2-ketogluconic Acid Research Articles

The Functional Characterization of the 6-Phosphogluconate Dehydratase Operon in 2-Ketogluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01.

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.

Exploring the response mechanism of Pseudomonas plecoglossicida to high-temperature stress by transcriptomic analyses for 2-keto gluconic acid production

High temperatures, particularly in summer, lead to decreased yields in the industrial application of Pseudomonas plecoglossicida for 2-keto gluconic acid (2KGA) fermentation. To address this, the alterations in the transcriptomics of P. plecoglossicida in response to high-temperature stress were examined at temperatures of 32 °C, 36 °C, and 40 °C. The analysis of differential expression revealed substantial discrepancies in the number of differentially expressed genes (DEGs) at 36 °C (357) and 40 °C (1,487), primarily affecting vital biological functions. Elevated temperatures resulted in a shift in the metabolic processing of glucose, transitioning from extracellular oxidation to intracellular phosphorylation. Notable changes were observed in metabolic pathways, including the pentose phosphate pathway and tricarboxylic acid cycle. A significant observation was the decline in the activity of genes associated with extracellular glucose oxidation, accompanied by an increase in the activity of genes involved in intracellular phosphorylation pathway. This indicates a prompt and dynamic response to high-temperature stress. The investigation revealed notable alterations in genes linked to glucose metabolism, emphasizing the strain's adaptive capabilities to endure high temperatures. The reveal of adaptations are crucial for optimizing 2KGA production in challenging industrial environments.

Can cadmium-contaminated rice be used to produce food additive sodium erythorbate?

Cadmium (Cd) in rice is a significant concern for its quality and safety. Currently, there is a crucial need to develop cost-effective and efficient ways to remove Cd or re-utilize Cd-contaminated rice. The food additive sodium erythorbate is produced via 2-ketogluconic acid (2KGA) fermentation by Pseudomonas plecoglossicida and lactonization using starch-rich raw materials, such as rice. We aimed to determine whether cadmium-contaminated rice can be used to produce sodium erythorbate. To achieve this aim, the migration of cadmium during the production of sodium erythorbate from Cd-contaminated rice was studied. Five rice varieties with different Cd contents from 0.10 to 0.68 mg/kg were used as raw materials. The results indicated the presence of Cd in rice and CaCO3 did not have a notable impact on the fermentation performance of 2KGA. The acidification of 2KGA fermentation broth, the addition of K4Fe(CN)6·3H2O and ZnSO4, and 2KGA purification using cation exchange effectively removed >98% of the Cd in the fermentation broth, but the 2KGA yield remained high at approximately 94%. The sodium erythorbate synthesized from Cd-contaminated rice was of high quality and free from Cd, meeting the requirements of the Chinese National Standard, GB 1886.28–2016. The study provided a safe and effective strategy for comprehensively utilizing Cd-contaminated rice to produce high value-added food additive.

Gene Expression and Metabolome Analysis Reveals Anti-Inflammatory Impacts of 11,17diHDoPE on PM10-Induced Mouse Lung Inflammation.

Oxylipins, the metabolites of polyunsaturated fatty acids, are vital in regulating cell proliferation and inflammation. Among these oxylipins, specialized pro-resolving mediators notably contribute to inflammation resolution. Previously, we showed that the specialized pro-resolving mediators isomer 11,17dihydroxy docosapentaenoic acid (11,17diHDoPE) can be synthesized in bacterial cells and exhibits anti-inflammatory effects in mammalian cells. This study investigates the in vivo impact of 11,17diHDoPE in mice exposed to particulate matter 10 (PM10). Our results indicate that 11,17diHDoPE significantly mitigates PM10-induced lung inflammation in mice, as evidenced by reduced pro-inflammatory cytokines and pulmonary inflammation-related gene expression. Metabolomic analysis reveals that 11,17diHDoPE modulates inflammation-related metabolites such as threonine, 2-keto gluconic acid, butanoic acid, and methyl oleate in lung tissues. In addition, 11,17diHDoPE upregulates the LA-derived oxylipin pathway and downregulates arachidonic acid- and docosahexaenoic acid-derived oxylipin pathways in serum. Correlation analyses between gene expression and metabolite changes suggest that 11,17diHDoPE alleviates inflammation by interfering with macrophage differentiation. These findings underscore the in vivo role of 11,17diHDoPE in reducing pulmonary inflammation, highlighting its potential as a therapeutic agent for respiratory diseases.

