Balderas-Hernández, Víctor E

Bolívar, Francisco

Báez-Viveros, José L

Cabrera-Valladares, Natividad

Gosset, Guillermo

Hernández-Chávez, Georgina

Martínez, Alfredo

Sabido-Ramos, Andrea

Silva, Patricia

Microbial Cell Factories

English

PubMed

Victor E. Balderas-Hernandez

Andrea Sabido-Ramos

Patricia Silva

Natividad Cabrera-Valladares

Georgina Hernandez-Chavez

Jose L. Baez-Viveros

Alfredo Martinez

Francisco Bolivar

Guillermo Gosset

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Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli

Abstract Background Anthranilate 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. Results Sequencing 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. Conclusion This 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.</p

Gosset Guillermo

Bolívar Francisco

Martínez Alfredo

Báez-Viveros José L

Hernández-Chávez Georgina

Cabrera-Valladares Natividad

Silva Patricia

Sabido-Ramos Andrea

Balderas-Hernández Víctor E

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Metabolic engineering for improving anthranilate synthesis from glucose in <it>Escherichia coli</it>

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Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli

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