WO2022228169A1 - 一种产乳酰-n-新四糖的基因工程菌及生产方法 - Google Patents

一种产乳酰-n-新四糖的基因工程菌及生产方法 Download PDF

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WO2022228169A1
WO2022228169A1 PCT/CN2022/087318 CN2022087318W WO2022228169A1 WO 2022228169 A1 WO2022228169 A1 WO 2022228169A1 CN 2022087318 W CN2022087318 W CN 2022087318W WO 2022228169 A1 WO2022228169 A1 WO 2022228169A1
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gene
neotetraose
genetically engineered
nucleotide sequence
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张涛
江波
胡苗苗
李梦丽
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江南大学
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Definitions

  • the invention relates to a genetically engineered bacterium for producing lactoyl-N-neotetraose and a production method, and belongs to the technical fields of metabolic engineering and food fermentation.
  • HMOs Human milk oligosaccharides
  • the content in mature milk is 12-13 g/L, while in colostrum can reach 22-13 g/L. 23g/L, which is unmatched by milk (milk oligosaccharide ⁇ 1g/L).
  • HMOs can withstand the hydrolysis of enzymes in the infant digestive tract, thereby resisting the infection of gastrointestinal pathogenic microorganisms and maintaining the gastrointestinal microecological balance.
  • lactoyl-N-neotetrasaccharide As an important oligosaccharide in human milk oligosaccharide, lactoyl-N-neotetrasaccharide has biological functions such as enhancing human immunity, regulating intestinal flora, promoting cell maturation and accelerating wound healing. In view of the important biological function and physiological activity of lactoyl-N-neotetraose, it has been allowed to be added into commercial infant formula. However, the amount obtained by separation and extraction from natural products is very small, which is far less than the needs of research. Therefore, it is the best choice to obtain such compounds by artificial synthesis.
  • lactoyl-N-neotetrasaccharide mainly include three kinds, namely chemical synthesis, enzymatic synthesis and fermentation synthesis.
  • the chemical synthesis method has problems such as complex reaction steps and expensive raw materials, resulting in high production costs and unfavorable industrial large-scale synthesis, and some toxic reagents used in chemical synthesis make the product unsuitable for food additives.
  • Enzymatic synthesis requires expensive nucleotide sugars as substrates, which is not conducive to the large-scale production of lactoyl-N-neotetraose.
  • the biological method can utilize cheap carbon and nitrogen sources and substrates to realize the synthesis of lactoyl N-neotetraose without affecting the environment. Therefore, the preparation of lactoyl N-neotetraose by biological methods has received more and more attention.
  • the present invention provides a genetically engineered bacterium producing lactoyl-N-neotetraose, wherein the genetically engineered bacterium knocks out the ⁇ -galactosidase gene lacZ and overexpresses ⁇ -1,3-acetylglucosamine transferase Gene lgtA, ⁇ -1,4-galactosyltransferase gene lgtB, phosphoglucomutase gene pgm, UDP-glucose 4-epimerase gene galE, galactose-1-phosphate uridyltransferase gene galT, the galactokinase gene galK and the beta-galactoside permease gene lacY.
  • the ⁇ -1,3-acetylglucosamine transferase gene IgtA and the ⁇ -1,4-galactosyltransferase gene IgtB are both derived from Neisseria meningitidis, and the ⁇ -1 , the nucleotide sequence of 3-acetylglucosamine transferase gene IgtA is shown in SEQ ID NO:1, and the nucleotide sequence of ⁇ -1,4-galactosyltransferase gene IgtB is shown in SEQ ID NO:2 .
  • the ⁇ -galactosidase permease gene lacY and the ⁇ -galactosidase gene lacZ are both derived from Escherichia coli K-12, and the Gene ID of the phosphoglucomutase gene pgm is 945271 (nucleotide sequence such as SEQ ID NO: 24), the Gene ID of the UDP-glucose 4-epimerase gene galE is 945354 (the nucleotide sequence is shown in SEQ ID NO: 25), the galactose-1-phosphate uridyltransferase gene galT The Gene ID is 945357 (the nucleotide sequence is shown in SEQ ID NO: 26), the Gene
  • the genetically engineered bacteria use the pETDuet-1 plasmid to express the gene lacY, the pRSFDuet-1 plasmid to sequentially express the genes pgm, galE, galT and galK, and the pCDFDuet-1 plasmid to sequentially express the genes lgtA and lgtB.
  • the present invention also provides a method for producing lactoyl-N-neotetrasaccharide, the method steps are:
  • IPTG with a final concentration of 0.4 mM and lactose with a final concentration of 8 g/L to 10 g/L are added for induction, and after induction, a fermentation broth containing lactoyl-N-neotetraose is obtained.
