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Gluconobacter oxydans:
Its Biotechnological Applications
Arun Gupta1, Vinay K. Singh1, G.N. Qazi2 bacterial rot of apples and pears accompanied by
and Anil Kumar1 various shades of browning. Several soluble and
particulate polyol dehydrogenases have been
1
School of Biotechnology, Devi Ahilya University, described. The organism brings about the incomplete
Khandwa Road, Indore-452017, MP, India oxidation of sugars, alcohols and acids. Incomplete
2
Dept. of Biotechnology, Regional Research Laboratory oxidation leads to nearly quantitative yields of the
(CSIR), Jammu, India oxidation products making G. oxydans important for
industrial use. Gluconobacter strains can be used
industrially to produce L-sorbose from D-sorbitol; D-
Abstract gluconic acid, 5-keto- and 2-ketogluconic acids from
D-glucose; and dihydroxyacetone from glycerol. It is
Gluconobacter oxydans is a gram-negative bacterium primarily known as a ketogenic bacterium due to 2,5-
belonging to the family Acetobacteraceae. G. oxydans diketogluconic acid formation from D-glucose.
is an obligate aerobe, having a respiratory type of Extensive fermentation studies have been performed
metabolism using oxygen as the terminal electron to characterize its direct glucose oxidation, sorbitol
acceptor. Gluconobacter strains flourish in sugary oxidation, and glycerol oxidation. The enzymes
niches e.g. ripe grapes, apples, dates, garden soil, involved have been purified and characterized, and
baker’s soil, honeybees, fruit, cider, beer, wine. molecular studies have been performed to understand
Gluconobacter strains are non-pathogenic towards these processes at the molecular level. Its possible
man and other animals but are capable of causing application in biosensor technology has also been
worked out. Several workers have explained its basic
and applied aspects. In the present paper, its different
Abbreviations biotechnological applications, basic biochemistry and
µg-microgram; µl-microlitre; 2,5-DKG-2,5-diketogluconic acid; 2,5-DKGR- molecular biology studies are reviewed.
2,5-diketo-D-gluconate reductase; 2KGADH-2-ketogluconate
dehydrogenase; 2kgadh-2-keto-gluconate dehydrogenase gene; 2KLG-2-
keto–L-gulonic acid; 5KDGR-5-keto-D-gluconate reductase; 5KGA-5-
Introduction
ketogluconic acid ; 2,5-DKGR-2,5-diketo-D-gluconate reductase. 5KDGR-
5-ketogluconate reductase; 2,5-DKGR-A - 2,5-diketo-D-gluconate reductase Gluconobacter oxydans is a gram-negative bacterium
product of gene yqfE of E. coli; 2,5-DKGR-B - 2,5-diketo-D-gluconate belonging to the family Acetobacteraceae. The shape of
reductase product of gene yqfB of E. coli; A. calcoaceticus-Acinetobacter
calcoaceticus; ADH-alcohol dehydrogenase; Adh-alcohol dehydrogenase the cells is ellipsoidal to rod shaped, 0.5-0.8 X 0.9-4.2 µm,
gene; ADP-adenosine diphosphate; ALDH-aldose-dehydrogenase; Asn- occurring singly and/or in pairs and rarely in chains. G.
asparagine; ATCC-American type culture collection; bp-base pair; CaCO3- oxydans is an obligate aerobe, having a strictly respiratory
calcium carbonate; Ca(OH)2-Calcium hydroxide; Cfu-colony formation unit;
CO 2 -carbon dioxide; Cyt C- cytochrome C; Da-dalton; DHA-
type of metabolism with oxygen as the terminal electron
dihydroxyacetone; DO-dissolved oxygen; E. coli- Escherichia coli; E. acceptor (De Ley and Swings, 1994).
herbicola-Erwinia herbicola; EC-Enzyme commission; FAD+-flavin adenine Gluconobacter is an industrially important genus for
dinucleotide; g-gram; G. oxydans-Gluconobacter oxydans; G. sacchari- the production of L-sorbose from D-sorbitol; D-gluconic
Gluconobacter sacchari ; GA-gluconic acid; GADH-gluconate
dehydrogenase; GADH--gluconate dehydrogenase deficient mutant; gadh- acid, 5-keto- and 2-ketogluconic acids from D-glucose; and
gluconate dehydrogenase gene; GDH-glucose dehydrogenase ; GDH- - dihydroxyacetone from glycerol. The present review deals
glucose dehydrogenase deficient mutant; gdh-glucose dehydrogenase with the fermentation studies, applications in Biotechnology
gene; GNO- gluconate NADP+ 5-oxidoreductase gene; GYC- glucose-yeast
extract-calcium carbonate medium; H2O-water; His-histidine; H2S-hydrogen
and genetic aspects of Gluconobacter oxydans.
disulfide; HPLC-high performance liquid chromatography; hr-hour; IFO-
Institute of Fermentation, Osaka; IS-insertion sequences; Kb-kilobase; Kda- Classification and Nomenclature of Gluconobacter
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- In 1935, a group of gram-negative bacteria related to
ampere; NADP+-nicotinamide adenine dinucleotide phosphate; NaOH- Acetobacter was renamed and put under the genus
sodium hydroxide; N-source-nitrogen source; O2-oxygen; OPV-oxidized Gluconobacter (Asai, 1935). Gluconobacter, earlier known
polyvinyl alcohol; P. fluorescens- Pseudomonas fluorescens ; PAGE-
polyacrylamide gel electrophoresis; PCR-Polymerase Chain Reaction;
as Acetobacter oxydans, has been characterized as having
PFGE-pulse field gel electrophoresis; PQQ-Pyrrolo Quinoline Quinone; pqq- the pronounced capability for the oxidation of glucose to
pyrrolo quinoline quinone genes; RibF-riboflavin kinase F gene; rpm- gluconate and a weak ability for the oxidation of ethanol to
revolutions per minute; SDH-sorbitol dehydrogenase; SDS-sodium dodecyl acetate (Kluyver and Boezaardt, 1938).
sulfate; SNDH-sorbosone dehydrogenase; Tn5-Transposon Tn5; vvm-
volumetric volume per mole. A review on the classification and nomenclature
problems related to the genus Gluconobacter has been
published (De Ley and Frateur, 1970). The following
Received February 29, 2000; revised April 18, 2000; accepted July 5, 2000;
final manuscript received February 16, 2001. *For correspondence. Email
biochemical tests are found uniformly negative for all strains
bioinfo@sancharnet.in; Fax. 91-731-470372. of Gluconobacter oxydans: glucose oxidase, reduction of
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nitrates, H2S formation and indole production (De Ley, purified and characterized. All Gluconobacter strains
1978). Loitsianskaya et al. (1979) proposed the existence require D-mannitol as a carbon source and pantothenic
of a single species within the genus on the basis of acid, niacin, thiamine and p-aminobenzoic acid as growth
numerical analyses of 136 phenotypic features of 56 strains factors (Gosselé et al., 1980). The other carbon sources
of acetic acid bacteria. The genus Gluconobacter belongs used for the growth are sorbitol, glycerol, D-fructose and
to the fourth rRNA superfamily and constitutes a separate D-glucose (Olijve and Kok, 1979). The bacteria may also
branch together with the genus Acetobacter (Gillis and De be maintained on MYP medium (mannitol, 25; yeast extract,
Ley, 1980). 5; peptone, 3 g/l, respectively). The bacteria grow optimum
Gluconobacter and Acetobacter are closely related to in temperature range 25-30°C and pH 5.5 – 6.0. The
each other and are classified as acetic acid bacteria (Asai, colonies of Gluconobacter strains are pale. Gluconobacter
1968). The rapid and accurate differentiation of strains are able to grow in a chemically defined medium
Gluconobacter from Acetobacter is based on ethanol and without amino acids using ammonium ions as a sole
lactate oxidation. Gluconobacter does not oxidize ethanol nitrogen source (Shamberger, 1960). No amino acid is
to CO2 and H2O via acetate and does not oxidize lactate essential for Gluconobacter oxydans (Gosselé et al., 1981).
