RESUMO
Gluconobacter strains perform incomplete oxidation of various sugars and alcohols, employing regio- and stereoselective membrane-bound dehydrogenases oriented toward the periplasmic space. This oxidative fermentation process is utilized industrially. The ketogluconate production pathway, characteristic of these strains, begins with the conversion of d-glucose to d-gluconate, which then diverges and splits into 2 pathways producing 5-keto-d-gluconate and 2-keto-d-gluconate and subsequently 2,5-diketo-d-gluconate. These transformations are facilitated by membrane-bound d-glucose dehydrogenase, glycerol dehydrogenase, d-gluconate dehydrogenase, and 2-keto-d-gluconate dehydrogenase. The variance in end products across Gluconobacter strains stems from the diversity of enzymes and their activities. This review synthesizes biochemical and genetic knowledge with biotechnological applications, highlighting recent advances in metabolic engineering and the development of an efficient production process focusing on enzymes relevant to the ketogluconate production pathway in Gluconobacter strains.
Assuntos
Biotecnologia , Gluconatos , Gluconobacter , Desidrogenase do Álcool de Açúcar , Gluconatos/metabolismo , Gluconobacter/metabolismo , Gluconobacter/enzimologia , Gluconobacter/genética , Biotecnologia/métodos , Fermentação , Engenharia Metabólica/métodos , Glucose/metabolismo , Glucose 1-Desidrogenase/metabolismo , Glucose 1-Desidrogenase/genéticaRESUMO
Gluconobacter is a potential strain for single-step production of 2-keto-L-gulonic acid (2-KLG), which is the direct precursor of vitamin C. Three dehydrogenases, namely, sorbitol dehydrogenase (SLDH), sorbose dehydrogenase (SDH), and sorbosone dehydrogenase (SNDH), are involved in the production of 2-KLG from D-sorbitol. In the present study, the potential SNDH/SDH gene cluster in the strain Gluconobacter cerinus CGMCC 1.110 was mined by genome analysis, and its function in transforming L-sorbose to 2-KLG was verified. Proteomic analysis showed that the expression level of SNDH/SDH had a great influence on the titer of 2-KLG, and fermentation results showed that SDH was the rate-limiting enzyme. A systematic metabolic engineering process, which was theoretically suitable for increasing the titer of many products involving membrane-bound dehydrogenase from Gluconobacter, was then performed to improve the 2-KLG titer in G. cerinus CGMCC 1.110 from undetectable to 51.9 g/L in a 5-L bioreactor after fermentation optimization. The strategies used in this study may provide a reference for mining other potential applications of Gluconobacter. KEY POINTS: ⢠The potential SNDH/SDH gene cluster in G. cerinus CGMCC 1.110 was mined. ⢠A systematic engineering process was performed to improve the titer of 2-KLG. ⢠The 2-KLG titer was successfully increased from undetectable to 51.9 g/L.
Assuntos
Gluconacetobacter , Gluconobacter , Proteômica , Açúcares Ácidos/metabolismo , Sorbose/metabolismo , Gluconobacter/metabolismo , Gluconacetobacter/metabolismoRESUMO
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 â¼4-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 , FotossínteseRESUMO
5-Ketogluconate (5KGA) is a precursor for synthesizing tartrate, a valuable compound used in several industries. In a previous study, Gluconobacter japonicus NBRC 3271 mutant strain D2, which lacks two membranous gluconate 2-dehydrogenases, was shown to produce 5KGA but not 2-ketogluconate from a mixture of glucose and gluconate. In this study, we aimed to develop an efficient 5KGA production system using G. japonicus D2 as the parental strain. D2 produced 5KGA from glucose in a jar fermentor culture; however, 5KGA levels were reduced during the late phase of cultivation. To increase the potential of D2 for 5KGA production, the cytoplasmic metabolism related to the utilization of 5KGA and gluconate was modified; the gno and gntK genes encoding 5KGA reductase and gluconokinase, respectively, were deleted from D2, generating D4. Improved 5KGA production was observed in D4 compared to that in D2, but a significant amount of gluconate remained at the end of cultivation, leading to an unsatisfied yield of 0.83 mol (mol glucose)-1. The conversion of gluconate to 5KGA is catalyzed by pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which easily forms an apoenzyme by releasing PQQ and calcium ions. Thus, the effects of CaCl2 addition to the culture medium on 5KGA production by D4 were investigated. We demonstrated that 1 mM CaCl2 addition positively affected the maintenance of the PQQ-GLDH activity toward gluconate and consequently enhanced 5KGA production, and the yield reached 0.97 mol (mol glucose)-1. KEY POINTS: ⢠An efficient 5KGA production system was developed with Gluconobacter japonicus. ⢠Deleting the gno and gntK genes blocked the catabolism of 5KGA and gluconate. ⢠The addition of 1 mM CaCl2 efficiently improved the conversion of glucose to 5KGA.
