Method for producing sialyllactose by co-fermentation of two strains

Abstract

A method for producing sialyllactose by co-fermentation of two strains is provided, belonging to the technical fields of synthetic biology and fermentation engineering. An engineered Escherichia coli strain was constructed for the co-expression of α-2,6-sialyltransferase (ST6) or α-2,3-sialyltransferase (ST3) and NeuA enzyme. A two-step fermentation process was adopted: in a first stage, the engineered strain was cultured; in a second stage, said strain was co-fermented with yeast of two strains, using lactose and sialic acid as substrates to achieve the synthesis of sialyllactose. Under a co-fermentation mode of the engineered strain and yeast, shake flask horizontal fermentation is performed for 30 hours, the concentration of 6'-sialyllactose being 35.58 g / L, and the conversion rate of sialic acid being 93.69%; shake flask horizontal fermentation is performed for 24 hours, an optimal concentration of 3'-sialyllactose production being 21.8 g / L. In a 7L fermenter, horizontal fermentation is performed for 16, the highest yield of 6'-sialyllactose reaching 52.29 g / L, and the conversion rate being 97.99%. These results lay a technical foundation for the large-scale production of sialyllactose.

Description

A method for producing sialyllactose by dual-bacteria coupled fermentation Technical Field

[0001] The present invention relates to the technical field of biological fermentation, in particular to a method for producing sialyllactose by dual-bacteria coupled fermentation. Background Art

[0002] Human milk oligosaccharides (HMOs) are a series of naturally occurring oligosaccharide molecules found in breast milk that play a crucial role in establishing the early intestinal microbiome. HMOs are composed of five structural units: glucose (Glc), galactose (Gal), acetylglucosamine (GlcNAc), fucose (Fuc), and N-acetylneuraminic acid (NeuAc). These units are linked by glycosidic bonds to form oligosaccharides with diverse properties. Sialyl lactose is an acidic oligosaccharide composed of sialic acid and lactose. Depending on the binding site, it can be further divided into 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL). Sialyl lactose is a potent prebiotic with unique effects on the proliferation of bifidobacteria, shaping the intestinal microbiome, and intestinal maturation. Sialyl lactose also exhibits antiviral and immunomodulatory activities, promoting brain development and improving cognition, demonstrating its significant commercial value in formula milk.

[0003] Due to its important biological activity and physiological functions, the synthesis of sialyllactose has attracted widespread attention and in-depth research. Research has shown that there are currently three main methods for its synthesis: chemical, enzymatic, and biological. Chemical methods are cumbersome and inefficient, and enzymatic methods involve toxic reagents that do not meet the requirements of clinical reactions and food formulations. Biological methods address these deficiencies, leading researchers to turn their attention to biological methods and invest significant effort and research in their synthesis.

[0004] Due to high production costs and low production efficiency, inexpensive, large-scale production of sialyllactose remains elusive. Sialic acid and cytidine triphosphate (CTP) are expensive, and the synthesis of sialyllactose requires NeuAc and large amounts of CTP, resulting in high production costs. Since yeast cells contain an enzyme system that catalyzes the conversion of CMP to CTP without consuming lactose, the idea of ​​integrating baker's yeast cells into a bioconversion system has been proposed. The regeneration of CMP to CTP by yeast cells effectively reduces the high costs associated with simply supplementing CTP.

[0005] To address these issues, a three-strain coupled fermentation strategy for 6′-SL conversion was proposed (Figure 1, A). Considering that the flow of substances into and out of the cell can affect 6′-SL conversion efficiency, this study also constructed a strategy for 6′-SL synthesis using a coupled co-culture of the engineered E. coli JM109(DE3) / pETDeut-1-neuA-plst6 strain and baker's yeast (Figure 1, B). By comparing the advantages and disadvantages of these two strategies, we aim to develop a more optimal fermentation strategy and process for 6′-SL production.

[0006] 6′-SL / 3′-SL plays an important physiological role in the growth and development of infants and young children, and has broad market prospects. The development of 6′-SL / 3′-SL synthesis processes has become a research hotspot in recent years. Due to the technical limitations of chemical and enzymatic methods, attention has turned to biological methods. In 2010, Drouillard et al. discovered that reducing the expression level of sialyltransferase and increasing the expression level of NeuABC could promote the synthesis of 6′-SL and effectively prevent the synthesis of 6,6'-disialyllactose in the presence of excessive lactose. Therefore, they employed a high-density fermentation strategy with the continuous addition of glycerol and lactose to synthesize 6′-SL, achieving final extracellular and intracellular concentrations of 6′-SL of 23 g / L and 11 g / L, respectively. In 2018, Beauprez et al. engineered Escherichia coli to synthesize 6′-SL and used a fed-batch culture strategy to achieve a final concentration of 30.5 g / L. Considering the problem of product accumulation in the cell during single-cell synthesis, resulting in product inhibition, we proposed a multi-strain coupling strategy to achieve the production of 6′-SL. Summary of the Invention

[0007] Since sialyllactose is the most abundant sialyllactose among human milk oligosaccharides (HMOs), it plays an important role in the growth and development of infants. Achieving efficient production of sialyllactose has always been a difficulty and challenge in the field of oligosaccharide synthesis. To address this issue, the present invention provides a method for producing sialyllactose by dual-bacterial coupled fermentation. The present invention constructs an engineered strain co-expressing sialyltransferase and CMP-Neu5Ac synthase, evaluates the effect of coupling it with baker's yeast to synthesize sialyllactose, and optimizes the results.

