1The field of metabolic engineering has the potential to produce a wide variety of 2 chemicals in both an inexpensive and ecologically-friendly manner. Heterologous 3 expression of novel combinations of enzymes promises to provide new or improved 4 synthetic routes towards a substantially increased diversity of small molecules. Recently, 5we constructed a synthetic pathway to produce D-glucaric acid, a molecule that has been 6 deemed a "top-value added chemical" from biomass, starting from glucose. Limiting 7 flux through the pathway is the second recombinant step, catalyzed by myo-inositol 8 oxygenase (MIOX), whose activity is strongly influenced by the concentration of the 9 myo-inositol substrate. To synthetically increase the effective concentration of myo-10 inositol, polypeptide scaffolds were built from protein-protein interaction domains to co-11 localize all three pathway enzymes in a designable complex as previously described 12 (Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number 13 of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of 14 increased numbers of MIOX-targeted domains was much less significant. We 15 determined that the scaffolds directly increased the specific MIOX activity and that 16 glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed 17 an approximately 5-fold improvement in product titers over the non-scaffolded control, 18 and a 50% improvement over the previously reported highest titers. These results further 19 validate the utility of these synthetic scaffolds as a tool for metabolic engineering. 20 21

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.

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.

Metabolic engineering focuses on controlling cellular metabolism and heterologous pathway flux to maximize the production of a product of interest. In recent years, various "devices" have begun to emerge from synthetic biology which could find widespread application in the field of metabolic engineering. In this review, we describe devices from synthetic biology and discuss the difficulties that may be encountered when using these devices as tools for metabolic engineering. Highlights • Synthetic biology devices may be useful tools for metabolic engineering. • The widespread application of synthetic biology devices is hindered by the complexity of biology. • Continued work with these devices may help elucidate design rules for future device engineering.