Reconstruction of a bacterial isoprenoid biosynthetic pathway in
Je ´ro ˆme Maurya,*, Mohammad A. Asadollahib, Kasper Møllerd, Michel Schalke, Anthony Clarke,
Luca R. Formentib, Jens Nielsenc
aCenter for Microbial Biotechnology, DTU-Biosys, Building 223, 2800 Kgs Lyngby, Denmark
bCenter for Microbial Biotechnology, DTU-Biosys, Lyngby, Denmark
cDepartment of Chemical and Biological Engineering, Chalmers University of Technology, Sweden
dCMC Biopharmaceuticals AS, Søborg, Denmark
eFirmenich SA, Geneva, Switzerland
Received 9 June 2008; revised 9 October 2008; accepted 28 October 2008
Available online 6 November 2008
Edited by Gianni Cesareni
expressed in Escherichia coli, and a mammalian hydrocortisone
biosynthetic pathway rebuilt in Saccharomyces cerevisiae are
examples showing that transferring metabolic pathways from
one organism to another can have a powerful impact on cell
properties. In this study, we reconstructed the E. coli isoprenoid
biosynthetic pathway in S. cerevisiae. Genes encoding the seven
enzymatic steps of the pathway were cloned and expressed in S.
cerevisiae. mRNA from the seven genes was detected, and the
pathway was shown able to sustain growth of yeast in conditions
of inhibition of its constitutive isoprenoid biosynthetic pathway.
? ? 2008 Federation of European Biochemical Societies. Pub-
lished by Elsevier B.V. All rights reserved.
A eukaryotic mevalonate pathway transferred and
Keywords: Isoprenoid; Yeast; 2-Methyl erythritol
4-phosphate; Mevalonate; Saccharomyces cerevisiae
Bridging between synthetic biology and metabolic engineer-
ing, researchers have been attempting to create organisms with
new properties of interest by transferring metabolic pathways
from one organism to another. Pioneering studies have demon-
strated the usefulness of reconstituting and expressing entire
heterologous pathways in non-native hosts for production pur-
poses. In a single yeast strain, Szczebara et al. brought to func-
tion a complex, artificial and self-sufficient pathway for the
biosynthesis of hydrocortisone, which is the major steroid in
mammals . After engineering 13 different enzymatic steps,
they successfully rerouted the native yeast sterol-biosynthetic
pathway, so that hydrocortisone became the major steroid
produced . This first report of the total biosynthesis of
hydrocortisone by a recombinant microorganism from a sim-
ple carbon source constitutes a solid basis for the development
of an environmentally friendly and low-cost industrial process
for the production of corticoid drugs . In another study, a
eukaryotic mevalonate pathway was transferred and engi-
neered in Escherichia coli with the aim to produce artemisinin,
an antimalarial drug usually isolated from plant material .
