Genome mining in streptomyces avermitilis: Cloning and characterization of sav-76, the synthase for a new sesquiterpene, avermitilol

Article abstract of DOI:10.1021/ja103087w

The terpene synthase encoded by the sav76 gene of Streptomyces avermtilis was expressed in Escherichia coli as an N-terminal-His₆-tag protein, using a codon-optimized synthetic gene. Incubation of the recombinant protein, SAV-76, with farnesyl diphosphate (1, FPP) in the presence of Mg²⁺ gave a new sesquiterpene alcohol avermitilol (2), whose structure and stereochemistry were determined by a combination of ¹H, ¹³C, COSY, HMQC, HMBC, and NOESY NMR, along with minor amounts of germacrene A (3), germacrene B (4), and viridiflorol (5). The absolute configuration of 2 was assigned by ¹H NMR analysis of the corresponding (R)- and (S)-Mosher esters. The steady state kinetic parameters were k_cat 0.040 ± 0.001 s^-1 and K_m 1.06 ± 0.11 μM. Individual incubations of recombinant avermitilol synthase with [1,1-²H₂]FPP (1a), (1S)-[1-²H]-FPP (1b), and (1R)-[1-²H]-FPP (1c) and NMR analysis of the resulting avermitilols supported a cyclization mechanism involving the loss of H-1 _re to generate the intermediate bicyclogermacrene (7), which then undergoes proton-initiated anti-Markovnikov cyclization and capture of water to generate 2. A copy of the sav76 gene was reintroduced into S. avermitilis SUKA17, a large deletion mutant from which the genes for the major endogenous secondary metabolites had been removed, and expressed under control of the native S. avermitilis promoter rpsJp (sav4925). The resultant transformants generated avermitilol (2) as well as the derived ketone, avermitilone (8), along with small amounts of 3, 4, and 5. The biochemical function of all four terpene synthases found in the S. avermtilis genome have now been determined.

