CN114426959A - 一种磷酸胆碱胞苷酰转移酶突变体及其应用 - Google Patents

一种磷酸胆碱胞苷酰转移酶突变体及其应用 Download PDF

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CN114426959A
CN114426959A CN202210067168.9A CN202210067168A CN114426959A CN 114426959 A CN114426959 A CN 114426959A CN 202210067168 A CN202210067168 A CN 202210067168A CN 114426959 A CN114426959 A CN 114426959A
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应汉杰
缪榕新
温庆仕
郑诚
王骏之
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Abstract

本发明基于NCBI序列号为NP_011718.1的野生型的磷酸胞苷胆碱酰转移酶的氨基酸进行改造，希望通过增加酸性氨基酸的数量，提高突变体的耐盐性，意外筛选得到了3种高耐盐性磷酸胞苷胆碱酰转移酶突变体，改造后的磷酸胆碱胞甘酰转移酶，相较于改造之前，具有更高的耐盐性，发酵过程中能提高CDP‑胆碱的产量，满足工业生产需求。

Description

一种磷酸胆碱胞苷酰转移酶突变体及其应用
技术领域
本发明涉及基因工程，具体涉及磷酸胆碱胞苷酰转移酶突变体的制备及应用。
背景技术
CDP-胆碱作为一种药物已被广泛用于治疗脑血管和心血管疾病。工业生产CDP-胆碱曾使用的是化学合成的方法，但是由于其底物转化率低、使用的试剂有毒性的等劣势，并不适合大批量生产。
在胞苷二磷酸胆碱的生产过程中，随着反应进行盐浓度逐渐升高，当盐浓度超过1200mM时，反应过程关键磷酸胆碱胞苷酰转移酶(来自酿酒酵母S288C NP_011718.1)活性降低了75％～80％。这种抑制会阻碍这一工艺在工业中的应用。因此，基于酿酒酵母S288C的NP_011718.1基因，构建改造的高耐盐性的磷酸胆碱胞苷酰转移酶。
发明内容
发明目的：为解决现有技术中存在的技术问题，本发明提供了改造的高耐盐性的磷酸胆碱胞苷酰转移酶的突变体。
为实现上述目的，本发明通过对NCBI序列号为NP_011718.1的磷酸胆碱胞苷酰转移酶进行突变，得到若干种磷酸胆碱胞苷酰转移酶突变体。、
其中，磷酸胞苷胆碱酰转移酶突变体M1的氨基酸序列与NCBI序列号NP_011718.1的磷酸胞苷胆碱酰转移酶的序列相比，将第14位赖氨酸突变为谷氨酸、将第25位赖氨酸突变为谷氨酸、将第68位赖氨酸突变为天冬氨酸、将第371位的亮氨酸突变为天冬氨酸、将第409位的精氨酸突变为天冬氨酸，所述磷酸胞苷胆碱酰转移酶突变体M1的氨基酸序列如SEQID NO:1所示。
磷酸胆碱胞苷酰转移酶突变体M2的氨基酸与磷酸胆碱胞苷酰转移酶突变体M1相比，将第28位的赖氨酸突变为谷氨酸、将第44位的氨基酸赖氨酸突变为天冬氨酸，将第62位的赖氨酸突变为天冬氨酸，将第82位的脯氨酸突变为天冬氨酸，将第415位的亮氨酸突变为天冬氨酸，所述磷酸胆碱胞苷酰转移酶突变体M2的氨基酸序列如SEQ ID NO:2所示。
磷酸胆碱胞苷酰转移酶突变体M3的氨基酸与磷酸胆碱胞苷酰转移酶突变体M2相比，将第9位的丝氨酸突变为谷氨酸，将第36位的苏氨酸突变为谷氨酸，将第304位的赖氨酸突变为天冬氨酸，将第316位的异亮氨酸突变为天冬氨酸，将第320位的亮氨酸突变为天冬氨酸，将第329位的天冬酰胺突变为谷氨酸，将第348位的丙氨酸突变为天冬氨酸，将第365位的脯氨酸突变为天冬氨酸，将第410位的亮氨酸突变为天冬氨酸，所述磷酸胆碱胞苷酰转移酶突变体M3的氨基酸序列如SEQ ID NO:3所示。
编码上述磷酸胆碱胞苷酰转移酶突变体的核苷酸序列也在本发明的保护范围之内。
具体地，SEQ ID NO:1对应的碱基序列如SEQ ID NO:4所示；SEQ NO.2对应的碱基序列如SEQ ID NO:5所示：SEQ ID NO:3对应的碱基序列如SEQ ID NO:6所示。
进一步地，本发明提出了包含上述编码磷酸胆碱胞苷酰转移酶突变体的核苷酸序列的表达载体，优选地，所述表达载体为pET-28a。
更进一步地，本发明提出了一种宿主细胞，所述宿主细胞包含上述表达载体。优选地，所述宿主细胞为大肠杆菌BL21(DE3)。
本发明进一步提出了上述磷酸胆碱胞苷酰转移酶突变体M1-M3在制备胞苷二磷酸胆碱上的应用。
有益效果：本发明通过对野生型的磷酸胆碱胞苷酰转移酶进行理性设计，意外的筛选得到3种高耐盐性的磷酸胆碱胞苷酰转移酶突变体，发酵过程中能提高CDP-胆碱的产量，满足工业生产需求。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
实施例1磷酸胞苷胆碱酰转移酶突变体M1-M3的构建。
本发明基于NCBI序列号为NP_011718.1的野生型的磷酸胞苷胆碱酰转移酶的氨基酸进行改造，希望通过增加酸性氨基酸的数量，提高突变体的耐盐性。
野生型的氨基酸序列为(SEQ ID NO:7)：
MANPTTGKSSIRAKLSNSSLSNLFKKNKNKRQREETEEQDNEDKDESKNQDENKDTQLTPRKRRRLTKEFEEKEARYTNELPKELRKYRPKGFRFNLPPTDRPIRIYADGVFDLFHLGHMKQLEQCKKAFPNVTLIVGVPSDKITHKLKGLTVLTDKQRCETLTHCRWVDEVVPNAPWCVTPEFLLEHKIDYVAHDDIPYVSADSDDIYKPIKEMGKFLTTQRTNGVSTSDIITKIIRDYDKYLMRNFARGATRQELNVSWLKKNELEFKKHINEFRSYFKKNQTNLNNASRDLYFEVREILLKKTLGKKLYSKLIGNELKKQNQRQRKQNFLDDPFTRKLIREASPATEFANEFTGENSTAKSPDDNGNLFSQEDDEDTNSNNTNTNSDSDSNTNSTPPSEDDDDNDRLTLENLTQKKKQSAN。
