Pentalenic acid is a shunt metabolite in the biosynthesis of the pentalenolactone family of metabolites: Hydroxylation of 1-deoxypentalenic acid mediated by CYP105D7 (SAV-7469) of Streptomyces avermitilis

Article abstract of DOI:10.1038/ja.2010.135

Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462-ptl- clusterΔptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Δsav7469 carrying pKU462-ptl-clusterΔptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.

Full text of DOI:10.1038/ja.2010.135

The Journal of Antibiotics (2011) 64, 65–71
2011 Japan Antibiotics Research Association All rights reserved 0021-8820/11 $32.00
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&
ORIGINAL ARTICLE
Pentalenic acid is a shunt metabolite in the
biosynthesis of the pentalenolactone family of
metabolites: hydroxylation of 1-deoxypentalenic
acid mediated by CYP105D7 (SAV_7469)
of Streptomyces avermitilis
Satoshi Takamatsu1,4, Lian-Hua Xu2,4, Shinya Fushinobu2, Hirofumi Shoun2, Mamoru Komatsu1,
David E Cane3 and Haruo Ikeda1
Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic
pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the
genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting
of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are
involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated
to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as
its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene
encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally
different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11
carrying pKU462Hptl-clusterDptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an
extra-copy of the sav7469 gene to SUKA11 Dsav7469 carrying pKU462Hptl-clusterDptlH restored the production of pentalenic
acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid
(5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and
in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid
(5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.
The Journal of Antibiotics (2011) 64, 65–71; doi:10.1038/ja.2010.135; published online 17 November 2010
Keywords: biosynthesis; cytochrome P450; pentalenolactone; sesquiterpene; Streptomyces avermitilis
INTRODUCTION
muscle cell proliferation.7 The first step in pentalenolactone biosynth-
Pentalenolactone is a sesquiterpenoid antibiotic first discovered in esis is the cyclization of farnesyl diphosphate, the universal precursor
1957 in the culture extracts of Streptomyces roseogriseus,1 and subse- of all sesquiterpenes, to pentalenene (2), which is the parent hydro-
quently isolated from numerous Streptomyces microorganisms.2–4 carbon of the pentalenolactone family of metabolites. From the results
Pentalenolactone is active against both Gram-positive and Gram- of feeding experiments for pentalenolactone biosynthesis, a variety of
negative strains of bacteria, as well as pathogenic and saprophytic plausible intermediates in the conversion of pentalenene to pentale-
fungi.5 Pentalenolactone is also a potent and specific antiviral agent, nolactone have been isolated, including 1-deoxypentalenic acid (5; as
inhibiting the replication of DNA viruses, such as the causal agent of glucuronate ester),4 as well as pentalenic acid (1),8,9 a demonstrated
herpes simplex HSV-1 and HSV-2,6 and can inhibit vascular smooth shunt metabolite of the main pentalenolactone biosynthetic pathway.
1Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, Minami-ku, Sagamihara, Kanagawa, Japan; 2Department of Biotechnology, Graduate
School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan and 3Department of Chemistry, Brown University, Providence, RI, USA
4These authors contributed equally to this work.
Correspondence: Professor H Ikeda, Laboratory of Microbial Engineering, Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara,
Kanagawa 252-0373, Japan.
E-mail: ikeda@ls.kitasato-u.ac.jp
Dedicated to the late Dr C Richard Hutchinson for his exceptional contributions to natural product biosynthesis, engineering, and drug discovery and in honor of his friendship.
