Method for producing GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc through double-bacterium coupling fermentation

Abstract

The invention relates to a method for producing 6 '-fucosylated chitosan disaccharide GlcNacbeta1-4 (Fucalpha1-6) GlcNAc through double-bacterium coupling fermentation, and belongs to the technical field of fermentation engineering. The method comprises the following steps: taking fucose and N-acetyl-D-glucosamine as substrates in a fermentation system; the 6 '-fucosylated chitosan disaccharide is produced through coupled fermentation of an engineering escherichia coli strain and yeast, and a GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc synthesis pathway including N-acetylhexosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucose kinase Fkp and N, N-diacetylchitosan phosphorylase Chbp is constructed in the engineering escherichia coli, so that the 6'-fucosylated chitosan disaccharide is obtained. According to the invention, the yeast is added to realize cyclic regeneration from GMP to GTP, so that not only is the problem of high oligosaccharide production cost caused by pure exogenous supplement of GTP effectively solved, but also efficient production of GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc is realized, and the yield of GlcNAcbeta1-4 (Fucalpha1-6) GlcNAc is increased to 25.50 g / L by an optimized coupling fermentation production technology. The invention provides a novel synthesis technology of complex N-glycans, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a method for producing GlcNAcβ1-4(Fucα1-6)GlcNAc by dual-bacteria coupled fermentation. Background Art

[0002] Asparagine-linked (N-link) glycosylation is one of the most common post-translational modifications of intracellular and extracellular proteins, which directly affects their biological functions, such as protein folding, stability, and intercellular traffic. The production of homogeneous N-glycans with clear structures is conducive to the comprehensive study of their biological effects and molecular basis. The natural content is very low and the purification process is complicated. The biosynthesis method has the advantages of low process production cost and low environmental pollution, and is the synthesis method used in commercial products today. GlcNAcβ1-4(Fucα1-6)GlcNAcβ1-4 is the core structure of complex N-glycans, which can lay the foundation for the further synthesis of complete complex N-glycans, create substrate conditions for the development of GlcNAcβ1-4(Fucα1-6)GlcNAcβ1-4 functions, and provide a new way for the synthesis of complex N-glycans.

[0003] The intermediate products of the reaction, such as chitobiose, GDP-Fucose, chitosan oligosaccharides and chitosan oligosaccharides, have a sweet taste and are soluble in water but have lower solubility than monosaccharides. Therefore, chitosan oligosaccharides have good hygroscopicity and moisture retention 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 intestines and promote the proliferation of beneficial bacteria in the intestines. Chitosan oligosaccharides and chitosan oligosaccharides can regulate the metabolic activities of microorganisms in the intestines of animals, improve the distribution of intestinal microbial flora, and thus enhance the body's immunity. In addition, chitosan oligosaccharides also have the effects of anti-cancer and activating the body's immune function, can induce pancreatic lymphocyte T cells to produce interleukins, enhance the body's immunity and are non-toxic or low-toxic to organisms. Therefore, chitosan oligosaccharides ((GIcNAc)n(10≥n≥2) include (GlcNAc) 2 It has huge market development potential in food development, antibacterial, immune regulation and anti-infection.

[0004] Due to the high production cost and low production efficiency, the large-scale production of 6'-fucosylated chitosan GlcNAcβ1-4(Fucα1-6)GlcNAc has not yet been achieved. Sialic acid and guanosine triphosphate (cytidinetriphosphate, GTP) are expensive, and the synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc requires the consumption of GlcNAc and a large amount of GTP, resulting in high synthesis cost of GlcNAcβ1-4(Fucα1-6)GlcNAc and low yield of 14.82mg / L. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for producing 6'-fucosylated chitobiose GlcNAcβ1-4 (Fucα1-6) GlcNAc by dual bacteria coupled fermentation, in which fucose and N-acetyl-D-glucosamine are used in the fermentation system, and a GlcNAcβ1-4 (Fucα1-6) GlcNAc synthesis pathway is constructed in genetically engineered bacteria, including N-acetylglucosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucokinase Fkp and N,N-diacetylchitosan phosphorylase Chbp, and the construction method of the expression vector in the engineered bacteria is adjusted, and then coupled fermentation is carried out with yeast. On this basis, the fermentation conditions are optimized, including adjusting the fermentation pH value, fermentation time, and adding metal ions and surfactants to increase the yield.

