RESUMO
BACKGROUND: Nematodes are parasitic animals that cause over 100 billion US dollars loss in agricultural business. The whole-genomes of two Streptomyces strains, Streptomyces spectabilis KCTC9218T and Streptomyces sp. AN091965, were sequenced. Both strains produce spectinabilin, an antinematode drug. Its secondary metabolism was examined to aid the development of an efficient nematicidal drug-producing host strain. RESULTS: The whole-genome sequences of S. spectabilis KCTC9218T and Streptomyces sp. AN091965 were analyzed using PacBio and Illumina sequencing platforms, and assembled using hybrid methodology. The total contig lengths for KCTC9218T and AN091965 were 9.97 Mb and 9.84 Mb, respectively. A total of 8,374 and 8,054 protein-coding genes, as well as 39 and 45 secondary metabolite biosynthetic gene clusters were identified in KCTC9218T and AN091965, respectively. 18.4 ± 6.45 mg/L and 213.89 ± 21.30 mg/L of spectinabilin were produced by S. spectabilis KCTC9218T and Streptomyces sp. AN091965, respectively. Pine wilt disease caused by nematode was successfully prevented by lower concentration of spectinabilin injection than that of abamectin recommended by its manufacturer. Production of multiple antinematode drugs, including spectinabilin, streptorubin B, and undecylprodigiosin was observed in both strains using high-resolution liquid chromatography mass spectrometry (LC-MS) analysis. CONCLUSIONS: Whole-genome sequencing of spectinabilin-producing strains, coupled with bioinformatics and mass spectrometry analyses, revealed the production of multiple nematicidal drugs in the KCTC9218T and AN091965 strains. Especially, Streptomyces sp. AN091965 showed high production level of spectinabilin, and this study provides crucial information for the development of potential nematicidal drug producers.
Assuntos
Antinematódeos , Metabolismo Secundário , Streptomyces , Animais , Antinematódeos/farmacologia , Família Multigênica , Nematoides/efeitos dos fármacos , Análise de Sequência de DNA , Streptomyces/genética , Streptomyces/metabolismo , Sequenciamento Completo do GenomaRESUMO
Mevalonate is an important platform compound for the biosynthesis of isoprenoids. It can be synthesized from acetyl-CoA in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) by the introduced mvaES operon in Escherichia coli. The influences of E. coli hosts, acetyl-CoA supply, and NADPH availability were assessed and engineered to improve the production titer and yield of mevalonate from glycerol. As a result, E. coli DH5α was found to be the best host with high specific capability and titer of mevalonate from glycerol. Through the engineering of phosphoketolase-phosphotransacetylase (xPK-PTA) bypass and NADPH availability, a final titer of 7.21 g/L with a specific capability of 1.36 g/g dry cell weight was gained in flask culture. Our work could offer new information to metabolically engineer the mevalonate pathway for the efficient production of isoprenoids.
Assuntos
Escherichia coli , Ácido Mevalônico , Acetilcoenzima A/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicerol/metabolismo , Engenharia Metabólica , Ácido Mevalônico/metabolismo , NADP/metabolismo , Terpenos/metabolismoRESUMO
A Gram-stain positive, aerobic, irregularly rod-shaped, non-spore-forming bacterium, designated as BN130099T, was isolated from farmland soil sampled in Goesan-gun, Chungbuk, Republic of Korea. Phylogenetic analysis of its 16S rRNA gene sequence showed that the strain is closely related to Nocardioides pelophilus KACC 19192T with 98.11â% similarity. The DNA G+C content of strain BN130099T was 68.84 mol% (draft genome sequence). The genome sequence of BN130099T displayed key enzymes involved in bioremediation of organic pollutants and biosynthetic clusters of saquayamycin. The strain contained ll-2,6-diaminopimelic acid in the cell-wall peptidoglycan and MK-8(H4) as the major respiratory quinone. The predominant fatty acid was iso-C16â:â0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. The results of physiological and biochemical characterization allowed the phenotypic differentiation of strain BN130099T from N. pelophilus KACC 19192T. The strain represents a novel species of the genus Nocardioides, for which we propose the name Nocardioides humilatus sp. nov. The type strain is BN130099T (=KCTC 49079T=CCTCC AB 2018135T).
