Optimization and hyper-expressed production of lysine through chemical mutagenesis of Brevibacterium flavum by N-nitroso-N-ethylurea

Abstract

One of best-known commercially produced amino acids is lysine, used as a feed additive to enhance its nutritional value. Some microorganisms have the capability of producing high amounts of lysine. Brevibacterium flavum was used for lysine production in this study. The bacterium was grown on nutrient agar medium. Following the optimization of culture conditions, the maximum growth was achieved after incubating the bacterial cells at 30°C (at pH 7.0), in the presence of 20 g/ corn steep liquor and glucose each, for 24 h. Incorporation of lactate, in the range between 2-12%, increased lysine production significantly (P>0.05). The mutagen, N-nitroso-N-ethylurea (ENU) was used for the hyper-production of lysine. Before initiating the mutation program, B. flavum was grown on suitable production medium. Cells were then exposed to 35 mM ENU for 5-30 min. After washing the bacterial pellet with sodium citrate buffer and growing on seed culture medium containing methionine and threonine as auxotrophic requirements, lysine production was enhanced up to 125 g/l in the culture exposed to the mutagen for 5 min as compared to 81 g/l, produced from wild type bacterial cells (P>0.001). These findings indicate the increased lysine production capacity of B. flavum in the presence of appropriate amounts of essential nutrients, added to the culture medium. Key words: Auxotrophs, Brevibacterium flavum, lysine, N-nitroso-N-ethylura, mutants.