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The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.
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Sistema Libre de Células , Metilación de ADN , Biosíntesis de Proteínas , Transcripción Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Transformación Bacteriana , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Metilasas de Modificación del ADN/metabolismo , Metilasas de Modificación del ADN/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Saccharomyces boulardii (Sb) is an emerging probiotic chassis for delivering biomolecules to the mammalian gut, offering unique advantages as the only eukaryotic probiotic. However, precise control over gene expression and gut residence time in Sb have remained challenging. To address this, we developed five ligand-responsive gene expression systems and repaired galactose metabolism in Sb, enabling inducible gene expression in this strain. Engineering these systems allowed us to construct AND logic gates, control the surface display of proteins, and turn on protein production in the mouse gut in response to dietary sugar. Additionally, repairing galactose metabolism expanded Sb's habitat within the intestines and resulted in galactose-responsive control over gut residence time. This work opens new avenues for precise dosing of therapeutics by Sb via control over its in vivo gene expression levels and localization within the gastrointestinal tract.
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Galactosa , Probióticos , Saccharomyces boulardii , Animales , Ratones , Galactosa/metabolismo , Saccharomyces boulardii/genética , Saccharomyces boulardii/metabolismo , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/metabolismo , DietaRESUMEN
Exploring the gastrointestinal role of hydrogen sulfide (H2S) is difficult because of its volatility and the absence of a precisely controllable model system for manipulating the gut environment. Hayes et al. address this issue by engineering Escherichia coli to titrate H2S levels in a gas-impermeable gut-on-chip device.
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Sulfuro de Hidrógeno , Tracto Gastrointestinal , Escherichia coli/genéticaRESUMEN
Carotenoids have been associated with risk reduction for several chronic diseases, including the association of their dietary intake/circulating levels with reduced incidence of obesity, type 2 diabetes, certain types of cancer, and even lower total mortality. In addition to some carotenoids constituting vitamin A precursors, they are implicated in potential antioxidant effects and pathways related to inflammation and oxidative stress, including transcription factors such as nuclear factor κB and nuclear factor erythroid 2-related factor 2. Carotenoids and metabolites may also interact with nuclear receptors, mainly retinoic acid receptor/retinoid X receptor and peroxisome proliferator-activated receptors, which play a role in the immune system and cellular differentiation. Therefore, a large number of downstream targets are likely influenced by carotenoids, including but not limited to genes and proteins implicated in oxidative stress and inflammation, antioxidation, and cellular differentiation processes. Furthermore, recent studies also propose an association between carotenoid intake and gut microbiota. While all these endpoints could be individually assessed, a more complete/integrative way to determine a multitude of health-related aspects of carotenoids includes (multi)omics-related techniques, especially transcriptomics, proteomics, lipidomics, and metabolomics, as well as metagenomics, measured in a variety of biospecimens including plasma, urine, stool, white blood cells, or other tissue cellular extracts. In this review, we highlight the use of omics technologies to assess health-related effects of carotenoids in mammalian organisms and models.
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Carotenoides , Diabetes Mellitus Tipo 2 , Animales , Humanos , Carotenoides/metabolismo , Inflamación , Antioxidantes/farmacología , Luteína , Mamíferos/metabolismoRESUMEN
Sustainably enhancing crop production is a global necessity to meet the escalating demand for staple crops while sustainably managing their associated carbon/nitrogen inputs. Leveraging plant-associated microbiomes is a promising avenue for addressing this demand. However, studying these communities and engineering them for sustainable enhancement of crop production have remained a challenge due to limited genetic tools and methods. In this work, we detail the development of the Maize Root Microbiome ToolKit (MRMTK), a rapid Modular Cloning (MoClo) toolkit that only takes 2.5 h to generate desired constructs (5400 potential plasmids) that replicate and express heterologous genes in Enterobacter ludwigii strain AA4 (Elu), Pseudomonas putida strain AA7 (Ppu), Herbaspirillum robiniae strain AA6 (Hro), Stenotrophomonas maltophilia strain AA1 (Sma), and Brucella pituitosa strain AA2 (Bpi), which comprise a model maize root synthetic community (SynCom). In addition to these genetic tools, we describe a highly efficient transformation protocol (107-109 transformants/µg of DNA) 1 for each of these strains. Utilizing this highly efficient transformation protocol, we identified endogenous Expression Sequences (ES; promoter and ribosomal binding sites) for each strain via genomic promoter trapping. Overall, MRMTK is a scalable and adaptable platform that expands the genetic engineering toolbox while providing a standardized, high-efficiency transformation method across a diverse group of root commensals. These results unlock the ability to elucidate and engineer plant-microbe interactions promoting plant growth for each of the 5 bacterial strains in this study.
