Escherichia coli that produces lacto-n-tetraose, and dual-strain coupled fermentation method based thereon

Abstract

An Escherichia coli that produces lacto-N-tetraose, and a dual-strain coupled fermentation method based thereon. Firstly, an LNT biosynthetic pathway is constructed in Escherichia coli; then combined optimization is performed on a dual-plasmid system, four engineered strains are constructed, and an EP3 strain (containing galT1-SPase and galE-Cvβ3GalT expression cassettes) is screened out, which strain has the optimal LNT production capacity in coupled fermentation with yeast, wherein 25.2 g / L LNT can be produced with a molar conversion (mol / mol LNT II) of 71.22%. Subsequently, the production system is optimized, and the effects of UTP and UMP as cofactors on LNT synthesis are compared, wherein only the addition of 20 mM UMP as a cofactor can produce 29.4 g / L LNT with a molar conversion (mol / mol LNT II) of 83.09%. Finally, the EP3 strain is cultured in a fed-batch mode, and 39.6 g of LNT is produced after whole-cell catalysis. The present invention provides a new strategy for efficient and inexpensive production of LNT.

Description

Escherichia coli for producing lacto-N-tetraose and double bacterial coupling fermentation method based thereon TECHNICAL FIELD

[0001] The present application relates to an Escherichia coli for producing lacto-N-tetraose and a double bacterial coupling fermentation method based thereon, belonging to the technical field of biological fermentation. BACKGROUND

[0002] Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk after lactose and fat, with concentrations of 5-25 g / L. HMOs have prebiotic properties, enriching the gut of infants with beneficial bacteria such as bifidobacteria, can interfere with the adhesion of pathogens as host cell receptors, modulate the expression of intestinal surface glycans, affect cell growth and differentiation, and provide nutrition for the brain development and cognition of infants. So far, more than 200 structurally different HMOs have been found in human milk, which constitute the main difference between human milk and animal milk. Currently, at least 7 different HMOs have been approved by the Food and Drug Administration (FDA) as Generally Recognized as Safe (GRAS), including 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL), 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), lacto-N-neotetraose (LNnT) and lacto-N-tetraose (LNT). Six of them have been commercialized and added to various brands of infant formula.

[0003] Lacto-N-tetraose (LNT) is composed of galactose, N-acetylglucosamine and glucose, with the structure of Galβ1,3GlcNAcβ1,3Galβ1,4Glc. LNT is not only a basic component of HMOs, accounting for about 6% (w / w) of the total amount of HMOs, but also an important core structure that can be further fucosylated and sialylated to produce longer core structure chains. This structural diversity gives HMOs great commercial value and application potential in the food and pharmaceutical industries. The physiological effects of LNT have been determined, including prebiotic effects, prevention of necrotizing enterocolitis, anti-adhesion / anti-microbial activity, and anti-viral protection. It has been added to infant formula as a functional fortifier.

[0004] Currently, a large number of structurally diverse HMOs are mainly extracted from breast milk through complex procedures, but the content is extremely low and the purification process is complex; although the chemical synthesis method has developed, there are still several obstacles in the industrial scale synthesis of HMOs, namely the cumbersome protection and deprotection group steps, low total yield and the use of toxic reagents unsuitable for food; the problems of enzyme synthesis such as unbalanced transglycosylation reaction, low regioselectivity and the need for relatively expensive donor substrates make it impossible to synthesize many HMOs at a reasonable price. Biological synthesis of HMOs has the advantages of environmental friendliness, high specificity, renewable substrate, low cost, etc. In addition, with the development of metabolic engineering strategies, whole-cell biosynthesis has relatively mature scale-up production technology and process, making the biosynthesis of HMOs receive attention.

