(175d) Improvement of D-Glucaric Acid Production From a Synthetic Pathway in Escherichia Coli

Improvement of D-Glucaric
Acid Production from a Synthetic Pathway in Escherichia
coli

Eric Shiue, Kristala Jones Prather

D-glucaric acid is a naturally occurring compound that has
been investigated for a number of potential uses, including cancer treatment,
cholesterol reduction, and production of biodegradable polymers.  Additionally, D-glucaric acid has been
labeled a ?top value-added chemical from biomass? by the US Department of
Energy.  A pathway for the biosynthesis
of D-glucaric acid from glucose has been constructed in E. coli, consisting of three heterologously expressed natural
enzymes: myo-inositol-1-phosphate synthase
(INO1) from Saccharomyces cerevisiae,
myo-inositol oxygenase (MIOX) from
mouse, and uronate dehydrogenase (Udh)
from Pseudomonas syringae.  Using this pathway, glucaric acid titers of
1.13 g/L were achieved following optimization of culture and induction conditions.[1]

Characterization of the pathway's constituent enzymes has
revealed MIOX to be the rate-limiting enzyme. 
Achievement of industrially relevant titers of D-glucaric acid therefore
hinges on improving the activity of MIOX. 
Various strategies have been explored to improve MIOX activity,
including timed induction of pathway enzymes, directed evolution of MIOX for
improved activity, and MIOX-MBP fusions for enhancement of soluble
expression.  We will present experimental
results on the application of these strategies towards enhancing MIOX activity
and glucaric acid productivity.