WO2020124830A1 - 基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统 - Google Patents

基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统 Download PDF

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WO2020124830A1
WO2020124830A1 PCT/CN2019/078809 CN2019078809W WO2020124830A1 WO 2020124830 A1 WO2020124830 A1 WO 2020124830A1 CN 2019078809 W CN2019078809 W CN 2019078809W WO 2020124830 A1 WO2020124830 A1 WO 2020124830A1
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gene
promoter
expression
bacillus subtilis
repressor protein
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French (fr)
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周哲敏
崔文璟
韩来闯
郝文亮
周丽
刘中美
燕宇
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江南大学
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Priority to US17/160,582 priority Critical patent/US12098373B2/en

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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

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  • the invention relates to a high-efficiency induction expression system of Bacillus subtilis based on artificial tandem promoter, which belongs to the technical field of genetic engineering.
  • Bacillus subtilis is a Gram-positive model strain widely used for the expression of foreign proteins. It is widely used in the production of industrial enzyme preparations because it can efficiently express foreign proteins.
  • the promoter is the most basic element for constructing a high-efficiency expression system, and the activity of the promoter directly determines the efficiency of the expression system. Promoters are mainly divided into two types: constitutive promoters and inducible promoters. Constitutive promoters can express foreign genes at all stages of bacterial growth, while inducible promoters show no or low activity when they are not induced. After the addition of an inducer, the activity of the promoter is greatly increased to efficiently express foreign genes. Source genes. Inducible promoters are widely used because of their controllable activity.
  • IPTG induction system and the xylose induction system are widely used in Bacillus subtilis.
  • the IPTG induction system is mainly constructed based on the pHT series of vectors, using an unnatural hybrid promoter and E. coli LacI repression Protein and binding site; and the xylose induction system usually uses the natural PxylA promoter and XylR repressor protein.
  • induction systems such as IPTG and xylose have been widely used, due to limited promoter activity, low expression of foreign proteins has always been a key issue limiting the application of the system.
  • highly active promoters are often not used to construct highly efficient inducible foreign gene expression systems, mainly because promoters and other elements of gene expression control systems are often incompatible, and highly active promoters cannot often be effectively inhibited by repressor proteins. . Therefore, how to construct a strictly regulated inducible exogenous gene expression system based on a high-efficiency promoter is a hot spot for the development of new expression systems.
  • the first object of the present invention is to provide an element for regulating gene expression, including: (1) a vector; (2) an artificial tandem promoter; (3) a repressor protein gene; (4) located at the transcription start site of the promoter Downstream DNA fragments that can bind to the repressor protein; the artificial tandem promoter includes PAWH-D30-106 , PAH-D75-106 or PAH-D75-106 , the nucleotide sequences are as SEQ ID NO.1, respectively , SEQ ID NO.2, SEQ ID NO.3 shown.
  • the repressor protein includes Lacl, XylR, AraC or TetR.
  • repressor protein when the repressor protein is Lacl, gene expression is induced by IPTG.
  • repressor protein when the repressor protein is XylR, gene expression is induced by xylose.
  • repressor protein when the repressor protein is AraC, gene expression is induced by arabinose.
  • repressor protein when the repressor protein is TetR, gene expression is induced by tetracycline.
  • the carrier includes pHT-01.
  • the expression host of the gene of interest includes Bacillus subtilis.
  • the expression host of the target gene includes Bacillus subtilis 168, Bacillus subtilis WB400, Bacillus subtilis WB600, or Bacillus subtilis WB800.
  • the target gene includes a foreign gene or an endogenous gene.
  • the target gene includes an enzyme gene or a non-enzyme gene.
  • the second object of the present invention is to provide a recombinant plasmid that expresses the above elements that regulate gene expression
  • the third object of the present invention is to provide a genetically engineered bacterium that expresses the above recombinant plasmid.
  • the fourth object of the present invention is to provide a method for regulating the expression of a target gene, co-expressing the above-mentioned elements that regulate gene expression and the target gene.
