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1.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1277-1285, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328484

RESUMO

Leaf water potential of peanut subjected to drought stress is positively related to the oil content of peanut kernels. The aim of this study was to directly screen the high oil mutants of peanut and create the new peanut varieties using hydroxyproline as water potential regulator. In vitro mutagenesis was carried out with the embryonic leaflets of peanut variety Huayu 20 as explants and pingyangmycin as a mutagen added into the somatic embryo formation medium. The formed somatic embryos were successively transferred to somatic embryo germination and selection medium containing 6 mmol/L hydroxyproline (at -2.079 MPa water potential ) to induce regeneration and directionally screen high oil content mutants. After that, these plantlets were grafted and transplanted to the experimental field and 132 high oil mutants with oil content over 55% were obtained from the offspring of regenerated plants. Finally, among them, the oil contents of 27 lines were higher than 58% and of 2 lines were higher than 60%. A new peanut variety Yuhua 9 with high yield and oil content was bred from the regenerated plant progenies combining the pedigree breeding method. The yield was 14.0% higher than that of the control cultivar in the testing new peanut varieties of Liaoning province, and also it has passed the national registration of non-major crop varieties. Yuhua 9 with an oil content of 61.05%, which was 11.55 percentage points higher than that of the parent Huayu 20, was the peanut cultivar with the highest oil content in the world. The result showed that it was an effective way for directional breeding of high oil peanut varieties by means of the three-step technique including in vitro mutagenesis, directional screening by reducing water potential in medium and pedigree selection of regenerated plant progenies.


Assuntos
Arachis , Germinação , Secas , Mutagênese , Melhoramento Vegetal
2.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1317-1325, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328488

RESUMO

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 µL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.


Assuntos
Microfluídica , Pichia , Mutagênese , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes
3.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1335-1347, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328490

RESUMO

Docosahexaenoic acid (DHA) has many unique physiological functions such as promoting the development of brain and retina in infants. Therefore, it is widely applied to food, pharmacy, breeding and other industries. To obtain engineered strains of Aurantiochytrium limacinum SR21 suitable for industrial application with increased lipid and DHA production, we designed a simple, fast, accurate and high-throughput screening method based on Nile red staining of oil droplets. First, ultraviolet C (UVC) mutagenesis was used to generate a random mutant library of A. limacinum. Second, screening conditions were optimized including staining conditions of Nile red and the sorting criterion. Thereby, three putative high-lipid mutants (D03432, D05106 and D01521) were selected from the mutant library containing 3 648 mutated clones. The three mutants grew faster and produced higher amounts of lipids and DHA compared to wild type (WT). In 100 mL cultures, the lipid content of D03432 and D05106 mutants reached 64.74% and 63.13% of dry cell weight respectively, whereas the wild strain exhibited only 43.19%. DHA yield in these two mutants were even 2.26-fold and 2.37-fold higher than that of the wild strain. Experiment with 5 L fermentor further confirmed that D03432 and D05106 mutants had better performance than the wild strain on DHA yield (45.51% and 66.46% more than that of the wild strain, respectively), and demonstrated their high potential for industrial application. This work not only generated several high-DHA content mutants with high potential for industrial use, but also provided vital guidance for high-throughput screening of lipid hyper-accumulating mutants in other oil-producing microorganisms.


Assuntos
Estramenópilas , Reatores Biológicos , Ácidos Docosa-Hexaenoicos , Mutagênese , Coloração e Rotulagem
4.
J Photochem Photobiol B ; 197: 111535, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31319267

