Method for producing glcnacβ1-4(fucα1-6)glcnac by dual-bacterium coupled fermentation

Abstract

Provided is a method for producing 6'-fucosylated chitobiose GlcNAcβ1-4(Fucα1-6)GlcNAc by dual-bacterium coupled fermentation, wherein fucose and N-acetyl-D-glucosamine are used as substrates, and 6'-fucosylated chitobiose is produced by coupled fermentation of an engineered Escherichia coli strain and a yeast; a synthetic pathway for GlcNAcβ1-4(Fucα1-6)GlcNAc comprising N-acetylhexosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucose kinase Fkp, and N,N'-diacetylchitobiose phosphorylase Chbp is constructed in the engineered Escherichia coli; the yeast is added to achieve recycling regeneration from GMP to GTP; and the yield is increased to 25.50 g / L.

Description

A method for producing GlcNAcβ1-4(Fucα1-6)GlcNAc by coupled fermentation of two bacteria Technical Field

[0001] This invention relates to the field of fermentation engineering technology, and in particular to a method for producing GlcNAcβ1-4(Fucα1-6)GlcNAc by coupled fermentation of two microorganisms. Background Technology

[0002] Glycosylation of asparagine-linked N-links is one of the most common co-translational modifications of proteins distributed both intracellularly and extracellularly, directly affecting their biological functions, such as protein folding, stability, and intercellular communication. The production of well-defined homogeneous N-glycans facilitates comprehensive research into their biological roles and molecular basis. Natural N-glycans are found in very low quantities and require complex purification processes. Biosynthesis offers advantages such as low production costs and minimal environmental pollution, making it the preferred synthetic method for commercial products today. GlcNAcβ1-4(Fucα1-6)GlcNAcβ1-4 is the core structure of complex N-glycans, laying the foundation for the further synthesis of complete complex N-glycans, creating substrate conditions for the development of the functions of GlcNAcβ1-4(Fucα1-6)GlcNAcβ1-4, and providing a new pathway for the synthesis of complex N-glycans.

[0003] Intermediate products of the reaction, such as chitobiose, GDP-Fucose, chitosan oligosaccharides, and chitosan oligosaccharides, have a sweet taste and are soluble in water, although their solubility is lower than that of monosaccharides. Therefore, chitosan oligosaccharides have good hygroscopic and moisturizing properties and can be used as flavoring agents and preservatives in food development. Chitosan oligosaccharides can inhibit the growth of Escherichia coli and pathogenic bacteria in the intestine and promote the proliferation of beneficial bacteria in the intestine. They can regulate the metabolic activities of microorganisms in the animal intestine through chitosan oligosaccharides and chitosan oligosaccharides, improve the distribution of the intestinal microbiota, and thus enhance the body's immunity. In addition, chitosan oligosaccharides also have anti-cancer and immune-activating effects, can induce pancreatic T lymphocytes to produce interleukins, enhance the body's immunity, and are non-toxic or have low toxicity to organisms. Therefore, chitosan oligosaccharides ((GIcNAc)n (10≥n≥2) including (GlcNAc)2 have huge market development potential in food development, antibacterial, immunomodulatory, and anti-infective applications.

[0004] Due to high production costs and low efficiency, large-scale production of 6'-fucosylated chitobiose GlcNAcβ1-4(Fucα1-6)GlcNAc is currently not feasible. Sialic acid and cytidine triphosphate (GTP) are expensive, and the synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc requires the consumption of GlcNAc and large amounts of GTP, resulting in high synthesis costs and low yields (14.82 mg / L). Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for producing 6'-fucosylated chitobiose GlcNAcβ1-4(Fucα1-6)GlcNAc via coupled fermentation. In the fermentation system, fucose and N-acetyl-D-glucosamine are used to construct the GlcNAcβ1-4(Fucα1-6)GlcNAc synthetic pathway in genetically engineered bacteria. This pathway includes N-acetylglucosamine 1-kinase (nahK), fucosyltransferase (nodZ), L-fucokinase (Fkp), and N,N-diacetylchitosan phosphorylase (Chbp). The construction method of the expression vector in the engineered bacteria is adjusted, followed by coupled fermentation with yeast. Furthermore, the fermentation conditions are optimized, including adjusting the fermentation pH and fermentation time, and adding metal ions and surfactants to increase yield.