  • the fifth object of the present invention is to provide the application of the above-mentioned elements for regulating gene expression in the preparation of a protein of interest.
  • the sixth object of the present invention is to provide the application of the above-mentioned elements for regulating gene expression in the fields of food, chemical or pharmaceutical.
  • the seventh object of the present invention is to provide the application of the above genetically engineered bacteria in the preparation of the target protein.
  • the eighth object of the present invention is to provide the application of the above genetically engineered bacteria in the fields of food, chemical or pharmaceutical.
  • the present invention utilizes an efficient artificial tandem constitutive promoter.
  • this promoter By designing this promoter in combination with operon related elements (repressor protein and its binding site), the activity of the constitutive promoter is regulated by the inducer , And finally constructed a highly efficient inducible expression system of Bacillus subtilis induced by the inducer.
  • the results show that the activity of the artificial tandem promoter in this system is about 15 times higher than that of the strong constitutive promoter of P43.
  • the promoter activity can be precisely controlled by adding different concentrations of inducers. Therefore, the high-efficiency expression system has a simple structure, high activity and strict regulation, and has broad application prospects in the efficient expression of heterologous proteins and synthetic biology research.
  • Figure 1 Comparison of P AWH-D30-106 , P AH-D75-106 and P AH-D75-106 and P43 promoter activities.
  • FIG. 2 IPTG induced expression system construction and characterization, a: IPTG induction system principle; b: IPTG induction system characterization; c: SDS-PAGE verification of sfGFP expression.
  • Figure 3 Construction and characterization of xylose-induced expression system, a: principle of xylose-induced system; b: characterization of xylose-induced system; c: SDS-PAGE verification of sfGFP expression.
  • Plasmid construction method design primers containing promoter sequences, use pHT01 plasmid as the backbone template, and use PrimeSTARMAX DNA polymerase (purchased from Takara, article number: R045Q) to perform whole plasmid PCR.
  • the PCR program is: pre-denaturation 98°C1min, The cycle is denaturation at 98°C for 30s, annealing at 50°C for 30s, extension at 72°C for 1min, a total of 30 cycles, and finally extension at 72°C for 10min.
  • the plasmid template was removed by digestion with restriction enzyme DpnI, and the PCR product was purified. Amplify the fragments to be cloned in the same way, and then use the Infusion recombination method to assemble multiple fragments and transform into E. coli JM109 competent cells.
  • the detection method of sfGFP fluorescence intensity the sample is centrifuged at 12000 ⁇ g for 2 minutes, the cells are collected, washed 3 times with PBS buffer, diluted with PBS to a certain concentration of the cell suspension, and 200 ⁇ L is taken to a 96-well microtiter plate and placed SynergyTM H4 fluorescence microplate reader detects fluorescence. Excitation light 485nm, absorption light 528nm, fluorescence detection.
  • Medium LB medium (L -1 ): Tryptone 10g, NaCl 10g, yeast extract 5g, pH 7.0, 20g agar powder is added when preparing the solid medium.
  • Transformation method of Bacillus subtilis 168 pick single colony Bacillus subtilis 168 inoculated into 2mL SPI medium, shake culture at 37°C for 12h-14h; take 100 ⁇ L from the culture, inoculate into 5mL SPI medium, 37 OD 600 was measured after 4-5h incubation on a shaker at °C.
  • SDS-PAGE detection take 200 ⁇ L of bacterial solution, centrifuge at 12000 ⁇ g for 2 min, and collect bacterial cells. Resuspend with 200 ⁇ L of 20 mM Tris-HCl (pH 8.0) buffer containing 20 ⁇ g/mL lysozyme, and incubate at 37°C for 30 min to lyse the cell wall. Then add 50 ⁇ L of 10 ⁇ Loading Buffer, boil for 10 minutes in boiling water and centrifuge at 12000 ⁇ g for 5 minutes. Take 30 ⁇ L supernatant samples for electrophoresis detection, analyze the electrophoresis results after staining with Coomassie Brilliant Blue R250 and decolorizing the decolorizing solution.