[0006] The first object of the present invention is to provide a method for producing GlcNAcβ1-4(Fucα1-6)GlcNAc by dual-bacteria coupled fermentation, wherein fucose and N-acetyl-D-glucosamine are used as substrates in the fermentation system, and the engineered Escherichia coli and yeast are coupled for fermentation;

[0007] The first engineering bacteria heterologously express N-acetylhexosamine 1-kinase nahK, fucosyltransferase nodZ, L-fucokinase Fkp and N,N-diacetylchitosan phosphorylase Chbp.

[0008] Furthermore, the first engineering bacteria comprises a first expression vector and a second expression vector, the first expression vector comprises a nahK encoding gene and a Fkp encoding gene, and the second expression vector comprises a nodZ encoding gene and a Chbp encoding gene.

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

[0010] Furthermore, the yeast is saccharomyces cerevisiae, beer yeast or baker's yeast.

[0011] Preferably, the yeast is Saccharomyces cerevisiae.

[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 to the yeast in the fermentation system is 1:1-3.

[0014] Preferably, the ratio of the first engineered bacteria to 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, metal ions are added to the fermentation system, wherein the metal ions are Mg 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 was 20 mM.

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

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

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

[0023] Beneficial effects of the present invention:

[0024] The method for producing GlcNAcβ1-4(Fucα1-6)GlcNAc by dual-bacteria coupled fermentation of the present invention constructs a synthetic pathway in the same engineering bacteria, thereby avoiding the influence on the product yield caused by the frequent entry and exit of substances in and out of the cell due to the large amount of engineering strains. In order to solve the problem of high cost caused by the need to consume a large amount of GTP in the synthesis process, yeast is added to the fermentation system to form a dual-bacteria coupled fermentation system, and the yeast realizes the cyclic regeneration of GMP to GTP. After dual-bacteria coupled fermentation, the yield reaches 18.42g / L, and the conversion rate is 63.52%. On this basis, the fermentation system is further optimized, the fermentation pH value, fermentation time, and carbon source type are adjusted, and metal ions and surfactants are added to further increase the yield to 25.50g / L, laying a good foundation for the industrial large-scale production of GlcNAcβ1-4(Fucα1-6)GlcNAc. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0026] Figure 1 A schematic diagram of the technical route for producing GlcNAcβ1-4 (Fucα1-6)GlcNAc by dual-bacteria coupled fermentation provided by the present invention;

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

[0028] Figure 3 The TLC images of the detection products in Example 1 of the present invention, wherein A is the TLC analysis of 1-P-GlcNAc produced by E. coli BL21 (DE3) / pET28a-nahK, band 1 is 1-P-GlcNAc, band 2 is GlcNAc, and band 3 is the reaction solution; B is the TLC analysis of E. coli BL21 (DE3) / pET28a-nahK and E. coli BL21 (DE3) / pET28a-Chbp double bacteria coupling production (GlcNAc) 2 TLC analysis of the band 1 is GlcNAc, and the band 2 is (GlcNAc) 2 , band 3 is the reaction solution; C is the TLC analysis of the four-bacteria coupled fermentation product, band 1 is Fucose, band 2 is GlcNAc, and band 3 is (GlcNAc) 2 , band 4 is the fermentation supernatant;

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

[0030] Figure 5 This is the hydrogen nuclear magnetic resonance spectrum of the product GlcNAcβ1-4(Fucα1-6)GlcNAc in Example 1 of the present invention;

[0031] Figure 6The figure is a liquid phase result diagram in Example 1 of the present invention, which includes, from top to bottom, a liquid phase diagram of the standard product GlcNAc, a liquid phase diagram of the product GlcNAcβ1-4(Fucα1-6)GlcNAc, and a liquid phase diagram of the fermentation product of the four bacteria coupling of 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 Schematic diagram of the construction process of 12 dual expression plasmids in Example 2 of the present invention, wherein A is a schematic diagram of plasmid construction using pET28a-nahK-Fkp as an example, and B is a schematic diagram of introducing dual plasmids to construct recombinant strains;

[0033] Figure 8 This is a graph showing the electrophoresis results of induced expression of the six dual-plasmid strains E1-E6 constructed in Example 2 of the present invention;

[0034] Fig. 9 The results of screening six double-plasmid strains in Example 2 of the present invention, wherein A is the TLC analysis chart of the products of the six double-plasmid strains, and B is the yield of the six double-plasmid strains;

[0035] Fig.10 This is the fermentation curve of the engineered bacteria E3 in a 7L fermenter;

