Bioengineered heparin is being investigated as a potential substitute for the animal-sourced anticoagulant drug. One step in the current process to prepare bioengineered heparin involves the conversion of N-sulfo heparosan, rich in -> aEuro parts per thousand 4)GlcNS(1 -> aEuro parts per thousand 4) GlcA(1 -> aEuro parts per thousand sequences (where S is sulfo, GlcN is alpha-d-glucosamine, and GlcA is beta-d-glucuronic acid), to a critical intermediate, rich in -> aEuro parts per thousand 4)GlcNS(1 -> aEuro parts per thousand 4) IdoA2S(1 -> aEuro parts per thousand sequences (where S is sulfo and IdoA is alpha-l-iduronic acid), using 2-O-sulfotransferase (2-OST) and C5 epimerase (C-5-epi). Until now, these heparan sulfate biosynthetic enzymes have been expressed in Escherichia coli grown in shake flask culture as fusion proteins. The current study is focused on the high cell density fed-batch cultivation of recombinant E. coli strains expressing both enzymes. We report the high productivity expression of active 2-OST and C-5-epi enzymes of 6.0 and 2.2 mg/g dry cell weight, respectively.