Construction and expression of a dual vector for chemo-enzymatic synthesis of plant indole alkaloids in Escherichia coli

Article abstract of DOI:10.1080/14786410903247304

A dual vector (pQE-70-STR1-SG) containing coding regions of strictosidine synthase (STR1, EC 4.3.3.2) and strictosidine glucosidase (SG, EC 3.2.1.105) from the Indian medicinal plant Rauvolfia serpentina was constructed. Functional expression of the vector in Escherichia coli cells (M15 strain) was proven by isolation of prepurified enzyme extracts, which show both STR1 and SG activities. Incubation of the enzyme in the presence of tryptamine and secologanin delivered the indole alkaloid cathenamine, demonstrating functional co-expression of both STR1- and SG-cDNAs. Cathenamine reduction by sodium borohydride leading to tetrahydroalstonine revealed the chemo-enzymatic indole alkaloid synthesis.

Full text of DOI:10.1080/14786410903247304

Natural Product Research
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Construction and expression of a dual vector
for chemo-enzymatic synthesis of plant indole
alkaloids in Escherichia coli
Joachim Stöckigt , Bodo Hammes & Martin Ruppert
To cite this article: Joachim Stöckigt , Bodo Hammes & Martin Ruppert (2010) Construction
and expression of a dual vector for chemo-enzymatic synthesis of plant indole alkaloids in
Escherichia coli , Natural Product Research, 24:8, 759-766, DOI: 10.1080/14786410903247304
To link to this article: http://dx.doi.org/10.1080/14786410903247304
Published online: 29 Apr 2010.
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Date: 05 November 2015, At: 19:59

Natural Product Research
Vol. 24, No. 8, 10 May 2010, 759–766
Construction and expression of a dual vector for chemo-enzymatic
synthesis of plant indole alkaloids in Escherichia coli
ab
b
b
Joachim Sto
¨
ckigt *, Bodo Hammes and Martin Ruppert
a
College of Pharmaceutical Sciences, Institute of Materia Medica, Zhejiang University,
b
Zijingang Campus, 388 Yu Hang Tang Road, Hangzhou 310058, P.R. China; Department of
Pharmaceutical Biology, Institute of Pharmacy, Johannes Gutenberg-University Mainz,
Staudinger Weg 5, D-55128 Mainz, Germany
(
Received 20 May 2009; final version received 10 August 2009)
A dual vector (pQE-70-STR1-SG) containing coding regions of stricto-
sidine synthase (STR1, EC 4.3.3.2) and strictosidine glucosidase (SG, EC
3
.2.1.105) from the Indian medicinal plant Rauvolfia serpentina was
constructed. Functional expression of the vector in Escherichia coli cells
M15 strain) was proven by isolation of prepurified enzyme extracts, which
(
show both STR1 and SG activities. Incubation of the enzyme in the
presence of tryptamine and secologanin delivered the indole alkaloid
cathenamine, demonstrating functional co-expression of both STR1- and
SG-cDNAs. Cathenamine reduction by sodium borohydride leading
to tetrahydroalstonine revealed the chemo-enzymatic indole alkaloid
synthesis.
Keywords: Rauvolfia serpentina; dual vector pQE-70-STR1-SG; engineered
Escherichia coli; chemo-enzymatic synthesis; indole alkaloids
1
. Introduction
The increasing number of well-known biosynthetic pathways, the concomitant
knowledge of participating enzymes and the availability of the relevant genes
(
cDNAs) have led to new opportunities for applying these to future production
strategies of natural products: for example, alkaloids (Kutchan, 1995; Loris et al.,
007; Ruppert, Ma, & Stockigt, 2005). In this context, the heterologous
2
¨
co-expression of plant genes in microbial hosts, such as Escherichia coli, may play
an important role (Chemler & Koffas, 2008; Minami et al., 2008). This article
describes the construction and the functional expression in E. coli of a dual vector
harbouring both cDNAs of the enzymes strictosidine synthase (STR1) (Kutchan,
Hampp, Lottspeich, Beyreuther, & Zenk, 1988) and strictosidine glucosidase (SG)
¨
(Gerasimenko, Sheludko, Ma, & Stockigt, 2002) from Rauvolfia serpentina (L.)
Benth. ex Kurz. Both the gene products can be used for the chemo-enzymatic
formation of indole alkaloids.
*Corresponding author. Email: joesto2000@yahoo.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410903247304
http://www.informaworld.com

