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1.
J Am Chem Soc ; 144(38): 17516-17521, 2022 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-36102697

RESUMO

Non-natural light-driven enzymatic reactivity was recently developed to perform the highly stereoselective reactions required for pharmaceutical synthesis. However, photoenzymes require high-intensity light to function because of the poor absorption properties of their photoactive intermediates. Inspired by the modular architecture of photosynthesis, we designed a conjugate composed of a covalently linked photoenzyme and a light antenna to separate light capture from catalysis. Spectroscopic characterization of the conjugate showed the presence of efficient energy transfer from the light-harvesting components to the photoenzyme. In the presence of energy transfer, a ∼4-fold increase in product yield was observed for intramolecular hydroalkylation of alkenes, and reactivity was enabled for intermolecular hydroalkylation of alkenes. These improvements establish the power of incorporating nature's design into non-natural photoenzymatic catalysis.


Assuntos
Gluconobacter , Oxirredutases , Alcenos , Catálise , Luz , Preparações Farmacêuticas , Fotossíntese
2.
Biosci Biotechnol Biochem ; 86(5): 681-690, 2022 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-35150230

RESUMO

We identified a novel flavoprotein-cytochrome c complex d-gluconate dehydrogenase (GADH) encoded by gndXYZ of Gluconobacter oxydans NBRC 3293, which is phylogenetically distinct from previously reported GADHs encoded by gndFGH and gndSLC of Gluconobacter spp. To analyze the biochemical properties of respective GADHs, Gluconobacter japonicus NBRC 3271 mutant strain lacking membranous d-gluconate dehydrogenase activity was constructed. All GADHs (GndFGH, GndSLC, and GndXYZ) were successfully overexpressed in the constructed strain. The optimal pH and KM value at that pH of GndFGH, GndSLC, and GndXYZ were 5, 6, and 4, and 8.82 ± 1.15, 22.9 ± 5.0, and 11.3 ± 1.5 m m, respectively. When the mutants overexpressing respective GADHs were cultured in d-glucose-containing medium, all of them produced 2-keto-d-gluconate, revealing that GndXYZ converts d-gluconate to 2-keto-d-gluconate as well as other GADHs. Among the recombinants, the gndXYZ-overexpressing strain accumulated the highest level of 2-keto-d-gluconate, suggesting its potential for 2-keto-d-gluconate production.


Assuntos
Gluconobacter oxydans , Gluconobacter , Gluconatos/química , Gluconobacter/genética , Gluconobacter oxydans/genética , Oxirredutases
3.
Biotechnol Appl Biochem ; 69(3): 1190-1198, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34009642

RESUMO

Glycerol is an abundant byproduct of biodiesel production that has significant industrial value and can be converted into dihydroxyacetone (DHA). DHA is widely used for the production of various chemicals, pharmaceuticals, and food additives. Gluconobacter can convert glycerol to DHA through two different pathways, including membrane-bound dehydrogenases with pyrroloquinoline quinone (PQQ) and NAD(P)+ -dependent enzymes. Previous work has indicated that membrane-bound dehydrogenases are present in Gluconobacter oxydans and Gluconobacter frateurii, but the metabolic mechanism of Gluconobacter thailandicus's glycerol conversion is still not clear. Through in-depth analysis of the G. thailandicus genome and annotation of its metabolic pathways, we revealed the existence of both PQQ and NAD(P)+ -dependent enzymes in G. thailandicus. In addition, this study provides important information related to the tricarboxylic acid cycle, glycerol dehydrogenase level, and phylogenetic relationships of this important species.


Assuntos
Genoma Bacteriano , Gluconobacter , Glicerol , Microrganismos Geneticamente Modificados , Ciclo do Ácido Cítrico/genética , Di-Hidroxiacetona/metabolismo , Engenharia Genética , Genoma Bacteriano/genética , Gluconobacter/genética , Gluconobacter/metabolismo , Glicerol/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , NAD/metabolismo , NADP/metabolismo , Cofator PQQ/metabolismo , Filogenia , Desidrogenase do Álcool de Açúcar/análise
4.
Biosensors (Basel) ; 11(11)2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34821682

RESUMO

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 µA mM-1cm-2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.


