文献类型：期刊文献

英文题名：Enzymatic Manufacture of Deoxythymidine-5′-Triphosphate with Permeable Intact Cells of E-coli Coexpressing Thymidylate Kinase and Acetate Kinase

作者：Zhang, Jiao[1];Qian, Yahui[1];Ding, Qingbao[1];Ou, Ling[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2015

卷号：25

期号：12

起止页码：2034

外文期刊名：JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000368425500009)】；

基金：This research was supported by Shanghai Sinobest Biotechnology Co., Ltd.

语种：英文

外文关键词：Deoxythymidine-5 '-triphosphate; thymidylate kinase; coexpression; permeabilization; ATP regeneration

摘要：A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25 degrees C for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'-triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.