Escherichia coli for producing lacto-n-tetraose and a double-bacterial coupling fermentation method based thereon

Abstract

The application discloses an Escherichia coli for producing lactose-N-tetrasaccharide and a double-bacterium coupling fermentation method based on the same. The application firstly constructs an LNT biosynthesis pathway in the Escherichia coli; then a four-strain engineering bacterium is constructed by combining and optimizing a double-plasmid system, and an EP3 strain (containing a galT1-SP ase and galE-Cv beta3GalT expression frame) is screened out, and the coupling fermentation of the EP3 strain with yeast has the best LNT production capacity, and 25.2 g / L LNT can be produced, and the molar conversion rate (mol / mol LNT II) is 71.22%. Then, the production system is optimized, and the influence of UTP and UMP as cofactors on LNT synthesis is compared, and when only 20 mM UMP is added as a cofactor, 29.4 g / L LNT can be generated, and the molar conversion rate (mol / mol LNT II) is 83.09%. Finally, the EP3 strain is subjected to fed-batch culture, and 39.6 g LNT is produced after whole-cell catalysis. The application provides a new strategy for efficiently and cheaply producing LNT.

Description

Technical Field

[0001] This invention relates to an Escherichia coli that produces lactose-N-tetrasaccharide and a dual-strain coupled fermentation method based thereon, belonging to the field of biofermentation technology. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after lactose and fat, with a concentration of 5-25 g / L. HMOs possess prebiotic properties, enriching beneficial bacteria such as Bifidobacterium infantis in the gut. They can act as host cell receptors to interfere with pathogen adhesion, regulate the expression of intestinal surface glycans, influence cell growth and differentiation, and provide nutrition for infant brain development and cognition. To date, more than 200 structurally different HMOs have been identified in breast milk, constituting the main difference between breast milk and animal milk. Currently, at least seven different HMOs have been approved as Generally Recognized As Safe (GRAS) by the Food and Drug Administration (FDA), including 2'-fucosylated lactose (2'-FL), 3-fucosylated lactose (3-FL), difucosylated lactose (DFL), 3'-sialylated lactose (3'-SL), 6'-sialylated lactose (6'-SL), lactose-N-neotetrasaccharide (LNnT), and lactose-N-tetrasaccharide (LNT). Six of these have been commercially added to various brands of infant formula.

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

[0004] Currently, many structurally diverse HMOs are mainly extracted from breast milk through complex procedures, but the content is extremely low and the purification process is complicated. Although chemical synthesis methods have been developed, several obstacles remain in the industrial-scale synthesis of HMOs: cumbersome protection and deprotection steps, low overall yield, and the use of toxic reagents unsuitable for food. Enzymatic synthesis suffers from problems such as unbalanced transglycosylation reactions, low regioselectivity, and the need for relatively expensive donor substrates, making it impossible to synthesize many HMOs at a reasonable price. Biosynthetic HMOs, on the other hand, offer advantages such as environmental friendliness, high specificity, renewable substrates, and low cost. Furthermore, with the development of metabolic engineering strategies, whole-cell biosynthesis has relatively mature large-scale production technologies and processes, making the biosynthesis of HMOs a focus of attention.

[0005] Several chemical synthesis methods for LNT and LNT derivatives have been reported. Mohamed REAly et al. designed a linkage between two disaccharides and introduced a dimethylmaleyl (DMM) group as a new amino protecting group for glucosamine. After multiple protection and deprotection steps, they synthesized 0.018 g of LNT with a yield of 78%. Wenlong Yao et al. reported a chemoenzymatic method, which first produced lact-N-biose (LNB) using a one-pot two-enzyme method, then acetylated LNB (Galβ1-3GlcNTFAβSEt), and then coupled it with readily available lactose to produce approximately 65.8 mg of LNT with a conversion rate of 89%. Both the reported chemical and chemoenzymatic methods can only synthesize LNT on a small scale and are cumbersome and expensive. Yingying Zhu et al., based on a previously constructed LNTII-producing strain, obtained 25.49 g / L LNT by enhancing UDP-Gal synthesis and introducing β-1,3-galactosyltransferase, achieving 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), obtained 219.1 mg / L LNT. Miaomiao Hu et al., by overexpressing the dual-plasmid system lgtA-wbgO and galE-galT-galK genes and knocking out the ugd gene associated with the UDP-Gal bypass, ultimately produced 31.56 g / L LNT. All the reported biosynthetic methods for LNT use glycerol as the carbon source, obtaining UDP-Gal through multiple intracellular metabolic pathways, which cannot guarantee a timely and large-scale supply of UDP-Gal.