本申请设计了若干定点改造方案，其中意外筛选得到了3中具有高耐盐性磷酸胞苷胆碱酰转移酶突变体：
磷酸胞苷胆碱酰转移酶突变体M1通过如下方法得到：将野生型磷酸胞苷胆碱酰转移酶的第14位赖氨酸突变为谷氨酸、将第25位赖氨酸突变为谷氨酸、将第68位赖氨酸突变为天冬氨酸、将第371位的亮氨酸突变为天冬氨酸、将第409位的精氨酸突变为天冬氨酸，得到的磷酸胞苷胆碱酰转移酶突变体M1的氨基酸序列如SEQ ID NO:1所示：
MANPTTGKSSIRAELSNSSLSNLFEKNKNKRQREETEEQDNEDKDESKNQDENKDTQLTPRKRRRLDKEFEEKEARYTNELPKELRKYRPKGFRFNLPPTDRPIRIYADGVFDLFHLGHMKQLEQCKKAFPNVTLIVGVPSDKITHKLKGLTVLTDKQRCETLTHCRWVDEVVPNAPWCVTPEFLLEHKIDYVAHDDIPYVSADSDDIYKPIKEMGKFLTTQRTNGVSTSDIITKIIRDYDKYLMRNFARGATRQELNVSWLKKNELEFKKHINEFRSYFKKNQTNLNNASRDLYFEVREILLKKTLGKKLYSKLIGNELKKQNQRQRKQNFLDDPFTRKLIREASPATEFANEFTGENSTAKSDDDNGNLFSQEDDEDTNSNNTNTNSDSDSNTNSTPPSEDDDDNDRLTLENLTQKKKQSAN(SEQ ID NO:1)。
进一步地，本申请在M1的基础上，增加5个突变位点，具体地，将M1氨基酸序列的第28位的赖氨酸突变为谷氨酸、将第44位的氨基酸赖氨酸突变为天冬氨酸，将第62位的赖氨酸突变为天冬氨酸，将第82位的脯氨酸突变为天冬氨酸，将第415位的亮氨酸突变为天冬氨酸，所述磷酸胆碱胞苷酰转移酶突变体M2的氨基酸序列如SEQ ID NO:2所示：
MANPTTGKSSIRAELSNSSLSNLFEKNENKRQREETEEQDNEDDDESKNQDENKDTQLTPDKRRRLDKEFEEKEARYTNEDPKELRKYRPKGFRFNLPPTDRPIRIYADGVFDLFHLGHMKQLEQCKKAFPNVTLIVGVPSDKITHKLKGLTVLTDKQRCETLTHCRWVDEVVPNAPWCVTPEFLLEHKIDYVAHDDIPYVSADSDDIYKPIKEMGKFLTTQRTNGVSTSDIITKIIRDYDKYLMRNFARGATRQELNVSWLKKNELEFKKHINEFRSYFKKNQTNLNNASRDLYFEVREILLKKTLGKKLYSKLIGNELKKQNQRQRKQNFLDDPFTRKLIREASPATEFANEFTGENSTAKSDDDNGNLFSQEDDEDTNSNNTNTNSDSDSNTNSTPPSEDDDDNDDLTLENLTQKKKQSAN(SEQ ID NO:2)。
更进一步地，本申请在M2的基础上，新增加8个改造位点，将M2的第9位的丝氨酸突变为谷氨酸，将第36位的苏氨酸突变为谷氨酸，将第304位的赖氨酸突变为天冬氨酸，将第316位的异亮氨酸突变为天冬氨酸，将第320位的亮氨酸突变为天冬氨酸，将第329位的天冬酰胺突变为谷氨酸，将第348位的丙氨酸突变为天冬氨酸，将第365位的脯氨酸突变为天冬氨酸，将第410位的亮氨酸突变为天冬氨酸，得到的磷酸胆碱胞苷酰转移酶突变体M3的氨基酸序列如SEQ ID NO:3所示：
MANPTTGKESIRAELSNSSLSNLFEKNENKRQREEEEEQDNEDDDESKNQDENKDTQLTPDKRRRLDKEFEEKEARYTNEDPKELRKYRPKGFRFNLPPTDRPIRIYADGVFDLFHLGHMKQLEQCKKAFPNVTLIVGVPSDKITHKLKGLTVLTDKQRCETLTHCRWVDEVVPNAPWCVTPEFLLEHKIDYVAHDDIPYVSADSDDIYKPIKEMGKFLTTQRTNGVSTSDIITKIIRDYDKYLMRNFARGATRQELNVSWLKKNELEFKKHINEFRSYFKKNQTNLNNASRDLYFEVRDILLKKTLGKKDYSKDIGNELKKQEQRQRKQNFLDDPFTRKLIDEASPATEFANEFTGENDTAKSDDDNGNLFSQEDDEDTNSNNTNTNSDSDSNTNSTPPSEDDDDNDDLTLENLTQKKKQSAN(SEQ ID NO:3)。
按照上述磷酸胞苷胆碱酰转移酶突变体的氨基酸序列人工合成对应的碱基序列，扩增构建表达载体，表达CDP-胆碱并进行含量检测。
按照上述磷酸胞苷胆碱酰转移酶突变体的氨基酸序列人工合成对应的碱基序列(SEQ ID NO:4至SEQ ID NO:6)，并插入pET-28a表达载体，转化至大肠杆菌BL21(DE3)细胞中。含有突变体表达载体的大肠杆菌在TB培养基中培养至OD600＝0.6-0.8时，加入终浓度为0.2mM的IPTG以诱导蛋白表达。培养24h后收集菌体，用于催化反应。
实验例2磷酸胞苷胆碱酰转移酶突变体M1-M3在不同盐浓度和盐溶液中活性的检测。
将本申请突变得到的的高耐盐性磷酸胞苷胆碱酰转移酶突变体M1、M2和M3以及野生型的磷酸胞苷胆碱酰转移酶WT(NCBI序列号：NP_011718.1)使用4种不同的盐KAc、LiAc、NaAc和NH4Ac，按照300mM，900mM，1500mM浓度梯度实验，在3个实验组中使用表1的方法配置500ul反应体系，pH＝7.5，37℃摇床150rpm反应30min，取样200ul灭活5分钟，使用HPLC进行产量检测，得到CDP-胆碱的产量，计算获得活性。
表1酶活反应体系
Figure BDA0003480681170000051
表2实验组设置
Figure BDA0003480681170000052
