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Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168. The application of EcΔ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in EcΔ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of BsΔ44 was due to the deletion of the phage SPP1 surface receptor gene yueB, resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain BsΔyueB exhibited a 61.99% higher acetoin titer compared to strain Bs168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain BsΔyueB achieved an 80.63% higher titer than Bs168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for E. coli and B. subtilis, highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.
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The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.
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Apoptosis , Aterosclerosis , Células Espumosas , Canales Iónicos , Macrófagos , Fagocitosis , Animales , Canales Iónicos/metabolismo , Canales Iónicos/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , Ratones , Células Espumosas/metabolismo , Células Espumosas/patología , Humanos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Tiofenos/farmacología , Masculino , Especies Reactivas de Oxígeno/metabolismo , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/genética , Mitocondrias/metabolismo , Pirazinas , TiadiazolesRESUMEN
Coexpressing multiple identical single guide RNAs (sgRNAs) in CRISPR-dependent engineering triggers genetic instability and phenotype loss. To provide sgRNA derivatives for efficient DNA digestion, we design a high-throughput digestion-activity-dependent positive screening strategy and astonishingly obtain functional nonrepetitive sgRNA mutants with up to 48 out of the 61 nucleotides mutated, and these nonrepetitive mutants completely lose canonical secondary sgRNA structure in simulation. Cas9-sgRNA complexes containing these noncanonical sgRNAs maintain wild-type level of digestion activities in vivo, indicating that the Cas9 protein is compatible with or is able to adjust the secondary structure of sgRNAs. Using these noncanonical sgRNAs, we achieve multiplex genetic engineering for gene knockout and base editing in microbial cell factories. Libraries of strains with rewired metabolism are constructed, and overproducers of isobutanol or 1,3-propanediol are identified by biosensor-based fluorescence-activated cell sorting (FACS). This work sheds light on the remarkable flexibility of the secondary structure of functional sgRNA.
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Citometría de Flujo , ARN Guía de Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Citometría de Flujo/métodos , Sistemas CRISPR-Cas/genética , Mutación/genética , Conformación de Ácido Nucleico , Ensayos Analíticos de Alto Rendimiento/métodos , Butanoles/metabolismo , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Proteína 9 Asociada a CRISPR/genéticaRESUMEN
OBJECTIVE: It was previously believed that atherosclerotic (AS) plaque starts to develop from the intima and that intraplaque vasa vasorum (VV) hyperplasia promotes adventitial VV (AVV) hyperplasia. However, recent studies have shown that arterial AVV hyperplasia precedes early intimal thickening, suggesting its possible role as an initiating factor of AS. To provide further insight into this process, in this study, we examine the evolution of AAV and VV development in a preclinical model of early AS with longitudinal ultrasound imaging. METHODS: Models of early AS were established. Duplex ultrasound scanning and contrast-enhanced ultrasound were performed for diagnosis. Pearson correlation tests were used to analyze the relationships between AVV hyperplasia and VV hyperplasia, or between AVV hyperplasia and intima-media thickness (IMT). RESULTS: During 0-12 wk of high-fat feeding, AVV gradually increased and intima-media thickened gradually in the observation area; in the 2nd wk of high-fat feeding, the observation area showed obvious AVV proliferation; at the 4th wk, the intima-media membrane became thicker; at the 12th wk, early plaque formation and intraplaque VV proliferation were observed. There was a strong positive correlation between AVV proliferation and IMT thickening and a strong negative correlation between AVV proliferation and the change rate of vessel diameter. CONCLUSION: This study demonstrated that AVV proliferation in the arteries occurred earlier than IMT thickening and was positively correlated with IMT. At present, the indicators of ultrasonic diagnosis of AS, such as IMT, Intraplaque VV, Echo property, all appear in the advanced stage of AS. The AVV may be an innovative diagnostic target for the early stage of AS plaque.