[0036] Fig.11 It is the whole-cell catalysis of the engineered bacteria E3 at the fermenter level;

[0037] Fig.12 The effect of fermentation conditions on trisaccharide production of the dual-bacteria strain in Example 3 of the present invention, where A is the effect of different pH, B is the effect of different Mg 2+ The influence of concentration is shown in Figure 1, C is the influence of different reaction times, D is the influence of different carbon sources, 1-glucose and glycerol, 2-glucose, 3-glycerol, 4-no additional carbon source added, E is the influence of different yeast biomass, and F is the influence of different concentrations of octadecylamine polyoxyethylene ether. DETAILED DESCRIPTION

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

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

[0040] Saccharomyces cerevisiae: The Saccharomyces cerevisiae has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on May 12, 2021, with the deposit number GDMCC No: 61663, and is disclosed in the method of large-scale production of CMP-sialic acid by coupled fermentation of genetically engineered bacteria and yeast in patent CN113881737A;

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

[0042] Vibrio furnissii gene, Bacteroides fragilis gene, Rhizobium sp gene, Bifidobacterium gene: obtained through artificial optimization and artificial synthesis.

[0043] The culture medium 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 ° C 20 min;

[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 ° C 20 min;

[0046] Fermentation medium (g / L): casein 10.0, yeast extract 5.0, NH 4 Cl 2.674, Na 2 HPO 4 3.549, KH 2 PO4 3.402, Na 2 SO 4 0.7102,MgSO 4 0.3244, glycerol 5.0, glucose 0.5, lactose 4.0, sterilization conditions 115℃ 30min;

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

[0048] Yeast basic fermentation medium (g / L): glucose 20, ammonium sulfate 8, KH 2 PO 4 2.5, MgSO 4 7H2 O 0.5, pH = 5.5.

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

[0050] The detection method involved in the embodiment is as follows:

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

[0052] Separation and purification method: Use HyperSep Hypercarb solid phase extraction cartridge (SPE cartridge) for purification, and the purification steps are as follows; activation: activate the SPE cartridge with 3 mL of methanol; balance: balance the SPE cartridge with 3 mL of ultrapure water and moisturize it; loading: take 300 μL of catalytic supernatant and evenly pass it through the SPE cartridge; elution: elute the SPE cartridge with 1 mL of ultrapure water, repeat three times; elution: elute the product with 0.5 mL of 80% acetonitrile, repeat 3 times, and freeze-dry the eluted liquid 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 thin layer chromatography plate is naturally dried, it is dyed with anisaldehyde dye and placed in a 160°C oven for 2-3 minutes for color development.

[0055] (3) Matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS): The molecular weight of the purified product was determined by mixing 1 μL of the purified product with 1 μL of the spotted matrix 2,5-dihydroxybenzoic acid containing Na + , respectively spot the samples at the same position of the target plate, mix well and dry before processing on the mass spectrometer. Mass spectrometry conditions: use reflectron cation mode; scan molecular weight range: 0-2000Da.

[0056] (4) H NMR spectroscopy ( 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; chromatographic column was Aminex HPX-87H Ion Exclusion Column (7.8 mm × 300 mm); mobile phase was 5 mM H 2 SO 4; Column temperature is 60℃; Injection volume is 10μL; Flow rate is 0.6mL / min.

[0058] (6) SDS-PAGE gel electrophoresis: Take 40 μL of bacterial solution before and after induction, add 10 μL of 5× protein loading buffer and mix evenly, boil in boiling water for 4 min, ice bath for 2 min, and take 10 μL of the mixture for SDS-PAGE gel electrophoresis. After protein electrophoresis, the bacteria that have been successfully induced to express the target protein are stored at -20°C for future use.

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

[0060] The sequence of the nahK gene (shown in SEQ ID NO.1) in Bifidobacteria was used as a template to synthesize the target gene fragment, and primers F-nahK-F (5'-CCATGGGCAAAAAAA TCCTGACTGTGCTGTCT-3') and F-nahK-R (5'-GGTGGTGCTCGAGTCACT TGGTCGTCTC-3') were designed to perform PCR amplification on the target gene nahK, and primers pet-F (5'-CATTGGTGTTGTTCATATGGCTGC-3') and pet-R (5'-GACGACCAAGTGA CTCGAGCACCAC-3') were designed to perform PCR amplification on the vector pET-28a. After verification and recovery, the nahK gene fragment after PCR was purified and connected with the vector fragment using ClonExpress technology to obtain the recombinant plasmid pET28a-nahK. The construction process is as follows: Figure 2 As shown in A. Then it was transformed into competent cells E.coliBL21 (DE3), plated with Kan as the screening marker, single colonies were picked for shake flask culture after colony PCR verification, plasmids in the bacterial solution were extracted for enzyme digestion verification, and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing and identification.

