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1.
Nucleic Acids Res ; 52(D1): D590-D596, 2024 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-37889041

RESUMEN

CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.


Asunto(s)
Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Bases de Datos Genéticas , Endodesoxirribonucleasas , Sistemas CRISPR-Cas/genética , Filogenia , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/clasificación , Proteínas Asociadas a CRISPR/genética , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/clasificación , Endodesoxirribonucleasas/genética , Enciclopedias como Asunto
2.
Cell ; 185(9): 1487-1505.e14, 2022 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-35366417

RESUMEN

Small molecules encoded by biosynthetic pathways mediate cross-species interactions and harbor untapped potential, which has provided valuable compounds for medicine and biotechnology. Since studying biosynthetic gene clusters in their native context is often difficult, alternative efforts rely on heterologous expression, which is limited by host-specific metabolic capacity and regulation. Here, we describe a computational-experimental technology to redesign genes and their regulatory regions with hybrid elements for cross-species expression in Gram-negative and -positive bacteria and eukaryotes, decoupling biosynthetic capacity from host-range constraints to activate silenced pathways. These synthetic genetic elements enabled the discovery of a class of microbiome-derived nucleotide metabolites-tyrocitabines-from Lactobacillus iners. Tyrocitabines feature a remarkable orthoester-phosphate, inhibit translational activity, and invoke unexpected biosynthetic machinery, including a class of "Amadori synthases" and "abortive" tRNA synthetases. Our approach establishes a general strategy for the redesign, expression, mobilization, and characterization of genetic elements in diverse organisms and communities.


Asunto(s)
Vías Biosintéticas , Interacciones Microbiota-Huesped , Microbiota , Biología Sintética/métodos , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Eucariontes/genética , Eucariontes/metabolismo , Ingeniería Genética , Humanos , Metabolómica
3.
Life (Basel) ; 9(3)2019 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-31284384

RESUMEN

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

4.
ACS Chem Biol ; 13(12): 3286-3293, 2018 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-30403848

RESUMEN

Colibactins are genotoxic secondary metabolites produced in select Enterobacteriaceae, which induce downstream DNA double-strand breaks (DSBs) in human cell lines and are thought to promote the formation of colorectal tumors. Although key structural and functional features of colibactins have been elucidated, the full molecular mechanisms regulating these phenotypes remain unknown. Here, we demonstrate that free model colibactins induce DSBs in human cell cultures and do not require delivery by host bacteria. Through domain-targeted editing, we demonstrate that a subset of native colibactins generated from observed module skipping in the nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) biosynthetic assembly line share DNA alkylation phenotypes with the model colibactins in vitro. However, module skipping eliminates the strong DNA interstrand cross-links formed by the wild-type pathway in cell culture. This product diversification during the modular NRPS-PKS biosynthesis produces a family of metabolites with varying observed mechanisms of action (DNA alkylation versus cross-linking) in cell culture. The presence of membranes separating human cells from model colibactins attenuated genotoxicity, suggesting that membrane diffusion limits colibactin activity and could account for the reported bacterium-human cell-to-cell contact phenotype. Additionally, extracellular supplementation of the colibactin resistance protein ClbS was able to intercept colibactins in an Escherichia coli-human cell transient infection model. Our studies demonstrate that free model colibactins recapitulate cellular phenotypes associated with module-skipped products in the native colibactin pathway and define specific protein domains that are required for efficient DNA interstrand cross-linking in the native pathway.


Asunto(s)
ADN/química , Mutágenos/farmacología , Péptidos/farmacología , Policétidos/farmacología , Alquilación/efectos de los fármacos , Línea Celular Tumoral , Reactivos de Enlaces Cruzados/farmacología , ADN/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Escherichia coli/genética , Humanos , Péptidos/genética
5.
J Immunol ; 201(12): 3569-3579, 2018 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-30446568

RESUMEN

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports, we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing, IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably, Bcl6hi B cells can form in the combined absence of IL-21R- and STAT6-derived signals; however, these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6, aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus, IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals.


Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Interleucina-4/metabolismo , Interleucinas/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Apoptosis , Diferenciación Celular , Autorrenovación de las Células , Células Cultivadas , Activación de Linfocitos , Ratones , Ratones Noqueados , Comunicación Paracrina , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Receptores de Interleucina-21/genética , Factor de Transcripción STAT6/metabolismo , Transducción de Señal
6.
Nat Commun ; 9(1): 1203, 2018 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-29572528

RESUMEN

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.


Asunto(s)
Aminoácidos/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Factores de Terminación de Péptidos/química , Sistema Libre de Células , Codón , Proteínas de Escherichia coli/genética , Ingeniería Genética , Genoma Bacteriano , Proteínas Fluorescentes Verdes/metabolismo , Espectrometría de Masas , Mutación , Factores de Terminación de Péptidos/genética , Péptidos/metabolismo , Fenilalanina/metabolismo , Plásmidos/metabolismo , Biosíntesis de Proteínas
7.
J Am Chem Soc ; 138(48): 15563-15570, 2016 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-27934011

RESUMEN

Precolibactins and colibactins represent a family of natural products that are encoded by the clb gene cluster and are produced by certain commensal, extraintestinal, and probiotic E. coli. clb+ E. coli induce megalocytosis and DNA double-strand breaks in eukaryotic cells, but paradoxically, this gene cluster is found in the probiotic Nissle 1917. Evidence suggests precolibactins are converted to genotoxic colibactins by colibactin peptidase (ClbP)-mediated cleavage of an N-acyl-d-Asn side chain, and all isolation efforts have employed ΔclbP strains to facilitate accumulation of precolibactins. It was hypothesized that colibactins form unsaturated imines that alkylate DNA by cyclopropane ring opening (2 → 3). However, as no colibactins have been isolated, this hypothesis has not been tested experimentally. Additionally, precolibactins A-C (7-9) contain a pyridone that cannot generate the unsaturated imines that form the basis of this hypothesis. To resolve this, we prepared 13 synthetic colibactin derivatives and evaluated their DNA binding and alkylation activity. We show that unsaturated imines, but not the corresponding pyridone derivatives, potently alkylate DNA. The imine, unsaturated lactam, and cyclopropane are essential for efficient DNA alkylation. A cationic residue enhances activity. These studies suggest that precolibactins containing a pyridone are not responsible for the genotoxicity of the clb cluster. Instead, we propose that these are off-pathway fermentation products produced by a facile double cyclodehydration route that manifests in the absence of viable ClbP. The results presented herein provide a foundation to begin to connect metabolite structure with the disparate phenotypes associated with clb+ E. coli.


Asunto(s)
Productos Biológicos/toxicidad , Escherichia coli/química , Mutágenos/toxicidad , Péptidos/toxicidad , Policétidos/toxicidad , Productos Biológicos/síntesis química , Productos Biológicos/química , División del ADN/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Estructura Molecular , Familia de Multigenes/genética , Mutágenos/síntesis química , Mutágenos/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Péptidos/síntesis química , Péptidos/química , Péptidos/genética , Fenotipo , Policétidos/síntesis química , Policétidos/química
9.
Curr Opin Chem Biol ; 28: 83-90, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26186264

RESUMEN

The translation system (the ribosome and associated factors) is the cell's factory for protein synthesis. The extraordinary catalytic capacity of the protein synthesis machinery has driven extensive efforts to harness it for novel functions. For example, pioneering efforts have demonstrated that it is possible to genetically encode more than the 20 natural amino acids and that this encoding can be a powerful tool to expand the chemical diversity of proteins. Here, we discuss recent advances in efforts to expand the chemistry of living systems, highlighting improvements to the molecular machinery and genomically recoded organisms, applications of cell-free systems, and extensions of these efforts to include eukaryotic systems. The transformative potential of repurposing the translation apparatus has emerged as one of the defining opportunities at the interface of chemical and synthetic biology.


