Bioresource Technology 101 (2010) 8294–8299

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Enhanced production of dihydroxyacetone from glycerol by overexpression

of glycerol dehydrogenase in an alcohol dehydrogenase-deficient mutant
of Gluconobacter oxydans
Ming-hua Li, Jian Wu, Xu Liu, Jin-ping Lin *, Dong-zhi Wei **, Hao Chen
State Key Laboratory of Bioreactor Engineering, Institute of Newworld Biotechnology, East China University of Science and Technology, Shanghai 200237, China

a r t i c l e i n f o a b s t r a c t

Article history: Gluconobacter oxydans can rapidly and incompletely oxidize glycerol to dihydroxyacetone (DHA), a ver-
Received 1 December 2009 satile product extensively used in cosmetic, chemical and pharmaceutical industries. To improve DHA
Received in revised form 11 May 2010 production, the glycerol dehydrogenase (GDH) responsible for DHA formation was overexpressed in G.
Accepted 21 May 2010
oxydans M5AM, in which the gene coding for the membrane-bound alcohol dehydrogenase (ADH) was
Available online 23 June 2010
interrupted. Real-time PCR and enzyme activity assay revealed that the absence of ADH together with
the overexpression of GDH gene resulted in an increased GDH activity in the resulting strain M5AM/
Keywords:
GDH, which led to a substantially enhanced production of DHA in a resting cell system. In a batch bio-
Gluconobacter oxydans
Glycerol dehydrogenase
transformation process, M5AM/GDH exhibited a 2.4-fold increased DHA productivity of 2.4 g/g CDW/h
Dihydroxyacetone from 1.0 g/g CDW/h, yielding 96 g/L DHA from 100 g/L glycerol. When 140 g/L glycerol was supplied, a
Biotransformation final DHA concentration of 134 g/L was accumulated within 14 h. In four repeated batch runs, 385 g
Glycerol DHA over a time period of 34 h was achieved from 400 g glycerol with an average productivity of
2.2 g/g CDW/h. These results indicated that this newly developed strain G. oxydans M5AM/GDH with high
productivity and increased tolerance against product inhibition has potential for DHA production in an
industrial bioconversion process.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction drogenase (GDH) which transfers electrons from glycerol to the fi-

As a by-product of biodiesel production, glycerol has become a
relatively cheap and readily available commodity for which new
used have to be explored. Conversion into higher-value products
such as 1,3-propanediol, 2,3-butanediol, succinic acid, citric acid,
ethanol, 3-hydroxypropionic acid and glyceric acid by microbial
fermentation has already been explored (Zhao et al., 2006; Zhang
et al., 2010; Imandi et al., 2007; Easterling et al., 2009; Mohan
Raj et al., 2009; Habe et al., 2009), and microbial production of
dihydroxyacetone (DHA) has also been described (Mishra et al.,
2008).
DHA, which serves as a sunless tanning agent, a precursor of
pharmaceuticals and a building block for the synthesis of various
fine chemicals (Hekmat et al., 2003; Mishra et al., 2008), is cur-
rently being produced via incomplete oxidation of glycerol by
Gluconobacter oxydans in a fermentation process (Claret et al.,
1994; Hekmat et al., 2003; Bauer et al., 2005; Gätgens et al.,
2007). The oxidative reaction is catalyzed by the membrane-
bound, pyrroloquinoline quinone (PQQ)-dependent glycerol dehy-
* Corresponding author. Tel.: +86 21 64252981; fax: +86 21 64250068.
** Corresponding author. Tel.: +86 21 64252981; fax: +86 21 64250068.
E-mail addresses: jplin@ecust.edu.cn (J.-p. Lin), dzhwei@ecust.edu.cn (D.-z. Wei).
nal acceptor, oxygen, via an electron transport chain without
NADH involvement (Claret et al., 1994; Shinjon et al., 2002). Since
the active site of the GDH is oriented towards the periplasm, glyc-
erol can be oxidized in the periplasmic space without the need to
enter the cytoplasm (Matsushita et al., 1994), which facilitates re-
lease and accumulation of DHA in the medium.
