The ugp promoter (P~w,) responsible for expression of the binding-protein-dependent sn-giycerol-3-phosphate transport system in Escherichia coil

The ugp promoter (P~w,) responsible for expression of the binding-protein-dependent sn-giycerol-3-phosphate transport system in Escherichia coil was cloned into a small multicopy plasmid pTER5, a derivative of pBR322, between the transcription terminators rpoCt and tLt. 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 pUCI9 plasmid. The efficiency and regulatory properties ofp,~, were measured using xy/E and lacZ as reporter genes, which code for the corresponding enzymes catechol-2,3-dioxygenase (C230) and p-galactosidase (~Gal), respectively. Enzyme activities were virtually completely repressed in the presence of excess inorganic phosphates (Pi) and high concentrations of glucose. Maximal induction was observed at limiting Pi (<0.1 mM) and normal levels of glucose (0.2-0.4~). The maximum expression of the p,~,-directed/~Gal synthesis was approx. 80~ of that directed by strong Ptoc. When the xylE gene was maximally expressed, the induced enzyme constituted approx. 50~0 of total cellular protein as judged by laser densitometry following sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. These results suggest the usefulness of the P~e, in expression vectors for strong, but controlled, expression of cloned genes in E. coll. This Pl controlled vector can be adapted to large-scale fermentation by using Pl-limiting growth conditions.