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1.
Mater Sci Eng C Mater Biol Appl ; 109: 110615, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32228941

RESUMO

Four nanostructured MnO2 with various controllable morphologies, including nanowires, nanorods, nanotubes and nanoflowers were synthesized, and then further composited with nitrogen-doped graphene (NG) with the assistance of ultrasonication. The surface morphologies, phase structures, and electrochemical performances of the proposed MnO2/NG nanohybrids were investigated by various techniques, and their catalytic activities on the electrooxidation of dopamine (DA) and uric acid (UA) were compared systematically. The sensing performances were found to be highly correlated with their morphologies. Among these morphologies, the nanoflower-like MnO2, composited with NG, displayed the most sensitive response signals for DA and UA. The boosted electrocatalytic activity was ascribed to the unique porous structure, large electroactive area, and low charge transfer resistance (Rct), which facilitated the electron transfer between electrode and analytes. Two linear response ranges (0.1 µM-10 µM and 10 µM-100 µM) were accompanied with very low detection limits of 34 nM and 39 nM for DA and UA, respectively. Moreover, the successful application of the MnO2NFs/NG composites for the simultaneous detection of DA and UA in human serum was realized using second-derivative linear sweep voltammetry (SDLSV). These findings give valuable insights for understanding the morphology-dependent sensing properties of MnO2 based nanomaterials, which is conducive to the rapid development of ubiquitous MnO2-based electrochemical sensors.

2.
Fitoterapia ; 142: 104531, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32114039

RESUMO

Nine new (1-9) and three known (10-12) sesquiterpenoids were isolated from the ethanol-water (7:3, v/v) extract of the Datura metel L. leaves. The structures of 1-9 were elucidated by detailed spectroscopic analyses, including 1D and 2D NMR, HR-ESI-MS. All isolates (1-12) were evaluated for anti-inflammatory activity against the production of nitrogen oxide in lipopolysaccharide-induced RAW264.7 cells and compound 5 possessed the best inhibitory effect among them, with the IC50 value reaching 9.33-11.67 µM, which was lower than positive control, L-NMMA, with IC50 range from 13.64 to 17.02 µM.

3.
ACS Chem Biol ; 15(1): 63-73, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31613083

RESUMO

Human rhinovirus 3C protease (HRV 3C-P) is a high-value commercial cysteine protease that could specifically recognize the short peptide sequence of LEVLFQ↓GP. In here, a strategy based on our previous Yeast Endoplasmic Reticulum Sequestration Screening (YESS) approach was developed in Saccharomyces cerevisiae, a model microorganism, to fully characterize the substrate specificity of a typical human virus protease, HRV 3C-P, in a quantitative and fast manner. Our results demonstrated that HRV 3C-P had very high specificity at P1 and P1' positions, only recognizing Gln/Glu at the P1 position and Gly/Ala/Cys/Ser at the P1' position, respectively. Comparably, it exhibited efficient recognition of most residues at the P2' position, except Trp. Further biochemical characterization through site mutagenesis, enzyme structural modeling, and comparison with other 3C proteases indicated that the S1 pocket of HRV 3C-P was constituted by neutral and basic amino acids, in which His160 and Thr141 specifically interacted with Gln or Glu residues at the substrate P1 position. Additionally, the stringent S1' pocket determined its unique property of only accommodating residues without or with short side chains. Based on our characterization, LEVLFQ↓GM was identified as a more favorable substrate than the original LEVLFQ↓GP at high temperature, which might be caused by the conversion of random coils to ß-turns in HRV 3C-P along with the temperature increase. Our studies prompted a further understanding of the substrate specificity and recognition mechanism of HRV 3C-P. Besides, the YESS-PSSC combined with the enzyme modeling strategy in this study provides a general strategy for deciphering the substrate specificities of proteases.

