WO2025218244A1 - 一种生产乳糖-n-四糖的大肠杆菌及基于其的双菌耦合发酵方法 - Google Patents

一种生产乳糖-n-四糖的大肠杆菌及基于其的双菌耦合发酵方法

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WO2025218244A1
WO2025218244A1 PCT/CN2024/142146 CN2024142146W WO2025218244A1 WO 2025218244 A1 WO2025218244 A1 WO 2025218244A1 CN 2024142146 W CN2024142146 W CN 2024142146W WO 2025218244 A1 WO2025218244 A1 WO 2025218244A1
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lnt
gene
glucose
escherichia coli
encoding gene
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张洪涛
赵建新
刘登峰
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Jiangnan University
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Definitions

  • the invention relates to an Escherichia coli for producing lactose-N-tetraose and a dual-bacteria coupled fermentation method based thereon, belonging to the technical field of biological fermentation.
  • HMOs Human milk oligosaccharides
  • Bifidobacterium infantis possess prebiotic properties, enriching beneficial bacteria in the gut, such as Bifidobacterium infantis. They can also act as host cell receptors to interfere with pathogen adhesion, regulate the expression of intestinal surface glycans, influence cell growth and differentiation, and provide nutrients for infant brain development and cognition.
  • HMOs have been identified in breast milk, constituting the primary difference between breast milk and animal milk.
  • HMOs Generally Recognized as Safe
  • FDA Food and Drug Administration
  • DFL difucosyllactose
  • 3'-SL 3'-sialyllactose
  • 6'-SL 6'-sialyllactose
  • LNnT lacto-N-neotetraose
  • LNT lacto-N-tetraose
  • Lactose-N-tetraose is composed of galactose, N-acetylglucosamine, and glucose, with the structure Gal ⁇ 1,3GlcNAc ⁇ 1,3Gal ⁇ 1,4Glc.
  • LNT is not only a fundamental component of HMOs, comprising approximately 6% (w/w) of the total HMO population, but also serves as a crucial core structure that can be further fucosylated and sialylated to produce longer core chains. This structural diversity gives HMOs enormous commercial value and potential for application in the food and pharmaceutical industries.
  • the physiological effects of LNT have been determined, including prebiotic effects, prevention of necrotizing enterocolitis, antiadhesion/antimicrobial activity, and antiviral protection. It has been added to infant formula as a functional enhancer.
  • HMOs a large number of structurally diverse HMOs are primarily extracted from breast milk through complex procedures, but their content is extremely low and the purification process is complex.
  • industrial-scale HMO synthesis still faces several obstacles, including tedious steps for protecting and deprotecting groups, low overall yields, and the use of toxic reagents unsuitable for food.
  • Enzymatic synthesis suffers from unbalanced transglycosylation reactions, low regioselectivity, and the requirement for relatively expensive donor substrates, making the synthesis of many HMOs uneconomical.
  • Biosynthetic HMOs offer advantages such as environmental friendliness, high specificity, renewable substrates, and low cost.
  • whole-cell biosynthesis has become a relatively mature, scalable production method, attracting significant attention to the biosynthesis of HMOs.
  • LNB was then acetylated (Gal ⁇ 1-3GlcNTFA ⁇ SEt) and coupled 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 the steps are cumbersome and expensive.
  • LNT glycosyltransferase-mediated glycosylation.
  • ⁇ -1,3-GalT has considerable practical application potential because its substrate, UDP-Gal, is an intermediate in cellular metabolism and can be easily accumulated through metabolic engineering approaches.
  • LNT synthesis can be achieved using ⁇ -1,3-GalT as a glycosyltransferase, UDP-Gal as a donor, and LNT II as an acceptor. Therefore, an adequate supply of UDP-Gal is crucial for efficient LNT synthesis.
  • Glc-1-P and UDP-Glc are produced through the coordinated catalysis of UDP-glucose:galactose-1-phosphate ureyltransferase (GalT) and UDP-glucose-4-epimerase (GalE); and (2) Glc-1-P and UTP are used as substrates and produced through a two-step catalysis of pyrophosphorylase (BLUSP) and UDP-glucose-4-epimerase (GalE).
  • BUSP pyrophosphorylase
  • GalE UDP-glucose-4-epimerase
  • the present invention adopts a dual-plasmid co-expression strategy to construct a single strain, obtains the optimal strain through optimization screening of the dual-plasmid combination, and conducts dual-bacteria coupled fermentation with yeast, which performs excellently in the production of LNT.
  • the first object of the present invention is to provide a recombinant Escherichia coli for producing lactose-N-tetraose, wherein the recombinant Escherichia 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 uridyl transferase encoding gene galT, and the second gene expression cassette contains a UDP-glucose-4-epimerase encoding gene galE and a ⁇ -1,3-galactosyltransferase encoding gene Cv ⁇ 3GalT.
  • the expression cassette contained in the recombinant Escherichia coli is any one of the following combinations:
  • the first gene expression frame contains the sucrose phosphorylase encoding gene SPase and the UDP-glucose-hexose-1-phosphate uridyl transferase encoding gene galT located after SPase
  • the second gene expression frame contains the UDP-glucose-4-epimerase encoding gene galE and the ⁇ -1,3-galactosyltransferase encoding gene Cv ⁇ 3GalT located after galE;
  • the first gene expression frame contains the sucrose phosphorylase encoding gene SPase and the UDP-glucose-hexose-1-phosphate uridyl transferase encoding gene galT located after the SPase gene
  • the second gene expression frame contains the UDP-glucose-4-epimerase encoding gene galE and the ⁇ -1,3-galactosyltransferase encoding gene Cv ⁇ 3GalT located before the galE gene
  • the first gene expression frame contains the sucrose phosphorylase encoding gene SPase and the UDP-glucose-hexose-1-phosphate uridyl transferase encoding gene galT located before the SPase gene
  • the second gene expression frame contains the UDP-glucose-4-epimerase encoding gene galE and the ⁇ -1,3-galactosyltransferase encoding gene Cv ⁇ 3GalT located after the galE gene;
  • the first gene expression frame contains the sucrose phosphorylase encoding gene SPase and the UDP-glucose-hexose-1-phosphate uridyl transferase encoding gene galT located before the gene SPase
  • the second gene expression frame contains the UDP-glucose-4-isomerase encoding gene galE and the ⁇ -1,3-galactosyl transferase encoding gene Cv ⁇ 3GalT located before the gene galE.