7
60
J. Sto¨ckigt et al.
2
2
. Material and methods
.1. Bacterial strains and culture conditions
Escherichia coli cultures were cultivated in Luria–Bertani (LB) medium (Bertani,
1
951, 2004) and, dependent on the strain, supplemented with ampicillin and/or
ꢀ
kanamycin at 37 C (100 rpm). TOP10 (Invitrogen, Karlsruhe, Germany) strains were
used for cloning experiments and M15 (Qiagen, Hilden, Germany) bacteria were
used for enzyme expressions.
2
.2. Cloning and expression of STR1 cDNA
Restriction enzymes were purchased from New England Biolabs (Frankfurt am
Main, Germany) and the T4 DNA ligase was bought from Promega (Mannheim,
Germany). The template used for primer design was the cDNA sequence of STR1
(GenBank: Y00756.1) from R. serpentina (Kutchan et al., 1988). pQE-70 (Qiagen,
Hilden, Germany) was selected as the host vector as it contains the SphI and BamHI
restriction sites. Polymerase chain reaction (PCR) was carried out with primers
0
(MWG, Ebersberg, Germany) STR1forSphI (5 -GCA TGC ATG GCC AAA CTT
TCT GAT TCG CAA AC-3 , forward) and STR1revBamHI (5 -GGA TCC TTA
0
0
0
ATG ACT TGA AAC AAA AGA ATT TCC-3 , reverse) in Mastercycler Personal
Õ
(Eppendorf, Hamburg, Germany) with Advantage Polymerase (Clontech,
Heidelberg, Germany) under the following conditions: 94 C for 1 min, followed by
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3
1
0 cycles of 94 C for 30 s, 58 C for 1 min, 72 C for 2 min, then held at 72 C for
0 min.
After ligation into pGEM-T vector (Promega), correct cDNA orientation was
confirmed by using SphI. As the STR1 cDNA contains an internal restriction site for
SphI, the resultant pattern of the fragments shows either one line (403 bp, correct
orientation) or two lines (403 and 658 bp, false orientation) on a 1% agarose gel.
Because of the internal restriction site, a partial digestion was necessary in order to
obtain the 1035 bp fragment. The cDNA was therefore double digested with SphI
ꢀ
and BamHI (30 min, 37 C). The designated fragment was detected after 10 min
incubation and extracted with the NucleoSpin Extract kit (Macherey & Nagel,
Du
¨
ren, Germany).
The fragment was then ligated into the SphI and BamHI sites of the expression
vector pQE-70 and transformed into strain TOP10 in order to prove its presence.
The vector including the STR1 cDNA was then transformed into M15 cells for
expression tests. For activity tests, 12 mL of overnight culture was inoculated into
ꢁ
1
ꢁ1
2
00 mL of LB-medium containing 50 mg L ampicillin and 25 mg L kanamycin
ꢀ
and incubated at 37 C up to OD ¼ 0.5. The production of STR1 was induced by
6
00
adding 1 M stock solution of isopropyl ꢀ-D-thiogalactopyranoside (IPTG) to a final
ꢀ
concentration of 0.6 mM for 16 h at 24 C and 100 rpm. A control culture was
incubated containing only vector pQE-70.
2
.3. Enzyme assay for STR1
ꢀ
Bacteria were harvested after 16 h by centrifugation (10 min at 5000 ꢂg, 4 C) and
then resuspended in 2.5 mL of lysis buffer (50 mM potassium phosphate buffer
(KPi) pH 7.0; 20 mM mercaptoethanol (MSH); 1 mg mL
ꢁ
1
lysozyme) per vial.

