Sequence Specific Modeling ofE. coliCell-Free Protein Synthesis

Abstract

Cell-free protein synthesis (CFPS) has become a widely used research tool in systems 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 1 . CC-BY-NC-ND4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/139774 doi: bioRxiv preprint first posted online May. 23, 2017; 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 suggested 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.

Integrated Constraint-Based Modeling ofE. coliCell-Free Protein Synthesis

“…In particular, these measurements constrain the permissible bounds that a metabolic flux can have in the process of interest. Previously, Vilkhovoy and coworkers used flux balance analysis, in combination with time series metabolite measurements, to predict the synthesis of green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT) under different promoters in different CFPS systems [22]. While the model predicted the overall protein synthesis rate well, there was significant uncertainty regarding the underlying metabolic flux distribution supporting protein expression.…”

“…Next, we explored an important mathematical question: the role of the objective function used in the flux balance analysis calculations. The previous study of Vilkhovoy and coworkers [22] assumed an objective of the maximization of the translation rate (which was also found to describe the data in the current study). However, in the cases of a perturbed network, e.g., protein production in the presence of inhibitors, optimal translation may no longer be a valid network objective.…”

“…A critical tool enabling this goal is mathematical modeling. Modeling the integration of cell-free transcription and translation processes with metabolism remains in its infancy, with few published mathematical models [21][22][23]. Horvath and coworkers developed an ensemble of dynamic E. coli CFPS models using kinetic parameter sets estimated from the metabolite and protein measurements [23].…”

“…Stoichiometric reconstructions have been expanded to include the integration of metabolism with detailed descriptions of gene expression (ME-Model) [28][29][30] and protein structures (GEM-PRO) [31,32]. Vilkhovoy and coworkers [22] developed an experimentally validated constraint-based model of CFPS, which integrated the expression of a protein product with the supply of metabolic precursors and energy. However, while the earlier Vilkhovoy model could reproduce (and in some cases predict) the expression of model proteins, several open questions were raised by the Vilkhovoy study.…”

“…In this study, we expanded on the previous sequence-specific constraint-based model of Vilkhovoy and coworkers [22] by integrating kinetic turnover rates, enzyme levels, kinetic descriptions of transcription/translation, enzyme activities, and absolute metabolite concentrations to constrain CFPS metabolic calculations. Metabolic flux estimated using flux balance analysis (FBA) and experimental inhibitor studies suggested that oxidative phosphorylation was active in the myTXTL system.…”