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

J Microbiol Biotechnol. 2015 Dec 28;25(12):2034-42. doi: 10.4014/jmb.1506.06020.

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°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.

Keywords: ATP-regeneration; Deoxythymidine-5′-triphosphate; Thymidylate kinase; coexpression; permeabilization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetate Kinase / genetics
  • Acetate Kinase / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Gene Expression
  • Metabolic Engineering / methods*
  • Nucleoside-Phosphate Kinase / genetics
  • Nucleoside-Phosphate Kinase / metabolism*
  • Organophosphates / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Temperature
  • Thymidine Monophosphate / metabolism
  • Thymine Nucleotides / metabolism*

Substances

  • Organophosphates
  • Recombinant Proteins
  • Thymine Nucleotides
  • Thymidine Monophosphate
  • acetyl phosphate
  • Acetate Kinase
  • Nucleoside-Phosphate Kinase
  • dTMP kinase
  • thymidine 5'-triphosphate