Analysis of gut target microbiota and species difference in patients with obstructive sleep apnea based on 16S rRNA sequencing

To explore the difference in gut microbiota composition between patients with obstructive sleep apnea (OSA) and healthy individuals and the role of gut microbiota in the pathogenesis of OSA. Thirty-nine patients with OSA admitted to our hospital between May and December, 2022 and 20 healthy individuals were enrolled in this study. Stool samples were collected from all the participants for analysis of microbiome composition using 16S rRNA high- throughput sequencing analysis. The alpha diversity, beta diversity, and species difference were determined between the two groups and marker species analysis and metabolic pathway function prediction analysis were performed. The species diversity (Shannon and Simpson) indexes, richness (observed species) and evenness (Pielou) of gut microbiota were significantly lower in OSA patients than in the healthy individuals (P < 0.05). The OSA patients had also a significantly lowered community diversity (P < 0.05) with different gut microbial communities from those of the healthy individuals shown by increased relative abundance of potentially pathogenic bacteria such as Pseudomonas and Monocytogenes (P < 0.05). LEfSe analysis showed that the abundance of 23 species of gut microbiota differed significantly between the two groups and the OSA patients had significant increases in the abundance of Pseudomonas, Meganomonas, and Fusobacterium (P < 0.05). The differential marker flora affected host homeostasis. Random Forest and ROC curve analyses confirmed that Pseudomonas could be used as important biomarkers for a differential diagnosis. Metabolic pathway function prediction analysis showed that biosynthesis function had the greatest contribution to maintaining gut microbiota homeostasis, and Pseudomonas affected the occurrence and progression of OSA by participating in aromatic bioamine degradation and ketogluconic acid metabolic pathway. OSA patients have obvious gut microbiota disturbances, and Pseudomonas may affect the development of OSA by participating in substance metabolism to serve as the potential target gut bacteria for OSA treatment.

Selective separation of vitamin C by reactive extraction using ionic liquid: Experimental and modelling

Vitamin C (ascorbic acid), is an important nutrient widely used as a dietary supplement in the food, beverage, cosmetic and feed industries or as a drug in the pharmaceutical industry. It can be produced by a traditional two-step fermentation or a novel one-step process, but its separation from the fermentation broth still needs improvement. This study investigated a reactive extraction method to selectively separate vitamin C from 2-ketogluconic acid using an ionic liquid as the extracting agent and heptane as diluent. The separation efficiency and selectivity factor, as well as the analysis of extraction conditions (type and ionic liquid concentration, pH of the aqueous phase, contact time, temperature) influence and extraction mechanism were discussed. Heptane as the solvent and 100 g/L [P6,6,6,14][Phos] as the extractant were the optimum mixture for reactive extraction, resulting in an extraction yield of 99.98 % for vitamin C. A maximum selectivity factor of 3.5 was obtained for ascorbic acid for 160 g/L [P6,6,6,14][Phos] and pH 3 of the aqueous phase. The process was modeled and optimized using an Artificial Neural Networks (ANNs) and Differential Evolution (DE) algorithm. Based on the performance parameters evaluated and extraction yields obtained, ionic liquids have great potential for vitamin C separation.

Low pH Stress Enhances Gluconic Acid Accumulation with Enzymatic Hydrolysate as Feedstock Using Gluconobacter oxydans

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.

Phosphate Solubilizing and Phytate Degrading Streptomyces Isolates Stimulate the Growth and P Accumulation of Maize (Zea mays) Fertilized with Different Phosphorus Sources

Actinobacteria are involved in many soil processes related to plant growth, including the secretion of various bioactive products and nutrient cycling. The aim of this study was to characterize the plant growth promoting traits (phosphate solubilizing activity, phytate degradation ability and indolic compounds production, and organic acids secretion) of seven actinobacteria isolates and evaluate the effect of two selected rhizospheric Streptomyces on growth and phosphorus content of maize in presence of different phosphorus (P) sources (tri-calcium phosphate, triple super phosphate and calcium phytate). Phylogenetic analysis of partial 16S rRNA sequences showed that the Streptomyces isolates were clustered in two distinct separated groups. All Streptomyces isolates produced 2-ketogluconic acid and oxalic acid in fermentation medium supplemented with inorganic P sources, while in the presence of phytate, all isolates secreted 2-ketogluconic acid and malic acid. All the isolates were able to release inorganic P from insoluble phosphate sources and this behavior was negatively correlated with pH reduction. Phytate degradation raged from 1.65% to 95.2%, and total indole compound production ranged from 6.3 to 27.8 µg ml−1. The shoot dry weight, root dry weight, plant height, chlorophyll content and tissue P concentration were significantly (p < 0.05) increased by both inoculations with Streptomyces isolates and by the addition of different phosphorus sources, compared to their appropriate controls. The inoculation of Streptomyces isolates resulted in a significant increase in plant phosphorus content from different phosphate sources. Streptomyces sp. UTMC 1478 (Streptomyces sp. 63) and Streptomyces sp. 47 were suitable inoculants for increasing maize growth and P from soil amended with different phosphorus sources, indicating their potential use as plant growth-promoting phospho-bacteria.

Cascading and precise regulation of the selective bio-production of 2- or 5-ketogluconic acid from glucose with whole-cell catalysis technology

Ketogluconic acid is widely applied in several industries as the bio-precursor of vitamin C and tartaric acid, while the selective production of 2-kGA or 5-kGA from glucose is so far still a challenge for chemical and biotechnical processes.

Genomic Analysis of Pseudomonas asiatica JP233: An Efficient Phosphate-Solubilizing Bacterium.

The bacterium Pseudomonas sp. strain JP233 has been reported to efficiently solubilize sparingly soluble inorganic phosphate, promote plant growth and significantly reduce phosphorus (P) leaching loss from soil. The production of 2-keto gluconic acid (2KGA) by strain JP233 was identified as the main active metabolite responsible for phosphate solubilization. However, the genetic basis of phosphate solubilization and plant-growth promotion remained unclear. As a result, the genome of JP233 was sequenced and analyzed in this study. The JP233 genome consists of a circular chromosome with a size of 5,617,746 bp and a GC content of 62.86%. No plasmids were detected in the genome. There were 5097 protein-coding sequences (CDSs) predicted in the genome. Phylogenetic analyses based on genomes of related Pseudomonas spp. identified strain JP233 as Pseudomonas asiatica. Comparative pangenomic analysis among 9 P. asiatica strains identified 4080 core gene clusters and 111 singleton genes present only in JP233. Genes associated with 2KGA production detected in strain JP233, included those encoding glucose dehydrogenase, pyrroloquinoline quinone and gluoconate dehydrogenase. Genes associated with mechanisms of plant-growth promotion and nutrient acquisition detected in JP233 included those involved in IAA biosynthesis, ethylene catabolism and siderophore production. Numerous genes associated with other properties beneficial to plant growth were also detected in JP233, included those involved in production of acetoin, 2,3-butanediol, trehalose, and resistance to heavy metals. This study provides the genetic basis to elucidate the plant-growth promoting and bio-remediation properties of strain JP233 and its potential applications in agriculture and industry.

Impact of temperature on gluconic acid production during acetification by Acetobacter aceti.

The current approach to gluconic acid production is acetification at 30°C, a temperature that can be difficult to maintain in tropical countries. This study investigated the production of gluconic acid during acetification by Acetobacter aceti WK at high temperatures. An acid-tolerant and thermotolerant species, A. aceti WK, was used for acetification at three different temperatures, namely, 30°C (normal temperature), 37°C, and 40°C (high temperature). Acetification was performed in a 100L bioreactor with 0.15% CaCl2 for protection of the cells against high temperatures. The production of the organic acids, that is, acetic acid, gluconic acid, 2-keto gluconic acid, glucuronic acid, citric acid, succinic acid, lactic acid, and formic acid, was analyzed. Under acetification in the target total concentration of 80g/L, the highest acetic acid content (39.3g/L) was obtained at 37°C with an acetification rate of 0.3013g/L/h, while the acetic acid content and acetification rate achieved at 30°C were 31g/L and 0.3089g/L/h, respectively. Additionally, gluconic acid presented at the highest concentration of 2.17g/L. The rate of production of gluconic acid was 0.0169g/L/h at 37°C. This acetification process at 37°C will be valuable as an alternative source for gluconic acid production for commercial applications.

Evaluation of the Ability of Rhizobacterial Isolates to Solubilize Sparingly Soluble Iron Under In-vitro Conditions

One of the problems of calcareous soils is the lack of absorbable iron for plants grown in these areas. Research by various researchers has shown that some rhizosphere-dwelling microorganisms have the ability to solubilize resources containing low-soluble iron and conversion it to soluble and absorbable for the plants. Different mechanisms such as proton secretion, production of organic acids, and production of siderophore can be effective in solubilizing low-soluble iron. Accordingly, in this study, the potential of twenty rhizobacteria for solubilizing low-soluble iron sources (eg, iron phosphate, hematite, and goethite) has been evaluated. The experiment was performed under in-vitro condition in a completely randomized design with three replications. According to the results, three isolates, namely, ZP15, ZC10, and ZP13 were characterized as promising isolates. In the presence of iron phosphate, the concentration of solubilized iron by isolates ZP15, ZP13, and ZC10 was 5.19, 4.35, and 4.27 mg/l, respectively. These values for the isolates ZC10 and ZP13 in the presence of hematite was recorded 6.49 and 5.35 mg/l, respectively, and in the presence of goethite was 7.02 and 6.70 mg/l, respectively. The amount of siderophore production for the three isolates ZP15, ZC10, and ZP13 was 16.47, 17.1, and 31.55 µM, respectively. The high-performance liquid chromatography analysis revealed that the most important acids produced by these isolates were 2-ketogluconic acid (303.8 µg/ml) for ZP15, oxalic acid (246.9 µg/ml) for ZC10, and gluconic acid (100.2 µg/ml) for ZP13, respectively. The molecular and biochemical identification of these three isolates ZP15, ZC10, and ZP13 showed that they belong to the genera Stenotrophomonas, Pantoea, and Agromyces, respectively.

Identification of the Phosphorus-Solubilizing Bacteria Strain JP233 and Its Effects on Soil Phosphorus Leaching Loss and Crop Growth.

Phosphorus (P) is one of the most limiting nutrients in global agricultural ecosystems, and phosphorus-solubilizing bacteria (PSB) can convert insoluble P into soluble P, thereby improving the absorption and use of soil P by plants. Increasing leaching loss of soil P due to PSB that could lead to water eutrophication is a major concern, although no direct experimental evidence is available to evaluate these effects. In this study, a highly efficient PSB strain, Pseudomonas sp. JP233, was isolated from soil and its P-solubilizing agent was identified by metabolomics and HPLC analyses. The effects of JP233 on P contents in soil leachates were also analyzed by microcosm leaching experiments in the absence and presence of maize. JP233 could solubilize insoluble P into soluble forms, and the molybdate reactive phosphorus (MRP) content reached 258.07 mg/L in NBRIP medium containing 5 g/L Ca3(PO4)2 within 48 h. Metabolomics analysis demonstrated that the organic acid involved in JP233 P solubilization was primarily 2-keto gluconic acid (2KGA). Further, HPLC analysis revealed that 2KGA contents rapidly accumulated to 19.33 mg/mL within 48 h. Microcosm leaching experiments showed that MRP and total phosphorus (TP) contents in soil leaching solutions were not significantly higher after JP233 inoculation. However, inoculation with JP233 into maize plant soils significantly decreased MRP and TP contents in the soil leaching solutions on days 14 (P < 0.01), 21 (P < 0.01), and 28 (P < 0.05). Inoculation with strain JP233 also significantly increased the biomass of maize aerial components and that of whole plants (P < 0.05). Thus, strain JP233 exhibited a significant plant-growth-promoting effect on maize development. In conclusion, the application of PSB into soils does not significantly increase P leachate loss. Rather, the application of PSB can help reduce P leachate loss, while significantly promoting plant absorption and use of soil P.

Phosphate Solubilizing Microorganism Bacillus sp. MVY-004 and Its Significance for Biomineral Fertilizers' Development in Agrobiotechnology.

