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1.
J Appl Microbiol ; 128(1): 171-181, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31559676

RESUMO

AIMS: We aimed to explore a new Methylobacterium isolate to produce polyhydroxybutyrate (PHB) by using methanol as a sole carbon resource and improve PHB production. METHODS AND RESULTS: A new PHB-producing isolate (Methylobacterium sp. 1805) was obtained from oil fields by using methanol as a sole carbon source. The fermentation situation of PHB production was further optimized by using Box-Behnken response surface methodology (RSM). Before optimization, the cell biomass was 0·6 g l-1 after 3-day culture and 0·3 g l-1 PHB was produced after 5-day methanol-inducing stage. The RSM growth medium was optimized as 15 g l-1 glycerol, 10 g l-1 beef extract and 0·65 g l-1 MgSO4 ·7H2 O. The RSM methanol-inducing medium was optimized as 0·65 g l-1 MgSO4 (metal ions), 20 mmol l-1 PBS pH 6·5 and final 2% methanol (v/v). The biomass and PHB production reached 1·0 and 0·55 g l-1 after 3-day culture, respectively. The PHB yield increased by about 80% when compared with before optimization. CONCLUSIONS: The optimization of a two-stage fermentation process improved PHB production from methanol by using Methylobacterium sp. 1805. SIGNIFICANCE AND IMPACT OF THE STUDY: A new Methylobacterium isolate was isolated and produced high-level PHB by using methanol as a sole carbon resource. The bacteria will provide a potential tool for C1 resource in producing PHB.


Assuntos
Hidroxibutiratos/metabolismo , Metanol/metabolismo , Methylobacterium/metabolismo , Campos de Petróleo e Gás/microbiologia , Poliésteres/metabolismo , Biomassa , Meios de Cultura , Fermentação , Methylobacterium/crescimento & desenvolvimento , Methylobacterium/isolamento & purificação , Microbiologia do Solo
2.
Chemosphere ; 239: 124686, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31494321

RESUMO

Oxidative degradation of aniline in aqueous solution was performed by the sono-activated peroxydisulfate coupled with PbO process, wherein a dramatic synergistic effect was found. Experiments were carried out in the batch-wise mode to investigate the influence of various operation parameters on the sonocatalytic behavior, such as ultrasonic power intensity, peroxydisulfate anion concentrations and PbO dosages. According to the scavenging effect of ethanol, methanol and tert-butyl alcohol, the principal oxidizing agents were presumed to be sulfate radicals descended from peroxydisulfate anions, activated via ultrasound or sonocatalysis of PbO. Based on the results attained from gas chromatograph-mass spectrometer, it was hypothesized that aniline was initially oxidized into iminobenzene radicals, followed with formation of nitrosobenzene, p-benzoquinonimine and nitrobenzene respectively. Condensation of nitrosobenzene with aniline generated azobenzene. Phenol was detected as one of degradation intermediates, which was sequentially converted into hydroquinone and p-benzoquinone.


Assuntos
Compostos de Anilina/química , Chumbo/química , Óxidos/química , Fenol/química , Sulfatos/química , Compostos Azo/síntese química , Benzoquinonas/síntese química , Etanol/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hidroquinonas/síntese química , Metanol/metabolismo , Nitrobenzenos/síntese química , Compostos Nitrosos/síntese química , Oxidantes , Oxirredução , Semicondutores , Ondas Ultrassônicas , terc-Butil Álcool/metabolismo
3.
Microb Cell Fact ; 18(1): 187, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675969

RESUMO

BACKGROUND: The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. RESULTS: The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. CONCLUSIONS: PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression-heterologous genes included-, thus directly influencing the production kinetics of recombinant protein.


Assuntos
Oxirredutases do Álcool/genética , Proteínas Fúngicas/metabolismo , Metanol/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Dosagem de Genes , Expressão Gênica , Regulação Fúngica da Expressão Gênica , Pichia/genética , Regiões Promotoras Genéticas
4.
PLoS Genet ; 15(11): e1008458, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31710603

