CN117568341A - 一种突变体启动子Pre10及其应用 - Google Patents
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Abstract
本发明属于微生物与基因工程技术领域,具体涉及一种突变启动子Pre10及其应用。所述启动子Pre10,是通过对枯草芽孢杆菌来源的启动子分析改造获得的,核苷酸序列如SEQ ID NO:2所示。本发明所述突变启动子Pre10实现了氨肽酶的高效异源表达,相较于原始启动子,氨肽酶酶活提高了106.5%,为进一步实现氨肽酶YwaD的高效表达提供了参考依据,同时也为氨肽酶的工业化生产奠定基础。
Description
技术领域:
本发明属于微生物与基因工程技术领域,具体涉及一种突变启动子Pre10及其应用。
背景技术:
氨肽酶(Aminopeptidase),作为一种外肽酶,能够特异性的识别蛋白和多肽链N末端的氨基酸残基,并将其逐个水解下来。并与碱性蛋白酶、羧肽酶等协同水解可有效减轻蛋白水解液中的苦味,制备出多种生物活性肽,在食品、医疗行业等具有广阔的应用前景。由于微生物蛋白酶均为胞外酶,与动植物源蛋白酶相比具有下游技术处理相对简单、价格低廉、来源广、菌体易于培养、产量高等优势,易于实现工业化生产。
解淀粉芽孢杆菌B.amyloliquefaciens具有所有革兰氏阳性细菌的特征,其安全无毒具有非致病性,在原核表达系统中被认为是良好的宿主之一,具有广阔的应用前景。近年来,随着分子生物学和基因工程技术的迅速发展,解淀粉芽孢杆菌底盘细胞从启动子工程、基因编辑、基因回路、辅因子工程以及途径酶组装等方面进行研究,从而实现外源蛋白的高效表达。
而实现外源蛋白的高效表达的关键因素之一是使用高强度且易控制的启动子。启动子是具有RNA聚合酶识别、结合和转录起始功能的一段特殊的非编码DNA序列。在转录起始时,细菌启动子是与RNA聚合酶结合的靶序列,是细菌中基因表达的必需调控元件,决定了细菌基因转录的起始时间和程度。但是天然启动子的局限性在于它们无法获得完整连续的转录调控强度,并且无法使生物体内的转录水平最大化。通过启动子的插入或缺失,可以改变细菌基因的表达,实现对菌体生长发育以及代谢调控的研究。
因此,通过对启动子上保守序列进行替换等方法,提高异源蛋白基因的转录水平,可以为进一步提高异源蛋白在B.amyloliquefaciens的表达水平提供参考依据。
发明内容:
为了解决上述技术问题,本发明将以枯草芽孢杆菌来源的α-淀粉酶启动子为基础,通过分子生物学手段对其进行突变,从而筛选出有利于氨肽酶外源表达的启动子突变体。
本发明提供的技术方案之一,是一种突变的启动子Pre10,所述启动子Pre10,其核苷酸序列如SEQ ID NO:2所示。
本发明提供的技术方案之二,是一种包含所述启动子Pre10的表达载体;所述表达载体的骨架可以是任何本领域已知的芽胞杆菌的表达载体,包括但不限于pHT01、pHT43、pHCMC05、pWB980等;
根据本发明一种优选的实施方式,所述表达载体是pWB980。
本发明提供的技术方案之三,是包含技术方案一所述启动子或技术方案二所述表达载体的表达系统,所述表达系统采用的宿主可以是适合于本发明的启动子或表达载体的任何宿主,例如解淀粉芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌。
根据本发明一种优选的实施方式,所述宿主是解淀粉芽胞杆菌(B.amyloliquefaciens)CGMCC No.11218、枯草芽孢杆菌(B.Subtilis)WB600、枯草芽孢杆菌(B.Subtilis)WB800。
