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1.
J Basic Microbiol ; 63(12): 1426-1439, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37821396

RESUMO

Careless handling of petroleum in petrochemical industries releases toxic hydrocarbons and metals to soil and water. The aim of the present study was to isolate hydrocarbon-utilizing and metal-tolerant bacteria. Hydrocarbon-utilizing bacteria from petroleum-contaminated soils were isolated on the Bushnell Hass medium. Hydrocarbon degradation by Pseudomonas taiwanensis strain YSA-17 was observed by gas chromatography-mass spectrometry. Bioaccumulation of metals by strain YSA-17 was assessed in nutrient broth. Among different strains, YSA-17 showed the highest potential for hydrocarbon utilization. After 20 days of incubation, YSA-17 completely degraded one compound and during its degradation, there was the formation of 13 new compounds which were absent in uninoculated control. Results of scanning electron microscope and Fourier transmission infrared (FTIR) indicated degradation of hydrocarbons. FTIR showed the formation of new functional groups in YSA-17 inoculated medium. Expression of the total quantity of hydrocarbon-degrading gene (AlkB and NehAc) in petroleum-amended nutrient broth inoculated with strain YSA-17 enhanced significantly during 20 days of incubation compared to control. YSA-17 also significantly removed metals. This study concluded that bioinoculant can be utilized for the bioremediation of pollutants cocontaminated with hydrocarbons and metals. Petroleum-contaminated soil will be remediated for pollutants.


Assuntos
Poluentes Ambientais , Petróleo , Poluentes do Solo , Petróleo/metabolismo , Solo/química , Poluentes do Solo/metabolismo , Microbiologia do Solo , Hidrocarbonetos/metabolismo , Biodegradação Ambiental , Poluentes Ambientais/metabolismo , Bactérias/genética
2.
J Biol Chem ; 296: 100698, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895142

RESUMO

Homologues of the Oscillatoria agardhii agglutinin (OAA) lectins contain a sequence repeat of ∼66 amino acids, with the number of tandem repeats varying across family members. OAA homologues bind high-mannose glycans on viral surface proteins, thereby interfering with viral entry into host cells. As such, OAA homologues have potential utility as antiviral agents, but a more detailed understanding of their structure-function relationships would enable us to develop improved constructs. Here, we determined the X-ray crystal structure of free and glycan-bound forms of Pseudomonas taiwanensis lectin (PTL), an OAA-family lectin consisting of two tandem repeats. Like other OAA-family lectins, PTL exhibited a ß-barrel-like structure with two symmetrically positioned glycan-binding sites at the opposite ends of the barrel. Upon glycan binding, the conformation of PTL undergoes a more significant change than expected from previous OAA structural analysis. Moreover, the electron density of the bound glycans suggested that the binding affinities are different at the two binding sites. Next, based on analysis of these structures, we used site-specific mutagenesis to create PTL constructs expected to increase the population with a conformation suitable for glycan binding. The engineered PTLs were examined for their antiviral activity against the influenza virus. Interestingly, some exhibited stronger activity compared with that of the parent PTL. We propose that our approach is effective for the generation of potential microbicides with enhanced antiviral activity.


Assuntos
Antivirais/metabolismo , Antivirais/farmacologia , Lectinas/metabolismo , Lectinas/farmacologia , Polissacarídeos/metabolismo , Engenharia de Proteínas , Antivirais/química , Cristalografia por Raios X , Lectinas/química , Lectinas/genética , Modelos Moleculares , Orthomyxoviridae/efeitos dos fármacos , Ligação Proteica , Conformação Proteica em Folha beta
3.
Int J Mol Sci ; 21(22)2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33207795

RESUMO

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.


Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional , Oryza/microbiologia , Doenças das Plantas/microbiologia , Pseudomonas , Controle Biológico de Vetores , Pseudomonas/genética , Pseudomonas/metabolismo , Sequenciamento Completo do Genoma , Xanthomonas/crescimento & desenvolvimento
4.
Ecotoxicol Environ Saf ; 170: 479-487, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30553926

RESUMO

Copper, a common heavy metal, may be beneficial for or poisonous to microbial activity. The objective of this study was to determine the effect of different copper ion concentrations on the nitrogen removal performance of Arthrobacter arilaitensis strain Y-10 and Pseudomonas taiwanensis strain J488. The non-competitive inhibition model was employed to evaluate the 50% inhibition concentrations (IC50 values) of copper ions toward the pure strains. In the absence of magnesium ions, a low concentration of copper (0.1 mg/L) significantly enhanced the ammonium removal ability of strain Y-10 and its removal efficiency increased by 10.88% compared with the control treatment. Copper ranging from 0 to 0.1 mg/L had no significant effect on the ammonium removal capacity of strain J488. After adding 9.90 mg/L of magnesium to the basal medium, the effects of copper on nitrification of ammonium or denitrification of nitrate or nitrite were also assessed. In these conditions, 0.25 mg/L copper ions could strongly inhibit the ammonium, nitrate and nitrite removal activities for strain Y-10. For strain J488, no clear deterioration in ammonium removal efficiency was observed at copper ion concentrations below 0.5 mg/L, but 0.25 mg/L copper ions significantly inhibited nitrate and nitrite removal efficiencies, which were only 45.88% and 6.35%, respectively. The IC50 values of copper ions for nitrate and nitrite removal by strain Y-10 were 0.195 and 0.090 mg/L respectively; for strain J488, the IC50 values were 0.175 and 0.196 mg/L. The magnesium ions could improve the cell growth, nitrogen removal efficiency and copper ion resistance of bacteria.


Assuntos
Cobre/química , Magnésio/química , Nitrogênio/isolamento & purificação , Compostos de Amônio/química , Compostos de Amônio/isolamento & purificação , Arthrobacter/efeitos dos fármacos , Arthrobacter/metabolismo , Biodegradação Ambiental , Desnitrificação , Concentração Inibidora 50 , Modelos Teóricos , Nitratos/química , Nitratos/isolamento & purificação , Nitrificação , Nitritos/química , Nitritos/isolamento & purificação , Nitrogênio/química , Pseudomonas/efeitos dos fármacos , Pseudomonas/metabolismo
5.
Biometals ; 31(5): 785-795, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29956040

RESUMO

The structures of three previously unknown siderophores produced by the fluorescent, biotechnologically relevant Pseudomonas taiwanensis (P. taiwanensis) VLB120 bacteria were elucidated by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated to high-resolution tandem mass spectrometry (HRMS/MS). They could be verified as iron(III)- and aluminum(III) complexes as well as the protonated molecules of the siderophores formed by in-source fragmentation. The siderophores were separated according to their different acyl side chains and additionally according their central ions. One of the siderophores was identified as pyoverdine with a malic acid (hydroxy succinic acid) amide side chain and a peptide moiety consisting of Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. The other analytes were assigned to an azotobactin with the identical peptide chain linked to the characteristic chromophoric unit and a pyoverdine with a variation in the amino acid sequence. Proline is directly linked to the pyoverdine chromophore instead of ornithine. Acidic and enzymatic hydrolyses were carried out to analyze the individual amino acids. Beside OHAsn, each amino acid of the peptide part was identified by HILIC-HRMS and comparison to authentic standards. Additionally, 15N-labeled cellular supernatants were analyzed by means of HRMS/MS. The results of the MS/MS experiments complemented by accurate mass data facilitated elucidation of the structures studied in this work and provided further confirmation of the three recently described pyoverdines of P. taiwanensis VLB120 (Baune et al. in Biometals 30:589-597, 2017. https://doi.org/10.1007/s10534-017-0029-7 ).


Assuntos
Nitrogênio/química , Pseudomonas/metabolismo , Sideróforos/análise , Marcação por Isótopo , Sideróforos/biossíntese , Espectrometria de Massas em Tandem
6.
Biometals ; 30(4): 589-597, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28631237

RESUMO

The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.


