RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019, constitutes an emerging human pathogen of zoonotic origin. A critical role in protecting the host against invading pathogens is carried out by interferon-stimulated genes (ISGs), the primary effectors of the type I interferon (IFN) response. All coronaviruses studied thus far have to first overcome the inhibitory effects of the IFN/ISG system before establishing efficient viral replication. However, whether SARS-CoV-2 evades IFN antiviral immunity by manipulating ISG activation remains to be elucidated. Here, we show that the SARS-CoV-2 main protease (Mpro) significantly suppresses the expression and transcription of downstream ISGs driven by IFN-stimulated response elements in a dose-dependent manner, and similar negative regulations were observed in two mammalian epithelial cell lines (simian Vero E6 and human A549). Our analysis shows that to inhibit the ISG production, Mpro cleaves histone deacetylases (HDACs) rather than directly targeting IFN signal transducers. Interestingly, Mpro also abolishes the activity of ISG effector mRNA-decapping enzyme 1a (DCP1A) by cleaving it at residue Q343. In addition, Mpro from different genera of coronaviruses has the protease activity to cleave both HDAC2 and DCP1A, even though the alphacoronaviruse Mpro exhibits weaker catalytic activity in cleaving HDAC2. In conclusion, our findings clearly demonstrate that SARS-CoV-2 Mpro constitutes a critical anti-immune effector that modulates the IFN/ISG system at multiple levels, thus providing a novel molecular explanation for viral immune evasion and allowing for new therapeutic approaches against coronavirus disease 2019 infection.
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COVID-19 , Interferón Tipo I , Animales , Humanos , SARS-CoV-2 , Histona Desacetilasas/genética , Interferón Tipo I/farmacología , Péptido Hidrolasas , Mamíferos , Endorribonucleasas , TransactivadoresRESUMEN
The development of oligomeric glucagon-like peptide-1 (GLP-1) and GLP-1-containing coagonists holds promise for enhancing the therapeutic potential of the GLP-1-based drugs for treating type 2 diabetes mellitus (T2DM). Here, we report a facile, efficient, and customizable strategy based on genetically encoded SpyCatcher-SpyTag chemistry and an inducible, cleavable self-aggregating tag (icSAT) scheme. icSAT-tagged SpyTag-fused GLP-1 and the dimeric or trimeric SpyCatcher scaffold were designed for dimeric or trimeric GLP-1, while icSAT-tagged SpyCatcher-fused GLP-1 and the icSAT-tagged SpyTag-fused GIP were designed for dual GLP-1/GIP (glucose-dependent insulinotropic polypeptide) receptor agonist. These SpyCatcher- and SpyTag-fused protein pairs were spontaneously ligated directly from the cell lysates. The subsequent icSAT scheme, coupled with a two-step standard column purification, resulted in target proteins with authentic N-termini, with yields ranging from 35 to 65 mg/L and purities exceeding 99%. In vitro assays revealed 3.0- to 4.1-fold increased activities for dimeric and trimeric GLP-1 compared to mono-GLP-1. The dual GLP-1/GIP receptor agonist exhibited balanced activity toward the GLP-1 receptor or the GIP receptor. All the proteins exhibited 1.8- to 3.0-fold prolonged half-lives in human serum compared to mono-GLP-1 or GIP. This study provides a generally applicable click biochemistry strategy for developing oligomeric or dual peptide/protein-based drug candidates.
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Química Clic , Péptido 1 Similar al Glucagón , Péptido 1 Similar al Glucagón/química , Humanos , Receptores de la Hormona Gastrointestinal/agonistas , Receptores de la Hormona Gastrointestinal/química , Receptores de la Hormona Gastrointestinal/metabolismo , Diseño de Fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Polipéptido Inhibidor Gástrico/química , Polipéptido Inhibidor Gástrico/farmacología , Receptor del Péptido 1 Similar al Glucagón/agonistasRESUMEN
BACKGROUND: Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release. RESULTS: In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72-97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%. CONCLUSION: Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.
