WO2023011577A1 - 一种高产乳酰-n-新四糖的微生物的构建方法及应用 - Google Patents

一种高产乳酰-n-新四糖的微生物的构建方法及应用 Download PDF

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WO2023011577A1
WO2023011577A1 PCT/CN2022/110193 CN2022110193W WO2023011577A1 WO 2023011577 A1 WO2023011577 A1 WO 2023011577A1 CN 2022110193 W CN2022110193 W CN 2022110193W WO 2023011577 A1 WO2023011577 A1 WO 2023011577A1
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gene
escherichia coli
encoding
recombinant escherichia
lactose
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沐万孟
张文立
朱莺莺
罗国聪
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江南大学
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Definitions

  • the invention relates to a construction method and application of a microorganism with high lactoyl-N-neotetrasaccharide production, and belongs to the field of microbial genetic engineering.
  • breast milk is generally considered the most important source of nutrition for infants.
  • the synthesis of breast milk oligosaccharides plays an important role in the growth of beneficial intestinal flora in infants and in preventing the adhesion of pathogenic bacteria to epithelial cells.
  • the non-fucosylated neutral breast milk oligosaccharides with the highest proportion mainly include lactoyl-N-tetraose and lactoyl-N-neotetraose, accounting for 6% of the total breast milk oligosaccharides respectively.
  • lactoyl-N-tetraose and lactoyl-N-neotetraose accounting for 6% of the total breast milk oligosaccharides respectively.
  • lactoyl-N-tetraose and lactoyl-N-neotetraose have been approved by the US FDA and EU as additives into infant formula. Therefore, it has received widespread attention.
  • the current production costs of lactoyl-N-tetraose and lactoyl-N-neotetraose are relatively high and the production methods have certain limitations.
  • lactoyl-N-neotetrasaccharide can be obtained through chemical synthesis and biosynthesis.
  • Chemical synthesis usually requires the introduction of protective groups, cumbersome steps, insufficient protection, incomplete subsequent removal, and other side reactions, and often requires the use of toxic and hazardous reagents.
  • biological synthesis is more suitable for large-scale industrial production due to the high specificity of enzymes and substrates, cheap substrates, simplified synthesis steps, fewer by-products, and greatly improved yields.
  • lactoyl-N-neotetrasaccharide is mainly synthesized by microbial fermentation.
  • lactoyl-N-neotetraose by microbial fermentation requires a key ⁇ -1,4-galactosyltransferase that can catalyze the conversion of the precursor lactoyl-N-triose II.
  • ⁇ -1,4-galactosyltransferase from Neisseria meningitidis has been studied and applied to the production of lacto-N-neotetrasaccharide.
  • Bacillus subtilis was engineered to produce lactoyl-N-neotetraose (LNnT) by chromosomal integration of two key genes lgtA (encoding ⁇ -1,3-N-acetylglucosamine aminotransferase) and lgtB , after the optimization steps, the engineered strain simultaneously produced lactoyl-N-neotetraose (LNnT) and lactoyl-N-triose II, and the titers in batch culture were 4.52-5.41 and 2.64-2.98 g, respectively /L(Dong XM, Li N, Liu ZM, et al.
  • the inventors screened a ⁇ -1,4-galactosyltransferase derived from Aggregatibacter actinomycetemcomitans, which uses lactoyl-N-triose II and UDP-galactose As a substrate, it produces lacto-N-neotetraose, which can significantly increase the production of lacto-N-neotetraose.
  • the invention provides a recombinant Escherichia coli, which expresses ⁇ -1,4-galactosyltransferase derived from Aggregatibacter actinomycetemcomitans NUM4039, and ⁇ -1,3-acetylglucose derived from Neisseria meningitidis Aminotransferase LgtA, UDP-glucose 4 epimerase GalE from Escherichia coli, and knockout the gene encoding UDP-N-acetylglucosamine-2-epimerase, encoding glucosamine-6 phosphate demerase
  • the genes of ammoniaase and ⁇ -galactosidase; the amino acid sequence of the ⁇ -1,4-galactosyltransferase is shown in SEQ ID NO.4.
  • pACYCDuet-1, pCDFDuet-1, pRSFDuet-1, pCOLADuet-1 or pETDuet-1 vectors are used to express the gene lgtA encoding ⁇ -1,3-acetylglucosamine transferase, and pACYCDuet-1, The pCDFDuet-1, pRSFDuet-1, pCOLADuet-1 or pETDuet-1 vectors simultaneously express the gene galE encoding UDP-glucose 4 epimerase and the gene Aa- ⁇ - 1,4-GalT.
  • the pRSFDuet-1 vector is used to express the gene lgtA encoding ⁇ -1,3-acetylglucosamine transferase, and the pRSFDuet-1 is used to simultaneously express the gene galE encoding UDP-glucose 4 epimerase and the gene encoding ⁇ -1,4-galactosyltransferase gene Aa- ⁇ -1,4-GalT.
