Abstract
BackgroundAnthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED) and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies.ResultsSequencing of the trpED genes from E. coli W3110 trpD9923 revealed a nonsense mutation in the trpD gene, causing the loss of anthranilate phosphoribosyl transferase activity, but maintaining anthranilate synthase activity, thus causing anthranilate accumulation. The effects of expressing genes encoding a feedback inhibition resistant version of the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (aroGfbr), transketolase (tktA), glucokinase (glk) and galactose permease (galP), as well as phosphoenolpyruvate:sugar phosphotransferase system (PTS) inactivation on anthranilate production capacity, were evaluated. In shake flask experiments with minimal medium, strains W3110 trpD9923 PTS- and W3110 trpD9923/pJLBaroGfbrtktA displayed the best production parameters, accumulating 0.70–0.75 g/L of anthranilate, with glucose-yields corresponding to 28–46% of the theoretical maximum. To study the effects of extending the growth phase on anthranilate production a fed-batch fermentation process was developed using complex medium, where strain W3110 trpD9923/pJLBaroGfbrtktA produced 14 g/L of anthranilate in 34 hours.ConclusionThis work constitutes the first example of a microbial system for the environmentally-compatible synthesis of anthranilate generated by metabolic engineering. The results presented here, including the characterization of mutation in the trpD gene from strain W3110 trpD9923 and the development of a fermentation strategy, establish a step forward towards the future improvement of a sustainable process for anthranilate production. In addition, the present work provides very useful data regarding the positive and negative consequences of the evaluated metabolic engineering strategies.
Highlights
Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products
Carbon flow into the common aromatic pathway starts with the condensation of D-erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) to yield 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP), in a reaction catalyzed by the enzyme DAHP synthase
Characterization of E. coli W3110 trpD9923 The W3110 trpD9923 strain belongs to a set of E. coli mutants obtained after random mutagenesis by UV light
Summary
Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Several microbial and plant species have the metabolic capacity to synthesize this aromatic compound, opening the possibility for generating sustainable technologies for anthranilate manufacture. This compound is a metabolic intermediate and it is normally not accumulated. In Escherichia coli, the first two reactions in the L-Trp biosynthetic pathway are catalyzed by the enzyme complex anthranilate synthase-phosphoribosyl transferase (TrpETrpD). It is a multifunctional and heterotetrameric complex composed of two TrpE and two TrpD polypeptides (component I and II, respectively).
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