Assuntos
Técnicas Biossensoriais , Grafite , Hexoses , Nanopartículas Metálicas , Reatores Biológicos , Desidrogenases de Carboidrato , Enzimas Imobilizadas , Frutose , Galactose , Gluconobacter/enzimologia , Ouro , Hexoses/análise
5.
Int J Biol Macromol ; 193(Pt A): 693-701, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34737079

RESUMO

A new strain of bacterial cellulose (BC)-producing Gluconobacter cerinus HDX-1 was isolated and identified, and a simple, low-cost complexation method was used to biosynthesis Lactobacillus paracasei 1∙7 bacteriocin BC (BC-B) nanofiber. The structure and antibacterial properties of the nanofibers were evaluated. Solid-state nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR) and x-ray diffraction (XRD) analysis showed that BC and BC-B nanofibers had typical crystalline form of the cellulose I. X-ray photoelectron spectrometer (XPS), scanning electron microscope (SEM) and atomic force microscopy (AFM) revealed that the bacteriocin and BC were successfully compounded, and the structure of BC-B nanofiber was tighter than BC nanofiber, with lower porosity, swelling ratio and water vapor transmission rate (WVTR). The tensile strength and Young's modulus of BC-B nanofibers were 13.28 ± 1.26 MPa and 132.10 ± 4.92 MPa, respectively, higher than that of BC nanofiber (6.12 ± 0.87 MPa and 101.59 ± 5.87 MPa), indicating that bacteriocin enhance the mechanical properties of BC nanofiber. Furthermore, the BC-B nanofibers exhibited significant thermal stability, antioxidant capacity and antibacterial activity than BC nanofiber. Therefore, bacteriocin-loaded BC nanofiber may be used as antimicrobial agents in active food packaging and medical material.


Assuntos
Bacteriocinas/química , Celulose/química , Gluconobacter/metabolismo , Química Verde , Antibacterianos/química , Antioxidantes , Bactérias/efeitos dos fármacos , Bacteriocinas/farmacologia , Celulose/isolamento & purificação , DNA Ribossômico , Módulo de Elasticidade , Gluconobacter/isolamento & purificação , Testes de Sensibilidade Microbiana , Nanofibras/química , Espectroscopia Fotoeletrônica , Espectroscopia de Infravermelho com Transformada de Fourier , Resistência à Tração , Difração de Raios X
6.
Sci Rep ; 11(1): 18792, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34552134

RESUMO

Kombucha is a beverage made by fermenting sugared tea using a symbiotic culture of bacteria belonging to the genus Acetobacter, Gluconobacter, and the yeasts of the genus Saccharomyces along with glucuronic acid, which has health-promoting properties. The paper presents the evaluation of ferments as a potential cosmetic raw material obtained from Yerba Mate after different fermentation times with the addition of Kombucha. Fermented and unfermented extracts were compared in terms of chemical composition and biological activity. The antioxidant potential of obtained ferments was analyzed by evaluating the scavenging of external and intracellular free radicals. Cytotoxicity was determined on keratinocyte and fibroblast cell lines, resulting in significant increase in cell viability for the ferments. The ferments, especially after 14 and 21 days of fermentation showed strong ability to inhibit (about 40% for F21) the activity of lipoxygenase, collagenase and elastase enzymes and long-lasting hydration after their application on the skin. Moreover, active chemical compounds, including phenolic acids, xanthines and flavonoids were identified by HPLC/ESI-MS. The results showed that both the analyzed Yerba Mate extract and the ferments obtained with Kombucha may be valuable ingredients in cosmetic products.


