704
Fig. 1. The nonmevalonate pathway for IPP biosynthesis, and the structure of a DXP reductoisomerase inhibitor, fosmidomycin
To elucidate the remaining reactions leading to 1 from 3, we initiated studies on the cloning of
the genes responsible for conversion of 3 into 1. To this end, we employed a unique strategy for
preparation of mutants of E. coli possessing a metabolic block(s) between 3 and 1. Since such mutations
would be lethal, we constructed an E. coli transformant possessing an additional biosynthetic pathway
for 1, the mevalonate pathway.‡ Using this transformant as the parent strain, we prepared mutants
with an obligatory requirement of mevalonate for growth and survival. Thirty-three mutants from ca.
60 000 colonies screened showed the expected phenotypes; addition of mevalonate to the minimal
medium M9 facilitated the growth of these mutants.5 Thus, these phenotypic features of the mutants
unequivocally demonstrated that these mutants have a defect(s) in the pathway leading to 1 from 3. With
these mutants in hand, we could clone several genes that complemented the defects of these blocked
mutants in synthesizing 1 from 3. To identify the functions of these genes, we constructed plasmids for
overexpression of these gene products and succeeded in preparation of purified enzymes in quantities
sufficient for their characterization.5 We found that one of these gene products converted 3 into an
unknown product in the presence of cytidine 50-triphosphate (CTP).§
Incubation of this enzyme (4 mg¶) with 3 (10 mM) in the presence of 10 mM CTP at 37°C for 4 h in 25
ml of 100 mM Tris–HCl (pH 8.0) containing 5 mM·MnCl2 resulted in appearance of a new peak in HPLC
with an Aminex HPX-87H column (7.8×300 mm, Bio-Rad).k The reaction product corresponding to this
peak was purified with the assistance and guidance of this HPLC. Thus, the reaction mixture was diluted
to 250 ml with H2O, subjected to Dowex 1-X8 (Cl− type, 2×6 cm) chromatography and eluted with
‡
This transformant with mevalonate kinase, phosphomevalonate kinase and pyrophosphomevalonate decarboxylase activities
can utilize the partial mevalonate pathway for IPP biosynthesis only in the presence of mevalonate. The genes encoding these
enzymes were cloned from Streptomyces sp. strain CL190.6 Thus mevalonate added into the growth medium is converted by
these three enzymes into IPP to be utilized as a precursor of terpenoids by the transformant.
§
This gene product can utilize ATP or UTP as well, but not GTP or TTP. ATP or UTP was less effective than CTP.
Protein expression and purification of the MEP cytidylyltransferase: The coding region of the E. coli MEP cytidylyltrans-
¶
ferase gene5 was cloned into the expression vector pQE30 (Qiagen) to give pQEMECT. E. coli M15 containing pREP4 [neo,
lacI] (Qiagen) was used as a host for expression of the MEP cytidylyltransferase gene. E. coli M15 (pREP4, pQEMECT) was
cultured at 37°C in 100 ml of Luria–Bertani medium containing 25 µg/ml kanamycin and 200 µg/ml ampicillin for 5 h with 0.1
mM isopropyl ß-D-thiogalactoside upon reaching an optical density at 660 nm of 0.8. Cells were harvested by centrifugation
and resuspended in 100 mM Tris–HCl (pH 8.0). After brief sonication, the lysate was centrifuged at 10 000×g for 20 min
and the supernatant was collected. A 50% slurry of Ni-nitrilotriacetic acid (NTA) agarose resin (Qiagen) was added into the
supernatant and stirred on ice for 60 min. The resin was washed with 50 mM imidazole in 100 mM Tris–HCl (pH 8.0) and then
the protein which bound to the Ni-NTA agarose resin was eluted with 200 mM imidazole in 100 mM Tris–HCl (pH 8.0). The
keluate was used as the purified MEP cytidylyltransferase in the subsequent experiments.
Formation of the reaction product was monitored at 270 nm with an Aminex HPX-87H column (Bio-Rad), eluted with 5
mM H2SO4 at the flow rate of 0.6 ml/min at room temperature.