CN116848252A - 用于组成型表达的新型启动子变体及其用途 - Google Patents
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- CN116848252A CN116848252A CN202180087513.9A CN202180087513A CN116848252A CN 116848252 A CN116848252 A CN 116848252A CN 202180087513 A CN202180087513 A CN 202180087513A CN 116848252 A CN116848252 A CN 116848252A
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Abstract
本发明提供一种新型启动子变体,其中缺失了大肠杆菌(Escherichia coli)gapA(3‑磷酸甘油醛脱氢酶(glyceraldehyde‑3‑phosphate dehydrogenase))基因启动子的部分核苷酸。本发明的新型启动子变体可以在大肠杆菌中组成型地高表达靶蛋白,特别是酶。因此,通过使用用包含本发明的新型启动子变体的表达载体转化的重组菌株,可以经济地大量生产靶蛋白,特别是酶。作为一例,通过使用用包含本发明的新型启动子变体的表达载体转化的重组菌株,可以经济地大量生产阿卢糖差向异构酶或者从果糖经济地大量生产阿卢糖。
Description
技术领域
本发明涉及一种新型启动子变体等,更详细地,涉及一种能够组成型地高表达靶蛋白的新型启动子变体及其各种用途。
背景技术
随着分子生物学的发展,已经查明了几种调节基因表达的机制。基因表达是指细胞内发生的根据通过转录(transcription)及翻译(translation)输入到基因的代码合成蛋白质的一系列过程。尤其,转录过程是基因表达的初始阶段,RNA聚合酶在各种辅助因子的帮助下与位于基因上位的启动子(promoter)序列结合而启动,转录因子(TF,transcription factor)是此类辅助因子之一,已知其可以直接与启动子序列结合。尤其,由于原核生物中基因表达的调节主要发生在转录阶段,因此研究人员不断发现新的转录因子及启动子。
为了在工业上生产酶等外源蛋白,主要将通过用含有外源蛋白基因的pET型表达载体转化大肠杆菌等原核生物而制备的转化体用作表达***。用pET型表达载体转化的原核生物表达***通常需要IPTG(异丙基-β-D-硫代吡喃半乳糖苷(isopropyl-β-D-thiogalactopyranoside))等昂贵的表达衍生物(inducer),而且存在需要细致地调节衍生物浓度或设备、表达诱导时间等的缺点。
另一方面,为了大量生产具有将果糖转化为阿卢糖(或阿洛酮糖)的活性的阿洛酮糖差向异构酶(或阿卢糖差向异构酶),提出了使用作为GRAS(公认安全(GenerallyRecognized As Safe))菌株的棒状杆菌属菌株作为宿主细胞的表达***。关于基于棒状杆菌属菌株的阿洛酮糖差向异构酶(或阿卢糖差向异构酶)表达***,韩国授权专利第10-1656063号公开了编码阿洛酮糖差向异构酶的核苷酸序列以及可操作地连接到其上游(upstream),并含有调节阿洛酮糖差向异构酶在棒状杆菌属菌株中表达的调节序列的基因表达盒,上述调节序列由转录启动子、第一核糖体结合区(ribosome binding region,RBS)序列、第一空间序列、接头序列以及第二RBS序列等组成。并且,韩国授权专利第10-1695830号公开了编码阿洛酮糖差向异构酶的核酸序列、可操作地连接到其上游(upstream)并含有调节上述阿洛酮糖差向异构酶在棒状杆菌属菌株中表达的调节序列的基因表达盒,上述调节序列包括大肠杆菌(E.coli)衍生的转录启动子(transcription promoter)。然而,基于棒状杆菌的阿洛酮糖差向异构酶(或阿卢糖差向异构酶)表达***由于可使用的启动子表达水平低且选择范围小,因而不适合大量的酶表达***。
因此,为了阿洛酮糖差向异构酶(或阿卢糖差向异构酶)的大量生产或由利用阿洛酮糖差向异构酶(或阿卢糖差向异构酶)的果糖阿卢糖的大量生产,需要开发组成型表达启动子以及包含其的组成型表达***,该组成型表达启动子能够在大肠杆菌宿主细胞的一般培养条件下稳定且高水平地表达外源蛋白而不抑制生长。
发明内容
技术问题
本发明是在传统技术背景下导出的,本发明的目的在于提供能够组成型地高表达靶蛋白的新型启动子变体。并且,本发明的目的在于提供上述新型启动子变体的各种用途。
解决问题的手段
本发明的发明人通过将作为用于表达大肠杆菌(Escherichia coli)gapA(3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase))基因的启动子的gapA基因启动子与编码阿卢糖差向异构酶的多核苷酸可操作地连接来制备重组表达载体,并将其引入大肠杆菌后进行转化。在此情况下,由于gapA启动子或阿卢糖差向异构酶基因序列发生随机变异,因此本发明的发明人获得了用与引入的重组表达载体不同的各种重组表达载体转化的重组大肠杆菌。在通过上述随机变异获得的重组表达载体中,对用阿卢糖差向异构酶基因序列未发生变异而只有gapA基因启动子发生变异的重组表达载体转化的重组大肠杆菌的阿卢糖差向异构酶表达活性进行了比较,结果确认缺失gapA基因启动子的碱基序列中第130个核苷酸的腺嘌呤(A)的启动子变体能够组成型地高表达阿卢糖差向异构酶,从而完成了本发明。
为了实现上述目的,本发明的一例提供一种由序列号9的碱基序列组成的启动子变体。并且,本发明的一例提供一种包含由序列号9的碱基序列组成的启动子变体的重组载体。并且,本发明的一例提供一种表达载体,其包含编码靶蛋白的多核苷酸以及可操作地与其连接并由序列号9的碱基序列组成的启动子变体。并且,本发明的一例提供一种用上述表达载体转化的重组菌株。并且,本发明的一例提供一种使用上述重组菌株生产靶蛋白的方法。并且,本发明的优选一例提供一种由底物制备酶转化反应产物的方法,其包括在含底物溶液中加入上述重组菌株并进行酶转化反应的步骤。
发明的效果
本发明的新型启动子变体可以在大肠杆菌中组成型地高表达靶蛋白,特别是酶。因此,通过使用用包含本发明的新型启动子变体的表达载体转化的重组菌株,可以经济地大量生产靶蛋白,特别是酶。作为一例,通过使用用包含本发明的新型启动子变体的表达载体转化的重组菌株,可以经济地大量生产阿卢糖差向异构酶或者可以从果糖经济地大量生产阿卢糖。
附图说明
图1为本发明实施例中制备的重组表达载体pPgapAm1-FpDPE[W29K/A77S/G216S/M234I]的酶切图谱。
图2为本发明实施例中制备的重组表达载体pPgapAm2-FpDPE[W29K/A77S/G216S/M234I]的酶切图谱。
图3为本发明实施例中制备的重组表达载体pPblma-FpDPE[W29K/A77S/G216S/M234I]的酶切图谱。
图4为示出当使用本发明实施例中制备的重组大肠杆菌进行果糖到阿卢糖的转化反应时根据反应时间的转化率。
