Coenzyme B12-dependent glycerol dehydratase deficiency in Klebsiella pneumoniae

Article abstract of DOI:10.14233/ajchem.2013.14581

The physiology and fermentation properties of coenzyme B12-dependent glycerol dehydratase deficient mutant Klebsiella pneumoniae B1.9131 were investigated. Compared with that of wild type strain, glycerol dehydratase activity decreased by 80 %. Activities of dha regulon coded two enzymes are influenced by inactivation of GDHt. 1,3-Propanediol oxidoreductase activity decreased by 62 % while glycerol dehydrogenase activity increased by 36 %. Fed-batch fermentation showed that more of the metabolic flux of glycerol was directed to lactate and ethanol in the mutant. Instead, lactate other than 1,3-propanediol was the main product and its final concentration increased from 41 to 76 g L-1 with mutant isolate. The concentration of 1,3-propanediol decreased from 91 to 40 g L-1. The results demonstrate that GDHt is not indispensable in glycerol metabolism but is crucial for the efficient synthesis of 1,3-propanediol.

Full text of DOI:10.14233/ajchem.2013.14581

Asian Journal of Chemistry; Vol. 25, No. 13 (2013), 7323-7326
http://dx.doi.org/10.14233/ajchem.2013.14581
Coenzyme B12-Dependent Glycerol Dehydratase Deficiency in Klebsiella pneumoniae
1,*
1
1
2
ZONG-MING ZHENG , TI-PENG WANG , YUN-ZHEN XU and DE-HUA LIU
1National Engineering Laboratory for Biomass Power Generation Equipment, School of Renewable Energy, North China Electric Power University,
Beijing 102206, P.R. China
2Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 100084, P.R. China
*Corresponding author: E-mail: zheng-zm05@mails.tsinghua.edu.cn
(Received: 15 September 2012;
Accepted: 24 June 2013)
AJC-13700
The physiology and fermentation properties of coenzyme B12-dependent glycerol dehydratase deficient mutant Klebsiella pneumoniae
B1.9131 were investigated. Compared with that of wild type strain, glycerol dehydratase activity decreased by 80 %. Activities of dha
regulon coded two enzymes are influenced by inactivation of GDHt. 1,3-Propanediol oxidoreductase activity decreased by 62 % while
glycerol dehydrogenase activity increased by 36 %. Fed-batch fermentation showed that more of the metabolic flux of glycerol was
directed to lactate and ethanol in the mutant. Instead, lactate other than 1,3-propanediol was the main product and its final concentration
increased from 41 to 76 g L-1 with mutant isolate. The concentration of 1,3-propanediol decreased from 91 to 40 g L-1. The results demonstrate
that GDHt is not indispensable in glycerol metabolism but is crucial for the efficient synthesis of 1,3-propanediol.
Key Words: Dha regulon, Dehydratase, Fermentation, Glycerol, Klebsiella pneumoniae, 1,3-Propanediol.
except for the diol dehydratase of Clostridium butyricum7. They
may work individually or together in several microorganisms,
INTRODUCTION
1,3-Propanediol (1,3-PD) is one of the platform chemicals
in the chemical industry and can be synthesized either from
such as Bacillus, Clostridia, Citrobactor, Lactobacillus and
Klebsiella8.
petrochemical or renewable resources. The value of 1,3-
propanediol lies predominantly in its use in polymers prepared
from 1,3-propanediol and terephthalic acid, demand of which
In order to investigate its metabolic role in K. pneumoniae
CGMCC 1.9131, GDHt mutant K. pneumoniae was constructed
via knocking out dhaB-α. we obtained a GDHt mutant isolate
is estimated to be one to two billion pounds per year in 10
and used it to study glycerol metabolism with fed-batch fermen-
tation. The time profiles of fermentation and phsiological
years1. The microbial approach to 1,3-propanediol production
becomes an emerging area of intellectual endeavor and indus-
change will be studied in this work.
trial practice with great promise in recent years2.
