RESUMEN
Commercial production of recombinant streptavidin (SAV) using soluble expression route is cost-prohibitive, resulting from its inherent toxicity toward commercially available Escherichia coli hosts (such as BL21) and low productivity of existing manufacturing processes. Quality challenges can also result from binding of streptavidin in the host cells. One way to overcome these challenges is to allow formation of inclusion bodies (IBs). Nevertheless, carried-over cellular contaminants during IBs preparation can hinder protein refolding and application of SAV in nucleic acid-based applications. Hence, removing associated contaminants in recombinant IBs is imperative for maximum product outcomes. In this study, the IBs isolation method from our group was improved to remove residual DNA found in refolded core SAV (cSAV). The improvements were attained by incorporating quantitative real-time polymerase chain reactions (qPCR) for residual DNA monitoring. We attained 99 % cellular DNA removal from cSAV IBs via additional wash and sonication steps, and the addition of benzonase nuclease during lysis. A 10 % increment of cSAV refolding yield (72 %) and 83 % reduction of residual DNA from refolding of 1 mg cSAV IBs were observed under extensive sonication. Refolding of cSAV was not affected and its activity was not compromised. The optimized process reported here highlights the importance of obtaining cSAV IBs with minimal contaminants prior to refolding to increase product yield, and the usefulness of the qPCR method to monitor nucleic acid removed from each step of the process.
Asunto(s)
Escherichia coli , Cuerpos de Inclusión , Replegamiento Proteico , Proteínas Recombinantes , Estreptavidina , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Estreptavidina/química , Estreptavidina/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesisRESUMEN
Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some ß-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.
Asunto(s)
Celulasa , Cryptococcus gattii , Escherichia coli , Cryptococcus gattii/genética , Cryptococcus gattii/enzimología , Cryptococcus gattii/química , Celulasa/genética , Celulasa/química , Celulasa/aislamiento & purificación , Celulasa/metabolismo , Celulasa/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/biosíntesis , Expresión Génica , Clonación Molecular , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
The human milk fat globule membrane (hMFGM) and Lactobacillus modulate the infant's gut and benefit health. Hence, the current study assesses the probiotic potential of Lactiplantibacillus plantarum (MRK3), Limosilactobacillus ferementum (MK1) isolated from infant feces, and its interaction with hMFGM during conditions mimicking infant digestive tract. Both strains showed high tolerance to gastrointestinal conditions, cell surface hydrophobicity, and strong anti-pathogen activity against Staphylococcus aureus. During digestion, hMFGM significantly exhibited xanthine oxidase activity, membrane roughness, and surface topography. In the presence of hMFGM, survival of MRK3 was higher than MK1, and electron microscopic observation revealed successful entrapment of MRK3 in the membrane matrix throughout digestion. Interestingly, probiotic-membrane matrix interaction showed significant synergy to alleviate oxidative stress and damage induced by cell-free supernatant of Escherichia coli in Caco-2 cells. Our results show that a probiotic-encapsulated membrane matrix potentially opens the functional infant formula development pathway.
Asunto(s)
Glucolípidos , Glicoproteínas , Gotas Lipídicas , Leche Humana , Estrés Oxidativo , Probióticos , Humanos , Probióticos/farmacología , Probióticos/química , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Glicoproteínas/química , Glicoproteínas/farmacología , Glicoproteínas/metabolismo , Células CACO-2 , Glucolípidos/química , Glucolípidos/farmacología , Glucolípidos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Leche Humana/química , Lactante , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Fórmulas Infantiles/química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Tracto Gastrointestinal/microbiología , Tracto Gastrointestinal/metabolismoRESUMEN
Signal peptide (SP) is required for secretion of recombinant proteins and typically cleaved by signal peptidase at its C-region to generate the mature proteins. Miscleavage of the SP is reported occasionally, resulting in a truncated- or elongated-terminal sequence. In the present work, we demonstrated that cation exchange (CEX) chromatography is an effective means for removing SP variants with a case study. With the selected resin/conditions, the chromatographic performance is comparable between runs performed at the low end and high end of load density and elution range. The procedure described in this work can be used as a general approach for resin selection and optimization of chromatographic conditions to remove byproducts that bind more strongly than the product to the selected resin.
