(513c) Sequence Specific Constraint-Based Modeling of E. coli Cell-Free Protein Synthesis
AIChE Annual Meeting
2018
2018 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Emerging Tools and Enabling Technologies in Synthetic Biology: Sensors and Actuators
Wednesday, October 31, 2018 - 1:06pm to 1:24pm
Cell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. Cell-free systems offer many advantages for the study, manipulation and modeling of metabolism compared to in vivo processes. Central amongst these is direct access to metabolites and the biosynthetic machinery without the interference of a cell wall or the complications associated with cell growth. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we have developed experimental and computational tools to model CFPS. First, we adopted a robust reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) method for identification and quantification of metabolites involved in glycolysis, pentose phosphate pathway, the tricarboxylic acid cycle, amino acids and coenzymes. This method is based on derivatization with an aniline-labeled tag and enables separation of structural isomer pairs such as glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. We used the RPLC-MS method to obtain a set of 40 time-resolved metabolite measurements from a TX/TL 2.0 cell free reaction producing green fluorescent protein (GFP). We then used this comprehensive dataset, along with qRT-PCR and fluorescence to quantify the protein product, as constraints for a sequence specific constraint-based model of E. coli cell free protein synthesis. A core E. coli metabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis and degradation was augmented with sequence specific descriptions of transcription and translation, and effective models of promoter function. Model parameters were largely taken from literature; thus, the constraint-based approach coupled the transcription and translation of the protein product, and the regulation of gene expression, with the availability of metabolic resources using only a limited number of adjustable model parameters. We investigated the biochemical processes to determine whether central catabolism, oxidative phosphorylation and protein synthesis could be co-activated in a single reaction system. The simultaneous activation of these complex processes would enhance protein synthesis yields and energy efficiency of the platform. Previous CFPS systems such as the Cytomim showed to be oxygen dependent with the hypothesis that membrane vesicles still have electron transport activity in their extract. The constraint-based model determined oxidative phosphorylation was not required to meet the observed protein productivity, instead energy could be produced via substrate level phosphorylation. We examined this prediction experimentally by comparing the protein productivity of the control reaction, with a CFPS reaction in the presence of thenoyltrifluoroacetone, an electron transport chain inhibitor, and 2-4-dinitrophenol, an uncoupling agent of phospholipid bilayer membrane vesicles. Taken together, we generated a comprehensive metabolomic dataset for a CFPS reaction producing a model protein. We used this data set, along with a genome scale constraint-based model of E. coli cell free protein synthesis, to determine whether central catabolism, oxidative phosphorylation and protein synthesis could be co-activated in a single reaction system. While experimentation is on-going, model simulations suggest the simultaneous activation of these complex processes would enhance protein synthesis yields and energy efficiency of the platform.