(139e) Model-Guided Design Strategies for Bioplastic Overproduction in Rhodopseudomonas Palustris

Rhodopseudomonas Palustris is a metabolically versatile Purple Non-Sulfur Bacterium (PNSB). Depending on growth conditions, R. palustris can operate on either one of the four different forms of metabolism: photoautotrophic, photoheterotrophic, chemoautotrophic, and chemoheterotrophic. R. palustris is also a facultative anaerobe, meaning it can operate both aerobically and anaerobically. Furthermore, the organism is capable of fixing nitrogen and subsequently producing hydrogen and the bioplastic precursor polyhydroxybuyrate (PHB). Recent experimental findings revealed that PHB yields in R. palustris were highly dependent on the characteristics of the utilized carbon source. PHB production significantly increased when grown on the carbon- and electron-rich lignin breakdown product p-coumarate (C₉H₈O₃) compared to acetate when the same amount of carbon was supplied. However, the maximum yield did not improve further when grown on coniferyl alcohol (C₁₀H₁₂O₃). To obtain a systems-level understanding of factors driving PHB yield, a model-driven investigation was performed. A thermo-kinetic analysis of the PHB synthesis pathway identified how the relative concentration of various metabolites in the pathway influenced overall productivity. These findings were incorporated into a recently constructed genome-scale metabolic model of the bacterium to understand how characteristics of the utilized carbon substrate affected PHB productivity. This model-guided approach yielded several engineering design strategies for PHB over-production, including utilizing reduced, high molecular weight substrates that bypass the thiolase reaction. Overall, these findings uncover key thermodynamic and enzyme saturation limitations controlling PHB production and lead to design strategies that can potentially be transferrable to other PHB producing bacteria.