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Modelos Animales de Enfermedad , Hiperplasia , Placa Aterosclerótica , Vasa Vasorum , Animales , Vasa Vasorum/diagnóstico por imagen , Vasa Vasorum/patología , Placa Aterosclerótica/diagnóstico por imagen , Conejos , Hiperplasia/diagnóstico por imagen , Masculino , Adventicia/diagnóstico por imagen , Adventicia/patología , Ultrasonografía/métodosRESUMEN
Native prokaryotic promoters share common sequence patterns, but are species dependent. For understudied species with limited data, it is challenging to predict the strength of existing promoters and generate novel promoters. Here, we developed PromoGen, a collection of nucleotide language models to generate species-specific functional promoters, across dozens of species in a data and parameter efficient way. Twenty-seven species-specific models in this collection were finetuned from the pretrained model which was trained on multi-species promoters. When systematically compared with native promoters, the Escherichia coli- and Bacillus subtilis-specific artificial PromoGen-generated promoters (PGPs) were demonstrated to hold all distribution patterns of native promoters. A regression model was developed to score generated either by PromoGen or by another competitive neural network, and the overall score of PGPs is higher. Encouraged by in silico analysis, we further experimentally characterized twenty-two B. subtilis PGPs, results showed that four of tested PGPs reached the strong promoter level while all were active. Furthermore, we developed a user-friendly website to generate species-specific promoters for 27 different species by PromoGen. This work presented an efficient deep-learning strategy for de novo species-specific promoter generation even with limited datasets, providing valuable promoter toolboxes especially for the metabolic engineering of understudied microorganisms.
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Bacillus subtilis , Aprendizaje Profundo , Escherichia coli , Regiones Promotoras Genéticas , Bacillus subtilis/genética , Escherichia coli/genética , Redes Neurales de la Computación , Especificidad de la EspecieRESUMEN
Nano-MoS2 exhibit oxidoreductase-like activities, and has been shown to effectively eliminate excessive intracellular ROS and inhibit Aß aggregation, thus demonstrating promising potential for anti-Alzheimer's disease (anti-AD) intervention. However, the low water dispersibility and high toxicity of nano-MoS2 limits its further application. In this study, we developed a chondroitin sulphate (CS)-modified MoS2 nanoenzyme (CS@MoS2) by harnessing the excellent biocompatibility of CS and the exceptional activities of nano-MoS2 to explore its potential in anti-AD research. Promisingly, CS@MoS2 significantly inhibited Aß1-40 aggregation and prevented toxic injury in SH-SY5Y cells caused by Aß1-40. In addition, CS@MoS2 protected these cells from oxidative stress damage by regulating ROS production, as well as promoting the activities of SOD and GSH-Px. CS@MoS2 also modulated the intracellular Ca2+ imbalance and downregulated Tau hyperphosphorylation by activating GSK-3ß. CS@MoS2 suppressed p-NF-κB (p65) translocation to the nucleus by inhibiting MAPK phosphorylation, and modulated the expression of downstream anti- and proinflammatory cytokines. Owing to its multifunctional activities, CS@MoS2 effectively improved spatial learning, memory, and anxiety in D-gal/AlCl3-induced AD mice. Taken together, these results indicate that CS@MoS2 has significant potential for improving the therapeutic efficacy of the prevention and treatment of AD, while also presenting a novel framework for the application of nanoenzymes.
