gluconobacter oxydans

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Biotechnological Applications of G. oxydans 445

Gluconobacter oxydans :Its Biotechnological Applications

Received February 29, 2000; revised April 18, 2000; accepted July 5, 2000;

final manuscript received February 16, 2001. *For correspondence. [email protected] ; Fax. 91-731-470372 .

J. Mol. Microbiol. Biotechnol. (2001) 3(3): 445-456.

2001 Horizon Scientific Press

JMMB Review

Arun Gupta 1, Vinay K. Singh 1, G.N. Qazi 2

and Anil Kumar 1

1School of Biotechnology, Devi Ahilya University,Khandwa Road, Indore-452017, MP, India2Dept. of Biotechnology, Regional Research Laboratory(CSIR), Jammu, India

Abstract

Gluconobacter oxydans is a gram-negative bacterium

belonging to the family Acetobacteraceae . G. oxydans is an obligate aerobe, having a respiratory type ofmetabolism using oxygen as the terminal electronacceptor. Gluconobacter strains flourish in sugaryniches e.g. ripe grapes, apples, dates, garden soil,bakers soil, honeybees, fruit, cider, beer, wine.Gluconobacter strains are non-pathogenic towardsman and other animals but are capable of causing

bacterial rot of apples and pears accompanied byvarious shades of browning. Several soluble andparticulate polyol dehydrogenases have beendescribed. The organism brings about the incompleteoxidation of sugars, alcohols and acids. Incompleteoxidation leads to nearly quantitative yields of theoxidation products making G. oxydans important forindustrial use. Gluconobacter strains can be usedindustrially to produce L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-ketogluconic acids fromD-glucose; and dihydroxyacetone from glycerol. It isprimarily known as a ketogenic bacterium due to 2,5-

diketogluconic acid formation from D-glucose.Extensive fermentation studies have been performedto characterize its direct glucose oxidation, sorbitoloxidation, and glycerol oxidation. The enzymesinvolved have been purified and characterized, andmolecular studies have been performed to understandthese processes at the molecular level. Its possibleapplication in biosensor technology has also beenworked out. Several workers have explained its basicand applied aspects. In the present paper, its differentbiotechnological applications, basic biochemistry andmolecular biology studies are reviewed.

Introduction

Gluconobacter oxydans is a gram-negative bacteriumbelonging to the family Acetobacteraceae . The shape ofthe cells is ellipsoidal to rod shaped, 0.5-0.8 X 0.9-4.2 m,occurring singly and/or in pairs and rarely in chains. G.oxydans is an obligate aerobe, having a strictly respiratorytype of metabolism with oxygen as the terminal electronacceptor (De Ley and Swings, 1994).

Gluconobacter is an industrially important genus forthe production of L-sorbose from D-sorbitol; D-gluconicacid, 5-keto- and 2-ketogluconic acids from D-glucose; anddihydroxyacetone from glycerol. The present review dealswith the fermentation studies, applications in Biotechnologyand genetic aspects of Gluconobacter oxydans .

Classification and Nomenclature of Gluconobacter

In 1935, a group of gram-negative bacteria related toAcetobacter was renamed and put under the genusGluconobacter (Asai, 1935). Gluconobacter, earlier knownas Acetobacter oxydans, has been characterized as havingthe pronounced capability for the oxidation of glucose togluconate and a weak ability for the oxidation of ethanol toacetate (Kluyver and Boezaardt, 1938).

A review on the classification and nomenclatureproblems related to the genus Gluconobacter has beenpublished (De Ley and Frateur, 1970). The followingbiochemical tests are found uniformly negative for all strainsof Gluconobacter oxydans : glucose oxidase, reduction of

Abbreviationsg-microgram; l-microlitre; 2,5-DKG-2,5-diketogluconic acid; 2,5-DKGR-2,5-diketo-D-gluconate reductase; 2KGADH-2-ketogluconatedehydrogenase; 2kgadh-2-keto-gluconate dehydrogenase gene; 2KLG-2-ketoL-gulonic acid; 5KDGR-5-keto-D-gluconate reductase; 5KGA-5-ketogluconic acid ; 2,5-DKGR-2,5-diketo-D-gluconate reductase. 5KDGR-5-ketogluconate reductase; 2,5-DKGR-A - 2,5-diketo-D-gluconate reductaseproduct of gene yqfE of E. coli ; 2,5-DKGR-B - 2,5-diketo-D-gluconatereductase product of gene yqfB of E. coli ; A. calcoaceticus -Acinetobacter calcoaceticus ; ADH-alcohol dehydrogenase; Adh-alcohol dehydrogenasegene; ADP-adenosine diphosphate; ALDH-aldose-dehydrogenase; Asn-asparagine; ATCC-American type culture collection; bp-base pair; CaCO 3-calcium carbonate; Ca(OH) 2-Calcium hydroxide; Cfu-colony formation unit;CO 2 -carbon dioxide; Cyt C- cytochrome C; Da-dalton; DHA-dihydroxyacetone; DO-dissolved oxygen; E. coli - Escherichia coli ; E.herbicola -Erwinia herbicola ; EC-Enzyme commission; FAD +-flavin adeninedinucleotide; g-gram; G. oxydans -Gluconobacter oxydans; G. sacchari -Gluconobacter sacchari ; GA-gluconic acid; GADH-gluconatedehydrogenase; GADH --gluconate dehydrogenase deficient mutant; gadh-gluconate dehydrogenase gene; GDH-glucose dehydrogenase ; GDH - -glucose dehydrogenase deficient mutant; gdh-glucose dehydrogenasegene; GNO- gluconate NADP + 5-oxidoreductase gene; GYC- glucose-yeast

extract-calcium carbonate medium; H 2O-water; His-histidine; H 2S-hydrogendisulfide; HPLC-high performance liquid chromatography; hr-hour; IFO-Institute of Fermentation, Osaka; IS-insertion sequences; Kb-kilobase; Kda-Kilodalton; Kg-kilogram; l-litre; m-meter; Mda- megadalton; mg-milligram;ml-millilitre; mM-millimolar; MIC-minimum inhibitory concentration; MW-molecular weight; MYP- mannitol-yeast extract-peptone medium; nA-nano-ampere; NADP +-nicotinamide adenine dinucleotide phosphate; NaOH-sodium hydroxide; N-source-nitrogen source; O 2-oxygen; OPV-oxidizedpolyvinyl alcohol; P. fluorescens -Pseudomonas fluorescens ; PAGE-polyacrylamide gel electrophoresis; PCR-Polymerase Chain Reaction;PFGE-pulse field gel electrophoresis; PQQ-Pyrrolo Quinoline Quinone; pqq-pyrrolo quinoline quinone genes; RibF-riboflavin kinase F gene; rpm-revolutions per minute; SDH-sorbitol dehydrogenase; SDS-sodium dodecylsulfate; SNDH-sorbosone dehydrogenase; Tn5-Transposon Tn5; vvm-volumetric volume per mole.


