期刊
BIOTECHNOLOGY JOURNAL
卷 13, 期 8, 页码 -出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201700529
关键词
aroL; biocatalysis; enzymatic phosphorylation; shikimate kinase; shikimic acid 3-phosphate
资金
- German Federal Ministry of Education and Research (BMBF) [P28]
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-1- and P-31-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.
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