  • the carbon source in the fermentation medium is one or more of glucose, galactose, and glycerol.
  • the carbon source is glycerol, or a mixture of glycerol and galactose.
  • the carbon source is 20 g/L glycerol, or a mixture of 10 g/L glycerol and 10 g/L galactose.
  • the fermentation system further contains 13.5g/L potassium dihydrogen phosphate, 4.0g/L diammonium hydrogen phosphate, 1.7g/L citric acid, 1.4g/L magnesium sulfate heptahydrate and 10ml/L L trace metal elements; the trace metal elements are 10g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 2.0g/L calcium chloride dihydrate, pH 6.8.
  • the seed medium in the method is LB liquid medium
  • the seed culture conditions are 35°C-40°C, 200-250 rpm, and shake flask culture for 10-14 hours.
  • the specific method for obtaining the fermentation broth in the method is to insert the seed liquor into the fermentation system, and at 35°C to 40°C and 200rpm to 250rpm, after culturing to an OD 600 of 0.6 to 0.8, add IPTG with a final concentration of 0.4 mM, and lactose with a final concentration of 8 g/L to 10 g/L was added at the same time, at 22 °C to 30 °C, and 200 rpm to 250 rpm for 42 h to 48 h.
  • the present invention also provides the application of the genetically engineered bacteria in the production of lactoyl-N-neotetraose.
  • the present invention regulates the overexpression of lacY, pgm, galE, galT and galK in the synthesis pathway of lactyl-N-neotetrasaccharide through the exogenous expression of IgtA and IgtB, and knocks out the lactyl-N-neotetrasaccharide of the Escherichia coli host.
  • lacZ in the sugar synthesis pathway and the optimization of the carbon source of the fermentation medium, so as to achieve the purpose of regulating the carbon flux of the metabolic pathway and increasing the production of lactoyl-N-neotetraose.
  • Fig. 1 is the metabolic pathway diagram of lactyl-N-neotetraose
  • Fig. 2A is the secondary mass spectrum of lactoyl-N-neotetrasaccharide standard sample
  • Figure 2B is a secondary mass spectrum of a lactoyl-N-neotetrasaccharide product sample.
  • LB liquid medium 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride.
  • LB solid medium 10g/L peptone, 5g/L yeast extract powder, 10g/L sodium chloride, 15g/L agar powder.
  • lactoyl-N-neotetrasaccharide used in the embodiment of the present invention uses HPLC, and is specifically:
  • HPLC detection conditions differential refractive index detector; chromatographic column is Rezex ROA-organic acid (Phenomenex, USA), column temperature is 50 °C; mobile phase is 5mM H2SO4 aqueous solution, flow rate is 0.6mL/min; injection volume to 10 ⁇ L.
  • Example 1 Knockout of Escherichia coli BL21 (DE3) genomic gene lacZ
  • 400ng pTargetF plasmid and 1000ng donor DNA fragment (that is, the template fragment of lacZ obtained in step 1) were electrotransferred to the Escherichia coli BL21/pCas competent state of step (4), and coated on LB plate (Kanamyces spp. and spectinomycin), cultured at 30°C for 24h, picked the positive colonies on the plate, cultured in LB for 10h, and sent to Tianlin Biotechnology (Shanghai) Co., Ltd. for sequencing verification.
  • step (6) Pick the positive clone colonies obtained in step (5) into a 4ml LB liquid test tube, add IPTG with a final concentration of 1mM and 30mg/L kanamycin, and cultivate at 30°C for 8-16h to remove the pTargetF plasmid, and then add After culturing at 42°C for 12h, the pCas plasmid was removed.
  • lacY gene fragment (the Gene ID of lacY is 949083): Using the genome of Escherichia coli K-12 (Escherichia coli) as a template, and using lacY-F/lacY-R as primers, PCR amplifies the lacY gene fragment , the DNA fragment was recovered by gel, and the recovered DNA gene fragment was connected to the BamHI and SalI restriction sites of the vector pETDuet-1 through a seamless cloning kit (Nanjing Novizan Life Technology Co., Ltd.), and the final plasmid obtained was pET -lacY;
  • the recovered DNA gene fragments were respectively connected to the BamHI, SalI, BgiII, XhoI restriction sites of the vector pRSFDuet-1 through a seamless cloning kit (Nanjing Novizan Life Technology Co., Ltd.), and the final plasmid pRSF-pgm was obtained.
  • pRSF-pgm-galE, pRSF-pgm-galET and pRSF-pgm-galETK
  • lactoyl-N-neotetraose The yields of lactoyl-N-neotetraose of different engineering strains after fermentation were 304 mg/L, 508 mg/L, 595 mg/L, 654 mg/L, 749 mg/L and 837 mg/L, respectively.