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Nocardioides/classificação , Filogenia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Fazendas , Ácidos Graxos/química , Nocardioides/isolamento & purificação , Fosfolipídeos/química , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
A light pink coloured bacterium, designated strain BN140002T, was isolated from a soil sample collected in Goesan-gun, Chungcheongbuk-do, Republic of Korea. Cells of strain BN140002T were Gram-stain-negative, aerobic, motile and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed 94.7, 94.7, 93.9, 93.3, 93.4 and 93.0% similarities to Salinarimonas rosea KCTC 22346T, Salinarimonas ramus DSM 22962T, Saliniramus fredricksonii HL-109T, Microvirga soli R491T, Chelatococcus caeni EBR-4-1T and Chelatococcus composti PC-2T, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylethanolamine. The major cellular fatty acids were summed feature 8 (C18â:â1 ω7c and/or C18â:â1 ω6c) and summed feature 1 (C12â:â0 aldehyde and/or unknown 10.98) and the predominant ubiquinone was Q-10. The genomic DNA G+C content of strain BN140002T was 70.1 mol%. The genomic orthoANI values between strain BN140002T and Salinarimonas rosea KCTC 22346T and Salinarimonas ramus DSM 22962T were 75.0 and 74.8â%, respectively. Strain BN140002T had a class I-C type CRISPR-Cas system (CRISPR-associated helicase Cas3, CRISPR-associated protein Cas8c, CRISPR-associated protein Cas7, CRISPR-associated RecB family exonuclease Cas4, CRISPR-associated protein 1, 2). Based on phenotypic, chemotaxonomic and phylogenetic data, strain BN140002T should be assigned as a novel species of the genus Salinarimonas, for which the name Salinarimonas soli sp. nov. is proposed. The type strain is BN140002T (=KCTC 42643T=CCTCC AB 2017173T).
Assuntos
Alphaproteobacteria/classificação , Filogenia , Microbiologia do Solo , Alphaproteobacteria/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Fosfolipídeos/química , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Ubiquinona/análogos & derivados , Ubiquinona/químicaRESUMO
BACKGROUND AND PURPOSE: Although it is not recognized as essential to test for antiphospholipid antibody (aPL) in stroke of unknown cause, aPL-related stroke may account for a considerable number of cryptogenic strokes. We aimed to assess the current status and diagnostic value of aPL testing in cryptogenic stroke patients. METHODS: Consecutive patients admitted with acute ischemic stroke were examined to confirm the factors associated with performing aPL testing and with positive aPL test results in real-world practice. Cryptogenic stroke patients were separately examined in the same manner. The antibody profiles of cryptogenic stroke patients with aPL positivity were compared by age. RESULTS: Among 2947 patients, 606 (20.6%) were tested for aPLs and 129 (21.3%) were positive. Physicians tended to perform aPL testing in patients aged <50 years and in cryptogenic stroke patients. Cryptogenic stroke was a strong predictor of positive aPL results (adjusted odds ratio 3.70, 95% confidence interval 2.38-5.76). However, aPL positivity did not differ by age in stroke patients. Among 283 cryptogenic stroke patients, 136 (48.1%) were tested for aPLs and 56 (41.2%) were positive. aPL tests were performed predominantly in patients aged <50 years rather than in older patients, even among cryptogenic stroke patients. The two age groups had similar positivity rates of >40% (<50 years: 43.2%; ≥50 years: 40.4%; p = 0.92) and their antibody profiles were similar. CONCLUSIONS: A significant number of patients with cryptogenic stroke had positive aPL results regardless of age. aPL testing may offer additional diagnostic opportunities in cryptogenic stroke patients, and thus may reduce the incidence of cryptogenic stroke.