  • the P AWH-D30-106 , P AH-D75-106 and P AH-D75-106 promoters are all artificially constructed tandem hybrid promoters.
  • sequences of the P AWH-D30-106 , P AH-D75-106 , and P AH-D75-106 promoters are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.
  • the expression boxes of the promoters P AWH-D30-106 and sfGFP were cloned into the pHT01 vector using the primers in Table 1, and the P spac promoter sequence on the original vector was replaced.
  • the expression vector was transformed into Bacillus subtilis 168, the recombinant bacteria were cultured for 20h, and the expression level of sfGFP was detected.
  • the expression levels of sfGFP in the culture medium of recombinant bacteria containing P AH-D75-106 and P WAH-D75-106 recombinant plasmid were detected in the same way.
  • the plasmid pHT-AWH-lac-sfGFP was constructed.
  • the recombinant plasmid was transformed into Bacillus subtilis 168, and the recombinant bacteria were cultured in LB medium at 37°C and 200 rpm. The expression level of sfGFP was detected 24 hours later.
  • the repressor protein LacI can effectively bind downstream of the transcription start site of the promoter, preventing the transcription of downstream genes by RNA polymerase, thereby inhibiting the expression of downstream genes.
  • IPTG IPTG
  • the activity of the promoter is released.
  • the higher the concentration of IPTG added (not more than 1 mM), the stronger the promoter activity. Therefore, the downstream gene expression intensities with different intensities can be obtained by adjusting the amount of IPTG ( Figure 2b and Table 4).
  • SDS-PAGE detection also proved that the addition of different concentrations of inducer IPTG obtained different strengths of sfGFP expression ( Figure 2c).
  • Example 3 Construction and characterization of xylose inducible expression system
  • the primer backbone PAWH-xylR-v1/PAWH-xylR-v2 (Table 5) was used to amplify the vector backbone, and the primer PAWH-xylR-i1/PAWH-xylR-i2 was used to amplify the xylR Gene, the two amplified fragments were recombined to obtain a plasmid with lacI replaced by xylR, and then the primers PAWH-xylO-1/PAWH-xylO-2 were used to replace the lacI binding site with the xylR binding by the method of whole plasmid PCR At site AGTTAGTTTATTGGATAAACAAACTAACT (SEQ ID NO. 5), the plasmid pHT-AWH-xyl-sfGFP was finally constructed.
  • the recombinant plasmid was transformed into Bacillus subtilis 168, and the recombinant bacteria were cultured in LB medium at 37°C and 200 rpm. The expression level of sfGFP was detected 24 hours later.
  • the repressor protein xylR can effectively bind downstream of the transcription start site of the promoter, preventing the transcription of the promoter by RNA polymerase, thereby inhibiting the expression of downstream genes ( Figure 3a).
  • Figure 3a When different concentrations of xylose are added, the activity of the promoter is gradually released, so the downstream gene expression intensity of different strengths can be obtained by adjusting the xylose concentration.

