Recombinant AroL-Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3-Phosphate.

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 H- and 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.