RESUMO

Measurement of Pulse-Amplitude-Modulated (PAM) chlorophyll a fluorescence is widely used method for obtaining information on the functional state of photosystem II (PSII). Recently, it has been shown that some of long-established fluorescence parameters must be interpreted with caution, when the light-induced chloroplast movements occur. In our work we have analyzed the effect of chloroplast movements on these parameters. We have derived new parameters that are independent of the change in PSII absorption occurring during measurement. To verify whether there is a need for new parameters or the difference between the parameters commonly used and the newly derived ones is insignificant, we conducted an experiment with Arabidopsis thaliana wild type plants and its phot1 phot2 mutant defective in chloroplast movement. Plants were exposed to light of different qualities (450, 470, 550 or 660 nm) and quantities (100, 400 or 1200 µmol m-2 s-1) for up to 40 min. Since the blue light-induced chloroplast avoidance reaction is a photoprotective mechanism, we expected that phot1 phot2 mutant will compensate the lack of this mechanism by increasing non-photochemical quenching. However, using the light at both 450 and 470 nm, the calculation of commonly used parameter, ΦNPQ (quantum yield of regulated light-induced thermal energy dissipation in PSII) based on Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73-81] showed the opposite. On the other hand, the results obtained using our newly proposed formulae to determine quantum yield of PSII thermal energy dissipation were in line with our assumption. Thus, the experimental data showed that some formulae of fluorescence parameters are dependent on the change in PSII absorption and need to be interpreted carefully. On the contrary, the formulae introduced by us can remove the effect of changes in PSII absorption that occur during measurement, without additional measurements, and give the real estimate of light-induced non-photochemical quenching.


Assuntos
Proteínas de Arabidopsis/metabolismo , Clorofila A/química , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/fisiologia , Luz , Modelos Teóricos , Mutagênese , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/química , Teoria Quântica , Termodinâmica
5.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(6): 557-562, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31292061

RESUMO

Objective To construct a random mutagenesis library of 3E1D7, a chimerical antibody against c-mesenchymal epithelial transition factor (c-Met), using mammalian cell surface display. Methods Antibody genes with randomly mutated complementarity-determining region 3 (CDR3) part were inserted into the mammalian expression plasmid pSZI-CD to construct the random mutagenesis library using double enzyme digestion. Reconstructed plasmids were then cloned into CHO cells by transfection. The expression level of antibodies on the surface of CHO cells was checked by C6 PLUS flow cytometry. Results 3E1D7 random mutagenesis library was successfully constructed with a volume of 5.52×106 in diversity on gene level. Sequence analysis showed that all 20 clones randomly picked from the library coded for 20 different mutated amino acid sequences in open reading frames. After transfection, the expression of full-length antibodies on CHO cell surfaces could be detected by flow cytometry. Conclusion A random mutagenesis library of a certain anti-c-Met antibody has been successfully constructed with an exhibitable diversity of 5.52×106, which would be a useful platform for further screening of therapeutic antibodies.


Assuntos
Biblioteca Gênica , Vetores Genéticos , Mutagênese , Sequência de Aminoácidos , Animais , Anticorpos/química , Cricetinae , Cricetulus , Fases de Leitura Aberta , Plasmídeos/genética , Transfecção
6.
Nat Commun ; 10(1): 2452, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31165728

RESUMO

3-ß-hydroxysteroid-Δ8, Δ7-isomerase, known as Emopamil-Binding Protein (EBP), is an endoplasmic reticulum membrane protein involved in cholesterol biosynthesis, autophagy, oligodendrocyte formation. The mutation on EBP can cause Conradi-Hunermann syndrome, an inborn error. Interestingly, EBP binds an abundance of structurally diverse pharmacologically active compounds, causing drug resistance. Here, we report two crystal structures of human EBP, one in complex with the anti-breast cancer drug tamoxifen and the other in complex with the cholesterol biosynthesis inhibitor U18666A. EBP adopts an unreported fold involving five transmembrane-helices (TMs) that creates a membrane cavity presenting a pharmacological binding site that accommodates multiple different ligands. The compounds exploit their positively-charged amine group to mimic the carbocationic sterol intermediate. Mutagenesis studies on specific residues abolish the isomerase activity and decrease the multidrug binding capacity. This work reveals the catalytic mechanism of EBP-mediated isomerization in cholesterol biosynthesis and how this protein may act as a multi-drug binder.


