Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region.

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P(vgb) of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P(vgb) was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P(vgb) provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P(vgb), while a construct containing P(vgb), the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P(vgb), vgb operon) or pKVpPV (P(vgb), vgb operon, par) slightly attenuated (approximately 30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P(vgb), egfp) or pKVpP-E (P(vgb), par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P(vgb), vgb operon, par, tod) or pKVpP-T (P(vgb), par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.