Biosynthesis of Terpenoids
J . Org. Chem., Vol. 66, No. 11, 2001 3951
1
8
-deoxy-D-xylulose were recorded in 90% H
, uncorrected glass electrode reading).
2
O/10% D
2
O (pH
was applied to a column of hydroxyapatite (2.5 × 6.0 cm) that
had been equilibrated with buffer A. The column was washed
with 30 mL of buffer A, followed by 60 mL of 20 mM potassium
phosphate, pH 6.8, and was then developed with a gradient
of 0-1 M potassium phosphate, pH 6.8.
Assa y of 1-Deoxy-D-xylu lose 5-P h osp h a te Syn th a se.
Assay mixtures contained 200 mM Tris hydrochloride, pH 8.0,
1 mM manganese sulfate, 5 mM sodium pyruvate (1), 10 mM
D,L-glyceraldehyde 3-phosphate (2), 1 mM thiamine pyrophos-
phate, 1 mM dithiothreitol, 0.3 mM NADPH, 42 µg (1 U) of
recombinant 1-deoxy-D-xylulose 5-phosphate reductoisomerase
from E. coli, and protein in a total volume of 1 mL. The mixture
was incubated at 37 °C, and the absorbance at 340 nm was
monitored for 10 min.
1
3
13
13
Ma ter ia ls. [U- C
6
]glucose, [2- C
1
]pyruvate, and [2,3- C
2
]-
1
4
pyruvate were obtained from Omicron, South Bend, IN. [2- C]-
pyruvate was obtained from Amersham, Braunschweig, Ger-
many. Sodium pyruvate, dihydroxyacetone phosphate (dilithium
salt), D,L-glyceraldehyde 3-phosphoric acid, thiamine pyro-
phosphate, ATP (disodium salt), phosphoenol pyruvate (potas-
sium salt, PEP), NADPH, 2,5-diethoxy-1,4-dioxane-2,5-di-
(
methyl phosphate) dibarium salt (dimeric dihydroxyacetone
phosphate acetal), and glycolytic enzymes (hexokinase, glucose
-phosphate isomerase, phosphofructokinase, aldolase, triose
6
phosphate isomerase), and pyruvate kinase were purchased
from Sigma Chemicals, Deisenhofen, Germany. Oligonucleo-
tides were custom-synthesized by MWG Biotech, Ebersberg,
Germany. T4 ligase was obtained from Gibco-BRL, Eggenstein,
Germany. Sepharose QFF and restriction enzymes were
purchased from Amersham Pharmacia Biotech, Freiburg,
Germany. DNase I was purchased from Roche Diagnostics,
Mannheim, Germany. Taq polymerase and isopropyl-â-D-
thiogalactopyranoside were supplied by Eurogentec, Seraing,
Belgium. RNase A and silica gel N-HR plates were from
Macherey-Nagel, D u¨ ren, Germany. ECTEOLA 23 cellulose was
obtained from Fluka, Deisenhofen, Germany. Macroprep hy-
droxyapatite columns, 40 µm, were supplied from Biorad,
Munich, Germany. The preparation of recombinant 1-deoxy-
D-xylulose 5-phosphate reductoisomerase will be published
elsewhere.
Micr oor ga n ism . The bacterial strain BR151,15 the high
copy expression vector pNCO113 (PTA-852, American type
culture collection, unrestricted patent deposit; see also ref 16)
and the expression vector construct for the dxs gene of B.
subtilis, PNCODXSBACSU were used in this study.
Con st r u ct ion of a n E xp r ession P la sm id for t h e d xs
gen e of B. su btilis. The dxs gene of B. subtilis (accession
number dbj D84432, base pair 193991-195892) was amplified
by PCR using chromosomal B. subtilis DNA as template and
the oligonucleotides 5′-tgatccgccatggatcttttatcaatacagg-3′ and
Un la beled 1-Deoxy-D-xylu lose 5-P h osp h a te (3) (P r o-
ced u r e 1). A solution of D,L-glyceraldehyde 3-phosphoric acid
(
1
2) (51 mg, 300 µmol) in 1 mL of water was neutralized with
M sodium hydroxide and added to a solution containing 150
mM Tris hydrochloride, pH 8.0, 10 mM magnesium chloride,
.50 mg (3.2 µmol) of dithiothreitol, 2.7 mg (6.4 µmol) of
0
thiamine pyrophosphate, 16.5 mg (150 µmol) of sodium pyru-
vate (1), and 2 U (0.6 mg) of recombinant 1-deoxy-D-xylulose
5
-phosphate synthase from B. subtilis in a total volume of 3.2
mL. The mixture was incubated at 37 °C for 6 h.
P u r ifica tion of 1-Deoxy-D-xylu lose 5-P h osp h a te (3).
Typically, 1 g of lyophilized reaction mixture was dissolved in
1
5 mL of water and applied to a column of ECTEOLA 23
cellulose (55 × 4 cm) at 4 °C. The column was developed with
a linear gradient of 0-0.3 M triethylammonium acetate (pH
-1
6
2
.0; total volume, 4.1 L; flow rate, 2 mL min ). Fractions of
5 mL were collected and analyzed by thin-layer chromatog-
raphy using silica gel N-HR plates, which were developed in
n-propanol/ethyl acetate/water (6:1:3, v/v). The plates were
dried, sprayed with anisaldehyde-sulfuric acid reagent,17 and
heated to 100 °C. The product was identified as blue-green
spots with a Rf
value of about 0.5. Fractions were combined
and lyophilized repeatedly to remove triethylammonium ac-
etate. Yield, 27.5 mg (67.5 µmol, 46%) as triethylammonium
salt.