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446 Gupta et al .

nitrates, H 2S formation and indole production (De Ley,1978). Loitsianskaya et al . (1979) proposed the existenceof a single species within the genus on the basis ofnumerical analyses of 136 phenotypic features of 56 strainsof acetic acid bacteria. The genus Gluconobacter belongs

to the fourth rRNA superfamily and constitutes a separatebranch together with the genus Acetobacter (Gillis and DeLey, 1980).

Gluconobacter and Acetobacter are closely related toeach other and are classified as acetic acid bacteria (Asai,1968). The rapid and accurate differentiation ofGluconobacter from Acetobacter is based on ethanol andlactate oxidation. Gluconobacter does not oxidize ethanolto CO 2 and H 2O via acetate and does not oxidize lactateto CO 2 and H 2O, whereas Acetobacter does (De Ley andSwings, 1994). Yamada et al . (1997) examined 36 strainsof Acetobacter , Gluconobacter and Acidomonas for theirpartial sequences in positions 1220 through 1375 of their16S RNAs and performed phylogenetic studies.

Frank et al . (2000) have developed a PCR basedmethod for the detection of Gluconobacter sacchari fromthe microenvironment of the sugarcane leaf sheath. G.sacchari specific 16S RNA-targeted oligonucleotide primerswere designed and used in PCR amplification of G. sacchari DNA directly from mealybug, and in a nested PCR to detectlow numbers of the bacteria from sugarcane leaf sheathfluid and cane internode scrapings.

Occurrence

Gluconobacter strains flourish in sugary niches such asflowers and fruits e.g. ripe grapes (Blackwood et al ., 1969;Passmore and Carr, 1975; and Ameyama, 1975); applesand dates, (Passmore and Carr, 1975). Gluconobacter strains also occur in garden soil, bakers soil, honeybees,fruits, cider, beer and wine (De Ley, 1961). Lambert et al .(1981) isolated 56 Gluconobacter oxydans strains and oneAcetobacter strain from honeybees and their environmentfrom three different regions in Belgium and found twodifferent patterns of proteins by polyacrylamide gelelectrophoresis. Gluconobacter strains are not found tohave any pathogenic effect towards man and other animals.However, they are capable of causing a bacterial rot ofapples and pears accompanied by various shades ofbrowning. Gluconobacter strains have been shown to sharea common antigen (McIntosh, 1962). Bacteria responsiblefor pineapple pink disease in Hawaii were identified asGluconobacter oxydans, Acetobacter aceti and Erwinia herbicola (Cho et al ., 1980).

Growth Conditions

Enrichment and isolation of Gluconobacter strains has beensummarized by Swings and De Ley (1981). Enrichment ofGluconobacter strains present in flowers, fruits or bees canbe set up using the following medium (g/l of distilled water):yeast extract, 5.0; D-glucose, 50; acitidione, 0.1; andbromophenol blue, 0.016. After incubation at 28C for 2-4days, tubes showing acidification are streaked onto platesof standard GYC medium (GYC contains - yeast extract

10.0, glucose 50.0, CaCO 3 30.0; and agar 25.0 g/l,respectively). Colonies that dissolve the CaCO 3 are further

purified and characterized. All Gluconobacter strainsrequire D-mannitol as a carbon source and pantothenicacid, niacin, thiamine and p-aminobenzoic acid as growthfactors (Gossel et al ., 1980). The other carbon sourcesused for the growth are sorbitol, glycerol, D-fructose and

D-glucose (Olijve and Kok, 1979). The bacteria may alsobe maintained on MYP medium (mannitol, 25; yeast extract,5; peptone, 3 g/l, respectively). The bacteria grow optimumin temperature range 25-30C and pH 5.5 6.0. Thecolonies of Gluconobacter strains are pale. Gluconobacter strains are able to grow in a chemically defined mediumwithout amino acids using ammonium ions as a solenitrogen source (Shamberger, 1960). No amino acid isessential for Gluconobacter oxydans (Gossel et al ., 1981).

Carbohydrate Metabolism in G. oxydans

The enzymes of Gluconobacter oxydans have been dividedinto two groups based on the location and function. One

group consists of the particulate enzymes tightly bound tothe bacterial membrane and linked to the cytochromesystems. Examples are D-glucose dehydrogenase(Cheldelin, 1961), D-gluconate dehydrogenase (Matsushitaet al ., 1979), glycerol dehydrogenase (Kersters and DeLey, 1963), and D-sorbitol dehydrogenase (Shinagawa et al ., 1982). The enzymes are flavoproteins and also containcytochrome. They catalyze the direct oxidation of thesubstrates and function primarily in the oxidative formationof acetic acid, D-gluconate, 2- or 5-keto-D-gluconate, L-sorbose, and dihydroxyacetone. The bacteria accumulatelarge amounts of oxidative products extracellularly. Thesecond group consists of enzymes located in the cytoplasmcatalyzing the intracellular metabolism of carbohydrates.It is well known that in G. oxydans , only the pentosephosphate pathway functions and the complete glycolyticand Krebs cycle enzyme sequences are absent (Hauge et al ., 1955; Stouthamer, 1959; De Ley, 1961). 6-Phosphogluconate is a key intermediate in this pathway.Adachi et al . (1979) described cyclic regeneration of NADP +

in Gluconobacter suboxydans IFO12528. Gluconobacter sp. oxidizes glucose via two alternate pathways. Oneoperates inside the cell followed by oxidation via thepentose phosphate pathway and second operates outsidethe cell and involves the formation of gluconic acid andketogluconic acid (Kulka and Walker, 1954; Olijve and Kok,1979). The former is carried by NADP +-dependent glucosedehydrogenase, and the latter is performed by NADP +

independent glucose dehydrogenase and is also called asdirect glucose oxidation pathway (Kitos et al ., 1958).Diagrammatic representation of the pathway is shown inFigure 1.