[0006] The first objective of this invention is to provide a method for producing GlcNAcβ1-4(Fucα1-6)GlcNAc by coupled fermentation of two bacteria, wherein fucose and N-acetyl-D-glucosamine are used as substrates and the fermentation is carried out by coupled fermentation of engineered Escherichia coli and yeast.

[0007] The first engineered bacteria heterologously expresses N-acetylglucosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucokinase Fkp, and N,N-diacetylchitosan phosphorylase Chbp.

[0008] Furthermore, the first engineered bacterium contains a first expression vector and a second expression vector, the first expression vector containing the nahK coding gene and the Fkp coding gene, and the second expression vector containing the nodZ coding gene and the Chbp coding gene.

[0009] Further, the nahK encoding gene is shown in SEQ ID NO.1, the Chbp encoding gene is shown in SEQ ID NO.2, the Fkp encoding gene is shown in SEQ ID NO.3, and the nodZ encoding gene is shown in SEQ ID NO.4.

[0010] Furthermore, the yeast is brewer's yeast, brewer's yeast, or baker's yeast.

[0011] Preferably, the yeast is brewer's yeast.

[0012] Furthermore, the first expression vector uses pRSFDuet-1 plasmid as a backbone, and the second expression vector uses pETDuet-1 plasmid as a backbone.

[0013] Furthermore, the ratio of the engineered Escherichia coli and yeast in the fermentation system is 1:1-3.

[0014] Preferably, the ratio of the first engineered bacteria and the second engineered bacteria in the fermentation system is 1:1.

[0015] Furthermore, the fermentation time is 36-60 hours.

[0016] Preferably, the fermentation time is 36 hours.

[0017] Furthermore, a metal ion, specifically Mg, is added to the fermentation system. 2+ .

[0018] Furthermore, the Mg 2+ The concentration in the fermentation system is 0-80 mM.

[0019] Preferably, the Mg 2+ The concentration in the fermentation system is 20 mM.

[0020] Furthermore, a surfactant, namely octadecylamine polyoxyethylene ether, is added to the fermentation system.

[0021] Furthermore, the concentration of the octadecylamine polyoxyethylene ether in the fermentation system is 8-16 g / L.

[0022] Preferably, the concentration of the octadecylamine polyoxyethylene ether in the fermentation system is 8 g / L.

[0023] The beneficial effects of this invention are:

[0024] This invention discloses a method for producing GlcNAcβ1-4(Fucα1-6)GlcNAc via dual-strain coupled fermentation. The synthetic pathway is constructed within a single engineered bacterial strain, avoiding the impact on product yield caused by frequent material entry and exit from the cell due to a large number of engineered strains. Furthermore, to address the high cost caused by the large consumption of GTP during synthesis, yeast is added to the fermentation system to form a dual-strain coupled fermentation system, where yeast achieves the cyclic regeneration of GMP to GTP. The yield after dual-strain coupled fermentation reached 18.42 g / L, with a conversion rate of 63.52%. Further optimization of the fermentation system, adjusting the fermentation pH, fermentation time, carbon source type, and adding metal ions and surfactants, further increased the yield to 25.50 g / L, laying a solid foundation for the large-scale industrial production of GlcNAcβ1-4(Fucα1-6)GlcNAc. Attached Figure Description

[0025] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0026] Figure 1 is a schematic diagram of the technical route for generating GlcNAcβ1-4(Fucα1-6)GlcNAc by dual-strain coupled fermentation provided by the present invention.