Associated with the expression of a synthetic amorpha-4,11-
diene synthase, it led to significant improvements in amor-
pha-4,11-diene production, an intermediate in the biosynthesis
of artemisinin . Biosynthesis of paclitaxel, a mitotic inhibitor
used in cancer chemotherapy, has been extensively studied and
partly transferred from Taxus species to E. coli or Saccharomy-
ces cerevisiae [3–4] in order to establish a microbial production
process. Ten enzymatic steps from the paclitaxel biosynthesis
of Taxus species were successfully and independently expressed
in yeast . Five sequential steps catalyzing the synthesis of the
intermediate taxadien-5a-acetoxy-10b-ol were installed in a
single yeast strain . However, the complete pathway from
the precursor geranylgeranyl diphosphate to paclitaxel in-
cludes nineteen steps in Taxus species . These examples
showed that transferring entire metabolic pathways into a
new host can result in tremendous improvements in the pro-
duction of desired compounds. Transferring a pathway to an-
other biological host, and making it functional in a non-native
environment can also provide substantial knowledge about the
mechanisms and regulations of the aforesaid heterologous
In this study, we investigated the biosynthesis of the isopren-
oid family of compounds in the yeast, S. cerevisiae. This is the
largest family of natural compounds with more than 40000 de-
scribed compounds [5–6]. Isoprenoids are involved in various
important cellular biological functions, and many isoprenoids
are of commercial interest due to their potent anticancer, anti-
tumor, cytotoxic, antiviral, or antibiotic properties or their
characteristic flavour or aroma. Here, the cloning and expres-
sion of seven enzymatic steps from a bacterial isoprenoid bio-
synthetic pathway into S. cerevisiae are reported. The seven
enzymatic steps of this pathway, the 2-C-methyl-D D-erythritol
4-phosphate (MEP) pathway have recently been unraveled
 (Fig. 1). Genes were amplified from E. coli, cloned on
self-replicating yeast plasmids, and were expressed in S. cerevi-
siae. Their expression and the ability of the bacterial MEP
pathway to sustain growth of S. cerevisiae, which otherwise de-
pends on another native biosynthetic pathway (the mevalonate
pathway) for the generation of isoprenoids, were investigated.
Abbreviations: DMAPP, dimethyl allyl diphosphate; G3P, glyceralde-
hyde 3-phosphate; GC–MS, gas chromatography–mass spectrometry;
IPP, isopentenyl diphosphate; MEP, 2-C-methyl-D D-erythritol 4-phos-
phate; MVA, mevalonate; RT-PCR, reverse transcriptase polymerase
E-mail address: firstname.lastname@example.org (J. Maury).
0014-5793/$34.00 ? 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS Letters 582 (2008) 4032–4038
2. Materials and methods
2.1. Strains and plasmids
Table 1 lists all strains and plasmids used in this study.
2.2. Construction of plasmids pIP001 and pIP002
2.2.1. Cloning of the MEP pathway encoding genes. E. coli genes
encoding Dxs (AAC73523), Dxr (AAC73284), IspD (AAC75789),
IspE (AAC74292), IspF (AAC75788), GcpE (AAC75568), and LytB
Fig. 1. Isoprenoid biosynthetic pathways. Mevalonate pathway from Saccharomyces cerevisiae (blue) and the Escherichia coli MEP pathway (dark
red) are presented. Intermediates: 1 acetyl-CoA, 2 acetoacetyl-CoA, 3 3-hydroxy-3-methylglutaryl-CoA, 4 mevalonate, 5 isopentenyl diphosphate, 6
dimethyl allyl diphosphate, 7 geranyl diphosphate, 8 farnesyl diphosphate, 9 D D-glyceraldehyde 3-phosphate, 10 pyruvate, 11 1-deoxy-D D-xylulose 5-
phosphate, 12 2-C-methyl-D D-erythritol 4-phosphate, 13 4-diphosphocytidyl-2-C-methyl-D D-erythritol, 14 2-phospho-4-diphosphocytidyl-2-C-methyl-
D D-erythritol, 15 2-C-methyl-D D-erythritol 2,4-cyclodiphosphate, 16 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate.
Strains and plasmids.
StrainGenotypePlasmid Plasmid description
MATa MAL2-8cSUC2 ura3-52 trp1-289
MATa MAL2-8cSUC2 ura3-52
MATa MAL2-8cSUC2 ura3-52
MATa MAL2-8cSUC2 ura3-52
pYX212 2l URA3 PTPI1-GFTpSD
pESC-URA 2l URA3 PGAL1- GFTpSD
F0u80lacZDM15 D(lacZYA-argF)U169 deoR
recA1 endA1 hsdR17(rK-mK+) phoA supE44 k- thi-1 gyrA96 relA1
Plasmid nameKey features
2l TRP1 PGAL1/TCYC1PGAL10/TADH1AmpR(bla)
2l URA3 PGAL1/TCYC1PGAL10/TADH1AmpR(bla)
J. Maury et al. / FEBS Letters 582 (2008) 4032–4038
(AAC73140) were amplified from E. coli genomic DNA by PCR (Sup-
plementary Table 1). Valencene synthase encoding gene (GFTpsD,
CQ813508) was amplified from plasmid pET11a or pET101 containing
this gene (Supplementary Table 1). Expand High Fidelity (Roche Ap-
plied Science) was used for the PCR. PCR fragments of expected size
were gel purified using High Pure PCR Product Purification Kit
(Roche Applied Science).
