A novel phosphate-regulated expression vector in Escherichia coli

The ugp promoter (p_ugp) responsible for expression of the binding-protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and t_L1. The resulting expression vector, pPH3, permits convenient insertion of structural genes containing their own translational-initiation regions, into the multiple-cloning site derived from the pUC19 plasmid. The efficiency and regulatory properties of p_ugp were measured using xylE and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C23O) and β-galactosidase (βGal), respectively. Enzyme activities were virtually completely repressed in the presencee of excess inorganic phosphates (P_i) and high concentrations of glucose. Maximal induction was observed at limiting P_i (<0.1 mM) and normal levels of glucose (0.2-0.4%). The maximum expression of the p_ugp -directed βGal synthesis was approx. 80% of that directed by directed by strong p_tac. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50% of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the p_ugp in expression vectors for strong, but controlled, expression of cloned genes in E. coli. This P_i controlled vector can be adapted to large-scale fermentation by using P_i-limiting growth conditions.