[0005] Several chemical syntheses of LNT and LNT derivatives have been reported, Mohamed R. E. Aly et al. designed two disaccharide linkages, introduced a dimethylmaleoyl (DMM) group as a new amino protecting group for glucosamine, and synthesized 0.018 g of LNT with a yield of 78% after multiple protection and deprotection steps; Wenlong Yao et al. reported a chemical enzymatic method, first using a one-pot two-enzyme method to produce lacto-N-biose (LNB), then acetylating LNB (Galβ1-3GlcNTFAβSEt), and then coupling with easily available lactose to produce about 65.8 mg of LNT with a conversion rate of 89%; both the reported chemical method and the chemical enzymatic method can only synthesize LNT in small scale and the steps are complicated and expensive. Yingying Zhu et al. based on the previously constructed LNT II producing strain, by strengthening the synthesis of UDP-Gal and introducing β-1,3-galactosyltransferase, 25.49 g / L of LNT was obtained with a molar conversion rate of 85.9%; Florian Baumgrtner et al. by integrating lgtA (β-1,3-n-acetylglucosamine transferase) and wbgO (β-1,3-galactosyltransferase), 219.1 mg / L of LNT was obtained; Miaomiao Hu et al. by overexpressing the double plasmid system lgtA-wbgO and galE-galT-galK genes and knocking out the UDP-Gal bypass related ugd gene, finally 31.56 g / L of LNT was produced. The above reported biological synthesis of LNT is to use glycerol as carbon source, through the intracellular multi-step metabolic pathway to obtain UDP-Gal, which cannot guarantee the timely and large supply of UDP-Gal.

[0006] An effective strategy for producing LNT is to conduct glycosylation mediated by glycosyltransferase. The practical application value of β-1, 3-GalT is good because the substrate UDP-Gal is an intermediate product of cell metabolism and is easy to accumulate through metabolic engineering. The synthesis of LNT can be realized by using β-1, 3-GalT as a glycosyltransferase, taking UDP-Gal as a donor and LNT II as a receptor. Therefore, sufficient supply of UDP-Gal is the key to realize the efficient synthesis of LNT. At present, there are usually two ways to obtain UDP-Gal: (1) Glc-1-P and UDP-Glc are obtained through the synergistic catalysis of UDP-glucose: galactose-1-phosphate uridyltransferase (GalT) and UDP-glucose-4-epimerase (GalE), (2) Glc-1-P and UTP are used as substrates to obtain through two-step catalysis of pyrophosphorylase (BLUSP) and UDP-glucose-4-epimerase (GalE), but which way is more suitable for the synthesis of LNT has not been reported so far. Therefore, the present application aims to explore how to produce LNT efficiently. SUMMARY

[0007] To solve the above problems, the present application adopts a double-plasmid co-expression strategy to construct a single strain, and the optimal strain is obtained through optimization and screening of the double-plasmid combination, and the strain is coupled with yeast bacteria for fermentation, and excellent performance is shown in the production of LNT.

[0008] The first object of the present application is to provide a recombinant Escherichia coli for producing lactose-N-tetrasaccharide, which contains a first gene expression frame and a second gene expression frame, the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridylyltransferase encoding gene galT, and the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT.

[0009] Further, the expression frame contained in the recombinant Escherichia coli is any one of the following combinations:

[0010] (1) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridylyltransferase encoding gene galT located after SPase, and the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located after galE;

[0011] (2) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT located after gene SPase, and the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located before gene galE;

[0012] (3) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT located before gene SPase, and the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located after gene galE;

[0013] (4) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT located before gene SPase, and the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located before gene galE.

[0014] Further, the nucleotide sequence of gene SPase is shown as SEQ ID NO. 1, the nucleotide sequence of gene galT is shown as SEQ ID NO. 2, the nucleotide sequence of gene galE is shown as SEQ ID NO. 5, and the nucleotide sequence of gene Cvβ3GalT is shown as SEQ ID NO. 6.

[0015] Further, the host bacteria of the recombinant E. coli can be common model strains or modified strains. For example, Escherichia coli BL21 (DE3), Escherichia coli JM109 (DE3), Escherichia coli K12 (DE3), Escherichia coli Nissle 1917 (DE3)△pMUT1-pMUT2△lacZ (GDMCC 63722), and the like.

[0016] Further, the genes in the first gene expression frame and / or the second gene expression frame are expressed by inducible promoters. For example, each gene is expressed by a T7 promoter.

