In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties

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

Biology by Design: From Top to Bottom and Back

Synthetic biology is a nascent technical discipline that seeks to enable the design and construction of novel biological systems to meet pressing societal needs. However, engineering biology still requires much trial and error because we lack effective approaches for connecting basic “parts” into higher-order networks that behave as predicted. Developing strategies for improving the performance and sophistication of our designs is informed by two overarching perspectives: “bottom-up” and “top-down” considerations. Using this framework, we describe a conceptual model for developing novel biological systems that function and interact with existing biological components in a predictable fashion. We discuss this model in the context of three topical areas: biochemical transformations, cellular devices and therapeutics, and approaches that expand the chemistry of life. Ten years after the construction of synthetic biology's first devices, the drive to look beyond what does exist to what can exist is ushering in an era of biology by design

The genome-scale metabolic model iIN800 of Saccharomyces cerevisiae and its validation: a scaffold to query lipid metabolism

Background: Up to now, there have been three published versions of a yeast genome-scale metabolic model: iFF708, iND750 and iLL672. All three models, however, lack a detailed description of lipid metabolism and thus are unable to be used as integrated scaffolds for gaining insights into lipid metabolism from multilevel omic measurement technologies (e.g. genome-wide mRNA levels). To overcome this limitation, we reconstructed a new version of the Saccharomyces cerevisiae genome-scale model, ilN800 that includes a more rigorous and detailed descrition of lipid metabolism. Results: The reconstructed metabolic model comprises 1446 reactions and 1013 metabolites. Beyond incorporating new reactions involved in lipid metabolism, we also present new biomass equations that improve the predictive power of flux balance analysis simulations. Predictions of both growth capability and large scale in silico single gene deletions by ilN800 were consistent with experimental data. In addition, 13C-labeling experiments validated the new biomass equations and calculated intracellular fluxes. To demonstrate the applicability of ilN800, we show that the model can be used as a scaffold to reveal the regulatory importance of lipid metabolism precursors and intermediates that would have been missed in previous models from transcriptome datasets. Conclusions: Performing integrated analyses using ilN800 as a network scaffold is shown to be a valuable tool for elucidating the behavior of complex metabolic networks, particularly for identifying regulatory targets in lipid metabolism that can be used for industrial applications or for understanding lipid disease states

Self-assembly of temperature-responsive protein–polymer bioconjugates

We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed “click” chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition

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

Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator

Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Δsnf1, Δsnf4, and Δsnf1Δsnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low-energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases

A rapid cell-free expression and screening platform for antibody discovery

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly

Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: A three factor design

<p>Abstract</p> <p>Background</p> <p>Characterization of cellular growth is central to understanding living systems. Here, we applied a three-factor design to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of <it>Saccharomyces cerevisiae</it>. The three factors we considered were specific growth rate, nutrient limitation, and oxygen availability.</p> <p>Results</p> <p>We identified 268 growth rate dependent genes, independent of nutrient limitation and oxygen availability. The transcriptional response was used to identify key areas in metabolism around which mRNA expression changes are significantly associated. Among key metabolic pathways, this analysis revealed <it>de novo </it>synthesis of pyrimidine ribonucleotides and ATP producing and consuming reactions at fast cellular growth. By scoring the significance of overlap between growth rate dependent genes and known transcription factor target sets, transcription factors that coordinate balanced growth were also identified. Our analysis shows that Fhl1, Rap1, and Sfp1, regulating protein biosynthesis, have significantly enriched target sets for genes up-regulated with increasing growth rate. Cell cycle regulators, such as Ace2 and Swi6, and stress response regulators, such as Yap1, were also shown to have significantly enriched target sets.</p> <p>Conclusion</p> <p>Our work, which is the first genome-wide gene expression study to investigate specific growth rate and consider the impact of oxygen availability, provides a more conservative estimate of growth rate dependent genes than previously reported. We also provide a global view of how a small set of transcription factors, 13 in total, contribute to control of cellular growth rate. We anticipate that multi-factorial designs will play an increasing role in elucidating cellular regulation.</p

Cell-free protein synthesis enables high yielding synthesis of an active multicopper oxidase

Multicopper oxidases (MCOs) are broadly distributed in all kingdoms of life and perform a variety of important oxidative reactions. These enzymes have potential biotechnological applications; however, the applications are impeded by low expression yields in traditional recombinant hosts, solubility issues, and poor copper cofactor assembly. As an alternative to traditional recombinant protein expression, we show the ability to use cell-free protein synthesis (CFPS) to produce complex MCO proteins with high soluble titers. Specifically, we report the production of MCOs in an Escherichia coli-based cell-free transcription-translation system. Total yields as high as 1.2 mg mL-1 were observed after a 20-h batch reaction. More than 95% of the protein was soluble and activity was obtained by simple post-CFPS addition of copper ions in the form of CuSO4. Scale-up reactions were achieved from 15 to 100 μL without a decrease in productivity and solubility. CFPS titers were higher than in vivo expression titers and more soluble, avoiding the formation of inclusion bodies. Our work extends the utility of the cell-free platform to the production of active proteins containing copper cofactors and demonstrates a simple method for producing MCOs