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Model-driven development and utilization of an oxygen-independent platform strain

  • Author(s): Portnoy, Vasiliy A.
  • Advisor(s): Palsson, Bernhard Ø
  • et al.

The microbial production of commodity chemicals is a promising avenue for the development of sustainable processes for the utilization of renewable resources and reducing our dependency on foreign oil. In order to become cost and energy effective, the process must utilize an organism that is optimized for production of a number of reduced by-products from variety of feedstocks. Escherichia coli is one of the most commonly used host organisms for metabolic engineering and overproduction of metabolites due to its metabolic versatility, amenability to genetic manipulation, and the ability to produce a wide variety of reduced by-products such as bio-ethanol and organic acids. E. coli has also been extensively characterized with respect to its metabolic physiology. It is capable of surviving in a variety of environmental conditions, such as oxic and anoxic; however the different growth rates and different secretion products under oxic anc anoxic conditions poses a significant challenge for metabolic engineering processes in which environmental perturbations will influence the outcome of the bio-catalytic process. Therefore, the utilization of the oxygen-independent strain for bio-catalysis eliminates the need for the stringent control over the fermentation environment with respect to oxygenation, thus significantly reducing the cost of the entire bio-catalytic process. Therefore, it is of interest to develop an E. coli strain incapable of oxygen utilization, to be used as a platform strain for metabolic engineering.

Here, I present the work aimed at the (i) development of an oxygen-independent platform strain, (ii) understanding of its physiological behavior, and (iii) utilization of this strain for metabolic engineering applications. Such a strain can be useful for the overproduction of commodity chemicals under various conditions independent of oxygen supply and optimization of anaerobic metabolic engineering designs using adaptive evolution under oxic conditions. The results show that upon the removal of the oxygen-utilization pathway, the ECOM4 (Escherichia coli Cytochrome Oxidase Mutant 4) strain was unable to undergo an aerobic-anaerobic shift and exhibited similar phenotypes under both conditions with D-lactic acid as a sole growth-associated by-product. Moreover, I show that the ECOM4 strain can be used for the overproduction of organic and amino acids from renewable resources.

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