IDENTIFICATION AND REMOVAL OF IMPEDIMENTS TO BIOCATALYTIC SYNTHESIS OF AROMATICS FROM D-GLUCOSE - RATE-LIMITING ENZYMES IN THE COMMON PATHWAY OF AROMATIC AMINO-ACID BIOSYNTHESIS

Increasing the flow of D-glucose equivalents into the common pathway of aromatic amino acid biosynthesis creates a metabolic situation where individual common pathway enzymes become rate-limiting. Such enzymes are unable to convert substrate to product at a rate sufficient to avoid intracellular accumulation of substrate. Export of the accumulating substrate by the host microbe into its culture supernatant results in lowered percent conversions and decreased purity of desired aromatic products. Methodology has now been developed which facilitates rapid identification and removal of impediments to biocatalytic synthesis of aromatics from D-glucose which are caused by rate-limiting, common pathway enzymes. An Escherichia coli mutant, D2704, incapable of L-tryptophan and L-tyrosine synthesis was transformed with a plasmid which increased the in vivo catalytic activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase and transketolase. Analysis by H-1 NMR of the culture supernatant of this construct revealed the accumulation of L-phenylalanine and phenyllactate along with common pathway enzyme substrates and related metabolites. Plasmid-borne genes encoding common pathway enzymes were then introduced into D2704 individually and in various combinations followed by H-1 NMR analysis of these constructs' culture supernatants. Progress toward increasing the catalytic activity of rate-limiting enzymes was indicated by a decrease in the number of accumulated enzyme substrates and increased L-phenylalanine synthesis and phenyllactate synthesis. In this fashion, 3-dehydroquinate (DHQ) synthase, shikimate kinase, 5-enolpyruvoylshikimate 3-phosphate (EPSP) synthase, and chorismate synthase were identified as rate-limiting enzymes. A feedback loop in E. coli was also identified involving inhibition of shikimate dehydrogenase by shikimic acid.