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1.
J Bacteriol ; : e0013324, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39012109

RESUMO

The peptidoglycan hydrolases responsible for the cell separation of Bacillus subtilis cells are collectively referred to as autolysins. However, the role of each autolysin in the cell separation of B. subtilis is not fully understood. In this study, we constructed a series of cell separation-associated autolysin deficient strains and strains overexpressing the transcription factors SlrR and SinR, and the morphological changes of these strains in liquid culture were observed. The results showed that the absence of D,L-endopeptidases CwlS and LytF only increased the cell chain length in the early exponential phase. The absence of D,L-endopeptidase LytE or N-acetylmuramyl-L-alanine amidase LytC can cause cells to form chains throughout the growth of B. subtilis, although the cell chain length was significantly shortened during the stationary phase. However, the absence of peptidoglycan N-acetylglucosaminidase LytD only caused minor defect in cell separation. Therefore, we concluded that LytE and LytC were the major autolysins that ensure the timely separation of B. subtilis daughter cells, whereas CwlS, LytF, and LytD were the minor autolysins. In addition, overexpression of the transcription factors SinR and SlrR in the cwlS lytF lytC lytE mutant enabled B. subtilis cells to form ultra-long chains in the vegetative phase, and its biomass level was basically the same as that of the wild type. This led to the conclusion that besides inhibiting the expression of lytC and lytF, the SinR-SlrR complex also has other potential mechanisms to inhibit cell separation.IMPORTANCEIn this study, the effects of CwlS, LytC, LytD, LytF, LytE, and SinR-SlrR complex on the cell separation of Bacillus subtilis at different growth phases were studied, and an ultra-long-chained B. subtilis strain was constructed. In microbial fermentation, due to its large cell size, this ultra-long-chained B. subtilis strain may be more likely to be precipitated or intercepted during the removal of bacterial process with centrifugation and membrane filtration as the main methods, which is crucial to improve the purity of the product.

2.
Anal Chem ; 95(2): 1454-1460, 2023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-36538530

RESUMO

Organic photoelectrochemical transistor (OPECT) bioanalytics has recently appeared as a promising route for biological measurements, which has major implications in both next-generation photoelectrochemical (PEC) bioanalysis and futuristic biorelated implementations. Via biological dissociation of materials, bioetching is a useful technique for bio-manufacturing and bioanalysis. The intersection of these two domains is expected to be a possible way to achieve innovative OPECT bioanalytics. Herein, we validate such a possibility, which is exemplified by alkaline phosphatase (ALP)-mediated bioetching of a CoOOH/BiVO4 gate for a signal-on OPECT immunoassay of human immunoglobulin G (HIgG) as the model target. Specifically, target-dependent bioetching of the upper CoOOH layer could result into an enhanced electrolyte contact and light accessibility to BiVO4, leading to the modulated response of the polymeric poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) channel that could be monitored by the channel current. The introduced biosensor achieves sensitive detection of HIgG with high selectivity and sensitivity. This work features bioetching-enabled high-efficacy OPECT bioanalysis and is anticipated to serve as a generic protocol, considering the diverse bioetching routes.


Assuntos
Fosfatase Alcalina , Técnicas Biossensoriais , Humanos , Fosfatase Alcalina/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Óxidos
3.
J Hum Genet ; 68(7): 477-484, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-36864287

RESUMO

Mutations in the FHL1 gene can be associated with a variety of X-linked myopathies and cardiomyopathies, among which X-linked dominant scapuloperoneal myopathy is a rare phenotype. We collected the clinical data of two unrelated Chinese patients with X-linked scapuloperoneal myopathy and analyzed their clinical, pathological, muscle imaging, and genetic features. Both patients were characterized by scapular winging, bilateral Achilles tendon contractures, and weakness in shoulder-girdle and peroneal muscles. Muscle biopsy revealed myopathic changes, and no reducing bodies were found. Muscle magnetic resonance imaging was dominated by fatty infiltration, with minor edema-like findings. Genetic analysis revealed two novel mutations in the FHL1 gene: c.380T > C (p.F127S) and c.802C > T (p.Q268*), which were located in the LIM2 domain and the C-terminal sequence, respectively. To our knowledge, this is the first report of X-linked scapuloperoneal myopathy in the Chinese population. Our findings broadened the genetic and ethnic spectrum of FHL1-related disorders and proposed to look for variants in the FHL1 gene when scapuloperoneal myopathy is observed in the clinical work.