1,3-Propanediol is excreted in a growth-associated manner
with glycerol by Klebsiella pneumoniae relying on dha
regulon3. 1,3-propanediol synthesis is carried out in two
enzymatic steps. Glycerol dehydratase (EC 4.2.1.30, GDHt)
removes a water molecule from glycerol in presence of coenzyme
vitamin B12 to form 3-hydroxypropionaldehyde (3-HPA)4.
Subsequently, 1,3-propanediol oxidoreductase (EC 1.1.1.202,
PDR) transfers a reducing equivalent from NADH to 3-
hydroxypropionaldehyde, giving 1,3-propanediol. The
isofunctional dehydratase diol dehydratase (EC 4.2.1.28,
DDHt) in K. pneumoniae could also convert glycerol, 1,2-
propanediol and 1,2-ethanediol to 3-hydroxypropionaldehyde,
propionaldehyde and acetaldehyde, respectively5. GDHt is
associated with the dha regulon, while DDHt is associated
with the pdu regulon. Both GDHt and DDHt have a subunit
composition of α2β2γ26 and are coenzyme B12-dependent,
EXPERIMENTAL
Klebsiella pneumoniae CGMCC 1.9131 was isolated from
campus soil in Tsinghua University. The strain is stored in the
China General Microbiological Culture Collection Center
(CGMCC, Beijing, China).
Construction of gene-specific mutants:An 1000-bp seg-
ment of truncated GDHt gene dhaB-α of K. pneumoniae was
polymerase chain reaction (PCR)-amplified with oligonucleo-
tides primer1 (5'- CGAATTCGTGATGGCGCAGATGAAC
GT-3') and primer 2 (reverse 5'- GTCTAGACGCATCTCGT
TGCTGCCGT-3'). The segment was cloned into pGEM-T easy
vector (Promega, USA) and then transferred to suicide vector
pGPCm after being digested with EcoRI and XbaI, resulting
in vector pGP-Ba. The pGP-Ba vector was then transformed
into E. coli SM109 and the resulting strain was used as donor

7324 Zheng et al.
Asian J. Chem.
in conjugation with K. pneumoniae. Transconjugants were
selected for both chloromycetin resistance from pGPCm
inserting and ampicillin resistance because the K. pneumoniae
strain is resistive to ampicillin while the E. coli SM10 is
sensitive.
Growth conditions: The basic culture media contained
(L-1): Glycerol 30 g, yeast extract 1.5 g, (NH4)2SO4 4.0 g,
K2HPO4 0.69 g, KH2PO4 0.25 g, MgSO4·7H2O 0.2 g, trace
element solution 1 mL and Fe2+ solution 1 mL. The trace
element solution contained (L-1): MgSO4·4H2O 100 mg, ZnCl2
70 mg, Na2MoO4·2H2O 35 mg, H3BO3 60 mg, CoCl2·6H2O
200 mg, CuSO4·5H2O 29.28 mg, NiCl2·6H2O 25 mg and HCl
(37 %) 0.9 mL.
Fig. 1. PCR detection of K. pneumoniae B1.9131 and CGMCC 1.9131.
Lanes 1-4: K. pneumoniae B1.9131-1,2,3,4; M: Marker; ck: K.
pneumoniae CGMCC 1.9131. The mutants were identified by PCR,
with forward (5'-GTCGACGGATCCCAAGCTT-3') and reverse (5'-
AGATCCTCCACCACGTTAC-3') primers
The fermentable cultivation was conducted in a 5 l
fermentor (B. Braun, Germany) with a working volume of 4 l.
The fed-batch fermentation was carried out at 37 ºC, pH 6.8,
250 rpm and 0.5 vvm air flow. The initial glycerol is 25 g L-1.
The pure glycerol was fed when glycerol concentration is
below 5 g L-1.
