Nitrite Inhibition of Vitreoscilla Hemoglobin (VHb) in Recombinant E. coli: Direct Evidence that VHb Enhances Recombinant Protein Production

Bacteria engineered with the gene (vgb) encoding Vitreoscilla hemoglobin (VHb) typically produce more protein than unengineered cells, and it has generally been assumed that VHb is responsible for this effect. Here, using matched strains of E. coli that bear a recombinant a-amylase gene (MK57) or the a-amylase gene and vgb (MK79), we provide evidence supporting this assumption. Sodium nitrite (which is known to inhibit heme proteins) was tested over a range of concentrations regarding effects on growth, a-amylase production, respiration, and VHb function in MK57 and MK79. Nitrite concentrations were identified at which respiration of cell membranes was inhibited only slightly and to approximately equal degrees in both strains, while whole cell respiration was inhibited to a greater extent and about twice as much in MK79 as MK57. This suggests that these concentrations inhibit VHb while having a much smaller effect on cytochrome oxidase. Direct measurements of VHb showed, in fact, that the same nitrite concentrations greatly decreased the levels of active (ferrous) and, to a somewhat lesser extent, total (ferrous plus ferric) VHb in MK79. Finally, these same nitrite concentrations reversed the advantage regarding a-amylase production of MK79 over MK57 seen at 0 mM nitrite, linking the presence of active VHb with the increase in a-amylase production.