具体实施方式
以下,将具体说明本发明。
本发明中使用的术语“启动子”是指与待转录的靶核苷酸序列可操作地连接并调节上述靶核苷酸序列的转录的最小核酸序列。并且,上述启动子可包含足以允许表达细胞类型特异性或外部信号或制剂诱导的可调节启动子依赖基因的启动子元件,这些元件可以位于基因的5'或3'部分。上述启动子包括保守型启动子以及诱导型启动子这两者。启动子序列可以源自原核生物、真核生物或病毒。原核生物中的启动子通常被定义为与RNA聚合酶结合的转录起始点(Transcription Start Site)紧邻的结合位点。
本发明中使用的术语“启动子变体”被定义为基本启动子的核酸序列中的一些核苷酸被缺失、添加或取代而具有与基本启动子不同或改进的靶蛋白表达活的启动子。
本发明中使用的术语“同源性”表示与野生型(wild type)或具有相同活性的变体的核酸序列的同一性,同源性比较可以用肉眼或使用易于购买的比较程序计算2个以上序列之间的同源性百分比(%)。
本发明中使用的术语“靶蛋白”是指通常不能存在于表达上述蛋白质作为外源蛋白的转化菌株(或宿主细胞)中的蛋白质。
本发明中使用的术语“多核苷酸”是指未修饰(non-modified)或修饰的(modified)所有多聚核糖核苷酸(RNA)或多聚脱氧核糖核苷酸(DNA)。上述多核苷酸包括单链或双链DNA、作为单链区及双链区的混合物的DNA、单链及双链RNA、作为单链区及双链区的混合物的RNA或它们的杂化分子,但不限于此。
本发明中使用的术语“可操作地连接(operably linked)”被定义为启动子序列和编码靶蛋白的核苷酸序列功能性地连接而使得启动子可以调节靶蛋白的表达的状态。例如,当启动子可以控制编码序列的表达时(即,当编码序列在启动子的转录调节下时),启动子与编码序列连接操作,或者若核糖体结合位点位于可促进翻译的位置,则核糖体结合位点与编码序列连接操作。编码序列可以在有义方向或反义方向上与调节序列连接操作。
本发明中使用的术语“重组载体”被定义为通过使用限制酶切除启动子变体或靶基因并将其***载体中来制备的重组DNA。
本发明中使用的术语“克隆载体”被定义为可以将DNA片段转运到宿主细胞中并进行再生产的物质。上述克隆载体可包括聚腺苷酸化信号(polyadenylation signal)、转录终止序列(transcription termination sequence)及多克隆位点(multiple cloningsite)。上述多克隆位点(multiple cloning site)包括至少一个内切核酸酶(endonuclease)限制酶限制位点(restriction site)。并且,克隆载体还可以包括启动子。并且,克隆载体中编码靶蛋白的多核苷酸可位于聚腺苷酸化信号(polyadenylationsignal)及转录终止序列(transcription termination sequence)的上游(upstream)。
本发明中使用的术语“表达载体”被定义为在适当宿主中转录和翻译被克隆的DNA所需的DNA序列,具体指包含可操作地连接到***体的必备基因调节要素的基因构建体,使得***体在存在于细胞中时被表达。可以使用标准重组DNA技术制备及纯化表达载体。上述表达载体的种类不受特别限制,只要具有在各种宿主细胞如原核细胞及真核细胞真核表达所需基因并生产所需蛋白质的功能即可。表达载体至少包括启动子、起始密码子、编码所需蛋白质的基因及终止密码子终止子。并且,表达载体还可以适当地包括编码信号肽的DNA、额外的表达调节序列、所需基因的5'侧及3'侧的非翻译区、选择标记区或可复制单元等。上述选择标记区可以是用于筛选靶载体的抗生物质的选择标记基因。
本发明中使用的术语“重组菌株”是指通过将编码一种以上靶蛋白的多核苷酸或具有该多核苷酸的表达载体引入宿主细胞而转化的细胞。将上述表达载体引入主细胞制备转化体的方法包括瞬时转染法(transient transfection)、显微注射法、转导法(transduction)、细胞融合法、磷酸钙沉淀法、脂质体介导的转染法(liposemmediatedtransfection)、DEAE葡聚糖介导的转染法(DEAE Dextran-mediated transfection)、聚凝胺介导的转染法(polybrene-mediated transfection),电穿孔法(electroporation)、电注射法(electroinjection)、PEG等化学处理方法、使用基因枪(gene gun)等的方法、热休克(heat shock)法等,但不限于此。
本发明中使用的术语“底物”是指通过酶的作用被转化或将被转化为其他化合物的任意物质或化合物。上述术语包括单一化合物以及化合物的组合及它们的衍生物,例如溶剂、混合物及含有至少一种底物的其他材料。
本发明的一实施方式涉及一种可以组成型地高表达靶蛋白的新型启动子变体。本发明一例的新型启动子变体由序列号9的碱基序列组成。本发明一例的新型启动子变体是源自由序列号1的碱基序列组成的大肠杆菌(Escherichia coli)的gapA(甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase))基因启动子变异的,具体地,缺失了作为序列号1的碱基序列中第130个核苷酸的腺嘌呤。上述作为序列号1的碱基序列中第130个核苷酸的腺嘌呤对应于核糖体结合位点(Ribosome-Binding Site,RBS)。包含本发明一例的新型启动子变体的表达***可以在大肠杆菌中组成型地高表达靶蛋白。因此,本发明一例的新型启动子变体可以用作用于组成型表达的启动子。本发明一例的新型启动子变体由序列号9的碱基序列组成,但本发明一例的新型启动子变体的等同范围并不一定限于此。例如,本发明一例的新型启动子变体的等同范围包括在维持组成型地高表达靶蛋白功能的范围内序列号9的碱基序列中一些核苷酸被取代、***和/或缺失的启动子。并且,本发明一例的新型启动子变体的等同范围包括与序列号9的碱基序列具有实质同一性的序列。上述实质同一性是指通过将序列号9的碱基序列与任意其他序列进行排列以尽可能对应,并分析序列,使得上述任意其他序列与序列号9的碱基序列具有70%以上、90%以上、或98%以上序列同源性。本领域普通技术人员可以容易理解可使用本领域已知的基因重组技术等取代、添加或缺失上述新型启动子变体的碱基序列中一个或一个以上碱基,从而制备在具有实质同源性的范围内具有相同或类似活性的多核苷酸。这种同源性的比较可通过使用市面上销售的计算机程序计算2个以上序列之间的同源性百分比(%)来进行。因此,本发明一例的新型启动子变体的等同范围可在维持组成型地高表达靶蛋白功能的范围内包括与序列号9的碱基序列具有70%以上、80%以上、90%以上、95%以上或99%以上同源性的碱基序列。
本发明的另一实施方式涉及一种本发明一例的新型启动子变体的各种用途。本发明一例的新型启动子变体的用途包括重组载体、表达载体、重组菌株、靶蛋白的生产方法以及使用重组菌株发挥靶蛋白功能的方法等,但并不一定限于此。