In the case of cloning via the classical digestion–ligation-transforma-
tion approach, purified PCR products and the cloning vector (pESC-
TRP or pESC-URA) were digested with suitable restriction enzymes
(Supplementary Table 1), and were purified with QIAEX?II Gel
extraction kit (Qiagen). In vitro ligation was performed as the standard
procedure given for T4 DNA ligase (Roche Applied Science) with an
insert/vector ratio above 3:1. After transformation , E. coli DH5a
transformants were selected on LB medium supplemented with ampi-
cillin. Constructed plasmids were purified and verified by restriction
profiling and sequencing.
In the case of construction via in vivo homologous recombination in
S. cerevisiae, both PCR products and digested plasmid were
transformed in S. cerevisiae YIP-00-02 using standard transformation
procedure . Transformants were selected on SC-trp medium or
SC-ura medium. Plasmids were directly purified from yeast . After
transformation into E. coli DH5a, transformants were selected on LB
medium supplemented with ampicillin and the plasmids were verified
as mentioned earlier. A series of four plasmids were obtained from
these initial clonings: pIP008, pIP015, pIP017, and pIP019 (Table 2).
2.2.2. Construction of the main plasmids to trigger MEP pathway
gene expression. The four abovementioned plasmids were combined
to end up with two main plasmids: pIP001 bearing dxs, dxr, ispD,
and ispE, and pIP002 bearing gcpE, lytB, ispF, and GFTpsD.
To construct plasmid pIP002, the DNA fragment containing ispF,
GFTpsD, GAL1, and GAL10 promoters, CYC1 and ADH1 termina-
tors from pIP015 was amplified by PCR using the couple of primers
named ‘‘fusion’’ (Supplementary Table 1). The resulting PCR products
were purified using High Pure PCR Product Purification Kit (Roche
Applied Science). pIP008 (Table 2) was digested by MfeI, and subse-
quently gel purified using QIAEX?II Gel Extraction Kit (Qiagen).
Purified and digested plasmid together with the PCR products was
transformed into S. cerevisiae YIP-00-03 (Table 1) . Transformants
were selected on plates containing SC-ura medium. Plasmids were iso-
lated and verified as mentioned earlier. The resulting plasmid, pIP002,
was obtained (Fig. 2, Table 2).
The same protocol was followed to construct pIP001, with an ampli-
fication by PCR of the DNA fragment containing ispD, ispE, GAL1,
and GAL10 promoters, CYC1 and ADH1 terminators from pIP017
(Table 2, Supplementary Table 1). In this case, the receiving vector
was pIP019 (Table 2) that was digested by XcmI, and S. cerevisiae
YIP-00-02 (Table 1) was used for transformation . Transformants
were selected on plates containing SC-trp medium. Plasmids were iso-
lated and verified as mentioned earlier. The resulting plasmid, pIP001,
was obtained (Fig. 2 and Table 2).
2.3. Transformation into S. cerevisiae
Purified pIP001 and pIP002 were transformed into S. cerevisiae YIP-
00-02 (Table 1) . Transformants were selected on plates containing
SC-trp-ura medium. The presence of the MEP pathway genes in the
resulting yeast strain was verified by PCR.