[0061] According to the above method, recombinant plasmids pET28a-Chbp, pET28a-Fkp, and pET28a-nodZ were constructed to obtain recombinant engineered bacteria E. coli BL21 (DE3) / pET28a-Chbp, E. coli BL21 (DE3) / pET28a-Fkp, and E. coli BL21 (DE3) / pET28a-nodZ, wherein 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. A single colony of the engineered strain was picked and inoculated into 10 mL LB medium with Kan resistance, and cultured overnight at 37°C to obtain seed liquid, and then the seed liquid was transferred to 200 mL LB medium with Kan resistance at an inoculation rate of 2%, and cultured in a shake flask at 37°C and 200 r / min. When the bacteria grew to OD 600 When the concentration of IPTG was 0.6-0.8, IPTG was added to induce protein expression at a final concentration of 0.3 mM. The cells were induced at 16°C for 24 h and centrifuged to collect the cells. The induced cells were verified by SDS-PAGE protein electrophoresis. The results were as follows: Figure 2 As shown in Figures B and C, it can be seen that there is an obvious protein band at around 40 kD, which is consistent with the reported target enzyme protein size. This indicates that nahK, Chbp, Fkp and nodZ are successfully expressed in the engineering 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] 50 g / L of 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 cultured in 250 mM KH 2 PO 4 / K 2 HPO 4 (pH=8.0), 80mM ATP, 25mM MgCl 2, 20mM Poly-P, 20mL / L alcohol, 10mmol / L DTT, 8g / L octadecylamine polyoxyethylene ether, 120mM GlcNAc, 50mM fucose, 50mM GTP, 25℃, 200r / min for 24h. After the reaction, the reaction system was centrifuged at 12000rpm / min for 2min, and the supernatant was collected for detection. The detection results are shown in Figure 3 shown.

[0063] Using MALDI-TOF-MS, it was found that there was a substance with the same molecular weight as trisaccharide in the fermentation broth ( Figure 4 ), we can preliminarily identify the target product GlcNAcβ1-4(Fucα1-6)GlcNAc. To further determine whether the synthetic product is a trisaccharide, 1 HNMR structural identification ( Figure 5 ), 1H NMR (400MHz, D2O) δ5.24 (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). By analyzing the NMR results, it was finally determined that the successfully synthesized target product was GlcNAcβ1-4 (Fucα1-6) GlcNAc, which also proved that the four enzymes expressed by the recombinant strain had catalytic activity.

[0064] After the target product GlcNAcβ1-4(Fucα1-6)GlcNAc was determined, the fermentation supernatant was quantitatively analyzed by HPLC. Figure 6 As shown, after 24 hours 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 best quality plasmids synthesizing GlcNAcβ1-4(Fucα1-6)GlcNAc by the engineered strain E. coli BL21(DE3)

[0066] Primers Frsf-F (5'-CGAATTCGGATCCTGGCTG-3') and Frsf-R (5'-AGCTCGGCGCGCCT-3') were designed to perform PCR amplification on the vector pRSFDuet-1. The gene fragment described in Example 1 was purified and connected 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] The pETDuet-1 vector was PCR amplified using the primers described in Example 1, and the gene fragment described in Example 1 was purified and connected 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 as follows: Figure 7 As shown in A.

[0068] like Figure 7 As shown in B, the recombinant plasmids with different vectors as the backbone were transformed into competent cells E. coli BL21 (DE3), and Kan and Amp were used as screening markers for plate coating. After colony PCR verification, single colonies were picked for shake flask culture, and the plasmids in the bacterial solution were extracted for restriction enzyme verification and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing and identification, and the engineered strains E1-E6 were obtained respectively. E1-E6 were induced to express, and the induced bacteria were verified by SDS-PAGE protein electrophoresis. The results of DS-PAGE protein electrophoresis are shown in Figure 8 As shown, the GlcNAcβ1-4(Fucα1-6)GlcNAc synthesis pathway was successfully constructed in six homologous recombination expression strains.

[0069] Results Fig. 9 As can be seen in A, there are obvious protein bands at around 40kD, 85kD, and 100kD, and after induced expression, trisaccharides are produced, proving that the engineering strains E1-E6 were successfully constructed.

