Dynamic Models of Metabolic Networks and Analysis of Cell-Free Protein Synthesis

253 pagesMetabolism is the central process through which cells manage their resources to survive, adapt and meet energetic demands. To implement these diverse functions, cells have complex and highly interconnected networks of chemical reactions between genes, RNA, proteins and metabolites. Systems modeling and metabolic engineering arose from the desire to harness the power of metabolism to produce products that benefit society. A primary challenge is the development of metabolic mathematical models that are able to describe the effect genetic perturbations have on cellular behavior. In this study, we first review metabolic modeling methods and go on to develop computational tools for the analysis and engineering of microbial systems with a focus on cell-free protein synthesis (CFPS). Cell-free protein expression has become a widely used research tool in systems and synthetic biology, and a promising technology for biomanufacturing of proteins. 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. Cell-free protein synthesis relies on transcription and translation machinery to produce a protein of interest. To fuel this process requires biochemical enzymes and reactions that are involved in complex metabolic pathways. Toward this, we developed dynamic mathematical models that describe CFPS metabolism and provide insights into improving these systems. We began with a sequence specific constraint based model that coupled transcription/translation processes and the regulation of gene expression with the availability of metabolic resources. Then, we developed a robust comprehensive analysis to quantify absolute levels of metabolites in CFPS using isotopically labeled standards. We expanded our modeling framework by integrating absolute metabolite measurements along with kinetic parameters, enzyme levels, and enzyme activity assays. The framework predicted the overall production of mRNA and protein along with changes in metabolic behavior with two different oxidative phosphorylation inhibitors. Taken together, we provided a comprehensive mathematical framework of CFPS metabolism that could be used to identify strategies for the improvement of CFPS productivity, yield and efficiency

Sequence Specific Modeling of <i>E. coli</i> 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 <i>E. coli</i> cell-free protein synthesis. A core <i>E. coli</i> 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 <i>a priori</i> 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

Sequence Specific Modeling of <i>E. coli</i> 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 <i>E. coli</i> cell-free protein synthesis. A core <i>E. coli</i> 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 <i>a priori</i> 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