Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6494617 | Metabolic Engineering | 2014 | 10 Pages |
Abstract
d-glucaric acid has been explored for a myriad of potential uses, including biopolymer production and cancer treatment. A biosynthetic route to produce d-glucaric acid from glucose has been constructed in Escherichia coli (Moon et al., 2009b), and analysis of the pathway revealed myo-inositol oxygenase (MIOX) to be the least active enzyme. To increase pathway productivity, we explored protein fusion tags for increased MIOX solubility and directed evolution for increased MIOX activity. An N-terminal SUMO fusion to MIOX resulted in a 75% increase in d-glucaric acid production from myo-inositol. While our directed evolution efforts did not yield an improved MIOX variant, our screen isolated a 941Â bp DNA fragment whose expression led to increased myo-inositol transport and a 65% increase in d-glucaric acid production from myo-inositol. Overall, we report the production of up to 4.85Â g/L of d-glucaric acid from 10.8Â g/L myo-inositol in recombinant E. coli.
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Authors
Eric Shiue, Kristala L.J. Prather,