Use of dihydroxyacetone synthase for the assimilation of methanol in E. coli.

Dihydroxyacetone synthase (Das) is a key enzyme of the xylulose monophosphate (XuMP) pathway, catalysing the assimilation of formaldehyde in methylotrophic yeasts. In this study, we first performed a systematic screening of twenty-five Das variants in combination with a methanol dehydrogenase (Mdh) from Acinetobacter gerneri to identify the Das variants supporting the most efficient methanol assimilation.13C-methanol labelling experiments revealed marked differences in 13C incorporation into central carbon metabolism among the various Das variants and enabled the identification of the Pichia angusta Das variant as the most efficient candidate. To further investigate Das activity in vitro, a coupled enzymatic assay was developed using glycerol dehydrogenase as a reporter via NADH oxidation. Initial enzymatic assays revealed significant background activity, prompting optimization of purification and assay conditions. Implementation of higher ionic strength buffers, protease inhibition, increased wash stringency, and stepwise imidazole elution substantially improved enzyme purity and assay specificity, enabling reliable measurement of P. angusta Das activity. Finally, to directly demonstrate the carbon transfer catalyzed by P. angusta Das, purified enzyme was assayed with 13C-formaldehyde and xylulose-5-phosphate, and reaction products were analyzed by 1H NMR spectroscopy. 13C incorporation was detected exclusively in dihydroxyacetone, confirming that Das catalyzes a thiamine pyrophosphate-dependent transfer of a C1 unit from formaldehyde to xylulose-5-phosphate, yielding equimolar amounts of dihydroxyacetone and glyceraldehyde-3-phosphate. This provides the first direct in vitro demonstration of Das transketolase activity using purified enzyme and native substrates, and establishes a robust framework for studying and engineering synthetic methanol assimilation pathways.