Production of L-arginine by genetically modified Escherichia coli

Abstract: In the recent years, the demand for environmental friendly produced L-arginine has risen with the increasing number of applications for this amino acid in pharmaceuticals, nutraceuticals, cosmetics, animal feed and fertilizers. Members of the Corynebacteriaceae family are usually used for microbial L-arginine production. However, Escherichia coli present the advantage of being able to utilize a wider range of substrates, including pentose sugars found in lignocellulosic feedstocks.  The present thesis illustrates the first steps in the development of a sustainable process to produce L-arginine using E. coli. It starts with the constructions of a L-arginine overproducing strain, followed by optimization of the nitrogen supply for the fermentations. The first part of this thesis aimed at engineering an E. coli train able to produce high level of L-arginine. Mutations on key genes of the L-arginine biosynthesis pathway were step-wisely done. The mutants obtained at each step were tested in bioreactor fermentations to assess the effect of each genetic modification. The final strain was able to produce almost 12 g/l during fermentation, at a productivity of 0.24 g/l/h. In comparison the starting strain, E. coli K12 C600, was not able to excrete any L-arginine.  To minimize nitrogen wastes and optimize the L-arginine production the impact of different nitrogen sources and concentration were investigated. It was shown that while ammonium phosphate dibasic was the most potent nitrogen source during cultivation on complex medium, all the sources were equivalent with minimal media; this probably reflected the phosphate deficiency of the complex medium used. In fermentation on minimal medium, a carbon to nitrogen ratio of 5 was demonstrated to be the most suitable, yielding up to 4.5 g/l L-arginine. At this ratio, both glucose and the nitrogen source were completely utilized during fermentation.

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