[0017] Further, the first gene expression frame is located on a pRSFDuet-1 vector, and the second gene expression frame is located on a pETDuet-1 vector.

[0018] A second object of the present application is to provide a multi-bacterial coupling system, which comprises the recombinant E. coli and a yeast.

[0019] Further, the yeast comprises but is not limited to Saccharomyces cerevisiae, Saccharomyces pastorianus, etc., and preferably is Saccharomyces cerevisiae (GDMCC 61663).

[0020] Further, in the multi-bacterial coupling system, the ratio of the recombinant E. coli and the yeast is 1:0.5-1.5.

[0021] A third object of the present application is to provide an application of the recombinant E. coli or the multi-bacterial coupling system in preparing lacto-N-tetraose.

[0022] A fourth object of the present application is to provide a method for producing lacto-N-tetraose, which comprises fermenting by using the recombinant E. coli or the multi-bacterial coupling system.

[0023] Further, the fermentation system further comprises at least sucrose, UTP and / or UMP, and lacto-N-triose LNT II.

[0024] Further, the fermentation system comprises 100-1000 mmol / L sucrose, 10-100 mmol / L UTP / UMP, 10-100 mmol / L LNT II, 100-1000 mmol / L phosphate buffer (pH 7.0), 10-100 mmol / L MgCl2, 10-100 mL / L alcohol, 10-100 mmol / L DTT, and 1-50 g / L octadecylamine polyoxyethylene ether.

[0025] Further, the fermentation condition is 18-37℃ and 100-300 r / min.

[0026] In the process of catalytic synthesis of LNT, it is crucial to achieve sufficient supply of UDP-Gal. At present, it is known that one molecule of UDP-Gal is synthesized in two known synthesis pathways of UDP-Gal, and one molecule of UTP is consumed. Although the demand for whole-cell catalytic synthesis of UDP-Gal can be met by adding exogenous UTP, since the price of UTP is expensive (800 RMB / KG), the production of LNT by adding a large amount of exogenous UTP will result in the problem of expensive LNT. In view of the problem, how to use cells to realize efficient recycling of UTP / UDP at low cost is the key to high yield and low price of LNT. The present application compares the effects of UTP and UMP as cofactors on the synthesis of LNT, and when only 20 mM UMP is added as a cofactor, 29.4 g / L of LNT is generated, and the molar conversion rate (mol / mol LNT II) reaches 83.09%.

[0027] Further, when the fermentation is scale-up fermentation, the fermentation comprises two stages: (1) recombinant E. coli fermentation stage; (2) co-fermentation stage of recombinant E. coli and yeast.

[0028] Further, the recombinant E. coli fermentation stage comprises the following steps: fermentation at 32-40℃, with dissolved oxygen maintained at 15-25%; when the glucose content is less than 5 g / L, supplement the carbon source glucose at a flow rate of 10%; after OD 600 reaches 9-11, induce expression, and replace glucose with glycerol as a carbon source, and adjust the temperature to 25-30℃, and control the pH between 6.8-7.2 during the whole fermentation process.

[0029] Further, the co-fermentation stage of recombinant E. coli and yeast comprises the following steps: adding recombinant E. coli and yeast into the above fermentation system and performing fermentation under the above fermentation conditions (18-37℃, 100-300 r / min).

[0030] The beneficial effects of the present application are:

[0031] (1) Using sucrose as a substrate, a large amount of Glc-1-P is supplied by sucrose phosphorylase;

[0032] (2) The abilities of different sources of UDP-glucose-galactose-1-phosphate uridyltransferase (GalT1, GalT2), pyrophosphorylase (BLUSP) to synthesize UDP-Gal in cooperation with sucrose phosphorylase (SPase) and UDP-glucose-4-epimerase (GalE) are compared, and a combination with strong ability to synthesize UDP-Gal in cooperation is screened;

[0033] (3) For the regeneration of UTP, the yeast's own UTP regeneration system is considered to be used to achieve efficient recycling of UTP through coupling fermentation with engineered bacteria;

[0034] (4) In order to realize the sufficient circulation of UDP / UTP, the strategy of exogenous trace supplementing cofactor UMP / UTP is adopted. Considering that UTP (8000 RMB / KG) is more expensive than UMP (2000 RMB / KG), and the yeast's regeneration system can directly convert UMP to UTP, the effects of adding UTP and UMP as exogenous cofactors on the synthesis of LNT are compared, and the most cost-effective cofactor is selected for the synthesis of LNT. The results show that under the condition of adding yeast and exogenous trace cofactor UTP, the molar conversion rate of LNT synthesized by the engineered bacteria is higher than that without adding yeast; the yield of LNT synthesized by adding equal amounts of UMP and UTP is approximately the same.

