CN113151232A - 忽地笑1-氨基环丙烷-1-羧酸合成酶及其编码基因与应用 - Google Patents
忽地笑1-氨基环丙烷-1-羧酸合成酶及其编码基因与应用 Download PDFInfo
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- CN113151232A CN113151232A CN202110358488.5A CN202110358488A CN113151232A CN 113151232 A CN113151232 A CN 113151232A CN 202110358488 A CN202110358488 A CN 202110358488A CN 113151232 A CN113151232 A CN 113151232A
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Abstract
本发明涉及石蒜属植物忽地笑1‑氨基环丙烷‑1‑羧酸合成酶及其编码基因与应用。本发明首次揭示了石蒜属植物忽地笑的1‑氨基环丙烷‑1‑羧酸合成酶,其具有良好的酶活性,可催化腺苷甲硫氨酸合成1‑氨基环丙烷‑1‑羧酸。本发明还揭示了编码所述1‑氨基环丙烷‑1‑羧酸合成酶的多核苷酸,表达所述1‑氨基环丙烷‑1‑羧酸合成酶的载体与宿主细胞,以及利用全细胞转化的方法生产1‑氨基环丙烷‑1‑羧酸的方法。
Description
技术领域
本发明涉及生物技术和植物生物学领域;更具体地,本发明涉及一种来源于石蒜属植物忽地笑的1-氨基环丙烷-1-羧酸合成酶及其编码基因与应用。
背景技术
乙烯作为一种植物内源激素,具有多种生理功能,主要表现在促进果实成熟和叶、花、果脱落等。但是,乙烯是气体,难于直接在田间应用,一般使用能够释放乙烯的生长调节剂。传统上使用的乙烯释放剂——乙烯利不能与碱、金属盐、金属铝铜铁等共存,且易受温度和pH值影响,过量使用时还具有一定的毒性和腐蚀性,因此被国家技术监督局划为第6.1类毒害品。
因此,农业上需要寻找安全实用的新型乙烯类植物生长调节剂。作为植物体内乙烯合成的直接前体,1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylic acid,ACC)与目前常用的生长调节剂乙烯利相比具有明显的优势。1-氨基环丙烷-1-羧酸是一种植物内源性的非蛋白氨基酸,可直接参与体内代谢,在植物体内运输,其合成受酶调节,不直接受pH及温度等外部条件的影响。1-氨基环丙烷-1-羧酸属于植物体内天然存在的非蛋白氨基酸,施用时更加安全可靠,因此其可为农业提供可安全实用的新型乙烯类植物生长调节剂。1-氨基环丙烷-1-羧酸还具有保护神经和降血压作用,在医药领域同样具有巨大的市场需求。
由于具有以上优点,1-氨基环丙烷-1-羧酸具有很好的应用前景。但是其在植物体内含量极低,难以通过直接从植物提取的方法获得。目前,国内外1-氨基环丙烷-1-羧酸的生产主要采用化学合成的方法,化学合成能耗高,原料昂贵,反应条件苛刻,合成过程复杂,污染环境,且收率不高,这也造成了1-氨基环丙烷-1-羧酸市场价格较高。
目前1-氨基环丙烷-1-羧酸售价约为1.5-2万元每公斤。1-氨基环丙烷-1-羧酸的售价过高造成其目前难以应用于实际生产。基于这一现实需要解决的问题,植物来源的1-氨基环丙烷-1-羧酸合成酶(1-aminocyclopropane-1-carboxylic acid synthease,ACS)及其编码基因可通过植物转基因和异源表达的方式进行天然产物的生物转化或生物合成1-氨基环丙烷-1-羧酸。本发明克隆了来源于石蒜属植物忽地笑(Lycoris aurea)的1-氨基环丙烷-1-羧酸合成酶(LaACS),该酶具有催化S-腺苷蛋氨酸(S-Adenosyl methionine,SAM)合成1-氨基环丙烷-1-羧酸的活性。