Received 15 August 2010; revised 16 October 2010; accepted 17 October 2010; published online 17 November 2010

Hydroxylation of 1-deoxypentalenic acid
S Takamatsu et al
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Figure 1 AseI-physical map of S. avermitilis and distribution of biosynthetic genes encoding terpenoid compounds (sav76, gene for avermitilol biosynthesis;
crt, genes for isoneriatene biosynthesis; hop, genes for squalene and hopanoid biosynthesis; sav2163, gene for germacradienol and geosmin biosynthesis;
sav3032, gene for epi-isozizaene biosynthesis) (a); and gene cluster for neopentalenoketolactone biosynthesis (sav2989, gene encoding MarR-family
transcriptional regulator; gap1, gene for pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase; ptlH, gene for 1-deoxypentalenic acid 11-b
hydroxylase; ptlG, gene for transmembrane efflux protein; ptlF, 1-deoxy-11b-hydroxypentalenic acid dehydrogenase; ptlE, gene for Baeyer-Villiger
monooxygenase; ptlD, gene for dioxygenase; ptlC, gene for hypothetical protein; ptlB, gene for farnesyl diphosphate synthase; ptlA, gene for pentalenene
synthase; ptlI, gene for pentalenene C13 hydroxylase CYP183A1; sav3000, gene for AraC-family transcriptional regulator; sav3001, gene for lyase; sav3002,
gene for hypothetical protein) (b). Two genes, sav7469 and sav7470, encode CYP105D7 and FdxH, respectively.
The fact that tritium label from (1R)-[1-3H] pentalenene (2) was lost
We have thus, elucidated all but the final step in the biosynthetic
upon formation of pentalenic acid (1), but was retained in the derived pathway for the pentalenolactone variant, neopentalenoketolactone
pentalenolactone has definitively ruled out 1 as an intermediate in the (9), whereas the genetic and biochemical basis for the accumulation of
formation of pentalenolactone.9 A plausible biosynthetic pathway for pentalenic acid (1) in S. avermitilis remained to be clarified. The shunt
pentalenolactone biosynthesis has been proposed on the basis of the product pentalenic acid (1) is thought to be formed by C-1 hydro-
structures of these isolated pentalenolactone metabolites, but until xylation of 1-deoxypentalenic acid (5), an intermediate in the bio-
recently, however, there had been no direct experimental evidence for synthesis of the pentalenolactone and neopentalenolactone family of
biosynthetic sequence leading from pentalenene (2) to the pentaleno- metabolites, presumably under the control of a suitable hydroxylase or
lactone family of metabolites.
cytochrome P450 (CYP). Significantly, however, no candidate gene
Streptomyces avermitilis is a Gram-positive soil bacterium respon- encoding such a hydroxylase is evident in the 13.4-kb ptl cluster. In
sible for the production of the anthelminthic macrocyclic lactone fact, several S. avermitilis CYP genes are located within the biosyn-
avermectins that are widely used in human and veterinary medicine. thetic clusters for avermectin, oligomycin, filipin, albaflavenone and
Sequencing of the complete 9.03-Mb linear genome of S. avermitilis neopentalenolactone biosynthesis. Other CYP genes are distributed
revealed at least 37 presumed biosynthetic gene clusters related independently throughout the linear chromosome of S. avermitilis.20
to secondary metabolism, with at least six of them encoding It is, therefore, of considerable interest to identify which S. avermitilis
putative terpenoid biosynthetic pathways as shown in Figure 1a.10–13 CYP gene is responsible for the conversion of 1-deoxypentalenic acid
Among the latter, a 13.4-kb gene cluster centered at 3.75Mb in the (5) to pentalenic acid (1). We now provide evidence that CYP105D7
S. avermitilis genome and containing 13 unidirectionally transcribed encoded by sav7469 is responsible for hydroxylation at the C-1
open reading frames (sav2990—sav3002) has been implicated in the position of 1-deoxypentalenic acid (5), resulting in accumulation of
biosynthesis of pentalenolactone-like metabolites (Figure 1b). pentalenic acid (1) as a shunt metabolite of pentalenolactone or
Although pentalenolactone itself has not been detected in the organic neopentalenolactone biosynthesis.
extract of S. avermitilis, we have isolated the common pentalenolac-
tone shunt metabolite pentalenic acid (1) from these cultures.14
MATERIALS AND METHODS
Bacterial strains
Deletion of the 13.4-kb operon from S. avermitilis abolished the
production of both neopentalenoketolactone (9) and pentalenic acid
(1), whereas transfer of the entire gene cluster to Streptomyces. lividans
1326, a strain that normally does not produce pentalenolactones,
resulted in generation of pentalenic acid (1). In parallel with these
molecular genetic investigations, we have been systematically expres-
sing in E. coli the individual genes from the 13.4-kb biosynthetic
cluster in order to identify the natural substrates and intrinsic
biochemical reactions for each open reading frame (Figure 2). Direct
evidence for the function of the 13.4-kb cluster came from the
expression of recombinant ptlA (sav2998),14 ptlI (sav2999),15 ptlH
(sav2991),16,17 ptlF (sav2993)18 and ptlE (sav2994),19 respectively.