  • the yield of lactoyl-N-neotetraose was increased by 175.3% in strain A5 relative to A0. Therefore, expressing the E. coli endogenous genes pgm and galE-galT-galK related to UDP-galactose synthesis can increase the production of lactoyl-N-neotetraose.
  • the lactoyl-N-neotetraose metabolic pathway diagram of strain A5 is shown in Figure 1.
  • the fermentation method is as follows: the constructed genetically engineered bacteria A0-A5 are inoculated in LB liquid medium, 37 ° C, 200 rpm, and shake flask culture for 12 h to obtain seed liquid; then the seed liquid is inserted into 50 ml fermentation culture at an inoculum volume of 2 mL/100 mL base, 37 °C, 200 rpm, shake flask to culture to OD 600 of 0.6; add IPTG with a final concentration of 0.4 mM, and at the same time add lactose to a lactose concentration of 10 g/L, induce culture for 48 h at 25 °C, 200 rpm, and obtain a fermentation broth .
  • composition of above-mentioned fermentation medium is 20g/L glycerol, 13.5g/L potassium dihydrogen phosphate, 4.0g/L dihydrogen phosphate, 1.7g/L citric acid, 1.4g/L magnesium sulfate heptahydrate and 10ml/L trace Metal elements; trace metal elements include: 10g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 2.0g/L calcium chloride dihydrate, pH 6.8.
  • Example 4 Screening of lactoyl-N-neotetrasaccharide produced by different carbon source combinations
  • the high-yielding lactoyl-N-neotetrasaccharide engineering bacteria A5 obtained in Example 3 is fermented by using the fermentation medium of different carbon source combinations, and the specific fermentation method is as follows:
  • the constructed genetically engineered bacteria A5 was inoculated into LB liquid medium at 37°C, 200rpm, and cultured in a shaker flask for 12h to obtain seed liquid; then the seed liquid was inoculated into 50ml fermentation medium at an inoculum volume of 2mL/100mL, 37°C, 200rpm , cultured in shake flask to OD 600 of 0.6; added IPTG with a final concentration of 0.4 mM, and at the same time added lactose to a lactose concentration of 10 g/L, induced and cultured for 48 h at 25 °C and 200 rpm to obtain a fermentation broth.
