpm) and the aeration rate (2 - 8 L / min).

[0114] After 124 h of fermentation, the yield of sialyllactosyl-N-tetraose a reached 8.178 g / L( Figure 6 ), and the OD 600 reached a maximum of 200.

[0115] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A recombinant Escherichia coli for synthesizing sialyllactosyl-N-tetraose a, which uses recombinant Escherichia coli BL21(DE3)ΔlacZΔwecBΔnagBΔugDΔnanAΔnanTΔnanK,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, characterized in that, Multiple-copy integration of β-1,3-galactosyltransferase wbgO under the control of the strong promoter P J23119 Free expression of UDP-N-acetylmannosamine epimerase neuC, N-acetylneuraminic acid synthase neuB, cytosine-5'-monophosphate-N-acetylneuraminic acid synthase neuA, and α2,3-sialyltransferase gene; regulation of the expression intensity of UDP-GlcNAc synthesis pathway genes glmM and glmUS through ribosome binding site engineering; introduction of CTP synthase pyrG to enhance CMP-Neu5Ac supply.

2. The recombinant Escherichia coli for synthesizing lactosyl-N-tetraose a of sialic acid according to claim 1, characterized in that, The β1,3-galactosyltransferase wbgO is derived from Escherichia coli O55:H7, and its nucleotide sequence is as shown in SEQ ID NO.

1.

3. A recombinant Escherichia coli for synthesizing sialyllactosyl-N-tetraose a according to claim 1, characterized in that, The genes neuB, neuC, and neuA are tandemly expressed to obtain the gene sequence neuBCA, which is derived from Campylobacter jejuni or N. meningitidis. Among them: The nucleotide sequence of neuBCA derived from Campylobacter jejuni is as shown in SEQ ID NO.2; The nucleotide sequence of neuBCA derived from N. meningitidis is as shown in SEQ ID NO.

4.

4. A recombinant Escherichia coli for synthesizing sialyllactosyl-N-tetraose a according to claim 1, characterized in that, The nucleotide sequence of the α2,3-sialyltransferase gene Nm3ST derived from Neisseria meningitidis is as shown in SEQ ID NO.

6.

5. The recombinant Escherichia coli for synthesizing sialyllactosyl-N-tetraose a according to claim 1, characterized in that Containing at least one of the UDP-GlcNAc synthesis pathway genes glmM, glmU, and glmS, and regulating the expression intensity through the combination of ribosome binding sites RBS. Among them: The nucleotide sequence of glmM is as shown in SEQ ID NO.7; The nucleotide sequence of glmU is as shown in SEQ ID NO.8; The nucleotide sequence of glmS is as shown in SEQ ID NO.9; The nucleotide sequence of RBS29 is as shown in SEQ ID NO.10; The nucleotide sequence of RBS31 is as shown in SEQ ID NO.11; The nucleotide sequence of RBS32 is as shown in SEQ ID NO.

12.

6. A recombinant Escherichia coli for synthesizing sialyllactosyl-N-tetraose a according to claim 1, characterized in that, The nucleotide sequence of the pyrG is as shown in SEQ ID NO.

15.

7. A method for constructing the recombinant Escherichia coli according to any one of claims 1-6, comprising the following steps: Using the CRISPR / Cas9 system to integrate multiple copies of wbgO into the non-essential gene loci poxB, pheA, ldhA, and arsB of the host bacterium chromosome; Constructing a plasmid pCDFDuet-1 expressing the α2,3-sialyltransferase gene derived from Neisseria meningitidis N. meningitidis; Constructing a plasmid pETDuet-1 with the CMP-Neu5Ac pathway genes neuC, neuB, and neuA from two different sources, Campylobacter jejuni and N. meningitidis, inserted into MCS1 respectively, and at least one of the UDP-GlcNAc synthesis pathway genes glmM, glmU, and glmS inserted into the MCS2 site, and optimizing the regulation of the expression of glmMUS through RBS; Constructing a plasmid pCOLADuet-1 expressing pyrG.

8. A method for producing sialyllactosyl-N-tetraose a, characterized in that, Using glycerol as the carbon source, lactose as the substrate, IPTG as the inducer, and the recombinant Escherichia coli according to any one of claims 1-6 as the fermentation strain to ferment and produce sialyllactosyl-N-tetraose a.

9. The method according to claim 8, wherein Add the recombinant Escherichia coli to a medium containing 25 g / L glycerol and 5 g / L yeast extract, and culture at 37°C and 200 rpm until the OD 600 reaches 0.6 - 0.8, then add IPTG with a final concentration of 0.5 mM and lactose with a concentration of 5 g / L, and induce the culture at 25°C and 200 rpm for at least 72 h.

10. Use of the recombinant Escherichia coli according to any one of claims 1-6 in the preparation of sialyllactosyl-N-tetraose a.

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