5
′-ttgaatagaggatccccgcc-3′ as primers. The primer sequence
was conducive to the exchange of the TTG start codon by ATG.
The amplificate was purified, treated with the restriction
endonucleases NcoI and BamHI, and ligated into the plasmid
vector pNCO113, which had been treated with the same
enzymes. The ligation mixture was electroporated into the
recombinant E. coli XL1-Blue cells affording the recombinant
strain XL1-pNCODXSBACSU.
Gr ow th of Recom bin a n t E. coli Cells. The recombinant
E. coli strain XL1-pNCODXSBACSU was grown in 500 mL of
Luria-Bertani (LB) medium containing ampicillin (150 mg L
and kanamycin (50 mg L ) in 2-L shake flasks. The flasks
were inoculated with an overnight culture at a ratio of 1:50
and were incubated with shaking at 37 °C. At an optical
density of 0.6 (600 nm), isopropyl-â-D-thiogalactopyranoside
was added to a final concentration of 2 mM, and incubation
was continued for 5 h. Cells were harvested by centrifugation
and stored at -20 °C.
13
13
[
1,2- C
2 2
]1-Deoxy-D-xylu lose 5-P h osp h a te ([1,2- C ]-3)
(
P r oced u r e 2). A solution containing 2.0 g (3.0 mmol) of
dimeric dihydroxyacetone phosphate acetal (11) in 50 mL of
water was added to a suspension of Dowex 50 WX8 (30 mL,
+
H
form). The mixture was incubated at 65 °C for 4 h. The
solid was filtered off and washed with water. The combined
solution was lyophilized. Dihydroxyacetone phosphate glass
(1.1 g, containing about 0.42 g (1.5 mmol) of 10) was dissolved
in 12 mL of water, and the pH was adjusted to 6.0 by the
addition of 8 M sodium hydroxide. A solution (6.6 mL)
containing 450 mM Tris hydrochloride, pH 8.0, 30 mM
magnesium chloride, 31 mg (73 µmol) of thiamine pyrophos-
phate, 0.50 mg (3.3 µmol) of dithiothreitol, and 0.17 g (1.5
-1
)
-
1
13
2
mmol) of [2,3- C ]pyruvate (sodium salt) (1) was added. The
pH was adjusted to 8.0, and 400 U (0.07 mg) of triose
phosphate isomerase and 9.6 U (3.0 mg) of 1-deoxy-D-xylulose
5
-phosphate synthase were added. The mixture was incubated
P u r ifica tion of 1-Deoxy-D-xylu lose 5-P h osp h a te Syn -
th a se. Frozen cell mass of recombinant E. coli strain XL1-
pNCODXSBACSU (3.7 g) was thawed in 40 mL of 50 mM Tris
hydrochloride, pH 7.8, containing 2 mM dithiothreitol and 4
mg of lysozyme. The suspension was incubated at 37 °C for
at 37 °C for 11 h. The product was purified as described above.
Yield, 0.25 g (0.60 mmol, 40%) as triethylammonium salt.
13
13
[
3,4,5- C
) (P r oced u r e 3). A solution containing 150 mM Tris hydro-
chloride, 10 mM magnesium chloride, 1.0 g (5.4 mmol) of
[
(
(
(
3 3
]1-Deoxy-D-xylu lose 5-P h osp h a te ([3,4,5- C ]-
3
3
0 min. Subsequently, it was cooled on ice, subjected to
13
6
U- C ]glucose (6), 0.23 g (1.5 mmol) of dithiothreitol, 0.3 g
ultrasonic treatment, and centrifuged. The supernatant was
applied to a Sepharose QFF column (4 × 7 cm) that had been
equilibrated with 20 mM Tris hydrochloride, pH 7.8, contain-
ing 2 mM dithiothreitol (buffer A). The column was washed
with 60 mL of buffer A and developed with a gradient of 0-1
M potassium chloride in 800 mL of buffer A. Fractions were
combined and dialyzed overnight against buffer A. The solution
0.7 mmol) of thiamine pyrophosphate, 0.1 g (0.2 mmol) of ATP
disodium salt), and 2.2 g (11 mmol) of phosphoenol pyruvate
12) (potassium salt) in a total volume of 300 mL was adjusted
to pH 8 by the addition of 8 M sodium hydroxide. A solution
containing 1,500 U (10 mg) of hexokinase, 300 U (0.4 mg) of
glucose 6-phosphate isomerase, 105 U (1 mg) of fructose
6
-phosphate kinase, 610 U (0.1 mg) of triose phosphate
isomerase, 57 U (0.01 mg) of aldolase, 403 U (2.8 mg) of
pyruvate kinase, and 8 U (2.5 mg) of 1-deoxy-D-xylulose
5-phosphate synthase in 4 mL of Tris hydrochloride, pH 8.0,
(
15) Williams, D. M.; Duvall E. J .; Lovett, P. S. J . Bacteriol. 1981,
46, 1162-1165.
16) Bacher, A.; Zenk, M.; Eisenreich, W.; Fellermeier, M.; Fischer,
1
(
M.; Hecht, S.; Herz, S.; Kis, K.; L u¨ ttgen, H.; Rohdich, F.; Sagner, S.;
Schuhr, C. A.; Wungsintaweekul, J . PCT Int. Appl. 2001, 194.
(17) Stahl, E.; Kaltenbach, U. J . Chromatogr. 1961, 5, 351.
Hecht