[0035] Biological material preservation

[0036] Saccharomyces cerevisiae xpli, which has been preserved in Guangdong Microbial Culture Collection Center on May 12, 2021, with the preservation number GDMCC No:61663 and the preservation address being No. 59 Building, 5th Floor, Guangdong Institute of Microbiology, 100 Middle Martyrs Road, Guangzhou.

[0037] Escherichia coli EcN 03, which has been preserved in Guangdong Microbial Culture Collection Center on August 7, 2023, with the preservation number GDMCC No:63722 and the preservation address being No. 59 Building, 5th Floor, Guangdong Institute of Microbiology, 100 Middle Martyrs Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 is the result of single engineered bacteria and engineered bacteria-yeast coupling fermentation for producing LNT. Among them, (A) is the construction diagram of co-expressed strain; (B) is the SDS-PAGE diagram of co-expressed strain (C) is the synthesis of LNT by single co-expressed strain; (D) is the synthesis of LNT by co-expressed strain coupled with yeast.

[0039] Figure 2 is the effect of different factors on the synthesis of LNT by strain EP3 and yeast coupling. Among them, A: temperature; B: yeast concentration; C: phosphate buffer concentration; D: LNT II concentration; E: sucrose concentration; F: LNT yield before and after optimization.

[0040] Figure 3 is the synthesis of LNT by optimizing cofactors UMP and UTP. Among them, (A): UMP as cofactor; (B): UTP as cofactor.

[0041] Figure 4 is a fermentation curve of LNT produced by two-stage fermentation strategy. Wherein, (A) fed-batch fermentation of EP3 strain in 5L fermenter; (B) coupled fermentation of EP3 strain and yeast to synthesize LNT, blue arrow indicates starting feeding glucose, purple arrow indicates switching to feeding glycerol and adding inducer IPTG.

[0042] Figure 5 is TLC detection of LNT. Wherein, Figure 5A is the result of four bacteria coupled fermentation, 1-LNT II standard; 2-LNT standard; 3-5 are 6h, 12, 24h fermentation broth respectively; Figure 5B is the fermentation result of EP1-EP4, 1-LNT II standard; 2-LNT standard; 3-6 are EP1, EP2, EP3, EP4 fermentation broth respectively. DETAILED DESCRIPTION

[0043] The present application will be further described below in conjunction with the drawings and specific examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not as a limitation on the present application.

[0044] The materials and methods involved in the present application are as follows:

[0045] (1) LNT standard was given by Professor Yin Jingyu of Dalian Institute of Chemical Physics, LNT II standard was purchased from Rongcheng Huaihai Chuanda Biological Technology Co., Ltd. (Shandong, China), sucrose was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China), UMP and UTP were purchased from Hangzhou Gailong Biotechnology Co., Ltd. (Zhejiang, China). Molecular biology reagents were from Vazyme Co., Ltd. (Nanjing, China). Other chemical reagents were of the highest purity.

[0046] (2) Medium composition

[0047] Luria-Bertani (LB) medium: LB medium contains 5 g / L yeast extract, 10 g / L tryptone and 10 g / L sodium chloride.

[0048] Fermentation medium: contains 10 g / L casein, 5 g / L yeast extract, 10 g / L glycerol, 4 g / L lactose, 20 g / L glucose, 3.55 g / L Na2HPO4, 3.4 g / L KH2PO4, 2.68 g / L NH4Cl, 0.17 g / L Na2SO4, 3.55 g / L MgSO4. Supplement with antibiotics ampicillin (Amp) or kanamycin (Km), final concentration is 100 and 50 μg / mL respectively.