实验结果如表3-1和表3-2所示，表中数值为该条件下酶活与无盐环境下酶活的比值(％)。(该处酶活定义为1分钟内，每1g含酶湿菌体催化生成CDPC的浓度)在4种不同的盐溶液中，所有改造的突变体M1、M2、M3在高盐浓度下活性均比WT高，当盐溶液浓度超过600mM时这种差异更明显，而且耐盐性随着突变体表面残基的突变数增加而增加。由此可见，经过改造的磷酸胞苷胆碱酰转移酶具有更高的耐盐性，能够满足工业生产的需求。
表3-1不同盐浓度下酶活
Figure BDA0003480681170000061
表3-2不同盐浓度下酶活
Figure BDA0003480681170000062
然而，申请人发现，对于野生型磷酸胞苷胆碱酰转移酶的突变具有不可预测性，申请人同样进行了下述改造，虽然同样是增加酸性氨基酸的个数，却未能提高耐盐性，反而失去了催化活性：
(1)对野生型进行T67S定点突变，得到的对应蛋白失去了催化活性；
(2)对野生型进行如下突变：F70S、R76H、F95S和I104N，得到的对应蛋白失去了催化活性；
(3)对野生型进行如下突变：I11F、N29S、T59A、G92C和Y107C，得到的对应蛋白失去了催化活性；
(4)对野生型进行如下突变：Q255D、N265D、E268G、E275G、K329R、E354D和E413A，得到的对应蛋白失去了催化活性。
本发明提供了高耐盐性磷酸胞苷胆碱转移酶的思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
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<110> 郑州大学
<120> 一种磷酸胆碱胞苷酰转移酶突变体及其应用
<160> 7
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<212> PRT
<213> 磷酸胞苷胆碱酰转移酶突变体M1氨基酸序列(Artificial Sequence)
<400> 1
Met Ala Asn Pro Thr Thr Gly Lys Ser Ser Ile Arg Ala Glu Leu Ser
1 5 10 15
Asn Ser Ser Leu Ser Asn Leu Phe Glu Lys Asn Lys Asn Lys Arg Gln
20 25 30
Arg Glu Glu Thr Glu Glu Gln Asp Asn Glu Asp Lys Asp Glu Ser Lys
35 40 45
Asn Gln Asp Glu Asn Lys Asp Thr Gln Leu Thr Pro Arg Lys Arg Arg
50 55 60
Arg Leu Asp Lys Glu Phe Glu Glu Lys Glu Ala Arg Tyr Thr Asn Glu
65 70 75 80
Leu Pro Lys Glu Leu Arg Lys Tyr Arg Pro Lys Gly Phe Arg Phe Asn
85 90 95
Leu Pro Pro Thr Asp Arg Pro Ile Arg Ile Tyr Ala Asp Gly Val Phe
100 105 110
Asp Leu Phe His Leu Gly His Met Lys Gln Leu Glu Gln Cys Lys Lys
115 120 125
Ala Phe Pro Asn Val Thr Leu Ile Val Gly Val Pro Ser Asp Lys Ile
130 135 140
Thr His Lys Leu Lys Gly Leu Thr Val Leu Thr Asp Lys Gln Arg Cys
145 150 155 160
Glu Thr Leu Thr His Cys Arg Trp Val Asp Glu Val Val Pro Asn Ala
165 170 175
Pro Trp Cys Val Thr Pro Glu Phe Leu Leu Glu His Lys Ile Asp Tyr
180 185 190
Val Ala His Asp Asp Ile Pro Tyr Val Ser Ala Asp Ser Asp Asp Ile
195 200 205
Tyr Lys Pro Ile Lys Glu Met Gly Lys Phe Leu Thr Thr Gln Arg Thr
210 215 220
Asn Gly Val Ser Thr Ser Asp Ile Ile Thr Lys Ile Ile Arg Asp Tyr
225 230 235 240
Asp Lys Tyr Leu Met Arg Asn Phe Ala Arg Gly Ala Thr Arg Gln Glu
245 250 255
Leu Asn Val Ser Trp Leu Lys Lys Asn Glu Leu Glu Phe Lys Lys His
260 265 270
Ile Asn Glu Phe Arg Ser Tyr Phe Lys Lys Asn Gln Thr Asn Leu Asn
275 280 285
Asn Ala Ser Arg Asp Leu Tyr Phe Glu Val Arg Glu Ile Leu Leu Lys
290 295 300
Lys Thr Leu Gly Lys Lys Leu Tyr Ser Lys Leu Ile Gly Asn Glu Leu
305 310 315 320
Lys Lys Gln Asn Gln Arg Gln Arg Lys Gln Asn Phe Leu Asp Asp Pro
325 330 335
Phe Thr Arg Lys Leu Ile Arg Glu Ala Ser Pro Ala Thr Glu Phe Ala
340 345 350
