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http://dx.doi.org/10.4014/jmb.1506.06020

Enzymatic Manufacture of Deoxythymidine-5'-Triphosphate with Permeable Intact Cells of E. coli Coexpressing Thymidylate Kinase and Acetate Kinase  

Zhang, Jiao (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Qian, Yahui (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Ding, Qingbao (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Ou, Ling (State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology)
Publication Information
Journal of Microbiology and Biotechnology / v.25, no.12, 2015 , pp. 2034-2042 More about this Journal
Abstract
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℃ 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.
Keywords
Deoxythymidine-5'-triphosphate; thymidylate kinase; coexpression; permeabilization; ATP regeneration;
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