The Kmvalues of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes.. Keywords: bifunctional enzym
Trang 1Fusion of farnesyldiphosphate synthase and epi -aristolochene
synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis
Maria Brodelius1, Anneli Lundgren1, Per Mercke2,†and Peter E Brodelius1
1
Department of Chemistry and Biomedical Sciences, University of Kalmar, Sweden;2Department of Plant Biochemistry,
Lund University, Sweden
A clone encoding farnesyl diphosphate synthase (FPPS)
was obtained by PCR from a cDNA library made from
young leaves of Artemisia annua A cDNA clone encoding
the tobacco epi-aristolochene synthase (eAS) was kindly
supplied by J Chappell (University of Kentucky,
Lex-ington, KY, USA) Two fusions were constructed, i.e FPPS/
eAS and eAS/FPPS The stop codon of the N-terminal
en-zyme was removed and replaced by a short peptide
(Gly-Ser-Gly) to introduce a linker between the two ORFs These two
fusions and the two single cDNA clones were separately
introduced into a bacterial expression vector (pET32)
Escherichia coliwas transformed with the expression vectors
and enzymatically active soluble proteins were obtained after
induction with isopropyl thio-b-D-thiogalactoside The
recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns The fusion enzymes produced epi-aristolochene from isopentenyl diphosphate through a coupled reaction The Kmvalues of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes The bifunctional enzymes showed
a more efficient conversion of isopentenyl diphosphate to epi-aristolochene than the corresponding amount of single enzymes
Keywords: bifunctional enzyme; epi-aristolochene synthase; farnesyl diphosphate synthase; gene fusion; recombinant expression
The enzymatic machinery of a living cell is very complex
Thousands of enzymes are present and the flow of
metabolites has to be tightly regulated Consequently,
enzymes are localized to different organelles and within a
specific organelle the enzymes are organized in different
ways They may be found as soluble, membrane-associated
or membrane-integrated enzymes In order to make
meta-bolism more efficient, enzymes catalysing sequential
reac-tions are often found in close proximity to each other They
may form aggregates, be immobilized close to each other by
adsorption to cellular structures or they may be organized in
bi- or multifunctional enzymes within one single
polypep-tide chain Such enzymes exhibit substrate channelling
which is a process by which two or more sequential enzymes
of a pathway interact to transfer a metabolite directly from
one active site to the next without allowing free diffusion of the intermediate [1,2] Channelling is believed to play an important role in metabolic regulation and cellular control
of enzymatic activities The three-dimensional structures of bifunctional enzymes indicate that channelling can be achieved in different manners In tryptophan synthase from Salmonella typhimurium, a hydrophobic 25 A˚ tunnel, which matches the dimensions of the intermediate indole, connects the two active sites [2] In the bifunctional enzyme thymidine synthase/dihydrofolate reductase from the protozoan Leish-mania major, the dihydrofolate intermediate is channelled
on the basis of electrostatic interactions at the protein surface [3] In abietadiene synthase from grand fir, two distinct active sites within a structural domain catalyse two sequential, mechanistically different cyclizations to form the tricyclic perhydrophenanthrene-type structure of abietadi-ene from the universal diterpabietadi-ene precursor geranylgeranyl diphosphate [4] The copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme
Artificial bi- or multi-functional enzymes may be obtained by fusion of two or more structural genes [5] The translational 3¢ terminus of the first gene is deleted along with any prosequence at the 5¢ terminus of the second gene and the genes are ligated in-frame A small linker sequence coding for a few amino acids is often introduced between the two structural genes This linker separates the two proteins in space by a small distance allowing each of them to fold properly without constrains from the other protein molecule Linkers of different length have been used but it has been shown that if a too long linker is used the proximity effect is abolished [6] Direct fusion of enzymes
Correspondence to P E Brodelius, Department of Chemistry and
Biomedical Sciences, University of Kalmar, S-39182 Kalmar, Sweden.
Fax: + 46 480 446262, Tel.: + 46 480 447358,
E-mail: peter.brodelius@hik.se
Abbreviations: ADS, amorpha-4,11-diene synthase; eAS,
epi-aristo-lochene synthase; FPP, farnesyl diphosphate; FPPS, farnesyl
diphos-phate synthase; GPP, geranyl diphosdiphos-phate; IPP, isopentenyl
diphosphate; IMAC, immobilized metal affinity chromatography;
IPTG, isopropyl thio-b- D -thiogalactoside.