Attempts at increasing DHA yield have been made by improving
oxygen supply (Hoist et al., 1985; Adlercreutz and Mattiasson,
1982; Leung et al., 1997), optimizing media components (Wethmar
and Deckwer, 1999; Wei et al., 2007a; Albin et al., 2007; Wei et al.,
2009) and modifying fermentation patterns (Hekmat et al., 2003;
Bauer et al., 2005). In addition, G. oxydans has been genetically
modified to enhance production of DHA (Gätgens et al., 2007). So
far, the highest DHA production of 220 g/L was achieved in a
two-stage repeated fed-batch fermentation process which lasted
as long as 77 h and gave a productivity of 2.9 g/L h (Bauer et al.,
2005).
Recently, Wei (2008) showed that alcohol dehydrogenase
(ADH) and acetaldehyde dehydrogenase (ALDH) mutants of G. oxy-
dans M5 displayed higher DHA production from glycerol than the
wild type. A similar observation was made with a gluconate-2-
dehydrogenase (GA2DH) mutant of G. oxydans DSM 2343 in shake
flask culture (Gätgens et al., 2007).

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.05.065
 M.-h. Li et al. / Bioresource Technology 101 (2010) 8294–8299
In the current study, glycerol dehydrogenase (GDH) was over-
expressed in the ADH-deficient mutant, G. oxydans M5AM, (Wei,
2008) and DHA production of this recombinant strain was deter-
mined in a resting cell system.
2. Methods
2.1. Strains, plasmids, and culture conditions
Escherichia coli DH5a (Hanahan, 1983) was used as a host for
plasmid construction and maintenance. E. coli HB101 (Boyer and
Roulland-Dussoix, 1969) harboring helper plasmid pRK2013 (Fig-
urski and Helinski, 1979) was used for transformation experi-
ments. G. oxydans M5 (Yang et al., 2008) and G. oxydans M5AM
were used for GDH expression and DHA production. The broad host
range vector pBBR1MCS-5 (Kovach et al., 1995) was used for
expression of GDH in G. oxydans.
All E. coli strains were cultivated in Luria–Bertani (LB) medium
(yeast extract, 5 g/L; tryptone, 10 g/L; NaCl, 10 g/L; pH 7.0) at 37 °C
with the addition of 10 lg/mL gentamicin or 50 lg/mL kanamycin
to select for the presence of plasmids when necessary. Recombi-
nant G. oxydans strains were cultivated at 30 °C in sorbitol medium
(sorbitol, 80 g/L; yeast extract, 20 g/L; (NH4)2SO4, 5 g/L; KH2PO4,
1.5 g/L; MgSO47H2O, 0.5 g/L) containing 50 lg/mL gentamicin.
2.2. Vector construction
General molecular biological techniques were used for the con-
struction, purification, and analysis of plasmid DNA and for the
transformation of E. coli (Sambrook and Russell, 2000). Based on
the available sequence information for G. oxydans ATCC 621H (Prust
et al., 2005), oligonucleotide primers were designed to amplify the
GDH gene by PCR. The GDH gene was amplified with LA Taq poly-
merase (TaKaRa, Dalian) from genomic DNA of G. oxydans M5, using
oligonucleotides (50 -CATAAGCTTGGCGTTTACGATGGTGC-30 ) as a
forward primer and (50 -CTAGGTACCGTGAACGAAAACAGCCTG-30 )
as a reverse primer. The resulting fragment was digested with
restriction enzymes Hind III and KpnI (MBI Fermentas) and ligated
into the prepared vector pBBR1MCS-5 using T4 DNA ligase (MBI Fer-
mentas), generating plasmid pBBR-GDH. The recombinant plasmid
was transformed into competent E. coli DH5a. Transformants were
selected on LB agar plates containing 10 lg/mL gentamicin, and fur-
ther confirmed by colony PCR and restriction enzyme digestions.