  • nucleotide sequence of gene SPase is shown as SEQ ID NO.1
  • nucleotide sequence of gene galT is shown as SEQ ID NO.2
  • nucleotide sequence of gene galE is shown as SEQ ID NO.5
  • nucleotide sequence of gene Cv ⁇ 3GalT is shown as SEQ ID NO.6.
  • the host bacteria of the recombinant Escherichia coli can be a common model strain or a modified strain, such as Escherichia coli BL21 (DE3), Escherichia coli JM109 (DE3), Escherichia coli K12 (DE3), Escherichia coli Nissle 1917 (DE3) ⁇ pMUT1-pMUT2 ⁇ lacZ (GDMCC 63722), etc.
  • genes in the first gene expression cassette and/or the second gene expression cassette are expressed by an inducible promoter, such as each gene is expressed by a T7 promoter.
  • the first gene expression cassette is located on the pRSFDuet-1 vector
  • the second gene expression cassette is located on the pETDuet-1 vector.
  • the second object of the present invention is to provide a multi-bacteria coupling system, wherein the multi-bacteria coupling system contains: the recombinant Escherichia coli and yeast.
  • yeast includes but is not limited to cerevisiae, brewer's yeast, etc., preferably cerevisiae (GDMCC 61663).
  • the usage ratio of the recombinant Escherichia coli and the yeast is 1:0.5-1.5.
  • the third object of the present invention is to provide the use of the recombinant Escherichia coli or the multi-bacteria coupling system in the preparation of lactose-N-tetraose.
  • the fourth object of the present invention is to provide a method for producing lactose-N-tetraose, which uses the recombinant Escherichia coli or multi-bacteria coupling system for fermentation production.
  • the fermentation system contains at least sucrose, UTP and/or UMP, and lactose-N-trisaccharide LNT II.
  • the fermentation system contains 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 MgCl 2 , 10-100 mL/L alcohol, 10-100 mmol/L DTT, and 1-50 g/L octadecylamine polyoxyethylene ether.
  • the fermentation conditions are 18-37° C., 100-300 r/min.
  • the present invention compares the effects of UTP and UMP as cofactors on LNT synthesis.
  • 20mM UMP is added as a cofactor, 29.4g/L LNT can be generated, and its molar conversion rate (mol/mol LNT II) reaches 83.09%.
  • the fermentation when the fermentation is an amplified fermentation, the fermentation includes two stages: (1) a recombinant E. coli fermentation stage; and (2) a recombinant E. coli and yeast co-fermentation stage.
  • the recombinant E. coli fermentation stage includes the following steps: fermentation at 32-40°C, with dissolved oxygen maintained at 15-25%; when the glucose content is lower than 5g/L, supplementing the carbon source glucose at a flow rate of 10%; inducing expression when the OD600 reaches 9-11, and replacing glucose with glycerol as the carbon source, adjusting the temperature to 25-30°C, and controlling the pH between 6.8-7.2 during the entire fermentation process.
  • the co-fermentation stage of the recombinant E. coli and yeast comprises the following steps: adding the recombinant E. coli and yeast into the above fermentation system and fermenting under the above fermentation conditions (18-37° C., 100-300 r/min).
  • sucrose phosphorylase was used to achieve a large supply of Glc-1-P;
  • Brewer's yeast (Saccharomyces cerevisiae) xpli was deposited in the Guangdong Provincial Microbial Strain Collection Center on May 12, 2021, with the deposit number GDMCC No: 61663, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong province.
  • Escherichia coli Escherichia coli
  • EcN 03 was deposited in Guangdong Provincial Microbiological Strain Collection Center on August 7, 2023, with the deposit number GDMCC No: 63722, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangdong province.
  • Figure 1 shows the results of LNT production using a single engineered bacterium and a coupled engineered bacterium-yeast fermentation.
  • A Construction diagram of the co-expression strain
  • B SDS-PAGE image of the co-expression strain
  • C LNT production using a single co-expression strain
  • D LNT production using a co-expression strain coupled with yeast.
  • Figure 2 shows the effects of different factors on the coupled LNT synthesis by strain EP3 and yeast.
  • Figure 3 shows the optimization of cofactors UMP and UTP for LNT synthesis.
  • A UMP as a cofactor
  • B UTP as a cofactor.
  • Figure 4 shows the fermentation curves for LNT production using a two-stage fermentation strategy.
  • A Fed-batch fermentation of the EP3 strain in a 5-L fermentor;
  • B LNT production by coupled fermentation of the EP3 strain and yeast.
  • the blue arrow indicates the start of glucose feeding, and the purple arrow indicates the switch to glycerol feeding and the addition of the inducer IPTG.
  • Figure 5 shows the TLC analysis of LNT.
  • Figure 5A shows the results of a four-bacteria coupled fermentation: 1: LNT II standard; 2: LNT standard; 3-5: fermentation broths from 6 h, 12 h, and 24 h, respectively.
  • Figure 5B shows the results of EP1-EP4 fermentations: 1: LNT II standard; 2: LNT standard; 3-6: fermentation broths from EP1, EP2, EP3, and EP4, respectively.
  • LNT standard was a gift from 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).
  • UMP and UTP were purchased from Hangzhou Kaipeng Biotechnology Co., Ltd. (Zhejiang, China).
  • Molecular biology reagents were obtained from Vazyme (Nanjing, China). All other chemical reagents were of the highest purity.
  • Luria-Bertani (LB) medium contains 5 g/L yeast extract, 10 g/L tryptone, and 10 g/L sodium chloride.
  • 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 Na 2 HPO 4 , 3.4 g/L KH 2 PO 4 , 2.68 g/L NH 4 Cl, 0.17 g/L Na 2 SO 4 , 3.55 g/LMgSO 4 . Supplemented with antibiotics ampicillin (Amp) or kanamycin (Km) to final concentrations of 100 and 50 ⁇ g/mL, respectively.