Asunto(s)
Aminoácidos/genética , Código Genético , Ingeniería Genética/métodos , Biosíntesis de Proteínas , Biología Sintética/métodos , Animales , Humanos
10.
Nucleic Acids Res ; 43(3): 1945-54, 2015 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-25567985

RESUMEN

Genetically modified organisms (GMOs) are commonly used to produce valuable compounds in closed industrial systems. However, their emerging applications in open clinical or environmental settings require enhanced safety and security measures. Intrinsic biocontainment, the creation of bacterial hosts unable to survive in natural environments, remains a major unsolved biosafety problem. We developed a new biocontainment strategy containing overlapping 'safeguards'-engineered riboregulators that tightly control expression of essential genes, and an engineered addiction module based on nucleases that cleaves the host genome-to restrict viability of Escherichia coli cells to media containing exogenously supplied synthetic small molecules. These multilayered safeguards maintain robust growth in permissive conditions, eliminate persistence and limit escape frequencies to <1.3 × 10(-12). The staged approach to safeguard implementation revealed mechanisms of escape and enabled strategies to overcome them. Our safeguarding strategy is modular and employs conserved mechanisms that could be extended to clinically or industrially relevant organisms and undomesticated species.


Asunto(s)
Escherichia coli/genética , Organismos Modificados Genéticamente/crecimiento & desarrollo , Clonación Molecular , Técnicas de Cocultivo , Medios de Cultivo , Escherichia coli/crecimiento & desarrollo , Recombinación Genética
11.
Nature ; 518(7537): 89-93, 2015 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-25607356

RESUMEN

Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations. Here we describe the construction of a series of genomically recoded organisms (GROs) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼10(11) cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.


Asunto(s)
Aminoácidos/síntesis química , Aminoácidos/farmacología , Contención de Riesgos Biológicos/métodos , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Viabilidad Microbiana/efectos de los fármacos , Biología Sintética/métodos , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Dominio Catalítico/genética , Codón/genética , Medios de Cultivo/química , Medios de Cultivo/farmacología , Ambiente , Escherichia coli/citología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Transferencia de Gen Horizontal/genética , Genes Esenciales/genética , Código Genético/genética , Ingeniería Genética/métodos , Genoma Bacteriano/genética , Viabilidad Microbiana/genética , Datos de Secuencia Molecular , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/crecimiento & desarrollo , Organismos Modificados Genéticamente/metabolismo , Factores de Terminación de Péptidos/genética , Fenilalanina/química , Fenilalanina/metabolismo , Multimerización de Proteína/genética , ARN de Transferencia/genética
12.
Immunity ; 34(6): 947-60, 2011 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-21636295

RESUMEN

We identify the interfollicular (IF) zone as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first 2 days postimmunization, antigen-specific T and B cells remained confined within the IF zone, formed long-lived interactions, and upregulated the transcriptional repressor Bcl6. T cells also acquired the Tfh cell markers CXCR5, PD-1, and GL7. Responding B and T cells migrated to the follicle interior directly from the IF zone, T cell immigration preceding B cells by 1 day. Notably, in the absence of cognate B cells, Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS, and GL7 were no longer expressed on follicular Bcl6(hi) T cells that nevertheless persisted in the follicle. Thus, Ag-specific B cells are required for the maintenance of the PD-1(hi)ICOS(hi)GL7(hi) Tfh cell phenotype within the follicle, but not for their initial differentiation in the IF zone.


Asunto(s)
Linfocitos B/inmunología , Diferenciación Celular , Centro Germinal/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos/inmunología , Linfocitos B/citología , Movimiento Celular , Centro Germinal/citología , Ratones , Fenotipo , Linfocitos T Colaboradores-Inductores/citología
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