本发明提供的技术方案之四,是启动子Pre10的应用,特别是在控制蛋白表达中的应用,更特别的是在控制氨肽酶表达中的应用,更特别的是应用于解淀粉芽孢杆菌为宿主的蛋白表达中的应用;
根据本发明一种优选的实施方式,所述启动子Pre10用于控制氨肽酶基因YwaD在解淀粉芽孢杆菌系统中的表达;
进一步地,所述氨肽酶的氨基酸序列如SEQ ID NO:4所示。
有益效果:
本发明通过对枯草芽孢杆菌来源的启动子分析改造获得了一种突变启动子Pre10。本发明所述突变启动子Pre10实现了氨肽酶的高效异源表达,相较于原始启动子,氨肽酶酶活提高了106.5%,为进一步实现氨肽酶YwaD的高效表达提供了参考依据,同时也为氨肽酶的工业化生产奠定基础。
附图说明:
图1氨肽酶的pNA标准曲线;
图2启动子和信号肽PCR验证结果图
其中,1、PamyE+SPamyE基因片段;2、Marker;
图3重组质粒PCR验证结果图
其中,1、YwaD基因片段;2、Marker;
图4重组菌株表达氨肽酶活性对比。
具体实施方式:
下面通过具体的实施方案进一步叙述本发明。除非特别说明,以下实施方案所涉及的技术手段、材料等均可以是本领域技术人员所公知的,可以在已知的能解决相应技术问题的手段和材料中选择合适的。另外,实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言,在不背离本发明实质和范围的前提下,对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
本发明中α-淀粉酶启动子PamyE的碱基序列突变位点如下:
以下将通过具体的实施例对本发明进行更详细描述。如无特别说明,本发明及实施例中涉及的部分培养基及酶活测定方法具体如下:
(1)部分培养基组成如下:
LB培养基:蛋白胨10g/L,酵母粉5g/L,NaCl 10g/L,固体培养基加入琼脂18g/L,其他成分一致。
发酵培养基:称取豆饼粉60g/L,氢氧化钠溶液调节溶液pH至12.5,50℃碱解120min,磷酸调节pH至7.3,称取葡萄糖按30g/L加入其中,121℃灭菌20min,获得葡萄糖为碳源的发酵培养基。
枯草芽胞杆菌感受态制备培养基:
SP-A盐溶液:(NH4)2SO4 4g/L,K2HPO4·3H2O 28g/L,KH2PO4 12g/L,柠檬酸钠2g/L;
SP-B盐溶液:MgSO4·7H2O 0.4g/L;
100×CAYE溶液:酪蛋白水解物20g/L,酵母粉100g/L;
SPI(200mL):SP-A盐溶液98mL,SP-B盐溶液98mL,50%葡萄糖2mL,100×CAYE 2mL;
SPII培养基(600mL):SPI 588mL,50mmol/L CaCl2 6mL,250mmol/L MgCl2 6mL;
100×EGTA溶液:10mmol/L EGTA溶液。
(2)氨肽酶YwaD酶活力的测定方法:
①标准曲线的绘制
先配制终浓度为40μg/mL的对硝基苯胺ρNA标准储备溶液,以其为母液进行稀释,具体配制方法如下:
测定氨肽酶的对硝基苯胺(ρNA)标准溶液配制表
以编号1为对照,使用紫外分光光度计将配置好的标准溶液在波长405nm条件下检测吸光度值,以对硝基苯胺的浓度为横坐标,吸光度值为纵坐标绘制标准曲线,如图1所示。
②样品的制备:对酶液进行稀释,浓度范围为酶活力10U/mL~20U/mL。
③酶活定义:即1个酶活力单位(U/mL)定义为1mL酶液在60℃、pH 9条件下反应1min水解亮氨酸对硝基苯胺产生1μg对硝基苯胺所需要的酶量。每一样品设三次重复,结果取平均值。
④氨肽酶的酶活力测定方法参照LNA法,测定步骤如下:
a.发酵液13000r/min离心2min取上清作为粗酶液,并使用pH 9的Tris-HCl缓冲液进行n倍稀释;