[0049] (3) Fermentation method

[0050] Shaking flask culture method of the engineering strain: the engineering E. coli cells were pre-cultured overnight at 37℃ in 10 mL LB liquid medium. Then the seed liquid was inoculated into 100 mL of LB medium at an inoculation amount of 2%, and cultured at 37℃ and 200 r / min. When the cell density (OD 600 ) reached 0.6 to 0.8, isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.3 mM, respectively. In addition, the culture was continued at 15℃ and 200 r / min for 20 hours. (This condition was used when all strains were cultured in a shaking flask)

[0051] (4) Detection method

[0052] TLC detection of Glc-1-P and UDP-Glc: developing agent: V(n-butanol):V(acetic acid):V(water)=2:1:1; TLC detection of LNT II and LNT: V(n-propanol):V(ammonia water):V(water)=5:2:1(35); color developing agent was anisaldehyde. The color developing method was: 165℃, 5 min.

[0053] HPLC detection of UDP-Glc and UDP-Gal conditions: chromatographic column was Inertil ODS-4 (3 μm, 4.6 x 150 mm), mobile phase was 100 mM potassium phosphate buffer (pH 6.4) containing 8 mM tetrabutylammonium hydrogen sulfate, flow rate was 1 mL / min, detection wavelength was 254 nm, column temperature was 40℃, injection volume was 10 μL.

[0054] HPLC quantitative analysis of LNT II and LNT conditions: chromatographic column was XAmide (5um, 4.6mm x 250mm), elution gradient: 0-20 min: 70% acetonitrile-60% acetonitrile, 20-30 min: 60% acetonitrile-70% acetonitrile, flow rate was 1 mL / min, detection wavelength was 214 nm, column temperature was 30℃, injection volume was 10 μL.

[0055] The molecular weight of LNT was analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0056] Example 1: Construction of recombinant strains

[0057] Table 1 lists all strains and plasmids used in the present application.

[0058] Table 1 Strains and plasmids

[0059] E. coli JM109(DE3) was used as the host for recombinant enzyme expression. Codon-optimized SPase (SEQ ID NO. 1), galT1 (SEQ ID NO. 2), galT2 (SEQ ID NO. 3), BLUSP (SEQ ID NO. 4), galE (SEQ ID NO. 5) and Cvβ3GalT (SEQ ID NO. 6) genes were constructed in the vector pET28a. Plasmids pET28a-SPase, pET28a-galT1, pET28a-galT2, pET28a-BLSUP, pET28a-galE, pET28a-Cvβ3GalT were transformed into E. coli JM109(DE3) respectively, and the engineering strains E1, E2, E3, E4, E5 and E6 were constructed. pRSFDuet-1 vector was used to construct recombinant plasmids for co-expression of SPase and galT encoding genes. pETDuet-1 vector was used to construct recombinant plasmids for co-expression of galE and Cvβ3GalT encoding genes. Recombinant plasmids pRSF-SPase-galT1, pRSF-galT1-SPase, pET-galE-Cvβ3GalT and pET-Cvβ3GalT-galE were constructed and transformed into E. coli JM109(DE3) respectively, and the engineering strains EP1, EP2, EP3 and EP4 were generated.

[0060] Example 2: Fermentation of LNT by recombinant strains EP1-EP4

[0061] The best engineering strain for LNT synthesis was obtained by comparing the LNT synthesis ability of each strain, and was used for subsequent fermentation optimization research. Fermentation conditions: 400 mmol / L sucrose, 50 mmol / L UTP, 50 mmol / L LNTII, 400 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L alcohol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L engineering bacteria (one of EP1, EP2, EP3 or EP4), 30°C, 200 r / min for 22 h. The results are shown in Figure 1C.

[0062] Example 3: Coupled fermentation for LNT production

[0063] The fermentation conditions of "engineered bacteria-yeast" coupling synthesis of LNT were as follows: 400 mmol / L sucrose, 50 mmol / L UTP, 50 mmol / L LNT II, 400 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L alcohol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L yeast (GDMCC 61663), 100 g / L engineered strain (one of EP1, EP2, EP3 or EP4), and fermentation was carried out at 30 °C and 200 r / min for 22 h. The results are shown in FIG. 1D.