[0006] An effective strategy for producing LNTs is glycosyltransferase-mediated glycosylation. β-1,3-GalT has good practical application value because its substrate UDP-Gal is an intermediate product of cell metabolism and is easily accumulated through metabolic engineering. Using β-1,3-GalT as a glycosyltransferase, UDP-Gal as a donor, and LNT II as an acceptor, LNTs can be synthesized. Therefore, a sufficient supply of UDP-Gal is the key to achieving efficient LNT synthesis. Currently, there are usually two pathways for obtaining UDP-Gal: (1) Glc-1-P and UDP-Glc are obtained through the synergistic catalysis of UDP-glucose:galactose-1-phosphouresyltransferase (GalT) and UDP-glucose-4-epimerase (GalE); (2) Glc-1-P and UTP are obtained through two-step catalysis by pyrophosphorylase (BLUSP) and UDP-glucose-4-epimerase (GalE). However, which pathway is more suitable for LNT synthesis has not yet been reported. Therefore, this invention aims to explore how to efficiently produce LNTs. Summary of the Invention

[0007] To address the aforementioned issues, this invention employs a dual-plasmid co-expression strategy to construct a single strain. The optimal strain is obtained through optimized screening of dual-plasmid combinations and then coupled with yeast for dual-strain fermentation, demonstrating excellent performance in LNT production.

[0008] The first objective of this invention is to provide a recombinant *Escherichia coli* that produces lactose-N-tetrasaccharide, wherein the recombinant *E. coli* contains a first gene expression cassette and a second gene expression cassette, wherein the first gene expression cassette contains a sucrose phosphorylase encoding gene SPase and a UDP-glucose-hexose-1-phosphate uridine transferase encoding gene galT, and the second gene expression cassette contains a UDP-glucose-4-epimerase encoding gene galE and a β-1,3-galactosyltransferase encoding gene Cvβ3GalT.

[0009] Furthermore, the expression cassettes contained in the recombinant Escherichia coli are any combination of the following:

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

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

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

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

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

[0015] Furthermore, the host bacterium of the recombinant Escherichia coli can be a common type strain or a modified strain, such as Escherichia coli BL21(DE3), Escherichia colijJM109(DE3), Escherichiacoli K12(DE3), Escherichia coli Nissle 1917(DE3)△pMUT1-pMUT2△lacZ(GDMCC63722), etc.

[0016] Furthermore, 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] Furthermore, the first gene expression cassette is located on the pRSFDuet-1 vector, and the second gene expression cassette is located on the pETDuet-1 vector.

[0018] A second objective of this invention is to provide a multi-bacterial coupling system containing the recombinant Escherichia coli and yeast.

[0019] Furthermore, the yeast includes, but is not limited to, brewer's yeast, brewer's yeast, etc., preferably brewer's yeast (GDMCC 61663).

[0020] Furthermore, in the multi-strain coupling system, the ratio of recombinant Escherichia coli to yeast is 1:0.5-1.5.

[0021] A third objective of this invention is to provide the application of the recombinant Escherichia coli or multi-strain coupling system in the preparation of lactose-N-tetrasaccharides.

[0022] The fourth objective of this invention is to provide a method for producing lactose-N-tetrasaccharide, using the recombinant Escherichia coli or a multi-strain coupling system for fermentation production.

[0023] Furthermore, the fermentation system also contains at least sucrose, UTP and / or UMP, and lactose-N-trisaccharide LNT II.

[0024] Furthermore, the fermentation system contains 100-1000 mmol / L sucrose, 10-100 mmol / L LUTP / 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 ethanol, 10-100 mmol / L DTT, and 1-50 g / L octadecylamine polyoxyethylene ether.