2.3. Conjugational plasmid transfer into G. oxydans
The expression vector pBBR-GDH was transferred into G. oxy-
dans M5 and G. oxydans M5AM by triparental mating as described
previously (Hölscher and Görisch, 2006). E. coli DH5a bearing the
expression vector pBBR-GDH and E. coli HB101 harboring the
mobilizing plasmid pRK2013 were used as the donor and helper
strain, respectively. The three strains were grown to late exponen-
tial phase, pelleted, washed and resuspended in sorbitol medium,
then mixed at a 1:1:1 ratio. 100 lL of this mixture was spreaded
onto sorbitol agar plates without antibiotics and incubated over-
night at 30 °C. The cultures were then scraped from the plates
and spread onto selective sorbitol agar plates containing cefoxitin
(50 lg/mL) and gentamicin (50 lg/mL). Plates were incubated at
30 °C for 2–4 days until cefoxitin- and gentamicin-resistant colo-
nies appeared. Because the recombinant G. oxydans strains exhib-
ited a slightly lower cell growth rate than the wild strain in
gentamicin-containing medium, we transformed the plasmid
pBBR1MCS-5 into G. oxydans M5 and G. oxydans M5AM to generate
M5/pBBR and M5AM/pBBR which were used as control strains dur-
ing the experiments.
8295
2.4. Preparation of membrane fractions
To prepare membrane fractions, single colonies of the G. oxy-
dans strains were inoculated into 20 mL sorbitol medium and
grown at 30 °C and 200 rpm for 20 h on a rotary shaker. Then 1%
(v/v) of the precultures were inoculated into 1000-mL shake flasks
containing 200 mL sorbitol medium and cultivated under the same
conditions for 24 h. The G. oxydans cells were harvested by centri-
fugation (8000g, 10 min, 4 °C) and then resuspended in 50 mM so-
dium phosphate buffer (pH 6) at a cell wet weight (CWW)
concentration of 0.1 g/mL. Cell disruption was carried out with
an ultrasonifier (JY92–2D, Xinzhi, Ningbo) for 50 cycles (3 s sonica-
tion, 5 s pause on ice). After centrifugation at 10,000g for 10 min to
remove the cell debris, the resulting supernatants were centrifuged
at 100,000g and 4 °C for 60 min. The sediments were collected and
designated the membrane fractions, which were resuspended into
the above sodium phosphate buffer on ice subsequently.
2.5. Enzyme activity assay and protein determination
The activity of membrane-bound GDH was determined at 30 °C
by measuring the initial reduction rate of 2,6-dichlorophenolindo-
phenol (2,6-DCIP, Sigma) at 600 nm as described by Sugisawa and
Hoshino (2002). The basal reaction mixture consisted of 50 mM so-
dium phosphate buffer (pH 6), 0.25 mM DCIP and 0.325 mM phen-
azine methosulphate (PMS, Sigma), which was prepared just before
the assay. A cuvette with 1 cm light path containing 0.8 mL basal
reaction mixture and 10 lL diluted enzyme was incubated at
30 °C for 5 min. The reaction was started by adding 200 lL of
0.2 M pre-warmed glycerol. One unit of GDH activity was defined
as the amount of the enzyme that catalyzes the reduction of
1 lM of DCIP per min at 30 °C. Enzyme activity was calculated
using the molar extinction coefficient of DCIP, which was 9.45,
10.8, 13.0, and 15.0 mM1 cm1 at pH 5.5, 6.0, 6.5, and 7.0–7.6,
respectively. Protein concentration was determined by the method
of Bradford (1976), using bovine serum albumin as a standard.
