title: Improving D-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport.
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 941 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 4.85 g/L of D-glucaric acid from 10.8 g/L myo-inositol in recombinant E. coli.
authors: Department of Chemical Engineering, Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors: Eric
authors: E
authors: Shiue
authors: Department of Chemical Engineering, Synthetic Biology Engineering Research Center (SynBERC), Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: kljp@mit.edu.
authors: Kristala L J
authors: KL
authors: Prather
mesh_terms: D001693:Biological Transport, Active / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005937:Glucaric Acid / Q000378:metabolism*; D007294:Inositol / Q000378:metabolism*; D050562:Inositol Oxygenase / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D007294:Inositol; D050562:Inositol Oxygenase; D005937:Glucaric Acid
keywords: Directed evolution; Metabolic engineering; Myo-inositol; Soluble protein fusions; Substrate transport; d-glucaric acid