[0025] Furthermore, the fermentation conditions are 18-37℃ and 100-300r / min.

[0026] Achieving a sufficient supply of UDP-Gal is crucial in the catalytic synthesis of LNTs. Currently, it is known that synthesizing one molecule of UDP-Gal requires consuming one molecule of UTP in both known pathways. While exogenous addition of UTP can meet the requirements for whole-cell catalytic synthesis of UDP-Gal, the high price of UTP (800 RMB / kg) leads to high LNT production costs. Therefore, utilizing cells to achieve efficient and inexpensive UTP / UDP recycling is key to high-yield, low-cost LNT production. This invention compares the effects of UTP and UMP as cofactors on LNT synthesis. Adding only 20 mM UMP as a cofactor yielded 29.4 g / L LNT, achieving a molar conversion rate (mol / mol LNT II) of 83.09%.

[0027] Furthermore, when the fermentation is a scaled-up fermentation, the fermentation includes two stages: (1) a recombinant Escherichia coli fermentation stage; and (2) a co-fermentation stage of recombinant Escherichia coli and yeast.

[0028] Furthermore, the fermentation stage of recombinant *E. coli* includes the following steps: fermentation at 32-40°C, with dissolved oxygen maintained at 15-25%; when the glucose content is below 5 g / L, glucose is added as a carbon source at a flow rate of 10%; when the OD... 600 After reaching 9-11, expression was induced, and glycerol was used instead of glucose as the carbon source. The temperature was adjusted to 25-30℃, and the pH was controlled between 6.8 and 7.2 throughout the fermentation process.

[0029] Furthermore, the co-fermentation stage of recombinant Escherichia coli and yeast includes the following steps: adding recombinant Escherichia coli and yeast to the above fermentation system and fermenting under the above fermentation conditions (18-37℃, 100-300r / min).

[0030] The beneficial effects of this invention are:

[0031] (1) Using sucrose as a substrate, a large supply of Glc-1-P was achieved by utilizing sucrose phosphorylase;

[0032] (2) The ability of UDP-glucose-galactose-1-phosphouresyltransferase (GalT1, GalT2) and pyrophosphorylase (BLUSP) from different sources to synthesize UDP-Gal in conjunction with sucrose phosphorylase (SPase) and UDP-glucose-4-epimerase (GalE) was compared, and combinations with strong ability to synergistically catalyze the synthesis of UDP-Gal were screened.

[0033] (3) For the regeneration of UTP, we consider using the yeast’s own UTP regeneration system to achieve efficient recycling and regeneration of UTP through coupled fermentation with engineered bacteria;

[0034] (4) To achieve a sufficient cyclic supply of UDP / UTP, a strategy of supplementing with exogenous trace amounts of cofactors UMP / UTP was adopted. Considering that UTP (8000 RMB / KG) is more expensive than UMP (2000 RMB / KG), and that the yeast regeneration system can directly convert UMP into UTP, the effects of adding UTP and UMP as exogenous cofactors on LNT synthesis were compared. The cofactor with the best cost-effectiveness was selected for LNT synthesis. The results showed that under the conditions of adding yeast and exogenous trace amount of cofactor UTP, the molar conversion rate of LNT synthesis by engineered bacteria was higher than that without yeast; the LNT synthesis yield was similar when exogenously adding equal amounts of UMP and UTP.

[0035] Preservation of biological materials

[0036] Saccharomyces cerevisiae xpli, which was deposited on May 12, 2021 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:61663, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province.

[0037] Escherichia coli EcN 03 was deposited on August 7, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 63722), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong Province. Attached Figure Description

[0038] Figure 1 The results show the production of LNTs by single engineered bacteria and engineered bacteria-yeast coupled fermentation. Among them, (A) is a diagram of the construction of the co-expression strain; (B) is an SDS-PAGE image of the co-expression strain; (C) LNT synthesis by a single co-expression strain; and (D) LNT synthesis by coupling the co-expression strain with yeast.

[0039] Figure 2 The effects of different factors on the coupled synthesis of LNT by strain EP3 and yeast were investigated. Where 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 3To optimize the synthesis of LNTs using cofactors UMP and UTP. Wherein, (A): UMP is used as a cofactor; (B): UTP is used as a cofactor.