Assuntos
Complexos de Coordenação/isolamento & purificação , Oligopeptídeos/isolamento & purificação , Pseudomonas putida/química , Pseudomonas/química , Sideróforos/isolamento & purificação , Alumínio/química , Cromatografia Líquida/métodos , Complexos de Coordenação/química , Ferro/química , Estrutura Molecular , Oligopeptídeos/química , Pseudomonas/metabolismo , Pseudomonas putida/metabolismo , Sideróforos/química , Extração em Fase Sólida/métodos
7.
Ecotoxicol Environ Saf ; 145: 8-15, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28689070

RESUMO

Dye dispersion and the interaction efficiency between azoreductases and dye molecules are rate-limiting steps for the decolorization of azo dyes. In this study, a biosurfactant-producing strain, Pseudomonas taiwanensis L1011, was isolated from crude oil. To increase the yield of the biosurfactant BS-L1011 from P. taiwanensis L1011, culture conditions were optimized including temperature, initial pH, carbon source, nitrogen source and C/N ratio. A maximum yield of 1.12g/L of BS-L1011 was obtained using D-mannitol as carbon source and yeast extract/urea as compound nitrogen source with C/N ratio of 10/4, pH 7.0 and 28°C. BS-L1011 exhibited a low critical micelle concentration (CMC) of 10.5mg/L and was able to reduce the surface tension of water to 25.8±0.1 mN/m. BS-L1011 was stable over a wide range of temperatures, pH values and salt concentrations. The biosurfactant is reported for the first time to accelerate chemical decolorization of Congo red by sodium hypochlorite, and biological decolorization of Amaranth by Bacillus circulans BWL1061, thus showing a potential in the treatment of dyeing wastewater.


Assuntos
Compostos Azo/análise , Corantes/análise , Pseudomonas/metabolismo , Tensoativos/química , Poluentes Químicos da Água/análise , Corante Amaranto/análise , Biodegradação Ambiental , Vermelho Congo/análise , NADH NADPH Oxirredutases/química , Nitrorredutases , Petróleo/microbiologia , Pseudomonas/crescimento & desenvolvimento , Pseudomonas/isolamento & purificação , Tensoativos/isolamento & purificação , Águas Residuárias/química
8.
Metab Eng Commun ; 18: e00241, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-39021639

RESUMO

The microbial production of value-added chemicals from renewable feedstocks is an important step towards a sustainable, bio-based economy. Therefore, microbes need to efficiently utilize lignocellulosic biomass and its dominant constituents, such as d-xylose. Pseudomonas taiwanensis VLB120 assimilates d-xylose via the five-step Weimberg pathway. However, the knowledge about the metabolic constraints of the Weimberg pathway, i.e., its regulation, dynamics, and metabolite fluxes, is limited, which hampers the optimization and implementation of this pathway for bioprocesses. We characterized the Weimberg pathway activity of P. taiwanensis VLB120 in terms of biomass growth and the dynamics of pathway intermediates. In batch cultivations, we found excessive accumulation of the intermediates d-xylonolactone and d-xylonate, indicating bottlenecks in d-xylonolactone hydrolysis and d-xylonate uptake. Moreover, the intermediate accumulation was highly dependent on the concentration of d-xylose and the extracellular pH. To encounter the apparent bottlenecks, we identified and overexpressed two genes coding for putative endogenous xylonolactonases PVLB_05820 and PVLB_12345. Compared to the control strain, the overexpression of PVLB_12345 resulted in an increased growth rate and biomass generation of up to 30 % and 100 %, respectively. Next, d-xylonate accumulation was decreased by overexpressing two newly identified d-xylonate transporter genes, PVLB_18545 and gntP (PVLB_13665). Finally, we combined xylonolactonase overexpression with enhanced uptake of d-xylonate by knocking out the gntP repressor gene gntR (PVLB_13655) and increased the growth rate and biomass yield by 50 % and 24 % in stirred-tank bioreactors, respectively. Our study contributes to the fundamental knowledge of the Weimberg pathway in pseudomonads and demonstrates how to encounter the metabolic bottlenecks of the Weimberg pathway to advance strain developments and cell factory design for bioprocesses on renewable feedstocks.