Asunto(s)
Señales de Clasificación de Proteína , Cromatografía por Intercambio Iónico/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Resinas de Intercambio de Catión/química , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the ß and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I , Humanos , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Fosfatidilinositol 3-Quinasa Clase Ia/química , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/química , Solubilidad , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Essential amino acid, tryptophan which intake from food plays a critical role in numerous metabolic functions, exhibiting extensive biological functions and applications. Tryptophan is beneficial for the food sector by enhancing nutritional content and promoting the development of functional foods. A putative gene encoding tryptophan synthase was the first identified in Sphingobacterium soilsilvae Em02, a cellulosic bacterium making it inherently more environmentally friendly. The gene was cloned and expressed in exogenous host Escherichia coli, to elucidate its function. The recombinant tryptophan synthase with a molecular weight 42 KDa was expressed in soluble component. The enzymatic activity to tryptophan synthase in vivo was assessed using indole and L-serine and purified tryptophan synthase. The optimum enzymatic activity for tryptophan synthase was recorded at 50 ºC and pH 7.0, which was improved in the presence of metal ions Mg2+, Sr2+ and Mn2+, whereas Cu2+, Zn2+ and Co2+ proved to be inhibitory. Using site-directed mutagenesis, the consensus pattern HK-S-[GGGSN]-E-S in the tryptophan synthase was demonstrated with K100Q, S202A, G246A, E361A and S385A as the active sites. Tryptophan synthase has been demonstrated to possess the defining characteristics of the ß-subunits. The tryptophan synthase may eventually be useful for tryptophan production on a larger scale. Its diverse applications highlight the potential for improving both the quality and health benefits of food products, making it an essential component in advancing food science and technology.
Asunto(s)
Escherichia coli , Mutagénesis Sitio-Dirigida , Triptófano Sintasa , Triptófano , Triptófano Sintasa/metabolismo , Triptófano Sintasa/genética , Triptófano Sintasa/química , Triptófano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Sphingomonadaceae/enzimología , Sphingomonadaceae/genética , Sphingomonadaceae/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Dominio Catalítico , Clonación Molecular , Concentración de Iones de Hidrógeno , Indoles/metabolismo , Catálisis , Serina/metabolismoRESUMEN
The cold-shock expression system in Escherichia coli was developed on a manual induction approach using optical density at 600 nm (OD600) measurements and isopropyl ß-D-1-thiogalactopyranoside (IPTG) addition. In this study, we show that cold-shock expression performs equally well using an autoinduction approach wherein OD600 measurements and IPTG addition may be eliminated. We further demonstrate that cold-shock expression with autoinduction can better facilitate high-throughput experiments.
Asunto(s)
Escherichia coli , Isopropil Tiogalactósido , Escherichia coli/genética , Escherichia coli/metabolismo , Isopropil Tiogalactósido/farmacología , Regulación Bacteriana de la Expresión Génica , Respuesta al Choque por Frío/genética , Frío , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The implementation of biocatalytic steroid hydroxylation processes plays a crucial role in the pharmaceutical industry due to a plethora of medicative effects of hydroxylated steroid derivatives and their crucial role in drug approval processes. Cytochrome P450 monooxygenases (CYP450s) typically constitute the key enzymes catalyzing these reactions, but commonly entail drawbacks such as poor catalytic rates and the dependency on additional redox proteins for electron transfer from NAD(P)H to the active site. Recently, these bottlenecks were overcome by equipping Escherichia coli cells with highly active variants of the self-sufficient single-component CYP450 BM3 together with hydrophobic outer membrane proteins facilitating cellular steroid uptake. The combination of the BM3 variant KSA14m and the outer membrane pore AlkL enabled exceptionally high testosterone hydroxylation rates of up to 45 U gCDW-1 for resting (i.e., living but non-growing) cells. However, a rapid loss of specific activity heavily compromised final product titers and overall space-time yields. In this study, several stabilization strategies were evaluated on enzyme-, cell-, and reaction level. However, neither changes in biocatalyst configuration nor variation of cultivation media, expression systems, or inducer concentrations led to considerable improvement. This qualified the so-far used genetic construct pETM11-ksa14m-alkL, M9 medium, and the resting-cell state as the best options enabling comparatively efficient activity along with fast growth prior to biotransformation. In summary, we report several approaches not enabling a stabilization of the high testosterone hydroxylation rates, providing vital guidance for researchers tackling similar CYP450 stability issues. A comparison with more stable natively steroid-hydroxylating CYP106A2 and CYP154C5 in equivalent setups further highlighted the high potential of the investigated CYP450 BM3-based whole-cell biocatalysts. The immense and continuously developing repertoire of enzyme engineering strategies provides promising options to stabilize the highly active biocatalysts.
