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Phosphoenolpyruvate Availability and the Biosynthesis of Shikimic Acid

The impact of increased availability of phosphoenolpyruvate during shikimic acid biosynthesis has been examined in Escherichia coliK‐12 constructs carrying plasmid‐localized aroFFBR and tktAinserts encoding, respectively, feedback‐insensitive 3‐deoxy‐d‐arabino‐heptulosonic acid 7‐phosphate synthase and transketolase. Strategies for increasing the availability of phosphoenolpyruvate were based on amplified expression of E. coli ppsA‐encoded phosphoenolpyruvate synthase or heterologous expression of the Zymomonas mobilis glf‐encoded glucose facilitator. The highest titers and yields of shikimic acid biosynthesized from glucose in 1 L fermentor runs were achieved using E. coli SP1.lpts/pSC6.090B, which expressed both Z. mobilis glf‐encoded glucose facilitator protein and Z. mobilis glk‐encoded glucose kinase in a host deficient in the phosphoenolpyruvate:carbohydrate phosphotransferase system. At 10 L scale with yeast extract supplementation, E. coli SP1.lpts/pSC6.090B synthesized 87 g/L of shikimic acid in 36% (mol/mol) yield with a maximum productivity of 5.2 g/L/h for shikimic acid synthesized during the exponential phase of growth.