  • the pETDuet-1 vector is used to express the gene lgtA encoding ⁇ -1,3-acetylglucosamine transferase
  • the pRSFDuet-1 is used to simultaneously express the gene galE encoding UDP-glucose 4 epimerase and the gene encoding ⁇ -1,4-galactosyltransferase gene Aa- ⁇ -1,4-GalT.
  • the pCDFDuet-1 vector is used to express the gene lgtA encoding ⁇ -1,3-acetylglucosamine transferase
  • the pETDuet-1 is used to express the gene galE encoding UDP-glucose 4 epimerase and the gene encoding ⁇ - 1,4-Galactosyltransferase gene Aa- ⁇ -1,4-GalT.
  • the pACYCDuet-1 vector is used to express the gene lgtA encoding ⁇ -1,3-acetylglucosamine transferase
  • the pCOLADuet-1 is used to simultaneously express the gene galE encoding UDP-glucose 4 epimerase and the gene encoding ⁇ -1,4-galactosyltransferase gene Aa- ⁇ -1,4-GalT.
  • sequence of the lgtA gene of Neisseria meningitidis is shown in SEQ ID NO.1.
  • nucleotide sequence of the gene Aa- ⁇ -1,4-GalT encoding ⁇ -1,4-galactosyltransferase is shown in SEQ ID NO.2.
  • the UDP-glucose 4 epimerase gene galE is derived from Escherichia coli K-12, and its nucleotide sequence is shown in SEQ ID NO.3.
  • the NCBI sequence number of the UDP-N-acetylglucosamine-2-epimerase WecB is YP_026253.1
  • the NCBI sequence number of the glucosamine-6 phosphate deaminase NagB is NP_415204. 1.
  • the NCBI sequence number of ⁇ -galactosidase LacZ is NP_414878.1.
  • the Escherichia coli includes but not limited to Escherichia coli BL21(DE3).
  • the invention provides a method for producing lactoyl-N-neotetrasaccharide.
  • the method uses the recombinant Escherichia coli to ferment and produce lactoyl-N-neotetraose.
  • the recombinant Escherichia coli is cultured at 35-40°C and 180-220rpm to obtain a seed liquid, and the seed liquid is added into a fermentation system containing glycerol in an amount of 2-5%, and cultivated until the OD600 is 0.6 -0.8, add IPTG and lactose so that the concentrations of IPTG and lactose in the reaction system are 0.1-0.5mM and 3-5g/L respectively, and induce culture for not less than 90h.
  • the recombinant Escherichia coli is cultured at 37°C and 200rpm to obtain a seed liquid, and the seed liquid is added to a fermentation system containing glycerol in an amount of 5%, cultivated to an OD600 of 0.6-0.8, and IPTG and Lactose, so that the concentrations of IPTG and lactose in the reaction system were 0.2mM and 5g/L, respectively, and induced for 96h.
  • the recombinant Escherichia coli is cultured at 35-40°C and 180-220rpm to obtain seed liquid, and the seed liquid is added to the fermentation system in an amount of 5-10%, and cultivated until the OD600 is 17 ⁇ 3 , adding IPTG and lactose so that the concentrations of IPTG and lactose in the reaction system are 0.1-0.5mM and 5-10g/L respectively, and the induction culture is not less than 45h.
  • the recombinant Escherichia coli is cultivated at 37°C and 200rpm to obtain seed liquid, and the seed liquid is added to the fermentation system in an amount of 10%, cultivated until the OD600 is 17 ⁇ 3, adding IPTG and lactose,
  • concentrations of IPTG and lactose in the reaction system were respectively 0.2mM and 10g/L, and induced for 47.5h.
  • lactose and glycerol are added during the reaction to maintain the concentration of glycerol not lower than 6g/L and the concentration of lactose not lower than 5g/L.
  • the concentration of glycerol in the reaction system is lower than 6g/L
  • the glycerin with a final concentration of 6g/L is added at one time
  • the concentration of lactose in the reaction system is lower than 5g/L
  • the final concentration of one-time addition is 5g/L of lactose.
  • the present invention provides the application of the ⁇ -1,4-galactosyltransferase whose amino acid sequence is shown in SEQ ID NO.4 in the preparation of lactoyl-N-neotetrasaccharide.
  • the ⁇ -1,4-galactosyltransferase is used to produce lactoyl-N-neotetraose by using lactoyl-N-triose II and UDP-galactose as substrates.
  • the invention provides the application of the recombinant Escherichia coli in the fields of food, chemical industry and medicine.
  • the invention provides the application of the recombinant Escherichia coli in the preparation of lactoyl-N-neotetrasaccharide and derivative products thereof.
  • the invention screens highly efficient ⁇ -1,4-galactosyltransferase and applies it to fermentation to produce lactyl-N-neotetrasaccharide.
  • the ⁇ -1,3-acetylglucosamine transferase gene lgtA and the screened Aa- ⁇ -1,4-GalT gene were overexpressed, and the precursor UDP-galactose supply, introduction of UDP-glucose 4 epimerase gene galE. Efficient production of lacto-N-neotetrasaccharide.