to CO2 and H2O, whereas Acetobacter does (De Ley and
Swings, 1994). Yamada et al. (1997) examined 36 strains Carbohydrate Metabolism in G. oxydans
of Acetobacter, Gluconobacter and Acidomonas for their
partial sequences in positions 1220 through 1375 of their The enzymes of Gluconobacter oxydans have been divided
16S RNAs and performed phylogenetic studies. into two groups based on the location and function. One
Frank et al. (2000) have developed a PCR based group consists of the particulate enzymes tightly bound to
method for the detection of Gluconobacter sacchari from the bacterial membrane and linked to the cytochrome
the microenvironment of the sugarcane leaf sheath. G. systems. Examples are D-glucose dehydrogenase
sacchari specific 16S RNA-targeted oligonucleotide primers (Cheldelin, 1961), D-gluconate dehydrogenase (Matsushita
were designed and used in PCR amplification of G. sacchari et al., 1979), glycerol dehydrogenase (Kersters and De
DNA directly from mealybug, and in a nested PCR to detect Ley, 1963), and D-sorbitol dehydrogenase (Shinagawa et
low numbers of the bacteria from sugarcane leaf sheath al., 1982). The enzymes are flavoproteins and also contain
fluid and cane internode scrapings. cytochrome. They catalyze the direct oxidation of the
substrates and function primarily in the oxidative formation
Occurrence of acetic acid, D-gluconate, 2- or 5-keto-D-gluconate, L-
sorbose, and dihydroxyacetone. The bacteria accumulate
Gluconobacter strains flourish in sugary niches such as large amounts of oxidative products extracellularly. The
flowers and fruits e.g. ripe grapes (Blackwood et al., 1969; second group consists of enzymes located in the cytoplasm
Passmore and Carr, 1975; and Ameyama, 1975); apples catalyzing the intracellular metabolism of carbohydrates.
and dates, (Passmore and Carr, 1975). Gluconobacter It is well known that in G. oxydans, only the pentose
strains also occur in garden soil, baker’s soil, honeybees, phosphate pathway functions and the complete glycolytic
fruits, cider, beer and wine (De Ley, 1961). Lambert et al. and Krebs cycle enzyme sequences are absent (Hauge et
(1981) isolated 56 Gluconobacter oxydans strains and one al ., 1955; Stouthamer, 1959; De Ley, 1961). 6-
Acetobacter strain from honeybees and their environment Phosphogluconate is a key intermediate in this pathway.
from three different regions in Belgium and found two Adachi et al. (1979) described cyclic regeneration of NADP+
different patterns of proteins by polyacrylamide gel in Gluconobacter suboxydans IFO12528. Gluconobacter
electrophoresis. Gluconobacter strains are not found to sp. oxidizes glucose via two alternate pathways. One
have any pathogenic effect towards man and other animals. operates inside the cell followed by oxidation via the
However, they are capable of causing a bacterial rot of pentose phosphate pathway and second operates outside
apples and pears accompanied by various shades of the cell and involves the formation of gluconic acid and
browning. Gluconobacter strains have been shown to share ketogluconic acid (Kulka and Walker, 1954; Olijve and Kok,
a common antigen (McIntosh, 1962). Bacteria responsible 1979). The former is carried by NADP+-dependent glucose
for pineapple pink disease in Hawaii were identified as dehydrogenase, and the latter is performed by NADP+
Gluconobacter oxydans, Acetobacter aceti and Erwinia independent glucose dehydrogenase and is also called as
herbicola (Cho et al., 1980). “direct glucose oxidation” pathway (Kitos et al., 1958).
Diagrammatic representation of the pathway is shown in
Growth Conditions Figure 1.
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).
2,5-Diketogluconic acid formation Gluconobacter oxydans Lockwood, 1954; Asai, 1971; Kita and
from 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., 1982
Single step production of 2KLG from D-glucose Recombinant Erwinia herbicola Anderson et al., 1985
Recombinant G. oxydans Saito et al., 1998
Ascorbic acid formation from Gulono-γ-lactone G. oxydans DSM 4025 Sugisawa et al., 1995
5-Ketogluconic acid formation from D-Glucose G. oxydans DSM 3503 Klasen et al., 1992;
G. oxydans NBIMCC 1043 Beschkov et al., 1995
Free D-Gluconic acid formation from D-Glucose Gluconobacter oxydans Meiberg et al., 1983
Gluconobacter oxydans NBIMCC 1043 Velizarov and Beschkov, 1994
L-Sorbose formation from D-Sorbitol Immobilized G. suboxydans ATCC 621 Stefanova et al., 1987
Immobilized G. oxydans NBIMCC 902 Trifov et al., 1991
G. oxydans Rosenberg et al., 1993
L-Sorbosone formation from L-Sorbose Immobilized G. melanogenus IFO 3293 Martin and Perlman, 1976
Dihydroxyacetone formation from D-Glucose G. oxydans ATCC 621 Bories et al., 1991
G. oxydans CCM 1783 (ATCC 621) Svitel and Sturdik, 1994a
G. oxydans Ohrem and Voss, 1995
Aldehyde formation from Alcohol G. oxydans isolated from Beer Molinari et al., 1995
Acetic acid,Propionic acid, Acetone, Butyric G. oxydans CCM 3607, 1783. Svitel and Kullnik, 1995
acid 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, 1994b
Propionic acid formation from n-Propanol G. oxydans CCM 1783 Svitel and Kullnik, 1995
Isovaleraldehyde 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
448 Gupta et al.
Table2. Enzymes purified and characterized from Gluconobacter oxydans/other ketogenic bacteria
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-γ-lactone dehydrogenase Gluconobacter oxydans DSM4025 - Sugisawa et al., 1995.
