CN116926036A - 一种丙酮酸激酶突变体、重组微生物及其构建方法与应用 - Google Patents
一种丙酮酸激酶突变体、重组微生物及其构建方法与应用 Download PDFInfo
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Abstract
本发明涉及微生物工程技术领域,具体公开了一种丙酮酸激酶突变体、重组微生物及其构建方法与应用。本发明的丙酮酸激酶突变体,以SEQ ID NO.1或2所示的氨基酸序列为参考序列,含有第1位缬氨酸突变为亮氨酸,或第4位甲硫氨酸突变为缬氨酸或亮氨酸。包含本发明丙酮酸激酶突变体的重组微生物可有效增加核苷产量,提高核苷产率,提升核苷工业化生产的水平。
Description
技术领域
本发明涉及微生物工程技术领域,具体地说,涉及一种丙酮酸激酶突变体、重组微生物及其构建方法与应用。
背景技术
核苷是一类糖苷的总称,是核酸和核苷酸的组成成分。核苷都是由D-核糖或D-2-脱氧核糖与嘧啶碱或嘌呤碱缩合而成。核苷一般为无色结晶,不溶于普通有机溶剂,易溶于热水,熔点为160~240℃。由D-核糖生成的核苷称核糖核苷,参与RNA组成,由D-α-脱氧核糖生成的核苷称脱氧核糖核苷,参与DNA组成。D-核糖与腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶缩合生成相应的腺嘌呤核糖核苷、鸟嘌呤核糖核苷、胞嘧啶核糖核苷、胸腺嘧啶核糖核苷和尿嘧啶核糖核苷,它们分别简称为腺苷(A)、鸟苷(G)、胞苷(C)、胸苷(T)和尿苷(U)。
腺苷即腺嘌呤核苷,化学名为6-氨基-9-β-D-呋喃核糖基-9-氢嘌呤,它是腺嘌呤核苷酸脱磷酸后的产物,属于重要的核苷酸衍生物。腺苷是一种遍布人体细胞的内源性核苷,可直接进入心肌经磷酸化生成腺苷酸,参与心肌能量代谢,同时还参与扩张冠脉血管,增加血流量。腺苷对心血管系统和肌体的许多其它系统及组织均有生理作用。腺苷除了可以用作治疗心脏的特效药物之外,还是用于合成三磷酸腺苷(ATP)、腺嘌呤、腺苷酸、阿糖腺苷的重要中间体,广泛应用于医药等行业。
鸟嘌呤核苷,又名9-β-D呋喃核糖鸟嘌呤,以下也称鸟苷,具有多种用途,在食品和医药行业有着广泛的作用。在食品领域,鸟苷是鸟苷酸二钠的重要前体,而鸟苷酸二钠与肌苷酸二钠组合使用作为食品增鲜剂,广泛应用于鸡精、酱油等调味品中。在医药领域,鸟苷可以作为多种抗病毒药物的医药中间体,如无环鸟苷、三氮唑核苷、三磷酸鸟苷钠等都需要鸟苷作为合成原料。
肌苷,化学名称:9-β-D-核糖次黄嘌呤。其是细胞代谢改善药,参与体内核酸代谢,在体内转变为肌苷酸及三磷酸腺苷,参与细胞的能量代谢和蛋白质合成,提高各种酶,特别是辅酶A与丙酮酸氧化酶的活性,从而使细胞在缺氧状态下继续进行代谢,活化肝脏功能,促进受损伤肝脏的恢复,可刺激体内产生抗体并促进肠道对铁的吸收。适用于各种慢性肝脏疾患、心脏疾患、白血球或血小板减少症、中心性视网膜炎等,并可预防接触锑剂引起的对肝脏心脏的副作用。
嘌呤核苷既可通过化学法合成,也可通过微生物发酵法合成,并且微生物发酵法由于其诸多的优势(条件温和、环境污染小),已成为主流生产方法。但发酵法的缺点则是成本高、转化率低。因此,亟待改善菌种性能。
发明内容
本发明的目的是提供一种可高效生产核苷及其衍生物的重组微生物及其构建方法与应用。
本发明的技术方案如下:
一种丙酮酸激酶突变体,以SEQ ID NO.1或2所示的氨基酸序列为参考序列,所述丙酮酸激酶突变体含有第1位缬氨酸突变为亮氨酸(PYKV1L),或第4位甲硫氨酸突变为缬氨酸(PYKM4V)或亮氨酸(PYKM4L)。
本发明中,编码SEQ ID NO.1或2所示的序列的核苷酸序列,相比于野生型丙酮酸激酶基因序列(可来自于ATCC13952或B.subtilis 168)还包含野生型基因上游紧邻的9个核苷酸。NCBI中ATCC13952的丙酮酸激酶编码基因为KS08_14090。NCBI中B.subtilis 168的丙酮酸激酶编码基因为BSU29180。
本发明研究发现,当将丙酮酸激酶进行上述突变后,可提升菌株的核苷生产效率,具有理想的工业应用前景。
本发明还提供一种编码上述丙酮酸激酶突变体的核酸。
本发明的核酸,具有如SEQ ID NO.3~8任一所示的核苷酸序列。
本发明另提供一种含有上述核酸的生物材料,所述生物材料为表达盒、载体或宿主细胞。
本发明再提供一种重组微生物,所述重组微生物表达丙酮酸激酶突变体,所述丙酮酸激酶突变体如上所述;所述重组微生物的出发菌株为芽孢杆菌或棒状杆菌。
优选,所述重组微生物的出发菌株为枯草芽孢杆菌(Bacillus subtilis)或解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。
本发明还提供一种上述丙酮酸激酶突变体,或核酸,或生物材料,或重组微生物的如下任一种应用:
(1)在发酵生产核苷及其衍生物中的应用;
(2)在用于生产核苷及其衍生物的微生物遗传育种中的应用;
(3)在提高发酵生产核苷及其衍生物产量上的应用。
本发明另提供一种核苷及其衍生物生产方法,其包括以重组微生物进行发酵培养的步骤,所述重组微生物如上所述。
本发明再提供一种构建重组微生物的方法,其包括使所述重组微生物表达丙酮酸激酶突变体的步骤,所述丙酮酸激酶突变体如上所述;所述重组微生物的出发菌株为芽孢杆菌或棒状杆菌,优选为枯草芽孢杆菌、解淀粉芽孢杆菌、短小芽孢杆菌或产氨棒杆菌。
本发明的有益效果至少在于:
含有本发明丙酮酸激酶突变体的重组微生物可减少副产物的积累,并有效增加核苷产量,提高核苷产率,提升核苷工业化生产的水平。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。下述实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件,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,均为可通过正规渠道商购买得到的常规产品。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明中。本发明的方法及产品已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合,来实现和应用本发明技术。
为了进一步理解本发明,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
如无特殊说明,本发明实施例中所涉及的试剂均为市售产品,均可以通过商业渠道购买获得。本发明所用到的原始菌株:ATCC13952、B.subtilis 168从公开渠道购买,B.subtilis A5已在中国专利CN 110257315中公开,B.subtilis 168(Δpyk)为本实验室构建,可通过代谢工程手段获得。本发明所用到的腺苷、鸟苷和肌苷标准品从Sigma公司(http://www.sigmaaldrich.com/sigma-aldrich)购买,所用DNA聚合酶、DNA纯化试剂盒、限制性内切酶、去磷酸化酶、DNA连接酶等分子生物学试剂从Thermo公司购买(http://www.thermoscientificbio.com/fermentas),所用其他生化试剂从生工生物工程(上海)股份有限公司购买(http://www.sangon.com/)。