Assuntos
Cosméticos/metabolismo , Ilex paraguariensis , Chá de Kombucha , Acetobacter/metabolismo , Cosméticos/farmacologia , Fármacos Dermatológicos/metabolismo , Fármacos Dermatológicos/farmacologia , Fermentação , Gluconobacter/metabolismo , Células HaCaT/efeitos dos fármacos , Humanos , Ilex paraguariensis/metabolismo , Concentração Inibidora 50 , Metaloproteinases da Matriz/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Saccharomyces/metabolismo , Fatores de Tempo
7.
Appl Microbiol Biotechnol ; 105(18): 6835-6852, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34448898

RESUMO

For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3' region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. KEY POINTS: • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.


Assuntos
Gluconobacter oxydans , Gluconobacter , Ácido Acético , Expressão Gênica , Gluconobacter oxydans/genética , Plasmídeos/genética
8.
Appl Microbiol Biotechnol ; 105(18): 6749-6758, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34453563

RESUMO

In contrast to D-glyceric acid (D-GA) production with 99% enantiomeric excess (ee) by Acetobacter tropicalis NBRC 16470, Gluconobacter sp. CHM43 produced 19.6 g L-1 of D-GA with 73.7% ee over 4 days of incubation in flask culture. To investigate the reason for this enantiomeric composition of GA, the genes encoding membrane-bound alcohol dehydrogenase (mADH) of A. tropicalis NBRC 16470, composed of three subunits (adhA, adhB, and adhS), were cloned using the broad-host-range vector pBBR1MCS-2 and heterologously expressed in Gluconobacter sp. CHM43 and its ΔadhAB ΔsldBA derivative TORI4. Reverse-transcription quantitative real-time polymerase chain reaction demonstrated that adhABS genes from A. tropicalis were expressed in TORI4 transformants, and their membrane fraction exhibited mADH activities of 0.13 and 0.31 U/mg with or without AdhS, respectively. Compared with the GA production of TORI4-harboring pBBR1MCS-2 (1.23 g L-1), TORI4 transformants expressing adhABS and adhAB showed elevated GA production of 2.46 and 3.67 g L-1, respectively, suggesting a negative effect of adhS gene expression on GA production as well as mADH activity in TORI4. Although TORI4 was found to produce primarily L-GA with 42.5% ee, TORI4 transformants expressing adhABS and adhAB produced D-GA with 27.6% and 49.0% ee, respectively, demonstrating that mADH of A. tropicalis causes a sharp increase in the enantiomeric composition of D-GA. These results suggest that one reason for D-GA production with 73.7% ee in Gluconobacter spp. might be a property of the host, which possibly produces L-GA intracellularly. KEY POINTS: • Membrane-bound ADH from Acetobacter tropicalis showed activity in Gluconobacter sp. • D-GA production from glycerol was performed using recombinant Gluconobacter sp. • Enantiomeric excess of D-GA was affected by both membrane and intracellular ADHs.


Assuntos
Gluconobacter , Acetobacter , Álcool Desidrogenase , Gluconobacter/genética , Ácidos Glicéricos
9.
J Bacteriol ; 203(19): e0055820, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34309403

RESUMO

Gluconobacter sp. strain CHM43 oxidizes mannitol to fructose and then oxidizes fructose to 5-keto-d-fructose (5KF) in the periplasmic space. Since NADPH-dependent 5KF reductase was found in the soluble fraction of Gluconobacter spp., 5KF might be transported into the cytoplasm and metabolized. Here, we identified the GLF_2050 gene as the kfr gene encoding 5KF reductase (KFR). A mutant strain devoid of the kfr gene showed lower KFR activity and no 5KF consumption. The crystal structure revealed that KFR is similar to NADP+-dependent shikimate dehydrogenase (SDH), which catalyzes the reversible NADP+-dependent oxidation of shikimate to 3-dehydroshikimate. We found that several amino acid residues in the putative substrate-binding site of KFR were different from those of SDH. Phylogenetic analyses revealed that only a subclass in the SDH family containing KFR conserved such a unique substrate-binding site. We constructed KFR derivatives with amino acid substitutions, including replacement of Asn21 in the substrate-binding site with Ser that is found in SDH. The KFR-N21S derivative showed a strong increase in the Km value for 5KF but a higher shikimate oxidation activity than wild-type KFR, suggesting that Asn21 is important for 5KF binding. In addition, the conserved catalytic dyad Lys72 and Asp108 were individually substituted for Asn. The K72N and D108N derivatives showed only negligible activities without a dramatic change in the Km value for 5KF, suggesting a catalytic mechanism similar to that of SDH. With these data taken together, we suggest that KFR is a new member of the SDH family. IMPORTANCE A limited number of species of acetic acid bacteria, such as Gluconobacter sp. strain CHM43, produce 5-ketofructose, a potential low-calorie sweetener, at a high yield. Here, we show that an NADPH-dependent 5-ketofructose reductase (KFR) is involved in 5-ketofructose degradation, and we characterize this enzyme with respect to its structure, phylogeny, and function. The crystal structure of KFR was similar to that of shikimate dehydrogenase, which is functionally crucial in the shikimate pathway in bacteria and plants. Phylogenetic analysis suggested that KFR is positioned in a small subgroup of the shikimate dehydrogenase family. Catalytically important amino acid residues were also conserved, and their relevance was experimentally validated. Thus, we propose KFR as a new member of shikimate dehydrogenase family.


