Efficient biocatalytic synthesis of D-tagatose 1,6-diphosphate by LacC-catalysed phosphorylation of D-tagatose 6-phosphate

Abstract

D-tagatose 1,6-diphosphate is an important metabolite which can be formed via the classical tagatose 6-phosphate pathway or via the tagatose 1-phosphate pathway discovered more recently. The chiral metabolite naturally occurs as an intermediate in the galactose metabolism of various organisms, where it is formed by a reversible condensation of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate, whereas D-tagatose 6-phosphate has been synthesized chemically, no chemical synthetic method for further phosphorylation to the D-tagatose 1,6-diphosphate has been described so far. Therefore, a site-specific biocatalytic phosphorylation of the 1-position in D-tagatose 6-phosphate has been chosen. The lacC gene from Lactococcus lactis subsp. lactis was synthesized, cloned into an appropriate expression vector to allow for the production of LacC, the D-tagatose 6-phosphate kinase in Escherichia coli. Subsequently, an efficient biocatalytic synthesis has been developed for the phosphorylation of D-tagatose 6-phosphate at its C1-position by using the recombinant D-tagatose 6-phosphate kinase and the phosphoenolpyruvate/pyruvate kinase-system for ATP regeneration. This straightforward and scalable one-step biocatalytic synthesis of D-tagatose 1,6-diphosphate was successfully scaled up to the gram scale.

Graphical Abstract

Keywords:

• Tagatose 6-phosphate pathway
• enzymatic phosphorylation
• D-tagatose 6-phosphate kinase
• LacC
• D-tagatose 1,6-diphosphate

Acknowledgements

We would like to thank the German Federal Ministry of Education and Research (BMBF) for the support of project P28 under the cluster of Biocatalysis 2021 as well as Andrea Weckbecker and Simon Esser from evoxx for their support within the review process.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the German Federal Ministry of Education and Research (BMBF).