其中,各实施例涉及的引物序列如表1所示。
表1引物序列(SEQ ID No.9-24)
引物名称 | 序列5'→3' |
pykV1L-S-1f | taatacgactcactatagggtcgaccaagaagctcacggatttcgagcac |
pykV1L-S-1r | tgaagatttcagaaggaattgaaccaaatgagaaaaactaaaattgtt |
pykV1L-S-2f | aattttagtttttctcatttggttcaattccttctgaaatcttcag |
pykV1L-S-2r | gatgaagattatttcttaatctagaattcgcgatacagcgacttctcatg |
pykM4V-S-1f | taatacgactcactatagggtcgaccaagaagctcacggatttcgagcac |
pykM4V-S-1r | atttcagaaggaagtgaaccaagtgagaaaaactaaaattgtttgtaccat |
pykM4V-S-2f | aaacaattttagtttttctcacttggttcacttccttctgaaatcttcag |
pykM4V-S-2r | gatgaagattatttcttaatctagaattcgcgatacagcgacttctcatg |
pykM4L-S-1f | taatacgactcactatagggtcgaccaagaagctcacggatttcgagcac |
pykM4L-S-1r | agaaggaagtgaaccaattgagaaaaactaaaattgtttgtaccatcggt |
pykM4L-S-2f | gtacaaacaattttagtttttctcaattggttcacttccttctgaaat |
pykM4L-S-2r | gatgaagattatttcttaatctagaattcgcgatacagcgacttctcatg |
pyk-S-f | aagcttttaaagaacgctcgcacggc |
pyk-S-r | ggatccaaggctgaagatttcagaagg |
pyk-N20-f | tacgcaattttagtttttctcatt |
pyk-N20-r | aaacaatgagaaaaactaaaattg |
实施例1丙酮酸激酶突变体PYKV1L、PYKM4V、PYKM4L酶活性对比
丙酮酸激酶酶活性的检测,首先将PYK突变体序列通过表达载体pHP13(GenBank:DQ297764.1),转化到B.subtilis 168(Δpyk)菌株中,再提取丙酮酸激酶蛋白检测酶活。详细操作如下:
步骤1:用引物PYK-S-f/PYKV1L-S-1r和PYKV1L-S-2f/PYK-S-r、PYK-S-f/PYKM4V-S-1r和PYKM4V-S-2f/PYK-S-r、PYK-S-f/PYKM4L-S-1r和PYKM4L-S-2f/PYK-S-r分别以B.subtilis168基因组为模板,得到PYKV1L、PYKM4V、PYKM4L上下同源臂片段。用引物PYK-S-f/PYK-S-r分别融合上下游同源臂片段,将得到的片段和pHP13载体用BamHI/HindIII同时酶切、纯化并使用T4连接酶连接后转化到B.subtilis 168(△PYK)中,制备表达载体pHP13-PYKV1L、pHP13-PYKM4V、pHP13-PYKM4L,分别转入B.subtilis 168(△pyk)感受态细胞中。
步骤2:将上述菌株和B.subtilis 168菌株培养一段时间后,将菌体离心后使用1xPBS溶液悬浮破碎。使用Bio-Scale Mini Profinity IMAC滤芯和Profinia蛋白质纯化系统(Bio-Rad)纯化丙酮酸激酶用于酶活测定。反应条件:将1.5mM磷酸烯醇丙酮酸,150uMNADH,4mM ADP,10mM MgCl4,0.1M KCl和2.64U乳酸脱氢酶溶于100uL pH 7.5的Tris-HCl缓冲液中,加入2μl酶样品,25℃反应10min,温度调至99℃反应2min以终止反应。最终加入NADH分析缓冲液27℃反应30min。使用微板分光光度计在波长340nm条件下测定NADH的浓度。测定NADH浓度在0,10,20,50,100,和200μM时的浓度标准曲线。
酶活测定结果见表2。从实验结果可以看出,同菌株B.subtilis 168(B.s 168)中丙酮酸激酶酶活相比,PYKV1L、PYKM4V、PYKM4L的丙酮酸激酶突变体活性显著降低。
表2工程菌株酶活测定
样品 | B.s 168 | PYKV1L | PYKM4V | PYKM4L |
酶活(μM) | 296 | 123 | 142 | 128 |
实施例2在B.subtilis 168中引入PYK突变体
使用芽孢杆菌CRISPR-Cas9工具(可参见Editing of the Bacillus subtilisGenome by the CRISPR-Cas9 System,Josef Altenbuchner,et al.Applied&Environmental Microbiology,2016)对菌株进行编辑,主要操作步骤包括:1.构建pJOE8999-PYK-N20质粒;2.在pJOE8999-PYK-N20质粒基础上,连接同源片段;3.电转芽孢杆菌感受态制备和电转化;4.转化子验证;5.pJOE8999工具质粒的消除。详细操作如下:
(1)pJOE8999-PYK-N20质粒构建
以引物PYK-N20-f和PYK-N20-r为模板,经过94℃到4℃的匀速退火后得到PYK-N20片段。将pJOE8999质粒(可参见Editing of the Bacillus subtilis Genome by theCRISPR-Cas9 System,Josef Altenbuchner,et al.Applied&EnvironmentalMicrobiology,2016)经BsaI酶进行酶切后,将得到的线性化质粒和PYK-N20片段经过T4酶4℃过夜连接,将连接产物通过化转的方法转化到BL21感受态中,获得pJOE8999-PYK-N20,并进行PCR鉴定及测序验证。
(2)pJOE8999-PYK-N20-LR质粒构建
步骤1:分别用引物PYKV1L-S-1f/1r和PYKV1L-S-2f/2r、PYKM4V-S-1f/1r和PYKM4V-S-2f/2r、PYKM4L-S-1f/1r和PYKM4L-S-2f/2r,以B.subtilis 168基因组为模板,使用pfu高保真DNA聚合酶扩增分别得到PYKV1L、PYKM4V、PYKM4L的上下游同源臂片段。分别用引物PYKV1L-S-1f/2r、PYKM4V-S-1f/2r、PYKM4L-S-1f/2r融合上下游同源臂片段得到PYKV1L、PYKM4V、PYKM4L同源片段(分别含有V1L、M4V、M4L突变,其核苷酸序列如SEQ ID No.3-5所示)。