  • composition of above-mentioned fermentation medium is carbon source (refer to Table 4 for each carbon source combination and content), 13.5g/L potassium dihydrogen phosphate, 4.0g/L diammonium hydrogen phosphate, 1.7g/L citric acid, 1.4g/L Magnesium sulfate heptahydrate and 10ml/L trace metal elements; trace metal elements include: 10g/L ferrous sulfate, 2.25g/L zinc sulfate heptahydrate, 1.0g/L anhydrous copper sulfate, 2.0g/L dihydrate chloride Calcium, pH 6.8.

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Abstract

一种产乳酰-N-新四糖的基因工程菌及生产方法，属于代谢工程和食品生物技术领域。为解决现有的利用微生物方法生产乳酰-N-新四糖产量较低的问题，通过对lgtA和lgtB的外源表达，合理组合调控乳酰-N-新四糖合成途径中lacY，pgm，galE，galT和galK的过表达，敲除大肠杆菌宿主中的lacZ表达，并且优化培养过程中的碳源配置，从而达到了调控代谢通路的碳通量，提高乳酰-N-新四糖的产量的目的。在摇瓶实验中，大肠杆菌生产乳酰-N-新四糖的能力由304mg/L提升至1031mg/L。

Description

一种产乳酰-N-新四糖的基因工程菌及生产方法 技术领域
本发明涉及一种产乳酰-N-新四糖的基因工程菌及生产方法，属于代谢工程和食品发酵技术领域。
背景技术
人乳寡糖(human milk oligosaccharides，HMOs)是母乳中仅次于乳糖和脂肪的第三大固体组分，在成熟乳中的含量为12-13g/L，而在初乳中可达22-23g/L，这是牛乳无法比拟的(牛乳寡糖＜1g/L)。HMOs可耐受婴儿消化道内酶的水解，进而抵御胃肠道病原微生物的感染和维持胃肠道微生态平衡。乳酰-N-新四糖作为人乳寡糖中的一种重要寡糖，具有增强人体免疫力，调节肠道菌群，促进细胞成熟和加速伤口愈合等生物学功能。鉴于乳酰-N-新四糖的重要生物功能和生理活性，已经允许添加进商品化婴幼儿配方奶粉中。然而从天然产物中分离提取所得到的量很少，远不及研究的需要，因此通过人工合成的方法获得该类化合物就成为最佳选择。
目前报道的乳酰-N-新四糖的合成方法主要包括三种，分别是化学合成、酶法合成和发酵法合成。化学合成法存在反应步骤复杂、原材料价格昂贵等问题，造成生产成本较高不利于工业的大规模合成，且化学合成中使用的部分有毒试剂，使产品不宜用于食品添加剂。酶合成法需要昂贵的核苷酸糖作为底物，不利于乳酰-N-新四糖的大规模生产。而生物法可以利用廉价碳氮源和底物，实现乳酰N-新四糖的合成，且对环境不造成影响。因此，通过生物法制备乳酰N-新四糖受到了越来越多的关注。
发明内容
本发明提供了一种产乳酰-N-新四糖的基因工程菌，所述基因工程菌敲除β-半乳糖苷酶基因lacZ，并过表达β-1，3-乙酰葡萄糖胺转移酶基因lgtA、β-1，4-半乳糖基转移酶基因lgtB、磷酸葡萄糖变位酶基因pgm、UDP-葡萄糖4-差向异构酶基因galE、半乳糖-1-磷酸尿苷酰转移酶基因galT、半乳糖激酶基因galK和β-半乳糖苷通透酶基因lacY。
在一种实施方式中，所述β-1，3-乙酰葡萄糖胺转移酶基因lgtA和β-1，4-半乳糖基转移酶基因lgtB均来源于脑膜炎奈瑟球菌，所述β-1，3-乙酰葡萄糖胺转移酶基因lgtA的核苷酸序列如SEQ ID NO:1所示，β-1，4-半乳糖基转移酶基因lgtB的核苷酸序列如SEQ ID NO:2所示。
在一种实施方式中，所述磷酸葡萄糖变位酶基因pgm、UDP-葡萄糖4-差向异构酶基因 galE、半乳糖-1-磷酸尿苷酰转移酶基因galT、半乳糖激酶基因galK、β-半乳糖苷通透酶基因lacY和β-半乳糖苷酶基因lacZ均来源于大肠杆菌K-12，磷酸葡萄糖变位酶基因pgm的Gene ID为945271(核苷酸序列如SEQ ID NO:24所示)，UDP-葡萄糖4-差向异构酶基因galE的Gene ID为945354(核苷酸序列如SEQ ID NO:25所示)，半乳糖-1-磷酸尿苷酰转移酶基因galT的Gene ID为945357(核苷酸序列如SEQ ID NO:26所示)，半乳糖激酶基因galK的Gene ID为945358(核苷酸序列如SEQ ID NO:27所示)，β-半乳糖苷通透酶基因lacY的Gene ID为949083(核苷酸序列如SEQ ID NO:28所示)，β-半乳糖苷酶基因lacZ的Gene ID为945006(核苷酸序列如SEQ ID NO:23所示)。
在一种实施方式中，所述基因工程菌以pETDuet-1质粒表达基因lacY，以pRSFDuet-1质粒依次表达基因pgm、galE、galT和galK，以pCDFDuet-1质粒依次表达基因lgtA和lgtB。
本发明还提供了一种生产乳酰-N-新四糖的方法，所述方法步骤为：
(1)将权利要求1～4任意一项所述的基因工程菌在种子培养基中培养获得种子液；
(2)再将种子液接种于发酵体系中，培养至OD 600为0.6～0.8；
(3)加入终浓度为0.4mM的IPTG，及终浓度为8g/L～10g/L的乳糖进行诱导，经诱导后，获得含乳酰-N-新四糖的发酵液。