[0027] Figure 2 is a flowchart of the construction of expression plasmids and a protein expression electrophoresis diagram in Example 1 of the present invention, wherein A is a flowchart of the construction of plasmid pET28a-nahK, B is an SDS-PAGE result of protein expression of recombinant plasmids pET28a-nahK and pET28a-Chbp, and C is an SDS-PAGE result of protein expression of recombinant plasmids pET28a-Fkp and pET28a-nodZ;

[0028] Figure 3 shows the TLC diagrams of the products detected in Example 1 of this invention. A is the TLC analysis diagram of 1-P-GlcNAc produced by E. coli BL21(DE3) / pET28a-nahK expression, where band 1 is 1-P-GlcNAc, band 2 is GlcNAc, and band 3 is the reaction solution. B is the TLC analysis diagram of (GlcNAc)2 generated by the coupling of E. coli BL21(DE3) / pET28a-nahK and E. coli BL21(DE3) / pET28a-Chbp, where band 1 is GlcNAc, band 2 is (GlcNAc)2, and band 3 is the reaction solution. C is the TLC analysis diagram of the fermentation products of four coupled bacteria, where band 1 is Fucose, band 2 is GlcNAc, band 3 is (GlcNAc)2, and band 4 is the fermentation supernatant.

[0029] Figure 4 shows the MALDI-TOF MS analysis results of the product GlcNAcβ1-4(Fucα1-6)GlcNAc in Example 1 of the present invention;

[0030] Figure 5 is the 1H NMR spectrum of the product GlcNAcβ1-4(Fucα1-6)GlcNAc in Example 1 of the present invention;

[0031] Figure 6 shows the liquid phase results in Example 1 of the present invention. From top to bottom, the liquid phase diagrams are: standard GlcNAc, product GlcNAcβ1-4(Fucα1-6)GlcNAc, and fermentation products of four coupled E. coli BL21(DE3) / pET28a-nahK, E. coli BL21(DE3) / pET28a-Chbp, E. coli BL21(DE3) / pET28a-Fkp, and E. coli BL21(DE3) / pET28a-nodZ.

[0032] Figure 7 is a schematic diagram of the construction process of 12 dual expression plasmids in Example 2 of the present invention, where A is a schematic diagram of plasmid construction using pET28a-nahK-Fkp as an example, and B is a schematic diagram of constructing recombinant strains by introducing dual plasmids.

[0033] Figure 8 shows the electrophoresis results of the induced expression of the six dual-plasmid strains E1-E6 constructed in Example 2 of this invention;

[0034] Figure 9 shows the screening results of six dual-plasmid strains in Example 2 of the present invention, where A is the TLC analysis diagram of the products of the six dual-plasmid strains and B is the yield of the six dual-plasmid strains.

[0035] Figure 10 shows the fermentation curve of engineered strain E3 in a 7L fermenter;

[0036] Figure 11 shows the whole-cell catalysis of engineered strain E3 at the fermenter level;

[0037] Figure 12 shows the effect of fermentation conditions on the trisaccharide yield of the dual-strain fermentation in Example 3 of the present invention, where A represents the effect of different pH values, and B represents the effect of different Mg values. 2+ The effects of concentration are shown in Figure 1, C represents the effect of different reaction times, D represents the effect of different carbon sources (1-glucose and glycerol, 2-glucose, 3-glycerol, 4-no additional carbon source), E represents the effect of different yeast biomass, and F represents the effect of different concentrations of octadecylamine polyoxyethylene ether. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0039] The materials involved in the embodiments are as follows:

[0040] Brewing yeast: The brewer's yeast was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 12, 2021, with the accession number GDMCC No:61663, and is disclosed in patent CN113881737A, "Method for Large-Scale Production of CMP-Sialic Acid by Coupled Fermentation of Genetically Engineered Bacteria and Yeast";

[0041] Escherichia coli JM109(DE3) was purchased from Tiangen Biotech (Beijing) Co., Ltd., and Escherichia coli BL21(DE3) was purchased from Vazyme.

[0042] The genes for Vibrio furnissii, Bacteroides fragilis, Rhizobium sp, and Bifidobacterium were artificially synthesized after optimization.

[0043] The culture media and reagents involved in the examples are as follows:

[0044] LB liquid medium (g / L): NaCl 10.0, tryptone 10.0, yeast extract 5.0, pH=7.0, sterilization conditions 121℃ 20min;

[0045] LB solid medium (g / L): NaCl 10.0, tryptone 10.0, yeast extract 5.0, agar 20.0, pH=7.0, sterilization conditions 121℃ 20min;

[0046] Fermentation medium (g / L): casein 10.0, yeast extract 5.0, NH4Cl 2.674, Na2HPO4 3.549, KH2PO4 3.402, Na2SO4 0.7102, MgSO4 0.3244, glycerol 5.0, glucose 0.5, lactose 4.0; sterilization conditions: 115℃ for 30 min.

