WO2021128464A1 - 一种转录因子SugR编码基因及其在N-乙酰氨基葡萄糖生产中的应用 - Google Patents

一种转录因子SugR编码基因及其在N-乙酰氨基葡萄糖生产中的应用 Download PDF

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WO2021128464A1
WO2021128464A1 PCT/CN2020/070593 CN2020070593W WO2021128464A1 WO 2021128464 A1 WO2021128464 A1 WO 2021128464A1 CN 2020070593 W CN2020070593 W CN 2020070593W WO 2021128464 A1 WO2021128464 A1 WO 2021128464A1
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corynebacterium glutamicum
acetylglucosamine
sugr
encoding gene
gene
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卢健行
刘龙
陈坚
刘长峰
吕雪芹
堵国成
李江华
邓琛
卢建功
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江南大学
山东润德生物科技有限公司
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Publication of WO2021128464A1 publication Critical patent/WO2021128464A1/zh

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    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C12R2001/15—Corynebacterium

Definitions

  • the invention relates to the field of metabolic engineering, in particular to a transcription factor SugR encoding gene and its application in the production of N-acetylglucosamine.
  • N-Acetyl Glucosamine is a derivative of Glucosamine, which has reducing properties and is also an important precursor substance for the synthesis of bifidus factor and hyaluronic acid, also known as 2-(acetylamino)-2-deoxy-glucose And N-acetylglucosamine, which is the basic unit of a variety of polysaccharides in the organism, has important physiological functions in the organism.
  • Corynebacterium glutamicum is a gram-positive soil bacterium with high GC content in the Actinomycota. It has been used in the industrial production of amino acids and is designed to produce various compounds, including polymer building blocks and biofuels.
  • transcription factors including two-component system and sigma factor
  • the current research on transcription factors (including two-component system and sigma factor) in bacteria has revealed the transcriptional regulatory link between metabolic pathways and stress response systems, forming a complex transcriptional regulatory network. Therefore, it is important to develop new recombinant bacteria and methods for increasing the production of N-acetylglucosamine in Corynebacterium glutamicum.
  • the purpose of the present invention is to provide a transcription factor SugR encoding gene and its application in the production of N-acetylglucosamine.
  • the present invention overexpresses the transcription factor SugR in Corynebacterium glutamicum, The recombinant Corynebacterium glutamicum with improved N-acetylglucosamine production was obtained.
  • the N-acetylglucosamine-producing recombinant Corynebacterium glutamicum of the present invention is obtained by overexpressing the transcription factor SugR of its own origin in the origin bacterium Corynebacterium glutamicum.
  • the present invention overexpresses SugR, a transcription factor related to carbon center metabolism in Corynebacterium glutamicum, thereby increasing the production of N-acetylglucosamine by Corynebacterium glutamicum.
  • SugR a transcription factor related to carbon center metabolism in Corynebacterium glutamicum
  • the starting bacterium Corynebacterium glutamicum knocked out the acetylglucosamine deacetylase encoding gene NagA, the acetylglucosamine deaminase encoding gene GamA, and the L-lactate dehydrogenase encoding gene ldh.
  • Corynebacterium glutamicum is Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA- ⁇ ldh.
  • Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA- ⁇ ldh is obtained from C. glutamicum S9114 ⁇ nagA- ⁇ gamA as the starting strain, and on the basis of which the gene ldh encoding L-lactate dehydrogenase is knocked out.
  • the encoding gene ldh of L-lactate dehydrogenase is shown in NCBI-Gene ID: 1020853.
  • Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA For the construction method of Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA, refer to CN 110195036 A.
  • the pathway of the host bacterium Corynebacterium glutamicum to synthesize the by-product lactic acid is blocked.
  • the coding gene of the transcription factor SugR is shown in NCBI-GeneID: 1019888, its nucleotide sequence is shown in SEQ ID NO. 8, and the amino acid sequence encoded by the SugR gene is shown in SEQ ID NO. 9.
  • SugR is a DeoR-type transcriptional regulator. It was originally identified as a repressor of the gene encoding the PTS component of sugar uptake in Corynebacterium glutamicum, which controls the gene encoding the fermentation lactate dehydrogenase and the gene encoding glycolytic enzymes. Transcription can make the carbon metabolism flow more to the GlcNAc production pathway, thereby improving the ability of Corynebacterium glutamicum to produce GlcNAc.
  • the gene encoding the transcription factor SugR is expressed by the expression vector pJYW-4-ceN-C.glglmS.
  • the specific construction process of the expression vector pJYW-4-ceN-C.glglmS can be found in the literature-Chen Deng, XueqinLv, Yanfeng Liu, Long Liu. Biotechnology, 2019.4: 120-129.
