Toward the Complete Functional Characterization of a Minimal Bacterial Proteome

Bianchi, David M.; Pelletier, James F.; Hutchison, Clyde A.; Glass, John I.; Luthey‐Schulten, Zaida

doi:10.1021/acs.jpcb.2c04188

Cited by 10 publications

(9 citation statements)

References 111 publications

(309 reference statements)

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“…This minimal genome has been extensively characterized, and only 91 genes remain without an annotated function. A recent study by Bianchi et al (2022) uses computational analyses to further elucidate the function of uncharacterized genes and work toward complete functional characterization of the proteome. By gaining a better understanding of the function of encoded proteins, we will be able to inform the spatial distribution of proteins in our whole-cell model.…”

Section: Building Cells With the Martini Ecosystemmentioning

confidence: 99%

Molecular dynamics simulation of an entire cell

et al. 2023

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The ultimate microscope, directed at a cell, would reveal the dynamics of all the cell’s components with atomic resolution. In contrast to their real-world counterparts, computational microscopes are currently on the brink of meeting this challenge. In this perspective, we show how an integrative approach can be employed to model an entire cell, the minimal cell, JCVI-syn3A, at full complexity. This step opens the way to interrogate the cell’s spatio-temporal evolution with molecular dynamics simulations, an approach that can be extended to other cell types in the near future.

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Section: Building Cells With the Martini Ecosystemmentioning

confidence: 99%

Molecular dynamics simulation of an entire cell

et al. 2023

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“…Despite the drastic genome reduction, JCVI-syn3a encodes 38 essential and quasi-essential membrane proteins for which it has not yet been possible to predict a function, that is, one-third of all membrane proteins; the same is true also for its soluble proteins. Computational efforts are ongoing to bridge the gap between these genes and the minimal proteome …”

Section: Membrane Modules For a Bottom-up Minimal Metabolismmentioning

confidence: 99%

“…JCVI-syn3a encodes the machinery for the cotranslational insertion of membrane proteins (secA, secE, secY, secG, secD, secF, and yidC), coupled to signalrecognition-particle docking (f tsY), quality-control (f tsH), and protein excretion (lspA). Other components involved in protein insertion/translocation in E. coli are missing in JCVI-syn3a (secB, yajC), 152 but JCVI-syn3a may use other chaperone-like proteins to substitute for SecB, similar to B. subtilis. 153 Uncharacterized processes.…”

Section: Membrane Modules For a Bottom-up Minimal Metabolismmentioning

confidence: 99%

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Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

et al. 2023

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Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.

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“…Another important application of the Syn3A CFE system is to study the minimal cellular metabolism, especially to determine the function of unannotated essential genes present in the Syn3A genome (90 out of 452 protein-coding genes). , To date, the elucidation of gene function has relied on metabolic modeling based on experimental information from closely related organisms (e.g., Mycoplasma mycoides), transposon experiments, and proteomic data. , The Syn3A CFE system can serve as a platform to test the function of a specific gene by reconstructing its immediate metabolic network in vitro . Therefore, Syn3A CFE can play an important role in unravelling the function of specific proteins that cannot be explored in vivo .…”

Section: Introductionmentioning

confidence: 99%

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium

et al. 2023

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Cell-free expression (CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the construction of a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms are necessary to understand the principles behind life-like behavior. Here, we report the successful creation of a CFE system derived from JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high ribonuclease activity in Syn3A lysates impeded the establishment of functional CFE systems. Now, we describe how an unusual cell lysis method (nitrogen decompression) yielded Syn3A lysates with reduced ribonuclease activity that supported in vitro expression. To improve the protein yields in the Syn3A CFE system, we optimized the Syn3A CFE reaction mixture using an active machine learning tool. The optimized reaction mixture improved the CFE 3.2-fold compared to the preoptimized condition. This is the first report of a functional CFE system derived from a minimal synthetic bacterium, enabling further advances in bottom-up synthetic biology.

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Toward the Complete Functional Characterization of a Minimal Bacterial Proteome

Cited by 10 publications

References 111 publications

Molecular dynamics simulation of an entire cell

Molecular dynamics simulation of an entire cell

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium

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