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Síndrome Antifosfolipídica , AVC Isquêmico , Acidente Vascular Cerebral , Idoso , Anticorpos Antifosfolipídeos , Síndrome Antifosfolipídica/complicações , Síndrome Antifosfolipídica/diagnóstico , Humanos , Pessoa de Meia-Idade , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologiaRESUMO
PURPOSE: The aim of this study is to investigate the effect of gradual dipyridamole titration and the incidence of dipyridamole-induced headache in patients with ischemic stroke or transient ischemic attack (TIA). METHODS: A randomized, double-blind, double-placebo, parallel group, phase 4 clinical trial (KCT0005457) was conducted between July 1, 2019, and February 25, 2020, at 15 medical centers in South Korea. The study included patients aged >19 years diagnosed with a noncardioembolic ischemic stroke or TIA within the previous 3 weeks. The participants were randomized 1:1:1 to receive Adinox® (aspirin 25 mg/dipyridamole 200 mg) and aspirin (100 mg) once daily for the first 2 weeks followed by Adinox® twice daily for 2 weeks (titration group), Adinox® twice daily for 4 weeks (standard group), and aspirin 100 mg once daily for 4 weeks (control group). The primary endpoint was incidence of headache over 4 weeks. The key secondary endpoint was mean cumulative headache. RESULTS: Ninety-six patients were randomized into the titration (n = 31), standard (n = 32), and control (n = 33) groups. The titration and standard groups (74.1% vs. 74.2%, respectively) showed no difference in the primary endpoint. However, the mean cumulated headache was significantly lower in the titration group than in the standard group (0.31 ± 0.46 vs. 0.58 ± 0.51, p = 0.023). Further, adverse drug reactions were more common in the standard group than in the titration group (28.1% vs. 9.7%, respectively, p = 0.054), although not significantly different. CONCLUSION: The titration strategy was effective in lowering the incidence of cumulative dipyridamole-induced headache.
Assuntos
Ataque Isquêmico Transitório , AVC Isquêmico , Acidente Vascular Cerebral , Aspirina/efeitos adversos , Dipiridamol/efeitos adversos , Método Duplo-Cego , Quimioterapia Combinada , Cefaleia/induzido quimicamente , Cefaleia/diagnóstico , Cefaleia/tratamento farmacológico , Humanos , Ataque Isquêmico Transitório/diagnóstico , Ataque Isquêmico Transitório/tratamento farmacológico , Inibidores da Agregação Plaquetária , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic has led to changes in stroke patients' healthcare use. This study evaluated changes in Korean stroke patients' health-seeking behaviors and stroke care services using data from the Korean Stroke Registry (KSR). METHODS: We reviewed data from patients with acute stroke and transient ischemic attack (TIA) during 2019 (before COVID-19 period) and 2020 (COVID-19 period). Outcomes included patient characteristics, time from stroke onset to hospital arrival, and in-hospital stroke pathways. Subgroup analyses were performed for an epidemic region (Daegu city and Gyeongsangbuk-do region, the D-G region). RESULTS: The study included 1,792 patients from the pre-COVID-19 period and 1,555 patients from the COVID-19 period who visited hospitals that contribute to the KSR. During the COVID-19 period, the D-G region had two-thirds the number of cases (vs. the pre-CO-VID-19 period) and a significant decrease in the proportion of patients with TIA (9.97%-2.91%). Unlike other regions, the median onset-to-door time increased significantly in the D-G region (361 min vs. 526.5 min, p = 0.016), and longer onset-to-door times were common for patients with mild symptoms and who were in their 60s or 70s. The number of patients who underwent intravenous thrombolysis also decreased during the COVID-19 period, although the treatment times were not significantly different between the 2 periods. DISCUSSION/CONCLUSION: Korean stroke patients in a CO-VID-19 epidemic region exhibited distinct changes in health-seeking behaviors. Appropriate triage system and public education regarding the importance of early treatment are needed during the COVID-19 pandemic.