  • Fig. 1 is a diagram of lactoyl-N-neotetrasaccharide metabolic pathway
  • Fig. 2 is the output figure of lactoyl-N-neotetrasaccharide under the regulation of different copy number plasmids of metabolic pathway;
  • Figures 3A to 3D are liquid phase diagrams and mass spectrograms of the product lactoyl-N-neotetrasaccharide standard sample and the product sample;
  • Fig. 4 is a graph showing the fermentation yield of lactoyl-N-neotetraose in a 3L fermenter.
  • the plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are carried out according to the kit instructions.
  • LB liquid medium peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L.
  • (2) LB solid medium 10g/L peptone, 5g/L yeast extract powder, 10g/L sodium chloride, 15g/L agar powder.
  • Fermentation medium 20g/L glucose, 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, 0.35g/L manganese sulfate monohydrate, 0.23g/L sodium borate decahydrate , 0.11g/L ammonium molybdate, 2.0g/L calcium chloride dihydrate.
  • Lactoyl-N-neotetrasaccharide fermentation process Inoculate the constructed strain in LB liquid medium, 37°C, 200rpm, and culture overnight for 12h to obtain seed liquid, and inoculate the seed liquid with an inoculation amount of 2mL/100mL 25ml of fermentation medium (containing 20g/L glycerol), cultured at 37°C, 200rpm until the OD600 was 0.6, added with a final concentration of 0.2mM IPTG, and 5g/L lactose at the same time, 25°C, 200rpm to continue induction culture for 96h. Take 1mL of fermentation broth, centrifuge at 10,000rpm for 10min, and take the supernatant for HPLC determination.
  • HPLC detection conditions high-efficiency ion exchange chromatography; chromatographic column: CarboPac PA10 (4mm ⁇ 250mm); detector: pulsed amperometric detector; mobile phase: A, ultrapure water; B, 1M sodium acetate; C, 250mM hydrogen Sodium oxide; flow rate: 1.0mL/min; injection volume: 20 ⁇ L.
  • Embodiment 1 Construction of recombinant vector
  • Neisseria meningitidis Neisseria meningitidis (N.meningitidis) lgtA gene sequence (nucleotide sequence as shown in SEQ ID NO.1) as template, lgtA-F/R as primers, PCR amplified lgtA gene fragment, gel recovery DNA fragments; use lgtA-V-F/R as primers and pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 vectors as templates to amplify the corresponding vector fragments respectively, and recover the DNA fragments from the gel.
  • the lgtA gene fragment amplified above and the corresponding vector fragment were ligated by Gibson kit (NEB Reagent Company, USA) to obtain plasmids pRSF-lgtA, pET-lgtA, pCDF-lgtA, pCO-lgtA and pAC-lgtA, respectively.
  • Aa- ⁇ -1,4-GalT was synthesized by Suzhou Weizhi after codon optimization (the nucleotide sequence is shown in SEQ ID NO.2), using the synthesized gene as a template and Aa-F/R as a primer,
  • the Aa- ⁇ -1,4-GalT gene fragment was amplified by PCR, and the DNA fragment was recovered from the gel; the genome of Escherichia coli K-12 (Escherichia coli) was used as a template, and Aa-GalE-F/R was used as a primer for PCR amplification
  • the galE gene fragment (the nucleotide sequence of the galE gene is shown in SEQ ID NO.3), and the DNA fragment was recovered by gel;
  • the plasmids pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1 were amplified, and the DNA fragments were
  • the amplified Aa- ⁇ -1,4-GalT gene fragment, galE gene fragment and the corresponding vector fragment were connected through the Gibson kit (NEB Reagent Company, USA) to obtain plasmids pAC-Aa-galE, pCO-Aa- galE, pCDF-Aa-galE, pET-Aa-galE, and pRSF-Aa-galE.
  • Embodiment 2 Construction of recombinant bacterial strain
  • the knockout method of the gene please refer to the patent with publication number CN111979168A to construct the recombinant bacteria.
  • the recombinant plasmid constructed in Example 1 was transferred into the above-mentioned recombinant bacteria of Escherichia coli in which the genes wecB, nagB, and lacZ were knocked out, and the key gene of lactoyl-N-neotetrasaccharide was expressed in combination to obtain Eighteen different engineering bacteria were identified, which were denoted as EA1-18 respectively.
  • the recombinant bacteria constructed are shown in Table 2.
  • Example 3 Fermentative production of lactoyl-N-neotetrasaccharide by recombinant bacteria
  • Plasmid pRSFDuet-1 has a high copy number
  • plasmid pETDuet-1 has an intermediate copy number
  • plasmids pCDFDuet-1 and pCOLADuet-1 have a low copy number
  • pACYCDuet-1 has the lowest level of copy number.
  • RSF, ColE1, CDF, ColA and P15A are the replicons of expression plasmids pRSFDuet-1, pETDuet-1, pCDFDuet-1, pCOLADuet-1 and pACYCDuet-1, respectively, representing different copy numbers, and the copy numbers of the five are respectively for 100, 40, 20-40, 20-40 and 10-12.
