Biocatalytic synthesis of 2'-deoxynucleotide 5'-triphosphates from bacterial genomic DNA: Proof of principle.


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Abstract

2'-deoxynucleoside 5'-triphosphates (dNTPs) are the building blocks of DNA and are key reagents which are incorporated by polymerase enzymes during nucleic acid amplification techniques, such as polymerase chain reaction (PCR). These techniques are of high importance, not only in molecular biology research, but also in molecular diagnostics. dNTPs are generally produced by a bottom-up technique which relies on synthesis or isolation of purified small molecules like deoxynucleosides. However, the disproportionately high cost of dNTPs in low- and middle-income countries (LMICs) and the requirement for cold chain storage during international shipping makes an adequate supply of these molecules challenging. To reduce supply chain dependency and promote domestic manufacturing in LMICs, a unique top-down biocatalytic synthesis method is described to produce dNTPs. Readily available bacterial genomic DNA provides a crude source material to generate dNTPs and is extracted directly from Escherichia coli (step 1). Nuclease enzymes are then used to digest the genomic DNA creating monophosphorylated deoxynucleotides (dNMPs) (step 2). Design and recombinant production and characterization of E. coli nucleotide kinases is presented to further phosphorylate the monophosphorylated products to generate dNTPs (step 3). Direct use of the in-house produced dNTPs in nucleic acid amplification is shown (step 4) and their successful use as reagents in the application of PCR, thereby providing proof of principle for the future development of recombinant nucleases and design of a recombinant solid-state bioreactor for on-demand dNTP production.

Description

Funder: Affordable Diagnostics for Low‐income Countries campaign including MRC‐Newton Fund MR/R025444/1


Funder: A Shuttleworth Foundation Fellowship has been awarded to Jennifer C. Molloy


Funder: Anna R. Bird has been funded by a Krishnan‐Ang studentship at Trinity College Cambridge.

Keywords
dNTP synthesis, nucleic acid test reagents, nucleotide synthesis, on-demand reagent synthesis, DNA, Bacterial, Escherichia coli, DNA, Nucleotides, Genomics
Journal Title
Biotechnol Bioeng
Conference Name
Journal ISSN
0006-3592
1097-0290
Volume Title
Publisher
Wiley
Sponsorship
MRC (MC_PC_17142)
Medical Research Council (MR/R025444/1)
MRC