Assuntos
Gluconobacter , Cloreto de Cálcio , Gluconatos/metabolismo , Cofator PQQ/metabolismo , Glucose/metabolismoRESUMO
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/análiseRESUMO
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 , OxirredutasesRESUMO
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 ProteicaRESUMO
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 GlicéricosRESUMO
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 , GlicerolRESUMO
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/genéticaRESUMO
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 , HumanosRESUMO
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/metabolismoRESUMO
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/análiseRESUMO
Two bacterial strains, isolates AC10T and AC20, which were reported in a previous study on the diversity of acetic acid bacteria in Thailand, were subjected to a taxonomic study. The phylogenetic analysis based on the 16S rRNA gene sequences showed that the two isolates were located closely to the type strains of Gluconobacter oxydans and Gluconobacter roseus. However, the two isolates formed a separate cluster from the type strains of the two species. The genomic DNA of isolate AC10T was sequenced. The assembled genomes of the isolate were analysed for average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH). The results showed that the highest ANI and dDDH values between isolate AC10T and G. oxydans DSM 3503T were 91.15 and 68.2â%, which are lower than the suggested values for species delineation. The genome-based tree was reconstructed and the phylogenetic lineage based on genome sequences showed that the lineage of isolate AC10T was distinct from G. oxydans DSM 3503T and its related species. The two isolates were distinguished from G. oxydans and their relatives by their phenotypic characteristics and MALDI-TOF profiles. Therefore, the two isolates, AC10T (=BCC 15749T=TBRC 11329T=NBRC 103576T) and AC20 (=BCC 15759=TBRC 11330=NBRC 103579), can be assigned to an independent species within the genus Gluconobacter, and the name Gluconobacter aidae sp. nov. is proposed for the two isolates.
Assuntos
Frutas/microbiologia , Gluconobacter/classificação , Filogenia , Ácido Acético , Ananas/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , Citrullus/microbiologia , DNA Bacteriano/genética , Gluconobacter/isolamento & purificação , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , TailândiaRESUMO
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/genéticaRESUMO
Many bacteria and archaea produce the polydisperse fructose polymer levan from sucrose upon biofilm formation via extracellular levansucrases (EC 2.4.1.10). We have investigated levansucrase-release and -activities as well as molecular size of the levan formed by the acetic acid bacterium Gluconobacter albidus TMW 2.1191 at varying environmental pH conditions to obtain insight in the ecological role of its constitutively expressed levansucrase and the produced levan. A buffer system was established enabling the recovery of levansucrase-containing supernatants from preincubated cell suspensions at pH 4.3-pH 5.7. The enzyme solutions were used to produce levans at different pH values and sucrose concentrations. Finally, the amounts and size distributions of the produced levans as well as the corresponding levansucrase activities were determined and correlated with each other. The data revealed that the levansucrase was released into the environment independently of its substrate sucrose, and that more levansucrase was released at pH ≥ 5.0. The glucose release and formation of high molecular weight levans (> 3.5 kDa) from 0.1 M initial sucrose was comparable between pH ~ 4.3-5.7 using equal amounts of released levansucrase. Hence, this type of levansucrase appears to be structurally adapted to changes in the extracellular pH and to exhibit a similar total activity over a wide acidic pH range, while it produced higher amounts of larger levan molecules at higher production pH and sucrose concentrations. These findings indicate the physiological adaptation of G. albidus TMW 2.1191 to efficient colonisation of sucrose-rich habitats via released levansucrases despite changing extracellular pH conditions in course of acid formation.
Assuntos
Frutanos/metabolismo , Gluconobacter/enzimologia , Gluconobacter/metabolismo , Hexosiltransferases/metabolismo , Sacarose/metabolismo , Metabolismo dos Carboidratos , Frutose/metabolismo , Hexosiltransferases/química , Concentração de Íons de Hidrogênio , Peso MolecularRESUMO
GLUCONOBACTER FRATEURII: CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.
