Protein Engineering of Isoprene Synthase Enables High Titer Isoprene Production Conference: Metabolic Engineering ConferenceYear: 2014Proceeding: Metabolic Engineering XGroup: General SubmissionsSession: Parts to Protein Scale Engineering Time: Monday, June 16, 2014 - 9:40am-10:05am Authors: Cervin, M., DuPont and The Goodyear Tire & Rubber Company are developing a BioIsoprene™ product alternative to petroleum derived isoprene. Isoprene is a key chemical required to produce a diverse range of industrial products including a wide variety of specialty elastomers used in surgical gloves, rubber bands, golf balls, shoes and tire manufacture. The annual world market potential for polymer grade isoprene is approximately 2 billion dollars. We envision the biological production of isoprene (BioIsopreneTM monomer) as a sustainable solution that can address the lack of supply and price instability of this valuable commodity. The biosynthetic production of isoprene presents significant metabolic engineering challenges that include overcoming pathway enzyme regulation, channeling carbon to product, balancing redox and energy requirements of the cell, and selection of appropriate enzymes. The key enzyme, isoprene synthase, has poor kinetic parameters and was a major hurdle to production of isoprene. Using rational protein engineering and PCR mutagenesis in combination with in vitro and in vivo screening techniques we identified variants that enabled isoprene to be produced at commercially relevant metrics. Analysis has demonstrated that the BioIsoprene™ product is >99% pure prior to recovery and purification. Pre-pilot work with the developing process has lead to the manufacture of prototype passenger car tires demonstrating the functional process from start to finish.