Enzymes: farnesyl diphosphate synthase (EC 2.5.1.10);
epi-aristolo-chene synthase (EC 4.1.99.7).
Present address: Plant Research International, Business Unit Cell
Cybernetics, PO Box16, 6700 AA Wageningen, the Netherlands.
(Received 18 February 2002, revised 13 May 2002,
accepted 13 June 2002)
Trang 2without a linker may also result in an active bifunctional
enzyme [7]
Fused genes may be expressed in a suitable host (e.g
Escherichia coli) and the recombinant bi- or
multi-functional enzyme used to study the effects of fusion
on enzyme kinetics and stability In a number of studies,
it has been shown that recombinant fused enzymes
exhibit a higher catalytic efficiency than the
correspond-ing mixture of scorrespond-ingle enzymes Some examples are
D-hydantoinase/N-carbamylase [7],
b-galactosidase/galac-tokinase [8], citrate synthase/malate dehydrogenase [9],
aminocyclopropane-carboxylic acid
synthase/aminocyclo-propane-carboxylic acid oxidase [10], and
trehalose-6-phosphate synthetase/trehalose-6-trehalose-6-phosphate phosphatase
[11] However, it has recently been argued, based on
kinetic studies, that these bifunctional enzymes do not
exhibit substrate channelling [12,13] The higher catalytic
efficiency, observed for these bifunctional enzymes, is
entirely due to proximity effects
The use of fused enzymes for metabolic engineering at
a branching point of a biosynthetic pathway is of great
potential However, so far this approach has been used
to a relatively limited extent The lactose utilization [6]
and the osmotolerance of E coli [14] have been
influenced by introduction of the bifunctional enzymes
b-galactosidase/galactokinase and c-glutamyl kinase/
c-glutamyl phosphate reductase, respectively The starch
degrading bifunctional enzyme a-amylase/glucose
isom-erase was expressed in potato tubers and upon heating
(65C for 45 min) of the crushed fresh tubers, glucose
and fructose was produced from the starch present in the
tubers [15] The a-amylase/glucose isomerase fusion is not
active at ambient temperatures and therefore it did not
have any adverse effects on plant development and
metabolism
We are involved in studies on the biosynthesis of
sesquiterpenoids in plants with the aim of improving by
metabolic engineering the amount of sesquiterpenes
pro-duced The common precursor for sesquiterpenoids is
farnesyl diphosphate (FPP), which is also a substrate for
the biosynthesis of other terpenoid metabolites such as
sterols (Fig 1) Sesquiterpene and sterol biosynthesis occurs
in the cytosol while the biosynthesis of mono- and
diterpenes take place in plastids
From studies with cell cultures of tobacco, it is well established that the sesquiterpene synthase, epi-aristolo-chene synthase (eAS), involved in biosynthesis of the phytoalexin capsidiol, is induced upon treatment of the culture with a fungal elicitor [16,17] A coordinated reduction in activity of squalene synthase (SS), an enzyme catalysing the formation of squalene from two molecules of FPP, is observed Obviously, a shift in flow of metabolites, i.e FPP, is achieved by these changes of enzyme activities and the conversion of FPP to epi-aristolochene is a regulatory step in sesquiterpene biosynthesis FPP is produced from isopentenyl diphosphate/dimethylallyl diphosphate by farnesyl diphosphate synthase (FPPS) Thus, fusion of the two enzymes, FPPS and eAS, would give a bifunctional enzyme that catalyses the conversion of the C5-substrate isopentenyl diphosphate (IPP) to the complex C15-product epi-aristolochene (Fig 1) The expression of this bifunctional enzyme in plant cells may result in an increased metabolic flow into sesquit-erpene biosynthesis This phenomenon may be even more pronounced as the enzymes are located on each side of
an important branching point of terpene metabolism Transformation of tobacco with a gene construct enco-ding the FPPS/eAS will lead to increased formation of epi-aristolochene and possibly the phytoalexin capsidiol with a simultaneous decrease in biosynthesis of sterols
We have constructed, expressed in E coli and partially characterized fusions of FPPS and eAS as a step in our efforts to increase the yield of sesquiterpenes in plants by metabolic engineering
M A T E R I A L S A N D M E T H O D S