2.6. Quantitative real-time PCR
For real-time reverse transcription PCR (RT-PCR) experiments,
1% (v/v) of the precultures of the G. oxydans strains described in Sec-
tion 2.4 were inoculated into 250-mL shake flasks containing 50 mL
sorbitol medium and grown to their late exponential phase (16 h) at
30 °C and 200 rpm on a rotary shaker. Then the cells were harvested
by centrifugation at 10,000g and 4 °C for 1 min for total RNA isola-
tion. Total RNA was extracted by RNAiso reagent (TaKaRa, Dalian)
and then treated with DNase I (TaKaRa, Dalian). Each RNA sample
was quantified using NanoDrop 2000 spectrophotometer (Thermo
Scientific) and adjusted to the same concentration based on the
absorbance value. For transcriptional analysis of GDH gene, RT-
PCR was carried out with a first-strand cDNA synthesis kit (Prome-
ga, USA), using total RNA as template. Quantitative gene expression
analysis was performed by quantitative real-time PCR, which was
performed with the StepOnePlus™ Real-Time PCR System (Applied
Biosystems, USA) using primers 50 -CCTGCGTAGCCCTGAAGAAAAC-
30 and 50 -CGAGCCGATGTCATAGTCCC-30 . All measurements were
done in two independent experiments. The 16S rRNA gene was
used as internal standard, which was obtained based on the primers
50 -GCGGTTGTTACAGTCAGATG-30 and 50 -GCCTCAGCGTCAGTATCG-
30 .
2.7. Preparation of resting cells
Cultivations of G. oxydans were performed either in 1000-mL
shake flasks containing 150 mL sorbitol medium at 30 °C and
200 rpm on a rotary shaker, or in a 5-L fermentor (Baoxing Biotech
8296 M.-h. Li et al. / Bioresource Technology 101 (2010) 8294–8299
Ltd., Shanghai) containing 2.5 L sorbitol medium with the pH and
temperature maintained at 6.0 and 30 °C automatically. Agitation
and aeration were controlled at 900 rpm and 2 vvm, respectively.
Cell cultivations were initiated by inoculating 1% (v/v) of 20-h pre-
cultures of G. oxydans strains. After 24 h, the cells reached early
stationary phase and were harvested by centrifugation at 8000g
and 4 °C for 10 min, washed twice with 50 mM sodium phosphate
buffer (pH 6), and resuspended in reaction solution at a final con-
centration of 20 g CWW/L [corresponding to 5.05 g/L cell dry
weight (CDW)] to start the biotransformation of glycerol to DHA.
2.8. Biotransformation of glycerol to DHA
Biotransformation of glycerol to DHA was performed in shake
flasks and a 5-L fermentor. For shake-flask experiments, resting
cells were resuspended in reaction solution containing 200 mM so-
dium phosphate buffer (pH 6), 40 g/L glycerol, and incubated at
30 °C and 250 rpm on a rotary shaker. To further investigate the
DHA production of different strains, experiments in a 5-L fermen-
tor with a working volume of 2.5 L were performed at 30 °C and
900 rpm. The reaction solution consisted of 100–180 g/L glycerol
dissolved in distilled water and the reaction was started by adding
the resting cells into the fermentor. Dissolved oxygen (DO) was
provided by injecting filtered air at a flow of 5 L/min and the pH
was controlled at 6.0 by automatic addition of an aqueous solution
of 2.0 M NaOH. Repeated-batch biotransformation was also carried
out in a 5-L fermentor supplied with 100 g/L glycerol under the
above conditions. When the reaction reached equilibrium, the cells
were recovered by centrifugation and resuspended in fresh reac-
tion buffer for next run of bioconversion. The composition of the
reaction buffer was the same as that used in the first cycle. Samples
were collected at regular intervals for concentration determination
of glycerol and DHA.
2.9. Quantification of glycerol and DHA
Glycerol and DHA concentrations were determined by high-per-
formance liquid chromatography (HPLC) after filtration and dilu-
tion of samples. HPX-87H (Bio-Rad, USA) column was employed
and maintained at 35 °C during the measurements. The mobile
phase was 5 mM H2SO4 pumped at a flow rate of 0.7 mL/min by
HPLC (HP1100, Agilent, USA) equipped with a refractive index
detector (RID-6A, Shimadzu, Japan).