Assuntos
Androstenos/metabolismo , Anticolesterolemiantes/metabolismo , Antagonistas de Estrogênios/metabolismo , Esteroide Isomerases/metabolismo , Tamoxifeno/metabolismo , Colesterol/biossíntese , Condrodisplasia Punctata , Resistencia a Medicamentos Antineoplásicos , Humanos , Simulação de Acoplamento Molecular , Mutagênese , Ligação Proteica , Estrutura Terciária de Proteína , Esteroide Isomerases/ultraestrutura
7.
Microbiol Res ; 223-225: 44-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178050

RESUMO

Classic genome editing tools including ZFN, TALEN, and CRISPR/Cas9 rely on DNA double-strand breaks for genome editing. To prevent the potential hazard caused by double-strand breaks (DSBs), a series of single base editing tools that convert cytidine (C) to thymine (T) without DSBs have been developed extensively in multiple species. Herein, we report for the first time that C was converted to T with a high frequency in the filamentous fungi Aspergillus niger by fusing cytidine deaminase and Cas9 nickase. Using the CRISPR/Cas9-dependent base editor and inducing nonsense mutations via single base editing, we inactivated the uridine auxotroph gene pyrG and the pigment gene fwnA with an efficiency of 47.36%-100% in A.niger. At the same time, the single-base editing results of the non-phenotypic gene prtT showed an efficiency of 60%. The editable window reached 8 bases (from C2 to C9 in the protospacer) in A. niger. Overall, we successfully constructed a single base editing system in A. niger. This system provides a more convenient tool for investigating gene function in A. niger, and provides a new tool for genetic modification in filamentous fungi.


Assuntos
Aspergillus niger/genética , Sistemas CRISPR-Cas , Citidina Desaminase/genética , Edição de Genes/métodos , Aspergillus niger/enzimologia , Sequência de Bases , Desoxirribonuclease I/genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes , Genes Fúngicos/genética , Mutagênese
8.
Chemosphere ; 231: 518-527, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31151012

RESUMO

Retene (RET) is the most abundant polycyclic aromatic hydrocarbon (PAH) released upon burning of cellulose, although it is not considered as one of the priority PAHs and is not included for risk assessments by the US Environmental Protection Agency (US-EPA). There are only a few studies concerning the toxic effects of RET. To the best of our knowledge, this study is the first one to examine whether RET, in an environmental concentration, plays a crucial role in the induction of oxidative stress in A549 lung cell line, and its consequence as such as mutagenicity and cell death. Our results revealed that RET was able to significantly decrease cell viability only at 72 h of exposure, increase oxidative stress, mitochondrial membrane potential and mitochondrial contents, leading an increased reactive oxygen species (ROS) production. Mutagenic activity was not detected in Salmonella strains, suggesting that RET does not induce base-pair substitution (TA100), frameshift (TA98 and TA97a) and transition/transversion (TA102) mutations. However, exposure to RET led to a significant increase in micronuclei (MN), nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) frequency, as well as cell death, mainly due to necrosis. Taken together, the results of our study provide new evidence suggesting that RET promotes oxidative stress, contributes to the processes of genomic instability, and favors necrosis. Thus, we highlight the importance of including RET in routine environmental analyses in the future as a potential risk factor involved in complex diseases and carcinogenesis.


Assuntos
Mutagênicos/toxicidade , Estresse Oxidativo , Fenantrenos/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Células A549 , Morte Celular , Humanos , Potencial da Membrana Mitocondrial , Mutagênese , Testes de Mutagenicidade/métodos , Mutação , Hidrocarbonetos Policíclicos Aromáticos/análise , Salmonella/efeitos dos fármacos
9.
Photochem Photobiol Sci ; 18(7): 1685-1699, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31166333