4.
Medicine (Baltimore) ; 98(44): e17854, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31689877

RESUMO

Breast cancer is the most common diagnosed malignancy in women. This study genotyped blood samples from 236 Han Chinese women with breast cancer and 128 healthy controls for single nucleotide polymorphisms (SNPs) rs2977537, rs2929970, rs2929973, rs2977530, and rs62514004, to determine whether these WNT1-inducible signaling pathway protein 1 (WISP-1) genetic polymorphisms increase the risk of developing breast cancer. Compared with wild-type (AA) carriers, those carrying the WISP1 rs62514004 AG or AG + GG genetic variants had a greater risk of developing breast cancer. In an evaluation of the association between clinicopathological aspects and the WISP1 SNP rs62514004 in the breast cancer cohort, patients with the GG genotype were less likely than those with the AA genotype to develop stage III/IV disease. Patients carrying the WISP1 rs2929973 GG + TT variant were almost twice as likely as those carrying the GT genotype to have estrogen receptor (ER)- and progesterone receptor (PR)-positive tumors, while those with the WISP1 rs62514004 AG + GG genetic variants were around twice as likely as those with the AA genotype to have HER2-positive tumors. This study details risk associations between WISP1 SNPs and breast cancer susceptibility in women of Han Chinese ethnicity.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Neoplasias da Mama/genética , Proteínas de Sinalização Intercelular CCN/genética , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas/genética , Adulto , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Pessoa de Meia-Idade , Receptor ErbB-2/análise , Receptores Estrogênicos/análise , Receptores de Progesterona/análise , Fatores de Risco , Transdução de Sinais
5.
Microb Cell Fact ; 18(1): 162, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581942

RESUMO

BACKGROUND: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. RESULTS: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. CONCLUSIONS: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.


Assuntos
Edição de Genes/métodos , Genoma Bacteriano , Zymomonas/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Francisella/enzimologia , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia/genética , RNA Guia/metabolismo , Zymomonas/metabolismo
6.
Nucleic Acids Res ; 47(21): 11461-11475, 2019 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-31647102

RESUMO

Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction-modification (R-M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R-M system. This study might shed light on exploiting and improving CRISPR-Cas systems in other microorganisms for genome editing and metabolic engineering practices.

7.
Artigo em Inglês | MEDLINE | ID: mdl-31626362

RESUMO

Zymomonas mobilis is a model bacterial ethanologen and has been engineered to produce lignocellulosic biofuels and biochemicals such as 2,3-butanediol. We have previously identified promoters of different strengths using systems biology datasets and characterized them using the flow cytometry-based dual reporter-gene system. Here, we further demonstrated the capability of applying the dual reporter-gene system and omics datasets on discovering inducible promoters. Ten candidate ethanol-inducible promoters were identified through omics datasets mining and clustering. Using the dual reporter-gene system, these promoters were characterized under natural growth, ethanol stress, and ethanol-induced condition to investigate the transcriptional strength and ethanol inducibility. The results demonstrated that three promoters of P0405, P0435, and P0038 driving the expression of native genes of ZMO0405, ZMO0435, and ZMO0038, correspondingly, are potential ethanol-responsive promoters and may be growth related. This study not only identified and verified three ethanol-inducible promoters as biological parts, which can be used to synchronize the expression of heterologous pathway genes with the ethanol production process of Z. mobilis, but also demonstrated the power of combining omics datasets and dual reporter-gene system to identify biological parts for metabolic engineering and synthetic biology applications in Z. mobilis and related microorganisms.

8.
Chemosphere ; 237: 124485, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31394453

RESUMO

Forward osmosis (FO) has been used in the wastewater treatment due to its advantages including low energy consumption and low membrane fouling. In this study, real municipal wastewater was concentrated by FO process using seawater concentrate as draw solution (DS). The influences of operating conditions such as temperature, flow velocity and sewage pre-filtration on water flux were investigated. Chemical oxygen demand, total nitrogen, ammonia nitrogen and total phosphorus could not be enriched by 4 times while sewage was reduced to 1/4 volume. Excitation and emission matrix fluorescence spectrum showed that a fraction of dissolved organic compounds in sewage transported across membrane into DS. Membrane fouling was evaluated by scanning electronic microscope analysis that a dense cake layer was formed on the membrane surface after sewage filtration. However, water flux of the fouled membrane was highly recovered after 1 h of physical cleaning.


Assuntos
Água do Mar/química , Eliminação de Resíduos Líquidos/métodos , Análise da Demanda Biológica de Oxigênio , Filtração , Membranas Artificiais , Nitrogênio , Compostos Orgânicos , Osmose , Fósforo , Esgotos , Soluções , Águas Residuárias/química , Água , Purificação da Água
9.
Nanomaterials (Basel) ; 9(6)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31159377