[0008] The present invention is achieved through the following technical solutions:

[0009] The first object of the present invention is to provide a method for producing sialyllactose by dual-bacteria coupled fermentation, wherein sialyllactose is synthesized by coupled fermentation of yeast and a recombinant bacterium co-expressing sialyltransferase and CMP-Neu5Ac synthase using sialic acid and lactose as substrates; the sialyltransferase is selected from α-2,6-sialyltransferase and / or α-2,3-sialyltransferase.

[0010] In one embodiment of the present invention, the recombinant bacteria uses Escherichia coli, yeast or Bacillus subtilis as a host.

[0011] In one embodiment of the present invention, the Escherichia coli is selected from Escherichia coli BL21 (DE3), Escherichia coli K12 MG1655, Escherichia coli JM109 (DE3) or Escherichia coli 1917 (DE3).

[0012] In one embodiment of the present invention, the plasmid used is pRSF-Duet-1, pET-Duet-1 or pCDF-Duet-1, preferably pETDeut-1.

[0013] In one embodiment of the present invention, the concentration of sialic acid is 60 mM-90 mM, preferably 60 mM-70 mM.

[0014] In one embodiment of the present invention, the concentration of lactose is 80 mM-200 mM.

[0015] In one embodiment of the present invention, the concentration of the exogenously added cofactor CMP is 10 mM-50 mM.

[0016] In one embodiment of the present invention, the fermentation reaction system further contains octadecylamine polyoxyethylene ether or octadecylamine polyoxyethylene ether at a concentration of 1 g / L-10 g / L.

[0017] In one embodiment of the present invention, the amount of the recombinant bacteria co-expressing sialyltransferase and CMP-Neu5Ac synthetase added is 50 g / L-100 g / L.

[0018] In one embodiment of the present invention, the added amount of the yeast is 50 g / L-250 g / L.

[0019] In one embodiment of the present invention, the yeast is selected from one or more of baker's yeast, brewer's yeast and cerevisiae yeast.

[0020] In one embodiment of the present invention, the fermentation temperature is 20°C-40°C, preferably 30°C-40°C; the fermentation time is 10h-30h, preferably 16h-28h.

[0021] In one embodiment of the present invention, the fermentation reaction system further contains 5mM-20mM Mg + , preferably 10mM-20mM.

[0022] The second object of the present invention is to provide the use of sialyllactose produced by the method in the preparation of milk powder or nutritional supplements for infants and the elderly.

[0023] The above technical solution of the present invention has the following advantages over the prior art:

[0024] The present invention provides a method for producing sialyllactose by dual-strain coupled fermentation. Currently, due to the large number of engineered strains, the fermentation process is relatively complex, and the movement of substances into and out of the cells can also affect the conversion rate of sialyllactose. The present invention constructs an engineered strain to achieve co-expression of sialyltransferase and NeuA, and then couples it with baker's yeast to achieve sialyllactose synthesis. In the dual-strain coupled fermentation mode, after 30 hours of shake flask fermentation, the concentration of 6′-sialyllactose reached 35.58 g / L, and the conversion rate of sialic acid was 93.69%. The optimal yield of 3′-SL in shake flask fermentation was 21.8 g / L. In a 7L fermentor, after 16 hours of fermentation, the 6′-sialyllactose yield reached a maximum of 51.29 g / L, with a conversion rate of 97.99%, the highest yield reported to date. The results of this invention lay the technical foundation for the large-scale and mass production of sialyllactose. 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 based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0026] Figure 1 is a technical roadmap for the synthesis of 6′-SL by multi-bacteria coupled fermentation according to the present invention; wherein A is a technical roadmap for the synthesis of 6′-S by three-bacteria coupled fermentation; and B is a technical roadmap for the synthesis of 6′-SL by two-bacteria coupled fermentation;

[0027] Figure 2 is a flow chart and electrophoresis diagram of the construction of the recombinant plasmid pET28a-plst6 of the present invention; wherein A is a flow chart of the construction of the recombinant plasmid pET28a-plst6; B is a double enzyme digestion diagram of the recombinant plasmid pET28a-plst6; C is an SDS-PAGE diagram of the recombinant plasmid pET28a-plst6;

[0028] Figure 3 is a diagram of the detection and analysis of 6′-SL of the present invention; A is a TLC analysis diagram of the three-bacteria coupled fermentation product: 1-Neu5Ac standard, 2-lactose standard; 3-6′-SL standard; 4-fermentation supernatant; B is a MALDI-TOF MS analysis of 6′-SL; C is a hydrogen nuclear magnetic resonance spectrum of 6′-SL; D is, from top to bottom, a liquid phase diagram of the standard 6′-SL, a liquid phase diagram of the standard Neu5Ac, and a liquid phase diagram of the three-bacteria coupled fermentation product;

[0029] Figure 4 shows the effects of different induction concentrations on the synthesis of 6′-SL by the engineered strains of the present invention; A shows the effects of different induction concentrations on the synthesis of 6′-SL by E. coli JM109 (DE3) / pET28a-neuA; B shows the effects of different induction concentrations on the synthesis of 6′-SL by E. coli JM109 (DE3) / pET28a-plst6;