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Escherichia coli , Péptidos , Humanos , Escherichia coli/metabolismo , Péptidos/metabolismo , Proteínas/metabolismo , Inteínas , Procesamiento Proteico-Postraduccional , Cloruro de Sodio/metabolismoRESUMEN
BACKGROUND: Compound-protein interaction site and binding affinity predictions are crucial for drug discovery and drug design. In recent years, many deep learning-based methods have been proposed for predications related to compound-protein interaction. For protein inputs, how to make use of protein primary sequence and tertiary structure information has impact on prediction results. RESULTS: In this study, we propose a deep learning model based on a multi-objective neural network, which involves a multi-objective neural network for compound-protein interaction site and binding affinity prediction. We used several kinds of self-supervised protein embeddings to enrich our protein inputs and used convolutional neural networks to extract features from them. Our results demonstrate that our model had improvements in terms of interaction site prediction and affinity prediction compared to previous models. In a case study, our model could better predict binding sites, which also showed its effectiveness. CONCLUSION: These results suggest that our model could be a helpful tool for compound-protein related predictions.
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Redes Neurales de la Computación , Proteínas , Proteínas/química , Secuencia de Aminoácidos , Sitios de Unión , Descubrimiento de DrogasRESUMEN
BACKGROUND: During fermentation, industrial microorganisms encounter multiple stresses that inhibit cell growth and decrease fermentation yields, in particular acid stress, which is due to the accumulation of acidic metabolites in the fermentation medium. Although the addition of a base to the medium can counteract the effect of acid accumulation, the engineering of acid-tolerant strains is considered a more intelligent and cost-effective solution. While synthetic biology theoretically provides a novel approach for devising such tolerance modules, in practice it is difficult to assemble stress-tolerance modules from hundreds of stress-related genes. RESULTS: In this study, we designed a set of synthetic acid-tolerance modules for fine-tuning the expression of multi-component gene blocks comprising a member of the proton-consuming acid resistance system (gadE), a periplasmic chaperone (hdeB), and reactive oxygen species (ROS) scavengers (sodB and katE). Directed evolution was used to construct an acid-responsive asr promoter library, from which four variants were selected and used in the synthetic modules. The module variants were screened in a stepwise manner under mild acidic conditions (pH 5-6), first by cell growth using the laboratory Escherichia coli strain MG1655 cultured in microplates, and then by lysine production performance using the industrial lysine-producing E. coli strain MG1655 SCEcL3 cultured first in multiple 10-mL micro-bioreactors, and then in 1.3-L parallel bioreactors. The procedure resulted in the identification of a best strain with lysine titer and yield at pH 6.0 comparable to the parent strain at pH 6.8. CONCLUSION: Our results demonstrate a promising synthetic-biology strategy to enhance the growth robustness and productivity of E. coli upon the mildly acidic conditions, in both a general lab strain MG1655 and an industrial lysine-producing strain SCEcL3, by using the stress-responsive synthetic acid-tolerance modules comprising a limited number of genes. This study provides a reliable and efficient method for achieving synthetic modules of interest, particularly in improving the robustness and productivity of industrial strains.