Asunto(s)
Biocatálisis , Sistema Enzimático del Citocromo P-450 , Escherichia coli , Hidroxilación , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Testosterona/metabolismo , Esteroides/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Estabilidad de EnzimasRESUMEN
Using CO2 as the primary feedstock offers the potential for high-value utilization of CO2 while forging sustainable pathways for producing valuable natural products, such as l-tyrosine. Cascade catalysis is a promising approach but limited by stringent purity demands of nexus molecules. We developed an abiotic/biotic cascade catalysis using blended nexus molecules for l-tyrosine synthesis. Specifically, we begin by constructing a solid-state reactor to reduce CO2 electrochemically, yielding a mixture of acetic acid and ethanol, which serves as the blended nexus molecules. Subsequently, we use genetic engineering to introduce an ethanol utilization pathway and a tyrosine producing pathway to Escherichia coli to facilitate l-tyrosine production. The ethanol pathway synergistically cooperated with the acetic acid pathway, boosting l-tyrosine production rate (nearly five times higher compared to the strain without ethanol utilization pathway) and enhancing carbon efficiency. Our findings demonstrate that using blended nexus molecules could potentially offer a more favorable strategy for the cascade catalysis aimed at producing valuable natural products.
Asunto(s)
Dióxido de Carbono , Escherichia coli , Etanol , Tirosina , Dióxido de Carbono/metabolismo , Dióxido de Carbono/química , Tirosina/metabolismo , Tirosina/química , Escherichia coli/metabolismo , Escherichia coli/genética , Etanol/metabolismo , Catálisis , Ácido Acético/metabolismo , Ácido Acético/químicaRESUMEN
The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli. Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.
Asunto(s)
Proteínas Bacterianas , Escherichia coli , Inteínas , Mycobacterium tuberculosis , Empalme de Proteína , Rec A Recombinasas , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Rec A Recombinasas/metabolismo , Rec A Recombinasas/genética , Inteínas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas/genética , Daño del ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Serina EndopeptidasasRESUMEN
This study was to investigate the antibacterial effects and metabolites derived from bifidobacterial fermentation of an exopolysaccharide EPS-LM produced by a medicinal fungus Cordyceps sinensis, Cs-HK1. EPS-LM was a partially purified polysaccharide fraction which was mainly composed of Man, Glc and Gal at 7.31:12.95:1.00 mol ratio with a maximum molecular weight of 360 kDa. After fermentation of EPS-LM in two bifidobacterial cultures, B. breve and B. longum, the culture digesta showed significant antibacterial activities, inhibiting the proliferation and biofilm formation of Escherichia coli. Based on untargeted metabolomic profiling of the digesta, the levels of short chain fatty acids, carboxylic acids, benzenoids and their derivatives were all increased significantly (p < 0.01), which probably contributed to the enhanced antibacterial activity by EPS-LM. Since EPS-LM was only slightly consumed for the bifidobacterial growth, it mainly stimulated the biosynthesis of bioactive metabolites in the bifidobacterial cells. The results also suggested that EPS-LM polysaccharide may have a regulatory function on the bifidobacterial metabolism leading to production of antibacterial metabolites, which may be of significance for further exploration.
Asunto(s)
Antibacterianos , Cordyceps , Escherichia coli , Fermentación , Polisacáridos Bacterianos , Antibacterianos/farmacología , Antibacterianos/química , Cordyceps/metabolismo , Cordyceps/química , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Polisacáridos Bacterianos/farmacología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/metabolismo , Biopelículas/efectos de los fármacos , Polisacáridos Fúngicos/farmacología , Polisacáridos Fúngicos/química , Pruebas de Sensibilidad MicrobianaRESUMEN
Background: Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK's catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. Methods: The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. Results: The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. Conclusion: The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.