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Enfermedad de Alzheimer , Sulfatos de Condroitina , Disulfuros , Molibdeno , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacología , Animales , Ratones , Humanos , Molibdeno/química , Molibdeno/farmacología , Disulfuros/química , Disulfuros/farmacología , Péptidos beta-Amiloides/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Oxidativo/efectos de los fármacos , Línea Celular Tumoral , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/química , Masculino , Modelos Animales de EnfermedadRESUMEN
OBJECTIVE: Arterial adventitial vasa vasorum (AVV) plays an important role in the occurrence and development of atherosclerotic (AS) disease. AS is a systemic disease, and plaque is not only a local vascular event, but also occurs at multiple sites throughout the vascular bed. Currently, effective anti-AVV therapies are lacking. Therefore, we posed the following scientific questions: "does human carotid adventitial vasa vasorum density reflect plaque neovascularization and intimal-media hyperplasia in carotid?"; and "is it possible to reduce human AVV density by sonodynamic therapy (SDT)?" METHODS: A retrospective study was conducted on 160 patients with carotid atherosclerosis. Duplex ultrasound scanning (DUS), contrast-enhanced ultrasound (CEUS), coronary angiography, and coronary CT angiography (CTA) were used for diagnosis and screening. Pearson correlation tests and Receiver operating characteristic (ROC) curve were used to analyze the relationships between AVV hyperplasia, vasa vasorum (VV) hyperplasia and the intima-media thickness (IMT). SDT was developed for the treatment of arterial AVV hyperplasia and AS plaques. RESULTS: The presence of local AVV in carotid unstable plaques correlated with the echogenic properties of the carotid plaque and the extent of plaque progression; Furthermore local AVV hyperplasia in patients with carotid atherosclerotic plaques was associated with acute coronary syndrome (ACS) events; Local AVV hyperplasia in patients with carotid atherosclerotic plaques was associated with coronary artery stenosis. Notably, SDT reduced local AVV hyperplasia and shrank the plaques in human femoral and carotid atherosclerotic lesions. CONCLUSIONS: The presence of AVV in human carotid arteries reflects the severity of carotid and coronary artery AS. Further, SDT can reduce the hyperplasia of local AVV in human femoral and carotid plaques.
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Aterosclerosis , Placa Aterosclerótica , Humanos , Placa Aterosclerótica/diagnóstico por imagen , Placa Aterosclerótica/patología , Estudios Retrospectivos , Vasa Vasorum/diagnóstico por imagen , Hiperplasia/patología , Grosor Intima-Media Carotídeo , Medios de ContrasteRESUMEN
BACKGROUND: Intra-plaque hemorrhage (IPH) leads to rapid plaque progression and instability through upregulation of matrix metalloproteinases (MMPs) and collagen degradation. Hemoglobin-derived hemin during IPH promotes plaque instability. We investigated whether hemin affects MMP overexpression in macrophages and explored the underlying mechanisms. MATERIAL AND METHODS: In vivo, hemorrhagic plaque models were established in rabbits and ApoE-/- mice. Ferrostatin-1 was used to inhibit ferroptosis. Plaque size, collagen, and MMP2/9 levels were evaluated using immunohistochemistry, H&E, Sirius Red, and Masson staining. In vitro, mouse peritoneal macrophages were extracted. Western blot and ELISA were used to measure MMP2/9 levels. Bioinformatics analysis investigated the association between MMPs and ferroptosis pathway genes. Macrophage ferroptosis was assessed by evaluating cell viability, lipid reactive oxygen species, mitochondrial ultrastructure, iron content, and COX2 levels after pretreatment with cell death inhibitors. Hemin's impact on ferroptosis and MMP expression was studied using Ferrostatin-1 and SB202190. RESULTS: In the rabbit hemorrhagic plaques, hemin deposition and overexpression of MMP2/9 were observed, particularly in macrophage-enriched regions. In vitro, hemin induced ferroptosis and MMP2/9 expression in macrophages. Ferrostatin-1 and SB202190 inhibited hemin-induced MMP2/9 overexpression. Ferrostatin-1 inhibited p38 phosphorylation in macrophages. Ferostatin-1 inhibits macrophage ferroptosis, reduces MMP2/9 levels in plaques, and stabilizes the hemorrhagic plaques. CONCLUSION: Our results suggested that hemin-induced macrophage ferroptosis promotes p38 pathway activation and MMP2/9 overexpression, which may play a crucial role in increasing hemorrhagic plaque vulnerability. These findings provide insights into the pathogenesis of hemorrhagic plaques and suggest that targeting macrophage ferroptosis may be a promising strategy for stabilizing vulnerable plaque.
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Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most popular flavors with wide applications in food, fragrance, and pharmaceutical industries. However, the high cost and limited yield of plant extraction failed to meet the vast market demand of natural vanillin. Vanillin biotechnology has emerged as a sustainable and cost-effective alternative to supply vanillin. In this review, we explored recent advances in vanillin biosynthesis and highlighted the potential of vanillin biotechnology. In particular, we addressed key challenges in using microorganisms and provided promising approaches for improving vanillin production with a special focus on chassis development, pathway construction and process optimization. Future directions of vanillin biosynthesis using inexpensive precursors are also thoroughly discussed.