本发明一例的重组载体包括由序列号9的碱基序列组成的启动子变体。
上述重组载体可以是克隆载体。上述克隆载体可包括复制起点、用于克隆靶蛋白基因的多克隆位点(Multi clonig site,MCS)、转录终止序列(transcriptiontermination sequence)及选择标记(selection marker)。上述选择标记用于筛选用载体转化的细胞,可以使用赋予可选择的表型例如耐药性、营养缺陷性、细胞毒性剂的抗性或表面蛋白表达等的标记。由于只有表达选择标记的细胞才能在用选择剂(selective agent)处理的环境中存活,因此可以筛选出转化的细胞。例如,上述选择标记可以是耐药性基因,例如卡那霉素(Kanamycin)抗生素抗性基因或氨苄青霉素(Ampicillin)抗生素抗性基因。
并且,上述重组载体可以是表达载体。上述表达载体包括编码靶蛋白的多核苷酸以及可操作地与其连接并由序列号9的碱基序列组成的启动子变体。上述启动子变体优选位于编码靶蛋白的多核苷酸的上游(upstream)。通过本发明一例的表达载体表达的靶蛋白的种类没有特别限制,例如,可以选自参与碳水化合物的生物合成或代谢的蛋白质、参与脂质及脂肪酸的生物合成或代谢的蛋白质、参与蛋白质及肽的生物合成或代谢的蛋白质等。并且,考虑到启动子变体的表达调节活性,上述靶蛋白优选为酶。上述酶的种类没有特别限制,可以选自D-阿卢糖3-差向异构酶(D-allulose3-epimerase)、D-塔格糖3-差向异构酶(D-tagatose 3-epimerase)、(1→4)-α-D-葡聚糖1-α-D-葡糖基变位酶[(1→4)-alpha-D-glucan 1-alpha-D-glucosylmutase]、4-α-D-{(1→4)-α-D-葡聚糖}海藻糖海藻糖水解酶[4-alpha-D-{(1→4)-alpha-D-glucano}trehalose trehalohydrolase]、L-鼠李糖异构酶(L-rhamnose isomerase)、果糖-6-磷酸-3-差向异构酶(fructose-6-phosphate-3-epimerase)等,优选地,选自D-阿卢糖3-差向异构酶(D-allulose 3-epimerase),(1→4)-α-D-葡聚糖1-α-D-葡糖基变位酶[(1→4)-alpha-D-glucan 1-alpha-D-glucosylmutase],4-α-D-{(1→4)-α-D-葡聚糖}海藻糖海藻糖水解酶[4-alpha-D-{(1→4)-alpha-D-glucano}trehalose trehalohydrolase]。尽管在本发明的实施例中没有具体描述,但是与源自由序列号1的碱基序列组成的大肠杆菌(Escherichia coli)的gapA(甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase))基因启动子相比,本发明的新型启动子变体以约1.4倍~1.5倍的水平表达了更多的(1→4)-α-D-葡聚糖1-α-D-葡糖基变位酶[(1→4)-alpha-D-glucan 1-alpha-D-glucosylmutase],4-α-D-{(1→4)-α-D-葡聚糖}海藻糖海藻糖水解酶[4-alpha-D-{(1→4)-alpha-D-glucano}trehalosetrehalohydrolase]等。上述阿卢糖差向异构酶的种类没有特别限制,只要具有将果糖转化为阿卢糖的活性的酶即可,例如可以由序列号3的氨基酸序列、序列号5的氨基酸序列或序列号7的氨基酸序列组成。并且,编码上述阿卢糖差向异构酶的多核苷酸可以由序列号4的碱基序列、序列号6的碱基序列或序列号8的碱基序列组成。并且,有关阿卢糖差向异构酶以及编码其的多核苷酸,本发明包括韩国授权专利公报第10-1919713号、韩国授权专利公报第10-2187354号、韩国授权专利公报第10-1656063号、韩国授权专利公报第10-1695830号、韩国授权专利公报第10-2189458号、韩国授权专利公报第10-1539097号、韩国授权专利公报第10-1539096号、韩国授权专利公报第10-1455759号、韩国授权专利公报第10-1318422号等中公开的内容。本发明优选一例的表达载体具有图1的酶切图谱。具有图1的酶切图谱的表达载体为源自pUC的复制起点(replication origin,ori)、由序列号9的碱基序列组成的启动子变体(PgapAm1)、由序列号8的碱基序列组成的编码阿卢糖差向异构酶的多核苷酸(FpDPE[W29K/A77S/G216S/M234I])、卡那霉素抗性基因标记(KanR)等依次连接的结构。
本发明一例的重组菌株是通过引入由编码靶蛋白的多核苷酸以及可操作地与其连接并由序列号9的碱基序列组成的启动子变体组成的表达盒或包括上述表达盒的表达载体而使宿主细胞转化而成的。在本发明中,可由表达载体转化的宿主细胞的种类没有特别限制,只要可使本发明一例的新型启动子变体顺利操作即可,优选原核生物,考虑到新型启动子变体的表达调节活性、DNA的引入效率、引入的DNA的表达效率等,更优选大肠杆菌。上述大肠杆菌包括BL21、JM109、K-12、LE392、RR1、DH5α或W3110等,但不限于此。本发明一例的重组菌株优选为国际保藏机构韩国微生物保藏中心保藏的重组大肠杆菌(Escherichiacoli)DS00004(保藏编号:KCCM13092P;保藏日期:2021年12月14日)。上述重组大肠杆菌(Escherichia coli)DS00004(保藏编号:KCCM13092P;保藏日期:2021年12月14日)是通过在作为宿主细胞的大肠杆菌DH5α中引入具有图1的酶切图谱的重组表达载体pPgapAm1-FpDPE[W29K/A77S/G216S/M234I]并转化而成的。
本发明一例的靶蛋白的制备方法包括:通过培养上述重组菌株来表达靶蛋白的步骤;以及从重组菌株的培养液或重组菌株的菌体中分离靶蛋白的步骤。根据种类,上述靶蛋白可在表达后存在于重组菌株的菌体中或分泌到重组菌株的菌体外。例如,当靶蛋白为阿卢糖差向异构酶时,由重组菌株生产的阿卢糖差向异构酶存在于重组菌株的菌体中。本发明优选一例的阿卢糖差向异构酶的制备方法包括:通过引入由编码阿卢糖差向异构酶的多核苷酸以及可操作地与其连接并由序列号9的碱基序列组成的启动子变体组成的表达盒或包括上述表达盒的表达载体来培养转化的重组菌株,从而表达阿卢糖差向异构酶的步骤;以及从表达上述阿卢糖差向异构酶的重组菌株的裂解物中分离阿卢糖差向异构酶的步骤。由于本发明一例的新型启动子变体为组成型表达载体,因此可以在不使用作为蛋白质表达诱导因子的IPTG(异丙基-1-硫代-β-D-吡喃半乳糖苷(isopropyl-1-thio-β-D-galactopyranoside))等的情况下诱导表达。在本发明中,阿卢糖差向异构酶可以从重组菌株的裂解物中回收。