[0047] Yeast seed culture medium (g / L): glucose 10, peptone 5, yeast extract 15, sodium chloride 4, pH=7.0.

[0048] Yeast basal fermentation medium (g / L): glucose 20, ammonium sulfate 8, KH2PO4 2.5, MgSO4·7H2O 0.5, pH=5.5.

[0049] Anisaldehyde dye: Dissolve 2 mL of sulfuric acid, 12 mL of acetic acid and 6 mL of anisaldehyde in 180 mL of 95% ethanol.

[0050] The detection methods involved in the embodiments are as follows:

[0051] (1) Isolation and purification of GlcNAcβ1-4(Fucα1-6)GlcNAc

[0052] Separation and purification method: The HyperSep Hypercarb solid-phase extraction column (SPE column) was used for purification. The purification steps are as follows: Activation: The SPE column was activated with 3 mL of methanol; Equilibration: The SPE column was equilibrated with 3 mL of ultrapure water and moistened; Sample loading: 300 μL of catalytic supernatant was passed evenly through the SPE column; Washing: The SPE column was washed with 1 mL of ultrapure water, repeated three times; Elution: The product was eluted with 0.5 mL of 80% acetonitrile, repeated three times. The eluent was then lyophilized under nitrogen to obtain the sample.

[0053] (2) Analysis and detection of GlcNAcβ1-4(Fucα1-6)GlcNAc

[0054] Thin-layer chromatography (TLC): The developing solvent is v(n-propanol):v(water):v(acetic acid) = 2:1:1. After the TLC plate is naturally dried, it is stained with anisaldehyde dye and placed in a 160℃ oven for 2-3 minutes for color development.

[0055] (3) Matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS): Molecular weight identification of the purified product. 1 μL of the purified product and 1 μL of the matrix 2,5-dihydroxybenzoic acid containing Na were spotted. + The samples were spotted at the same location on the target plate, mixed thoroughly, dried, and then processed by mass spectrometry. Mass spectrometry conditions: reflectance cation mode; molecular weight range: 0-2000 Da.

[0056] (4) Proton nuclear magnetic resonance spectrum (NMR) 1 HNMR: The purified product was lyophilized, dissolved in 600 μL of deuterated water (D2O), and collected using a nuclear magnetic resonance spectrometer at a resonance frequency of 400 MHz. 1 H spectrum.

[0057] (5) High performance liquid chromatography (HPLC): UV detection wavelength was 210 nm; the chromatographic column was an Aminex HPX-87H Ion Exclusion Column (7.8 mm × 300 mm); the mobile phase was 5 mM H2SO4; the column temperature was 60 ℃; the injection volume was 10 μL; and the flow rate was 0.6 mL / min.

[0058] (6) SDS-PAGE gel electrophoresis: Take 40 μL of bacterial culture before and after induction, add 10 μL of 5× protein loading buffer, mix well, boil in water for 4 min, then in an ice bath for 2 min, and take 10 μL of the mixture for SDS-PAGE gel electrophoresis. After protein electrophoresis, the bacterial cells that were successfully induced to express the target protein were stored at -20℃ for later use.

[0059] Example 1: Synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc by four bacterial couplings

[0060] The target gene fragment was synthesized using the sequence of the nahK gene (SEQ ID NO.1) in Bifidobacteria as a template. Primers F-nahK-F (5'-CCATGGGCAAAAAAATCCTGACTGTGCTGTCT-3') and F-nahK-R (5'-GGTGGTGCTCGAGTCACTTGGTCGTCTC-3') were designed to amplify the target gene nahK by PCR. Primers pet-F (5'-CATTGGTGTTGTTCATATGGCTGC-3') and pet-R (5'-GACGACCAAGTGACTCGAGCACCAC-3') were designed to amplify the vector pET-28a by PCR. After verification and recovery, the PCR-absorbed nahK gene fragment was purified and ligated with the vector fragment using ClonExpress technology to obtain the recombinant plasmid pET28a-nahK. The construction process is shown in Figure 2A. The cells were then transformed into competent E.coli BL21(DE3) cells, plated with Kan as a selection marker, and after colony PCR verification, single colonies were picked for shake-flask culture. Plasmids were extracted from the bacterial culture for enzyme digestion verification and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing identification.

