RESUMEN
Can a heterotrophic organism be evolved to synthesize biomass from CO2 directly? So far, non-native carbon fixation in which biomass precursors are synthesized solely from CO2 has remained an elusive grand challenge. Here, we demonstrate how a combination of rational metabolic rewiring, recombinant expression, and laboratory evolution has led to the biosynthesis of sugars and other major biomass constituents by a fully functional Calvin-Benson-Bassham (CBB) cycle in E. coli. In the evolved bacteria, carbon fixation is performed via a non-native CBB cycle, while reducing power and energy are obtained by oxidizing a supplied organic compound (e.g., pyruvate). Genome sequencing reveals that mutations in flux branchpoints, connecting the non-native CBB cycle to biosynthetic pathways, are essential for this phenotype. The successful evolution of a non-native carbon fixation pathway, though not yet resulting in net carbon gain, strikingly demonstrates the capacity for rapid trophic-mode evolution of metabolism applicable to biotechnology. PAPERCLIP.
Asunto(s)
Dióxido de Carbono/metabolismo , Evolución Molecular Dirigida , Escherichia coli/genética , Escherichia coli/metabolismo , Gluconeogénesis , Redes y Vías Metabólicas , Procesos Autotróficos , Carbohidratos/biosíntesis , Escherichia coli/crecimiento & desarrollo , Espectrometría de MasasRESUMEN
Autotrophy is the basis for complex life on Earth. Central to this process is rubisco-the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s-1 median versus <7 s-1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature.
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Ribulosa-Bifosfato Carboxilasa , Ribulosa-Bifosfato Carboxilasa/metabolismo , Ribulosa-Bifosfato Carboxilasa/genética , Cinética , Dióxido de Carbono/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cianobacterias/metabolismo , Cianobacterias/enzimología , Cianobacterias/genética , Bacterias/enzimología , Bacterias/metabolismo , Bacterias/genéticaRESUMEN
CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast-carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form-II and form-II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s-1 -sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.
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Minería de Datos , Bases de Datos de Ácidos Nucleicos , Ribulosa-Bifosfato Carboxilasa , Isoenzimas/clasificación , Isoenzimas/genética , Ribulosa-Bifosfato Carboxilasa/clasificación , Ribulosa-Bifosfato Carboxilasa/genéticaRESUMEN
The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.
RESUMEN
Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.
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Genoma Fúngico , Biblioteca Genómica , Proteoma/genética , Saccharomyces cerevisiae/genética , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Lugares Marcados de SecuenciaRESUMEN
Enzymes catalyze a vast range of reactions. Their catalytic performances, mechanisms, global folds, and active-site architectures are also highly diverse, suggesting that enzymes are shaped by an entire range of physiological demands and evolutionary constraints, as well as by chemical and physicochemical constraints. We have attempted to identify signatures of these shaping demands and constraints. To this end, we describe a bird's-eye view of the enzyme space from two angles: evolution and chemistry. We examine various chemical reaction parameters that may have shaped the catalytic performances and active-site architectures of enzymes. We test and weigh these considerations against physiological and evolutionary factors. Although the catalytic properties of the "average" enzyme correlate with cellular metabolic demands and enzyme expression levels, at the level of individual enzymes, a multitude of physiological demands and constraints, combined with the coincidental nature of evolutionary processes, result in a complex picture. Indeed, neither reaction type (a chemical constraint) nor evolutionary origin alone can explain enzyme rates. Nevertheless, chemical constraints are apparent in the convergence of active-site architectures in independently evolved enzymes, although significant variations within an architecture are common.
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Enzimas/química , Enzimas/fisiología , Evolución Molecular , Animales , Archaea/enzimología , Bacterias/enzimología , Catálisis , Dominio Catalítico , Difusión , Hongos/enzimología , Humanos , Cinética , Conformación Proteica , Virus/enzimologíaRESUMEN
Rubisco is the primary carboxylase of the Calvin cycle, the most abundant enzyme in the biosphere, and one of the best-characterized enzymes. On the basis of correlations between Rubisco kinetic parameters, it is widely posited that constraints embedded in the catalytic mechanism enforce trade-offs between CO2 specificity, SC/O, and maximum carboxylation rate, kcat,C. However, the reasoning that established this view was based on data from ≈20 organisms. Here, we re-examine models of trade-offs in Rubisco catalysis using a data set from ≈300 organisms. Correlations between kinetic parameters are substantially attenuated in this larger data set, with the inverse relationship between kcat,C and SC/O being a key example. Nonetheless, measured kinetic parameters display extremely limited variation, consistent with a view of Rubisco as a highly constrained enzyme. More than 95% of kcat,C values are between 1 and 10 s-1, and no measured kcat,C exceeds 15 s-1. Similarly, SC/O varies by only 30% among Form I Rubiscos and <10% among C3 plant enzymes. Limited variation in SC/O forces a strong positive correlation between the catalytic efficiencies (kcat/KM) for carboxylation and oxygenation, consistent with a model of Rubisco catalysis in which increasing the rate of addition of CO2 to the enzyme-substrate complex requires an equal increase in the O2 addition rate. Altogether, these data suggest that Rubisco evolution is tightly constrained by the physicochemical limits of CO2/O2 discrimination.
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Modelos Biológicos , Ribulosa-Bifosfato Carboxilasa/metabolismo , Dióxido de Carbono/metabolismo , Cinética , Oxígeno/metabolismo , TermodinámicaRESUMEN
Turnover numbers, also known as kcat values, are fundamental properties of enzymes. However, kcat data are scarce and measured in vitro, thus may not faithfully represent the in vivo situation. A basic question that awaits elucidation is: how representative are kcat values for the maximal catalytic rates of enzymes in vivo? Here, we harness omics data to calculate kmax(vivo), the observed maximal catalytic rate of an enzyme inside cells. Comparison with kcat values from Escherichia coli, yields a correlation ofr(2)= 0.62 in log scale (p < 10(-10)), with a root mean square difference of 0.54 (3.5-fold in linear scale), indicating that in vivo and in vitro maximal rates generally concur. By accounting for the degree of saturation of enzymes and the backward flux dictated by thermodynamics, we further refine the correspondence between kmax(vivo) and kcat values. The approach we present here characterizes the quantitative relationship between enzymatic catalysis in vitro and in vivo and offers a high-throughput method for extracting enzyme kinetic constants from omics data.
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Enzimas/metabolismo , CatálisisRESUMEN
Genetically identical cells exposed to the same environment display variability in gene expression (noise), with important consequences for the fidelity of cellular regulation and biological function. Although population average gene expression is tightly coupled to growth rate, the effects of changes in environmental conditions on expression variability are not known. Here, we measure the single-cell expression distributions of approximately 900 Saccharomyces cerevisiae promoters across four environmental conditions using flow cytometry, and find that gene expression noise is tightly coupled to the environment and is generally higher at lower growth rates. Nutrient-poor conditions, which support lower growth rates, display elevated levels of noise for most promoters, regardless of their specific expression values. We present a simple model of noise in expression that results from having an asynchronous population, with cells at different cell-cycle stages, and with different partitioning of the cells between the stages at different growth rates. This model predicts non-monotonic global changes in noise at different growth rates as well as overall higher variability in expression for cell-cycle-regulated genes in all conditions. The consistency between this model and our data, as well as with noise measurements of cells growing in a chemostat at well-defined growth rates, suggests that cell-cycle heterogeneity is a major contributor to gene expression noise. Finally, we identify gene and promoter features that play a role in gene expression noise across conditions. Our results show the existence of growth-related global changes in gene expression noise and suggest their potential phenotypic implications.
