title: ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR.
abstract: E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.
authors.affiliation: Research School of Chemistry, Australian National University , Canberra ACT 2601, Australia.
authors.forename: Apostolos
authors.identifier: 0000-0002-0177-3617
authors.initials: A
authors.lastname: Alissandratos
authors.affiliation: Research School of Chemistry, Australian National University , Canberra ACT 2601, Australia.
authors.forename: Karine
authors.initials: K
authors.lastname: Caron
authors.affiliation: Research School of Chemistry, Australian National University , Canberra ACT 2601, Australia.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Loan
authors.affiliation: Research School of Chemistry, Australian National University , Canberra ACT 2601, Australia.
authors.forename: James E
authors.initials: JE
authors.lastname: Hennessy
authors.affiliation: Research School of Chemistry, Australian National University , Canberra ACT 2601, Australia.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Easton
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods; D010755:Organophosphates / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015854:Up-Regulation; D014529:Uridine / Q000378:metabolism; D014541:Uridine Kinase / Q000235:genetics / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism
chemical_list: D010755:Organophosphates; C011632:acetyl phosphate; D000255:Adenosine Triphosphate; D014541:Uridine Kinase; D014544:Uridine Triphosphate; D014529:Uridine