Hydroaromatic equilibration during shikimic acid and quinic acid biosynthesis

The expense and limited availability of shikimic acid and quinic acid isolated from plants has impeded utilization of these hydroaromatics as synthetic starting materials. The microbial biocatalyses reported in this account could supplant the tedious, multi-step natural product isolation of shikimic acid and quinic acid. Recombinant Escherichia coli biocatalysts genetically engineered to biosynthesize shikimic acid from glucose accumulated not only shikimic acid, but sizable concentrations of quinic acid and 3-dehydroshikimic acid byproducts. 3-Dehydroshikimic acid accumulation results from the feedback inhibition of shikimate dehydrogenase by shikimic acid. The source of quinic acid formation is less clear however. Kinetic experiments revealed shikimate dehydrogenase was capable of accepting both 3-dehydroshikimic acid and 3-dehydroquinic acid as substrates for reduction. Fed-batch fermentor conditions which employed unlimited glucose availability shifted the typical 48 h glucose-limited E. coli SP1.1/pKD12.138A equilibrium from 28 g/L shikimic acid in 13% yield (mol/mol) from glucose as a 1.6:1.0:0.65 (mol/mol/mol) shikimate:quinate:3-dehydroshikimate mixture to 58 g/L shikimic acid in 23% yield (mol/mol) from glucose as a 18:1.0:4.9 (mol/mol/mol) mixture in 60 h.; Homologous quinic acid biosynthesis was investigated by evaluating E. coli QP1.1/pKD12.138A under glucose-limited fed-batch fermentor conditions. QP1.1/pKD12.138A synthesized 49 g/L of quinic acid from glucose in 20% (mol/mol) yield as a 15:1.0 (mol/mol) quinate:3-dehydroquinate mixture and established a hightiter, homologous route for quinic acid biosynthesis. Fed-batch fermentor conditions unlimited in glucose decreased the quinate:3-dehydroquinate ratio of QP1.1/pKD12.138A to 0.74 however.; Physiological State (PS) variable monitoring and control was applied to QP1.1/pKD12.138A within the framework of a Knowledge-Based (KB), intelligent control system. The KB control system consisted of four phases. The novel attributes of the KB control system included phase three manipulation of the specific oxygen uptake rate (SOUR) to approximate the oxygen transfer rate (OTR) increases observed during unlimited glucose availability, and phase four control of the carbon dioxide evolution rate (CER) by manipulating the glucose feed rate to the reactor. Reactor studies revealed that perturbations in the pseudo-steady-state glucose concentration of less than 1 mM could shift the quinate:3-dehydroquinate equilibrium ten-fold.; An online stoichiometric model (SM) was constructed using reactor mass balances and pseudo-online artificial neural network predictions as inputs. The SM predicted phosphoenolpyruvate (PEP) limitations occurred under high glucose uptake rate fermentation conditions during peak growth rates. The predicted PEP limitations were consistent with literature precedent and suggest that pps-encoded PEP synthase overexpression might alleviate QP1.1/pKD12.138A intracellular PEP limitations.