Assuntos
População do Leste Asiático , Doenças Musculares , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas Musculares/genética , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Doenças Musculares/genética , Mutação
4.
J Med Genet ; 59(4): 351-357, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-33811136

RESUMO

BACKGROUND: Progressive cavitating leukoencephalopathy (PCL) is thought to result from mutations in nuclear genes affecting mitochondrial function and energy metabolism. To date, mutations in two subunits of complex I, NDUFS1 and NDUFV1, have been reported to be related to PCL. METHODS: Patients underwent clinical examinations, brain MRI, skin biopsy and muscle biopsy. Whole-genome or whole-exome sequencing was performed on the index patients from two unrelated families with PCL. The effects of the mutations were examined through complementation of the NDUFV2 mutation by cDNA expression. RESULTS: The common clinical features of the patients in this study were recurring episodes of acute or subacute developmental regression that appeared in the first years of life, followed by gradual remissions and prolonged periods of stability. MRI showed leukoencephalopathy with multiple cavities. Three novel NDUFV2 missense mutations were identified in these families. Complex I deficiency was confirmed in affected individuals' fibroblasts and a muscle biopsy. Functional and structural analyses revealed that these mutations affect the structural stability and function of the NDUFV2 protein, indicating that defective NDUFV2 function is responsible for the phenotypes in these individuals. CONCLUSIONS: Here, we report the clinical presentations, neuroimaging and molecular and functional analyses of novel mutations in NDUFV2 in two sibling pairs of two Chinese families presenting with PCL. We hereby expand the knowledge on the clinical phenotypes associated with mutations in NDUFV2 and the genotypes causative for PCL.


Assuntos
Leucoencefalopatias , Doenças Mitocondriais , NADH Desidrogenase , Exoma , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/genética , Doenças Mitocondriais/genética , Mutação , NADH Desidrogenase/genética , Sequenciamento do Exoma
5.
Biotechnol Lett ; 44(8): 991-1000, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35767162

RESUMO

OBJECTIVE: To construct a Bacillus subtilis strain for improved purity of poly-γ-glutamic acid. RESULTS: The construction of strain GH16 was achieved by knocking out five genes encoding extracellular proteins and an operon from Bacillus subtilis G423. We then analyzed the amount of protein impurities in the γ-PGA produced by the resulting strain GH16/pHPG, which decreased from 1.48 to 1.39%. Subsequently the fla-che operon, PBSX, as well as the yrpD, ywoF and yclQ genes were knocked out successively, resulting in the mutant strains GH17, GH18 and GH19. Ultimately, the amount of protein impurities was reduced from 1.48 to 0.83%. In addition, the amount of polysaccharide impurities in the γ-PGA was also decreased from 2.21 to 1.93% after knocking out the epsA-O operon. CONCLUSIONS: The high purity γ-PGA producer was constructed, and the resulting strain was a promising platform for the manufacture of other highly pure extracellular products and secretory proteins.


Assuntos
Bacillus subtilis , Ácido Glutâmico , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ácido Glutâmico/metabolismo , Óperon/genética , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/metabolismo
6.
Clin Neuropathol ; 40(2): 93-97, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32831159

RESUMO

AIMS: Lambert-Eaton myasthenic syndrome (LEMS) is a kind of autoimmune disease of the neuromuscular junction that is often misdiagnosed as a peripheral nerve disease or myopathy. For some difficult cases, muscle biopsy examination is useful for differential diagnosis. However, studies about the pathological findings of LEMS patients are rare, especially of patients who were diagnosed with small cell lung carcinoma (SCLC). This study aimed to describe several pathological muscle features in patients with LEMS associated with SCLC. MATERIALS AND METHODS: The 5 patients enrolled in this study were diagnosed with LEMS associated with SCLC by clinical manifestation, electromyography, and lung biopsy. Muscle biopsies were performed for the patients, and enzyme histopathology was assessed. RESULTS: The hematoxylin and eosin (H & E) stain showed different sizes of fibers, and the shape of atrophic fibers were angular or polygonal. The adenosine triphosphatase (ATPase) stain demonstrated that the majority of atrophic fibers were type II. Type II fiber predominance was observed in case 1 and case 5. Also, scattered muscle fiber necrosis was shown in case 3. CONCLUSION: Type II fiber atrophy and type II fiber predominance may often be found in patients with LEMS associated with SCLC. Also, scattered fiber necrosis may appear in this disease.