[0064] As can be seen from FIGS. 1C and 1D, the LNT production of all four strains in the co-culture fermentation mode with yeast was higher than that of single strain fermentation, and the LNT production of strains EP1 and EP3 in the coupling fermentation with yeast was much higher than that of strains EP2 and EP4 in the coupling fermentation. It is worth mentioning that the LNT production of single strain EP1 was the highest, reaching 17.46 g / L, but after coupling fermentation with yeast, the LNT production of strain EP3 (containing plasmids pRSF-galT1-SPase and pET-galE-Cvβ3GalT) was higher than that of strain EP1, and 25.2 g / L of LNT was synthesized, with a molar conversion rate of 71.22%.

[0065] Example 4: Optimization of fermentation conditions for yeast-strain EP3 coupling fermentation for synthesis of LNT

[0066] In order to further improve the LNT production of the optimal strain EP3, the strain EP3 was re-induced, and the substrate sucrose and LNT II concentration, phosphate buffer concentration, yeast cell addition amount and temperature in the yeast-strain EP3 coupling fermentation mode were optimized by single factor optimization.

[0067] Temperature directly affects enzyme activity, and thus affects the synthesis of LNT. Under the determined optimal yeast cell addition amount, the LNT synthesis amount was determined at different reaction temperatures, and it was found that the LNT synthesis amount at 18 °C and 37 °C was not much different, but the LNT synthesis amount at 30 °C was the highest, reaching 22.4 g / L (2A).

[0068] The effect of yeast cell concentration on the LNT production in the reaction system was studied, and the LNT production increased with the increase of yeast strain concentration, which indicated that the regeneration and synthesis ability of yeast UTP directly affected the LNT yield. When the yeast addition amount in the co-culture system reached 100 g / L, the LNT production no longer increased significantly (2B), and in order to reduce the load of the fermentation system and realize the economic production of LNT, 100 g / L of yeast (GDMCC 61663) was selected as the optimal yeast concentration, and the highest LNT production reached 20.8 g / L.

[0069] After determining the amount of yeast and temperature conditions, the effect of phosphate concentration on LNT production was studied. When the concentration of added phosphate buffer was 500 mmol / L, the yield of LNT was the highest, reaching 30.4 g / L, so 500 mmol / L was selected as the optimal reaction concentration of phosphate buffer (Figure 2C).

[0070] Based on the optimized conditions, the effects of substrate sucrose and LNT II concentration on LNT synthesis were further explored. During the process of increasing the concentration of LNT II from 10 mmol / L to 90 mmol / L in the reaction system, the yield of LNT first increased and then decreased, with the highest yield of 31.8 g / L at an LNT II concentration of 50 mmol / L (Figure 2D). To achieve economic production of LNT, the optimal concentration of substrate LNT II was determined to be 50 mmol / L. The yield of LNT increased with increasing sucrose concentration, and when the sucrose concentration in the system reached 400 mmol / L, the yield of LNT no longer increased significantly (Figure 2E), with a yield of 33.45 g / L. Therefore, a sucrose concentration of 400 mmol / L was selected for LNT production. After optimization of the fermentation conditions, the yield of LNT increased from 20.8 g / L before optimization to 33.45 g / L (Figure 2F).

[0071] Example 5: Optimization of cofactors in coupled fermentation system

[0072] Considering that the price of UMP is lower than that of UTP, under the same conditions, the effects of adding equal amounts of UTP and UMP as exogenous cofactors on LNT synthesis were compared (Figure 3). Catalytic conditions: 400 mmol / L sucrose, UMP / UTP (10 mmol / L-40 mmol / L, 50 mmol / L LNT II, 500 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L alcohol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L yeast, 100 g / L engineered strain, fermentation at 30°C, 200 r / min for 22 h.