b.对照组:取0.2mL稀释后的酶液,加入到含有2.6mL的pH 9的Tris-HCl缓冲液的酶活试管,60℃条件下预热5min,加入1mL 40%的乙酸溶液,反应10min,再加入50mM LNA溶液0.20ml,混匀后静置10min;
c.实验组:取0.2mL稀释后的酶液,加入到含有2.6mL pH 9的Tris-HCl缓冲液的酶活试管中,60℃条件下预热5min,向试管中加入50mM的亮氨酸对硝基苯胺(LNA)溶液0.20ml,反应10min,加入1mL 40%的乙酸终止反应,混匀后静置10min;
d.使用可见光分光光度计测定波长在405nm条件下的吸光度。
⑤计算公式
酶活按照下面公式计算:
式中:
ΔOD—实验组吸光值与对照组吸光值之差;
n—稀释倍数;
4—反应试剂的总体积;
0.2—加入反应体系的酶液的体积;
K-对硝基苯胺标准曲线的斜率0.0411;
1/10—反应时间10,以1min计。
(3)本发明中,涉及的部分序列如下:
野生型α-淀粉酶启动子PamyE的碱基序列如SEQ ID NO.1所示:
CATTATGTTTGAATTTCCGTTTAAAGAATGGGCTGCAAGCCTTGTGTTTTTGTTCATCATTATCTTATATTACTGCATCAGGGCTGCGGCATCCGGAATGCTCATGCCGAGAATAGACACCAAAGAAGAACTGCAAAAACGGGTGAAGCAGCAGCGAATAGAATCAATTGCGGTCGCCTTTGCGGTAGTGGTGCTTACGATGTACGACAGGGGGATTCCCCATACATTCTTCGCTTGGCTGAAAATGATTCTTCTTTTTATCGTCTGCGGCGGCGTTCTGTTTCTGCTTCGGTATGTGATTGTGAAGCTGGCTTACAGAAGAGCGGTAAAAGAAGAAATAAAAAAGAAATCATCTTTTTTGTTTGGAAAGCGAGGGAAGCGTTCACAGTTTCGGGCAGCTTTTTTTATAGGAACATTGATTTGTATTCACTCTGCCAAGTTGTTTTGATAGAGTGATTGTGATAATTTTAAATGTAAGCGTTAACAAAATTCTCCAGTCTTCACATCGGTTTGAAAGGAGGAAGCGGAAGAATGAAGTAAGAGGGATTTTTGACTCCGAAGTAAGTCTTCAAAAAATCAAATAAGGAGTGTCAAGA;
突变的启动子Pre10的碱基序列如SEQ ID NO.2所示:
CATTATGTTTGAATTTCCGTTTAAAGAATGGGCTGCAAGCCTTGTGTTTTTGTTCATCATTATCTTATATTACTGCATCAGGGCTGCGGCATCCGGAATGCTCATGCCGAGAATAGACACCAAAGAAGAACTGCAAAAACGGGTGAAGCAGCAGCGAATAGAATCAATTGCGGTCGCCTTTGCGGTAGTGGTGCTTACGATGTACGACAGGGGGATTCCCCATACATTCTTCGCTTGGCTGAAAATGATTCTTCTTTTTATCGTCTGCGGCGGCGTTCTGTTTCTGCTTCGGTATGTGATTGTGAAGCTGGCTTACAGAAGAGCGGTAAAAGAAGAAATAAAAAAGAAATCATCTTTTTTGTTTGGAAAGCGAGGGAAGCGTTCACAGTTTCGGGCAGCTTTTTTTATAGGAACATTGATTTGTATTCACTCTGCCAAGTTGTTTTGATAGAGTGATTGTGATAATTTTAAATGTAAACAAAAGATAAATTCTCCAGTCTTCACATCGGTTTGAAAGGAGGAAGCGGAAGAATGAAGTAAGAGGGATTTTTGACTCCGAAGTAAGTCTTCAAAAAATCAAATAAGGAGTGTCAAGA。