D-Sorbitol dehydrogenase (SDH) G. suboxydans var. α 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.
Extensive fermentation studies have been performed et al., 1979). However, E. coli GDH does not have any
to develop the process for 2,5-diketogluconic acid bound cofactor (Hommes et al., 1984). NADP+- dependent
production at the industrial scale. Weenk et al. (1984) GDH, a dimer having two identical subunits of MW 54 Kda,
showed 2- and 5-ketogluconates production from G. showed no significant homology to membrane bound GDH
oxydans ATCC621-H in batch culture using glucose-yeast (Jansen et al., 1988).
extract medium in a 2-l fermentor. They also showed Matsushita et al. (1986) showed the immunocross-
production of gluconic acid, if G. oxydans 621-H is grown reactivity among the membrane bound GDHs isolated from
without pH control. However, if the pH is controlled by the several Gram-negative bacteria. The immunocross-
addition of CaCO3, then ketogluconates formation takes reactivity was examined by the immunoblotting technique
place. Many G. oxydans strains grown under standard using antibodies raised against GDH of Pseudomonas
conditions in a pH controlled batch culture, produce fluorescens. Membranes prepared from E. coli, Klebsiella
ketogluconates. The regulation of gluconate and pneumoniae, G. suboxydans, Acinetobacter calcoaceticus
ketogluconate formation in G. oxydans ATCC 621-H has are found to have a polypeptide cross reacting with the
been studied by Levering et al. (1988). Qazi et al. (1991) antibodies specific for GDH of P. fluorescens indicating
compared the growth and 2,5-DKG production by G. close homology to each other.
oxydans ATCC 9937 in a 10 l stirred tank fermentor and in
an ideally mixed air lift fermentor and they showed that the Pyrrolo Quinoline Quinone
oxidation of gluconate to 2,5-diketogluconate took place 2,7,9 Tricarboxy pyrrolol[2,3 f]quinoline dione is a
through an intermediate 2KGA rather than 5-KGA. There compound having a pyrrole ring fused to a quinoline ring
are two reaction phases - direct glucose oxidation and with an o-quinone group in it. The representatives of this
gluconate oxidation. The positive influence of continuous group are found among the NAD(P) + independent,
availability of O2 causes induction of membrane bound periplasmic bacterial dehydrogenases (Duine et al., 1979).
dehydrogenases involved in the direct glucose oxidation.
Buse et al. (1992) found DO of 30% relative to air at 01 bar D-Gluconate Dehydrogenase
as threshold level for optimum production of keto acids in Matsushita et al. (1979) isolated and purified membrane
airlift batch and stirred tank chemostat cultures of G. bound D-gluconate dehydrogenase (EC 1.1.99.3) from
oxydans ATCC 9937. Pseudomonas aeruginosa. The enzyme showed single
band on polyacrylamide gel electrophoresis. The MW of
Enzymology of Industrially Important Pathways the enzyme protein is found to be 138 Kda. In the presence
of SDS, the enzyme dissociated into three subunits having
The important enzymes involved in different oxidative MWs 66 Kda, 50 Kda and 22 Kda. The subunits of 66 Kda
pathways are listed in Table 2. and 50 Kda are found to be flavo-protein and cytochrome
C, respectively. Flavo-protein helps in transferring hydrogen
Glucose Dehydrogenase atoms from gluconate, and cytochrome C acts as an
Glucose dehydrogenase (EC 1.1.99.17), a membrane acceptor for the electron generated during the reaction.
bound quino-protein having bound pyrroloquinoline However, role of the subunit of MW 22 Kda in enzyme
quinone (PQQ) catalyzes the direct oxidation of D-glucose catalysis is not clear.
