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Cryptococcal infection of central nervous system commonly involves meningitis or meningoencephalitis, but rarely mimics inflammatory myelitis. We present short segment myelitis as a dominant manifestation caused by Cryptococcus neoformans in a patient with nephrotic syndrome under immunosuppressive therapy. This case report highlights Cryptococcus neoformans as a potential etiological factor for short segment myelitis in immunocompromised hosts.
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Criptococose , Cryptococcus neoformans , Mielite , Humanos , Mielite/microbiologia , Mielite/tratamento farmacológico , Mielite/diagnóstico , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Criptococose/diagnóstico , Cryptococcus neoformans/isolamento & purificação , Masculino , Hospedeiro Imunocomprometido , Pessoa de Meia-Idade , Síndrome Nefrótica/complicações , Imunossupressores/uso terapêutico , Imageamento por Ressonância MagnéticaRESUMO
BACKGROUND: Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation. RESULTS: This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively. CONCLUSION: This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.
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Escherichia coli , Fermentação , Glucose , Glicólise , Engenharia Metabólica , Escherichia coli/metabolismo , Escherichia coli/genética , Engenharia Metabólica/métodos , Glucose/metabolismo , Vias Biossintéticas , Regiões Promotoras Genéticas , MalatosRESUMO
BACKGROUND: Percutaneous coronary intervention (PCI) with primary stenting, which stands for stent implantation regardless of obtaining satisfactory results with balloon angioplasty, has superseded conventional plain old balloon angioplasty with provisional stenting. With drug-coated balloon (DCB), primary DCB angioplasty with provisional stenting has shown non-inferiority to primary stenting for de novo coronary small vessel disease. However, the long-term efficacy and safety of such a strategy to the primary stenting on clinical endpoints in de novo lesions without vessel diameter restrictions remain uncertain. STUDY DESIGN: The REC-CAGEFREE I is an investigator-initiated, multicenter, randomized, open-label trial aimed to enroll 2270 patients with acute or chronic coronary syndrome from 43 interventional cardiology centers in China to evaluate the non-inferiority of primary paclitaxel-coated balloons angioplasty to primary stenting for the treatment of de novo, non-complex lesions without vessel diameter restrictions. Patients who fulfill all the inclusion and exclusion criteria and have achieved a successful lesion pre-dilatation will be randomly assigned to the two arms in a 1:1 ratio. Protocol-guided DCB angioplasty and bailout stenting after unsatisfactory angioplasty are mandatory in the primary DCB angioplasty group. The second-generation sirolimus-eluting stent will be used as a bailout stent in the primary DCB angioplasty group and the treatment device in the primary stenting group. The primary endpoint is the incidence of Device-oriented Composite Endpoint (DoCE) within 24 months after randomization, including cardiac death, target vessel myocardial infarction, and clinically and physiologically indicated target lesion revascularization. DISCUSSION: The ongoing REC-CAGEFREE I trial is the first randomized trial with a clinical endpoint to assess the efficacy and safety of primary DCB angioplasty for the treatment of de novo, non-complex lesions without vessel diameter restrictions. If non-inferiority is shown, PCI with primary DCB angioplasty could be an alternative treatment option to primary stenting. TRIAL REGISTRATION: Registered on clinicaltrial.gov (NCT04561739).
