Fig. 6.

Real-time observation of glucarate production. (A) Glucarate is produced from glucose with the expression of three heterologous enzymes of differing activities: Udh has high activity, MIOX has low activity, and Ino1 competes with glycolysis for glucose-6-phosphate. The biosensor monitors the presence of glucarate while ignoring pathway intermediates. (B) Fluorescence is observed over time as pathway intermediates are converted to glucarate. Biosensor activation by glucuronate (blue line) lags behind activation by glucarate (green line). Activation of the biosensor by glucarate or glucuronate occurs more rapidly than activation by either myo-inositol (purple line) or glucose (tan line). This result reflects the relative activities of the enzymes in the glucarate biosynthesis pathway. End-point fluorescence measured after 8 h trends well with the glucarate titers determined by LC/MS. Error bars and confidence bands represent the 95% confidence interval (n = 3).