  • the bacterial strains constructed in Example 2 were respectively inoculated in LB liquid medium, 37° C., 200 rpm, and cultivated overnight for 12 hours to obtain seed liquid.
  • the seed liquid was inserted into 25 mL of fermentation medium (containing 20 g/L Glycerol), 37°C, 200rpm, cultured to OD600 of 0.6, adding a final concentration of 0.2mM IPTG, while adding 5g/L lactose, 25°C, 200rpm to continue induction culture for 96h. Take 1mL of fermentation broth, centrifuge at 10,000rpm for 10min, and take the supernatant for HPLC determination.
  • the engineered bacterium containing recombinant plasmid pAC-lgtA and pCO-Aa-galE obtained the highest yield of 0.91g/L (the yield of lactoyl-N-neotetraose of each engineered strain is shown in Figure 2). Therefore, the lgtA gene expressing a relatively low gene dosage and the relatively high gene dosage genes Aa- ⁇ -1,4-GalT and galE can have higher lacto-N-neotetraose production.
  • Example 4 Efficient production of engineering bacteria fermenter to produce lactyl-N-neotetrasaccharide
  • the yield of lactoyl-N-neotetraose was increased. Inoculate the seed solution of recombinant Escherichia coli EA18 with 10% inoculum amount into the fermentation medium whose working volume is 1L, the fermentation temperature of the fermenter is 37°C, the stirring speed is 800r/min, the ventilation rate is 1vvm, and the pH is 7.0 (automatically controlled by adding ammonia water) ). Ferment for 12.5 hours (OD 600 is about 17.6), add lactose with a final concentration of 10g/L and IPTG with a final concentration of 0.2mM, and culture at 25°C.

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Abstract

一种高产乳酰-N-新四糖的微生物的构建方法及应用，属于微生物基因工程领域。在敲除相关基因的菌株基础上，过表达β-1,3-乙酰葡萄糖胺转移酶，β-1,4-半乳糖基转移酶和/或UDP-葡萄糖4差向异构酶的编码基因，使其拥有生产乳酰-N-四糖的合成能力。通过筛选高效的β-1,4-半乳糖基转移酶基因，并通过组合调控乳酰-N-新四糖合成途径中lgtA，Aa-β-1,4-GalT和galE的表达，从而精确调控代谢通路的碳通量，缓解代谢压力。在摇瓶实验中，大肠杆菌生产乳酰-N-新四糖的能力为0.91g/L，在3L发酵罐中，乳酰-N-新四糖的产量达到12.14g/L。

Description

一种高产乳酰-N-新四糖的微生物的构建方法及应用 技术领域
本发明涉及一种高产乳酰-N-新四糖的微生物的构建方法及应用，属于微生物基因工程领域。
背景技术
母乳通常被认为是提供婴儿营养的最重要的来源。作为母乳中含量仅次于乳糖和脂肪的第三大固体组分，对于婴幼儿有益肠道菌群的生长以及预防病原菌与上皮细胞的粘附，母乳寡糖的合成发挥着重要的作用。已经报道的母乳寡糖的种类有两百多种，主要分为三大类，唾液酸化，岩藻糖基化和非岩藻糖基化的中性母乳寡糖，并分别占比12-14％，35-50％和42-55％。其中占比最高的非岩藻糖基化的中性母乳寡糖，主要包括乳酰-N-四糖和乳酰-N-新四糖，分别占总的母乳寡糖的6％。大量文献集中报道这两种中性四糖对婴幼儿的健康具有明显的促进作用，且乳酰-N-四糖和乳酰-N-新四糖都已经被美国FDA和EU批准作为添加剂添加到婴幼儿奶粉中。因此受到广泛关注。但是目前乳酰-N-四糖和乳酰-N-新四糖的生产成本较高且生产方法有一定局限性。
