CN110627880B - 分离的多肽及其应用 - Google Patents
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Abstract
本发明公开了分离的多肽及其应用。其中,该分离的多肽包含nisB序列和前体肽序列,其中,所述nisB序列与SEQ ID NO:2所示的氨基酸序列具有至少90%一致性,且部分序列高度保守,且具有催化所述前体肽序列形成的肽段脱水的活性;所述前体肽序列与SEQ ID NO:3所示的氨基酸序列具有至少90%一致性,且部分序列高度保守,且具有与所述nisB序列形成的肽段结合并被脱水修饰的活性。通过表达nisin的产量依赖敏感的NisB序列和前体肽序列,使nisin的产量显著提高。
Description
技术领域
本发明涉及生物工程领域,具体地,涉及分离的多肽及其应用,更具体地,涉及分离的多肽、核酸、重组载体、重组细胞和制备乳酸链球菌素的方法。
背景技术
乳酸链球菌素(Nisin)是乳酸菌产生的一种天然活性抑菌多肽,属于羊毛硫抗生素家族。成熟的Nisin由34个氨基酸构成,对于大多数革兰氏阳性菌及其芽孢具有强烈的抑制作用,在与EDTA等共同作用时对革兰氏阴性菌也具有杀伤作用,是唯一被批准用于食品防腐的细菌素。Nisin对蛋白酶特别敏感,在消化道中能很快被α-胰凝乳蛋白酶分解而不影响肠道内正常菌群,对人体基本无毒性,也不与医用抗生素产生交叉抗药性。现已成为一种极具前景的绿色食品添加剂和潜在的抗生素替代品,被广泛应用于食品工业及生物医药领域。目前可通过乳酸菌发酵获得Nisin,但产量较低,无法满足生产需要。
由此,高产量的制备乳酸链球菌素的方法有待研究。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。发明人通过体外重建体系探明nisin合成的关键限速步骤是NisB的脱水步骤,并通过过表达nisin合成的底物前体肽和nisB,使发酵产生的nisin的产量显著提高。
需要说明的是,本发明是基于发明人的下列工作而完成的:
Nisin的生物合成途径如图1所示,前体肽nisZ或nisA经过NisB脱水,NisC环化产生羊毛硫氨酸环,再经由NisP去除前导肽,最终形成成熟的nisin。
发明人通过体外重建体系,研究图1所示的nisin的生物合成途径中各步骤对nisin产量的影响。研究发现,在限定了体系内供给的能量、还原力等条件下,当前体肽的浓度在0至0.4nM范围内,乳酸链球菌素产量随着前体肽浓度的增加而显着增加并且在0.4至1.3nM之间达到稳定,随着前体肽浓度的增加缓慢增加,并且,当前体肽的浓度在0.4至1.3nM之间,nisin产量高。而NisP编码质粒浓度对乳酸链球菌肽生产没有明显影响。但当NisB的浓度从10至1500nM变化,活性乳酸链球菌素Z的产量随着NisB浓度显着提高。而活性Nisin Z的产生在1至100nM的相当低的浓度范围内对NisC含量敏感。在1nM至100nM NisC之间观察到超过10倍的乳链菌肽生产变化,而较高的NisC浓度(从100至10000nM)不明显影响乳链菌肽的产生。由此,在发酵制备nisin的过程中,NisB和前体肽的浓度直接影响nisin的产量,发明人通过使乳酸菌过表达NisB和前体肽,提高乳酸菌的nisin的产量,实验发现,过表达NisB和前体肽的乳酸菌相对于原始的乳酸菌产量提高达60%。
因而,根据本发明的第一方面,本发明提供了一种分离的多肽。根据本发明的实施例,该分离的多肽包含nisB序列和前体肽序列,其中,
所述nisB序列与SEQ ID NO:2所示的氨基酸序列具有至少90%一致性,并在SEQID NO:2所示的氨基酸序列的第83位为精氨酸、第87位为精氨酸、第89位为苏氨酸、第121位为天冬氨酸、第154位为精氨酸、第171位为异亮氨酸、第176位为缬氨酸、第198位为缬氨酸、第202位为酪氨酸、第209位为亮氨酸、第213位为酪氨酸、第217位为亮氨酸、第299位为天冬氨酸、第464位为精氨酸、第786位为精氨酸、第826位为精氨酸和第961位为组氨酸,且具有催化所述前体肽序列形成的肽段脱水的活性;
所述前体肽序列与SEQ ID NO:3所示的氨基酸序列具有至少90%一致性,并在SEQID NO:3所示的氨基酸序列的第6位至第9位为苯丙氨酸-天冬酰胺-亮氨酸-天冬氨酸,第26位为丝氨酸,第28-34位为丝氨酸-亮氨酸-半胱氨酸-苏氨酸-脯氨酸-甘氨酸-半胱氨酸,第36位为苏氨酸,第37位甘氨酸,第39位为亮氨酸,第42位为半胱氨酸,第46位为苏氨酸,第48位为苏氨酸,第49位为半胱氨酸,第51位为半胱氨酸,且具有与所述nisB序列形成的肽段结合并被脱水修饰的活性,其中,SEQ ID NO:2和SEQ ID NO:3的序列如下:
MIKSSFKAQPFLVRNTILSPNDKRSFTEYTQVIETVSKNKVFLEQLLLANPKLYNVMQKYNAGLLKKKRVKKLFESIYKYYKRSYLRSTPFGLFSETSIGVFSKSSQYKLMGKTTKGIRLDTQWLIRLVHKMEVDFSKKLSFTRNNANYKFGDRVFQVYTINSSELEEVNIKYTNVYQIISEFCENDYQKYEDICETVTLCYGDEYRELSEQYLGSLIVNHYLISNLQKDLLSDFSWDTFLTKVEAIDEDKKYIIPLKKVQKFIQEYSEIEIGEGIEKLKEIYQEMSQILENDNYIQIDLISDSEINFDVKQKQQLEHLAEFLGNTTKSVRRTYLDDYKDKFIEKYGVDQEVQITELFDSTFGIGAPYNYNHPRNDFYESEPSTLYYSEEEREKYLSMYVEAVKNHNVINLDDLESHYQKMDLEKKSELQGLELFLNLAKEYEKDIFILGDIVGNNNLGGASGRFSALSPELTSYHRTIVDSVERENENKEITSCEIVFLPENIRHANVMHTSIMRRKVLPFFTSTSHNEVQLTNIYIGIDEKEKFYARDISTQEVLKFYITSMYNKTLFSNELRFLYEISLDDKFGNLPWELIYRDFDYIPRLVFDEIVISPAKWKIWGRDVNNKMTIRELIQSKEIPKEFYIVNGDNKVYLSQENPLDMEILESAIKKSSKRKDFIELQEYFEDENIINKGQKGRVADVVVPFIRTRALGNEGRAFIREKRVSVERREKLPFNEWLYLKLYISINRQNEFLLSYLPDIQKIVANLGGKLFFLRYTDPKPHIRLRIKCSDLFLAYGSILEILKRSQKNRIMSTFDISIYDQEVERYGGFDTLELSEAIFCADSKIIPNLLTLIKDTNNDWKVDDVSILVNYLYLKCFFQNDNKKILNFLNLVSPKKVKENVNEKIEHYLKLLKVDNLGDQIFYDKNFKELKHAIKNLFLKMIAQDFELQKVYSIIDSIIHVHNNRLIGIERDKEKLIYYTLQRLFVSEEYMK(SEQ ID NO:2)
MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCNCSIHVSK(SEQ ID NO:3)
根据本发明实施例的分离的多肽,通过表达nisin的产量依赖敏感的nisB和前体肽,使nisin的产量显著提高。
根据本发明的第二方面,本发明提供了一种分离的核酸。根据本发明的实施例,所述核酸编码前述的多肽。该核酸具有前述多肽的全部技术特征和技术效果,在此不再赘述。
根据本发明的实施例,所述核酸进一步包括:Pnis启动子序列,所述Pnis启动子序列具有SEQ ID NO:1所示的核苷酸序列,其中Pnis启动子序列的核苷酸序列具体如下:
TAATATCTTGATTTTCTAGTTCCTGAATAATATAGATATAGGTTTATTGAGTCTTAGACATAATTGAATGACCTAGTCTTATAACTATACTGACAATAGAAACATTAACAAATCTAAAACAGTCTTAATTCTATCTTGAGAAAGTATTGGCAATAATATTATTGTCGATAACGCGATCATAATAAACGGCTCTGATTAAATTCTGAAGTTTGTTAGATACAATGATTTCGTTCGAAGGAACTACAAAATAAATTATAAGGAGGCACTCAAA(SEQ ID NO:1)
根据本发明的第三方面,本发明提供了一种重组载体。根据本发明的实施例,该重组载体含有前述的核酸。该载体可以通过例如将上述核苷酸序列插入克隆载体或表达载体而得到,或者可以通过人工合成得到。例如,该载体可以是质粒。
根据本发明的第四方面,本发明提供了一种重组细胞。根据本发明的实施例,该重组细胞含有前述的重组载体。该重组细胞具有前述核酸的全部技术特征和技术效果,在此不再赘述。
根据本发明的实施例,该重组细胞可以通过将前述载体转化至宿主细胞而得到。
根据本发明的实施例,该重组细胞为乳酸菌。
根据本发明的第五方面,本发明提供了一种制备乳酸链球菌素的方法。根据本发明的实施例,工程细胞过表达nisB序列或共表达nisB序列和前体肽序列。由于在发酵制备nisin的过程中,nisB和前体肽的浓度直接影响nisin的产量,发明人通过使工程细胞过表达nisB和前体肽,提高nisin的产量。
根据本发明的实施例,所述工程细胞为前述的重组细胞。根据本发明的优选实施例,所述工程细胞为含有前述的重组载体的乳酸菌。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1显示了Nisin的生物合成途径示意图;
图2显示了多种前体肽氨基酸序列的示意图;
图3显示了根据本发明一个实施例的乳酸链球菌肽Z的离子流色谱示意图;
图4显示了根据本发明一个实施例的乳酸链球菌肽Z的产量示意图;
图5显示了根据本发明一个实施例的乳酸链球菌肽的产量示意图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
需要说明的是,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地,在本发明的描述中,除非另有说明,“多个”的含义是两个或两个以上。
分离的多肽、核酸、重组载体、重组细胞
根据本发明的第一方面,本发明提供了一种分离的多肽。根据本发明的实施例,
该分离的多肽包含nisB序列和前体肽序列,其中,所述nisB序列与SEQ ID NO:2所示的氨基酸序列具有至少90%一致性,并在SEQ ID NO:2所示的氨基酸序列的第83位为精氨酸、第87位为精氨酸、第89位为苏氨酸、第121位为天冬氨酸、第154位为精氨酸、第171位为异亮氨酸、第176位为缬氨酸、第198位为缬氨酸、第202位为酪氨酸、第209位为亮氨酸、第213位为酪氨酸、第217位为亮氨酸、第299位为天冬氨酸、第464位为精氨酸、第786位为精氨酸、第826位为精氨酸和第961位为组氨酸,且具有催化所述前体肽基因形成的肽段脱水的活性;所述前体肽序列与SEQ ID NO:3所示的氨基酸序列具有至少90%一致性,并在SEQ IDNO:3所示的氨基酸序列的第6位至第9位为苯丙氨酸-天冬酰胺-亮氨酸-天冬氨酸,第26位为丝氨酸,第28-34位为丝氨酸-亮氨酸-半胱氨酸-苏氨酸-脯氨酸-甘氨酸-半胱氨酸,第36位为苏氨酸,第37位甘氨酸,第39位为亮氨酸,第42位为半胱氨酸,第46位为苏氨酸,第48位为苏氨酸,第49位为半胱氨酸,第51位为半胱氨酸,且具有与所述nisB序列形成的肽段结合并被脱水修饰的活性。
其中,需要说明的是,本领域技术人员可以根据需要对SEQ ID NO:2所示的氨基酸序列和SEQ ID NO:3所示的氨基酸序列中的个别氨基酸进行修饰、替换、增加和删除,但是,修改后的序列与原氨基酸序列具有至少90%一致性,例如,可以是91%、92%、93%、94%、95%、96%、97%、98%或99%的一致性。
具体地,以前体肽序列进行说明,前体肽有多种,但多种前体肽具有相似的序列,其中,SEQ ID NO:3所示的氨基酸序列为nisZ多肽的氨基酸序列,通过对第50位氨基酸进行替换,将天冬酰胺(N)替换为组氨酸(H),即获得了另一种前导肽nisA,具体序列如下所示:
MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCHCSIHVSK(SEQ ID NO:4)
前导肽nisA与SEQ ID NO:3所示的氨基酸序列具有至少90%一致性,也可以与nisB结合,并在nisB的催化下进行修饰和脱水。
同时,对前导肽的其它位点进行替换或删除还可以获得其它的前导肽,具体详见图2,但需要说明的是,各种修饰后的前导肽,部分关键的活性位点是不变的,即在SEQ IDNO:2所示的氨基酸序列的第83位为精氨酸、第87位为精氨酸、第89位为苏氨酸、第121位为天冬氨酸、第154位为精氨酸、第171位为异亮氨酸、第176位为缬氨酸、第198位为缬氨酸、第202位为酪氨酸、第209位为亮氨酸、第213位为酪氨酸、第217位为亮氨酸、第299位为天冬氨酸、第464位为精氨酸、第786位为精氨酸、第826位为精氨酸和第961位为组氨酸,也就是说,在对氨基酸序列中的个别氨基酸进行修饰、替换、增加和删除时,上述氨基酸始终保持不变。
根据本发明实施例的分离的多肽,通过表达nisin的产量依赖敏感的nisB和前体肽,使nisin的产量显著提高;同时,以Pnis启动子序列作为nisB和前体肽的启动子,进一步提供nisB和前体肽表达,使nisin的产量也相应进一步地提高。根据本发明的实施例,过表达nisB和前体肽的乳酸菌相对于原始的乳酸菌产量提高达60%。
根据本发明的实施例,该多肽表达nisB蛋白和前体肽是以Pnis启动子为启动子进行的。
根据本发明的第二方面,本发明提供了一种分离的核酸。根据本发明的实施例,所述核酸编码前述的多肽。该核酸具有前述多肽的全部技术特征和技术效果,在此不再赘述。
在本发明中所使用的术语“核酸”可以是任何包含脱氧核糖核苷酸或者核糖核苷酸的聚合物,包括但不限于经过修饰的或者未经修饰的DNA、RNA,其长度不受任何特别限制。
根据本发明的实施例,所述核酸进一步包括:Pnis启动子基因,所述Pnis启动子基因具有SEQ ID NO:1所示的核苷酸序列,并且,Pnis启动子基因、nisB基因(编码前述nisB序列的基因)和前体肽基因(编码前述前体肽序列的基因)任意相连。也就是说,以Pnis启动子基因为启动子表达nisB基因和前体肽基因,使nisB基因和前体肽基因的表达量上调,nisin的产量更高。
其中,Pnis启动子基因、NisB基因和前体肽基因可以直接相连,即SEQ ID NO:1序列的3’末端和SEQ ID NO:2序列的5’端直接连接,依次类推;也可以间接相连,例如Pnis启动子基因和NisB基因之间可以有其它的核苷酸序列,只要不影响NisB基因和前体肽基因的表达即可。而且,Pnis启动子基因、NisB基因和前体肽基因也可以以任意顺序连接,即可以是Pnis启动子基因-NisB基因-前体肽基因,也可以是Pnis启动子基因-前体肽基因-NisB基因。
根据本发明的第三方面,本发明提供了一种重组载体。根据本发明的实施例,该重组载体含有前述的核酸。
本发明中所使用的术语“重组载体”是指这样的一种遗传载体,包含特定的核酸序列,并且能够将目的核酸序列转入宿主细胞中,以获得重组细胞。根据本发明的实施例,重组载体的形式不受特别限制。