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Ambiente , Regulación Fúngica de la Expresión Génica , Expresión Génica , Interacción Gen-Ambiente , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Modelos Biológicos , Regiones Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Análisis de la Célula Individual , Activación TranscripcionalRESUMEN
Bacterial growth depends crucially on metabolic fluxes, which are limited by the cell's capacity to maintain metabolic enzymes. The necessary enzyme amount per unit flux is a major determinant of metabolic strategies both in evolution and bioengineering. It depends on enzyme parameters (such as kcat and KM constants), but also on metabolite concentrations. Moreover, similar amounts of different enzymes might incur different costs for the cell, depending on enzyme-specific properties such as protein size and half-life. Here, we developed enzyme cost minimization (ECM), a scalable method for computing enzyme amounts that support a given metabolic flux at a minimal protein cost. The complex interplay of enzyme and metabolite concentrations, e.g. through thermodynamic driving forces and enzyme saturation, would make it hard to solve this optimization problem directly. By treating enzyme cost as a function of metabolite levels, we formulated ECM as a numerically tractable, convex optimization problem. Its tiered approach allows for building models at different levels of detail, depending on the amount of available data. Validating our method with measured metabolite and protein levels in E. coli central metabolism, we found typical prediction fold errors of 4.1 and 2.6, respectively, for the two kinds of data. This result from the cost-optimized metabolic state is significantly better than randomly sampled metabolite profiles, supporting the hypothesis that enzyme cost is important for the fitness of E. coli. ECM can be used to predict enzyme levels and protein cost in natural and engineered pathways, and could be a valuable computational tool to assist metabolic engineering projects. Furthermore, it establishes a direct connection between protein cost and thermodynamics, and provides a physically plausible and computationally tractable way to include enzyme kinetics into constraint-based metabolic models, where kinetics have usually been ignored or oversimplified.
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Proteínas Bacterianas/fisiología , Metabolismo Energético/fisiología , Enzimas/fisiología , Escherichia coli/metabolismo , Análisis de Flujos Metabólicos/métodos , Modelos Biológicos , Simulación por Computador , Activación Enzimática/fisiologíaRESUMEN
Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held desire to measure absolute protein abundances on a genome-wide scale. However, can this knowledge be translated into a clearer picture of how cells invest their protein resources? This article aims to give a broad perspective on the composition of proteomes as gleaned from recent quantitative proteomics studies. We describe proteomaps, an approach for visualizing the composition of proteomes with a focus on protein abundances and functions. In proteomaps, each protein is shown as a polygon-shaped tile, with an area representing protein abundance. Functionally related proteins appear in adjacent regions. General trends in proteomes, such as the dominance of metabolism and protein production, become easily visible. We make interactive visualizations of published proteome datasets accessible at www.proteomaps.net. We suggest that evaluating the way protein resources are allocated by various organisms and cell types in different conditions will sharpen our understanding of how and why cells regulate the composition of their proteomes.
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Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Transducción de Señal , Bases de Datos de Proteínas , Escherichia coli/citología , Escherichia coli/metabolismo , Humanos , Internet , Modelos Biológicos , Mycoplasma pneumoniae/citología , Mycoplasma pneumoniae/metabolismo , Proteínas/clasificación , Proteínas/metabolismo , Proteoma/clasificación , Proteoma/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Especificidad de la EspecieRESUMEN
Plaque analysis allows the determination of phage titer and multiplicity of infection. Yet, this overnight assay provides only endpoint results, ignoring kinetic aspects. We introduce an alternative high-throughput and rapid method for kinetic analysis of lytic coliphage activity. Escherichia coli was infected with serial dilutions of MS2 coliphage, and bacterial growth was monitored using a multi-well plate reader providing within hours the equivalent data as obtained overnight. Additional information is yielded, including phage replication rate, progeny size per cycle, and viral propagation during bacterial growth. This method offers further insights into physicochemical mechanisms of lytic coliphage infection and temporal control. It also provides a virus-host interaction acumen.
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Escherichia coli/virología , Ensayos Analíticos de Alto Rendimiento , Levivirus/fisiología , Escherichia coli/crecimiento & desarrollo , Cinética , Levivirus/crecimiento & desarrollo , Levivirus/patogenicidad , Factores de TiempoRESUMEN
BACKGROUND: The oligomeriztion of α-synuclein (α-syn) into ordered assemblies is associated with the symptoms of Parkinson's Disease (PD). Yet, it is still debatable whether oligomers are formed as part of a multistep process towards amyloid fibril formation or alternatively as "off-pathway" aggregates. METHODS: 100µM α-syn was incubated with decreasing amounts of cinnamon extract precipitation (CEppt). The fibril formation was measured using spectroscopy and microscopy analyses and oligomers were detected using western blot analysis. The secondary structure of the protein was analyzed using CD. Drosophila brains were studied using immunostaining and confocal microscopy. RESULTS: Here we probed the inhibition pattern of oligomeric and fibrillar forms of α-syn, using a natural substance, CEppt which was previously shown to effectively inhibit aggregation of ß-amyloid polypeptide. We demonstrated that CEppt has a differential inhibitory effect on the formation of soluble and insoluble aggregates of α-synuclein in vitro. This inhibition pattern revokes the possibility of redirection to "off-pathway" oligomers. When administering to Drosophila fly model expressing mutant A53T α-syn in the nervous system, a significant curative effect on the behavioral symptoms of the flies and on α-syn aggregation in their brain was observed. CONCLUSIONS: We conclude that CEppt affects the process of aggregation of α-syn without changing its secondary structure and suggest that increasing amounts of CEppt slow this process by stabilizing the soluble oligomeric phase. When administered to Drosophila fly model, CEppt appears to have a curative effect on the defective flies. GENERAL SIGNIFICANCE: Our results indicate that CEppt can be a potential therapeutic agent for PD.