RESUMO

Various morphologies of iron oxide nanoparticles (Fe2O3 NPs), including cubic, thorhombic and discal shapes were synthesized by a facile meta-ion mediated hydrothermal route. To further improve the electrochemical sensing properties, discal Fe2O3 NPs with the highest electrocatalytic activity were coupled with graphene oxide (GO) nanosheets. The surface morphology, microstructures and electrochemical properties of the obtained Fe2O3 NPs and Fe2O3/GO nanohybrids were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. As expected, the electrochemical performances were found to be highly related to morphology. The discal Fe2O3 NPs coupled with GO showed remarkable electrocatalytic activity toward the oxidation of dopamine (DA) and uric acid (UA), due to their excellent synergistic effect. The electrochemical responses of both DA and UA were linear to their concentrations in the ranges of 0.02-10 µM and 10-100 µM, with very low limits of detection (LOD) of 3.2 nM and 2.5 nM for DA and UA, respectively. Moreover, the d-Fe2O3/GO nanohybrids showed good selectivity and reproducibility. The proposed d-Fe2O3/GO/GCE realized the simultaneous detection of DA and UA in human serum and urine samples with satisfactory recoveries.

10.
Nanomaterials (Basel) ; 9(6)2019 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-31159490

RESUMO

This study reports facile synthesis of MnO2 nanoflowers/N-doped reduced graphene oxide (MnO2NFs/NrGO) composite and its application on the simultaneous determination of dopamine (DA) and uric acid (UA). The microstructures, morphologies, and electrochemical performances of MnO2NFs/NrGO were studied using X-ray diffraction (XRD), scanning electron microscopy (SEM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS), respectively. The electrochemical experiments showed that the MnO2NFs/NrGO composites have the largest effective electroactive area and lowest charge transfer resistance. MnO2NFs/NrGO nanocomposites displayed superior catalytic capacity toward the electro-oxidation of DA and UA due to the synergistic effect from MnO2NFs and NrGO. The anodic peak currents of DA and UA increase linearly with their concentrations varying from 0.2 µM to 6.0 µM. However, the anodic peak currents of DA and UA are highly correlated to the Napierian logarithm of their concentrations ranging from 6.0 µM to 100 µM. The detection limits are 0.036 µM and 0.029 µM for DA and UA, respectively. Furthermore, the DA and UA levels of human serum samples were accurately detected by the proposed sensor. Combining with prominent advantages such as facile preparation, good sensitivity, and high selectivity, the proposed MnO2NFs/NrGO nanocomposites have become the most promising candidates for the simultaneous determination of DA and UA from various actual samples.

11.
Biotechnol Biofuels ; 12: 126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31139256

RESUMO

Background: Fast, complete, and ultimate removal of inhibitory compounds derived from lignocellulose pretreatment is the prerequisite for efficient production of cellulosic ethanol and biochemicals. Biodetoxification is the most promising method for inhibitor removal by its unique advantages. The biodetoxification mechanisms of a unique diploid fungus responsible for highly efficient biodetoxification in solid-state culture was extensively investigated in the aspects of cellular structure, genome sequencing, transcriptome analysis, and practical biodetoxification. Results: The inborn heterozygous diploid structure of A. resinae ZN1 uniquely contributed to the enhancement of inhibitor tolerance and conversion. The co-expression of gene pairs contributed to the enhancement of the degradation of lignocellulose-derived model inhibitors. The ultimate inhibitors degradation pathways and sugar conservation were elucidated by microbial degradation experimentation as well as the genomic and transcriptomic sequencing analysis. Conclusions: The finding of the heterozygous diploid structure in A. resinae ZN1 on biodetoxification took the first insight into the global overview of biodetoxification mechanism of lignocellulose-derived inhibitors. This study provided a unique and practical biodetoxification biocatalyst of inhibitor compounds for lignocellulose biorefinery processing, as well as the synthetic biology tools on biodetoxification of biorefinery fermenting strains.

12.
Appl Microbiol Biotechnol ; 103(13): 5231-5241, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31028436

RESUMO

Cold-active enzymes have become attractive biocatalysts in biotechnological applications for their ability to retain high catalytic activity below 30 °C, which allows energy reduction and cost saving. Here, a 1041 bp gene pel1 encoding a 34.7 KDa pectate lyase was cloned from a facultatively psychrophilic Antarctic bacterium Massilia eurypsychrophila and heterologously expressed in Escherichia coli. PEL1 presented the highest 66% identity to the reported mesophilic pectate lyase PLXc. The purified PEL1 exhibits the optimum temperature and pH of 30 °C and 10 toward polygalacturonic acid, respectively. PEL1 is a cold-active enzyme that can retain 60% and 25% relative activity at 10 °C and 0 °C, respectively, while it loses most of activity at 40 °C for 10 min. PEL1 has the highest specific activity (78.75 U mg-1) than all other reported cold-active pectinase, making it a better choice for use in industry. Based on the detailed sequence and structure comparison between PEL1 and PLXc and mutation analysis, more flexible structure and some loop regions may contribute to the cold activity and thermal instability of PEL1. Our investigations of the cold-active mechanism of PEL1 might guide the rational design of PEL1 and other related enzymes.


