Department of Biochemistry and Cell Biology,
Rice University, Houston, Texas 77005-1892
A gene encoding a putative 150-amino-acid methylglyoxal synthase
was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified.
Methylglyoxal synthase has a native molecular mass of 60 kDa and an
optimum pH of 7.5. The Km and
Vmax values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min
1
µg1, respectively. When E. coli glycerol
dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.
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TEXT |
Methylglyoxal synthase, which
catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to
methylglyoxal and inorganic phosphate, has been found in many
organisms, including enteric bacteria (6, 8, 11), some
gram-positive bacteria (7), a number of archaebacteria
(18), several yeast species (1), and goat liver
(19). This enzyme provides bacteria with an alternative to
triosphosphate isomerase for metabolizing DHAP. Phosphate acts as an
allosteric inhibitor of the enzyme, which suggests that the
methylglyoxal bypass may have significant activity under phosphate starvation conditions; methylglyoxal is also known to be cytotoxic, and
it has been suggested that methylglyoxal is a growth regulator. In
bacteria, this compound may function as an antibiotic; in mammals, it
has been implicated in diabetic complications (3). We are interested in the use of metabolic engineering for developing processes
for microbial conversion of sugars to diols, such as 1,2-propanediol,
in Clostridium acetobutylicum ATCC 824, which is a
gram-positive, spore-forming, saccharolytic bacterium that is capable
of fermenting a wide variety of sugars. Figure
1 shows the metabolic pathway to
1,2-propanediol from DHAP, an intermediate in sugar metabolism. A
number of bacteria, such as Clostridium pasteurianum
(17), Klebsiella spp. (4, 21), and
Lactobacillus species (24), ferment glycerol and
produce many fermentation products, including 1,2-propanediol and
1,3-propanediol. In this study, we overexpressed, purified, and
characterized methylglyoxal synthase from C. acetobutylicum
ATCC 824 and produced 1,2-propanediol in recombinant Escherichia
coli by coexpression of E. coli glycerol dehydrogenase.
Amino acid sequence analysis.
Newly available sequence data
from the C. acetobutylicum genome sequence database
(9a) made possible identification of a gene coding for a
putative methylglyoxal synthase. The amino acids of the predicted gene
products deduced from the DNA sequence were similar to the amino acids
of E. coli methylglyoxal synthase, which was recently cloned
and expressed (22, 27), and other putative methylglyoxal
synthases (Fig. 2). A comparison of the sequences of methylglyoxal synthase and related open reading frames from other bacterial species revealed that four aspartic acids (Asp-17,
Asp-68, Asp-88, and Asp-98), which were suggested to be involved in
protecting the enzyme from the substrate DHAP or reactive intermediates
in the catalytic pathway (22), are highly conserved.
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FIG. 2.
Alignment of the amino acid sequences of C. acetobutylicum ATCC 824, E. coli, Bacillus
subtilis, Haemophilus influenzae,
Synechocystis, and Bacillus abortus methylglyoxal
synthases. Positions at which more than 50% of the sequences have
identical amino acids are shaded.
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Cloning and expression of the methylglyoxal synthase gene.
The
methylglyoxal synthase gene was cloned by PCR amplification
(15) by using a set of appropriate primers. C. acetobutylicum chromosomal DNA was the template used, the
oligonucleotide 5'-GAATTCATATGGCACTTATAATGAATAGT was the forward primer, and the oligonucleotide
5'-CCGCTCGAGTTAAAAATTCTGTTTTCTAAT was the reverse
primer. An NdeI site 5' to the methylglyoxal synthase gene and an XhoI site 3' to the methylglyoxal synthase gene
were introduced. The resulting 450-bp DNA fragment was ligated to the corresponding cloning sites of plasmids pET30a and pET15b after digestion with NdeI and XhoI to form pMGS1 and
pMGS2, respectively. Sequence integrity was confirmed by DNA
sequencing, and no change due to PCR amplification was observed. In
plasmid pMGS1, methylglyoxal synthase gene transcription is under
control of an isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible T7 promoter, and the gene contains the affinity His-tag coding region of the plasmid (the molecular weight of the
His-tag fusion methylglyoxal synthase deduced from the methylglyoxal synthase sequence plus the His-tag DNA sequence is 21,969). In pMGS2,
methylglyoxal synthase gene transcription is under control of an
IPTG-inducible T7 promoter, and the gene has no His-tag (the molecular
weight deduced from the methylglyoxal synthase DNA sequence is 15,165).