  • Amicillin ampicillin
  • Km kanamycin
  • E. coli cells were pre-cultured in 10 mL of LB liquid medium at 37°C overnight. The seed solution was then inoculated into 100 mL of LB medium at a 2% inoculum and cultured at 37°C and 200 r/min. When the cell density (OD 600 ) reached 0.6 to 0.8, isopropyl- ⁇ -D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.3 mM. In addition, the culture was continued at 15°C and 200 r/min for 20 hours. (These conditions were used for all strains when shake flask cultured)
  • IPTG isopropyl- ⁇ -D-1-thiogalactopyranoside
  • TLC Thin layer chromatography
  • HPLC detection conditions for UDP-Glc and UDP-Gal chromatographic column: Inertil ODS-4 (3 ⁇ m, 4.6x 150mm), mobile phase: 100mM potassium phosphate buffer (pH6.4) containing 8mM tetrabutylammonium hydrogen sulfate, flow rate: 1mL/min, detection wavelength: 254nm, column temperature: 40°C, injection volume: 10 ⁇ L.
  • the molecular weight of LNT was analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF-MS).
  • Table 1 lists all strains and plasmids used in the present invention.
  • 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 Cv ⁇ 3GalT (SEQ ID NO. 6) genes were constructed in the pET28a vector. Plasmids pET28a-SPase, pET28a-galT1, pET28a-galT2, pET28a-BLSUP, pET28a-galE, and pET28a-Cv ⁇ 3GalT were transformed into E.
  • 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 Cv ⁇ 3GalT genes.
  • Recombinant plasmids pRSF-SPase-galT1, pRSF-galT1-SPase, pET-galE-Cv ⁇ 3GalT, and pET-Cv ⁇ 3GalT-galE were constructed and transformed into Escherichia coli JM109(DE3) to generate engineered strains EP1, EP2, EP3, and EP4, respectively.
  • Example 2 Fermentation production of LNT by recombinant strains EP1 to EP4
  • the engineered strain with the best LNT-synthesis ability was identified and used for subsequent fermentation optimization studies.
  • 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 ethanol, 20 mmol/L DTT, 8 g/L octadecylamine polyoxyethylene ether, and 100 g/L engineered bacteria (one of strains EP1, EP2, EP3, or EP4). Fermentation was performed at 30°C and 200 rpm for 22 hours. The results are shown in Figure 1C.
  • the fermentation conditions for LNT synthesis using the engineered bacteria-yeast coupling 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 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). Fermentation was performed at 30°C and 200 rpm for 22 h. The results are shown in Figure 1D.
  • strain EP1 produced the highest LNT yield of 17.46 g/L when fermented alone.
  • strain EP3 containing the plasmids pRSF-galT1-SPase and pET-galE-Cv ⁇ 3GalT
  • strain EP1 produced the highest LNT yield of 17.46 g/L when fermented alone.
  • strain EP3 (containing the plasmids pRSF-galT1-SPase and pET-galE-Cv ⁇ 3GalT) produced even more LNT than strain EP1 when coupled with yeast, producing 25.2 g/L of LNT with a molar conversion rate of 71.22%.
  • Example 4 Optimization of fermentation conditions for LNT synthesis by yeast-strain EP3 coupled fermentation
  • strain EP3 was re-induced, and the single-factor conditions of substrate sucrose and LNT II concentrations, phosphate buffer concentration, yeast cell addition amount and temperature in the yeast-strain EP3 coupled fermentation mode were optimized.
  • LNT synthesis was measured at different reaction temperatures. It was found that LNT synthesis at 18°C and 37°C was similar, but LNT synthesis peaked at 30°C, reaching 22.4 g/L (2A).
  • LNT production increased with increasing yeast strain concentration, indicating that the yeast's ability to regenerate UTP directly influences LNT yield.
  • yeast addition level reached 100 g/L in the co-culture system, LNT production no longer significantly increased (2B).
  • an optimal yeast addition level of 100 g/L was selected, achieving a maximum LNT yield of 20.8 g/L.
  • Example 6 Synthesis of LNT by two-stage horizontal fermentation in a 7 L fermenter
  • LNT production To achieve large-scale LNT synthesis, a two-stage fermentation strategy was employed for LNT production. In the first stage, a high-density fermentation strategy was used to achieve high-density fermentation of the engineered strain for LNT synthesis. In the second stage, LNT synthesis was achieved by co-culturing the engineered strain EP3 with Saccharomyces cerevisiae.
  • Phase I Cultivation of the engineered strain: A single colony of the engineered strain was inoculated into 10 ml of LB medium supplemented with the appropriate antibiotic and incubated 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 supplemented with the appropriate antibiotic and incubated 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 fermentation medium had a working volume of 2.5 liters and was maintained at 37°C throughout the entire fermentation process.
  • the dissolved oxygen in the fermentor was maintained at 20%, and the fermentor was automatically agitated at a maximum speed of 600 rpm.
  • glucose was added as a carbon source at a 10% flow rate (i.e., a 1-minute feed interval every 10 minutes).
  • Induction of the engineered strain was achieved by adding exogenous inducer IPTG (final concentration 0.3 mmol/L) during the late logarithmic growth phase.
  • Glycerol was used as the carbon source instead of glucose, and the temperature was maintained at 28°C.
  • the pH was maintained at 7.0 with 50% ammonia throughout the fermentation process.
  • the second stage is the co-cultivation of engineered bacteria and yeast to produce LNT: the enriched bacteria are used for whole-cell fermentation.
  • the fermentation conditions are: 400mmol/L sucrose, UMP (20mmol/L), 50mmol/L LNT II, 500mmol/L phosphate buffer (pH 7.0), 20mmol/ LMgCl2 , 20mL/L alcohol, 20mmol/L DTT, 8g/L octadecylamine polyoxyethylene ether, 100g/L yeast, 100g/L engineered strain, and fermentation is carried out at 30°C and 200r/min for 48h.
  • the engineered strain EP3 was fermented in a 7L bioreactor using an intermittent feeding strategy.
  • 300g/L glucose was used for feeding during the growth phase, and glucose feeding was started when the glucose concentration fell below 5g/L.