9.
Bioresour Technol ; 388: 129741, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37717703

RESUMO

Polyketides from (hydroxy)benzoates are an interesting group of plant polyphenolic compounds, whose biotechnological production is so far underrepresented due to their challenging heterologous biosynthesis. Efficient heterologous production of 2,4,6-tri- and 2,3',4,6-tetrahydroxybenzophenone, 3,5-dihydroxybiphenyl, and 4-hydroxycoumarin by whole-cell biocatalysis in combination with in situ product extraction with an organic solvent was demonstrated. Production was highly dependent on the used CoA ligase and polyketide synthase type III. Therefore, different combinations of polyketide synthases and benzoate-CoA ligases were evaluated for their biosynthesis performance in the solvent-tolerant Pseudomonas taiwanensis VLB120. A solvent screening yielded 2-undecanone as biocompatible, extraction-efficient solvent with good phase separation. In aqueous-organic two-phase cultivations, this solvent extraction circumvents product instability in the aqueous cultivation medium, and it increases yields by reducing inhibitory effects. Complete de novo synthesis from glucose of all (hydroxy)benzoate-derived polyketides was achieved in two-phase cultivations with metabolically engineered strains. Additionally, mutasynthesis was applied to obtain fluorinated benzophenone derivatives.


Assuntos
Policetídeos , Benzoatos , Plantas , Pseudomonas , Solventes
10.
Bioresour Technol ; 350: 126912, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35231598

RESUMO

To overcome a large amount of nitrite accumulation and poor removal rate for hydroxylamine, a simultaneous nitrification and denitrification (SND) bacterium was isolated and identified as Pseudomonas taiwanensis EN-F2 by DNA sequencing. Strain EN-F2 could remove 100% of ammonium (52.90 mg/L), 100% of hydroxylamine (23.32 mg/L), 86.99% of nitrite (56.32 mg/L) and 89.21% of nitrate (56.18 mg/L) with a maximum removal rate of 8.72, 2.12, 4.55 and 5.80 mg/L/h, respectively. Ammonium and hydroxylamine could be preferentially removed during the SND process. The nitrite removal rate and cell growth were substantially enhanced by 2.10 mg/L/h and 0.45 after supplementation of hydroxylamine. The specific activities of ammonia monooxygenase (AMO), hydroxylamine oxidoreductase (HAO), nitrate reductase (NR), nitrite reductase (NIR) were successfully detected as 0.95, 0.31, 0.42 and 0.03 U/mg protein, respectively. All results demonstrated that strain EN-F2 could perform SND to remove multiple nitrogen sources from wastewater.


Assuntos
Compostos de Amônio , Nitrificação , Aerobiose , Compostos de Amônio/metabolismo , Desnitrificação , Processos Heterotróficos , Hidroxilamina , Hidroxilaminas , Nitritos/metabolismo , Nitrogênio , Pseudomonas
11.
Environ Sci Pollut Res Int ; 29(4): 5526-5540, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34424469

RESUMO

There are many studies on the treatment of heavy metals by manganese-oxidizing bacteria and the reaction is good; the problem of compound pollution of heavy metals in soil has been difficult to solve. In this study, the application of manganese-oxidizing bacteria in soil was studied. The tolerance of manganese-oxidizing strains (Pseudomonas taiwanensis) to environmental conditions and the treatment effect of heavy metals As, Pb, and Cd in aqueous solution were investigated, and the effect of iron-manganese ratio on the treatment effect was discussed. The results showed that the suitable pH conditions for the growth of P. taiwanensis were 5-9, and the salt tolerance was 6% (by sodium chloride). The tolerant concentrations for heavy metals As(V) and Mn(II) were 500 mg L-1 and 120 mg L-1, respectively. The strains were enriched by nutrient broth medium. After the logarithmic phase, the bacterial suspension was mixed with ATCC#279 medium at a ratio of 1:10, and a certain amount (10 mg L-1) of Mn(II) was added. The results of As, Pb, and Cd removal in the composite polluted water phase were 22.09%, 30.75%, and 35.33%, respectively. The molar ratio of manganese and iron affected the removal efficiency of single arsenic, the highest efficiency is 68%, and the ratio of iron to manganese is 1:5. However, when the soil was treated by the same method, the results showed that not all metals were passivated, such as Cu. At the same time, for As, Pb, and Cd, the treatment effects in soil were worse than those in water, perhaps more consideration should be given to environmental conditions, such as soil moisture and temperature, when manganese-oxidizing bacteria are used to treat soil.