to D-gluconic acid in many bacterial strains. Ameyama et
al. (1981a) have isolated and characterized GDH from G. 2-Ketogluconate Dehydrogenase
oxydans ATCC 9937. The thermal stability of water-soluble Shinagawa et al. (1981) solubilized and purified 2-keto-D-
PQQ dependent glucose dehydrogenase can be increased gluconate dehydrogenase (EC.1.1.99.4) from
by single amino acid replacement using site directed Gluconobacter melanogenus. The purified enzyme is found
mutagenesis technique (Sode et al., 2000). The enzyme to have tightly bound cytochrome C. The MW of the enzyme
is a monomeric protein having the MW 87 Kda. Most is found to be 133 Kda. SDS-PAGE showed the presence
investigations of GDH have been performed using oxidative of three subunits having MWs of 61 Kda (flavo protein), 47
bacteria such as Gluconobacter oxydans and Acinetobacter Kda (cytochrome C) and 25 Kda (with unknown function).
calcoaceticus. GDHs are found to have bound PQQ (Duine
Biotechnological Applications of G. oxydans 449
2,5-DKG Reductase
2,5-Diketo-Gluconic Acid → 2-Keto-L-Gulonic Acid
450 Gupta et al.
using G. oxydans CCM 1783 (ATCC621). An increase in acid. Svitel and Sturdik (1995) examined the conversion
the O 2 consumption rate was observed at glycerol of n-propanol to propionic acid by G. oxydans CCM 1783
concentration upto 300g/l. Batch cultures incubated with (ATCC 621) using fed batch conversion at pH 6.0. Propanol
gassing by air and/or O2, yielded upto 94% DHA. Sodium feeding was started after the growth phase. In the fed-
glycerate was identified in the fermentation broth (after batch conversion, calcium propionate 37g/l, formed within
neutralization with NaOH). Ohrem and Voss (1995) 30 hr (when Ca(OH)2 was used for the neutralization), and
reported production of 220 g/l DHA (96-98% production) sodium propionate, 56g/l, within 70 hr (when NaOH was
after G. oxydans fermentation for 30 hr. The Maximum used for the neutralization), was obtained.
productivity reached after 6-10 hr. The fermentation broth
pH was maintained between 3.8-4.8 using Ca(OH)2. Ohrem Applications of Gluconobacter in Biosensor
and Voss (1996) studied effect of glycerol on the growth Technology
and DHA production in Gluconobacter oxydans. They
showed that neither glycerol oxidation nor metabolism of Gluconobacter oxydans as whole cells as well as its
DHA was inhibited by high glycerol concentrations. They enzymes have been examined for its use as biosensor for
showed that DHA damaged the cells irreversibly and the the identification of various compounds. Applications of
viability of Gluconobacter decreased exponentially with Gluconobacter oxydans in biosensor technology are listed
time. in Table 3.
Smolander et al. (1993) purified a membrane bound
Aldehyde Formation xylose oxidizing PQQ-dependent dehydrogenase from
Gluconobacter oxydans ATCC 621 and studied its possible
The aldehyde formation by alcohol oxidation with G. applications in biosensor technology. Dimethyl and
oxydans submerged cultures and resting cells, carboxylic acid derivatives of ferrocene were able to
resuspended in different phosphate buffers (0.1M) has mediate electron transfer in xylose oxidation using enzyme
been studied by Molinari et al. (1995). Large-scale cultures immobilized on graphite electrode. Smolander et al. (1995)
were grown using stirred fed-batch reactors (1.5 l working developed an amperometric enzyme electrode biosensor
volume, 250rpm and/or 2vvm air), bubble columns (500 for analysis of xylose and glucose in fermentation samples
ml working volume, 0.5vvm/1vvm air) and airlift reactors using PQQ-dependent ALDH from G. oxydans ATCC621.
(1l working volume, 0.5 vvm/1vvm air). New G. oxydans The best electrode performance was obtained when ALDH
strain (from beer) oxidized various short chain aliphatic was adsorbed on the surface of a carbon-paste electrode.
alcohols to their corresponding aldehydes. Molinari et al. The lowest working potential and highest catalytic current
(1996) reported 3-isoamylalcohol as the best substrate were obtained with dimethylferrocene as a mediator.