  • the inducer IPTG was added, and at this time, 300g/L glycerol was used for feeding.
  • the entire fermentation process curve is shown in Figure 4A. As can be seen from Figure 4A, the glucose concentration began to fall below 5g/L at 6h, and when the fermentation continued for 12h, the bacterial concentration OD600 reached 10. After 32h of fermentation, the OD600 reached its maximum value, reaching 31, corresponding to a bacterial cell volume of 42g/L.
  • LNT production gradually increased from the start of the co-culture, reaching a maximum of 13.2 g/L at 42 hours (a total of 39.6 g LNT).
  • LNT concentration began to decrease from 42 to 48 hours, and LNT II consumption also gradually slowed after 42 hours.
  • the trend of LNT molar conversion was consistent with the LNT synthesis trend, reaching a maximum of 37.31%.
  • LNT was synthesized by coupled fermentation using the four bacteria 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 MgCl 2 , 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). Fermentation was carried out for 24 h (30° C., 200 r/min).

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Abstract

一种生产乳糖-N-四糖的大肠杆菌及基于其的双菌耦合发酵方法。首先在大肠杆菌中构建了LNT生物合成途径；然后对双质粒系统进行组合优化，构建了四株工程菌，并筛选出EP3菌株(含有galT1-SP ase和galE-Cvβ3GalT表达框)与酵母耦合发酵产LNT能力最优，可产生25.2g/L LNT，其摩尔转化率(mol/mol LNT II)为71.22％。接着对生产体系进行优化，比较UTP和UMP作为辅因子对LNT合成的影响，在仅添加20mM UMP作为辅因子时，即可生成29.4g/L LNT，其摩尔转化率(mol/mol LNT II)为83.09％。最后补料分批培养EP3菌株，经全细胞催化后产生39.6g LNT。为高效廉价生产LNT提供了一种新的策略。

Description

一种生产乳糖-N-四糖的大肠杆菌及基于其的双菌耦合发酵方法 技术领域
本发明涉及一种生产乳糖-N-四糖的大肠杆菌及基于其的双菌耦合发酵方法，属于生物发酵技术领域。
背景技术
母乳低聚糖(HMOs)是母乳中仅次于乳糖和脂肪的第三大固体成分，浓度为5-25g/L。HMOs具有益生元特性，可丰富肠道内婴儿双歧杆菌等有益菌，可作为宿主细胞受体干扰病原体粘附，调节肠道表面聚糖的表达，影响细胞生长和分化，并为婴儿的大脑发育和认知提供营养。到目前为止，已在母乳中发现了200多种结构不同的HMOs，它们构成了母乳和动物奶之间的主要差异。目前，至少有7种不同的HMOs已被食品和药物管理局(FDA)批准为公认的安全(GRAS)，包括2’-岩藻糖基乳糖(2’-FL)、3-岩藻糖基乳糖(3-FL)、二岩藻糖基乳糖(DFL)、3’-唾液酸乳糖(3’-SL)、6’-唾液酸乳糖(6’-SL)、乳糖-N-新四糖(LNnT)和乳糖-N-四糖(LNT)。其中六种已经商业化地添加到各种品牌的婴儿配方奶粉中。
乳糖-N-四糖(LNT)由半乳糖、N-乙酰氨基葡萄糖和葡萄糖组成，结构为Galβ1,3GlcNAcβ1,3Galβ1,4Glc。LNT不仅是HMOs的基本成分，约占HMOs总量的6％(w/w)，还是一种重要的核心结构，可以进一步岩藻糖化和唾液酸化产生更长的核心结构链。这种结构的多样性使HMOs在食品和制药行业具有巨大的商业价值和应用潜力。LNT的生理作用已被测定，包括益生元作用、预防坏死性小肠结肠炎、抗黏附/抗微生物活性、和抗病毒保护。它已被作为功能性强化剂添加到婴儿配方奶粉中。
目前，大量结构不同的HMOs主要是通过复杂的程序从母乳中提取，但含量极低且纯化工艺复杂；尽管化学合成方法有所发展，但工业规模的HMOs合成仍然存在几个障碍，即繁琐的保护和去保护基团步骤、总收率低和使用不适合食品的有毒试剂；酶法合成存在转糖基化反应不平衡、区域选择性低和需要相对昂贵的供体底物等问题，使许多HMOs无法以合理的价格合成。而生物合成HMOs具有环境友好、专一性高、底物可再生、成本低等优点。此外，随着代谢工程策略的发展，全细胞生物合成具有相对成熟的规模化生产技术和工艺，使得HMOs的生物合成受到了关注。
已报道了几种LNT和LNT衍生物的化学合成，Mohamed R.E.Aly et al.设计了两个二糖的连接，引入了二甲基马来酰(DMM)基团作为葡萄糖胺新的氨基保护基团，经过多步保护和去保护步骤合成了0.018g LNT，产率为78％；Wenlong Yao et al.报道了一种化学酶法，先用一锅双酶法产生乳-N-二糖(LNB)，然后LNB被乙酰化(Galβ1-3GlcNTFAβSEt)，再与容易获得的乳糖进行偶联，产生了约65.8mg LNT，转化率为89％；报道的化学法和化学酶法均只能小规模的合成LNT且步骤繁琐，价格昂贵。Yingying Zhu et al.在先前构建的产LNT II的菌株基础上通过加强UDP-Gal合成和引入β-1,3-半乳糖基转移酶从而得到25.49g/LLNT，摩尔转化率为85.9％；FlorianBaumgrtner et al.通过整合lgtA(β-1,3-n-乙酰氨基葡萄糖转移酶)和wbgO(β-1,3-半乳糖转移酶)，得到219.1mg/L LNT；Miaomiao Hu et al.通过过表达双质粒系统lgtA-wbgO和galE-galT-galK基因和敲除UDP-Gal旁路相关的ugd基因，最终产生了31.56g/LLNT。以上报道的生物法合成LNT均是以甘油为碳源，通过细胞内的多步代谢途径获得UDP-Gal，无法保证UDP-Gal的及时、大量供应。