Asn Glu Phe Thr Gly Glu Asn Ser Thr Ala Lys Ser Asp Asp Asp Asn
355 360 365
Gly Asn Leu Phe Ser Gln Glu Asp Asp Glu Asp Thr Asn Ser Asn Asn
370 375 380
Thr Asn Thr Asn Ser Asp Ser Asp Ser Asn Thr Asn Ser Thr Pro Pro
385 390 395 400
Ser Glu Asp Asp Asp Asp Asn Asp Arg Leu Thr Leu Glu Asn Leu Thr
405 410 415
Gln Lys Lys Lys Gln Ser Ala Asn
420
<210> 2
<211> 424
<212> PRT
<213> 磷酸胞苷胆碱酰转移酶突变体M2氨基酸序列(Artificial Sequence)
<400> 2
Met Ala Asn Pro Thr Thr Gly Lys Ser Ser Ile Arg Ala Glu Leu Ser
1 5 10 15
Asn Ser Ser Leu Ser Asn Leu Phe Glu Lys Asn Glu Asn Lys Arg Gln
20 25 30
Arg Glu Glu Thr Glu Glu Gln Asp Asn Glu Asp Asp Asp Glu Ser Lys
35 40 45
Asn Gln Asp Glu Asn Lys Asp Thr Gln Leu Thr Pro Asp Lys Arg Arg
50 55 60
Arg Leu Asp Lys Glu Phe Glu Glu Lys Glu Ala Arg Tyr Thr Asn Glu
65 70 75 80
Asp Pro Lys Glu Leu Arg Lys Tyr Arg Pro Lys Gly Phe Arg Phe Asn
85 90 95
Leu Pro Pro Thr Asp Arg Pro Ile Arg Ile Tyr Ala Asp Gly Val Phe
100 105 110
Asp Leu Phe His Leu Gly His Met Lys Gln Leu Glu Gln Cys Lys Lys
115 120 125
Ala Phe Pro Asn Val Thr Leu Ile Val Gly Val Pro Ser Asp Lys Ile
130 135 140
Thr His Lys Leu Lys Gly Leu Thr Val Leu Thr Asp Lys Gln Arg Cys
145 150 155 160
Glu Thr Leu Thr His Cys Arg Trp Val Asp Glu Val Val Pro Asn Ala
165 170 175
Pro Trp Cys Val Thr Pro Glu Phe Leu Leu Glu His Lys Ile Asp Tyr
180 185 190
Val Ala His Asp Asp Ile Pro Tyr Val Ser Ala Asp Ser Asp Asp Ile
195 200 205
Tyr Lys Pro Ile Lys Glu Met Gly Lys Phe Leu Thr Thr Gln Arg Thr
210 215 220
Asn Gly Val Ser Thr Ser Asp Ile Ile Thr Lys Ile Ile Arg Asp Tyr
225 230 235 240
Asp Lys Tyr Leu Met Arg Asn Phe Ala Arg Gly Ala Thr Arg Gln Glu
245 250 255
Leu Asn Val Ser Trp Leu Lys Lys Asn Glu Leu Glu Phe Lys Lys His
260 265 270
Ile Asn Glu Phe Arg Ser Tyr Phe Lys Lys Asn Gln Thr Asn Leu Asn
275 280 285
Asn Ala Ser Arg Asp Leu Tyr Phe Glu Val Arg Glu Ile Leu Leu Lys
290 295 300
Lys Thr Leu Gly Lys Lys Leu Tyr Ser Lys Leu Ile Gly Asn Glu Leu
305 310 315 320
Lys Lys Gln Asn Gln Arg Gln Arg Lys Gln Asn Phe Leu Asp Asp Pro
325 330 335
Phe Thr Arg Lys Leu Ile Arg Glu Ala Ser Pro Ala Thr Glu Phe Ala
340 345 350
Asn Glu Phe Thr Gly Glu Asn Ser Thr Ala Lys Ser Asp Asp Asp Asn
355 360 365
Gly Asn Leu Phe Ser Gln Glu Asp Asp Glu Asp Thr Asn Ser Asn Asn
370 375 380
Thr Asn Thr Asn Ser Asp Ser Asp Ser Asn Thr Asn Ser Thr Pro Pro
385 390 395 400
Ser Glu Asp Asp Asp Asp Asn Asp Asp Leu Thr Leu Glu Asn Leu Thr
405 410 415
Gln Lys Lys Lys Gln Ser Ala Asn
420
<210> 3
<211> 424
<212> PRT
<213> 磷酸胞苷胆碱酰转移酶突变体M3氨基酸序列(Artificial Sequence)
<400> 3
Met Ala Asn Pro Thr Thr Gly Lys Glu Ser Ile Arg Ala Glu Leu Ser
1 5 10 15