氨肽酶YwaD的氨基酸序列如SEQ ID NO:4所示:
MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHAVQISNSERELPFKAKHAYSTISQLSEAIGPRIAGTAAEKKSALLIASSMRKLKLDVKVQRFNIPDRLEGTLSSAGRDILLQAASGSAPTEEQGLTAPLYNAGLGYQKDFTADAKGKIALISRGDLTYYEKAKNAEAAGAKAVIIYNNKESLVPMTPNLSGNKVGIPVVGIKKEDGEALTQQKEATLKLKAFTNQTSQNIIGIKKPKNIKHPDIVYVTAHYDSVPFSPGANDNGSGTSVMLEMARVLKSVPSDKEIRFIAFGAEELGLLGSSHYVDHLSEKELKRSEVNFNLDMVGTSWEKASELYVNTLDGQSNYVWESSRTAAEKIGFDSLSLTQGGSSDHVPFHEAGIDSANFIWGDPETEEVEPWYHTPEDSIEHISKERLQQAGDLVTAAVYEAVKKEKKPKTIKKQMKAKASDIFEDIK
以下通过具体实施例对本发明作进一步地解释说明。
实施例1:野生型启动子PamyE片段的获得
引物PamyE-P1(F):
TGGTCGGCACTGAATTCGAGCATTATGTTTGAATTTCCGTTTAAAGAATGG
引物PamyE-P2(R):
gacagtcaaaagctttttcatTGTAAGCTCATTCGATTTGTTCGC
以P1和P2作为上、下游引物,以枯草芽孢杆菌168基因组为模板进行PCR扩增,获得野生型启动子PamyE;
其扩增的反应体系为:
上游引物PamyE-P1 | 1.5μL |
下游引物PamyE-P2 | 1.5μL |
DNA模板 | 2μL |
PrimeSTAR酶 | 25μL |
ddH2O | 20μL |
扩增程序的设置为:预变性:95℃,5min;变性:98℃,10s;退火:56℃,20s;延伸:72℃,60s;反应31个循环;延伸:72℃,10min,得到淀粉酶启动子和信号肽片段:PamyE+SPamyE片段,将此片段进行进行切胶回收,大小为725bp,如图2所示。
实施例2:重组氨肽酶基因工程菌
2.1重组质粒构建
(1)目的基因和载体的扩增
根据SEQ ID NO:3由苏州金唯智生物科技有限公司合成氨肽酶基因的核苷酸序列;并使用引物YwaD-P1和YwaD-P2对该氨肽酶基因进行扩增;使用引物pWB980-P1和pWB980-P2对pWB980质粒进行反向扩增;
YwaD-P1:ACAAATCGAATGAGCTTACAatgaaaaagcttttgactgtcatg;
YwaD-P2:CAGGTCGACTCTAGAGGATCcttatttgatatcttcaaaaatgtcagatgc;
pWB980-P1:ttttgaagatatcaaataagGATCCTCTAGAGTCGACCTG;
pWB980-P2:ACGGAAATTCAAACATAATGCTCGAATTCAGTGCCGACCA;
扩增的反应体系均为:
上游引物P1 | 1.5μL |
下游引物P2 | 1.5μL |
DNA模板 | 2μL |
PrimeSTAR酶 | 25μL |
ddH2O | 20μL |
扩增程序的设置为:预变性:95℃,5min;变性:98℃,10s;退火:56℃,20s;延伸:72℃,60s;反应31个循环;延伸:72℃,10min,分别获得ywad基因片段和pWB980质粒片段,并对其进行纯化回收。