在一种实施方式中，发酵培养基中的碳源为葡萄糖、半乳糖和甘油中的一种或多种。
优选地，碳源为甘油，或为甘油和半乳糖的混合物。
更优选地，碳源为20g/L的甘油，或10g/L甘油和10g/L半乳糖的混合物。
在一种实施方式中，所述发酵体系中还含有13.5g/L磷酸二氢钾，4.0g/L磷酸氢二氨，1.7g/L柠檬酸，1.4g/L七水硫酸镁和10ml/L微量金属元素；所述微量金属元素为10g/L硫酸亚铁，2.25g/L七水硫酸锌，1.0g/L无水硫酸铜，2.0g/L二水氯化钙，pH为6.8。
在一种实施方式中，所述方法中的种子培养基为LB液体培养基，种子培养的条件为35℃～40℃，200rpm～250rpm，摇瓶培养10h～14h。
在一种实施方式中，所述方法中发酵液的具体获得方法是将种子液接入发酵体系，于35℃～40℃，200rpm～250rpm条件下，培养至OD 600为0.6～0.8后，加入终浓度为0.4mM的IPTG，同时加入终浓度为8g/L～10g/L的乳糖，22℃～30℃，200rpm～250rpm诱导培养42h～48h。
本发明还提供了所述基因工程菌在生产乳酰-N-新四糖中的应用。
本发明的有益效果：
本发明通过lgtA和lgtB的外源表达，合理组合调控乳酰-N-新四糖合成途径中lacY，pgm，galE，galT和galK的过表达并敲除大肠杆菌宿主乳酰-N-新四糖合成途径中的lacZ表达，以 及对发酵培养基碳源的优化，从而达到了调控代谢通路的碳通量，提高乳酰-N-新四糖的产量的目的，在摇瓶实验中，大肠杆菌生产乳酰-N-新四糖的能力由304mg/L提升至1031mg/L，，为乳酰-N-新四糖的工业化生产奠定了基础。
附图说明
图1为乳酰-N-新四糖代谢通路图；
图2A为乳酰-N-新四糖标样的二级质谱图；
图2B为乳酰-N-新四糖产物样品的二级质谱图。
具体实施方式
以下结合实例与附图对本发明的具体实施作进一步的说明，以下实例中所使用的质粒、PCR试剂、限制性内切酶、质粒抽提试剂盒、DNA胶回收试剂盒等采用商业产品，具体操作按照试剂盒说明书进行。本发明的实施方式不限于此，其他未注明的实验操作和工艺参数按照常规技术进行。
质粒和DNA产物的测序工作由天霖生物科技(上海)有限公司完成。
大肠杆菌感受态的制备：上海生工生物工程公司试剂盒。
LB液体培养基：10g/L蛋白胨，5g/L酵母提取物，10g/L氯化钠。
LB固体培养基：10g/L蛋白胨，5g/L酵母浸粉，10g/L氯化钠，15g/L琼脂粉。
本发明实施例中所述的乳酰-N-新四糖的测定方法使用HPLC，具体为：
1mL发酵液于100℃煮沸10min，13400rpm离心10min，上清液经0.22μm膜过滤处理，利用HPLC检测乳酰-N-新四糖的生成量。HPLC检测条件：示差折光检测器；色谱柱为Rezex ROA-organic acid(Phenomenex，USA)，柱温为50℃；流动相为5mM的H 2SO 4水溶液，流速为0.6mL/min；进样量为10μL。
实施例1：大肠杆菌BL21(DE3)染色体组基因lacZ的敲除
重利用CRISPR-Cas9基因敲除系统敲除大肠杆菌BL21中lacZ(lacZ的Gene ID为945006，SEQ ID NO:23)，具体步骤如下(所涉及到的引物序列见表1)：
(1)以大肠杆菌BL21基因组为模板，使用lacZ-up-F/R和lacZ-down-F/R通过PCR分别扩增出lacZ，的上下游片段，胶回收。再分别以lacZ上下游片段为模板，采用lacZ-up-F/lacZ-down-R引物通过重叠PCR得到完整的lacZ模板，胶回收DNA片段。
(2)以原始pTargetF质粒为模板，lacZ-sg-F/R为引物，采用PCR扩增将原始质粒上的N20序列分别替换为与lacZ序列互补的N20序列，得到带有靶向lacZ的pTargetF质粒(构建的质粒名称为pTargetF-lacZ，靶向质粒信息为带有lacZ特异性N20序列的靶向质粒pTargetF)。 转化大肠杆菌DH5α感受态，涂布LB平板(含壮观霉素)，37℃扩大培养提取质粒并测序。
(3)取pCas质粒及大肠杆菌BL21感受态，冰上放置5min至感受态融化，取5μL质粒加入100μL感受态细胞中，轻轻混匀。冰浴30min，42℃热激90s，立即置于冰上5min。加入1mL LB培养基，30℃，180rpm培养1h。取200μL浓缩菌液，均匀涂布于LB平板(含卡那霉素)上，30℃倒置培养过夜至长出大肠杆菌BL21/pCas的单菌落。
(4)挑取大肠杆菌BL21/pCas单菌落于LB培养基中，30℃培养1.0h，加入终浓度为30mM的L-阿拉伯糖以诱导pCas-λ-red系统表达。当OD 600达到0.6-0.8时，制备大肠杆菌BL21/pCas感受态。
(5)将400ng pTargetF质粒和1000ng的供体DNA片段(即步骤1得到的lacZ的模板片段)，电转至步骤(4)的大肠杆菌BL21/pCas感受态，涂布于LB平板(卡那霉素和壮观霉素)，30℃培养24h，挑取平板上的阳性菌落于LB中培养10h，送天霖生物科技(上海)有限公司测序验证。
(6)将步骤(5)得到的阳性克隆菌落挑至4ml LB液体试管，加入终浓度为1mM的IPTG和30mg/L卡那霉素，30℃培养8-16h，以去除pTargetF质粒，再在42℃培养12h，去除pCas质粒。
表1.lacZ敲除的引物序列
Figure PCTCN2022087318-appb-000001