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Proteínas de Escherichia coli , Escherichia coli , Ácidos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentación , Concentración de Iones de Hidrógeno , Lisina/metabolismo , Ingeniería Metabólica/métodosRESUMEN
Acid tolerance of microorganisms is a desirable phenotype for many industrial fermentation applications. In Escherichia coli, the stress response sigma factor RpoS is a promising target for engineering acid-tolerant phenotypes. However, the simple overexpression of RpoS alone is insufficient to confer these phenotypes. In this study, we show that the simultaneous overexpression of the noncoding small RNA (sRNA) DsrA and the sRNA chaperone Hfq, which act as RpoS activators, significantly increased acid tolerance in terms of cell growth under modest acidic pH, as well as cell survival upon extreme acid shock. Directed evolution of the DsrA-Hfq module further improved the acid tolerance, with the best mutants showing a 51 to 72% increase in growth performance at pH 4.5 compared with the starting strain, MG1655. Further analyses found that the improved acid tolerance of these DsrA-Hfq strains coincided with activation of genes associated with proton-consuming acid resistance system 2 (AR2), protein chaperone HdeB, and reactive oxygen species (ROS) removal in the exponential phase. This study illustrated that the fine-tuning of sRNAs and their chaperones can be a novel strategy for improving the acid tolerance of E. coliIMPORTANCE Many of the traditional studies on bacterial acid tolerance generally focused on improving cell survival under extreme-pH conditions, but cell growth under less harsh acidic conditions is more relevant to industrial applications. Under normal conditions, the general stress response sigma factor RpoS is maintained at low levels in the growth phase through a number of mechanisms. This study showed that RpoS can be activated prior to the stationary phase via engineering its activators, the sRNA DsrA and the sRNA chaperone Hfq, resulting in significantly improved cell growth at modest acidic pH. This work suggests that the sigma factors and likely other transcription factors can be retuned or retimed by manipulating the respective regulatory sRNAs along with the sufficient supply of the respective sRNA chaperones (i.e., Hfq). This provides a novel avenue for strain engineering of microbes.
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Proteínas de Escherichia coli/fisiología , Escherichia coli/fisiología , Proteína de Factor 1 del Huésped/fisiología , ARN Pequeño no Traducido/fisiología , Adaptación Fisiológica , Concentración de Iones de HidrógenoRESUMEN
Human growth hormone (hGH) plays an important role in growth control, growth promotion, cell development, and regulation of numerous metabolic pathways in the human body and has been approved by the U.S. FDA for the treatment of several human dysfunctions. Over-expression of recombinant hGH (rhGH) affords a misfolded form in cytoplasm of Escherichia coli, and the refolding step required to obtain active rhGH greatly affects its production costs. Herein, the cleavable self-aggregating tag (cSAT) scheme was used for the expression and purification of rhGH in E. coli. Four aggregating tags (L6KD/α3-peptide/EFK8/ELK16) successfully drove rhGH into active protein aggregates. After the Mxe GyrA intein-mediated cleavage, 2.8-21.4 µg rhGH/mg wet cell weight was obtained at laboratory scale, of which the L6KD fusion achieved the highest rhGH yield. The further refined rhGH maintained 92% of the bioactivity compared to commercial rhGH. The self-assembling of the aggregating tag might physically separate the hGH polypeptide chains, which in turn was beneficial to its folding into the active form. This study provided a simple and cost-effective approach for active rhGH production, and suggested an opportunity for improve folding of recombinant proteins in E. coli.
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Expresión Génica , Hormona de Crecimiento Humana/genética , Inteínas/genética , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Cromatografía de Afinidad , Cromatografía en Gel , Clonación Molecular , Girasa de ADN/genética , Girasa de ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hormona de Crecimiento Humana/biosíntesis , Hormona de Crecimiento Humana/aislamiento & purificación , Humanos , Péptidos/genética , Péptidos/metabolismo , Agregado de Proteínas , Pliegue de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Site-directed protein immobilization allows the homogeneous orientation of proteins with high retention of activity, which is advantageous for many applications. Here, we report a facile, specific, and efficient strategy based on the SpyTag-SpyCatcher chemistry. Two SpyTag-fused model proteins, that is, the monomeric red fluorescent protein (RFP) and the oligomeric glutaryl-7-aminocephalosporanic acid acylase, were easily immobilized onto a SpyCatcher-modified resin directly from cell lysates, with activity recoveries in the range of 85-91%. This strategy was further adapted to protein purification, which proceeded through the selective capture of the SpyCatcher-fused target proteins by a SpyTag-modified resin, with the aid of an intein to generate authentic N-termini. For two model proteins, that is, RFP and a variable domain of a heavy chain antibody, the yields were â¼3-7 mg/L culture with >90% purities. This approach could provide a versatile tool for producing high-performance immobilized protein devices and proteins for industrial and therapeutic uses.