[0030] Figure 5-A shows the effect of different sialic acid concentrations on the synthesis of 6′-SL by three-bacteria coupled fermentation;

[0031] Figure 5-B shows the effect of different lactose concentrations on the conversion and synthesis of 6′-SL by three-bacteria coupled fermentation;

[0032] Figure 5-C shows the effect of different temperatures on the conversion and synthesis of 6′-SL by three-bacteria coupled fermentation;

[0033] Figure 5-D shows different concentrations of Mg 2+ Effects on the synthesis of 6′-SL by three-bacteria coupled fermentation;

[0034] Figure 5-E shows the effect of different yeast biomasses on the synthesis of 6′-SL by three-bacteria coupled fermentation;

[0035] Figure 6-A shows the fermentation curve of the engineered strain E. coli JM109(DE3) / pET28a-neuA using lactose as the only inducer;

[0036] Figure 6-B shows the fermentation curve of the engineered strain E. coli JM109(DE3) / pET28a-plst6 using lactose as the only inducer;

[0037] Figure 6-C shows the yield and conversion efficiency of 6′-SL synthesized by three-bacteria coupling in 30 h using lactose as the only inducer;

[0038] FIG7-A is a fermentation curve of the engineered strain E. coli JM109(DE3) / pET28a-neuA using lactose and IPTG as inducers;

[0039] FIG7-B is a fermentation curve of the engineered strain E. coli JM109(DE3) / pET28a-plst6 using lactose and IPTG as inducers;

[0040] FIG7-C is a curve diagram of the three-bacteria coupled fermentation process using lactose and IPTG as inducers;

[0041] Figure 8 is a 6'-SL detection analysis diagram of the present invention; wherein A is a recombinant plasmid pETDeut-1-plst6-neuA plasmid map; B is a recombinant plasmid pETDuet-1-plst6-neuA SDS-PAGE diagram; C is a TLC analysis diagram of the dual-bacteria coupled fermentation product: 1-lactose standard; 2-6'-SL standard; 3-fermentation supernatant; D is, from top to bottom, a liquid phase diagram of the standard Neu5Ac, a liquid phase diagram of the standard 6'-SL, and a liquid phase diagram of the dual-bacteria coupled fermentation product;

[0042] Figure 9 is a fermentation curve of the engineered strain of the present invention using lactose and IPTG as inducers simultaneously; wherein A is the fermentation curve of the engineered strain E. coli JM109 (DE3) / pETDeut-1-neuA-plst6; B is a curve diagram of the dual-bacteria coupled fermentation process;

[0043] Figure 10 is a flow chart of the construction of the recombinant plasmid pCDF Deut-1-neuA-nst3 of the present invention;

[0044] FIG11-A is an SDS-PAGE image of the recombinant plasmid pCDF Deut-1-neuA-nst3 of the present invention;

[0045] FIG11-B is a TLC analysis of the dual-bacteria coupled fermentation product of the present invention: 1-sialic acid standard; 2-3′-SL standard; 3-fermentation supernatant; 4-blank reaction solution;

[0046] FIG12 is a gene structure constructed by connecting gene fragments through homologous recombination of the present invention;

[0047] FIG13 is a graph showing the yield of 3′-SL synthesized by whole-cell catalysis of four engineered bacterial strains of the present invention and baker's yeast in co-culture. DETAILED DESCRIPTION

[0048] 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 the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0049] The sources of the materials described in the present invention are as follows:

[0050] (1) Baker's yeast; obtained by fermentation culture of GDMCC61663. Escherichia coli JM109 (DE3) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; engineered strain E. coli JM109 (DE3) / pET28a-neuA (synthesis method reference patent CN113444756A). All other reagents used were commercially available.

[0051] (2) Source of genes required for the experiment: Photobacterium leiognathi JT-SHIZ-145 genes, obtained through artificial synthesis after manual optimization;

[0052] (3) Culture medium and main solutions

[0053] LB liquid medium (g / L): NaCl 10.0, tryptone 10.0, yeast extract 5.0, pH 7.0; sterilization conditions: 121°C for 20 min.

[0054] 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 for 20 min.

[0055] 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°C for 30 min.

[0056] Yeast seed medium: glucose 10 g / L, peptone 5 g / L, yeast extract 15 g / L, sodium chloride 4 g / L, pH 7.0.

[0057] Yeast basal fermentation medium: glucose 20 g / L, ammonium sulfate 8 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 0.5 g / L, pH 5.5.

[0058] Diphenylamine-aniline-phosphoric acid solution: 4 g diphenylamine, 4 mL aniline and 20 mL 85% phosphoric acid are dissolved in 200 mL acetone.

[0059] The detection method used in the present invention is as follows:

[0060] (1) Separation and purification of sialyllactose

[0061] Isolation and purification method: Purification was performed using a HyperSep Hypercarb solid-phase extraction cartridge (SPE cartridge), and the purification steps were as follows: activation: the SPE cartridge was activated with 3 mL of methanol; equilibration: the SPE cartridge was equilibrated and moistened with 3 mL of ultrapure water; loading: 300 μL of the catalytic supernatant was evenly passed through the SPE cartridge; rinsing: the SPE cartridge was rinsed with 1 mL of ultrapure water, repeated three times; elution: the product was eluted with 0.5 mL of 80% acetonitrile, repeated three times.

