Metabolic Engineering of Escherichia Coli K5 for Production of Heparosan, a Precursor to the Anticoagulant Pharmaceutical Heparin

Heparosan is a key precursor of the anticoagulant heparin (a drug with a market size greater than $10 billion annually) that is currently derived from porcine intestinal extract. Animal-sourced heparin production has several disadvantages and imposes quality concerns on the product. This issue was brought to international attention in 2008 when a heparin contamination crisis resulted in hundreds of deaths. To overcome the drawbacks of the current heparin production process, we are utilizing stoichiometric-based modeling to improve the production of heparosan in Escherichia coli K5. We have developed a multilevel optimization algorithm capable of predicting combinations of genetic manipulations for increased production of target metabolites. The algorithm builds upon the success of Cipher of Evolutionary Design (CiED), a genetic algorithm developed in our lab for predicting gene knockouts, to allow for prediction of sets of gene overexpressions and deletions consistent with superior cellular phenotype. Here we present experimental validation of computational predictions leading to an engineered E. coli K5 strain that achieves improved heparosan titers. In conjunction with computational predictions, traditional fermentation optimization strategies make heparosan production in E. coli K5 a safe and viable heparin production route.