Full text of DOI:10.1021/ja103087w

Published on Web 06/10/2010
Genome Mining in Streptomyces avermitilis: Cloning and Characterization of
SAV_76, the Synthase for a New Sesquiterpene, Avermitilol
Wayne K. W. Chou,† Immacolata Fanizza,† Takuma Uchiyama,‡ Mamoru Komatsu,‡ Haruo Ikeda,‡
and David E. Cane*,†
Department of Chemistry, Box H, Brown UniVersity, ProVidence, Rhode Island 02912-9108 and Laboratory of
Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato UniVersity, 1-15-1 Kitasato, Sagamihara,
Kanagawa 228-8555, Japan
Received April 12, 2010; E-mail: David_Cane@brown.edu
Streptomyces are gram-positive bacteria known for their produc-
tion of an enormous variety of biologically active secondary
metabolites.1 The growing number of completed Streptomyces
genome sequences has revealed that only a fraction of the bio-
synthetic potential of these versatile bacteria has been uncovered.1-3
Genome mining has thus provided a powerful new tool for the
discovery of both known and previously unknown natural products
and the elucidation of new biochemical transformations and
biosynthetic pathways.3
36.8 ppm) protons shielded by the cyclopropyl ring. Four methyl
signals were also observed, corresponding to H-12 (δ 0.99, s; C-12
29.8 ppm), H-13 (δ 0.91, s; C-13 15.6 ppm), H-14 (δ 0.87, s; C-14
14.6 ppm), and H-15 (δ 1.00, d, J ) 7.5 Hz; C-15 15.4 ppm), in
addition to the alcohol (δ 1.24) and carbinyl protons, H-1 (δ 3.13;
C-1 79.7 ppm). The absence of any olefinic protons or allylic methyl
groups indicated that 2 was a tricyclic, cyclopropane-containing,
secondary alcohol.
Scheme 1. Cyclization of FPP (1) to Avermitilol (2)
Streptomyces aVermitilis, a well-studied member of this genus,
is used for the industrial production of the important anthelminthic
macrolide avermectin.2,4,5 Sequencing of the S. aVermitilis genome
has revealed four presumptive terpene synthase genes.4 One of
these, ptlA (saV2998), encodes a pentalenene synthase,6 a second,
geoA (sco6073), is a germacradienol/geosmin synthase,7 and a third,
saV3032, encodes an epi-isozizaene synthase.8 The function of the
remaining putative terpene synthase gene, saV76, has not previously
been established. Two highly conserved Mg2+-binding motifs,
characteristic of essentially all terpene cyclases, are evident in the
predicted SAV_76 protein as an aspartate-rich 80DDQFD motif and
the “NSE” triad motif, 239NDVYSLEKE.9 We now report that
SAV_76 catalyzes the cyclization of farnesyl diphosphate (1, FPP)
to a previously unknown tricyclic sequiterpene alcohol, which we
have named avermitilol (2) (Scheme 1).
A synthetic saV76 gene, with codons optimized for expression in E.
coli, was cloned into the pET28a(+) expression vector. The resultant
plasmid was transformed into E. coli BL21(DE3) and used for high-level
expression of recombinant SAV_76 protein carrying an N-terminal His6-
tag, which was affinity-purified using Ni-NTA chromatography.
Incubation of purified recombinant SAV_76 with FPP in the
presence of MgCl2 gave a sesquiterpene alcohol (2) (m/z 222) as the
major enzymatic reaction product (85%,), accompanied by germacrene
A (3) (1%), germacrene B (4) (5%), and the known tricyclic
sesquiterpene alcohol, viridiflorol (5) (3%), as well as several unidenti-
fied minor sesquiterpene products, as determined by capillary GC-
MS analysis. The steady-state kinetic parameters for the SAV_76-
catalyzed reaction, measured by monitoring the formation of 2 using
[1-3H]FPP, were kcat 0.040 ( 0.001 s-1 and Km 1.06 ( 0.11 µM,
comparable to those for typical terpene synthases.6-9
The 1H-13C connectivity in 2 was established by a combination
of HMQC and HMBC spectroscopy (Figure 1A). Long-range
1H-13C correlations defining the cyclopropane ring were observed
between the methyl H-13 protons and C-6, C-7, and C-11 of the
cyclopropane ring, in addition to the reciprocal crosspeaks between
the protons and carbons of the geminal methyl pair. Pairwise 2-
and 3-bond correlations between H-5 and C-1, C-4, C-6, C-9, C-10,
C-11, C-14, and C-15 established the central position of the
bridgehead H-5 proton. Additional crosspeaks between H-1 and
C-2, C-3, C-9, C-10, and C-14 as well as between H-9ax and C-1,
C-5, C-8, C-10, and C-14 established the remaining connectivity.
The structure of sesquiterpene alcohol 2 was assigned by a
combination of 1-D and 2-D NMR spectroscopy. Key resonances
observed in the 1H NMR and HMQC spectra were the two upfield
methine proton signals corresponding to H-6 (δ 0.47; C-6 22.2 ppm)
and H-7 (δ 0.53; C-7 19.3 ppm) attached to a cyclopropane ring,
as well as the H-5 (δ 0.94; C-5 40.5 ppm) and H-9ax (δ 0.63; C-9
Figure 1. (A) HMBC and (B) NOESY correlations for 2.
The relative stereochemistry of sesquiterpene 2 was readily
deduced from the NOESY NMR spectrum. Major NOESY
† Brown University.
‡ Kitasato University.
9
8850 J. AM. CHEM. SOC. 2010, 132, 8850–8851
10.1021/ja103087w  2010 American Chemical Society