Reagents Restriction enzymes, [1-14C]IPP (55 mCiÆmmol)1) and [1-3H]FPP (16 CiÆmmol)1) were from Amersham-Pharma-cia Biotech Isopropyl thio-b-D-thiogalactopyranoside (IPTG), IPP, geranyl diphosphate (GPP) and FPP were from Sigma The tobacco eAS cDNA clone (TEAS) was kindly provided by J Chappell, University of Kentucky, Lexington
PCR cloning of FPPS fromArtemisia annua
A cDNA library, previously constructed from poly(A+) RNA extracted from young leaves of A annua, was u sed to amplify a fragment encoding FPPS by PCR [18] Primers for the PCR reaction were designed according to a published sequence of FPPS from A annua [19] Primers P1 (forward) and P2 (reverse) contained an NcoI and a XhoI restriction site, respectively (Table 1)
PCR was carried out in a total volume of 50 lL with the following reagents: 1· cloned Pfu polymerase buffer (Stratagene), 0.2 mMdNTPs (Pharmacia), 20 pmol of each primer and 1 U Turbo Pfu polymerase (Stratagene) PCR cycling was: two cycles of 94C (0.5 min), 50 C (1.0 min),
72C (1.5 min); 29 cycles of 94 C (0.5 min), 56 C (1 min),
72C (1.5 min); 72 C (5 min) The amplified fragment was resolved on a 1% agarose gel and visualized by staining with ethidium bromide
The FPPS-wild type fragment was digested with NcoI and XhoI The bacterial expression vector pET32c (Novagen)
Fig 1 Biosynthetic pathway from IPP to the sesquiterpene
epi-aristo-lochene and squalene The reaction carried out by the bifunctional
enzymes described here is depicted within the shaded area.
Trang 3was cleaved with the same enzymes and treated with alkaline
phosphatase Vector and the fragment were isolated from
agarose gel bands using the JETQUICK gel extraction spin
kit (Genomed) Ligation of the fragment in frame with a
multifunctional tag (including a hexa-His) in the vector was
carried out according to standard procedures using T4 DNA
ligase (Boehringer) The plasmid obtained, pET32FPPS,
was transformed into E coli NovaBlue (Novagen) Colonies
were analysed by PCR using primers P1 and P2 to confirm
the presence of the FPPS gene Cells were grown in Luria–
Bertani medium containing 50 lgÆmL)1ampicillin and the
plasmid was purified using the JETQUICK plasmid
puri-fication spin kit (Genomed) and used as template for PCR
amplification as described below
PCR amplification of FPPS andeAS
For fusions of FPPS and eAS a number of DNA fragments
were prepared by PCR amplification using the same
conditions as above Two sets of primers (P6/P2 and P1/
P5) were used for amplification of FPPS using the
pET32FPPS as template (Table 1) In a similar manner
three sets of primers (P3/P4, P3/P7 and P8/P4) were used to
amplify the eAS gene using the TEAS cDNA as template
(Table 1)
Construction of expression vectors for production
of single and fused enzymes
Wild type eAS was digested and cloned into pET32c as
described for FPPS above The resulting plasmid
pET32eAS was transformed into E coli NovaBlue
Ligation of FPPS and eAS was achieved by sequential
cloning into the bacterial expression vector pET32c Two
fused enzymes, i.e FPPS/eAS and eAS/FPPS, were
con-structed First, fragments FPPS-nsc and eAS-nsc were
digested with NcoI and BamHI and separately cloned into
pET32c as described above yielding the plasmids
pET32FPPS-nsc and pET32eAS-nsc These two plasmids
were transformed into E coli NovaBlue for production of
the plasmids The plasmids were purified using the
JETQUICK plasmid purification spin kit
Subsequently the fragments FPPS-L and eAS-L were
digested with BamHI and XhoI and cloned into the plasmid
pET32eAS-nsc and pET32FPPS-nsc, respectively, as
des-cribed above yielding plasmids pET32FPPS/eAS and
pET32eAS/FPPS These two plasmids were transformed
into E coli NovaBlue
DNA sequencing DNA sequencing of cloned PCR fragments was performed using a DNA BigDyeTMTerminator Cycle Sequencing Kit (Perkin Elmer) for the labelling of the sequencing reactions Analyses were then carried out on an ABI PRISMTM310 Genetic Analyzer Oligonucleotides (15-mers) were synthes-ized according to sequence information and were used as primers for sequencing
Expression of the recombinant proteins and preparation of bacterial extracts NovaBlue cells carrying the plasmids pET32FPPS, pET32eAS, pET32FPPS/eAS and pET32eAS/FPPS were grown overnight in Luria–Bertani medium containing ampicillin (50 lgÆmL)1) at 37C The plasmids were purified using the JETQUICK plasmid purification spin kit and transferred into E coli strain BL21(DE3) pLysS The BL21 cells were grown at 37C in 20 mL Lu ria– Bertani medium containing ampicillin (50 lgÆmL)1) to an