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Benzaldehídos , Biotecnología , Benzaldehídos/metabolismoRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been regarded as a persistent challenge for the swine farms worldwide. microRNAs (miRNAs) play key roles in regulating almost every important biological process, including virus-host interaction. In this study, we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection. Subsequently, we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages (PAMs). Through bioinformatic analysis, we found that there existed a potential binding site of miR-204 on the 3'UTR of microtubule associated protein 1 light chain 3B (MAP1LC3B, LC3B), a hallmark of autophagy. Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay, we demonstrated that miR-204 directly targeted LC3B, thereby downregulating autophagy. Meanwhile, we investigated the interplay between autophagy and PRRSV replication in PAMs, confirming that PRRSV infection induces autophagy, which in turn facilitates viral replication. Overall, we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs. These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.
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MicroARNs , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Porcinos , Animales , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Síndrome Respiratorio y de la Reproducción Porcina/genética , Línea Celular , MicroARNs/genética , MicroARNs/metabolismo , Replicación Viral , Autofagia/genéticaRESUMEN
The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter (pAOX1). However, it is difficult to use P. pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination (HR) efficiency and the limitation of handy selective markers, especially in the condition of multistep biosynthetic pathways. Hence, we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker (HiEE-ReSM) to facilitate cell factory in P. pastoris. Firstly, we improved the HR rates of P. pastoris through knocking out the non-homologous-end-joining gene (Δku70) and overexpressing HR-related proteins (RAD52 and RAD59), resulting in higher positive rate compared to the basal strain, achieved 97%. Then, we used the uracil biosynthetic genes PpURA3 as the reverse screening marker, which can improve the recycling efficiency of marker. Meanwhile, the HR rate is still 100% in uracil auxotrophic yeast. Specially, we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter (scFUR4) to increase the uptake of exogenous uracil from medium. Meanwhile, we explored the optimal concentration of uracil (90 mg/L) for strain growth. In the end, the HiEE-ReSM system has been applied for the inositol production (250 mg/L) derived from methanol in P. pastoris. The systems will contribute to P. pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon (C1) bio-utilization.
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The UAG-based genetic code expansion (GCE) enables site-specific incorporation of noncanonical amino acids (ncAAs) harboring novel chemical functionalities in specific target proteins. However, most GCE studies were done in several whole-genome engineered chassis cells whose hundreds of UAG stop codons were systematically edited to UAA to avoid readthrough in protein synthesis in the presence of GCE. The huge workload of removing all UAG limited the application of GCE in other microbial cell factories (MCF) such as Bacillus subtilis, which has 607 genes ended with UAG among its 4245 coding genes. Although the 257 essential genes count only 6.1% of the genes in B. subtilis, they transcribe 12.2% of the mRNAs and express 52.1% of the proteins under the exponential phase. Here, we engineered a strain named Bs-22 in which all 22 engineerable UAG stop codons in essential genes were edited to UAA via CRISPR/Cas9-mediated multiple-site engineering to minimize the negative effect of GCE on the expression of essential genes. Besides the process of constructing GCE-compatible B. subtilis was systematically optimized. Compared with wild-type B. subtilis (Bs-WT), the fluorescence signal of the eGFP expression could enhance 2.25-fold in Bs-22, and the production of protein tsPurple containing l-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) was increased 2.31-fold in Bs-22. We verified that all purified tsPurple proteins from Bs-22 contained Cou, indicating the excellent fidelity of the strategy. This proof-of-concept study reported efficient overexpression of ncAA-rich proteins in MCF with minimized engineering, shedding new light on solving the trade-off between efficiency and workload.