目前乳酰-N-新四糖可以通过化学合成和生物合成获得。化学法合成通常需要引入保护基团，步骤繁冗，且存在保护不到位，后续脱除不彻底及发生其他副反应的问题，而且常需要使用有毒有害试剂。相比之下，生物法合成由于酶与底物的特异性高，底物廉价，合成步骤简化，副产物少，产率大大提高，更适合大规模的工业化生产。目前主要是采用微生物发酵法来合成乳酰-N-新四糖。微生物发酵法合成乳酰-N-新四糖需要一个关键的能催化前体乳酰-N-三糖II转化的β-1,4-半乳糖基转移酶。目前仅有一个来源于脑膜炎双球菌(Neisseria meningitidis)的编码β-1,4-半乳糖基转移酶的基因(lgtB)被研究并应用于乳酰-N-新四糖的生产。最近，通过对两个关键基因lgtA(编码β-1,3-N-乙酰氨基葡萄糖氨基转移酶)和lgtB进行染色体整合，改造了枯草芽孢杆菌以产生乳酰-N-新四糖(LNnT)，经过优化步骤后，工程菌株同时生产了乳酰-N-新四糖(LNnT)和乳酰-N-三糖II，在分批培养中的滴度分别为4.52-5.41和2.64-2.98g/L(Dong XM,Li N,Liu ZM,et al.Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis.Biotechnol Biofuels.2019；12(1):212.和Dong XM,Li N,Liu ZM,et al.CRISPRi-Guided multiplexed fine-tuning of metabolic flux for enhanced lacto-N-neotetraose production in Bacillus subtilis.J Agric Food Chem.2020；68(8):2477-2484.)。但目前使用的微生物方法合成的乳酰-N-新四糖产量均较低，目前已 报道的β-1,4-半乳糖基转移酶可能是一个限制因素，都不能达到工业化大规模生产的要求，因此，为了解决目前微生物生产的瓶颈，打造更高效的生产菌株是非常必要的。
发明内容
针对现有的技术难点及存在的问题，发明人筛选到了一种来源于Aggregatibacter actinomycetemcomitans的β-1,4-半乳糖基转移酶，此酶以乳酰-N-三糖II和UDP-半乳糖为底物，生产乳酰-N-新四糖，可显著提升乳酰-N-新四糖的产量。
本发明提供了一种重组大肠杆菌，所述大肠杆菌表达了来源于Aggregatibacter actinomycetemcomitans NUM4039的β-1,4-半乳糖基转移酶，来源于脑膜炎奈瑟球菌的β-1,3-乙酰葡萄糖胺转移酶LgtA，来源于大肠杆菌的UDP-葡萄糖4差向异构酶GalE，并且敲除编码UDP-N-乙酰葡萄糖胺-2-差向异构酶的基因、编码葡萄糖胺-6磷酸脱氨酶和β-半乳糖苷酶的基因；所述β-1,4-半乳糖基转移酶的氨基酸序列如SEQ ID NO.4所示。
在一种实施方式中，利用pACYCDuet-1、pCDFDuet-1、pRSFDuet-1，pCOLADuet-1或pETDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pACYCDuet-1、pCDFDuet-1、pRSFDuet-1，pCOLADuet-1或pETDuet-1载体同时表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
在一种实施方式中，利用pRSFDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pRSFDuet-1同时表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
在一种实施方式中，利用pETDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pRSFDuet-1同时表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
在一种实施方式中，利用pCDFDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pETDuet-1表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
在一种实施方式中，利用pACYCDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pCOLADuet-1同时表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
在一种实施方式中，所述脑膜炎奈瑟球菌的lgtA基因的序列如SEQ ID NO.1所示。
在一种实施方式中，编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT的核苷酸序列如 SEQ ID NO.2所示。
在一种实施方式中，所述UDP-葡萄糖4差向异构酶基因galE来源于大肠杆菌K-12，核苷酸序列如SEQ ID NO.3所示。
在一种实施方式中，所述UDP-N-乙酰葡萄糖胺-2-差向异构酶WecB的NCBI序列号为YP_026253.1，葡萄糖胺-6磷酸脱氨酶NagB的NCBI序列号为NP_415204.1，β-半乳糖苷酶LacZ的NCBI序列号为NP_414878.1。
在一种实施方式中，所述大肠杆菌包括但不限于大肠杆菌BL21(DE3)。
