Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG.

To elucidate the possible biosynthetic pathway of a precursor UDP-glucose of the sphingan WL gum produced by Sphingomonas sp. WG, two enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase) were bioinformatically analysed, expressed in Escherichia coli BL21 (DE3) and characterized. PGM was in the phosphoglucomutase/phosphomannomutase subclass and UGPase was predicted to be a UDP-glucose pyrophosphatase in a tetrameric structure. Both enzymes were expressed in soluble form, purified to near homogeneity with high activity at 1159 and 796 U/mg, exhibited folding with reasonable secondary structures, and existed as monomer and tetramer, respectively. The optimal pH and temperature of PGM were 9.0 and 50 °C, respectively, and this protein was stable at pH 8.0 and at temperatures ranging from 40 to 50 °C. The optimal pH and temperature of UGPase were 9.0 and 45 °C, respectively, and the protein was stable at pH 8.0 and at temperatures ranging from 30 to 55 °C. A small-scale one-pot biosynthesis of UDP-glucose by combining PGM and UGPase using glucose-6-phosphate and UTP as substrates was also performed, and formation of UDP-glucose was observed by HPLC detection, which confirmed the biosynthetic pathway of UDP-glucose in vitro. PGM and UGPase will be ideal targets for the metabolic engineering to improve WL gum yields in industrial production.