Asn Ser Ser Leu Ser Asn Leu Phe Glu Lys Asn Glu Asn Lys Arg Gln
20 25 30
Arg Glu Glu Glu Glu Glu Gln Asp Asn Glu Asp Asp Asp Glu Ser Lys
35 40 45
Asn Gln Asp Glu Asn Lys Asp Thr Gln Leu Thr Pro Asp Lys Arg Arg
50 55 60
Arg Leu Asp Lys Glu Phe Glu Glu Lys Glu Ala Arg Tyr Thr Asn Glu
65 70 75 80
Asp Pro Lys Glu Leu Arg Lys Tyr Arg Pro Lys Gly Phe Arg Phe Asn
85 90 95
Leu Pro Pro Thr Asp Arg Pro Ile Arg Ile Tyr Ala Asp Gly Val Phe
100 105 110
Asp Leu Phe His Leu Gly His Met Lys Gln Leu Glu Gln Cys Lys Lys
115 120 125
Ala Phe Pro Asn Val Thr Leu Ile Val Gly Val Pro Ser Asp Lys Ile
130 135 140
Thr His Lys Leu Lys Gly Leu Thr Val Leu Thr Asp Lys Gln Arg Cys
145 150 155 160
Glu Thr Leu Thr His Cys Arg Trp Val Asp Glu Val Val Pro Asn Ala
165 170 175
Pro Trp Cys Val Thr Pro Glu Phe Leu Leu Glu His Lys Ile Asp Tyr
180 185 190
Val Ala His Asp Asp Ile Pro Tyr Val Ser Ala Asp Ser Asp Asp Ile
195 200 205
Tyr Lys Pro Ile Lys Glu Met Gly Lys Phe Leu Thr Thr Gln Arg Thr
210 215 220
Asn Gly Val Ser Thr Ser Asp Ile Ile Thr Lys Ile Ile Arg Asp Tyr
225 230 235 240
Asp Lys Tyr Leu Met Arg Asn Phe Ala Arg Gly Ala Thr Arg Gln Glu
245 250 255
Leu Asn Val Ser Trp Leu Lys Lys Asn Glu Leu Glu Phe Lys Lys His
260 265 270
Ile Asn Glu Phe Arg Ser Tyr Phe Lys Lys Asn Gln Thr Asn Leu Asn
275 280 285
Asn Ala Ser Arg Asp Leu Tyr Phe Glu Val Arg Asp Ile Leu Leu Lys
290 295 300
Lys Thr Leu Gly Lys Lys Asp Tyr Ser Lys Asp Ile Gly Asn Glu Leu
305 310 315 320
Lys Lys Gln Glu Gln Arg Gln Arg Lys Gln Asn Phe Leu Asp Asp Pro
325 330 335
Phe Thr Arg Lys Leu Ile Asp Glu Ala Ser Pro Ala Thr Glu Phe Ala
340 345 350
Asn Glu Phe Thr Gly Glu Asn Asp Thr Ala Lys Ser Asp Asp Asp Asn
355 360 365
Gly Asn Leu Phe Ser Gln Glu Asp Asp Glu Asp Thr Asn Ser Asn Asn
370 375 380
Thr Asn Thr Asn Ser Asp Ser Asp Ser Asn Thr Asn Ser Thr Pro Pro
385 390 395 400
Ser Glu Asp Asp Asp Asp Asn Asp Asp Leu Thr Leu Glu Asn Leu Thr
405 410 415
Gln Lys Lys Lys Gln Ser Ala Asn
420
<210> 4
<211> 1272
<212> DNA
<213> 磷酸胞苷胆碱酰转移酶突变体M1核苷酸序列(Artificial Sequence)
<400> 4
atggcgaacc cgaccaccgg caaaagcagc attcgcgcgg aactgagcaa cagcagcctg 60
agcaacctgt ttgaaaaaaa caaaaacaaa cgccagcgcg aagaaaccga agaacaggat 120
aacgaagata aagatgaaag caaaaaccag gatgaaaaca aagataccca gctgaccccg 180
cgcaaacgcc gccgcctgga taaagaattt gaagaaaaag aagcgcgcta taccaacgaa 240
ctgccgaaag aactgcgcaa atatcgcccg aaaggctttc gctttaacct gccgccgacc 300
gatcgcccga ttcgcattta tgcggatggc gtgtttgatc tgtttcatct gggccatatg 360
aaacagctgg aacagtgcaa aaaagcgttt ccgaacgtga ccctgattgt gggcgtgccg 420
agcgataaaa ttacccataa actgaaaggc ctgaccgtgc tgaccgataa acagcgctgc 480