[0061] Recombinant plasmids pET28a-Chbp, pET28a-Fkp, and pET28a-nodZ were constructed according to the above method, resulting in recombinant engineered bacteria E. coli BL21(DE3) / pET28a-Chbp, E. coli BL21(DE3) / pET28a-Fkp, and E. coli BL21(DE3) / pET28a-nodZ. The nucleotide sequence of Chbp is shown in SEQ ID NO.2, the nucleotide sequence of Fkp is shown in SEQ ID NO.3, and the nucleotide sequence of nodZ is shown in SEQ ID NO.4. Single colonies of the engineered strains were picked and inoculated into 10 mL of LB medium containing Kan resistance. The culture was carried out overnight at 37°C to obtain a seed culture. The seed culture was then transferred at a 2% inoculation rate to 200 mL of LB medium containing Kan resistance and cultured in shake flasks at 37°C and 200 rpm. When the bacterial cells grew to the OD... 600 When the saturation level was 0.6-0.8, IPTG was added to a final concentration of 0.3 mM for protein expression induction. The cells were induced at 16℃ for 24 h, and then collected by centrifugation. SDS-PAGE protein electrophoresis was performed on the induced cells for verification. The results are shown in Figures 2B and 2C. A clear protein band was observed at approximately 40 kDa, consistent with the reported size of the target enzyme protein. This indicates that nahK, Chbp, Fkp, and nodZ were successfully expressed in the engineered strains E. coli BL21(DE3) / pET28a-nahK, E. coli BL21(DE3) / pET28a-Chbp, E. coli BL21(DE3) / pET28a-Fkp, and E. coli BL21(DE3) / pET28a-nodZ, respectively.

[0062] The engineered strains E. coli BL21(DE3) / pET28a-nahK, E. coli BL21(DE3) / pET28a-Chbp, E. coli BL21(DE3) / pET28a-Fkp, and E. coli BL21(DE3) / pET28a-nodZ were reacted at 25℃ and 200 rpm for 24 h using 250 mM KH2PO4 / K2HPO4 (pH=8.0), 80 mM ATP, 25 mM MgCl2, 20 mM Poly-P, 20 mL / L ethanol, 10 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 120 mM GlcNAc, 50 mM fucose, and 50 mM GTP. After the reaction, the reaction system was centrifuged at 12000 rpm for 2 min, and the supernatant was collected for detection. The results are shown in Figure 3.

[0063] Preliminary detection using MALDI-TOF-MS revealed the presence of a substance with the same molecular weight as the trisaccharide in the fermentation broth (Figure 4), preliminarily identifying the target product as GlcNAcβ1-4(Fucα1-6)GlcNAc. To further confirm whether the synthesized product is indeed a trisaccharide, further analysis was conducted... 1 The structure was identified by ¹H NMR (Figure 5). ¹H NMR (400MHz, D₂O) δ 5.24 (dd, J = 13.5, 3.7Hz, 0.4H), 4.68 (d, J = 8.0Hz, 0.6H), 4.44 (d, J = 7.8Hz, 1H), 3.33 (t, J = 8.4Hz, 0.6H), 2.73 (dd, J = 12.4, 4.7Hz, 1H), 2.05 (s, 3H), 1.76 (t, J = 12.2Hz, 1H). Analysis of the NMR results confirmed the successful synthesis of the target product as GlcNAcβ1-4(Fucα1-6)GlcNAc, thus demonstrating that all four enzymes expressed by the recombinant strain possess catalytic activity.

[0064] After identifying the target product GlcNAcβ1-4(Fucα1-6)GlcNAc, the supernatant of the fermentation broth was quantitatively analyzed by HPLC. The results are shown in Figure 6. After 24 h of reaction, the concentration of GlcNAcβ1-4(Fucα1-6)GlcNAc was 11.53 g / L, and the conversion rate of fucose was 39.74%.