Asunto(s)
Amidohidrolasas/metabolismo , Biotecnología/métodos , Enzimas Inmovilizadas/metabolismo , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Proteínas Luminiscentes/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/metabolismo , Amidohidrolasas/genética , Enzimas Inmovilizadas/química , Humanos , Cadenas Pesadas de Inmunoglobulina/metabolismo , Proteínas Luminiscentes/genética , Proteínas Recombinantes de Fusión/genética , Proteína Fluorescente RojaRESUMEN
A Gram-positive, aerobic, non-spore-forming, non-pigmented and non-motile actinobacterial strain was isolated from a soil sample collected in Guangzhou, China. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain 130T is most closely related to the type strain Nocardioides iriomotensis NBRC 105384T, with a sequence similarity of 97.69â%. The isolate was distinguished from this phylogenetically related type strain by DNA-DNA hybridization (33.3â%), digital DNA-DNA hybridization (21.2â%), average nucleotide identity (75.7â%) and by a range of physiological and biochemical characteristics. Strain 130T contained MK-8(H4) and MK-7 as the major menaquinones, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol as the main polar lipids, and iso-C16â:â0, C17â:â1ω8c, anteiso-C17â:â0, C18â:â1ω9c, C17â:â0 and iso-C14â:â0 as the major cellular fatty acids. The novel strain grew at 20-36 °C, at pH 6.0-8.0 and in the presence of 0-6â% (w/v) NaCl. The genomic DNA G+C content was 72.9 mol%. The genome contained 4817 putative protein-coding sequences, and 45 tRNA and three rRNA genes. Phylogenomic analysis confirmed that strain 130T belongs to the genus Nocardioides and distinguished it from recognized Nocardioides species with available genomes. Based on these polyphasic taxonomic data, strain 130T represents a new species of the genus Nocardioides, for which the name Nocardioidesguangzhouensis sp. nov. is proposed. The type strain is 130T (=CICC 24668T=JCM 33269T).
Asunto(s)
Actinobacteria/clasificación , Filogenia , Microbiología del Suelo , Actinobacteria/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Composición de Base , China , ADN Bacteriano/genética , Ácidos Grasos/química , Hibridación de Ácido Nucleico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/químicaRESUMEN
BACKGROUND: The GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. RESULTS: In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of ß-ionone of 3.5 mg/L. To further improve the ß-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of ß-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a ß-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. CONCLUSIONS: An efficient ß-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest ß-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.
Asunto(s)
Ingeniería Metabólica , Norisoprenoides/metabolismo , Yarrowia/metabolismo , Acetilcoenzima A/metabolismo , Fermentación , Microbiología Industrial , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Oxígeno/metabolismo , Yarrowia/genéticaRESUMEN
BACKGROUND: Acid stress is often encountered during industrial fermentation as a result of the accumulation of acidic metabolites. Acid stress increases the intracellular acidity and can cause DNA damage and denaturation of essential enzymes, thus leading to a decrease of growth and fermentation yields. Although acid stress can be relieved by addition of a base to the medium, fermentations with acid-tolerant strains are generally considered much more efficient and cost-effective. RESULTS: In this study, the global regulator H-NS was found to have significant influence on the acid tolerance of E. coli. The final OD600 of strains overexpressing H-NS increased by 24% compared to control, when cultured for 24 h at pH 4.5 using HCl as an acid agent. To further improve the acid tolerance, a library of H-NS was constructed by error-prone PCR and subjected to selection. Five mutants that conferred a significant growth advantage compared to the control strain were obtained. The final OD600 of strains harboring the five H-NS mutants was enhanced by 26-53%, and their survival rate was increased by 10- to 100-fold at pH 2.5. Further investigation showed that the improved acid tolerance of H-NS mutants coincides with the activation of multiple acid resistance mechanisms, in particular the glutamate- and glutamine-dependent acid resistance system (AR2). The improved acid tolerance of H-NS mutants was also demonstrated in media acidified by acetic acid and succinic acid, which are common acidic fermentation by-products or products. CONCLUSIONS: The results obtained in this work demonstrate that the engineering of H-NS can enhance the acid tolerance of E. coli. More in general, this study shows the potential of the engineering of global regulators acting as repressors, such as H-NS, as a promising method to obtain phenotypes of interest. This approach could expand the spectrum of application of global transcription machinery engineering.
