A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-d-gluconic acid

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO₂, 0.05% pCO₂) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.     

High-yield 5-keto-d-gluconic acid formation is mediated by soluble and membrane-bound gluconate-5-dehydrogenases of Gluconobacter oxydans

Gluconobacter oxydans DSM 2343 is known to catalyze the oxidation of glucose to gluconic acid, and subsequently, to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA), by membrane-bound and soluble dehydrogenases. In G. oxydans MF1, in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated, formation of the undesired 2-KGA was absent. This mutant strain uniquely accumulates high amounts of 5-KGA in the culture medium. To increase the production rate of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, we equipped G. oxydans MF1 with plasmids allowing the overproduction of the soluble and the membrane-bound 5-KGA-forming enzyme. Whereas the overproduction of the soluble gluconate:NADP 5-oxidoreductase resulted in the accumulation of up to 200 mM 5-KGA, the detected 5-KGA accumulation was even higher when the gene coding for the membrane-bound gluconate-5-dehydrogenase was overexpressed (240 to 295 mM 5-KGA). These results provide a basis for designing a biotransformation process for the conversion of glucose to 5-KGA using the membrane-bound as well as the soluble enzyme system.The corresponding author contributed equally to the first author.     

Biotransformation of glucose to 5-keto-<Emphasis Type="SmallCaps">d</Emphasis>-gluconic acid by recombinant<Emphasis Type="Italic"> Gluconobacter oxydans</Emphasis> DSM 2343

For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a precursor of the industrially important l-(+)-tartaric acid, Gluconobacter strains were genetically engineered. In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G. oxydans strain DSM 2434. This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA. As the 5-KGA reductase activity depends on the cofactor NADP⁺, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G. oxydans strain. Growth of the sthA-carrying strains was indistinguishable from the G. oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno. G. oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA.     

Genetic analysis of D-xylose metabolism pathways in Gluconobacter oxydans 621H

D-xylose is one of the most abundant carbohydrates in nature. This work focuses on xylose metabolism of Gluconobacter oxydans as revealed by a few studies conducted to understand xylose utilization by this strain. Interestingly, the G. oxydans 621H Δmgdh strain (deficient in membrane-bound glucose dehydrogenase) was greatly inhibited when grown on xylose and no xylonate accumulation was observed in the medium. These experimental observations suggested that the mgdh gene was responsible for the conversion of xylose to xylonate in G. oxydans, which was also verified by whole-cell biotransformation. Since 621H Δmgdh could still grow on xylose in a very small way, two seemingly important genes in the oxo-reductive pathway for xylose metabolism, a xylitol dehydrogenase-encoding gox0865 (xdh) gene and a putative xylulose kinase-encoding gox2214 (xk) gene, were knocked out to investigate the effects of both genes on xylose metabolism. The results showed that the gox2214 gene was not involved in xylose metabolism, and there might be other genes encoding xylulose kinase. Though the gox0865 gene played a less important role in xylose metabolism compared to the mgdh gene, it was significant in xylitol utilization in G. oxydans, which meant that gox0865 was a necessary gene for the oxo-reductive pathway of xylose in vivo. To sum up, when xylose was used as the carbon source, the majority of xylose was directly oxidized to xylonate for further metabolism in G. oxydans, whereas only a minor part of xylose was metabolized by the oxo-reductive pathway.     

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens,Phlebia radiata

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12 days gained the highest laccase activity (9.42 ± 0.73 U mg^?1). Combined with the data of laccase and manganese peroxidase, oxalate and H₂O₂ were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6–114.1 % with the selected fungi, and total pore volume was improved by 90.2 % with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H₂O₂ and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.     

Genetic engineering of Ketogulonigenium vulgare for enhanced production of 2-keto-L-gulonic acid

Folate derivatives are crucial growth factors for Ketogulonigenium vulgare which is used in mixed culture with Bacillus megaterium for the industrial production of 2-keto-l-gulonic acid (2-KGA), the precursor of l-ascorbic acid (l-AA) or vitamin C (Vc). To improve the growth and 2-KGA production, five genes involved in folate biosynthesis identified in a folate gene cluster from Lactococcus lactis MG1363, including folB, folKE, folP, folQ and folC, were over-expressed in K. vulgare. Intracellular folate concentration in the recombinant strain harboring folate biosynthesis genes cluster under the control of P_sdh (sorbose dehydrogenase gene sdh promoter from K. vulgare) was 8 times higher than that of the wildtype K. vulgare DSM 4025 (P < 0.001). In shake flask studies, the cell density and 2-KGA production of the recombinant K. vulgare Rif (pMCS2PsdhfolBC) were increased by 18% (P < 0.001) and 14% (P < 0.001), respectively, under a relatively stable pH 7 condition. In fermentor studies, enhancements around 25% cell density (P < 0.001) and approximately 35% 2-KGA productivity (P < 0.001) were observed in comparison with the controls without over-expressing the folate biosynthesis genes. This was the first successful study of metabolic engineering on K. vulgare for enhanced 2-KGA production.     

Effects of membrane-bound glucose dehydrogenase overproduction on the respiratory chain of Gluconobacter oxydans

The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265, was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.     

