Abstract
Cell-free protein synthesis (CFPS) has become a widely used research tool in sys-tems and synthetic biology. In this study, we used sequence specific constraint based modeling to evaluate the performance of an E.coli cell-free protein synthesis system. A core E. coli metabolic model, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis and degradation was augmented with sequence specific descriptions of transcription and translation with effective models of promoter function. Thus, sequence specific constraint based modeling explicitly couples transcription and translation processes and the regulation of gene expression with the availability of metabolic resources. We tested this approach by simulating the expression of two model proteins: chloramphenicol acetyltransferase and dual emission green fluorescent protein, for which we have training data sets; we then expanded the simulations to a range of therapeutically relevant proteins. Protein expression simulations were consistent with measurements for a variety of cases. We then compared optimal and experimentally constrained CFPS reactions, which sug-gested the experimental system over-consumed glucose and had suboptimal oxidative phosphorylation activity. Lastly, global sensitivity analysis identified the key metabolic processes that controlled the CFPS productivity, energy efficiency, and carbon yield. In summary, sequence specific constraint based modeling of CFPS offered a novel means to a priori estimate the performance of a cell-free system, using only a limited number of of adjustable parameters. In this study we modeled the production of a single protein, however this approach could be extended to multi-protein synthetic circuits, RNA circuits or small molecule production.