  • the present invention also provides a method for constructing the above-mentioned N-acetylglucosamine-producing recombinant Corynebacterium glutamicum, which comprises the following steps:
  • the pJYW-4-ceN-C.glglmS-SugR expression vector is transferred into the host bacteria to obtain the N-acetylglucosamine-producing recombinant Corynebacterium glutamicum.
  • the host bacterium is Corynebacterium glutamicum in which the acetylglucosamine deacetylase encoding gene NagA, the acetylglucosamine deaminase encoding gene GamA, and the L-lactate dehydrogenase encoding gene ldh have been knocked out.
  • the host bacteria is Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA- ⁇ ldh, and its construction method includes the following steps:
  • knockout box of the acetylglucosamine deacetylase encoding gene NagA Use the knockout box of the acetylglucosamine deacetylase encoding gene NagA, the knockout box of the acetylglucosamine deaminase encoding gene GamA, and the knockout box of the L-lactate dehydrogenase encoding gene ldh to knock out the valleys in turn
  • the acetylglucosamine deacetylase encoding gene NagA, the acetylglucosamine deaminase encoding gene GamA and the L-lactate dehydrogenase encoding gene ldh in Corynebacterium glutamicum S9114.
  • Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA- ⁇ ldh is obtained from C. glutamicum S9114 ⁇ nagA- ⁇ gamA as the starting strain, and on the basis of which the gene encoding ldh for L-lactate dehydrogenase is knocked out.
  • For the construction method of Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA refer to CN 110195036 A. On this basis, continue to construct the knockout box of the gene encoding ldh of L-lactate dehydrogenase, and after homologous recombination, the kanamycin resistance gene kana in the knockout box of ldh is used to replace histone glutamate.
  • Corynebacterium C.glutamicum S9114 ⁇ nagA- ⁇ gamA The gene encoding L-lactate dehydrogenase ldh in the genome.
  • step (2) the rapid cloning reagent is ClonExpress II One Step Cloning Kit of Novoz Biotechnology Co., Ltd.
  • the invention also discloses the application of the above-mentioned N-acetylglucosamine-producing recombinant Corynebacterium glutamicum in the production of N-acetylglucosamine.
  • the recombinant Corynebacterium glutamicum seed liquid cultured at 28-30°C and 220 rpm for 16 hours is transferred into the fermentation medium so that the initial OD 562 of the fermentation medium is 1.6. Incubate at 28-30°C and 220rpm for 72-100h.
  • the present invention has at least the following advantages:
  • the present invention provides a method for constructing a genetically engineered strain that can increase the yield of N-acetylglucosamine. It is used to knock out the coding gene NagA of acetylglucosamine deacetylase, the coding gene GamA of acetylglucosamine deaminase and L-lactate dehydrogenase.
  • PCR was used to amplify the gene encoding the transcription factor SugR, a global regulatory factor for carbon metabolism of Corynebacterium glutamicum, and the gene was linked to the shuttle expression vector pJYW- of Corynebacterium glutamicum and Escherichia coli.
  • 4-ceN-C.glglmS affects the distribution of intracellular carbon metabolism, increases the accumulation of N-acetylglucosamine outside the cell, and its concentration can reach up to 26g/L, which is used for further metabolic engineering of Corynebacterium glutamicum to produce amino Glucose laid the foundation.
  • the method for constructing recombinant Corynebacterium glutamicum provided by the invention is simple, convenient to use, and has good application prospects.
  • Figure 1 is a plasmid map of pJYW-4-ceN-C.glglmS.
  • Figure 2 is a plasmid map for constructing recombinant pJYW-4-ceN-C.glglmS-SugR.
  • Figure 3 is a graph showing the yield of GlcNAc in shake flask fermentation supernatants of different strains.
  • High performance liquid chromatography (HPLC) detection method Agilent 1260, RID detector, HPX-87H column (Bio-Rad Hercules, CA), mobile phase: 5mM H 2 S0 4 , flow rate 0.6mL/min, column temperature 35°C, The injection volume is 10 ⁇ L.
  • Seed activation medium liquid (LBG) (g/L): peptone 10.0, yeast extract 5.0, NaCl 10.0, glucose 5.0, filling volume 20ml per 250ml Erlenmeyer flask.
  • Seed activation medium solid (LBG solid) (g/L): peptone 10.0, yeast powder 5.0, NaCl 10.0, glucose 5.0, nutrient agar 15.0-20.0.
  • Competent Medium Peptone 10.0, Yeast Extract 5.0, NaCl 10.0, Glycine 30.0, Isoniazid 4.0, and 10ml Tween80 is added at the same time, and the filling volume is 50ml per 500ml Erlenmeyer flask.
  • Recovery medium LBHIS (g/L) after electric shock transformation peptone 5.0, yeast extract 2.5, NaCl 5.0, brain heart infusion 18.5, sorbitol 91.0.
