Attempt of Increasing Yield of Hyaluronan by Randomly Mutating Streptococcus pyogenes Hyaluronan Synthase Expressed in E. coli

Hyaluronan (HA), a linear polysaccharide composed of β(1,3)-GlcNAc-β(1,4)- GlcA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. HAs are synthesized by HA synthases (HASs), which are dual-action glycosyltransferases that catalyze the addition of two different sugars GlcA and GlcNAc from UDP-linked precursors to the growing HA chain. Since the recent medical and cosmetic uses of HA have largely increased its demand, as described in this invention, we have overexpressed spHAS from Gram positive Streptococcus pyogenes c203S on the membrane of E. coli to produce and secrete HA with high molecular weight into the media. Random mutagenesis by error-prone PCR was performed attempting to isolate the mutants with improved yield of HA.