N-脱氧核糖转移酶Ⅱ催化合成克拉屈滨及其定向进化
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摘要
目的:使用表达N-脱氧核糖转移酶Ⅱ (Nucleoside deoxyribose transferase, NDT)的大肠杆菌重组工程菌E.coli BL21/pET-NDT,作为催化剂,合成克拉屈滨(Cladribine)。同时构建高通量的酶活筛选体系,利用其改造NDT,以期提高克拉屈滨的合成效率。方法:首先用野生型NDT催化克拉屈滨的合成。接着以黄嘌呤氧化酶和辣根过氧化物酶联合作用来检测NDT酶活。最后构建NDT随机突变体库,并筛选出突变体。结果:在10%DMSO的体系中,野生型NDT催化2-氯腺嘌呤合成克拉屈滨的转化率达到93%。同时使用构建的筛选体系在突变体库中筛选到了酶活发生改变的突变体。结论:本研究使用NDT作为催化剂,成功地一步合成了克拉屈滨。同时本研究构建的高通量筛选方法成功应用于改造NDT的酶学性质,为拓展NDT合成核苷类似物的能力提供了一种新的方法。
Objective: N-deoxyribosyltransferase II(NDT) was produced by recombinant strain E.coli BL21/pET-NDT. This recombinant strain was used as catalysis to synthesize cladribine. High-throughput screening system was established to engineer NDT, in order to increase the yield of cladribine. Methods: First, synthesize cladribine with wild NDT. Then combine Xanthine Oxidase with Horseradish Peroxidase to assay the activity of NDT in a high-throughput manner. Finally, construct a random mutant library of NDT,and screen the mutants of interest. Results: In the reaction mixture of 10 % DMSO, wild NDT catalysed the synthesis of cladribine, and the yield was 93%. The constructed random library was screened, and a mutant with altered activity was found. Conclusion: In this study,NDT was employed as a catalyst, and cladribine was synthesized successfully in one step. And the high-throughput screening system constructed in this study was successfully applied to the alternation of the NDT activity, which offers a new way to expand the ability of NDT in preparation of unnatural nucleoside analogs.
Objective: N-deoxyribosyltransferase II(NDT) was produced by recombinant strain E.coli BL21/pET-NDT. This recombinant strain was used as catalysis to synthesize cladribine. High-throughput screening system was established to engineer NDT, in order to increase the yield of cladribine. Methods: First, synthesize cladribine with wild NDT. Then combine Xanthine Oxidase with Horseradish Peroxidase to assay the activity of NDT in a high-throughput manner. Finally, construct a random mutant library of NDT,and screen the mutants of interest. Results: In the reaction mixture of 10 % DMSO, wild NDT catalysed the synthesis of cladribine, and the yield was 93%. The constructed random library was screened, and a mutant with altered activity was found. Conclusion: In this study,NDT was employed as a catalyst, and cladribine was synthesized successfully in one step. And the high-throughput screening system constructed in this study was successfully applied to the alternation of the NDT activity, which offers a new way to expand the ability of NDT in preparation of unnatural nucleoside analogs.
