Directing cell catalysis of glucose to 2-keto-d-gluconic acid using Gluconobacter oxydans NL71

2-Keto-d-gluconic acid (2-KGA) is a high-value-added product from lignocellulosic straw-based biorefining process obtained via cell catalysis using Gluconobacter oxydans (G. oxydans); nonetheless, the economical production has some limitations because of the formation of undesirable mixture products of keto-gluconic acid from glucose. In this study, it was demonstrated that pulse addition of hydrogen peroxide (H₂O₂) to synthetic media was beneficial to improve the conversion rate of glucose to 2-KGA by 1.47-fold in G. oxydans. Moreover, some lignocellulosic degradation compounds served as substitute and played the role of H₂O₂ on corn stover hydrolysate for the production of 2-KGA. By conducting qRT-PCR analysis, eight dehydrogenase genes were identified in G. oxydans, which were responsible for regulating 2-KGA production. Moreover, most of the identified genes were found to produce membrane-bound glucose dehydrogenase and gluconate 2-dehydrogenase, and were up-regulated by adding H₂O₂ or degradation compounds. These up-regulated genes could be responsible for directing cell catalysis of glucose to 2-KGA in G. oxydans.     

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.     

Incapability of Gluconobacter oxydans to produce tartaric acid

The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that trataric acid could only be produced in a medium containing vanadate (NH(4)VO(3)). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5-ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5-ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L-(+)-type. Oxalic acid was formed from 5-ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.     

Hydrogen peroxide as an oxygen source for immobilized Gluconobacter oxydans converting glycerol to dihydroxyacetone

Summary A flow injection analysis (FIA) system with amperometric detection was developed for measuring hydrogen peroxide which was used as an oxygen source for immobilized cells. A constant concentration of peroxide in the reactor was maintained by processing the analytical signal in a computer programmed as a PI-regulator. The concentration of dissolved oxygen was followed using a commercial Clark-electrode. The simultaneous measurements of hydrogen peroxide and dissolved oxygen are discussed with respect to process control.Conversion of glycerol to dihydroxyacetone by Gluconobacter oxydans immobilized in calcium alginate was used as a model system.Initial specific productivity increased with increasing hydrogen peroxide concentration. However, decreases in viable counts, enzymatic activities and overall productivities were noted. Various techniques for improving operational stability are discussed.     

Acetate production from lactate and glucose fermentation by Gluconobacter oxydans

Summary The production of acetate from the fermentation of lactate by Gluconobacter oxydans was studied. Batch experiments showed that glucose was the preferred substrate compared to lactate. A fed-batch culture was fed with a mixture of glucose and lactate followed by periodic addition of lactate. The maximum productivity of acetate was 0.16 g/l h but this value decreased during the fedbatch culture due to growth inhibition by acetate.     

A single amino acid substitution changes the substrate specificity of quinoprotein glucose dehydrogenase in Gluconobacter oxydans.

Summary Gluconobacter oxydans contains pyrroloquinoline quinone-dependent glucose dehydrogenase (GDH). Two isogenic G. oxydans strains, P1 and P2, which differ in their substrate specificity with respect to oxidation of sugars have been analysed. P1 can oxidize only d-glucose, whereas P2 is also capable of the oxidation of the disaccharide maltose. To investigate the nature of this maltose-oxidizing property we cloned the gene encoding GDH from P2. Expression of P2 gdh in P1 enables the latter strain to oxidize maltose, indicating that a mutation in the P2 gdh gene is responsible for the change in substrate specificity. This mutation could be ascribed to a 1 by substitution resulting in the replacement of His 787 by Asn.     

Electron transport system associated with direct glucose oxidation in Gluconobacter oxydans

Summary The mode of electron transport associated with the dehydrogenase enzymes located on the cytoplasmic membrane inGluconobacter oxydans (ATCC 9937) has been postulated. High turnover of dehydrogenases under oxygen enrichment conditions is explained on the basis of a simplistic electron transport chain comprising cytochrome c553 (MW 23000) as a subunit of dehydrogenase and a cytochrome b562. The electron transport chain under low dissolved oxygen tension (DOT) is shown to comprise a number of cytochrome c species with very low midpoint potential difference.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Direct fermentation of 2-keto-L-gulonic acid in recombinant Gluconobacter oxydans

We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from D-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of L-sorbosone and 2-KLGA, L-sorbose dehydrogenase (SDH) and L-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine resulted in improvement of the production level.     

Macrokinetic model for Gluconobacter oxydans in 2-keto-L-gulonic acid mixed culture

A set of kinetic models have been developed for the production of 2-keto-L-gulonic acid from L-sorbose by a mixed culture of Gluconobacter oxydans and Bacillus megaterium. A metabolic pathway is proposed for Gluconobacter oxydans, and a macrokinetic model has been developed for Gluconobacter oxydans, where the balances of some key metabolites, ATP and NADH are taken into account. An unstructured model is proposed for concomitant bacterium Bacillus megaterium. In the macrokinetic model and unstructured model, the mechanism of interaction between Gluconobacter oxydans and Bacillus megaterium is investigated and modeled. The specific substrate uptake rate and the specific growth rate obtained from the macrokinetic model are then coupled into a bioreactor model such that the relationship between the substrate feeding rate and the main state variables, such as the medium volume, the biomass concentrations, the substrate, and the is set up. A closed loop regulator model is introduced to approximate the induction of enzyme pool during lag phase after inoculation. Experimental results demonstrate that the model is able to describe 2-keto-L-gulonic acid fermentation process with reasonable accuracy.     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Complete genome sequence of the acetic acid bacterium Gluconobacter oxydans