[0073] The results show that: the yield of LNT increases with the increase of UTP concentration, when the UTP concentration in the system reaches 30 mmol / L, the yield of LNT no longer increases obviously, the highest yield of LNT reaches 31.4 g / L, and the molar conversion rate is 88.75% (Figure 3B); in the fermentation system, replace UTP with UMP, when the UMP concentration increases from 10 mmol / L to 40 mmol / L, the yield of LNT first increases and then decreases, when the UMP concentration is 20 mmol / L, the yield of LNT is the highest, and the highest yield reaches 29.4 g / L, and the molar conversion rate is 83.09% (Figure 3A). Considering that the price of UMP is only 1 / 4 of UTP, and the effect of synthesizing LNT by adding UMP and UTP is similar, therefore, 20 mmol / L of UMP is selected as the optimal concentration of exogenous cofactor for the synthesis of LNT.

[0074] Example 6: Two-stage fermentation for synthesizing LNT in 7L fermentation tank

[0075] In order to realize large-scale synthesis of LNT, two-stage fermentation strategy was used to study the fermentation production of LNT. In the first stage, high-density fermentation strategy was used to realize high-density fermentation of LNT engineering strain; the second stage was LNT synthesis stage, and through co-culture of engineering strain EP3 and Saccharomyces cerevisiae, the fermentation synthesis of LNT was realized.

[0076] The specific steps are as follows:

[0077] The first stage of engineering strain culture: pick single colony of engineering strain and inoculate into 10 ml LB medium with corresponding antibiotic, cultivate at 37℃ for 12 h to obtain primary seed liquid, then inoculate the primary seed liquid into 250 ml LB medium with corresponding antibiotic at 2% inoculation amount, cultivate at 37℃ overnight to obtain secondary seed liquid, inoculate the secondary seed liquid into fermentation tank at 10% inoculation amount. The working volume of fermentation medium is 2.5 liters, the whole fermentation process is cultured at 37℃, the dissolved oxygen in the fermentation tank is maintained at 20%, and the stirrer with the maximum rotation speed of 600 r / min is automatically controlled. When the glucose content is less than 5 g / L, supplement the carbon source glucose at a flow rate of 10% (i.e. 1 min of feeding time per 10 min); add exogenous inducer IPTG (final concentration of 0.3 mmol / L) for induction after the logarithmic growth of engineering strain, at this time, replace glucose with glycerol as carbon source, and adjust the temperature to 28℃. The pH value is adjusted to 7.0 by 50% ammonia water during the whole fermentation process.

[0078] Second stage engineering bacteria-yeast co-culture production of LNT: the enrichment of the bacterial cells for whole-cell fermentation, fermentation conditions: 400 mmol / L sucrose, UMP (20 mmol / L), 50 mmol / L LNT II, 500 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L alcohol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L yeast, 100 g / L engineering strains, under the condition of 30℃, 200 r / min for 48 h.

[0079] The results are as follows:

[0080] The first stage, in 7L bioreactor using intermittent feeding strategy for fermentation of engineering strains EP3, in the growth phase using 300 g / L glucose for feeding, when the glucose concentration is less than 5 g / L start glucose feeding. In the induction expression control, when OD 600 10, add inducer IPTG, at this time use 300 g / L glycerol for feeding, the whole fermentation process curve as shown in Figure 4A. As can be seen from Figure 4A, 6h when the glucose concentration begins to be less than 5 g / L, fermentation continued to 12h, the bacterial concentration OD 600 10. After 32h fermentation, OD 600 reached a maximum, up to 31, corresponding to the amount of bacterial 42 g / L.

[0081] In the second stage of LNT fermentation synthesis phase, engineering bacteria and yeast co-culture fermentation time for 48h, sampling at different time points, centrifugation after taking the supernatant, using HPLC detection of LNT synthesis and LNT II consumption in fermentation (Figure 4B). From the co-culture fermentation LNT production gradually increased, at 42h LNT production reached a maximum, the maximum value of 13.2 g / L (total production of 39.6 g LNT), from 42h to 48h LNT concentration began to decrease, for the consumption of LNT II also gradually slowed after 42h. The molar conversion rate of LNT trend consistent with the synthesis of LNT trend, the maximum value of 37.31%.