having over 90% yields of 3-isoamylaldehyde without any Hikuma et al. (1995) prepared a biosensor consisting
by-product formation. of an anode mounted with immobilized alcohol
dehydrogenase (EC 1.1.1.1) from G. oxydans IFO 3172,
Other Fermentation Processes and a cathode containing hexacyanoferrate (III). The
surface of the biosensor was covered with a gas-permeable
Svitel and Kullnik (1995) studied the potentials of acetic membrane and measurements were made without reagent
acid bacteria for oxidation of low MW monoalcohols. consumption by pumping a sample solution through the
Acetobacter aceti CCM 3620, Acetobacter liquefaciens flow cell for 1 or 2 minutes. Alcohol contents of the alcoholic
CCM 3621, Acetobacter pasteurians CCM 2374, G. drinks determined using the sensor as well as by gas
oxydans CCM 3607 and CCM 1783 (ATCC 621) were used chromatography were coincided indicating high sensitivity
for the oxidation of acids and ketones. The oxidations were of the sensor. Reshetilov et al. (1998a) used G. oxydans
performed in fed-batch cultures using a 5 l reactor (LF-2) strains as microbial electrode biosensor for xylose analysis
filled with 2.7 l of sterile medium at 500rpm and 1vvm using clark-type oxygen electrodes. Reshetilov et al.
aeration. The pH during acid production was maintained (1998b) described the use of Gluconobacter oxydans whole
at 6.0. Ethanol, propanol, isopropanol, butanol and 2- cells for determination of sugars, alcohols, and polyols.
butanol were converted to acetic acid, propionic acid, Lusta and Reshetilov (1998) reviewed the prospects of
acetone, butyric acid and 2-butanone, respectively. Svitel Gluconobacter oxydans for its use in Biotechnology as
and Sturdik (1994b) reported galactonic acid formation from biosensor system. Svitel et al . (1998) described the
D-galactose in batch culture of 32 hr. During transformation, microbial cell-based biosensor for sensing glucose, sucrose
96% of D-galactose (100g/l) got converted to D-galactonic or lactose. The glucose–sensing membrane was prepared
Xylose and Glucose Aldose dehydrogenase G. oxydans ATCC621 Smolander et al., 1995
Alcohol 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
452 Gupta et al.
with intact cells of Gluconobacter oxydans immobilized in substitution resulting in the substitution of His 787 by Asn
gelatin cross-linked with glutaraldehyde. The disaccharide– in an open reading frame of 2424 bp. The putative protein
sensing membranes were prepared by co-immobilization is of 808 amino acids with a MW of 87,587 Da. Riboflavin
of G. oxydans either with cells of Saccharomyces cerevisiae kinase (rib F) gene has been identified from G. oxydans
containing invertase for sucrose determination, or with ATCC9937 that is involved in FAD+ synthesis for GADH
permeabilized cells of Kluyveromyces marxianus enzyme (Gupta et al., 1999). FAD+ is a cofactor for GADH
containing ß-galactosidase for lactose determination. The enzyme (McIntire et al., 1985). The other genes namely
strain of G. oxydans was able to oxidize both anomers of gadh and 2kgadh involved in 2,5-dkg pathway from G.
glucose at the same rate. Therefore, there was no need oxydans could not be detected yet. Xba I restriction map
for mutarotase co-immobilization in disaccharide-sensing of the genome from G. oxydans ATCC 9937 has been
membranes. The sensitivity of glucose sensor was 50 nA/ developed using Pulse field gel electrophoresis studies
mM and the range of the calibration curve was 0-0.8 mM (Verma et al., 1997). Southern hybridization and PFGE
with response time of 2 min. The response after 1 week of showed single copy of pqq and ribF genes in G. oxydans
storage was 62% of the initial one. The detection linear ATCC 9937 and IFO 3293 present on different locations
range of disaccharide sensor was found to be upto 4mM (A.Gupta, unpublished data).
with response time of 5min. The activities of the sensors
after 1 week of storage at ambient temperature were in Identification of Genes Involved in 2,5-dkg Pathway/
the range of 50-65% of the initial activity. Other Pathways from Other Bacteria
The genes of 2,5-dkg pathway/other pathways reported
Genetic Analyses of G. oxydans Strains from G. oxydans /other bacteria like Acinetobacter
calcoaceticus, Erwinia herbicola are listed in Table 4.