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Aminoácidos , Bacillus subtilis , Aminoácidos/genética , Aminoácidos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Codón de Terminación , Proteínas/metabolismo , Biosíntesis de Proteínas/genéticaRESUMEN
Objective@#To understand the potential categories of harmful behaviors of college students in Wuling Mountain Area and its relationship with campus bullying, so as to provide a theoretical basis for promoting the development of college students physical and mental health.@*Methods@#The stratified random cluster sampling method was used to select 3 042 college students from six universities in Wuling Mountain Area from October to December, 2022. The "Chinese Youth Health Related/Risk Behaviors Questionnaire" (University Edition) compiled by the National Youth Health Related/Risk Behaviors Survey Group was used to investigate the health risk behaviors. The potential category analysis method was used to analyze the food preference, insecurity, fighting, loneliness, depression, insomnia, heartbreak, suicidal ideation, smoking, drinking, game addiction, Internet addiction of health risk behaviors were used to further analyze the relationship between different categories and campus bullying by using multi category Logistic regression method.@*Results@#College students in Wuling Mountain Area were classified into low risk group of category 1 (44.2%), category 2 (5.4%) substance dependent group, category 3(50.4%) emotional disorder group. The distribution of potential categories of health risk behaviors among college students was statistically significant by gender( χ 2=31.44, 11.69, P < 0.05 ). Logistic regression analysis showed that after controlling demographic variables, campus bullying was the risk factor of category 3 emotional disorder group( OR =1.88, P <0.01).@*Conclusion@#bullying. Colleges and universities should attach great importance to the occurrence of campus bullying, formulate intervention programs for different categories of health hazard behaviors, and promote the healthy development of college students physical and mental health.
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We study a minimal extension of the standard model by introducing three right-handed neutrinos and a new scotogenic scalar doublet, in which the mass splittings between neutral and charged components are responsible for the W-boson mass newly measured by the CDF Collaboration. This model can not only generate non-vanishing Majorana neutrino masses via the interaction of right-handed neutrinos and scotogenic scalars, but also explain the Universe's missing matter in the form of FIMP dark matter. We also study the influence of the mass splitting on the first order electroweak phase transition, and find that it can further enhance the transition strength and thus induce gravitational waves during the phase transition, which may be detected in the forthcoming detectors such as U-DECIGO.
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BACKGROUND: Ticagrelor provides more rapid, potent, and consistent anti-platelet efficacy than clopidogrel. This randomized trial aimed to evaluate the anti-inflammation effects of ticagrelor versus clopidogrel on thrombus aspirated from the ST-elevation myocardial infarction (STEMI) patients. METHOD: A total of 98 patients with STEMI and intended percutaneous coronary intervention (PCI) were randomly assigned to receive clopidogrel (600-mg loading dose) or ticagrelor (180-mg loading dose), of whom 55 with large thrombus burden underwent thrombus aspiration during PCI. Thrombus specimens were successfully aspirated from 49 patients. Finally, 24 patients in the clopidogrel group and 23 in the ticagrelor group completed the study. Inflammatory cells within thrombi were assessed by hematoxylin-eosin and immunohistochemistry stainings. RESULTS: Compared with the clopidogrel group, the number of total inflammatory cells per mm2 thrombus area in the ticagrelor group was decreased by 28% (P = 0.009). The numbers of neutrophils and myeloperoxidase-positive cells per mm2 thrombus area in the ticagrelor group were respectively decreased by 35% (P = 0.016) and 28% (P = 0.047), as compared with those in the clopidogrel group. Moreover, ticagrelor treatment reduced the ratio of monocytes number higher than 250 per mm2 thrombus area compared with clopidogrel treatment (4% versus 29%, P = 0.048). CONCLUSION: In patients with undergoing PCI for STEMI, the loading dose ticagrelor regimen was associated with a reduction in inflammatory cell infiltration within thrombus compared with the loading dose clopidogrel regimen.