[0062] (2) Analysis and detection of sialyllactose

[0063] Thin layer chromatography (TLC): The developing solvent was v (n-propanol): v (water): v (25% ammonia water) = 37.5:15:10. After the TLC plate was naturally dried, it was impregnated with diphenylamine-aniline-phosphoric acid solution and placed in a 105°C oven for 1 min for color development.

[0064] (3) Matrix-assisted laser ionization time-of-flight mass spectrometry (MALDI-TOF-MS): The molecular weight of the purified product was determined. 1 μL of the purified product and 1 μL of the spotted matrix 2,5-dihydroxybenzoic acid containing Na + , spot the samples at the same position on the target plate, mix well, dry, and process on the mass spectrometer. Mass spectrometry conditions: reflectron cation mode; scan molecular weight range: 0-2000Da.

[0065] (4) H NMR spectroscopy ( 1 HNMR): The purified product was lyophilized and dissolved in 600 μL of deuterated water (D2O). The HNMR spectrum was acquired using a nuclear magnetic resonance spectrometer at a resonance frequency of 400 MHz. 1 H spectrum.

[0066] (5) High-performance liquid chromatography (HPLC): UV detection wavelength, 210 nm; chromatographic column, Aminex HPX-87H Ion Exclusion Column (7.8 mm × 300 mm); mobile phase, 5 mM H2SO4; column temperature, 60°C; injection volume, 10 μL; flow rate, 0.6 mL / min.

[0067] (6) SDS-PAGE gel electrophoresis: 40 μL of the bacterial solution before and after induction was added to 10 μL of protein loading buffer and mixed evenly. The mixture was incubated in a boiling water bath for 4 min and then in an ice bath for 2 min. 10 μL of the mixture was then subjected to SDS-PAGE gel electrophoresis. After protein electrophoresis, the bacteria that were successfully induced to express the target protein were stored at -20°C until further use.

[0068] Example 1 Construction of the engineered strain E. coli JM109 (DE3) / pET28a-plst6.

[0069] The target gene fragment was synthesized using the sequence of the plst6 gene (GenBank accession number MN721873.1) in Photobacterium leiognathi JT-SHIZ-145 as a template, and primers plst6F

[0070] (5'-CCATGGGCAAAAAAATCCTGACTGTGCTGTCT-3', underlined for the Nco I restriction enzyme site) and plst6R (5'-GGATCCTTAGTCAGCCCAAAACAGAACGTCT-3', underlined for the BamHI restriction enzyme site). The target gene plst6 was PCR amplified using primers plst6F and plst6R. After verification and recovery, the plst6 gene fragment and the pET28a empty vector were double-digested with Nco I and BamHI. The plst6 gene fragment after enzyme digestion was purified and connected with the vector fragment to obtain the recombinant plasmid pET28a-plst6. The construction process is shown in A in Figure 2. It was then transformed into competent cells E. coli JM109 (DE3), plated with Kan as a screening marker, and after colony PCR verification, a single colony was picked for shake flask culture. The plasmid in the bacterial solution was extracted for enzyme digestion verification and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing and identification.

[0071] The results of enzyme digestion verification of pET28a-plst6 are shown in Figure 2B. The resulting fragments are approximately 5300 bp and 1600 bp in length, consistent with the pET28a vector and the target gene plst6, respectively. Further sequencing verification confirmed that the sequencing results were consistent with the template gene sequence, confirming the successful construction of the recombinant plasmid pET28a-plst6. The nucleotide sequence of the gene plst6 is shown in SEQ ID NO. 1.

[0072] Example 2 Expression and culture of the engineered strain E. coli JM109 (DE3) / pET28a-plst6.

[0073] A single colony of the engineered strain was inoculated into 10 mL LB medium with Kan resistance, and cultured overnight at 37°C to obtain seed solution. The seed solution was then transferred to 200 mL LB medium with Kan resistance at a 2% inoculum volume and incubated at 37°C and 200 rpm for 1 h. -1 Under the conditions of shake flask culture, when the bacteria grow to OD 600 When the pH value was between 0.6 and 0.8, IPTG was added to a final concentration of 0.1 mM to induce protein expression. The temperature was lowered to 16°C for 20 hours of induction, and the cells were collected by centrifugation. The induced cells were then subjected to SDS-PAGE protein electrophoresis verification, as shown in Figure 2, C. Figure 2, C, shows a clear protein band between 55 kD and 72 kD, consistent with the reported size of the target enzyme protein. This indicates that α-2,6-sialyltransferase ST6 was successfully expressed in the engineered E. coli JM109(DE3) / pET28a-plst6 strain.

[0074] Fermentation culture: Pick a single colony of the engineered strain and inoculate it into 10 mL of LB medium with Kan resistance. Culture it at 37°C overnight to obtain a seed solution. Transfer the seed solution to 200 mL of fermentation medium with Kan resistance at an inoculum volume of 2%. Culture it in a shake flask at 37°C, 200 rpm for 10 h, and collect the bacteria by centrifugation.