The fact that the DptlD mutant of S. avermitilis accumulates neo-
pentalenolactone D (8) suggests that 8 is likely to be the natural
substrate for the ptlD gene product in the formation of neopentale-
noketolactone (9).20
The large-deletion mutants of S. avermitilis, SUKA5, SUKA7 (SUKA5
Dsav7469), SUKA11 (SUKA5 Dptl-clusterHermE) and SUKA13 (SUKA11
Dsav7469),21 were used for production of neopentalenolactone and related
compounds. Culture conditions for the sporulation and regeneration of
protoplasts after PEG-mediated protoplast transformation have been pre-
viously described.22,23
Construction of DptlH-deletion mutant
The DptlH-deletion mutants were prepared using the entire 14.9-kb
S. avermitlis ptl-cluster clone (pKU462Hptl-cluster), in which ptlA had been
placed under the control of the ermE promoter, as previously described,14,19
using as host strains S. avermitilis SUKA11 and SUKA13. In-frame DptlH-
deletion mutant: forward primer, 5¢-ACCACGGCCACTGAGGTCTCACAAGG
AGATGAATCTGTGGCCAGTGAGTTCGAGCGACT-3¢ (bold characters indi-
cate the upstream region of ptlH on the chromosome and underlined
characters correspond to the start codon of ptlH) and the reverse primer
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Hydroxylation of 1-deoxypentalenic acid
S Takamatsu et al
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Figure 2 Proposed neopentalenoketolactone biosynthetic pathway in S. avermitilis. a-KG indicates a-ketoglutarate.
5¢-GCCCGCCGGGGAAAATCTCCGGGTGGGGGTTGGTCGTCACCCCGGG
of the sav7469 gene, the two genes appear to belong to a single transcriptional
TACCGAGCGAAC-3¢ (bold characters indicate the downstream region of ptlH unit. An operon containing both sav7469 and sav7470 was amplified by PCR
on the chromosome and underlined characters correspond to the stop codon of from template DNA from cosmid CL_228_E11 using the primer pair, forward:
ptlD). This primer pair was used for the00amplification of the loxP-aad(300)-loxP 5¢-GCCCATATGACAGAGCCCGGTACGTCCGT-3¢ (underlined characters
segment using pKU473 (pULwLHaad(3 )) as template.19 The initial denatura- indicate the NdeI site and bold characters correspond to the start codon of
tion step (95 1C, 5 min) was followed by five cycles of amplification (95 1C, sav7469) and reverse: 5¢-GCACTAGTTCAGCTTGCCGACCGCGGGAC-3¢
30 s; 501C, 30s; 721C, 60s) followed by 25 cycles using an annealing (underlined characters indicate the SpeI site and bold characters correspond
temperature of 65 1C, and then a final incubation at 721C (10 min). The to the stop codon of sav7470). The amplified DNA segment was digested with
desired in-frame deletion plasmid, pKU462Hptl-clusterDptlH, was constructed NdeI and SpeI and the digested segment was ligated with the NdeI/XbaI
using the l recombination system and Cre/loxP-based site-specific excision, as fragment of pKU460HxylAp-sav3129-sav5675.24 The resultant recombinant
previously described.19,21
plasmid pKU460HxylAp-sav7469-sav7470-sav3129-sav5675 was digested with
NdeI/HindIII and the resultant NdeI/HindIII segment sav7469-sav7470-
sav3129-sav5675 was ligated with the large segment of NdeI/HindIII
pKU460HrpsJp.21 The plasmid isolated from the resulting transformants was
digested with NheI/HindIII and the NheI/HindIII segment, rpsJp-sav7469-
sav7470-sav3129-sav5675, was ligated with the large segment of NheI/HindIII
pKU490Hhph and the ligation product was used to transform E. coli DH5a.
