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Tryptophan biosynthesis by genetically engineered Escherichia coli utilizing different carbon sources
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The commercial production of L-uWtophan, an essential ainino acid for animal species and humans, depends largely on microbial processes due to drawback of chemical synthesis: the formation of a racemic mixture of DL-tryptophan. Biotransforrnation of chemically synthesized precursors or production by fermentation from inexpensive carbon and nitrogen sources are the major microbial production techniques used. Unfortunately, high production costs of both of these processes due to expensive precursors in the former and low yield in the latter hamper the expected large market of L-tryptophan. Most of the previous studies to improve the yield in fermentative processes have focused on varying the specific activity of key enzymes involved in L-tryptophan biosynthesis, in order to optimize production. However, very few attempts were made to optimize precursor levels in vivo . While investigation along this line was in progress in our laboratory, positive effects of transketolase (TktA) activity on aromatics production in Escherichia coli was reported by Draths et. al. In this study, we investigate the effects of overexpressing the tryptophan operon (TrpAE), 2-keto-3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (AroG), TktA, phosphoenolpyruvate carboxykinase (Pck), and phosphoenolpyruvate synthase (Pps) in a suitable E. coli host cell. While overexpression of AroG alone with TrpAE improved L-tryptophan biosynthesis 28% over the basal level obtained from TrpAE overexpression only, overexpression of TktA together with AroG and TrpAE showed a 90% increase over the basal level. The overexpression of neither Pps, nor Pck did not improve Ltryptophan biosynthesis either with TrpAE or AroG and TktA. The effect of glutamine as an additional nitrogen source was studied: glutamine was not found to improve neither plasmid stability, nor tryptophan biosynthesis. We also compared the efficiency of glucose, xylose, 0.3% xylose+0.7% pyruvic acid and 0.3% xylose+0.7% glycerol as carbon sources for L-tryptophan biosynthesis. The experimental yield obtained from glucose, of 10% increased to 14% when xylose was used. In the overexpression of TrpAE only, the specific uwtophan concentration in the medium increased by 28% in xylose compared to glucose. Overexpression of either AroG or AroG with TktA had a negative effect on L-tryptophan biosynthesis in xylose medium. Use of 0.3% xylose+0.7% pyruvic acid and 0.3% xylose+0.7% glycerol as carbon sources did not improve tryptophan biosynthesis. We conclude that xylose, abundant in nature as a major component of lignocellulosic biomass, can be used more efficiently than glucose with overexpression of TzpAE only. On the other hand, TktA and AroG can be overexpressed together with TrpAE to get higher yields of tryptophan where glucose is favored as a carbon source.
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Ongay, Reyhan (1994). Tryptophan biosynthesis by genetically engineered Escherichia coli utilizing different carbon sources. Master's thesis, Texas A&M University. Available electronically from
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