[0075] (2) Fermentation tank culture

[0076] A single colony of the engineered strain was inoculated into 10 mL of LB medium containing Kan-resistant strains and cultured at 37°C for 12 hours to obtain a primary seed solution. This primary seed solution was then transferred at a 2% inoculum into 250 mL of LB medium containing Kan-resistant strains and cultured overnight at 37°C to obtain a secondary seed solution. This secondary seed solution was then inoculated into a fermentor at a 10% inoculum. The culture temperature was 37°C, the aeration rate was 2 vvm, and the initial working volume in the fermentor was 2.5 L. The stirring speed was coupled to the dissolved oxygen to maintain a constant DO of 20%. When the glucose content fell below 5 g / L, glucose was supplemented at a concentration of 300 g / L at a flow rate of 0.6 mL / min. The pH was maintained at 7.0 throughout the fermentation process using 50% ammonia water. An exogenous inducer was added to induce the engineered strain at the late logarithmic growth stage, replacing glucose with 300 g / L glycerol as the carbon source. After fermentation, the fermentation broth was centrifuged at 8000 rpm for 5 minutes, and the cells were harvested.

[0077] Example 3 Synthesis of 6'-sialyllactose by three-strain co-culture strategy.

[0078] Synthesis of 6′-SL by three-bacterial coupled catalytic conversion in shake flasks: 50 g / L of the engineered strains E. coli JM109(DE3) / pET28a-neuA (synthesis method referenced in patent CN113444756A), E. coli JM109(DE3) / pET28a-plst6, and 100 g / L baker's yeast were reacted with 60 mM Neu5Ac and 80 mM lactose as substrates in a reaction system containing 70 mM CMP, 300 mM glucose, 1 mM DTT, 20 mL / L glycerol, 10 g / L xylene, 5 g / L acetaldehyde, 20 mM MgCl2, 248.3 mM KH2PO4, 150 mM Tris, and 4 g / L octadecylamine polyoxyethylene ether. The reaction was carried out at 30°C and 200 rpm for 30 h. After completion of the reaction, the reaction system was centrifuged at 12,000 rpm for 2 min, and the supernatant was collected for analysis.

[0079] Preliminary TLC detection revealed the presence of a component in the fermentation broth with the same relative migration value as the standard 6′-SL (Figure 3A), preliminarily confirming that the target product 6′-SL was synthesized. To further confirm whether the synthesized product was 6′-SL, the molecular weight was determined by MALDI-TOF-MS (Figure 3B). The molecular ion peak corresponding to the product 6′-SL was at m / z = 656.174 [M+Na] + ,m / z=678.159[M+2Na-H] + , indicating that the purified product contains a component with the same molecular weight as the standard 6′-SL, which is consistent with the results reported by Priem, B (Priem, B., et al., A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology, 2002.12(4): p.235-240). 1 The structure was identified by HNMR (Figure 3C). 1 HNMR(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), and Drouillard (Drouillard, S., et al. al., Efficient synthesis of 6′-sialyllactose,6,6′-disialyllactose, and 6′-KDO-lactose by metabolically engineered E.coli expressing a multifunctional sialyllactase from the Photobacterium sp.JT-ISH-224.

[0080] The results were consistent with those reported by others (Carbohydrate Research, 2010.345(10): p.1394-1399), and the target product 6′-SL was successfully synthesized. This also proved that the ST6 enzyme expressed by the recombinant strain had catalytic activity.

[0081] After confirming the synthesis of the target product 6′-SL, the fermentation supernatant was quantitatively analyzed by HPLC. After 30 h of reaction, the concentration of 6′-SL was 23.31 g / L and the conversion rate of sialic acid was 61.32% ( FIG. 3D ).

[0082] 6′-SL was synthesized by three-strain coupling catalysis in a 7-L fermentor: 50 g / L engineered strains E. coli JM109 (DE3) / pET28a-neuA, E. coli JM109 (DE3) / pET28a-plst6 and 150 g / L baker's yeast were fermented with 60 mM Neu5Ac and 160 mM lactose as substrates in a conversion system containing 70 mM CMP, 300 mM glucose, 1 mM DTT, 20 mL / L glycerol, 10 g / L xylene, 5 g / L acetaldehyde, 20 mM MgCl2, 248.3 mM KH2PO4 and 150 mM Tris, and 4 g / L octadecylamine polyoxyethylene ether. After 20 hours of fermentation, a mixed solution of 90 mM sialic acid and 90 mM lactose was supplemented at a flow rate of 0.6 mL / min. The stirring speed was set to be coupled with the dissolved oxygen to control the dissolved oxygen at 20%, the temperature was set to 30°C, and the ventilation volume was 1 vvm. After the reaction was completed, the fermentation broth was centrifuged at 10,000 r / min for 5 minutes, and the supernatant was collected for detection.

[0083] Example 4 Optimization of the three-bacteria coupled synthesis of 6′-SL.

[0084] (1) Optimization of lactose concentration as an inducer in fermentation medium

[0085] Lactose can be used as a good protein expression inducer in fermentation media. Appropriate lactose concentrations are beneficial for efficient expression of target proteins. Therefore, it is crucial to investigate the effects of different lactose concentrations on protein expression in the engineered strains E. coli JM109(DE3) / pET28a-neuA and E. coli JM109(DE3) / pET28a-plst6.