Assuntos
COVID-19 , Acidente Vascular Cerebral , Humanos , Pandemias , Aceitação pelo Paciente de Cuidados de Saúde , Sistema de Registros , República da Coreia/epidemiologia , Estudos Retrospectivos , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/terapiaRESUMO
BACKGROUND: Leukotriene receptor antagonists are recommended to treat asthma and allergic rhinitis. Although they had been used for a long time, recent studies have reported neuropsychiatric adverse drug reactions are associated with montelukast. OBJECTIVE: This study analyzed the adverse drug reactions of montelukast and pranlukast, which are the two most frequently prescribed leukotriene receptor antagonists, respectively in Korea. METHODS: This study retrospectively reviewed ADRs of 5,426 montelukast and 1,146 pranlukast reported in the Korea Adverse Event Reporting System between January 2014 and December 2018. RESULTS: When both drugs are classified by system organ class, the most adverse drug reactions were related to the gastro-intestinal system, followed by psychiatric events. The reported adverse drug reactions for both drugs were more common in women, and the ratio of adverse drug reactions to prescriptions was highest in the elderly. Women aged 19 to 64 years reported more than twice as many adverse drug reactions than men of the same age, and more than 5 times in insomnia. CONCLUSIONS: When prescribing montelukast and pranlukast, attention would need to digestive and sleep disorders, especially women aged 19 to 64. After prescribing montelukast, physicians would need to pay more attention to agitation (5/396378 vs 0/82475), bad or vivid dreams (6/396378 vs 0/82475), anxiety (11/396378 vs 0/82475), depression (14/396378 vs 1/82475), tremor (53/396378 vs 7/82475), irritability (5/396378 vs 1/82475), insomnia (159/396378 vs 25/82475), and headache (68/396378 vs 10/82475), compared to when prescribing pranlukast. Further prospective research needs to elucidate the relationship between neuropsychiatric events and montelukast.
RESUMO
BACKGROUND: Until recently, it is generally considered safe to perform magnetic resonance imaging (MRI) in patients with an intrathecal drug administration system (ITDAS) device. In this study, we presented a case of morphine overdose due to ITDAS malfunction during MRI evaluation for the diagnosis of stroke. CASE PRESENTATION: A 58-year-old woman was referred to the emergency department for left-sided hemiparesis and dysarthria. She had undergone ITDAS implantation 4 years ago because of intractable back pain. Her brain MRI examination did not show any abnormalities except an old hemorrhagic infarction in the right basal ganglia. After MRI was performed, her symptoms completely resolved. Approximately 3 h after the MRI scan, the patient showed progressive stuporous consciousness and decreased respiration with decreased peripheral oxygen saturation of 80%. Initial arterial blood gas analysis revealed respiratory acidosis with hypoxia and hypercapnia. We suspected the opioid overdose for her unconciousness, small and sluggish pupils, and slow respiration. The patient regained consciousness within 3 min after the administration of naloxone with severe anxiety and irritability, without any respiratory symptoms or focal neurological deficits. In the pump interrogation and actual reservoir checks performed 6 h after the MRI scan, there was no significant difference between the expected reservoir volume and actual reservoir volume. Follow-up MRI performed to rule out posterior circulation infarction showed no structural lesions. The patient was eventually discharged without further neurologic or functional deterioration, with diagnosis of transient ischemia attack for initial symptoms of focal neurologic deficits. CONCLUSION: Although both ex vivo and in vivo studies have provided evidence that ITDAS devices are MRI-compatible, the pump is made of titanium and has ferromagnetic components. Since misdiagnosis of overinfusion could lead to mortality, early awareness of overinfusion of the intrathecal drug is needed to all clinicians in case of performing MRI in ITDAS implanted patients.
