(626e) Improving UDP-Sugar Precursor Availability for Heparosan Biosynthesis in Escherichia Coli

E. coli Nissle 1917, an archetypal probiotic agent, naturally produces and secretes heparosan as the primary component of its cell capsule. Heparosan is an unsulfated polysaccharide precursor to the glycosaminoglycan heparin, the oldest and most successful polysaccharide pharmaceutical of the 20th century that is still in widespread use today. Due to an international crisis stemming from adulteration of the feed stream of this highly sulfated anticoagulant pharmaceutical, increasing costs and concern over quality control led our group to develop a process for producing commercially relevant quantities of bioengineered heparin starting from E. coli derived heparosan. Supplementing E. coli cultures with the nucleotide activated sugar precursors UDP-N-acetylglucosamine and UDP-glucuronic acid improved heparosan titers, suggesting that intracellular availabilities of these valuable metabolites were limiting. We have recently sequenced the genomes of Nissle 1917 and related strain K5 and constructed genome-scale models enabling computational predictions for simultaneous gene overexpressions, downregulations, and deletions to improve availability of UDP-sugar precursors. In conjunction with metabolic remodeling, transcription of the heparosan biosynthetic pathway was modulated using ePathBrick, a combinatorial pathway assembly method developed in our lab to evaluate the effect of regulatory control element configuration. We demonstrate that coupling computational modeling with transcriptional modulation is an effective strategy to explore metabolic space for production of microbial polysaccharides. Engineered strains capable of generating high intracellular concentrations of UDP-activated sugar donors will be critical for in vivo glycosylation of natural products and for production of other medically important glycosaminoglycans like chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan.