Biochemistry and molecular genetics of the biosynthesis of the earthy odorant methylisoborneol in Streptomyces coelicolor

Article abstract of DOI:10.1021/ja803639g

Methylisoborneol (2) is a volatile organic compound produced by a wide variety of Actinomycete soil organisms, myxobacteria, and cyanobacteria. It has an unusually low odor threshold and, together with geosmin, is responsible for the characteristic smell of moist soil as well as unpleasant taste and odor episodes associated with public water supplies and contamination of various foodstuffs, including fish, wine, and beer. Despite considerable interest in detection and remediation of methylisoborneol, the biosynthesis of this methylated monoterpene has been obscure. In Streptomyces coelicolor, the sco7700 and sco7701 genes are shown to correspond to a two-gene operon responsible for methylisoborneol biosynthesis. Both genes have been amplified by PCR and the resulting DNA has been cloned and expressed in Escherichia coli. Incubation of recombinant SCO7701 protein, annotated as a possible C-methyltransferase, with geranyl diphosphate (1) and S-adenosylmethionine gave the previously unknown compound, (E)-2-methylgeranyl diphosphate (3). Incubation of 3 in the presence of Mg²⁺ with recombinant SCO7700, previously annotated only as a possible metal-binding protein or terpenoid synthase, resulted in the formation of 2-methylisoborneol (2). The steady-state kinetic parameters for both biochemical reactions have been determined. Incubation of geranyl diphosphate and S-adenosylmethionine with a mixture of both SCO7700 and SCO7701 resulted in formation of methylisoborneol (2). Cyclization of 2-methylgeranyl diphosphate (3) to methylisoborneol (2) likely involves the intermediacy of 2-methyllinalyl diphosphate. Copyright

Full text of DOI:10.1021/ja803639g

Published on Web 06/19/2008
Biochemistry and Molecular Genetics of the Biosynthesis of the Earthy
Odorant Methylisoborneol in Streptomyces coelicolor
Chieh-Mei Wang and David E. Cane*
Department of Chemistry, Brown UniVersity, Box H, ProVidence, Rhode Island 02912-9108
Received May 15, 2008; E-mail: David_Cane@brown.edu
Streptomyces are ubiquitous, gram-positive soil bacteria that are
Scheme 1. Biosynthesis of Methylisoborneol
known to produce an enormously wide range of pharmaceutically
1
useful compounds. The 8.7-Mb linear chromosome of S. coelicolor
A3(2), the most thoroughly genetically characterized representative
1
of the genus, harbors 7825 predicted genes that include open-
reading frames (ORFs) for at least three known or presumptive
terpene synthases: sco6073 encoding germacradienol/geosmin
2
a
3
synthase; sco5222, encoding epi-isozizaene synthase; and a third
gene of unassigned function, sco7700. The predicted SCO7700
protein contains a DDCYCED motif, an unusual variant of the
2+
universally conserved Mg -binding, aspartate-rich DDXXD char-
acteristic of terpene synthases, as well as a second conserved
4
2
+
4
Mg -binding “NSE” triad, NDLYSYTKE, located 148 amino
acids downstream of the aspartate-rich motif. We now report the
biochemical characterization of the S. coelicolor terpene synthase,
SCO7700, and the demonstration that the encoded protein works
in combination with the C-methyl transferase SCO7701 to catalyze
the two-step conversion of geranyl diphosphate (GPP) (1) to
Scheme 2. Cyclization of GPP by SCO7700
2
-methyl-isoborneol (2) by way of 2-methylgeranyl diphosphate
(
3) (Scheme 1).
7
b
The 1.3-kb sco7700 gene, encoding a protein of 440 amino acids
products. Little was known about the biosynthesis of 2-methyl-
isoborneol, other than the 1981 report that feeding of either labeled
acetate or methionine to cultures of S. antibioticus gave labeled
methylisoborneol, indicating that methylisoborneol is probably a
was amplified by polymerase chain reaction (PCR) from S.
coelicolor A3(2) genomic DNA and ligated into the pET28a
expression vector. The derived construct, pET28a-sco7700, was
transformed into Escherichia coli BL21(DE3). After induction with
IPTG, the expressed soluble SCO7700 protein, carrying an N-
8
methylated monoterpene. We therefore were intrigued that the 3′-
end of the S. coelicolor sco7700 gene is only16 nucleotides
upstream of the sco7701 ORF, which is annotated only as a putative
C-methyltransferase. We thus speculated that sco7700 and sco7701
might together comprise a two-gene operon for the biosynthesis of
2-methylisoborneol from geranyl diphosphate.
To test this hypothesis, we used PCR to amplify the 879-bp
sco7701 gene from S. coelicolor genomic DNA, ligated the resulting
DNA into pET28a, and used the derived pET28a-sco7701 to
transform E. coli BL21(DE3). After induction with IPTG, the
terminal His
Ni-NTA chromatography. MALDI-TOF analysis gave a M
9937 ( 25 (calcd, 49955). Incubation of recombinant SCO7700
6
-tag, was purified to homogeneity (>95% pure) by
D
of
4
with farnesyl diphosphate or geranylgeranyl diphosphate did not
result in the formation of any detectable sesquiterpene or diterpene
products. By contrast, GC-MS analysis of the products resulting
from the incubation of 60 µM GPP (2) with 25 µM recombinant
SCO7700 revealed the formation of a complex mixture of cyclic
monoterpenes, consisting of R-pinene (4) (6%), ꢀ-pinene (5) (23%),
limonene (6) (32%), γ-terpinene (7) (29%), and δ-terpinene (8)
expressed soluble N-terminal His -tagged SCO7701 protein was
6
purified (>95%) by Ni-NTA affinity chromatography. The protein
(
10%) accompanied by traces of the monoterpene alcohols ꢀ-ter-
had a MD by MALDI-TOF of 34993 ( 17 (calcd, 34987).
Incubation of recombinant SCO7701 (25 µM) with 60 µM GPP
and 120 µM S-adenosyl-L-methionine (SAM) in 4 mL of assay
pineol, 4-terpineol, and R-terpineol (Scheme 2). Although similar
monoterpene mixtures have been reported for several plant mono-
terpene synthases and the K
m
value of 2.8 ( 0.5 µM for cyclization
buffer (50 mM PIPES, 10 mM MgCl , 100 mM NaCl, pH 7.0;
2
of GPP by SCO7700 was well within the range typical for such
18 h, 37 °C) was followed by the addition of 25 µM of purified
recombinant SCO7700 and further incubation (18 h, 30 °C). After
5
-5 -1
synthases, the unusually low kcat of 3.0 ( 0.2 × 10
s
suggested
that GPP might possibly be a surrogate substrate rather than the
natural substrate for the SCO7700 protein.
extraction with pentane/Et O, the combined, concentrated organic
2
extracts were analyzed by capillary GC-MS, revealing formation
Although few microorganisms are reported to produce mono-
terpenes, 2-methylisoborneol (2) is a common volatile organic
of 2-methylisoborneol (2, C11H20O, m/z 168) as the major product,
identical by GC retention time and mass spectrum with an authentic
6
metabolite of many actinomycetes, as well as cyanobacteria and
myxobacteria, with an extremely low threshold for detection (parts
per trillion). Together with geosmin, methylisoborneol is respon-
sible for the characteristic odor of moist soil and can also impart
an unpleasant musty or muddy off-taste to drinking water and food
sample of 2, accompanied by a trace amount of 2-methylbornene
(C11H18, m/z 150).
We next examined the SCO7701-catalyzed reaction directly.
Following incubation of SCO7701 (25 µM) with GPP (60 µM)
and SAM (25 µM) (18 h, 37 °C), the reaction mixture was treated
7a
8
908 9 J. AM. CHEM. SOC. 2008, 130, 8908–8909
10.1021/ja803639g CCC: $40.75  2008 American Chemical Society

