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

Review Biotechnological Applications JMMB of G. oxydans 445

Gluconobacter oxydans: Its Biotechnological Applications

Arun Gupta1, Vinay K. Singh1, G.N. Qazi2 and Anil Kumar1
1 School of Biotechnology, Devi Ahilya University, Khandwa Road, Indore-452017, MP, India 2 Dept. 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 of metabolism using oxygen as the terminal electron acceptor. Gluconobacter strains flourish in sugary niches e.g. ripe grapes, apples, dates, garden soil, bakers soil, honeybees, fruit, cider, beer, wine. Gluconobacter strains are non-pathogenic towards man and other animals but are capable of causing

Abbreviations g-microgram; l-microlitre; 2,5-DKG-2,5-diketogluconic acid; 2,5-DKGR2,5-diketo-D-gluconate reductase; 2KGADH-2-ketogluconate dehydrogenase; 2kgadh-2-keto-gluconate dehydrogenase gene; 2KLG-2ketoL-gulonic acid; 5KDGR-5-keto-D-gluconate reductase; 5KGA-5ketogluconic acid ; 2,5-DKGR-2,5-diketo-D-gluconate reductase. 5KDGR5-ketogluconate reductase; 2,5-DKGR-A - 2,5-diketo-D-gluconate reductase product of gene yqfE of E. coli; 2,5-DKGR-B - 2,5-diketo-D-gluconate reductase product of gene yqfB of E. coli; A. calcoaceticus-Acinetobacter calcoaceticus; ADH-alcohol dehydrogenase; Adh-alcohol dehydrogenase gene; ADP-adenosine diphosphate; ALDH-aldose-dehydrogenase; Asnasparagine; ATCC-American type culture collection; bp-base pair; CaCO3calcium carbonate; Ca(OH)2-Calcium hydroxide; Cfu-colony formation unit; CO 2 -carbon dioxide; Cyt C- cytochrome C; Da-dalton; DHAdihydroxyacetone; DO-dissolved oxygen; E. coli- Escherichia coli; E. herbicola-Erwinia herbicola; EC-Enzyme commission; FAD+-flavin adenine dinucleotide; g-gram; G. oxydans-Gluconobacter oxydans; G. sacchariGluconobacter sacchari ; GA-gluconic acid; GADH-gluconate dehydrogenase; GADH--gluconate dehydrogenase deficient mutant; gadhgluconate dehydrogenase gene; GDH-glucose dehydrogenase ; GDH- glucose dehydrogenase deficient mutant; gdh-glucose dehydrogenase gene; GNO- gluconate NADP+ 5-oxidoreductase gene; GYC- glucose-yeast extract-calcium carbonate medium; H2O-water; His-histidine; H2S-hydrogen disulfide; HPLC-high performance liquid chromatography; hr-hour; IFOInstitute of Fermentation, Osaka; IS-insertion sequences; Kb-kilobase; KdaKilodalton; Kg-kilogram; l-litre; m-meter; Mda- megadalton; mg-milligram; ml-millilitre; mM-millimolar; MIC-minimum inhibitory concentration; MWmolecular weight; MYP- mannitol-yeast extract-peptone medium; nA-nanoampere; NADP+-nicotinamide adenine dinucleotide phosphate; NaOHsodium hydroxide; N-source-nitrogen source; O2-oxygen; OPV-oxidized polyvinyl alcohol; P. fluorescens- Pseudomonas fluorescens ; PAGEpolyacrylamide gel electrophoresis; PCR-Polymerase Chain Reaction; PFGE-pulse field gel electrophoresis; PQQ-Pyrrolo Quinoline Quinone; pqqpyrrolo quinoline quinone genes; RibF-riboflavin kinase F gene; rpmrevolutions per minute; SDH-sorbitol dehydrogenase; SDS-sodium dodecyl sulfate; SNDH-sorbosone dehydrogenase; Tn5-Transposon Tn5; vvmvolumetric volume per mole.

bacterial rot of apples and pears accompanied by various shades of browning. Several soluble and particulate polyol dehydrogenases have been described. The organism brings about the incomplete oxidation of sugars, alcohols and acids. Incomplete oxidation leads to nearly quantitative yields of the oxidation products making G. oxydans important for industrial use. Gluconobacter strains can be used industrially to produce L-sorbose from D-sorbitol; Dgluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose; and dihydroxyacetone from glycerol. It is primarily known as a ketogenic bacterium due to 2,5diketogluconic acid formation from D-glucose. Extensive fermentation studies have been performed to characterize its direct glucose oxidation, sorbitol oxidation, and glycerol oxidation. The enzymes involved have been purified and characterized, and molecular studies have been performed to understand these processes at the molecular level. Its possible application in biosensor technology has also been worked out. Several workers have explained its basic and applied aspects. In the present paper, its different biotechnological applications, basic biochemistry and molecular biology studies are reviewed. Introduction

Gluconobacter oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae. The shape of the 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 respiratory type of metabolism with oxygen as the terminal electron acceptor (De Ley and Swings, 1994). Gluconobacter is an industrially important genus for the production of L-sorbose from D-sorbitol; D-gluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose; and dihydroxyacetone from glycerol. The present review deals with the fermentation studies, applications in Biotechnology and genetic aspects of Gluconobacter oxydans.
Classification and Nomenclature of Gluconobacter In 1935, a group of gram-negative bacteria related to Acetobacter was renamed and put under the genus Gluconobacter (Asai, 1935). Gluconobacter, earlier known as Acetobacter oxydans, has been characterized as having the pronounced capability for the oxidation of glucose to gluconate and a weak ability for the oxidation of ethanol to acetate (Kluyver and Boezaardt, 1938). A review on the classification and nomenclature problems related to the genus Gluconobacter has been published (De Ley and Frateur, 1970). The following biochemical tests are found uniformly negative for all strains of Gluconobacter oxydans: glucose oxidase, reduction of

Received February 29, 2000; revised April 18, 2000; accepted July 5, 2000; final manuscript received February 16, 2001. *For correspondence. Email bioinfo@sancharnet.in; Fax. 91-731-470372.