Plasmids pMGS1 and pMGS2 were transformed into E. coli
BL21(DE3) (26) and were selected on Luria broth (23) supplemented with kanamycin (50 µg/ml). Addition of
0.4 mM IMPG to liquid cultures resulted in intense protein bands in crude cell extracts that migrated at molecular masses of 25 kDa for
BL21(DE3)/pMGS1 cultures and 15 kDa for BL21(DE3)/pMGS2 cultures (Fig.
3) during sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5%
acrylamide gels (12) which were stained with Coomassie blue
R-250. Thus, both construct pMGS1 and construct pMGS2 resulted in
overexpression of the methylglyoxal synthase gene in E. coli.
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FIG. 3.
SDS-PAGE analysis of fractions from each purification
step and total protein. Samples were electrophoresed on a 12%
acrylamide gel. The resulting gel was subjected to protein staining.
(A) Lane M, molecular weight standards, lane 1, total protein from
induced E. coli BL21(DE3)/pMGS1 (12 µg after 4 h of
induction with 0.4 mM IPTG); lane 2, soluble protein from E. coli BL21(DE3)/pMGS1 (14 µg); lanes 3 and 4, purified
methylglyoxal synthase (1 and 2 µg, respectively). (B) Lane M,
molecular weight standards; lane 1, total protein from E. coli BL21(DE3)/pMGS1; lane 2, total protein from E. coli BL21(DE3)/pMGS2; lane 3, total protein from E. coli BL21(DE3)/pGLDH.
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Purification of methylglyoxal synthase.
Recombinant
methylglyoxal synthase was purified by using the procedure described in
the Talon metal affinity resin manual (Clontech, Palo Alto, Calif.).
The purification data were based on analysis of a 50-ml culture.
Protein concentrations were determined by the method of Bradford
(2) by using a Bio-Rad protein assay kit and bovine serum
albumin as the standard. E. coli BL21(DE3)/pMGS1 cultures
were harvested 4 h after induction with IPTG by centrifugation for
10 min at 4,000 × g. The pellet from a 50-ml culture
was resuspended in 4 ml of lysis buffer containing 100 mM Tris-HCl (pH,
8.0), 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 100 mM NaCl and incubated for 30 min at 4°C in the presence of 0.75 mg of
lysozyme per ml. The cells were lysed by sonication (four times,
15 s each) at full power by maintaining the temperature below
8°C with an ice bath. Unlysed cells and cell debris were removed by
centrifugation at 12,000 × g for 15 min. The
supernatant was mixed with Talon metal affinity resin, gently agitated
at 4°C for 30 min, and centrifuged at 700 × g for 5 min. The supernatant was discarded, and the resin was washed four times
with 15 ml of lysis buffer. The washed resin was then resuspended in
lysis buffer and transferred to a gravity column. The column was washed with 10 ml of washing buffer (0.1 M Tris-HCl, 10 mM imidazole, 10%
glycerol, 1 mM PMSF), and the methylglyoxal synthase was eluted with 3 ml of elution buffer (0.1 M Tris-HCl, 10% glycerol, 0.1 M imidazole, 1 mM PMSF). The fractions collected were judged to be homogeneous based
on SDS-PAGE results (Fig. 3). Methylglyoxal synthase activity was
assayed by using the procedure described previously (8). The
recombinant methylglyoxal synthase was readily purified to homogeneity
(>90%) (Table 1). The overall yield of
the purification procedure was 85%, which corresponded to 1.4 mg of
purified methylglyoxal synthase obtained from a 50-ml culture. Purified
methylglyoxal synthase was reasonably stable when it was stored in
elution buffer at 
20°C for at least 1 month. This property was
different from the storage properties of E. coli
methylglyoxal synthase, which lost activity quickly when the
Pi concentration decreased, requiring 1 mM Pi
in the buffer to stabilize E. coli methylglyoxal synthase
during purification (11).
Physical properties of methylglyoxal synthase activity.