gaaaccctga cccattgccg ctgggtggat gaagtggtgc cgaacgcgcc gtggtgcgtg 540
accccggaat ttctgctgga acataaaatt gattatgtgg cgcatgatga tattccgtat 600
gtgagcgcgg atagcgatga tatttataaa ccgattaaag aaatgggcaa atttctgacc 660
acccagcgca ccaacggcgt gagcaccagc gatattatta ccaaaattat tcgcgattat 720
gataaatatc tgatgcgcaa ctttgcgcgc ggcgcgaccc gccaggaact gaacgtgagc 780
tggctgaaaa aaaacgaact ggaatttaaa aaacatatta acgaatttcg cagctatttt 840
aaaaaaaacc agaccaacct gaacaacgcg agccgcgatc tgtattttga agtgcgcgaa 900
attctgctga aaaaaaccct gggcaaaaaa ctgtatagca aactgattgg caacgaactg 960
aaaaaacaga accagcgcca gcgcaaacag aactttctgg atgatccgtt tacccgcaaa 1020
ctgattcgcg aagcgagccc ggcgaccgaa tttgcgaacg aatttaccgg cgaaaacagc 1080
accgcgaaaa gcgatgatga taacggcaac ctgtttagcc aggaagatga tgaagatacc 1140
aacagcaaca acaccaacac caacagcgat agcgatagca acaccaacag caccccgccg 1200
agcgaagatg atgatgataa cgatcgcctg accctggaaa acctgaccca gaaaaaaaaa 1260
cagagcgcga ac 1272
<210> 5
<211> 1272
<212> DNA
<213> 磷酸胞苷胆碱酰转移酶突变体M2核苷酸序列(Artificial Sequence)
<400> 5
atggcgaacc cgaccaccgg caaaagcagc attcgcgcgg aactgagcaa cagcagcctg 60
agcaacctgt ttgaaaaaaa cgaaaacaaa cgccagcgcg aagaaaccga agaacaggat 120
aacgaagatg atgatgaaag caaaaaccag gatgaaaaca aagataccca gctgaccccg 180
gataaacgcc gccgcctgga taaagaattt gaagaaaaag aagcgcgcta taccaacgaa 240
gatccgaaag aactgcgcaa atatcgcccg aaaggctttc gctttaacct gccgccgacc 300
gatcgcccga ttcgcattta tgcggatggc gtgtttgatc tgtttcatct gggccatatg 360
aaacagctgg aacagtgcaa aaaagcgttt ccgaacgtga ccctgattgt gggcgtgccg 420
agcgataaaa ttacccataa actgaaaggc ctgaccgtgc tgaccgataa acagcgctgc 480
gaaaccctga cccattgccg ctgggtggat gaagtggtgc cgaacgcgcc gtggtgcgtg 540
accccggaat ttctgctgga acataaaatt gattatgtgg cgcatgatga tattccgtat 600
gtgagcgcgg atagcgatga tatttataaa ccgattaaag aaatgggcaa atttctgacc 660
acccagcgca ccaacggcgt gagcaccagc gatattatta ccaaaattat tcgcgattat 720
gataaatatc tgatgcgcaa ctttgcgcgc ggcgcgaccc gccaggaact gaacgtgagc 780
tggctgaaaa aaaacgaact ggaatttaaa aaacatatta acgaatttcg cagctatttt 840
aaaaaaaacc agaccaacct gaacaacgcg agccgcgatc tgtattttga agtgcgcgaa 900
attctgctga aaaaaaccct gggcaaaaaa ctgtatagca aactgattgg caacgaactg 960
aaaaaacaga accagcgcca gcgcaaacag aactttctgg atgatccgtt tacccgcaaa 1020
ctgattcgcg aagcgagccc ggcgaccgaa tttgcgaacg aatttaccgg cgaaaacagc 1080
accgcgaaaa gcgatgatga taacggcaac ctgtttagcc aggaagatga tgaagatacc 1140
aacagcaaca acaccaacac caacagcgat agcgatagca acaccaacag caccccgccg 1200
agcgaagatg atgatgataa cgatgatctg accctggaaa acctgaccca gaaaaaaaaa 1260
cagagcgcga ac 1272
<210> 6
<211> 1272
<212> DNA
<213> 磷酸胞苷胆碱酰转移酶突变体M3核苷酸序列(Artificial Sequence)
<400> 6