根据本发明的实施例,重组载体可以为质粒。质粒作为遗传载体,具有操作简单,可以携带较大片段的性质,便于操作和处理。质粒的形式也不受特别限制,既可以是环形质粒,也可以是线性质粒,即可以是单链的,也可以是双链的。病毒很容易转染到受体细胞中。本领域技术人员可以根据需要进行选择。对于用于构建重组细胞的重组载体,优选所述核酸为DNA,因为DNA相对于RNA而言,其更稳定,并且易于操作。
根据本发明的第四方面,本发明提供了一种重组细胞。根据本发明的实施例,该重组细胞含有前述的重组载体。
根据本发明的实施例,该重组细胞可以通过将前述载体转化至宿主细胞而得到。
目前,乳酸链球菌素主要通过乳酸菌发酵进行制备,产量高,成本低。相应地,根据本发明的实施例,该重组细胞为乳酸菌。由此,利用乳酸菌作为重组细胞,细胞的活性高,并且乳酸链球菌素的产量高,成本低。
制备乳酸链球菌素的方法
根据本发明的第五方面,本发明提供了一种制备乳酸链球菌素的方法。根据本发明的实施例,工程细胞过表达nisB序列或共表达nisB序列和前体肽序列。由于在发酵制备nisin的过程中,NisB和前体肽的浓度直接影响nisin的产量,发明人通过使工程细胞过表达NisB和前体肽,提高nisin的产量。
根据本发明的实施例,所述工程细胞为前述的重组细胞。该重组细胞含有nisB基因和前体肽基因,并且启动子为Pnis启动子,nisB基因和前体肽基因的表达量上调,有利于提高乳酸链球菌素的产量。由于现有的乳酸链球菌素通常利用乳酸菌发酵获得的,优选地,可以采用乳酸菌作为重组细胞,也就是说,该重组细胞为含有前述的重组载体的乳酸菌。由此,乳酸链球菌素的产量更高。
下面参考具体实施例,对本发明进行说明,需要说明的是,这些实施例仅仅是说明性的,而不能理解为对本发明的限制。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,第三版,科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品,例如可以采购自Sigma公司。
实施例1
在本实施例中,构建调节乳酸链球菌素制备的几种关键组分的质粒,具体方法如下
(1)使用Blood&Cell Culture DNA Mini试剂盒(QIAGEN,Hileden,Germany)根据说明获得来自乳酸乳球菌(Lactococcus lactis)的基因组DNA。
(2)通过PCR从基因组中扩增nisB(SEQ ID No:6)、nisC(SEQ ID No:7)和nisP(SEQID No:8)基因,通过基因合成nisZ序列nisZ(SEQ ID No:5),然后通过适合的酶切位点将nisZ亚克隆到pJL1中,nisB,nisC,nisP克隆到pET28a上,构建nisZ,nisB,nisC和nisP表达质粒,其中,所用引物及相关质粒信息分别见表1和表2。利用表1和表2的引物从ATCC11454酵母菌基因组上通过PCR扩增相应的基因,将PCR扩增片段采用表2中所对应的酶切位点进行酶切,同时将表2所对应的质粒也用相同的酶切位点进行切割后,采用T4连接酶进行酶连后,挑选正确的克隆,并将阳性克隆进行测序,正确的进行依次命名。其中,SEQ ID NO5-8的核苷酸序列如下:
nisZ:ATGAGTACAAAAGATTTTAACTTGGATTTGGTATCTGTTTCGAAGAAAGATTCAGGTGCATCACCACGCATTACAAGTATTTCGCTATGTACACCCGGTTGTAAAACAGGAGCTCTGATGGGTTGTAACATGAAAACAGCAACTTGTAATTGTAGTATTCACGTAAGCAAATAA(SEQ ID No:5)
nisB:ATGATAAAAAGTTCATTTAAAGCTCAACCGTTTTTAGTAAGAAATACAATATTATCTCCAAACGATAAACGGAGTTTTACTGAATATACTCAAGTCATTGAGACTGTAAGTAAAAATAAAGTTTTTTTGGAACAGTTACTACTAGCTAATCCTAAACTCTATAATGTTATGCAGAAATATAATGCTGGTCTGTTAAAGAAGAAAAGGGTTAAAAAATTATTTGAATCTATTTACAAGTATTATAAGAGAAGTTATTTACGATCAACTCCATTTGGATTATTTAGTGAAACTTCAATTGGTGTTTTTTCGAAAAGTTCACAGTACAAGTTAATGGGAAAGACTACAAAGGGTATAAGATTGGATACTCAGTGGTTGATTCGCCTAGTTCATAAAATGGAAGTAGATTTCTCAAAAAAGTTATCATTTACTAGAAATAATGCGAATTATAAGTTTGGAGATCGAGTTTTTCAAGTTTATACCATAAATAGTAGTGAGCTTGAAGAAGTAAATATTAAATATACGAATGTTTATCAAATTATTTCTGAATTTTGTGAGAATGACTATCAAAAATATGAAGATATTTGTGAAACTGTAACCCTTTGCTATGGAGACGAATATAGAGAACTATCGGAACAATATCTTGGCAGTCTGATAGTTAATCATTATTTGATCTCTAATTTACAAAAAGATTTGTTGTCAGATTTTTCTTGGGACACTTTTTTGACTAAAGTTGAAGCAATAGATGAAGATAAAAAATATATAATTCCTCTGAAAAAAGTTCAAAAGTTTATTCAAGAATACTCAGAAATAGAAATTGGTGAAGGTATTGAGAAACTGAAAGAAATATATCAGGAAATGTCACAAATTCTTGAGAATGATAATTATATTCAAATTGATTTAATTAGTGATAGTGAAATAAATTTTGATGTTAAACAAAAGCAACAATTAGAACATTTAGCTGAGTTTTTAGGAAATACGACAAAATCTGTAAGAAGAACATATTTGGATGACTATAAGGATAAATTTATCGAAAAATATGGTGTAGATCAAGAAGTACAAATAACAGAATTATTTGATTCCACATTTGGCATAGGAGCTCCATATAATTATAATCATCCTCGAAATGACTTTTATGAGTCCGAACCGAGTACTCTATACTATTCAGAAGAGGAGAGAGAAAAGTACCTCAGCATGTATGTAGAAGCCGTTAAAAATCATAATGTAATTAATCTTGACGACTTAGAGTCTCATTACCAAAAAATGGACTTAGAAAAGAAAAGTGAACTTCAAGGGTTAGAATTATTTTTGAATTTGGCAAAGGAGTATGAAAAAGATATTTTTATTTTAGGGGATATCGTTGGAAATAATAATTTGGGAGGGGCATCAGGTAGATTTTCTGCACTCTCTCCGGAGTTAACAAGTTATCATAGAACGATAGTAGATTCTGTCGAAAGAGAAAATGAGAATAAAGAAATTACATCGTGTGAAATAGTATTTCTTCCAGAAAATATCAGACATGCTAACGTTATGCATACATCAATTATGAGGAGGAAAGTACTTCCATTTTTTACAAGTACAAGTCACAATGAAGTTCAGTTAACTAATATCTATATTGGAATAGACGAAAAAGAAAAATTTTATGCACGAGACATTTCAACTCAAGAGGTATTGAAATTCTACATTACAAGCATGTACAATAAAACGTTATTCAGTAATGAGCTAAGATTTCTTTACGAAATTTCATTAGATGACAAGTTTGGTAATTTACCTTGGGAACTTATTTACAGAGACTTTGATTATATTCCACGTTTAGTATTTGACGAAATAGTAATATCTCCTGCTAAATGGAAAATTTGGGGAAGGGATGTAAATAATAAGATGACAATAAGAGAACTTATTCAAAGCAAAGAAATTCCCAAAGAGTTTTATATTGTCAATGGAGATAATAAAGTTTATTTATCACAGGAAAACCCATTGGATATGGAAATTTTAGAGTCGGCGATAAAGAAGAGCTCAAAAAGAAAAGATTTTATAGAGCTACAAGAATATTTTGAAGATGAAAATATCATAAATAAAGGACAAAAGGGGAGAGTTGCCGATGTTGTAGTGCCTTTCATTAGAACGAGAGCATTAGGTAATGAAGGGAGAGCATTTATAAGAGAGAAAAGAGTTTCGGTTGAACGGCGTGAAAAACTGCCCTTTAACGAGTGGCTTTATCTCAAGTTGTACATTTCTATAAATCGTCAAAATGAATTTTTACTGTCGTATCTTCCAGATATTCAGAAAATAGTAGCAAACCTGGGTGGAAAACTATTCTTCCTAAGATATACTGATCCTAAACCACATATTAGATTGCGTATAAAATGTTCAGATTTATTTTTAGCTTACGGATCTATTCTTGAAATCTTAAAAAGGAGTCAGAAAAATAGGATAATGTCAACTTTTGATATTTCTATTTATGATCAAGAAGTAGAAAGATATGGTGGATTTGATACTTTAGAGTTATCCGAAGCAATATTTTGTGCCGATTCTAAAATTATTCCAAATTTGCTTACATTGATAAAAGATACTAATAATGATTGGAAAGTCGATGATGTATCAATCTTGGTGAATTATTTATATCTGAAATGCTTCTTTCAGAATGATAACAAAAAGATTCTTAATTTTTTGAATTTAGTTAGTCCTAAAAAGGTTAAAGAAAATGTCAATGAAAAGATTGAACATTATCTTAAACTTCTGAAAGTTGATAATCTAGGTGACCAAATTTTTTATGACAAGAATTTTAAAGAATTAAAGCATGCCATAAAAAATTTATTTTTAAAAATGATAGCTCAAGATTTTGAACTTCAGAAAGTTTATTCAATTATTGACAGTATCATTCATGTCCATAATAACCGACTAATTGGTATTGAACGAGATAAAGAGAAATTAATTTATTACACACTTCAAAGGTTGTTTGTTTCGGAAGAATACATGAAATGA(SEQ ID NO:6)
nisC:ATGAATAAAAAAAATATAAAAAGAAATGTTGAAAAAATTATTGCTCAATGGGATGAGAGAACTAGAAAAAATAAAGAAAACTTCGATTTCGGAGAGTTGACTCTCTCTACAGGATTGCCTGGTATAATTTTAATGTTAGCGGAGTTAAAAAATAAAGATAACTCAAAGATATATCAGAAAAAGATAGACAATTATATTGAATATATTGTTAGCAAACTTTCAACATATGGGCTTTTAACAGGATCGCTTTATTCGGGAGCAGCTGGCATTGCATTAAGTATCCTACATTTACGAGAAGATGACGAAAAATATAAGAATCTTCTTGACAGCCTAAATAGATATATCGAATATTTCGTCATAGAAAAAATTGAAGGGTTTAATTTGGAAAACATTACTCCTCCTGATTATGACGTGATTGAAGGTTTATCTGGGATACTTTCCTATCTATTATTAATCAACGACGAGCAATATGATGATTTGAAAATACTCATTATCAATTTTTTATCAAATCTGACTAAAGAAAACAAAGGACTAATATCGCTTTACATCAAATCGGAGAATCAGATGTCTCAATCAGAAAGTGAGATGTATCCACTAGGCTGTTTGAATATGGGATTAGCACATGGACTTGCTGGAGCGGGCTGTATCTTAGCTTATGCCCACATAAAAGGATATAGTAATGAAGCCTCGTTGTCAGCTTTGCAAAAAATTATTTTTATTTATGAAAAGTTTGAACTTGAAATTAAAAATCAGTTTCTATGGAAAGATGGACTTGTAGCAGATGAATTAAAAAAAGAGAAAGTAATTAGGGAAGCAAGTTTCATTAGAGATGCATGGTGCTATGGAGGTCCAGGTATTAGTCTGCTATACTTATACGGAGGATTAGCACTGGATAATGACTATTTTGTAGATAAAGCAGAAAAAATATTAGAGTCAGCTATGCAAAGAAAACTTGGTATTGATTCATATATGATTTGCCATGGCTATTCTGGTTTAATAGAAATTTGTTCTTTATTTAAGCGGCTATTAAATACAAAAAAGTTTGATTCATACATAGAAGAATTTAATGTTAATAGTGAGCAAATTCTTGAAGAATACGGAGATGAAAGTGGCACGGGTTTTCTTGAAGGAATAAGTGGCTGTATACTGGTATTATCGAAATTTGAATATTCAATCAATTTTACTTATTGGAGACAAGCACTGTTACTTTTTGATGATTTTTTGAAAGGAGGGAAGAGGAAATGA(SEQ ID 7)
nisP:GTGAAAAAAATACTAGGTTTCCTTTTTATCGTTTGTTCGTTGGGTTTATCAGCAACTGTGCATGGGGAGACAACAAATTCACAACAGTTACTCTCAAATAATATTAATACGGAATTAATTAATCATAATTCTAATGCAATTTTATCTTCAACAGAGGGATCAACGACTGATTCGATTAATCTAGGGGAGCAGTCAACTGCAGTAAAATCGACAACAAGGACTGAATTGGATGTAACTGGTGCTGCTAAAACTTTATTACAGACATCAGCTGTTCAAAAAGAAATGAAAGTTTCGTTGCAAGAAACTCAAGTTAGTTCTGAATTCAGTAAGAGAGATAGCGTTACAAATAAAGAAGCAGTTCCAGTATCTAAGGATGAGCTACTTGAGCAAAGTGAAGTAGTCGTTTCAACATCATCGATTCAAAAAAATAAAATCCTCGATAATAAGAAGAATAGAGCTAACTTTGTTACTTCCTCTCAGCTTATTAAGGAAAAACCATCAAATTCTAAAGATGCATCTGGTGTAATTGATAATTCTGCTTCTCCTCTATCTTATCGTAAAGCTAAGGAAGTGGTATCTCTTAGACAACCTTTAAAAAATCAAAAAGTAGAGGCACAACCTCTATTGATAAGTAATTCTTCTGAAAAGAAAGCAAGTGTTTATACAAATTCACATGATTTTTGGGATTATCAGTGGGATATGAAATATGTGACAAATAATGGAGAAAGCTATGCGCTCTACCAGCCCTCAAAGAAAATTTCTGTTGGAATTATTGATTCAGGAATCATGGAAGAACACCCTGATTTGTCAAATAGTTTAGGAAATTATTTTAAAAATCTTGTTCCTAAGGGAGGGTTTGATAATGAAGAACCTGATGAAACTGGAAATCCAAGTGATATTGTGGACAAAATGGGACACGGGACGGAAGTCGCAGGTCAGATTACAGCAAATGGTAATATTTTAGGAGTAGCACCAGGGATTACTGTAAATATATACAGAGTATTTGGTGAAAATCTTTCGAAATCGGAATGGGTAGCTAGAGCAATAAGAAGAGCTGCGGATGATGGGAACAAGGTCATCAATATAAGTGCTGGACAGTATCTTATGATTTCAGGATCGTATGATGATGGAACAAATGATTATCAAGAGTATCTTAATTATAAGTCAGCAATAAATTATGCAACAGCAAAAGGAAGTATTGTTGTCGCAGCTCTTGGTAATGATAGTTTAAACATACAAGATAACCAAACAATGATAAACTTTCTTAAGCGTTTCAGAAGTATAAAGGTTCCGGGAAAAGTTGTAGATGCACCGAGTGTATTTGAGGATGTAATAGCCGTAGGTGGAATAGATAGTTATGGTAATATTTCTGATTTTAGTAATATTGGAGCGGATGCAATTTATGCTCCTGCTGGCACAACGGCCAATTTTAAAAAATATGGGCAAGATAAATTTGTCAGTCAGGGTTATTATTTGAAAGATTGGCTTTTTACAACTACTAATACTGGCTGGTACCAATATGTTTATGGCAACTCATTTGCTGCTCCTAAAGTATCTGGGGCACTGGCATTAGTAGTTGATAAATATGGAATAAAGAATCCTAACCAACTAAAAAGGTTTCTTCTAATGAATTCTCCAGAAGTTAATGGGAATAGAGTATTGAATATTGTTGATTTATTGAATGGGAAAAATAAAGCTTTTAGCTTAGATACAGATAAAGGTCAGGATGATGCTATTAACCATAAATCGATGGAGAATCTTAAAGAGTCTAGGGATACAATGAAACAGGAACAAGATAAAGAAATTCAAAGAAATACAAATAACAATTTTTCTATCAAAAATGATTTTCATAACATTTCAAAAGAAGTAATTTCAGTTGATTATAATATTAATCAAAAAATGGCTAATAATCGAAATTCGAGAGGTGCTGTTTCTGTACGAAGTCAAGAAATTTTACCTGTTACTGGAGATGGAGAAGATTTTTTACCTGCTTTAGGTATAGTGTGTATCTCAATCCCTGGTATATTGAAAAGGAAGACTAAAAATTGA(SEQ ID 8)
表1.所用引物信息
表2.所构建质粒信息
实施例2
利用实施例1制备的nisZ、nisB、nisC和nisP表达质粒重建nisin体外合成途径,具体方法如下:
1、重建nisin体外反应体系(CFPS)的主要成分的制备
(1)NisB和NisC蛋白:
(a)用质粒pYL02和pYL03新鲜转化大肠杆菌BL21(DE3)细胞,然后单菌落转化体在50mL补充有50μg/mL卡那霉素的培养基中37℃下生长过夜。
(b)按照1%的接种量转接2L LB培养基直到OD600达到0.6-0.8,然后将培养物冷却至18℃,加入IPTG至终浓度0.5mM(NisB)或0.2mM(NisC)诱导,其中,对于NisC过表达,需加入另外的100μM ZnCl2以确保其活性。
(c)步骤(b)的培养物继续培养生长20小时后,通过在4℃以5000g离心20分钟收获细胞,并重新悬浮于缓冲液A(20mM Tris,pH 7.6,500mMNaCl,10%甘油)中。
(d)通过在10000-15000psig的可变压力下均质化裂解细胞悬浮液,并在4℃以25000g离心1小时,去上清,得到裂解后的细胞。
(e)将裂解后的细胞加入Ni-NTA柱(GE Healthcare,Marlborough,MA,USA)并用2倍柱体积(CV)的缓冲液A洗涤,然后将过滤的上清液施加到柱上,树脂用含有0,25,50,100,200和500mM咪唑的2CV各缓冲液洗涤,得到纯化后的蛋白洗脱液。
(f)将纯化后的蛋白洗脱液利用Amicon Ultra-15离心过滤装置(Millipore,Darmstadt,德国)浓缩含有所需蛋白质的洗脱液,并通过PD-10柱(GE)将缓冲液交换至储存缓冲液(100mM磷酸盐缓冲液,10%甘油,pH 7.6),得到纯化后的蛋白。
(g)用Pierce BCA蛋白质测定试剂盒(Thermo Fisher Scientific,Waltham,MA)测量步骤(f)得到的蛋白质的浓度,并在液氮中快速冷冻后,将蛋白质在-80℃储存。
(2)细胞提取物的制备:
(a)将大肠杆菌BL21接单克隆到10ml无抗LB中,37度摇床过夜培养,将10ml培养物全部转接到1L TYPG中,37度摇床培养至OD=0.6-0.8后,终浓度为1mMIPTG诱导,放回37度摇床继续培养至OD=6-94度,3500转,10分钟收集菌体。
(b)用S30Buffer(10mM Tris-醋酸,14mM醋酸镁,60mM谷氨酸钾,2mM DTT PH 8.2)重悬菌体,离心3次称量,最后一次离心完去掉上清的细胞湿重,液氮速冻,可先放到-80度保存。使用前冻融,用1ml S30Buffer/1g细胞湿重重悬菌体,使用高压冷冻破碎仪破碎细胞(800-900bar,10分钟)4度,30000rcf,30分钟收集上清,冰上分装,液氮速冻,-80保存。
(3)4×Premix(10mL):1.6mL的2mM氨基酸混合液(该氨基酸混合液含有精胺酸、缬胺酸、色氨酸、苯丙氨酸、异亮氨酸、亮氨酸、半胱氨酸、甲硫氨酸、丙氨酸、天冬酰胺、天门冬氨酸、甘氨酸、谷氨酰胺、谷氨酸、组氨酸、赖氨酸、脯氨酸、丝氨酸和苏氨酸),0.4mL的100×NTPmix(NTPmix含有ATP 120mM,GTP 85mM,UTP 85mM和CTP 85mM),4mL的10×盐溶液(盐溶液含有谷氨酸钾1.26M,谷氨酸钠0.04M,谷氨酸镁0.12M,醋酸铵0.1M和草酸钾0.04M),0.8mL大肠杆菌的tRNAmix混合液,132μL的100mMNAD+保存液,216μL的50mMCoA保存液,1.32mL1MPEP保存液PH7.4,68μL20mg/mL叶酸保存液,160μL250mM腐胺保存液,240μL250mM亚精胺保存液,milli-Q水1.064mL,将上述成份充分混匀后用离心管分装成500μL每管,保存在-80℃。
2、nisin体外反应体系(CFPS)的构建
按照下表3体系加入以下成分,在30℃下进行6小时。通过在85℃温育10分钟终止反应,并且通过以10000g离心5分钟沉淀蛋白质,取上清液进行下游分析。
表3.体外重建反应体系
3、结果分析
以10000g离心10分钟后获得CFPS反应混合物的上清液,并进行LC-MS分析。使用装备有LCQ-Fleet ESI-Iontrap MS(Thermo fisher)的HPLC系统进行LC-MS分析,色谱条件如下:
色谱柱:Hypersil GOLD TM aQ C18柱(2.1mm×100mm,3μm粒径,175孔径,Thermofisher);
流动相:A-0.01%(v/v)三氟乙酸水溶液(TFA);B-100%乙腈(B);
洗脱液流程:0-10分钟,10%B;10-35分钟,10-80%B;35-40分钟80%B;