[0070] At the shake flask level, the six engineered strains that were successfully induced were co-cultured with Saccharomyces cerevisiae, and the two bacteria were coupled to catalyze the synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc, and the catalytic reaction lasted for 24 hours. The TLC results of the fermentation products of the six engineered strains are as follows Fig. 9 As shown in A, the fermentation broth supernatant was quantitatively analyzed by HPLC. The results are shown in Fig. 9 As shown in B, after 24 hours of fermentation, the engineered strain E. coli BL21 (DE3) / pRSFDuet-1-nodZ-Chbp / pETDuet-1-nahK-Fkp (E3) had the best yield, with a concentration of 18.09 g / L and a conversion rate of 62.38%.

[0071] Pick a single colony of the engineering strain E3 and inoculate it into 10mL LB medium with Kan and Amp resistance, culture it at 37℃ for 12h to obtain the first-level seed solution, then transfer the first-level seed solution to 250mL LB medium with Kan and Amp resistance at a 2% inoculation amount, culture it at 37℃ overnight to obtain the second-level seed solution, inoculate the second-level seed solution into the fermentation tank at a 10% inoculation amount, the culture temperature is 37℃, the ventilation volume is 2.5vvm, and the initial working volume in the tank is 2.5L. Set the stirring speed to couple with dissolved oxygen to control the dissolved oxygen at 30%. When the glucose content is lower than 0.1g / L, supplement the concentration of 300g / L glucose at a flow rate of 0.6mL / min; use 25% ammonia water to adjust the pH value to maintain at 6.9 during the entire fermentation process; add exogenous inducer to induce the engineering strain in the late logarithmic growth period, that is, when the OD value reaches 30. After the fermentation is completed, centrifuge the fermentation broth at 8000r / min for 5min, collect the bacteria, and then add the exogenous inducer to induce the engineered strain. Fig.10 As shown, the final OD 600 is 71.1, and the biomass is 96.4 g / L (wet weight).

[0072] Example 3: Dual-bacteria strategy coupled synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc

[0073] Synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc by dual bacterial coupling catalytic conversion at shake flask level: 100 g / L engineered strain E. coli BL21(DE3) / pRSFDuet1-nahK-Fkp / pETDuet1-nodZ-Chbp and 100 g / L Saccharomyces cerevisiae were added in 250 mM KH 2 PO 4 / K 2 HPO 4 (pH8.0), 80mM ATP, 25mM MgCl 2, 20mM Poly-P, 20mL / L alcohol, 10mmol / LDTT, 8g / L octadecylamine polyoxyethylene ether, 120mM GlcNAc, 50mM fucose, 50mM GMP, 25℃, 200r / min for 24h. After the reaction, the reaction system was centrifuged at 12000rpm / min for 2min, and the supernatant was collected for detection.

[0074] The fermentation supernatant was quantitatively analyzed by HPLC. After 24 hours of reaction, the concentration of GlcNAcβ1-4(Fucα1-6)GlcNAc was 18.09 g / L and the conversion rate was 62.38%.

[0075] Co-culture of engineered strains and yeast to produce GlcNAcβ1-4(Fucα1-6)GlcNAc 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 cultured in 250 mM KH 2 PO 4 / K 2 HPO 4 (pH=8.0), 80mM ATP, 25mM MgCl 2 , 20mM Poly-P, 20mL / L alcohol, 10mmol / L DTT, 8g / L octadecylamine polyoxyethylene ether, 120mM GlcNAc, 50mM fucose, 50mM GMP, 20g / L glycerol. Fermented for 24h, pH was adjusted to 7.2 with 25% ammonia water, the temperature was set to 25°C, the ventilation volume was 1vvm, after the reaction, the fermentation liquid was centrifuged at 8000r / min for 5min, and the supernatant was collected for detection. The supernatant of the fermentation liquid was quantitatively analyzed by HPLC, and the results are as follows Fig.11 As shown, after 36 hours of reaction, the concentration of GlcNAcβ1-4(Fucα1-6)GlcNAc was 18.42 g / L, and the conversion rate was 63.52%.