2.4. Verification of gene expression by reverse transcriptase polymerase
chain reaction (RT-PCR)
While cultivating strains YIP-DV-02 and YIP-0V-02 in shake flasks
(defined minimal medium , 20 g L?1galactose; 30 ?C, 150 rpm),
total RNA were extracted from samples taken in late galactose growth
phase using FastRNA?Pro Red kit (Qbiogene). RNA samples were
subsequently treated with Turbo DNase? (Ambion, Foster City,
CA). After verifying concentration and quality of the RNA samples,
RT-PCR was performed as follows: reverse transcription for synthesis
of the first single-stranded DNA was realized according to Expand
Reverse Transcriptase standard conditions (Roche Applied Science)
using specific primers for each gene (Supplementary Table 2). The
reaction was stopped by placing the tubes on ice. Then, a PCR reaction
was realized using Taq DNA polymerase in standard conditions.
Annealing temperatures were 55 ?C for dxs, ispD, ispE, and ispF,
and 57 ?C for dxr, gcpE, and lytB.
2.5. Cultivation of S. cerevisiae in the presence of lovastatin
Lovastatin was hydrolyzed in ethanolic sodium hydroxide (15% (v/v)
ethanol, 0.25% (w/v) NaOH) at 60 ?C for 1 h. After cooling down to
room temperature, the 20 mg/ml stock solution was filter sterilized
and stored at ?20 ?C. Test tubes containing 5 mL of defined minimal
medium , different amounts of lovastatin, and 20 g L?1galactose as
carbon source were inoculated from an overnight pre-culture at an
OD600of 0.01, and were incubated at 30 ?C and 150 rpm. As control
experiments, cultivations in minimal medium supplemented with either
the hydrolytic solution (15% (v/v) ethanol, 0.25% (w/v) NaOH) (i.e. the
highest volume of hydrolytic solution applied, 500 lL, devoid of any
lovastatin) or the non-hydrolyzed lovastatin were also prepared. Cell
growth was followed by measuring OD600.
Plasmids constructed during this study.
PlasmidMEP pathway genes Genetic marker
Fig. 2. Plasmids pIP001 and pIp002.
J. Maury et al. / FEBS Letters 582 (2008) 4032–4038
3. Results and discussion
3.1. Cloning of the E. coli MEP pathway genes into S. cerevisiae
The seven genes encoding the E. coli MEP pathway, i.e. dxs,
dxr, ispD, ispE, ispF, gcpE, and lytB were amplified by PCR
using specific primers (Supplementary Table 1). The specific
primers were designed for the amplification of each open read-
ing frame with 50and 30overhangs allowing for the transfer
into the expression plasmid either by restriction digestion–liga-
tion or by in vivo homologous recombination (Supplementary
Table 1, Section 2.2). pESC Yeast Epitope Tagging Vectors
(Stratagene?) were chosen as yeast expression vectors. This
choice was motivated by some of their interesting features:
potent expression of two genes per vector, tight control of gene
expression by GAL1 and GAL10 promoters, strong expression
levels achieved, and high plasmid copy number. A sesquiter-
pene synthase originating from plant, valencene synthase en-
coded by GFTpsD, was also cloned as a biological reporter.
From a first cloning phase, four vectors were obtained:
pIP019 (dxs and dxr), pIP017 (ispD and ispE), pIP015 (ispF
and GFTpsD), and pIP008 (gcpE and lytB) (Table 2).
3.2. Plasmid-based expression of the seven MEP pathway genes
In order to assemble the MEP pathway, it is of course nec-
essary to express all seven enzymatic steps to ensure the pro-
duction ofdimethyl allyl
isopentenyl diphosphate (IPP) from the central metabolism
intermediates glyceraldehyde 3-phosphate (G3P) and pyruvate
(Fig. 1). Each pESC vector can originally trigger the expres-
sion of two genes which would imply in total the use of four
different pESC vectors to sustain the expression of all the
MEP pathway encoding genes. In order to reduce the number
of different plasmids that have to be maintained in the final
yeast strain and to minimize the number of genetic markers
to be used, pESC-URA and pESC-TRP vectors were further
modified to trigger the expression of four genes each instead
of two initially. Constructions were realized using in vivo
homologous recombination in S. cerevisiae (Section 2.2). The
design of this fusion event was carefully elaborated. First an
insertion site placing the selective marker (URA3 or TPI1) in
between the two potential PGAL1/PGAL10expression cassettes
was sought: XcmI for pESC-TRP and MfeI for pESC-URA.