TABLE-1
ACTIVITY OF GLYCEROL DEHYDRATASE (GDHt)
AND DIOL DEHYDRATASE (DDHt) IN K. pneumoniae
CGMCC 1.9131 AND THE MUTANTS
GDHt activity
DDHt activity
Protein (U mg-1)
Strains
Protein (U mg-1)
0.93 0.03
0.19 0.01
0.22 0.01
0.17 0.01
0.19 0.01
CGMCC 1.91316
B1.9131-1a
B1.9131-2
0.25 0.01
Substrate and products analysis: Dry cell mass was
computed from the optical density calibration curved deter-
mined at 650 nm (OD650). Glycerol, 1,3-propanediol, 2,3-
butanediol (2,3-BD), lactate, succinate, acetate and ethanol
were determined by a Shimadzu 10AVP HPLC system using
an Aminex HPX-87H column (300×7.8mm) (Bio-Rad, Palo
AHO, Ca, USA) as described10. 3-HPA was determined using
the colorimetric method described by Circle et al.11.
Measurement of enzyme activities: GDHt and DDHt
activities were determined by the 3-methyl-2-benzothia-
zolinone hydrazone method12, in which glycerol or 1,2-
propanediol was used as the respective substrates. PDR and
glycerol dehydrogenase (GDH) activities were determined
according to the method of Xu et al.13.
0.18 0.01
0.18 0.002
0.14 0.01
B1.9131-3
B1.9131-4
0.15 0.003
Bacteria were cultivated in the basic culture media as described in
“Growth conditions” section at 37 ºC and 150 rpm for 12 h. Enzyme
assays were determined according to the method described by Xu et al.
13. The average data of three individual experiments are presented.
aB1.9131-1, 2, 3 and 4 are four GDHt-deficient mutant isolates.
76 to 82 % in comparison with that of its wild type. DDHt
may contribute to the residual activities. Mutant B1.9131-3
had the lowest GDHt activity amongst the four mutants. This
isolate was selected for the subsequent fermentation work.
Effect of GDHt deficiency on PDR and GDH expre-
ssion: Activities of PDR and GDH from CGMCC 1.9131 and
B1.9131-3 were determined (Table-2). Compared with that of
wild strain, the specific activities of GDHt and PDR decreased
by 79.36 and 61.74 %, respectively, with K. pneumoniae
B1.9131, whereas the specific activity of GDH increased by
36.45 %.
RESULTS AND DISCUSSION
Construction and assay of GDHt deficient mutants:
The dha regulon enables microorganisms such as Klebsiella
pneumoniae or Citrobacter freundii to grow on glycerol. Four
enzymes are encoded by this regulon: glycerol dehydrogenase
(GDH), dihydroxyacetone kinase (DHK), GDHt and PDR.
GDHt and PDR are naturally under the control of two different
promoters and are transcribed in different directions.An 1000-
bp DNA fragment from the GDHt-encoding region of K.
pneumoniae was amplified by PCR. The resulting sequence
was cloned into the suicide vector to construct pGP-Bα
harboring chloromycetin and ampicillin resistance. pGP-Bα
was then introduced by conjugational transfer into K.
pneumoniae, giving rise to chloromycetin-resistant isolate.
Four GDHt deficient isolates, K. pneumoniae B1.9131-1,2,3
and 4, were obtained. The occurrence of an insertion event
was checked by PCR as mentioned in experimental section.
The 1160-bp DNA fragment was observed when genomic
DNA from B1.9131-1, 2, 3 and 4 was used as a template
(Fig. 1, lane 1, 2, 3 and 4) whereas no fragment was amplified
with genomic DNA from the wild strain B1.9131 (lane CK).
The occurrence of the insertion in dhaB-α was further
confirmed by the GDHt enzymatic activity assay. As shown
in Table-1, GDHt activities of 4 isolates samples decreased by
TABLE-2
EFFECT OF KNOCKING OUT THE GLYCEROL
DEHYDRATASE (GDHt) GENE ON THE EXPRESSION
OF 1,3-PROPANEDIOL OXIDOREDUCTASE (PDR)
AND GLYCEROL DEHYDROGENASE (GDH)
Activity (U mg-1)
Strains
GDHt
PDR
GDH
CGMCC 1.9131
B1.9131-3
0.93 0.04
0.19 0.01
1.1 0.03
0.43 0.01
1.4 0.06
1.9 0.02
Bacteria were cultivated in the basic culture media as described in
“Growth conditions” section at 37 °C and 150 rpm. The samples were
obtained in 9 h. Enzyme assays were determined according to the
method described by Xu et al. 13. The average data of three individual
experiments are presented.