  • the transformant detected medium solids g/L: peptone 5.0, yeast extract 2.5, NaCl 5.0, brain heart extract 18.5, sorbitol 91.0, nutrient agar 15.0-20.0.
  • Seed medium (g/L): glucose 25.0, corn steep liquor 20.0, KH 2 PO 4 1.0, (NH 4 ) 2 SO 4 0.5, urea 1.25, pH 7.0.
  • Fermentation medium (g/L): glucose 40.0, corn steep liquor 20.0, KH 2 PO 4 1.0, (NH 4 ) 2 SO 4 20.0, MgSO 40.5, CaCO 3 20.0, pH 7.0.
  • Optimized fermentation medium glucose 100.0, corn steep liquor 10.0, KH 2 PO 4 1.0, (NH 4 ) 2 SO 4 20.0, MgSO 40.5, CaCO 3 20.0, FeSO 4 0.18, pH 7.0.
  • Sterilization conditions 115°C, 20min, add 25mg/L thiokanamycin when all the media are used for transformant detection or for recombinant bacteria culture.
  • L-lactate dehydrogenase encoding gene (nucleotide sequence shown in SEQ ID NO.1) published on NCBI, the knockout homology arm was designed Amplification primers, the left arm upper and downstream primers are LdhloxPUF (nucleotide sequence shown in SEQ ID NO. 2) and LdhloxPUR (nucleotide sequence shown in SEQ ID NO.
  • right arm upper and downstream primers They are LdhloxPDF (nucleotide sequence shown in SEQ ID NO.4) and LdhloxPDR (nucleotide sequence shown in SEQ ID NO.5); respectively, using the genomic DNA of Corynebacterium glutamicum S9114 strain as a template, by PCR Amplify the left and right arms.
  • the right arm was digested with EcoRI for 2 hours, and the plasmid pBluescriptIISK(+) (provided by Dr. Xiaoyuan Wang of Jiangnan University) digested with XhoI/EcoRI for 2 hours was ligated with T4 ligase overnight at 16°C.
  • the recombinant plasmid pBluescriptIISK(+)-ldh was electroporated into Corynebacterium glutamicum S9114- ⁇ NagA-GamA, and the kanamycin resistance plate was screened and colony PCR was verified to confirm that both the left and right arms of the knockout box were bound to the S9114 genome.
  • the gene encoding L-lactate dehydrogenase ldh was knocked out to obtain Corynebacterium glutamicum S9114- ⁇ NagA-GamA- ⁇ ldh. After 72h, the GlcNAc yield of this strain was 24.7g/L.
  • PCR conditions are: 95°C pre-denaturation 10min; 98°C denaturation 1min; 55°C annealing 1min; 72°C extension 1min, The reaction was carried out for 30 cycles; the final extension was at 72°C for 10 minutes, and the PCR product was recovered with DNA purification kit.
  • the SugR gene was amplified from the Corynebacterium glutamicum S9114 genome, and the SugR gene was amplified using LA Taq HS DNA polymerase.
  • the plasmid pJYW-4-ceN-C.glglmS constructed in the preliminary work of the laboratory was used as an expression vector to express the SugR gene.
  • the specific construction process of the pJYW-4-ceN-C.glglmS plasmid please refer to the literature——Chen Deng, XueqinLv, Yanfeng Liu, Long Liu. Metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis. Synthetic and Systems Biotechnology, 2019.4: 120-129.
  • PCR conditions are: 95°C pre-denaturation for 3 min; 98°C denaturation for 1 min; 55°C annealing for 1 min; 72°C extension for 5 min , React for 30 cycles; finally extend at 72°C for 10 min.
  • the PCR product was recovered with a DNA purification kit to obtain the linearized plasmid pJYW-4-ceN-C.glglmS.
  • the transformants with the correct colony PCR were selected, and then sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing verification, and the recombinant expression vector pJYW-4-ceN-C.glglmS-SugR was obtained.
  • the plasmid pJYW-4-ceN-C.glglmS-SugR was transformed into Corynebacterium glutamicum S9114 ⁇ nagA- ⁇ gamA- ⁇ ldh strain by electroporation transformation.
  • C. glutamicum is inoculated on LBG medium (it needs to be selected on a freshly cultivated slope, otherwise it will affect the growth of the bacteria), placed on a circulatory shaker (200rpm), cultivated at 30°C for 16 hours, and the OD 562 reaches 3.0 .
  • Competent Corynebacterium glutamicum stored at -80°C will melt in an ice bath.
  • Competent efficiency verification add 5.0 ⁇ L sterile ddH 2 O as a negative control, sterile colony, positive control add 1-5 ⁇ L plasmid pXMJl9 (total DNA is about 1.0 ⁇ g), grow a large number of colonies, the sequence is correct. It is a recombinant Corynebacterium glutamicum.