3. Results
3.1. Effect of ADH absence on the expression of GDH gene in G. oxydans
To compare the expression levels of GDH in G. oxydans strains,
membrane fractions were prepared and the GDH activities were
determined in vitro. As shown in Table 1, a slight increase in
GDH activity from 1.7 to 1.9 U/mg protein was noticed in M5AM/
pBBR in comparison to M5/pBBR. Similarly, the overexpression of
GDH gene in the wild strain did not lead to a significantly en-
hanced GDH activity and only a small increase of 29.4% from 1.7
Table 1
Activities of the membrane-bound GDH of different G. oxydans strains.
Strains GDH activity (U/mg protein)
G. oxydans M5/pBBR 1.7 ± 0.1
G. oxydans M5/GDH 2.2 ± 0.2
G. oxydans M5AM/pBBR 1.9 ± 0.1
G. oxydans M5AM/GDH 3.0 ± 0.3
Each value represents the average value and standard deviation of three indepen-
dent experiments.
to 2.2 U/mg protein was observed. When the GDH-expressing plas-
mid pBBR-GDH was introduced into M5AM, however, the resultant
strain M5AM/GDH displayed a significant increase in GDH activity
which was 3.0 U/mg protein, 57.9% and 76.5% more than those of
M5AM/pBBR and M5/pBBR. These results implied that there were
synergistic effects between the ADH absence and GDH overexpres-
sion on the improvement of GDH activity. Similar effects were ob-
served in GA2DH disruptant G. oxydans MF1 when the gene coding
for glucose dehydrogenase or gluconate-5-dehydrogenase was
overexpressed (Merfort et al., 2006).
3.2. Real-time RT-PCR analysis of GDH gene transcription in different
G. oxydans strains
To demonstrate the expression of GDH gene in different G. oxy-
dans strains, detection of the mRNA was carried out with real-time
RT-PCR. The absence of ADH showed no effects on cell growth of G.
oxydans, as well as the overexpression of GDH gene (Fig. 1). As gene
expression often varied significantly in different growth phases
(Quintero et al., 2009), all the cells used for RNA isolation were cul-
tured for constant period to minimize its effect on RT-PCR analysis.
Taking 16S rRNA as the internal standard, it was clearly observed
that the transcription level of GDH gene in M5AM/pBBR was much
higher than that of the control strain M5/pBBR (Fig. 1). The overex-
pression of GDH exhibited a similar improvement in GDH gene
transcription to that of ADH disruption. In M5AM/GDH, the tran-
script of GDH gene was about 120 times more abundant than that
in M5/pBBR, which was due to both the ADH disruption and GDH
overexpression. However, the increase in GDH activity was much
less distinct in contrast to the transcript level (Table 1).
3.3. Biotransformation of glycerol to DHA in shake flasks
As the GDH activity was increased when the GDH gene was
overexpressed, a detailed study of the time course of glycerol oxi-
dation by M5/GDH and M5AM/GDH as well as their corresponding
control strains was carried out in shake flasks. Differences in DHA
production among the four strains are depicted in Fig. 2. The DHA
production of all strains reached approximately the maximum
yields after 24 h of biotransformation when the pH of the reaction
solution decreased below 4.0. At this low pH, oxidation of glycerol
was barely observable and DHA yields remained approximately
Fig. 1. Comparison of cell growth (open) and GDH expression (filled) in different G.
oxydans strains. All cells were cultured in sorbitol medium for 16 h and harvested
for total RNA isolation. Relative expression of GDH gene was determined by real-
time PCR. Cell growth was determined by measuring optical density at 600 nm
(OD600). Each point represents the average value of two independent experiments.
 M.-h. Li et al. / Bioresource Technology 101 (2010) 8294–8299 8297

Fig. 2. Comparison of DHA production among different G. oxydans strains in a

resting cell system in shake flasks. Error bars represent standard deviation from
mean for three batches.

constant thereafter. Results also indicated that the amounts of DHA

accumulated by the four tested strains were in good agreement
with the results obtained from the GDH activity measurements.