D660of 0.6 IPTG was then added to the final concen-tration of 1 mM The cells were harvested after 4 h of cultivation at 30C by centrifugation at 200 g for 10 min at room temperature and the pellet was resuspended in 2 mL extraction buffer (50 mM Tris/HCl pH 8.0, containing
15 mMMgCl2and 20% glycerol) The cells were disrupted
by sonication (Braun-Sonic 2000 microprobe at maximum power for 3· 20 s bursts with 0.5 min chilling period on ice between bursts) The extract was centrifuged at 10 000 g for
15 min at 4C The supernatant was collected and analysed
Optimization of expression Optimization of expression was carried out for the pET32eAS/FPPS construct The parameters investigated were IPTG concentration (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mM) used for induction, induction temperature (12, 22 and
30C) and time of harvest after induction (90, 135, 210, 295 and 330 min) BL21 cells carrying the pET32eAS/FPPS plasmid were grown at 37C in 20 mL Luria–Bertani medium containing ampicillin (50 lgÆmL)1) to an D660of
0.6 Induction under various conditions was subsequently carried out The cells were harvested by centrifugation at
200 g for 10 min at room temperature and the pellet was resuspended in 2 mL extraction buffer The cells were disrupted by sonication The extract was centrifuged at
Table 1 Primers used in cloning Restriction sites are underlined.
Trang 410 000 g for 15 min at 4C The supernatant was collected
and enzyme activities determined
Production and purification of recombinant proteins
BL21 cells were grown and induced (0.4 mM IPTG) as
described above in 600 mL Luria–Bertani medium
contain-ing ampicillin Proteins were extracted with 40 mL buffer
Purification of recombinant proteins was carried out in a
single step using immobilized metal affinity
chromato-graphy (IMAC) The supernatant was applied to a 5-mL
HiTrap Chelating HP column (Amersham Pharmacia
Biotech) loaded with Co2+ Nonbound proteins were
removed by washing with buffer Elution of adsorbed
recombinant proteins was achieved with extraction buffer
containing 500 mM imidazole Fractions of 0.5 mL were
collected and fractions showing enzyme activity were pooled
and frozen in liquid nitrogen in aliquots (0.5 mL) and stored
at)80 C until used
Electrophoresis and Western blotting
SDS/PAGE electrophoresis was carried out on 4–20% Tris/
glycine gels or on NuPAGE 4–12% Bis/Tris gels from
Invitrogen according to instructions supplied by the
manu-facturer After electrophoresis, the gels were either stained
with Coomassie blue or the proteins transferred to
nitro-cellulose membranes Blotted proteins were detected with
the Stag Alkaline Phosphatase Western blot kit (Novagen)
according to the instructions supplied by the manufacturer
Enzyme assays
FPPS An aliquot of bacterial extract (10–46 lL) or
purified enzyme (2–10 lL) was assayed in 50 mM Tris/
HCl pH 8.0, containing 15 mM MgCl2, 10 mM
2-mercap-toethanol, 20% glycerol, 55 lMGPP and 50 lM[1-14C]IPP
(1.8 lCiÆmmol)1) in a total volume of 50 lL The
samples were incubated for 10 min at 30C and
subse-quently an aliquot of 6MHCl (5 lL) was added to stop the
reaction and incubation was continued for 30 min to
hydrolyse FPP formed to extractable farnesol (the substrate
IPP is stable under these conditions) Neutralization was
performed with 6MNaOH (7.5 lL) The mixture was first
extracted with hexane (400 lL) and subsequently the
hexane phase (350 lL) was removed and extracted with
water (300 lL) An aliquot (200 lL) of the hexane phase
was taken into a scintillation vial and measured in a
scintillation counter
eAS An aliquot of bacterial extract (10–48 lL) or purified
enzyme (2–10 lL) was assayed in 50 mMTris/HCl pH 8.0,
containing 15 mMMgCl2, 10 mM2-mercaptoethanol, 20%
glycerol and 20 lM [1-3H]FPP (0.1 CiÆmmol)1) in a total
volume of 50 lL After a 10-min incubation at 30C, the
reactions were stopped by addition of an equal volume of
0.2M KOH containing 0.1M EDTA Subsequently, the
reaction mixture was extracted with hexane (2· 0.4 mL)
and the hexane extracts were passed over a small column
filled with 200–250 mg silica (Merck; size: 0.2–0.5 lm) and
the column was rinsed with additional hexane (1.0 mL) The