Assuntos
Morfina/efeitos adversos , Naloxona/administração & dosagem , Acidente Vascular Cerebral/diagnóstico , Feminino , Humanos , Imageamento por Ressonância Magnética , Pessoa de Meia-Idade , Morfina/administração & dosagem , Dor Intratável/tratamento farmacológicoRESUMO
A Gram-positive, aerobic, rod-shaped, non-spore-forming bacterium, designated as BN140041T, was isolated from cave soil at Gubyeongsan Mountain, Boeun-gun, Chungbuk province in Republic of Korea. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain is closely related to Nocardioides silvaticus S-34 T, N. pelophilus THG-T63T, and N. immobilis FLL521T with 97.4%, 97.1%, and 96.8% similarity. The draft genome length was 4.27 Mb containing 424 contigs with a DNA G + C content of 70.5 mol%. The ANI value between strain BN140044T and its closely related species N. silvaticus S-34 T was 82.6%. The genome sequence of BN140041T displayed a key enzyme involved in the bioremediation of organic pollutants. The diagnostic diamino acid of peptidoglycan was LL-2,6-diaminopimelic acid. The major respiratory quinone was MK-8(H4), and the major fatty acids (> 5% of the total fatty acids) were iso-C16:0 (55.3%), C18:1ω9c (7.7%) and iso-C17:0 (5.7%). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and phosphatidylinositol. The results of genotypical, physiological, and biochemical characterization allow the phenotypic differentiation of strain BN140041T from related the Nocardioides strains. Therefore, strain BN140041T represents a novel species of the genus Nocardioides, for which we propose the name Nocardioides antri sp. nov. The type strain is BN140041T (= KCTC 49080 T = CCTCC AB 2018226 T).
Assuntos
Actinomycetales , Actinomycetales/genética , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Ácidos Graxos , Nocardioides , Fosfolipídeos , Filogenia , RNA Ribossômico 16S/genética , República da Coreia , Análise de Sequência de DNA , Solo , Microbiologia do SoloRESUMO
This study evaluated the potential of iron oxide nanoparticle-loaded human embryonic stem cell (ESC)-derived spherical neural masses (SNMs) to improve the transportation of stem cells to the brain, ameliorate brain damage from intracerebral hemorrhage (ICH), and recover the functional status after ICH under an external magnetic field of a magnet attached to a helmet. At 24 h after induction of ICH, rats were randomly separated into three experimental groups: ICH with injection of phosphate-buffered saline (PBS group), ICH with intravenous injection of magnetosome-like ferrimagnetic iron oxide nanocubes (FION)-labeled SNMs (SNMs* group), and ICH with intravenous injection of FION-labeled SNMs followed by three days of external magnetic field exposure for targeted delivery by a magnet-embedded helmet (SNMs*+Helmet group). On day 3 after ICH induction, an increased Prussian blue-stained area and decreased swelling volume were observed in the SNMs*+Helmet group compared with that of the other groups. A significantly decreased recruitment of macrophages and neutrophils and a downregulation of pro-inflammatory cytokines followed by improved neurological function three days after ICH were observed in the SNMs*+Helmet group. Hemispheric atrophy at six weeks after ICH was significantly decreased in the SNMs*+Helmet group compared with that of the PBS group. In conclusion, we have developed a targeted delivery system using FION tagged to stem cells and a magnet-embedded helmet. The targeted delivery of SNMs might have the potential for developing novel therapeutic strategies for ICH.
Assuntos
Encéfalo/efeitos dos fármacos , Hemorragia Cerebral/tratamento farmacológico , Células-Tronco Embrionárias Humanas/metabolismo , Magnetoterapia/métodos , Nanopartículas Magnéticas de Óxido de Ferro/química , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Escala de Avaliação Comportamental , Encéfalo/patologia , Encéfalo/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Hemorragia Cerebral/radioterapia , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/radioterapia , Injeções Intravenosas , Masculino , Células-Tronco Neurais/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/fisiologia , Esferoides Celulares/metabolismoRESUMO
BackgroundThere are few data on the relationship between acute hypersensitivity reactions and the dose and injection rate of iodinated contrast material for CT.PurposeTo determine the relationship between lower dose and injection speed of iodinated contrast material for CT and the rate of acute hypersensitivity reactions.Materials and MethodsThis retrospective study included adults (age ≥18 years) undergoing nonionic iodinated contrast material-enhanced abdominal CT between August 2016 and January 2017 (control period) and between August 2017 and January 2018 (intervention period); all examinations were conducted in an outpatient setting. Compared with CT during the control period, CT during the intervention period involved a reduced dose of contrast material achieved by lowering the CT tube voltage. CT examinations in the control period were performed with 120 kVp, a contrast material dose of 2 mL/kg (maximum, 150 mL), and an injection speed of 3 or 4 mL/sec. CT examinations in the intervention period were performed