本发明提供了一种生产乳酰-N-新四糖的方法，所述方法是利用所述重组大肠杆菌发酵生产乳酰-N-新四糖。
在一种实施方式中，将所述重组大肠杆菌在35～40℃、180～220rpm培养得到种子液，将种子液按2～5％量加入含有甘油的发酵体系中，培养至OD 600为0.6-0.8，加入IPTG和乳糖，使得IPTG和乳糖在反应体系中的浓度分别为0.1～0.5mM、3～5g/L，诱导培养不少于90h。
在一种实施方式中，将所述重组大肠杆菌在37℃、200rpm培养得到种子液，将种子液按5％量加入含有甘油的发酵体系中，培养至OD 600为0.6-0.8，加入IPTG和乳糖，使得IPTG和乳糖在反应体系中的浓度分别为0.2mM、5g/L，诱导培养96h。
在一种实施方式中，将所述重组大肠杆菌在35～40℃、180～220rpm培养得到种子液，将种子液按5～10％的量加入发酵体系中，培养至OD 600为17±3，加入IPTG和乳糖，使得IPTG和乳糖在反应体系中的浓度分别为0.1～0.5mM、5～10g/L，诱导培养不少于45h。
在一种实施方式中，将所述重组大肠杆菌在37℃、200rpm培养得到种子液，将种子液按10％的量加入发酵体系中，培养至OD 600为17±3，加入IPTG和乳糖，使得IPTG和乳糖在反应体系中的浓度分别为0.2mM、10g/L，诱导培养47.5h。
在一种实施方式中，反应过程中补加乳糖和甘油，以维持甘油浓度不低于6g/L，乳糖浓度不低于5g/L。
在一种实施方式中，当反应体系中甘油浓度低于6g/L时，一次性添加终浓度为6g/L的甘油；当反应体系中乳糖浓度低于5g/L，一次性添加终浓度为5g/L的乳糖。
本发明提供了氨基酸序列如SEQ ID NO.4所示的β-1,4-半乳糖基转移酶在制备乳酰-N-新四糖中的应用。
在一种实施方式中，以乳酰-N-三糖II和UDP-半乳糖为底物，利用所述β-1,4-半乳糖基转移酶，生产乳酰-N-新四糖。
本发明提供了所述重组大肠杆菌在食品、化工、医药领域中的应用。
本发明提供了所述重组大肠杆菌在制备乳酰-N-新四糖及其衍生产品中的应用。
本发明的有益效果：
本发明通过筛选高效的β-1,4-半乳糖基转移酶，并应用于发酵生产乳酰-N-新四糖。在本团队前期构建的敲除相关基因的大肠杆菌宿主基础上，过表达β-1,3-乙酰葡萄糖胺转移酶基因lgtA和筛选的Aa-β-1,4-GalT基因，并强化前体UDP-半乳糖的供应，引入UDP-葡萄糖4差向异构酶基因galE。高效生产乳酰-N-新四糖。在摇瓶实验中，大肠杆菌生产乳酰-N-新四糖的能力为0.91g/L，在3L发酵罐中，乳酰-N-新四糖的产量达到12.14g/L，具备工业应用的前景。
附图说明
图1为乳酰-N-新四糖代谢通路图；
图2为代谢通路在不同拷贝数质粒调节下的乳酰-N-新四糖的产量图；
图3A～3D为产物乳酰-N-新四糖标样和产物样品液相图和质谱图；
图4为3L发酵罐中乳酰-N-新四糖的发酵产量结果图。
具体实施方式
1、以下实例中所使用的质粒，内切酶，PCR酶，柱式DNA抽提试剂盒和DNA凝胶回收试剂盒等采用商用产品，具体操作按照试剂盒说明书进行。
2、菌落PCR，核酸琼脂糖凝胶电泳，蛋白质SDS-PAGE凝胶电泳，热击转化，电转化和感受态细胞的制备和细菌基因组的提取保存等常规操作方法根据Molecular Cloning:A Laboratory Manual(Fourth Edition)进行。
3、质粒和DNA产物的测序工作交予上海生工生物工程公司完成。
4、大肠杆菌感受态的制备：TAKARA试剂盒。
5、乳酰-N-四糖发酵过程及检测：
(1)LB液体培养基：蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L。
(2)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。
(3)发酵培养基：20g/L葡萄糖，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，0.35g/L一水硫酸锰，0.23g/L十水硼酸钠，0.11g/L钼酸铵，2.0g/L二水氯化钙。
(4)乳酰-N-新四糖发酵过程：将构建的菌株接种于LB液体培养基，37℃，200rpm，过 夜培养12h，得到种子液，将种子液以2mL/100mL的接种量接入25ml发酵培养基(含20g/L甘油)，37℃，200rpm，培养至OD 600为0.6，加入终浓度为0.2mM IPTG，同时加入5g/L乳糖，25℃，200rpm继续诱导培养96h。取1mL发酵液，10,000rpm，离心10min，取上清，用于HPLC测定。
(5)HPLC检测条件：高效离子交换色谱；色谱柱：CarboPac PA10(4mm×250mm)；检测器：脉冲安培检测器；流动相：A，超纯水；B，1M醋酸钠；C，250mM氢氧化钠；流速：1.0mL/min；进样量：20μL。
实施例1：重组载体的构建
重组表达载体构建具体步骤如下(所涉及到的引物序列见表1)：
(1)lgtA基因片段的获得以及质粒pAC-lgtA，pCO-lgtA，pCDF-lgtA，pET-lgtA和pRSF-lgtA的构建：
以脑膜炎奈瑟球菌(N.meningitidis)lgtA基因序列(核苷酸序列如SEQ ID NO.1所示)为模板，以lgtA-F/R为引物，PCR扩增出lgtA基因片段，胶回收DNA片段；以lgtA-V-F/R为引物，以pRSFDuet-1，pETDuet-1，pCDFDuet-1，pCOLADuet-1和pACYCDuet-1载体为模板，分别扩增出相应的载体片段，胶回收DNA片段。