Regulation of gluconate and ketogluconate production in Gluconobacter oxydans ATCC 621-H

Gluconobacter spp. possess the enzymic potential for two pathways of direct glucose oxidation. It has been proposed that the major part of glucose is oxidized to gluconate via NADP-dependent glucose dehydrogenase and that reoxidation of NADPH under these conditions proceeds via recycling of gluconate through ketogluconates. This hypothesis was tested in experiments in which Gluconobacter oxydans ATCC 621-H was grown in glucose-yeast extract medium containing [¹⁴C]2-ketogluconate. As expected, glucose was almost quantitatively oxidized to gluconate, without further accumulation of 2- and 5-ketogluconate. Interestingly, the total amount of neither [¹⁴C]2-ketogluconate nor [¹⁴C]gluconate did change significantly during this oxidation phase, indicating that recycling of gluconate through ketogluconates did not occur. An analysis of enzyme activities in cell-free extracts of glucose-grown cells of G. oxydans ATCC 621-H showed that the membrane-bound glucose dehydrogenase was far more active than the NADP-linked glucose dehydrogenase. The activity of the latter enzyme constituted only 10–15% of that of quinoprotein glucose dehydrogenase and was far too low to match the in vivo rates of gluconate production in batch cultures of G. oxydans. It is concluded that under these conditions glucose is mainly oxidized to gluconate via the membrane-bound glucose dehydrogenase. Implications of these results for the regulation of ketogluconate formation are discussed.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PQQ pyrrolo-quinoline quinone     

Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans

The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NAD/NADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.     

Biochemistry of the oxidation of lignin by Phangerochate chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus . We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H₂O₂) and possibly superoxide (O₂ ·^-) in lignin degradation. Involvement of hydroxyl radical (·OH) or singlet oxygen (¹O₂) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H₂O₂ for activity.     

Corynebacterium glutamicum tailored for efficient isobutanol production

We recently engineered Corynebacterium glutamicum for aerobic production of 2-ketoisovalerate by inactivation of the pyruvate dehydrogenase complex, pyruvate:quinone oxidoreductase, transaminase B, and additional overexpression of the ilvBNCD genes, encoding acetohydroxyacid synthase, acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. Based on this strain, we engineered C. glutamicum for the production of isobutanol from glucose under oxygen deprivation conditions by inactivation of l-lactate and malate dehydrogenases, implementation of ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 (ADH2) from Saccharomyces cerevisiae, and expression of the pntAB transhydrogenase genes from Escherichia coli. The resulting strain produced isobutanol with a substrate-specific yield (Y_P/S) of 0.60 ± 0.02 mol per mol of glucose. Interestingly, a chromosomally encoded alcohol dehydrogenase rather than the plasmid-encoded ADH2 from S. cerevisiae was involved in isobutanol formation with C. glutamicum, and overexpression of the corresponding adhA gene increased the Y_P/S to 0.77 ± 0.01 mol of isobutanol per mol of glucose. Inactivation of the malic enzyme significantly reduced the Y_P/S, indicating that the metabolic cycle consisting of pyruvate and/or phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme is responsible for the conversion of NADH+H⁺ to NADPH+H⁺. In fed-batch fermentations with an aerobic growth phase and an oxygen-depleted production phase, the most promising strain, C. glutamicum ΔaceE Δpqo ΔilvE ΔldhA Δmdh(pJC4ilvBNCD-pntAB)(pBB1kivd-adhA), produced about 175 mM isobutanol, with a volumetric productivity of 4.4 mM h⁻¹, and showed an overall Y_P/S of about 0.48 mol per mol of glucose in the production phase.     

Enhanced thermo-tolerance in transgenic potato (Solanum tuberosum L.) overexpressing hydrogen peroxide-producing germin-like protein (GLP)

Germins and germin-like proteins (GLPs) were reported to participate in plant response to biotic and abiotic stresses involving hydrogen peroxide (H₂O₂) production, but their role in mitigating heat stress is poorly understood. Here, we investigated the ability of a Solanum tuberosum L. GLP (StGLP) gene isolated from the yeast cDNA library generated from heat-stressed potato plants and characterized its role in generating innate and/or acquired thermo-tolerance to potato via genetic transformation. The transgenic plants exhibited enhanced tolerance to gradual heat stress (GHS) compared with sudden heat shock (SHS) in terms of maximal cell viability, minimal ion leakage and reduced chlorophyll breakdown. Further, three StGLP transgenic lines (G9, G12 and G15) exhibited enhanced production of H₂O₂, which was either reduced or blocked by inhibitors of H₂O₂ under normal and heat stress conditions. This tolerance was mediated by up-regulation of antioxidant enzymes (catalase, ascorbate peroxidase and glutathione reductase) and other heat stress-responsive genes (StHSP70, StHSP20 and StHSP90) in transgenic potato plants. These results demonstrate that H₂O₂ produced by over-expression of StGLP in transgenic potato plants triggered the reactive oxygen species (ROS) scavenging signaling pathways controlling antioxidant and heat stress-responsive genes in these plants imparting tolerance to heat stress.     

AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: Implication of the cell survival in mitochondrial diseases

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H₂O₂. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H₂O₂-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H₂O₂ in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H₂O₂ was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H₂O₂-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H₂O₂-treated normal skin fibroblasts to oxidative stress.     

Ginkgo biloba responds to herbivory by activating early signaling and direct defenses

Background

Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses.

Methodology/Principal Findings

Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca²⁺]_cyt) and H₂O₂ production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca²⁺]_cyt and H₂O₂. Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides.

Conclusions/Significance

By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this “living fossil” plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.     

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6 g/l yeast extract, 4.7 g/l glycerol, 42.1 g/l mannitol, 0.5 g/l K₂HPO₄, 0.5 g/l KH₂PO₄, 0.1 g/l MgSO₄·7H₂O, and 2.0 g/l CaCO₃ with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8 ± 2.4% of conversion rate of glycerol to DHA was achieved after 72 h of biotransformation using 10 g/l resting cells at 30 °C, pH 5.0 and 1.5 vvm of aeration rate.     

Cloning of a gluconate/polyol dehydrogenase gene from <Emphasis Type="Italic">Gluconobacter suboxydans</Emphasis> IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain

A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.