The desired plasmid, pKU490HrpsJp-sav7469-sav7470-sav3129-sav5675,
obtained by selection for hygromycin B resistance, was transferred into
S. avermitilis SUKA13 carrying pKU462Hptl-clusterDptlH by conjugation, as
previously described.25
Isolation of products from S. avermitilis and its deletion mutants
The cultivation of in-frame deletion mutants was performed as previously
described.14,19 After cultivation, an equal volume of methanol was added and
mixed for 10min. The mycelia were sedimented by centrifugation, the super-
natant was evaporated under reduced pressure to remove methanol. The
aqueous extract was diluted with a half volume of water, adjusted to pH 2
with 2 N HCl and extracted twice with a half volume of EtOAc. The resulting
organic extracts were combined, dried under Na2SO4 and evaporated to
dryness. The brownish oily residue was dissolved in 0.1 ml of benzene-
methanol (9:1) and the products were methylated by adding 0.02 ml of
trimethylsilyldiazomethane10% in n-hexane) for 15 min at ambient tempera-
ture. After removal of solvent by evaporation, the methylated products were
re-dissolved in a small amount of methanol and a portion of the extract was
subjected to gas chromatography-MS (GC-MS) analysis (Shimadzu GC-17A,
70 eV, electron impact, positive ion mode; 30mꢀ0.25mm f Neutra Bond-5
capillary column (5% phenylmethylsilicon) using a temperature program of
50–280 1C and temperature gradient of 201C minꢁ1, inlet temperature 280 1C;
run time 20 min).
Heterologous expression and purification of CYP105D7
(SAV_7469)
The sav7469 gene (8895 895–8 897103 nt in S. avermitilis) was amplified by
PCR with template DNA from cosmid CL_228_E11 using the primer pair,
forward: 5¢-CGGAATTCCATATGACAGAGCCCGGTACGTCCGTGTC-3¢ (under-
lined characters indicate the NdeI site) and reverse: 5¢-GGACTAGTTCAGTGGT
GGTGGTGCCAGGTCACGGGGAGTTCCAGCATC-3¢ (underlined characters
indicate the SpeI site and bold characters represent His4 tag). The PCR program
employed was as follows: initial denaturation step (96 1C, 2 min), followed by 30
cycles of amplification (96 1C, 30 s; 65 1C, 30 s; and 72 1C, 60 s), and then a final
Construction of the sav7469 expression cassette
As the sav7470 gene (8897 117–8 894401 nucleotide in the S. avermitilis incubation at 72 1C (5 min). The resultant amplicon and pET17b vector were each
genome) encoding a ferredoxin (FdxH) is located 14-basepairs downstream then double-digested with NdeI and SpeI. The digested PCR product was ligated
The Journal of Antibiotics

Hydroxylation of 1-deoxypentalenic acid
S Takamatsu et al
68
with the large NdeI/SpeI fragment of pET17b by T4 DNA ligase. The ligation
mixture was used to transform competent cells of E. coli JM109. The resultant
plasmid, pET17bHsav7469, isolated from cultures grown on LA medium contain-
ing 50 mgmlꢁ1 of ampicillin, was used to transform E. coli BL21 CodonPlus (DE3).
Expression of the sav7469 gene and purification of C-terminal His4-tagged
CYP105D7 was performed by procedures similar to those described previously.26
The purified CYP105D7 (10 mg mlꢁ1) was flash-frozen in small aliquots in liquid
N2 and stored at ꢁ80 1C. The measurement of UV-visible absorption spectrum
under oxidized and reduced conditions, and CO-difference spectrum was per-
formed by procedures described previously.27
Preparation of 1-deoxypentalenic acid (5)
The culture conditions for the production of 1-deoxypentalenic acid by S.
avermitilis SUKA13 carrying pKU462Hptl-clusterDptlH were described pre-
viously.19 Around 2 l of the culture supernatant was acidified to pH 2.5 with
2N HCl and the organic products were extracted twice with 1 l of EtOAc. The
organic extracts were combined, dried under Na2SO4 and evaporated to dryness.
The brownish oily material was dissolved in 2 ml of benzene–methanol (9:1) and
treated with 0.1 ml of trimethylsilyldiazomethane for 15 min at room tempera-
ture. After removal of solvent, methylated products were subjected to silica gel
column chromatography using CHCl3 as eluent with collection of the (5) methyl
ester-rich fraction. Pure (5)-methyl ester was obtained by preparative HPLC
developing with 90% acetonitrile in water. The resulting (5)-methyl ester (27 mg)
was hydrolyzed with 5mM K2CO3 in 30% (v/v) aqueous methanol (reflux, 24 h).