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Microbiota , Zea mays , Zea mays/genética , Zea mays/microbiología , Bacterias/genética , Plásmidos/genética , Ingeniería Genética/métodos , Microbiota/genéticaRESUMEN
Infections by Clostridioides difficile, a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff. infection. We describe an approach that combines a Peptide Binding Design (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a KD of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).
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Clostridioides difficile , Humanos , Colon , Algoritmos , Biocatálisis , Péptidos/farmacologíaRESUMEN
The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.
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Probióticos , Saccharomyces boulardii , Saccharomyces , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces boulardii/genética , Saccharomyces boulardii/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Probióticos/metabolismo , Endopeptidasas/metabolismoRESUMEN
Carotenoids have been related to a number of health benefits. Their dietary intake and circulating levels have been associated with a reduced incidence of obesity, diabetes, certain types of cancer, and even lower total mortality. Their potential interaction with the gut microbiota (GM) has been generally overlooked but may be of relevance, as carotenoids largely bypass absorption in the small intestine and are passed on to the colon, where they appear to be in part degraded into unknown metabolites. These may include apo-carotenoids that may have biological effects because of higher aqueous solubility and higher electrophilicity that could better target transcription factors, i.e., NF-κB, PPARγ, and RAR/RXRs. If absorbed in the colon, they could have both local and systemic effects. Certain microbes that may be supplemented were also reported to produce carotenoids in the colon. Although some bactericidal aspects of carotenoids have been shown in vitro, a few studies have also demonstrated a prebiotic-like effect, resulting in bacterial shifts with health-associated properties. Also, stimulation of IgA could play a role in this respect. Carotenoids may further contribute to mucosal and gut barrier health, such as stabilizing tight junctions. This review highlights potential gut-related health-beneficial effects of carotenoids and emphasizes the current research gaps regarding carotenoid-GM interactions.
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Carotenoides , Microbioma Gastrointestinal , Humanos , Carotenoides/farmacología , Carotenoides/metabolismo , Colon/metabolismo , Prebióticos , Suplementos DietéticosRESUMEN
Clostridioides difficile ( C. diff .) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of C. diff . infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines a pep tide b inding d esign algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a K D of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR). Significance Statement: Infections by Clostridioides difficile , a bacterium that targets the large intestine (colon), impact a significant number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can inhibit the biocatalytic activity of these toxins represent a promising strategy to prevent and treat C. diff . infection. We describe an approach that combines a Peptide B inding D esign (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in small intestinal and colon epithelial cells. Importantly, our designed peptide, SA1, bound toxin A with nanomolar affinity and blocked toxicity in colon cells.
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Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge for most existing methods. Since many important microbial phenotypes are encoded by multigene pathways, they are usually improved via adaptive laboratory evolution (ALE), which while straightforward to implement can suffer from off-target and hitchhiker mutations that can adversely affect the fitness of the evolved strain. We have therefore developed a new directed evolution method (Inducible Directed Evolution, IDE) that combines the specificity and throughput of recent continuous directed evolution methods with the ease of ALE. Here, we present detailed methods for operating Inducible Directed Evolution (IDE), which enables long (up to 85kb) DNA sequences to be mutated in a high throughput manner via a simple series of incubation steps. In IDE, an intracellular mutagenesis plasmid (MP) tunably mutagenizes the pathway of interest, located on the phagemid (PM). MP contains a mutagenic operon ( danQ926, dam, seqA, emrR, ugi , and cda1 ) that can be expressed via the addition of a chemical inducer. Expression of the mutagenic operon during a cell cycle represses DNA repair mechanisms such as proofreading, translesion synthesis, mismatch repair, and base excision and selection, which leads to a higher mutation rate. Induction of the P1 lytic cycle results in packaging of the mutagenized phagemid, and the pathway-bearing phage particles infect naïve cells, generating a mutant library that can be screened or selected for improved variants. Successive rounds of IDE enable optimization of complex phenotypes encoded by large pathways (as of this writing up to 36 kb), without requiring inefficient transformation steps. Additionally, IDE avoids off-target genomic mutations and enables decoupling of mutagenesis and screening steps, establishing it as a powerful tool for optimizing complex phenotypes in E. coli .
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G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker's yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.