Assuntos
Metais Pesados , Poluentes do Solo , Bactérias , Manganês/análise , Metais Pesados/análise , Oxirredução , Solo , Poluentes do Solo/análise
12.
Trends Biotechnol ; 40(4): 385-397, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34482995

RESUMO

Developing cost-efficient biotechnological processes is a major challenge in replacing fossil-based industrial production processes. The remarkable progress in genetic engineering ensures efficient and fast tailoring of microbial metabolism for a wide range of bioconversions. However, improving intrinsic properties such as tolerance, handling, growth, and substrate consumption rates is still challenging. At the same time, synthetic biology tools are becoming easier applicable and transferable to nonmodel organisms. These trends have resulted in the exploitation of new and unconventional microbial systems with sophisticated properties, which render them promising hosts for the bio-based industry. Here, we highlight the metabolic and cellular capabilities of representative prokaryotic newcomers and discuss the potential and drawbacks of these hosts for industrial application.


Assuntos
Biotecnologia , Biologia Sintética , Biotecnologia/métodos , Engenharia Genética , Engenharia Metabólica/métodos
13.
Bioresour Technol ; 341: 125785, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34455248

RESUMO

The nitrogen metabolic pathways of Pseudomonas taiwanensis J488 have not been confirmed from genomic function analysis and its divalent metal ion resistance remains poorly understood. In this study, the key denitrifying gene of Pseudomonas taiwanensis J488, nirB, was determined by draft genome sequencing. The nitrification of ammonium was insensitive to high concentrations of Ca(II), Mn(II), Zn(II), and Cd(II). Similarly, complete nitrite removal was achieved despite Mn(II) and Zn(II) reaching concentrations up to 30 mg/L. Furthermore, the efficiency of nitrate removal was significantly enhanced by 1.33%, 3.33%, 5.99%, and 1.53% with the addition of 0.5 mg/L Ca(II), 20 mg/L Mn(II), 5 mg/L Zn(II), and 2 mg/L Cd(II), respectively, comparison with the control. The bacterial growth in both nitrifying and denitrifying processes was substantially promoted by various dosages of divalent metal ions. These results indicate that divalent metal ions would not severely limit the capacity of strain J488 to purify nitrogen-polluted wastewater.


Assuntos
Compostos de Amônio , Hipotermia , Aerobiose , Desnitrificação , Processos Heterotróficos , Humanos , Nitrificação , Nitritos , Nitrogênio , Pseudomonas , Águas Residuárias
14.
J Hazard Mater ; 388: 122114, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31962213

RESUMO

A hypothermia aerobic denitrifying bacterium, Pseudomonas taiwanensis strain J488, can effectively remove multiple nitrogen sources from wastewater at 15 °C. The ammonium, nitrate and nitrite removal efficiencies were 100 %, 92.61 % and 92.49 %, respectively. Strain J488 could survive with hydroxylamine as sole nitrogen source and its removal efficiency was 97.71 %. The removal efficiency of ammonium was 100 % even in the presence of the classical inhibitors of nitrification allylthiourea and diethyldithiocarbamate. These findings fundamentally changed the picture that the ammonia monooxygenase could be inhibited by the copper chelators of allylthiourea or diethyldithiocarbamate. Similarly, the nitrite removal capacity of strain J488 was not sensitive to inhibition by Pb2+, and its removal efficiency was also 100 %. Additionally, by identifying the intermediates accumulation of nitrification and denitrification, using nitrification and denitrification inhibitors, measuring enzyme activities and determining N2O concentrations, it was demonstrated that N2O could be produced directly from ammonium, nitrate and nitrite.


Assuntos
Compostos de Amônio/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Óxido Nitroso/metabolismo , Pseudomonas/metabolismo , Poluentes Químicos da Água/metabolismo , Desnitrificação , Nitrificação , Águas Residuárias , Purificação da Água
15.
Artigo em Inglês | MEDLINE | ID: mdl-31245364