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Abstract

本发明公开了基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统，属于基因工程技术领域。本发明利用一种高效的人工串联组成型启动子，通过将该启动子与操纵子相关元件(阻遏蛋白及其结合位点)组合设计，进而使该组成型启动子的活性受到诱导剂的调控，最终构建出受诱导剂诱导的枯草芽孢杆菌高效诱导型表达系统。结果表明，与P43强组成型启动子相比，本系统中的人工串联启动子活性高出15倍左右。并且可以通过添加不同浓度的诱导剂精准控制启动子活性。因此本高效表达系统结构简单、活性高、调控严谨，在异源蛋白高效表达和合成生物学研究中有广阔的应用前景。

Description

基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统 技术领域
本发明涉及基于人工串联启动子的枯草芽孢杆菌高效诱导表达系统，属于基因工程技术领域。
背景技术
枯草芽孢杆菌是广泛应用于表达外源蛋白的革兰氏阳性模式菌株，其因能够高效表达外源蛋白在工业酶制剂生产方面有广泛应用。启动子是构建高效表达系统的最基本的元件，启动子的活性直接决定了表达系统的效率。启动子从功能上主要分为组成型启动子和诱导型启动子两类。组成型启动子在菌体的生长各个阶段都可以表达外源基因，而诱导型启动子在没有被诱导时表现无活性或者低活性，在加入诱导剂之后启动子的活性大幅提高进而高效表达外源基因。诱导型启动子由于其活性可控而被广泛应用，人们可以通过调控加入诱导剂的试剂和浓度控制外源基因的表达时间和强度。目前在枯草芽孢杆菌当中应用较为广泛的主要有IPTG诱导系统和木糖诱导系统，其中，IPTG诱导系统主要基于pHT系列载体构建而成，使用的是非天然的杂合启动子和大肠杆菌的LacI阻遏蛋白和结合位点；而木糖诱导系统通常使用天然的PxylA启动子和XylR阻遏蛋白。
虽然IPTG、木糖等诱导系统已经广泛使用，但是由于启动子活性受限，外源蛋白的表达量低始终是限制系统应用的一个关键问题。人们通常采用改造天然启动子和重新设计人工启动子两种策略来提高启动子的活性和稳定性。但是高活性启动子往往无法用于构建高效诱导型外源基因表达系统，主要是由于启动子和基因表达调控系统的其他元件往往不兼容，高活性的启动子往往不能被阻遏蛋白有效地抑制活性。因此如何基于高效启动子构建严谨调控的诱导型外源基因表达系统是目前新型表达系统的开发热点。
发明内容
本发明的第一个目的是提供一种调控基因表达的元件，包括：(1)载体；(2)人工串联启动子；(3)阻遏蛋白基因；(4)位于启动子转录起始位点下游的能与阻遏蛋白结合的DNA片段；所述人工串联启动子包括P AWH-D30-106、P AH-D75-106或P AH-D75-106，核苷酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。
在本发明的一种实施方式，所述阻遏蛋白包括LacI、XylR、AraC或TetR。
在本发明的一种实施方式，所述阻遏蛋白为LacI时，基因表达受到IPTG的诱导作用。
在本发明的一种实施方式，所述阻遏蛋白为XylR时，基因表达受到木糖的诱导作用。
在本发明的一种实施方式，所述阻遏蛋白为AraC时，基因表达受到阿拉伯糖的诱导作用。
在本发明的一种实施方式，所述阻遏蛋白为TetR时，基因表达受到四环素的诱导作用。
在本发明的一种实施方式，所述载体包括pHT-01。
在本发明的一种实施方式，目的基因的表达宿主包括枯草芽孢杆菌。
在本发明的一种实施方式，目的基因的表达宿主包括Bacillus subtilis 168、Bacillus subtilis WB400、Bacillus subtilis WB600或Bacillus subtilis WB800。
在本发明的一种实施方式，目的基因包括外源基因或内源基因。
在本发明的一种实施方式，目的基因包括酶基因或非酶基因。
本发明的第二个目的是提供一种重组质粒，表达上述调节基因表达的元件
本发明的第三个目的是提供一种基因工程菌，表达上述重组质粒。
本发明的第四个目的是提供一种调控目的基因表达的方法，将上述调节基因表达的元件与目的基因共表达。
本发明的第五个目的是提供上述调控基因表达的元件在制备目的蛋白中的应用。