[0076] Example 4: Optimization of the dual-bacteria strategy for coupling synthesis of GlcNAcβ1-4(Fucα1-6)GlcNAc

[0077] (1) Effect of reaction pH on dual-bacteria coupled fermentation conversion; Environmental pH directly affects the dissociation state of enzyme and substrate, thereby affecting the binding of enzyme and substrate and the reaction rate. Therefore, the most suitable temperature for the overall reaction system was explored to achieve the maximum yield of GlcNAcβ1-4(Fucα1-6)GlcNAc. The results are shown in Fig.12As shown in A, when the reaction system has a pH of 7.5, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc reaches a maximum of 19.41 g / L, and the conversion rate is 66.93%.

[0078] (2) Cofactor Mg 2+ Effect of concentration on dual-bacteria coupled fermentation conversion: Studies have shown that the catalytic reactions of nahK enzymes and Fkp enzymes and the regeneration of the GMP-GTP cycle require the participation of metal ions, especially Mg 2+ The participation of 2+ It is also important to explore the concentration of Fig.12 As shown in B, Mg 2+ When the concentration was 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 dual-bacteria coupled fermentation conversion: Studies have shown that too low a reaction time will result in 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 of benefits. Therefore, the most suitable time for the overall reaction system was explored to achieve the maximum yield of GlcNAcβ1-4(Fucα1-6)GlcNAc. The results are as follows Fig.12 As shown in C, the yield of trisaccharide in the reaction system increased with time within 12-72 hours, but the growth rate dropped significantly after 36 hours. Considering the load and cost of the catalytic system, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc at 36 hours was selected to reach 24.19 g / L, and the conversion rate was 83.41%.

[0080] (4) Effects of carbon from different sources on the three-bacteria coupled fermentation conversion: The carbon source not only provides C elements for the whole-cell catalytic reaction, but also plays a role in energy supply, so the selection of a suitable carbon source also has a great influence on the reaction. The results show that Fig.12 As shown in D, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc using glycerol alone as the carbon source was high, reaching 24.38 g / L, and the conversion rate at this time was 84.07%.

[0081] (5) Effect of yeast cell biomass on dual-bacteria coupled fermentation conversion: Since yeast cells participate in the GMP-GTP cycle regeneration, the yeast cell biomass was optimized on the basis of the above optimization. Fig.12As shown in Figure E, with the increase of yeast cell biomass, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc is also increasing. When the yeast cell biomass is 300g / L, the yield reaches 26.2g / L, but when the biomass is 100g / L, the conversion rate is 85.43% and the yield is 24.35g / L. Because the yield difference is small, considering the catalytic system load and cost issues, 100g / L is selected as the optimal yeast cell biomass in the system for subsequent fermentation conversion.

[0082] (6) Effect of surfactant octadecylamine polyoxyethylene ether on dual-bacteria coupled fermentation conversion: Octadecylamine polyoxyethylene ether, as a surfactant, promotes the reaction by reducing the activation energy of the reactants, increasing the reaction activity, changing the hydrophobicity of the reaction system and increasing the active center. Therefore, the most suitable concentration of octadecylamine polyoxyethylene ether in the total reaction system was explored to achieve the maximum yield of GlcNAcβ1-4(Fucα1-6)GlcNAc. The results are shown in Fig.12 As shown in F, when the concentration of octadecylamine polyoxyethylene ether in the reaction system is 8 g / L, the yield of GlcNAcβ1-4(Fucα1-6)GlcNAc reaches a maximum of 25.50 g / L, and the conversion rate is 87.94%.

[0083] After the optimization of the above conversion system, the yield of trisaccharide 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 examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Production of 6'-fucosylated chitobiose by dual bacterial coupling fermentation The method of GlcNAcβ1-4(Fucα1-6)GlcNAc is characterized by: In the fermentation system, fucose and N-acetyl-D-glucosamine were used as substrates and fermented by coupled engineering Escherichia coli and yeast; The engineered Escherichia coli heterologously expresses N-acetylhexosamine 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 comprises a first expression vector and a second expression vector, wherein the first expression vector comprises a nahK encoding gene and a Fkp encoding gene, and the second expression vector comprises a nodZ encoding gene and a Chbp encoding 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 the pRSFDuet-1 plasmid as a backbone, and the second expression vector uses the pETDuet-1 plasmid as a backbone.

5. The method according to claim 1, characterized in that: The ratio of the engineered Escherichia coli to the 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: Adding metal ions to the fermentation system, wherein the metal ions are Mg 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 is added to the fermentation system, wherein the surfactant is octadecylamine polyoxyethylene ether, characterized in that the concentration of the octadecylamine polyoxyethylene ether in the fermentation system is 2-16 g / L.

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

Images