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761
After cracking with sonication (Sonoplus HD 2070, 3 ꢂ 10 s, 75 W), the suspension
ꢀ
was centrifuged again (30 min, 12,000 ꢂg, 4 C). The supernatant was taken for
enzyme assays.
An assay (100 mL) contained 6.25 mg tryptamine HCl, 12 mg secologanin and
ꢀ
8
1.75 mL (500 mg) enzyme solution. Incubation was for 30 min at 30 C and 500 rpm.
The reaction was stopped by adding 100 mL MeOH. Following centrifugation (5 min
ꢀ
at 14,000 ꢂ g, 4 C), the supernatant was analysed by high-performance liquid chro-
Õ
Õ
matography (HPLC) using a LiChroCART 250-4 LiChrospher 60 RP-select B
(
5 mm) column (Merck, Darmstadt, Germany), solvent system : acetonitrile/H O pH
2
2
8
1
.3 (adjusted with concentrated H PO ), gradient 10 : 90 ! 50 : 50 within 8 min, !
3
4
0 : 20 within 3 min, ! 10 : 90 within 0.5 min, ! 10 : 90 for 4.5 min, flow rate
ꢁ1
.5 mL min , detection at 250 nm, 40 mL injection volume. Retention times (R )
t
were 4.7 min for tryptamine HCl, 5.8 min for secologanin and 8.0 min for
strictosidine.
2
.4. Construction of the dual vector pQE-70-STR1-SG
.4.1. Introduction of an additional multiple cloning site
2
The initial system for construction of the gene cluster was the pQE-70 vector
containing STR1 cDNA. In order to potentiate a vector system that can harbour
more than one gene, the pQE-70-STR1-system was supplemented with new
restriction sites. Therefore, a portion of the multiple cloning site (MCS) from the
pSE280 vector (Invitrogen) was subjected to a restriction digest with BamHI and
HindIII. In between these two sites, there are 28 other possible restriction sites that
can be used for ligation of additional cDNAs.
Isolation of the MCS from the pSE280 vector was carried out by digesting the
ꢀ
plasmid extract with HindIII and BamHI for 1.5 h at 37 C. The resulting 253 bp
fragment was isolated from agarose gel (NucleoSpin Extract kit) and ligated into
BamHI and HindIII restriction sites of pQE-70-STR1. Following the digestion of
the plasmid extract, the presence of the MCS adjacent to the STR1 cDNA in pQE-
7
0-STR1 was verified.
2
.4.2. Cloning of strictosidine ꢀ-D-glucosidase cDNA with a ribosome binding site
A PCR template pTYB1 vector harbouring the SG cDNA (Gerasimenko et al., 2002)
was extracted from E. coli strain ER2566 by the use of the NucleoSpin Plasmid kit
(Macherey & Nagel). Primer design was based on the SG sequence (GenBank:
AJ302044.1) from R. serpentina (Gerasimenko et al., 2002). In order to ensure that
the enzyme was also expressed after its ligation upstream of the STR1 gene, the
sequence of the ribosome binding site (RBS) from pQE-70 vector was transferred
into the SG forward primer. PCR was performed under the following conditions:
ꢀ
ꢀ
ꢀ
ꢀ
9
2
4 C for 1 min, followed by five cycles of 94 C for 30 s, 45 C for 1 min, 72 C for
ꢀ
ꢀ
ꢀ
ꢀ
.5 min, 25 cycles of 94 C for 30 s, 58 C for 1 min, 72 C for 2.5 min then held at 72 C
0
for 10 min. By using the primers SG þ RBSforBamHI (5 -GGA TCC GAA TTC
0
ATT AAA GAG GAG AAA TTA ATG GAC AAT ACT CAA GCT G-3 ) and
0
SGrevKpnI (5 -GGT ACC TTA GGT TTT TTG CCT CTT GAC TAA CTC-32), a
1
633 bp fragment was obtained.