Industrial Importance

Prescott and Dunn (1959) showed that G. oxydans is welladapted for industrial use. G. oxydans generally bringsabout the incomplete oxidation of sugars, alcohols andacids even in the presence of excess oxygen. Therefore,Gluconobacter strains can be used industrially to produceproducts such as L-sorbose from D-sorbitol, D-gluconic

acid, 5-keto- and 2-ketogluconic acids from D-glucose, anddihydroxyacetone from glycerol. Direct incomplete


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oxidation of sugars, aliphatic and cyclic alcohols, andsteroids proceed through one or two discrete steps andlead to nearly quantitative yields of the oxidation products.Several soluble and particulate polyol dehydrogenaseshave been described (Kersters and De Ley, 1963; De Leyand Kersters, 1964; and Asai, 1968). More than 85% ofGluconobacter strains form acid from n-propanol, n-butanol, glycerol, m-erythritol, D-mannitol, D-arabinose, D-ribose, D-fructose, D-mannose and maltose (Asai, 1971).

Different applications of Gluconobacter oxydans inbiotechnology have been summarized in Table 1.

Fermentation Studies

2, 5-Diketogluconic Acid FermentationGluconobacter oxydans is predominantly known as aketogenic bacterium (Boutroux, 1886). The first study ofthe bacterial production of ketogluconates was publishedin 1940 (Stubbs et al ., 1940). Conversion of D-glucose into2,5-diketogluconic acid is mediated by membrane boundNADP +-independent three dehydrogenases [glucose

dehydrogenase (GDH), gluconate dehydrogenase (GADH)and 2-ketogluconate dehydrogenase (2KGADH)].

Figure 1. Diagrammatic representation of the direct glucose oxidation metabolism in Gluconobacter oxydans . Enzymes involved are membrane bound. Thepathway works only in the presence of >15 mM glucose in the culture medium (Weenk et al., 1984).

Table1. Biotechnological Applications of Gluconobacter oxydans

Application Organism Reference

2,5-Diketogluconic acid formation Gluconobacter oxydans Lockwood, 1954; Asai, 1971; Kita andfrom D-Glucose Fenton, 1982; Weenk et al., 1984; Qazi et al.,

1991; Buse et al., 1990 and 1992.2-keto-L-gulonate formation from 2,5-DKG Erwinia herbicola and Corynebacterium SHS 7522001 Sonoyama et al., 1982Single step production of 2KLG from D-glucose Recombinant Erwinia herbicola Anderson et al., 1985

Recombinant G. oxydans Saito et al., 1998Ascorbic acid formation from Gulono- -lactone G. oxydans DSM 4025 Sugisawa et al., 19955-Ketogluconic acid formation from D-Glucose G. oxydans DSM 3503 Klasen et al., 1992;

G. oxydans NBIMCC 1043 Beschkov et al., 1995Free D-Gluconic acid formation from D-Glucose Gluconobacter oxydans Meiberg et al., 1983

Gluconobacter oxydans NBIMCC 1043 Velizarov and Beschkov, 1994L-Sorbose formation from D-Sorbitol Immobilized G. suboxydans ATCC 621 Stefanova et al., 1987

Immobilized G. oxydans NBIMCC 902 Trifov et al., 1991G. oxydans Rosenberg et al., 1993

L-Sorbosone formation from L-Sorbose Immobilized G. melanogenus IFO 3293 Martin and Perlman, 1976Dihydroxyacetone formation from D-Glucose G. oxydans ATCC 621 Bories et al., 1991

G. oxydans CCM 1783 (ATCC 621) Svitel and Sturdik, 1994aG. oxydans Ohrem and Voss, 1995

Aldehyde formation from Alcohol G. oxydans isolated from Beer Molinari et al., 1995Acetic acid,Propionic acid, Acetone, Butyric G. oxydans CCM 3607, 1783. Svitel and Kullnik, 1995acid and 2-Butanone formation from Ethanol,Propanol, Isopropanol, Butanol and 2-Butanol,respectively.D-Galactonic acid formation from D-Galactose. G. oxydans Svitel and Sturdik, 1994bPropionic acid formation from n-Propanol G. oxydans CCM 1783 Svitel and Kullnik, 1995Isovaleraldehyde formation from Isoamyl alcohol Hydrophobic hollow fibre membrane Molinari et al., 1996

reactor of G. oxydans Continuous production of Isovaleraldehyde G. oxydans in isooctane solvent Cabral et al., 1997


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Extensive fermentation studies have been performedto develop the process for 2,5-diketogluconic acidproduction at the industrial scale. Weenk et al . (1984)showed 2- and 5-ketogluconates production from G.

oxydans ATCC621-H in batch culture using glucose-yeastextract medium in a 2-l fermentor. They also showedproduction of gluconic acid, if G. oxydans 621-H is grownwithout pH control. However, if the pH is controlled by theaddition of CaCO 3, then ketogluconates formation takesplace. Many G. oxydans strains grown under standardconditions in a pH controlled batch culture, produceketogluconates. The regulation of gluconate andketogluconate formation in G. oxydans ATCC 621-H hasbeen studied by Levering et al . (1988). Qazi et al . (1991)compared the growth and 2,5-DKG production by G.oxydans ATCC 9937 in a 10 l stirred tank fermentor and inan ideally mixed air lift fermentor and they showed that theoxidation of gluconate to 2,5-diketogluconate took placethrough an intermediate 2KGA rather than 5-KGA. Thereare two reaction phases - direct glucose oxidation andgluconate oxidation. The positive influence of continuousavailability of O 2 causes induction of membrane bounddehydrogenases involved in the direct glucose oxidation.Buse et al . (1992) found DO of 30% relative to air at 01 baras threshold level for optimum production of keto acids inairlift batch and stirred tank chemostat cultures of G.oxydans ATCC 9937.

Enzymology of Industrially Important Pathways

The important enzymes involved in different oxidativepathways are listed in Table 2.

