Preparative in vitro biosynthesis of complex polyketides

My Dean’s Scholars thesis project concerns the development of an enzymatic system for the in vitro production of polyketides, which we hope will lead to the discovery of new molecules and new medicines. Modular polyketide synthases (PKSs) are massive enzymes that act as molecular assembly lines to elegantly transform small carboxylic acids into complex polyketides. Many natural polyketides are currently used for their medicinal effects, including the antibiotic erythromycin, the immunosuppressant rapamycin, and the anticancer agent epothilone. A long-term goal of the Keatinge-Clay group is to use modified PKSs to create synthetic polyketides, which we hope will lead to new pharmaceutical drugs and valuable chiral intermediates for organic synthesis. Unfortunately, both the study and practical utility of PKSs are hampered by the low yield of their polyketide products – yields from the current protocol rarely exceed one milligram per liter of culture. To create a more efficient, reduced-component platform for studying engineered PKSs and to increase their yield, we devised a system for the preparative in vitro production of complex polyketides. This system includes enzymes necessary for the production of polyketide intermediates from propionate, a modified PKS to form a synthetic polyketide, and enzymes that aid in the regeneration of ATP and NADPH from polyphosphate and glucose, respectively. Currently, we have isolated and assayed most enzymes in the system and are optimizing the system conditions for polyketide yield. We hypothesize that gram quantities of complex polyketides will be produced using the in vitro system, greatly increasing the time and energy efficiency of polyketide synthesis.