The disruption of the GDH gene in G. oxydans M5 increased DHA
production to some extent, as observed in the GA2DH disruptant
G. oxydans MF1 which displayed an enhanced production of DHA
compared to its parental strain DSM2343 (Gätgens et al., 2007). Be-
sides, the overexpression of GDH gene also improved the oxidation
of glycerol to DHA in M5/GDH, leading to a slight increase in DHA
production compared with M5/pBBR. However, when the GDH
gene was overexpressed in M5AM, the resulting strain M5AM/
GDH produced the highest level of DHA which reached up to
26.3 g/L after 24 h, 42.2% and 60.4% more than those of M5AM/
pBBR (18.5 g/L) and M5/pBBR (16.4 g/L), respectively. Therefore,
it was the absence of ADH gene and the GDH overexpression that
together resulted in the distinct improvement of DHA in M5AM/
GDH.

3.4. Biotransformation of glycerol to DHA in a 5-L fermentor

To assess the potential use of M5AM/GDH for industrial produc-
tion of DHA, scaled-up biotransformations were performed in a
pH-controlled conversion process in combination with a continu-
ous high oxygen supply. As can be seen in Fig. 3, glycerol oxidation
started immediately at a high rate whereas the DO concentration
dropped sharply from 100% air saturation to less than 23% when
the cells were added to the fermentor. For M5AM/GDH, there
was only 10 g/L glycerol left in the reaction solution and 88 g/L
DHA was produced after 6 h of biotransformation. After a further
2 h of bioconversion, all of the supplied glycerol (100 g/L) was ex-
hausted and 96 g/L DHA was obtained, giving a yield of 96%
(Fig. 3a). In contrast to M5AM/GDH, M5/pBBR showed a much
slower bioconversion rate when incubated in the same reaction
solution. As can be calculated from the time course of glycerol oxi-
dation, the DHA productivity of M5AM/GDH was significantly in-
creased to 2.4 g/g CDW/h, whereas it was only 1.0 g/g CDW/h for
M5/pBBR. Thus, overexpression of GDH in M5AM accelerated the
oxidative reaction and resulted in an increase in DHA productivity
of about 140% compared with M5/pBBR. In addition, the productiv-
ity achieved by M5AM/GDH cells was much higher than that by
immobilized cells (Wei et al., 2007b) and by growing cells in a
batch and fed-batch fermentation process (Bories et al., 1991).
Taking into account its high productivity and yield of DHA,
M5AM/GDH was used for further batch biotransformation experi-
ments with initial glycerol concentrations of 140 and 180 g/L. As
shown in Fig. 3b, 140 g/L glycerol was completely exhausted and
134 g/L DHA was accumulated by M5AM/GDH within 14 h, giving
a yield of 96% and a productivity of 1.9 g/g CDW/h. In a previous
Fig. 3. (a) Comparison of DHA production between G. oxydans M5/pBBR (open) and
G. oxydans M5AM/GDH (filled) in a 5-L fermentor supplied with 100 g/L glycerol. (b)
Biotransformation of DHA by G. oxydans M5AM/GDH in a 5-L fermentor supplied
with 140 g/L (filled) and 180 g/L (open) glycerol. The profiles of DHA (circle),
glycerol (triangle) and DO (square) are shown. Each point represents the average
value of two independent experiments.
study, G. oxydans cells were able to tolerate DHA concentrations
in the range of 80–120 g/L for several hours without irreversible
damage (Hekmat et al., 2003). In the present work, M5AM/GDH
exhibited an increased tolerance against higher DHA concentration
for a prolonged time period. When incubated in reaction solution
containing 180 g/L glycerol, M5AM/GDH resting cells accumulated
an approximately maximum DHA concentration of 153 g/L after
18 h, and extended incubation for another 10 h had few effects
on DHA yield. These results indicated that the GDH overexpressing
strain M5AM/GDH could tolerate no less than 130 g/L DHA and the
DHA productivity decreased with the increase in the initial glycerol
concentration in a batch biotransformation.