Assuntos
Síndrome Miastênica de Lambert-Eaton , Neoplasias Pulmonares , Doenças Musculares , Carcinoma de Pequenas Células do Pulmão , Diagnóstico Diferencial , Humanos , Síndrome Miastênica de Lambert-Eaton/diagnóstico , Carcinoma de Pequenas Células do Pulmão/complicações
7.
Clin Neuropathol ; 40(6): 319-327, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34236310

RESUMO

OBJECTIVE: To analyze muscle histopathology of myasthenia gravis (MG) patients and further explore the underlying mechanism comparing with previous literature. MATERIALS AND METHODS: We analyzed the clinicopathological features of 8 MG patients who had muscle biopsy examinations. RESULTS: Eight patients with a diagnosis of MG were retrospectively recruited from the Chinese PLA General Hospital. One patient had positive anti-MuSK antibodies, 5 patients had positive anti-AChR antibodies (1 of whom had additional positive anti-Titin antibodies), and 2 patients were seronegative. Seronegative-MG presented normal muscle histology, occasionally with lipid deposition. Small angular atrophy (mainly in type II fibers) and necrosis in H & E stain were found in AChR-MG, furthermore, patterns of polymyositis (PM) could be found in AChR-MG with anti-Titin antibodies. Mitochondrial abnormalities were found only in MuSK-MG. CONCLUSION: Muscle histological abnormalities mimicking myopathy may be found in MG patients. Patients with different antibodies present with different muscle histopathology. PM pattern pathology is a special pattern of muscle histology in MG that should not be misdiagnosed. Our study has extended the muscle pathological features of MG in addition to deepening the understanding of MG.


Assuntos
Miastenia Gravis , Receptores Colinérgicos , Autoanticorpos , Humanos , Miastenia Gravis/diagnóstico , Receptores Proteína Tirosina Quinases , Estudos Retrospectivos
8.
Mikrochim Acta ; 188(8): 243, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34231032

RESUMO

The design and development of a 3D hierarchical CdS/NiO heterojunction and its application in a self-powered cathodic photoelectrochemical (PEC) bioanalysis is introduced. Specifically, NiO nanoflakes (NFs) were in situ formed on carbon fibers via a facile liquid-phase deposition method followed by an annealing step and subsequent integration with CdS quantum dots (QDs). The glucose oxidase (GOx) was then coated on the photocathode to allow the determination of glucose. Under 5 W 410 nm LED light and at a working voltage of 0.0 V (vs. Ag/AgCl), this method can assay glucose concentrations down to 1.77×10-9 M. The linear range was 5×10-7 M to 1×10-3 M, and the relative standard deviation (RSD) was below 5%. The photocathodic biosensor achieved target detection with high sensitivity and selectivity. This work is expected to stimulate more passion in the development of innovative hierarchical heterostructures for advanced self-powered photocathodic bioanalysis. Design of 3D hierarchical CdS/NiO heterojunction and its application in a self-powered cathodic photoelectrochemical (PEC) bioanalysis.


Assuntos
Compostos de Cádmio/química , Glucose Oxidase/metabolismo , Glucose/análise , Nanocompostos/química , Níquel/química , Sulfetos/química , Técnicas Biossensoriais , Fibra de Carbono , Técnicas Eletroquímicas , Limite de Detecção , Processos Fotoquímicos , Pontos Quânticos
9.
J Clin Rheumatol ; 27(8S): S771-S772, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32732524

RESUMO

ABSTRACT: Dystrophia myotonica type 1 (DM1) and DM2 are the most common muscular dystrophies. In both diseases, the skeletal muscle is the most severely affected. Additional symptoms are also involved in the eye, heart, brain, endocrine glands, gastrointestinal tract, skin, skeleton, and peripheral nerves. Skeletal muscle pathology is mainly manifested as myopathic changes including internal nuclei, sarcoplasmic masses, preferential type 1 fiber atrophy, and so on. Rimmed vacuoles (RVs) seen on muscle biopsy are areas of muscle destruction with an accumulation of autophagic vacuoles. However, there is no report about RVs in skeletal muscle of myotonic dystrophy patients. Here, we describe the first case of DM1 with RVs in skeletal muscle pathology.