流速:0.15毫升/分钟;
温度:30℃。
在m/z 200至1800的质量范围内取总离子流色谱图(TIC),并采用提取离子色谱图(XIC)定性鉴定在相应的m/z为1111.7的无细胞生产的乳链菌肽Z,乳酸链球菌肽Z的[M+3H]3+,结果如图3所示,证明在体外重建了nisin的合成体系。
实施例3
对nisin进行定量检测,方法如下:
(1)通过琼脂扩散法测定乳酸链球菌素活性,具体如下,通过将50mg市售乳链菌肽106I U/g(Sigma-Aldrich)加入到50mL无菌0.02mol/L HCl中来制备储备乳链菌肽溶液(2000IU/mL)。
(2)使用乳链菌肽原液制备1000、500、250、200、100、20和5IU/mL的标准乳链菌肽溶液,并用0.02mol/L HCl稀释并用于构建标准曲线。
(3)配制生物测定培养基,该生物测定培养基含有1.2%胰蛋白胨,0.75%酵母提取物,0.75%NaCl,0.3%NaH2PO4和2%琼脂,向该生物测定培养基中加入无菌0.75%葡萄糖和0.5%Tween 20后,得到琼脂培养基,将该琼脂培养基冷却至50℃,并接种1.5%指示菌株藤黄微球菌NCIB 8166的过夜培养物后倒平板,在充分凝固后在每个板上钻几个孔,然后将2μl体外重建反应的混合物的上清液和等体积的乳链菌肽Z标准溶液分别分配到孔中。
(4)将接种后的培养基在30℃温育18小时后,使用数字卡尺(TAJIMA Tool Co.,Ltd.,Shanghai,China)测量区域直径,水平和垂直地绘制乳链菌肽抑制区,绘制乳链菌肽标准溶液单位的标准曲线,利用该标准曲线可以估算每种体外重建反应(CFPS)混合物的乳链菌肽浓度。
实施例4
利用实施例2体外重建的nisin合成体系,研究nisin合成的关键限速步骤,该合成速率限速步骤通过系统性替换编码表达差的蛋白质和相应的纯化酶的质粒进行研究,本实施例中,通过系统滴定实验来研究各组分浓度的影响。
首先,通过改变其浓度来检测编码前体肽nisZ的质粒pJL1-nisZ的浓度,同时固定0.5μMNisB,0.5μM的NisC和0.5nM的pET28-nisP。滴定结果显示,乳酸链球菌素产量随着nisZ在0至0.4nM范围内的增加而显着增加并且在0.4至1.3nM之间略微增加,而在适当范围的nisZ浓度在0.4至1.3nM之间可以获得最高的乳链菌肽Z产量,结果详见图4A。
我们按照上述方法改变nisP的浓度,结果显示NisP编码质粒浓度对乳酸链球菌肽生产没有明显影响,结果如图4B所示,表明微量表达的nisP可以实现前导肽去除程序。基于nisZ和nisP滴定研究的结果,我们推断它们的优选浓度分别为1.3nM和0.1nM。
接下来,我们检测两种负责乳酸链球菌肽后修饰的酶。在这些测定中,将pJL1-nisZ和pET28-nisP的浓度分别设定为1.3nM和0.1nM。NisB的浓度从10至1500nM变化,而NisC的浓度从1至10000nM变化。当测试脱水过程时,NisC设定为500nM,而NisB浓度变化直接影响活性乳酸链球菌素Z的产量,NisB浓度在1000nM以下,活性乳酸链球菌素Z的产量随着nisB浓度的增加显着提高,然后在nisB浓度高于1000nM时发生饱和,结果详见图4C。在NisC的滴定中,nisB设定为500nM,活性Nisin Z的产生在1至100nM的相当低的浓度范围内对NisC量敏感。在1nM至100nM NisC之间观察到超过10倍的乳链菌肽生产变化,而较高的NisC浓度(从100至10000nM)不明显影响乳链菌肽Z的产生,结果如图4D。
实验结果表明,在nisin合成过程中编码前体肽nisZ的基因和编码脱水酶的基因nisB表达量越高,nisin的产量也越高。
实施例5
构建nisin高产菌株,也就是nisA和nisB高表达菌株,方法如下:
1、菌株构建
ATCC 11454是nisin发酵菌株。通过Gibson组装方法以pMG36e质粒为骨架构建在Pnis启动子的控制下过表达nisA基因质粒pRL414。同样也使用Gibson组装方法按照nisABTCIP操纵子其原始顺序将nisA和nisB一起扩增在Pnis启动子控制下过表达nisA和nisB的基因片段,并将其构建在以pMG36e质粒为骨架的质粒上形成重组质粒pRL424。通过电穿孔将得到的质粒pRL414和pRL424分别转化到ATCC 11454菌株中,分别产生工程菌株RL410和RL411。该部分中使用的引物示于表4中。
表4.构建pRL415和pRL423所用引物
2、菌株培养
根据文献报道方法(Zhang,J.,Caiyin,Q.,Feng,W.,Zhao,X.,Qiao,B.,Zhao,G.,Qiao,J.,2016.Enhance nisin yield via improving acid-tolerant capability ofLactococcus lactis F44.Scientific reports.6,27973.)对ATCC11454,RL410和RL411进行发酵,发酵16h后取出10mL发酵样品,离心后,将上清液进行定量生物测定以确定乳链菌肽产量,结果如图5所示,亲代菌株ATCC11454在发酵16小时后产生554.9±31.63IU/mLnisin,菌株RL410产生647.9±44.3IU/mLnisin,比原始菌株高16.8%,而RL411菌株的nisin产量增加至882.8±33.62IU/mL,比原始菌株增加近60%,与菌株RL410相比,产量提高了36.2%。该结果进一步验证了实施例4的体外滴定结果,即NisB在乳酸链球菌素生物合成机制中发挥着至关重要的作用。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例,本领域的普通技术人员可以理解:在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由权利要求及其等同物限定。
SEQUENCE LISTING
<110> 武汉臻智生物科技有限公司
<120> 分离的多肽及其应用
<130> PIDC3182875
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aagttgtaca tttctataaa tcgtcaaaat gaatttttac tgtcgtatct tccagatatt 2280
cagaaaatag tagcaaacct gggtggaaaa ctattcttcc taagatatac tgatcctaaa 2340
ccacatatta gattgcgtat aaaatgttca gatttatttt tagcttacgg atctattctt 2400
gaaatcttaa aaaggagtca gaaaaatagg ataatgtcaa cttttgatat ttctatttat 2460
gatcaagaag tagaaagata tggtggattt gatactttag agttatccga agcaatattt 2520
tgtgccgatt ctaaaattat tccaaatttg cttacattga taaaagatac taataatgat 2580
tggaaagtcg atgatgtatc aatcttggtg aattatttat atctgaaatg cttctttcag 2640
aatgataaca aaaagattct taattttttg aatttagtta gtcctaaaaa ggttaaagaa 2700
aatgtcaatg aaaagattga acattatctt aaacttctga aagttgataa tctaggtgac 2760