Assuntos
Angioplastia Coronária com Balão , Cateteres Cardíacos , Fármacos Cardiovasculares , Materiais Revestidos Biocompatíveis , Doença da Artéria Coronariana , Paclitaxel , Humanos , Angioplastia Coronária com Balão/instrumentação , Angioplastia Coronária com Balão/efeitos adversos , Angioplastia Coronária com Balão/mortalidade , Resultado do Tratamento , Fármacos Cardiovasculares/administração & dosagem , Fármacos Cardiovasculares/efeitos adversos , China , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Doença da Artéria Coronariana/terapia , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/mortalidade , Fatores de Tempo , Feminino , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Stents , Idoso , Stents Farmacológicos , Estudos de Equivalência como Asunto , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Cetuximab resistance has been a major challenge for head and neck squamous cell carcinoma (HNSCC) patients receiving targeted therapy. However, the mechanism that causes cetuximab resistance, especially microRNA (miRNA) regulation, remains unclear. Growing evidence suggests that miRNAs may act as "nuclear activating miRNAs" for targeting promoter regions or enhancers related to target genes. This study elucidates a novel mechanism underlying cetuximab resistance in HNSCC involving the nuclear activation of KDM7A transcription via miR-451a. Herein, small RNA sequencing, quantitative real-time polymerase chain reaction (qRTâPCR) and fluorescence in situ hybridization (FISH) results provided compelling evidence of miR-451a nuclear enrichment in response to cetuximab treatment. Chromatin isolation via RNA purification, microarray analysis, and bioinformatic analysis revealed that miR-451a interacts with an enhancer region in KDM7A, activating its expression and further facilitating cetuximab resistance. It has also been demonstrated that the activation of KDM7A by nuclear miR-451a is induced by cetuximab treatment and is AGO2 dependent. Logistic regression analyses of 87 HNSCC samples indicated the significance of miR-451a and KDM7A in the development of cetuximab resistance. These discoveries support the potential of miR-451a and KDM7A as valuable biomarkers for cetuximab resistance and emphasize the function of nuclear-activating miRNAs.
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Cetuximab , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço , MicroRNAs , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Animais , Camundongos , Núcleo Celular/metabolismo , Núcleo Celular/genética , Feminino , Camundongos NusRESUMO
The spontaneous solid-state stacking process (SSSP) of Baijiu is an environmentally friendly and cost-effective process for enriching and assembling environmental microorganisms to guarantee the subsequent fermentation efficiency. In this study, how SSSP create spatial heterogeneity of stacking piles were found through spatiotemporal sampling. The degree of difficulty in oxygen exchange categorizes the stacking pile into depleted (≤4%), transitional (4 %-17 %), and enriched (≥17 %) oxygen-defined layers. This results in variation in succession rates (Vdepleted > Vtransitional > Venriched), which accelerates spatial heterogeneity during SSSP. As a dominant species (65 %-99 %) in depleted and transitional layers, Acetilactobacillus jinshanensis can rapidly reduce oxygen disturbance by upregulating poxL and catE, that sustains spatial heterogeneity. The findings demonstrated the value of oxygen control in shaping spatial heterogeneity during SSSP processes, which can create specific functional microbiome. Adding spatial heterogeneity management will help achieve more precise control of such solid-state fermentation systems.
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Fermentação , Oxigênio , Oxigênio/metabolismoRESUMO
Network estimation has been a critical component of high-dimensional data analysis and can provide an understanding of the underlying complex dependence structures. Among the existing studies, Gaussian graphical models have been highly popular. However, they still have limitations due to the homogeneous distribution assumption and the fact that they are only applicable to small-scale data. For example, cancers have various levels of unknown heterogeneity, and biological networks, which include thousands of molecular components, often differ across subgroups while also sharing some commonalities. In this article, we propose a new joint estimation approach for multiple networks with unknown sample heterogeneity, by decomposing the Gaussian graphical model (GGM) into a collection of sparse regression problems. A reparameterization technique and a composite minimax concave penalty are introduced to effectively accommodate the specific and common information across the networks of multiple subgroups, making the proposed estimator significantly advancing from the existing heterogeneity network analysis based on the regularized likelihood of GGM directly and enjoying scale-invariant, tuning-insensitive, and optimization convexity properties. The proposed analysis can be effectively realized using parallel computing. The estimation and selection consistency properties are rigorously established. The proposed approach allows the theoretical studies to focus on independent network estimation only and has the significant advantage of being both theoretically and computationally applicable to large-scale data. Extensive numerical experiments with simulated data and the TCGA breast cancer data demonstrate the prominent performance of the proposed approach in both subgroup and network identifications.