Positioning the genetic marker in between the two PGAL1/
PGAL10 expression cassettes allows the use of the plasmids
for potential future genomic integration of the MEP pathway
encoding genes using the bipartite gene targeting method .
The homologous regions for insertion of the second PGAL1/
PGAL10 expression cassette were designed, so that the two
PGAL1and the two PGAL10situated on both sides of the genetic
marker are inverted one to the other, thereby avoiding poten-
tial loss of part of the plasmid due to homologous recombina-
tion between directly repeated regions (Fig. 2).
After in vivo homologous recombination, two main multi-
copy plasmids, pIP001 and pIP002, were obtained. These
Fig. 3. Detection of mRNA from Escherichia coli MEP pathway in Saccharomyces cerevisiae. A: YIP-DV-02 (A) and YIP-0V-02 (B) were tested.
ACT1 mRNA is used as an internal control. The primer set used here for amplification of dxr is dxr1. GeneRuler? 50 bp DNA ladder from
Fermentas Life Science (L). B: RT-PCR on dxr with a second primer set called dxr2 (see Supplementary Table 2). Duplicate cultivations of YIP-DV-
02 (A1 and A2) and of YIP-0V-02 (B1 and B2) were tested. FlashGel?RNA marker (L2).
J. Maury et al. / FEBS Letters 582 (2008) 4032–4038
two plasmids trigger the expression of the seven MEP pathway
encoding genes from S. cerevisiae GAL1 and GAL10 promot-
ers (Fig. 2).
3.3. Expression of the plasmid-borne E. coli MEP pathway in
Purified pIP001 and pIP002 were transformed into S. cerevi-
siae strain YIP-00-02. Strain YIP-DV-02, characterized by the
presence of the MEP pathway genes and GFTpsD, as verified
by PCR (data not shown), was selected for further investiga-
In order to assess the functionality of the E. coli MEP path-
way in S. cerevisiae, a first qualitative experiment aiming at
detecting specific mRNA for the seven bacterial genes was de-
signed. RT-PCR realized on total RNA extracted from late
exponential growth phase with galactose as carbon and energy
source showed that all the seven E. coli genes are eventually
transcribed in S. cerevisiae (Fig. 3). Specific and intense signals
were observed, when RT-PCR was realized on a yeast strain
transformed with the MEP pathway for all the genes except
for dxr (Fig. 3A). A control PCR was performed in the same
conditions as for the RT-PCR but omitting the reverse tran-
scription step and this revealed no DNA contamination (data
not shown). The result obtained for dxr was surprising as an
amplification of this gene was often observed, both for the
MEP pathway expressing S. cerevisiae and for a ‘‘wild-type’’
