Biology subjects

Vilkhovoy, M.

Publications and source records attributed to Vilkhovoy, M..

5 recordsLinked to original sources

bioRxiv2023

Integrated Constraint-Based Modeling of E. coli Cell-Free Protein Synthesis

Cell-free protein expression has become a widely used research tool in systems and synthetic biology and a promising technology for protein biomanufacturing. Cell-free protein synthesis relies on in-vitro transcription and translation processes to produce a protein of interest. However, transcription and translation depend upon the operation of complex metabolic pathways for precursor and energy regeneration. Toward understanding the role of metabolism in a cell-free system, we developed a dynamic constraint-based simulation of protein production in the myTXTL E. coli cell-free system with and without electron transport chain inhibitors. Time-resolved absolute metabolite measurements for [M] = 63 metabolites, along with absolute concentration measurements of the mRNA and protein abundance and measurements of enzyme activity, were integrated with kinetic and enzyme abundance information to simulate the time evolution of metabolic flux and protein production with and without inhibitors. The metabolic flux distribution estimated by the model, along with the experimental metabolite and enzyme activity data, suggested that the myTXTL cell-free system has an active central carbon metabolism with glutamate powering the TCA cycle. Further, the electron transport chain inhibitor studies suggested the presence of oxidative phosphorylation activity in the myTXTL cell-free system; the oxidative phosphorylation inhibitors provided biochemical evidence that myTXTL relied, at least partially, on oxidative phosphorylation to generate the energy required to sustain transcription and translation for a 16-hour batch reaction.

synthetic biology↗

bioRxiv2022

Hyaluronic acid biosynthesis promotes an invasive, stem-like cancer cell phenotype by broadly altering metabolism

Metastasis is the leading cause of breast cancer-related deaths and often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion have been associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process remains unclear due in part to the lack of convergent approaches that integrate engineered model systems, computational tools, and cancer biology. Using microfluidic invasion models, metabolomics, computational flux balance analysis (FBA), and bioinformatic analysis of patient data we investigated the functional links between the stem-like, invasive, and metabolic phenotype of breast cancer cells as a function of HA biosynthesis. Our results suggest that CSCs are more invasive than non-CSCs and that broad metabolic changes caused by overproduction of HA play a role in this process. Accordingly, overexpression of hyaluronic acid synthases (HAS) 2 or 3 induced a metabolic phenotype that promoted breast cancer cell stemness and invasion in vitro and upregulated a transcriptomic signature that was predictive of increased invasion and worse survival in patients. Collectively, this study suggests that HA overproduction leads to metabolic adaptations that help satisfy the energy demands necessary for 3D invasion of breast cancer stem cells further highlighting the importance of engineered model systems and multidisciplinary approaches in cancer research.

cancer biology↗

bioRxiv2020

Effective Biophysical Modeling of Cell Free Transcription and Translation Processes

Transcription and translation are at the heart of metabolism and signal transduction. In this study, we developed an effective biophysical modeling approach to simulate transcription and translation processes. We tested this approach by simulating the dynamics of two cell free synthetic circuits. First, we considered a simple circuit in which sigma factor 70 induced the expression of green fluorescent protein. This relatively simple case was then followed by a more complex negative feedback circuit in which two control genes were coupled to the expression of a third reporter gene, green fluorescent protein. While many of the model parameters were estimated from previous biophysical literature, the remaining unknown model parameters for each circuit were estimated from messenger RNA (mRNA) and protein measurements using multi-objective optimization. In particular, either the literature parameter estimates were used directly in the model simulations, or characteristic literature values were used to establish feasible ranges for the multiobjective parameter search. Next, global sensitivity analysis was used to determine the influence of individual model parameters on the expression dynamics. Taken together, the effective biophysical modeling approach captured the expression dynamics, including the transcription dynamics, for the two synthetic cell free circuits. While we considered only two circuits here, this approach could potentially be extended to simulate other genetic circuits in both cell free and whole cell biomolecular applications. The model code, parameters, and analysis scripts are available for download under an MIT software license from the Varnerlab GitHub repository.

systems biology↗

bioRxiv2017

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.

synthetic biology↗

bioRxiv2017

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 training 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 protein length 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 multi-protein synthetic circuits, RNA circuits or the cell free production of small molecule products.

synthetic biology↗