将上述扩增得到的lgtA基因片段和对应的载体片段通过Gibson试剂盒(美国NEB试剂公司)连接，分别获得质粒pRSF-lgtA，pET-lgtA，pCDF-lgtA，pCO-lgtA和pAC-lgtA。
(2)Aa-β-1,4-GalT和galE基因片段的获得以及质粒pAC-Aa-galE，pCO-Aa-galE，pCDF-Aa-galE，pET-Aa-galE，和pRSF-Aa-galE的构建：
Aa-β-1,4-GalT由苏州唯智通过密码子优化后合成(核苷酸序列如SEQ ID NO.2所示)，以该合成的基因为模板，以Aa-F/R为引物，PCR扩增出Aa-β-1,4-GalT基因片段，胶回收DNA片段；以大肠杆菌K-12(Escherichia coli)的基因组为模板，以Aa-GalE-F/R为引物，PCR扩增出galE基因片段(galE基因的核苷酸序列如SEQ ID NO.3所示)，胶回收DNA片段；利用Aa-GalE-V 1-F/R和Aa-GalE-V 2-F/R两对引物，分别对质粒pRSFDuet-1，pETDuet-1，pCDFDuet-1，pCOLADuet-1和pACYCDuet-1进行扩增，并胶回收DNA片段。
将上述扩增得到的Aa-β-1,4-GalT基因片段，galE基因片段和对应载体片段通过Gibson试剂盒(美国NEB试剂公司)连接，分别获得质粒pAC-Aa-galE，pCO-Aa-galE，pCDF-Aa-galE，pET-Aa-galE，和pRSF-Aa-galE。
表1质粒构建引物
Figure PCTCN2022110193-appb-000001
实施例2：重组菌株的构建
敲除大肠杆菌BL21中编码UDP-N-乙酰葡萄糖胺-2-差向异构酶WecB(NCBI序列号为YP_026253.1)的基因wecB、编码葡萄糖胺-6磷酸脱氨酶NagB(NCBI序列号为NP_415204.1)的基因nagB以及编码β-半乳糖苷酶LacZ(NCBI序列号为NP_414878.1)的基因lacZ，基因的敲除方法具体参见公开号为CN111979168A的专利，构建得到重组菌。
在上述重组菌的基础上，将实施例1所构建得到的重组质粒转入上述敲除了基因wecB，nagB，lacZ的大肠杆菌重组菌中，组合表达乳酰-N-新四糖关键基因，得到了18个不同的工程菌，分别表示为EA1～18。构建得到的重组菌如表2所示。
实施例3：重组菌发酵生产乳酰-N-新四糖
质粒pRSFDuet-1具有较高的拷贝数，质粒pETDuet-1有中等的拷贝数，而质粒pCDFDuet-1和pCOLADuet-1具有较低拷贝数，pACYCDuet-1具有最低水平的拷贝数。其中RSF，和ColE1，CDF，ColA和P15A分别是表达质粒pRSFDuet-1，pETDuet-1，pCDFDuet-1，pCOLADuet-1和pACYCDuet-1的复制子，代表不同的拷贝数，五者的拷贝数分别为100，40，20-40，20-40和10-12。
将实施例2中构建的菌株分别接种于LB液体培养基，37℃，200rpm，过夜培养12h，得 到种子液，将种子液以2mL/100mL的接种量接入25mL发酵培养基(含20g/L甘油)，37℃，200rpm，培养至OD 600为0.6，加入终浓度为0.2mM IPTG，同时加入5g/L乳糖，25℃，200rpm继续诱导培养96h。取1mL发酵液，10,000rpm，离心10min，取上清，用于HPLC测定。
结果如表2所示：发酵后不同工程菌株乳酰-N-新四糖的产量分别为0.78g/L，0.13g/L，0.01g/L，0.61g/L，0.58g/L，0.43g/L，0.42g/L，0.02g/L，0.64g/L，0.17g/L，0.15g/L，0.47g/L，0.23g/L，0.44g/L，0.11g/L，0.35g/L，0.24g/L和0.91g/L。其中含有重组质粒pAC-lgtA和pCO-Aa-galE的工程菌(即菌株EA18)获得了0.91g/L的最高产量(各工程菌株乳酰-N-新四糖产量参见图2)。因此，表达相对较低的基因剂量的lgtA基因同时相对较高基因剂量的基因Aa-β-1,4-GalT和galE可以有更高的乳酰-N-新四糖产量。
表2各工程菌摇瓶发酵详细信息
Figure PCTCN2022110193-appb-000002
Figure PCTCN2022110193-appb-000003
实施例4：高效生产工程菌发酵罐生产乳酰-N-新四糖
为了进一步验证乳酰-N-新四糖合成方法的有效性，提高乳酰-N-新四糖的产量。将重组大肠杆菌EA18种子液以10％的接种量接种到工作体积为1L的发酵培养基中，发酵罐发酵温度37℃，搅拌转速800r/min，通气量1vvm，pH 7.0(补加氨水自动控制)。发酵12.5h(OD 600约为17.6)，加入终浓度为10g/L乳糖和终浓度为0.2mM的IPTG，在25℃下培养。期间手动补料甘油和乳糖：当反应体系中甘油浓度低于6g/L的时补加30mL母液(母液甘油浓度600g/L)，乳糖浓度低于5g/L时进补加25mL母液(母液乳糖浓度200g/L)，以维持菌体的生长以及乳酰-N-新四糖的合成。培养整个过程达到47.5h后，菌体OD 600达到127，乳酰-N-新四糖的产量达到最高，达到12.14g/L(图4)。
表3发酵过程中菌和乳酰-N-新四糖合成量动态变化表
Figure PCTCN2022110193-appb-000004
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 (19)