发明内容
本发明的目的在于提供一种石蒜属植物忽地笑的1-氨基环丙烷-1-羧酸合成酶,所述的酶选自:
(a)氨基酸序列如SEQ ID NO:1所示的蛋白质;或
(b)将SEQ ID NO:1氨基酸序列经过一个或多个(如1-90个)氨基酸残基的取代、缺失或添加而形成的,且具有1-氨基环丙烷-1-羧酸合成酶活性的由(a)衍生的蛋白质;或
(c)与SEQ ID NO:1氨基酸序列有至少90%以上同源性,且具有1-氨基环丙烷-1-羧酸合成酶催化活性的由(a)衍生的蛋白质。
本发明SEQ ID NO:1所示的蛋白质是从石蒜属植物忽地笑(Lycoris aurea)中分离得到的一种新的1-氨基环丙烷-1-羧酸合成酶。为便于表述,将SEQ ID NO:1所示的蛋白质命名为LaACS。
在一个优选例中,所述的1-氨基环丙烷-1-羧酸合成酶活性是指催化S-腺苷蛋氨酸(S-Adenosyl methionine,SAM)合成产物1-氨基环丙烷-1-羧酸。
在另一个优选例中,所述的序列(c)还包括:由(a)或(b)添加了标签序列、信号序列或分泌信号序列后所形成的融合蛋白。
考虑到密码子的简并性以及不同物种密码子的偏好性,本领域技术人员可以根据需要使用合适特定物种表达的密码子。因而,编码本发明1-氨基环丙烷-1-羧酸合成酶的多核苷酸还包括由SEQ ID NO:2所示,核苷酸序列经取代、缺失和/或增加一个或几个核苷酸,得到的编码具有活性的1-氨基环丙烷-1-羧酸合成酶的核苷酸序列。
本发明的又一目的是提供一种载体,它含有如SEQ ID NO:2所示的多核苷酸。所述的载体是将编码本发明所述的1-氨基环丙烷-1-羧酸合成酶的多核苷酸与表达载体可操作地连接,得到能够表达本发明所述1-氨基环丙烷-1-羧酸合成酶的重组表达载体或抑制本发明所述1-氨基环丙烷-1-羧酸合成酶编码多核苷酸表达的基因沉默载体。
在一个优选例中,该载体是含有编码所述1-氨基环丙烷-1-羧酸合成酶的SEQ IDNO:2所示序列的重组表达载体,所述的表达载体pECXK-99E和pET29a(+)。
本发明的又一目的是提供一种宿主细胞,它含有所述的重组表达载体或基因组中整合有所述的多核苷酸。所述的宿主细胞是原核细胞或真核细胞。常用的原核宿主细胞包括大肠杆菌、枯草杆菌、棒杆菌等;常用的真核宿主细胞包括真菌细胞、植物细胞、昆虫细胞和哺乳动物细胞等,所述的真菌细胞包括酵母细胞。将所述的重组表达载体或者基因沉默载体导入所述的适当宿主细胞中,获得表达本发明所述酶的基因工程菌株、转基因细胞系、转基因愈伤、转基因组织、转基因植株或者基因工程植株。
本发明的又一目的是提供所述的1-氨基环丙烷-1-羧酸合成酶的用途,在于用于合成产物1-氨基环丙烷-1-羧酸。
本发明的又一目的是提供一种表达构建物。所述表达构建物包括以下酶的编码基因和/或基因表达盒。
所述基因表达盒是酶在宿主细胞中表达与调控所需要的生物学元件,包括启动子、增强子、衰减子、核糖体结合位点、Kozak序列、内含子和/或转录终止子等;此外,还可包括标签编码序列和/或信号(肽)编码序列等。
在一个优选例中,所述的表达构建物还包括:1-氨基环丙烷-1-羧酸合成酶编码基因。
在另一个优选例中,当转化大肠杆菌细胞时,所述的表达构建物中,还包含大肠杆菌启动子、大肠杆菌核糖体结合位点和/或大肠杆菌转录终止子等基因表达盒。
在另一个优选例中,当转化产氨棒杆菌细胞时,所述的表达构建物中,还包含色氨酸启动子、核糖体结合位点和/或转录终止子等基因表达盒。
