Production of L-arginine by Escherichia coli : Impact of genetic modifications, carbon and nitrogen sources

• 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.Microbial production of L-arginine usually relies on Corynebacterium glutam-icum and Corynebacterium crenatum strains. However, Escherichia coli presents 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 sustain-able process to produce L-arginine using E. coli. It starts with the construction of an L-arginine overproducing strain, followed by an investigation into adequate nitrogen and carbon sources for cell growth and L-arginine production.The first part of this thesis aimed at engineering an E. coli strain able to produce high level of L-arginine. Mutations on key genes of the L-arginine biosynthesis pathway were stepwisely 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 fer-mentation, 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. Besides, one mutant from each step was further examined in a metabolomic study in order to gain deeper insight into the effects of the various genetic modifications performed.To minimize nitrogen waste and optimize the L-arginine production the impact of different nitrogen sources and concentrations were then investigated. Ammonium phosphate dibasic, ammonium sulfate and ammonia solution were the best nitrogen sources for L-arginine production. In minimal medium, the optimum carbon to nitrogen ratio was 6, yielding about 4 g/L L-arginine from 30 g/L glucose. At this ratio, both glucose and the nitrogen source were com-pletely utilized during fermentation.Finaly various carbon courses commonly found in lignocellulosic feedstocks were tested. D-glucose and D-xylose were the most suitable carbon sources followed by L-arabinose. D-galactose and D-mannose resulted in significantly less arginie formation. However, a mixture of all five sugars yielded a higher production than any individual sugar.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Bioprocessteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Bioprocess Technology (hsv//eng)

Nyckelord

Biokemisk processteknik

Biochemical Process Engineering

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