3.5. Repeated-batch biotransformation of glycerol to DHA by
G. oxydans M5AM/GDH
In repetitive batch experiments with M5AM/GDH, four runs
were repeated in a 5-L fermentor under defined conditions. As re-
vealed in Fig. 4, the cells could be recycled three times without
obvious decline in the DHA productivity, and only a small decrease
in the fourth cycle was observed. Within this period, the specific
productivity of DHA decreased from 2.5 g/g CDW/h to 2.0 g/g
8298 M.-h. Li et al. / Bioresource Technology 101 (2010) 8294–8299
Fig. 4. DHA production (circle) and specific productivity (diamond) of G. oxydans
M5AM/GDH in repeated biotransformations. Temperature, pH, aeration and agita-
tion were controlled at 30 °C, 6.0, 2.0 vvm, and 900 rpm, respectively, in all the four
biotransformation cycles. Each point represents the average value of two indepen-
dent experiments.
CDW/h, showing 25% loss of productivity after four rounds, which
was probably due to cell inactivation by shear forces, DHA damage
or the deletion of the essential cofactor (PQQ) for GDH. The stabil-
ity of free M5AM/GDH cells was lower than that of immobilized
cells which could recover 90% of initial activity after five cycles
of biotransformation in reaction solution containing 60 g/L glycerol
(Wei et al., 2007b). However, M5AM/GDH displayed a much higher
conversion rate and productivity at an initial glycerol concentra-
tion of 100 g/L. During the repeated-batch experiments, about
96 g/L DHA was accumulated in each round of biotransformation
with an increase in reaction time from the first to the fourth cycle
(Fig. 4). In total, 385 g DHA was formed within 34 h in the four runs
of reaction, giving an average DHA productivity of 2.2 g/g CDW/h.
From an economic viewpoint, the results were of great interest
for DHA production by resting cells of G. oxydans M5AM/GDH on
an industrial scale.
4. Discussion
We have demonstrated that overexpression of GDH in a strain
lacking the gene encoding for ADH led to a significant improve-
ment of GDH activity and DHA production. The increased activity
led to a 2.4-fold increase in productivity compared with M5/pBBR
in a batch biotransformation when 100 g/L glycerol was supplied
and shortened the reaction time (Fig. 3a). It is also possible that
higher levels of GDH increased the tolerance of the strain against
DHA inhibition, as suggested by Gätgens et al. (2007). Since in
the presence of higher levels of glycerol, a relatively low yield
was (Fig. 3b), possibly due to inhibition by high concentrations of
DHA, further improvements in yield will have to involve changes
in fermentation procedures such as those described by Hekmat
et al. (2003) who designed a reactor system consisting of a shaking
tank and a permeable column harboring immobilized cells.
In addition, more biotransformation cycles may be realized in
repeated-batch production of DHA with immobilized M5AM/GDH
cells which could maintain consistent oxidative activity more eas-
ily than free cells during the oxidation process (Adlercreutz et al.,
1985; Wei et al., 2007b).
The current experiments were carried out with pure glycerol. In
a commercial setting, most likely biodiesel-derived glycerol would
have to be used. This type of glycerol typically contains various
contaminants that could cause severe inhibition at high concentra-
tions, or even are toxic to the cells (Sabourin-Provost and Hallen-
beck, 2009). It remains to be determined if our recombinant
strain will be able to provide high yields under those conditions.
5. Conclusion
A promising strain for industrial production of DHA was devel-
oped by overexpressing the GDH gene in the ADH-negative strain
G. oxydans M5AM. Taking advantage of the elevated activity of
GDH, the M5AM/GDH strain produced DHA at a high concentration
and displayed a substantially increased DHA productivity in a rest-
ing cell system. In repetitive batch biotransformation, the cells
could be used for several cycles without significant loss of produc-
tivity. The recombinant strain thus has potential for industrial pro-
duction of DHA.
Acknowledgements
This work was financially supported by the National Key Basic
Research Development Program of China (‘‘973” Program, No.
2009CB724703), and the National Special Fund for State Key Labo-
ratory of Bioreactor Engineering, Grant No. 2060204. We also
thank professor Yang Susheng (China Agricultural University) for
kindly providing the plasmid pBBR1MCS-5.
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