L-fuculokinase-catalyzed phosphorylation and its use for the straightforward synthesis of L-fuculose-1-phosphate

Abstract

L-Fuculokinases play a key role in both natural and synthetic metabolic pathways, not only for the utilization of L-fucose, but also for related monosaccharides, and catalyze selective phosphorylation of L-fuculose in the 1-position or of D-ribulose in the 5-position. Therefore, L-fuculokinases have been selected, the fucK gene from Escherichia coli K12 has been synthesized and cloned into an appropriate expression vector for the transformation of E. coli BL21(DE3). The cultivation of the transformed strain and the overexpression of the fucK gene has led to highly active L-fuculokinase after straightforward downstream processing. The soluble fraction from the crude cell extract could be directly applied in the enzymatic phosphorylation of L-fuculose. Recycling of the ATP cofactor was done with pyruvate kinase (PK) and the high energy phosphoenolpyruvate (PEP) as donor. The time course of the phosphorylation reactions has been monitored by ³¹P-NMR and ¹H-NMR spectroscopy and L-fuculose could be phosphorylated with nearly 100% conversion. After standard workup, the L-fuculose-1-phosphate has been obtained in high yield and purity.

Graphical Abstract

Keywords:

• L-fuculokinase
• enzymatic phosphorylation
• L-fuculose-1-phosphate
• anomeric sugar kinases
• ATP recycling

Acknowledgements

We would like to thank Dr. Christine Hellriegel for her support with NMR-experiments and the German Federal Ministry of Education and Research (BMBF) for the support of project P28 under the cluster of Biocatalysis2021.

Disclosure statement

No potential conflict of interest was reported by the author(s).