  • Example 3 The effect of overexpression of SugR gene in recombinant Corynebacterium glutamicum on the production of N-acetylglucosamine
  • the recombinant Corynebacterium glutamicum strain containing the plasmid pJYW-4-ceN-C.glglmS-SugR with the correct sequencing result was inoculated into a streaked LBG plate (sulfokanamycin 25mg/L) in a glycerol tube at 30°C After 18 hours of culture at 220 rpm, single colonies were selected and re-streaked on the LBG plate until a large number of colonies grew.
  • LBG plate sulfokanamycin 25mg/L
  • the seed culture solution was inoculated into the fermentation medium at an inoculum of 10%, and cultured at 30°C at 220 rpm for 72 hours. Measure the amount of GlcNAc produced.
  • the GlcNAc yield was 24.7g/L after 72h (CK in Figure 3), while the plasmid pJYW overexpressing the SugR gene
  • the GlcNAc yield of the strain of -4-ceN-C.glglmS-SugR 72h was 26g/L (SugR in Figure 3).

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Abstract

提供了一种生产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌及其应用。该重组谷氨酸棒杆菌通过在谷氨酸棒杆菌中过表达自身来源的转录因子SugR得到。该重组谷氨酸棒杆菌提高了乙酰氨基葡萄糖的产量，产量达到26g/L。

Description

一种转录因子SugR编码基因及其在N-乙酰氨基葡萄糖生产中的应用 技术领域
本发明涉及代谢工程领域，尤其涉及一种一种转录因子SugR编码基因及其在N-乙酰氨基葡萄糖生产中的应用。
背景技术
N-乙酰氨基葡萄糖(GlcNAc)是氨基葡萄糖的一种衍生物，具有还原性，也是合成双岐因子和透明质酸的重要前体物质，又称2-(乙酰氨基)-2-脱氧-葡萄糖及N-乙酰葡萄糖胺，是生物体内多种多糖的基本组成单位，在生物体内具有重要的生理功能。谷氨酸棒杆菌是放线菌门中高GC含量的革兰氏阳性土壤细菌，已被用于氨基酸的工业生产，并被设计用于生产各种化合物，包括聚合物结构单元和生物燃料。自其基因组序列首次发表以来，其多功能代谢途径及其遗传成分和调控机制已得到广泛研究。为了提高生物技术生产的效率，基于基因组序列信息开发了遗传工具和基于组学的分析方法，包括转录组学，蛋白质组学，代谢组学和流变学，并广泛用于了解代谢途径及其在转录后的调控。
目前对细菌中转录因子(包括双组分系统和σ因子)的研究揭示了代谢途径与应激反应系统之间的转录调控联系，形成了复杂的转录调控网络。因此，开发新的提高谷氨酸棒杆菌中N-乙酰氨基葡萄糖产量的重组菌及方法即为重要。
发明内容
为解决上述技术问题，本发明的目的是提供一种一种转录因子SugR编码基因及其在N-乙酰氨基葡萄糖生产中的应用，本发明通过在谷氨酸棒杆菌中过表达转录因子SugR，而得到了N-乙酰氨基葡萄糖产量提高的重组谷氨酸棒杆菌。
本发明的技术方案如下：
本发明的一种产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌，该重组谷氨酸棒杆菌通过在出发菌谷氨酸棒杆菌中过表达自身来源的转录因子SugR得到。
为了重新分配谷氨酸棒杆菌中的碳代谢流，本发明过表达了谷氨酸棒杆菌中一个与碳中心代谢相关的转录因子SugR，从而提高了谷氨酸棒杆菌生产N-乙酰氨基葡萄糖的能力。
进一步地，出发菌谷氨酸棒杆菌敲除了乙酰氨基葡萄糖脱乙酰酶编码基因NagA、乙酰氨基葡萄糖脱氨基酶编码基因GamA及L-乳酸脱氢酶的编码基因ldh。