本发明的第六个目的是提供上述调控基因表达的元件在食品、化工或制药领域的应用。
本发明的第七个目的是提供上述基因工程菌在制备目的蛋白中的应用。
本发明的第八个目的是提供上述基因工程菌在食品、化工或制药领域的应用。
本发明利用一种高效的人工串联组成型启动子，通过将该启动子与操纵子相关元件(阻遏蛋白及其结合位点)组合设计，进而使该组成型启动子的活性受到诱导剂的调控，最终构建出受诱导剂诱导的枯草芽孢杆菌高效诱导型表达系统。结果表明，与P43强组成型启动子相比，本系统中的人工串联启动子活性高出15倍左右。并且可以通过添加不同浓度的诱导剂精准控制启动子活性。因此本高效表达系统结构简单、活性高、调控严谨，在异源蛋白高效表达和合成生物学研究中有广阔的应用前景。
附图说明
图1：P AWH-D30-106、P AH-D75-106和P AH-D75-106与P43启动子活性比较。
图2：IPTG诱导表达系统构建和表征，a：IPTG诱导系统原理；b：IPTG诱导系统表征；c：SDS-PAGE验证sfGFP表达。
图3：xylose诱导表达系统构建和表征，a：xylose诱导系统原理；b：xylose诱导系统表征；c：SDS-PAGE验证sfGFP表达。
具体实施方式
1、质粒构建方法：设计包含启动子序列的引物，以pHT01质粒作为骨架模板，用PrimeSTARMAX DNA聚合酶(购自Takara，货号：R045Q)进行全质粒PCR，PCR程序为：预变性98℃ 1min，循环为变性98℃ 30s，退火50℃ 30s，延伸72℃ 1min，共30个循环，最后72℃延伸10min。之后用限制性内切酶DpnI消化去除质粒模板，将PCR产物纯化。同样方法扩增需要克隆的片段，之后用Infusion重组方法将多个片段进行组装，转化大肠杆菌JM109感受态细胞。
2、sfGFP荧光强度的检测方法：样品12000×g离心2min，收集菌体，PBS缓冲液洗3次，用PBS稀释到一定浓度的菌体悬液，取200μL至96孔酶标板，放入SynergyTM H4荧光酶标仪检测荧光。激发光485nm，吸收光528nm，检测荧光。
3、培养基：LB培养基(L -1)：胰蛋白胨10g，NaCl 10g，酵母提取物5g，pH 7.0，配制固体培养基时添加琼脂粉20g。
4、枯草芽孢杆菌168转化方法：挑单菌落枯草芽孢杆菌168接种至2mL的SPI培养基中，37℃摇床培养12h-14h；从培养物中取100μL，接种至5mL SPI培养基中，37℃摇床培养4-5h后开始测OD 600。当OD 600约为1.0时，移取200μL菌液转接至2mL的SPII培养基中，于37℃、100r·min -1摇床孵育1.5h；向管中加入20μL l00×EGTA(乙二醇双(α-氨基乙基醚)四乙酸)溶液，于37℃、100r·min -1摇床中培养10min后分装500μL每l.5mL离心管；向管中加入经过测序验证正确的适量质粒，吹吸混匀放置于37℃、100r·min -1的摇床中培养2h；培养结束，吸取菌液约200μL均匀涂相应的选择性平板，37℃培养12h-14h。
5、SDS-PAGE检测：取200μL菌液，12000×g离心2min，收集菌体。用200μL含有20μg/mL浓度溶菌酶的20mM Tris-HCl(pH8.0)缓冲液重悬浮，37℃孵育30min以裂解细胞壁。之后加入50μL 10×Loading Buffer，沸水煮10min后12000×g离心5min。取30μL上清样品电泳检测，经考马斯亮蓝R250染色与脱色液脱色后分析电泳结果。
实施例1：人工串联启动子活性鉴定
P AWH-D30-106、P AH-D75-106和P AH-D75-106启动子均为人工构建的核心区串联杂合启动子。
P AWH-D30-106、P AH-D75-106、P AH-D75-106启动子的序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。
将启动子P AWH-D30-106与sfGFP(Genbank ID：AVR55189.1)表达框用表1中的引物克隆至pHT01载体上，替换原载体上的P spac启动子序列。将该表达载体转化至枯草芽孢杆菌168中，培养重组菌20h，检测sfGFP的表达水平。以相同的方式分别检测含P AH-D75-106和含P WAH-D75-106重组质粒的重组菌培养液的sfGFP的表达水平。
