(586d) Building Bottom-up Kinetic Models for Optimizing Cell-Free Lignocellulose Degradation Systems

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is difficult (and cost prohibitive) to use in conventional processes. A promising route for lignocellulose bioprocessing is the cell-free cellulose decomposition as high product yields and selectivity as well as greater process control is possible than in bioprocessing. Using a modified glycolytic pathway based on the lignocellulolytic bacterium, Clostridium thermocellum, a cell-free ligocellulolytic degradation system was designed. This system is modeled using Elementary Decomposition (ED), which provides a way to generate mechanistic kinetic models from reaction stoichiometry and a framework for kinetic parameter determination. Existing approaches are somewhat limited in that the kinetic parameters estimated are based holistic, top-down network information and relative enzyme and metabolite concentrations, often using lumped reactions and kinetic parameters. For small cell-free systems, where more accurate kinetic knowledge is desired, we use the ED approach for estimating kinetic parameters from progress-curve data for individual enzymes (i.e. a bottom-up approach). Using spectroscopy data, our approach minimizes error between the measured and calculated concentrations of one reaction participant by solving a system of equations describing the ED kinetics with variable kinetic parameters and participating species concentrations. The process as a whole then performs a bottom-up and absolute estimation of kinetic parameters. To validate our system, we are testing it on formate dehydrogenase (EC 1.2.1.2), a commercially available enzyme that is commonly used for recycling of NAD⁺ to NADH in redox reactions.