本发明的又一目的是提供一种宿主细胞。所述的宿主细胞中包括所述的表达构建物。所述的宿主细胞是原核细胞或真核细胞。常用的原核宿主细胞包括大肠杆菌、枯草杆菌、棒杆菌等;常用的真核宿主细胞包括真菌细胞、植物细胞、昆虫细胞和哺乳动物细胞等。较佳地,所述的宿主细胞是内源存在1-氨基环丙烷-1-羧酸合成酶的底物(腺苷甲硫氨酸)或其底物前体(甲硫氨酸)的细胞。
本发明的又一目的是提供所述的表达构建物的用途,用于生产1-氨基环丙烷-1-羧酸。
本发明的又一目的是提供一种生产1-氨基环丙烷-1-羧酸的方法。所述方法包括:利用所述的1-氨基环丙烷-1-羧酸合成酶生产1-氨基环丙烷-1-羧酸。
在一个优选例中,所述方法包括:以所述的表达构建物转化宿主细胞,催化腺苷甲硫氨酸生成1-氨基环丙烷-1-羧酸;所述的宿主细胞是原核细胞或真核细胞。常用的原核宿主细胞包括大肠杆菌、枯草杆菌、运动假单胞菌和乳酸菌等;常用的真核宿主细胞包括真菌细胞、植物细胞、昆虫细胞和哺乳动物细胞等。所述的真菌细胞包括酵母细胞。
在另一个优选例中,所述方法包括:以所述的表达构建物转化大肠杆菌细胞或产氨棒杆菌,通过生物转化的方法合成1-氨基环丙烷-1-羧酸。较佳地,该宿主细胞是内源腺苷甲硫氨酸含量较高的细胞。
本发明首次揭示了石蒜属植物忽地笑来源的1-氨基环丙烷-1-羧酸合成酶LaACS。本发明还揭示了编码所述1-氨基环丙烷-1-羧酸合成酶的多核苷酸、表达所述1-氨基环丙烷-1-羧酸合成酶LaACS的表达载体及宿主细胞。本发明应用石蒜属植物来源的1-氨基环丙烷-1-羧酸合成酶,进而实现1-氨基环丙烷-1-羧酸的生物转化以及生物合成。
附图说明
图1是引物对SEQ ID NO:3和SEQ ID NO:4的聚合酶链式反应(PCR)扩增产物的琼脂糖凝胶电泳检测结果。
图2是克隆载体pMD19-T-LaACS的菌落PCR验证电泳图。
图3是重组表达载体pECXK-LaACS的菌落PCR验证电泳图。
图4是重组表达载体pET29a-LaACS的菌落PCR验证电泳图。
图5是1-氨基环丙烷-1-羧酸标品HPLC检测绘制的标准曲线。
图6是转化液中检测到的1-氨基环丙烷-1-羧酸HPLC图谱。
具体实施方式
以下结合具体实施例并附图,进一步阐述本发明。
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
实施例1、1-氨基环丙烷-1-羧酸合成酶LaACS编码基因的克隆
合成两条引物分别具有序列表中SEQ ID NO:3、SEQ ID NO:4的核苷酸序列。
以从忽地笑中提取的RNA反转录获得的cDNA为模板,利用如上两条引物SEQ IDNO:3和SEQ ID NO:4进行PCR。DNA聚合酶选用南京诺唯赞生物科技有限公司的Super-FidelityDNA聚合酶。PCR扩增程序为:95℃ 5min;94℃ 10s,56℃ 10s,72℃ 1min,运行32个循环;72℃ 10min,10℃保温。PCR产物经琼脂糖凝胶电泳检测,扩增结果如图1。切下与目标大小一致的DNA条带。采用DNA纯化试剂盒(北京百泰克生物技术有限公司)从琼脂糖凝胶中回收DNA。利用宝生物工程(大连)有限公司(TaKaRa)的pMD19-T克隆试剂盒,将回收的PCR产物克隆到pMD19-T载体,载体的验证见图2,所构建的载体命名为pMD19-T-LaACS。经测序获得LaACS基因的序列。
LaACS基因具有序列表中SEQ ID NO:2的核苷酸序列。自SEQ ID NO:2的5’端第1-1473位核苷酸为基因LaACS的开放阅读框(Open Reading Frame,ORF),自SEQ ID NO:2的5’端的第1-3位核苷酸为LaACS基因的起始密码子ATG,自SEQ ID NO:2的5’端的第1471-1473位核苷酸为LaACS基因的终止密码子TAA。基因LaACS编码一个含有490个氨基酸的蛋白质LaACS,具有SEQ ID NO:1的氨基酸序列,软件预测该蛋白质的理论分子量大小为54954.97Da,等电点pI为8.65。