7
62
J. Sto¨ckigt et al.
1
2
3
Marker
6
5
000 bp
000 bp
2
2
500 bp
000 bp
1500 bp
1000 bp
8
6
00 bp
00 bp
4
2
00 bp
00 bp
Figure 1. Agarose gel (1%) after digestion of pQE-70-STR1-SG to prove for successful
ligation of SG cDNA with the RBS into pQE-70-STR1.
Notes: Line 1: Digestion with BamHI, fragments with the size of 725 and 5500 bp appeared;
line 2: digestion with SphI and BamHI, fragments with the size of 404, 635, 725 and 4461 bp
are visible; line 3: digestion with SphI and KpnI, resulting in fragments of 404, 2268 and
¨
3553 bp; marker: SmartLadder 200 (Eurogentec, Koln, Germany).
2
.4.3. Completion of the dual vector pQE-70-STR1-SG
The pQE-70-STR1 vector could be completely digested with KpnI and BamHI at
ꢀ
3
ligation of SG cDNA. The linearised SG cDNA was digested with KpnI (2 h at 37 C)
7 C for 2 h. The resulting band of 4592 bp was extracted and then used as a host for
ꢀ
ꢀ
followed by partial digestion with BamHI for 5, 10, 15, 20, 30 and 45 min (37 C,
tot. vol. 150 mL). The desired band of the 1633 bp fragment appeared in each
incubation, even though the intensity decreased with incubation time, and was
extracted from gel.
The presence of SG cDNA as well as the short MCS was proved by three
digestions containing different combinations of restriction endonucleases:
(
(
(
1) 10 U BamHI with expected fragments of 725/5500 bp.
2) 10 U BamHI þ 10 U SphI with expected fragments of 404/635/725/4461 bp.
3) 10 U SphI þ 10 U KpnI with expected fragments of 404/2268/3553 bp.
ꢀ
After incubation for 1.5 h at 37 C, each digestion yielded the expected fragments
(see Figure 1). Additionally, 100 mL control cultures containing pQE-70 and pQE-
7
0-STR1 vectors were treated analogously.
2
.5. Product identification
The alkaloids formed were identified by R values (TLC), R (HPLC), mass
f
t
spectrometry (Finnigan MAT44S quadrupole instrument, 70 eV) and derivatisation
reduction of cathenamine), and comparison with standard samples.
(