进一步地，出发菌谷氨酸棒杆菌为谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA-Δldh。谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA-Δldh是以谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA为出发菌株，在其基础上敲除了L-乳酸脱氢酶的编码基因ldh得到。其中，L-乳酸脱氢酶的编码基因ldh如NCBI-Gene ID:1020853所示。谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA的构建方法参照CN 110195036 A。通过敲除编码催化由丙酮酸形成乳酸的L-乳酸脱氢酶的ldh基因，来阻断宿主菌谷氨酸棒杆菌合成副产物乳酸的途径。
进一步地，转录因子SugR的编码基因如NCBI-GeneID:1019888所示，其核苷酸序列如SEQ ID NO.8所示，SugR基因所编码的氨基酸序列如SEQ ID NO.9所示。
SugR是一种DeoR型转录调节因子，最初被鉴定为编码谷氨酸棒杆菌中糖摄取的PTS成分的基因的阻遏物，控制编码发酵乳酸脱氢酶的基因和编码糖酵解酶的基因的转录，可以使得碳代谢流更多地流向生产GlcNAc的途径，从而提高了谷氨酸棒杆菌生产GlcNAc的能力。
进一步地，转录因子SugR的编码基因通过表达载体pJYW-4-ceN-C.glglmS表达。表达载体pJYW-4-ceN-C.glglmS具体构建过程参见文献——Chen Deng,XueqinLv,Yanfeng Liu,Long Liu.Metabolic engineering of Corynebacteriumglutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis.Synthetic and Systems Biotechnology,2019.4:120-129。
本发明还提供了一种上述产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌的构建方法，包括以下步骤：
将pJYW-4-ceN-C.glglmS-SugR表达载体转入宿主菌，得到所述产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌。
进一步地，宿主菌为敲除了乙酰氨基葡萄糖脱乙酰酶编码基因NagA、乙酰氨基葡萄糖脱氨基酶编码基因GamA和L-乳酸脱氢酶的编码基因ldh的谷氨酸棒杆菌。
进一步地，宿主菌为谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA-Δldh，其构建方法包括以下步骤：
利用乙酰氨基葡萄糖脱乙酰酶编码基因NagA的基因敲除框、乙酰氨基葡萄糖脱氨基酶编码基因GamA的基因敲除框和L-乳酸脱氢酶的编码基因ldh的基因敲除框依次敲除谷氨酸棒杆菌C.glutamicum S9114中的乙酰氨基葡萄糖脱乙酰酶编码基因NagA、乙酰氨基葡萄糖脱氨基酶编码基因GamA及L-乳酸脱氢酶的编码基因ldh。
谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA-Δldh是以谷氨酸棒杆菌C.glutamicum  S9114 ΔnagA-ΔgamA为出发菌株，在其基础上敲除了L-乳酸脱氢酶的编码基因ldh得到。谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA的构建方法参照CN 110195036 A。在此基础上，继续构建L-乳酸脱氢酶的编码基因ldh的基因敲除框，经同源重组，用ldh的基因敲除框中的卡那霉素抗性基因kana代替组谷氨酸棒杆菌C.glutamicum S9114 ΔnagA-ΔgamA基因组中的L-乳酸脱氢酶编码基因ldh。
更具体地，pJYW-4-ceN-C.glglmS-SugR的构建方法，步骤如下：
(1)按照S9114基因组设计扩增引物扩增SugR
上游引物FragmentSugR.FOR：
5’——CCGTCGAATAAAAGAAATTCGGACATATTTAGTAAATTGGCTTTT——3’；
下游引物FragmentSugR.REV：
5’——CTTTGCTAGTCGGACTTGCAGTGACTGTAAGAATCA——3’；
同时设计载体pJYW-4-ceN-C.glglmS线性化引物
上游引物VectorSugR.FOR：
5’——TGCAAGTCCGACTAGCAAAGGAGAAGAAAAGCCG——3’；
下游引物VectorSugR.REV：
5’——TCCGAATTTCTTTTATTCGACGGTGACAGACTTTGC——3’；
(2)将PCR得到的线性化载体以及带有载体同源性末端的目的基因片段胶回收后以3：1的摩尔比采用快速克隆试剂盒进行连接，构建重组表达载体pJYW-4-ceN-C.glglmS-SugR。
进一步地，在步骤(2)中，快速克隆试剂为诺唯赞生物科技有限公司的ClonExpress II One Step Cloning Kit。
本发明还公开了上述产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌在生产N-乙酰氨基葡萄糖中的应用。
进一步地，采用摇瓶法发酵培养生产N-乙酰氨基葡萄糖。