Assuntos
Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/metabolismo , Gluconobacter/enzimologia , Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/classificação , Desidrogenases de Carboidrato/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Gluconobacter/genética , Gluconobacter/metabolismo , Modelos Moleculares , Filogenia , Conformação Proteica
10.
Biosci Biotechnol Biochem ; 85(4): 998-1004, 2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33686415

RESUMO

We characterized the pyrroloquinoline quinone (PQQ)-dependent dehydrogenase 9 (PQQ-DH9) of Gluconobacter sp. strain CHM43, which is a homolog of PQQ-dependent glycerol dehydrogenase (GLDH). We used a plasmid construct to express PQQ-DH9. The expression host was a derivative strain of CHM43, which lacked the genes for GLDH and the membrane-bound alcohol dehydrogenase and consequently had minimal ability to oxidize primary and secondary alcohols. The membranes of the transformant exhibited considerable d-arabitol dehydrogenase activity, whereas the reference strain did not, even if it had PQQ-DH9-encoding genes in the chromosome and harbored the empty vector. This suggests that PQQ-DH9 is not expressed in the genome. The activities of the membranes containing PQQ-DH9 and GLDH suggested that similar to GLDH, PQQ-DH9 oxidized a wide variety of secondary alcohols but had higher Michaelis constants than GLDH with regard to linear substrates such as glycerol. Cyclic substrates such as cis-1,2-cyclohexanediol were readily oxidized by PQQ-DH9.


Assuntos
Gluconobacter/metabolismo , Oxirredutases/metabolismo , Cofator PQQ/metabolismo , Álcool Desidrogenase/metabolismo , Genoma Bacteriano , Plasmídeos , Álcoois Açúcares/metabolismo
11.
Rev Argent Microbiol ; 53(4): 343-348, 2021.
Artigo em Espanhol | MEDLINE | ID: mdl-33618898

RESUMO

Microbial food alterations lead to unfit products for consumption, and their discarding, to significant economic losses for the food industry. During storage, fresh foods offer available niches for the survival and growth of undesirable microorganisms. In dairy products, data regarding spoilage and/or pathogenic bacteria is better documented than those for molds and yeasts. Dairy products are less susceptible to mold's contamination than products such as fruits and vegetables, due to their refrigerated storage; their elaboration from heat-treated milk and, for fermented ones, the dominant microbiota that acidifies the medium. However, even cheeses and yogurts may be susceptible to mold contamination. Atypical cases of yogurt samples containing spoilage microorganisms not previously reported (molds producing gas and bacteria of the genus Gluconobacter) in Argentinean fermented milks are presented here. For yogurt, in particular, the "classic" altering organisms were always being yeasts, and in other countries, molds belonging to the genus Aspergillus.