S. cerevisiae, even after varying the parameters of the reaction
or using a different set of primers (Fig. 3B). No significant
match in the S. cerevisiae DNA sequence was found either
for the sequences of the primers used for RT-PCR or for the
entire dxr sequence (data not shown). PCR products obtained
in both cases, YIP-DV-02 and YIP-0V-02, were eventually
purified and sequenced. The fragment amplified from RNA
extracted from the ‘‘wild-type’’ S. cerevisiae was shown to
originate from S. cerevisiae gene CKA1 (GenBank accession
number: M22473). CKA1 encodes the alpha subunit of yeast
casein kinase II and has no apparent link with isoprenoid
biosynthesis . In the case of RNA extracted from strain
YIP-DV-02, the amplified fragment was confirmed to originate
from E. coli dxr mRNA (data not shown). Having shown that
all the MEP pathway encoding genes are efficiently transcribed
in S. cerevisiae, the functionality of the whole pathway itself in
S. cerevisiae still remained to be addressed.
3.4. E. coli MEP pathway can sustain growth of S. cerevisiae in
the presence of lovastatin
YIP-DV-02 was shown to produce valencene by gas chroma-
tography–mass spectrometry (GC–MS) analysis in batch culti-
vation using galactose as carbon source, both in shake flasks
 and in 5 l bioreactor (data not shown). This strain was able
to produce up to 1 mg L?1of valencene (data not shown).
However this in itself did not prove that the bacterial MEP
pathway was functional in this strain. To demonstrate this,
growth of YIP-DV-02 in the presence of a potent and well-
known mevalonate pathway inhibitor – mevinolin or lova-
statin – was characterized (Fig. 4). This experiment was meant
to assess the functionality of the MEP pathway in S. cerevisiae
and more specifically its capacity to sustain growth of yeast
cells under conditions where the native mevalonate pathway
is inhibited. Lovastatin specifically acts at the level of HMG-
CoA reductase, which is a major flux controlling step of the
mevalonate pathway, and leads to a reduced flow of interme-
diates through the sterol-biosynthetic pathway and to reduced
amounts of both steryl esters and free sterols . As a conse-
quence, concentrations of lovastatin above 150 lg mL?1lead
to reduced cell growth rate and cell yield . Inhibition of
the mevalonate pathway by lovastatin is, therefore, expected
to lead to growth impairment in a wild-type yeast background.
In the presence of lovastatin, only a yeast strain expressing a
functional heterologous MEP pathway, ensuring the supply
of essential intermediates of the isoprenoid biosynthetic path-
way, would be able to continue to grow. While YIP-0V-01
stopped growing at an OD600of 9.6 ± 0.6 in the presence of
the highest concentration of lovastatin, YIP-DV-02 carried
on growing till an optical density of approximately 18 after
73 h of cultivation (Fig. 4). Growth of YIP-0V-01 was neither
Fig 4. Growth of Saccharomyces cerevisiae strains YIP-DV-02 and YIP-0V-01 in the presence of lovastatin. The effect of the presence of inhibitory
concentrations of lovastatin on strain YIP-DV-02 (expressing the MEP pathway and valencene synthase) was determined (B) and compared to the
effect of lovastatin on strain YIP-0V-01 (only expressing valencene synthase) (A). Six different cultivation conditions are presented: minimal medium
(M.M.) (d), M.M. + 500 lL of hydrolytic solution (15% ethanol (v/v), 0.25% NaOH (w/v)) devoid of lovastatin (s), M.M. + 1g L?1non-hydrolyzed
lovastatin (.), M.M + 2g L?1non-hydrolyzed lovastatin (.), M.M. + 1g L?1hydrolyzed lovastatin (j), M.M + 2g L?1hydrolyzed lovastatin (h).
The cultivation volume was 5 mL. The figure does not include the first 24 h of cultivation.
J. Maury et al. / FEBS Letters 582 (2008) 4032–4038
affected by the presence of the hydrolysis solution nor affected
by the presence of non-hydrolyzed lovastatin as such (Fig. 4).
This experiment confirms that a yeast strain expressing the
E. coli MEP pathway is able to thwart the specific effect of
an inhibitory concentration of lovastatin and sustain growth.
Cultivation in the presence of a combination of lovastatin
and of a specific inhibitor of the MEP pathway, e.g. fosmido-
mycin, may further validate our observations. Growth inhibi-
tion of strain YIP-DV-02 is expected in these conditions.