在本发明的一种实施方式中，将28～30℃,220rpm下培养16h的重组谷氨酸棒杆菌种子液以使得发酵培养基的初始OD 562为1.6的接种量转入发酵培养基，于28～30℃，220rpm条件下培养72～100h。
借由上述方案，本发明至少具有以下优点：
本发明提供了能提高N-乙酰氨基葡萄糖产量的基因工程菌株的构建方法，在敲除了乙酰氨基葡萄糖脱乙酰酶编码基因NagA、乙酰氨基葡萄糖脱氨基酶编码基因GamA和L-乳酸脱氢酶的编码基因ldh的谷氨酸棒杆菌中，利用PCR扩增谷氨酸棒杆菌碳代谢全局调控因子转录因子SugR编码基因，将该基因连接在谷氨酸棒杆菌与大肠杆菌的穿梭表达载体 pJYW-4-ceN-C.glglmS，影响细胞内碳代谢分布，提高了N-乙酰氨基葡萄糖在胞外的积累量，其浓度最高可达26g/L，为进一步代谢工程改造谷氨酸棒杆菌生产氨基葡萄糖奠定了基础。本发明提供的重组谷氨酸棒杆菌构建方法简单，便于使用，具有很好地应用前景。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1为pJYW-4-ceN-C.glglmS质粒图谱。
图2为构建重组pJYW-4-ceN-C.glglmS-SugR质粒图谱。
图3为不同菌株摇瓶发酵上清液中GlcNAc的产量图。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
(一)本发明以下实施例中，N-乙酰氨基葡萄糖的测定方法如下：
高效液相色谱(HPLC)检测法：Agilent 1260,RID检测器，HPX-87H柱(Bio-Rad Hercules,CA)，流动相:5mM H 2S0 4，流速0.6mL/min，柱温35℃，进样体积为10μL。
(二)本发明以下实施例中，所使用的培养基如下：
种子活化培养基液体(LBG)(g/L)：蛋白胨10.0，酵母膏5.0，NaCl 10.0,葡萄糖5.0，装液量20ml每250ml三角瓶。
种子活化培养基固体(LBG固体)(g/L)：蛋白胨10.0，酵母粉5.0，NaCl 10.0,葡萄糖5.0，营养琼脂15.0-20.0。
感受态培养基(g/L):：蛋白胨10.0，酵母膏5.0，NaCl 10.0,甘氨酸30.0，异烟肼4.0，同时加入10ml的Tween80,装液量50ml每500ml三角瓶。
电击转化后恢复培养基LBHIS(g/L)：蛋白胨5.0，酵母膏2.5，NaCl 5.0,脑心浸液18.5，山梨醇91.0。
转化子检出培养基固体(g/L)：蛋白胨5.0，酵母膏2.5，NaCl 5.0,脑心浸液18.5，山梨醇91.0,营养琼脂15.0-20.0。
种子培养基(g/L)：葡萄糖25.0,玉米浆20.0，KH 2PO 4 1.0，(NH 4) 2SO 4 0.5，尿素1.25，pH 7.0。
发酵培养基(g/L)：葡萄糖40.0，玉米浆20.0,KH 2PO 4 1.0，(NH 4) 2SO 4 20.0，MgSO40.5， CaCO 3 20.0,pH 7.0。
优化发酵培养基(g/L)：葡萄糖100.0，玉米浆10.0,KH 2PO 4 1.0，(NH 4) 2SO 4 20.0，MgSO40.5，CaCO 3 20.0,FeSO 4 0.18,pH 7.0。
灭菌条件：115℃，20min,所有培养基用于转化子检出或用于重组菌培养时加入25mg/L硫卡那霉素。
实施例1 敲除L-乳酸脱氢酶编码基因(ldh)
根据NCBI上公布的谷氨酸棒杆菌(Corynebacteriumglutamicum ATCC 13032)L-乳酸脱氢酶编码基因(ldh)(核苷酸序列如SEQ ID NO.1所示)上下游序列，设计敲除同源臂扩增引物，左臂上、下游引物分别为LdhloxPUF(核苷酸序列如SEQ ID NO.2所示)和LdhloxPUR(核苷酸序列如SEQ ID NO.3所示)；右臂上、下游引物分别为LdhloxPDF(核苷酸序列如SEQ ID NO.4所示)和LdhloxPDR(核苷酸序列如SEQ ID NO.5所示)；分别以谷氨酸棒杆菌S9114菌株基因组DNA为模板，通过PCR将左臂和右臂扩增下来。
根据质粒pDTW-202(由江南大学王小元博士提供)上loxp-kana-loxp基因的核苷酸系列设计引物KanloxpldhF(核苷酸序列如SEQ ID NO.6所示)和KanloxpldhR(核苷酸序列如SEQ ID NO.7所示)，以质粒pDTW-202为模板将loxp-kana-loxp基因扩增loxp基因和卡那霉素抗性基因。通过酶切连接的方法，用快切酶XhoI/XbaI酶切2小时后的左臂、用快切酶XbaI/BamHI酶切2小时后的loxp-kana-loxp基因片段、用快切酶BamHI/EcoRI酶切2小时的右臂、用快切酶XhoI/EcoRI酶切2小时的质粒pBluescriptIISK(+)(由江南大学王小元博士提供)用T4连接酶于16℃过夜连接。
