YIELD AND TITER CONSIDERATIONS
GS4104 is a neuraminidase inhibitor that is effective as an anti-influenza drug.
3 Currently marketed by Roche as Tamiflu, the key problem with the synthesis of this drug was the availability of the hydroaromatic starting material. Shikimic acid is an obvious candidate as a starting material; it is a cyclohexene carboxylic acid possessing the appropriate asymmetric centers. However, only limited quantities of shikimic acid were available because its isolation from the seeds of Illicium plants is expensive.
4 The unfavorable price and limited availability of shikimic acid have been major impediments to its use by chemists as a chiral synthon. Quinic acid, which is isolated from Cinchona bark,
4 is less expensive and more available. This hydroaromatic has been widely employed by chemists as a chiral synthon,
5 although considerably more steps are required to convert quinic acid into GS4104 (
Figure 12.1) relative to the use of shikimic acid as starting material.
3 The scarcity and expense of shikimic acid are similar to those of numerous natural products that are important pharmaceutical building blocks because they too have to be isolated from plants whose cultivation does not benefit from large-scale monoculture.
Our task was to design and construct a microbe that would synthesize shikimic acid.
6 Escherichia coli was selected as the starting point for construction of a shikimate-synthesizing biocatalyst. Shikimic acid is an intermediate in the common pathway of aromatic amino acid biosynthesis (
Figure 12.2), which begins with the condensation of phosphoenolpyruvic acid with d-erythrose 4-phosphate catalyzed by 3-deoxy-d-arabino-heptulosonic acid 7-phosphate (DAHP,
Figure 12.2) synthase. DAHP is then cyclized to 3-dehydroquinic acid (DHA) by aroB-encoded 3-dehydroquinate synthase. Dehydration of 3-dehydroquinic acid catalyzed by aroD-encoded 3-dehydroquinate dehydratase is followed by the reduction of the resulting 3-dehydroshikimic acid (DHS) to the desired shikimic acid by aroE-encoded shikimate dehydrogenase. To prevent the conversion of shikimic acid into shikimate 3-phosphate, both isozymes of shikimate kinase were inactivated by transduction using P1 phage of aroL478::Tn10 and aroK17::CmR. The next task was to increase the flow of carbon down the common pathway. This was accomplished by plasmid localization of a mutated isozyme of DAHP synthase encoded by aroFFBR that is insensitive to feedback inhibition by aromatic amino acids. By blocking the conversion of shikimic acid into shikimate 3-phosphate, de novo biosynthesis of aromatic amino acid is blocked. 1-Phenylalanine, 1-tyrosine, 1-tryptophan, and aromatic vitamins must be added to cultures to enable the microbial construct to grow, but they inhibit DAHP synthase activity and thus the flow of carbon into the common pathway. Employment of aroFFBR and the encoded DAHP synthase that is insensitive to feedback inhibition is thus critical to directing increased carbon flow into aromatic amino acid biosynthesis.
Individual common pathway enzymes become impediments to increased carbon flow due to their inability to convert substrate into product at a rate sufficiently rapid to avoid cytoplasmic accumulation and subsequent export of the substrate into the culture medium.
7 3-Dehydroquinate synthase and shikimate dehydrogenase are both impediments to increased carbon flow that result, respectively, in accumu-
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FIGURE 12.1 Starting materials for the synthesis of the neuraminidase inhibtor GS4104.