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Probióticos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Reproducción/genética , Receptores Acoplados a Proteínas G/metabolismo , Feromonas/metabolismo , Receptores de Superficie Celular/metabolismo , Diferenciación Celular , Comunicación Celular , LigandosRESUMEN
BACKGROUND: Terpenes are one of the most diverse and abundant classes of natural biomolecules, collectively enabling a variety of therapeutic, energy, and cosmetic applications. Recent genomics investigations have predicted a large untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivated organisms living in the soil, indicating a vast array of putative terpenoids waiting to be discovered. RESULTS: We aimed to develop a high-throughput functional metagenomic screening system for identifying novel terpene synthases from bacterial metagenomes by relieving the toxicity of terpene biosynthesis precursors to the Escherichia coli host. The precursor toxicity was achieved using an inducible operon encoding the prenyl pyrophosphate synthetic pathway and supplementation of the mevalonate precursor. Host strain and screening procedures were finely optimized to minimize false positives arising from spontaneous mutations, which avoid the precursor toxicity. Our functional metagenomic screening of human fecal metagenomes yielded a novel ß-farnesene synthase, which does not show amino acid sequence similarity to known ß-farnesene synthases. Engineered S. cerevisiae expressing the screened ß-farnesene synthase produced 120 mg/L ß-farnesene from glucose (2.86 mg/g glucose) with a productivity of 0.721 g/Lâh. CONCLUSIONS: A unique functional metagenomic screening procedure was established for screening terpene synthases from metagenomic libraries. This research proves the potential of functional metagenomics as a sequence-independent avenue for isolating targeted enzymes from uncultivated organisms in various environmental habitats.
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Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5-10 kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution. Next, we evolved a 15.4 kb, 10-gene pathway from Bifidobacterium breve UC2003 that aids E. coli's utilization of melezitose. After three rounds of IDE, we isolated evolved pathways that both reduced lag time by more than 2-fold and enabled 150% higher final optical density. Taken together, this work enhances the capacity and utility of a whole pathway directed evolution approach in E. coli.
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Bacterias/genética , Evolución Molecular Dirigida , Bacterias/metabolismo , Bacterias/virología , Bacteriófagos/genética , Evolución Molecular Dirigida/métodos , Mutagénesis , Fenotipo , Plásmidos/genéticaRESUMEN
Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.
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Toxinas Bacterianas/antagonistas & inhibidores , Simulación por Computador , Enterotoxinas/antagonistas & inhibidores , Péptidos/farmacología , Toxinas Bacterianas/química , Diseño de Fármacos , Enterotoxinas/química , Modelos Moleculares , Péptidos/química , Unión Proteica , Conformación Proteica , Dominios Proteicos , Reproducibilidad de los ResultadosRESUMEN
Genetically engineered microbes that secrete therapeutics, sense and respond to external environments, and/or target specific sites in the gut fall under an emergent class of therapeutics, called live biotherapeutic products (LBPs). As live organisms that require symbiotic host interactions, LBPs offer unique therapeutic opportunities, but also face distinct challenges in the gut microenvironment. In this review, we describe recent approaches (often demonstrated using traditional probiotic microorganisms) to discover LBP chassis and genetic parts utilizing omics-based methods and highlight LBP delivery strategies, with a focus on addressing physiological challenges that LBPs encounter after oral administration. Finally, we share our perspective on the opportunity to apply an integrated approach, wherein discovery and delivery strategies are utilized synergistically, towards tailoring and optimizing LBP efficacy.
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Probióticos , Administración Oral , Ingeniería Genética , Probióticos/uso terapéuticoRESUMEN
Fabrics are widely used in hospitals and many other settings for bedding, clothing, and face masks; however, microbial pathogens can survive on surfaces for a long time, leading to microbial transmission. Coatings of metallic particles on fabrics have been widely used to eradicate pathogens. However, current metal particle coating technologies encounter numerous issues such as nonuniformity, processing complexity, and poor adhesion. To overcome these issues, an easy-to-control and straightforward method is reported to coat a wide range of fabrics by using gallium liquid metal (LM) particles to facilitate the deposition of liquid metal copper alloy (LMCu) particles. Gallium particles coated on the fabric provide nucleation sites for forming LMCu particles at room temperature via galvanic replacement of Cu2+ ions. The LM helps promote strong adhesion of the particles to the fabric. The presence of the LMCu particles can eradicate over 99% of pathogens (including bacteria, fungi, and viruses) within 5 min, which is significantly more effective than control samples coated with only Cu. The coating remains effective over multiple usages and against contaminated droplets and aerosols, such as those encountered in facemasks. This facile coating method is promising for generating robust antibacterial, antifungal, and antiviral fabrics and surfaces.
