E. coli strain engineering approaches for improving glucaric acid production

Co-expression of myo-Inositol 1-phosphate synthase (MIPS), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) in an E. coli host results in the production of glucaric acid from glucose. Glucose-6-phosphate (G6P) is the key central metabolite for this glucaric acid biosynthetic pathway that competes with glycolysis and the pentose phosphate pathways for this intermediate. A host strain with deletions in zwf (G6P dehydrogenase, pentose phosphate pathway) and pgi (phosphoglucoisomerase, glycolysis) evaluated in benchtop bioreactors showed a five-fold increase in glucaric acid yield from glucose (Y_GA/G) compared to the unmodified host. A pgi single deletion host strain (zwf+) resulted in a three-fold increase in Y_GA/G. Catabolite repression was relieved in these modified host strains as glucose and xylose, added as a co-substrate, were utilized simultaneously. However, titers were limited by a dramatic reduction in glucose uptake rates. Strain engineering strategies to restore glucose uptake rates in these central metabolism deletion strains included: 1) co-expressing a fragment from the manX gene as a means of titrating out the sgrS gene product, known to regulate pts glucose transport system via a double-stranded mRNA mechanism, and 2) deleting the sgrS gene directly to eliminate regulation.