经过与常用的强组成型启动子P43表达sfGFP的情况作比较，结果显示，P AWH-D30-106、P AH-D75-106、P WAH-D75-106启动子表达sfGFP的水平远比P43启动子高，达到P43的15倍左右(图1和表2)。说明启动子P AWH-D30-106、P AH-D75-106、P WAH-D75-106是活性非常高的强组成型启动子。
表1 表达载体构建引物
引物 序列(5’-3’) a
PpHT-AWH-i1 CTAACGGAAAAGGGATTTTTGAGTGATCTTCTCAAAAAATAC
PpHT-AWH-i2 CCTCGTATGTTTCAAAGAGTGCACCATATGCGG
PpHT-AWH-v1 CATATGGTGCACTCTTTGAAACATACGAGGCTAATATCGG
PpHT-AWH-v2 AAGATCACTCAAAAATCCCTTTTCCGTTAGCTTTTTC
表2 人工串联启动子活性
启动子 荧光强度(a.u./OD 600)
P43 5370
P AWH-D30-106 75340
P AH-D75-106 77014
P WAH-D75-106 77334
实施例2：IPTG诱导型表达系统的构建和表征
lacI的结合位点序列为GGAATTGTGAGCGGATAACAATTCC(SEQ ID NO.4)，将其设计在表3中的引物P AWH-lacO-1/P AWH-lacO-2上，利用全质粒PCR方法将LacI蛋白结合位点克隆至启动子P AWH-D30-106转录起始位点的下游，同时利用pHT01载体骨架上本身有的lacI模块表达阻遏蛋白LacI，这样就构建出IPTG诱导型的表达系统(图2a)。
以sfGFP作为目的蛋白表征表达量高低，构建得到质粒pHT-AWH-lac-sfGFP。将该重组质粒转化至枯草芽孢杆菌168中，在LB培养基中37℃，200rpm下培养重组菌，24h后检测sfGFP的表达水平。
在没有IPTG诱导剂时，阻遏蛋白LacI能有效结合在启动子转录起始位点下游，阻止RNA聚合酶对下游基因的转录，从而抑制下游基因的表达。当加入IPTG时，启动子的活性被释放。加入IPTG浓度越高(不超过1mM)，启动子活性越强。因此，可以通过调节IPTG的用 量获得不同强度的下游基因表达强度(图2b和表4)。SDS-PAGE检测也证明了不同浓度诱导剂IPTG的添加获得了不同强度的sfGFP表达(图2c)。
表3 IPTG诱导表达载体构建引物
引物 序列(5’-3’) a
PAWH-lacO-1 GGAATTGTGAGCGGATAACAATTCCATGCTTTTATTCGAACATCATATTTAAAG
PAWH-lacO-2 GGAATTGTTATCCGCTCACAATTCCTAGTGTATCAATTCCACGATTTTTTC
表4 不同浓度的IPTG诱导下，sfGFP表达情况
条件 荧光强度(a.u./OD600)
pHT-01 179
pHT-AWH(未组合操纵子) 6635
0mM IPTG 516
0.01mM IPTG 929
0.05mM IPTG 1993
0.1mM IPTG 2665
0.5mM IPTG 4132
1mM IPTG 4163
实施例3：xylose诱导型表达系统的构建和表征
利用表3中的引物将阻遏蛋白XylR结合位点克隆至启动子P AWH-D30-106转录起始位点的下游，同时将pHT01载体骨架上的lacI基因替换为xylR基因，构建XylR表达模块，这样就构建出xylose诱导型的表达系统。
以质粒pHT-AWH-lac-sfGFP为模板，用引物PAWH-xylR-v1/PAWH-xylR-v2(表5)扩增载体骨架，用引物PAWH-xylR-i1/PAWH-xylR-i2扩增xylR基因，将扩增的两片段重组，获得lacI被xylR替换的质粒，再利用引物PAWH-xylO-1/PAWH-xylO-2，通过全质粒PCR的方法将lacI的结合位点替换为xylR的结合位点AGTTAGTTTATTGGATAAACAAACTAACT(SEQ ID NO.5)，最终构建得到质粒pHT-AWH-xyl-sfGFP。
将该重组质粒转化至枯草芽孢杆菌168中，在LB培养基中37℃，200rpm下培养重组菌，24h后检测sfGFP的表达水平。