(2)利用从北京全式金生物技术有限公司购买的无缝克隆酶将氨肽酶基因ywaD回收片段、PamyE+SPamyE片段及pWB980质粒片段连接获得重组质粒PamyE-SPamyE-ywaD-pWB980;连接体系如下:
PamyE+SPamyE回收片段 | 1μL |
氨肽酶基因回收片段 | 1μL |
pWB980质粒片段 | 3μL |
无缝克隆酶 | 5μL |
(3)将步骤(2)中的反应体系50℃反应15min后化转到枯草芽孢杆菌WB600中,方法如下所述;
①分别挑取新活化的枯草芽孢杆菌WB600单菌于5mL LB液体培养基中,37℃,220r/min,过夜培养;
②取100μL培养液转接至5mL SPI培养基中,37℃,220r/min培养至对数生长末期OD600=1.2(约3-4h);
③取200μL生长至对数期末的培养液至2mL SPII培养基中,37℃,100r/min培养1.5h;
④在上述SPII培养基的菌体中加入20μL 10mmol/L EGTA,37℃,100r/min培养10min;
⑤加入20μL连接产物:重组质粒PamyE+SPamyE-ywaD-pWB980,37℃,100r/min培养30min;
调节转速至220r/min,继续培养1.5h,取菌液涂布于含有100μg/mL卡那霉素的LB筛选平板,37℃培养12h,筛选阳性转化子,用引物YwaD-P1、YwaD-P2进行PCR验证,如图3所示。
实施例3:启动子突变体的获得
3.1设计原理
α-淀粉酶启动子PamyE作为常用的表达元件,已在枯草芽胞杆菌及其亲缘关系较近的底盘菌株中实现多种工业酶的发酵生产,例如,蛋白酶、淀粉酶、磷脂酶、果聚糖酶等。然而,葡萄糖介导的碳分解代谢物抑制(CCR)是阻遏启动子PamyE在表达系统中应用的主要问题,参与这一全局调控的联级反应包括:特征性顺式活性序列(CRE,分解代谢物反应元件)和反式作用蛋白(CcpA,分解代谢物控制蛋白)识别CRE元件,负调控相应基因的转录。有报道称,通过CcpA蛋白上结合启动子的结合基序进而达到调节碳源代谢的目的。例如,在地衣芽孢杆菌中突变CcpA蛋白的关键氨基酸Lys31,Ile42和Leu56,在葡萄糖存在的前提下,提高了木糖的利用率;通过随机突变CcpA蛋白,系统的重新连接全局碳、氮代谢的调控网络,在枯草芽孢杆菌中实现最佳的营养摄入并提高异源蛋白的产量;这些结果为CcpA介导调节的分子基础提供了新的见解。尽管CcpA蛋白的突变或缺失可以缓解或解除CCR的阻遏作用,但却对生长可能产生不利影响,这是由于CcpA蛋白不仅对碳源代谢进行调控,而且在氮代谢、磷代谢、脂代谢等都有重要的调控作用。
相较于分子水平上调整CcpA蛋白,通过改造启动子的CRE元件,对于缓解全局调控因子CcpA蛋白介导的CCR效应是一种最直接、最有效的策略。所以CRE元件介导的CCR效应是微生物适应不断变化环境时最重要的机制之一,并且在枯草芽孢中,已经鉴定出50多个CRE序列,提出了一个共识的CRE序WTGNAANCGNWNNCW(W:A/T,N:A/G/C/T)。因此,上述共识的CRE序列作为参考模板,在启动子PamyE的序列中寻找假定的CRE元件。并设计突变引物P1/P2,以PamyE-SPamyE-ywaD-pWB980质粒为模板,对α-淀粉酶启动子PamyE的CRE元件进行PCR扩增和突变,获得突变质粒Pre10-SPamyE-ywaD-pWB980(其中包含了突变启动子Pre10,经过测序确定核苷酸序列如SEQ ID NO.2所示),并按照枯草芽胞杆菌划转的方法进行划转,筛选阳性转化子进行验证。
突变扩增体系如下:
上游引物P1 | 1.5μL |