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Amiloide/antagonistas & inhibidores , Cinnamomum zeylanicum , Drosophila , Enfermedad de Parkinson/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/metabolismo , Amiloide/genética , Amiloide/metabolismo , Animales , Animales Modificados Genéticamente , Células CHO , Cinnamomum zeylanicum/química , Cricetinae , Cricetulus , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Drosophila/genética , Femenino , Humanos , Masculino , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fitoterapia/métodos , Multimerización de Proteína/genética , alfa-Sinucleína/genéticaRESUMEN
MOTIVATION: The laws of thermodynamics describe a direct, quantitative relationship between metabolite concentrations and reaction directionality. Despite great efforts, thermodynamic data suffer from limited coverage, scattered accessibility and non-standard annotations. We present a framework for unifying thermodynamic data from multiple sources and demonstrate two new techniques for extrapolating the Gibbs energies of unmeasured reactions and conditions. RESULTS: Both methods account for changes in cellular conditions (pH, ionic strength, etc.) by using linear regression over the ΔG(â) of pseudoisomers and reactions. The Pseudoisomeric Reactant Contribution method systematically infers compound formation energies using measured K' and pK(a) data. The Pseudoisomeric Group Contribution method extends the group contribution method and achieves a high coverage of unmeasured reactions. We define a continuous index that predicts the reversibility of a reaction under a given physiological concentration range. In the characteristic physiological range 3µM-3mM, we find that roughly half of the reactions in Escherichia coli's metabolism are reversible. These new tools can increase the accuracy of thermodynamic-based models, especially in non-standard pH and ionic strengths. The reversibility index can help modelers decide which reactions are reversible in physiological conditions. AVAILABILITY: Freely available on the web at: http://equilibrator.weizmann.ac.il. Website implemented in Python, MySQL, Apache and Django, with all major browsers supported. The framework is open-source (code.google.com/p/milo-lab), implemented in pure Python and tested mainly on Linux. CONTACT: ron.milo@weizmann.ac.il SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Metabolismo Energético , Escherichia coli/metabolismo , Programas Informáticos , Termodinámica , Biología Computacional , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
Importance: Optimal quarantine length for COVID-19 infection is unclear, in part owing to limited empirical data. Objective: To assess postquarantine transmission risk for various quarantine lengths and potential associations between quarantine strictness and transmission risk. Design, Setting, and Participants: Retrospective cohort study in 4 US universities from September 2020 to February 2021, including 3641 university students and staff who were identified as close contacts to individuals who tested positive for SARS-CoV-2 infection. Individuals were tested throughout the 10 to 14-day quarantine, and follow-up testing continued at least weekly throughout the 2020-2021 academic year. Exposures: Strict quarantine, including designated housing with a private room, private bathroom, and meal delivery, vs nonstrict, which potentially included interactions with household members. Main Outcomes and Measures: Dates of last known exposure, last negative test result, and first positive test result during quarantine. Results: This study included 301 quarantined university students and staff who tested SARS-CoV-2-positive (of 3641 quarantined total). These 301 individuals had a median (IQR) age of 22.0 (20.0-25.0) years; 131 (43.5%) identified as female; and 20 (6.6%) were staff. Of the 287 self-reporting race and ethnicity according to university-defined classifications, 21 (7.3%) were African American or Black, 60 (20.9%) Asian, 17 (5.9%) Hispanic or Latinx, 174 (60.6%) White, and 15 (5.2%) other (including multiracial and/or multiethnic). Of the 301 participants, 40 (13.3%; 95% CI, 9.9%-17.6%) had negative test results and were asymptomatic on day 7 compared with 15 (4.9%; 95% CI, 3.0%-8.1%) and 4 (1.4%; 95% CI, 0.4%-3.5%) on days 10 and 14, respectively. Individuals in strict quarantine tested positive less frequently than those in nonstrict quarantine (10% vs 12%; P = .04). Conclusions and Relevance: To maintain the 5% transmission risk used as the basis for US Centers for Disease Control and Prevention's 7-day test-based quarantine guidance, our data suggest that quarantine with quantitative polymerase chain reaction testing 1 day before intended release should be 10 days for nonstrict quarantine and 8 days for strict quarantine, as ongoing exposure during quarantine may be associated with the higher rate of positive test results following nonstrict quarantine.