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Ácido Acético/farmacología , Escherichia coli/efectos de los fármacos , Ácido Succínico/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentación/efectos de los fármacos , Reacción en Cadena de la PolimerasaRESUMEN
The rapid growth of the therapeutic protein market calls for more efficient purification methods. Various aggregating tags have recently emerged as simple, fast, cost-effective and column-free technologies for protein (and peptide) purification. In general, these column-free protein purification technologies involve the use of aggregating tags that induce the target protein into insoluble aggregates. These aggregates can be easily separated from soluble impurities and the target protein or peptide is then liberated by a cleavage process. This review summarizes the current state-of-the-art in using aggregating tags for protein purification. The methods are here categorized as follows: (1) tags that allow soluble expression of target protein in vivo and induce aggregation in vitro; (2) tags that induce soluble expression and self-assembling of target protein on insoluble biological polyester beads in vivo; (3) tags that induce formation of inactive aggregates in vivo; (4) tags that induce formation of active aggregates in vivo.
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Proteínas/química , Proteínas/aislamiento & purificación , Humanos , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/uso terapéutico , Agregado de Proteínas , Ingeniería de Proteínas/métodos , Proteínas/uso terapéutico , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/uso terapéuticoRESUMEN
BACKGROUND: Peptides have recently become attractive for therapeutic applications. However, efficient production of medium- to large-sized peptides (30-100 amino acids [aa]) remains challenging both by recombinant and chemical synthesis. We previously reported the formation of active enzyme aggregates in Escherichia coli cells induced by the short ß-structured peptide ELK16 (LELELKLKLELELKLK) and developed a streamlined protein expression and purification approach. In this approach, a cleavable self-aggregating tag (cSAT) consisting of an intein molecule and ELK16 was used to release the recombinant peptides with reasonable purity from active aggregates. RESULTS: In this work, we extended the cSAT approach to a generalized expression and purification solution for a set of medium- to large-sized peptides with important therapeutic uses, including human glucagon-like peptide 1 (31 aa), B-type natriuretic peptide (32 aa), exendin 4 (39 aa), chemokine (C-C motif) ligand 5 (also known as RANTES, 66 aa), stromal cell-derived factor 1α (67 aa), insulin-like growth factor 1 (70 aa), and leptin (146 aa). After intein-mediated cleavage, the soluble peptides were released directly into the supernatant while insoluble peptides could be refolded and purified by reverse phase high-performance liquid chromatography. Additionally, an N-terminal thioredoxin tag was added upstream of the target peptides, which can be removed by enterokinase cleavage, generating native N-terminus for target peptides. Final yields of the peptides ranged from 0.1 to 1.8 µg/mg wet cell weight at laboratory scale. CONCLUSIONS: The approach described in this study provides a fast and efficient route to express and purify peptides that are difficult or expensive to produce by chemical synthesis or by ordinary recombinant methods. It is particularly well suited for large peptides, peptides likely to be degraded, and peptides that have toxic effects on the host. It can greatly reduce the cost and time of downstream processing, and thus may be useful for both industrial manufacture and laboratory applications.
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Escherichia coli/genética , Microbiología Industrial/métodos , Biosíntesis de Péptidos , Péptidos/metabolismo , Proteínas Recombinantes/biosíntesis , Escherichia coli/enzimología , Escherichia coli/metabolismo , Humanos , Inteínas , Péptidos/genética , Péptidos/aislamiento & purificación , Agregado de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems.
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Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Marinobacter/enzimología , Ácido Oléico/metabolismo , Ingeniería de Proteínas , Transporte de Electrón , Ferredoxinas/metabolismo , Hidroxilación , Marinobacter/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , NADH NADPH Oxidorreductasas/metabolismoRESUMEN
BACKGROUND: In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. RESULTS: In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 µg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. CONCLUSIONS: The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with reasonable quantity and purity from active aggregates. Owing to the structural simplicity, strong hydrophobicity, and high aggregating efficiency, these peptides can be further explored for enzyme production and immobilization.
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Escherichia coli/metabolismo , Cuerpos de Inclusión/metabolismo , Péptidos/metabolismo , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Péptidos/química , Péptidos/genética , Ingeniería de Proteínas , Esterol Esterasa/genética , Esterol Esterasa/metabolismoRESUMEN
Cephalosporin C (CPC) acylase is important for the one-step production of 7-aminocephalosporanic acid (7-ACA), a key intermediate for cephalosporin antibiotics. However, its application is hampered by the low activity, substrate inhibition, and product inhibition. In this study, two rounds of combinatorial active-site saturation testing (CASTing) were carried out on the CPC acylase acyII from Pseudomonas SE83, and one mutant H57ßA/H70ßY with no substrate inhibition was obtained. For further engineering to reduce the product inhibition, a quick pH indicator assay was developed, allowing for real-time monitoring of the product inhibition in the presence of added 7-ACA. The utility of the assay was demonstrated by screening six libraries of site-directed saturation mutagenesis libraries of H57ßA/H70ßY. A new mutant H57ßA/H70ßY/I176ßN was obtained, which showed a k cat 3.26-fold and a K IP 3.08-fold that of the wild type, respectively. Given the commercial value of the enzyme, both this pH indicator assay and the triple mutant should be useful for further engineering of the enzyme to increase the specific activity and to decrease the product inhibition.
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Cefalosporinas/biosíntesis , Cefalosporinas/metabolismo , Penicilina Amidasa/genética , Penicilina Amidasa/metabolismo , Ingeniería de Proteínas , Dominio Catalítico , Cefalosporinas/química , Pruebas de Enzimas , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Penicilina Amidasa/química , Pseudomonas/enzimologíaRESUMEN
In industrial fermentation processes, microorganisms often encounter acid stress, which significantly impact their productivity. This study focused on the acid-resistant module composed of small RNA (sRNA) DsrA and the sRNA chaperone Hfq. Our previous study had shown that this module improved the cell growth of Escherichia coli MG1655 at low pH, but failed to obtain this desired phenotype in industrial strains. Here, we performed a quantitative analysis of DsrA-Hfq module to determine the optimal expression mode. We then assessed the potential of the CymR-based negative auto-regulation (NAR) circuit for industrial application, under different media, strains and pH levels. Growth assay at pH 4.5 revealed that NAR-05D04H circuit was the best acid-resistant circuit to improve the cell growth of E. coli MG1655. This circuit was robust and worked well in the industrial lysine-producing strain E. coli SCEcL3 at a starting pH of 6.8 and without pH control, resulting in a 250 % increase in lysine titer and comparable biomass in shaking flask fermentation compared to the parent strain. This study showed the practical application of NAR circuit in regulating DsrA-Hfq module, effectively and robustly improving the acid tolerance of industrial strains, which provides a new approach for breeding industrial strains with tolerance phenotype.
RESUMEN
Vast shotgun metagenomics data remain an underutilized resource for novel enzymes. Artificial intelligence (AI) has increasingly been applied to protein mining, but its conventional performance evaluation is interpolative in nature, and these trained models often struggle to extrapolate effectively when challenged with unknown data. In this study, we present a framework (DeepMineLys [deep mining of phage lysins from human microbiome]) based on the convolutional neural network (CNN) to identify phage lysins from three human microbiome datasets. When validated with an independent dataset, our method achieved an F1-score of 84.00%, surpassing existing methods by 20.84%. We expressed 16 lysin candidates from the top 100 sequences in E. coli, confirming 11 as active. The best one displayed an activity 6.2-fold that of lysozyme derived from hen egg white, establishing it as the most potent lysin from the human microbiome. Our study also underscores several important issues when applying AI to biology questions. This framework should be applicable for mining other proteins.
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Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR-Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.
Asunto(s)
Bacteriófagos , Bacteriófagos/genética , ARN , Transposasas/genética , Sistemas CRISPR-Cas , Genes Virales , Bacterias/genéticaRESUMEN
D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.