Assuntos
Doenças Musculares , Distrofia Miotônica , Encéfalo , Humanos , Músculo Esquelético , Distrofia Miotônica/complicações , Distrofia Miotônica/diagnóstico , Vacúolos
10.
Biotechnol Lett ; 42(4): 551-555, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31993847

RESUMO

OBJECTIVES: The metabolic pathway related to uridine production was modified in Bacillus subtilis in order to increase the production of uridine. RESULTS: Decreasing the relative transcriptional level of pur operon in Bacillus subtilis TD300 to 80%, and the production of the derived strain TD312 was increased to 11.81 g uridine/l and the yield was increased to 270 mg uridine/g glucose. The expression of pucR gene in situ by PccpA resulting in a 194.01-fold increase in the relative transcriptional level of pucR gene and 349.71-fold increase in the relative transcriptional level of ure operon, respectively. Furthermore, the production of TD314 reached 13.06 g uridine/l, while the yield reached 250 mg uridine/g glucose. CONCLUSION: This is the first report that more than 13 g uridine/l with a yield of 250 mg uridine/g glucose is produced in shake flask fermentation of genetically engineered Bacillus subtilis.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Regulação para Baixo , Redes e Vias Metabólicas , Uridina/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Técnicas de Cultura Celular por Lotes/instrumentação , Fermentação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Mutagênese Sítio-Dirigida , Óperon
11.
J Pak Med Assoc ; 70(5): 913-916, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32400752

RESUMO

GNE myopathy is an adult-onset muscle disorder featuring distal muscle atrophy and weakness. Rimmed vacuoles found in the muscle biopsies and gene mutations lead to the diagnosis of GNE myopathy. We collected clinical information, performed muscle biopsies and genetic testing on three patients. These cases developed typical disease presentations with distal muscle weakness at the ages of 26, 23, and 37 years. Their muscle pathologies revealed rimmed vacuoles. Genetic analysis led to the findings which included, c.1543-1544delGA (p.D515QfsX2)/c.38G>C (p.C13S) compound heterozygous mutation, c.733A>G (p.K245E) homozygous mutation and c.527A>T (p.D176V)/c.1634-1G>C (splicing) ; in which c.1543-1544 del GA (p.D515QfsX2), c.733A>G (p.K245E) and c.1634-1G>C (splicing) are three de novo mutations that have never been reported before. In conclusion, this study broadens the mutational spectrum of the GNE gene.


Assuntos
Miopatias Distais , Complexos Multienzimáticos/genética , Músculo Esquelético , Adulto , Biópsia/métodos , China , Miopatias Distais/diagnóstico , Miopatias Distais/genética , Miopatias Distais/fisiopatologia , Feminino , Testes Genéticos/métodos , Humanos , Masculino , Debilidade Muscular/etiologia , Debilidade Muscular/patologia , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Mutação , Exame Neurológico/métodos
12.
Protein Expr Purif ; 142: 81-87, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28963005

RESUMO

Lipases are among the most versatile biocatalysts, and are used in a range of industrially relevant bioconversion reactions. However, the production of LipA in recombinant Bacillus subtilis is still limited, due to unresolved issues surrounding the regulation of the expression and secretion systems. In this study, the gene encoding LipA from B. subtilis 168 was expressed in BNA under the control of the P43 and the PAE promoter. The extracellular lipase activity of the resulting strains BNACL and BNAAL was 7.8 U ml-1 and 12.6 U ml-1, respectively. To further enhance the expression of LipA, pHP13L was constructed by inserting the PAE-lip into the shuttle vector pHP13, which produced an extracellular lipase activity of 180.5 U ml-1 of BNA/pHP13L. The strain BNAY8 described in Supplement data which lacks eight extracellular proteins was constructed and the deletion a few of the much weaker secreting proteins had no significant effect on the secretion of LipA. Moreover, the four Sec pathway components, secA-prfB, secDF, secYEG, prsA, were individually overexpressed in BNA. The overexpression of secDF and prsA enhanced the production of LipA by 28% and 49%, respectively. Furthermore, the co-overexpression of secDF with prsA improved the extracellular amount of LipA by 59% over that of BNA/pHP13L, reaching 287.8 U ml-1. It can therefore be said that both regulatory elements and secretion pathway had an impact on the production of secreted LipA. Their optimization and modification is a useful strategy to improve the homologous overproduction of other extracellular proteins in B. subtilis.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/química , Lipase/genética , Via Secretória , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Primers do DNA/síntese química , Primers do DNA/metabolismo , Vetores Genéticos/metabolismo , Cinética , Lipase/metabolismo , Regiões Promotoras Genéticas
14.
Biotechnol Lett ; 40(1): 151-155, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29038923