1,3-Propanediol was not a substrate for the reactions
catalyzed by coenzyme B12-dependent dehydratases but mani-
fested a competitive inhibition towards the substrates dehy-
drated by GDHt. The activities of GDHt were determined in
the presence of increasing 1,3-propanediol concentrations.

Vol. 25, No. 13 (2013)
Coenzyme B12-Dependent Glycerol Dehydratase Deficiency in Klebsiella pneumoniae 7325
6
GDHt activity decreased by 44.84 and 88.64 % in the presence
of 1.0 and 2.0 mol L-1 1,3-propanediol (Fig. 2), respectively
which was similar to our previous studies with K. pneumoniae
CGMCC 1.636614.
30
20
10
0
8
4
4
2
0
3
0
6
100
80
60
40
20
0
30
20
10
0
2
1
0
4
2
0
0
2
4
6
8
10
12
Time (h)
Fig. 3. Accumulation of 3-HPA in the early phase of batch fermentation
by K. pneumoniae CGMCC 1.9131 (top entry) and B1.9131 (bottom
entry): glycerol (open circles), cell mass (open triangles), 3-HPA
(filled squares). Fermentation was carried out in a 5 l fermentor at
37 ºC, pH 6.8, 250 rpm and 0.5 vvm air flow. The initial 25 g glycerol
L-1 was used in cultures. The average data of three individual
experiments are presented
0
0.5
1
1.5
2
Amount of added 1,3-PDO (M)
Fig. 2. Effect of 1,3-propanediol (1,3-PD) on the activity of glycerol
dehydratases. The glycerol dehydratase (GDHt) activity was
measured in the presence of 0-2 M 1,3-propanediol with MBTH
method as described by Xu et al.13
80
60
40
20
0
30
20
10
3-HPA accumulation in fed-batch fermentation: 3-HPA
is identified as a toxic intermediary metabolite in the 1,3-
propanediol synthesis pathway14,15. High level of 3-HPA in
the culture may cause the cessation of 1,3-propanediol
production and cell growth. 3-HPAmax of 8.94 and 1.23 mM
was detected in the batch fermentations with CGMCC 1.9131
and B1.9131, respectively (Fig. 3). It was found that a high
activity ratio of GDHt over PDR contributed to 3-HPA accumu-
lation in the early period of K. pneumoniae fermentation in
our previous works10,13. The low level of 3-HPA agreed with
the enzymatic activities (Table-2). However, the carbon flux
to 1,3-propanediol pathway decreases with isolate B1.9131,
although the fermentation could be sustained at higher glycerol
concentration.
0
0
20
40
60
100
80
60
40
20
0
30
A
20
10
0
0
16
32
Time (h)
48
64
1,3-Propanediol production with CGMCC 1.9131 and
B1.9131: The batch fermentations were conducted by CGMCC
1.9131 and 4 isolates in the shaker flasks to investigate the
product spectrum (Table-3). As expected, 1,3-propanediol
synthesis decreased remarkably with 4 mutant isolates. It is
mentioned that the ethanol production was enhanced in all
mutants cultures. The fed-batch fermentations in the 5 l fermen-
tor were performed with CGMCC 1.9131 and B1.9131(Fig.
