Biocatalytic conversion of D-glucose into aromatics

Increased carbon flow directed into the common pathway of aromatic amino acid biosynthesis does not necessarily translate into increased synthesis of end product aromatic amino acids. This is due to the rate-limiting character of common pathway enzymes which are unable to catalyze conversion of substrate into product at a rate sufficient to avoid accumulation of substrate. Methods for identifying and removing rate-limiting common pathway enzymes are described in the following thesis. In addition, methodology is elaborated for conversion of 3-dehydroshikimate (DHS) into gallic acid and pyrogallol. Analysis of cell supernatants by $\sp1$H NMR spectroscopy has identified 3-dehydroquinate (DHQ) synthase, shikimate kinase, 5-enolpyruvoylshikimate 3-phosphate (EPSP) synthase, and chorismate synthase as metabolic blocks which impede the flow of carbon through the common pathway in E. coli. Plasmid-based amplification of these enzymes results in removal of the majority of pathway intermediates from the culture broth as well as a twofold increase in end product phenylalanine and phenyllactate accumulation. $\sp1$H NMR analysis of culture supernatants has also identified a feedback loop involving product inhibition of shikimate dehydrogenase by shikimate. Minimal amplification of rate-limiting enzyme expression is needed to yield increased product suggesting plasmid-based expression is above and beyond what is required. Construction and insertion of a synthetic cassette containing the loci for aroA, aroC, and aroB into the genome of E. coli provided sufficient amplification to remove the rate-limiting character of EPSP synthase, chorismate synthase, and DHQ synthase, respectively. Increasing the expression levels of shikimate kinase such that its rate-limiting character was no longer apparent was accomplished by transduction of a tyrR mutation into the E. coli genome. Common pathway enzyme substrates accumulated by appropriately constructed microbes provide novel starting points for the synthesis of organic chemicals. This thesis details an abiotic route to gallic acid from DHS. Stirring of DHS in phosphate buffer achieves a 40% conversion of starting material into gallic acid. Treatment of the crude gallic acid with crude protocatechuate decarboxylase affords a quantitative conversion of the gallic acid into pyrogallol