hexane extract was examined for radioactivity by
scintilla-tion counting
Coupled enzyme assay The coupled enzyme reaction was analysed for the bifunctional enzymes An aliquot of bacterial extract (10–46 lL) or purified enzyme (2–10 lL) was assayed in 50 mMTris/HCl pH 8.0, containing 15 mM MgCl2, 10 mM 2-mercaptoethanol, 20% glycerol, 55 lM GPP and 50 lM [1-14C]IPP (1.8 lCiÆmmol)1) in a total volume of 50 lL The samples were incubated for 10 min at
30C Two sets of samples were made In one set, the reaction was stopped with 0.2MKOH and the final product was analysed according to the eAS assay description To the other set, HCl was added and the evaluation of the FPPS activity was performed as described for the FPPS assay
Protein concentrations Protein concentrations of extracts and partly purified recombinant enzymes were determined according to Bradford [20] using BSA as standard
R E S U L T S A N D D I S C U S S I O N
Expression of recombinant enzymes For production of single and fused enzymes the bacterial expression vector pET32c was used This expression vector was selected because the target protein is expressed as a fusion with a tag containing the thioredoxin for increased protein solubility [21], a histidine tag sequence facilitating protein purification and a Stag sequence for sensitive protein quantification and detection The sequence also contains an enterokinase cleavage sites for removal of the fusion tag
The four plasmids pET32FPPS, pET32eAS, pET32eAS/FPPS and pET32FPPS/eAS were isolated and transferred into the E coli BL21(DE3)pLysS for production of the recombinant enzymes The transformed bacteria were grown and induced with IPTG (1 mM), and cell-free extracts were prepared and evaluated for FPPS and/or eAS activity All extracts exhibited the expected activities Recombinant proteins of the predicted molecu-lar mass were detected in Western blots u sing Stag alkaline phosphatase staining The recombinant bifunc-tional enzymes show both activities and obviously the linker (Gly-Ser-Gly) is sufficiently long to permit the two enzymes to fold properly The three-dimensional structure
of eAS has been reported [22] but so far no structure for a plant FPPS has been published However, the three-dimensional structure of chicken FPPS, which shows high amino acid identity (46.6%) and similarity (67.2%) to the Artemisia enzyme, has been reported [23] Assuming a conserved structure between the two FPPSs, these two structures may be used to model the fusion enzyme It is evident from such models that the linker used is sufficiently long to permit proper folding of the fused enzymes Linkers of different length have been used in constructs of bifunctional enzymes [6,24,25] However, long linkers may be exposed and sensitive to proteolytic attack and a too long distance between the two active sites may lead to reduced or no channelling of substrate as has been reported for the bifunctional b-galactosidase/galacto-kinase [6] The Gly-Ser-Gly linker is relatively short and
is convenient to use as the corresponding nucleotide
Trang 5sequence contains a BamHI site, which may be used for the
fusion of two genes The Gly-Ser-Gly linker was used in a
functional fusion of citrate synthase and malate
dehydrog-enase [9]
Optimization of expression was carried out for one of
the constructs, i.e pET32 eAS/FPPS The amount of eAS
activity was determined as a function of incubation time
after addition of IPTG, concentration of IPTG and
induction temperature The FPPS activity showed the
same pattern Based on these results the following
conditions were selected for large-scale induction of all
four recombinant proteins: incubation time, 4 h; IPTG
concentration, 0.4 mM; induction temperature, 30C
These conditions are consistent with the conditions used
for the production of a number of other recombinant
proteins in our laboratory
It is interesting to note that the highest activity of the
relatively large recombinant protein (119.9 kDa) is
observed at 30C We assume this to be due to the fact
that the fused enzyme is expressed as a fusion with the
thioredoxin protein, which is improving the solubility of the
expressed protein [21] In fact, no recombinant protein
could be detected in the insoluble fraction by SDS/PAGE
For expression in E coli of eAS without any tag a
significant part of the recombinant protein was found in
