Vitreoscilla Haemoglobin. Genetic Fusions and Metabolic Characterisation

Sammanfattning: Vitreoscilla is an obligate aerobic bacterium living in oxygen-poor environments such as stagnant ponds and decaying vegetable matter. Vitreoscilla expresses a homodimeric haemoglobin (VHb) when subjected to oxygen-limited conditions. In several organisms, expression of VHb has been shown to increase microaerobic cell growth and enhance oxygen-dependent cell metabolism. Moreover, the presence of VHb has been shown to enhance recombinant protein synthesis, as demonstrated by both total protein content and the activity of expressed enzymes. It has been hypothesised that VHb may facilitate oxygen transport and/or storage. Furthermore, it has been shown that VHb interacts specifically with subunit I of the cytochrome o complex from Vitreoscilla and Escherichia coli, thereby suggesting that VHb provides oxygen directly to the terminal oxidase. This thesis describes the results of two different fusion approaches that were applied to VHb in order to improve and elucidate its properties. In the first approach, short random polypeptide tags were added to the N-terminus of VHb. The results clearly demonstrated the large impact a small N-terminal polypeptide extension can have on the properties of a protein; the library of randomly hexapeptide-tagged VHb displayed a wide variety of properties, e.g. a broad range of expression levels, cell colours and VHb activities. Furthermore, the results indicate the importance of mRNA secondary structure predictions when optimising expression levels of recombinant proteins. In the second approach, a VHb-VHb fusion protein was created (termed double VHb) which displayed enhanced growth properties when expressed in E. coli compared with those of native VHb. Higher final cell densities were obtained under all cultivation conditions tested, i.e. growth under low and high aeration conditions as well as growth in high-salinity media. Furthermore, enhanced levels of ribosomes and tRNA were observed in cells expressing double VHb. DNA chip analysis revealed mRNA levels that indicated an increased TCA cycle, respiration and ATP synthesis rate of double-VHb-expressing cells.