2001 Horizon Scientific Press

446 Gupta et al. purified and characterized. All Gluconobacter strains require D-mannitol as a carbon source and pantothenic acid, niacin, thiamine and p-aminobenzoic acid as growth factors (Gossel et al., 1980). The other carbon sources used for the growth are sorbitol, glycerol, D-fructose and D-glucose (Olijve and Kok, 1979). The bacteria may also be maintained on MYP medium (mannitol, 25; yeast extract, 5; peptone, 3 g/l, respectively). The bacteria grow optimum in temperature range 25-30C and pH 5.5 6.0. The colonies of Gluconobacter strains are pale. Gluconobacter strains are able to grow in a chemically defined medium without amino acids using ammonium ions as a sole nitrogen source (Shamberger, 1960). No amino acid is essential for Gluconobacter oxydans (Gossel et al., 1981). Carbohydrate Metabolism in G. oxydans The enzymes of Gluconobacter oxydans have been divided into two groups based on the location and function. One group consists of the particulate enzymes tightly bound to the bacterial membrane and linked to the cytochrome systems. Examples are D-glucose dehydrogenase (Cheldelin, 1961), D-gluconate dehydrogenase (Matsushita et al., 1979), glycerol dehydrogenase (Kersters and De Ley, 1963), and D-sorbitol dehydrogenase (Shinagawa et al., 1982). The enzymes are flavoproteins and also contain cytochrome. They catalyze the direct oxidation of the substrates and function primarily in the oxidative formation of acetic acid, D-gluconate, 2- or 5-keto-D-gluconate, Lsorbose, and dihydroxyacetone. The bacteria accumulate large amounts of oxidative products extracellularly. The second group consists of enzymes located in the cytoplasm catalyzing the intracellular metabolism of carbohydrates. It is well known that in G. oxydans, only the pentose phosphate pathway functions and the complete glycolytic and Krebs cycle enzyme sequences are absent (Hauge et al ., 1955; Stouthamer, 1959; De Ley, 1961). 6Phosphogluconate 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. One operates inside the cell followed by oxidation via the pentose phosphate pathway and second operates outside the cell and involves the formation of gluconic acid and ketogluconic acid (Kulka and Walker, 1954; Olijve and Kok, 1979). The former is carried by NADP+-dependent glucose dehydrogenase, and the latter is performed by NADP+ independent glucose dehydrogenase and is also called as direct glucose oxidation pathway (Kitos et al., 1958). Diagrammatic representation of the pathway is shown in Figure 1. Industrial Importance Prescott and Dunn (1959) showed that G. oxydans is well adapted for industrial use. G. oxydans generally brings about the incomplete oxidation of sugars, alcohols and acids even in the presence of excess oxygen. Therefore, Gluconobacter strains can be used industrially to produce products such as L-sorbose from D-sorbitol, D-gluconic acid, 5-keto- and 2-ketogluconic acids from D-glucose, and dihydroxyacetone from glycerol. Direct incomplete

nitrates, H2S formation and indole production (De Ley, 1978). Loitsianskaya et al. (1979) proposed the existence of a single species within the genus on the basis of numerical analyses of 136 phenotypic features of 56 strains of acetic acid bacteria. The genus Gluconobacter belongs to the fourth rRNA superfamily and constitutes a separate branch together with the genus Acetobacter (Gillis and De Ley, 1980). Gluconobacter and Acetobacter are closely related to each other and are classified as acetic acid bacteria (Asai, 1968). The rapid and accurate differentiation of Gluconobacter from Acetobacter is based on ethanol and lactate oxidation. Gluconobacter does not oxidize ethanol to CO2 and H2O via acetate and does not oxidize lactate to CO2 and H2O, whereas Acetobacter does (De Ley and Swings, 1994). Yamada et al. (1997) examined 36 strains of Acetobacter, Gluconobacter and Acidomonas for their partial sequences in positions 1220 through 1375 of their 16S RNAs and performed phylogenetic studies. Frank et al. (2000) have developed a PCR based method for the detection of Gluconobacter sacchari from the microenvironment of the sugarcane leaf sheath. G. sacchari specific 16S RNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybug, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. Occurrence

Gluconobacter strains flourish in sugary niches such as flowers and fruits e.g. ripe grapes (Blackwood et al., 1969; Passmore and Carr, 1975; and Ameyama, 1975); apples and 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 one Acetobacter strain from honeybees and their environment from three different regions in Belgium and found two different patterns of proteins by polyacrylamide gel electrophoresis. Gluconobacter strains are not found to have any pathogenic effect towards man and other animals. However, they are capable of causing a bacterial rot of apples and pears accompanied by various shades of browning. Gluconobacter strains have been shown to share a common antigen (McIntosh, 1962). Bacteria responsible for pineapple pink disease in Hawaii were identified as Gluconobacter oxydans, Acetobacter aceti and Erwinia herbicola (Cho et al., 1980).
Growth Conditions Enrichment and isolation of Gluconobacter strains has been summarized by Swings and De Ley (1981). Enrichment of Gluconobacter strains present in flowers, fruits or bees can be set up using the following medium (g/l of distilled water): yeast extract, 5.0; D-glucose, 50; acitidione, 0.1; and bromophenol blue, 0.016. After incubation at 28C for 2-4 days, tubes showing acidification are streaked onto plates of 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 CaCO3 are further

Biotechnological Applications of G. oxydans 447

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

oxidation of sugars, aliphatic and cyclic alcohols, and steroids proceed through one or two discrete steps and lead to nearly quantitative yields of the oxidation products. Several soluble and particulate polyol dehydrogenases have been described (Kersters and De Ley, 1963; De Ley and Kersters, 1964; and Asai, 1968). More than 85% of Gluconobacter strains form acid from n-propanol, nbutanol, glycerol, m-erythritol, D-mannitol, D-arabinose, Dribose, D-fructose, D-mannose and maltose (Asai, 1971). Different applications of Gluconobacter oxydans in biotechnology have been summarized in Table 1.

Fermentation Studies 2, 5-Diketogluconic Acid Fermentation Gluconobacter oxydans is predominantly known as a ketogenic bacterium (Boutroux, 1886). The first study of the bacterial production of ketogluconates was published in 1940 (Stubbs et al., 1940). Conversion of D-glucose into 2,5-diketogluconic acid is mediated by membrane bound NADP + -independent three dehydrogenases [glucose dehydrogenase (GDH), gluconate dehydrogenase (GADH) and 2-ketogluconate dehydrogenase (2KGADH)].