Assuntos
Gluconobacter , Iogurte , Bactérias , Microbiologia de Alimentos , Fungos , Leveduras , Iogurte/análise
12.
Appl Microbiol Biotechnol ; 105(6): 2441-2453, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33616697

RESUMO

There is an increasing public awareness about the danger of dietary sugars with respect to their caloric contribution to the diet and the rise of overweight throughout the world. Therefore, low-calorie sugar substitutes are of high interest to replace sugar in foods and beverages. A promising alternative to natural sugars and artificial sweeteners is the fructose derivative 5-keto-D-fructose (5-KF), which is produced by several Gluconobacter species. A prerequisite before 5-KF can be used as a sweetener is to test whether the compound is degradable by microorganisms and whether it is metabolized by the human microbiota. We identified different environmental bacteria (Tatumella morbirosei, Gluconobacter japonicus LMG 26773, Gluconobacter japonicus LMG 1281, and Clostridium pasteurianum) that were able to grow with 5-KF as a substrate. Furthermore, Gluconobacter oxydans 621H could use 5-KF as a carbon and energy source in the stationary growth phase. The enzymes involved in the utilization of 5-KF were heterologously overproduced in Escherichia coli, purified and characterized. The enzymes were referred to as 5-KF reductases and belong to three unrelated enzymatic classes with highly different amino acid sequences, activities, and structural properties. Furthermore, we could show that 15 members of the most common and abundant intestinal bacteria cannot degrade 5-KF, indicating that this sugar derivative is not a suitable growth substrate for prokaryotes in the human intestine. KEY POINTS: • Some environmental bacteria are able to use 5-KF as an energy and carbon source. • Four 5-KF reductases were identified, belonging to three different protein families. • Many gut bacteria cannot degrade 5-KF.


Assuntos
Bactérias , Edulcorantes , Bactérias/genética , Clostridium , Frutose/análogos & derivados , Gammaproteobacteria , Gluconobacter , Humanos
13.
J Oleo Sci ; 70(3): 289-295, 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33583924

RESUMO

Glyceric acid (GA) is an oxidative product of glycerol, and its d-isomer is obtained as a phytochemical from tobacco leaves and fruits of some plants. However, the production and applications of GA have not yet been fully investigated. In this review, recent developments in the microbial production of GA and its application to bio-related materials are summarized. The sodium salt of diacylated GA showed superior surface tension-lowering activity and antitrypsin activity. GA and its glucosyl derivative had positive effects on the viability and collagen production of skin cells in vitro, respectively. Glucosyl derivatives of GA showed protective effects against heat-induced protein aggregation. In addition, the microbial production of GA using raw glycerol as the starting material was investigated. The effect of methanol, a major impurity in raw glycerol, on GA production was investigated, and mutant strains to tolerate methanol in the culture were constructed. Enantioselective production of GA using newly isolated microbial strains has also been developed.


Assuntos
Acetobacter/metabolismo , Gluconobacter/metabolismo , Ácidos Glicéricos/metabolismo , Antituberculosos , Biocombustíveis , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Fermentação , Ácidos Glicéricos/química , Ácidos Glicéricos/farmacologia , Glicerol , Isomerismo , Oxirredução , Agregação Patológica de Proteínas/prevenção & controle , Pele/citologia , Pele/metabolismo , Tensoativos
14.
Appl Microbiol Biotechnol ; 105(3): 1227-1236, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33475798