4). Although GDHt was deficient, 40 g 1,3-propanediol L-1
was obtained in cultures with B1.9131, confirming the expre-
ssion of DDHt during the fermentation as mentioned above in
Fig. 4. Time course of fed-batch fermentation by K. pneumoniae CGMCC
1.9131 (bottom entry) and B1.9131 (top entry): 1,3-propanediol
(filled circles), lactate (filled squares), ethanol (filled triangles),
succinate (open triangles), acetate (open circles), 2,3-butanediol
(open squares). Fed-batch fermentation was carried out in a 5 l
fermentor at 37 ºC, pH 6.8, 250 rpm and 0.5 vvm air flow. The
initial 25 g glycerol L-1 was used in betach cultures. The addition
of pure glycerol into the fed-batch cultures was started when the
glycerol concentration in the broth was below 5 g L-1. The glycerol
level in subsequent cultures was controlled at approximately 5-10
g L-1. The average data of three individual experiments are presented
TABLE-3
FERMENTATIONS BY K. pneumoniae CGMCC 1.9131 AND THE MUTANTS
Product (g L-1)
Strains
Succinate
0.06 0.01
0.29 0.01
0.17 0.004
0.19 0.01
0
Lactate
0
Acetate
1,3-Propanediol
13 0.3
2,3-Butanediol
2.2 0.08
3.9 0.17
1.9 0.01
3.0 0.13
1.5 0.07
Ethanol
CGMCC 1.9131
B1.9131-1
0.58 0.02
1.1 0.05
0.55 0.01
0.54 0.02
1.3 0.02
1.5 0.06
3.8 0.13
6.5 0.08
2.5 0.08
4.8 0.11
2.5 0.04
0
4.2 0.09
6.4 0.23
1.8 0.08
2.8 0.09
B1.9131-2
B1.9131-3
1.9 0.05
1.3 0.05
B1.9131-4
Products assay ware carried out as described in “Materials and methods” section. The average data of three individual experiments are presented.

7326 Zheng et al.
Asian J. Chem.
Table-1. The fermentations with wild and mutant isolates
shared the similar cell growth and glycerol uptake pattern (Figs.
3 and 4), indicating that knocking out of GDHt resulted in the
redistribution of the carbon flux. K. pneumoniae has the
sophisticated mechanism to maintain the substrate uptake and
cell growth. The product spectra may change under the diffe-
rent genetic or chemical conditions. 75.61 g lactate L-1 was
obtained in fed-batch with B1.9131, whereas only 40.99 g L-1
lactate was produced with CGMCC 1.9131. Ethanol formation
was also greatly enhanced in the first 24 h. Succinate production
remained almost at the same level, while 1,3-propanediol and
2,3-butanediol production decreased by about 56 and 75 %,
respectively.
Conclusion
This work attempts to understand GDHt's role in glycerol
consumption and 1,3-propanediol synthesis with a GDHt-
deficient mutant. GDHt-deficiency influence the activities of
other two dha regulon coding enzyme, 1,3-propanediol oxido-
reductase and glycerol dehydrogenase. 1,3-Propanediol
oxidoreductase activity deceased by 62 % in cultures with
mutant isolate, while the glycerol dehydrogenase activity
increased by 36 %. This is accompanied by that more carbon
is redistributed to lactate and ethanol formation. Cell growth
and glycerol uptake were not influnced during the culture with
GDHt-deficient isolate, suggesting K. pneumoniae metabolic
pathway is flexible to adapt genetic perturbation. However,
1,3-propanediol and 2,3-butanediol production decreased by
56 and 75 %, respectively. Therefore, GDHt is not indispens-
able in glycerol metabolism but is crucial for 1,3-propanediol
efficient production. Blocking of lactate and ethanol formation
may be a plausible approach to promoting 1,3-propanediol
production.
The gdh and gdhr genes encode GDHt and DDHt in K.
pneumoniae, respectively. K. pneumoniae ATCC 25955 can
produce both GDHt and DDHt, which are distinguishable in
vitro, whereas K. pneumoniaeATCC 8724 forms only DDHt5.
Although the two types of dehydratases have high sequence
homology, GDHt is a soluble cytosolic enzyme, whereas DDHt
is a low-solubility enzyme associated with carboxysome-like
polyhedral organelles6. To broaden knowledge on the diversity
of glycerol dehydratases, comprehensive sequence and mole-
cular modelling analyses of these enzymes were performed16.
The sequence analysis showed that GDHt and DDHt are not
related, suggesting that they evolved from different ancestors.
A gene fusion event occurred between α and β subunits of
GDHt in several bacteria during enzyme evolution. This is
confirmed by characterizing of GDHt expressed relying on
fusion of α- and β-subunits17. The fusion protein GDHALB/C
had the greatest catalytic activity.
ACKNOWLEDGEMENTS
This study was supported by National Science and
Technology Support Program (2012BAA09B01), National
Natural Science Foundation of China (50976032), Natural
Science Foundation of Beijing (3101001) and the Fundamental
Research Funds for the Central Universities (12MS43).
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