将构建好的带有ldh敲除框的pBluescriptIISK(+)连接体系转化E.coli JM109感受态细胞(感受态制备方法详见Takara大肠杆菌感受态试剂盒说明书；货号：9128)，挑选菌落PCR正确的转化子进行测序验证，得到重组质粒pBluescriptIISK(+)-ldh。提取重组质粒pBluescriptIISK(+)-ldh电转化谷氨酸棒杆菌S9114-ΔNagA-GamA，通过卡那霉素抗性平板筛选，菌落PCR验证，确认敲除框左右臂均结合到S9114基因组上，确认L-乳酸脱氢酶编码基因ldh敲除，得到谷氨酸棒杆菌S9114-ΔNagA-GamA-Δldh，经过72h后该菌株的GlcNAc产量为24.7g/L。
实施例2 重组质粒pJYW-4-ceN-C.glglmS-SugR的构建及重组谷氨酸棒杆菌的构建
(1)按照S9114基因组设计扩增引物扩增SugR
上游引物FragmentSugR.FOR：
5’——CCGTCGAATAAAAGAAATTCGGACATATTTAGTAAATTGGCTTTT——3’
下游引物FragmentSugR.REV：
5’——CTTTGCTAGTCGGACTTGCAGTGACTGTAAGAATCA——3’
同时设计载体pJYW-4-ceN-C.glglmS线性化引物
上游引物VectorSugR.FOR：
5’——TGCAAGTCCGACTAGCAAAGGAGAAGAAAAGCCG——3’
下游引物VectorSugR.REV：
5’——TCCGAATTTCTTTTATTCGACGGTGACAGACTTTGC——3’
使用引物FragmentSugR.FOR和FragmentSugR.REV，以本实验室储存的谷氨酸棒杆菌S9114为模板，PCR条件为：95℃预变性10min；98℃变性1min；55℃退火1min；72℃延伸1min，反应30个循环；最后72℃延伸l0min，PCR产物用DNA纯化试剂盒回收。将SugR基因基因从谷氨酸棒杆菌S9114基因组上扩增下来，使用LA Taq HS DNA聚合酶扩增SugR基因。
使用实验室前期工作中构建的质粒pJYW-4-ceN-C.glglmS作为表达载体来表达SugR基因，pJYW-4-ceN-C.glglmS质粒的具体构建过程参见文献——Chen Deng,XueqinLv,Yanfeng Liu,Long Liu.Metabolic engineering of Corynebacteriumglutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis.Synthetic and Systems Biotechnology,2019.4:120-129.
使用引物VectorSugR.FOR和VectorSugR.REV，以提取的质粒pJYW-4-ceN-C.glglmS为模板，PCR条件为：95℃预变性3min；98℃变性1min；55℃退火1min；72℃延伸5min，反应30个循环；最后72℃延伸l0min。PCR产物用DNA纯化试剂盒回收，获得线性化的质粒pJYW-4-ceN-C.glglmS。
(2)采用诺唯赞生物科技有限公司的ClonExpress II One Step Cloning Kit快速克隆试剂盒进行连接，PCR得到的线性化载体以及带有载体同源性末端的目的基因片段胶回收后以3：1的摩尔比混合，同时加入5×CE II Buffer 4μL，Exnase II 2μL，然后加入ddH 2O使得连接体系总体积达到20μL，37℃反应30min，降低至4℃保温。然后取10μL连接体系转化E.coli.BL21(DE3)感受态细胞(感受态制备方法详见Takara大肠杆菌感受态试剂盒说明书)。挑选菌落PCR正确的转化子，然后送往苏州金唯智生物科技有限公司进行测序验证，得到重组表达载体pJYW-4-ceN-C.glglmS-SugR。
将质粒pJYW-4-ceN-C.glglmS-SugR通过电击转化法转化到谷氨酸棒杆菌S9114ΔnagA-ΔgamA-Δldh菌株中。
谷氨酸棒杆菌电转感受态的制备：
(1)C.glutamicum接种于LBG培养基(需要新鲜培养的斜面上进行挑选，否则会影响菌体的生长)，置于巡回式摇床(200rpm)上，30℃培养16h,OD 562达到3.0。
(2)10％转接入感受态培养基中OD 562达到0.3，置于巡回式摇床(200rpm)上，30℃培养至OD 562达到0.9(培养约3-5h，处于对数生长期即可，一般如果菌浓的持续较低约0.6左右也可以继续后续操作)。需要保证菌体浓度尽量要浓，一般浓缩倍数为100倍(50mL感受态培养基浓缩至0.5mL制备5管感受态细胞)。
(3)菌液冰水浴15min,4,000rpm,4℃离心10min，小心的弃去上清。
(4)用30mL预冷10％甘油充分悬浮菌体，4,000rpm，4℃离心10min，小心的弃去上清，重复洗涤四次。
(5)用500μL预冷10％甘油重悬细胞(浓缩100倍)，1.5mL无菌离心管分装，每管100μL。
(6)-80℃保存待用，为保证感受态的转化效率最好现用现做，不能放置超过1周，否则由于感受态细胞裂解细胞内容物释放，在后续电击转化过程中造成电转杯的击穿，同时影响转化效率。
谷氨酸棒杆菌的电击转化：
(1)-80℃保存的谷氨酸棒状杆菌感受态，冰浴中融化。
