New insights into transport capability of sugars and its impact on growth from novel mutants of Escherichia coli.

The fast-growing capability of Escherichia coli strains used to produce industrially relevant metabolites relies on their capability to transport efficiently glucose or potential industrial feedstocks such as sucrose or xylose as carbon sources. E. coli imports extracellular glucose into the periplasmic space across the outer membrane porins: OmpC, OmpF, and LamB. As the internal membrane is an impermeable barrier for sugars, the cell employs several primary and secondary active transport systems, and the phosphoenolpyruvate (PEP)-sugar phosphotransferase (PTS) system for glucose transport. PTS:glucose is the preferred system by E. coli to transport and phosphorylate the periplasmic glucose; nevertheless, PTS imposes a strict metabolic control mechanism on the preferential consumption of glucose over other carbon sources in sugar mixtures such as glucose and xylose resulting from the hydrolysis of lignocellulosic biomass, by the carbon catabolite repression. In this contribution, we summarize the major sugar transport systems for glucose and disaccharide transport, the exhibited substrate plasticity, and their impact on the growth of E. coli, highlighting the relevance of PTS in the control of the expression of genes for the transport and catabolism of other sugars as xylose. We discuss the strategies developed by evolved mutants of E. coli during adaptive laboratory evolution experiments to overcome the nutritional stress condition imposed by inactivation of PTS as a strategy for the selection of fast-growing derivatives in glucose, xylose, or mixtures of glucose:xylose. This approach results in the recruitment of other primary and secondary active transporters, demonstrating relevant sugar plasticity in derivative-evolved mutants. Elucidation of the molecular and biochemical basis of sugar-transport substrate plasticity represents a consistent approach for sugar-transport system engineering for the design of efficient E. coli derivative strains with improved substrate assimilation for biotechnological purposes.

Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli.

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.