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

Abstract

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.

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

“…The analysis of chemical and biological routes for the synthesis of metabolites often shows large differences in the number of reaction steps required to construct highly and differentially functionalized small molecules. Designing highly selective and straightforward biocatalytic one-step synthesis methods instead of lengthy and challenging chemical routes has been established as the preferred method for preparative access to a large number of valuable metabolites (Richter et al, 2009;Schell et al, 2009;Matsumi et al, 2014;Schoenenberger et al, 2017a;Hardt et al, 2017;Schoenenberger et al, 2018;Krevet et al, 2020;Schoenenberger et al, 2020;Sun et al, 2021). As the finding and selection of routes is a key task in target-oriented synthesis the development of appropriate methodologies has attracted much interest in both chemistry and biology.…”

Biocatalysis in the Swiss Manufacturing Environment

“…The exploration of the biocatalytic space has been very useful for the synthesis of metabolites by a systems biocatalysis approach [41]. Selective enzyme-catalyzed Oand N-phosphorylations have been demonstrated as versatile platform technologies and provide various advantages over classical chemical phosphorylations as demonstrated by Sigma-Aldrich/Merck KGaA for the manufacturing of a large variety of phosphorylated metabolites [42][43][44][45][46][47][48][49]. Selective enzymatic water elimination reactions exhibiting full conversion enabled a simple synthetic access to 2-keto-3-deoxysugar acids from easily accessible sugar acids using dehydratases [50][51][52].…”

Key advances in biocatalytic phosphorylations in the last two decades: Biocatalytic syntheses in vitro and biotransformations in vivo (in humans)

“…[85,86] The high selectivity of shikimate kinase-catalyzed phosphorylations of cyclic trihydroxy-carboxylic acids is demonstrated by the simple synthesis of shikimic acid 3-phosphate from shikimic acid catalyzed by recombinant E. coli K12 shikimate kinase AroL. [87] This biocatalytic route shortens tedious multi-step synthesis, reduces costs and working time, and avoids toxic heavy metal reagents. [87] Lengthy synthetic routes can also be substituted in the preparation of enantiomerically pure (R)-mevalonate-5-phosphate, which can be synthesized by phosphorylation of the pure R-enantiomer of mevalolactone catalyzed by mevalonate kinase.…”

“…[ 87 ] This biocatalytic route shortens tedious multi‐step synthesis, reduces costs and working time, and avoids toxic heavy metal reagents. [ 87 ] Lengthy synthetic routes can also be substituted in the preparation of enantiomerically pure ( R )‐mevalonate‐5‐phosphate, which can be synthesized by phosphorylation of the pure R ‐enantiomer of mevalolactone catalyzed by mevalonate kinase. [ 88 ] Another approach is the mevalonate kinase‐catalyzed kinetic resolution of racemic mevalolactone and subsequent separation of the ( R )‐mevalonate‐5‐phosphate from the non‐converted ( S )‐mevalonate.…”