[0065] Example 2: Construction and screening of the highest quality GlcNAcβ1-4(Fucα1-6)GlcNAc synthesized from engineered strain E. coli BL21(DE3)

[0066] Primers Frsf-F (5'-CGAATTCGGATCCTGGCTG-3') and Frsf-R (5'-AGCTCGGCGCGCCT-3') were designed to amplify the vector pRSFDuet-1 by PCR. The gene fragment described in Example 1 was purified and ligated with the vector fragment using ClonExpress technology to construct recombinant plasmids pRSFDuet-1-nodZ-Fkp, pRSFDuet-1-nahK-Chbp, pRSFDuet-1-nodZ-Chbp, pRSFDuet-1-nahK-Fkp, pRSFDuet-1-Chbp-Fkp, and pRSFDuet-1-nodZ-nahK.

[0067] PCR amplification was performed on the pETDuet-1 vector using the primer pair described in Example 1. The gene fragment described in Example 1 was purified and ligated with the vector fragment using ClonExpress technology to construct recombinant plasmids pETDuet-1-nahK-Chbp, pETDuet-1-nodZ-Fkp, pETDuet-1-nahK-Fkp, pETDuet-1-nodZ-Chbp, pETDuet-1-nodZ-nahK, and pETDuet-1-Chbp-Fkp. The construction process is shown in Figure 7A.

[0068] As shown in Figure 7B, recombinant plasmids with different vectors as backbones were transformed into competent E. coli BL21(DE3) cells. Using Kan and Amp as selection markers, the cells were plated and, after colony PCR verification, single colonies were picked for shake-flask culture. Plasmids were extracted from the bacterial culture for enzyme digestion verification and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing identification, yielding engineered strains E1-E6. E1-E6 were induced to express proteins, and the induced cells were verified by SDS-PAGE protein electrophoresis. The SDS-PAGE results are shown in Figure 8. The GlcNAcβ1-4 (Fucα1-6) GlcNAc synthesis pathway was successfully constructed in all six homologous recombinant expression strains.

[0069] The results are shown in Figure 9A. It can be seen that there are obvious protein bands at around 40kD, 85kD, and 100kD, and the inducible expression shows the production of trisaccharides, proving that the engineered strain E1-E6 was successfully constructed.

[0070] At the shake-flask level, six successfully induced engineered strains were co-cultured with *Saccharomyces cerevisiae*, and the two strains were coupled to catalyze the synthesis of *GlcNAcβ1-4(Fucα1-6)GlcNAc*, with a catalytic reaction time of 24 h. The TLC results of the fermentation products from the six engineered strains are shown in Figure 9A. Quantitative analysis of the fermentation supernatant was performed using HPLC, and the results are shown in Figure 9B. After 24 h of fermentation, the engineered strain *E. coli* BL21(DE3) / pRSFDuet-1-nodZ-Chbp / pETDuet-1-nahK-Fkp(E3)* showed the best yield, with a concentration of 18.09 g / L and a conversion rate of 62.38%.

[0071] Single colonies of engineered strain E3 were inoculated into 10 mL of LB medium containing Kan and Amp resistance and cultured at 37°C for 12 h to obtain primary seed culture. The primary seed culture was then transferred to 250 mL of LB medium containing Kan and Amp resistance at a 2% inoculation rate and cultured overnight at 37°C to obtain secondary seed culture. The secondary seed culture was then inoculated into a fermenter at a 10% inoculation rate. The fermentation temperature was 37°C, the aeration rate was 2.5 vvm, and the initial working volume of the fermenter was 2.5 L. The stirring speed was set in conjunction with dissolved oxygen to control dissolved oxygen at 30%. When the glucose content was below 0.1 g / L, glucose was added at a flow rate of 0.6 mL / min to a concentration of 300 g / L. Throughout the fermentation process, the pH was maintained at 6.9 using 25% ammonia. At the late logarithmic growth stage of the engineered strain, when the OD value reached 30, an exogenous inducer was added for induction. After fermentation, the fermentation broth was centrifuged at 8000 r / min for 5 min, and the cells were collected, as shown in Figure 10. The final OD value... 600 The value was 71.1, and the biomass at this point was 96.4 g / L (wet weight).