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COVID-19/epidemiología , COVID-19/transmisión , Cuarentena/estadística & datos numéricos , Adulto , Femenino , Humanos , Masculino , Estudios Retrospectivos , Estudiantes/estadística & datos numéricos , Universidades , Adulto JovenRESUMEN
The kinetic parameters of enzymes are key to understanding the rate and specificity of most biological processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an analysis of k(cat) and K(M) values of several thousand enzymes collected from the literature. We found that the "average enzyme" exhibits a k(cat) of ~0 s(-1) and a k(cat)/K(M) of ~10(5) s(-1) M(-1), much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies? Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metabolism are, on average, ~30-fold slower than those of central metabolism. We also find indications that the physicochemical properties of substrates affect the kinetic parameters. Specifically, low molecular mass and hydrophobicity appear to limit K(M) optimization. In accordance, substitution with phosphate, CoA, or other large modifiers considerably lowers the K(M) values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochemical constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or laboratory evolution.
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Evolución Biológica , Enzimas/metabolismo , Cinética , PresiónRESUMEN
Asymptomatic surveillance testing together with COVID-19-related research can lead to positive SARS-CoV-2 tests resulting not from true infections, but non-infectious, non-hazardous by-products of research (amplicons). Amplicons can be widespread and persistent in lab environments and can be difficult to distinguish for true infections. We discuss prevention and mitigation strategies.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Laboratorios , Prueba de COVID-19RESUMEN
While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on function would promote accurate use of tags in experimental setups. Here we examine the effect of green fluorescent protein tagging at either the N or C terminus of budding yeast proteins on subcellular localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides a step toward broad investigation of protein fusion libraries.
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Proteínas Fúngicas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
How does environmental complexity affect the evolution of single genes? Here, we measured the effects of a set of Bacillus subtilis glutamate dehydrogenase mutants across 19 different environments-from phenotypically homogeneous single-cell populations in liquid media to heterogeneous biofilms, plant roots and soil populations. The effects of individual gene mutations on organismal fitness were highly reproducible in liquid cultures. However, 84% of the tested alleles showed opposing fitness effects under different growth conditions (sign environmental pleiotropy). In colony biofilms and soil samples, different alleles dominated in parallel replica experiments. Accordingly, we found that in these heterogeneous cell populations the fate of mutations was dictated by a combination of selection and drift. The latter relates to programmed prophage excisions that occurred during biofilm development. Overall, for each condition, a wide range of glutamate dehydrogenase mutations persisted and sometimes fixated as a result of the combined action of selection, pleiotropy and chance. However, over longer periods and in multiple environments, nearly all of this diversity would be lost-across all the environments and conditions that we tested, the wild type was the fittest allele.
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Ambiente , Pleiotropía Genética , Variación Genética/genética , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Biopelículas/crecimiento & desarrollo , Evolución Molecular , Flujo Genético , Aptitud Genética , Glutamato Deshidrogenasa/genética , Mutación , Selección Genética , Activación ViralRESUMEN
Enzyme kinetics are fundamental to an understanding of cellular metabolism and for crafting synthetic biology applications. For decades, enzyme characterization has been based on in vitro enzyme assays. However, kinetic parameters are only available for <10% of reactions, and this data scarcity limits the predictive power of metabolic models. Here we review recent studies that leverage quantitative proteomics to gain insight into in vivo enzyme kinetics. We discuss findings on the relationship between in vivo and in vitro enzyme catalysis and show how proteomics can be used to characterize the efficiency of enzyme utilization across conditions. Lastly, the efficient use of enzymes is shown to rationalize preference for low energy-yield metabolic strategies, such as aerobic fermentation at high growth rate.