步骤2:将pJOE8999-PYK-N20质粒使用SalI/XbalI进行双酶切,得到的线性化片段和上述同源片段分别通过无缝组装Clone Express试剂盒进行组装,随后通过化转的方法将连接产物转化到BL21感受态细胞中,获得pJOE8999-PYK-N20-LR(PYKV1L)、pJOE8999-PYK-N20-LR(PYKM4V)、pJOE8999-PYK-N20-LR(PYKM4L),并进行PCR鉴定及测序验证。
(3)感受态细胞制备及电转化
步骤1:取B.subtilis 168甘油菌涂到LB平板上,37℃静置过夜培养;转接单菌落到10ml LB中,37℃200rpm过夜培养,之后1%接种量接种到100ml LB中,37℃200rpm培养到OD562为0.1后制备电转感受态细胞。
步骤2:将上述(2)中步骤2得到的pJOE8999-PYK-N20-LR(PYKV1L)、pJOE8999-PYK-N20-LR(PYKM4V)、pJOE8999-PYK-N20-LR(PYKM4L)质粒分别电转化至B.subtilis 168的感受态细胞中,30℃复苏3h后,涂布到含有5μg/mL卡那霉素和0.2%甘露糖的LB平板上,在30℃下静置培养。将长出的单菌落转接到含有5μg/mL卡那霉素的LB平板上,30℃下静置培养至长出菌落,经过PCR验证及测序确认,即为目的改造株。
(4)质粒消除
步骤1:挑取测序正确的单菌落接种到含有5‰鸟嘌呤的5ml LB试管中,37℃200rpm过夜培养;之后转接至5‰鸟嘌呤的5ml LB试管中,在37℃200rpm过夜培养后稀释涂布到含有5μg/mL卡那霉素的LB平板上,37℃静置培养直至长出单菌落。
步骤2:挑取单菌落对点到含有卡那霉素和不含抗性的LB平板上,37℃过夜培养,若在含有卡那霉素的LB平板上不能生长,在无抗LB平板上能够生长,则表明pJOE8999-PYK-N20-LR质粒已经丢失,得到的菌株命名为B.subtilis P1(PYKV1L)、B.subtilis P2(PYKM4V)、B.subtilis P3(PYKM4L)。
实施例3工程菌株B.subtilis P1、P2、P3和B.subtilis 168核苷合成能力对比
1.培养基:
(1)种子培养基配方(g/L):葡萄糖20,酵母粉5,玉米浆干粉5,磷酸二氢钾3,硫酸镁0.5,硫酸亚铁0.02,硫酸锰0.01,pH 7.0~7.2,121℃下灭菌20min。
(2)发酵培养基配方(g/L):葡萄糖60,酵母粉3.5,磷酸二氢钾3,硫酸铵25,硫酸锰0.01,硫酸镁5,味精10,玉米浆干粉15,碳酸钙25,pH 7.0~7.2,121℃下灭菌20min。
2.培养方法
(1)将菌株三区划线LB平板,37℃过夜培养;
(2)挑单菌落接种至30mL种子培养基中,110rpm,34℃培养7~8h;
(3)按10%接种量转接至30ml发酵培养基中,摇床转速130rpm,35.5℃培养46h;
发酵结果见表3(取3个平行试验的平均值),从结果可以看出,同出发菌株B.subtilis 168相比,工程菌的肌苷、鸟苷和腺苷积累量均有不同程度的提高。P1(PYKV1L)、P2(PYKM4V)、P3(PYKM4L)产苷量如下:肌苷产量分别为0.8g/L、1.2g/L、1.3g/L;鸟苷产量分别是1.1g/L、1.3g/L、1.3g/L;腺苷产量分别是1.3g/L、1.5g/L、1.4g/L,这说明PYK突变体可以显著提升芽孢杆菌中核苷的积累量。PYK突变体明显减弱了丙酮酸激酶的酶活,导致了磷酸烯醇式丙酮酸更少的生成丙酮酸,让其更多的流向核苷生成途径中,促进了腺苷等嘌呤核苷酸的合成。
表3工程菌株产核苷检测
菌株 | B.s 168 | P1 | P2 | P3 |
肌苷(g/L) | 0.15 | 0.8 | 1.2 | 1.3 |
鸟苷(g/L) | 0.1 | 1.1 | 1.3 | 1.3 |
腺苷(g/L) | 0.12 | 1.3 | 1.5 | 1.4 |
本发明就此推理,该发明内容适用于枯草芽孢杆菌的其他株系。因此,本发明对实验室保藏的一株来源于枯草芽孢杆菌168的腺苷生产菌B.subtilis A5和肌苷生产菌ATCC13952进行了丙酮酸激酶修饰改造,同样达到了提高核苷产率的目的。
实施例4丙酮酸激酶突变体PYKV1L、PYKM4V、PYKM4L和B.subtilis A5中丙酮酸激酶活性对比
酶活的检测原理及方法同实施例1。表达菌株为B.subtilis A5(△pyk)。酶活测定结果见表4。从实验结果可以看出,同B.subtilis A5(B.s A5)中的丙酮酸激酶相比,PYKV1L、PYKM4V、PYKM4L的丙酮酸激酶突变体酶活显著降低。
表4工程菌株酶活测定
菌株 | B.s A5 | PYKV1L | PYKM4V | PYKM4L |
酶活(μM) | 259 | 120 | 115 | 123 |
实施例5在B.subtilis A5中引入PYK突变
构建原理及方法同实施例2,出发菌株为B.subtilis A5,获得含有目的突变的菌株:1、丙酮酸激酶第1位缬氨酸变为亮氨酸,命名为B.subtilis E1(PYKV1L);2、丙酮酸激酶第4位甲硫氨酸变为缬氨酸,命名为B.subtilis E2(PYKM4V);3、丙酮酸激酶第4位甲硫氨酸变为亮氨酸,命名为B.subtilis E3(PYKM4L)。其中,本实施例中获得的PYKV1L、PYKM4V、PYKM4L同源片段的核苷酸序列如实施例2中所述。
实施例6工程菌株菌株B.subtilis E1、E2、E3和B.subtilis A5核苷合成能力对比
培养基及发酵方法同实施例3。
发酵结果见表5(取3个生物学平行试验的平均值),从结果可以看出,同出发菌株B.subtilis A5相比,工程菌的核苷积累量均有不同程度的提高。E1(PYKV1L)、E2(PYKM4V)、E3(PYKM4L)产苷量如下:肌苷产量分别是1.03g/L、1.3g/L、1.44g/L;鸟苷产量分别是0.96g/L、1.47g/L、1.38g/L;腺苷产量分别是5.31g/L、6.9g/L、6.86g/L。PYK基因的突变体可以显著提升芽孢杆菌中核苷积累量。
表5工程菌株产核苷检测
实施例7丙酮酸激酶突变体PYKV1L、PYKM4V、PYKM4L和ATCC 13952中丙酮酸激酶活性对比
酶活的检测原理及方法同实施例1。表达菌株为ATCC 13952。酶活测定结果见表6。从实验结果可以看出,同ATCC 13952中的丙酮酸激酶相比,PYKV1L、PYKM4V、PYKM4L的丙酮酸激酶突变体酶活显著降低。
表6工程菌株酶活测定
菌株 | ATCC 13952 | PYKV1L | PYKM4V | PYKM4L |
酶活(μM) | 276 | 112 | 133 | 103 |
实施例8在ATCC 13952中引入PYK突变
构建原理及方法同实施例2。
获得含有丙酮酸激酶突变的菌株:1、丙酮酸激酶第1位缬氨酸变为亮氨酸,命名为B.a C1(PYKV1L);2、丙酮酸激酶第4位甲硫氨酸变为缬氨酸,命名为B.a C2(PYKM4V);3、丙酮酸激酶第4位甲硫氨酸变为亮氨酸,命名为B.a C3(PYKM4L)。
其中,本实施例中获得的PYKV1L、PYKM4V、PYKM4L同源片段的核苷酸序列如SEQ IDNo.6-8所示。
实施例9工程菌株B.a C1、C2、C3和ATCC 13952核苷合成能力对比
培养基及发酵方法同实施例3。