C O M M U N I C A T I O N S
with a mixture of apyrase and acid phosphatase (2 h, 30 °C) to
hydrolyze the diphosphate esters. GC-MS analysis of the ether-
extractable products confirmed the formation of (E)-2-methylge-
all consist of ∼435 amino acids, about 100 amino acids larger than
typical microbial terpene synthases. Each of these proteins carries
a ∼100-amino acid peptide leader sequence of unknown function
with an unusually high (25-30%) proline content. The biosynthesis
of the volatile metabolite methylisoborneol appears to be highly
raniol (9) (C11H20O, m/z 168), identical in retention time and mass
9
spectrum to an authentic sample of 9. To further characterize the
1
2
reaction products, a preparative-scale incubation (100 mL, 18 h,
conserved among actinomycetes carrying this pathway.
3
7 °C) was carried out with 6 µmol of GPP, 12 µmol of SAM, and
a total of 0.2 µmol of SCO7700 protein, added in equal batches at
, 6, and 12 h. After washing with Et O, the water-soluble products
Acknowledgment. This research was supported by National
Institutes of Health Grant GM30301 to D.E.C. We thank Tiangang
Liu for assistance with the cloning of the MIB synthase gene.
0
2
were conveniently purified by absorption onto a CHP-20P column
which was then washed with water followed by 5% aq acetonitrile
to elute 2-methylgeranyl diphosphate (3). The H-decoupled
NMR spectrum of 3 showed a pair of doublets (J ) 21 Hz)
corresponding to the presence of the expected diphosphate mono-
Supporting Information Available: Sequence comparisons, ex-
perimental methods, GC-MS data, and full citation for ref 1. This
material is available free of charge via the Internet at http://pubs.acs.org.
1
31
P
ester. Hydrolysis of 3 with apyrase/phosphatase then yielded
1
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13
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13
10
raniol (9), which was also labeled by [methyl- C]-Met. It is thus
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(
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5
diphosphate, except that the penultimate bornyl cation is quenched
1
1
on the exo face by water, analogous to the formation of fenchol,
rather than by internal return of the pyrophosphate to the endo face.
Close homologues of the sco7700/sco7701 two-gene operon can
be found in several streptomycetes, including S. griseus, S.
ambofaciens, and S. scabies, as well as Saccharopolyspora eryth-
raea. Interestingly, the predicted methylisoborneol synthase proteins
JA803639G
J. AM. CHEM. SOC. 9 VOL. 130, NO. 28, 2008 8909