Improving product yields on D-glucose in Escherichia coli via knockout of pgi and zwf and feeding of supplemental carbon sources.

The use of lignocellulosic biomass as a feedstock for microbial fermentation processes presents an opportunity for increasing the yield of bioproducts derived directly from glucose. Lignocellulosic biomass consists of several fermentable sugars, including glucose, xylose, and arabinose. In this study, we investigate the ability of an E. coli Δpgi Δzwf mutant to consume alternative carbon sources (xylose, arabinose, and glycerol) for growth while reserving glucose for product formation. Deletion of pgi and zwf was found to eliminate catabolite repression as well as the ability of E. coli to consume glucose for biomass formation. In addition, the yield from glucose of the bioproduct D-glucaric acid was significantly increased in a Δpgi Δzwf strain.

Improving D-glucaric acid production from myo-inositol in E. coli by increasing MIOX stability and myo-inositol transport.

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.

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli.

The field of metabolic engineering has the potential to produce a wide variety of chemicals in both an inexpensive and ecologically-friendly manner. Heterologous expression of novel combinations of enzymes promises to provide new or improved synthetic routes towards a substantially increased diversity of small molecules. Recently, we constructed a synthetic pathway to produce d-glucaric acid, a molecule that has been deemed a "top-value added chemical" from biomass, starting from glucose. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To synthetically increase the effective concentration of myo-inositol, polypeptide scaffolds were built from protein-protein interaction domains to co-localize all three pathway enzymes in a designable complex as previously described (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of increased numbers of MIOX-targeted domains was much less significant. We determined that the scaffolds directly increased the specific MIOX activity and that glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed an approximately 5-fold improvement in product titers over the non-scaffolded control, and a 50% improvement over the previously reported highest titers. These results further validate the utility of these synthetic scaffolds as a tool for metabolic engineering.