atggcgaacc cgaccaccgg caaagaaagc attcgcgcgg aactgagcaa cagcagcctg 60
agcaacctgt ttgaaaaaaa cgaaaacaaa cgccagcgcg aagaagaaga agaacaggat 120
aacgaagatg atgatgaaag caaaaaccag gatgaaaaca aagataccca gctgaccccg 180
gataaacgcc gccgcctgga taaagaattt gaagaaaaag aagcgcgcta taccaacgaa 240
gatccgaaag aactgcgcaa atatcgcccg aaaggctttc gctttaacct gccgccgacc 300
gatcgcccga ttcgcattta tgcggatggc gtgtttgatc tgtttcatct gggccatatg 360
aaacagctgg aacagtgcaa aaaagcgttt ccgaacgtga ccctgattgt gggcgtgccg 420
agcgataaaa ttacccataa actgaaaggc ctgaccgtgc tgaccgataa acagcgctgc 480
gaaaccctga cccattgccg ctgggtggat gaagtggtgc cgaacgcgcc gtggtgcgtg 540
accccggaat ttctgctgga acataaaatt gattatgtgg cgcatgatga tattccgtat 600
gtgagcgcgg atagcgatga tatttataaa ccgattaaag aaatgggcaa atttctgacc 660
acccagcgca ccaacggcgt gagcaccagc gatattatta ccaaaattat tcgcgattat 720
gataaatatc tgatgcgcaa ctttgcgcgc ggcgcgaccc gccaggaact gaacgtgagc 780
tggctgaaaa aaaacgaact ggaatttaaa aaacatatta acgaatttcg cagctatttt 840
aaaaaaaacc agaccaacct gaacaacgcg agccgcgatc tgtattttga agtgcgcgat 900
attctgctga aaaaaaccct gggcaaaaaa gattatagca aagatattgg caacgaactg 960
aaaaaacagg aacagcgcca gcgcaaacag aactttctgg atgatccgtt tacccgcaaa 1020
ctgattgatg aagcgagccc ggcgaccgaa tttgcgaacg aatttaccgg cgaaaacgat 1080
accgcgaaaa gcgatgatga taacggcaac ctgtttagcc aggaagatga tgaagatacc 1140
aacagcaaca acaccaacac caacagcgat agcgatagca acaccaacag caccccgccg 1200
agcgaagatg atgatgataa cgatgatctg accctggaaa acctgaccca gaaaaaaaaa 1260
cagagcgcga ac 1272
<210> 7
<211> 424
<212> PRT
<213> 磷酸胞苷胆碱酰转移酶野生型氨基酸序列(Artificial Sequence)
<400> 7
Met Ala Asn Pro Thr Thr Gly Lys Ser Ser Ile Arg Ala Lys Leu Ser
1 5 10 15
Asn Ser Ser Leu Ser Asn Leu Phe Lys Lys Asn Lys Asn Lys Arg Gln
20 25 30
Arg Glu Glu Thr Glu Glu Gln Asp Asn Glu Asp Lys Asp Glu Ser Lys
35 40 45
Asn Gln Asp Glu Asn Lys Asp Thr Gln Leu Thr Pro Arg Lys Arg Arg
50 55 60
Arg Leu Thr Lys Glu Phe Glu Glu Lys Glu Ala Arg Tyr Thr Asn Glu
65 70 75 80
Leu Pro Lys Glu Leu Arg Lys Tyr Arg Pro Lys Gly Phe Arg Phe Asn
85 90 95
Leu Pro Pro Thr Asp Arg Pro Ile Arg Ile Tyr Ala Asp Gly Val Phe
100 105 110
Asp Leu Phe His Leu Gly His Met Lys Gln Leu Glu Gln Cys Lys Lys
115 120 125
Ala Phe Pro Asn Val Thr Leu Ile Val Gly Val Pro Ser Asp Lys Ile
130 135 140
Thr His Lys Leu Lys Gly Leu Thr Val Leu Thr Asp Lys Gln Arg Cys
145 150 155 160
Glu Thr Leu Thr His Cys Arg Trp Val Asp Glu Val Val Pro Asn Ala
165 170 175
Pro Trp Cys Val Thr Pro Glu Phe Leu Leu Glu His Lys Ile Asp Tyr
180 185 190
Val Ala His Asp Asp Ile Pro Tyr Val Ser Ala Asp Ser Asp Asp Ile
195 200 205
Tyr Lys Pro Ile Lys Glu Met Gly Lys Phe Leu Thr Thr Gln Arg Thr
