Vitreoscilla hemoglobin. In vitro mutagenesis and transcript profiling to improve its biotechnological usefulness

Sammanfattning: Vitreoscilla is an obligate aerobic bacterium found in oxygen-poor environments such as stagnant ponds and decaying vegetable matter. Vitreoscilla induces the production of a homodimeric hemoglobin (VHb) when subjected to oxygen-limiting conditions. It is thought that VHb facilitates oxygen delivery to the respiratory apparatus of its host by increasing the local oxygen concentration under oxygen-limiting conditions. Furthermore, VHb may increase the level and activity of terminal oxidases, preferably o, thus enhancing respiration and thereby ATP formation, or alternatively act as an alternative terminal oxidase itself. VHb has been shown to have numerous positive effects on diverse cellular processes which are affected by the availability of oxygen or subjected to oxygen-poor conditions. Heterologous VHb expression has led to higher cell density and faster growth, higher yield of recombinant proteins or higher production of secondary metabolites, altered by-product formation, and enhanced bioremediation of toxic wastes. This thesis focuses on development of novel, improved forms of VHb, a set of point mutated VHb forms and a VHb-VHb fusion protein, and on the characterization of their growth properties in Escherichia coli. We have thus demonstrated that error-prone PCR can be used to generate VHb mutants able to further promote growth rates in microaerobic E. coli cultures. In addition, these mutants produce higher levels of active ribosomes in their host cells. The VHb-VHb fusion protein also enhances E. coli cell growth under fully aerobic conditions. In order to delineate the metabolic consequences of VHb expression, transcript profiling using high-density DNA arrays has been carried out. These data have revealed that previously obtained metabolic results using e.g. NMR methodologies, are supported by global gene expression measurements.