用于蛋白质表达的细胞可以通过各种物理或化学手段裂解,例如反复冷冻-解冻、超声波处理、机械裂解或细胞崩解剂等,可以通过常规生化分离技术分离或纯化(Sambrook et al.,Molecular Cloning:A laborarory Manual,2nd Ed.,Cold SpringHarbor Laboratory Press,1989;Deuscher,M.,Guide to Protein PurificationMethods Enzymology,Vol.182.Academic Press.Inc.,San Diego,CA,1990)。例如,分离或纯化由宿主细胞表达的蛋白质的方法包括电泳、离心、凝胶过滤、沉淀、透析、层析(离子交换层析、亲和层析、免疫吸附亲和层析、反相HPLC、凝胶渗透HPLC)、等电聚焦和各种变化或复合方法,但不限于此。另一方面,在本发明中,从重组菌株的裂解物中分离阿卢糖差向异构酶的步骤可以优选地通过使用肽标签的亲和层析(affinity chromatography)来进行。作为上述肽标签,可以使用HA标签、FLAG标签、His标签、BCCP(生物素羧基载体蛋白(biotincarboxyl carrier protein))、c-myc标签、V5标签、谷胱甘肽-S-转移酶(GST)或MBP(麦芽糖结合蛋白(maltose binding protein))等已知的各种标签,其中优选His标签。His-标记的蛋白质被特异性捕获在Ni-NTA(镍-次氮基三乙酸)树脂柱上,可以用EDTA或咪唑洗脱。
本发明一例的重组菌株除了用于制备靶蛋白,还可以用于间接发挥靶蛋白的功能。例如,当上述靶蛋白为酶时,本发明一例的重组菌株可用于通过底物的酶转化反应制备产物。具体地,当靶蛋白为阿卢糖差向异构酶时,本发明提供一种从果糖制备阿卢糖的方法,其包括将重组菌株加入到含果糖的溶液中并进行反应的步骤。上述重组菌株是通过引入编码阿卢糖差向异构酶的多核苷酸以及可操作地与其连接并由序列号9的碱基序列组成的启动子变体组成的表达盒或包括上述表达盒的表达载体来转化的宿主细胞,优选为重组大肠杆菌(Escherichia coli)DS00004(保藏编号:KCCM13092P;保藏日期:2021年12月14日)。并且,上述含果糖的溶液还可以包含Ca2+、Mn2+等金属离子,以促进阿卢糖差向异构酶的活性。并且,在上述从果糖制备阿卢糖的方法中,反应温度为60℃~70℃,优选为60℃~67℃,考虑到重组菌株的酶的顺利表达,酶的稳定性及最大活性,更优选为60℃~65℃范围,反应pH为6.5~8,优选为6.5~7.5,更优选为6.5~7范围。并且,在上述从果糖制备阿卢糖的方法中,果糖的浓度没有特别限制,但考虑到生产性和经济性,以总反应物为计,优选为1%~75%(w/w),更优选为4%~35%(w/w)。
以下,将通过实施例更具体地说明本发明。但一下实施例仅用于清楚地说明本发明的技术特征,并非用于限制本发明的保护范围。
实施例1:获得用于表达酶基因的启动子
1.1.获得gapA(甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphatedehydrogenase))基因启动子
gapA(甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase))基因启动子是用于表达大肠杆菌(Escherichia coli)gapA(3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase))基因的启动子,由序列号1的碱基序列组成,已知其为在大肠杆菌中诱导强组成型表达的启动子[参照Thouvenot et al(2004)Thestrong efficiency of the Escherichia coli gapA P1 promoter depends on acomplex combination of functional determinants,Biochemical Journal 383:371-382.DOI:10.1042/BJ20040792]。
为了从大肠杆菌获得对应于gapA(甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase))基因的启动子位点的多核苷酸片段,使用大肠杆菌MG1655的基因组DNA(genomic DNA)作为模板并使用下表1所述的引物组进行PCR。将得到的扩增产物克隆到pGEM-Teasy载体(vector)(Promega Co.,USA)中并分析碱基序列,结果确认多核苷酸片段的长度为137bp,由序列号1的碱基序列组成。
表1
引物名称 | 引物描述 | 引物碱基序列(5'→3') |
PgapA-F | gapA启动子正向引物 | AGGCGAGTCAGTCGCGTAAT |
PgapA-R | gapA启动子反向引物 | ATATTCCTCCAGCTATTTGTTAG |
1.2.获得BLMA基因启动子
BLMA基因启动子是用于表达源自地衣芽孢杆菌(Bacillus licheniformis)的麦芽糖淀粉酶(maltogenic amylase)基因的启动子,由序列号2的碱基序列组成,已知其为在大肠杆菌中诱导外源基因的稳定高表达的启动子[参照TAE-JIP KIM et al(1999)Modesof Action of Acarbose Hydrolysis and Transglycosylation Catalyzed by aThermostable Maltogenic Amylase,the Gene for Which Was Cloned from a Therm usStrain]。
为了获得对应于源自地衣芽孢杆菌(Bacillus licheniformis)的麦芽糖淀粉酶(m altogenic amylase)基因的启动子位点的多核苷酸片段,使用地衣芽孢杆菌(Bacilluslicheniformis)ATCC 14580的基因组DNA(genomic DNA)作为模板并使用下表2所述的引物组进行PCR。将得到的扩增产物克隆到pGEM-Teasy载体(vector)(Pro mega Co.,USA)中并分析碱基序列,结果该多核苷酸片段的长度为115bp,由序列号2的碱基序列组成。
表2
引物名称 | 引物描述 | 引物碱基序列(5'→3') |
Pblma-F | BLMA启动子正向引物 | GGTGTCTCATTCTGTTACCG |
Pblma-R | BLMA启动子反向引物 | GTTTCCCCCTTTTGGTTGTC |
实施例2:获得阿卢糖差向异构酶变体克隆载体
本发明的申请人通过韩国授权专利公报第10-14739180号公开了一种源自普氏黄杆菌(Flavonifractor plautii)的野生型D-阿卢糖差向异构酶以及编码其的多核苷酸。上述野生型D-阿卢糖差向异构酶由序列号3的氨基酸序列组成,编码其的多核苷酸由序列号4的碱基序列组成。