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Intervención Coronaria Percutánea , Infarto del Miocardio con Elevación del ST , Trombosis , Clopidogrel/uso terapéutico , Humanos , Intervención Coronaria Percutánea/efectos adversos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Infarto del Miocardio con Elevación del ST/tratamiento farmacológico , Trombosis/etiología , Ticagrelor/uso terapéutico , Resultado del TratamientoRESUMEN
Bacillus subtilis is a versatile microbial cell factory that can produce valuable proteins and value-added chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable host strains. Herein, we develop an efficient CRISPR-Cas9 method for large-scale and scarless genome engineering in the Bacillus subtilis genome, which can delete up to 134.3 kb DNA fragments, 3.5 times as long as the previous report, with a positivity rate of 100%. The effects of using a heterologous NHEJ system, linear donor DNA, and various donor DNA length on the engineering efficiencies were also investigated. The CRISPR-Cas9 method was then utilized for Bacillus subtilis genome simplification and construction of a series of individual and cumulative deletion mutants, which are further screened for overproducer of isobutanol, a new generation biofuel. These results suggest that the method is a powerful genome engineering tool for constructing and screening engineered host strains with enhanced capabilities, highlighting the potential for synthetic biology and metabolic engineering.
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Bacillus subtilis , Edición Génica , Bacillus subtilis/genética , Biocombustibles , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Genoma Bacteriano , Ingeniería MetabólicaRESUMEN
Nonmodel microbes with unique and diverse metabolisms have become rising stars in synthetic biology; however, the lack of efficient gene engineering techniques still hinders their development. Recently, the use of base editors has emerged as a versatile method for gene engineering in a wide range of organisms including nonmodel microbes. This method is a fusion of impaired CRISPR/Cas9 nuclease and base deaminase, enabling the precise point mutation at the target without inducing homologous recombination. This review updates the latest advancement of base editors in microbes, including the conclusion of all microbes that have been researched by base editors, the introduction of newly developed base editors, and their applications. We provide a list that comprehensively concludes specific applications of BEs in nonmodel microbes, which play important roles in industrial, agricultural, and clinical fields. We also present some microbes in which BEs have not been fully established, in the hope that they are explored further and so that other microbial species can achieve arbitrary base conversions. The current obstacles facing BEs and solutions are put forward. Lastly, the highly efficient BEs and other developed versions for genome-wide reprogramming of cells are discussed, showing great potential for future engineering of nonmodel microbes.
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Native transcription factor-based biosensors (TFBs) have the potential for the in situ detection of value-added chemicals or byproducts. However, their industrial application is limited by their ligand promiscuity, low sensitivity, and narrow detection range. Alcohols exhibit similar structures, and no reported TFB can distinguish a specific alcohol from its analogues. Here, we engineered an alcohol-regulated transcription factor, BmoR, and obtained various mutants with remarkable properties. For example, the generated signal-molecule-specific BmoRs could distinguish the constitutional isomers n-butanol and isobutanol, with insensitivity up to an ethanol concentration of 800 mM (36.9 g/L). Linear detection of 0-60 mM of a specific higher alcohol could be achieved in the presence of up to 500 mM (23.0 g/L) ethanol as background noise. Furthermore, we obtained two mutants with raised outputs and over 107-fold higher sensitivity and one mutant with an increased upper detection limit (14.8 g/L n-butanol or isobutanol). Using BmoR as an example, this study systematically explored the ultimate detection limit of a TFB toward its small-molecule ligands, paving the way for in situ detection in biofuel and wine industries.
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1-Butanol , Técnicas Biosensibles , Biocombustibles , Butanoles , Etanol , Factores de Transcripción/genéticaRESUMEN
AIM: To prepare several novel microcapsules using chitosan (Cs) and Alginate (Alg) as coating materials, and nano-ZnO, nano-SiO2, nano-TiO2 as UV protective agents for improving UV resistance of Cry1Ac. METHODS: Microcapsules were prepared by the layer-by-layer (LbL) self-assembly technique and electrostatic adsorption. The morphologies were observed by scanning electron microscopy (SEM), and the stability under UV radiation was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bioassay. RESULTS: SEM showed that nano-ZnO and nano-TiO2 could be adsorbed on the negatively charged MC with the outermost layer being Alg, while nano-SiO2 could be adsorbed on the positively charged MC with Cs as the outermost layer. SDS-PAGE and bioassay showed that nano-ZnO and nano-SiO2 could provide effective UV protection after 8 h UV irradiation (p > 0.05), and nano-TiO2 could provide effective UV protection after 4 h UV irradiation (p > 0.05). CONCLUSION: The microcapsules loaded with nanoparticles provided excellent UV resistance for Cry1Ac.