Table1. Biotechnological Applications of Gluconobacter oxydans Application 2,5-Diketogluconic acid formation from D-Glucose Organism Reference Lockwood, 1954; Asai, 1971; Kita and Fenton, 1982; Weenk et al., 1984; Qazi et al., 1991; Buse et al., 1990 and 1992. Sonoyama et al., 1982 Anderson et al., 1985 Saito et al., 1998 Sugisawa et al., 1995 Klasen et al., 1992; Beschkov et al., 1995 Meiberg et al., 1983 Velizarov and Beschkov, 1994 Stefanova et al., 1987 Trifov et al., 1991 Rosenberg et al., 1993 Martin and Perlman, 1976 Bories et al., 1991 Svitel and Sturdik, 1994a Ohrem and Voss, 1995 Molinari et al., 1995 Svitel and Kullnik, 1995

Gluconobacter oxydans

2-keto-L-gulonate formation from 2,5-DKG Erwinia herbicola and Corynebacterium SHS 7522001 Single step production of 2KLG from D-glucose Recombinant Erwinia herbicola Recombinant G. oxydans Ascorbic acid formation from Gulono--lactone G. oxydans DSM 4025 5-Ketogluconic acid formation from D-Glucose G. oxydans DSM 3503 G. oxydans NBIMCC 1043 Free D-Gluconic acid formation from D-Glucose Gluconobacter oxydans Gluconobacter oxydans NBIMCC 1043 L-Sorbose formation from D-Sorbitol Immobilized G. suboxydans ATCC 621 Immobilized G. oxydans NBIMCC 902 G. oxydans L-Sorbosone formation from L-Sorbose Immobilized G. melanogenus IFO 3293 Dihydroxyacetone formation from D-Glucose G. oxydans ATCC 621 G. oxydans CCM 1783 (ATCC 621) G. oxydans Aldehyde formation from Alcohol G. oxydans isolated from Beer Acetic acid,Propionic acid, Acetone, Butyric G. oxydans CCM 3607, 1783. acid and 2-Butanone formation from Ethanol, Propanol, Isopropanol, Butanol and 2-Butanol, respectively. D-Galactonic acid formation from D-Galactose. G. oxydans Propionic acid formation from n-Propanol G. oxydans CCM 1783 Isovaleraldehyde formation from Isoamyl alcohol Hydrophobic hollow fibre membrane reactor of G. oxydans Continuous production of Isovaleraldehyde G. oxydans in isooctane solvent

Svitel and Sturdik, 1994b Svitel and Kullnik, 1995 Molinari et al., 1996 Cabral et al., 1997

448 Gupta et al.

Table2. Enzymes purified and characterized from Gluconobacter oxydans/other ketogenic bacteria Enzyme Glucose dehydrogenase (GDH) Gluconic acid dehydrogenase (GADH) 2-Ketogluconic acid dehydrogenase (2KGADH) L-Gulono--lactone dehydrogenase D-Sorbitol dehydrogenase (SDH) L-Sorbose dehydrogenase D-Fructose dehydrogenase Aldehyde dehydrogenase L-Sorbose reductase Xylitol dehydrogenases Organism Co-enzyme PQQ FAD+ FAD+ NAD+ PQQ. PQQ NADPH NAD+ Reference Ameyama et al., 1981 a. Matsushita et al., 1979 and Mclntire et al., 1985. Shinagawa et al., 1981 and Mclntire et al., 1985. Sugisawa et al., 1995. Shinagawa et al., 1982. Fujiwara et al., 1987. Ameyama et al., 1981 b. Ameyama et al., 1981 c. Adachi et al., 1999. Masakazu et al., 2000.

Gluconobacter oxydans ATCC 9937 Pseudomonas aeruginosa Gluconobacter melanogenus Gluconobacter oxydans DSM4025 G. suboxydans var. IFO 3250 G. oxydans UV-10 G. industrius Acetobacter aceti Gluconobacter melanogenus IFO 3294 Gluconobacter oxydans

Extensive fermentation studies have been performed to develop the process for 2,5-diketogluconic acid production at the industrial scale. Weenk et al. (1984) showed 2- and 5-ketogluconates production from G. oxydans ATCC621-H in batch culture using glucose-yeast extract medium in a 2-l fermentor. They also showed production of gluconic acid, if G. oxydans 621-H is grown without pH control. However, if the pH is controlled by the addition of CaCO3, then ketogluconates formation takes place. Many G. oxydans strains grown under standard conditions in a pH controlled batch culture, produce ketogluconates. The regulation of gluconate and ketogluconate formation in G. oxydans ATCC 621-H has been 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 in an ideally mixed air lift fermentor and they showed that the oxidation of gluconate to 2,5-diketogluconate took place through an intermediate 2KGA rather than 5-KGA. There are two reaction phases - direct glucose oxidation and gluconate oxidation. The positive influence of continuous availability of O2 causes induction of membrane bound dehydrogenases involved in the direct glucose oxidation. Buse et al. (1992) found DO of 30% relative to air at 01 bar as threshold level for optimum production of keto acids in airlift batch and stirred tank chemostat cultures of G. oxydans ATCC 9937. Enzymology of Industrially Important Pathways The important enzymes involved in different oxidative pathways are listed in Table 2. Glucose Dehydrogenase Glucose dehydrogenase (EC 1.1.99.17), a membrane bound quino-protein having bound pyrroloquinoline quinone (PQQ) catalyzes the direct oxidation of D-glucose to 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-soluble PQQ dependent glucose dehydrogenase can be increased by single amino acid replacement using site directed mutagenesis technique (Sode et al., 2000). The enzyme is a monomeric protein having the MW 87 Kda. Most investigations 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 any bound cofactor (Hommes et al., 1984). NADP+- dependent GDH, 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 immunocrossreactivity among the membrane bound GDHs isolated from several Gram-negative bacteria. The immunocrossreactivity was examined by the immunoblotting technique using 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 the antibodies specific for GDH of P. fluorescens indicating close homology to each other.
Pyrrolo Quinoline Quinone 2,7,9 Tricarboxy pyrrolol[2,3 f]quinoline dione is a compound having a pyrrole ring fused to a quinoline ring with an o-quinone group in it. The representatives of this group are found among the NAD(P) + independent, periplasmic bacterial dehydrogenases (Duine et al., 1979). D-Gluconate Dehydrogenase Matsushita et al. (1979) isolated and purified membrane bound D-gluconate dehydrogenase (EC 1.1.99.3) from Pseudomonas aeruginosa. The enzyme showed single band on polyacrylamide gel electrophoresis. The MW of the enzyme protein is found to be 138 Kda. In the presence of SDS, the enzyme dissociated into three subunits having MWs 66 Kda, 50 Kda and 22 Kda. The subunits of 66 Kda and 50 Kda are found to be flavo-protein and cytochrome C, respectively. Flavo-protein helps in transferring hydrogen atoms from gluconate, and cytochrome C acts as an acceptor for the electron generated during the reaction. However, role of the subunit of MW 22 Kda in enzyme catalysis is not clear. 2-Ketogluconate Dehydrogenase Shinagawa et al. (1981) solubilized and purified 2-keto-Dgluconate dehydrogenase (EC.1.1.99.4) from Gluconobacter melanogenus. The purified enzyme is found to have tightly bound cytochrome C. The MW of the enzyme is found to be 133 Kda. SDS-PAGE showed the presence of three subunits having MWs of 61 Kda (flavo protein), 47 Kda (cytochrome C) and 25 Kda (with unknown function).