实施例2、LaACS基因的大肠杆菌及棒杆菌重组表达载体的构建
(1)表达载体pECXK-LaACS的构建
合成分别具有序列表中SEQ ID NO:5和SEQ ID NO:6核苷酸序列的两条引物。在合成的引物SEQ ID NO:5和SEQ ID NO:6的5’端分别设置BamHI和SalI两个酶切位点及同源重组序列,并在N端引入6个组氨酸,以pMD19-T-LaACS为模板进行PCR扩增。PCR扩增程序同实施例1。PCR扩增产物经琼脂糖凝胶电泳检测、分离、切胶回收后,利用同源重组,连接至经BamHI和SalI双酶切的pECXK-99E载体中,N端引入了His表达标签用于后续蛋白纯化。连接产物转化大肠杆菌(E.coli)DH5α感受态细胞,并涂布于添加25μg/mL卡那霉素的LB平板上。通过菌落PCR验证获得阳性转化子,菌落PCR验证的琼脂糖电泳结果如图3。所获得的重组质粒命名为pECXK-His-LaACS。通过测序进一步验证重组质粒pECXK-His-LaACS构建成功,在BamHI和SalI酶切位点之间含有SEQ ID NO:2的全长多核苷酸序列。
(2)表达载体pET29a-LaACS的构建
合成分别具有序列表中SEQ ID NO:7和SEQ ID NO:8核苷酸序列的两条引物。在合成的引物SEQ ID NO:7和SEQ ID NO:8的5’-端分别设置NdeI和XhoI两个酶切位点及其同源重组序列,以pMD19-T-LaACS为模板进行PCR扩增。PCR扩增程序同实施例1。PCR扩增产物经琼脂糖凝胶电泳检测、分离、切胶回收后,利用同源重组,连接至NdeI和XhoI双酶切的pET29a载体中。连接产物转化大肠杆菌(E.coli)DH5α感受态细胞,并涂布于添加卡那霉素(终浓度为25μg/mL)的LB平板上。通过菌落PCR验证阳性转化子,菌落PCR产物的琼脂糖电泳结果如图4。所获得的重组质粒命名为pET29a-LaACS。测序进一步验证重组质粒pET29a-LaACS构建成功,并在NdeI和XhoI酶切位点之间含有SEQ ID NO:2的全长多核苷酸序列。
实施例3、LaACS蛋白的诱导表达
蛋白的原核表达会受到诱导时间、诱导温度、启动子强度和宿主等的影响。因此可以通过改变诱导条件,利用不同表达载体和宿主菌等方法对蛋白的原核表达进行优化。将上述构建成功的重组表达载体pECXK-His-LaACS分别转化大肠杆菌BL21(DE3)和产氨棒杆菌21170,将重组表达载体pET29a-LaACS转化大肠杆菌BL21(DE3)。获得重组菌株BL21(DE3)/pECXK-His-LaACS、BL21(DE3)/pET29a-LaACS和产氨棒杆菌ATCC21170/pECXK-His-LaACS保存于-80℃冰箱,备用。
(1)大肠杆菌培养基配方:选用的诱导培养基配方(1L)为23g Na2HPO4,5g KH2PO4,2.5g NaCl,5.0g(NH4)2SO4,0.5g MgSO4,0.01g CaCl2,30g葡萄糖,微量元素终浓度为5mgFeSO4·7H2O,5mg MnSO4·7H2O和5mg ZnSO4。
(2)大肠杆菌蛋白诱导条件优化:将鉴定成功的单菌落接种到含相应抗生素(25mg/L卡那霉素)的LB的液体培养基中,37℃,200rpm振荡培养过夜;过夜培养的菌液按1%的接种量接入50mL诱导培养基中。37℃培养4-6小时,当培养液OD600达到0.8左右时,加入IPTG至终浓度为1mM进行诱导表达,以不加IPTG的作为对照。进行3组实验,分别放入25℃(6h)、30℃(6h)、37℃(6h),振荡培养,对诱导后的全菌蛋白进行SDS-PAGE凝胶电泳检测,结果发现30℃条件下,转化质粒pET29a-LaACS的大肠杆菌BL21(DE3)的LaACS蛋白表达量最高。在诱导温度(30℃)和诱导时间(6h)不变的前提下调整IPTG的诱导浓度,分别选取0.05mM、0.1mM、0.2mM、0.5mM和1mM浓度的IPTG加入OD600达到0.8左右的培养液中,发现转化质粒pET29a-LaACS的大肠杆菌BL21(DE3)菌株在30℃、选用0.1mM的IPTG时诱导效果最好。