[0041] Figure 4 Fermentation curves for LNT production using a two-stage fermentation strategy are shown. (A) EP3 strain was fed-batch fermented in a 5L fermenter; (B) EP3 strain and yeast were coupled for LNT synthesis. Blue arrows indicate the start of fed-batch glucose, and purple arrows indicate the switch to glycerol with the addition of the inducer IPTG.

[0042] Figure 5 For TLC detection of LNTs. Among them, Figure 5 A represents the results of four-strain coupled fermentation: 1-LNT II standard; 2-LNT standard; 3-5 are fermentation broths after 6h, 12h, and 24h, respectively. Figure 5 B represents the fermentation results of EP1 to EP4, 1-LNT II standard; 2-LNT standard; 3-6 represent the fermentation broths of EP1, EP2, EP3, and EP4, respectively. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0044] The materials and methods involved in this invention are as follows:

[0045] (1) LNT standard was donated by Professor Yan Jingyu of Dalian Institute of Chemical Physics; LNT II standard was purchased from Rongcheng Huihai Chuangda Biotechnology Co., Ltd. (Shandong, China); sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); and UMP and UTP were purchased from Hangzhou Kaipeng Biotechnology Co., Ltd. (Zhejiang, China). Molecular biology manipulation reagents were from Vazyme (Nanjing, China). All other chemical reagents were of the highest purity.

[0046] (2) Culture 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: containing 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, and 3.55 g / L MgSO₄. Supplemented with antibiotics ampicillin (Amp) or kanamycin (Km) at final concentrations of 100 and 50 μg / mL, respectively.

[0049] (3) Fermentation method

[0050] Erlenmeyer flask culture method for engineered strains: Engineered *E. coli* cells were pre-cultured overnight at 37°C in 10 mL LB broth. Then, the seed culture was inoculated at a rate of 2% into 100 mL LB broth and cultured at 37°C and 200 rpm. When the cell density (OD) reached... 600 When the concentration reached 0.6 to 0.8, isopropyl-β-D-1-thiogalactoside (IPTG) was added to a final concentration of 0.3 mM. Furthermore, the cultures were continued for 20 hours at 15°C and 200 rpm. (These conditions were used for all strains during shake-flask culture.)

[0051] (4) Detection method

[0052] Thin-layer chromatography (TLC) detection of Glc-1-P and UDP-Glc: developing solvent: V(n-butanol):V(acetic acid):V(water) = 2:1:1; TLC detection of LNT II and LNT: V(n-propanol):V(ammonia):V(water) = 5:2:1 (35); colorimetric reagent was anisaldehyde. Colorimetric method: 165℃, 5min.

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

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

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

[0056] Example 1: Construction of recombinant strains

[0057] Table 1 lists all the strains and plasmids used in this invention.

[0058] Table 1. Strains and plasmids

[0059]

[0060] Escherichia coli JM109(DE3) was used as the host for recombinase 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 pET28a vector. The plasmids pET28a-SPase, pET28a-galT1, pET28a-galT2, pET28a-BLSUP, pET28a-galE, and pET28a-Cvβ3GalT were transformed into E. coli JM109(DE3) to construct engineered strains E1, E2, E3, E4, E5, and E6. The pRSFDuet-1 vector was used to construct recombinant plasmids co-expressing the SPase and galT encoding genes. The pETDuet-1 vector was used to construct recombinant plasmids co-expressing the 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) to generate engineered strains EP1, EP2, EP3, and EP4, respectively.

[0061] Example 2: Fermentation production of LNT by recombinant strains EP1-EP4

[0062] By comparing the LNT synthesis capabilities of different strains, the engineered strain with the best LNT synthesis capability was obtained and used for subsequent fermentation optimization studies. Fermentation conditions: 400 mmol / L sucrose, 50 mmol / L LUTP, 50 mmol / L LNTII, 400 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L ethanol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L engineered bacteria (one of EP1, EP2, EP3, or EP4), fermented at 30℃ and 200 rpm for 22 h. Results are shown below. Figure 1 C.