发酵结果见表7(取3个生物学平行试验的平均值),从结果可以看出,同出发菌株ATCC 13952相比,工程菌的核苷积累量均有不同程度的提高。C1(PYKV1L)、C2(PYKM4V)、C3(PYKM4L)产苷量如下:肌苷产量分别是5.5g/L、6.27g/L、6.16g/L;鸟苷产量分别是1.2g/L、1.6g/L、1.68g/L;腺苷产量分别是1.3g/L、1.5g/L、1.7g/L。PYK基因的突变体可以显著提升芽孢杆菌中核苷积累量。
表7工程菌株产核苷检测
菌株 | ATCC 13952 | C1 | C2 | C3 |
肌苷(g/L) | 3.4 | 5.5 | 6.27 | 6.16 |
鸟苷(g/L) | 0.2 | 1.2 | 1.6 | 1.68 |
腺苷(g/L) | 0.24 | 1.3 | 1.5 | 1.7 |
本发明的菌株的构建,其步骤的前后顺序不限定,本领域的技术人员按本发明公开的内容达到本发明的目的均属于本发明的保护范围。
本发明中的菌株代号如B.subtilis P1和B.subtilis E2等是为了方便描述,但不应理解为对本发明的限定。上述方法构建的包含芽孢杆菌丙酮酸激酶突变基因PYKV1L、PYKM4V、PYKM4L的工程菌的用途,包括但是不局限于生产核苷。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 廊坊梅花生物技术开发有限公司
<120> 一种丙酮酸激酶突变体、重组微生物及其构建方法与应用
<130> KHP221111657.6
<160> 24
<170> SIPOSequenceListing 1.0
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Ser Glu Ser Ile Glu Met Leu Thr Lys Leu Met Glu Ser Gly Met Asn
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Arg Ile Lys Asn Ile Arg Glu Ala Ser Lys Lys Leu Gly Lys Asn Val
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Gly Leu Val His Asp Val Glu Gln Gly Ser Thr Ile Leu Leu Asp Asp
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Val Asn Val Pro Gly Val Ser Val Asn Leu Pro Gly Ile Thr Glu Lys
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Ala Pro Ser Phe Ile Arg Arg Ser Thr Asp Val Leu Glu Ile Arg Glu
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Glu Asn Gln Glu Gly Val Asp Asn Ile Asp Ala Ile Leu Glu Val Ser
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Asp Gly Leu Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Ala
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Glu Glu Val Pro Leu Val Gln Lys Glu Leu Ile Lys Lys Cys Asn Ala
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Val Asn Glu Met Lys Lys Thr Lys Ile Val Cys Thr Ile Gly Pro Ala
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ttgaactaaa tgagaaaaac taaaattgtt tgtaccatcg gtccggcaag tgaaagtatt 60
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acggatgtgc tcgaaatccg tgagcttctt gaagagcaca acgctcagga tattcaaatc 660
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acacaaatgc ttgacagcat gcagcgcaac ccgcgtccga ctcgtgcgga agcaagtgac 900
gttgcaaacg cgatcttcga cggaacagat gcgatcatgc tttctggtga aactgctgcc 960
ggaagttacc cggttgaagc agttcaaaca atgcataaca tcgcgtcccg ttctgaagaa 1020
gcattaaatt ataaagaaat tctctcaaaa cgcagagacc aagtgggcat gacaattaca 1080
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attgttgcgg ttactgtaaa tgactctatt tccagaaagc ttgccctcgt atctggcgta 1260
ttcgcggaaa gcggccaaaa tgcgagctca acagatgaga tgcttgagga tgctgtccaa 1320
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gtgaactaag tgagaaaaac taaaattgtt tgtaccatcg gtccggcaag tgaaagtatt 60
gaaatgctta cgaaattaat ggagtcagga atgaacgtgg ctcgattaaa cttttctcac 120
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gaaaacggcg gtattgagct tgaaacaggc aaagagctca ttatttcaat ggacgaggtt 300
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ggttcaacga ttctgttaga tgacggcctt atcggtcttg aggtacttga tgtagatgcc 420
gctaaacgcg aaatcaaaac aaaagtatta aacaacggaa cactcaaaaa taaaaaaggt 480
gttaacgtac cgggcgtaag tgtcaatctt ccggggatta ctgaaaagga tgcgcgagac 540
atcgttttcg gtattgagca aggagtagac ttcatcgcac catctttcat tcgacgttct 600
