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

Schroeder, W. - Presenter, The Pennsylvania State University
Olson, D., Dartmouth College
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, a commercially available enzyme that is commonly used for recycling of NAD+ to NADH in redox reactions.