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Sph I
Sph I
4
6
04 bp
35 bp
pQE-70-STR1-SG
6 22 22 55 bb pp ))
BamH I
2
268 bp
(
RBS
7
25 bp
BamH I
Kpn I
STR1((1035 bp)
RBS
SG((1599 b)p)
MCS
t0
Figure 2. Expression vector map of the pQE-70-STR1-SG plasmid with recognition sequences
for the restriction enzymes SphI, BamHI and KpnI (colour online).
Notes: The new cloned cDNAs and MCS are also shown linearised. Light green: SphI
recognition sequence; blue: BamHI recognition sequence; dark green: KpnI recognition
sequence; purple: STR1; orange: SG; red: RBS; light blue: MCS; t : stop codons.
0
3
3
. Results and discussion
.1. Functional co-expression of STR1 and SG cDNAs
In order to test the enzyme activity of the protein synthesised by the vector construct
pQE-70-STR1-SG (see Figure 2), the construct was transformed into E. coli strain
M15. When an overnight culture of M15 cells (6 ꢂ 10 mL) containing the vector
ꢁ1
construct was inoculated into 6 ꢂ 600 mL LB-medium (50 mg L ampicillin and
ꢁ
1
ꢀ
2
two enzymes was induced by addition of IPTG to a final concentration of 0.6 mM
(
5 mg L kanamycin) and incubated at 37 C up to OD ¼ 0.5, expression of the
6
00
ꢀ
100 rpm, 25 C).
3
.2. Assay for functional co-expression of STR1 and SG cDNAs
ꢀ
Following a 22 h incubation (25 C), E. coli transformants were harvested by
ꢀ
centrifugation (10 min at 5000 ꢂg and 4 C). Following resuspension of each pellet in
ꢁ1
4 mL lysis buffer (50 mM KPi buffer pH 6.0; 20 mM MSH; 1 mg mL lysozyme), the
cells were cracked by sonication (10 s, 75 W). After incubation on ice (30 min),
bacteria were cracked again by sonication (3 ꢂ 10 s, 75 W). Cell fragments were
ꢀ
separated (30 min at 12,000 ꢂg, 4 C) and the supernatant was taken for precipitation
ꢀ
between 35 and 70% (NH ) SO . After centrifugation (30 min, 9000 ꢂg, 4 C) the
4
2
4
pellet containing the expressed enzymes was resuspended in 1 mL 50 mM KPi buffer
(pH 6.0, 150 mM NaCl, 20 mM MSH) and the solution was centrifuged as before.
The supernatant was purified on a HiTrap desalting column (General Electric,
Freiburg, Germany) to remove molecules 55000 Da. After centrifugation, the
supernatant was used for activity tests. Assays were performed with enzyme
preparations obtained from pQE-70 vectors without insert, pQE-70-STR1 and pQE-
7
0-STR1-SG.

7
64
J. Sto¨ckigt et al.
Incubations carried out to determine the single activity of STR1, as well as the
detection of both enzyme activities in the dual expression system, were carried out as
described for STR1-activity above. Assays for testing the single activity of SG
contained 8.5 mg strictosidine and 91.5 mL (729 mg) protein solution. Each reaction
ꢀ
was incubated at 35 C. Reactions were terminated after 1.5 h by addition of MeOH
(
0.1 mL), followed by centrifugation (14,000 ꢂg, 5 min). HPLC analysis (same
column as above) was performed using the following solvent system (Gao, von
Schumann, & Stockigt, 2002): acetonitrile/25 mM K HPO pH 2.3 (adjusted with
¨
2
4
conc. H PO ), gradient 28 : 72 ! 30 : 70 within 4 min, ! 35 : 65 within 2 min, !
3
4
ꢁ
1
2
2
8 : 72 within 0.1 min, ! 28 : 72 for 3.9 min, flowrate 1.68 mL min , detection at
80 nm, 60 mL injection volume.
HPLC analyses showed that single enzymes as well as the enzymes obtained by
co-expression of the dual vector were active. STR1 and SG activities detected under
described conditions indicated cathenamine to be the major product (R 8.1 min) of
t
the co-expression system. Strictosidine as an intermediate could, however, not be
detected, due to its immediate SG-catalysed conversion into cathenamine.
3
.3. Identification of cathenamine as a major product of STR1-SG-co-expression
by dual vector
In order to ascertain that cathenamine was the reaction product of co-expressed
STR1 and SG (see Figure 3), a tenfold incubation (pH 6.9) was made (tot. vol. 1 mL).
Previous tests to determine the occurrence of by-products based on long-term
incubations (0.5–3.0 h) demonstrated that additional peaks in HPLC-chromato-
grams were caused by degradation of cathenamine (data not shown). Enzyme
reactions for identification of cathenamine were therefore incubated for 15 min at
ꢀ
3
5 C. After adjusting the pH to 8.0 with 1 M KPi buffer (pH 8.0) reaction products
were extracted with 2 ꢂ 0.6 mL ethyl acetate followed by evaporation under nitrogen
at room temperature. The dried yellow residue was dissolved in 50 mL mixture of
CHCl /MeOH (9 : 1) and examined via TLC (solvent system : petrolether/acetone/
diethylamine 7 : 2 : 1, TLC plates (Merck)). The major product (absorption at
3
2
54 nm, R 0.46) co-migrated with an authentic cathenamine sample.
f
4
Figure 3. Chemo-enzymatic synthesis of tetrahydroalstonine from tryptamine and
secologanin.
Notes: An enzyme extract from E. coli harbouring the dual vector pQE-70-STR1-SG
expressing STR1 and SG followed by chemical reaction with borohydride was used. Due to
simultaneous expression of STR1 and SG, the intermediate strictosidine does not accumulate,
but is immediately converted into cathenamine.