产氨棒杆菌培养基:种子培养基(1L):葡萄糖20g,蛋白胨10g,酵母粉10g,NaCl3g,pH7.2。产氨棒杆菌发酵基本培养基(1L):葡萄糖60g,酵母粉10g,CaCl2 0.1g,MgSO4 2g,尿素20g和K2HPO4·3H2O 10g,KH2PO4 10g。产氨棒杆菌蛋白诱导检测显示蛋白表达在IPTG终浓度为1mM,OD600为1.0左右诱导效果最好。
实施例4、LaACS蛋白的纯化
(1)收集菌体:将菌株接入3mL LB液体培养基中(含50mg/L卡那霉素),过夜培养,再将过夜活化的菌液按照1%的接种量接入50mL(250mL三角瓶)发酵培养基中(含25mg/L卡那霉素),37℃,200rpm振荡培养,检测发酵液的OD600,当OD600到0.8-1.0左右,加入IPTG(终浓度0.1mM),在30℃诱导培养12h(设置未诱导组作为对照),诱导结束后于4℃,4000rpm离心20min,收集菌体。
(2)超声裂解菌体:收集菌体后加入8mL平衡缓冲液(50mM Na2HPO4,0.3M NaCl,pH=8.0)重新悬浮细胞,如有需要可加入适量的PMSF或者其他蛋白抑制剂。重悬菌液于超声细胞破碎仪在冰上进行超声破碎。探头伸到液面以下,注意不与管壁接触,破碎1秒,停3秒。超声后观察菌液是否澄清,若澄清则表示超声裂解充分。超声完成后,取出20μL作为全细胞裂解液待检测,剩下的裂解液4℃,12000rpm离心15分钟,取上清,即为裂解上清液。向沉淀中加入上清同等体积的平衡缓冲液,重悬沉淀,即为沉淀重悬液。上述样品立即用于后续电泳样品的制备或-80℃冰箱中保存待用。
(3)层析柱的填充:吸取一定量的Ni-NTA树脂加入到柱子中,让其自由沉降,并放干储存液,加入4倍柱体积的平衡缓冲液平衡柱子,或者待流出液的紫外吸光度A280值达到最低且稳定。
(4)过柱纯化:将菌体裂解上清液样品加入事先平衡好的Ni-NTA树脂中,让样品缓慢地流出,流速大约为0.5-1mL/min。收集流出液,待后续分析;以流速为1mL/min的洗涤缓冲液(50mM Na2HPO4,0.3M NaCl,10mM咪唑,pH=8.0)洗涤柱子以去除杂蛋白,一般用量为8倍柱体积,或者直到流出液的A280值达到最低且稳定;用5-10倍柱体积的洗脱缓冲液(50mMNa2HPO4,0.3M NaCl,250mM咪唑,pH=8.0)以0.5-1mL/min的流速洗脱,收集洗脱液,或根据流出液A280值判断,当数值陡然上升时开始接收洗脱液,直到A280数值降至最低且稳定停止收集。使用20mM Tris-HCl(pH 8.0)进行透析。
分别将全细胞裂解液、裂解上清液、沉淀重悬液和过柱洗脱液进行SDS-PAGE电泳,检测蛋白可溶性表达情况和过柱纯化收集效果。
实施例5、LaACS体外催化活性验证
将获得的LaACS酶透析液进行浓缩,目标蛋白最后浓缩于50mM MOPS缓冲液(pH8.0,含200mM NaCl和10%甘油),蛋白保存于-80℃冰箱。
酶活检测体系为50mM HEPES(pH 8.2),NaCl终浓度为25mM,此外加入400μM底物腺苷甲硫氨酸(SAM)和50μM PLP(磷酸吡哆醛),加入适量LaACS蛋白。加入煮沸过的蛋白或不添加底物腺苷甲硫氨酸(SAM)的反应体系作为负对照。所有的体系均在30℃反应10min。