Biotechnological Applications of G. oxydans 449

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

2,5-Diketogluconic Acid Pathway in Other Organisms Direct glucose oxidative reactions (Figure 2) are present in wide variety of micro- organisms 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 bound cofactor but gets activated by the addition of exogenous PQQ cofactor. Physiological Significance of Direct Glucose Oxidation Pathway In many gram-negative bacteria, glucose oxidation via glucose dehydrogenase, quinoprotein leads to the generation of a proton motive force, which can be coupled effectively to the energization of various cellular processes (Schie et al., 1985). Neijssel et al. (1989) showed that oxidation of glucose by membrane particles of G. oxydans could be coupled with the phosphorylation of ADP. Growth comparison between wild type and GDH- mutant of G. oxydans revealed that GDH helps the organism to attain fast growth in a glucose enriched medium. Lag phase of wild type in enriched glucose medium is 2 hr whereas GDHmutant has 8 hr lag phase (A.Gupta, unpublished data). Acinetobacter calcoaceticus utilizes glucose only in the form of gluconic acid. Glucose is first oxidized to gluconic acid by extracellular glucose dehydrogenase and gluconic acid is absorbed by the cell and further metabolized by Entner-Doudoroff pathway (Schie et al., 1985). Importance of 2,5-Diketogluconic Acid Pathway 2,5-Diketogluconate may be converted into 2-KLG by stereospecific reduction using 2,5-diketogluconic acid reductase (Sonoyama et al., 1982). 2-KLG is a penultimate intermediate in industrial production of ascorbic acid, known as vitamin C (Reichstein, et al ., 1934). The other intermediates 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 in Corynebacterium sp. SHS 752001 (Sonoyama et al., 1982). A two-stage fermentation process has been proposed for 2-keto-L-gulonate production. The glucose is converted into 2,5-DKG by Erwinia sp. After 26 hr cultivation, 328.6 mg of calcium 2,5-diketogluconate per ml with 94.5% yield from D-glucose has been reported. The broth was directly used for the next conversion without removal of the cells. The stereospecific reduction of calcium 2,5-diketogluconate to calcium 2-keto-L-gulonate was performed with mutant strain of Corynebacterium . The results of two-stage fermentation in 10-m3 conventional fermentors showed formation of an average of 106.3 mg of calcium 2-keto-Lgulonate per ml. The traits of two microorganisms (Erwinia herbicola and Corynebacterium sp) have been combined in a single cell by the gene manipulation techniques to simplify the conversion of D-glucose to 2-KLG (Anderson et al., 1985). They engineered E. herbicola ATCC 21988 by transforming 2,5-DKG reductase gene isolated from Corynebacterium sp. ATCC 31090. The engineered E. herbicola culture is able to produce upto 1 g/l 2-KLG in one step process. Direct production 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 the promoter of E. coli tufB1 gene and reintroduced into Gluconobacter oxydans T-100 to improve its efficiency for the production of 2-KLGA from D-sorbitol. Such a biological process is of industrial importance as it may replace presently used complex, multistep Reichsteins process. Reichstein-Grssner synthesis of L-ascorbic acid serves the basis for modern industrial production of ascorbic acid. The main steps in the Reichstein-Grssner process are chemical conversion of D-glucose into D-sorbitol followed by its microbial biooxidation to L-sorbose. L-Sorbose is converted to 2-KLG by chemical reactions. D-Sorbitol is oxidized to L-sorbose using Acetobacter suboxydans/ Gluconobacter melanogenus.

450 Gupta et al.

5-Ketogluconic Acid Production 5-Ketogluconic acid is a precursor of tartaric acid (Kazumi et al., 1984). G. oxydans DSM 3503 is capable of converting glucose into 5KGA via gluconic acid in a NADP+-dependent reaction (Klasen et al., 1992). Beschkov et al. (1995) studied kinetics and modelling of biotransformation of Dglucose to 5-keto-D-gluconates by G. oxydans NBIMCC1043. They found that Ca +2 had a central importance in the ketogluconic acid formation and addition of CaCO3 at the start of bioconversion was essential for high production rates. A simple mathematical model was presented showing an apparent lag phase in ketogluconic acid production necessary for accumulation of biomass as a biocatalyst and gluconic acid as a substrate. The maximum yield of 5-ketogluconic acid was 70% with respect to initial glucose concentration, and 15% 2-ketogluconic acid was accumulated as by-product. D-Gluconic Acid Fermentation D-Gluconic acid is an oxidation product of D-glucose. Annon (1980) has reviewed applications of gluconic acid and its salts. D-Gluconic acid, in its free form, calcium or sodium salts or as a lactone, is used as an additive in pharmaceutical, food, fodder and concrete industries. Gluconate is employed as the anion in pharmaceutical preparations containing nitrogen bases. Being an excellent metal sequestering agent, gluconate is also used for precious metal cleaning. Ferrous gluconate is often used to supplement iron during anemia. Similarly, chlorohexidine gluconate, a derivative of gluconate, is used as disinfectant. The market for gluconic acid has been developing since last 40 years and now amounts to about 50,000 tons per annum. Presently, gluconic acid is produced commercially by converting sodium gluconate into its free form. Sodium gluconate is produced using Aspergillus niger fermentation (May et al., 1929). Meiberg et al. (1983) reported formation of gluconates from D-glucose using Gluconobacter oxydans. Velizarov and Beschkov (1994) studied the microbial oxidation of glucose to free gluconic acid using batch cultures of G. oxydans NBIMCC1043. Cultivation was carried out on a medium containing either 10-90 g/l glucose or 210g/l glucose at 32C, with shaking at 1000 rpm and an aeration rate of 2vvm. Glucose with initial concentration 10 to 90g/l was almost quantitatively oxidized to gluconic acid, whereas with initial 210g/l glucose concentration, only 65% of glucose was converted into gluconic acid. In the presence of high glucose concentration, the pH of the culture medium dropped to about 2.0 and below this pH, growth and gluconic acid production were almost totally repressed. Velizarov and Beschkov (1998) reported inhibition of growth of G. oxydans by glucose at 0.5M concentration. L-Sorbose and L-Sorbosone Production by Gluconobacter Strains L-Sorbose is useful in industrial ascorbic acid production. L-Sorbose is the oxidation product of D-sorbitol, produced by the action of membrane bound SDH (Shingawa et al.,

1982). L-Sorbose is further converted to sorbosone by SNDH.