RESUMO

The UVR8 photoreceptor in Arabidopsis thaliana is specific for ultraviolet-B (UV-B; 280-315 nm) radiation and its activation leads to a number of UV-B acclimation responses, including the accumulation of flavonoids. UVR8 participates in a signaling cascade involving COP1 and HY5 so that the absence of any of these components results in a reduction in the ability of a plant to accumulate flavonoids in response to UV; Cop1 mutants show high dropouts and hy5-ks50 hyh double mutants show very low levels of flavonoids. The predominant phenolics in Arabidopsis thaliana are sinapic acid derivatives as well as non-aclyated quercetin and kaempferol di- and triglycosides containing glucose and rhamnose as glycosylated sugar moieties. How this flavonoid profile in Arabidopsis thaliana is affected by UV radiation, how rapidly these changes occur in changing UV conditions, and which components of the UV-B signalling pathway are involved in rapid UV acclimatization reactions is poorly understood. In the present study, we examined these questions by characterizing the flavonoid profiles of Arabidopsis thaliana signalling mutants and wild types grown under different UV levels of constant UV-B+PAR ratios and then transferring a subset of plants to alternate UV conditions. Results indicate that flavonoid accumulation in Arabidopsis thaliana is triggered by UV and this response is amplified by higher levels of UV but not by all compounds to the same extent. The catechol structure in quercetin seems to be less important than the glycosylation pattern, e.g. having 2 rhamnose moieties in determining responsivity. At low UV+PAR intensities the introduction of UV leads to an initial tendency of increase of flavonoids in the wild types that was detected after 3 days. It took 7 days for these changes to be detected in plants grown under high UV+PAR intensities suggesting a priming of PAR. Thus, the flavonoid profile in Arabidopsis thaliana is altered over time following exposure to UV and PAR, but the functional significance of these changes is currently unclear.


Assuntos
Arabidopsis/efeitos da radiação , Flavonoides/metabolismo , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cromatografia Líquida de Alta Pressão , Proteínas Cromossômicas não Histona/metabolismo , Flavonoides/análise , Mutagênese , Folhas de Planta/química , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Espectrometria de Massas por Ionização por Electrospray , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
10.
Vet Microbiol ; 233: 28-38, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176409

RESUMO

The anti-phagocytic abilities of bacteria often affect bacterial pathogenicity. Here, random mutant library of Streptococcus equi subsp. zooepidemicus (SEZ) was constructed using transposon mutagenesis. After careful screening, 30 transposon mutants with different transposon insertion sites were identified by conducting quantitative phagocytosis and insertion-site confirmation assays, whose anti-phagocytic abilities were significantly reduced relative to the wild-type strain. Insertion sites of 19 strains were monocistronic, including genes coding membrane proteins, transporters, and enzymes with unknown pathological function, such as sadM, adhP, purD, guaA, alpha-galactosidase coding gene, ABC transporter permease coding gene, metallo-beta-lactamase coding gene, and three secreted enzyme coding genes spuZ, slaB, and endoS, as well as known virulence factor coding genes, such as hasA and szM. The insertion sites of another 11 strains were polycistronic. We focused on four monocistronic-mutant strains: MhtpZ, MspuZ, MslaB, and MendoS. The anti-phagocytic abilities of not only the mutants that were precoincubated with the recombinant proteins, but also the complement strains were significantly more pronounced than those of all four corresponding mutants. The polyclonal antiserum against SlaB or EndoS also significantly decreased the anti-phagocytic capacity of wild-type SEZ. All four mutants exhibited significantly decreased viability in whole blood and reduced lethality in mice relative to the wild-type strain. Thus, we identified a variety of new anti-phagocytic factors, particularly multiple SEZ secreted enzymes. These factors are instrumental in the phagocytic resistance of SEZ in the absence of opsonin. Our results provide a framework for further studies of SEZ pathogenesis and relevant vaccine development for novel potential targets.