采用PITC柱前衍生的方法测定转化液中1-氨基环丙烷-1-羧酸的含量。反应液经过PITC柱前衍生后上样高效液相色谱仪(HPLC)进行分析。衍生方法如下:A液(PITC乙腈溶液,0.1M)和B液(三乙胺乙腈溶液,1M)等体积混合,取转化液200μL,与AB混合液等体积充分混匀,超声10min,室温放置1h,400μL正己烷震荡萃取,取下层溶液,滤膜过滤后备用。分析条件为:采用LC-20A高效液相色谱仪(岛津,日本),C18色谱柱(5μm,4.6mm×250mm),柱温箱温度40℃,二极管阵列检测器,254nm波长,20μl进样量,10mM磷酸钠水溶液为流动相A,100%乙腈为流动相B,1mL/min流速,梯度洗脱,洗脱程序如下:0-8min流动相B 5%,8-30min流动相B从5%升至33%,30-35min流动相B 33%,35-40min流动相B 90%,40-50min流动相B从90%降至5%,50-55min流动相B 5%。1-氨基环丙烷-1-羧酸液相标准曲线如图5。
LaACS蛋白的酶动力学分析使用如下的反应体系(40μL):50mM HEPES(pH 8.2),NaCl终浓度为25mM,蛋白量为50nM,底物腺苷甲硫氨酸(SAM)浓度为2.5-150μM,PLP终浓度为5μM。腺苷甲硫氨酸(SAM)终浓度为2.5μM,5μM,10μM,20μM,30μM,40μM,80μM,100μM和150μM,30℃反应10min后加入40μL MeCN终止反应。离心后,取30μL上清液直接进行HPLC分析。Km和Vmax值按照米氏方程(Michaelis-Menten equation):v=Vmax×[S]/(Km+[S])计算。结果显示,LaACS的酶反应的Km值为15±1.2μM,kcat值为2.1±0.02S-1。
实施例6、LaACS生物转化腺苷甲硫氨酸合成1-氨基环丙烷-1-羧酸
(1)配制培养基。大肠杆菌发酵培养基配方(1L):23g Na2HPO4,7g KH2PO4,2.5gNaCl,5.0g(NH4)2SO4,0.5g MgSO4,0.01g CaCl2,30g葡萄糖,1mL微量元素母液。微量元素母液配方为(1L):10g FeSO4·7H2O,2.25g ZnSO4·7H2O,1g CuSO4·5H2O,3.5g MnSO4·8H2O,溶解于0.1N HCl。产氨棒杆菌种子培养基(1L):葡萄糖20g,蛋白胨10g,酵母粉10g,NaCl3g,pH7.2。产氨棒杆菌发酵基本培养基(1L):葡萄糖60g,酵母粉10g,CaCl2 0.1g,MgSO4 2g,尿素20g和K2HPO4·3H2O 10g,KH2PO4 10g。
生物转化液组成:菌体40g/L,腺苷甲硫氨酸400μM,葡萄糖30g/L,Tris-HCl 50mM,磷酸盐240mM(KH2PO4和K2HPO4各120mM),MgCl2 20mM,二甲苯10mL/L,以上均为终浓度。
(2)将重组菌株活化后,分别将大肠杆菌菌株各挑取单克隆接种添加卡那霉素(终浓度为25mg/L)的3mL LB液体培养基于37℃、200rpm培养过夜。挑取产氨棒杆菌21170/pECXK-His-LaACS接种添加卡那霉素(终浓度为25mg/L)的装有产氨棒杆菌种子培养基的试管中,30℃、200rpm培养过夜。
(3)将过夜培养大肠杆菌按1%的接种量接种至于含卡那霉素(终浓度为25mg/L)的50mL发酵培养基中,37℃、200rpm振荡培养,待菌液生长至600nm波长下吸光度为0.8左右时,加入诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)终浓度为0.1mM,进行诱导培养,诱导时间为12h,诱导温度为30℃。将过夜培养产氨棒杆菌按1%的接种量接种至于含卡那霉素(终浓度为25mg/L)的50mL棒杆菌发酵基本培养基中,30℃、200rpm振荡培养,待菌液生长至600nm波长下吸光度为1左右时,加入诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)终浓度为1mM,进行诱导培养,诱导时间为12h,诱导温度为30℃。