acggatgtgc tcgaaatccg tgagcttctt gaagagcaca acgctcagga tattcaaatc 660
atccctaaaa tcgaaaacca agagggcgtt gacaacatcg atgcgattct cgaagtgtct 720
gacggcttaa tggttgcacg cggagactta ggtgtggaaa ttccagctga agaagtgccg 780
cttgtgcaaa aagaactgat caaaaaatgc aacgcgctgg gcaaacctgt tattacagcg 840
acacaaatgc ttgacagcat gcagcgcaac ccgcgtccga ctcgtgcgga agcaagtgac 900
gttgcaaacg cgatcttcga cggaacagat gcgatcatgc tttctggtga aactgctgcc 960
ggaagttacc cggttgaagc agttcaaaca atgcataaca tcgcgtcccg ttctgaagaa 1020
gcattaaatt ataaagaaat tctctcaaaa cgcagagacc aagtgggcat gacaattaca 1080
gacgcaattg gacaatctgt cgcacatacg gcgattaacc tgaatgctgc tgcgatcgta 1140
acgccgacag aaagcggcca tacagcacgt atgattgcaa aataccgtcc gcaggctccg 1200
attgttgcgg ttactgtaaa tgactctatt tccagaaagc ttgccctcgt atctggcgta 1260
ttcgcggaaa gcggccaaaa tgcgagctca acagatgaga tgcttgagga tgctgtccaa 1320
aaatcattga acagcggaat tgtaaaacac ggcgatctta tcgttattac agctggcact 1380
gtcggtgagt ccggcactac gaacttaatg aaggttcata ctgtcggcga tatcatcgct 1440
aaaggccaag gcattggacg caaatcagct tacggtccgg ttgtcgttgc acaaaatgca 1500
aaagaagctg agcaaaaaat gactgacggt gcggtacttg ttaccaaaag cactgaccgt 1560
gatatgattg catcccttga aaaagcgtct gctcttatta cagaagaagg cggtttgact 1620
agccatgctg cggtagtcgg attaagcctt ggcatcccgg ttatcgtggg tctggaaaat 1680
gcgacatcta ttttgacaga tggccaggat attacagttg acgcttccag aggcgcagtc 1740
tatcaaggcc gtgcgagcgt tctttaa 1767
<210> 5
<211> 1767
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gtgaactaat tgagaaaaac taaaattgtt tgtaccatcg gtccggcaag tgaaagtatt 60
gaaatgctta cgaaattaat ggagtcagga atgaacgtgg ctcgattaaa cttttctcac 120
ggagattttg aggagcacgg tgcaagaatt aaaaatatcc gcgaagcaag taaaaaactt 180
ggcaagaacg ttggaattct gcttgataca aaaggtcctg aaatccgcac acatacaatg 240
gaaaacggcg gtattgagct tgaaacaggc aaagagctca ttatttcaat ggacgaggtt 300
gtaggaacaa cagataaaat ttcagtgaca tatgaaggtt tagtccatga cgttgaacaa 360
ggttcaacga ttctgttaga tgacggcctt atcggtcttg aggtacttga tgtagatgcc 420
gctaaacgcg aaatcaaaac aaaagtatta aacaacggaa cactcaaaaa taaaaaaggt 480
gttaacgtac cgggcgtaag tgtcaatctt ccggggatta ctgaaaagga tgcgcgagac 540
atcgttttcg gtattgagca aggagtagac ttcatcgcac catctttcat tcgacgttct 600
acggatgtgc tcgaaatccg tgagcttctt gaagagcaca acgctcagga tattcaaatc 660
atccctaaaa tcgaaaacca agagggcgtt gacaacatcg atgcgattct cgaagtgtct 720
gacggcttaa tggttgcacg cggagactta ggtgtggaaa ttccagctga agaagtgccg 780
cttgtgcaaa aagaactgat caaaaaatgc aacgcgctgg gcaaacctgt tattacagcg 840
acacaaatgc ttgacagcat gcagcgcaac ccgcgtccga ctcgtgcgga agcaagtgac 900
gttgcaaacg cgatcttcga cggaacagat gcgatcatgc tttctggtga aactgctgcc 960
ggaagttacc cggttgaagc agttcaaaca atgcataaca tcgcgtcccg ttctgaagaa 1020
gcattaaatt ataaagaaat tctctcaaaa cgcagagacc aagtgggcat gacaattaca 1080
gacgcaattg gacaatctgt cgcacatacg gcgattaacc tgaatgctgc tgcgatcgta 1140
acgccgacag aaagcggcca tacagcacgt atgattgcaa aataccgtcc gcaggctccg 1200
attgttgcgg ttactgtaaa tgactctatt tccagaaagc ttgccctcgt atctggcgta 1260
ttcgcggaaa gcggccaaaa tgcgagctca acagatgaga tgcttgagga tgctgtccaa 1320
aaatcattga acagcggaat tgtaaaacac ggcgatctta tcgttattac agctggcact 1380
gtcggtgagt ccggcactac gaacttaatg aaggttcata ctgtcggcga tatcatcgct 1440
