Modulation of Phosphoenolpyruvate Synthase Expression Increases Shikimate Pathway Product Yields in E. coli Jian Yi, Kai Li, K. M. Draths, and J. W. Frost* Department of Chemistry, Michigan State University, East Lansing, Michigan 48824 Product yields in microbial synthesis are ultimately limited by the mechanism utilized for glucose transport. Altered expression of phosphoenolpyruvate synthase was examined as a method for circumventing these limits. Escherichia coli KL3/pJY1.216A was cultured under fed-batch fermentor conditions where glucose was the only source of carbon for the formation of microbial biomass and the synthesis of product 3-dehydroshikimic acid. Shikimate pathway byproducts 3-deoxy-D-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid were also generated. An optimal expression level of phosphoenolpyruvate synthase was identified, which did not correspond to the highest expression levels of this enzyme, where the total yield of 3-dehydroshikimic acid and shikimate pathway byproducts synthesized from glucose was 51% (mol/mol). For comparison, the theoretical maximum yield is 43% (mol/mol) for synthesis of 3-dehydroshikimic acid and shikimate pathway byproducts from glucose in lieu of amplified expression of phosphoenolpyruvate synthase. An extensive range of chemicals can be synthesized by exploiting microbial catalysis and the shikimate pathway (1). Escherichia coli catalyzed syntheses of the amino acids L-phenylalanine (2) and L-tryptophan (3), as well as the chiral synthon shikimic acid (4, 5), are already practiced industrially. Syntheses of commodity chemicals [adipic acid (6), phenol (7)], pseudocommodity chemicals [catechol (8), hydroquinone (9), p-hydroxybenzoic acid (10, 11)], fine chemicals [vanillin (12), indigo (13)], and ultrafine chemicals [gallic acid (14, 15), pyrogallol (14)] have also been reported. 3-Dehydroshikimic acid (Figure 1) is the most advanced shikimate pathway precursor common to the synthesis of all of the chemicals cited above. Strategies elaborated to increase the yield and concentration of 3-dehydroshikimic acid synthesized from glucose are thus applicable to the microbial synthesis of a wide range of molecules. The mechanism employed to transport carbohydrate starting material from the culture medium into the cytoplasm is now recognized to be an important deter- minant of the yield of a microbially synthesized product (16-18). In a wide variety of microbes that employ the phosphoenolpyruvate:carbohydrate phosphotransferase system (19), one molecule of phosphoenolpyruvate is converted into pyruvate for each molecule of glucose transported from the culture medium into the cytoplasm and phosphorylated to form glucose 6-phosphate (Figure 1). Subsequent conversion of pyruvate to CO 2 results in a maximum theoretical yield of 43% (mol/mol), based on a stoichiometric analysis, for the conversion of glucose into 3-dehydroshikimic acid (20). In this account, the impact of recycling pyruvate generated by the phospho- enolpyruvate:carbohydrate phosphotransferase system back to phosphoenolpyruvate (21, 22) on the yield of 3-dehydroshikimic acid synthesized by E. coli from glucose is examined under fermentor-controlled condi- tions. Conversion of phosphotransferase-generated pyru- * To whom correspondence should be addressed. Tel: (517) 355- 9715 extension 115. Fax: (517) 432-3873. E-mail: frostjw@ argus.cem.msu.edu. Figure 1. Intermediates (abbreviations): D-glucose 6-phos- phate (G6P), phosphoenolpyruvate (PEP), D-erythrose 4-phos- phate (E4P), 3-deoxy-D-arabino-heptulosonic acid (DAH) 7-phos- phate (DAHP), 3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), gallic acid (GA), shikimic acid (SA). Enzymes (genes): (a) PEP:carbohydrate phosphotransferase system, (b) PEP synthase (ppsA), (c) DAHP synthase (aroF FBR ), (d) DHQ synthase (aroB), (e) DHQ dehydratase (aroD), (f) shikimate dehydrogenase (aroE), (g) uncharacterized. 1141 Biotechnol. Prog. 2002, 18, 1141-1148 10.1021/bp020101w CCC: $22.00 © 2002 American Chemical Society and American Institute of Chemical Engineers Published on Web 10/31/2002