RESUMO

While microbiologists often make the simplifying assumption that genotype determines phenotype in a given environment, it is becoming increasingly apparent that phenotypic heterogeneity (in which one genotype generates multiple phenotypes simultaneously even in a uniform environment) is common in many microbial populations. The importance of phenotypic heterogeneity has been demonstrated in a number of model systems involving binary phenotypic states (e.g., growth/non-growth); however, less is known about systems involving phenotype distributions that are continuous across an environmental gradient, and how those distributions change when the environment changes. Here, we describe a novel instance of phenotypic diversity in tolerance to a metabolic toxin within wild-type populations of Methylobacterium extorquens, a ubiquitous phyllosphere methylotroph capable of growing on the methanol periodically released from plant leaves. The first intermediate in methanol metabolism is formaldehyde, a potent cellular toxin that is lethal in high concentrations. We have found that at moderate concentrations, formaldehyde tolerance in M. extorquens is heterogeneous, with a cell's minimum tolerance level ranging between 0 mM and 8 mM. Tolerant cells have a distinct gene expression profile from non-tolerant cells. This form of heterogeneity is continuous in terms of threshold (the formaldehyde concentration where growth ceases), yet binary in outcome (at a given formaldehyde concentration, cells either grow normally or die, with no intermediate phenotype), and it is not associated with any detectable genetic mutations. Moreover, tolerance distributions within the population are dynamic, changing over time in response to growth conditions. We characterized this phenomenon using bulk liquid culture experiments, colony growth tracking, flow cytometry, single-cell time-lapse microscopy, transcriptomics, and genome resequencing. Finally, we used mathematical modeling to better understand the processes by which cells change phenotype, and found evidence for both stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic shifts, depending on the growth substrate and the presence of toxin.


Assuntos
Heterogeneidade Genética , Variação Genética/genética , Metanol/metabolismo , Methylobacterium extorquens/genética , Tolerância a Medicamentos/genética , Formaldeído/química , Formaldeído/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genótipo , Methylobacterium extorquens/metabolismo , Fenótipo , Folhas de Planta/química
5.
Curr Microbiol ; 76(12): 1520-1524, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31515628

RESUMO

In this study, a wild type Methylophilus sp. strain D22 belonging to the family Methylophilus was isolated and characterized, which shows high tolerance towards methanol, as it can grow under 50 g/L of methanol. Methylophilus sp. strain D22 was isolated from the lake sludge in Nanjing Tech University, China. The assembled draft genome contains one circular chromosome with 3,004,398 bp, 49.7% of GC content, and 2107 predicted encoding proteins. Sequence-based genomic analysis demonstrates that the assimilation pathway of ribulose monophosphate (RuMP) pathway and dissimilation pathway of tetrahydromethanopterin (H4MPT) pathway are co-existing and contribute to the high methanol utilization efficiency.


Assuntos
Genoma Bacteriano , Metanol/metabolismo , Methylophilus/genética , Composição de Bases , Sequência de Bases , China , DNA Bacteriano/genética , Metanol/análise , Methylophilus/crescimento & desenvolvimento , Methylophilus/metabolismo , Pentoses/metabolismo , Filogenia
6.
Appl Microbiol Biotechnol ; 103(18): 7805-7817, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31414164

RESUMO

The application of lipases in liquid formulation instead of immobilized forms in the enzymatic biodiesel synthesis can make the process cost-efficient, more competitive, and sustainable. However, despite the benefits, the long reaction times required to achieve satisfactory yields is still a drawback of this biotechnological process. In this sense, employing the novel low-cost soluble NS40116 lipase, this paper proposes an innovative two-step hydroesterification reaction (TSHR) system as a technique of improving the reaction rate of an enzymatic biodiesel production. With the employment of two central composite statistical design to optimize the parameters of each of the reactions involved, the influence of the parameters "water concentration added to the reaction," "methanol-to-oil molar ratio," and "lipase load" on the process yield, besides the acid value of the samples, was investigated. After only 8 h of reaction, the highest fatty acid methyl ester yield reached was 97.1% with an acid value of 4.62 mg KOH g-1 utilizing a total of 8 wt% water, methanol-to-oil molar ratio of 6.3:1, and 0.70 wt% of lipase. Furthermore, the statistical models for both reactions indicated to be significant with 95% of reliability. Considering that the papers published using soluble lipases in a one-step batch process normally reach similar yields to those obtained in this research after 16 h to 24 h of reaction, the proposed system demonstrated to be a promising option of process configuration for the enzymatic production of biodiesel.