(2)加入1-5.0μL质粒混匀(DNA总量约为1.0μg)，冰浴5-10min。
(3)加入于预冷的0.1cm电击杯中，1.8KV电压5ms电击2次。
(4)迅速加入预热的恢复用培养基(LBWS)1.0mL混匀并转移到新的1.5mL无菌离心管中，46℃水浴6min，后放入冰浴中。
(5)将菌体置于巡回式摇床(100rpm)上，30℃后培养2h。
(6)6,000rpm，常温离心1min，涂布到加入对应抗性的转化子检出平板中，于30℃恒温培养箱，培养2-3天。
(7)感受态效率验证:加入5.0μL无菌ddH 2O作为阴性对照，无菌落，阳性对照加入1-5μL质粒pXMJl9(DNA总量约为1.0μg)，长出大量菌落，测序正确的即为重组谷氨酸棒杆菌。
实施例3 重组谷氨酸棒杆菌中过量表达SugR基因对N-乙酰氨基葡萄糖产量的影响
将测序结果正确的含有将质粒pJYW-4-ceN-C.glglmS-SugR的重组谷氨酸棒杆菌菌株于甘油管接种划线LBG平板(添加硫卡那霉素25mg/L),30℃下220rpm培养18h后再挑选单菌落重新划线LBG平板直至长出大量菌落。
接种一环单菌落至种子培养基，30℃下220rpm培养16至18h至细胞生长对数前期。
按10％的接种量将种子培养液接种至发酵培养基中，30℃220rpm培养72h。测量GlcNAc生成量。
以含有质粒pJYW-4-ceN-C.glglmS的重组菌为对照，在相同条件下培养、发酵，72h后GlcNAc产量为24.7g/L(图3中CK)，而过量表达SugR基因的质粒pJYW-4-ceN-C.glglmS-SugR的菌株72h GlcNAc产量为26g/L(图3中SugR)。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2020070593-appb-000001
Figure PCTCN2020070593-appb-000002
Figure PCTCN2020070593-appb-000003
Figure PCTCN2020070593-appb-000004
Figure PCTCN2020070593-appb-000005

Claims (10)

  1. 一种产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌，其特征在于：所述重组谷氨酸棒杆菌通过在谷氨酸棒杆菌中过表达自身来源的转录因子SugR得到。
  2. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于：所述谷氨酸棒杆菌敲除了乙酰氨基葡萄糖脱乙酰酶编码基因NagA、乙酰氨基葡萄糖脱氨基酶编码基因GamA及L-乳酸脱氢酶的编码基因ldh。
  3. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于：所述谷氨酸棒杆菌为谷氨酸棒杆菌C.glutamicum S9114ΔnagA-ΔgamA-Δldh。
  4. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于：所述转录因子SugR的编码基因序列如NCBI-GeneID:1019888所示。
  5. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于：所述转录因子SugR的编码基因通过表达载体pJYW-4-ceN-C.glglmS表达。
  6. 一种权利要求1-5中任一项所述的产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌的构建方法，其特征在于，包括以下步骤：
    将pJYW-4-ceN-C.glglmS-SugR表达载体转入宿主菌，得到所述产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌。
  7. 根据权利要求6所述的构建方法，其特征在于：所述宿主菌为敲除了乙酰氨基葡萄糖脱乙酰酶编码基因NagA、乙酰氨基葡萄糖脱氨基酶编码基因GamA和L-乳酸脱氢酶的编码基因ldh的谷氨酸棒杆菌。
  8. 根据权利要求7所述的构建方法，其特征在于，所述宿主菌为谷氨酸棒杆菌C.glutamicum S9114ΔnagA-ΔgamA-Δldh，其构建方法包括以下步骤：
    利用乙酰氨基葡萄糖脱乙酰酶编码基因NagA的基因敲除框、乙酰氨基葡萄糖脱氨基酶编码基因GamA的基因敲除框和L-乳酸脱氢酶的编码基因ldh的基因敲除框依次敲除谷氨酸棒杆菌C.glutamicum S9114中的乙酰氨基葡萄糖脱乙酰酶编码基因NagA、乙酰氨基葡萄糖脱氨基酶编码基因GamA及L-乳酸脱氢酶的编码基因ldh。
  9. 权利要求1-5中任一项所述的产N-乙酰氨基葡萄糖的重组谷氨酸棒杆菌在生产N-乙酰氨基葡萄糖中的应用。
  10. 根据权利要求9所述的应用，其特征在于：采用摇瓶法培养生产N-乙酰氨基葡萄糖。
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