并且,本发明的申请人通过韩国公开授权公报第10-2021-0132405号公开了一种提高果糖转化为阿卢糖的转化率以及热稳定性的D-阿卢糖差向异构酶变体W29K/G216S/M234I以及编码其的多核苷酸。在上述D-阿卢糖差向异构酶变体W29K/G216S/M234I中,源自普氏黄杆菌(Flavonifractor plautii)的野生型D-阿卢糖差向异构酶的氨基酸序列中位于第29位的色氨酸(Trp)被赖氨酸(Lys)取代,位于第216位的甘氨酸(Gly)被丝氨酸(Ser)取代,同时位于第234位的甲硫氨酸(Met)被异亮氨酸(Ile)取代,由序列号5的氨基酸序列组成,编码其的多核苷酸由序列号6的碱基序列组成。
并且,本发明的申请人导出在高温条件下热稳定性非常优异的D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I,并于2021年12月14日提出申请(韩国专利申请第10-2021-0178690号,未公开状态)。在上述D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I中,源自普氏黄杆菌(Flavonifractor plautii)的野生型D-阿卢糖差向异构酶的氨基酸序列中位于第29位的色氨酸(Trp)被赖氨酸(Lys)取代,位于第77位的丙氨酸(Als)被丝氨酸(Ser)取代,位于第216位的甘氨酸(Gly)被丝氨酸(Ser)取代,同时位于第234位的甲硫氨酸(Met)被异亮氨酸(Ile)取代,由序列号7的氨基酸序列组成,编码其的多核苷酸由序列号8的碱基序列组成。
基于D-阿卢糖差向异构酶变体W29K/G216S/M234I的多核苷酸(序列号:6),使用重叠延伸聚合酶链式反应(overlap extension polymerase chain reaction)方法制备出编码D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的氨基酸序列的多核苷酸片段。具体地,向加入100μM的三磷酸脱氧核苷酸(dATP、dCTP、dGTP、dTTP)的反应液中混合1pM的下表3的寡核苷酸引物(A77S正向引物、A77S反向引物)、100ng的用作模板(template)的D-阿卢糖差向异构酶变体W29K/G216S/M234I的多核苷酸(序列号:6),并使用热循环仪(Thermocycler)(TP600,TAKARA BIO Inc.,JAPAN)在1单位pfu-X DNA聚合酶混合物(Bioneer)存在下进行25~30循环的P CR反应。通过引物组合扩增变体片段之后,以扩增的片段为模板,使用引入了下表3的NdeI和XhoI限制酶识别位点序列的寡核苷酸引物(NdeI正向引物、XhoI反向引物)通过重叠延伸(overlap extentention)PCR最终制备出编码D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的氨基酸序列的多核苷酸片段(序列号:8)。然后,使用限制酶NdeI和XhoI将制备的多核苷酸片段***到pET28a载体(vector)(Nova gen)的相同限制酶位点来获得克隆载体。
表3
引物描述 | 引物碱基序列(5'→3') |
A77S正向引物 | AATACGACCTGAGCAGCGACGATCCGGCGGTG |
A77S反向引物 | GATCGTCGCTGCTCAGGTCGTATTTGGCCTCCA |
NdeI正向引物 | GCATGCCATATGAACCCGATTGGAATGCA |
XhoI反向引物 | GCATGCCTCGAGCGCGGTCAGCTCCTTGAGGA |
实施例3:启动子与阿卢糖差向异构酶变体基因的连接片段的制备
3.1.gapA启动子与阿卢糖差向异构酶变体基因连接的DNA片段的制备
为了扩增gapA启动子,使用实施例1中获得的gapA启动子被克隆的pGEM-Teasy载体(vector)作为模板,并使用下表4的引物组(XhoI-PgapA,PgapA-FpDPE_R)进行PCR。
并且,为了扩增源自普氏黄杆菌(Flavonifractor plautii)的D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的基因,使用实施例2中获得的D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的基因被克隆的pET28a载体(vector)(Novagen)作为模板,并使用下表4的引物组(PgapA-FpDPE_F、PstI-FpDPE)进行PCR。
表4
由于扩增时使用的引物的互补序列,以如上所述的方式扩增的gapA启动子片段和D-阿卢糖差向异构酶变体基因片段可以连接成一个片段。使用两个片段作为模板,并使用引入了上述表4的XhoI和PstI限制酶识别位点序列的引物(XhoI-PgapA、Ps tI-FpDPE)进行重叠延伸(overlap extension)PCR来获得一个扩增片段。将获得的g apA启动子与阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的基因连接的DNA片段命名为“PgapA-FpDPE”。
3.2.BLMA启动子与阿卢糖差向异构酶基因连接的DNA片段的制备
为了扩增BLMA启动子,使用实施例1中获得的BLMA启动子被克隆的pGEM-Teasy载体(vector)作为模板,并使用下表5的引物组(XhoI-Pblma,Pblma-FpDP E_R)进行PCR。
并且,为了扩增源自普氏黄杆菌(Flavonifractor plautii)的D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的基因,使用实施例2中获得的D-阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的基因被克隆的pET28a载体(vector)(Novagen)作为模板,并使用下表5的引物组(Pblma-FpDPE_F、PstI-FpDPE)进行PCR。
表5
由于扩增时使用的引物的互补序列,以如上所述的方式扩增的BLMA启动子片段和D-阿卢糖差向异构酶变体基因片段可以连接成一个片段。使用两个片段作为模板,并使用引入了上述表5的XhoI和PstI限制酶识别位点序列的引物(XhoI-Pblma,PstI-FpDPE)进行重叠延伸(overlap extension)PCR来获得一个扩增片段。将获得的BLMA启动子与阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的基因连接的D NA片段命名为“Pblma-FpDPE”。
实施例4:D-阿卢糖差向异构酶变体表达载体的制备
4.1.具有gapA启动子的D-阿卢糖差向异构酶变体表达载体的制备