After removal of the methanol under reduced pressure, the resultant aqueous
solution was adjusted to pH 2.5 with 2 N HCl and extracted with equal volume of
EtOAc to give 17 mg of 1-deoxypentalenic acid (5) as a colorless oil. The
Figure 3 GC-MS analysis of EtOAc extracts treated with trimethylsilyl-
diazomethane of S. avermitilis SUKA5 (a) and SUKA7 (SUKA5 Dsav7469H
aadA) (b). The two strains were cultured at 28 1C with shaking at 200r.p.m.
for 96h.
we have termed neopentalenolactone.19 One of the large-deletion
mutants of S. avermitilis, SUKA5, produced pentalenic acid (1) and
neopentalenoketolactone (9), which underwent facile conversion to
the hydroxyketo seco acid form (10) (Figure 3a). We had indepen-
dently constructed sav7469-deletion mutants for the bioconversion
experiments. The mutant SUKA7, which harbors a deletion of the
gene encoding CYP105D7 (SAV_7469), still produced neopentaleno-
ketolactone (9) and its hydrolyzed derivative (10) but did not produce
pentalenic acid (1) (Figure 3b). This result indicates that CYP105D7
is most likely involved in the formation of pentalenic acid (1) in
S. avermitilis. To clarify the role of CYP105D7 in the hydroxylation of
1
structure of 5 was confirmed by the H and 13C NMR spectra.
In vitro conversion of 1-deoxypentalenic acid (5) to pentalenic acid
(1) by CYP105D7
The catalytic reaction of CYP105D7 was performed using the following
reaction mixture: 0.4 mM 1-deoxypentalenic acid (5) in DMSO, 4.4 mM spinach
Fdx, 0.05U of spinach Fdx-NADP+ reductase, 0.5 mM NADP+, 0.5 mM glucose-
6-phosphate, 0.5 U of glucose-6-phosphate dehydrogenase, 50mM Tris-HCl
(pH 8.0), 10% (v/v) of glycerol and 1.1 mM purified C-terminal His4-tagged
CYP105D7, in a total volume of 200ml. The reaction mixture without
CYP105D7 was pre-incubated for 15 s at 25 1C before addition of CYP105D7 1-deoxypentalenic acid (5) at C-1, a biosynthetically blocked mutant
with a disrupted ptlH (sav2991) gene,16 encoding a non-heme iron,
a-ketoglutarate-dependent hydroxylase that catalyzes the hydroxy-
lation of 1-deoxypentalenic acid (5) at C-11 to generate 1-deoxy-
11b-hydroxypentalenic acid (6), was constructed by in-frame deletion
using the l recombination and Cre/loxP excision systems.19 As the
C-11 hydroxylation was not functional in SUKA11 carrying pKU462H
ptl-clusterDptlH, this deletion mutant produced 1-deoxypentalenic
acid (5) accompanied by relatively large amounts of pentalenic acid
(1) (Figure 4a). Furthermore, when the sav7469-deletion was intro-
and incubation for 2 h at 301C with shaking. The enzymatic reaction was
terminated by adding 20ml of 1 N HCl and the product was extracted using
2ꢀ100 ml of EtOAc. The combined organic extracts were dried over Na2SO4
and evaporated to dryness. The residue was dissolved in 100 ml of benzene–
methanol (9:1) and 20ml of trimethylsilyldiazomethane was added. After
15 min at room temperature, the reaction mixture was evaporated to dryness
and the residue was dissolved in 20ml methanol. A 5-ml portion of the
methylated product was analyzed by GC-MS. For kinetic measurements, assays
were performed under the above conditions over a range of substrate
concentrations. The steady-state kinetic parameters, Km, Vmax and kcat, were
calculated by fitting the observed GC-MS TIC values to the Michaelis–Menten duced into the SUKA11 carrying pKU462Hptl-clusterDptlH mutant
equation by non-linear, least-squares regression. Reported standard deviations
correspond to the statistical errors limits of the data fit. The dissociation
the resultant double-deletion mutant, SUKA13 carrying pKU462H
ptl-clusterDptlH, accumulated only 1-deoxypentalenic acid (5)
constant KD of CYP105D7 and 1-deoxypentalenic acid (5) was obtained as
(Figure 4b). Pentalenic acid (1) production could be restored by
previously described.15 1-Deoxypentalenic acid (5) dissolved in DMSO was
introduction of an extra copy of sav7469, which was co-expressed
added at increasing concentrations to a stock solution of 1.1mM of CYP105D7
along with two genes encoding the S. avermitilis electron-transport
in 50mM Tris-HCl (pH 8.0) and 10% (v/v) glycerol. The concentration of
partners, Fdx (SAV_3129; FdxD) and Fdx reductase (SAV_5675;
FprD) into the double-deletion mutant (Figure 4c), because the
DMSO was adjusted to 0.1% (v/v). UV difference spectra were recorded from 0
to 100 mM of 1-deoxypentalenic acid (5). The dissociation constant KD was
production of oxidative product(s) of epi-isozizaene derivatives were
enhanced by co-expression of these two genes with sav3032-sav3031.24
These results indicate that pentalenic acid (1) is generated in
S. avermitilis by hydroxylation of 1-deoxypentalenic acid (5) at C-1
catalyzed by CYP105D7.
