Khosla, Chaitan (1990) Vitreoscilla hemoglobin: gene structure and regulation functions, and applications to aerobic bioprocesses. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-04212004-154615
Vitreoscilla sp. is a Gram-negative obligately aerobic bacterium, which is capable of synthesizing a soluble, homodimeric hemoglobin-like molecule (VHb) in response to hypoxic environments. Although the mechanism of action of VHb is not understood, it has been hypothesized that the heme-protein enables the bacterium to survive in oxygen-limited environments. The functional role of this bacterial globin has been studied with the aim of exploiting such a naturally evolved strategy to improve oxygen-limited bioprocesses. Furthermore, the regulation of expression of the VHb gene in response to changes in environmental conditions has also been investigated. This has provided insights into mechanisms of microaerobic gene regulation, and has enabled the development of oxygen-dependent expression systems for high-level synthesis of recombinant proteins.
The gene encoding the VHb polypeptide was isolated from a Vitreoscilla genomic library. The nucleotide sequence of the gene and its flanking regions was determined and analyzed. Synthesis of active VHb was shown to occur in E. coli from the natural expression signals of the VHb gene.
Studies in fed-batch fermentations demonstrated that under oxygen limitation, the presence of the VHb gene on a multicopy plasmid enhanced the growth and respiratory characteristics of a recombinant E. coli host as compared to equivalent plasmid-carrying and plasmid-free cells. These results illustrated the possibility of studying the mechanism of VHb action in E. coli as a surrogate host, and were also indicative of the potential applicability of such a genetic strategy in organisms other than Vitreoscilla. Furthermore, it was observed that VHb expression is under oxygen-dependent control in E. coli, suggesting that the mechanism of regulation of the gene in Vitreoscilla is also functional in E. coli.
Biochemical studies revealed that a considerable fraction, but not all, of the intracellular VHb is localized in the periplasm of E. coli and Vitreoscilla. The activity of the two fractions was identical, as judged by visible spectroscopy. Genetic evidence for the role of the N-terminal domain of the VHb polypeptide in protein translocation was also obtained; however no cleavage was detected at this end as a result of translocation. Based on available biochemical and biophysical data, it was suggested on theoretical grounds that periplasmic VHb is capable of supporting an additional oxygen flux to the respiratory chain, which may be physiologically significant (the facilitated diffusion hypothesis).
In E. coli strains containing VHb integrated into the chromosome in single copy, the presence of VHb improved cellular energetics under oxygen-limiting, but not oxygen-excess conditions. Indirect evidence was obtained, suggesting that the net effect of VHb in E. coli is to improve the efficiency, rather than the kinetics of oxygen-limited aerobic metabolism. Although the facilitated diffusion hypothesis could not be confirmed or ruled out, an alternative hypothesis (the intracellular redox effector hypothesis) was also proposed. This implies that oxygenated VHb influences the activity of a key redox-sensitive cell function, which is affected under hypoxic conditions.
Genetic studies demonstrated the presence of an oxygen-responsive element (ORE) upstream of the VHb gene. Gene expression is maximal under microaerobic conditions, and is also influenced by catabolite repression. Mechanisms responsible for oxygen-dependent regulation act at the level of transcription initiation from two overlapping promoters within the region upstream of the VHb gene.
Protocols were developed for the use of ORE-based expression systems for regulated, high level synthesis of recombinant proteins in high cell density fermentations. A 30-fold modulation of promoter activity could be achieved in these experiments. By simply decreasing the air supply to cells carrying a VHb-lacZ fusion, [Beta]-galactosidase was expressed to a level of about 10% of total cellular protein in a culture containing 20 g dry cell weight/L.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||8 December 1989|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||23 Apr 2004|
|Last Modified:||26 Dec 2012 02:38|
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