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BACKGROUND: Microbial organisms hold significant potential for converting renewable substrates into valuable chemicals. Low pH fermentation in industrial settings offers key advantages, including reduced neutralizer usage and decreased wastewater generation, particularly in the production of amino acids and organic acids. Engineering acid-tolerant strains represents a viable strategy to enhance productivity in acidic environments. Synthetic biology provides dynamic regulatory tools, such as gene circuits, facilitating precise expression of acid resistance (AR) modules in a just-in-time and just-enough manner. RESULTS: In this study, we aimed to enhance the robustness and productivity of Escherichia coli, a workhorse for amino acid and organic acid production, in industrial fermentation under mild acidic conditions. We employed an Esa-type quorum sensing circuit to dynamically regulate the expression of an AR module (DsrA-Hfq) in a just-in-time and just-enough manner. Through careful engineering of the critical promoter PesaS and stepwise evaluation, we developed an optimal Esa-PBD(L) circuit that conferred upon an industrial E. coli strain SCEcL3 comparable lysine productivity and enhanced yield at pH 5.5 compared to the parent strain at pH 6.8. CONCLUSIONS: This study exemplifies the practical application of gene circuits in industrial environments, which present challenges far beyond those of well-controlled laboratory conditions.
Assuntos
Escherichia coli , Percepção de Quorum , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Redes Reguladoras de Genes , Fermentação , Microbiologia IndustrialRESUMO
This study introduces an improved Ski Climate Index (SCI) designed to assess skiing suitability in China by applying fuzzy logic. Using daily meteorological data from 733 weather stations for the periods 1961-1990 and 1991-2020, the study identifies significant changes in SCI distribution over time. Additionally, a coupled analysis is performed, integrating the SCI results with the distribution and spatial vitality of 389 ski resorts in China. This analysis provides a comprehensive understanding of the interplay between actual ski resources and the ongoing evolution of the skiing industry in China and three significant results:1) The snow module has a major impact on SCI distribution, while other non-snow natural elements, such as sunshine duration, wind speed, and thermal comfort, influence the overall SCI assessment less; 2) High SCI values are concentrated in Northwestern and Northeastern China, with increased ski climate resources being observed in Shaanxi-Gansu-Ningxia, Southwest Tibet, and Sichuan due to climate change and noticeable declines in the Southern regions of Northeast China.; 3) In terms of the distribution and vitality of ski resorts, the SCI also partially reflects the development of ski resorts. This skiing suitability model uses climate resources to offer valuable insights for key decision-making in resort development and operation, thereby supporting advancement of the ice-snow economy.
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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.
Assuntos
Bacteriófagos , Microbiota , Humanos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Mineração de Dados , Proteínas Virais/metabolismo , Redes Neurais de Computação , Animais , Muramidase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
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.
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Estabilidade Enzimática , Engenharia de Proteínas , Ruminococcus , Ruminococcus/enzimologia , Ruminococcus/genética , Engenharia de Proteínas/métodos , Peptídeos/química , Peptídeos/metabolismo , Carboidratos Epimerases/química , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Cinética , Modelos Moleculares , Frutose/metabolismo , Frutose/químicaRESUMO
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 Click , Peptídeo 1 Semelhante ao Glucagon , Peptídeo 1 Semelhante ao Glucagon/química , Humanos , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores dos Hormônios Gastrointestinais/química , Receptores dos Hormônios Gastrointestinais/metabolismo , Desenho de Fármacos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Polipeptídeo Inibidor Gástrico/química , Polipeptídeo Inibidor Gástrico/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistasRESUMO
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.
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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.
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Bacteriófagos , Bacteriófagos/genética , RNA , Transposases/genética , Sistemas CRISPR-Cas , Genes Virais , Bactérias/genéticaRESUMO
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 , Peptídeos , Humanos , Escherichia coli/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Inteínas , Processamento de Proteína Pós-Traducional , Cloreto de Sódio/metabolismoRESUMO
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 , Interferon Tipo I , Animais , Humanos , SARS-CoV-2 , Histona Desacetilases/genética , Interferon Tipo I/farmacologia , Peptídeo Hidrolases , Mamíferos , Endorribonucleases , TransativadoresRESUMO
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 Neurais de Computação , Proteínas , Proteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Descoberta de DrogasRESUMO
Mitigating unintended interferences between circuits and host cells is key to realize applications of synthetic regulatory systems both for bacteria and mammalian cells. Here, we demonstrated that growth burden and circuit dysregulation occurred in a concentration-dependent manner for specific transcription factors (CymR*/CymR) in E.coli, and direct negative feedback modules were able to control the concentration of CymR*/CymR, mitigate growth burden, and restore circuit functions. A quantitative design scheme was developed for circuits embedded with autorepression modules. Four key parameters were theoretically identified to determine the performance of autoregulated switches and were experimentally modified by fine-tuning promoter architectures and cooperativity. Using this strategy, we synthesized a number of switches and demonstrated its improvement of product titers and host growth controlling the complex deoxyviolacein biosynthesis pathway. Furthermore, we restored functions of a dysregulated multilayer NOR gate by integrating autorepression modules. Our work provides a blueprint for engineering host-adaptable synthetic systems.