caaatttttt atgacaagaa ttttaaagaa ttaaagcatg ccataaaaaa tttattttta 2820
aaaatgatag ctcaagattt tgaacttcag aaagtttatt caattattga cagtatcatt 2880
catgtccata ataaccgact aattggtatt gaacgagata aagagaaatt aatttattac 2940
acacttcaaa ggttgtttgt ttcggaagaa tacatgaaat ga 2982
<210> 7
<211> 1245
<212> DNA
<213> Artificial
<220>
<223> nisC
<400> 7
atgaataaaa aaaatataaa aagaaatgtt gaaaaaatta ttgctcaatg ggatgagaga 60
actagaaaaa ataaagaaaa cttcgatttc ggagagttga ctctctctac aggattgcct 120
ggtataattt taatgttagc ggagttaaaa aataaagata actcaaagat atatcagaaa 180
aagatagaca attatattga atatattgtt agcaaacttt caacatatgg gcttttaaca 240
ggatcgcttt attcgggagc agctggcatt gcattaagta tcctacattt acgagaagat 300
gacgaaaaat ataagaatct tcttgacagc ctaaatagat atatcgaata tttcgtcata 360
gaaaaaattg aagggtttaa tttggaaaac attactcctc ctgattatga cgtgattgaa 420
ggtttatctg ggatactttc ctatctatta ttaatcaacg acgagcaata tgatgatttg 480
aaaatactca ttatcaattt tttatcaaat ctgactaaag aaaacaaagg actaatatcg 540
ctttacatca aatcggagaa tcagatgtct caatcagaaa gtgagatgta tccactaggc 600
tgtttgaata tgggattagc acatggactt gctggagcgg gctgtatctt agcttatgcc 660
cacataaaag gatatagtaa tgaagcctcg ttgtcagctt tgcaaaaaat tatttttatt 720
tatgaaaagt ttgaacttga aattaaaaat cagtttctat ggaaagatgg acttgtagca 780
gatgaattaa aaaaagagaa agtaattagg gaagcaagtt tcattagaga tgcatggtgc 840
tatggaggtc caggtattag tctgctatac ttatacggag gattagcact ggataatgac 900
tattttgtag ataaagcaga aaaaatatta gagtcagcta tgcaaagaaa acttggtatt 960
gattcatata tgatttgcca tggctattct ggtttaatag aaatttgttc tttatttaag 1020
cggctattaa atacaaaaaa gtttgattca tacatagaag aatttaatgt taatagtgag 1080
caaattcttg aagaatacgg agatgaaagt ggcacgggtt ttcttgaagg aataagtggc 1140
tgtatactgg tattatcgaa atttgaatat tcaatcaatt ttacttattg gagacaagca 1200
ctgttacttt ttgatgattt tttgaaagga gggaagagga aatga 1245
<210> 8
<211> 2049
<212> DNA
<213> Artificial
<220>
<223> nisP
<400> 8
gtgaaaaaaa tactaggttt cctttttatc gtttgttcgt tgggtttatc agcaactgtg 60
catggggaga caacaaattc acaacagtta ctctcaaata atattaatac ggaattaatt 120
aatcataatt ctaatgcaat tttatcttca acagagggat caacgactga ttcgattaat 180
ctaggggagc agtcaactgc agtaaaatcg acaacaagga ctgaattgga tgtaactggt 240
gctgctaaaa ctttattaca gacatcagct gttcaaaaag aaatgaaagt ttcgttgcaa 300
gaaactcaag ttagttctga attcagtaag agagatagcg ttacaaataa agaagcagtt 360
ccagtatcta aggatgagct acttgagcaa agtgaagtag tcgtttcaac atcatcgatt 420
caaaaaaata aaatcctcga taataagaag aatagagcta actttgttac ttcctctcag 480
cttattaagg aaaaaccatc aaattctaaa gatgcatctg gtgtaattga taattctgct 540
tctcctctat cttatcgtaa agctaaggaa gtggtatctc ttagacaacc tttaaaaaat 600
caaaaagtag aggcacaacc tctattgata agtaattctt ctgaaaagaa agcaagtgtt 660
tatacaaatt cacatgattt ttgggattat cagtgggata tgaaatatgt gacaaataat 720
ggagaaagct atgcgctcta ccagccctca aagaaaattt ctgttggaat tattgattca 780
ggaatcatgg aagaacaccc tgatttgtca aatagtttag gaaattattt taaaaatctt 840
gttcctaagg gagggtttga taatgaagaa cctgatgaaa ctggaaatcc aagtgatatt 900
gtggacaaaa tgggacacgg gacggaagtc gcaggtcaga ttacagcaaa tggtaatatt 960
ttaggagtag caccagggat tactgtaaat atatacagag tatttggtga aaatctttcg 1020
aaatcggaat gggtagctag agcaataaga agagctgcgg atgatgggaa caaggtcatc 1080
aatataagtg ctggacagta tcttatgatt tcaggatcgt atgatgatgg aacaaatgat 1140
tatcaagagt atcttaatta taagtcagca ataaattatg caacagcaaa aggaagtatt 1200
gttgtcgcag ctcttggtaa tgatagttta aacatacaag ataaccaaac aatgataaac 1260