Assuntos
Proteínas de Bactérias/metabolismo , Desidrogenases de Carboidrato/genética , Fermentação/genética , Frutose/análogos & derivados , Gluconobacter/enzimologia , Manitol Desidrogenases/metabolismo , Proteínas de Bactérias/genética , Desidrogenases de Carboidrato/metabolismo , Membrana Celular/enzimologia , Membrana Celular/genética , Frutose/biossíntese , Frutose/isolamento & purificação , Expressão Gênica , Gluconobacter/genética , Humanos , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Manitol/metabolismo , Manitol Desidrogenases/genética , EstereoisomerismoRESUMO
Few data have been published on the occurrence and functional role of acetic acid bacteria (AAB) in lambic beer production processes, mainly due to their difficult recovery and possibly unknown role. Therefore, a novel aseptic sampling method, spanning both the spatial and temporal distributions of the AAB and their substrates and metabolites, was combined with a highly selective medium and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a high-throughput dereplication method followed by comparative gene sequencing for their isolation and identification, respectively. The AAB (Acetobacter species more than Gluconobacter species) proliferated during two phases of the lambic beer production process, represented by Acetobacter orientalis during a few days in the beginning of the fermentation and Acetobacter pasteurianus from 7 weeks until 24 months of maturation. Competitive exclusion tests combined with comparative genomic analysis of all genomes of strains of both species available disclosed possible reasons for this successive dominance. The spatial analysis revealed that significantly higher concentrations of acetic acid (from ethanol) and acetoin (from lactic acid) were produced at the tops of the casks, due to higher AAB counts and a higher metabolic activity of the AAB species at the air/liquid interface during the first 6 months of lambic beer production. In contrast, no differences in AAB species diversity occurred throughout the casks.IMPORTANCE Lambic beer is an acidic beer that is the result of a spontaneous fermentation and maturation process. Acidic beers are currently attracting attention worldwide. Part of the acidity of these beers is caused by acetic acid bacteria (AAB). However, due to their difficult recovery, they were never investigated extensively regarding their occurrence, species diversity, and functional role in lambic beer production. In the present study, a framework was developed for their isolation and identification using a novel aseptic sampling method in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry as a high-throughput dereplication technique followed by accurate molecular identification. The sampling method applied enabled us to take spatial differences into account regarding both enumerations and metabolite production. In this way, it was shown that more AAB were present and more acetic acid was produced at the air/liquid interface during a major part of the lambic beer production process. Also, two different AAB species were encountered, namely, Acetobacter orientalis at the beginning and Acetobacter pasteurianus in a later stage of the production process. This developed framework could also be applied for other fermentation processes.
Assuntos
Ácido Acético/metabolismo , Acetobacter/metabolismo , Cerveja/microbiologia , Gluconobacter/metabolismo , Fermentação , MicrobiotaRESUMO
Thermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adapted Gluconobacter frateurii strain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsAB strain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsAB mutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP+ ratio was higher in strain Wild-G, and much higher in the ΔotsAB strain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance in G. frateuriiIMPORTANCE The biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show that Gluconobacterfrateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP+ ratios and reductions in ROS levels. We concluded that in G. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP+ ratio to enhance thermotolerance.
Assuntos
Proteínas de Bactérias/genética , Gluconobacter/genética , Gluconobacter/metabolismo , Mutagênese Insercional , NADP/metabolismo , Ácido Acético/metabolismo , Proteínas de Bactérias/metabolismo , Temperatura Alta , Mutagênese Sítio-Dirigida , Fenótipo , Sorbose/metabolismo , Termotolerância , Trealose/metabolismoRESUMO
Background and Aims: Flowers can be highly variable in nectar volume and chemical composition, even within the same plant, but the causes of this variation are not fully understood. One potential cause is nectar-colonizing bacteria and yeasts, but experimental tests isolating their effects on wildflowers are largely lacking. This study examines the effects of dominant species of yeasts and bacteria on the hummingbird-pollinated shrub, Mimulus aurantiacus, in California. Methods: Wildflowers were inoculated with field-relevant titres of either the yeast Metschnikowia reukaufii or the bacterium Neokomagataea sp. (formerly Gluconobacter sp.), both isolated from M. aurantiacus nectar. Newly opened flowers were bagged, inoculated, harvested after 3 d and analysed for microbial abundance, nectar volume, and sugar and amino acid concentration and composition. Key Results: Yeast inoculation reduced amino acid concentration and altered amino acid composition, but had no significant effect on nectar volume or sugar composition. In contrast, bacterial inoculation increased amino acid concentration, enhanced the proportion of nectar sugars comprised by monosaccharides, and reduced nectar volume. Conclusions: The results presented suggest that microbial inhabitants of floral nectar can make nectar characteristics variable among flowers through divergent effects of yeasts and bacteria on nectar chemistry and availability, probably modifying plant-pollinator interactions.