Glucose DehydrogenaseGlucose dehydrogenase (EC 1.1.99.17), a membranebound quino-protein having bound pyrroloquinolinequinone (PQQ) catalyzes the direct oxidation of D-glucoseto D-gluconic acid in many bacterial strains. Ameyama et al . (1981a) have isolated and characterized GDH from G.oxydans ATCC 9937. The thermal stability of water-solublePQQ dependent glucose dehydrogenase can be increasedby single amino acid replacement using site directedmutagenesis technique (Sode et al ., 2000). The enzymeis a monomeric protein having the MW 87 Kda. Mostinvestigations of GDH have been performed using oxidative

bacteria such as Gluconobacter oxydans and Acinetobacter calcoaceticus . GDHs are found to have bound PQQ (Duine

et al ., 1979). However, E. coli GDH does not have anybound cofactor (Hommes et al ., 1984). NADP +- dependentGDH, a dimer having two identical subunits of MW 54 Kda,showed no significant homology to membrane bound GDH

(Jansen et al ., 1988).Matsushita et al . (1986) showed the immunocross-reactivity among the membrane bound GDHs isolated fromseveral Gram-negative bacteria. The immunocross-reactivity was examined by the immunoblotting techniqueusing antibodies raised against GDH of Pseudomonas fluorescens . Membranes prepared from E. coli, Klebsiella pneumoniae, G. suboxydans, Acinetobacter calcoaceticus are found to have a polypeptide cross reacting with theantibodies specific for GDH of P. fluorescens indicatingclose homology to each other.

Pyrrolo Quinoline Quinone2,7,9 Tricarboxy pyrrolol[2,3 f]quinoline dione is acompound having a pyrrole ring fused to a quinoline ringwith an o-quinone group in it. The representatives of thisgroup are found among the NAD(P) + independent,periplasmic bacterial dehydrogenases (Duine et al ., 1979).

D-Gluconate DehydrogenaseMatsushita et al . (1979) isolated and purified membranebound D-gluconate dehydrogenase (EC 1.1.99.3) fromPseudomonas aeruginosa . The enzyme showed singleband on polyacrylamide gel electrophoresis. The MW ofthe enzyme protein is found to be 138 Kda. In the presenceof SDS, the enzyme dissociated into three subunits havingMWs 66 Kda, 50 Kda and 22 Kda. The subunits of 66 Kdaand 50 Kda are found to be flavo-protein and cytochromeC, respectively. Flavo-protein helps in transferring hydrogenatoms from gluconate, and cytochrome C acts as anacceptor for the electron generated during the reaction.However, role of the subunit of MW 22 Kda in enzymecatalysis is not clear.

2-Ketogluconate DehydrogenaseShinagawa et al . (1981) solubilized and purified 2-keto-D-gluconate dehydrogenase (EC.1.1.99.4) fromGluconobacter melanogenus . The purified enzyme is foundto have tightly bound cytochrome C. The MW of the enzymeis found to be 133 Kda. SDS-PAGE showed the presenceof three subunits having MWs of 61 Kda (flavo protein), 47

Kda (cytochrome C) and 25 Kda (with unknown function).

Table2. Enzymes purified and characterized from Gluconobacter oxydans /other ketogenic bacteria

Enzyme Organism Co-enzyme Reference

Glucose dehydrogenase (GDH) Gluconobacter oxydans ATCC 9937 PQQ Ameyama et al., 1981 a.Gluconic acid dehydrogenase (GADH) Pseudomonas aeruginosa FAD+ Matsushita et al., 1979

and Mclntire et al., 1985.2-Ketogluconic acid dehydrogenase Gluconobacter melanogenus FAD+ Shinagawa et al., 1981(2KGADH) and Mclntire et al., 1985.L-Gulono- -lactone dehydrogenase Gluconobacter oxydans DSM4025 - Sugisawa et al., 1995.D-Sorbitol dehydrogenase (SDH) G. suboxydans var. IFO 3250 NAD + Shinagawa et al., 1982.L-Sorbose dehydrogenase G. oxydans UV-10 - Fujiwara et al., 1987.D-Fructose dehydrogenase G. industrius PQQ. Ameyama et al., 1981 b.Aldehyde dehydrogenase Acetobacter aceti PQQ Ameyama et al., 1981 c.L-Sorbose reductase Gluconobacter melanogenus IFO 3294 NADPH Adachi et al., 1999.Xylitol dehydrogenases Gluconobacter oxydans NAD+ Masakazu et al., 2000.


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2,5-Diketogluconic Acid Pathway in Other Organisms

Direct glucose oxidative reactions (Figure 2) are presentin wide variety of microorganisms like Erwinia herbicola (Sonoyama et al ., 1982) , Pseudomonas aeroginosa (Matsushita et al ., 1979), Acinetobacter calcoaceticus (Duine et al ., 1979), and Escherichia coli (Hommes et al .,1984). However, in E. coli , GDH does not have any boundcofactor but gets activated by the addition of exogenousPQQ cofactor.

Physiological Significance of Direct Glucose OxidationPathway

In many gram-negative bacteria, glucose oxidation viaglucose dehydrogenase, quinoprotein leads to thegeneration of a proton motive force, which can be coupledeffectively to the energization of various cellular processes(Schie et al ., 1985). Neijssel et al . (1989) showed thatoxidation of glucose by membrane particles of G. oxydans could be coupled with the phosphorylation of ADP. Growthcomparison between wild type and GDH - mutant of G.oxydans revealed that GDH helps the organism to attainfast growth in a glucose enriched medium. Lag phase ofwild type in enriched glucose medium is 2 hr whereas GDH -

mutant has 8 hr lag phase (A.Gupta, unpublished data).Acinetobacter calcoaceticus utilizes glucose only in

the form of gluconic acid. Glucose is first oxidized togluconic acid by extracellular glucose dehydrogenase andgluconic acid is absorbed by the cell and furthermetabolized by Entner-Doudoroff pathway (Schie et al .,1985).

Importance of 2,5-Diketogluconic Acid Pathway

2,5-Diketogluconate may be converted into 2-KLG bystereospecific reduction using 2,5-diketogluconic acidreductase (Sonoyama et al ., 1982). 2-KLG is a penultimateintermediate in industrial production of ascorbic acid, knownas vitamin C (Reichstein, et al ., 1934). The otherintermediates of the pathway like gluconate and 2-keto and/ or 5-keto gluconates are also of industrial importance.