Assuntos
Biocombustíveis , Lipase/metabolismo , Metanol/metabolismo , Óleos Vegetais/metabolismo , Biotecnologia , Catálise , Esterificação , Ácidos Graxos/metabolismo , Reprodutibilidade dos Testes
7.
Enzyme Microb Technol ; 130: 109366, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31421726

RESUMO

This study investigates how sorbitol/methanol mixed induction affects fermentation performance, dewatering characteristics of cells during harvesting and the profile of host cell proteins (HCP) in the process fluid when producing the target recombinant protein aprotinin. Compared to standard methanol induction, sorbitol/methanol (1:1, C-mol/C-mol) mixed induction improved cellular viability from 92.8 ±â€¯0.3% to 97.7 ±â€¯0.1% although resulted in a reduced product yield from 1.65 ±â€¯0.03 g L-1 to 1.12 ±â€¯0.07 g L-1. On the other hand, average oxygen consumption rate (OUR) dropped from 241.4 ±â€¯21.3 mmol L-1 h-1 to 145.5 ±â€¯6.7 mmol L-1 h-1. Cell diameter decreased over time in the mixed induction, resulting in a D50 value of 3.14 µm at harvest compared to 3.85 µm with methanol. The reduction in cell size enhanced the maximum dewatering efficiency from 78.1 ±â€¯3.9% to 84.5 ±â€¯3.3% as evaluated by using an established ultra scale-down methodology that models pilot and industrial scale disc stack centrifugation. Seventy host cell proteins (HCPs) were identified in clarified supernatant when using sorbitol/methanol mixed induction regimen. The total number of HCPs identified with standard methanol induction was nearly one hundred. The downstream process advantage of the mixed induction lies in improved product purity by reducing both cell mortality and level of released whole cell proteins. This needs to be balanced and optimised against the observed reduction in product yield during fermentation.


Assuntos
Centrifugação , Metanol/metabolismo , Pichia/metabolismo , Sorbitol/metabolismo , Biomassa , Sobrevivência Celular , Fermentação , Oxigênio/metabolismo , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes
8.
Genes (Basel) ; 10(8)2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31409011

RESUMO

Hirudin and its variants, as strong inhibitors against thrombin, are present in the saliva of leeches and are recognized as potent anticoagulants. However, their yield is far from the clinical requirement up to now. In this study, the production of hirudin variant 3 (HV3) was successfully realized by cultivating the recombinant Pichia pastoris GS115/pPIC9K-hv3 under the regulation of the promoter of AOX1 encoding alcohol oxidase (AOX). The antithrombin activity in the fermentation broth reached the maximum value of 5000 ATU/mL. To explore an effective strategy for improving HV3 production in the future, we investigated the influence of methanol assimilation on the general gene expression in this recombinant by transcriptomic study. The results showed that methanol was partially oxidized into CO2, and the rest was converted into glycerone-P which subsequently entered into central carbon metabolism, energy metabolism, and amino acid biosynthesis. However, the later metabolic processes were almost all down-regulated. Therefore, we propose that the up-regulated central carbon metabolism, energy, and amino acid metabolism should be beneficial for methanol assimilation, which would accordingly improve the production of HV3.


Assuntos
Hirudinas/genética , Metanol/metabolismo , Pichia/genética , Transcriptoma , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hirudinas/metabolismo , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
BMC Biotechnol ; 19(1): 37, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208387

RESUMO

BACKGROUD: Fusaruside is an immunomodulatory fungal sphingolipid which has medical potentials for treating colitis and liver injury, but its poor natural abundance limits its further study. RESULTS: In this study, we described a synthetic biology approach for fusaruside production by engineered Pichia pastoris that was based on polycistronic expression. Two fusaruside biosynthesis genes (Δ3(E)-sd and Δ10(E)-sd), were introduced into P. pastoris to obtain fusaruside producing strain FUS2. To further enhance the yield of fusaruside, three relevant biosynthetic genes (Δ3(E)-sd, Δ10(E)-sd and gcs) were subsequently introduced into P. pastoris to obtain FUS3. All of the biosynthetic genes were successfully co-expressed in FUS2 and FUS3. Compared to that produced by FUS2, fusaruside achieved from FUS3 were slightly increased. In addition, the culture conditions including pH, temperature and methanol concentration were optimized to improve the fusaruside production level. CONCLUSIONS: Here a novel P. pastoris fusaruside production system was developed by introducing the biosynthetic genes linked by 2A peptide gene sequences into a polycistronic expression construct, laying a foundation for further development and application of fusaruside.