Gluconobacter oxydans is unsurpassed by other organisms in its ability to incompletely oxidize a great variety of carbohydrates, alcohols and related compounds. Furthermore, the organism is used for several biotechnological processes, such as vitamin C production. To further our understanding of its overall metabolism, we sequenced the complete genome of G. oxydans 621H. The chromosome consists of 2,702,173 base pairs and contains 2,432 open reading frames. In addition, five plasmids were identified that comprised 232 open reading frames. The sequence data can be used for metabolic reconstruction of the pathways leading to industrially important products derived from sugars and alcohols. Although the respiratory chain of G. oxydans was found to be rather simple, the organism contains many membrane-bound dehydrogenases that are critical for the incomplete oxidation of biotechnologically important substrates. Moreover, the genome project revealed the unique biochemistry of G. oxydans with respect to the process of incomplete oxidation.     

Kinetic aspects of glucose oxidation by Gluconobacter oxydans cells immobilized in calcium alginate

Summary Gluconobacter oxydans subspecies suboxydans (ATCC 621 H), when growing at high glucose concentrations, oxidizes this substrate incompletely and gluconic acid accumulates in the medium in almost stoichiometric amounts. Such cells were harvested and entrapped in various alginate gels. The preparation with the highest retention of glucose oxidizing activity was used in further studies with the aim of developing an efficient process for continuous gluconic acid production.The retention of activity increases (up to 95%) as the alginate concentration in the gel decreases or the cell/alginate weight ratio is enhanced. In the latter case, however, transport of oxygen to and inside the biocatalyst beads rapidly becomes rate-limiting and thus lowers the efficiency of the biocatalyst. Similarly, the efficiency decreases as the size of the biocatalyst beads increases. In no case rate-limitation by transport of glucose was found. Thus, biocatalyst activity per unit volume of support, diameter of the biocatalyst beads, and aeration efficiency are important parameters for reactor design.     

Continuous production of gluconic acid by immobilized Gluconobacter oxydans cell bioreactor

Summary Living Gluconobacter oxydans cells were attached on fibrous nylon carrier. Free gluconic acid was directly continuously produced in an aerated tubular immobilized-cell bioreactor for at least 6 months, with a volumetric productivity of at least 5 g/lh at 100 g/l substrate glucose and about 80 g/l product gluconic acid concentrations. The highest volumetric productivity in respect to glucose concentration was obtained with 175 g/l glucose, with about 120 g/l product gluconic acid level. With self-directing optimization procedure in respect to maximum product gluconic acid level, productivities as high as about 12–15 g/lh were obtained at relatively high substrate feed rate of 0.166 l/lh and relatively low aeration rate of 0.5 l/lmin. The highest glucose conversion of about 96% was obtained with a long residence time, at the lowest substrate feed rate used at a relatively low aeration rate, resulting however in a significant increase in ketogluconic acid 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.     

Gluconobacter oxydans NH-10中NAD+型阿拉伯糖醇脱氢酶基因敲除菌株的构建

氧化葡萄糖酸杆菌Gluconobacter oxydans NH-10能够转化D-阿拉伯糖醇,经木酮糖生成木糖醇,但该菌中存在的NAD+型D-阿拉伯糖醇脱氢酶可将中间产物D-木酮糖还原成D-阿拉伯糖醇,从而影响木糖醇的积累.利用同源重组基因敲除的方法构建G.oxydans NH-10 NAD+型D-阿拉伯糖醇脱氢酶( sArDH)基因敲除突变株.PCR结果显示:sArDH基因在1株重组菌中完全被卡那抗性基因替代,表明sArDH基因敲除突变体构建成功.生物学特性鉴定显示:突变菌在菌落形态,生长状态方面与原始菌无明显差异.静息细胞转化D-阿拉伯糖醇结果显示,突变株不存在还原D-木酮糖产D-阿拉伯糖醇的逆反应,终产物木糖醇的产量有所提高.     

Scale-down and optimization studies of the gluconic acid fermentation by Gluconobacter oxydans

To simulate production-scale conditions of gluconic acid fermentation by Gluconobacter oxydans, different experimental setups are presented in this study. From the determination of the time constants of a production-scale reactor, it can be concluded that mixing and oxygen transfer are the rate-limiting mechanisms. This results in oxygen concentration gradients which were simulated in a one-compartment reactor in which the oxygen concentration was fluctuated by a fluctuated gassing with air and nitrogen. It could be concluded that only very long periods of absence of oxygen (ca. 180 s) results in lower specific oxygen uptake rates by Gluconobacter oxydans. From scale-down studies carried out in a two-compartment system to simulate a production-scale reactor more accurately, it could be concluded that not only the residence time in the aerated part of the system is important, but the liquid flow in between the different parts of the reactor is also an essential parameter. It could also be concluded that the microorganisms are not influenced negatively by the fluctuated oxygen concentrations with respect to their maximal oxidation capacity. The two-compartment system can also be used for optimization experiments in which the "aerated" compartment was gassed with pure oxygen. From these experiments it was concluded that also a short residence of the cells at high oxygen concentrations diminished the growth and product formation rates. These experiments show the necessity of the scale-down experiments if optimization is carried out. The two-compartment system presented in this study is a very attractive tool for reliable scale-down experiments.