[0063] Example 3: Coupled fermentation for LNT production

[0064] Fermentation conditions for LNT synthesis via engineered bacteria-yeast coupling: 400 mmol / L sucrose, 50 mmol / L LUTP, 50 mmol / L LNT II, ​​400 mmol / L phosphate buffer (pH 7.0), 20 mmol / L MgCl2, 20 mL / L ethanol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L yeast (GDMCC 61663), and 100 g / L engineered strain (one of EP1, EP2, EP3, or EP4), fermented at 30℃ and 200 rpm for 22 h. Results are shown below. Figure 1 D.

[0065] from Figure 1 As shown in C and 1D, the LNT synthesis yield of all four strains in the co-culture fermentation mode with yeast was higher than that of single-strain fermentation. Among them, the LNT synthesis yield of strains EP1 and EP3 coupled with yeast fermentation was much higher than that of strains EP2 and EP4 coupled with yeast fermentation. It is worth mentioning that the single-strain EP1 fermentation had the highest LNT synthesis yield of 17.46 g / L, but after coupled fermentation with yeast, strain EP3 (containing plasmids pRSF-galT1-SPase and pET-galE-Cvβ3GalT) produced a higher amount of LNT than strain EP1, synthesizing 25.2 g / L of LNT, with a molar conversion rate of 71.22%.

[0066] Example 4: Optimization of fermentation conditions for LNT synthesis via coupled fermentation of yeast strain EP3

[0067] To further improve the amount of LNT synthesized by the optimal strain EP3, strain EP3 was re-induced, and single-factor optimization was performed on the substrate sucrose and LNT II concentrations, phosphate buffer concentration, yeast cell addition amount, and temperature in the yeast-strain EP3 coupled fermentation mode.

[0068] Temperature directly affects enzyme activity, which in turn affects LNT synthesis. Based on the determined optimal yeast cell addition amount, the LNT synthesis amount was measured at different reaction temperatures. It was found that the LNT synthesis amount at 18℃ and 37℃ was not significantly different, but the LNT synthesis amount was the highest at 30℃, reaching 22.4 g / L (2A).

[0069] The effect of yeast cell concentration on LNT yield in the reaction system was studied. LNT yield increased with increasing yeast cell concentration, indicating that the regeneration and synthesis capacity of yeast UTP directly affects the LNT yield. When the yeast addition in the co-culture system reached 100 g / L, LNT yield no longer increased significantly (2B). To reduce the fermentation system load and achieve economical LNT production, an optimal yeast addition concentration of 100 g / L yeast (GDMCC 61663) was selected, at which the highest LNT yield reached 20.8 g / L.

[0070] After determining the yeast addition amount and temperature conditions, the effect of phosphate concentration on LNT production was studied. The highest LNT yield (30.4 g / L) was achieved when the added phosphate buffer concentration was 500 mmol / L. Therefore, 500 mmol / L was selected as the optimal reaction concentration for the phosphate buffer. Figure 2 C).

[0071] Based on optimized conditions, the effects of substrate sucrose and LNT II concentration on LNT synthesis were further explored. As the LNT II concentration added to the reaction system increased from 10 mmol / L to 90 mmol / L, the LNT yield first increased and then decreased, reaching its highest at a concentration of 50 mmol / L, with a maximum yield of 31.8 g / L. Figure 2 D) To achieve economical LNT production, the optimal concentration of substrate LNT II was determined to be 50 mmol / L. LNT yield increased with increasing sucrose concentration, but when the sucrose concentration reached 400 mmol / L, LNT yield no longer increased significantly. Figure 2 E), the yield reached 33.45 g / L, therefore a sucrose concentration of 400 mmol / L was selected for LNT production. After optimizing the fermentation conditions, the LNT yield increased from 20.8 g / L to 33.45 g / L. Figure 2 F).

[0072] Example 5: Optimization of cofactors in coupled fermentation systems

[0073] Considering that UMP is cheaper than UTP, the effects of adding equal amounts of UTP and UMP as exogenous cofactors on LNT synthesis were compared under the same conditions. Figure 3Catalytic 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 ethanol, 20 mmol / L LTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L yeast, 100 g / L engineered strain, fermented at 30℃ and 200 rpm for 22 h.