[0082] Comparative Example 1 four bacteria coupling fermentation production of LNT

[0083] LNT was synthesized by four bacteria coupling fermentation of E1, E2 (E3 or E4), E5 and E6 constructed in Example 1: 100 mmol / L sucrose, 50 mmol / L UTP, 100 mmol / L phosphate buffer (pH 7.0), 50 mmol / L LNT II, 20 mmol / L MgCl2, 20 mL / L alcohol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 50 g / L engineering bacteria (E1, E2 / E3 / E4, E5 and E6), fermentation for 24 h (30℃, 200 r / min).

[0084] After the completion of coupling fermentation, the supernatant was obtained by centrifugation and subjected to TLC detection (E1, E2, E5 and E6 combination with the best performance in four bacteria coupling, and EP1, EP2, EP3, EP4 single bacteria fermentation), and the results are shown in Figure 5. In Figure 5, 3-5 in A are E1, E2, E5 and E6 four bacteria fermentation liquid at 6h, 12h and 24h, respectively, and 3-6 in B are EP1, EP2, EP3, EP4 single bacteria fermentation liquid, respectively. As can be seen from Figure 5, there is a substance with the same Rf as the standard LNT in the reaction liquid, which indicates that LNT is successfully synthesized by four bacteria coupling fermentation using sucrose, UDP-Glc and LNT II as substrates. However, the yield of LNT synthesized by four bacteria coupling fermentation strategy is not good, and the remaining amount of substrate LNT II is too much (Figure 5A), which is worse than the results of EP1-EP4 single bacteria fermentation (Figure 5B).

[0085] Obviously, the above examples are only examples for the sake of clarity, and are not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A recombinant Escherichia coli producing lacto-N-tetraose, characterized in that, The recombinant E. coli contains a first gene expression frame and a second gene expression frame, the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT, the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT.

2. The recombinant E. coli of claim 1, wherein, The expression frame contained in the recombinant E. coli is any one of the following combinations: (1) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT located after SPase, the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located after galE; (2) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT located after gene SPase, the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located before gene galE; (3) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT located before gene SPase, the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located after gene galE; (4) the first gene expression frame contains sucrose phosphorylase encoding gene SPase and UDP-glucose-hexose-1-phosphate uridyltransferase encoding gene galT located before gene SPase, the second gene expression frame contains UDP-glucose-4-epimerase encoding gene galE and β-1, 3-galactosyltransferase encoding gene Cvβ3GalT located before gene galE.

3. A multi-bacterial coupling system, characterized in that, The multi-microbial coupling system contains the recombinant E. coli of claim 1 or 2, and a yeast.

4. The multi-bacterial coupling system of claim 3, wherein, The yeast includes Saccharomyces cerevisiae and / or Saccharomyces pastorianus.

5. The multi-bacterial coupling system of claim 4, wherein, In the multi-microbial coupling system, the ratio of the recombinant E. coli to the yeast is 1:0.5-1.

5.

6. Use of the recombinant E. coli of claim 1 or 2 or the multi-microbial coupling system of any one of claims 3-5 in the preparation of lacto-N-tetraose.

7. A method for producing lacto-N-tetraose, characterized by, The method comprises the following steps: fermentation production by using the recombinant E. coli of claim 1 or 2 or the multi-microbial coupling system of any one of claims 3-5.

8. The method of claim 7, wherein, The fermentation system further contains at least sucrose, UTP and / or UMP, and lacto-N-triose.

9. The method of claim 7, wherein, The fermentation comprises two stages: a recombinant E. coli fermentation stage, and a co-fermentation stage of recombinant E. coli and yeast.

10. The method of claim 9, wherein, The fermentation stage of recombinant E. coli includes the following steps: fermentation at 32-40°C, dissolved oxygen maintained at 15-25%, when the glucose content is less than 5 g / L, supplement carbon source glucose at a flow rate of 10%; when OD 600 After reaching 9-11, induce expression, and replace glucose with glycerol as carbon source, temperature adjusted to 25-30°C, control pH at 6.8-7.2 throughout the fermentation process.

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