Kara Calhoun

An integrated cell‐free metabolic platform for protein production and synthetic biology

Cell‐free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell‐free systems, however, have been limited by their inability to co‐activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell‐free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co‐activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell‐free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.

An Economical Method for Cell‐Free Protein Synthesis using Glucose and Nucleoside Monophosphates

Cell‐free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell‐free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell‐free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3‐h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and β‐galactosidase (472 kDa). The glucose/NMP cell‐free reaction system dramatically reduces reagent costs while supplying high protein yields.

Energy systems for ATP regeneration in cell-free protein synthesis reactions.

Supplying energy for cell-free protein synthesis reactions is one of the biggest challenges to the success of these systems. Oftentimes, short reaction duration is attributed to an unstable energy source. Traditional cell-free reactions use a compound with a high-energy phosphate bond, such as phosphoenolpyruvate, to generate the ATP required to drive transcription and translation. However, recent work has led to better understanding and activation of the complex metabolism that can occur during cell-free reactions. We are now able to generate ATP using energy sources that are less expensive and more stable. These energy sources generally involve multistep enzymatic reactions or recreate entire energy-generating pathways, such as glycolysis and oxidative phosphorylation. We describe the various types of energy sources used in cell-free reactions, give examples of the major classes, and demonstrate protocols for successful use of three recently developed energy systems: PANOxSP, cytomim, and glucose.

Sequence Specific Modeling of E. coli Cell-Free Protein Synthesis

Cell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. 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 used sequence specific constraint based modeling to evaluate the performance 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 tested this approach by simulating the expression of two model proteins: chloramphenicol acetyltransferase and dual emission green fluorescent protein, for which we have data sets; we then expanded the simulations to a range of additional proteins. Protein expression simulations were consistent with measurements for a variety of cases. The constraint based simulations confirmed that oxidative phosphorylation was active in the CAT cell-free extract, as without it there was no feasible solution within the experimental constraints of the system. We then compared the metabolism of theoretically optimal and experimentally constrained CFPS reactions, and developed parameter free correlations which could be used to estimate productivity as a function of carbon number and promoter type. Lastly, global sensitivity analysis identified the key metabolic processes that controlled CFPS productivity and energy efficiency. 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 adjustable parameters. While we modeled the production of a single protein in this study, the approach could easily be extended to multiprotein synthetic circuits, RNA circuits, or the cell-free production of small molecule products.

Toward a Genome Scale Sequence Specific Dynamic Model of Cell-Free Protein Synthesis in Escherichia coli

Cell-free protein expression systems have become widely used in systems and synthetic biology. In this study, we developed an ensemble of dynamic E. coli cell-free protein synthesis (CFPS) models. Model parameters were estimated from a training dataset for the cell-free production of a protein product, chloramphenicol acetyltransferase (CAT). The dataset consisted of measurements of glucose, organic acids, energy species, amino acids, and CAT. The ensemble accurately predicted these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the optimality of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity and the metabolism as a whole depend most on oxidative phosphorylation and glycolysis and gluco-neogenesis. Amino acid biosynthesis is also important for productivity, while the overflow metabolism and TCA cycle affect the overall system state. In addition, the translation rate is shown to be more important to productivity than the transcription rate. Finally, CAT production was robust to allosteric control, as was most of the network, with the exception of the organic acids in central carbon metabolism. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and should provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.