Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of gluco se into aromatic bioproducts and a variety of industrial chemicals. Microbi al synthesis of DHS, like other intermediates in the common pathway of arom atic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Esch erichia cell constructs is examined in a fed-batch fermenter where glucose availability, oxygenation revels, and solution pH are controlled. DHS yield s and titers are also determined by the activity of 3-deoxy-D-arabino-heptu losonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its i n vivo activity. By combining fed-batch fermenter control with amplified ex pression of a feedback-insensitive isozyme of DAHP synthase and amplified e xpression of transketolase, DHS titers of 69 g/L were synthesized in 30% yi eld (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to D HS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermenter conditions employed. (C) 1999 John Wiley & Sons, Inc.