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Alcohol intake provokes severe organ injuries including alcoholic cardiomyopathy with hallmarks of cardiac remodeling and contractile defects. This study examined the toxicity of facilitated ethanol metabolism in alcoholism-evoked changes in myocardial morphology and contractile function, insulin signaling and various cell death domains using cardiac-selective overexpression of alcohol dehydrogenase (ADH). WT and ADH mice were offered an alcohol liquid diet for 12 weeks prior to assessment of cardiac geometry, function, ER stress, apoptosis and ferroptosis. Alcohol intake provoked pronounced glucose intolerance, cardiac remodeling and contractile anomalies with apoptosis, ER stress, and ferroptosis, the effects were accentuated by ADH with the exception of global glucose intolerance. Hearts from alcohol ingesting mice displayed dampened insulin-stimulated phosphorylation of insulin receptor (tyr1146) and IRS-1 (tyrosine) along with elevated IRS-1 serine phosphorylation, the effect was augmented by ADH. Alcohol challenge dampened phosphorylation of Akt and GSK-3ß, and increased phosphorylation of c-Jun and JNK, the effects were accentuated by ADH. Alcohol challenge promoted ER stress, FK506 binding protein 5 (FKBP5), YAP, apoptosis and ferroptosis, the effects were exaggerated by ADH. Using a short-term ethanol challenge model (3 g/kg, i.p., twice in three days), we found that inhibition of FKBP5-YAP signaling or facilitated ethanol detoxification by Alda-1 alleviated ethanol cardiotoxicity. In vitro study revealed that the ethanol metabolite acetaldehyde evoked cardiac contractile anomalies, lipid peroxidation, and apoptosis, the effects of which were mitigated by Alda-1, inhibition of ER stress, FKBP5 and YAP. These data suggest that facilitated ethanol metabolism via ADH exacerbates alcohol-evoked myocardial remodeling, functional defects, and insulin insensitivity possibly through a FKBP5-YAP-associated regulation of ER stress and ferroptosis.
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Alcoolismo , Ferroptose , Intolerância à Glucose , Proteínas de Ligação a Tacrolimo , Camundongos , Animais , Etanol/farmacologia , Álcool Desidrogenase/metabolismo , Álcool Desidrogenase/farmacologia , Intolerância à Glucose/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Remodelação Ventricular , Camundongos Transgênicos , Alcoolismo/complicações , Alcoolismo/metabolismo , Contração Miocárdica , Insulina/metabolismo , Miócitos Cardíacos/metabolismoRESUMO
Global concern exists regarding the contamination of food and animal feed with aflatoxin B1 (AFB1), which poses a threat to the health of both humans and animals. Previously, we found that a laccase from Bacillus subtilis (BsCotA) effectively detoxified AFB1 in a reaction mediated by methyl syringate (MS), although the underlying mechanism has not been determined. Therefore, our primary objective of this study was to explore the detoxification mechanism employed by BsCotA. First, the enzyme and mediator dependence of AFB1 transformation were studied using the BsCotA-MS system, which revealed the importance of MS radical formation during the oxidation process. Aflatoxin Q1 (AFQ1) resulting from the direct oxidation of AFB1 by BsCotA, was identified using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results of UPLC-MS/MS and density functional theory calculations indicated that the products included AFQ1, AFB1-, and AFD1-MS-coupled products in the BsCotA-MS system. The toxicity evaluations revealed that the substances derived from the transformation of AFB1 through the BsCotA-MS mechanism exhibited markedly reduced toxicity compared to AFB1. Finally, we proposed a set of different AFB1-transformation pathways generated by the BsCotA-MS system based on the identified products. These findings greatly enhance the understanding of the AFB1-transformation mechanism of the laccase-mediator system.