[0072] Example 3: Coupling synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc using a dual-strain strategy

[0073] The horizontal two-strain coupled catalytic transformation of GlcNAcβ1-4(Fucα1-6)GlcNAc in shake flasks was performed as follows: 100 g / L of engineered strain E. coli BL21(DE3) / pRSFDuet1-nahK-Fkp / pETDuet1-nodZ-Chbp and 100 g / L Saccharomyces cerevisiae were reacted at 250 mM KH2PO4 / K2HPO4 (pH 8.0), 80 mM ATP, 25 mM MgCl2, 20 mM Poly-P, 20 mL / L ethanol, 10 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 120 mM GlcNAc, 50 mM fucose, and 50 mM GMP at 25 °C and 200 rpm for 24 h. After the reaction, the reaction system was centrifuged at 12000 rpm for 2 min, and the supernatant was collected for analysis.

[0074] Quantitative analysis of the fermentation broth supernatant was performed using HPLC. After 24 h of reaction, the concentration of GlcNAcβ1-4(Fucα1-6)GlcNAc was 18.09 g / L, and the conversion rate was 62.38%.

[0075] Production of GlcNAcβ1-4(Fucα1-6)GlcNAc by co-culturing engineered strains and yeast in a 7L fermenter: 100 g / L engineered strain E. coli BL21(DE3) / pET Deut-1-plst6-neuA and 100 g / L Saccharomyces cerevisiae were fermented in a conversion system of 250 mM KH2PO4 / K2HPO4 (pH=8.0), 80 mM ATP, 25 mM MgCl2, 20 mM Poly-P, 20 mL / L ethanol, 10 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 120 mM GlcNAc, 50 mM fucose, 50 mM GMP, and 20 g / L glycerol. Fermentation was carried out for 24 hours, with the pH maintained at 7.2 using 25% ammonia. The temperature was set at 25℃, and the aeration rate was 1 vvm. After the reaction, the fermentation broth was centrifuged at 8000 r / min for 5 min, and the supernatant was collected for analysis. Quantitative analysis of the fermentation broth supernatant was performed using HPLC. The results are shown in Figure 11. After 36 hours of reaction, the concentration of GlcNAcβ1-4(Fucα1-6)GlcNAc was 18.42 g / L, at which point the conversion rate was 63.52%.

[0076] Example 4: Optimization of the synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc using a dual-strain strategy

[0077] (1) Effect of reaction pH on dual-strain coupled fermentation conversion: Environmental pH directly affects the dissociation state of enzymes and substrates, thus affecting the binding of enzymes and substrates and the reaction rate. Therefore, the optimal temperature for the overall reaction system was investigated to achieve the maximum yield of GlcNAcβ1-4(Fucα1-6)GlcNAc. The results are shown in Figure 12A. Under the condition of pH 7.5, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc reached the maximum of 19.41 g / L, and the conversion rate was 66.93%.

[0078] (2) Cofactor Mg 2+ Effect of concentration on dual-strain coupled fermentation transformation: Studies have shown that the catalytic reactions of nahK and Fkp enzymes, as well as the GMP-GTP cycle regeneration, require the participation of metal ions, especially Mg. 2+ The participation of Mg, therefore, for optimal Mg 2+ Investigating the concentration of Mg is also crucial, and the results are shown in Figure 12B. 2+ At a concentration of 20 mM, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc reached a maximum of 22.37 g / L, and the conversion rate of GlcNAcβ1-4(Fucα1-6)GlcNAc was correspondingly increased to 77.16%.

[0079] (3) Effect of reaction time on the conversion of dual-strain coupled fermentation: Studies have shown that too short a reaction time will lead to incomplete reaction and waste of raw materials, while too long a reaction time will result in excessive by-products, and the reaction process is not conducive to the production efficiency. Therefore, the optimal reaction time of the overall reaction system was investigated to achieve the maximum yield of GlcNAcβ1-4(Fucα1-6)GlcNAc. The results are shown in Figure 12C. The yield of trisaccharides in the reaction system increased with time from 12 to 72 h, but the growth rate decreased significantly after 36 h. Considering the load of the catalytic system and cost issues, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc was selected at 36 h, at which time the yield reached 24.19 g / L, and the conversion rate was 83.41%.

