Modeling of Cell-Free Glycoprotein Production in Escherichia coli | AIChE

Modeling of Cell-Free Glycoprotein Production in Escherichia coli

Authors 

Horvath, N. G. - Presenter, West Virginia University
Vilkhovoy, M. - Presenter, University of Massachusetts
Varner, J. D., Purdue University
Swartz, J., Stanford University
Calhoun, K. A., Stanford University

Cell-free systems offer many advantages for the study, manipulation and modeling of metabolism compared to in vivo processes. Many of the challenges confronting genome-scale kinetic modeling can potentially be overcome in a cell-free system. For example, there is no complex transcriptional regulation to consider, transient metabolic measurements are easier to obtain, and we no longer have to consider cell growth. Thus, cell-free operation holds several significant advantages for model development, identification and validation. In this study, we constructed an ensemble of genome-scale kinetic models of E. coli cell-free synthesis of the chloramphenicol acetyl transferase (CAT) protein. The model, which described 234 species and 276 interactions in central carbon and amino acid biosynthesis, was identified using dynamic CAT, energy, amino acid, glucose and organic acid measurements. Allosteric interactions were described using a novel rules-based approach which integrated complex allosteric regulation with traditional kinetic pathway modeling using non-linear transfer functions. Next, we used global sensitivity analysis to estimate which experimentally controllable parameters (e.g., metabolite or enzyme initial conditions) most influenced culture performance (e.g., rate of CAT production and CAT yield). From this analysis we identified a subset of metabolite and enzyme initial conditions that improved CAT protein production. Taken together, we developed a genome-scale kinetic model describing the cell-free production of a model protein. This model was identified using a comprehensive dynamic data set, and analyzed using global sensitivity analysis to determine experimentally adjustable factors which influenced culture performance. This study represents a first step toward the rational design of the dynamic operation of cell-free protein production.