实施例2：重组菌的构建和质粒组合的筛选
重组菌的构建具体步骤如下(所涉及到的引物序列见表2)：
(1)lacY基因片段的获得(lacY的Gene ID为949083)：以大肠杆菌K-12(Escherichia coli)的基因组为模板，以lacY-F/lacY-R为引物，PCR扩增出lacY基因片段，胶回收DNA片段，将回收的DNA基因片段通过无缝克隆试剂盒(南京诺唯赞生命科技有限公司)连接到载体pETDuet-1的BamHI和SalI酶切位点上，最终获得的质粒是pET-lacY；
(2)pgm，galE，galE-galT和galE-galT-galK基因片段的获得(pgm的Gene ID为945271，galE的Gene ID为945354，galT的Gene ID为945357，galK的Gene ID为945358)：以大肠 杆菌K-12(Escherichia coli)的基因组为模板，以pgm-F/pgm-R为引物，PCR扩增出pgm基因片段，胶回收DNA片段；以galE-F/galE-R为引物，PCR扩增出galE基因簇片段，胶回收DNA片段；再以galET-F/galET-R为引物，PCR扩增出galE-galT基因簇片段，胶回收DNA片段；再以galETK-F/galETK-R为引物，PCR扩增出galE-galT-galK基因簇片段，胶回收DNA片段。将回收的DNA基因片段通过无缝克隆试剂盒(南京诺唯赞生命科技有限公司)分别连接到载体pRSFDuet-1的BamHI、SalI、BgiII、XhoI酶切位点上，最终获得的质粒pRSF-pgm、pRSF-pgm-galE、pRSF-pgm-galET和pRSF-pgm-galETK；
(3)lgtA和lgtB基因片段的获得：找出脑膜炎奈瑟球菌(Neisseria meningitidis)的lgtA和lgtB的基因序列(lgtA的核苷酸序列如SEQ ID NO:1所示，lgtB的核苷酸序列如SEQ ID NO:2所示)，并委托天霖生物科技(上海)有限公司进行合成，将合成后的基因片段通过酶切位点BamHI、SalI、BgiII、XhoI，通过无缝克隆试剂盒(南京诺唯赞生命科技有限公司)连接到载体pCDFDuet-1的BamHI、SalI、BgiII、XhoI酶切位点上，最终获得的质粒为pCDF-lgtA-lgtB。
表2.质粒构建引物
Figure PCTCN2022087318-appb-000002
Figure PCTCN2022087318-appb-000003
(4)采用上述步骤获得的质粒pET-lacY、pRSF-pgm、pRSF-pgm-galE、pRSF-pgm-galET、pRSF-pgm-galETK和pCDF-lgtA-lgtB，通过组合乳酰-N-新四糖合成通路中的关键基因的数量，得到了5个不同的工程菌，分别表示为A0、A1、A2、A3、A4和A5(见表3)。
发酵后不同工程菌株乳酰-N-新四糖的产量分别为304mg/L、508mg/L、595mg/L、654mg/L、749mg/L和837mg/L。菌株A5相对于A0，乳酰-N-新四糖的产量增加了175.3％。因此，表达与UDP-半乳糖合成相关的大肠杆菌内源性基因pgm和galE-galT-galK，能够增加乳酰-N-新四糖产量。菌株A5的乳酰-N-新四糖代谢通路图如图1所示。
发酵方法如下：将构建的基因工程菌A0～A5接种于LB液体培养基，37℃，200rpm，摇瓶培养12h，得到种子液；再将种子液以2mL/100mL的接种量接入50ml发酵培养基，37℃，200rpm，摇瓶培养至OD 600为0.6；加入终浓度为0.4mM的IPTG，同时加入乳糖至乳糖浓度为10g/L，25℃，200rpm的条件下诱导培养48h，获得发酵液。
上述发酵培养基的组成为20g/L甘油，13.5g/L磷酸二氢钾，4.0g/L磷酸氢二氨，1.7g/L柠檬酸，1.4g/L七水硫酸镁和10ml/L微量金属元素；微量金属元素包括：10g/L硫酸亚铁，2.25g/L七水硫酸锌，1.0g/L无水硫酸铜，2.0g/L二水氯化钙，pH 6.8。
表3.各工程菌详细信息
Figure PCTCN2022087318-appb-000004
Figure PCTCN2022087318-appb-000005
实施例4：不同碳源组合产乳酰-N-新四糖的筛选
采用不同碳源组合的发酵培养基对实施例3获得的高产乳酰-N-新四糖的工程菌A5进行发酵，具体发酵方法如下：
将构建的基因工程菌A5接种于LB液体培养基，37℃，200rpm，摇瓶培养12h，得到种子液；再将种子液以2mL/100mL的接种量接入50ml发酵培养基，37℃，200rpm，摇瓶培养至OD 600为0.6；加入终浓度为0.4mM的IPTG，同时加入乳糖至乳糖浓度为10g/L，25℃，200rpm的条件下诱导培养48h，获得发酵液。
上述发酵培养基的组成为碳源(各碳源组合及含量参见表4)，13.5g/L磷酸二氢钾，4.0g/L磷酸氢二氨，1.7g/L柠檬酸，1.4g/L七水硫酸镁和10ml/L微量金属元素；微量金属元素包括：10g/L硫酸亚铁，2.25g/L七水硫酸锌，1.0g/L无水硫酸铜，2.0g/L二水氯化钙，pH 6.8。
菌株A5在不同碳源组合的发酵培养基中乳酰-N-新四糖的产量检测的结果如表4所示，由该表可以看出，当培养基碳源组合为10g/L半乳糖+10g/L甘油时，相对于最开始使用的20g/L甘油为碳源的发酵生产乳酰-N-新四糖产量提高了23.2％。
