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Applied and Environmental Microbiology, January 2000, p. 98-104, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Expression of Alcaligenes eutrophus Flavohemoprotein and Engineered Vitreoscilla Hemoglobin-Reductase Fusion Protein for Improved Hypoxic Growth of Escherichia coli

Alexander D. Frey, James E. Bailey, and Pauli T. Kallio*

Institute of Biotechnology, ETH-Zürich, CH-8093 Zürich, Switzerland

Received 19 July 1999/Accepted 20 October 1999

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 amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in 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 the vhb gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical 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-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant beta -lactamase relative to the VHb-expressing control.

* Corresponding author. Mailing address: Institute of Biotechnology, ETH-Zürich, CH-8093 Zürich, Switzerland. Phone: 41 1 633 3446. Fax: 41 1 633 1051. E-mail: kallio{at}biotech.biol.ethz.ch.

Applied and Environmental Microbiology, January 2000, p. 98-104, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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