[0074] The results showed that LNT production increased with increasing UTP concentration. When the UTP concentration reached 30 mmol / L, LNT production no longer increased significantly. The highest LNT production reached 31.4 g / L, with a molar conversion rate of 88.75%. Figure 3 B); In the fermentation system, when UTP was replaced with UMP, the LNT yield first increased and then decreased as the UMP concentration increased from 10 mmol / L to 40 mmol / L. The highest LNT yield was achieved at a UMP concentration of 20 mmol / L, reaching 29.4 g / L, with a molar conversion rate of 83.09%. Figure 3 A). Considering that UMP is only 1 / 4 the price of UTP, and that the effects of exogenous addition of UMP and UTP in the synthesis of LNT are similar, 20 mmol / L of UMP was chosen as the optimal concentration of exogenous cofactor for the synthesis of LNT.

[0075] Example 6: Horizontal two-stage fermentation synthesis of LNT in a 7L fermenter

[0076] To achieve large-scale synthesis of LNT, a two-stage fermentation strategy was adopted for LNT fermentation production research. In the first stage, a high-density fermentation strategy was used to achieve high-density fermentation of the engineered strain for LNT synthesis; the second stage was the LNT synthesis stage, which was achieved by co-culturing the engineered strain EP3 with Saccharomyces cerevisiae to achieve LNT fermentation synthesis.

[0077] The specific steps are as follows:

[0078] The first stage of engineered strain cultivation: Single colonies of the engineered strain were inoculated into 10 ml of LB medium containing the corresponding antibiotic and cultured at 37°C for 12 h to obtain the primary seed culture. The primary seed culture was then transferred at a 2% inoculation rate to 250 ml of LB medium containing the corresponding antibiotic and cultured overnight at 37°C to obtain the secondary seed culture. The secondary seed culture was then inoculated into the fermenter at a 10% inoculation rate. The working volume of the fermentation medium was 2.5 liters. The entire fermentation process was carried out at 37°C, with dissolved oxygen maintained at 20%. The fermenter was automatically controlled by a stirrer with a maximum speed of 600 rpm. When the glucose content was below 5 g / L, glucose was added as the carbon source at a flow rate of 10% (i.e., a 1-minute feed interval every 10 minutes). During the late logarithmic growth phase of the engineered strain, exogenous inducer IPTG (final concentration 0.3 mmol / L) was added for induction. At this time, glycerol was used instead of glucose as the carbon source, and the temperature was adjusted to 28°C. Throughout the fermentation process, the pH was maintained at 7.0 using 50% ammonia.

[0079] The second stage involves co-culturing engineered bacteria and yeast to produce LNT: the enriched bacterial cells are used for whole-cell fermentation. The fermentation conditions are: 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 ethanol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, 100 g / L yeast, and 100 g / L engineered bacterial strain. Fermentation is carried out at 30℃ and 200 r / min for 48 h.

[0080] The results are as follows:

[0081] In the first stage, the engineered strain EP3 was fermented in a 7L bioreactor using a fed-batch strategy. During the growth phase, 300 g / L glucose was fed, and glucose feeding was initiated when the glucose concentration fell below 5 g / L. Regarding the control of induced expression, when OD... 600 Once the concentration reaches 10, add the inducing agent IPTG. At this point, switch to feeding with 300 g / L glycerol. The entire fermentation process curve is shown below. Figure 4 As shown in A. From Figure 4 As can be seen from A, the glucose concentration began to fall below 5 g / L at 6 h, and when fermentation continued until 12 h, the bacterial concentration OD 600 The OD reached 10. After fermentation for 32 hours, the OD... 600 It reached its maximum value, which was 31, corresponding to a bacterial count of 42 g / L.