Asunto(s)
Alcohol Deshidrogenasa , Escherichia coli , Hierro , Zinc , Zinc/metabolismo , Alcohol Deshidrogenasa/metabolismo , Alcohol Deshidrogenasa/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Hierro/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Sitios de Unión , Unión Proteica , Mutagénesis Sitio-DirigidaRESUMEN
To withstand their internal turgor pressure and external threats, most bacteria have a protective peptidoglycan (PG) cell wall. The growth of this PG polymer relies on autolysins, enzymes that create space within the structure. Despite extensive research, the regulatory mechanisms governing these PG-degrading enzymes remain poorly understood. Here, we unveil a novel and widespread control mechanism of lytic transglycosylases (LTs), a type of autolysin responsible for breaking down PG glycan chains. Specifically, we show that LD-crosslinks within the PG sacculus act as an inhibitor of LT activity. Moreover, we demonstrate that this regulation controls the release of immunogenic PG fragments and provides resistance against predatory LTs of both bacterial and viral origin. Our findings address a critical gap in understanding the physiological role of the LD-crosslinking mode in PG homeostasis, highlighting how bacteria can enhance their resilience against environmental threats, including phage attacks, through a single structural PG modification.
Asunto(s)
Pared Celular , N-Acetil Muramoil-L-Alanina Amidasa , Peptidoglicano , Peptidoglicano/metabolismo , Pared Celular/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Escherichia coli/metabolismo , Glicosiltransferasas/metabolismo , Bacillus subtilis/metabolismoRESUMEN
The inner capsid protein of rotavirus, VP6, emerges as a promising candidate for next-generation vaccines against rotaviruses owing to its abundance in virion particles and high conservation. However, the formation of inclusion bodies during prokaryotic VP6 expression poses a significant hurdle to rotavirus research and applications. Here, we employed experimental and computational approaches to investigate inclusion body formation and aggregation-prone regions (APRs). Heterologous recombinant VP6 expression in Escherichia coli BL21(DE3) cells resulted in inclusion body formation, confirmed by transmission electron microscopy revealing amorphous aggregates. Thioflavin T assay demonstrated incubation temperature-dependent aggregation of VP6 inclusion bodies. Computational predictions of APRs in rotavirus A VP6 protein were performed using sequence-based tools (TANGO, AGGRESCAN, Zyggregator, Waltz, FoldAmyloid, ANuPP, Camsol intrinsic) and structure-based tools (SolubiS, CamSol structurally corrected, Aggrescan3D). A total of 24 consensus APRs were identified, with 21 of them being surface-exposed in VP6. All identified APRs display a predominance of hydrophobic amino acids, ranging from 33 to 100%. Computational identification of these APRs corroborates our experimental observation of VP6 inclusion body or aggregate formation. Characterization of VP6's aggregation propensity facilitates understanding of its behaviour during prokaryotic expression and opens avenues for protein engineering of soluble variants, advancing research on rotavirus VP6 in pathology, therapy, and diagnostics.
Asunto(s)
Antígenos Virales , Proteínas de la Cápside , Escherichia coli , Cuerpos de Inclusión , Rotavirus , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Antígenos Virales/genética , Antígenos Virales/metabolismo , Cuerpos de Inclusión/metabolismo , Rotavirus/genética , Rotavirus/metabolismo , Agregado de Proteínas , Simulación por Computador , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The Ames test is used worldwide to initially screen the mutagenic potential of new chemicals. In the standard Ames test, S. typhimurium strains (TA100, TA98, TA1535, and TA1537) and Escherichia coli (WP2uvrA) are treated with substances with/without cytochrome P450s (CYPs)-induced rat S9 fractions for identifying mutagens and pro-mutagens. However, many substances show completely different toxicity patterns depending on whether the liver S9 fraction belongs to rats or humans. The natural product Polygoni Multiflori Radix (PMR) can also show bacterial reverse mutation, followed by the rat or human liver S9 fraction. While PMR elicits reverse mutations in the TA1537 strain in rat liver S9 but not in human liver S9, this mechanism has not been verified yet. To explain this, the differences in metabolic enzymes compositions commonly observed between rats and humans have been implicated. This study aimed to explore the key factors that cause differences in the genotoxicity of PMR between rat and human liver S9 metabolic enzymes. The results of next-generation sequencing (NGS) analysis showed that both rat and human metabolic enzymes caused similar mutations in TA1537. However, when the metabolic enzymes in each S9 fraction were analyzed using ion mobility tandem mass spectrometry (IM-MS), rat- and human-specific enzymes were identified among the cytochrome (CYP) family, especially aryl hydrocarbon receptor (AHR)-related CYPs. These findings suggest that CYP1A1 isoforms contribute to the mechanism of PMR in the Ames test. Therefore, an in vitro Ames test might be more reliable in predicting genotoxicity for both rodents and humans. This will also help overcome the limitations of laboratory animal-based toxicity evaluations, which provide unreliable results due to interspecies differences between humans and rodents.