210 215 220
Asn Gly Val Ser Thr Ser Asp Ile Ile Thr Lys Ile Ile Arg Asp Tyr
225 230 235 240
Asp Lys Tyr Leu Met Arg Asn Phe Ala Arg Gly Ala Thr Arg Gln Glu
245 250 255
Leu Asn Val Ser Trp Leu Lys Lys Asn Glu Leu Glu Phe Lys Lys His
260 265 270
Ile Asn Glu Phe Arg Ser Tyr Phe Lys Lys Asn Gln Thr Asn Leu Asn
275 280 285
Asn Ala Ser Arg Asp Leu Tyr Phe Glu Val Arg Glu Ile Leu Leu Lys
290 295 300
Lys Thr Leu Gly Lys Lys Leu Tyr Ser Lys Leu Ile Gly Asn Glu Leu
305 310 315 320
Lys Lys Gln Asn Gln Arg Gln Arg Lys Gln Asn Phe Leu Asp Asp Pro
325 330 335
Phe Thr Arg Lys Leu Ile Arg Glu Ala Ser Pro Ala Thr Glu Phe Ala
340 345 350
Asn Glu Phe Thr Gly Glu Asn Ser Thr Ala Lys Ser Pro Asp Asp Asn
355 360 365
Gly Asn Leu Phe Ser Gln Glu Asp Asp Glu Asp Thr Asn Ser Asn Asn
370 375 380
Thr Asn Thr Asn Ser Asp Ser Asp Ser Asn Thr Asn Ser Thr Pro Pro
385 390 395 400
Ser Glu Asp Asp Asp Asp Asn Asp Arg Leu Thr Leu Glu Asn Leu Thr
405 410 415
Gln Lys Lys Lys Gln Ser Ala Asn
420

Claims (7)

1.一种磷酸胆碱胞苷酰转移酶突变体M3，其特征在于，所述磷酸胆碱胞苷酰转移酶突变体M3的氨基酸与权利要求2所述的磷酸胆碱胞苷酰转移酶突变体M2相比，将第9位的丝氨酸突变为谷氨酸，将第36位的苏氨酸突变为谷氨酸，将第304位的赖氨酸突变为天冬氨酸，将第316位的异亮氨酸突变为天冬氨酸，将第320位的亮氨酸突变为天冬氨酸，将第329位的天冬酰胺突变为谷氨酸，将第348位的丙氨酸突变为天冬氨酸，将第365位的脯氨酸突变为天冬氨酸，将第410位的亮氨酸突变为天冬氨酸，所述磷酸胆碱胞苷酰转移酶突变体M3的氨基酸序列如SEQ ID NO:3所示。
2.一种磷酸胆碱胞苷酰转移酶突变体M2，其特征在于，所述磷酸胆碱胞苷酰转移酶突变体M2的氨基酸与权利要求1所述的磷酸胆碱胞苷酰转移酶突变体M1相比，将第28位的赖氨酸突变为谷氨酸、将第44位的氨基酸赖氨酸突变为天冬氨酸，将第62位的赖氨酸突变为天冬氨酸，将第82位的脯氨酸突变为天冬氨酸，将第415位的亮氨酸突变为天冬氨酸，所述磷酸胆碱胞苷酰转移酶突变体M2的氨基酸序列如SEQ ID NO:2所示。
3.一种磷酸胆碱胞苷酰转移酶突变体M1，其特征在于，所述磷酸胞苷胆碱酰转移酶突变体M1的氨基酸序列与NCBI 序列号NP_011718.1的磷酸胞苷胆碱酰转移酶的序列相比，将第14位赖氨酸突变为谷氨酸、将第25位赖氨酸突变为谷氨酸、将第68位赖氨酸突变为天冬氨酸、将第371位的亮氨酸突变为天冬氨酸、将第409位的精氨酸突变为天冬氨酸，所述磷酸胞苷胆碱酰转移酶突变体M1的氨基酸序列如SEQ ID NO:1所示。
4.编码权利要求1-3任一项所述的磷酸胆碱胞苷酰转移酶突变体的核苷酸序列。
5.一种表达载体，其特征在于，所述表达载体包含权利要求4所述的核苷酸序列中的任意一种。
6.一种宿主细胞，其特征在于，所述宿主细胞包含权利要求5所述的表达载体。
7.权利要求1-3任一项所述的磷酸胆碱胞苷酰转移酶突变体在制备胞苷二磷酸胆碱上的应用。
CN202210067168.9A 2022-01-20 2022-01-20 一种磷酸胆碱胞苷酰转移酶突变体及其应用 Pending CN114426959A (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101709304A (zh) * 2009-11-20 2010-05-19 上海市农业科学院 突变的家蝇乙酰胆碱酯酶基因及其表达载体
CN111808899A (zh) * 2020-08-31 2020-10-23 宁波酶赛生物工程有限公司 一种胞磷胆碱钠的合成方法
CN112574970A (zh) * 2020-12-22 2021-03-30 江苏诚信药业有限公司 一种烟酰胺单核苷酸腺苷转移酶突变体及其应用

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Publication number Priority date Publication date Assignee Title
CN101709304A (zh) * 2009-11-20 2010-05-19 上海市农业科学院 突变的家蝇乙酰胆碱酯酶基因及其表达载体
CN111808899A (zh) * 2020-08-31 2020-10-23 宁波酶赛生物工程有限公司 一种胞磷胆碱钠的合成方法
CN112574970A (zh) * 2020-12-22 2021-03-30 江苏诚信药业有限公司 一种烟酰胺单核苷酸腺苷转移酶突变体及其应用

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