生产LNT的有效策略是进行糖基转移酶介导的糖基化。β-1，3-GalT具有较好的实际应用价值，因其底物UDP-Gal是细胞代谢的中间产物，容易通过代谢工程的方法积累。利用β-1，3-GalT作为糖基转移酶，以UDP-Gal为供体，LNT II为受体，可以实现LNT的合成。因此UDP-Gal的充足供给是实现LNT高效合成的关键。目前，对于UDP-Gal的获取，通常有两条途径：(1)、Glc-1-P和UDP-Glc通过UDP-葡萄糖:半乳糖-1-磷酸尿基转移酶(GalT)和UDP-葡萄糖-4-差向异构酶(GalE)协同催化得到，(2)、以Glc-1-P和UTP为底物，经过焦磷酸化酶(BLUSP)和UDP-葡萄糖-4-差向异构酶(GalE)两步催化得到，但是哪条途径更适合LNT的合成，至今尚未报道。因此本发明旨在探究如何高效生产LNT。
发明内容
为解决上述问题，本发明采用双质粒共表达策略构建单菌株，通过双质粒组合的优化筛选得到最优菌株，并将其与酵母菌进行双菌耦合发酵，在生产LNT中表现优异。
本发明的第一个目的是提供一种生产乳糖-N-四糖的重组大肠杆菌，所述重组大肠杆菌中含有第一基因表达框和第二基因表达框，所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和β-1,3-半乳糖基转移酶编码基因Cvβ3GalT。
进一步地，所述重组大肠杆菌中含有的表达框为以下任一种组合：
(1)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于SPase之后的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于galE之后的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT；
(2)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于基因SPase之后的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于基因galE之前的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT；
(3)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于基因SPase之前的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于基因galE之后的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT；
(4)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于基因SPase之前的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于基因galE之前的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT。
进一步地，基因SPase的核苷酸序列如SEQ ID NO.1所示，基因galT的核苷酸序列如SEQ ID NO.2所示，基因galE的核苷酸序列如SEQ ID NO.5所示，基因Cvβ3GalT的核苷酸序列如SEQ ID NO.6所示。
进一步地，所述重组大肠杆菌的宿主菌可为常见的模式菌株，也可为经过改造的菌株。如Escherichia coli BL21(DE3)、Escherichia colijJM109(DE3)、Escherichia coli K12(DE3)、Escherichia coli Nissle 1917(DE3)△pMUT1-pMUT2△lacZ(GDMCC 63722)等。
进一步地，所述第一基因表达框和/或第二基因表达框中的基因由诱导启动子启动表达。如各基因分别由T7启动子启动表达。
进一步地，所述第一基因表达框位于pRSFDuet-1载体上，所述第二基因表达框位于pETDuet-1载体上。
本发明的第二个目的是提供一种多菌耦合体系，所述多菌耦合体系中含有：所述重组大肠杆菌和酵母菌。
进一步地，所述酵母菌包括但不限于酿酒酵母、啤酒酵母等，优选为酿酒酵母(GDMCC 61663)。
进一步地，所述多菌耦合体系中，重组大肠杆菌和酵母菌的用量比为1:0.5-1.5。
本发明的第三个目的是提供所述重组大肠杆菌或多菌耦合体系在制备乳糖-N-四糖中的应用。
本发明的第四个目的是提供一种生产乳糖-N-四糖的方法，采用所述重组大肠杆菌或多菌耦合体系进行发酵生产。
进一步地，发酵体系中至少还含有蔗糖、UTP和/或UMP、乳糖-N-三糖LNT II。
进一步地，发酵体系中含有100-1000mmol/L蔗糖、10-100mmol/LUTP/UMP、10-100mmol/L LNT II、100-1000mmol/L磷酸缓冲液(pH7.0)、10-100mmol/L MgCl2、10-100mL/L酒精、10-100mmol/L DTT、1-50g/L十八胺聚氧乙烯醚。
进一步地，发酵条件为18-37℃，100-300r/min。
在催化合成LNT的过程中，实现充足的UDP-Gal供给是关键。目前，已经知道合成UDP-Gal的两条途径中合成一分子的UDP-Gal均需要消耗一分子的UTP。虽然可以通过外源添加UTP满足全细胞催化合成UDP-Gal的需求，但由于UTP的价格昂贵800RMB/KG，因此通过外源大量添加UTP来用于LNT的生产，将会产生LNT价格昂贵的问题，针对该问题，如何利用细胞来廉价实现UTP/UDP的高效循环再生是LNT高产低价格的关键。而本发明比较了UTP和UMP作为辅因子对LNT合成的影响，在仅添加20mM UMP作为辅因子时，即可生成29.4g/L LNT，其摩尔转化率(mol/mol LNT II)达到83.09％。