Asunto(s)
Pruebas de Mutagenicidad , Mutágenos , Salmonella typhimurium , Animales , Humanos , Pruebas de Mutagenicidad/métodos , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Mutágenos/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Activación Metabólica , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Mutación , Daño del ADN/efectos de los fármacos , Fallopia multiflora/química , MasculinoRESUMEN
The arsRBC operon encodes a three-protein arsenic resistance system. ArsR regulates the transcription of the operon, while ArsB and ArsC are involved in exporting trivalent arsenic and reducing pentavalent arsenic, respectively. Previous research into Agrobacterium tumefaciens 5A has demonstrated that ArsR has regulatory control over a wide range of metal-related proteins and metabolic pathways. We hypothesized that ArsR has broad regulatory control in other Gram-negative bacteria and set out to test this. Here, we use differential proteomics to investigate changes caused by the presence of the arsR gene in human microbiome-relevant Escherichia coli during arsenite (AsIII) exposure. We show that ArsR has broad-ranging impacts such as the expression of TCA cycle enzymes during AsIII stress. Additionally, we found that the Isc [Fe-S] cluster and molybdenum cofactor assembly proteins are upregulated regardless of the presence of ArsR under these same conditions. An important finding from this differential proteomics analysis was the identification of response mechanisms that were strain-, ArsR-, and arsenic-specific, providing new clarity to this complex regulon. Given the widespread occurrence of the arsRBC operon, these findings should have broad applicability across microbial genera, including sensitive environments such as the human gastrointestinal tract.
Asunto(s)
Arsenitos , Proteínas de Escherichia coli , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Operón , Proteómica , Estrés Fisiológico , Arsenitos/toxicidad , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteómica/métodos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Operón/genética , Metaloproteínas/metabolismo , Metaloproteínas/genética , HumanosRESUMEN
The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the ß-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Sitios de Unión , Chaperonas Moleculares/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Unión Proteica , Adenosina Trifosfato/metabolismo , Dominios Proteicos , Pliegue de Proteína , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Modelos Moleculares , Proteínas PortadorasRESUMEN
The outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded ß-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear. Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane. Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Pliegue de Proteína , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/genética , Microscopía por Crioelectrón , Unión Proteica , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/química , Mutación , Proteínas Portadoras , Isomerasa de PeptidilprolilRESUMEN
BACKGROUND: Benzyl acetate is an aromatic ester with a jasmine scent. It was discovered in plants and has broad applications in food, cosmetic, and pharmaceutical industries. Its current production predominantly relies on chemical synthesis. In this study, Escherichia coli was engineered to produce benzyl acetate. RESULTS: Two biosynthetic routes based on the CoA-dependent ß-oxidation pathway were constructed in E. coli for benzyl acetate production. In route I, benzoic acid pathway was extended to produce benzyl alcohol by combining carboxylic acid reductase and endogenous dehydrogenases and/or aldo-keto reductases in E. coli. Benzyl alcohol was then condensed with acetyl-CoA by the alcohol acetyltransferase ATF1 from yeast to form benzyl acetate. In route II, a plant CoA-dependent ß-oxidation pathway via benzoyl-CoA was assessed for benzyl alcohol and benzyl acetate production in E. coli. The overexpression of the phosphotransacetylase from Clostridium kluyveri (CkPta) further improved benzyl acetate production in E. coli. Two-phase extractive fermentation in situ was adopted and optimized for benzyl acetate production in a shake flask. The most optimal strain produced 3.0 ± 0.2 g/L benzyl acetate in 48 h by shake-flask fermentation. CONCLUSIONS: We were able to establish the whole pathway for benzyl acetate based on the CoA-dependent ß-oxidation in single strain for the first time. The highest titer for benzyl acetate produced from glucose by E. coli is reported. Moreover, cinnamyl acetate production as an unwanted by-product was very low. Results provided novel information regarding the engineering benzyl acetate production in microorganisms.
Asunto(s)
Escherichia coli , Glucosa , Ingeniería Metabólica , Ingeniería Metabólica/métodos , Escherichia coli/metabolismo , Escherichia coli/genética , Glucosa/metabolismo , Fermentación , Acetatos/metabolismo , Oxidación-Reducción , Acetilcoenzima A/metabolismo , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , Compuestos de Bencilo/metabolismoRESUMEN
Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi- assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.