表4.各培养基碳源组合详细信息
Figure PCTCN2022087318-appb-000006
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (10)

  1. 一种产乳酰-N-新四糖的基因工程菌，其特征在于，所述基因工程菌敲除β-半乳糖苷酶基因lacZ，并过表达β-1，3-乙酰葡萄糖胺转移酶基因lgtA、β-1，4-半乳糖基转移酶基因lgtB、磷酸葡萄糖变位酶基因pgm、UDP-葡萄糖4-差向异构酶基因galE、半乳糖-1-磷酸尿苷酰转移酶基因galT、半乳糖激酶基因galK和β-半乳糖苷通透酶基因lacY。
  2. 根据权利要求1所述的基因工程菌，其特征在于，所述β-1，3-乙酰葡萄糖胺转移酶基因lgtA和β-1，4-半乳糖基转移酶基因lgtB均来源于脑膜炎奈瑟球菌(Neisseriaceae meningitidis)，所述β-1，3-乙酰葡萄糖胺转移酶基因lgtA的核苷酸序列如SEQ ID NO:1所示，β-1，4-半乳糖基转移酶基因lgtB的核苷酸序列如SEQ ID NO:2所示。
  3. 根据权利要求1所述的基因工程菌，其特征在于，所述磷酸葡萄糖变位酶基因pgm、UDP-葡萄糖4-差向异构酶基因galE、半乳糖-1-磷酸尿苷酰转移酶基因galT、半乳糖激酶基因galK、β-半乳糖苷通透酶基因lacY和β-半乳糖苷酶基因lacZ均来源于大肠杆菌(Escherichia coli)K-12；所述磷酸葡萄糖变位酶基因pgm的核苷酸序列如SEQ ID NO:24所示，UDP-葡萄糖4-差向异构酶基因galE的核苷酸序列如SEQ ID NO:25所示，半乳糖-1-磷酸尿苷酰转移酶基因galT的核苷酸序列如SEQ ID NO:26所示，半乳糖激酶基因galK的核苷酸序列如SEQ ID NO:27所示，β-半乳糖苷通透酶基因lacY的核苷酸序列如SEQ ID NO:28所示，β-半乳糖苷酶基因lacZ的核苷酸序列如SEQ ID NO:23所示。
  4. 根据权利要求1～3任意一项所述的基因工程菌，其特征在于，所述基因工程菌以pETDuet-1质粒表达基因lacY，以pRSFDuet-1质粒依次表达基因pgm、galE、galT和galK，以pCDFDuet-1质粒依次表达基因lgtA和lgtB。
  5. 一种生产乳酰-N-新四糖的方法，其特征在于，所述方法步骤为：
    (1)将权利要求1～4任意一项所述的基因工程菌在种子培养基中培养获得种子液；
    (2)再将种子液接种于发酵体系中，培养至OD 600为0.6～0.8；
    (3)加入终浓度为0.4mM的IPTG，及终浓度为8g/L～10g/L的乳糖进行诱导，经诱导后，获得含乳酰-N-新四糖的发酵液。
  6. 根据权利要求5所述的方法，其特征在于，所述发酵体系中的碳源为葡萄糖、半乳糖和甘油中的一种或多种。
  7. 根据权利要求6所述的方法，其特征在于，所述碳源为20g/L甘油，或为10g/L甘油和10g/L半乳糖的混合物；所述发酵体系中还含有13.5g/L磷酸二氢钾，4.0g/L磷酸氢二氨，1.7g/L柠檬酸，1.4g/L七水硫酸镁和10ml/L微量金属元素；所述微量金属元素为10g/L硫酸亚铁，2.25g/L七水硫酸锌，1.0g/L无水硫酸铜，2.0g/L二水氯化钙。
  8. 根据权利要求5所述的方法，其特征在于，所述方法中的种子培养基为LB液体培养 基；所述种子培养的条件为35℃～40℃，200rpm～250rpm，摇瓶培养10h～14h。
  9. 根据权利要求5所述的方法，其特征在于，将种子液接入发酵体系，于35℃～40℃，200rpm～250rpm条件下，培养至OD 600为0.6～0.8；并在22℃～30℃，200rpm～250rpm下诱导培养42h～48h。
  10. 权利要求1～4任意一项所述的基因工程菌在生产乳酰-N-新四糖中的应用。
PCT/CN2022/087318 2021-04-25 2022-04-18 一种产乳酰-n-新四糖的基因工程菌及生产方法 WO2022228169A1 (zh)

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Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109735480B (zh) * 2019-02-27 2021-11-12 光明乳业股份有限公司 一种合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN109825465B (zh) * 2019-02-27 2021-11-12 光明乳业股份有限公司 基于平衡udp-糖供给合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN113136357B (zh) * 2021-04-25 2022-10-11 江南大学 一种产乳酰-n-新四糖的基因工程菌及生产方法
CN113684163B (zh) * 2021-08-04 2023-07-25 江南大学 一种提高乳酰-n-四糖产量的基因工程菌及其生产方法