下游引物P2 | 1.5μL |
PamyE-SPamyE-ywaD-pWB980质粒为模板 | 2μL |
PrimeSTAR酶 | 25μL |
ddH2O | 20μL |
突变扩增程序如下:
扩增程序的设置为:预变性:95℃,5min;变性:98℃,10s;退火:56℃,20s;延伸:72℃,60s;反应31个循环;延伸:72℃,10min,
引物P1:F 5’-AACAAAAGAT AAATTCTCCAGTCTTCACATCGGTT-3’;
引物P2:R 5’-TACATTTAAAATTATCACAATCACTCTATCAAAAC-3’。
3.2解淀粉芽孢杆菌重组菌株构建
提取实施例2中在WB600中的重组质粒PamyE-SPamyE--ywaD-pWB980、以及突变质粒Pre10-SPamyE-ywaD-pWB980,分别电转到解淀粉芽孢杆菌CGMCC No.11218中。感受态的制备及电转方法如下:
1)75%酒精清洗电转杯,在紫外下照射20min以上,并在冰上预冷;
2)将100μL感受态和10ng质粒DNA混合加入电转杯,冰上放置2min;
3)2500V电击,电击时间一般为4-6ms;
4)电击后立即加入1ml复苏培养基,37℃,复苏3h。涂板,37℃培养12h,筛选阳性转化子,并进行测序(金唯智生物科技有限公司),结果表明,扩增得到了YwaD基因核苷酸序列与模板序列一致并将重组菌株分别命名为PamyE-AP(含有重组质粒PamyE-SPamyE--ywaD-pWB980)、Pre10-AP(含有质粒Pre10-SPamyE-ywaD-pWB980)。
实施例4:重组氨肽酶基因工程菌的表达及分析
将新鲜平板上的重组基因工程菌Pre10-AP、PamyE-AP单菌落接入50mL卡那霉素抗性种子培养基中,37℃、220rpm振荡培养12h,以相同的2%接种量转接于发酵培养基中摇瓶发酵,于37℃、220rpm发酵培养。
氨肽酶的酶活,分别取重组菌发酵培养36h、48h、60h的发酵液进行氨肽酶酶活测定,经测定60h时包含本发明突变启动子Pre10的重组菌Pre10-AP发酵上清液中重组氨肽酶活力达到最高为956.2U/mL,是对照菌PamyE-AP重组氨肽酶活力的2.06倍(如图4所示)。可见本发明提供了一种强度更高的启动子Pre10,利用该启动子可显著提高氨肽酶在芽孢杆菌系统中的表达活性。
虽然本发明已经以较佳实施例公开如上,但其并非用以限定本发明,任何本领域技术人员,在不脱离本发明的精神和原理的情况下,可以对这些实施例进行各种形式和细节的变化、修改、替换和变型,本发明的范围由权利要求及其等同物所限定。
Claims (9)
1.一种启动子,其特征在于,所述启动子为启动子Pre10,核苷酸序列如SEQ ID NO:2所示。
2.包含权利要求1所述启动子Pre10的重组质粒。
3.如权利要求2所述的重组质粒,其特征在于,所述重组质粒的骨架包括但不限于pHT01、pHT43、pHCMC05、pWB980等。
4.包含权利要求1所述启动子Pre10,或包含权利要求2所述重组质粒的表达系统。
5.如权利要求4所述的表达系统,其特征在于,所述表达系统采用的宿主包括:解淀粉芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌。
6.权利要求1所述启动子Pre10的应用。
7.如权利要求6所述的应用,其特征在于,是在控制蛋白表达中的应用。
8.如权利要求7所述的应用,其特征在于,所述蛋白为氨肽酶。
9.如权利要求8所述的应用,其特征在于,所述氨肽酶基酸序列如SEQ ID NO:4所示。
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