从pTrc99A载体(vector)(Pharmacia,US)通过基因操作制备出包含可在大肠杆菌中复制的源自pUC的复制起点(replication origin)、限制酶XhoI位点和PstI位点等多克隆位点(Multi clonig site,MCS)、转录终止子(transcription terminator)及卡那霉素(Kanamycin)抗生素抗性基因的重组质粒载体。然后,将实施例3中制备的多核苷酸片段PgapA-FpDPE用限制酶XhoI和PstI酶切后,与具有相同限制酶位点的上述重组质粒载体结扎(ligation),得到D-阿卢糖差向异构酶变体表达载体pPg apA-FpDPE[W29K/A77S/G216S/M234I]。然后,将D-阿卢糖差向异构酶变体表达载体通过热休克(heat shock)方法(参照Sambrook and Russell:Molecular Cloning)转化到大肠杆菌DH5α来获得具有卡那霉素抗性的菌落,选择6个菌落,回收6种重组表达载体pPgapA-FpDPE[W29K/A77S/G216S/M234I]之后分析了碱基序列。pPgapA-F pDPE[W29K/A77S/G216S/M234I]的6种重组表达载体的序列分析结果,在引入的ga pA启动子或阿卢糖差向异构酶变体W29K/A77S/G216S/M234I的基因序列中分别确认到随机变异,随机变异内容示于下表6中。
表6
如上表6所示,在从菌落2、3、4至6回收的重组表达载体的情况下,阿卢糖差向异构酶变体基因序列发生变异,预计将无法进行用于制备所需的阿卢糖差向异构酶变体的翻译(translation)。相反,在从菌落1或5回收的重组表达载体的情况下,尽管gapA启动子的核糖体结合位点(Ribosome-Binding Site,RBS)序列发生变异,但酶基因序列匹配,因此判断为有可能表达所需的酶。将从菌落1回收的重组表达载体中的变异启动子命名为“gapAm1,”将从菌落5回收的重组表达载体中的变异启动子命名为“gapAm2”。gapAm1启动子由序列号9的碱基序列组成,gapAm2启动子由序列号10的碱基序列组成。并且,将从菌落1回收的重组表达载体更名为“pPgapAm1-FpDPE[W29K/A77S/G216S/M234I],”将从菌落5回收的重组表达载体更名为“pPgapA m2-FpDPE[W29K/A77S/G216S/M234I]”。图1为本发明实施例中制备的重组表达载体pPgapAm1-FpDPE[W29K/A77S/G216S/M234I]的酶切图谱。图2为本发明实施例中制备的重组表达载体pPgapAm2-FpDPE[W29K/A77S/G216S/M234I]的酶切图谱。
4.2.具有BLMA启动子的D-阿卢糖差向异构酶变体表达载体的制备
从pTrc99A载体(vector)(Pharmacia,US)通过基因操作制备出包含可在大肠杆菌中复制的源自pUC的复制起点(replication origin)、限制酶XhoI位点和PstI位点等多克隆位点(Multi clonig site,MCS)、转录终止子(transcription terminator)及卡那霉素(Kanamycin)抗生素抗性基因的重组质粒载体。然后,将实施例3中制备的多核苷酸片段Pblma-FpDPE用限制酶XhoI和PstI酶切后,与具有相同限制酶位点的上述重组质粒载体结扎(ligation),得到D-阿卢糖差向异构酶变体表达载体pPbl ma-FpDPE[W29K/A77S/G216S/M234I]。然后,将D-阿卢糖差向异构酶变体表达载体通过热休克(heat shock)方法(参照Sambrook and Russell:Molecular Cloning)转化到大肠杆菌DH5α来获得具有卡那霉素抗性的菌落,选择6个菌落,回收6种重组表达载体pPblma-FpDPE[W29K/A77S/G216S/M234I]之后分析了碱基序列。6种重组表达载体pPblma-FpDPE[W29K/A77S/G216S/M234I]的序列分析结果,确认均按预期存在BLMA启动子以及阿卢糖差向异构酶变体基因序列。图3为本发明实施例中制备的重组表达载体pPblma-FpDPE[W29K/A77S/G216S/M234I]的酶切图谱。
实施例5:通过D-阿卢糖差向异构酶变体表达载体制备转化体
分别将实施例4中制备的重组表达载体pPgapAm1-FpDPE[W29K/A77S/G216S/M234I]、pPgapAm2-FpDPE[W29K/A77S/G216S/M234I]以及pPblma-FpDPE[W29K/A77S/G216S/M234I]通过热休克(heat shock)方法引入大肠杆菌(E.coli)W3110中。然后,确认卡那霉素抗生素抗性并筛选用上述重组表达载体转化的重组菌株。在制备的重组大肠杆菌中加入甘油溶液至终浓度为20%(v/v),在进行酶表达培养前,于-70℃冷冻保存。
实施例6:通过重组菌株使果糖转化为阿卢糖的转化率测定以及启动子的酶表达强度比较
由于D-阿卢糖差向异构酶可将果糖转化为阿卢糖,因此通过测定与重组菌株的酶表达量成正比的果糖转化为阿卢糖的转化率,比较了重组菌株中各启动子的酶表达诱导强度。
为培养用重组表达载体转化的重组大肠杆菌,在1L容量的烧瓶中收容含有终浓度为50μg/ml的卡那霉素的LB培养基100ml,其中接种实施例5中制备的重组大肠杆菌1ml。然后,将烧瓶转移到振荡培养箱中,将重组大肠杆菌在保持30℃的温度和140rpm的振荡条件下培养14小时,离心分离培养液并回收菌体。然后,将回收的细胞以1mg/ml的浓度添加到含有30%(w/w)果糖以及1mM硫酸锰(MnSO4)金属离子的50mM PIPES缓冲液(pH 7.0)中,并在62℃下反应规定时间后,将反应生成液的温度降至4℃以终止反应,在16600×g以及4℃条件下通过离心分离回收上清液。然后,使用高效液相色谱法(HPLC)测定上清液中的阿卢糖浓度以及果糖浓度,并根据测定结果计算果糖转化为阿卢糖的转化率,然后将转化率用作酶活性指标。图4为示出当使用本发明实施例中制备的重组大肠杆菌进行果糖到阿卢糖的转化反应时根据反应时间的转化率。在图4中,“PgapAm1”表示用重组表达载体pPgapAm1-FpDP E[W29K/A77S/G216S/M234I]转化的重组大肠杆菌,“PgapAm2”表示用重组表达载体pPgapAm2-FpDPE[W29K/A77S/G216S/M234I]转化的大肠杆菌,“Pblma”表示用重组表达载体pPblma-FpDPE[W29K/A77S/G216S/M234I]转化的大肠杆菌。如图4所示,与引入gapAm2启动子或BLMA启动子的重组大肠杆菌相比,引入gapAm1启动子的重组大肠杆菌表现出非常高的果糖转化为阿卢糖的转化率,从这种结果可以看出,与gapAm2启动子或BLMA启动子相比,gapAm1启动子的酶表达诱导效果非常强。
实施例7:重组菌株的保藏
将上述实施例6中果糖转化为阿卢糖的转化活性最高且用重组表达载体pPgapAm1-FpDPE[W29K/A77S/G216S/M234I]转化的重组大肠杆菌命名为“DS00004”。于2021年12月14日将上述重组大肠杆菌DS00004转移至公认保藏机构韩国微生物保藏中心(KoreanCulture Center of Microorganisms,KCCM;地址:韩国首尔特别市西大门区弘济内二街45YOULIM大厦3F),根据布达佩斯条约进行国际专利保藏,并授予保藏编号KCCM13092BP。上述重组大肠杆菌(Escherichia coli)DS00004(保藏编号:KCCM13092P)可以按照布达佩斯条约规定的程序分发给第三方。
