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 glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic 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 in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (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 DHS. 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 fermentor conditions employed.
Li
K
K
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Mikola
M R
MR
Draths
K M
KM
Worden
R M
RM
Frost
J W
JW
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D005707:Gallic Acid / Q000378:metabolism; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010957:Plasmids; D011801:Quinic Acid / Q000378:metabolism; D011995:Recombination, Genetic; D012765:Shikimic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism
D013403:Sugar Phosphates; D011801:Quinic Acid; C096387:3-dehydroshikimate; D012765:Shikimic Acid; C026959:erythrose 4-phosphate; D005707:Gallic Acid; D010728:Phosphoenolpyruvate; D010723:3-Deoxy-7-Phosphoheptulonate Synthase