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Recombinant AroL‐Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3‐Phosphate
Author(s) -
Schoenenberger Bernhard,
Wszolek Agata,
Meier Roland,
Brundiek Henrike,
Obkircher Markus,
Wohlgemuth Roland
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700529
Subject(s) - shikimic acid , shikimate pathway , biochemistry , enzyme , phosphorylation , chemistry , kinase , stereochemistry , biosynthesis
Shikimic acid 3‐phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5‐enolpyruvoyl‐shikimate 3‐phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3‐phosphate is only accessible via a chemical multi‐step route, a new straightforward preparative one‐step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3‐position. Highly active shikimate kinase is produced by straightforward expression of a synthetic aroL gene in Escherichia coli . The time course of the shikimate kinase‐catalyzed phosphorylation is investigated by 1 H‐ and 31 P‐NMR, using the phosphoenolpyruvate/pyruvate kinase system for the regeneration of the ATP cofactor. This enables the development of a quantitative biocatalytic 3‐phosphorylation of shikimic acid. After a standard workup procedure, a good yield of shikimic acid 3‐phosphate, with high HPLC‐ and NMR purity, is obtained. This efficient biocatalytic synthesis of shikimic acid 3‐phosphate is superior to any other method and has been successfully scaled up to multi‐gram scale.