Assuntos
Cerebrosídeos/metabolismo , Proteínas Fúngicas/metabolismo , Fatores Imunológicos/metabolismo , Engenharia Metabólica/métodos , Pichia/metabolismo , Cerebrosídeos/química , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metanol/metabolismo , Metanol/farmacologia , Modelos Químicos , Estrutura Molecular , Pichia/genética , Esfingolipídeos/metabolismo , Temperatura
10.
Nat Commun ; 10(1): 2675, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209220

RESUMO

Aerobic methane oxidation is catalyzed by particulate methane monooxygenase (pMMO), a copper-dependent, membrane metalloenzyme composed of subunits PmoA, PmoB, and PmoC. Characterization of the copper active site has been limited by challenges in spectroscopic analysis stemming from the presence of multiple copper binding sites, effects of detergent solubilization on activity and crystal structures, and the lack of a heterologous expression system. Here we utilize nanodiscs coupled with native top-down mass spectrometry (nTDMS) to determine the copper stoichiometry in each pMMO subunit and to detect post-translational modifications (PTMs). These results indicate the presence of a mononuclear copper center in both PmoB and PmoC. pMMO-nanodisc complexes with a higher stoichiometry of copper-bound PmoC exhibit increased activity, suggesting that the PmoC copper site plays a role in methane oxidation activity. These results provide key insights into the pMMO copper centers and demonstrate the ability of nTDMS to characterize complex membrane-bound metalloenzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Espectrometria de Massas/métodos , Methylococcaceae/metabolismo , Modelos Moleculares , Oxigenases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cobre/química , Cobre/metabolismo , Microscopia Crioeletrônica , Metano/metabolismo , Metanol/metabolismo , Methylococcaceae/química , Methylococcaceae/ultraestrutura , Oxirredução , Oxigenases/química , Oxigenases/ultraestrutura , Processamento de Proteína Pós-Traducional
11.
Mol Biotechnol ; 61(9): 663-673, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31228008

RESUMO

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.


Assuntos
Hidrolases de Éster Carboxílico/genética , Proteínas Fúngicas/genética , Gossypium/genética , Larva/efeitos dos fármacos , Metanol/toxicidade , Mariposas/efeitos dos fármacos , Proteínas de Plantas/genética , Agrobacterium/genética , Agrobacterium/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Parede Celular/parasitologia , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Gossypium/parasitologia , Herbivoria/efeitos dos fármacos , Herbivoria/fisiologia , Inseticidas/química , Inseticidas/toxicidade , Larva/patogenicidade , Metanol/metabolismo , Mariposas/patogenicidade , Células Vegetais/metabolismo , Células Vegetais/parasitologia , Folhas de Planta/genética , Folhas de Planta/parasitologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transgenes
12.
ISME J ; 13(8): 2107-2119, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31040382

RESUMO

Methyl substrates are important compounds for methanogenesis in marine sediments but diversity and carbon utilization by methylotrophic methanogenic archaea have not been clarified. Here, we demonstrate that RNA-stable isotope probing (SIP) requires 13C-labeled bicarbonate as co-substrate for identification of methylotrophic methanogens in sediment samples of the Helgoland mud area, North Sea. Using lipid-SIP, we found that methylotrophic methanogens incorporate 60-86% of dissolved inorganic carbon (DIC) into lipids, and thus considerably more than what can be predicted from known metabolic pathways (~40% contribution). In slurry experiments amended with the marine methylotroph Methanococcoides methylutens, up to 12% of methane was produced from CO2, indicating that CO2-dependent methanogenesis is an alternative methanogenic pathway and suggesting that obligate methylotrophic methanogens grow in fact mixotrophically on methyl compounds and DIC. Although methane formation from methanol is the primary pathway of methanogenesis, the observed high DIC incorporation into lipids is likely linked to CO2-dependent methanogenesis, which was triggered when methane production rates were low. Since methylotrophic methanogenesis rates are much lower in marine sediments than under optimal conditions in pure culture, CO2 conversion to methane is an important but previously overlooked methanogenic process in sediments for methylotrophic methanogens.