[委托信息]
保藏机构名称:韩国微生物保藏中心(国外)
保藏编号:KCCM13092P
保藏日期:20211214
如上所述,通过上述实施例对本发明进行了说明,但本发明并非仅限定于此,当然,可以在不脱离本发明的范畴和思想的范围内进行各种变形实施。因此,本发明的保护范围应当被解释为包括落入本发明所附权利要求范围内的所有实施方式。
序列表
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<120> 用于组成型表达的新型启动子变体及其用途
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Ser Met Pro Lys Val Gly Ala Ala Ile Leu Asn Gly Val Ser Tyr Ala
100 105 110
Gly Trp Gln Ala Leu Pro Asp His Gly Ile Thr Leu Asp Glu Lys Arg
115 120 125
Arg Lys Glu Glu Leu Ala Leu Glu Ser Met Ser Arg Leu Met Lys Val
130 135 140
Ala Glu Asp Cys Gly Val Leu Tyr Cys Cys Glu Val Val Asn Arg Phe
145 150 155 160
Glu Gln Tyr Leu Leu Asn Thr Ala Lys Glu Gly Val Glu Phe Val Lys
165 170 175
Arg Leu Gly Ser Pro Asn Ala Arg Val Leu Leu Asp Thr Phe His Met
180 185 190
Asn Ile Glu Glu Asp Ser Met Val Asp Ala Ile Leu Glu Ala Gly Pro
195 200 205
Trp Leu Gly His Phe His Val Ser Glu Asn Asn Arg Arg Pro Ala Gly
210 215 220
Ser Thr Asn Arg Leu Pro Trp Lys Asp Ile Ala Ala Ala Leu Lys Gln
225 230 235 240
Val Asn Tyr Gln Gly Ala Ile Val Met Glu Pro Phe Val Leu Met Gly
245 250 255
Gly Thr Ile Pro Tyr Asp Ile Lys Val Trp Arg Asp Leu Ser Gly Gly
260 265 270
Ala Gly Glu Ala Gly Leu Asp Glu Met Ala Gly Arg Ala Cys Arg Phe
275 280 285
Leu Lys Glu Leu Thr Ala
290
<210> 6
<211> 885
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaacccga ttggaatgca ctacggcttc tggagccaca actgggacga gattgcatac 60
atacccctga tggagaagct ggccaaactg ggctttgaca tctgcgaggt ggcctccgcc 120
gagtggggct attacgacga cgccaggctg cgggagctga aggcctgcgc cgatcacaac 180
ggcctgggca tcacctattc catcggcctg gaggccaaat acgacctggc cagcgacgat 240
ccggcggtgc gggagaacgg catccgccat gtcacccgca tcctggagag catgcccaag 300
gtgggggcgg ccatcctcaa cggcgtgtcc tacgccgggt ggcaggccct gcccgaccac 360
ggaatcaccc tggacgagaa gcgccgcaag gaggagcttg ccctggagtc catgtcccgg 420
ctcatgaagg tggcggagga ctgcggcgtg ctctactgct gcgaggtggt caaccgcttc 480
gagcagtacc tgctcaacac cgccaaagag ggcgtggagt ttgtcaagcg cctgggcagt 540
cccaacgccc gggtgctgct ggataccttc cacatgaaca tcgaggagga cagcatggtg 600
gacgccattc tggaggcggg cccctggctg gggcatttcc acgtgagcga gaacaaccgc 660
cgccccgccg gctccaccaa ccgcctgccc tggaaggaca ttgccgccgc cctcaagcag 720
gtgaactacc agggggccat tgtgatggag cccttcgtgc tcatgggggg taccattccc 780
tatgatatca aggtctggcg ggatctcagc ggcggggccg gggaggccgg gctggacgag 840
atggcgggcc gggcctgccg gttcctcaag gagctgaccg cgtaa 885
<210> 7
<211> 294
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Asn Pro Ile Gly Met His Tyr Gly Phe Trp Ser His Asn Trp Asp
1 5 10 15
Glu Ile Ala Tyr Ile Pro Leu Met Glu Lys Leu Ala Lys Leu Gly Phe
20 25 30
Asp Ile Cys Glu Val Ala Ser Ala Glu Trp Gly Tyr Tyr Asp Asp Ala
35 40 45
Arg Leu Arg Glu Leu Lys Ala Cys Ala Asp His Asn Gly Leu Gly Ile
50 55 60
Thr Tyr Ser Ile Gly Leu Glu Ala Lys Tyr Asp Leu Ser Ser Asp Asp
65 70 75 80
Pro Ala Val Arg Glu Asn Gly Ile Arg His Val Thr Arg Ile Leu Glu
85 90 95
Ser Met Pro Lys Val Gly Ala Ala Ile Leu Asn Gly Val Ser Tyr Ala