RESUMO

Dihydroxyacetone (DHA), a chemical suntan agent, is produced by the regiospecific oxidation of glycerol with Gluconobacter thailandicus NBRC3255. However, this microorganism consumes DHA produced in the culture medium. Here, we attempted to understand the pathway for DHA metabolism in NBRC3255 to minimize DHA degradation. The two gene products, NBRC3255_2003 (DhaK) and NBRC3255_3084 (DerK), have been annotated as DHA kinases in the NBRC 3255 draft genome. Because the double deletion derivative for dhaK and derK showed ATP-dependent DHA kinase activity similar to that of the wild type, we attempted to purify DHA kinase from ∆dhaK ∆derK cells to identify the gene for DHA kinase. The identified gene was NBRC3255_0651, of which the product was annotated as glycerol kinase (GlpK). Mutant strains with several combinations of deletions for the dhaK, derK, and glpK genes were constructed. The single deletion strain ∆glpK showed approximately 10% of wild-type activity and grew slower on glycerol than the wild type. The double deletion strain ∆derK ∆glpK and the triple deletion strain ∆dhaK ∆derK ∆glpK showed DHA kinase activity less than a detection limit and did not grow on glycerol. In addition, although ΔderK ΔglpK consumed a small amount of DHA in the late phase of growth, ∆dhaK ΔderK ΔglpK did not show DHA consumption on glucose-glycerol medium. The transformants of the ∆dhaK ΔderK ΔglpK strain that expresses one of the genes from plasmids showed DHA kinase activity. We concluded that all three DHA kinases, DhaK, DerK, and GlpK, are involved in DHA metabolism of G. thailandicus. KEY POINTS: • Dihydroxyacetone (DHA) is produced but degraded by Gluconobacter thailandicus. • Phosphorylation rather than reduction is the first committed step in DHA metabolism. • Three kinases are involved in DHA metabolism with the different properties.


Assuntos
Di-Hidroxiacetona , Gluconobacter , Trifosfato de Adenosina , Glicerol
15.
Carbohydr Polym ; 255: 117384, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436213

RESUMO

Prebiotics are known for their ability to modulate the composition of the human microbiome and mediate health-promoting benefits. Endo-levanases, which hydrolyze levan into short-chain FOS, could be used for the production of levan-based prebiotics. The novel endo-levanase (LevB2286) from Azotobacter chroococcum DSM 2286, combines an exceptionally high specific activity with advantageous hydrolytic properties. Starting from levan isolated from Timothy grass, LevB2286 produced FOS ranging from DP 2 - 8. In contrast to endo-levanases described in the literature, LevB2286 formed minor amounts of fructose and levanbiose, even with greatly extended incubation. The combined activity of LevB2286 and the levansucrase LevS1417 from Gluconobacter japonicus LMG 1417 led to a one-step synthesis of levan-type FOS from sucrose. 387.4 ± 17.3 g L-1 FOS were produced within 48 h by the production strategy based on crude cell extract of recombinant Escherichia coli expressing levS1417 and levB2286 simultaneously.


Assuntos
Azotobacter/enzimologia , Proteínas de Bactérias/metabolismo , Gluconobacter/enzimologia , Glicosídeo Hidrolases/metabolismo , Hexosiltransferases/metabolismo , Oligossacarídeos/biossíntese , Prebióticos/análise , Azotobacter/genética , Proteínas de Bactérias/genética , Dissacarídeos/química , Dissacarídeos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Frutanos/química , Frutanos/metabolismo , Frutose/química , Frutose/metabolismo , Expressão Gênica , Gluconobacter/genética , Glicosídeo Hidrolases/genética , Hexosiltransferases/genética , Humanos , Hidrólise , Oligossacarídeos/química , Phleum/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sacarose/química , Sacarose/metabolismo
16.
Biochim Biophys Acta Gen Subj ; 1865(1): 129740, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32956752

RESUMO

BACKGROUND: In acetic acid bacteria such as Gluconobacter oxydans or Gluconobacter cerinus, pyrroloquinoline quinone (PQQ) in the periplasm serves as the redox cofactor for several membrane-bound dehydrogenases that oxidize polyhydric alcohols to rare sugars, which can be used as a healthy alternative for traditional sugars and sweeteners. These oxidation reactions obey the generally accepted Bertrand Hudson's rule, in which only the polyhydric alcohols that possess cis d-erythro hydroxyl groups can be oxidized to 2-ketoses using PQQ as a cofactor, while the polyhydric alcohols excluding cis d-erythro hydroxyl groups ruled out oxidation by PQQ-dependent membrane-bound dehydrogenases. METHODS: Membrane fractions of G. oxydans were prepared and used as a cell-free catalyst to oxidize galactitol, with or without PQQ as a cofactor. RESULTS: In this study, we reported an interesting oxidation reaction that the polyhydric alcohols galactitol (dulcitol), which do not possess cis d-erythro hydroxyl groups, can be oxidized by PQQ-dependent membrane-bound dehydrogenase(s) of acetic acid bacteria at the C-3 and C-5 hydroxyl groups to produce rare sugars l-xylo-3-hexulose and d-tagatose. CONCLUSIONS: This reaction may represent an exception to Bertrand Hudson's rule. GENERAL SIGNIFICANCE: Bertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria. In this study, galactitol oxidation by a PQQ-dependent membrane-bound dehydrogenase represents an exception to the Bertrand Hudson's rule. Further identification of the associated enzymes and deciphering the explicit enzymatic mechanism will prove this theory.