2,5-DKG Reductase 2,5-Diketo-Gluconic Acid 2-Keto-L-Gulonic Acid

Bioproduction of 2-keto-L-Gulonic Acid

2,5-Diketogluconate reductase has been identified inCorynebacterium sp. SHS 752001 (Sonoyama et al ., 1982).A two-stage fermentation process has been proposed for2-keto-L-gulonate production. The glucose is converted into2,5-DKG by Erwinia sp. After 26 hr cultivation, 328.6 mg ofcalcium 2,5-diketogluconate per ml with 94.5% yield fromD-glucose has been reported. The broth was directly usedfor the next conversion without removal of the cells. Thestereospecific reduction of calcium 2,5-diketogluconate tocalcium 2-keto-L-gulonate was performed with mutantstrain of Corynebacterium . The results of two-stagefermentation in 10-m 3 conventional fermentors showedformation of an average of 106.3 mg of calcium 2-keto-L-gulonate per ml.

The traits of two microorganisms ( Erwinia herbicola and Corynebacterium sp) have been combined in a singlecell by the gene manipulation techniques to simplify theconversion of D-glucose to 2-KLG (Anderson et al ., 1985).They engineered E. herbicola ATCC 21988 by transforming2,5-DKG reductase gene isolated from Corynebacterium sp. ATCC 31090. The engineered E. herbicola culture isable to produce upto 1 g/l 2-KLG in one step process. Directproduction of 2-keto-L-gulonic acid using recombinant G.oxydans T-100 has also been reported (Saito et al ., 1998).The promoters of SDH and SNDH are replaced with thepromoter of E. coli tufB1 gene and reintroduced intoGluconobacter oxydans T-100 to improve its efficiency forthe production of 2-KLGA from D-sorbitol. Such a biologicalprocess is of industrial importance as it may replacepresently used complex, multistep Reichsteins process.Reichstein-Grssner synthesis of L-ascorbic acid servesthe basis for modern industrial production of ascorbic acid.The main steps in the Reichstein-Grssner process arechemical conversion of D-glucose into D-sorbitol followedby its microbial biooxidation to L-sorbose. L-Sorbose isconverted to 2-KLG by chemical reactions. D-Sorbitol isoxidized to L-sorbose using Acetobacter suboxydans/ Gluconobacter melanogenus .

Figure 2. Diagrammatic representation of keto acids pathways in different organisms.


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using G. oxydans CCM 1783 (ATCC621). An increase inthe O 2 consumption rate was observed at glycerolconcentration upto 300g/l. Batch cultures incubated withgassing by air and/or O 2, yielded upto 94% DHA. Sodiumglycerate was identified in the fermentation broth (after

neutralization with NaOH). Ohrem and Voss (1995)reported production of 220 g/l DHA (96-98% production)after G. oxydans fermentation for 30 hr. The Maximumproductivity reached after 6-10 hr. The fermentation brothpH was maintained between 3.8-4.8 using Ca(OH) 2. Ohremand Voss (1996) studied effect of glycerol on the growthand DHA production in Gluconobacter oxydans . Theyshowed that neither glycerol oxidation nor metabolism ofDHA was inhibited by high glycerol concentrations. Theyshowed that DHA damaged the cells irreversibly and theviability of Gluconobacter decreased exponentially withtime.

Aldehyde Formation

The aldehyde formation by alcohol oxidation with G.oxydans submerged cultures and resting cells,resuspended in different phosphate buffers (0.1M) hasbeen studied by Molinari et al . (1995). Large-scale cultureswere grown using stirred fed-batch reactors (1.5 l workingvolume, 250rpm and/or 2vvm air), bubble columns (500ml working volume, 0.5vvm/1vvm air) and airlift reactors(1l working volume, 0.5 vvm/1vvm air). New G. oxydans strain (from beer) oxidized various short chain aliphaticalcohols to their corresponding aldehydes. Molinari et al .(1996) reported 3-isoamylalcohol as the best substratehaving over 90% yields of 3-isoamylaldehyde without anyby-product formation.

Other Fermentation Processes

Svitel and Kullnik (1995) studied the potentials of aceticacid bacteria for oxidation of low MW monoalcohols.Acetobacter aceti CCM 3620, Acetobacter liquefaciens CCM 3621, Acetobacter pasteurians CCM 2374, G.oxydans CCM 3607 and CCM 1783 (ATCC 621) were usedfor the oxidation of acids and ketones. The oxidations wereperformed in fed-batch cultures using a 5 l reactor (LF-2)filled with 2.7 l of sterile medium at 500rpm and 1vvmaeration. The pH during acid production was maintainedat 6.0. Ethanol, propanol, isopropanol, butanol and 2-butanol were converted to acetic acid, propionic acid,acetone, butyric acid and 2-butanone, respectively. Sviteland Sturdik (1994b) reported galactonic acid formation fromD-galactose in batch culture of 32 hr. During transformation,96% of D-galactose (100g/l) got converted to D-galactonic

acid. Svitel and Sturdik (1995) examined the conversionof n-propanol to propionic acid by G. oxydans CCM 1783(ATCC 621) using fed batch conversion at pH 6.0. Propanolfeeding was started after the growth phase. In the fed-batch conversion, calcium propionate 37g/l, formed within

30 hr (when Ca(OH) 2 was used for the neutralization), andsodium propionate, 56g/l, within 70 hr (when NaOH wasused for the neutralization), was obtained.

Applications of Gluconobacter in BiosensorTechnology

Gluconobacter oxydans as whole cells as well as itsenzymes have been examined for its use as biosensor forthe identification of various compounds. Applications ofGluconobacter oxydans in biosensor technology are listedin Table 3.

Smolander et al . (1993) purified a membrane boundxylose oxidizing PQQ-dependent dehydrogenase from

Gluconobacter oxydans ATCC 621 and studied its possibleapplications in biosensor technology. Dimethyl andcarboxylic acid derivatives of ferrocene were able tomediate electron transfer in xylose oxidation using enzymeimmobilized on graphite electrode. Smolander et al . (1995)developed an amperometric enzyme electrode biosensorfor analysis of xylose and glucose in fermentation samplesusing PQQ-dependent ALDH from G. oxydans ATCC621.The best electrode performance was obtained when ALDHwas adsorbed on the surface of a carbon-paste electrode.The lowest working potential and highest catalytic currentwere obtained with dimethylferrocene as a mediator.