Assuntos
Dióxido de Carbono/metabolismo , Euryarchaeota/fisiologia , Metano/metabolismo , Methanosarcinaceae/fisiologia , Biomassa , Carbono/metabolismo , Euryarchaeota/crescimento & desenvolvimento , Sedimentos Geológicos , Metanol/metabolismo , Methanosarcinaceae/crescimento & desenvolvimento , Mar do Norte
13.
Science ; 364(6440): 566-570, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31073062

RESUMO

Bacteria that oxidize methane to methanol are central to mitigating emissions of methane, a potent greenhouse gas. The nature of the copper active site in the primary metabolic enzyme of these bacteria, particulate methane monooxygenase (pMMO), has been controversial owing to seemingly contradictory biochemical, spectroscopic, and crystallographic results. We present biochemical and electron paramagnetic resonance spectroscopic characterization most consistent with two monocopper sites within pMMO: one in the soluble PmoB subunit at the previously assigned active site (CuB) and one ~2 nanometers away in the membrane-bound PmoC subunit (CuC). On the basis of these results, we propose that a monocopper site is able to catalyze methane oxidation in pMMO.


Assuntos
Cobre/química , Metano/metabolismo , Metanol/metabolismo , Methylococcus capsulatus/enzimologia , Oxigenases/química , Domínio Catalítico , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Oxirredução , Conformação Proteica
14.
Appl Microbiol Biotechnol ; 103(14): 5879-5889, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139899

RESUMO

Bacillus methanolicus is a thermophilic, Gram-positive, rod-shaped bacterium. It is a facultative methylotroph which can use carbon and energy sources including mannitol and the one-carbon (C1) and non-food substrate methanol for growth and overproduction of amino acids, which makes it a promising candidate for biotechnological applications. Despite a growing tool box for gene cloning and expression, tools for targeted chromosomal gene knockouts and gene repression are still missing for this organism. Here, the CRISPRi-dCas9 technique for gene repression was established in B. methanolicus MGA3. Significantly reduced spore formation on the one hand and increased biofilm formation on the other hand could be demonstrated when the stage zero sporulation protein A gene spo0A was targeted. Furthermore, when the mannitol-1-phosphate 5-dehydrogenase gene mtlD was targeted by CRISPRi, mtlD RNA levels, and MtlD specific activities in crude extracts were decreased to about 50 % which resulted in reduced biomass formation from mannitol. As a third target, the catalase gene katA was chosen. Upon targeting katA by CRISPRi, catalase activity was decreased to about 25 % as shown in H2O2 drop assays and by determination of specific catalase activity in crude extracts. Our results support the predicted functions of Spo0A in sporulation and biofilm formation, of MtlD for mannitol catabolism, and of catalase in hydrogen peroxide dismutation. Thus, CRISPR interference as developed here serves as basis for the functional characterization of B. methanolicus physiology as well as for its application in biotechnology.


Assuntos
Bacillus/genética , Bacillus/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Peróxido de Hidrogênio/metabolismo , Manitol/metabolismo , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Catalase/genética , Catalase/metabolismo , Clonagem Molecular , Expressão Gênica , Inativação Gênica , Metanol/metabolismo , Análise de Sequência de DNA , Esporos/fisiologia
15.
ISME J ; 13(8): 2005-2017, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30952993

RESUMO

Lanthanides (Ln3+), known as rare earth elements, have recently emerged as enzyme cofactors, contrary to prior assumption of their biological inertia. Several bacterial alcohol dehydrogenases have been characterized so far that depend on Ln3+ for activity and expression, belonging to the methanol dehydrogenase clade XoxF and the ethanol dehydrogenase clade ExaF/PedH. Here we compile an inventory of genes potentially encoding Ln3+-dependent enzymes, closely related to the previously characterized XoxF and ExaF/PedH enzymes. We demonstrate their wide distribution among some of the most numerically abundant and environmentally important taxa, such as the phylogenetically disparate rhizobial species and metabolically versatile bacteria inhabiting world's oceans, suggesting that reliance on Ln3+-mediated biochemistry is much more widespread in the microbial world than previously assumed. Through protein expression and analysis, we here more than double the extant collection of the biochemically characterized Ln3+-dependent enzymes, demonstrating a range of catalytic properties and substrate and cofactor specificities. Many of these enzymes reveal propensity for oxidation of methanol. This observation, in combination with genome-based reconstruction of methylotrophy pathways for select species suggests a much wider occurrence of this metabolic capability among bacterial species, and thus further suggests the importance of methylated compounds as parts of the global carbon cycling.


