Callinstr. 5, 30167 Hannover, Germany

Center of Applied Chemistry
Institute of Technical Chemistry
Innovation cluster
Refinement of plant resources
Callinstr. 5, 30167 Hannover, Germany
Fusion protein and solubility enhancing strategies for heterologous
expression of novel plant sesquiterpene synthases
S. Hartwig , T. Frister , T. Scheper , S. Beutel
1
1
Introduction
1
Institute of Technical Chemistry, Leibniz University of Hannover, Germany
Cloning of protein expression plasmids
The plant Vetiveria zizanoides grows natively in Madagascar and is reknown
for its pleasant vetiver oil, consisting mainly of khusimol and a-/b-vetivone.
Although no terpene cyclase catalyzing the formation of these two components is known to date, a cDNA sequence coding for their precursor (+)-zizaene (GenBank HI931360) was identified earlier
[2], but no further data describing the enzyme was
published up to date. The sequence was carefully codon optimized, synthesized as two indepent
double-stranded DNA strings, and cloned into expression vectors using a modified Gibson-assembly
[3] approach. Constructs were transformed into E.
coli BL21(DE3) competent cells to utilize the T7Promoter and cspA-Promoter (cold shock protein
A) driven protein expression.
Fig. 3 Root systems of two cultivars of V. zizanoides,
grown and harvested in Madagascar.
1
);D?VLWH
]L]DHQHV\QWKDVHFR
(FR 5,
&OD ,
%OS ,
%DP +,
;KR ,
3VS ;,
&
1
SRO\+,6
);D?VLWH
]L]DHQHV\QWKDVHFR
ODF?,
6IR ,
1DU ,
.DV ,
S(7EKLV);D=,=$FR
ESV
$FF,
%VW,
7WK ,
7?SURPRWHU
6QD %,
$OZ 1,
(DJ ,
$PS?UHVLVWDQFH
%VP ,
(FR 2,
0OX ,
%VW (,,
3VS 20,
$SD ,
%VV +,,
+SD ,
)VS $,
%JO ,
1UX ,
(DJ ,
3VK $,
Fig. 4 Plasmid map and schematic representation of the pET
vector construct utilizing T7 promoter for protein expression.
%JO ,
3SX 0,
1FR ,
6W\,
)VS $,
6DS ,
6802?IXVLRQ
3VL ,
(FR N,
6DF,
$FF,
.SQ ,
$EV,
3VS ;,
;KR ,
%DP +,
(FR 5,
+LQ G,,,
$FF,
6DO ,
%VS 0,
;ED ,
1FR ,
S(76802KLV6802FY=,=$FR
ESV
6QD %,
(DJ ,
Enzymatic production
of (+)-zizaene
Fig. 2 Chemical structure of the sesquiterpene (+)-zizaene
Expression and purification of the recombinant synthase
Expression experiments using the His-tagged construct
(pET16b) yielded no detectable soluble protein production,
even during low temperature cultivations. In contrast, both
the cspA-Promoter driven induction as well as fusion to
a ubiquitin-modifier moeity resulted in strong and efficient soluble expression of the plant enzyme in E. coli. The
relatively low cultivation temperatures (15 °C) needed for
cspA induction slow down the protein translation machinery in the organism, so
that proper folding is possible. The SUMO domain is
highly soluble in E. coli and was shown previously to enable and enhance the solubility of
fusion partners attached to the N-terminus
[4]. Purification was performed on a sepharose column decorated with Ni2+ (GE Healthcare
HiTrap™ IMAC FF 5 ml), using a two elution
step method. Both enzymes were successfully
purified as shown by SDS-PAGE analysis and
western blots of the corresponding fractions.
Fig. 8
Western Blot using His-epitope
antibody, (A) elution fraction of purified
pColdI::Ziz(co) raw extract. (B) elution
fraction of purified pETSUMO::Ziz(co) raw
extract.
Fig. 7 SDS-PAGE showing purification steps of zizaene synthase by use of
Ni2+ based IMAC method. Using (A) pColdI-construct (theor. MW = 66 kDa).
(B) pETSUMO-construct (theor. MW = 77.4 kDa). RE = raw extract, FT = flow
through frac., WF = wash fractions, EF = elution fractions,
UF = after dialysis/ultrafiltration using a MWCO of 10,000 Da
7?WHUPLQDWRU
.DQ?UHVLVWDQFH
'UD ,,,
3VL ,
$OZ 1,
$VL 6,
3YX ,
$YD ,
6PD ,
;PD ,
Fig. 6 Plasmid map and schematic representation of the
SUMO fusion construct used in this study.