with 100 kVp, a contrast material dose of 1.5 mL/kg (maximum, 130 mL), and an injection speed of 2.5 or 3 mL/sec. Per-examination rates of acute hypersensitivity reactions to iodinated contrast material were compared between the control and intervention periods with use of a multivariable Poisson regression model, the parameters of which were estimated by using generalized estimating equations with an independence correlation structure.ResultsA total of 21947 adults (mean age ± standard deviation, 59 years ± 12; 8797 women [40%]) underwent 25119 CT examinations during the control period; 23019 adults (mean age, 59 years ± 12; 9538 women [41%]) underwent 26491 CT examinations during the intervention period. The rate of acute hypersensitivity reactions was 1.42% (376 of 26491 examinations; 95% confidence interval [CI]: 1.28%, 1.57%) in the intervention period and 1.86% (468 of 25119 examinations; 95% CI: 1.70%, 2.04%) in the control period, with a multivariable-adjusted relative risk of 0.85 (95% CI: 0.74, 0.99; P = .03).ConclusionReduction in the dose and injection speed of iodinated contrast material for CT was associated with a lower rate of acute hypersensitivity reactions to iodinated contrast material.© RSNA, 2019Online supplemental material is available for this article.
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Meios de Contraste/administração & dosagem , Meios de Contraste/efeitos adversos , Hipersensibilidade a Drogas , Compostos de Iodo/administração & dosagem , Compostos de Iodo/efeitos adversos , Tomografia Computadorizada por Raios X , Adulto , Feminino , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Radiografia Abdominal , República da Coreia , Estudos Retrospectivos , Fatores de RiscoRESUMO
Terpenoids are the most diverse natural products with many industrial applications and are all synthesized from simple precursors, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized by two distinct metabolic pathways - cytosolic mevalonate (MVA) pathway for C15 sesquiterpene and C30 triterpene, and plastidic methylerythritol phosphate (MEP) pathway for C10 monoterpene and C20 diterpene. A number of studies have altered the metabolic gene expressions in either the MVA or MEP pathway to increase terpene production; however, it remains unknown if the alteration of the acetyl-CoA pool in plastid fatty acid biosynthesis can influence terpenoid flux. Here, we focused on the fact that acetyl-CoA is the precursor for both fatty acid biosynthesis in plastid and terpene biosynthesis in cytosol, and the metabolic impact of increased plastidic acetyl-CoA level on the cytosolic terpene biosynthesis was investigated. In tobacco leaf infiltration studies, the acetyl-CoA carboxylase complex (the enzyme supplying malonyl-CoA in plastid) was partially inhibited by overexpressing the inactive form of biotin carboxyl carrier protein (BCCP) by a negative dominant effect. Overexpression of BCCP showed 1.4-2.4-fold increase of sesquiterpenes in cytosol; however, surprisingly overexpression of BCCP linked to truncated HMG-CoA reductase (tHMGR) by a cleavable peptide 2A showed 20-40-fold increases of C15 sesquiterpenes (α-bisabolol, amorphadiene, and valerenadiene) and a 6-fold increase of C30 ß-amyrin. α-Bisabolol and ß-amyrin production reached 28.8â¯mgâ¯g-1 and 9.8â¯mgâ¯g-1 dry weight, respectively. Detailed analyses showed that a large increase in flux was achieved by the additive effect of BCCP- and tHMGR-overexpression, and an enhanced tHMGR activity by 2A peptide tag. Kinetic analyses showed that tHMGR-2A has a three-fold higher kcat value than tHMGR. The tHMGR-2A-BCCP1 co-expression strategy in this work provides a new insight into metabolic cross-talks and can be a generally applicable approach to over-produce sesqui- and tri-terpene in plants.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Proteínas de Transporte/metabolismo , Hidroximetilglutaril-CoA Redutases/metabolismo , Nicotiana/metabolismo , Sesquiterpenos/metabolismo , Triterpenos/metabolismo , Acetilcoenzima A/metabolismo , Citosol/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , Ácidos Graxos/biossíntese , Hidroximetilglutaril-CoA Redutases/genética , Malonil Coenzima A/metabolismo , Sesquiterpenos Monocíclicos , Folhas de Planta/metabolismo , Nicotiana/genéticaRESUMO
Membrane proteins are involved in a diverse array of cellular functions and part of many important metabolic pathways. As such, they are attractive targets in the pharmaceutical and bio-based chemical industries. Despite their great potential, many challenges remain before membrane proteins gain widespread success in biotechnology. The two biggest issues are that expression of membrane proteins leads to inhibition of cellular growth and metabolism, and native membrane proteins often lack a desired function or specificity for use in engineered processes. To address these issues, protein engineering and synthetic biology approaches are leading the charge to develop membrane proteins for biotechnological applications. Here, we describe current methods for engineering membrane proteins and optimizing their expression levels in bacteria. We highlight success stories and describe challenges that still face this growing field.