[0082] In the second stage of LNT fermentation and synthesis, the engineered bacteria and yeast were co-cultured for 48 hours. Samples were taken at different time points, centrifuged, and the supernatant was collected. HPLC was used to detect the synthesis of LNT and the consumption of LNT II during fermentation. Figure 4 (B) From the start of co-culture fermentation, LNT production gradually increased, reaching a maximum of 13.2 g / L at 42 h (producing a total of 39.6 g of LNT). From 42 h to 48 h, the LNT concentration began to decrease, and the consumption of LNT II also gradually slowed down after 42 h. The trend of LNT molar conversion was consistent with the trend of LNT synthesis, with a maximum value of 37.31%.

[0083] Comparative Example 1: Production of LNT via Four-Strain Coupled Fermentation

[0084] LNT was synthesized by coupled fermentation of four strains (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 ethanol, 20 mmol / L DTT, 8 g / L octadecylamine polyoxyethylene ether, and 50 g / L engineered bacteria (E1, E2 / E3 / E4, E5 and E6), fermented for 24 h (30 °C, 200 r / min).

[0085] After the coupled fermentation was completed, the reaction solution was centrifuged, and the supernatant was collected for TLC analysis (the optimal performance of the four-strain coupled combinations E1, E2, E5, and E6, as well as single-strain fermentation of EP1, EP2, EP3, and EP4). The results are as follows: Figure 5 As shown, in section A, 3-5 represent the fermentation broths of four bacteria (E1, E2, E5, and E6) at 6h, 12h, and 24h, respectively; in section B, 3-6 represent the single-culteria fermentation broths of EP1, EP2, EP3, and EP4, respectively. Figure 5 It can be seen that the reaction solution contains substances identical to the standard LNT Rf. This result indicates that LNT was successfully synthesized using sucrose, UDP-Glc, and LNT II as substrates via four-strain coupled fermentation. However, the four-strain coupled fermentation strategy resulted in poor LNT yield, with excessive residual amount of substrate LNT II. Figure 5 A), worse than the results of single-strain fermentation of EP1-EP4 ( Figure 5 B).

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A co-fermentation microbial ensemble with multiple coupled microorganisms, characterized in that, The co-fermentation bacteria combination contains: recombinant Escherichia coli and yeast; The recombinant Escherichia coli contains a first gene expression cassette and a second gene expression cassette. The first gene expression frame contains a gene encoding sucrose phosphorylase. SPase and located in genes SPase The previous UDP-glucose-hexose-1-phosphate uridine transferase encoding gene galT The second gene expression cassette contains a gene encoding UDP-glucose-4-epimerase. galE and located in genes galE The subsequent β-1,3-galactosyltransferase encoding gene Cvβ3GalT, Gene SPase The nucleotide sequence is shown in SEQ ID NO.1, gene. galT The nucleotide sequence is shown in SEQ ID NO.2, gene. galE The nucleotide sequence is shown in SEQ ID NO.5, gene. Cvβ3GalT The nucleotide sequence is shown in SEQ ID NO. 6, and the yeast is Saccharomyces cerevisiae.

2. The co-fermentation microbial combination according to claim 1, characterized in that, In the co-fermentation bacterial combination, the ratio of recombinant Escherichia coli to yeast is 1:0.5-1.

5.

3. The use of the co-fermentation bacteria combination according to claim 1 or 2 in the preparation of lactose-N-tetrasaccharide.

4. A method for producing lactose-N-tetrasaccharide, characterized in that, The process includes the following steps: fermentation production using the co-fermentation bacteria combination described in claim 1 or 2.

5. The method according to claim 4, characterized in that, The fermentation system also contains at least sucrose, UTP and / or UMP, and lactose-N-trisaccharide.

6. The method according to claim 4, characterized in that, The fermentation process includes two stages: a recombinant Escherichia coli fermentation stage and a co-fermentation stage of recombinant Escherichia coli and yeast.

7. The method according to claim 6, characterized in that, The fermentation stage of recombinant *E. coli* includes the following steps: fermentation at 32-40°C, dissolved oxygen maintained at 15-25%, and when the glucose content is below 5 g / L, glucose is added as a carbon source at a flow rate of 10%; when OD... 600 After reaching 9-11, expression was induced, and glycerol was used instead of glucose as the carbon source. The temperature was adjusted to 25-30 °C, and the pH was controlled between 6.8 and 7.2 throughout the fermentation process.

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