Assuntos
Ácido Acético/metabolismo , Galactitol/metabolismo , Gluconobacter/metabolismo , Hexoses/metabolismo , Cetoses/metabolismo , Proteínas de Bactérias/metabolismo , Gluconobacter/enzimologia , Oxirredução , Oxirredutases/metabolismo , Cofator PQQ/metabolismo
17.
Appl Microbiol Biotechnol ; 104(21): 9267-9282, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32974745

RESUMO

The acetic acid bacterium (AAB) Gluconobacter oxydans incompletely oxidizes a wide variety of carbohydrates and is therefore used industrially for oxidative biotransformations. For G. oxydans, no system was available that allows regulatable plasmid-based expression. We found that the L-arabinose-inducible PBAD promoter and the transcriptional regulator AraC from Escherichia coli MC4100 performed very well in G. oxydans. The respective pBBR1-based plasmids showed very low basal expression of the reporters ß-glucuronidase and mNeonGreen, up to 480-fold induction with 1% L-arabinose, and tunability from 0.1 to 1% L-arabinose. In G. oxydans 621H, L-arabinose was oxidized by the membrane-bound glucose dehydrogenase, which is absent in the multi-deletion strain BP.6. Nevertheless, AraC-PBAD performed similar in both strains in the exponential phase, indicating that a gene knockout is not required for application of AraC-PBAD in wild-type G. oxydans strains. However, the oxidation product arabinonic acid strongly contributed to the acidification of the growth medium in 621H cultures during the stationary phase, which resulted in drastically decreased reporter activities in 621H (pH 3.3) but not in BP.6 cultures (pH 4.4). These activities could be strongly increased quickly solely by incubating stationary cells in D-mannitol-free medium adjusted to pH 6, indicating that the reporters were hardly degraded yet rather became inactive. In a pH-controlled bioreactor, these reporter activities remained high in the stationary phase (pH 6). Finally, we created a multiple cloning vector with araC-PBAD based on pBBR1MCS-5. Together, we demonstrated superior functionality and good tunability of an AraC-PBAD system in G. oxydans that could possibly also be used in other AAB. KEY POINTS: • We found the AraC-PBAD system from E. coli MC4100 was well tunable in G. oxydans. • In the absence of AraC or l-arabinose, expression from PBAD was extremely low. • This araC-PBAD system could also be fully functional in other acetic acid bacteria.


Assuntos
Gluconobacter oxydans , Gluconobacter , Ácido Acético , Arabinose , Escherichia coli/genética , Gluconobacter oxydans/genética , Plasmídeos/genética
18.
Int J Food Microbiol ; 333: 108797, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-32738750

