Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens
A newly isolated gene dxs11 from Agrobacterium tumefaciens (KCCM 10413), an organism with potential for the industrial production of ubiquinone-10 (UbiQ10), encoding a 1-deoxy-d-xylulose 5-phosphate synthase (Dxs), was cloned in Escherichia coli and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1920 bp, capable of encoding a polypeptide of 640 amino acids residues with a calculated isoelectric point of pH 5.63 and a molecular mass of 68,054 Da. The homodimeric enzyme was overexpressed in E. coli and purified as an active soluble form. The enzyme required thiamine diphosphate and a divalent metal ion, either Mg2+ or Mn2+, for enzymatic activity. The enzyme had an optimal pH and temperature of 8.0 and 37 °C, respectively, with a kcat of 26.8 s−1 and a kcat/Km of 0.67 and 1.17 s−1 M−1 for pyruvate and d-glyceraldehyde 3-phosphate, respectively. A. tumefaciens Dxs showed a comparable catalytic efficiency to other Dxs proteins. The dxs11 gene was transformed into A. tumefaciens KCCM 10413, and the resulting recombinant, A. tumefaciens pGX11, showed higher UbiQ10 production (502.4 mg/l) and content (8.3 mg/g DCW) than A. tumefaciens KCCM 10413, by 21.9 and 23.9%, respectively. This work describes Dxs from A. tumefaciens, an organism with the potential for industrial UbiQ10 production, and the first metabolic engineering study with the non-mevalonate pathway enzyme in A. tumefaciens.
Journal: Journal of Biotechnology - Volume 128, Issue 3, 20 February 2007, Pages 555–566