Simple SummaryAgriculture is one of the most significant and important sectors globally. Soil fertility and agricultural production are highly dependent on which plant protection and growth promotion substance is used. Although applying mineral fertilizers can promote rapid crop growth, extensive mineral fertilizer use has reduced soil quality worldwide. Such environmental aspects have led to the search for more sustainable methods to provide plants with nutrients, especially phosphorus. Phosphorus is one of the macronutrients that is indispensable for plant development and growth. Phosphate solubilizing microorganisms are widely applied in agronomic practices to increase the productivity of crops while maintaining the health of soils. Nowadays, special attention is focused globally on ecological farming. One of the new technologies in agriculture is the mixing of agricultural bio-stimulants with mineral fertilizers. Agricultural bio-stimulants are bioproducts that contain substances of natural origin along with beneficial microorganisms. They can be used to activate seeds, plants, and soil. Bio-stimulants are used in plants to improve nutritional efficiency, cause changes in vital and structural processes to affect plant growth, enhance abiotic and biotic stress tolerance and increase the yield and quality of products.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%).

Directional enhancement of 2-keto-gluconic acid production from enzymatic hydrolysate by acetic acid-mediated bio-oxidation with Gluconobacter oxydans

An acetic acid-mediated bio-oxidation strategy with Gluconobacter oxydans was developed to produce valuable 2-ketogluconic acid from lignocellulosic biomass. Metabolically, glucose is firstly oxidized to gluconic acid and further oxidized to 2-keto-gluconic acid by Gluconobacter oxydans. As a specific inhibitor for microbial fermentation generated from pretreatment, acetic acid was validated to have a down-regulated effect on bio-oxidizing glucose to gluconic acid. Nevertheless, it significantly facilitated 2-keto-gluconic acid accumulation and improved gluconate dehydrogenase activity. In the presence of 5.0 g/L acetic acid, the yield of 2-keto-gluconic acid increased from 38.0% to 80.5% using pure glucose as feedstock with 1.5 g/L cell loading. Meanwhile, 44.6 g/L 2-keto-gluconic acid with a yield of 83.5% was also achieved from the enzymatic hydrolysate. 2-keto-gluconic acid production, found in this study, laid a theoretical foundation for the industrial production of 2-keto-gluconic acid by Gluconobacter oxydans using lignocellulosic materials.

An Actinobacterium Strain From Soil of Cerrado Promotes Phosphorus Solubilization and Plant Growth in Soybean Plants.

The huge biological diversity of the Brazilian Cerrado is an important source of economically interesting microbial agents. The phylum Actinobacteria plays an important role in nutrient cycling, potentially improving their availability to plants. In this study, we isolated an actinobacteria (strain 3AS4) from wheat rhizospheres of crops cultivated in the Cerrado biome. Strain 3AS4 was identified as belonging to the genus Streptomyces and had phosphorus mobilization ability, mineralizing approximately 410 μg ml–1 from phytate, 300 μg ml–1 from calcium phosphate, and 200 μg ml–1 from rock phosphate. The analysis of the actinobacteria crude extract by spectrometric techniques revealed the presence of gluconic and 2-ketogluconic acid, and a greenhouse experiment was carried out to evaluate its plant growth promotion activity in soybean. Soil in its natural condition (with no phosphorus addition), 40 kg ha–1 rock phosphate from Bayovar (RP) added to soil, and triple super phosphate (SPT) added to soil were used. Significant differences in plant height were observed at 6 weeks when the plants were inoculated with the 3AS4 strain. The growth of inoculated plants in natural condition was promoted in 17% compared with the RP and SPT non-inoculated conditions, suggesting that inoculation can enable plants to grow with lower chemical P fertilizers. In the plants that were inoculated with the 3AS4 strain in the RP condition, the plant height increased by approximately 80% and the shoot:root ratio was approximately 30% higher compared to control conditions (non-inoculated plants in natural conditions). 3AS4 has P-solubilizing potential and can be exploited as an inoculant for soybean cultivation. These results suggest that this actinobacterium is a valuable resource for sustainable agriculture and will allow the reduction of phosphate fertilization in the future.