Assuntos
Genes Bacterianos , Óperon , Fagócitos/microbiologia , Fagocitose , Streptococcus equi/genética , Animais , Elementos de DNA Transponíveis , Feminino , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos ICR , Mutagênese , Mutação , Células RAW 264.7 , Streptococcus equi/patogenicidade , Fatores de Virulência/genética
11.
Biochemistry (Mosc) ; 84(4): 398-406, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228931

RESUMO

To identify Yersinia pestis genes involved in the microbe's resistance to cationic antimicrobial peptides, the strategy of random transposon mutagenesis with a Tn5 minitransposon was used, and the library was screened for detecting polymyxin B (PMB) susceptible mutants. The mutation responsible for PMB-sensitive phenotype and the lipopolysaccharide (LPS) structure were characterized for the Y. pestis strain KM218-A3. In this strain the mini-Tn5 was located in an open reading frame with the product homologous to the E. coli protein GmhB (82% identity) functioning as d-glycero-d-manno-heptose-1,7-diphosphate phosphatase. ESI FT ICR mass spectrometry of anions was used to study the structure of the unmodified LPS of Y. pestis KM218-A3, and molecules were revealed with the full-size LPS core or with two types of an incomplete core: consisting of Kdo-Kdo or Ko-Kdo disaccharides and Hep-(Kdo)-Kdo or Hep-(Ko)-Kdo trisaccharides. The performed complementation confirmed that the defect in the biological properties of the mutant strain was caused by inactivation of the gmhB gene. These findings indicated that the gmhB gene product of Y. pestis is essential for production of wild-type LPS resistant to antimicrobial peptides and serum.


Assuntos
Elementos de DNA Transponíveis/genética , Yersinia pestis/metabolismo , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Carboidratos , Farmacorresistência Bacteriana/genética , Lipopolissacarídeos/análise , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Mutagênese , Polimixina B/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Yersinia pestis/efeitos dos fármacos , Yersinia pestis/genética
12.
Nat Commun ; 10(1): 2747, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227715

RESUMO

Many intracellular bacteria, including Chlamydia, establish a parasitic membrane-bound organelle inside the host cell that is essential for the bacteria's survival. Chlamydia trachomatis forms inclusions that are decorated with poorly characterized membrane proteins known as Incs. The prototypical Inc, called IncA, enhances Chlamydia pathogenicity by promoting the homotypic fusion of inclusions and shares structural and functional similarity to eukaryotic SNAREs. Here, we present the atomic structure of the cytoplasmic domain of IncA, which reveals a non-canonical four-helix bundle. Structure-based mutagenesis, molecular dynamics simulation, and functional cellular assays identify an intramolecular clamp that is essential for IncA-mediated homotypic membrane fusion during infection.


Assuntos
Proteínas de Bactérias/ultraestrutura , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/patogenicidade , Corpos de Inclusão/microbiologia , Fusão de Membrana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Cristalografia por Raios X , Técnicas de Inativação de Genes , Células HeLa , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas SNARE/química
13.
Nature ; 571(7764): 275-278, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181567

RESUMO

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.


Assuntos
DNA/genética , Edição de Genes/métodos , Mutagênese , Mutação , Nucleosídeo Desaminases/genética , Engenharia de Proteínas , RNA/genética , Adenina/metabolismo , Aminoidrolases/genética , Aminoidrolases/metabolismo , Citosina/metabolismo , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Células HEK293 , Humanos , Nucleosídeo Desaminases/metabolismo , Especificidade por Substrato , Transfecção
14.
Biochemistry (Mosc) ; 84(Suppl 1): S206-S224, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213203

RESUMO

Reactive carbonyl compounds (RCC) are a group of compounds with clearly pronounced electrophilic properties that facilitate their spontaneous reactions with numerous nucleophilic reaction sites in proteins, lipids, and nucleic acids. The biological functions of RCC are determined by their concentration and governed by the hormesis (biphasic reaction) principle. At low concentrations, RCC act as signaling molecules activating defense systems against xenobiotics and oxidizers, and at high concentrations, they exhibit the cytotoxic effect. RCC participate in the formation of cell adaptive response via intracellular signaling pathways involving regulation of gene expression and cytoplasmic mechanisms related to the structure-functional rearrangements of proteins. Special attention in this review is given to the functioning of electrophiles as mediators of cell general adaption syndrome manifested as the biphasic response. The hypothesis is proposed that electrophilic signaling can be a proto-signaling system.


