Sequence Specific Modeling of <i>E. coli</i> Cell-Free Protein Synthesis

Vilkhovoy, Michael; Horvath, Nicholas; Shih, Che-Hsiao; Wayman, Joseph A.; Calhoun, Kara; Swartz, James R.; Varner, Jeffrey D.

doi:10.1021/acssynbio.7b00465

Cited by 30 publications

(52 citation statements)

References 53 publications

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“…The dynamic CFPS model equations were formulated using the hybrid cell-free modeling framework of Wayman et al [21]. An ensemble of model parameter sets (N = 3,000) was estimated from measurements of glucose, CAT, organic acids (pyruvate, lactate, acetate, succinate, malate), energy species (A(x)P, G(x)P, C(x)P, U(x)P), and 18 of the 20 proteinogenic amino acids [25] using a constrained Markov Chain Monte Carlo (MCMC) approach.…”

Section: Resultsmentioning

confidence: 99%

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

Horvath

Vilkhovoy²,

Wayman

et al. 2017

Preprint

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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 gluconeogenesis. 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.Introduction 1 Cell-free protein expression has become a widely used research tool in 2 systems and synthetic biology, and a promising technology for personalized 3 point of use biotechnology [1]. Cell-free systems offer many advantages for 4 the study, manipulation and modeling of metabolism compared to in vivo 5 processes. Central amongst these, is direct access to metabolites and the 6 biosynthetic machinery without the interference of a cell wall, or complica-7 tions associated with cell growth. This allows us to interrogate (and po-8 tentially manipulate) the chemical microenvironment while the biosynthetic 9 machinery is operating, potentially at a fine time resolution. Cell-free pro-10 tein synthesis (CFPS) systems are arguably the most prominent examples 11 of cell-free systems used today [2]. However, CFPS is not new; CFPS in 12 crude E. coli extracts has been used since the 1960s to explore fundamental 13 biological mechanisms. For example, Matthaei and Nirenberg used E. coli 14 cell-free extract in ground-breaking experiments to decipher the sequencing 15 of the genetic code [3, 4]. Spirin and coworkers later improved protein pro-16 duction in cell free extracts by continuously exchanging reactants and prod-17 ucts; however, while these extracts could run for tens of hours, they could 18 only synthesize a single product and were energy limited [5]. More recently, 19 energy and cofactor regeneration in CFPS has been significantly improved; 20 for example ATP can be regenerated using substrate level pho...

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Section: Resultsmentioning

confidence: 99%

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

Horvath

Vilkhovoy²,

Wayman

et al. 2017

Preprint

Self Cite

View full text Add to dashboard Cite

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“…Furthermore, such proteomic analyses might contribute to describing and enhancing reproducibility of CFPS extract preparation. Next to proteomic-based analyses, modeling approaches for simulating the synthesis of target proteins enable one to reveal system limitations, and furthermore offer the possibility to estimate and optimize the CFPS performance in silico [120].…”

Section: Limitations and Challenges Of Cfpsmentioning

confidence: 99%

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

2019

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Cell-free protein synthesis (CFPS) has become an established tool for rapid protein synthesis in order to accelerate the discovery of new enzymes and the development of proteins with improved characteristics. Over the past years, progress in CFPS system preparation has been made towards simplification, and many applications have been developed with regard to tailor-made solutions for specific purposes. In this review, various preparation methods of CFPS systems are compared and the significance of individual supplements is assessed. The recent applications of CFPS are summarized and the potential for biocatalyst development discussed. One of the central features is the high-throughput synthesis of protein variants, which enables sophisticated approaches for rapid prototyping of enzymes. These applications demonstrate the contribution of CFPS to enhance enzyme functionalities and the complementation to in vivo protein synthesis. However, there are different issues to be addressed, such as the low predictability of CFPS performance and transferability to in vivo protein synthesis. Nevertheless, the usage of CFPS for high-throughput enzyme screening has been proven to be an efficient method to discover novel biocatalysts and improved enzyme variants.

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“…We first selected a model enzyme, chloramphenicol acetyltransferase (CAT), the enzyme responsible for chloramphenicol resistance in E. coli. CAT expression has been demonstrated previously via plasmid-based CFPS (Vilkhovoy et al, 2018). We expressed three 15 µl reactions containing RCA amplified CAT template and three control reactions without template in 24 hr and then added them after expression (no purification) to an established assay by Shaw (1975) to measure CAT units (U) of activity (the rate [nmol/min]) of conversion of chloramphenicol and acetyl-CoA to chloramphenicol 3-acetate and CoA at pH 7.8 at 25°C (Figure 4a).…”

Section: Enzymesmentioning

confidence: 96%

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

Dopp

Rothstein

Mansell

et al. 2019

Biotech & Bioengineering

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In this study, we present a minimal template design and accompanying methods to produce assayable quantities of custom sequence proteins within 24 hr from receipt of inexpensive gene fragments from a DNA synthesis vendor. This is done without the conventional steps of plasmid cloning or cell‐based amplification and expression. Instead the linear template is PCR amplified, circularized, and isothermally amplified using a rolling circle polymerase. The resulting template can be used directly with cost‐optimized, scalably‐manufactured Escherichia coli extract and minimal supplement reagents to perform cell‐free protein synthesis (CFPS) of the template protein. We demonstrate the utility of this template design and 24 hr process with seven fluorescent proteins (sfGFP, mVenus, mCherry, and four GFP variants), three enzymes (chloramphenicol acetyltransferase, a chitinase catalytic domain, and native subtilisin), a capture protein (anti‐GFP nanobody), and 2 antimicrobial peptides (BP100 and CA(1–7)M(2–9)). We detected each of these directly from the CFPS reaction using colorimetric, fluorogenic, and growth assays. Of especial note, the GFP variant sequences were found from genomic screening data and had not been expressed or characterized before, thus demonstrating the utility of this approach for phenotype characterization of sequenced libraries. We also demonstrate that the rolling circle amplified version of the linear template exhibits expression similar to that of a complete plasmid when expressing sfGFP in the CFPS reaction. We evaluate the cost of this approach to be $61/mg sfGFP for a 4 hr reaction. We also detail limitations of this approach and strategies to overcome these, namely proteins with posttranslational modifications.

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

Cited by 30 publications

References 53 publications

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

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

Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development

Rapid prototyping of proteins: Mail order gene fragments to assayable proteins within 24 hours

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