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Escherichia coli , Redes Reguladoras de Genes , Escherichia coli/genética , Redes Reguladoras de Genes/genética , Engenharia Genética , Regiões Promotoras Genéticas/genética , Biologia Sintética , Fatores de Transcrição/genéticaRESUMO
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 , Fermentação , Concentração de Íons de Hidrogênio , Lisina/metabolismo , Engenharia Metabólica/métodosRESUMO
Synthetic genomics will advance our understanding of life and allow us to rebuild the genomes of industrial microorganisms for enhancing performances. Corynebacterium glutamicum, a Gram-positive bacterium, is an important industrial workhorse. However, its genome synthesis is impeded by the low efficiencies in DNA delivery and in genomic recombination/replacement. In the present study, we describe a genomic iterative replacement system based on RecET recombination for C. glutamicum, involving the successive integration of up to 10 kb DNA fragments obtained in vitro, and the transformants are selected by the alternative use of kanR and speR selectable markers. As a proof of concept, we systematically redesigned and replaced a 54.3 kb wild-type sequence of C. glutamicumATCC13032 with its 55.1 kb synthetic counterpart with several novel features, including decoupled genes, the standard PCRTags, and 20 loxPsym sites, which was for the first time incorporated into a bacterial genome. The resulting strain semi-synCG-A1 had a phenotype and fitness similar to the wild-type strain under various stress conditions. The stability of the synthetic genome region faithfully maintained over 100 generations of nonselective growth. Genomic deletions, inversions, and translocations occurred in the synthetic genome region upon induction of synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE), revealing potential genetic flexibility for C. glutamicum. This strategy can be used for the synthesis of a larger region of the genome and facilitate the endeavors for metabolic engineering and synthetic biology of C. glutamicum.
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Corynebacterium glutamicum , Corynebacterium glutamicum/metabolismo , Genoma Bacteriano/genética , Genômica , Engenharia Metabólica/métodos , Biologia SintéticaRESUMO
Efficient protein and peptide expression and purification technologies are highly needed in biotechnology, especially in light of the increasing number of proteins and peptides that are being exploited for therapeutic use, which are inherently difficult to produce via biological means. In this chapter, we describe a facile, reliable, and cost-effective peptide production and purification strategy based on short self-assembling peptides (e.g., L6KD (LLLLLLKD)) and a C-terminal cleavage intein (e.g., Mtu ΔI-CM). This cleavable self-aggregating tag (cSAT) scheme depends on the in vivo formation of aggregates of the fusion protein containing the target peptide, which is induced during the expression by the presence of the self-assembling peptide in the construct. After a simple separation of the aggregates by centrifugation, the purified target peptide with authentic N-terminus is released in solution by pH-induced intein self-cleavage. As an example, a yield of about 4.4 µg/mg wet cell pellet was obtained when the cSAT scheme was used for the expression and purification of the therapeutic peptide GLP-1. This strategy provides a viable approach for preparing peptides with authentic N-termini, especially those in the range of 30 ~ 100 amino acids in size that are typically unstable or susceptible to degradation in Escherichia coli.
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Inteínas , Peptídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos/química , Proteínas/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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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Expressão Gênica , Hormônio do Crescimento Humano/genética , Inteínas/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , DNA Girase/genética , DNA Girase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hormônio do Crescimento Humano/biossíntese , Hormônio do Crescimento Humano/isolamento & purificação , Humanos , Peptídeos/genética , Peptídeos/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.
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Proteínas de Bactérias/antagonistas & inibidores , Sistemas CRISPR-Cas , Edição de Genes , Genoma Bacteriano , Pseudomonas aeruginosa/genética , Proteínas de Bactérias/genética , Plasmídeos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismoRESUMO
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/fisiologia , Escherichia coli/fisiologia , Fator Proteico 1 do Hospedeiro/fisiologia , Pequeno RNA não Traduzido/fisiologia , Adaptação Fisiológica , Concentração de Íons de HidrogênioRESUMO
The tumor microenvironment is a complex ecosystem formed by distinct and interacting cell populations, and its composition is related to cancer prognosis and response to clinical treatment. In this study, we have taken the advantage of two single-cell RNA sequencing technologies (Smart-seq2 and DNBelab C4) to generate an atlas of 15,115 immune and nonimmune cells from primary tumors and hepatic metastases of 18 colorectal cancer (CRC) patients. We observed extensive changes in the proportions and functional states of T cells and B cells in tumor tissues, compared to those of paired non-tumor tissues. Importantly, we found that B cells from early CRC tumor were identified to be pre-B like expressing tumor suppressors, whereas B cells from advanced CRC tumors tended to be developed into plasma cells. We also identified the association of IgA+ IGLC2+ plasma cells with poor CRC prognosis, and demonstrated a significant interaction between B-cell and myeloid-cell signaling, and found CCL8+ cycling B cells/CCR5+ T-cell interactions as a potential antitumoral mechanism in advanced CRC tumors. Our results provide deeper insights into the immune infiltration within CRC, and a new perspective for the future research in immunotherapies for CRC.
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Neoplasias Colorretais/imunologia , Neoplasias Colorretais/fisiopatologia , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
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.
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Amidoidrolases/metabolismo , Biotecnologia/métodos , Enzimas Imobilizadas/metabolismo , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Proteínas Luminescentes/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Amidoidrolases/genética , Enzimas Imobilizadas/química , Humanos , Cadeias Pesadas de Imunoglobulinas/metabolismo , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Proteína Vermelha FluorescenteRESUMO
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.