aaaggccaag gcattggacg caaatcagct tacggtccgg ttgtcgttgc acaaaatgca 1500
aaagaagctg agcaaaaaat gactgacggt gcggtacttg ttaccaaaag cactgaccgt 1560
gatatgattg catcccttga aaaagcgtct gctcttatta cagaagaagg cggtttgact 1620
agccatgctg cggtagtcgg attaagcctt ggcatcccgg ttatcgtggg tctggaaaat 1680
gcgacatcta ttttgacaga tggccaggat attacagttg acgcttccag aggcgcagtc 1740
tatcaaggcc gtgcgagcgt tctttaa 1767
<210> 6
<211> 1767
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttgaacgagt tgaaaaaaac aaaaatcgtt tgtacaatcg gtccggccag tgaaagtgta 60
gaaatgctga caaagttgat ggaagcgggt atgaacgttg ctcgtctgaa tttttcacac 120
ggtgatttcg aagagcacgg cgcaagaatc aaaaacatca gagaagcaag caaaaagctc 180
ggcaaaaacg tgggaattct tcttgatacg aaaggcccgg aaatccgcac gcacgatatg 240
gaaaacggcg agcttgagct tcaggcggga aatgaaatta tcgtttctac gaaacaggtg 300
ctgggaacac ttgaaaagtt ctctgtttct tatgaaggcc tggcagacga cgtgtcagca 360
ggatctatca tcctgttgga tgacggttta atcggattgg aagtgcttga atcaaatccg 420
gaaaaacacg aaatcaaaac aaaaatctta aacagcggca cgctgaaaaa taaaaaaggc 480
gtaaacgttc cgggtgtcag cgtcaacctt ccgggaatta ctgaaaaaga cgccaaagat 540
atcgtattcg gtattgagca aggcgtagat ttcatcgctg cgtcatttgt gcgccgttct 600
acggatgtcc tggaaatccg cgaactgctt gaagagcaca acgcgtctga cattcaaatc 660
atccctaaaa tcgagaacca ggaaggcgtg gacaacctgg atgcgatcct ggaagtttct 720
gacggcttaa tggtagcgcg cggagacctc ggcgttgaga ttccggctga agaagtgccg 780
ctcgtgcaaa aagaaatgat caaaaaatgc aacgcgctcg gcaaaccggt tatcactgcg 840
acacaaatgc ttgacagcat gcagcgcaac cctcgtccga cacgcgctga agcaagtgac 900
gttgccaacg ccattttcga cggcacagat gctatcatgc tttccggtga aacagctgcg 960
ggacaatatc ctgttgaggc ggttcaaaca atgtttaaca tcgccacacg cacggaagaa 1020
tctctgaact acaaagaaat tctgtctaaa cgcagagatc aggtcggcat gacgattacc 1080
gatgcgatcg gccaatcagt tgcgcatacg gcgatcaacc tgaatgcagc agctatcgtt 1140
acgcctacag aaagcggcca tacggcgcgt atgatcgcga aataccgtcc tcaggctccg 1200
attgtagccg ttacggtgaa cgagtctgta tcacgcaaac tcggtcttgt attcggtgta 1260
ttcccagcaa gcggacaaaa cgcaaattca acagatgaaa tgcttgaaga tgcggtacaa 1320
aaatcacttg acagcggcat cgtgaaacgc ggtgatctga ttgtcattac agccggttct 1380
gtcggtgaat caggcacgac gaatctgatg aaagtccaca ctgtcggcag cattgtcgca 1440
aaagggcagg gcatcggacg caaatccgct ttcggtcctg tcgtagttgc aaaaaatgcg 1500
aaagaagctg aacaaaaaat gacagacggc gcggtattgg ttgtctcaag cactgaccgc 1560
gacatgatcg cttcacttga aaaagcatcc gcgctcatca ctgaagaagg cggtctgaca 1620
agccatgctg ccgtggtcgg tttaagcttg ggcattccgg ttatcgtagg cctggagaac 1680
gcaacttcag ttttaacaga aggacaggtt atcacagttg acgctgcccg cggcgctgtg 1740
tatgaaggcc gcgcaagcgt tctttaa 1767
<210> 7
<211> 1767
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtgaacgagg tgaaaaaaac aaaaatcgtt tgtacaatcg gtccggccag tgaaagtgta 60
gaaatgctga caaagttgat ggaagcgggt atgaacgttg ctcgtctgaa tttttcacac 120
ggtgatttcg aagagcacgg cgcaagaatc aaaaacatca gagaagcaag caaaaagctc 180
ggcaaaaacg tgggaattct tcttgatacg aaaggcccgg aaatccgcac gcacgatatg 240
gaaaacggcg agcttgagct tcaggcggga aatgaaatta tcgtttctac gaaacaggtg 300
ctgggaacac ttgaaaagtt ctctgtttct tatgaaggcc tggcagacga cgtgtcagca 360
ggatctatca tcctgttgga tgacggttta atcggattgg aagtgcttga atcaaatccg 420
gaaaaacacg aaatcaaaac aaaaatctta aacagcggca cgctgaaaaa taaaaaaggc 480