[0080] (4) Effects of carbon from different sources on the three-strain coupled fermentation conversion: The carbon source not only provides C for the whole-cell catalytic reaction, but also plays an energy role. Therefore, the selection of a suitable carbon source has a significant impact on the reaction. The results show that, as shown in Figure 12 (D), the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc is relatively high when using glycerol as the carbon source, reaching 24.38 g / L, with a conversion rate of 84.07%.

[0081] (5) Effect of yeast cell biomass on dual-strain coupled fermentation conversion: Since yeast cells participate in the GMP-GTP cycle regeneration, the yeast cell biomass was optimized based on the above optimization. The results are shown in E of Figure 12. With the increase of yeast cell biomass, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc also increased. When the yeast cell biomass was 300 g / L, the yield reached 26.2 g / L. However, when the biomass was 100 g / L, the conversion rate was 85.43% and the yield was 24.35 g / L. Because the difference in yield was small, considering the catalytic system load and cost, 100 g / L was selected as the optimal yeast cell biomass in the system for subsequent fermentation conversion.

[0082] (6) Effect of surfactant octadecylamine polyoxyethylene ether on the conversion of dual-strain coupled fermentation: As a surfactant, octadecylamine polyoxyethylene ether promotes the reaction by lowering the activation energy of the reactants, increasing the reactivity, changing the hydrophobicity of the reaction system, and increasing the number of active sites. Therefore, the optimal concentration of octadecylamine polyoxyethylene ether in the total reaction system was investigated to achieve the maximum yield of GlcNAcβ1-4(Fucα1-6)GlcNAc. The results are shown in F in Figure 12. Under the condition that the concentration of octadecylamine polyoxyethylene ether is 8 g / L, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc reaches the maximum of 25.50 g / L, and the conversion rate is 87.94%.

[0083] After optimizing the above conversion system, the yield of trisaccharides increased from 19.41 g / L to 25.50 g / L, and the conversion rate of fucose increased from 66.93% to 87.94%.

[0084] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A dual-strain coupled fermentation method for producing 6'-fucosylated chitobiose. The method of GlcNAcβ1-4(Fucα1-6)GlcNAc is characterized by: In the fermentation system, fucose and N-acetyl-D-glucosamine are used as substrates, and fermentation is carried out by engineered Escherichia coli and yeast in a coupled manner. The engineered Escherichia coli heterologously expresses N-acetylglucosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucokinase Fkp, and N,N-diacetylchitosan phosphorylase Chbp; 2. The method according to claim 1, characterized in that: The engineered Escherichia coli contains a first expression vector and a second expression vector. The first expression vector contains the nahK coding gene and the Fkp coding gene, and the second expression vector contains the nodZ coding gene and the Chbp coding gene.

3. The method according to claim 1, characterized in that: The yeast is brewer's yeast or baker's yeast.

4. The method according to claim 2, characterized in that: The first expression vector uses pRSFDuet-1 plasmid as a backbone, and the second expression vector uses pETDuet-1 plasmid as a backbone.

5. The method according to claim 1, characterized in that: The ratio of engineered Escherichia coli and yeast in the fermentation system is 1:1-3.

6. The method according to claim 1, characterized in that: The fermentation time is 36-60 hours.

7. The method according to claim 1, characterized in that: A metal ion, Mg, is added to the fermentation system. 2+ .

8. The method according to claim 7, characterized in that: The Mg 2+ The concentration in the fermentation system is 0-80 mM.

9. The method according to claim 1, characterized in that: A surfactant, namely octadecylamine polyoxyethylene ether, is added to the fermentation system. The octadecylamine polyoxyethylene ether has a concentration of 2-16 g / L in the fermentation system.

10. The method according to claim 1, characterized in that: In the fermentation system, GlcNAcβ1-4(Fucα1-6)GlcNAc is produced using glycerol as the carbon source.

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