The
mobilities of the recombinant methylglyoxal synthases from E. coli BL21(DE3)/pMGS1 and BL21(DE3)/pMGS2 during SDS-PAGE corresponded to Mr of 25,000 and 15,000, respectively (Fig. 3). The Mr of partially
purified methylglyoxal synthase (the fraction collected from a DEAE-52
column) from BL21(DE3)/pMGS2 was estimated to be 60,000 by gel
filtration high-performance liquid chromatography (HPLC) on a Bio-Sil
SEC 125 column (Bio-Rad) (data not shown). We suggest that the native
enzyme is a tetramer, like the E. coli methylglyoxal
synthase (22, 27), which is a tetramer of a total molecular
size of about 69 kDa. The reason why the His-tag (the molecular weight
of the His-tag and amino acids encoded as part of the multicloning site
sequences was only 5,000) affected the migration of methylglyoxal
synthase obtained from pMGS1 on SDS-PAGE gels so strongly is not known.
To compare the activity of our methylglyoxal synthase with the activity
of E. coli methylglyoxal synthase, we examined the activity
of the enzyme under various conditions. A standard methylglyoxal synthase activity test was used; each reaction mixture contained 0.14 µg of purified protein per ml, and the temperature used was 30°C.
The pH of the reaction buffer (Tris-HCl buffer) was adjusted with
either HCl or NaOH. When DHAP (0.375 mM) was the substrate, the pH
profile was essentially symmetrical, and optimal activity occurred at
pH 7.5. These conditions were used to examine the effects of possible
physiological inhibitors. Phosphate compounds have been found to
inhibit bacterial methylglyoxal synthase (6, 7). Phosphate,
pyrophosphate, phosphoenolpyruvate, and ADP were found to be modestly
inhibitory for C. acetobutylicum methylglyoxal synthase
(Table 2). This result was similar to the
results obtained with goat methylglyoxal synthase (19). ATP
resulted in 50% inhibition of the methylglyoxal synthase of C. acetobutylicum but had little effect on other methylglyoxal
synthases (7, 19).
Substrate specificity and kinetic parameters of methylglyoxal
synthase.
Methylglyoxal formation with purified methylglyoxal
synthase was examined by using DHAP, glyceraldehyde, and
glyceraldehyde-3-phosphate as substrates. None of these compounds
except DHAP could be converted to methylglyoxal, although
glyceraldehyde and glyceraldehyde-3-phosphate have been reported to be
possible precursors of methylglyoxal (20).
The Michaelis constant for methylglyoxal synthase was determined at pH
7.5 with imidazole-HCl buffer. The substrate saturation curve followed
typical Michaelis-Menten kinetics. The Km as
determined from a Lineweaver-Burk plot was 0.53 mM, and the
Vmax was 1.56 mmol min1
µg1 (Fig. 4). The
Km value is similar to the value recently
reported for the E. coli recombinant methylglyoxal synthase
(0.20 ± 0.03 mM) (22) and the value originally
reported for the methylglyoxal synthase isolated from E. coli (0.47 mM) (11).
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FIG. 4.
Determination of the apparent Michaelis constant for
DHAP. The assay mixtures (total volume, 100 µl) contained 50 mM
imidazole buffer (pH 7.5), different amounts of purified enzyme, and
different amounts of DHAP. After 5 min of incubation at 30°C, the
amount of methylglyoxal formed was measured colorimetrically
(8).
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Methylglyoxal synthase activity in C. acetobutylicum.
Methylglyoxal synthase activity was detected in cells of C. acetobutylicum that produced acetate, butyrate, and butanol as major products before they were harvested; considerable activity was
found in solvent-producing cells (36-h culture) (1.8 U/mg), and a lower
level of activity was detected in acid-producing cells (18-h culture)
(0.6 U/mg). The specific activity of methylglyoxal synthase from
C. acetobutylicum was higher than the specific activities of
E. coli methylglyoxal synthase (0.3 U/mg) (11)
and Clostridium sphenoides methylglyoxal synthase (0.1 U/mg)
(28).
Metabolic engineering of 1,2-propanediol production by coexpression
of E. coli glycerol dehydrogenase and methylglyoxal
synthase in E. coli BL21(DE3).
We improved solvent
production by C. acetobutylicum ATCC 824 by using genetic
engineering strategies (10, 16). These results motivated us
to investigate metabolic engineering approaches for production of other
useful solvents, such as 1,2-propanediol. Since E. coli
naturally produces some methylglyoxal and overexpression of C. acetobutylicum methylglyoxal synthase resulted in an increase in
the production of methylglyoxal, we thought that 1,2-propanediol might
be produced if the appropriate reductase was provided. E. coli contains a glycerol dehydrogenase, which is an NADH-linked reductase that is known to have broad substrate specificity and is
able to reduce hydroxyacetone (14). Thus, methylglyoxal
synthase and glycerol dehydrogenase were coexpressed to see if
1,2-propanediol could be produced.
