culture broth of S. lividans strain harboring pHSA81–cnsA. The or-
ganic layer was dried with Na2SO4 and then analyzed by GC-MS.
1,8-cineole is converted into an as yet unidentified product by
S. clavuligerus.
In conclusion, we have identified the SSCG_00536 gene
product (CnsA) as a 1,8-cineole synthase. Importantly, CnsA,
which shows significant homology to bacterial SCs, is the first
bacterial monoterpene cyclase to be identified that can effi-
ciently catalyze the direct conversion of GPP. This suggests
that bacterial SC homologues can include monoterpene cyclas-
es, similar to plant SC homologues. Thus, several monoter-
penes found in bacteria[23] could be synthesized by SC homo-
logues. Recent genome sequencing projects have revealed a
large number of potential bacterial terpenoid cyclase genes,
most of which encode SC homologues. The identification of
CnsA as a monoterpene cyclase allows us to speculate that the
diversity of bacterial SC homologues means a diversity of not
only sesquiterpenes but also monoterpenes in bacteria. There-
fore, terpenoids are significantly more widely distributed in
nature than has been previously appreciated, and are wide-
spread among not only eukaryotes, but also bacteria.
Production and purification of the recombinant CnsA: For the
production of N-terminally His10-tagged CnsA, E. coli BL21(DE3)
strain harboring pET16b–cnsA was inoculated into LB medium
(200 mL) containing ampicillin (50 mgmLÀ1) and grown at 26.58C
for 24 h. Cells were harvested by centrifugation and resuspended
in buffer A (50 mm Tris-HCl, 150 mm NaCl, 5 mm imidazole, 10%
glycerol, pH 8.0, 40 mL), then disrupted by sonication. The crude
cell lysate was prepared by removal of cell debris by centrifugation
at 10000g for 20 min. Recombinant protein with a His-tag was pu-
rified by using Ni-NTA superflow (Qiagen) according to the manu-
facturer’s instructions, with the exception that glycerol (10%) was
added to all buffers. The purified CnsA protein was dialyzed
against buffer B (10 mm Tris-HCl, 10% glycerol, pH 8.0). The purity
of the recombinant proteins was checked by SDS-PAGE. The pro-
tein concentration was measured with a NanoDrop 2000 spectro-
photometer (Thermo Scientific, Wilmington, DE, USA).
In vitro enzyme assay: The standard reaction mixture consisted of
Tris-HCl (50 mm, pH 8.0), MgCl2 (5 mm), mercaptoethanol (5 mm),
GPP (5.5 mm) or FPP (4.6 mm), and CnsA (0.5 mm) in a total volume
of 2.5 mL. The reaction mixture, overlaid with hexane (1 mL), was
incubated at 308C for 4 h. The reaction was terminated by the ad-
dition of EDTA (0.5m, pH 8.0, 200 mL) followed by immediate vor-
texing for 30 s. The product was extracted with hexane (1 mL) and
subjected to GC-MS analysis.
Experimental Section
Bacterial strains, plasmids, media and chemicals: E. coli strains
JM109 and BL21(DE3), plasmid pUC19, restriction enzymes and
other DNA-modifying enzymes were purchased from Takara Bio-
chemicals (Shiga, Japan). Plasmid pET16b was purchased from No-
vagen (Darmstadt, Germany). S. clavuligerus ATCC 27064 was ob-
tained from the American Type Culture Collection. S. lividans TK21
was obtained from David A. Hopwood (John Innes Centre, Nor-
wich, UK). Plasmid pHSA81, used for heterologous expression of
cnsA in S. lividans, was obtained from Michihiko Kobayashi (Univer-
sity of Tsukuba, Ibaraki, Japan). S. lividans was cultured in yeast
extract–malt extract (YEME) medium (1% glucose, 34% sucrose,
0.3% yeast extract, 0.3% malt extract, 0.5% bactopeptone, 0.1%
MgCl2·6H2O, pH 7.0). S. clavuligerus was cultured in soy medium
(1.5% soybean flour, 0.47% soluble starch, 0.01% KH2PO4, 0.02%
FeSO4·7H2O, pH 6.8). GPP and FPP were purchased from Sigma.
1,8-Cineole was purchased from Wako (Osaka, Japan).
Kinetic analysis: The reaction mixture for CnsA consisted of HEPES
(50 mm, pH 7.5), MgCl2 (10 mm), mercaptoethanol (5 mm), GPP
(0.05 to 2.7 mm) and CnsA (3.4 nm). The total volume of each reac-
tion mixture was 5 mL. Each reaction mixture, overlaid with hexane
(1 mL), was incubated at 308C for 3 min. Each reaction was termi-
nated by the addition of EDTA (0.5m; pH 8.0, 400 mL) followed by
immediate vortexing for 30 s. The product was extracted with
hexane (1 mL) and subjected to GC-MS analysis. We confirmed that
product formation was linear throughout this period. Steady-state
parameters were determined by fitting the curve to v=
Vmax[S]/(KM+[S]). Data were obtained from three independent ex-
periments.
Examination of terpenoid production in S. clavuligerus: S. clavuli-
gerus was grown in soy medium (100 mL) at 308C for 72 h. Low
molecular weight molecules were extracted with ethyl acetate
(5 mL) from a portion (50 mL) of the culture broth. The organic
layer was dried with Na2SO4 and then analyzed by GC-MS.
Construction of plasmids: All PCR experiments were conducted
by using the S. clavuligerus genomic DNA as a template. The ab-
sence of undesired alterations during PCR was confirmed by nu-
cleotide sequencing. A 1.0 kb DNA fragment containing the cnsA-
coding sequence was amplified by PCR with primer I, 5’-GGA
GAATTC CATATG CCCGCC GGCCAC GAAGAG TTCGAC ATAC-3’
(EcoRI site underlined, NdeI site italicized and the start codon of
cnsA in boldface) and primer II, 5’-CGG AAGCTT GGATCC TGGGGG
CGGAGG GACGGG CGGGTC ACCAAG GGGTGG-3’ (HindIII site un-
derlined and BamHI site italicized) and cloned between the EcoRI
and HindIII sites of pUC19, resulting in pUC19–cnsA. The NdeI–
HindIII fragment excised from pUC19–cnsA was cloned between
the NdeI and HindIII sites of pHSA81, resulting in pHSA81–cnsA.
The NdeI–BamHI fragment excised from pUC19–cnsA was cloned
between the NdeI and BamHI sites of pET-16b, resulting in
pET16b–cnsA.
General analytical methods: GC-MS analysis was performed by
using
a GCMS-QP2010 instrument (SHIMADZU, Kyoto, Japan)
equipped with an Intercap 5MS/Sil capillary column (30 mꢁ
0.25 mm ID, 0.25 mm film thickness, GL Science) by using the EI
mode operated at 70 eV. A temperature gradient of 40 (8 min
hold) to 2608C (208CminÀ1) was used.
Acknowledgements
This work was supported in part by the Targeted Proteins Re-
search Program (TPRP) of the Ministry of Education, Culture,
Sports, Science, and Technology of Japan and a Funding Pro-
gram for Next Generation World-Leading Researchers from the
Bureau of Science, Technology, and Innovation Policy, Cabinet
Office, Government of Japan.
Analysis of terpenoids produced by recombinant S. lividans
strains: S. lividans strain harboring pHSA81–cnsA was inoculated
into YEME medium (100 mL) containing thiostrepton (5 mgmLÀ1
)
and grown at 308C for 72 h. Low molecular weight molecules were
extracted with ethyl acetate (5 mL) from a portion (40 mL) of the
1990
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ChemBioChem 2011, 12, 1988 – 1991