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Aflatoxina B1 , Ácido Gálico/análogos & derivados , Lacase , Humanos , Aflatoxina B1/toxicidade , Aflatoxina B1/química , Cromatografia Líquida , Espectrometria de Massas em TandemRESUMO
The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3 g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: ⢠Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. ⢠Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. ⢠Spraying cello-oligosaccharides promoted tomato growth.
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Celobiose , Celulase , Zea mays , Oligossacarídeos/química , FosforilasesRESUMO
The development of highly efficient photocatalysts for visible-light-driven degradation of organic pollution is of great interest for wastewater purification. In this work, a sulfur vacancy-rich (α/ß-CdS)/SiO2 (α: hexagonal & ß: cubic) photocatalyst with a high catalytic activity was novelly synthesized on a nano-SiO2 carrier by the reaction of Cd2+ with a CS2 storage material (CS2SM) as sulfur source and crystalline modifiers. The dispersion of α/ß-CdS on the nano-SiO2 carrier significantly enhanced the visible-light-driven catalytic activity of (α/ß-CdS)/SiO2 photocatalyst, and 93.37 % rhodamine B (RhB) conversion was determined over 50 mg (α/ß-CdS)/SiO2 photocatalyst for 30 mL 400 mg/L RhB solution at light intensity of 150 mW/cm2 and 298.15 K. After five cycle tests, the (α/ß-CdS)/SiO2 photocatalyst still owned excellent visible-light-driven catalytic degradation stability (>90 %). The characterizations of morphology, functional groups, and photo-electrochemistry of (α/ß-CdS)/SiO2 photocatalyst demonstrated that nano-SiO2 as a carrier played meaningful role in dispersing α/ß-CdS and reducing agglomeration, thus increasing the active site of photocatalytic degradation reaction, and the presence of α/ß hetero-phase junctions and sulfur vacancies allows the rapid separation of photo-generated carriers and inhibits photo-generated electron-holes recombination. Meanwhile, the electron paramagnetic resonance (EPR) and free radical masking test have also proved that the main active species is ·O2- for the oxidation of RhB. Therefore, the work is providing a new reference to the visible-light-driven degradation of wastewater with high RhB concentration at room temperature.
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Luz , Dióxido de Silício , Dióxido de Silício/química , Rodaminas , EnxofreRESUMO
INTRODUCTION: Liver fibrosis is a life-threatening pathological anomaly which usually evolves into advanced liver cirrhosis and hepatocellular carcinoma although limited therapeutic option is readily available. FUN14 domain containing 1 (FUNDC1) is a mitophagy receptor with little information in liver fibrosis. OBJECTIVE: This study was designed to examine the role for FUNDC1 in carbon tetrachloride (CCl4)-induced liver injury. METHODS: GEO database analysis and subsequent validation of biological processes including western blot, immunofluorescence, and co-immunoprecipitation were applied to clarify the regulatory role of FUNDC1 on mitophagy and ferroptosis. RESULTS: Our data revealed elevated FUNDC1 levels in liver tissues of patients with liver fibrotic injury and CCl4-challenged mice. FUNDC1 deletion protected against CCl4-induced hepatic anomalies in mice. Moreover, FUNDC1 deletion ameliorated CCl4-induced ferroptosis in vivo and in vitro. Mechanically, FUNDC1 interacted with glutathione peroxidase (GPx4), a selenoenzyme to neutralize lipid hydroperoxides and ferroptosis, via its 96-133 amino acid domain to facilitate GPx4 recruitment into mitochondria from cytoplasm. GPx4 entered mitochondria through mitochondrial protein import system-the translocase of outer membrane/translocase of inner membrane (TOM/TIM) complex, prior to degradation of GPx4 mainly through mitophagy along with ROS-induced damaged mitochondria, resulting in hepatocyte ferroptosis. CONCLUSION: Taken together, our data favored that FUNDC1 promoted hepatocyte injury through GPx4 binding to facilitate its mitochondrial translocation through TOM/TIM complex, where GPx4 was degraded by mitophagy to trigger ferroptosis. Targeting FUNDC1 may be a promising therapeutic approach for liver fibrosis.
