Search Results (11)

Ascorbic acid (ASC) is a furan-based lactone derived from 2-ketogluconic acid that functions as a major antioxidant and redox buffer in mature plant tissues, although its content is lower in meristematic cells. ASC is commonly considered a reactive oxygen species (ROS) scavenger; however, its role in the regulation of plant development remains unclear. Additionally, the chemical behavior of ascorbate warrants special attention during ASC supplementation in in vitro plant culture. In this study, I investigated in detail the behavior of ascorbate in plant tissue culture medium and its uptake by plants. As a secondary objective, the role of ascorbate in root growth regulation was evaluated. The effects of low ASC levels on root architecture and its interaction with auxin signaling were studied using the vtc1 and vtc2 mutants of Arabidopsis thaliana, as well as through external ascorbate supplementation. Several marker lines for auxin response/distribution were used, along with direct ascorbate measurement via HPLC. Reducing ascorbate content through mutations had no significant effect on root development or auxin signaling, whereas high-concentration ASC supplementation inhibited both auxin signaling and root development, as demonstrated using auxin response and transport markers. At the organ level, ASC supplementation significantly downregulated auxin response-mediated cell cycle activation during lateral root induction. Based on the data presented, exogenous ascorbate may regulate root development through its interaction with auxin signaling pathways. Full article

The GntR is a transcriptional regulator generally known as a gluconate-operon repressor to specifically regulate the transportation and phosphorylation of gluconate. In the present study we report the cloning of the GntR-encoding gene of the industrial 2-ketogluconate (2KGA)-producer Pseudomonas plecoglossicida JUIM01, which is involved in the regulation of gluconate metabolism, along with the identification of some of its target genes and its operator sequence. GntR is a 36.36-kDa cytoplasmic and hydrophobic DNA-binding transcriptional regulator belonging to the LacI family. The knockout of gntR resulted in the significant upregulation of the transcription of the gluconate kinase gene gntK and, to a lesser extent, the permease gene gntP, as well as downregulation of genes involved in glucose uptake (oprB-1, gltB, gltF, gltG, and gltK) and those involved in 2-ketogluconate (2KGA) transport (kguT) and catabolism (kguE, kguK, and kguD). These results indicated that GntR positively regulated glucose and 2KGA transport and catabolism, while negatively affecting GntP-mediated gluconate uptake and gluconate phosphorylation by GntK. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting analyses confirmed that GntR interacted with operator sequences in the divergent promoter regions of gntK and gntP, as well as in the gntR promoter region. A putative operator sequence (consensus 5′-AG-N₂-AGCGCT-N-TCT-3′) was identified. These data suggest that GntR positively regulates genes involved in glucose uptake/transport and 2KGA transport/catabolism, while repressing its own expression as well as that of genes involved in gluconate transport/catabolism. These findings not only elucidate the regulation of GntR and its target genes in P. plecoglossicida, but also provide valuable insights for optimizing industrial 2KGA production. Full article

Pseudomonas plecoglossicida JUIM01 is an industrial 2-keto-d-gluconate (2KGA)-producing strain. However, its regulation mechanism of 2KGA metabolism remains to be clarified. Among other reported Pseudomonas species, the 2-ketogluconate utilization operon (kgu operon) plays key roles in 2KGA catabolism. In this study, the structural genes of the kgu operon and its promoter in P. plecoglossicida JUIM01 were identified using reverse transcription PCR and lacZ reporter gene fusion. The results showed the kgu operon in P. plecoglossicida was composed of four structural genes: kguE, kguK, kguT, and kguD. The ptxS gene located upstream of kguE was excluded from the kgu operon. Then, the knockout and corresponding complementation strains of kguE, kguK, kguT, and kguD were constructed, respectively, to prove the kgu operon was involved in 2KGA catabolism of P. plecoglossicida. The knockout stains, especially JUIM01ΔkguE, showed potential as industrial production strains for 2KGA. Moreover, the transcriptional regulation mechanism of PtxS on the kgu operon was elucidated using multiple methods. In P. plecoglossicida, the LacI-family transcription regulator PtxS could recognize a 14 bp palindrome (5′-TGAAACCGGTTTCA-3′) within the promoter region of the kgu operon and specifically bind to a 26 bp region where the palindrome was located. As the binding sites overlapped with the transcription start site of the kgu operon, the binding of PtxS possibly hindered the binding of RNA polymerase, thus repressing the transcription of the kgu operon and further regulating 2KGA catabolism. 2KGA bound to PtxS as an effector to dissociate it from the kgu operon promoter region, so as to relieve the transcription repression. The results will provide strategies for improving the product accumulation in 2KGA industrial production and theoretical bases for the construction of a Pseudomonas chassis. Full article