Natural Product Research
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For MS analysis, the product was eluted from the TLC-plate and could clearly be
identified as cathenamine, showing identical mass fragmentation (major fragments:
3
50, 249, 225, 169 m/z) compared to a reference cathenamine. In order to further
identify cathenamine produced by the enzyme extract of the engineered bacteria and
also to extend the enzymatic synthesis to a chemo-enzymatic approach, reduction
with borohydride was performed, delivering the alkaloid tetrahydroalstonine (THA).
3
.4. Identification of THA as a reduction product
After enzymatic formation of cathenamine (see Figure 3) from tryptamine and
secologanin using the dual vector, incubation mixtures were adjusted to pH 9–10,
and extracted twice with 0.6 mL ethyl acetate. Ethyl acetate fractions were pooled,
evaporated to dryness and resolved in 0.15 mL MeOH followed by incubation at
room temperature for 15 min after supplementing with ꢃ1 mg NaBH . The reduction
4
was terminated by adding 20 mL acetone and the reaction mixture analysed by TLC,
solvent system (1)–(3): (1) petrolether/acetone/diethylamine 7 : 2 : 1. Other solvent
systems confirmed THA as product; (2) n-hexane/diethylether/xylol/ethyl acetate
9 : 8 : 3 : 1, R 0.35; (3) n-hexane/ethyl acetate/diethylether R 0.29. All R values
f f f
coincided with those of the authentic THA sample.
Moreover, elution of the compound at R 0.48 from TLC and its measurement by
f
+
direct-inlet EI-MS gave the characteristic mass spectrum of THA (352 M , 351, 337,
2
23, 184, 169, 156, 143 m/z) and identical fragmentation pattern of reference THA,
which finally allowed verification of its overall chemo-enzymatic synthesis
from tryptamine and secologanin through strictosidine and cathenamine based on
co-expression of STR1 and SG using the newly constructed dual vector pQE-70-
STR1-SG.
4
. Conclusions and prospects
Expression of STR1 and SG cDNAs using the newly constructed vector pQE-70-
STR1-SG in E. coli M15 cells allowed enzymatic formation of cathenamine from
tryptamine and secologanin and proved the functionality of this dual vector
construct. The combination with chemical reduction (borohydride) led to the chemo-
enzymatic synthesis of the alkaloid THA. Thanks to the structure-based mutation of
STR1 (Loris et al., 2007) and to the insertion of additional restriction sites, cDNAs
encoding other alkaloid-biosynthesising enzymes, for example cathenamine reduc-
tase, may extend the use of the vector for multi-step in vitro enzymatic and chemo-
enzymatic approaches of indole alkaloid synthesis. Such a strategy will provide an
important future tool for developing appropriate in vivo production strategies for
pharmacologically interesting indole alkaloids.
Acknowledgements
The authors are grateful to the Deutsche Forschungsgemeinschaft (Bad-Godesberg,
Germany), Fonds der Chemischen Industrie (Frankfurt/Main, Germany) and Zhejiang
University K.P. Chao Foundation (Hangzhou, China) for support. The original STR1 cDNA-
clone was kindly provided by Prof. Kutchan, T.M. (Danforth Center, St Louis, USA).

7
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J. Sto¨ckigt et al.
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