进一步地，当所述发酵为放大发酵时，发酵包括两个阶段：(1)重组大肠杆菌发酵阶段；(2)重组大肠杆菌和酵母菌共发酵阶段。
进一步地，重组大肠杆菌发酵阶段包括以下步骤：于32-40℃下发酵，溶氧保持在15-25％；当葡萄糖含量低于5g/L时，以10％流速补充碳源葡萄糖；当OD600达到9-11后诱导表达，并用甘油替代葡萄糖作为碳源，温度调节至25-30℃，整个发酵过程中控制pH在6.8-7.2之间。
进一步地，重组大肠杆菌和酵母菌共发酵阶段包括以下步骤：将重组大肠杆菌和酵母菌加入上述发酵体系中于上述发酵条件(18-37℃，100-300r/min)下进行发酵。
本发明的有益效果：
(1)以蔗糖为底物，利用蔗糖磷酸化酶实现了Glc-1-P的大量供给；
(2)比较了不同来源的UDP-葡萄糖-半乳糖-1-磷酸尿基转移酶(GalT1、GalT2)、焦磷酸化酶(BLUSP)分别与蔗糖磷酸化酶(SPase)、UDP-葡萄糖-4-差向异构酶(GalE)协同合成UDP-Gal的能力，筛选得到协同催化合成UDP-Gal的能力较强的组合；
(3)对于UTP的再生，考虑利用酵母自身的UTP再生系统，通过与工程菌耦合发酵实现UTP的高效循环再生；
(4)为了实现UDP/UTP充足循环供给，采用外源微量补充辅因子UMP/UTP策略。考虑到UTP(8000RMB/KG)比UMP(2000RMB/KG)价格较昂贵，并且酵母的再生系统可以直接将UMP转化为UTP，比较了分别添加UTP和UMP作为外源辅因子，对合成LNT的影响，选择性价比最优的辅因子用于LNT的合成，结果表明：在添加酵母和外源微量辅因子UTP的条件下，工程菌合成LNT的摩尔转化率均高于未添加酵母的；外源添加等量的UMP和UTP，其合成LNT的产量近似。
生物材料保藏
酿酒酵母(Saccharomyces cerevisiae)xpli，所述酿酒酵母(Saccharomyces cerevisiae)xpli已于2021年05月12日保藏于广东省微生物菌株保藏中心，保藏编号为GDMCC No:61663，保藏地址为广东省先烈中路100号大院59号楼5楼。
大肠杆菌(Escherichia coli)EcN 03，所述大肠杆菌(Escherichia coli)EcN 03已于2023年08月07日保藏于广东省微生物菌株保藏中心，保藏编号为GDMCC No:63722，保藏地址为广东省先烈中路100号大院59号楼5楼。
附图说明
图1为单一工程菌和工程菌-酵母耦合发酵生产LNT的结果。其中，(A)共表达菌株的构建图；(B)共表达菌株的SDS-PAGE图(C)单一共表达菌株合成LNT；(D)共表达菌株与酵母耦合合成LNT。
图2为不同因素对菌株EP3和酵母耦合合成LNT的影响。其中，A：温度；B：酵母菌浓度；C：磷酸缓冲液浓度；D：LNT II浓度；E：蔗糖浓度；F：优化前后LNT产量。
图3为优化辅因子UMP和UTP合成LNT。其中，(A)：UMP作为辅因子；(B)：UTP作为辅因子。
图4为两阶段发酵策略生产LNT发酵曲线。其中，(A)EP3菌株在5L发酵罐中补料发酵；(B)EP3菌株和酵母耦合发酵合成LNT，蓝色箭头表示开始补料葡萄糖，紫色箭头表示换成甘油进行补料并且加入诱导剂IPTG。
图5为TLC检测LNT。其中，图5A为四菌耦合发酵结果，1-LNT II标品；2-LNT标品；3-5分别是6h、12、24h发酵液；图5B为EP1～EP4发酵结果，1-LNT II标品；2-LNT标品；3-6分别是EP1、EP2、EP3、EP4发酵液。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明涉及的材料和方法如下：
(1)LNT标准品由大连化物所闫竞宇教授赠与，LNT II标准品购自荣成市慧海创达生物科技有限公司(中国山东)，蔗糖购自国药集团化学试剂有限公司(中国上海),UMP和UTP购自杭州铠朋生物技术有限公司(中国浙江)。分子生物学操作试剂来自Vazyme公司(中国南京)。其他化学试剂均为最高纯度。
(2)培养基组成
Luria-Bertani(LB)培养基：LB培养基含有5克/升酵母提取物、10克/升胰蛋白胨和10克/升氯化钠。
发酵培养基：含有10g/L酪蛋白，5g/L酵母提取物，10g/L甘油，4g/L乳糖，20g/L葡萄糖，3.55g/LNa2HPO4，3.4g/LKH2PO4，2.68g/LNH4Cl，0.17g/LNa2SO4,3.55g/LMgSO4。补充抗生素氨苄(Amp)或卡那霉素(Km)，最终浓度分别为100和50μg/mL。
(3)发酵方法
工程菌株的三角瓶摇瓶培养方法：将工程大肠杆菌细胞在10mL LB液体培养基中于37℃预培养过夜。然后将种子液以2％的接种量接种到100mL的LB培养基中，并在37℃和200r/min下培养。当细胞密度(OD600)达到0.6至0.8时，分别加入终浓度为0.3mM的异丙基-β-D-1-硫代半乳糖苷(IPTG)。此外，在15℃和200r/min条件下继续培养20小时。(所有菌株摇瓶培养时均使用此条件)
(4)检测方法
薄层层析(TLC)检测Glc-1-P和UDP-Glc：展开剂：V(正丁醇):V(乙酸):V(水)＝2:1:1；TLC检测LNT II和LNT：V(正丙醇):V(氨水):V(水)＝5:2:1(35)；显色剂为茴香醛。显色方法为：165℃，5min。
HPLC检测UDP-Glc和UDP-Gal条件：色谱柱为Inertil ODS-4(3μm，4.6x 150mm)，流相为100mM磷酸钾缓冲液(pH6.4)，含有8mM四丁基硫酸氢铵，流速1mL/min，检测波长254nm，柱温40℃，进样量10μL。
HPLC定量分析LNT II和LNT条件：色谱柱XAmide(5um，4.6mm×250mm)，洗脱梯度：0-20min:70％乙腈-60％乙腈、20-30min:60％乙腈-70％乙腈，流速1mL/min，检测波长214nm，柱温30℃，进样量10μL。
利用基质辅助激光解析串联飞行时间质谱(MALDI-TOF-MS)分析LNT的分子量。
实施例1：重组菌株的构建
表1列出了本发明中使用的所有菌株和质粒。
表1菌株和质粒