gtaaacgttc cgggtgtcag cgtcaacctt ccgggaatta ctgaaaaaga cgccaaagat 540
atcgtattcg gtattgagca aggcgtagat ttcatcgctg cgtcatttgt gcgccgttct 600
acggatgtcc tggaaatccg cgaactgctt gaagagcaca acgcgtctga cattcaaatc 660
atccctaaaa tcgagaacca ggaaggcgtg gacaacctgg atgcgatcct ggaagtttct 720
gacggcttaa tggtagcgcg cggagacctc ggcgttgaga ttccggctga agaagtgccg 780
ctcgtgcaaa aagaaatgat caaaaaatgc aacgcgctcg gcaaaccggt tatcactgcg 840
acacaaatgc ttgacagcat gcagcgcaac cctcgtccga cacgcgctga agcaagtgac 900
gttgccaacg ccattttcga cggcacagat gctatcatgc tttccggtga aacagctgcg 960
ggacaatatc ctgttgaggc ggttcaaaca atgtttaaca tcgccacacg cacggaagaa 1020
tctctgaact acaaagaaat tctgtctaaa cgcagagatc aggtcggcat gacgattacc 1080
gatgcgatcg gccaatcagt tgcgcatacg gcgatcaacc tgaatgcagc agctatcgtt 1140
acgcctacag aaagcggcca tacggcgcgt atgatcgcga aataccgtcc tcaggctccg 1200
attgtagccg ttacggtgaa cgagtctgta tcacgcaaac tcggtcttgt attcggtgta 1260
ttcccagcaa gcggacaaaa cgcaaattca acagatgaaa tgcttgaaga tgcggtacaa 1320
aaatcacttg acagcggcat cgtgaaacgc ggtgatctga ttgtcattac agccggttct 1380
gtcggtgaat caggcacgac gaatctgatg aaagtccaca ctgtcggcag cattgtcgca 1440
aaagggcagg gcatcggacg caaatccgct ttcggtcctg tcgtagttgc aaaaaatgcg 1500
aaagaagctg aacaaaaaat gacagacggc gcggtattgg ttgtctcaag cactgaccgc 1560
gacatgatcg cttcacttga aaaagcatcc gcgctcatca ctgaagaagg cggtctgaca 1620
agccatgctg ccgtggtcgg tttaagcttg ggcattccgg ttatcgtagg cctggagaac 1680
gcaacttcag ttttaacaga aggacaggtt atcacagttg acgctgcccg cggcgctgtg 1740
tatgaaggcc gcgcaagcgt tctttaa 1767
<210> 8
<211> 1767
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtgaacgagt taaaaaaaac aaaaatcgtt tgtacaatcg gtccggccag tgaaagtgta 60
gaaatgctga caaagttgat ggaagcgggt atgaacgttg ctcgtctgaa tttttcacac 120
ggtgatttcg aagagcacgg cgcaagaatc aaaaacatca gagaagcaag caaaaagctc 180
ggcaaaaacg tgggaattct tcttgatacg aaaggcccgg aaatccgcac gcacgatatg 240
gaaaacggcg agcttgagct tcaggcggga aatgaaatta tcgtttctac gaaacaggtg 300
ctgggaacac ttgaaaagtt ctctgtttct tatgaaggcc tggcagacga cgtgtcagca 360
ggatctatca tcctgttgga tgacggttta atcggattgg aagtgcttga atcaaatccg 420
gaaaaacacg aaatcaaaac aaaaatctta aacagcggca cgctgaaaaa taaaaaaggc 480
gtaaacgttc cgggtgtcag cgtcaacctt ccgggaatta ctgaaaaaga cgccaaagat 540
atcgtattcg gtattgagca aggcgtagat ttcatcgctg cgtcatttgt gcgccgttct 600
acggatgtcc tggaaatccg cgaactgctt gaagagcaca acgcgtctga cattcaaatc 660
atccctaaaa tcgagaacca ggaaggcgtg gacaacctgg atgcgatcct ggaagtttct 720
gacggcttaa tggtagcgcg cggagacctc ggcgttgaga ttccggctga agaagtgccg 780
ctcgtgcaaa aagaaatgat caaaaaatgc aacgcgctcg gcaaaccggt tatcactgcg 840
acacaaatgc ttgacagcat gcagcgcaac cctcgtccga cacgcgctga agcaagtgac 900
gttgccaacg ccattttcga cggcacagat gctatcatgc tttccggtga aacagctgcg 960
ggacaatatc ctgttgaggc ggttcaaaca atgtttaaca tcgccacacg cacggaagaa 1020
tctctgaact acaaagaaat tctgtctaaa cgcagagatc aggtcggcat gacgattacc 1080
gatgcgatcg gccaatcagt tgcgcatacg gcgatcaacc tgaatgcagc agctatcgtt 1140
acgcctacag aaagcggcca tacggcgcgt atgatcgcga aataccgtcc tcaggctccg 1200
attgtagccg ttacggtgaa cgagtctgta tcacgcaaac tcggtcttgt attcggtgta 1260
ttcccagcaa gcggacaaaa cgcaaattca acagatgaaa tgcttgaaga tgcggtacaa 1320