The gene for E. coli glycerol dehydrogenase
(gldA) has been cloned and mapped (29). We
amplified this gene by PCR by using two primers (forward primer
5'-GCGGAATTCAGGAGGAATTTAAAATGCCGCATTTGGCACTACTCATCT CTAAAGG-3'
and reverse primer
5'-CGCGGATCCTTATTCCCACTCTTGCAGGAAAGCCTG-3'; E. coli wild-type genomic DNA was the template. A 1,143-bp PCR fragment was cut with EcoRI and BamHI and
inserted into the corresponding sites in pEXT (9), a vector
containing an R100 origin and a tac promoter, which resulted
in plasmid pGLDH. Plasmid pGLDH was transformed into E. coli
BL21(DE3) and was selected on Luria broth (23) supplemented
with kanamycin (50 mg/ml). Adding 0.4 mM IPTG to liquid cultures
resulted in an intense protein band in crude cell extracts that
migrated at a molecular mass of 39 kDa (29) on 12.5%
acrylamide SDS-PAGE gels (12) which were stained with Coomassie blue R-250 (Fig. 3). Thus, the construct pGLDH resulted in
overexpression of the glycerol dehydrogenase gene in E. coli. Glycerol dehydrogenase activity was assayed by using a
previously described procedure (29). The extracts obtained
from BL21(DE3)/pGLDH exhibited a 17-fold increase in activity compared
with the activity of the host with a vector lacking the insert
[BL21(DE3)/pEXT] (data not shown).
In order to select transformants with different antibiotics, two
plasmids, pGLDH and pMGS2, were cotransformed into E. coli BL21(DE3), and colonies were selected by using ampicillin and kanamycin. Fermentation by this recombinant E. coli strain
was carried out in a 5-ml anaerobic culture tube at 37°C on M9 medium supplemented with 5 g of yeast extract per liter, 5 g of
fructose per liter, 2 mM MgSO4, 0.1 M CaCl2,
100 µg of ampicillin per ml, and 50 µg of kanamycin per ml. After
24 h of growth, 0.4 mM IPTG was added. The amount of
1,2-propanediol in the medium was measured by HPLC 72 h after
induction. Some 1,2-propanediol was produced in the uninduced cultures
(due to the strength of T7 and the tac promoter, we detected
expression of some methylglyoxal synthase and glycerol dehydrogenase
even without induction [data not shown]). 1,2-Propanediol (3.9 mM,
0.3 g/liter) was produced by induced cultures containing pMGS2 and
pGLDH. A lower concentration, 2.7 mM 1,2-propanediol, was produced when
the host culture contained either pMGS2 or pGLDH. We did not detect
1,2-propanediol production in a control experiment in which we used a
host containing a plasmid(s) without the insert. The 1,2-propanediol
concentrations were low, but they were higher than the concentrations
observed in recombinant E. coli cultures that reportedly
fermented sugars to 1,2-propanediol (0.2 g/liter) (4) and
recombinant organisms that reportedly fermented sugars to
1,3-propanediol (less than 0.1 g/liter) (13). Although
methylglyoxal synthase and glycerol dehydrogenase were expressed very
well, the level of production of 1,2-propanediol was not as high as
desired. The possible limitations include the possibility that the
formation of methylglyoxal is metabolically regulated because this
compound is toxic and the possibility that the substrate specificity
and efficiency of the glycerol dehydrogenase may not be optimal. Other
reductases, such as human aldose reductases, which have been reported
to exhibit high substrate specificity for methylglyoxal (5, 25,
30), may be useful. We also plan to investigate the possibility
of 1,2-propanediol production by C. acetobutylicum ATCC 824 by using constructs that express methylglyoxal synthase and an
appropriate reductase.
This research was supported by grant MCB 9604562 from the National
Science Foundation and by grant 3604-051 from the Texas Advanced
Technology Program.
We thank T. Linn (University of Western Ontario, London, Ontario,
Canada) for providing plasmid pEXT and Yea-Tyng Yang (Department of
Bioengineering, Rice University) for performing the HPLC analysis.
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