C O M M U N I C A T I O N S
correlations established the presence of a trans-decalin ring system
with a cis-fused dimethylcyclopropane ring as deduced from
NOESY crosspeaks among the H-6 and H-7 methine protons and
the H-12 methyl signals (Figure 1B). The H-6 proton also displayed
a cross peak to the H-15 secondary methyl, which itself had a
crosspeak to the H-14 methyl at the ring junction. Additional
NOESY correlations were also observed between H-1 and each of
its 1,3-diaxial partners H-3ax, H-5, and H-9ax. This H-9ax proton
signal also displayed crosspeaks with both H-5 and the H-13 methyl.
The absolute configuration of 2 was assigned by 1H NMR
analysis of the derived (R)- and (S)-Mosher esters of the C-1
secondary alcohol,10 leading to the assignment of the absolute
configuration of 2 as 1S, 4R, 5S, 6R, 7R, 10S. Avermitilol (2) is a
new tricyclic sesquiterpene alcohol whose isolation has not been
previously reported.11
aVermitilis FPP synthase, along with saV76 increased the titers of
both 2 and 8. The formation of avermitilone (8) may result from
adventitious oxidation of 2 by an endogeneous dehydrogenase, since
no dehydrogenase gene is evident in the genome of S. aVermitilis
immediately upstream or downstream of the native saV76 cyclase gene.
To probe the stereochemical course of the SAV_76-catalyzed
reaction, recombinant SAV_76 was incubated in separate experi-
ments with [1,1-2H2]FPP (1a), (1S)-[1-2H]FPP (1b), and (1R)-
[1-2H]FPP (1c) (Scheme 1). Unlabeled 2 resulted from the incuba-
tion with (1R)-[1-2H]FPP, while [6-2H]-2a was formed in incuba-
tions with [1,1-2H2]FPP and with (1S)-[1-2H]FPP. The GC-mass
spectra of 2a and 2b had parent peaks m/z 223, corresponding to
[M+1]+, indicating that the H-1si proton of FPP is retained while
the H-1re of FPP is lost during formation of the cycloproprane ring
of avermitilol (2). The position of the deuterium label in [6-2H]-2a
was established by the absence of the normal H-6 proton signal at
δ 0.47. The concomitant loss of the vicinal couplings between H-6
and both H-5 and H-7 in [6-2H]-2a also supported the assigned
position of the deuterium label.
These labeling experiments are consistent with a mechanism for
the formation of avermitilol (2) in which FPP undergoes initial
ionization with electrophilic attack on the si-face of the distal double
bond to form a germacradienyl cation (6) (Scheme 1). Insertion of
the 2-propyl cation into the C-H bond with loss of the original
H-1re proton of FPP would result in formation of the enzyme-bound
bicyclogermacrene (7). Proton-initiated anti-Markovnikov cycliza-
tion of 7 and quenching of the tricyclic secondary carbocation by
water would yield avermitilol (2). Consistent with this proposed
mechanism is the observed formation of the minor products
germacrene A (3) and B (4) by alternative deprotonation of the
germacradienyl cation. The coproduction of the isomeric viridiflorol
(5) can be explained by competing proton-initiated Markovnikov
cyclization of bicyclogermacrene to form the cis-fused 5,7-ring
system followed by capture of water.
Although, avermitilol (2) was not detected in extracts of wild-
type S. aVermitilis, the in ViVo activity of the saV76 gene could be
directly demonstrated using a genome-minimized mutant, S. aVer-
mitilis SUKA17, from which >1-Mb of DNA had been deleted,
including the genes for the major endogenous secondary metabolites
produced by the parent strain.12 GC-MS analysis of hexane extracts
of cultures of S. aVermitilis SUKA17 harboring saV76 under control
of the native S. aVermitilis promoter rpsJp (saV4925) showed the
presence of avermitilol (2, 15%,), accompanied by small quantities
of germacrene A (3, 10%), germacrene B (4, 5%), and viridiflorol
(5, 2%) (Figure 2). The major component of the mixture was ketone
avermitilone (8, 67%, m/z 220), whose structure was confirmed by
1H and 13C NMR and direct comparison with a reference sample
prepared by oxidation of 2 with pyridinium chlorochromate.
Cointroduction of the ptlB gene (saV2997), encoding the native S.
Figure 2. GC-MS analysis of production of avermitilol (2) and avermitilone
(8) along with viridiflorol (5), germacrene B (4), and germacrene A (3) by
transformed S. aVermitilis SUKA17. (A) Control with plasmid pKU460.
(B) pKU460-rpsJp::saV76. C. pKU460-rpsJp::saV76-ptlB.
We have now assigned the biochemical functions of all four
terpene synthases originally revealed by the sequencing of the S.
aVermitilis genome.6-8 Avermitilol (2) is a new sequiterpene whose
isolation has not previously been reported. The saV76 gene product
has one close orthologue, SSAG_00457 (Uniprot ID B4UXV1)
which is found in Streptomyces sp. Mg1, with 78% identity and
85% positive matches over 334 amino acids.
Acknowledgment. This work was supported by NIH grant
GM30301 (D.E.C.), and by Grants-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan (MEXT) and from the Japan Society for the
Promotion of Science (JSPS) 20310122 (H.I.). We thank Tun-Li
Shen for assistance with mass spectrometry and Russell Hopson
for assistance with NMR.
Supporting Information Available: Sequence comparisons, ex-
perimental methods, and NMR and GC-MS data. This material is
available free of charge via the Internet at http://pubs.acs.org.
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