aaatcacttg acagcggcat cgtgaaacgc ggtgatctga ttgtcattac agccggttct 1380
gtcggtgaat caggcacgac gaatctgatg aaagtccaca ctgtcggcag cattgtcgca 1440
aaagggcagg gcatcggacg caaatccgct ttcggtcctg tcgtagttgc aaaaaatgcg 1500
aaagaagctg aacaaaaaat gacagacggc gcggtattgg ttgtctcaag cactgaccgc 1560
gacatgatcg cttcacttga aaaagcatcc gcgctcatca ctgaagaagg cggtctgaca 1620
agccatgctg ccgtggtcgg tttaagcttg ggcattccgg ttatcgtagg cctggagaac 1680
gcaacttcag ttttaacaga aggacaggtt atcacagttg acgctgcccg cggcgctgtg 1740
tatgaaggcc gcgcaagcgt tctttaa 1767
<210> 9
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
taatacgact cactataggg tcgaccaaga agctcacgga tttcgagcac 50
<210> 10
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tgaagatttc agaaggaatt gaaccaaatg agaaaaacta aaattgtt 48
<210> 11
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aattttagtt tttctcattt ggttcaattc cttctgaaat cttcag 46
<210> 12
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gatgaagatt atttcttaat ctagaattcg cgatacagcg acttctcatg 50
<210> 13
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
taatacgact cactataggg tcgaccaaga agctcacgga tttcgagcac 50
<210> 14
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atttcagaag gaagtgaacc aagtgagaaa aactaaaatt gtttgtacca t 51
<210> 15
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aaacaatttt agtttttctc acttggttca cttccttctg aaatcttcag 50
<210> 16
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gatgaagatt atttcttaat ctagaattcg cgatacagcg acttctcatg 50
<210> 17
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
taatacgact cactataggg tcgaccaaga agctcacgga tttcgagcac 50
<210> 18
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
agaaggaagt gaaccaattg agaaaaacta aaattgtttg taccatcggt 50
<210> 19
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gtacaaacaa ttttagtttt tctcaattgg ttcacttcct tctgaaat 48
<210> 20
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gatgaagatt atttcttaat ctagaattcg cgatacagcg acttctcatg 50
<210> 21
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
aagcttttaa agaacgctcg cacggc 26
<210> 22
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ggatccaagg ctgaagattt cagaagg 27
<210> 23
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tacgcaattt tagtttttct catt 24
<210> 24
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aaacaatgag aaaaactaaa attg 24
Claims (10)
1.一种丙酮酸激酶突变体,其特征在于,以SEQ ID NO.1或2所示的氨基酸序列为参考序列,所述丙酮酸激酶突变体含有第1位缬氨酸突变为亮氨酸,或第4位甲硫氨酸突变为缬氨酸或亮氨酸。
2.编码权利要求1所述的丙酮酸激酶突变体的核酸。
3.根据权利要求2所述的核酸,其特征在于,具有如SEQ ID NO.3~8任一所示的核苷酸序列。
4.含有权利要求2或3所述的核酸的生物材料,所述生物材料为表达盒、载体或宿主细胞。
5.一种重组微生物,其特征在于,所述重组微生物表达丙酮酸激酶突变体,所述丙酮酸激酶突变体如权利要求1所述;所述重组微生物的出发菌株为芽孢杆菌或棒状杆菌。
6.根据权利要求5所述的重组微生物,其特征在于,所述重组微生物的出发菌株为枯草芽孢杆菌、解淀粉芽孢杆菌、短小芽孢杆菌或产氨棒杆菌。
7.根据权利要求6所述的重组微生物,其特征在于,所述重组微生物的出发菌株为枯草芽孢杆菌或解淀粉芽孢杆菌。
8.权利要求1所述的丙酮酸激酶突变体,或权利要求2或3所述的核酸,或权利要求4所述的生物材料,或权利要求5、6或7所述的重组微生物的如下任一种应用:
(1)在发酵生产核苷及其衍生物中的应用;
(2)在用于生产核苷及其衍生物的微生物遗传育种中的应用;
(3)在提高发酵生产核苷及其衍生物产量上的应用。
9.一种核苷及其衍生物生产方法,其特征在于,包括以重组微生物进行发酵培养的步骤,所述重组微生物如权利要求5、6或7所述。
10.一种构建重组微生物的方法,其特征在于,包括使所述重组微生物表达丙酮酸激酶突变体的步骤,所述丙酮酸激酶突变体如权利要求1所述;所述重组微生物的出发菌株为芽孢杆菌或棒状杆菌,优选为枯草芽孢杆菌或解淀粉芽孢杆菌。
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