RESUMO

Aromatic compounds such as 4-hydroxybenzoic acid are broadly applied in industry for a myriad of applications used in everyday life. However, their industrial production currently relies heavily on fossil resources and involves environmentally unfriendly production conditions, thus creating the need for more sustainable biotechnological alternatives. In this study, synthetic biology was applied to metabolically engineer Pseudomonas taiwanensis VLB120 to produce 4-hydroxybenzoate from glucose, xylose, or glycerol as sole carbon sources. Genes encoding a 4-hydroxybenzoate production pathway were integrated into the host genome and the flux toward the central precursor tyrosine was enhanced by overexpressing genes encoding key enzymes of the shikimate pathway. The flux toward tryptophan biosynthesis was decreased by introducing a P290S point mutation in the trpE gene, and degradation pathways for 4-hydroxybenzoate, 4-hydroxyphenylpyruvate and 3-dehydroshikimate were knocked out. The resulting production strains were tailored for the utilization of glucose and glycerol through the rational modification of central carbon metabolism. In batch cultivations with a completely mineral medium, the best strain produced 1.37 mM 4-hydroxybenzoate from xylose with a C-mol yield of 8% and 3.3 mM from glucose with a C-mol yield of 19.0%. Using glycerol as a sole carbon source, the C-mol yield increased to 29.6%. To our knowledge, this is the highest yield achieved by any species in a fully mineral medium. In all, the efficient conversion of bio-based substrates into 4-hydroxybenzoate by these deeply engineered P. taiwanensis strains brings the renewable production of aromatics one step closer.

16.
Artigo em Inglês | MEDLINE | ID: mdl-32039175

RESUMO

High gene expression of enzymes partaking in recombinant production pathways is a desirable trait among cell factories belonging to all different kingdoms of life. High enzyme abundance is generally aimed for by utilizing strong promoters, which ramp up gene transcription and mRNA levels. Increased protein abundance can alternatively be achieved by optimizing the expression on the post-transcriptional level. Here, we evaluated protein synthesis with a previously proposed optimized gene expression architecture, in which mRNA stability and translation initiation are modulated by genetic parts such as self-cleaving ribozymes and a bicistronic design, which have initially been described to support the standardization of gene expression. The optimized gene expression architecture was tested in Pseudomonas taiwanensis VLB120, a promising, novel microbial cell factory. The expression cassette was employed on a plasmid basis and after single genomic integration. We used three constitutive and two inducible promoters to drive the expression of two fluorescent reporter proteins and a short acetoin biosynthesis pathway. The performance was confronted with that of a traditional expression cassette harboring the same promoter and gene of interest but lacking the genetic parts for increased expression efficiency. The optimized expression cassette granted higher protein abundance independently of the expression basis or promoter used proving its value for applications requiring high protein abundance.

17.
Environ Pollut ; 251: 555-563, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31108288

RESUMO

In the present study, the 16S-rRNA sequencing of heavy metal-resistant and susceptible bacterial strains isolated from the industrial and agriculture soil showed resemblance with Pseudomonas taiwanensis. Based on the growth rate, two bacterial strains SJPS_KUD54 and KUD-MBBT4 exhibited 10 ppm tolerance to Arsenic and Cadmium. These two heavy metals caused, a significant increase in stress enzymes like superoxide dismutase, catalase and glutathione S-transferase activities in SJPS_KUD54 when compared to KUD-MBBT4. Following heavy metal treatment, the atomic-force-microscopy observations showed no change in the cell-wall of SJPS_KUD54, whereas the cell-wall of KUD-MBBT4 got ruptured. Moreover, the protein-profile of SJPS_KUD54 treated with heavy metals exhibited varied patterns in comparison with untreated control. In addition, the accumulation of hydroxyl, thiol and amides were found in the SJPS_KUD54 relative to its control. Furthermore, the resistant SJPS_KUD54 strain showed a remarkable bioaccumulation properties to both Arsenic and Cadmium. Thus, it is inferred that the growth rate, stress enzymes and functional-groups play a significant role in the physiological-adaption of SJPS_KUD54 during stress conditions, which is positively involved in the prevention or repair mechanism for reducing the risks caused by heavy metal stress.


Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Arsênio/toxicidade , Cádmio/toxicidade , Pseudomonas/efeitos dos fármacos , Poluentes do Solo/toxicidade , Arsênio/metabolismo , Cádmio/metabolismo , Farmacorresistência Bacteriana/efeitos dos fármacos , Pseudomonas/enzimologia , Pseudomonas/fisiologia , Solo/química , Microbiologia do Solo , Poluentes do Solo/metabolismo
18.
Biotechnol Biofuels ; 11: 199, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30034525

RESUMO

BACKGROUND: Bio-conversion of lignocellulosic biomass to high-value products offers numerous benefits; however, its development is hampered by chemical inhibitors generated during the pretreatment process. A better understanding of how microbes naturally respond to those inhibitors is valuable in the process of designing microorganisms with improved tolerance. Pseudomonas taiwanensis VLB120 is a natively tolerant strain that utilizes a wide range of carbon sources including pentose and hexose sugars. To this end, we investigated the tolerance and metabolic response of P. taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors including organic acids (acetic acid, formic acid, and levulinic acid), furans (furfural, 5-hydroxymethylfurfural), and phenols (vanillin). RESULTS: The inhibitory effect of the tested compounds varied with respect to lag phase, specific growth rate, and biomass yield compared to the control cultures grown under the same conditions without addition of inhibitors. However, P. taiwanensis was able to oxidize vanillin and furfural to vanillic acid and 2-furoic acid, respectively. Vanillic acid was further metabolized, whereas 2-furoic acid was secreted outside the cells and remained in the fermentation broth without further conversion. Acetic acid and formic acid were completely consumed from the fermentation broth, while concentration of levulinic acid remained constant throughout the fermentation process. Analysis of free intracellular metabolites revealed varying levels when P. taiwanensis VLB120 was exposed to inhibitory compounds. This resulted in increased levels of ATP to export inhibitors from the cell and NADPH/NADP ratio that provides reducing power to deal with the oxidative stress caused by the inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of P. taiwanensis in the presence of biomass-derived inhibitors. CONCLUSIONS: In this study, the tolerance and metabolic response of P. taiwanensis VLB120 to biomass hydrolysate-derived inhibitors was investigated. P. taiwanensis VLB120 showed high tolerance towards biomass hydrolysate-derived inhibitors compared to most wild-type microbes reported in the literature. It adopts different resistance mechanisms, including detoxification, efflux, and repair, which require additional energy and resources. Thus, targeting redox and energy metabolism in strain engineering may be a successful strategy to overcome inhibition during biomass hydrolysate conversion and lead to development of more robust strains.

19.
Braz J Microbiol ; 48(2): 187-188, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-26991304

RESUMO

Pseudomonas taiwanensis strain SJ9 is a caprolactam degrader, isolated from industrial wastewater in South Korea and considered to have the potential for caprolactam bioremediation. The genome of this strain is approximately 6.2 Mb (G+C content, 61.75%) with 6,010 protein-coding sequences (CDS), of which 46% are assigned to recognized functional genes. This draft genome of strain SJ9 will provide insights into the genetic basis of its caprolactam-degradation ability.


Assuntos
Caprolactama/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Pseudomonas/genética , Pseudomonas/metabolismo , Análise de Sequência de DNA , Composição de Bases , Biotransformação , Resíduos Industriais , Coreia (Geográfico) , Anotação de Sequência Molecular , Fases de Leitura Aberta , Pseudomonas/isolamento & purificação , Microbiologia da Água
20.
Braz. j. microbiol ; Braz. j. microbiol;48(2): 187-188, April.-June 2017.
Artigo em Inglês | LILACS | ID: biblio-839392

RESUMO

Abstract Pseudomonas taiwanensis strain SJ9 is a caprolactam degrader, isolated from industrial wastewater in South Korea and considered to have the potential for caprolactam bioremediation. The genome of this strain is approximately 6.2 Mb (G + C content, 61.75%) with 6,010 protein-coding sequences (CDS), of which 46% are assigned to recognized functional genes. This draft genome of strain SJ9 will provide insights into the genetic basis of its caprolactam-degradation ability.


Assuntos
Pseudomonas/genética , Pseudomonas/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/química , Caprolactama/metabolismo , Genoma Bacteriano , Análise de Sequência de DNA , Pseudomonas/isolamento & purificação , Composição de Bases , Microbiologia da Água , Biotransformação , Fases de Leitura Aberta , Anotação de Sequência Molecular , Resíduos Industriais , Coreia (Geográfico)
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