Hikuma et al . (1995) prepared a biosensor consistingof an anode mounted with immobilized alcoholdehydrogenase (EC 1.1.1.1) from G. oxydans IFO 3172,and a cathode containing hexacyanoferrate (III). Thesurface of the biosensor was covered with a gas-permeablemembrane and measurements were made without reagentconsumption by pumping a sample solution through theflow cell for 1 or 2 minutes. Alcohol contents of the alcoholicdrinks determined using the sensor as well as by gaschromatography were coincided indicating high sensitivityof the sensor. Reshetilov et al . (1998a) used G. oxydans strains as microbial electrode biosensor for xylose analysisusing clark-type oxygen electrodes. Reshetilov et al .(1998b) described the use of Gluconobacter oxydans wholecells for determination of sugars, alcohols, and polyols.Lusta and Reshetilov (1998) reviewed the prospects ofGluconobacter oxydans for its use in Biotechnology asbiosensor system. Svitel et al . (1998) described themicrobial cell-based biosensor for sensing glucose, sucroseor lactose. The glucosesensing membrane was prepared

Table 3. Application of Gluconobacter oxydans in Biosensor Technology

Detection of the Substrate Enzyme/system Organism Reference

Xylose and Glucose Aldose dehydrogenase G. oxydans ATCC621 Smolander et al., 1995Alcohol Alcohol dehydrogenase G. oxydans IFO3172 Hikuma et al., 1995.Xylose Clark type-oxygen electrode G. oxydans Reshetilov et al., 1998a.Sugars, Alcohols and Polyols G. oxydans whole cells. Reshetilov et al., 1998b.Glucose, Sucrose or Lactose Glucose sensing membrane of intact cells of G. oxydans Svitel et al., 1998.

coimmobilized with Kluyeromyces marxians


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452 Gupta et al .

with intact cells of Gluconobacter oxydans immobilized ingelatin cross-linked with glutaraldehyde. The disaccharide sensing membranes were prepared by co-immobilizationof G. oxydans either with cells of Saccharomyces cerevisiae containing invertase for sucrose determination, or with

permeabilized cells of Kluyveromyces marxianus containing -galactosidase for lactose determination. Thestrain of G. oxydans was able to oxidize both anomers ofglucose at the same rate. Therefore, there was no needfor mutarotase co-immobilization in disaccharide-sensingmembranes. The sensitivity of glucose sensor was 50 nA/ mM and the range of the calibration curve was 0-0.8 mMwith response time of 2 min. The response after 1 week ofstorage was 62% of the initial one. The detection linearrange of disaccharide sensor was found to be upto 4mMwith response time of 5min. The activities of the sensorsafter 1 week of storage at ambient temperature were inthe range of 50-65% of the initial activity.

Genetic Analyses of G. oxydans Strains

Native Plasmids and Phages of G. oxydans Fukaya et al ., (1985a) detected plasmid DNAs in 23 out ofthe 36 strains of Gluconobacter sp. and most of them hadMW of more than 5 Mda. Verma et al . (1994) characterizedplasmids of G. oxydans strains ATCC 9937 and IFO3293,and found three native plasmids in ATCC 9937 having thesizes 27.7 kb (pVJ1), 12.3 kb (pVJ2) and 18 kb (pVJ4) andone native plasmid of 9.5 kb size (pVJ3) in IFO3293. Twophages have been isolated from 54 different strains ofGluconobacter (Robakis et al ., 1985). These two phageshave been reported to have different sized double strandedDNAs (approximately 37 and 250-300 kb sizes). Therestriction map of bacteriophage revealed ring like DNAcontaining cohesive ends.

Identification of Genes Involved in 2,5-DKG Pathwayfrom Gluconobacter oxydans Glucose dehydrogenase gene has been identified andsequenced from G. oxydans P1 and P2 strains (Jansen et al ., 1991). P1 and P2 are two isogenic forms of G. oxydans .P1 can oxidize only D-glucose, whereas P2 is also capableof oxidizing sucrose. The DNA sequence of both gdh genesfrom P1 and P2 are found identical except one nucleotide

substitution resulting in the substitution of His 787 by Asnin an open reading frame of 2424 bp. The putative proteinis of 808 amino acids with a MW of 87,587 Da. Riboflavinkinase (rib F) gene has been identified from G. oxydans ATCC9937 that is involved in FAD + synthesis for GADH

enzyme (Gupta et al ., 1999). FAD+

is a cofactor for GADHenzyme (McIntire et al ., 1985). The other genes namelygadh and 2kgadh involved in 2,5-dkg pathway from G.oxydans could not be detected yet. Xba I restriction mapof the genome from G. oxydans ATCC 9937 has beendeveloped using Pulse field gel electrophoresis studies(Verma et al ., 1997). Southern hybridization and PFGEshowed single copy of pqq and ribF genes in G. oxydans ATCC 9937 and IFO 3293 present on different locations(A.Gupta, unpublished data).

Identification of Genes Involved in 2,5-dkg Pathway/Other Pathways from Other BacteriaThe genes of 2,5-dkg pathway/other pathways reported

from G. oxydans /o ther bacter ia like Acinetobacter calcoaceticus, Erwinia herbicola are listed in Table 4.

Jansen et al . (1988, 1990) have reported the gdh genesfrom A. calcoaceticus and E. coli . The gdh genes isolatedfrom A. calcoaceticus, E. coli and G. oxydans showed highhomology at C-terminal ends (Jansen et al ., 1991). Cha et al . (1997) identified and characterized a gene encodingglucose dehydrogenase from Pantoea citrea and showedits involvement in causing Pineapple pink disease.

The pqq genes of A. calcoaceticus, Methylobacterium extorquens AMI and Klebsiella pneumoniae have beencloned and sequenced (Goosen et al ., 1987; Toyama et al ., 1997; Meulenberg et al ., 1992).