Assuntos
Aldeídos/metabolismo , Radicais Livres/metabolismo , Cetonas/metabolismo , Mutagênese/fisiologia , Estresse Oxidativo/fisiologia , Transdução de Sinais/fisiologia , Animais , Humanos , Oxirredução , Processamento de Proteína Pós-Traducional
15.
Microb Cell Fact ; 18(1): 106, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186003

RESUMO

BACKGROUND: Late-stage fermentation broth contains high concentrations of target chemicals. Additionally, it contains various cellular metabolites which have leaked from lysed cells, which would exert multifactorial stress to industrial hyperproducers and perturb both cellular metabolism and product formation. Although adaptive laboratory evolution (ALE) has been wildly used to improve stress tolerance of microbial cell factories, single-factor stress condition (i.e. target product or sodium chloride at a high concentration) is currently provided. In order to enhance bacterial stress tolerance to actual industrial production conditions, ALE in late-stage fermentation broth is desired. Genome replication engineering assisted continuous evolution (GREACE) employs mutants of the proofreading element of DNA polymerase complex (DnaQ) to facilitate mutagenesis. Application of GREACE coupled-with selection under stress conditions is expected to accelerate the ALE process. RESULTS: In this study, GREACE was first modified by expressing a DnaQ mutant KR5-2 using an arabinose inducible promoter on a temperature-sensitive plasmid, which resulted in timed mutagenesis introduction. Using this method, tolerance of a lysine hyperproducer E. coli MU-1 was improved by enriching mutants in a lysine endpoint fermentation broth. Afterwards, the KR5-2 expressing plasmid was cured to stabilize acquired genotypes. By subsequent fermentation evaluation, a mutant RS3 with significantly improved lysine production capacity was selected. The final titer, yield and total amount of lysine produced by RS3 in a 5-L batch fermentation reached 155.0 ± 1.4 g/L, 0.59 ± 0.02 g lysine/g glucose, and 605.6 ± 23.5 g, with improvements of 14.8%, 9.3%, and 16.7%, respectively. Further metabolomics and genomics analyses, coupled with molecular biology studies revealed that mutations SpeBA302V, AtpBS165N and SecYM145V mainly contributed both to improved cell integrity under stress conditions and enhanced metabolic flux into lysine synthesis. CONCLUSIONS: Our present study indicates that improving a lysine hyperproducer by GREACE-assisted ALE in its stressful living environment is efficient and effective. Accordingly, this is a promising method for improving other valuable chemical hyperproducers.


Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/metabolismo , Lisina/metabolismo , Engenharia Metabólica/métodos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fermentação , Mutagênese
16.
Nat Commun ; 10(1): 2113, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068592

RESUMO

Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.


Assuntos
Animais Geneticamente Modificados/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila/genética , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Feminino , Masculino , Corpos Pedunculados/metabolismo , Mutagênese , Sistema Nervoso/crescimento & desenvolvimento , Plasticidade Neuronal/genética , Neurônios/fisiologia , Plasmídeos/genética , RNA Guia/genética
17.
Nat Commun ; 10(1): 2334, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133642

RESUMO

Pseudomonas aeruginosa, a significant opportunistic pathogen, can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals leads to altered biofilm formation and increased tolerance to various antibiotics, and requires the histidine kinase PA1396. Here, we show that the membrane-associated sensory input domain of PA1396 has five transmembrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds to develop novel adjuvants improving the efficacy of existing antibiotics.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Ácidos Graxos Insaturados/metabolismo , Histidina Quinase/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Animais , Antibacterianos/uso terapêutico , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/imunologia , Histidina Quinase/genética , Humanos , Lipossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Mutagênese , Fosforilação , Polimixinas/farmacologia , Polimixinas/uso terapêutico , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
18.
Enzyme Microb Technol ; 127: 22-31, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088613