RESUMO

Acetic acid bacteria form a complex microbiota that plays a fundamental role in the industrial production of vinegar through the incomplete oxidation reaction from ethanol to acetic acid. The organoleptic properties and the quality of vinegar are influenced by many factors, especially by the raw material used as acetification substrate, the microbial diversity and the technical methods employed in its production. The metaproteomics has been considered, among the new methods employed for the investigation of microbial communities, since it may provide information about the microbial biodiversity and behaviour by means of a protein content analysis. In this work, alcohol wine vinegar was produced through a submerged culture of acetic acid bacteria using a pilot acetator, operated in a semi-continuous mode, where the main system variables were monitored and the cycle profile throughout the acetification was obtained. Through a first approach, at qualitative level, of a metaproteomic analysis performed at relevant moments of the acetification cycle (end of fast and discontinuous loading phases and just prior to unloading phase), it is aimed to investigate the microbiota existent in alcohol wine vinegar as well as its changes during the cycle; to our knowledge, this is the first metaproteomics report carried out in this way on this system. A total of 1723 proteins from 30 different genera were identified; 1615 out of 1723 proteins (93.73%) belonged to the four most frequent (%) genera: Acetobacter, Gluconacetobacter, Gluconobacter and Komagataeibacter. Around 80% of identified proteins belonged to the species Komagataeibacter europaeus. In addition, GO Term enrichment analysis highlighted the important role of catalytic activity, organic cyclic compound binding, metabolic and biosynthesis processes throughout acetic acid fermentation. These findings provide the first step to obtain an AAB profile at omics level related to the environmental changes produced during the typical semi-continuous cycles used in this process and it would contribute to the optimization of operating conditions and improving the industrial production of vinegar.


Assuntos
Ácido Acético/metabolismo , Acetobacter/metabolismo , Reatores Biológicos/microbiologia , Gluconacetobacter/metabolismo , Gluconobacter/metabolismo , Acetobacter/genética , Biodiversidade , Etanol/metabolismo , Fermentação/fisiologia , Gluconacetobacter/genética , Gluconobacter/genética , Microbiota/genética , Vinho/microbiologia
19.
Int J Biol Macromol ; 164: 295-303, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32679320

RESUMO

Levan, a ß-2,6-glycosidic linked fructan, is a promising alternative for the inulin dominated fructan market. Although levan is already used in some cosmetic products, the commercial availability of the fructan is still limited. Here we show that Gluconobacter japonicus LMG 1417 is a potent levan-forming organism and a promising platform for the industrial production of levan. The levansucrase LevS1417, which is produced by G. japonicus LMG 1417 and secreted by a signal-peptide-independent pathway, exhibited extraordinary high activity (4726 ±â€¯821 U mg-1 at 50 °C). A cell-free levan production based on the supernatant of the investigated strain led to a final levan yield of 157.9 ±â€¯7.6 g L-1. The amount of secreted levansucrase was more than doubled by plasmid-mediated homologous overproduction of LevS1417 in G. japonicus LMG 1417. Accordingly, the space-time yield of cell-free levan production was doubled using the plasmid-bearing mutant.


Assuntos
Frutanos/biossíntese , Gluconobacter/metabolismo , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Fibras na Dieta , Ativação Enzimática , Escherichia coli , Frutanos/isolamento & purificação , Expressão Gênica , Gluconobacter/enzimologia , Hexosiltransferases/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Plasmídeos/genética , Prebióticos , Espectroscopia de Infravermelho com Transformada de Fourier
20.
Cell Rep ; 32(3): 107938, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32698005

RESUMO

Necrotic cells elicit an inflammatory response through their endogenous factors with damage-associated molecular patterns. Blocking apoptosis in Drosophila wings leads to the necrosis-driven systemic immune response by unknown mechanisms. Here, we demonstrate that immune activation in response to necrotic cells is mediated by commensal gut microbiota. Removing the microbiome attenuates hyperactivation of the innate immune signaling IMD pathway in necrosis-induced flies. Necrotic cells in wings trigger Gluconobacter expansion in the gut. An isolated Gluconobacter sp. strain is sufficient for pathological IMD activation in necrosis-induced flies, while it is not inflammatory for control animals. In addition, bacterial colonization shifts the host metabolome and shortens the lifespan of necrosis-induced flies. This study shows that local necrosis triggers a pathological systemic inflammatory response through interaction between the host and the dysbiotic gut microbiome.


Assuntos
Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Disbiose/imunologia , Disbiose/patologia , Microbioma Gastrointestinal/imunologia , Animais , Contagem de Colônia Microbiana , Gluconobacter/crescimento & desenvolvimento , Necrose , Transdução de Sinais , Asas de Animais/imunologia
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