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Proteínas de Membrana/biossíntese , Engenharia de Proteínas/métodos , Bactérias/genética , Dosagem de Genes , Proteínas de Membrana/fisiologia , Regiões Promotoras Genéticas , Dobramento de Proteína , Biologia SintéticaRESUMO
Microbial synthesis of medium chain aliphatic hydrocarbons, attractive drop-in molecules to gasoline and jet fuels, is a promising way to reduce our reliance on petroleum-based fuels. In this study, we enabled the synthesis of straight chain hydrocarbons (C7-C13) by yeast Saccharomyces cerevisiae through engineering fatty acid synthases to control the chain length of fatty acids and introducing heterologous pathways for alkane or 1-alkene synthesis. We carried out enzyme engineering/screening of the fatty aldehyde deformylating oxygenase (ADO), and compartmentalization of the alkane biosynthesis pathway into peroxisomes to improve alkane production. The two-step synthesis of alkanes was found to be inefficient due to the formation of alcohols derived from aldehyde intermediates. Alternatively, the drain of aldehyde intermediates could be circumvented by introducing a one-step decarboxylation of fatty acids to 1-alkenes, which could be synthesized at a level of 3mg/L, 25-fold higher than that of alkanes produced via aldehydes.
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Alcanos/metabolismo , Alcenos/metabolismo , Ácidos Graxos , Engenharia Metabólica , Peroxissomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Peroxissomos/genética , Peroxissomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Low catalytic activities of pathway enzymes are often a limitation when using microbial based chemical production. Recent studies indicated that the enzyme activity of aldehyde decarbonylase (AD) is a critical bottleneck for alkane biosynthesis in Saccharomyces cerevisiae. We therefore performed functional screening to identify efficient ADs that can improve alkane production by S. cerevisiae. RESULTS: A comparative study of ADs originated from a plant, insects, and cyanobacteria were conducted in S. cerevisiae. As a result, expression of aldehyde deformylating oxygenases (ADOs), which are cyanobacterial ADs, from Synechococcus elongatus and Crocosphaera watsonii converted fatty aldehydes to corresponding Cn-1 alkanes and alkenes. The CwADO showed the highest alkane titer (0.13 mg/L/OD600) and the lowest fatty alcohol production (0.55 mg/L/OD600). However, no measurable alkanes and alkenes were detected in other AD expressed yeast strains. Dynamic expression of SeADO and CwADO under GAL promoters increased alkane production to 0.20 mg/L/OD600 and no fatty alcohols, with even number chain lengths from C8 to C14, were detected in the cells. CONCLUSIONS: We demonstrated in vivo enzyme activities of ADs by displaying profiles of alkanes and fatty alcohols in S. cerevisiae. Among the AD enzymes evaluated, cyanobacteria ADOs were found to be suitable for alkane biosynthesis in S. cerevisiae. This work will be helpful to decide an AD candidate for alkane biosynthesis in S. cerevisiae and it will provide useful information for further investigation of AD enzymes with improved activities.
