Multiple Metabolic Engineering Strategies to Improve Shikimate Titer in Escherichia coli

Shikimate is a valuable chiral precursor for synthesizing oseltamivir (Tamiflu^®) and other chemicals. High production of shikimate via microbial fermentation has attracted increasing attention to overcome the unstable and expensive supply of shikimate extracted from plant resources. The current cost of microbial production of shikimate via engineered strains is still unsatisfactory, and thus more metabolic strategies need to be investigated to further increase the production efficiency. In this study, we first constructed a shikimate E. coli producer through the application of the non-phosphoenolpyruvate: carbohydrate phosphotransferase system (non-PTS) glucose uptake pathway, the attenuation of the shikimate degradation metabolism, and the introduction of a mutant of feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase. Inspired by the natural presence of bifunctional 3-dehydroquinate dehydratase (DHD)-shikimate dehydrogenase (SDH) enzyme in plants, we then designed an artificial fusion protein of DHD-SDH to decrease the accumulation of the byproduct 3-dehydroshikimate (DHS). Subsequently, a repressed shikimate kinase (SK) mutant was selected to promote shikimate accumulation without the supplementation of expensive aromatic substances. Furthermore, EsaR-based quorum sensing (QS) circuits were employed to regulate the metabolic flux distribution between cell growth and product synthesis. The final engineered strain dSA10 produced 60.31 g/L shikimate with a yield of 0.30 g/g glucose in a 5 L bioreactor.