在没有诱导剂xylose时，阻遏蛋白xylR能有效结合在启动子转录起始位点下游，阻止RNA聚合酶对启动子的转录，从而抑制下游基因的表达(图3a)。当加入不同浓度的xylose 时，启动子的活性逐渐被释放，因此可以通过调节xylose浓度获得不同强度的下游基因表达强度。随着xylose的浓度的逐步提升，sfGFP表达产生的荧光信号随之增强，当xylose浓度达到或超过1％(W/V)时，sfGFP的表达水平甚至高于组成型表达sfGFP的对照pHT-AWH(图3b和表6)。进一步的SDS-PAGE检测进一步证明了不同浓度诱导剂xylose的添加获得了不同强度的sfGFP表达(图3c)。
表5 xylose诱导表达载体构建引物
Figure PCTCN2019078809-appb-000001
表4 不同木糖浓度下，sfGFP表达情况
条件 荧光强度(a.u./OD 600)
pHT-01 179
pHT-AWH(未组合操纵子) 6635
0％木糖 393
0.01％木糖 2263
0.05％木糖 3808
0.1％木糖 4322
0.5％木糖 5480
1％木糖 7326
2％木糖 9556
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。
Figure PCTCN2019078809-appb-000002
Figure PCTCN2019078809-appb-000003
Figure PCTCN2019078809-appb-000004
Figure PCTCN2019078809-appb-000005
Figure PCTCN2019078809-appb-000006

Claims (15)

  1. 一种调控目的基因表达的方法，其特征在于，将调控元件与目的基因共表达，所述调控元件中包括：(1)载体；(2)人工串联启动子；(3)阻遏蛋白基因；(4)位于启动子转录起始位点下游的能与阻遏蛋白结合的DNA片段；所述人工串联启动子包括P AWH-D30-106、P AH-D75-106或P AH-D75-106，核苷酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。
  2. 如权利要求1所述的方法，其特征在于，所述阻遏蛋白包括LacI、XylR、AraC或TetR。
  3. 如权利要求1所述的方法，其特征在于，所述载体包括pHT-01。
  4. 如权利要求2所述的方法，其特征在于，所述阻遏蛋白为LacI时，基因表达受到IPTG的诱导作用。
  5. 如权利要求2所述的方法，其特征在于，所述阻遏蛋白为XylR时，基因表达受到木糖的诱导作用。
  6. 如权利要求2所述的方法，其特征在于，所述阻遏蛋白为AraC时，基因表达受到阿拉伯糖的诱导作用。
  7. 如权利要求2所述的方法，其特征在于，所述阻遏蛋白为TetR时，基因表达受到四环素的诱导作用。
  8. 如权利要求1所述的方法，其特征在于，目的基因的表达宿主包括枯草芽孢杆菌。
  9. 如权利要求1所述的方法，其特征在于，目的基因的表达宿主包括Bacillus subtilis 168、Bacillus subtilis WB400、Bacillus subtilis WB600或Bacillus subtilis WB800。
  10. 如权利要求1所述的方法，其特征在于，目的基因包括外源基因或内源基因。
  11. 如权利要求1所述的方法，其特征在于，目的基因包括酶基因或非酶基因。
  12. 如权利要求1所述的方法，其特征在于，应用于食品或保健品领域。
  13. 如权利要求1所述的方法，其特征在于，应用于制药领域。
  14. 一种调控基因表达的元件，其特征在于，包括：(1)载体；(2)人工串联启动子；(3)阻遏蛋白基因；(4)位于启动子转录起始位点下游的能与阻遏蛋白结合的DNA片段；所述人工串联启动子包括P AWH-D30-106、P AH-D75-106或P AH-D75-106，核苷酸序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示。
  15. 表达权利要求14所述元件的基因工程菌。
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CN111269926B (zh) * 2019-12-31 2021-09-28 江南大学 一种钴离子诱导型蛋白表达系统及应用
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