Assuntos
Álcool Desidrogenase/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Coenzimas/metabolismo , Elementos da Série dos Lantanídeos/metabolismo , Álcool Desidrogenase/genética , Bactérias/classificação , Bactérias/metabolismo , Proteínas de Bactérias/genética , Metanol/metabolismo , Filogenia
16.
Appl Environ Microbiol ; 85(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31003982

RESUMO

Mycobacterium smegmatis and several other mycobacteria are able to utilize methanol as the sole source of carbon and energy. We recently showed that N,N-dimethyl-p-nitrosoaniline (NDMA)-dependent methanol dehydrogenase (Mno) is essential for the growth of M. smegmatis on methanol. Although Mno from this bacterium shares high homology with other known methanol dehydrogenases, methanol metabolism in M. smegmatis differs significantly from that of other described methylotrophs. In this study, we dissect the regulatory mechanism involved in the methylotrophic metabolism in M. smegmatis We identify a two-component system (TCS), mnoSR, that is involved in the regulation of mno expression. We show that the MnoSR TCS is comprised of a sensor kinase (MnoS) and a response regulator (MnoR). Our results demonstrate that MnoS undergoes autophosphorylation and is able to transfer its phosphate to MnoR by means of phosphotransferase activity. Furthermore, MnoR shows specific binding to the putative mno promoter region in vitro, thus suggesting its role in the regulation of mno expression. Additionally, we find that the MnoSR system is involved in the regulation of MSMEG_6239, which codes for a putative 1,3-propanediol dehydrogenase. We further show that M. smegmatis lacking mnoSR is unable to utilize methanol and 1,3-propanediol as the sole carbon source, which confirms the role of MnoSR in the regulation of alcohol metabolism. Our data, thus, suggest that the regulation of mno expression in M. smegmatis provides new insight into the regulation of methanol metabolism, which furthers our understanding of methylotrophy in mycobacteria.IMPORTANCE Methylotrophic metabolism has gained huge attention considering its broad application in ecology, agriculture, industries, and human health. The genus Mycobacterium comprises both pathogenic and nonpathogenic species. Several members of this genus are known to utilize methanol as the sole carbon source for growth. Although various pathways underlying methanol utilization have been established, the regulation of methylotrophic metabolism is not well studied. In the present work, we explore the regulation of methanol metabolism in M. smegmatis and discover a dedicated two-component system (TCS), MnoSR, that is involved in its regulation. We show that the loss of MnoSR renders the bacterium incapable of utilizing methanol and 1,3-propanediol as the sole carbon sources. Additionally, we establish that MnoS acts as the common sensor for the alcohols in M. smegmatis.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Metanol/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas Quinases/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/genética , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Proteínas Quinases/genética
17.
mBio ; 10(2)2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967465

RESUMO

Methylomicrobium buryatense 5GB1 is an obligate methylotroph which grows on methane or methanol with similar growth rates. It has long been assumed that the core metabolic pathways must be similar on the two substrates, but recent studies of methane metabolism in this bacterium suggest that growth on methanol might have significant differences from growth on methane. In this study, both a targeted metabolomics approach and a 13C tracer approach were taken to understand core carbon metabolism in M. buryatense 5GB1 during growth on methanol and to determine whether such differences occur. Our results suggest a systematic shift of active core metabolism in which increased flux occurred through both the Entner-Doudoroff (ED) pathway and the partial serine cycle, while the tricarboxylic acid (TCA) cycle was incomplete, in contrast to growth on methane. Using the experimental results as constraints, we applied flux balance analysis to determine the metabolic flux phenotype of M. buryatense 5GB1 growing on methanol, and the results are consistent with predictions based on ATP and NADH changes. Transcriptomics analysis suggested that the changes in fluxes and metabolite levels represented results of posttranscriptional regulation. The combination of flux balance analysis of the genome-scale model and the flux ratio from 13C data changed the solution space for a better prediction of cell behavior and demonstrated the significant differences in physiology between growth on methane and growth on methanol.IMPORTANCE One-carbon compounds such as methane and methanol are of increasing interest as sustainable substrates for biological production of fuels and industrial chemicals. The bacteria that carry out these conversions have been studied for many decades, but gaps exist in our knowledge of their metabolic pathways. One such gap is the difference between growth on methane and growth on methanol. Understanding such metabolism is important, since each has advantages and disadvantages as a feedstock for production of chemicals and fuels. The significance of our research is in the demonstration that the metabolic network is substantially altered in each case and in the delineation of these changes. The resulting new insights into the core metabolism of this bacterium now provide an improved basis for future strain design.