Assuntos
Temperatura Baixa , Oxalobacteraceae/enzimologia , Polissacarídeo-Liase/metabolismo , Regiões Antárticas , Biocatálise , Clonagem Molecular , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Oxalobacteraceae/genética , Polissacarídeo-Liase/genética , Especificidade por Substrato
13.
Biotechnol Biofuels ; 12: 60, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30923568

RESUMO

Background: Biological routes for utilizing both carbohydrates and lignin are important to reach the ultimate goal of bioconversion of full carbon in biomass into biofuels and biochemicals. Recent biotechnology advances have shown promises toward facilitating biological transformation of lignin into lipids. Natural and engineered Rhodococcus strains (e.g., R. opacus PD630, R. jostii RHA1, and R. jostii RHA1 VanA-) have been demonstrated to utilize lignin for lipid production, and co-culture of them can promote lipid production from lignin. Results: In this study, a co-fermentation module of natural and engineered Rhodococcus strains with significant improved lignin degradation and/or lipid biosynthesis capacities was established, which enabled simultaneous conversion of glucose, lignin, and its derivatives into lipids. Although Rhodococci sp. showed preference to glucose over lignin, nearly half of the lignin was quickly depolymerized to monomers by these strains for cell growth and lipid synthesis after glucose was nearly consumed up. Profiles of metabolites produced by Rhodococcus strains growing on different carbon sources (e.g., glucose, alkali lignin, and dilute acid flowthrough-pretreated poplar wood slurry) confirmed lignin conversion during co-fermentation, and indicated novel metabolic capacities and unexplored metabolic pathways in these organisms. Proteome profiles suggested that lignin depolymerization by Rhodococci sp. involved multiple peroxidases with accessory oxidases. Besides the ß-ketoadipate pathway, the phenylacetic acid (PAA) pathway was another potential route for the in vivo ring cleavage activity. In addition, deficiency of reducing power and cellular oxidative stress probably led to lower lipid production using lignin as the sole carbon source than that using glucose. Conclusions: This work demonstrated a potential strategy for efficient bioconversion of both lignin and glucose into lipids by co-culture of multiple natural and engineered Rhodococcus strains. In addition, the involvement of PAA pathway in lignin degradation can help to further improve lignin utilization, and the combinatory proteomics and bioinformatics strategies used in this study can also be applied into other systems to reveal the metabolic and regulatory pathways for balanced cellular metabolism and to select genetic targets for efficient conversion of both lignin and carbohydrates into biofuels.

14.
Biotechnol Biofuels ; 12: 52, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30911332

RESUMO

Background: Zymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production. Results: In this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R 2 values more than 0.7 and 0.9, respectively. Conclusions: This study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.

15.
Artigo em Inglês | MEDLINE | ID: mdl-30847341

RESUMO

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately -7°C) and cold-acclimated (CA; leaf LT50 approximately -50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

16.
Trends Biotechnol ; 37(9): 960-972, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30876702

RESUMO

Great effort has been devoted to engineering Saccharomyces cerevisiae with pentose metabolism through the oxido-reductase pathway for cellulosic ethanol production, but intrinsic cofactor imbalance is observed, which substantially compromises ethanol yield. Zymomonas mobilis not only can be engineered for pentose metabolism through the isomerase pathway without cofactor imbalance but also metabolizes sugar through the Entner-Doudoroff pathway with less ATP and biomass produced for more sugar to be used for ethanol production. Moreover, the availabilities of genome sequence information for multiple Z. mobilis strains and advanced genetics tools have laid a solid foundation for engineering this species, and the self-flocculation of the bacterial cells also presents significant advantages for bioprocess engineering. Here, we highlight some of recent advances in these aspects.

