Getting around flu drug shortage

Scientists explore alternative means to produce key ingredient that could limit oseltamivir quantity during epidemic

By | January 3, 2006

Getting around flu drug shortage Scientists explore alternative means to produce key ingredient that could limit oseltamivir quantity during epidemic Researchers worldwide are exploring alternative ways to make sure there's enough shikimic acid -- the key ingredient in the anti-influenza drug oseltamivir (Tamiflu) -- to meet the world's needs in the event of the much-feared bird flu pandemic. Currently, shikimic acid is mostly obtained from a seasonal plant that grows in four mountain provinces in China through a multi-step, low-yielding process of chemical extraction ? which has some scientists concerned. "You can't rely on star anise for the amounts of shikimic acid that the world needs to produce Tamiflu," John Frost of Michigan State University in East Lansing told The Scientist. The extraction of shikimic acid is a complex process that starts with dried star anise, or Illicium verum, Martina Rupp, a spokesperson for Roche, the company that manufactures oseltamivir, told The Scientist in an Email. The process "results in crude shikimic acid, which is purified by several crystallizations to yield pure shikimic acid," she added. "Extraction of 30 kilograms of star anise provides a yield of 1 kilogram of shikimic acid." The main alternative to this process is fermentation using genetically engineered strains of E. coli. According to a 2003 review, fermentation of shikimic acid could provide, in principle, an unlimited supply of the raw material. And researchers are hard at work to improve the fermentation process, making it even easier to generate the drug's key ingredient. In October, a report compared a shikimic acid producing strain of E. coli in which the arol (shikimic acid kinase ii) gene had been as deleted to that of a control strain under carbon- and phosphate-limited conditions. For the shikimic acid-producing strain, phosphate limitation resulted in a higher yield of shikimic acid and a lower yield of by-products from the shikimate pathway, but those advantages were counteracted by an increased cell lysis - which could make downstream processing more difficult, the authors caution. Frost, one of the designers of the fermentation technique, noted that the mere idea of extracting shikimic acid from a plant is outmoded. "This is the 21st century, and you make shikimic acid by fermentation. Anyone who's not doing it is really lacking in a fundamental understanding of the scaling-up process, or is not willing to have shikimic acid widely available," he said. Still, Roche, the maker of oseltamivir, says that for now, fermentation is still an alternative way to produce shikimic acid, taking a back seat to the traditional technique. "Right now, we derive two thirds of the shikimic acid from star anise and one third from fermentation," Darien Wilson, director of public affairs at Roche Pharmaceuticals, told The Scientist. "We look at fermentation as an alternative, to be able to ramp up production if needed, but right now it's not an issue for us to get enough star anise." But researchers are already struggling to improve fermentation using microorganisms, making it even easier to generate the drug's key ingredient. In 1999, Frost and his colleagues modified the E. coli shikimate pathway, a synthetic pathway that generates aromatic amino acids. The researchers disrupted the aroL and aroK loci in the genome of E. coli. These genes encode two isozymes that act on shikimic acid, and disrupting them stopped the pathway at the shikimic acid step. "As a result, the cells accumulate shikimic acid," said Karen Frost?formerly Karen Draths?the lead author of the paper that first proposed replacing the isolation of shikimic acid with fermentation. "The bacterium exports shikimic acid into the culture media and we extract it from there." In recent years, a few research groups have concentrated on the physiology of E. coli in an attempt to increase the efficiency of shikimic acid fermentation. Louise Johansson of Lund University in Sweden is studying the cell physiology of a modified strain of E. coli using fermentation technology and metabolic engineering methods, including mathematical modeling and transcriptome analysis. In a recent paper, John Frost and his group describe the impact of increasing the availability of phosphoenolpyruvate (pep) during shikimic acid biosynthesis, and pinpointed which strains of E.coli produce the highest titers and yields of shikimic acid from glucose. Links within this article John W. Frost Martina Rupp M. Kramer et al., "Metabolic engineering for microbial production of shikimic acid," Metabolic Engineering, October, 2003. PM_ID: 14642355. L. Johansson et al., "Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate-limited and carbon-limited conditions," Biotechnol Bioeng, October 20, 2005. Darien E. Wilson Karen M. Frost K.M. Draths et al., "Shikimic acid and quinic acid: Replacing isolation from plant sources with recombinant microbial biocatalysis," J. Am. Chem. Soc. 121: 1603-1604, 1999. J. Bongaerts et al., "Metabolic engineering for microbial production of aromatic amino acids and derived compounds," Metabolic Engineering, October, 2001. PM_ID: 11676565. Louise Johansson S.S. Chandran et al., "Phosphoenolpyruvate availability and the biosynthesis of shikimic acid," Biotechnology Progress, May-June, 2003. PM_ID: 12790643.

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