Gluconate dehydrogenase encoding genes have beenisolated and characterized from Erwinia cypripedii ATCC29267 (Yum et al ., 1997). The 2,5 diketo-D-gluconatereductase of E. coli has homology with 2,5-diketo-D-gluconate reductases of Corynebacterium sp., andGluconobacter oxydans (Yum et al ., 1999). 2,5-DKGR ofCornyebacterium showed 49.8% homology to yqhE geneproduct; and 2,5KDGR of Gluconobacter oxydans showed42% homology with yafB gene product. The gene productsof yqfE and yafB were identified as 2,5DKGR-A and2,5DKGR-B, respectively catalyzing the reduction of 2,5-DKG to 2-KLG.

Table 4. Genes identified from Gluconobacter oxydans / other ketogenic bacteria involved in 2,5-diketogluconic acid pathway/ other pathways.

Gene Organism Reference

gdh G. oxydans P1 & P2 Jansen et al., 1991RibF G. oxydans ATCC 9937 Gupta et al., 1999gdh Acinetobacter calcoaceticus Jansen et al., 1988gdh Escherichia coli Jansen et al., 1990gdh Pantoea citra Cha et al., 1997pqq Acinetobacter calcoaceticus Goosen et al., 1987pqq Methylaobacterium extorquens AMI Toyama et al., 1997pqq Klebsiella pneumonniae Meulenberg et al., 1992gadh Erwinia cypripedii ATCC 29267 Yum et al., 1997L-Sorbosone dehydrogenase (SNDH) gene Acetobacter liquefaciens IFO12258 Shinjoh et al., 1995D-Sorbitol dehydrogenase (SDH) and sorbosone dehydrogenase gene G. oxydans G 624 Saito et al., 1997Gluconate NADP +5-oxidoreductase (GNO) gene G. oxydans subsp. 3503 Klasen et al., 1995IS element G. suboxydans Kondo and Horinouchi, 1997RecA G. oxydans LCC 99 Liu et al., 1998Xylitol dehydrogenase genes (XDH1 & XDH2) Gluconobacter oxydans Masakazu et al., 2000.


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Identification of Genes Involved in Sorbitol OxidationShinjoh et al . (1995) cloned and sequenced the membranebound SNDH gene from Acetobacter liquefaciens IFO12258 and got it expressed in a 2-KLG producingmutant (strain OX4) of G. oxydans IFO3293. The open

reading frame of the gene is found to be 1,347 bp encodinga polypeptide of 449 amino acids, with a MW 48,223 Da.The cloned SNDH is located in the membrane of G.oxydans IFO3293. The recombinant cells produced 2KLGefficiently using L-sorbosone or L-sorbose in the medium.The yield of ascorbic acid with recombinant G. oxydans IFO3293 strain OX4 using L-sorbosone was improved fromabout 25 to 83%, whereas using L-sorbose was increasedfrom 68 to 81%.

Identification of Genes Involved in Other Pathwaysfrom G. oxydans Klasen et al . (1995) reported Gluconate NADP + 5-oxidoreductase gene from Gluconobacter oxydans sub sp.

DSM 3503. The enzyme oxidizes gluconate to 5-ketogluconate and is localized in the cytoplasm. The MWof the enzyme is reported to be 75 Kda. The enzymeshowed single band on SDS-PAGE having MW 33 Kdaindicating two identical subunits in the protein. Sequencingof the gene revealed an open reading frame of 771 bp,encoding a protein of 257 amino acids. Kondo andHorinouchi (1997) reported a novel insertion sequenceelement IS 12528 from G. suboxydans associated withinactivation of the alcohol dehydrogenase by insertion inthe adhA gene, that encodes the primary dehydrogenasesubunit of the three-component membrane-bound alcoholdehydrogenase complex in Gluconobacter suboxydans .Cloning and sequencing analyses revealed that IS 12528was 905 bp in length and had a terminal inverted repeat of18 bp. Liu et al . (1998) reported a recA gene from G.oxydans and they expressed it in E. coli recA - mutant. TheG. oxydans recA protein efficiently functions in homologousrecombination and DNA repair. The deduced amino acidsequence of recA gene revealed a protein of 344 aminoacids with a MW of 38 Kda.

Vector System for G. oxydans The construction of shuttle vectors for Gluconobacter oxydans and Escherichia coli is a further step in theapplication of recombinant DNA technology for this groupof microorganisms. Fukaya et al . (1985b) constructed ashuttle vector pMG101 for Gluconobacter oxydans . Theshuttle vector, pMG101, for Gluconobacter oxydans andEscherichia coli is constructed by ligation of a crypticplasmid, pMV201, found in G. suboxydans IFO 3130 withE. coli plasmid pACYC177. The chimeric plasmid namedpMG101 carries the ampicillin resistance gene derived fromplasmid ACYC177. The transformation efficiency is foundto be 10 2 per g DNA.

Palleroni (1986) showed that Gluconobacter oxydans IFO 3293 and ATCC 9937 could accept plasmid RSF 1010both by transformation and conjugation. Condon et al .(1991) reported conjugal transfer of a series of incompatiblegroup P and Q plasmids in Gluconobacter oxydans NCIB2008. Transfer frequency for the Inc P/Q vectors

ranges from 10-5

to 10-9

exconjugants per recipient cell.They constructed gentamycin resistant pRK290 vector as

a potential versatile gene delivery system for Gluconobacter oxydans .

Creaven et al . (1994) developed an efficientelectroporation system for transformation of the plasmidpMP220 in G. oxydans NCIB2008. The optimum

electroporation conditions were 12.5 KV/cm field strength,400 ohm parallel resistor setting, 0.4-0.5g DNA, 100l cellsuspension and electroporation buffer containing Mg +2 and10 11 cfu/ml of early to mid-log phase cells. Under theseconditions, transformation efficiencies of 10 5 /g DNA werereproducibly obtained. Storage for 5 days at -20C in a buffercontaining 1% glycerol decreased transforming efficiencyby 86% owing to a loss of cell viability.