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Ferroptose , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Mitofagia , Glutationa Peroxidase , Cirrose Hepática/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene (gadz11) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability, a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5'-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration, cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.
Assuntos
Escherichia coli , Glutamato de Sódio , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamato de Sódio/metabolismo , Fosfato de Piridoxal/metabolismo , Ácido gama-Aminobutírico , Glutamato Descarboxilase/genéticaRESUMO
OBJECTIVES: To develop a machine learning algorithm with prognosis data to identify different clinical phenotypes of biliary atresia (BA) and provide instructions for choosing treatment schemes. METHODS: Six hundred thirty-nine cases of type III BA were retrospectively collected from the Children's Hospital of Fudan University from Jan 1st, 2017 to Dec 1st, 2019 as a training dataset, and a survival-based forward clustering method, which can also be used to predict the subtype of a new patient was developed to identify BA subtypes. RESULTS: A total of 2 clusters were identified (cluster 1 = 324 and cluster 2 = 315), where cluster 2 had a lower 2 y native liver survival post-Kasai rate. The infant patients in cluster 2 have higher weight, liver, and spleen volume, wider portal vein width, and older operative age; worse coagulation and liver function results; higher grade of liver fibrosis and detection rate of hepatic portal fibrous mass, and higher recent infection detection rate of herpes simplex virus type I. With the proposed prognostic classification system, the authors predicted the subtypes of the 187 cases of type III BA in a testing dataset collected from the whole year of 2020. The p-value computed from the log-rank testing for the Kaplan-Meier survival curves of the predicted two testing groups was 0.0113. CONCLUSIONS: This classification system would be a convenient tool to choose appropriate treatment and accelerate the choice-making between clinicians and infant patients.
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Atrial fibrillation (AF) is a cardiovascular epidemic that occurs primarily in the elderly with primary cardiovascular diseases, leading to severe consequences such as stroke and heart failure. The heart is an energy-consuming organ, which requires a high degree of metabolic flexibility to ensure a quick switch of metabolic substrates to meet its energy needs in response to physiological and pathological stimulation. Metabolism is closely related to the occurrence of AF, and AF patients manifest metabolic inflexibility, such as insulin resistance and the metabolic shift from aerobic metabolism to anaerobic glycolysis. Moreover, our research group and the others have shown that metabolic inflexibility is a crucial pathologic mechanism for AF. Energy metabolism is closely linked to the aging process and aging-related diseases, and impaired metabolic flexibility is considered as an essential driver of aging. Therefore, this review focuses on the alteration of metabolic flexibility in the elderly and reveals that impaired metabolic flexibility may be an important driver for the high prevalence of AF in the elderly, hoping to provide intervention strategies for the prevention and treatment of AF in the elderly.
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Fibrilação Atrial , Insuficiência Cardíaca , Acidente Vascular Cerebral , Humanos , Idoso , Fibrilação Atrial/epidemiologia , Anticoagulantes , EnvelhecimentoRESUMO
BACKGROUND: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. RESULTS: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. CONCLUSIONS: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.