其中，大肠杆菌JM109(DE3)被用作重组酶表达的宿主。在载体pET28a中构建了密码子优化的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)和Cvβ3GalT(SEQ ID NO.6)基因。将质粒pET28a-SPase、pET28a-galT1、pET28a-galT2、pET28a-BLSUP、pET28a-galE、pET28a-Cvβ3GalT分别转化到大肠杆菌JM109(DE3)，构建了工程菌株E1、E2、E3、E4、E5和E6。pRSFDuet-1载体用于构建共表达SPase和galT编码基因的重组质粒。pETDuet-1载体用于构建共表达galE和Cvβ3GalT编码基因的重组质粒。分别构建重组质粒pRSF-SPase-galT1、pRSF-galT1-SPase、pET-galE-Cvβ3GalT和pET-Cvβ3GalT-galE并转化至大肠杆菌JM109(DE3)，分别生成工程菌株EP1、EP2、EP3和EP4。
实施例2：重组菌株EP1～EP4发酵生产LNT
通过比较各菌株合成LNT的能力，获得合成LNT能力最佳工程菌株，并用于后续发酵优化研究。发酵条件：400mmol/L蔗糖、50mmol/LUTP、50mmol/L LNTII、400mmol/L磷酸缓冲液(pH7.0)、20mmol/LMgCl2、20mL/L酒精、20mmol/L DTT、8g/L十八胺聚氧乙烯醚、100g/L工程菌(EP1、EP2、EP3或EP4中的一株)，30℃、200r/min条件下发酵22h。结果见图1C。
实施例3：耦合发酵生产LNT
“工程菌—酵母”耦合合成LNT发酵条件：400mmol/L蔗糖、50mmol/LUTP、50mmol/L LNT II、400mmol/L磷酸盐缓冲液(pH 7.0)、20mmol/LMgCl2、20mL/L酒精、20mmol/L DTT、8g/L十八烷基胺聚氧乙烯醚、100g/L酵母菌(GDMCC 61663)、100g/L工程菌株(EP1、EP2、EP3或EP4中的一株)，在30℃和200r/min下发酵22h。结果见图1D。
从图1C和1D可以看出，所有四个菌株与酵母共培养发酵模式下合成LNT产量均高于单一菌株发酵，其中菌株EP1和EP3耦合酵母菌发酵合成LNT产量远高于EP2和EP4菌株耦合发酵。值得一提的是，单菌株EP1发酵时合成LNT产量最高，为17.46g/L，但与酵母耦合发酵后，菌株EP3(含有质粒pRSF-galT1-SPase和pET-galE-Cvβ3GalT)与酵母耦合发酵产生LNT的量高于EP1菌株，合成了25.2g/L LNT，摩尔转化率为71.22％。
实施例4：酵母-菌株EP3耦合发酵合成LNT的发酵条件优化
为了进一步提高最优菌株EP3合成LNT的量，重新诱导菌株EP3，从酵母-菌株EP3耦合发酵模式中底物蔗糖和LNT II浓度、磷酸缓冲液浓度、酵母细胞添加量和温度进行单因素条件优化。
温度会直接影响酶活性，进而影响LNT的合成。在确定的最佳酵母细胞添加量基础上测定不同反应温度下LNT的合成量，发现18℃和37℃下LNT合成量相差不大，但是在30℃下LNT合成量最高，达到22.4g/L(2A)。
研究了酵母菌体浓度对反应体系中LNT产量的影响，LNT产量随着酵母菌株浓度增加而增加，这表明，酵母UTP的再生合成能力直接影响了LNT的产率。共培养体系中酵母添加量达到100g/L时，LNT产量不再明显增加(2B)，为降低发酵系统负荷，实现LNT的经济生产，选用100g/L酵母(GDMCC 61663)添加量为最佳添加酵母浓度，此时LNT最高产量达到20.8g/L。
在确定酵母添加量和温度条件后，研究磷酸盐浓度对LNT生产影响，添加的磷酸缓冲液浓度为500mmol/L时，LNT的产量最高，达到30.4g/L，因此选取500mmol/L为磷酸缓冲液的最佳反应浓度(图2C)。
以优化的条件为基础，进一步探索底物蔗糖和LNT II浓度对LNT合成的影响。在反应体系中添加的LNT II浓度从10mmol/L增加到90mmol/L过程中，LNT产量先增加后减少，其中LNT II浓度为50mmol/L的达到最高，最高产量达到31.8g/L(图2D)，为实现LNT经济生产，确定底物LNT II的最优浓度为50mmol/L。LNT产量随着蔗糖浓度增加而增加，体系中蔗糖浓度达到400mmol/L时，LNT产量不再明显增加(图2E)，产量达到33.45g/L，因此选取400mmol/L蔗糖浓度用于LNT的生产。经过发酵条件优化后，LNT的产量由优化前的20.8g/L增加至33.45g/L(图2F)。
实施例5：耦合发酵体系的辅因子优化
考虑到UMP的价格低于UTP，因此，在相同条件下比较了分别添加等量的UTP和UMP作为外源辅因子对LNT合成的影响(图3)。催化条件：400mmol/L蔗糖、UMP/UTP(10mmol/L-40mmol/L、50mmol/L LNT II、500mmol/L磷酸盐缓冲液(pH 7.0)、20mmol/LMgCl2、20mL/L酒精、20mmol/LDTT、8g/L十八胺聚氧乙烯醚、100g/L酵母菌、100g/L工程菌株，在30℃、200r/min条件下发酵22h。
结果显示：LNT产量随着UTP浓度增加而增加，体系中UTP浓度达到30mmol/L时，LNT产量不再明显增加，LNT的最高产量达到31.4g/L，摩尔转化率为88.75％(图3B)；在发酵体系中将UTP替换为UMP，UMP浓度从10mmol/L增加到40mmol/L过程中，LNT产量先增加后减少，UMP浓度20mmol/L时LNT产量最高，最高产量达到29.4g/L，摩尔转化率为83.09％(图3A)。考虑到UMP价格只有UTP的1/4，并且外源添加UMP和UTP合成LNT的效果近似，所以选择20mmol/L的UMP作为外源辅因子用于LNT的合成的最佳浓度。
实施例6：7L发酵罐水平两阶段发酵合成LNT
为了实现大规模合成LNT，采用两阶段发酵策略来进行LNT的发酵生产研究。第一阶段，通过高密度发酵策略，实现合成LNT工程菌株的高密度发酵；第二阶段为LNT合成阶段，通过工程菌株EP3和酿酒酵母共培养，实现LNT的发酵合成。
具体步骤如下：
第一阶段工程菌株培养：挑取工程菌株的单菌落接种至带有相应抗生素的10ml LB培养基中，37℃培养12h得到一级种子液，再将一级种子液以2％的接种量转接至带有相应抗生素的250ml LB培养基中，37℃过夜培养得到二级种子液，按10％接种量将二级种子液于接种于发酵罐中。发酵培养基的工作容积为2.5升，整个发酵过程在37℃下培养，发酵罐中的溶解氧保持在20％，发酵罐由最高转速为600r/min的搅拌器自动控制。当葡萄糖含量低于5g/L时，以10％流速(即每10min内流加补料时间为1min)补充碳源葡萄糖；在工程菌株对数生长后期添加外源诱导剂IPTG(终浓度为0.3mmol/L)进行诱导，此时用甘油替代葡萄糖作为碳源，温度调节至28℃。整个发酵过程中用50％氨水调控pH值为7.0。