RESUMO

The recombinant rAgaZC-1 was a family GH50 ß-agarase from Vibrio sp. ZC-1 (CICC 24670). In this paper, the mutant D622G (i.e., mutate the aspartic acid at position 622 to glycine) had better thermo-stability than rAgaZC-1, showing 1.5℃ higher T5010 (the temperature at which the half-time is 10 min) and 4-folds of half-time at 41℃, while they had almost same optimum temperature (38.5℃), optimum pH (pH6.0) and catalytic efficiency. Thermal deactivation kinetical analysis showed that D622G had higher activation energy for deactivation, enthalpy and Gibbs free energy than rAgaZC-1, indicating that more energy is required by D622G for deactivation. Substrate can protect agarase against thermal inactivation, especially D622G. Hence the yield of agarose hydrolysis catalyzed by D622G was higher than that by rAgaZC-1. The models of D622G and rAgaZC-1 predicted by homology modeling were compared to find that it is the improved distribution of surface electrostatic potential, great symmetric positive potential and more hydrophobic interactions of D622G that enhance the thermo-stability.


Assuntos
Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Temperatura Alta , Mutagênese , Vibrio/enzimologia , Estabilidade Enzimática , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Hidrólise , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Conformação Proteica , Estabilidade Proteica , Sefarose/metabolismo
19.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067626

RESUMO

The occurrence of damage on bacterial DNA (mediated by antibiotics, for example) is intimately associated with the activation of the SOS system. This pathway is related to the development of mutations that might result in the acquisition and spread of resistance and virulence factors. The inhibition of the SOS response has been highlighted as an emerging resource, in order to reduce the emergence of drug resistance and tolerance. Herein, we evaluated the ability of betulinic acid (BA), a plant-derived triterpenoid, to reduce the activation of the SOS response and its associated phenotypic alterations, induced by ciprofloxacin in Staphylococcus aureus. BA did not show antimicrobial activity against S. aureus (MIC > 5000 µg/mL), however, it (at 100 and 200 µg/mL) was able to reduce the expression of recA induced by ciprofloxacin. This effect was accompanied by an enhancement of the ciprofloxacin antimicrobial action and reduction of S. aureus cell volume (as seen by flow cytometry and fluorescence microscopy). BA could also increase the hyperpolarization of the S. aureus membrane, related to the ciprofloxacin action. Furthermore, BA inhibited the progress of tolerance and the mutagenesis induced by this drug. Taken together, these findings indicate that the betulinic acid is a promising lead molecule in the development helper drugs. These compounds may be able to reduce the S. aureus mutagenicity associated with antibiotic therapies.


Assuntos
Farmacorresistência Bacteriana/efeitos dos fármacos , Recombinases Rec A/genética , Staphylococcus aureus/genética , Triterpenos/farmacologia , Ciprofloxacino/efeitos adversos , Ciprofloxacino/farmacologia , DNA Bacteriano/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Resposta SOS (Genética)/efeitos dos fármacos , Resposta SOS (Genética)/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Fatores de Virulência/genética
20.
Hum Genet ; 138(6): 563-590, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31115652

RESUMO

Since its discovery, the Clustered Regularly Interspaced Short Palindromic Repeat (the CRISPR) system has been increasingly applied to therapeutic genome editing. Employment of several viral and non-viral vectors has enabled efficient delivery of the CRISPR system to target cells or tissues. In addition, the CRISPR system is able to modulate the target gene's expression in various ways, such as mutagenesis, gene integration, epigenome regulation, chromosomal rearrangement, base editing and mRNA editing. However, there are still limitations hindering an ideal application of the system: inefficient delivery, dysregulation of the delivered gene, the immune response against the CRISPR system, the off-target effects or the unintended on-target mutations. In addition, there are recent discoveries that have not been yet applied to CRISPR-mediated therapeutic genome editing. Here, we review the overall principles related to the therapeutic application of the CRISPR system, along with new strategies for the further application and prospects to overcome the limitations.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Transferência de Genes , Mutagênese , Epigenômica/métodos , Rearranjo Gênico , Terapia Genética/métodos , Humanos , Reprodutibilidade dos Testes
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