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Lacase , Sordariales , Lacase/genética , Lacase/metabolismo , Regiões 5' não Traduzidas/genética , Regiões Promotoras Genéticas , Sordariales/genética , Sordariales/metabolismoRESUMO
Capsaicinoids are mostly derived from chili peppers and have widespread applications in food, feed, and pharmacology. Compared with plant extraction, the use of microbial cell factories for capsaicinoids production is considered as a more efficient approach. Here, the biotransformation of renewable plant oil and vanillylamine into capsaicinoid nonivamide was investigated. Nonivamide biosynthesis using nonanoic acid and vanillylamine as substrates was achieved in Escherichia coli by heterologous expression of genes encoding amide-forming N-acyltransferase and CoA-ligase. Through increasing nonanoic acid tolerance of chassis cell, screening key enzymes involved in nonivamide biosynthesis and optimizing biotransformation conditions, the nonivamide titer reached 0.5 g/L. By further integrating a route for conversion of oleic acid to nonanoic acid, nonivamide biosynthesis was finally achieved using olive oil and vanillylamine as substrates, yielding a titer of approximately 10.7 mg/L. Results from this study provide valuable information for constructing highly efficient cell factories for the production of capsaicinoid compounds.
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Frutas , Óleos de Plantas , Óleos de Plantas/metabolismo , Biotransformação , Frutas/metabolismoRESUMO
BACKGROUND: A cost-effective pretreatment and saccharification process is a necessary prerequisite for utilizing lignocellulosic biomass (LCB) in biofuel and biomaterials production. Utilizing a multifunctional enzyme with both pretreatment and saccharification functions in a single step for simultaneous biological pretreatment and saccharification process (SPS) will be a green method of low cost and high efficiency. Manganese peroxidase (MnP, EC 1.11.1.13), a well-known lignin-degrading peroxidase, is generally preferred for the biological pretreatment of biomass. However, exploring the role and performance of MnP in LCB conversion will promote the application of MnP for lignocellulose-based biorefineries. RESULTS: In this study, we explored the ability of an MnP from Moniliophthora roreri, MrMnP, in LCB degradation. With Mn2+ and H2O2, MrMnP decomposed 5.0 g/L carboxymethyl cellulose to 0.14 mM of reducing sugar with a conversion yield of 5.0 mg/g, including 40 µM cellobiose, 70 µM cellotriose, 20 µM cellotetraose, and 10 µM cellohexaose, and degraded 1.0 g/L mannohexaose to 0.33 µM mannose, 4.08 µM mannotriose, and 4.35 µM mannopentaose. Meanwhile, MrMnP decomposed 5.0 g/L lichenan to 0.85 mM of reducing sugar with a conversion yield of 30.6 mg/g, including 10 µM cellotriose, 20 µM cellotetraose, and 80 µM cellohexose independently of Mn2+ and H2O2. Moreover, the versatility of MrMnP in LCB deconstruction was further verified by decomposing locust bean gum and wheat bran into reducing sugars with a conversion yield of 54.4 mg/g and 29.5 mg/g, respectively, including oligosaccharides such as di- and tri-saccharides. The catalytic mechanism underlying MrMnP degraded lignocellulose was proposed as that with H2O2, MrMnP oxidizes Mn2+ to Mn3+. Subsequently, it forms a complex with malonate, facilitating the degradation of CMC and mannohexaose into reducing sugars. Without H2O2, MrMnP directly oxidizes malonate to hydroperoxyl acetic acid radical to form compound I, which then attacks the glucosidic bond of lichenan. CONCLUSION: This study identified a new function of MrMnP in the hydrolysis of cellulose and hemicellulose, suggesting that MrMnP exhibits its versatility in the pretreatment and saccharification of LCB. The results will lead to an in-depth understanding of biocatalytic saccharification and contribute to forming new enzymatic systems for using lignocellulose resources to produce sustainable and economically viable products and the long-term development of biorefinery, thereby increasing the productivity of LCB as a green resource.