Genus Pseudomonas bacteria mainly consume glucose through the Entner–Doudoroff (ED) route due to a lack of a functional Embden–Meyerhof–Parnas (EMP) pathway. In the present study, a 6-phosphogluconate dehydratase (edd) operon in the ED route was well investigated to find its structural characteristics and roles in the regulation of glucose consumption and 2-ketogluconic acid (2KGA) metabolism in the industrial 2KGA-producer P. plecoglossicida JUIM01. The edd operon contained four structural genes of edd, glk, gltR, and gtrS, encoding 6-PG dehydratase Edd, glucokinase Glk, response regulatory factor GltR, and histidine kinase GtrS, respectively. A promoter region was observed in the 5′-upstream of the edd gene, with a transcriptional start site located 129 bp upstream of the edd gene and in a pseudo-palindromic sequence of 5′-TTGTN₇ACAA-3′ specifically binding to the transcription factor HexR. The knockout of the edd gene showed a remarkably negative effect on cell growth and re-growth using 2KGA as a substrate, beneficial to 2KGA production, with an increase of 8%. The deletion of glk had no significant effect on the cell growth or glucose metabolism, while showing an adverse impact on the 2KGA production, with a decrease of 5%. The outputs of the present study would provide a theoretical basis for 2KGA-producer improvement with metabolic engineering strategies and the development and optimization of P. plecoglossicida as the chassis cells. Full article

Rahnella aquatilis AZO16M2, was characterized for its phosphate solubilization capacity to improve the establishment and survival of Musa acuminata var. Valery seedlings under ex-acclimation. Three phosphorus sources (Rock Phosphate (RF), Ca₃(PO₄)₂ and K₂HPO₄) and two types of substrate (sand:vermiculite (1:1) and Premix N°8) were selected. The factorial analysis of variance (p < 0.05) showed that R. aquatilis AZO16M2 (OQ256130) solubilizes Ca₃(PO₄)₂ in solid medium, with a Solubilization Index (SI) of 3.77 at 28 °C (pH 6.8). In liquid medium, it was observed that R. aquatilis produced 29.6 mg/L soluble P (pH 4.4), and synthesized organic acids (oxalic, D-gluconic, 2-ketogluconic and malic), Indole Acetic Acid (IAA) (33.90 ppm) and siderophores (+). Additionally, acid and alkaline phosphatases (2.59 and 2.56 µg pNP/mL/min) were detected. The presence of the pyrroloquinoline-quinone (PQQ) cofactor gene was confirmed. After inoculating AZO16M2 to M. acuminata in sand:vermiculite with RF, the chlorophyll content was 42.38 SPAD (Soil Plant Analysis Development). Aerial fresh weight (AFW), aerial dry weight (ADW) and root dry weight (RDW) were superior to the control by 64.15%, 60.53% and 43.48%, respectively. In Premix N°8 with RF and R. aquatilis, 8.91% longer roots were obtained, with 35.58% and 18.76% more AFW and RFW compared with the control as well as 94.45 SPAD. With Ca₃(PO₄)₂, values exceeded the control by 14.15% RFW, with 45.45 SPAD. Rahnella aquatilis AZO16M2 favored the ex-climatization of M. acuminata through improving seedling establishment and survival. Full article

Gluconic acid has been increasingly in demand in recent years due to the wide applications in the food, healthcare and construction industries. Plant-derived biomass is rich in biopolymers that comprise glucose as the monomeric unit, which provide abundant feedstock for gluconic acid production. Gluconobacter oxydans can rapidly and incompletely oxidize glucose to gluconic acid and it is regarded as ideal industrial microorganism. Once glucose is depleted, the gluconic acid will be further bio-oxidized to 2-ketogluconic acid by Gluconobacter oxydans. The endpoint is difficult to be controlled, especially in an industrial fermentation process. In this study, it was found that the low pH environment (2.5~3.5) could limit the further metabolism of gluconic acid and that it resulted in a yield over 95%. Therefore, the low pH stress strategy for efficiently producing gluconic acid from biomass-derived glucose was put forward and investigated with enzymatic hydrolysate. As a result, 98.8 g/L gluconic acid with a yield of 96% could be obtained from concentrated corncob enzymatic hydrolysate that initially contained 100 g/L glucose with 1.4 g/L cells loading of Gluconobacter oxydans. In addition, the low pH stress strategy could effectively control end-point and decrease the risk of microbial contamination. Overall, this strategy provides a potential for industrial gluconic acid production from lignocellulosic materials. Full article