CN113684164B (zh) * 2021-08-06 2023-08-25 江南大学 一种高产乳酰-n-新四糖的微生物的构建方法及应用
CN113652385B (zh) * 2021-08-06 2023-10-03 江南大学 一种高产乳酰-n-四糖的微生物的构建方法及应用
CN113957027B (zh) * 2021-10-20 2023-10-03 江南大学 一种提高乳酰-n-岩藻六糖产量的基因工程菌及其生产方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020001831A1 (en) * 1998-11-18 2002-01-03 Defrees Shawn Low cost manufacture of oligosaccharides
US20080145899A1 (en) * 2004-09-17 2008-06-19 Neose Technologies Inc Production of Oligosaccharides By Microorganisms
CN108410787A (zh) * 2018-03-13 2018-08-17 光明乳业股份有限公司 一种合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN109735480A (zh) * 2019-02-27 2019-05-10 光明乳业股份有限公司 一种合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN109825465A (zh) * 2019-02-27 2019-05-31 光明乳业股份有限公司 基于平衡udp-糖供给合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN111548979A (zh) * 2020-05-25 2020-08-18 江南大学 合成乳酰n-新四糖的重组大肠杆菌及其构建方法与应用
CN112654697A (zh) * 2018-09-06 2021-04-13 詹尼温生物技术有限责任公司 通过利用混合原料的全发酵进行的寡糖的发酵生产
CN113136357A (zh) * 2021-04-25 2021-07-20 江南大学 一种产乳酰-n-新四糖的基因工程菌及生产方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111979168B (zh) * 2020-08-17 2022-07-22 江南大学 一种提高乳酰-n-三糖ii产量的基因工程菌及生产方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020001831A1 (en) * 1998-11-18 2002-01-03 Defrees Shawn Low cost manufacture of oligosaccharides
US20080145899A1 (en) * 2004-09-17 2008-06-19 Neose Technologies Inc Production of Oligosaccharides By Microorganisms
CN108410787A (zh) * 2018-03-13 2018-08-17 光明乳业股份有限公司 一种合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN112654697A (zh) * 2018-09-06 2021-04-13 詹尼温生物技术有限责任公司 通过利用混合原料的全发酵进行的寡糖的发酵生产
CN109735480A (zh) * 2019-02-27 2019-05-10 光明乳业股份有限公司 一种合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN109825465A (zh) * 2019-02-27 2019-05-31 光明乳业股份有限公司 基于平衡udp-糖供给合成乳酰-n-新四糖的重组枯草芽孢杆菌及其构建方法与应用
CN111548979A (zh) * 2020-05-25 2020-08-18 江南大学 合成乳酰n-新四糖的重组大肠杆菌及其构建方法与应用
CN113136357A (zh) * 2021-04-25 2021-07-20 江南大学 一种产乳酰-n-新四糖的基因工程菌及生产方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DONG XIAOMIN, LI NAN, LIU ZHENMIN, LV XUEQIN, LI JIANGHUA, DU GUOCHENG, WANG MIAO, LIU LONG: "Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis", BIOTECHNOLOGY FOR BIOFUELS, vol. 12, no. 1, 1 December 2019 (2019-12-01), pages 1 - 11, XP055980699, DOI: 10.1186/s13068-019-1551-3 *

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