calculated from the saturation curve of the measured delta value
(DAU¼A364nm—A416 nm) versus substrate concentration. All kinetics and
dissociation experiments were run in triplicate.
RESULTS
Production of pentalenic acid (1) in mutants of S. avermitilis
S. avermitilis harbors a biosynthetic gene cluster for a newly discovered
branch of the classical pentalenolactone family tree, with the micro-
organism producing a new type of pentalenolactone derivative, which
Characterictics of CYP105D7
From a BLAST homology search using the NCBI nr database,
S. avermitilis CYP105D7 was found to be similar at the amino-acid
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Hydroxylation of 1-deoxypentalenic acid
S Takamatsu et al
69
Figure 4 GC-MS analysis of EtOAc extracts treated with trimethylsilyl-
diazomethane of SUKA11 carrying pKU462Hptl-clusterDptlH (a), SUKA13
(SUKA11 Dsav7469HaadA) carrying pKU462Hptl-clusterDptlH (b) and
SUKA13 carrying pKU462Hptl-clusterDptlH and pKU493HrpsJp-sav7469-
sav7470-sav3129-sav5675 (c).
Figure 5 UV spectra of oxidized, reduced and CO-bound forms of C-terminal
His4-tagged CYP105D7 (a) and its CO-difference spectrum (b). UV differ-
ence binding spectra of CYP105D7 and 1-deoxypentalenic acid (5) binding
(c), saturation curve for CYP105D7 and 1-deoxypentalenic acid (5) (d).
(aa) sequence level to ZP_05504847 of Streptomyces sp. C (395aa, 77%
identity and 86% positive matches), BAG55292 of Streptomyces sp.
A-1544 (409 aa, 75% identity and 87% positive matches), ZP_05536090
of S. viridochromogenes DSM 40736 (398aa, 75% identity and 86%
positive matches), ZP_05021349 of S. sviceus Americam Type Culture
Collection 29083 (413aa, 75% identity and 85% positive matches), the protein was correctly folded (Figure 5b). The difference spectra
ZP_04683686 of S. ghanaensis Americam Type Culture Collection showed a typical type I spectral shift and positive peaks were observed
14672 (401 aa, 73% identity and 84% positive matches), ZP_05528097 in the difference binding spectra at 364 and 416 nm recorded at
(CYP105D4) of S. lividans TK24 (406aa, 74% identity and 85% increasing concentrations of 5 (Figure 5c). The plot of the measured
positive matches), NP_625076 (CYP105D5) of S. coelicolor A3(2) delta value (DAU¼A364 nm–A416nm) versus substrate concentration
(412 aa, 74% identity and 85% positive matches), P26911 is shown in Figure 5d. The DAUmax and a KD values were calculated as
(CYP105D1) of S. griseus (412aa, 71% identity and 80% positive 0.067 0.001 and 7.3 0.7 mM, respectively.
matches) and BAG50412 of S. griseolus (395aa, 69% identity and 80%
positive matches).