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Materiales Biocompatibles Revestidos/química , Cobre/química , Galio/química , Textiles/análisis , Aleaciones/química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Antivirales/química , Antivirales/farmacología , Bacterias/efectos de los fármacos , Materiales Biocompatibles Revestidos/farmacología , Hongos/efectos de los fármacos , Virus/efectos de los fármacosRESUMEN
The infant gut microbiota is shaped by diverse environmental exposures that alter its composition and can enrich antimicrobial resistance genes (ARGs). In this issue of Cell Host & Microbe, Li et al. (2021) studied the causes, spread, and dynamics of ARGs and their relationship with asthma-associated microbiota in Danish children.
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Asma , Microbioma Gastrointestinal , Antibacterianos/farmacología , Niño , Exposición a Riesgos Ambientales , Escherichia coli , Humanos , Lactante , Estilo de Vida , HermanosRESUMEN
Saccharomyces boulardii is a probiotic yeast that exhibits rapid growth at 37 °C, is easy to transform, and can produce therapeutic proteins in the gut. To establish its ability to produce small molecules encoded by multigene pathways, we measured the amount and variance in protein expression enabled by promoters, terminators, selective markers, and copy number control elements. We next demonstrated efficient (>95%) CRISPR-mediated genome editing in this strain, allowing us to probe engineered gene expression across different genomic sites. We leveraged these strategies to assemble pathways enabling a wide range of vitamin precursor (ß-carotene) and drug (violacein) titers. We found that S. boulardii colonizes germ-free mice stably for over 30 days and competes for niche space with commensal microbes, exhibiting short (1-2 day) gut residence times in conventional and antibiotic-treated mice. Using these tools, we enabled ß-carotene synthesis (194 µg total) in the germ-free mouse gut over 14 days, estimating that the total mass of additional ß-carotene recovered in feces was 56-fold higher than the ß-carotene present in the initial probiotic dose. This work quantifies heterologous small molecule production titers by S. boulardii living in the mammalian gut and provides a set of tools for modulating these titers.
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Antineoplásicos/metabolismo , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Indoles/metabolismo , Ingeniería Metabólica/métodos , Probióticos/metabolismo , Provitaminas/biosíntesis , Saccharomyces boulardii/metabolismo , beta Caroteno/biosíntesis , Animales , Sistemas CRISPR-Cas , Heces/química , Femenino , Microbioma Gastrointestinal , Edición Génica/métodos , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Microorganismos Modificados Genéticamente , Familia de Multigenes , Plásmidos/genética , Regiones Promotoras Genéticas , Saccharomyces boulardii/genética , Saccharomyces cerevisiae/genéticaRESUMEN
The development of robust engineered probiotic therapies demands accurate knowledge of genetic construct expression in the gut. However, the monetary and ethical costs of testing engineered strains in vertebrate hosts are incompatible with current high-throughput design-build-test cycles. To enable parallel measurement of multiple construct designs, we placed unique DNA barcodes in engineered transcripts and measured barcode abundances via sequencing. In standard curve experiments, the barcode sequences exhibited consistent relationships between input and measured abundances, which allowed us to use transcript barcoding to measure expression levels of 30 GFP-expressing strains of E. coli Nissle in parallel. Applying this technology in culture and in the mouse gut, we found that GFP expression in the gut could often be predicted from expression levels in culture, but several strains exhibited gut-specific expression. This work establishes the experimental design parameters and advantages of transcript barcoding to measure the performance of many engineered probiotic designs in mammalian hosts.
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Código de Barras del ADN Taxonómico/métodos , Escherichia coli/metabolismo , Microbioma Gastrointestinal , Animales , Escherichia coli/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Probióticos , Regiones Promotoras GenéticasRESUMEN
Genome editing is essential for probing genotype-phenotype relationships and for enhancing chemical production and phenotypic robustness in industrial bacteria. Currently, the most popular tools for genome editing couple recombineering with DNA cleavage by the CRISPR nuclease Cas9 from Streptococcus pyogenes. Although successful in some model strains, CRISPR-based genome editing has been slow to extend to the multitude of industrially relevant bacteria. In this review, we analyze existing barriers to implementing CRISPR-based editing across diverse bacterial species. We first compare the efficacy of current CRISPR-based editing strategies. Next, we discuss alternatives when the S. pyogenes Cas9 does not yield colonies. Finally, we describe different ways bacteria can evade editing and how elucidating these failure modes can improve CRISPR-based genome editing across strains. Together, this review highlights existing obstacles to CRISPR-based editing in bacteria and offers guidelines to help achieve and enhance editing in a wider range of bacterial species, including non-model strains.