[0086] After adjusting the lactose concentration in the fermentation medium, the two engineered strains were cultured. By comparing the final yields of the three-bacteria coupled fermentation conversion reactions, it was found that the conversion efficiency of E. coli JM109(DE3) / pET28a-neuA was highest at a lactose concentration of 4 g / L, and the 6′-SL concentration in the fermentation broth reached 10.17 g / L (Figure 4A); the conversion efficiency of E. coli JM109(DE3) / pET28a-plst6 was highest at a lactose concentration of 3 g / L, and the concentration in the fermentation broth reached 4.96 g / L (Figure 4B).

[0087] (2) Effects of different substrate concentrations on the three-bacteria coupled fermentation conversion; Since NeuA enzyme first utilizes sialic acid in the technical route, the effects of different sialic acid concentrations on 6′-SL synthesis were first explored under conditions of excess lactose; the synthesis of 6′-SL showed a trend of first increasing and then decreasing (Figure 5-A). When the sialic acid concentration was 60 mM, the yield reached a maximum of 5.43 g / L, and the sialic acid conversion rate was 14.28% at this time; on this basis, the effects of different lactose concentrations on 6′-SL synthesis were further explored (Figure 5-B). In the process of increasing the lactose concentration from 80 mM to 200 mM in the conversion system, the yield of 6′-SL reached a maximum of 12.00 g / L at a lactose concentration of 160 mM, and the sialic acid conversion rate was 27.24%.

[0088] (3) Effect of reaction temperature on the conversion of three-bacteria coupled fermentation: It is well known that the catalytic activity of enzymes is greatly affected by temperature. Since this reaction system is a mixed-bacteria coupled fermentation involving the catalytic reactions of multiple enzymes, it is impossible to explore the optimal temperature for each individual enzyme. Therefore, the most suitable temperature for the overall reaction system was explored to achieve the maximum yield of 6′-SL. The results (Figure 5-C) show that at 30°C, the yield of 6′-SL reached a maximum of 13.71 g / L, and the conversion rate was 36.06%.

[0089] (4) Cofactor Mg 2+ Effect of concentration on the conversion of three-bacteria coupled fermentation: Studies have shown that the catalytic reaction of NeuA enzyme and ST6 enzyme and the regeneration of CMP-CTP cycle require the participation of metal ions, especially Mg 2+ The participation of 2+ It is also crucial to explore the concentration of Mg 2+ When the concentration was 20 mM, the yield of 6′-SL was further increased to 27.48 g / L, and the NeuAc conversion rate was correspondingly increased to 72.30%.

[0090] (5) Effect of yeast cell biomass on the three-bacteria coupled fermentation conversion: Since yeast cells participate in the CMP-CTP cycle regeneration, the yeast cell biomass was optimized based on the above optimization. The results showed (Figure 5-E) that with the increase of yeast cell biomass, the yield of 6′-SL also continued to increase. When the yeast cell biomass was 250 g / L, the yield reached 37.55 g / L. However, when the biomass was 150 g / L, the conversion rate was the highest at 97.97% and the yield was 35.52 g / L. Because the difference in yield was small, considering the catalytic system load and cost issues, 150 g / L was selected as the optimal yeast cell biomass in the system for subsequent fermentation conversion.

[0091] After optimization of the above transformation system, the yield of 6′-SL increased from 4.96 g / L to 35.52 g / L, and the sialic acid conversion rate increased from 13.04% to 97.97%.

[0092] Example 5: Lactose was used as the only inducer to culture the engineered strain and perform three-bacteria coupling.

[0093] E. coli JM109(DE3) / pET28a-neuA and E. coli JM109(DE3) / pET28a-plst6 were cultured in 7 L fermentors. Due to the glucose effect, 4 g / L lactose was added at the beginning of the fermentation to induce the autoinduction of E. coli JM109(DE3) / pET28a-neuA (Figure 6-A). The final OD 600 was 13.02, and the biomass was 39.8 g / L (wet weight); 3 g / L lactose was added to the E. coli JM109 (DE3) / pET28a-plst6 for autoinduction culture (Figure 6-B), and the final OD 600 is 10.08, and the biomass is 37.5 g / L (wet weight).

[0094] Based on the optimization of the above-mentioned three-bacteria coupled fermentation conversion system, the two engineered strains cultured in a fermenter were coupled with yeast in a fermenter for three-bacteria production. 6′-SL was quantitatively analyzed by HPLC. The yield of 6′-SL after 28 h of fermentation was 11.95 g / L ( Figure 6-C ). At this time, the molar conversion rate of the substrate Neu5Ac was 25.11%, and the three-bacteria coupled fermentation production of 6′-SL was preliminarily achieved at the fermenter level.

[0095] Example 6: Lactose and IPTG were used as inducers to culture the engineered strain and perform three-bacteria coupling.

[0096] Considering that the three-bacteria coupling yield was only 4.8 g / L using lactose as the only inducer, it was speculated that the protein induction effect of using lactose as the inducer alone was poor. Therefore, a strategy of using lactose and IPTG as inducers was adopted to culture the engineered strains E. coli JM109 (DE3) / pET28a-neuA and E. coli JM109 (DE3) / pET28a-plst6 in fermenters.