To confirm the predicted function of CYP105D7, the recombinant acid (1) catalyzed by recombinant CYP105D7
In vitro hydroxylation of 1-deoxypentalenic acid (5) to pentalenic
protein was expressed in the T7-RNA polymerase-based expression The reaction mixture was incubated in the presence of 2 mM recombi-
host E. coli BL21 CodonPlus (DE3). As induction with IPTG (0.1– nant CYP105D7 for 2 h at 301C with shaking. The substrate 1-
0.5 mM) was not effective for the expression of sav7469, the cultivation deoxypentalenic acid (5) was almost completely consumed, based
for the E. coli transformant carrying pET17bHsav7469 was performed on the disappearance of the GC-MS peak (ret. time 8.458 min)
without IPTG induction. The soluble C-terminal His4-tagged corresponding to 5-methyl ester, whereas the formation of the product
CYP105D7 was purified by sequential Ni2+-affinity chromatography, pentalenic acid (1) was evident from the appearance of a methyl ester
ion-exchange chromatography and gel filtration chromatography. peak (1-methyl ester), retention time 9.883 min. In a parallel control
The recovered deep red recombinant CYP105D7 (15mg from 2 l of incubation carried out in the absence of CYP105D7, there was no
culture) was judged to be more than 90% pure by SDS–PAGE.
observable hydroxylation of 5 to 1. (Figure 6a). The EI-MS fragmen-
The UV-visible absorption spectrum of the oxidized-form of tation pattern and the retention time of the peak corresponding to
recombinant CYP105D7 showed a Soret peak plus b- and a-bands pentalenic acid (1) methyl ester were identical to those of an authentic
at 418, 534 and 568 nm, respectively, suggesting a typical low-spin sample (Figure 6b).
state spectrum (Figure 5a). The relatively sharp peak at 447 nm in the The pH-dependence of the CYP105D7-catalyzed reaction was
CO-difference spectrum of recombinant CYP105D7 confirmed that measured at pH 6.0, 6.5, 7.0, 7.5 and 8.0. The maximum rate of
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Hydroxylation of 1-deoxypentalenic acid
S Takamatsu et al
70
Figure 6 GC-MS analysis of EtOAc extracts treated with trimethylsilyldiazomethane from incubation of 1-deoxypentalenic acid (5) with (b) or without (a)
CYP105D7 protein in the presence of a NADPH-generating system, electron-transport partners (spinach Fdx and Fdx reductase) and Michaelis–Menten plot
of initial velocity of formation of pentalenic acid (1) (c).
formation of pentalenic acid (1) was observed at pH 8.0 (data not we sought to determine which of the 33 CYP genes that have been
shown). The steady-state kinetic parameters of the CYP105D7- identified in the genome of S. avermitilis would be responsible for the
catalyzed hydroxylation reaction were Km(5) of 27.2 2.5 mM, Vmax generation of pentalenic acid (1). The altered product profile in the
0.0172 0.003 mM sꢁ1 and kcat 0.116 0.003 sꢁ1 (Figure 6c). To exam- SUKA7 large-deletion mutant that lacks the sav7469 gene encoding
ine the substrate specificity of CYP105D7, several intermediates of CYP105D7 indicates that production of pentalenic acid (1) depends
pentalenolactone biosynthesis were incubated with CYP105D7. The on the presence of sav7469 gene. This relationship was further
recombinant CYP105D7 protein could not catalyze the C-1 hydro- examined and supported using biosynthetically blocked mutants of
xylation of pentalenene (2), 1-deoxy-11b-hydroxypentalenic acid (6), the large-deletion mutants SUKA5 and SUKA7.