(5)分别收集诱导12h的细菌培养液于6000rpm、4℃离心5min,弃上清。菌体用20mL生物转化液重悬后全部转移到250mL无菌三角瓶中,于30℃,250rpm振荡培养48h。
(6)取生物转化液1mL,12000rpm,离心5min,收集上清液。将上清液进行PITC衍生化后,进行液相检测。
生物转化液HPLC检测分析结果如图6,结果表明:LaACS能够催化底物腺苷甲硫氨酸合成产物1-氨基环丙烷-1-羧酸(出峰时间与标品一致,在20min左右)。转化空载体的菌株转化液中未检测到相应出峰。重组菌株BL21(DE3)/pECXK-His-LaACS、BL21(DE3)/pET29a-LaACS和产氨棒杆菌ATCC21170/pECXK-His-LaACS都具有生物转化合成1-氨基环丙烷-1-羧酸的能力。其中,产氨棒杆菌21170/pECXK-His-LaACS的转化液中1-氨基环丙烷-1-羧酸量最高,可达到986mg/L,这可能与产氨棒杆菌胞内ATP含量较高有关。
序列表
<110> 江苏省中国科学院植物研究所
<120> 忽地笑1-氨基环丙烷-1-羧酸合成酶及其编码基因与应用
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Claims (10)
1.一种来源于石蒜属植物忽地笑的1-氨基环丙烷-1-羧酸合成酶,其特征在于,所述的1-氨基环丙烷-1-羧酸合成酶的氨基酸序列如SEQ ID NO:1所示的蛋白质。
2.编码权利要求1所述的1-氨基环丙烷-1-羧酸合成酶的多核苷酸,其特征在于,该多核苷酸的核苷酸序列如SEQ ID NO:2所示。
3.一种载体,其特征在于,所述载体含有权利要求2所述的多核苷酸。
4.权利要求3所述的载体的用途,其特征在于:表达权利要求1所述的1-氨基环丙烷-1-羧酸合成酶。
5.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求3所述的载体或基因组中整合有权利要求2所述的多核苷酸。
6.权利要求5所述的宿主细胞,其特征在于,所述的宿主细胞是原核细胞或真核细胞;所述的原核宿主细胞包括大肠杆菌、枯草杆菌、棒杆菌和乳酸菌等,所述的真核宿主细胞包括真菌细胞、植物细胞、昆虫细胞和哺乳动物细胞等,所述的真菌细胞包括酵母细胞。
7.权利要求1所述的1-氨基环丙烷-1-羧酸合成酶的用途,其特征在于,转化底物腺苷甲硫氨酸合成1-氨基环丙烷-1-羧酸。
8.权利要求5和6所述的宿主细胞的用途,其特征在于,生产1-氨基环丙烷-1-羧酸。
9.一种生产1-氨基环丙烷-1-羧酸的方法,其特征在于,所述方法包括:以权利要求5和6所述的宿主细胞合成1-氨基环丙烷-1-羧酸,利用权利要求1所述的1-氨基环丙烷-1-羧酸合成酶将腺苷甲硫氨酸转化为1-氨基环丙烷-1-羧酸。
10.权利要求9所述的生产1-氨基环丙烷-1-羧酸的方法,其特征在于,所述方法以权利要求5和6所述的宿主细胞,在生物转化液(含腺苷甲硫氨酸 400 μM,葡萄糖30 g/L,Tris-HCl 50 mM,120 mM KH2PO4,120 mM K2HPO4,MgCl2 20 mM,二甲苯 10 mL/L)中通过全细胞转化的方法合成1-氨基环丙烷-1-羧酸。
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