[0097] In order to avoid the influence of large-scale protein expression in the early stage of bacterial growth, IPTG with a final concentration of 0.15mM was added to induce the engineered strain in the late logarithmic period. 600The OD value of the engineered strain E. coli JM109 (DE3) / pET28a-plst6 reached 15.835, at which point the biomass was 56.25 g / L (wet weight). After 27 h of fermentation (Figure 7-B), the OD 600 It reached 16.723, at which time the biomass was 50.02 g / L (wet weight).

[0098] Sialic acid and lactose were added during the fermentation conversion process. After the fermentation, 6′-SL was quantitatively analyzed by HPLC. According to the HPLC results (Figure 7-C), the 6′-SL production showed a trend of first increasing and then decreasing. At 24 hours, the maximum yield was 43.79 g / L, and the conversion rate was 72.66%.

[0099] Example 7 Construction of the engineered strain E. coli JM109 (DE3) / pETDeut-1-plst6-neuA and protein induced expression.

[0100] The recombinant plasmid pETDeut-1-plst6-neuA was constructed using homologous recombination. The plasmid fragment pETDeut-1, the neuA and plst6 gene fragments obtained by PCR amplification, were verified by nucleic acid electrophoresis and then recovered by gel excision. The nucleotide sequence of the plst6 gene fragment is shown in SEQ ID NO. 1, and the nucleotide sequence of the neuA gene fragment is shown in SEQ ID NO. 2. These fragments were ligated by homologous recombination. The construction process is shown in Figure 8A.

[0101] The successfully constructed recombinant plasmid pETDeut-1-plst6-neuA was transformed into competent E. coli JM109 (DE3) cells and plated with Amp as the selection marker. After colony PCR verification, single colonies were picked for shake flask culture. The plasmid in the bacterial solution was extracted for PCR verification and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing and identification.

[0102] The engineered strain E. coli JM109 (DE3) / pETDeut-1-plst6-neuA was induced for expression, and SDS-PAGE ( FIG8B ) showed that the size was consistent with the actual molecular weight, indicating that the target protein was successfully expressed.

[0103] In shake flasks, the successfully induced strain was co-cultured with baker's yeast to generate 6′-SL in whole-cell catalysis. The reaction lasted 30 hours. TLC analysis revealed the presence of a component in the fermentation broth with the same transfer ratio as the standard 6′-SL (Figure 8, C), confirming the synthesis of the target product 6′-SL. Quantitative analysis of the fermentation supernatant by HPLC revealed a 6′-SL concentration of 35.58 g / L after 30 hours of fermentation, and a sialic acid conversion rate of 93.69% (Figure 8, D).

[0104] Example 8 Synthesis of 6′-sialyllactose by the Second Strain Co-cultivation Strategy

[0105] Synthesis of 6′-SL by dual-bacterial coupling catalytic conversion in shake flasks: 50 g / L of the engineered E. coli JM109(DE3) / pET Deut-1-plst6-neuA and 100 g / L of baker's yeast were reacted with 60 mM Neu5Ac and 80 mM lactose as substrates in a reaction system containing 70 mM CMP, 300 mM glucose, 1 mM DTT, 20 mL / L glycerol, 10 g / L xylene, 5 g / L acetaldehyde, 20 mM MgCl2, 248.3 mM KH2PO4, 150 mM Tris, and 4 g / L octadecylamine polyoxyethylene ether. The reaction was carried out at 30°C and 200 rpm for 30 h. After completion of the reaction, the reaction system was centrifuged at 3000 rpm for 2 min, and the supernatant was collected for analysis.

[0106] 6′-SL was produced by co-cultivation of engineered strains and yeast in a 7-L fermentor: 50 g / L of the engineered strain E. coli JM109 (DE3) / pET Deut-1-plst6-neuA and 100 g / L of baker's yeast were fermented with 60 mM Neu5Ac and 160 mM lactose as substrates in a conversion system containing 70 mM CMP, 300 mM glucose, 1 mM DTT, 20 mL / L glycerol, 10 g / L xylene, 5 g / L acetaldehyde, 20 mM MgCl2, 248.3 mM KH2PO4, 150 mM Tris, and 4 g / L octadecylamine polyoxyethylene ether. After 6 h of fermentation, a mixed solution of 165 mM sialic acid and 250 mM lactose was added at a flow rate of 1.2 mL / min. The stirring speed was coupled with the dissolved oxygen to control the dissolved oxygen at 25%, the temperature was set at 30°C, and the ventilation volume was 1 vvm. After the reaction was completed, the fermentation broth was centrifuged at 3000 r / min for 5 min, and the supernatant was collected for detection.

[0107] Example 9: The engineered strain E. coli JM109 (DE3) / pETDeut-1-plst6-neuA was cultured in a fermenter and then fermented by coupling the two strains to synthesize 6′-SL.

[0108] The engineered strain E. coli JM109 (DE3) / pETDeut-1-plst6-neuA was cultured in a 7 L fermentor. IPTG with a final concentration of 0.15 mM was added to induce the engineered strain in the late logarithmic phase. After 28 h of fermentation (Figure 9A), OD 600 Reaching 16.10, the biomass was 41.75 g / L.