Native Plasmids and Phages of G. oxydans Jansen et al. (1988, 1990) have reported the gdh genes
Fukaya et al., (1985a) detected plasmid DNAs in 23 out of from A. calcoaceticus and E. coli. The gdh genes isolated
the 36 strains of Gluconobacter sp. and most of them had from A. calcoaceticus, E. coli and G. oxydans showed high
MW of more than 5 Mda. Verma et al. (1994) characterized homology at C-terminal ends (Jansen et al., 1991). Cha et
plasmids of G. oxydans strains ATCC 9937 and IFO3293, al. (1997) identified and characterized a gene encoding
and found three native plasmids in ATCC 9937 having the glucose dehydrogenase from Pantoea citrea and showed
sizes 27.7 kb (pVJ1), 12.3 kb (pVJ2) and 18 kb (pVJ4) and its involvement in causing Pineapple pink disease.
one native plasmid of 9.5 kb size (pVJ3) in IFO3293. Two The pqq genes of A. calcoaceticus, Methylobacterium
phages have been isolated from 54 different strains of extorquens AMI and Klebsiella pneumoniae have been
Gluconobacter (Robakis et al., 1985). These two phages cloned and sequenced (Goosen et al., 1987; Toyama et
have been reported to have different sized double stranded al., 1997; Meulenberg et al., 1992).
DNAs (approximately 37 and 250-300 kb sizes). The Gluconate dehydrogenase encoding genes have been
restriction map of bacteriophage revealed ring like DNA isolated and characterized from Erwinia cypripedii ATCC
containing cohesive ends. 29267 (Yum et al., 1997). The 2,5 diketo-D-gluconate
reductase of E. coli has homology with 2,5-diketo-D-
Identification of Genes Involved in 2,5-DKG Pathway gluconate reductases of Corynebacterium sp., and
from Gluconobacter oxydans Gluconobacter oxydans (Yum et al., 1999). 2,5-DKGR of
Glucose dehydrogenase gene has been identified and Cornyebacterium showed 49.8% homology to yqhE gene
sequenced from G. oxydans P1 and P2 strains (Jansen et product; and 2,5KDGR of Gluconobacter oxydans showed
al., 1991). P1 and P2 are two isogenic forms of G. oxydans. 42% homology with yafB gene product. The gene products
P1 can oxidize only D-glucose, whereas P2 is also capable of yqfE and yafB were identified as 2,5DKGR-A and
of oxidizing sucrose. The DNA sequence of both gdh genes 2,5DKGR-B, respectively catalyzing the reduction of 2,5-
from P1 and P2 are found identical except one nucleotide DKG to 2-KLG.
Table 4. Genes identified from Gluconobacter oxydans/ other ketogenic bacteria involved in 2,5-diketogluconic acid pathway/ other pathways.
Identification of Genes Involved in Sorbitol Oxidation a potential versatile gene delivery system for Gluconobacter
Shinjoh et al. (1995) cloned and sequenced the membrane oxydans.
bound SNDH gene from Acetobacter liquefaciens Creaven et al . (1994) developed an efficient
IFO12258 and got it expressed in a 2-KLG producing electroporation system for transformation of the plasmid
mutant (strain OX4) of G. oxydans IFO3293. The open pMP220 in G. oxydans NCIB2008. The optimum
reading frame of the gene is found to be 1,347 bp encoding electroporation conditions were 12.5 KV/cm field strength,
a polypeptide of 449 amino acids, with a MW 48,223 Da. 400 ohm parallel resistor setting, 0.4-0.5g DNA, 100µl cell
The cloned SNDH is located in the membrane of G. suspension and electroporation buffer containing Mg+2 and
oxydans IFO3293. The recombinant cells produced 2KLG 1011 cfu/ml of early to mid-log phase cells. Under these
efficiently using L-sorbosone or L-sorbose in the medium. conditions, transformation efficiencies of 105/µg DNA were
The yield of ascorbic acid with recombinant G. oxydans reproducibly obtained. Storage for 5 days at -20C in a buffer
IFO3293 strain OX4 using L-sorbosone was improved from containing 1% glycerol decreased transforming efficiency
about 25 to 83%, whereas using L-sorbose was increased by 86% owing to a loss of cell viability.