E. coli BL21(DE3)
pColdI::Ziza(co)
50 µM FPP
1 h @ 30 °C
intensity [mV]
Bioconversions of the substrate farnesyl
pyrophosphate (FPP) were carried out in
ml-scale batch reactions using purified
zizaene synthase enzyme (elution fractions). The liquid phase consisting of buffer, enzyme, and substrate were overlaid
by isooctane to yield a two-phase system.
Optimal reaction conditions were pH 7.0,
1h @ 30 °C. After a short extraction process, the upper organic phase was analyzed by GC-FID and compared to standard
sesquiterpene compounds. Both recombinant enzymes were active and produced (+)-zizaene from FPP.
+LQ G,,,
7WK ,
%VW ,
$FF ,
6DS ,
3FL ,
6FD ,
Fig. 5 Plasmid map and schematic representation of the
construct utilizing the cspA promoter for cold shock induction.
%VU *,
%VD ,
=,=BFR?&'6
3VK $,
S&ROG,KLV);D=,=$FR
ESV
3FL ,
ODF?RSHUDWRU
ODF?,
=L]BFR?&'6
&
$IO ,,
(FR 5,
0IH ,
$OZ1,
]L]DHQHV\QWKDVHFR
+,6?WDJ
%VU *,
$PS?UHVLVWDQFH
&9
1UX ,
7((
ODF?,
6802
0OX ,
3VS 20,
$SD ,
)DFWRU;D?VLWH
3VS 20,
$SD ,
6SK ,
SRO\+,6
7?SURPRWHU
0OX ,
$IO ,,,
6FD ,
=,=FR?&'6
+LV?WDJ
1
+LV?WDJ
(FR 1,
$DW,,
=UD ,
%VD ,
$KG ,
)DFWRU;D?VLWH
&
FVS$?SURPRWHU
7?WHUPLQDWRU
1
Plant essential oils consist mainly of terpenoids, which are used extensively by the fragrance
industry in everyday personal care products and costly perfumes. The extraction process of the
relevant plant source material is often laborious, unreliable and cost demanding. Biotechnology
enables new approaches to interesting terpene compounds. We over-expressed a plant enzyme
catalyzing the synthesis of (+)-zizaene, an interesting and valuable precursor to a-vetivone, in
a recombinant E. coli host. As the class of plant sesquiterpene synthases are considered hard-to
-express in a soluble form, different solubility enhancing strategies were evaluated in this study.
The recombinant enzyme catalyzed the production of (+)-zizaene from farnesyl pyrophosphate.
Fig. 1 Putative structure of recombinant
zizaene synthase, modelled by use of the
I-TASSER algorithm [1]
SRO\+,6
1
E. coli BL21(DE3)
pETSUMO::Ziza(co)
50 µM FPP
1 h @ 30 °C
Discussion and Outlook
This study shows, how important the choice of strain and fusion tag strategy
is when expressing eukaryotic genes in a prokaryotic host. Fusing a modified
ubiquitin moeity to the sesquiterpene synthase significantly increased the
yield of soluble enzyme while retaining its activity. In addition, use of a cspA
controlled induction at very low temperatures was able to increase expression levels. An IMAC-based Ni2+ chromatography step, exploiting a N-terminal
His-Tag epitope, produced high amounts of purified (> 90 %) enzyme. In GCFID analysis of batch bioactivity assays, (+)-zizaene could be identified as the
only product resulting from cyclization of FPP. Further studies will be undertaken to characterize kinetic parameters of the novel synthase.
zizaene
Literature
E. coli BL21(DE3)
no insert control
50 µM FPP
1 h @ 30 °C
retention time [min]
Fig. 9 GC-FID chromatograms showing bioactivity assays of the two different
zizaene synthase constructs.
QR Code for direct
PDF download:
[1] Roy et al., Nat. Protoc. (2010), 725-738
[2] Schalk et al., Patent US2012/0021475A1
[3] Gibson et al., Nat. Methods (2009), 343-345
[4] Marblestone et al., Protein Sci. (2006), 182-189
Acknowledgements
The authors would like to thank Prof. Berger and Dr. Krings (Institute of Food Chemistry, University Hannover) for assistance with terpene analytics. This work was funded by the European Union as part of the
EFRE (European Regional Development Fund) project „Refinement of plant resources“ (ZW 8-80130940).

Download Report