  1. 一种重组大肠杆菌，其特征在于，表达了来源于Aggregatibacter actinomycetemcomitans NUM4039的β-1,4-半乳糖基转移酶，来源于脑膜炎奈瑟球菌的β-1,3-乙酰葡萄糖胺转移酶，来源于大肠杆菌的UDP-葡萄糖4差向异构酶，并且敲除编码UDP-N-乙酰葡萄糖胺-2-差向异构酶的基因、编码葡萄糖胺-6磷酸脱氨酶的基因和编码β-半乳糖苷酶的基因；所述β-1,4-半乳糖基转移酶的氨基酸序列如SEQ ID NO.4所示。
  2. 根据权利要求1所述的重组大肠杆菌，其特征在于，利用pACYCDuet-1、pCDFDuet-1、pRSFDuet-1，pCOLADuet-1或pETDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因，利用pACYCDuet-1、pCDFDuet-1、pRSFDuet-1，pCOLADuet-1或pETDuet-1载体共同表达编码UDP-葡萄糖4差向异构酶的基因和编码β-1,4-半乳糖基转移酶的基因。
  3. 根据权利要求2所述的重组大肠杆菌，其特征在于，利用pRSFDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pRSFDuet-1同时表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
  4. 根据权利要求2所述的重组大肠杆菌，其特征在于，利用pETDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pRSFDuet-1同时表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
  5. 根据权利要求2所述的重组大肠杆菌，其特征在于，利用pCDFDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pETDuet-1表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
  6. 根据权利要求2所述的重组大肠杆菌，其特征在于，利用pACYCDuet-1载体表达编码β-1,3-乙酰葡萄糖胺转移酶的基因lgtA，利用pCOLADuet-1同时表达编码UDP-葡萄糖4差向异构酶的基因galE和编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT。
  7. 根据权利要求1～6任一所述的重组大肠杆菌，其特征在于，所述脑膜炎奈瑟球菌的lgtA基因的序列如SEQ ID NO.1所示。
  8. 根据权利要求7所述的重组大肠杆菌，其特征在于，编码β-1,4-半乳糖基转移酶的基因Aa-β-1,4-GalT的核苷酸序列如SEQ ID NO.2所示。
  9. 根据权利要求8所述的重组大肠杆菌，其特征在于，所述UDP-葡萄糖4差向异构酶基因galE来源于大肠杆菌K-12，核苷酸序列如SEQ ID NO.3所示。
  10. 根据权利要求9所述的重组大肠杆菌，其特征在于，所述UDP-N-乙酰葡萄糖胺-2-差向异构酶WecB的NCBI序列号为YP_026253.1，葡萄糖胺-6磷酸脱氨酶NagB的NCBI序列号为NP_415204.1，β-半乳糖苷酶LacZ的NCBI序列号为NP_414878.1。
  11. 根据权利要求10所述的重组大肠杆菌，其特征在于，所述大肠杆菌包括但不限于大 肠杆菌BL21(DE3)。
  12. 一种生产乳酰-N-新四糖的方法，其特征在于，利用权利要求1～11任一所述重组大肠杆菌发酵生产乳酰-N-新四糖。
  13. 根据权利要求12所述的方法，其特征在于，将所述重组大肠杆菌在35～40℃、180～220rpm培养得到种子液，将种子液按2～5％量加入含有甘油的发酵体系中，培养至OD 600为0.6～0.8，加入IPTG和乳糖，使得IPTG和乳糖在反应体系中的浓度分别为0.1～0.5mM、3～5g/L，诱导培养不少于90h。
  14. 根据权利要求13所述的方法，其特征在于，将所述重组大肠杆菌在35～40℃、180～220rpm培养得到种子液，将种子液按5～10％的量加入发酵体系中，培养至OD 600为17±3，加入IPTG和乳糖，使得IPTG和乳糖在反应体系中的浓度分别为0.1～0.5mM、5～10g/L，诱导培养不少于45h。
  15. 根据权利要求12～14所述的方法，其特征在于，将所述重组大肠杆菌在37℃、200rpm培养得到种子液，将种子液按10％的量加入发酵体系中，培养至OD 600为17±3，加入IPTG和乳糖，使得IPTG和乳糖在反应体系中的浓度分别为0.2mM、10g/L，诱导培养47.5h。
  16. 根据权利要求16所述的方法，其特征在于，反应过程中补加乳糖和甘油，以维持甘油浓度不低于6g/L，乳糖浓度不低于5g/L。
  17. 根据权利要求15或16所述的方法，其特征在于，当反应体系中甘油浓度低于6g/L时，一次性添加终浓度为6g/L的甘油；当反应体系中乳糖浓度低于5g/L，一次性添加终浓度为5g/L的乳糖。
  18. 氨基酸序列如SEQ ID NO.4所示的β-1,4-半乳糖基转移酶在制备乳酰-N-新四糖中的应用。
  19. 权利要求1～4任一所述重组大肠杆菌在食品、化工、医药领域中的应用。
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