[0109] The successfully induced engineered strain was coupled with yeast according to the method of Example 8. Sialic acid and lactose were added during the fermentation and conversion process. After fermentation, 6′-SL was quantitatively analyzed by HPLC (Figure 9B). As can be seen, 6′-SL production initially increased, then decreased, and finally stabilized, reaching a maximum yield of 51.29 g / L at 16 hours, with a conversion rate of 97.99%. Therefore, 16 hours was selected as the optimal fermentation time.

[0110] Example 10 Construction of the engineered strain E. coli JM109 (DE3) / pETDeut-1-neuA-nst3 and protein induced expression.

[0111] The recombinant plasmid pETDeut-1-neuA-nst3 was constructed using homologous recombination. The plasmid fragment pETDeut-1, along with the neuA and nst3 gene fragments, obtained by PCR amplification, was verified by electrophoresis and then recovered from the gel. The nucleotide sequence of the nst3 gene fragment is shown in SEQ ID NO. 3. These fragments were ligated by homologous recombination, as shown in Figure 10 (the construction process for pCDF Deut-1-neuA-nst3 is the same, so the construction flow chart is omitted).

[0112] The successfully constructed recombinant plasmid pETDeut-1-neuA-nst3 was transformed into competent E. coli JM109 (DE3) cells and plated with Amp as the selection marker. After colony PCR verification, single colonies were picked for shake flask culture. The plasmid in the bacterial solution was extracted for PCR verification and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing and identification.

[0113] The engineered strain E. coli JM109 (DE3) / pETDeut-1-neuA-nst3 was induced for expression, and SDS-PAGE ( FIG. 11-A ) showed that the size was consistent with the actual molecular weight, indicating that the target protein was successfully expressed.

[0114] In shake flasks, the successfully induced strain was co-cultured with baker's yeast for whole-cell catalytic synthesis of 3′-SL. The reaction lasted 24 hours. TLC analysis revealed the presence of a component in the fermentation broth with an identical relative transfer value to the standard 3′-SL (Figure 11-B), confirming the synthesis of the target product, 3′-SL.

[0115] Example 11 Construction and screening of the best quality plasmids for synthesizing 3′-SL by the engineered strain E. coli JM109 (DE3).

[0116] Recombinant plasmids pETDeut-1-neuA-nst3, pETDeut-1-nst3-neuA, pCDFDeut-1-neuA-nst3, and pCDFDeut-1-nst3-neuA were constructed using homologous recombination. The plasmid fragments pETDeut-1 and pCDFDeut-1, and the gene fragments neuA and nst3, obtained by PCR amplification, were verified by nucleic acid electrophoresis and then recovered by gel excision. These fragments were then ligated by homologous recombination to construct the gene structure shown in Figure 12.

[0117] The four successfully constructed recombinant plasmids were transformed into competent cells E. coli JM109 (DE3) respectively. The plasmid vector pETDeut-1 used Amp as the screening marker, and the plasmid vector pCDFDeut-1 used Str as the screening marker. The plates were spread and verified by colony PCR. Single colonies were picked for shake flask culture. The plasmids in the bacterial solution were extracted for PCR verification and sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing and identification.

[0118] In shake flasks, four successfully induced engineered strains were co-cultured with baker's yeast to catalyze the production of 3′-SL. The reaction lasted for 24 hours. Quantitative analysis of the fermentation supernatant by HPLC revealed that the engineered strain E. coli JM109(DE3) / pETDeut-1-nst3-neuA produced the highest 3′-SL yield after 24 hours of fermentation, reaching a concentration of 21.8 g / L and a sialic acid conversion rate of 47.46% (Figure 13).

[0119] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for producing sialyllactose by dual-bacteria coupled fermentation, characterized in that: Sialyl lactose is synthesized by coupling fermentation of yeast with recombinant bacteria that co-express sialyltransferase and CMP-Neu5Ac synthase using sialic acid and lactose as substrates; the sialyltransferase is selected from α-2,6-sialyltransferase and / or α-2,3-sialyltransferase.

2. The method according to claim 1, characterized in that The recombinant bacteria uses Escherichia coli, yeast or Bacillus subtilis as a host.

3. The method according to claim 1, characterized in that The concentration of sialic acid is 60 mM-90 mM.

4. The method according to claim 1, wherein The concentration of the lactose is 80 mM-200 mM.

5. The method according to claim 1, wherein The amount of the recombinant bacteria co-expressing sialyltransferase and CMP-Neu5Ac synthetase added is 50 g / L-100 g / L.

6. The method according to claim 1, characterized in that The added amount of the yeast is 50g / L-250g / L.

7. The method according to claim 1, characterized in that The yeast is selected from one or more of baker's yeast, brewer's yeast and brewer's yeast.

8. The method according to claim 1, characterized in that The fermentation temperature is 20° C.-40° C.; the fermentation time is 10 h-30 h.

9. The method according to claim 1, characterized in that One or more of the following conditions are met: (1) The fermentation reaction system also contains 5mM-20mM Mg + ; (2) The fermentation reaction system also contains exogenously added cofactor CMP at a concentration of 10 mM to 50 mM; (3) The fermentation reaction system also contains octadecylamine polyoxyethylene ether or octadecylamine polyoxyethylene ether.

10. Use of sialyllactose produced by the method according to any one of claims 1 to 9 in the preparation of milk powder or nutritional supplements for infants and the elderly.

Images