Characterization of a transcriptional regulator PtxS from Pseudomonas plecoglossicida for regulating 2-ketogluconic acid metabolism

Homologs of PtxS are ubiquitous transcriptional regulators controlling the expression of the glucose dehydrogenase and kgu operon to globally regulate the 2-ketogluconic acid (2KGA) metabolism in Pseudomonas. In the present study, a PtxS from a 2KGA industrial producer Pseudomonas plecoglossicida JUIM01 (PpPtxS) was heterologously expressed in E. coli BL21(DE3), then structurally and functionally characterized. The obtained results showed that PpPtxS was a 36.65-kDa LacI-family transcriptional regulator. 2KGA was the sole effector of PpPtxS. Glucose negatively affected the molecular binding of PpPtxS and 2KGA, and gluconic acid inhibited the PpPtxS-2KGA binding reaction. PpPtxS in water solution mainly existed as a dimer and bound to two molecules of 2KGA. The effector 2KGA mainly bound to the region close to the C-terminal of PpPtxS by interacting with the 299th to the 301st amino acids (Ala, Gln, Pro, Thr, Glu and Arg). PpPtxS specifically recognized and bound to a 14-bp palindrome sequence (5′-TGAAACCGGTTTCA-3′) due to its conserved HTH motif at the N-terminal. The characterization of PpPtxS in this study would provide a theoretical guidance for the industrial production of 2KGA.

The Auxiliary NADH Dehydrogenase Plays a Crucial Role in Redox Homeostasis of Nicotinamide Cofactors in the Absence of the Periplasmic Oxidation System in Gluconobacter oxydans NBRC3293.

Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydansIMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.

Mechanism, influencing factors exploration and modelling on the reactive extraction of 2-ketogluconic acid in presence of a phase modifier

Taking into account that limited research has been carried out on the reactive extraction of 2-ketogluconic acid, this study was focused on analyzing the pH dependent extraction performance and the molar ratios of acid and extractant (Amberlite LA-2) dissolved in three solvents with 1-octanol as phase modifier. Back extraction was successfully performed using NaOH solutions. The mechanism of the interfacial reaction in the presence of 1-octanol, pointed out that, indifferent of the pH value and solvent polarity, only one molecule of 2-ketogluconic acid and one of extractant react at the interface. The positive effect of 1-octanol on extraction efficiency was quantified by means of the amplification factor, its maximum values being 2.43 for dichloromethane, 3.67 for butyl acetate and 3.64 for n-heptane. In addition, the process was modelled using statistical regression and Artificial Neural Networks (ANNs) determined with chaos based Differential Evolution algorithm. The best ANN model had a mean squared error for the testing phase of 0.19 and modeled the process with an acceptable error.

SO046REGULATION OF GUT MICROBIOTA AND HOST CO-METABOLISM BY POTASSIUM HOMEOSTASIS IN PATIENTS ON HEMODIALYSIS

Abstract Background and Aims Gut microbiota composition is dysregulated in hemodialysis patients. However, the impact of potassium homeostasis on the gut microbiota and their metabolites has not been studied. Patiromer is a potassium binding polymer that exchanges calcium for potassium in the gastrointestinal (GI) tract, thereby increasing fecal potassium excretion and reducing serum potassium levels. Method In this non-randomized, open label, 3-period crossover trial with repeated measures within each period, we investigated the effect of 12 weeks of treatment with patiromer on the microbiome profile and microbiota-related metabolites in 27 hemodialysis patients using multi-omics integration of data from shotgun metagenomic sequencing and untargeted and targeted metabolomic profiling. Data from hemodialysis patients at baseline was first compared with individuals without kidney disease (n=20). Results We found that 18 bacterial species and 49 plasma chemical clusters were significantly different between patients and controls. Serum potassium decreased significantly from baseline (5.62±0.65 mEq/L) with patiromer treatment (4.80±0.46 meq/L) and increased during the post-treatment phase (5.45 ± 0.67 meq/L), (p&amp;lt;0.001). Three bacterial species and one bacterial pathway were altered by patiromer. We identified 52 serum metabolites, which were significantly different in patients at baseline compared with controls, and were partially reversed with patiromer treatment. Specifically, the serum levels of polyphenols such as vanillic acid, gallic acid and benzoic acid, and other microbiota-related metabolites such as 2,6-diaminopimelic acid, 2-ketogluconic acid, indole-3-carboxaldehyde and 3-hydroxyphenylacetic acid were reduced by patiromer treatment. Conclusion Our study suggests that control of hyperkalemia by patiromer alters the gut microbial composition and host co-metabolism in hemodialysis patients.