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Insulin-like growth factor-1 (IGF-1) is a pleiotropic protein hormone and has become an attractive therapeutic target because of its multiple roles in various physiological processes, including growth, development, and metabolism. However, its production is hindered by low heterogenous protein expression levels in various expression systems and hard to meet the needs of clinical and scientific research. Here, we report that human IGF-1 and its analog Long R3 IGF-1 (LR3 IGF-1) are recombinant expressed and produced in the Pichia pastoris (P. pastoris) expression system through being fused with highly expressed xylanase XynCDBFV. Furthermore, purified IGF-1 and LR3 IGF-1 display excellent bioactivity of cell proliferation compared to the standard IGF-1. Moreover, higher heterologous expression levels of the fusion proteins XynCDBFV-IGF-1 and XynCDBFV-LR3 IGF-1 are achieved by fermentation in a 15-L bioreactor, reaching up to about 0.5 g/L XynCDBFV-IGF-1 and 1 g/L XynCDBFV-TEV-LR3 IGF-1. Taken together, high recombinant expression of bioactive IGF-1 and LR3 IGF-1 is acquired with the assistance of xylanase as a fusion partner in P. pastoris, which could be used for both clinical and scientific applications. KEY POINTS: ⢠Human IGF-1 and LR3 IGF-1 are produced in the P. pastoris expression system. ⢠Purified IGF-1 and LR3 IGF-1 show bioactivity comparable to the standard IGF-1. ⢠High heterologous expression of IGF-1 and LR3 IGF-1 is achieved by fermentation in a bioreactor.
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Fator de Crescimento Insulin-Like I , Saccharomycetales , Humanos , Proteínas Recombinantes/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Pichia/genética , Pichia/metabolismo , Saccharomycetales/metabolismoRESUMO
In contrast to O2, H2O2 as the cosubstrate for lytic polysaccharide monooxygenases (LPMOs) exhibits great advantages in industrial settings for cellulose degradation. However, H2O2-driven LPMO reactions from natural microorganisms have not been fully explored and understood. Herein, secretome analysis unraveled the H2O2-driven LPMO reaction in the efficient lignocellulose-degrading fungus Irpex lacteus, including LPMOs with different oxidative regioselectivities and various H2O2-generating oxidases. Biochemical characterization of H2O2-driven LPMO catalysis showed orders of magnitude improvement in catalytic efficiency compared to that of O2-driven LPMO catalysis for cellulose degradation. Significantly, H2O2 tolerance of LPMO catalysis in I. lacteus was an order of magnitude higher than that in other filamentous fungi. In addition, natural reductants, gallic acid, in particular, presented in lignocellulosic biomass could sufficiently maintain LPMO catalytic reactions. Moreover, the H2O2-driven LPMO catalysis exhibited synergy with canonical endoglucanases for efficient cellulose degradation. Taken together, these findings demonstrate the great application potential of the H2O2-driven LPMO catalysis for upgrading cellulase cocktails to further improve cellulose degradation efficiency.
Assuntos
Basidiomycota , Polyporales , Peróxido de Hidrogênio/metabolismo , Polissacarídeos/metabolismo , Polyporales/metabolismo , Oxigenases de Função Mista/metabolismo , Basidiomycota/metabolismoRESUMO
The self-cleavage properties of proteases result in low activity and instability, which limit their industrial application. In this study, the serine protease ThAPT3 from Torrubiella hemipterigena was successfully expressed in Komagataella phaffii. We investigated the self-degradation mechanism of ThAPT3 and presented a rational strategy to alleviate self-cleavage. A major self-degradation site (Leu238-Met239) and a primary autolysis region were identified. The autolysis regions (loop18, α8-helix, and loop19) were redesigned and optimized using loop transplantation, energy calculations, surface cavity optimization, and loop anchoring. A triple-superposition mutant, ThAPT3-M9 (M239GKDGAVAAGLC250 â M239TLNRTTAANAC250/A251E/A254Q/R259L/A267E/S280N), was obtained. Compared to the wild type, the autolysis of M9 was significantly alleviated, and its half-life at 60 °C was increased approximately 39-fold (from 1.6 to 62.4 min). The optimal temperature and specific activity of M9 increased by 5 °C (from 60 to 65 °C) and 62% (4985 vs 3078 U/mg), respectively. M9 showed significant advantages in shrimp shell deproteinization.