tttcttaagc gtttcagaag tataaaggtt ccgggaaaag ttgtagatgc accgagtgta 1320
tttgaggatg taatagccgt aggtggaata gatagttatg gtaatatttc tgattttagt 1380
aatattggag cggatgcaat ttatgctcct gctggcacaa cggccaattt taaaaaatat 1440
gggcaagata aatttgtcag tcagggttat tatttgaaag attggctttt tacaactact 1500
aatactggct ggtaccaata tgtttatggc aactcatttg ctgctcctaa agtatctggg 1560
gcactggcat tagtagttga taaatatgga ataaagaatc ctaaccaact aaaaaggttt 1620
cttctaatga attctccaga agttaatggg aatagagtat tgaatattgt tgatttattg 1680
aatgggaaaa ataaagcttt tagcttagat acagataaag gtcaggatga tgctattaac 1740
cataaatcga tggagaatct taaagagtct agggatacaa tgaaacagga acaagataaa 1800
gaaattcaaa gaaatacaaa taacaatttt tctatcaaaa atgattttca taacatttca 1860
aaagaagtaa tttcagttga ttataatatt aatcaaaaaa tggctaataa tcgaaattcg 1920
agaggtgctg tttctgtacg aagtcaagaa attttacctg ttactggaga tggagaagat 1980
tttttacctg ctttaggtat agtgtgtatc tcaatccctg gtatattgaa aaggaagact 2040
aaaaattga 2049
<210> 9
<211> 32
<212> DNA
<213> Artificial
<220>
<223> nisB 引物
<400> 9
gggtttcata tgataaaaag ttcatttaaa gc 32
<210> 10
<211> 34
<212> DNA
<213> Artificial
<220>
<223> nisB 引物
<400> 10
gcgcggatcc tcatttcatg tattcttccg aaac 34
<210> 11
<211> 33
<212> DNA
<213> Artificial
<220>
<223> nisC 引物
<400> 11
gcgcggatcc atgaataaaa aaaatataaa aag 33
<210> 12
<211> 32
<212> DNA
<213> Artificial
<220>
<223> nisC 引物
<400> 12
atataagctt tcatttcctc ttccctcctt tc 32
<210> 13
<211> 31
<212> DNA
<213> Artificial
<220>
<223> nisP引物
<400> 13
gggtttcata tgaaaaaaat actaggtttc c 31
<210> 14
<211> 32
<212> DNA
<213> Artificial
<220>
<223> nisP 引物
<400> 14
atatggatcc tcaattttta gtctttcttt tc 32
<210> 15
<211> 50
<212> DNA
<213> Artificial
<220>
<223> 414-F
<400> 15
gtagcttttt aaatatgggt cgatctaata tcttgatttt ctagttcctg 50
<210> 16
<211> 51
<212> DNA
<213> Artificial
<220>
<223> 414-R
<400> 16
tccaagttaa aatcttttgt actcattttg agtgcctcct tataatttat t 51
<210> 17
<211> 51
<212> DNA
<213> Artificial
<220>
<223> 414-VF
<400> 17
aataaattat aaggaggcac tcaaaatgag tacaaaagat tttaacttgg a 51
<210> 18
<211> 50
<212> DNA
<213> Artificial
<220>
<223> 414-VR
<400> 18
caggaactag aaaatcaaga tattagatcg acccatattt aaaaagctac 50
<210> 19
<211> 50
<212> DNA
<213> Artificial
<220>
<223> 424-F
<400> 19
gtagtattca cgtaagcaaa taaccaaatc aaaggatagt attttgttag 50
<210> 20
<211> 50
<212> DNA
<213> Artificial
<220>
<223> 424-R
<400> 20
cttgcatgcc tgcaggtcga ctctagtcat ttcatgtatt cttccgaaac 50
<210> 21
<211> 50
<212> DNA
<213> Artificial
<220>
<223> 424-VF
<400> 21
gtttcggaag aatacatgaa atgactagag tcgacctgca ggcatgcaag 50
<210> 22
<211> 50
<212> DNA
<213> Artificial
<220>
<223> 424-VR
<400> 22
ctaacaaaat actatccttt gatttggtta tttgcttacg tgaatactac 50
Claims (1)
1.一种制备乳酸链球菌素的方法,其特征在于,工程细胞过表达nisB序列和前体肽序列,所述工程细胞为含有重组载体的乳酸菌,所述重组载体含有核酸,所述核酸编码nisB序列或nisB序列和前体肽序列,
其中,所述nisB序列为SEQ ID NO:2所示的氨基酸序列,并在SEQ ID NO:2所示的氨基酸序列的第83位为精氨酸、第87位为精氨酸、第89位为苏氨酸、第121位为天冬氨酸、第154位为精氨酸、第171位为异亮氨酸、第176位为缬氨酸、第198位为缬氨酸、第202位为酪氨酸、第209位为亮氨酸、第213位为酪氨酸、第217位为亮氨酸、第299位为天冬氨酸、第464位为精氨酸、第786位为精氨酸、第826位为精氨酸和第961位为组氨酸,且具有催化所述前体肽序列形成的肽段脱水的活性;
所述前体肽序列为SEQ ID NO:3所示的氨基酸序列,并在SEQ ID NO:3所示的氨基酸序列的第6位至第9位为苯丙氨酸-天冬酰胺-亮氨酸-天冬氨酸,第26位为丝氨酸,第28-34位为丝氨酸-亮氨酸-半胱氨酸-苏氨酸-脯氨酸-甘氨酸-半胱氨酸,第36位为苏氨酸,第37位甘氨酸,第39位为亮氨酸,第42位为半胱氨酸,第46位为苏氨酸,第48位为苏氨酸,第49位为半胱氨酸,第51位为半胱氨酸,且具有与所述nisB序列形成的肽段结合并被脱水修饰的活性。
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