Increasing the carbon flow into the aromatic common pathway: Biosynthesis of 3-dehydroshikimic acid from D-glucose

Metabolic engineering, DNA microarray and recombinant DNA technology were used to develop strategies to increase the carbon flow directed into the shikimate pathway. 3-Dehydroshikimic acid (DHS) is the most advanced intermediate shared by both biosynthesis of aromatic amino acids and biocatalytic syntheses of a variety of value-added chemicals. Strategies elaborated to increase the yield and concentration of DHS synthesized from glucose are thus applicable to the microbial synthesis of a wide range of chemicals.; Shikimate pathway product yields in microbial synthesis are ultimately limited by the glucose transport mechanism. To increase the availability of phosphoenolpyruvate (PEP), overexpression of PEP synthase or alteration of glucose transport by Glf-mediated facilitated diffusion or the Ga1P galactose permease in E. coli constructs led to increased yields of DHS and shikimate pathway byproducts. Overexpression of PEP synthase was currently leading to the synthesis of higher yields of DHS and shikimate pathway byproducts relative to alteration of glucose transport. In addition, the production of DHS can be enhanced by changing fermentor-controlled pH from 7 to 6.; DNA microarray technology was used to study gene expression profiles under fed-batch fermentor conditions. The results showed that a simple overexpression of PEP synthase led to dramatic expression changes of many genes. Based on the microarray study, a more stable DAHP synthase (AroGFBR) was isolated by PCR mutagenesis and has its advantage over previously used DAHP synthase (AroFFBR) because of its stability over the course of a fermentation run.; The export of shikimic acid or DHS was also studied. Two approaches to identify the shikimate export protein were attempted without success. The first approach attempted to identify a mutant deficient in shikimate export while the second approach was based on the overexpression of a shikimate export protein. The failure of both approaches is consistent with the possibility that multiple proteins exist for the export of shikimic acid. Furthermore, the determination that intracellular shikimic acid concentrations are much lower than extracellular concentrations suggests that export of this hydroaromatic is not a limiting factor during microbial synthesis.