17.
Environ Sci Pollut Res Int ; 26(9): 8585-8593, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30710329

RESUMO

In this study, trisodium citrate was used as draw solute in forward osmosis (FO) due to its biodegradability and easy reuse after FO dilution. The effect of operating conditions on FO performance was investigated. The study focused on the long-term flux performance and membrane fouling when surface water was used as feed solution. A water flux of 9.8 LMH was observed using 0.5 M trisodium citrate as draw solution in PRO mode. In the long-term FO process, trisodium citrate showed a slight decrease in total flux loss (13.06%) after 20 h of operation. The membrane fouling was significantly reduced after a two-step physical cleaning. A considerable flux recovery (> 95%) of the fouled membrane was finally obtained. Therefore, this study proves the superiority of trisodium citrate as draw solution and paves a new way in applying FO directly for surface water reclamation.


Assuntos
Citratos/química , Ácido Cítrico/química , Purificação da Água/métodos , Membranas Artificiais , Osmose , Soluções
18.
Nucleic Acids Res ; 47(7): e40, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30767015

RESUMO

Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria.


Assuntos
Bacillus subtilis/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Regulação Bacteriana da Expressão Gênica , Proteína 9 Associada à CRISPR/metabolismo , Genes Bacterianos/genética , Chaperonas Moleculares/metabolismo , Mutação , Regiões Promotoras Genéticas/genética , Dobramento de Proteína , RNA Guia/genética , Transcrição Genética , Transformação Genética
19.
Biotechnol Biofuels ; 12: 10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30627218

RESUMO

Background: Cellulosic biofuels are sustainable compared to fossil fuels. However, inhibitors, such as acetic acid generated during lignocellulose pretreatment and hydrolysis, would significantly inhibit microbial fermentation efficiency. Microbial mutants able to tolerate high concentration of acetic acid are needed urgently to alleviate this inhibition. Results: Zymomonas mobilis mutants AQ8-1 and AC8-9 with enhanced tolerance against acetic acid were generated via a multiplex atmospheric and room temperature plasma (mARTP) mutagenesis. The growth and ethanol productivity of AQ8-1 and AC8-9 were both improved in the presence of 5.0-8.0 g/L acetic acid. Ethanol yield reached 84% of theoretical value in the presence of 8.0 g/L acetic acid (~ pH 4.0). Furthermore, a mutant tolerant to pH 3.5, named PH1-29, was generated via the third round of ARTP mutagenesis. PH1-29 showed enhanced growth and ethanol production under both sterilized/unsterilized conditions at pH 4.0 or 3.5. Intracellular NAD levels revealed that mARTP mutants could modulate NADH/NAD+ ratio to respond to acetic acid and low pH stresses. Moreover, genomic re-sequencing revealed that eleven single nucleic variations (SNVs) were likely related to acetic acid and low pH tolerance. Most SNVs were targeted in regions between genes ZMO0952 and ZMO0956, ZMO0152 and ZMO0153, and ZMO0373 and ZMO0374. Conclusions: The multiplex mutagenesis strategy mARTP was efficient for enhancing the tolerance in Z. mobilis. The ARTP mutants generated in this study could serve as potential cellulosic ethanol producers.

20.
Biotechnol Biofuels ; 11: 306, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30455735

RESUMO

Background: Poly-γ-glutamic acid (γ-PGA) is a natural polymer with great potential applications in areas of agriculture, industry, and pharmaceutical. The biodiesel-derived glycerol can be used as an attractive feedstock for γ-PGA production due to its availability and low price; however, insufficient production of γ-PGA from glycerol is limitation. Results: The metabolic pathway of Bacillus licheniformis WX-02 was rewired to improve the efficiency of glycerol assimilation and the supply of NADPH for γ-PGA synthesis. GlpK, GlpX, Zwf, and Tkt1 were found to be the key enzymes for γ-PGA synthesis using glycerol as a feedstock. Through combinational expression of these key enzymes, the γ-PGA titer increased to 19.20 ± 1.57 g/L, which was 1.50-fold of that of the wild-type strain. Then, we studied the flux distributions, gene expression, and intracellular metabolites in WX-02 and the recombinant strain BC4 (over-expression of the above quadruple enzymes). Our results indicated that over-expression of the quadruple enzymes redistributed metabolic flux to γ-PGA synthesis. Furthermore, using crude glycerol as carbon source, the BC4 strain showed a high productivity of 0.38 g/L/h, and produced 18.41 g/L γ-PGA, with a high yield of 0.46 g γ-PGA/g glycerol. Conclusions: The approach to rewiring of metabolic pathways enables B. licheniformis to efficiently synthesize γ-PGA from glycerol. The γ-PGA productivity reported in this work is the highest obtained in glutamate-free medium. The present study demonstrates that the recombinant B. licheniformis strain shows significant potential to produce valuable compounds from crude glycerol.

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