SDH SNDH D-Sorbitol L-Sorbose L-Sorbosone

Shinagawa et al. (1982) described the solubilization, purification and characterization of D-sorbitol dehydrogenase from the membrane fraction of Gluconobacter suboxydans var. IFO3254. The production of L-sorbose by cells of Gluconobacter suboxydans ATCC621 immobilized in polyacrylamide gel has been carried out in a continuous process. The entrapped cells almost completely converted D-sorbitol into L-sorbose at a rate of about 7kg/m3 per hr over a long period of time (Stefanova et al., 1987). Trifov et al. (1991) immobilized G. oxydans NBIMCC902 cells in sodium alginate gel crosslinked with oxidized polyvinyl alcohol (OPV), glutaraldehyde and Ca+2. High oxidation activity was retained for more than 80 cycles of 22 hr each, with a production rate of 4.5g L-sorbose/l/hr. Doherty (1991) compared the physiology of free and immobilized cells of G. oxydans. A mass transfer model was applied to G. oxydans cells immobilized in calcium alginate gel beads and grown in a medium containing sorbitol. The bead radius affected the extent of both the internal and external transfer resistances. Rosenberg et al . (1993) studied the parameters influencing the conversion of sorbitol to sorbose by stationary phase cultures of an industrial strain of G. oxydans from Farmakon, Czechoslovakia. The sorbitol concentration influenced the O 2 consumption rate according to Michaelis Menton Kinetics. Sorbitol at 20-200 g/l had no inhibitory effect. However, high concentration of sorbose inhibited the O2 consumption rate. Optimum sorbitol conversion occurred at pH 5.0 and 35-40C. Sorbose formation occurred in a batch culture lasting 34 hr (sorbitol concentration 200g/l), and the yield was 96%. In the fed-batch cultures (30C, 1vvm aeration, 0.1mM pressure, 5% vol. inoculum) having a sorbitol concentration of 410g/l, the yield was found to be 92%. Dihydroxyacetone Fermentation The DHA is useful in chemical and pharmaceutical industries. DHA is an oxidation product of glycerol produced by the action of membrane bound glycerol dehydrogenase (Kersters and De Ley, 1963).
Glycerol Dehydrogenase Glycerol Dihydroxyacetone

The kinetic studies and optimization for the production of DHA from glycerol have been done using G. oxydans ATCC 621 (Bories et al., 1991). G. oxydans was cultured (batch or fed batch cultures) in a 6l LSL Biolafitte fermentor containing 4 l growth medium and 25-100g/l carbon source (glycerol or mannitol) and 10g/l nitrogen-source (yeast extract) at pH 6.0. The incubation was performed at 28C, 800rpm, 1vvm aeration and O2 partial pressure above 10%. The rate of growth decreased with increasing DHA concentration and finally ceased at DHA concentration of 61g/l. Glycerol oxidation into DHA ceased at 108 g/l. Svitel and Sturdik (1994a) studied the product yield and byproduct formation during conversion of glycerol into DHA

Biotechnological Applications of G. oxydans 451

using G. oxydans CCM 1783 (ATCC621). An increase in the O 2 consumption rate was observed at glycerol concentration upto 300g/l. Batch cultures incubated with gassing by air and/or O2, yielded upto 94% DHA. Sodium glycerate 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 Maximum productivity reached after 6-10 hr. The fermentation broth pH was maintained between 3.8-4.8 using Ca(OH)2. Ohrem and Voss (1996) studied effect of glycerol on the growth and DHA production in Gluconobacter oxydans. They showed that neither glycerol oxidation nor metabolism of DHA was inhibited by high glycerol concentrations. They showed that DHA damaged the cells irreversibly and the viability of Gluconobacter decreased exponentially with time. Aldehyde Formation The aldehyde formation by alcohol oxidation with G. oxydans submerged cultures and resting cells, resuspended in different phosphate buffers (0.1M) has been studied by Molinari et al. (1995). Large-scale cultures were grown using stirred fed-batch reactors (1.5 l working volume, 250rpm and/or 2vvm air), bubble columns (500 ml 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 aliphatic alcohols to their corresponding aldehydes. Molinari et al. (1996) reported 3-isoamylalcohol as the best substrate having over 90% yields of 3-isoamylaldehyde without any by-product formation. Other Fermentation Processes Svitel and Kullnik (1995) studied the potentials of acetic acid 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 used for the oxidation of acids and ketones. The oxidations were performed in fed-batch cultures using a 5 l reactor (LF-2) filled with 2.7 l of sterile medium at 500rpm and 1vvm aeration. The pH during acid production was maintained at 6.0. Ethanol, propanol, isopropanol, butanol and 2butanol were converted to acetic acid, propionic acid, acetone, butyric acid and 2-butanone, respectively. Svitel and Sturdik (1994b) reported galactonic acid formation from D-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 conversion of n-propanol to propionic acid by G. oxydans CCM 1783 (ATCC 621) using fed batch conversion at pH 6.0. Propanol feeding was started after the growth phase. In the fedbatch conversion, calcium propionate 37g/l, formed within 30 hr (when Ca(OH)2 was used for the neutralization), and sodium propionate, 56g/l, within 70 hr (when NaOH was used for the neutralization), was obtained. Applications of Gluconobacter in Biosensor Technology

