Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli

Expression of the vhb gene encoding hemoglobin from Vitreoscilla sp, (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The aminoterminal hemoglobin d omain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. Howeve r, like other flavohemoglobins and unlike VHb, FHP possesses a second (carb oxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) red uctase activities. To examine whether the carboxy-terminal redox-active sit e of flavohemoproteins can be used to improve the positive effects of VHb i n microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A. eutrophus in frame after th e vhb gene. Similarly, the gene for FHP was modified, and expression casset tes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP a ctivities were constructed. Biochemically active heme proteins were produce d from all of these constructions in Escherichia coli, as indicated by thei r ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NA D reductase increased the final cell density of transformed wild-type E. co li cells approximately 50 and 75% respectively, for hypoxic fed-batch cultu re relative to the control synthesizing VHb. Approximately the same final o ptical densities were achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-espressing control under the same cultivation conditions, The expression of VHb-NAD or FHPg-NAD fusion proteins reduced t he final cell densities approximately 20% relative to the VHb-espressing co ntrol. The VHb-FAD-NAD reductase-expressing strain was also able to synthes ize 2.3-fold more recombinant beta-lactamase relative to the VHb-espressing control.