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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
bioinfo@sancharnet.in; Fax. 91-731-470372.
J. Mol. Microbiol. Biotechnol. (2001) 3(3): 445-456.
© 2001 Horizon Scientific Press
JMMB Review
Arun Gupta1, Vinay K. Singh1, G.N. Qazi2
and Anil Kumar1
1School of Biotechnology, Devi Ahilya University,
Khandwa Road, Indore-452017, MP, India
2Dept. 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,
baker’s soil, honeybees, fruit, cider, beer, wine.
Gluconobacter
strains are non-pathogenic towards
man and other animals but are capable of causing
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; D-
gluconic acid, 5-keto- and 2-ketogluconic acids from
D-glucose; and dihydroxyacetone from glycerol. It is
primarily known as a ketogenic bacterium due to 2,5-
diketogluconic 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 µm,
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
Abbreviations
µg-microgram; µl-microlitre; 2,5-DKG-2,5-diketogluconic acid; 2,5-DKGR-
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-
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
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; Asn-
asparagine; ATCC-American type culture collection; bp-base pair; CaCO3-
calcium carbonate; Ca(OH)2-Calcium hydroxide; Cfu-colony formation unit;
CO2-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 adenine
dinucleotide; g-gram;
G. oxydans
-
Gluconobacter oxydans; G. sacchari
-
Gluconobacter sacchari
; GA-gluconic acid; GADH-gluconate
dehydrogenase; GADH--gluconate dehydrogenase deficient mutant; gadh-
gluconate 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; 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; O2-oxygen; OPV-oxidized
polyvinyl 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 dodecyl
sulfate; SNDH-sorbosone dehydrogenase; Tn5-Transposon Tn5; vvm-
volumetric volume per mole.
446 Gupta
et al
.
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, baker’s 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 28°C for 2-4
days, tubes showing acidification are streaked onto plates
of standard GYC medium (GYC contains - yeast extract
10.0, glucose 50.0, CaCO3 30.0; and agar 25.0 g/l,
respectively). Colonies that dissolve the CaCO3 are further
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 (Gosselé
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 25-30°C 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
(Gosselé
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, L-
sorbose, 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). 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. 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
Biotechnological Applications of G. oxydans 447
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, n-
butanol, glycerol, m-erythritol, D-mannitol, D-arabinose, D-
ribose, 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)].
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).
Table1. Biotechnological Applications of
Gluconobacter oxydans
Application Organism Reference
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
.
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 immunocross-
reactivity among the membrane bound GDHs isolated from
several Gram-negative bacteria. The immunocross-
reactivity 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-D-
gluconate 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).
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-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.
Biotechnological Applications of G. oxydans 449
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 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 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-L-
gulonate 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 Reichstein’s process.
Reichstein-Grüssner synthesis of L-ascorbic acid serves
the basis for modern industrial production of ascorbic acid.
The main steps in the Reichstein-Grüssner 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
.
Figure 2. Diagrammatic representation of keto acids pathways in different organisms.
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 D-
glucose 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-keto-
gluconic 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 32°C, 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 O2 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 35-40°C.
Sorbose formation occurred in a batch culture lasting 34
hr (sorbitol concentration 200g/l), and the yield was 96%.
In the fed-batch cultures (30°C, 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 28°C,
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 by-
product formation during conversion of glycerol into DHA
Biotechnological Applications of G. oxydans 451
using
G. oxydans
CCM 1783 (ATCC621). An increase in
the O2 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 2-
butanol 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 fed-
batch 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 glucose–sensing 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.,
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
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 ß-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-D-
gluconate 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 2,5-
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.,
1991
RibF
G. oxydans
ATCC 9937 Gupta
et al.,
1999
gdh
Acinetobacter calcoaceticus
Jansen
et al.,
1988
gdh
Escherichia coli
Jansen
et al.,
1990
gdh
Pantoea citra
Cha
et al.,
1997
pqq
Acinetobacter calcoaceticus
Goosen
et al.,
1987
pqq
Methylaobacterium extorquens
AMI Toyama
et al.,
1997
pqq
Klebsiella pneumonniae
Meulenberg
et al.,
1992
gadh
Erwinia cypripedii
ATCC 29267 Yum
et al.,
1997
L-Sorbosone dehydrogenase (SNDH) gene
Acetobacter liquefaciens
IFO12258 Shinjoh
et al.,
1995
D-Sorbitol dehydrogenase (SDH) and sorbosone dehydrogenase gene
G. oxydans
G 624 Saito
et al.,
1997
Gluconate NADP+5-oxidoreductase (GNO) gene
G. oxydans
subsp. 3503 Klasen
et al.,
1995
IS element
G. suboxydans
Kondo and Horinouchi, 1997
RecA
G. oxydans
LCC 99 Liu
et al.,
1998
Xylitol dehydrogenase genes (XDH1 & XDH2)
Gluconobacter oxydans
Masakazu
et al.,
2000.
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+ 5-
oxidoreductase gene from
Gluconobacter oxydans
sub sp.
DSM 3503. The enzyme oxidizes gluconate to 5-
ketogluconate 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 µg 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, 100µl 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/µg 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 L-
sorbose. 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
T-
100 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 C-
553 (CO) gene in
Gluconobacter suboxydans
subsp. α 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. α 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 anti-
cytochrome 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,
Xba
I 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 µg/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.
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 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 GADH-
mutant 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.
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.
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