from 68 to 81%. Shinjoh and Hoshino (1995) developed a shuttle vector
(plasmid pGE1, 11.9kb) and conjugative transfer system
Identification of Genes Involved in Other Pathways for G. oxydans. Transformed G. oxydans were shown to
from G. oxydans be capable of producing 2-keto-L-gulonic acid from L-
Klasen et al . (1995) reported Gluconate NADP + 5- sorbose. The plasmid pGE1 was constructed from cryptic
oxidoreductase gene from Gluconobacter oxydans sub sp. G. oxydans IFO 3293 plasmid pGO32938 (9.9kb, relaxed
DSM 3503. The enzyme oxidizes gluconate to 5- type), E. coli conjugative plasmid pSUP301 (5kb, Kmr and
ketogluconate and is localized in the cytoplasm. The MW ampr, relaxed type), plasmid pACYC 177 and the plasmid
of the enzyme is reported to be 75 Kda. The enzyme RP4 mob region. The pGE1 was transferred by conjugation
showed single band on SDS-PAGE having MW 33 Kda to G. oxydans IFO 3293 with high frequency (0.1
indicating two identical subunits in the protein. Sequencing transconjugants/recipient), and maintained stability without
of the gene revealed an open reading frame of 771 bp, antibiotic selection. It was also maintained in other strains
encoding a protein of 257 amino acids. Kondo and of G. oxydans like IFO 3172, IFO 3268, IFO 3271 and ATCC
Horinouchi (1997) reported a novel insertion sequence 9937; and G. frateurii IFO 3268, IFO 3129, IFO 3170,
element IS 12528 from G. suboxydans associated with IFO3223 and IFO 3225. The presence of pGE1 did not
inactivation of the alcohol dehydrogenase by insertion in inhibit growth or 2KLG productivity of G. oxydans IFO 3293.
the adhA gene, that encodes the primary dehydrogenase The vector was tested by subcloning gene of Acetobacter
subunit of the three-component membrane-bound alcohol liquefaciens IFO 12258 in G. oxydans IFO 3293. The
dehydrogenase complex in Gluconobacter suboxydans. plasmid pGE1 could be shortened further to plasmid pGE2
Cloning and sequencing analyses revealed that IS 12528 (9.8 kb).
was 905 bp in length and had a terminal inverted repeat of
18 bp. Liu et al. (1998) reported a recA gene from G. Gene Expression in Gluconobacter oxydans
oxydans and they expressed it in E. coli recA- mutant. The Saito et al . (1997) constructed a shuttle vector for
G. oxydans recA protein efficiently functions in homologous expression of sorbitol dehydrogenase and sorbosone
recombination and DNA repair. The deduced amino acid dehydrogenase genes in G. oxydans G624. A plasmid of
sequence of recA gene revealed a protein of 344 amino 4.4 kb designated as pF4 was isolated from G. oxydans T-
acids with a MW of 38 Kda. 100 and ligated with pHSG298 at the Hind III site to
construct a shuttle vector, designated as pFG15A. The
Vector System for G. oxydans plasmid pFG15A carrying SDH and SNDH genes was
The construction of shuttle vectors for Gluconobacter transformed into G. oxydans G624. The recombinant
oxydans and Escherichia coli is a further step in the Gluconobacter oxydans G624 was 2-3 fold more active
application of recombinant DNA technology for this group for the production of 2-KLGA than G. oxydans T-100;
of microorganisms. Fukaya et al. (1985b) constructed a however, G. oxydans G624 itself showed no ability to
shuttle vector pMG101 for Gluconobacter oxydans. The produce 2-KLGA.
shuttle vector, pMG101, for Gluconobacter oxydans and Takeda and Toshio (1992) expressed cytochrome C-
Escherichia coli is constructed by ligation of a cryptic 553 (CO) gene in Gluconobacter suboxydans subsp. α that
plasmid, pMV201, found in G. suboxydans IFO 3130 with complemented the second subunit deficiency of
E. coli plasmid pACYC177. The chimeric plasmid named membrane-bound alcohol dehydrogenase. The gene has
pMG101 carries the ampicillin resistance gene derived from been subcloned into a shuttle vector pGEA1 and the
plasmid ACYC177. The transformation efficiency is found recombinant was designated as pGEAC1. The gene has
to be 102 per µg DNA. been shown to replicate in Gluconobacter suboxydans and
Palleroni (1986) showed that Gluconobacter oxydans Escherichia coli. The pGEA1 transformed G. suboxydans
IFO 3293 and ATCC 9937 could accept plasmid RSF 1010 subsp. α showed low ethanol oxidation activity due to
both by transformation and conjugation. Condon et al. second subunit deficiency of membrane-bound ADH. The
(1991) reported conjugal transfer of a series of incompatible transformants harboring pGEAC1 expressed ethanol
group P and Q plasmids in Gluconobacter oxydans oxidation activity and showed cross-reactivity with anti-
NCIB2008. Transfer frequency for the Inc P/Q vectors cytochrome C-553 antibody. Furthermore, expression of
ranges from 10-5 to 10-9 exconjugants per recipient cell. cytochrome C-553 (CO) gene resulted in elevated levels
They constructed gentamycin resistant pRK290 vector as of heme C and CO-reactive cytochrome C complemented
454 Gupta et al.
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