引文
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[16]Liang C,Zhang Y,Jia Y,et al.Engineering a carbohydrate-processing transglycosidase into glycosyltransferase for natural product glycodiversification[J].Scientific Reports,2016,(6):21051
[17]Crespo N,Sánchez-Murcia P A,Gago F,et al.2'-Deoxyribosyltransferase from Leishmania mexicana,an efficient biocatalyst for one-pot,one-step synthesis of nucleosides from poorly soluble purine bases[J].Applied Microbiology and Biotechnology,2017,101(19):7187-7200
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[19]王洁,余磊,杨东,等.基于酵母表面展示技术的胸苷磷酸化酶全细胞催化剂的构建[J].生物技术通报,2016,32(1):201-206
[20]余磊,李骥璇,王忆茗,等.蔗糖磷酸化酶全细胞催化AA-2G的条件优化[J].现代生物医学进展,2017,17(14):2601-2605
[2]Jacobs B M,Ammoscato F,Giovannoni G,et al.Cladribine:mechanisms and mysteries in multiple sclerosis[J].Journal of Neurology,Neurosurgery&Psychiatry,2018,0:1-6
[3]李立威,古冬云,程志刚,等.克拉屈滨的合成工艺改进及其主要杂质研究[J].中国新药杂志,2015,24(05):565-569
[4]夏然,孙莉萍,渠桂荣,等.抗白血病药物克拉屈滨的合成[J].应用化学,2016,33(11):1274-1278
[5]Taran S A,Verevkina K N,Feofanov S A,et al.Enzymatic transglycosylation of natural and modified nucleosides by immobilized thermostable nucleoside phosphorylases from Geobacillus stearothermophilus[J].Bioorg Khim,2009,35(6):822-829
[6]Fernandez-Lucas J,Acebal C,Sinisterra J V,et al.Lactobacillus reuteri 2'-deoxyribosyltransferase,a novel biocatalyst for tailoring of nucleosides[J].Applied and Environmental Microbiology,2010,76(5):1462-1470
[7]Kaminski P A,Labesse G.Phosphodeoxyribosyltransferases,designed enzymes for deoxyribonucleotides synthesis[J].Journal of Biological Chemistry,2013,288(9):6534-6541
[8]Kaminski P A,Dacher P,DuguéL,et al.In vivo reshaping the catalytic site of nucleoside 2'-deoxyribosyltransferase for dideoxy and didehydronucleosides via a single amino acid substitution[J].Journal of Biological Chemistry,2008,283(29):20053-20059
[9]王玺,段胜林,熊舒莉,等.自诱导系统在酶促合成2'-脱氧胞苷中的应用[J].生物技术通报,2014,(11):225-232
[10]李忠琴,许小平,杨海麟,等.辣根过氧化物酶分光光度法测定黄嘌呤氧化酶的活性[J].分析化学,2006,34(06):821-824
[11]Fresco-Taboada A,de la Mata I,Arroyo M,et al.New insights on nucleoside 2'-deoxyribosyltransferases:a versatile Biocatalyst for one-pot one-step synthesis of nucleoside analogs[J].Applied Microbiology and Biotechnology,2013,97(9):3773-3785
[12]Sarkisjan D,Julsing J R,Smid K,et al.The cytidine analog fluorocyclopentenylcytosine(RX-3117)is activated by uridine-cytidine kinase 2[J].PLOS ONE,2016,11(9):e162901
[13]Muthu P,Chen H X,Lutz S.Redesigning human 2'-deoxycytidine kinase enantioselectivity for L?nucleoside analogues as reporters in positron emission tomography[J].ACS Chemical Biology,2014,9(10):2326-2333
[14]Muthu P,Lutz S.Quantitative detection of nucleoside analogues by multi-enzyme biosensors using time-resolved kinetic measurements[J].ChemMedChem,2016,11(7):660-666
[15]Li Y,Ding Q,Ou L,et al.One-pot process of 2'-deoxyguanylic acid catalyzed by a multi-enzyme system[J].Biotechnology and Bioprocess Engineering,2015,20(1):37-43
[16]Liang C,Zhang Y,Jia Y,et al.Engineering a carbohydrate-processing transglycosidase into glycosyltransferase for natural product glycodiversification[J].Scientific Reports,2016,(6):21051
[17]Crespo N,Sánchez-Murcia P A,Gago F,et al.2'-Deoxyribosyltransferase from Leishmania mexicana,an efficient biocatalyst for one-pot,one-step synthesis of nucleosides from poorly soluble purine bases[J].Applied Microbiology and Biotechnology,2017,101(19):7187-7200
[18]Arco J D,Fernandez-Lucas J.Purine and pyrimidine phosphoribosyltransferases:A versatile tool for enzymatic synthesis of nucleoside-5'-monophosphates[J].Current Pharmaceutical Design,2018,23(45):6898-6912
[19]王洁,余磊,杨东,等.基于酵母表面展示技术的胸苷磷酸化酶全细胞催化剂的构建[J].生物技术通报,2016,32(1):201-206
[20]余磊,李骥璇,王忆茗,等.蔗糖磷酸化酶全细胞催化AA-2G的条件优化[J].现代生物医学进展,2017,17(14):2601-2605
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