RESUMO

OBJECTIVES: To construct a Bacillus subtilis strain for improved uridine production. RESULTS: The AAG2846-2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation. CONCLUSION: This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.


Assuntos
Bacillus subtilis/metabolismo , Engenharia Metabólica/métodos , Uridina/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Deleção de Genes , Expressão Gênica , Glucose/metabolismo , Redes e Vias Metabólicas/genética
15.
Inorg Chem ; 56(10): 5498-5501, 2017 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-28437099

RESUMO

A zero-dimensional N,N'-dibutyl-4,4'-dipyridinium bromoplumbate, [BV]6[Pb9Br30], with unusual discrete [Pb9Br30]12- anionic clusters was prepared via a facile surfactant-mediated solvothermal process. This bromoplumbate exhibits a narrower optical band gap relative to the congeneric one-dimensional viologen bromoplumbates.

16.
Wei Sheng Wu Xue Bao ; 57(1): 54-65, 2017 Jan 04.
Artigo em Zh | MEDLINE | ID: mdl-29746760

RESUMO

Objective: We aimed at co-expressing heterologous D-hydantoinase and N-carbamoylase in Bacillus subtilis, and evaluating the feasibility of producing D-p-hydroxyphenylglycine by the recombinant B. subtilis whole-cell catalysis. Methods: The Paco expression cassette was combined with the coding sequence of hyd or sd1 gene as an artificial gene to express D-hydantoinase. The PAE expression cassette was combined with the coding sequence of adc gene as an artificial gene to express N-carbamoylase. The D-hydantoinase and N-carbamoylase co-expression plasmids pHCS(sd1+adc) and pHCY(hyd+adc) were constructed, using plasmid pHP13 as carrier; the co-expression plasmids pUCS(sd1+adc) was constructed, using plasmid pUB110 as carrier. The additional copy of acoR and sigL gene was integrated at chromosome. The skf and sdp gene were knocked out in B. subtilis. All recombinant strains bearing co-expression plasmid were characterized by analyzing whole-cell catalysis activity. Results: In the recombinant strains with plasmid pHCY and with pHCS, the whole-cell catalytic activity reached 0.21 U/mL and 0.31 U/mL, respectively. After the over-expression of acoR, sigL, and high-copy-number pUCS, the whole-cell catalytic activity reached 1.0 U/mL. Conclusion: Overexpression of acoR, sigL and the deletion of skf, sdp genes had significant effects on the catalysis activity of recombinant whole-cell.


Assuntos
Amidoidrolases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Glicina/análogos & derivados , Amidoidrolases/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Geobacillus stearothermophilus/enzimologia , Glicina/biossíntese , Engenharia Metabólica , Plasmídeos/genética , Plasmídeos/metabolismo , Rhodobacteraceae/enzimologia
18.
Wei Sheng Wu Xue Bao ; 56(1): 56-67, 2016 Jan 04.
Artigo em Zh | MEDLINE | ID: mdl-27305780