1-deoxy-11-oxopentalenic acid (7) or the corresponding methyl esters
It is well-known that CYP105 family monooxygenases have a
of 6, 7 and 5, respectively. CYP105D7 is, therefore, specific for the C-1 remarkable capacity for broad-based xenobiotic metabolism, utilizing
hydroxylation of 1-deoxypentalenic acid (1).
compounds of diverse structure and chemistry.20,29 In the ptl cluster
for the biosynthesis of neopentalenoketolactone, the only CYP gene
involved in the formation of an intermediate for neopentalenolac-
DISCUSSION
The wild-type strain of S. avermitilis and its large-deletion derivative tones, is ptlI (sav2999) the gene product (CYP183A1) of which has
SUKA5, as well as S. lividans 1326 carrying the entire ptl cluster of been shown to oxidize the C-13 methyl of pentalenene (2) to give 1-
S. avermitilis, each produce pentalenic acid (1), a known shunt deoxypentalenic acid (5).15 As no further metabolites of pentalenic
metabolite in the biosynthesis of the pentalenolactone family of acid (1) have been isolated from S. avermitilis, pentalenic acid (1) was
antibiotics.9 In principle, pentalenic acid (1) might be formed by thought to be formed by direct hydroxylation of 1-deoxypentalenic
the hydroxylation of 1-deoxypentalenic acid (5) at C-1 or the oxida- acid (5) catalyzed by CYP105D7. Notably, this C-1 oxidation involves
tion of pentalenene (2) at both C-1 and C-13. Pentalenic acid (1) is removal of a different diastereotopic proton from that which is lost in
normally isolated as a common co-metabolite of pentalenolactone and the final oxidative rearrangement that results in the formation of
its derivatives, including the biosynthetic intermediate, 1-deoxypenta- pentalenolactone.9
lenic acid (5).28 Although at least four genes in the S. avermitilis ptl
The in vitro enzymatic reaction unambiguously established that
cluster have been shown to encode oxygenases implicated in the CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid
biosynthesis of neopentalenoketolactone biosynthesis, the gene res- (5) to pentalenic acid (1). Although CYP105D7 has been shown to
ponsible for the formation of pentalenic acid (1) by hydroxylation of have a relatively broad substrate range, being able to catalyze the
1-deoxypentalenic acid (5) is not present in the ptl cluster. Therefore, hydroxylation of milbemycin A5, compactin, diclofenac, lauric acid
The Journal of Antibiotics

Hydroxylation of 1-deoxypentalenic acid
S Takamatsu et al
71
and narigenin (unpublished data), no pentalenolactone-related com-
pounds, other than 1-deoxypentalenic acid (5), could be hydroxylated
by CYP105D7. As pentalenene (2) was not oxidized by CYP105D7, the
formation of the shunt metabolite pentalenic acid (1) in S. avermitilis
is conclusively shown to involve sequence conversion of farnesyl
diphosphate to pentalenene (2) by pentalenene synthase, PtlI-cata-
lyzed oxidation of 2 to 1-deoxypentalenic acid (5), and finally
CYP105D7-catalyzed hydroxylation of 5 to pentalenic acid (1). We
have previously demonstrated that exoconjugants of S. lividans 1326
carrying the S. avermitilis ptl cluster produced only pentalenic acid
(1).14 As S. lividans also possesses a gene encoding a member of the
CYP105D subfamily of monooxygenases, CYP105D4,20 the S. lividans
exoconjugant carrying the ptl cluster most likely generated 1-deoxy-
pentalenic acid (5), which presumably served as the substrate for
hydroxylation by CYP105D4 to pentalenic acid (1). Genes enco-
ding CYP105D subfamily monooxygenases have been found in all
genome-sequenced Streptomyces, including draft genome sequenced
microorganisms. It is, therefore, likely that other pentalenolactone-
producing Streptomyces strains might possess the similar genes
encoding members of the CYP105D subfamily of monooxygenases.
Accumulation of the shunt product pentalenic acid (1) in pentaleno-
lactone-producing microorganisms would, therefore, result from the
abortive hydroxylation of the normal 1-deoxypentalenic acid (5)
intermediate by CYP105D subfamily monooxygenase in the produ-
cing microorganism. Streptomyces exfoliates UC5319 also accumulates
pentalenolactone H as a minor component, derived by C-1 hydro-
xylation of pentalenolactone F.9 It is likely that a specific CYP105D
subfamily monooxygenase in this microorganism might be able to
catalyze the requisite oxidation of pentalenolactone F.
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ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid for Scientific Research on
Innovative Areas from the Ministry of Education, Culture, Sports, Science and
Technology, Japan, a Grant-in-Aid from the Japan Society for the Promotion of
Science (JSPS) 20310122 and a research grant of the Institute for Fermentation,
Osaka, Japan (HI) and by NIH Grant GM30301 (DEC).
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