Gluconobacter oxydans as whole cells as well as its enzymes have been examined for its use as biosensor for the identification of various compounds. Applications of Gluconobacter oxydans in biosensor technology are listed in Table 3. Smolander et al. (1993) purified a membrane bound xylose oxidizing PQQ-dependent dehydrogenase from Gluconobacter oxydans ATCC 621 and studied its possible applications in biosensor technology. Dimethyl and carboxylic acid derivatives of ferrocene were able to mediate electron transfer in xylose oxidation using enzyme immobilized on graphite electrode. Smolander et al. (1995) developed an amperometric enzyme electrode biosensor for analysis of xylose and glucose in fermentation samples using PQQ-dependent ALDH from G. oxydans ATCC621. The best electrode performance was obtained when ALDH was adsorbed on the surface of a carbon-paste electrode. The lowest working potential and highest catalytic current were obtained with dimethylferrocene as a mediator. Hikuma et al. (1995) prepared a biosensor consisting of an anode mounted with immobilized alcohol dehydrogenase (EC 1.1.1.1) from G. oxydans IFO 3172, and a cathode containing hexacyanoferrate (III). The surface of the biosensor was covered with a gas-permeable membrane and measurements were made without reagent consumption by pumping a sample solution through the flow cell for 1 or 2 minutes. Alcohol contents of the alcoholic drinks determined using the sensor as well as by gas chromatography were coincided indicating high sensitivity of the sensor. Reshetilov et al. (1998a) used G. oxydans strains as microbial electrode biosensor for xylose analysis using clark-type oxygen electrodes. Reshetilov et al. (1998b) described the use of Gluconobacter oxydans whole cells for determination of sugars, alcohols, and polyols. Lusta and Reshetilov (1998) reviewed the prospects of Gluconobacter oxydans for its use in Biotechnology as biosensor system. Svitel et al . (1998) described the microbial cell-based biosensor for sensing glucose, sucrose or lactose. The glucosesensing membrane was prepared

Table 3. Application of Gluconobacter oxydans in Biosensor Technology Detection of the Substrate Xylose and Glucose Alcohol Xylose Sugars, Alcohols and Polyols Glucose, Sucrose or Lactose Enzyme/system Organism Reference Smolander et al., 1995 Hikuma et al., 1995. Reshetilov et al., 1998a. Reshetilov et al., 1998b. Svitel et al., 1998.

Aldose dehydrogenase G. oxydans ATCC621 Alcohol dehydrogenase G. oxydans IFO3172 Clark type-oxygen electrode G. oxydans G. oxydans whole cells. Glucose sensing membrane of intact cells of G. oxydans coimmobilized with Kluyeromyces marxians

452 Gupta et al. with intact cells of Gluconobacter oxydans immobilized in gelatin cross-linked with glutaraldehyde. The disaccharide sensing membranes were prepared by co-immobilization of 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. The strain of G. oxydans was able to oxidize both anomers of glucose at the same rate. Therefore, there was no need for mutarotase co-immobilization in disaccharide-sensing membranes. The sensitivity of glucose sensor was 50 nA/ mM and the range of the calibration curve was 0-0.8 mM with response time of 2 min. The response after 1 week of storage was 62% of the initial one. The detection linear range of disaccharide sensor was found to be upto 4mM with response time of 5min. The activities of the sensors after 1 week of storage at ambient temperature were in the 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 of the 36 strains of Gluconobacter sp. and most of them had MW of more than 5 Mda. Verma et al. (1994) characterized plasmids of G. oxydans strains ATCC 9937 and IFO3293, and found three native plasmids in ATCC 9937 having the sizes 27.7 kb (pVJ1), 12.3 kb (pVJ2) and 18 kb (pVJ4) and one native plasmid of 9.5 kb size (pVJ3) in IFO3293. Two phages have been isolated from 54 different strains of Gluconobacter (Robakis et al., 1985). These two phages have been reported to have different sized double stranded DNAs (approximately 37 and 250-300 kb sizes). The restriction map of bacteriophage revealed ring like DNA containing cohesive ends. Identification of Genes Involved in 2,5-DKG Pathway from Gluconobacter oxydans Glucose dehydrogenase gene has been identified and sequenced 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 capable of oxidizing sucrose. The DNA sequence of both gdh genes from P1 and P2 are found identical except one nucleotide

substitution resulting in the substitution of His 787 by Asn in an open reading frame of 2424 bp. The putative protein is of 808 amino acids with a MW of 87,587 Da. Riboflavin kinase (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 GADH enzyme (McIntire et al., 1985). The other genes namely gadh and 2kgadh involved in 2,5-dkg pathway from G. oxydans could not be detected yet. Xba I restriction map of the genome from G. oxydans ATCC 9937 has been developed using Pulse field gel electrophoresis studies (Verma et al., 1997). Southern hybridization and PFGE showed 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 Bacteria The genes of 2,5-dkg pathway/other pathways reported from G. oxydans /other bacteria like Acinetobacter calcoaceticus, Erwinia herbicola are listed in Table 4. Jansen et al. (1988, 1990) have reported the gdh genes from A. calcoaceticus and E. coli. The gdh genes isolated from A. calcoaceticus, E. coli and G. oxydans showed high homology at C-terminal ends (Jansen et al., 1991). Cha et al. (1997) identified and characterized a gene encoding glucose dehydrogenase from Pantoea citrea and showed its involvement in causing Pineapple pink disease. The pqq genes of A. calcoaceticus, Methylobacterium extorquens AMI and Klebsiella pneumoniae have been cloned and sequenced (Goosen et al., 1987; Toyama et al., 1997; Meulenberg et al., 1992). Gluconate dehydrogenase encoding genes have been isolated and characterized from Erwinia cypripedii ATCC 29267 (Yum et al., 1997). The 2,5 diketo-D-gluconate reductase of E. coli has homology with 2,5-diketo-Dgluconate reductases of Corynebacterium sp., and Gluconobacter oxydans (Yum et al., 1999). 2,5-DKGR of Cornyebacterium showed 49.8% homology to yqhE gene product; and 2,5KDGR of Gluconobacter oxydans showed 42% homology with yafB gene product. The gene products of yqfE and yafB were identified as 2,5DKGR-A and 2,5DKGR-B, respectively catalyzing the reduction of 2,5DKG to 2-KLG.

Table 4. Genes identified from Gluconobacter oxydans/ other ketogenic bacteria involved in 2,5-diketogluconic acid pathway/ other pathways. Gene gdh RibF gdh gdh gdh pqq pqq pqq gadh L-Sorbosone dehydrogenase (SNDH) gene D-Sorbitol dehydrogenase (SDH) and sorbosone dehydrogenase gene Gluconate NADP+5-oxidoreductase (GNO) gene IS element RecA Xylitol dehydrogenase genes (XDH1 & XDH2) Organism Reference Jansen et al., 1991 Gupta et al., 1999 Jansen et al., 1988 Jansen et al., 1990 Cha et al., 1997 Goosen et al., 1987 Toyama et al., 1997 Meulenberg et al., 1992 Yum et al., 1997 Shinjoh et al., 1995 Saito et al., 1997 Klasen et al., 1995 Kondo and Horinouchi, 1997 Liu et al., 1998 Masakazu et al., 2000.