Shinjoh and Hoshino (1995) developed a shuttle vector(plasmid pGE1, 11.9kb) and conjugative transfer systemfor G. oxydans . Transformed G. oxydans were shown tobe capable of producing 2-keto-L-gulonic acid from L-sorbose. The plasmid pGE1 was constructed from crypticG. oxydans IFO 3293 plasmid pGO32938 (9.9kb, relaxed

type), E. coli conjugative plasmid pSUP301 (5kb, Kmr and

amp r, relaxed type), plasmid pACYC 177 and the plasmidRP4 mob region. The pGE1 was transferred by conjugationto G. oxydans IFO 3293 with high frequency (0.1transconjugants/recipient), and maintained stability withoutantibiotic selection. It was also maintained in other strainsof G. oxydans like IFO 3172, IFO 3268, IFO 3271 and ATCC9937; and G. frateurii IFO 3268, IFO 3129, IFO 3170,IFO3223 and IFO 3225. The presence of pGE1 did notinhibit growth or 2KLG productivity of G. oxydans IFO 3293.The vector was tested by subcloning gene of Acetobacter liquefaciens IFO 12258 in G. oxydans IFO 3293. Theplasmid pGE1 could be shortened further to plasmid pGE2(9.8 kb).

Gene Expression in Gluconobacter oxydans Saito et al . (1997) constructed a shuttle vector forexpression of sorbitol dehydrogenase and sorbosonedehydrogenase genes in G. oxydans G624. A plasmid of4.4 kb designated as pF4 was isolated from G. oxydans T-100 and ligated with pHSG298 at the Hind III site toconstruct a shuttle vector, designated as pFG15A. Theplasmid pFG15A carrying SDH and SNDH genes wastransformed into G. oxydans G624. The recombinantGluconobacter oxydans G624 was 2-3 fold more activefor the production of 2-KLGA than G. oxydans T-100;however, G. oxydans G624 itself showed no ability toproduce 2-KLGA.

Takeda and Toshio (1992) expressed cytochrome C-553 (CO) gene in Gluconobacter suboxydans subsp. thatcomplemented the second subunit deficiency ofmembrane-bound alcohol dehydrogenase. The gene hasbeen subcloned into a shuttle vector pGEA1 and therecombinant was designated as pGEAC1. The gene hasbeen shown to replicate in Gluconobacter suboxydans andEscherichia coli . The pGEA1 transformed G. suboxydans subsp. showed low ethanol oxidation activity due tosecond subunit deficiency of membrane-bound ADH. Thetransformants harboring pGEAC1 expressed ethanoloxidation activity and showed cross-reactivity with anti-cytochrome C-553 antibody. Furthermore, expression of

cytochrome C-553 (CO) gene resulted in elevated levelsof heme C and CO-reactive cytochrome C complemented


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454 Gupta et al .

the deficiency of the second ADH subunit and restoredethanol oxidase activity.

Heterogeneity among G. oxydans StrainsVerma et al . (1997) reported genetic heterogeneity among

keto-acid-producing strains of G. oxydans ATCC9937, IFO3293, IFO 12258 and DSM 2343. The genomes of fourketo-acid-producing G. oxydans strains were analyzed bypulse field gel electrophoresis. Xba I was chosen forrestriction fragment analysis of the genomes. The genomesizes of the four strains were estimated to be between 2240kb and 3787 kb. Although these strains are physiologicallysimilar, Xba I restricted PFGE of the four strains showedno homology.

Mutagenic Studies in G. oxydans Qazi et al . (1989) reported mitomycin C as an effectivemutagenic agent for G. oxydans ATCC 9937. The MIC ofmitomycin for G. oxydans has been found to be 40 g/ml.

Mitomycin C is shown as an effective curing agent for G.oxydans ATCC9937.

Transposon MutagenesisKahn and Manning (1988) obtained a patent onGluconobacter oxydans strains having capability of higherproduction of 2-KLG from L-sorbose. They carried outadditional mutation in G. oxydans UV-10 mutant strain byinserting a transposon (PI::Tn5) in the 2-KLG reductase.The mutant strain, M23-15, produced more 2-KLG (33.3g/l) compared to the parent strain (19.6 g/l) due to reduced2-KLG-reductase activity.Gupta et al . (1997) have developed a protocol forTransposon Tn5 mutagenesis in Gluconobacter oxydans using pSUP2021 vector system. Simon et al . (1983)constructed pSUP2021 to perform Tn5 mutagenesis ingram negative bacteria. Tn5 transpogenesis frequency wasfound to be 10 -7 to 10 -8 /cell and 10 -5 to 10 -6 /cell in G.oxydans ATCC 9937 and G. oxydans IFO 3293,respectively. Using this technique, a library of Tn5transposed cells of G. oxydans ATCC 9937 was developedand screened for specific mutants blocked in direct glucoseoxidation pathway. Two different mutants of G. oxydans having defective GDH - and GADH -, respectively, wereisolated.

GDH - GADH - 2KGADH

Glucose ---X---Gluconic acid ---X---- 2-Ketogluconicacid----- 2,5- Diketogluconic acid

A GDH - mutant G. oxydans AG-a was selected on the basisof non-acid zone formation in P1 production medium plate(Gupta et al ., 1997). In this mutant, Tn5 insertion was foundin its PQQ cofactor synthesizing genes (pqq). A GADH -

mutant of G. oxydans ATCC 9937 was selected on thebasis of non-melanin formation in P1 production medium.The specific blockage at gluconate conversion point wasconfirmed by HPLC analysis (Gupta et al ., 1999). In thismutant, Tn5 insertion was found in the Rib F gene. Liu et al . (1999) constructed a recA deficient mutant ofGluconobacter oxydans called as LCC99, that could beused as a host to take up the recombinant plasmid for genemanipulation. The mutant was constructed by allelicexchange using the cloned recA gene that had beeninactivated by inserting a kanamycin-resistance cassette.

Conclusion

G. oxydans is a simple, non-pathogenic, industriallyimportant bacteria. The bacteria has a number ofmembrane bound dehydrogenases which are involved in

many oxidation reactions. These oxidation reactions canbe exploited for commercial purposes. Geneticunderstanding of the organism may be fruitful for thegenetic exploitation. Apart from its oxidative reactions, itcan also be manipulated for the expression of foreigngenes.

Acknowledgements

The Authors wish to acknowledge the facilities of Distributed Bioinformaticsubcentres of Devi Ahilya University, Indore; Regional Research Laboratory,Jammu; and Guru Nanak Dev University, Amritsar for providing necessaryfacilities for literature survey. The authors thank to Prof. Milton H. Saier,Dept. of Biology, University of California, San Diego, USA and Dr. SubratoGuha, Vaishnav College, Indore for critical review and correcting the Englishusage errors in the manuscript.

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