Assuntos
Aldeído Liases/metabolismo , Alcanos/metabolismo , Cianobactérias/enzimologia , Engenharia Metabólica , Saccharomyces cerevisiae/enzimologia , Aldeído Liases/genética , Aldeídos/metabolismo , Alcenos/metabolismo , Biocombustíveis , Vias Biossintéticas , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Álcoois Graxos/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
Advancement in metabolic engineering of microorganisms has enabled bio-based production of a range of chemicals, and such engineered microorganism can be used for sustainable production leading to reduced carbon dioxide emission there. One area that has attained much interest is microbial hydrocarbon biosynthesis, and in particular, alkanes and alkenes are important high-value chemicals as they can be utilized for a broad range of industrial purposes as well as 'drop-in' biofuels. Some microorganisms have the ability to biosynthesize alkanes and alkenes naturally, but their production level is extremely low. Therefore, there have been various attempts to recruit other microbial cell factories for production of alkanes and alkenes by applying metabolic engineering strategies. Here we review different pathways and involved enzymes for alkane and alkene production and discuss bottlenecks and possible solutions to accomplish industrial level production of these chemicals by microbial fermentation.
Assuntos
Alcanos/metabolismo , Alcenos/metabolismo , Fermentação , Engenharia Metabólica , Biocombustíveis/provisão & distribuição , Ácidos Graxos/metabolismoRESUMO
Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11 ± 0.004 h(-1) and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.
Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Biologia Sintética/métodos , Xilose/metabolismo , DNA Bacteriano/genética , Genes Reporter , Vetores Genéticos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Proteína Vermelha FluorescenteRESUMO
Pinene is a monoterpenes (C10) that is produced in a genetically-engineered microbial host for its industrial applications in fragrances, flavoring agents, pharmaceuticals, and biofuels. Herein, we have metabolically-engineered Corynebacterium glutamicum, to produce pinene and studied its toxicity in C. glutamicum. Geranyl diphosphate synthases (GPPS) and pinene synthases (PS), obtained from Pinus taeda and Abies grandis, were co-expressed with over-expressed native 1-deoxy-d-xylulose-5-phosphate synthase (Dxs) and isopentenyl diphosphate isomerase (Idi) from C. glutamicum using CoryneBrick vector. Most strains expressing PS-GPPSs produced detectable amounts of pinene, but co-expression of DXS and IDI with PS (P. taeda) and GPPS (A. grandis) resulted in 27 µg ± 7 α-pinene g(-1) cell dry weight, which is the first report in C. glutamicum. Further engineering of PS and GPPS in the C. glutamicum strain may increase pinene production.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/genética , Glucose/metabolismo , Engenharia Metabólica , Monoterpenos/metabolismo , Proteínas de Bactérias/genética , Monoterpenos Bicíclicos , Corynebacterium glutamicum/efeitos dos fármacos , Corynebacterium glutamicum/metabolismo , Monoterpenos/toxicidade , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND: Placement of self-expandable nitinol stents is useful for the treatment of esophageal and upper gastrointestinal (GI) obstruction. However, complications such as stent migration, tumor overgrowth, and bleeding occur. Although stent migration and tumor overgrowth are well documented in previous studies, the occurrence of bleeding has not been fully evaluated. PURPOSE: To evaluate the incidence, management strategies, and predictors of bleeding after placement of self-expandable nitinol stents in patients with esophageal and upper GI obstruction. MATERIAL AND METHODS: We retrospectively reviewed the medical records and results of computed tomography and endoscopy of 1485 consecutive patients with esophageal and upper GI obstructions who underwent fluoroscopically guided stent placement. RESULTS: Bleeding occurred in 25 of 1485 (1.7%) patients 0 to 348 days after stent placement. Early stent-related bleeding occurred in 10 patients (40%) and angiographic embolization was used for 5/10. Late bleeding occurred in 15 patients (60%) and endoscopic hemostasis was used for 7/15. Twenty-two of 25 (88%) patients with bleeding had received prior radiotherapy and/or chemotherapy. CONCLUSION: Bleeding is a rare complication after placement of expandable nitinol stents in patients with esophageal and upper GI obstruction, but patients with early bleeding may require embolization for control. Care must be exercised on placing stents in patients who have received prior radiotherapy or chemotherapy.