(228f) Reaction Compartmentalization Leads to Enhanced Production of PHA in Yarrowia Lipolytica

Making the most efficient use of the finite amount of carbon and energy present in feedstocks is a common goal for the microbial production of commodity chemicals, thus improving the yield of reactions and improving process economics. A well-studied method to improve reaction efficiency is to increase the local concentration of the reactants. To this end, we have focused on increasing the efficiency of reactions building off the beta-oxidation cycle of Yarrowia lipolytica as a means of improving production of the bioplastic polyhydroxyalkanoate (PHA). Local concentration changes are achieved by compartmentalizing key enzymes within the peroxisome, a membrane-enclosed organelle where beta-oxidation occurs. Import into this organelle is accomplished through recognition of some form of peroxisomal targeting signal (PTS) and mediated by a cascade of peroxisome-associated (PEX) enzymes. Manipulation of this tag and the associated enzymes resulted in a greater than three-fold increase in PHA production from the same carbon feed conditions. In an effort to further improve production, expansion of the total peroxisome volume was investigated through knockout and overexpression of specific PEX enzymes. Finally, improvements in carbon flux into the peroxisome were sought via traditional metabolic engineering strategies including carbon-sink knockouts, transporter manipulation, and pathway overexpression.