Assuntos
Regulação Bacteriana da Expressão Gênica , Metano/metabolismo , Metanol/metabolismo , Methylococcaceae/genética , Methylococcaceae/metabolismo , Isótopos de Carbono/análise , Perfilação da Expressão Gênica , Marcação por Isótopo , Análise do Fluxo Metabólico , Redes e Vias Metabólicas/genética , Metabolômica , Methylococcaceae/crescimento & desenvolvimento
18.
Prep Biochem Biotechnol ; 49(6): 606-615, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929565

RESUMO

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (kLa) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by MutS P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2 kW m-3. Specific growth rate, and then dextranase production, increased as kLa and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2 L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut+ strain. After a scale-up process, at constant kLa, the specific growth rate was kept at 30 L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30 L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.


Assuntos
Dextranase/biossíntese , Pichia/genética , Proteínas Recombinantes/biossíntese , Oxirredutases do Álcool/genética , Biomassa , Reatores Biológicos , Dextranase/genética , Fermentação , Proteínas Fúngicas/análise , Glicerol/análise , Glicerol/metabolismo , Engenharia Metabólica/métodos , Metanol/análise , Metanol/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Talaromyces/enzimologia , Talaromyces/genética
19.
Chemistry ; 25(37): 8760-8768, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-30908783

RESUMO

We report the first electrochemical study of a lanthanoid-dependent methanol dehydrogenase (Eu-MDH) from the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV with its own physiological cytochrome cGJ electron acceptor. Eu-MDH harbours a redox active 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) cofactor which is non-covalently bound but coordinates trivalent lanthanoid elements including Eu3+ . Eu-MDH and the cytochrome were co-adsorbed with the biopolymer chitosan and cast onto a mercaptoundecanol (MU) monolayer modified Au working electrode. Cyclic voltammetry of cytochrome cGJ reveals a well-defined quasi-reversible FeIII/II redox couple at +255 mV vs. NHE at pH 7.5 and this response is pH independent. The reversible one-electron response of the cytochrome cGJ transforms into a sigmoidal catalytic wave in the presence of Eu-MDH and its substrates (methanol or formaldehyde). The catalytic current was pH-dependent and pH 7.3 was found to be optimal. Kinetic parameters (pH dependence, activation energy) obtained by electrochemistry show the same trends as those obtained from an artificial phenazine ethosulfate/dichlorophenol indophenol assay.


Assuntos
Oxirredutases do Álcool/metabolismo , Citocromos c/química , Európio/química , Oxirredutases do Álcool/química , Biocatálise , Domínio Catalítico , Citocromos c/metabolismo , Técnicas Eletroquímicas , Eletrodos , Cinética , Metanol/química , Metanol/metabolismo , Oxirredução , Cofator PQQ/química , Cofator PQQ/metabolismo , Espectrofotometria , Especificidade por Substrato , Temperatura , Verrucomicrobia/enzimologia
20.
FEMS Microbiol Lett ; 366(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30869784

RESUMO

In this study, production of fungal phytase in thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC656 employing methanol-inducible OtAOX promoter and sucrose-inducible OtMal promoter was investigated in a high cell density fed-batch fermentation. Although a similar maximum cell concentration was obtained in both expression systems, the OtMal system gave ~2-fold higher phytase activity, specific yield, production yield, volumetric productivity and specific productivity rate compared with the OtAOX system. In addition to being more efficient, the OtMal system is more flexible because sucrose or sugarcane molasses can be utilized as less expensive carbon sources instead of glycerol in batch and fed-batch stages. Phytase yields from the OtMal system produced using sucrose or sugarcane molasses are comparable with those obtained with glycerol. We estimate the cost of phytase production by the OtMal system using sucrose or sugarcane molasses to be ~85% lower than the OtAOX system.


Assuntos
6-Fitase/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Microbiologia Industrial/métodos , Proteínas Recombinantes/genética , Saccharomycetales/genética , Sacarose/farmacologia , 6-Fitase/metabolismo , Aspergillus niger/enzimologia , Aspergillus niger/genética , Contagem de Células , Fermentação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Metanol/metabolismo , Melaço , Regiões Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Sacarose/metabolismo , Termotolerância
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