However, as observed in our study, very high concentrations
of lovastatin had to be used to affect yeast cell growth
(4.9 mM). When compared to previously reported HMG-
CoA reductase inhibition constants – Ki= 3.5 nM for a par-
tially purified yeast HMG-CoA reductase  or Ki= 2.5 nM
for the microsomal HMG-CoA reductase of etiolated radish
seedlings  – these very high concentrations required suggest
a very low penetration of this drug in yeast cells. Therefore, to
avoid using lovastatin type of drug, another experiment
involving a mutant of an essential step of the mevalonate path-
way, e.g. a null or a thermosensitive allele of ERG10, a double
mutant hmg1 hmg2, or a ERG13 deletion which have been de-
scribed earlier [18–20], and that would be transformed with the
plasmids triggering expression of the MEP pathway genes
could be realized. It would aim at showing that the plasmid-
borne MEP pathway can alleviate the mevalonate dependency
of these mutants.
The achievement of getting a S. cerevisiae strain to express
a chimeric isoprenoid biosynthetic pathway holds promises
for several reasons. One can imagine applying the S. cerevisi-
ae strain developed here in a screening for potential inhibitors
of the MEP pathway. Analysis of phylogenetic distribution of
the mevalonate (MVA) and MEP pathways suggests the
MEP pathway as a promising new target for the development
of herbicides and agents against microbial pathogens, e.g.
Plasmodium falciparum, the causal agent of Malaria .
Since the enzymes of the MEP pathway are highly conserved
but show no homology to mammalian proteins, the finding of
specific inhibitors should result in novel antimicrobial drugs.
Furthermore, all the enzymes of the MEP pathway represent
potential targets, but specific inhibitors are so far only known
for the enzymatic step catalyzed by 1-deoxy-D D-xylulose 5-
phosphate reductoisomerase . However, for this type of
application, further genetic modifications are necessary to
physically turn the MEP pathway essential as the use of
lovastatin in the screening is unlikely due to the high concen-
tration required to affect the cell growth. Mutant strains of
an essential step of the MVA pathway and mentioned further
above, combined with the galactose inducible plasmid-borne
MEP pathway, would constitute a suitable system for the
suggested screening. Of course one may observe a similar
problem as with lovastatin that specific drugs that inhibit
the MEP pathway are poorly taken up by the yeast cells,
but this may in fact be used to an advantage, as one can
compare the sensitivity of growth towards different drugs
for both pathogenic bacterial strains and an eukaryotic cell
having the same pathway. Of course it is desirable if the
eukaryotic cell is less sensitive, as this will indicate poorer
uptake of the drug.
The pioneering works of Szczebara et al. and Martin et al.
demonstrated the feasibility of shifting metabolic pathways be-
tween organisms and its potential benefits [1,2]. Our study
points in the same direction, with the establishment of an en-
tire bacterial pathway transferred into yeast. An interesting
application of our yeast strain is for biotechnological produc-
tion of isoprenoids in yeast, as through expression of the bac-
terial pathway it is possible to direct carbon towards
isoprenoids and bypass the highly regulated MVA pathway.
In order to evaluate this we expressed, together with the heter-
ologous MEP pathway, valencene synthase. The resulting
strain could produce up to 1 mg/L of valencene in a batch
fermentation (data not shown), which shows that it is possible
to couple the heterologous expression of the MEP pathway to
direct production of FPP-derived products. The level of iso-
prenoid production is lower than what has been reported in
other studies, but through optimized expression of the MEP
pathway we are confident that it will be possible to use the here
reported yeast strain as a platform for production of isopre-
noids, especially complex isoprenoids that require a biosyn-
thetic step catalyzed by cytochrome P450 enzymes which are
often difficult to express in bacteria. Efficient yeast expression
systems for P450s and engineered yeast strains which co-ex-
press appropriate redox partners, e.g. S. cerevisiae WAT11
or WAT21, have been described, and are currently available
Acknowledgements: The authors gratefully acknowledge Firmenich SA
for funding the project. We also wish to acknowledge Dr. Uffe H. Mor-
tensen for fruitful discussions.
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