In this study, a phosphate solubilizing microorganism was isolated from the soil of an agricultural field in Lithuania. Based on 16S rRNA gene sequence analysis, the strain was identified as Bacillus sp. and submitted to the NCBI database, Sector of Applied Bio-catalysis, University Institute of Biotechnology, Vilnius, Lithuania and allocated the accession number KY882273. The Bacillus sp. was assigned with the number MVY-004. The culture nutrient medium and growth conditions were optimized: molasses was used as a carbon source; yeast extract powder was used as an organic source; NH₄H₂PO₄ was used as a nitrogen source; the culture growth temperature was 30 ± 0.5 °C; the initial value of pH was 7.0 ± 0.5; the partial pressure of oxygen (pO₂) was 60 ± 2.0; the mixer revolutions per minute (RPM) were 25–850, and the incubation and the fermentation time was 48–50 h. Analysis using Liquid Chromatography Time-of-Flight Mass Spectrometry (LC-TOF/MS) results showed that Bacillus sp. MVY-004 produced organic acids such as citric, succinic, 2-ketogluconic, gluconic, malic, lactic, and oxalic acids. Furthermore, the experiment showed that Bacillus sp. MVY-004 can also produce the following phytohormones: indole-3-acetic (IAA), jasmonic (JA), and gibberellic (GA3) acids. In the climate chamber, the experiment was performed using mineral fertilizer (NPS-12:40:10 80 Kg ha⁻¹) and mineral fertilizers in combination with Bacillus sp. MVY-004 cells (NPS-12:40:10 80 Kg ha⁻¹ + Bacillus sp. MVY-004) in loamy soil. Analysis was performed in three climate conditions: normal (T = 20 °C; relative humidity 60%); hot and dry (T = 30 °C; relative humidity 30%); hot and humid (T = 30 °C; relative humidity 80%). Full article

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time. Full article

2-Ketogluconate (2KGA) is an organic acid that is important for pharmaceutical, cosmetic, and environmental applications. Pseudomonas plecoglossicida JUIM01 strain is an important industrial 2KGA producer in China. In this paper, we found that P. plecoglossicida JUIM01 could convert glucose to 2KGA extracellularly, and the formed 2KGA was subsequently consumed after glucose was exhausted during the fermentation process. Experiments of glucose and 2KGA supplementation during fermentation process revealed that, only when glucose was exhausted, the strain started to consume the product 2KGA. Then, the mechanism of this phenomenon was investigated at transcription and protein levels, and the results indicated that P. plecoglossicida JUIM01 possesses carbon catabolite repression of 2KGA metabolism by glucose. Next, increasing the supply of glucose could attenuate 2KGA consumption and enhance the 2KGA yield from glucose. Finally, fed-batch fermentation of P. plecoglossicida JUIM01 resulted in 205.67 g/L of 2KGA with a productivity of 6.86 g/L/h and yield of 0.953 g/g glucose. These results can provide references for the industrial fermentation production of 2KGA and other fermentation products. Full article

Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner–Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically ¹³C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from ¹³C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with ¹³C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments. Full article

Glucose dehydrogenase (GlcDH) is the rate-limiting catalyst for microbial conversion of glucose to the important organic acid 2-ketogluconic acid (2KGlcA). In this study, a D-glucose dehydrogenase was purified from the industrial 2KGlcA producer Arthrobacter globiformis C224. After four purification steps, the GlcDH was successfully purified over 180 folds and specific activity of 88.1 U/mg. A single protein band of 87 kDa was detected by SDS-PAGE. The purified GlcDH had the broad substrate specificity with the K_m values for D-glucose, D-xylose, D-galactose and maltose of 0.21 mM, 0.34 mM, 0.46 mM and 0.59 mM, respectively. The kinetic studies proved that A. globiformis GlcDH followed the ping-pong kinetic mechanism. The GlcDH showed an optimum catalytic activity at pH 5.0 and 45 °C with the stable activity at temperature of 20–40 °C and pH of 6.0–7.0. Organic solvents, metal ions or EDTA could significantly influence the GlcDH activity to different degrees. Full article