G. oxydans P1 & P2 G. oxydans ATCC 9937 Acinetobacter calcoaceticus Escherichia coli Pantoea citra Acinetobacter calcoaceticus Methylaobacterium extorquens AMI Klebsiella pneumonniae Erwinia cypripedii ATCC 29267 Acetobacter liquefaciens IFO12258 G. oxydans G 624 G. oxydans subsp. 3503 G. suboxydans G. oxydans LCC 99 Gluconobacter oxydans

Biotechnological Applications of G. oxydans 453

Identification of Genes Involved in Sorbitol Oxidation Shinjoh et al. (1995) cloned and sequenced the membrane bound SNDH gene from Acetobacter liquefaciens IFO12258 and got it expressed in a 2-KLG producing mutant (strain OX4) of G. oxydans IFO3293. The open reading frame of the gene is found to be 1,347 bp encoding a 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 2KLG efficiently 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 from about 25 to 83%, whereas using L-sorbose was increased from 68 to 81%. Identification of Genes Involved in Other Pathways from G. oxydans Klasen et al . (1995) reported Gluconate NADP + 5oxidoreductase gene from Gluconobacter oxydans sub sp. DSM 3503. The enzyme oxidizes gluconate to 5ketogluconate and is localized in the cytoplasm. The MW of the enzyme is reported to be 75 Kda. The enzyme showed single band on SDS-PAGE having MW 33 Kda indicating two identical subunits in the protein. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids. Kondo and Horinouchi (1997) reported a novel insertion sequence element IS 12528 from G. suboxydans associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, that encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS 12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. Liu et al. (1998) reported a recA gene from G. oxydans and they expressed it in E. coli recA- mutant. The G. oxydans recA protein efficiently functions in homologous recombination and DNA repair. The deduced amino acid sequence of recA gene revealed a protein of 344 amino acids 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 the application of recombinant DNA technology for this group of microorganisms. Fukaya et al. (1985b) constructed a shuttle vector pMG101 for Gluconobacter oxydans. The shuttle vector, pMG101, for Gluconobacter oxydans and Escherichia coli is constructed by ligation of a cryptic plasmid, pMV201, found in G. suboxydans IFO 3130 with E. coli plasmid pACYC177. The chimeric plasmid named pMG101 carries the ampicillin resistance gene derived from plasmid ACYC177. The transformation efficiency is found to be 102 per g DNA. Palleroni (1986) showed that Gluconobacter oxydans IFO 3293 and ATCC 9937 could accept plasmid RSF 1010 both by transformation and conjugation. Condon et al. (1991) reported conjugal transfer of a series of incompatible group 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 efficient electroporation system for transformation of the plasmid pMP220 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 cell suspension and electroporation buffer containing Mg+2 and 1011 cfu/ml of early to mid-log phase cells. Under these conditions, transformation efficiencies of 105/g DNA were reproducibly obtained. Storage for 5 days at -20C in a buffer containing 1% glycerol decreased transforming efficiency by 86% owing to a loss of cell viability. Shinjoh and Hoshino (1995) developed a shuttle vector (plasmid pGE1, 11.9kb) and conjugative transfer system for G. oxydans. Transformed G. oxydans were shown to be capable of producing 2-keto-L-gulonic acid from Lsorbose. The plasmid pGE1 was constructed from cryptic G. oxydans IFO 3293 plasmid pGO32938 (9.9kb, relaxed type), E. coli conjugative plasmid pSUP301 (5kb, Kmr and ampr, relaxed type), plasmid pACYC 177 and the plasmid RP4 mob region. The pGE1 was transferred by conjugation to G. oxydans IFO 3293 with high frequency (0.1 transconjugants/recipient), and maintained stability without antibiotic selection. It was also maintained in other strains of G. oxydans like IFO 3172, IFO 3268, IFO 3271 and ATCC 9937; and G. frateurii IFO 3268, IFO 3129, IFO 3170, IFO3223 and IFO 3225. The presence of pGE1 did not inhibit 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. The plasmid pGE1 could be shortened further to plasmid pGE2 (9.8 kb). Gene Expression in Gluconobacter oxydans Saito et al . (1997) constructed a shuttle vector for expression of sorbitol dehydrogenase and sorbosone dehydrogenase genes in G. oxydans G624. A plasmid of 4.4 kb designated as pF4 was isolated from G. oxydans T100 and ligated with pHSG298 at the Hind III site to construct a shuttle vector, designated as pFG15A. The plasmid pFG15A carrying SDH and SNDH genes was transformed into G. oxydans G624. The recombinant Gluconobacter oxydans G624 was 2-3 fold more active for the production of 2-KLGA than G. oxydans T-100; however, G. oxydans G624 itself showed no ability to produce 2-KLGA. Takeda and Toshio (1992) expressed cytochrome C553 (CO) gene in Gluconobacter suboxydans subsp. that complemented the second subunit deficiency of membrane-bound alcohol dehydrogenase. The gene has been subcloned into a shuttle vector pGEA1 and the recombinant was designated as pGEAC1. The gene has been shown to replicate in Gluconobacter suboxydans and Escherichia coli. The pGEA1 transformed G. suboxydans subsp. showed low ethanol oxidation activity due to second subunit deficiency of membrane-bound ADH. The transformants harboring pGEAC1 expressed ethanol oxidation activity and showed cross-reactivity with anticytochrome C-553 antibody. Furthermore, expression of cytochrome C-553 (CO) gene resulted in elevated levels of heme C and CO-reactive cytochrome C complemented

454 Gupta et al.

the deficiency of the second ADH subunit and restored ethanol oxidase activity. Heterogeneity among G. oxydans Strains Verma et al. (1997) reported genetic heterogeneity among keto-acid-producing strains of G. oxydans ATCC9937, IFO 3293, IFO 12258 and DSM 2343. The genomes of four keto-acid-producing G. oxydans strains were analyzed by pulse field gel electrophoresis. Xba I was chosen for restriction fragment analysis of the genomes. The genome sizes of the four strains were estimated to be between 2240 kb and 3787 kb. Although these strains are physiologically similar, XbaI restricted PFGE of the four strains showed no homology. Mutagenic Studies in G. oxydans Qazi et al. (1989) reported mitomycin C as an effective mutagenic agent for G. oxydans ATCC 9937. The MIC of mitomycin 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 Mutagenesis Kahn and Manning (1988) obtained a patent on Gluconobacter oxydans strains having capability of higher production of 2-KLG from L-sorbose. They carried out additional mutation in G. oxydans UV-10 mutant strain by inserting a transposon (PI::Tn5) in the 2-KLG reductase. The mutant strain, M23-15, produced more 2-KLG (33.3 g/l) compared to the parent strain (19.6 g/l) due to reduced 2-KLG-reductase activity. Gupta et al . (1997) have developed a protocol for Transposon Tn5 mutagenesis in Gluconobacter oxydans using pSUP2021 vector system. Simon et al. (1983) constructed pSUP2021 to perform Tn5 mutagenesis in gram negative bacteria. Tn5 transpogenesis frequency was found 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 Tn5 transposed cells of G. oxydans ATCC 9937 was developed and screened for specific mutants blocked in direct glucose oxidation pathway. Two different mutants of G. oxydans having defective GDH- and GADH-, respectively, were isolated.
GDHGADH2KGADH