100 105 110
Gly Trp Gln Ala Leu Pro Asp His Gly Ile Thr Leu Asp Glu Lys Arg
115 120 125
Arg Lys Glu Glu Leu Ala Leu Glu Ser Met Ser Arg Leu Met Lys Val
130 135 140
Ala Glu Asp Cys Gly Val Leu Tyr Cys Cys Glu Val Val Asn Arg Phe
145 150 155 160
Glu Gln Tyr Leu Leu Asn Thr Ala Lys Glu Gly Val Glu Phe Val Lys
165 170 175
Arg Leu Gly Ser Pro Asn Ala Arg Val Leu Leu Asp Thr Phe His Met
180 185 190
Asn Ile Glu Glu Asp Ser Met Val Asp Ala Ile Leu Glu Ala Gly Pro
195 200 205
Trp Leu Gly His Phe His Val Ser Glu Asn Asn Arg Arg Pro Ala Gly
210 215 220
Ser Thr Asn Arg Leu Pro Trp Lys Asp Ile Ala Ala Ala Leu Lys Gln
225 230 235 240
Val Asn Tyr Gln Gly Ala Ile Val Met Glu Pro Phe Val Leu Met Gly
245 250 255
Gly Thr Ile Pro Tyr Asp Ile Lys Val Trp Arg Asp Leu Ser Gly Gly
260 265 270
Ala Gly Glu Ala Gly Leu Asp Glu Met Ala Gly Arg Ala Cys Arg Phe
275 280 285
Leu Lys Glu Leu Thr Ala
290
<210> 8
<211> 885
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgaacccga ttggaatgca ctacggcttc tggagccaca actgggacga gattgcatac 60
atacccctga tggagaagct ggccaaactg ggctttgaca tctgcgaggt ggcctccgcc 120
gagtggggct attacgacga cgccaggctg cgggagctga aggcctgcgc cgatcacaac 180
ggcctgggca tcacctattc catcggcctg gaggccaaat acgacctgag cagcgacgat 240
ccggcggtgc gggagaacgg catccgccat gtcacccgca tcctggagag catgcccaag 300
gtgggggcgg ccatcctcaa cggcgtgtcc tacgccgggt ggcaggccct gcccgaccac 360
ggaatcaccc tggacgagaa gcgccgcaag gaggagcttg ccctggagtc catgtcccgg 420
ctcatgaagg tggcggagga ctgcggcgtg ctctactgct gcgaggtggt caaccgcttc 480
gagcagtacc tgctcaacac cgccaaagag ggcgtggagt ttgtcaagcg cctgggcagt 540
cccaacgccc gggtgctgct ggataccttc cacatgaaca tcgaggagga cagcatggtg 600
gacgccattc tggaggcggg cccctggctg gggcatttcc acgtgagcga gaacaaccgc 660
cgccccgccg gctccaccaa ccgcctgccc tggaaggaca ttgccgccgc cctcaagcag 720
gtgaactacc agggggccat tgtgatggag cccttcgtgc tcatgggggg taccattccc 780
tatgatatca aggtctggcg ggatctcagc ggcggggccg gggaggccgg gctggacgag 840
atggcgggcc gggcctgccg gttcctcaag gagctgaccg cgtaa 885
<210> 9
<211> 136
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aggcgagtca gtcgcgtaat gcttaggcac aggattgatt tgtcgcaatg attgacacga 60
ttccgcttga cgctgcgtaa ggtttttgta attttacagg caacctttta ttcactaaca 120
aatagctggg gaatat 136
<210> 10
<211> 132
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aggcgagtca gtcgcgtaat gcttaggcac aggattgatt tgtcgcaatg attgacacga 60
ttccgcttga cgctgcgtaa ggtttttgta attttacagg caacctttta ttcactaaca 120
aatagctgga gt 132
Claims (10)
1.一种启动子变体,其特征在于,由序列号9的碱基序列组成。
2.一种重组载体,其特征在于,包含根据权利要求1所述的启动子变体。
3.一种表达载体,其特征在于,包含编码靶蛋白的多核苷酸以及与其可操作地连接的根据权利要求1所述的启动子变体。
4.根据权利要求3所述的表达载体,其特征在于,上述靶蛋白为酶。
5.根据权利要求4所述的表达载体,其特征在于,上述酶为阿卢糖差向异构酶。
6.根据权利要求5所述的表达载体,其特征在于,上述阿卢糖差向异构酶由序列号3的氨基酸序列、序列号5的氨基酸序列或序列号7的氨基酸序列组成。
7.根据权利要求5所述的表达载体,其特征在于,编码上述阿卢糖差向异构酶的多核苷酸由序列号4的碱基序列、序列号6的碱基序列或序列号8的碱基序列组成。
8.一种重组菌株,其特征在于,由根据权利要求3所述的表达载体转化而成。
9.根据权利要求8所述的重组菌株,其特征在于,上述重组菌株是保藏编号为KCCM13092P的重组大肠杆菌DS00004。
10.一种从果糖制备阿卢糖的方法,其特征在于,包括在含果糖的溶液中添加根据权利要求9所述的重组菌株并进行反应的步骤。
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