RESUMO

OBJECTIVE: We studied several crucial factors influencing the uridine biosynthesis in Bacillus subtilis, including mutations of phosphoribosylpyrophosphate synthetase (PRPP synthetase) (prs) and carbamyl phosphate synthetase (pyrAA/pyrAB), and overexpression of heterologous 5'-nucleotidase (sdt1). METHODS: According to the inferred allosteric sites, we introduced point mutation into coding sequences of prs and pyrAB. The mutated prs gene was integratedly expressed in the xylR locus of the chromosome and the pyrAB gene was modified in-situ. The sdt1 gene was overexpressed in the saB locus of the chromosome. The effect of the genetic modification on uridine biosynthesis was characterized by the analysis of uridine, cytidine and uracil in the fermentation broth. RESULTS: The mutations of Asn120Ser, Leu135Ile, Glu52Gly or Val312Ala on PRPP synthase resulted in an increase of uridine production by 67% and 96%, respectively. The mutations of Ser948Phe, Thr977Ala and Lys993Ile on carbamyl phosphate synthase resulted in a 182% increase of uridine yield to 6.97 g/L. The overexpression of heterologous 5'-nucleotidase resulted in a 17% increase of uridine yield to 8.16 g/L. CONCLUSION: The activity and regulation mechanism of PRPP synthase and carbamyl phosphate synthase was an important factor to limit the excessive synthesis of uridine. Asn120Ser and Leu135Ile mutations of PRPP synthase and Ser948Phe, Thr977Ala and Lys993Ile mutations of carbamyl phosphate synthase will facilitate the biosynthesis of uridine. The additional Glu52Gly and Val312Ala mutations of PRPP synthase were beneficial for uridine biosynthesis. The reaction from UMP to uridine also limited the biosynthesis of uridine in B. subtilis.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Uridina/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Clonagem Molecular , Ribose-Fosfato Pirofosfoquinase/genética , Ribose-Fosfato Pirofosfoquinase/metabolismo
19.
Microb Cell Fact ; 14: 54, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25890046

RESUMO

BACKGROUND: Cytidine and uridine are produced commercially by Bacillus subtilis. The production strains of cytidine and uridine were both derivatives from mutagenesis. However, the exact metabolic and genetic factors affecting the productivity remain unknown. Genetic engineering may be a promising approach to identify and confirm these factors. RESULTS: With the deletion of the cdd and hom genes, and the deregulation of the pyr operon in Bacillus subtilis168, the engineered strain produced 200.9 mg/L cytidine, 14.9 mg/L uridine and 960.1 mg/L uracil. Then, the overexpressed prs gene led to a dramatic increase of uridine by 25.9 times along with a modest increase of cytidine. Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively. Moreover, the overexpression of the pyrH gene increasesd the yield of cytidine by 40%, along with a modest augments of uridine and uracil. Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L. CONCLUSIONS: The deregulation of the pyr operon and the overexpression of the prs, pyrG and pyrH genes all contribute to the accumulation of pyrimidine nucleoside compounds in the medium. Among these factors, the overexpression of the pyrG and pyrH genes can particularly facilitate the production of cytidine. Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Citidina/biossíntese , Uridina/biossíntese , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Fermentação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Homosserina Desidrogenase/genética , Homosserina Desidrogenase/metabolismo , Engenharia Metabólica/métodos , Mutagênese , Óperon/genética , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Analyst ; 140(4): 1046-53, 2015 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-25563509

RESUMO

A novel luminescence probe based on mono-6-amino-ß-cyclodextrin (NH2-ß-CD) functionalised gold nanoclusters (ß-CD-AuNC) was designed for dopamine (DA) detection. The NH2-ß-CD molecules were conjugated onto the surface of 11-mercaptoundecanoic acid capped AuNCs (11-MUA-AuNC) via a carbodiimide coupling reaction. The integrity of the ß-CD cavities was preserved on the surface of AuNCs and they retained their capability for molecular DA host-guest recognition. DA could be captured by the ß-CD cavities to form an inclusion complex in which the oxidised DA could quench the fluorescence of the ß-CD-AuNC probe by electron transfer. The probe could be used to quantify DA in the range of 5-1000 nM with a detection limit of 2 nM. This sensitivity was 1-2 orders of magnitude higher than that in previously reported methods. Interference by both ascorbic acid (AA) and uric acid (UA) was not observed. Therefore, the ß-CD-AuNC probe could be directly used to determine the DA content in biological samples without further separation. This strategy was successfully applied to a DA assay in spiked human serum samples and it exhibited remarkable accuracy, sensitivity and selectivity.


Assuntos
Dopaminérgicos/sangue , Dopamina/sangue , Ouro/química , Substâncias Luminescentes/química , Nanopartículas Metálicas/química , beta-Ciclodextrinas/química , Ácidos Graxos/química , Humanos , Limite de Detecção , Luminescência , Medições Luminescentes/métodos , Nanopartículas Metálicas/ultraestrutura , Compostos de Sulfidrila/química
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