Conclusion

G. oxydans is a simple, non-pathogenic, industrially important bacteria. The bacteria has a number of membrane bound dehydrogenases which are involved in many oxidation reactions. These oxidation reactions can be exploited for commercial purposes. Genetic understanding of the organism may be fruitful for the genetic exploitation. Apart from its oxidative reactions, it can also be manipulated for the expression of foreign genes.
Acknowledgements The Authors wish to acknowledge the facilities of Distributed Bioinformatic subcentres of Devi Ahilya University, Indore; Regional Research Laboratory, Jammu; and Guru Nanak Dev University, Amritsar for providing necessary facilities for literature survey. The authors thank to Prof. Milton H. Saier, Dept. of Biology, University of California, San Diego, USA and Dr. Subrato Guha, Vaishnav College, Indore for critical review and correcting the English usage errors in the manuscript. References Adachi, O., Matsushita, K., Shinagawa, E., and Ameyama, M., 1979. Occurrence of old yellow enzyme in Gluconobacter suboxydans and the cyclic regeneration of NADP+. J. Biochem. 86:699-709. Adachi, O., Ano, Y., Moonmangmee, D., Shinagawa, E., Toyama, H., Theeragool, G., Lotong, N., and Matsushita, K. 1999. Crystallization and properties of NADPH-dependent L-sorbose reductase from Gluconobacter melanogenus IFO 3294. Biosci., Biotechnol., Biochem. 63: 2137-2143. Ameyama, M. 1975. Gluconobacter oxydans subsp. sphaericus, new subspecies isolated from grapes. Int. Syst. Bacteriol. 25: 365-370. Ameyama, M., Shinagawa, E., Matsushita, K., and Adachi, O. 1981a. DGlucose dehydrogenase of Gluconobacter suboxydans: solubilization, purification and characterization. Agric. Biol. Chem. 45: 851-861. Ameyama, M., Shinagawa, E., Matsushita, K., and Adachi, O. 1981b. DFructose dehydrogenase of Gluconobacter industrius. J. Bacteriol. 145: 814-823. Ameyama, M., Shinagawa, E., Matsushita, K., and Adachi, O. 1981c. Purification and characterization of aldehyde dehydrogenase of Acetobacter aceti. Agric. Biol. Chem. 45: 1889-1890. Anderson, S., Maraks, C.B., Lazarus, R., Miller, J., Staffed, K., Sayour, J., Light, D., Rastetter, W., and Estell, D. 1985. Production of 2-keto-Lgulonate an intermediate in L-ascorbate synthesis by a genetically modified Erwinia herbicola. Science. 230: 144-149. Annon, 1980. Sodium gluconate as speciality in chemical industry. Indian chem. Manuf. 18: 27-32. Asai, T. 1935. Taxonomic studies on acetic acid bacteria and allied oxidative bacteria isolated from fruits. A new classification of the oxidative bacteria. J. Agr. Chem. Soc. Jpn. 11: 499-513, 610-620, 674-708. Asai, T. 1968. Classification and Biochemical activities. In: Acetic acid bacteria. University of Tokyo Press, Tokyo, Japan. Asai, T. 1971. The classification and biochemistry of acetic acid bacteria. In: Biochemical and Industrial aspects of fermentation. K. Sakaguchi, T. Vemuna, S. Kinoshito, eds. Kadansha Ltd., Tokyo, Japan. p. 201-232. Beschkov, V., Velizarov, S., and Peevva, L. 1995. Some Kinetic aspects and modelling of biotransformation of D-glucose to keto-D-gluconate. Bioprocess Eng. 13: 301-305. Blackwood, A.C., Guimberteau, and Peynaud, E. 1969. Sur les bactries acetiques isolies de raisins. R. Hebd Sances Acad. Sci. Ser. D. 269: 802-804. Bories, A., Claret, C., and Soucaille, P. 1991. Kinetic study and optimization of the production of dihydroxyacetone from glycerol using Gluconobacter oxydans. Process Biochem. 26: 243-248. Boutroux, L. 1886. sur une fermentation acids du glucose. C.R. Hebd. Seances Acad. Sci. 102:924-947. Buse, R., Qazi, G.N., and Onken, U. 1992. Influence of constant and oscillating oxygen concentration on keto acid production by Gluconobacter oxydans subsp. Melanogenus. J. Biotechnol. 26: 231-244. Buse, R., Qazi, G.N., Trger, M., and Onken, U. 1990. Influence of dissolved oxygen tension on the production rate of 2,5-diketogluconic acid by Gluconobacter oxydans. Biotechnol. Lett. 12: 111-116. Cabral, J.M., Barros, M.R., Pinheiro, H., and Prazeres, D.M. 1997. Biotransformation in organic media by enzymes and whole cells. J. Biotechnol. 59: 133-143 Cha, J.S., Pujol, C., and Kado, C.I. 1997. Identification and characterization

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

A GDH- mutant G. oxydans AG-a was selected on the basis of non-acid zone formation in P1 production medium plate (Gupta et al., 1997). In this mutant, Tn5 insertion was found in its PQQ cofactor synthesizing genes (pqq). A GADHmutant of G. oxydans ATCC 9937 was selected on the basis of non-melanin formation in P1 production medium. The specific blockage at gluconate conversion point was confirmed by HPLC analysis (Gupta et al., 1999). In this mutant, Tn5 insertion was found in the Rib F gene. Liu et al . (1999) constructed a recA deficient mutant of Gluconobacter oxydans called as LCC99, that could be used as a host to take up the recombinant plasmid for gene manipulation. The mutant was constructed by allelic exchange using the cloned recA gene that had been inactivated by inserting a kanamycin-resistance cassette.

Biotechnological Applications of G. oxydans 455

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