inclusion bodies [26] However, a twofold increase in
recombinant eAS activity was obtained when the induction
temperature was lowered from 37 to 27C Similarly, an
increased amount of recombinant epi-cedrol synthase was
obtained by lowering the induction temperature to 20C
[18] The high activity of the bifunctional enzyme in extracts
from cells grown at 30C is reflected in a higher total
protein content The specific activity in extracts obtained at
30C is lower than for extracts prepared from cells induced
at lower temperatures However, for large-scale production
of the recombinant proteins an induction temperature of
30C was used
Production and purification of recombinant proteins
The four recombinant proteins were produced on a large
scale (2· 600 mL cultures) using the conditions established
above The proteins carrying a (His)6-tag were purified in a
one-step procedure by chromatography on IMAC-columns
This convenient procedure is widely used to purify
recom-binant proteins [27] Columns charged with three different
ions, i.e Ni2+, Zn2+ and Co2+, were tested Some
unspecific binding of E coli proteins appeared to occur on
all three ions, which may be due to the presence of metal
binding sites in some proteins In fact, on SDS/PAGE the
same contaminating proteins appeared to be present in all
four purified proteins The least unspecific binding was
observed for columns charged with Co2+, which were used
for the large-scale purifications Analysis by SDS/PAGE of
protein pu rified on a Co2+column showed that they were at
least 95% pure (Fig 2) No attempts were made to remove
the impurities by other purification steps
During prolonged incubations with enterokinase at 30C
for removal of the affinity tag, a significant loss of enzyme
activity was observed for the recombinant proteins
There-fore, the characterization of the recombinant proteins was
carried out on enzymes containing the
thioredoxin-Stag-His-tag as an N-terminal fusion
Kinetic properties of recombinant enzymes The IMAC-purified enzymes were used to determine Km values according to standard techniques The Kmfor IPP was determined to be 3.3 for recombinant Artemisia FPPS
No significant difference in Kmvalues was observed for the fused and single enzymes The Km values for IPP was calculated to be 3.8 and 4.0 lMfor the two fusion enzymes eAS/FPPS and FPPS/eAS, respectively These Kmvalues are similar to those reported for FPPS from other sources [28–31]
The Kmvalue for FPP with the recombinant tobacco eAS was estimated to be 1.7 lM, which is the same as the 2–5 lM reported for the purified wild-type tobacco eAS [32] The Km values for FPP were calculated to be 1.6 and 2.6 lMfor the two fusion enzymes eAS/FPPS and FPPS/eAS, respectively The Kmvalues for FPP of other wild-type and recombinant plant sesquiterpene synthases have been reported to be in the range 0.5–7 lM[18,33–37]
In conclusion, essentially the same Km values were obtained for the single FPPS and eAS as for the two fusion enzymes FPPS/eAS and eAS/FPPS and these Km were similar to those reported previously for FPPS and sesquiterpene synthases from other sources Apparently, the fusion of the two enzymes does not affect the affinity for the substrates Folding of recombinant single and bifunc-tional enzymes is appropriate Furthermore, these results indicate that an N terminal tag does not affect the catalytic properties of the recombinant enzymes
Coupled activity The fused enzymes FPPS/eAS and eAS/FPPS convert IPP
to epi-aristolochene via FPP (Fig 1) With increasing recombinant enzyme amount an increased formation of epi-aristolochene from IPP is obtained (Fig 3) It is evident from Fig 3 that the amount of enzyme used in an assay must be carefully adjusted for linearity of the assay and that the incubation time should not be too long under the conditions used
Fig 2 SDS/PAGE of recombinant enzymes produced in E coli Lane 1, molecular mass standards; lane 2, crude extract FPPS/eAS (14.0 lg protein); lane 3, purified FPPS/eAS (5.1 lg); lane 4, cru de extract eAS/ FPPS (11.2 lg); lane 5, purified eAS/FPPS (4.4 lg); lane 6, crude extract FPPS (13.4 lg); lane 7, pu rified FPPS (3.3 lg); lane 8, crude extract eAS (13.0 lg); lane 9, purified eAS (4.2 lg) The calculated molecular weights of FPPS, eAS and the fusion enzymes are 57, 80 and
120 kDa, respectively.