第二阶段工程菌-酵母共培养生产LNT：将富集得到的菌体用于全细胞发酵，发酵条件：400mmol/L蔗糖、UMP(20mmol/L)、50mmol/LLNT II、500mmol/L磷酸盐缓冲液(pH 7.0)、20mmol/LMgCl2、20mL/L酒精、20mmol/LDTT、8g/L十八胺聚氧乙烯醚、100g/L酵母菌、100g/L工程菌株，在30℃、200r/min条件下发酵48h。
结果如下：
第一阶段，在7L生物反应器中采用间歇补料策略对工程菌株EP3进行发酵，在生长阶段使用300g/L葡萄糖进行补料，当葡萄糖浓度低于5g/L就开启葡萄糖补料。在诱导表达控制方面，当OD600达到10，加入诱导剂IPTG，此时换用300g/L甘油进行补料，整个发酵过程曲线如图4A所示。从图4A可以看出，6h时葡萄糖浓度开始低于5g/L，发酵持续到12h时，菌浓OD600达到10。发酵32h后，OD600达到最大值，最高达为31，对应菌体量为42g/L。
在第二阶段LNT发酵合成的阶段，工程菌与酵母共培养发酵的时间为48h，在不同时间点取样，离心后取上清液，利用HPLC检测发酵中LNT的合成情况和LNT II消耗情况(图4B)。从共培养发酵开始LNT的产量逐渐增加，在42h时LNT产量达到最大值，最大值为13.2g/L(共产生39.6g LNT)，从42h到48h LNT的浓度开始降低，对于LNT II的消耗也在42h后逐渐减缓。LNT的摩尔转化率变化趋势与LNT的合成趋势相一致，最大值为37.31％。
对比例1四菌耦合发酵生产LNT
采用实施例1中构建的E1、E2(E3或E4)、E5和E6四菌耦合发酵合成LNT：100mmol/L蔗糖、50mmol/L UTP、100mmol/L磷酸缓冲液(pH7.0)、50mmol/L LNT II、20mmol/L MgCl2、20mL/L酒精、20mmol/L DTT、8g/L十八胺聚氧乙烯醚、50g/L工程菌(E1，E2/E3/E4，E5和E6)，发酵24h(30℃，200r/min)。
耦合发酵完成后，反应液离心取上清进行TLC检测(四菌耦合中表现最优的E1，E2，E5和E6组合，以及EP1、EP2、EP3、EP4单菌发酵)，结果如图5所示，其中，A中3-5分别是E1，E2，E5和E6四菌在6h、12、24h发酵液，B中3-6分别是EP1、EP2、EP3、EP4单菌发酵液。从图5可以看出，反应液中有与标准品LNT Rf相同的物质存在，这果表明，以蔗糖、UDP-Glc和LNT II为底物，利用四菌耦合发酵成功合成了LNT，但是四菌株耦合发酵策略合成LNT产量不佳，底物LNT II的剩余量过多(图5A)，差于EP1～EP4单菌发酵的结果(图5B)。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种生产乳糖-N-四糖的重组大肠杆菌，其特征在于，所述重组大肠杆菌中含有第一基因表达框和第二基因表达框，所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和β-1,3-半乳糖基转移酶编码基因Cvβ3GalT。
  2. 根据权利要求1所述的重组大肠杆菌，其特征在于，所述重组大肠杆菌中含有的表达框为以下任一种组合：
    (1)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于SPase之后的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于galE之后的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT；
    (2)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于基因SPase之后的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于基因galE之前的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT；
    (3)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于基因SPase之前的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于基因galE之后的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT；
    (4)所述第一基因表达框中含有蔗糖磷酸化酶编码基因SPase和位于基因SPase之前的UDP-葡萄糖-己糖-1-磷酸尿苷酰转移酶编码基因galT，所述第二基因表达框中含有UDP-葡萄糖-4-差向异构酶编码基因galE和位于基因galE之前的β-1,3-半乳糖基转移酶编码基因Cvβ3GalT。
  3. 一种多菌耦合体系，其特征在于，所述多菌耦合体系中含有：权利要求1或2所述重组大肠杆菌，以及酵母菌。
  4. 根据权利要求3所述的多菌耦合体系，其特征在于，所述酵母菌包括酿酒酵母和/或啤酒酵母。
  5. 根据权利要求4所述的多菌耦合体系，其特征在于，所述多菌耦合体系中，重组大肠杆菌和酵母菌的用量比为1:0.5-1.5。
  6. 权利要求1或2所述重组大肠杆菌或权利要求3-5任一项所述多菌耦合体系在制备乳糖-N-四糖中的应用。
  7. 一种生产乳糖-N-四糖的方法，其特征在于，包括以下步骤：采用权利要求1或2所述重组大肠杆菌或权利要求3-5任一项所述多菌耦合体系进行发酵生产。
  8. 根据权利要求7所述的方法，其特征在于，发酵体系中至少还含有蔗糖、UTP和/或UMP、乳糖-N-三糖。
  9. 根据权利要求7所述的方法，其特征在于，所述发酵包括两个阶段：重组大肠杆菌发酵阶段、重组大肠杆菌和酵母菌共发酵阶段。
  10. 根据权利要求9所述的方法，其特征在于，重组大肠杆菌发酵阶段包括以下步骤：于32-40℃下发酵，溶氧保持在15-25％，当葡萄糖含量低于5g/L时，以10％流速补充碳源葡萄糖；当OD600达到9-11后诱导表达，并用甘油替代葡萄糖作为碳源，温度调节至25-30℃，整个发酵过程中控制pH在6.8-7.2之间。
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