Trang 6It is interesting to note that the relative amount of
epi-aristolochene formed by the bifunctional enzymes increases
at higher enzyme activities, i.e a larger portion of the FPP
produced by the FPPS part of the enzyme is converted to
final product by eAS (Fig 4) This may be expected as the
building-up of the intermediate FPP is more rapid at higher
amounts of FPPS and the subsequent eAS experiences a
higher substrate concentration, i.e a steady-state condition
is approached No difference can be observed between the
two bifunctional enzymes Obviously, the order in which the
two enzymes are fused does not influence the activity of the
enzymes This is also reflected in the Kmvalues determined
for the two bifunctional enzymes
Finally, to further evaluate the performance of the
bifunctional enzymes, the level of epi-aristolochene
pro-duced from IPP by the eAS/FPPS was compared with that
produced by the corresponding amounts of the two single
enzymes As shown in Fig 5 the amount of FPP produced
is essentially the same for the two systems However, the
amount of epi-aristolochene produced is considerably
higher for the fusion enzyme than for the mixture of single
enzymes Apparently, a proximity effect or substrate
channelling operates in the fusion enzyme and increases
the overall catalytic activity of the reaction The FPP
produced by the first enzyme is transferred to the active site
of eAS with limited diffusion into the surrounding solution
Similar substrate channelling has been observed for a
number of artificial bifunctional enzymes [7–11]
C O N C L U D I N G R E M A R K S
The fused enzymes described above are fully active when expressed in E coli Next these gene constructs will be transferred to a plant transformation vector Transgenic tobacco plants producing the bifunctional enzymes will be established and the effects on sesquiterpene and sterol biosynthesis investigated We are involved in studies on the
Fig 3 Time course for formation of FPP and epi-aristolochene from
IPP and GPP as function of the amount of purified recombinant
bifunctional enzymes (A) FPPS/eAS (B) eAS/FPPS Open symbols,
FPP; solid symbols, epi-aristolochene s, d, 2 lL purified enzyme; n,
m, 4 lL purified enzyme; h, j, 6 lL purified enzyme Each point is
the mean of two determinations The protein concentrations of the
purified enzyme preparations were 1.9 and 3.0 mg proteinÆmL)1for
FPPS/eAS and eAS/FPPS, respectively.
Fig 4 Amount of epi-aristolochene formed as function of FPP produced
by the purified bifunctional enzymes FPPS/eAS and eAS/FPPS using IPP and GPP as substrates Each point corresponds to a separate enzymatic assay containing either different amount of enzyme or being incubation for different times d, FPPS/eAS; j, eAS/FPPS.
Fig 5 Time course for formation of FPP and epi-aristolochene from IPP and GPP by the two purified recombinant single enzymes (open symbols) or the purified recombinant bifunctional eAS/FPPS (solid symbols) as a function of incubation time The activities of the single enzymes in the assay were carefully adjusted to the corresponding activities of the bifunctional enzyme h, j, FPP; s, d, epi-aristo-lochene Each point is the mean of two determinations.
Trang 7biosynthesis of the antimalarial sesquiterpene artemisinin in
A annua The sesquiterpene cyclase, amorpha-4,11-diene
synthase (ADS), converting FPP to the first intermediate of
artemisinin biosynthesis was recently cloned in our
labor-atory [33] We will make fusions of FPPS and ADS and
introduce the bifunctional enzyme into plants of A annua
We expect to obtain an increased biosynthesis of artemisinin
in transgenic plants of A annua expressing the FPPS/ADS
fusion
A C K N O W L E D G E M E N T S
The financial support to P.E.B from the Swedish Research Council for
Engineering Sciences and the Swedish Council for Forestry and
Agricultural Research During a part of this work M.B received a
Marie Curie Scholarship from the European Union We thank
professor J Chappell for the kind gift of the TEAS cDNA clone.
R E F E R E N C E S
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