Cycloheximide and actiphenol production in streptomyces sp. YIM56141 governed by single biosynthetic machinery featuring an acyltransferase-less type i polyketide synthase

Article abstract of DOI:10.1021/ol501179w

Cycloheximide (1) and actiphenol (2) have been isolated from numerous Streptomyces species. Cloning, sequencing, and characterization of a gene cluster from Streptomyces sp. YIM65141 now establish that 1 and 2 production is governed by single biosynthetic machinery. Biosynthesis of 1 features an acyltransferase-less type I polyketide synthase to construct its carbon backbone but may proceed via 2 as a key intermediate, invoking a provocative reduction of a phenol to a cyclohexanone moiety in natural product biosynthesis.

Full text of DOI:10.1021/ol501179w

Letter
pubs.acs.org/OrgLett
Cycloheximide and Actiphenol Production in Streptomyces sp.
YIM56141 Governed by Single Biosynthetic Machinery Featuring an
Acyltransferase-less Type I Polyketide Synthase
Min Yin,†,‡,# Yijun Yan,†,§,# Jeremy R. Lohman,† Sheng-Xiong Huang,† Ming Ma,† Guang-Rong Zhao,∥
Li-Hua Xu,‡ Wensheng Xiang,§ and Ben Shen*,†,∥,⊥,¶
†Department of Chemistry, ⊥Department of Molecular Therapeutics, and ¶Natural Products Library Initiative, The Scripps Research
Institute, Jupiter, Florida 33458, United States
‡Yunnan Institute of Microbiology and School of Medicine, Yunnan University, Kunming, Yunnan 650091, China
§School of Life Sciences, Northeast Agricultural University, Harbin, Heilongjiang 150030, China
∥Division of Pharmaceutical Sciences, University of Wisconsin−Madison, Madison, Wisconsin 53705, United States
S
* Supporting Information
ABSTRACT: Cycloheximide (1) and actiphenol (2) have
been isolated from numerous Streptomyces species. Cloning,
sequencing, and characterization of a gene cluster from
Streptomyces sp. YIM65141 now establish that 1 and 2
production is governed by single biosynthetic machinery.
Biosynthesis of 1 features an acyltransferase-less type I
polyketide synthase to construct its carbon backbone but
may proceed via 2 as a key intermediate, invoking a
provocative reduction of a phenol to a cyclohexanone moiety
in natural product biosynthesis.
ycloheximide (1) is one of the most well-known members
of the glutarimide-containing polyketide family of natural
C
products and has been used for decades as an inhibitor of
eukaryotic translation.1 Actiphenol (2), sharing the same
carbon skeleton as 1 but having a phenol in place of a
cyclohexanone moiety, exhibits weak translation inhibiton
activity.1c,2 Other members of this family include streptimidone
(3), 9-methylstreptimidone (4), iso-migrastatin (5), migrasta-
tin, and lactimidomycin (6) (Figure 1A). Whereas 1 inhibits
translation globally, 6 inhibits preferentially translation
initiation but not elongation, a property that has been exploited
recently in the development of the global translation initiation
sequencing (GTI-seq) technology that enables high-resolution
mapping of translation initiation sites across the entire
transcriptome.3 Members of this family have also been pursued
as promising anti-metastatic drug leads for their potent cell
migration inhibiton activity and cytotoxcicity.4
Figure 1. Cloning of the chx biosynthetic gene cluster from
Streptomyces sp. YIM56141. (A) Structures of 1, 2, and selected
glutarimide-containing polyketides (3−6). (B) Cloning of the chx
cluster from Streptomyces sp. YIM56141 using a probe encoding
glutarimide moiety biosynthesis as represented by two overlapping
cosmids. (C) The chx cluster spanning ∼35 kb and consisting of 10
genes with their predicted functions color-coded.
During our recent efforts toward discovering inhibitors of
eukaryotic translation, we rediscovered 1 from two Streptomyces
species, YIM56141 and YIM56132.1c Interestingly, both species
also produced 2, along with other congeners. Upon delving into
the literature, we found that many of the strains reported to
produce 1 also produced 2, including Streptomyces griseus,2a
Streptomyces albulus,2b,c and Streptomyces noursei.2d Co-
production of 1 and 2 raises an interesting question if they
are biosynthetically related, and if true, the biosynthetic
relationship between the phenol moiety of 2 and cyclo-
hexanone moiety of 1 are fascinating and cannot be readily
predicted a priori according to current knowledge of natural
product biosynthesis.
Received: April 23, 2014
Published: May 9, 2014
© 2014 American Chemical Society
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Here we report the cloning, sequencing, and characterization
of a gene cluster from Streptomyces sp. YIM56141. In vivo and
in vitro studies establish that the production of 1 and 2 is
governed by single biosynthetic machinery, featuring an
acyltransferase (AT)-less type I polyketide synthase (PKS) to
construct their carbon backbones and a provocative phenol-to-
cyclohexanone reduction in 1 biosynthesis.
enoylreductase (ChxG), a ketoreductase (ChxH), a cytochrome
P450 oxidoreductase (ChxI), and a three-domain carboxylic
acid reductase (ChxJ) for converting the nascent glutarimide-
containing polyketide intermediate to 1 or 2, respectively, and
(iv) two genes encoding regulator proteins (ChxA and ChxF)
for pathway regulation (Figure 1C and Table S3 in SI). The
genetic organization of the chx cluster, as well as the deduced
functions thereof, shows high similarity to gene clusters known
for biosynthesis of other glutarimide-containing polyketides,
including 4,5 5,6 and 6,7 but also features several distinct
features (Figure S3 in SI). While the similarities among the
different pathways support the biosynthesis of a common
glutarimide-containing polyketide intermediate, the variations
among the tailoring enzymes account for channeling of the
common intermediate into the various end products (Figure 3).
We next carried out in vivo experiments establishing the
cloned chx gene cluster encoding the biosynthesis of both 1 and
2. Central to the chx cluster is chxE, which encodes a five-
module AT-less type I PKS that is highly homologous to the
AT-less type I PKSs for 4,5 5,6 and 67 biosynthesis. ChxE and
SmdI share an identical architecture with the exception of SmdI
lacking the C-terminal thioesterase (TE) domain. ChxE and
SmdI appear to be the result of a fusion between MgsE/LtmE
module-3 and MgsF/LtmF module-4, which terminates after
MgsF/LtmF module-6 with the TE domain from MgsG/LtmG
(Figure S3 in SI). With the clear homology among ChxE, SmdI,
MgsEFG, and LtmEFG (Table S3 in SI), we propose that they
produce a common intermediate at module-6 (Figures 3 and
Figure S3 in SI). Thus, in a biosynthetic analogy to 4, 5, and 6,
ChxC, ChxD, and ChxE consist of a six-module AT-less type I
PKS, with ChxB loading the extender unit of malonyl-CoA to
each of the six modules in trans, to biosynthesize the nascent
glutarimide-containing polyketide intermediate (7) from six
molecules of malonyl-CoA, two molecules of S-adenosylme-
thionine (SAM) (for the two CH3 groups at C-11 and C-13),
and an amino acid (as a donor for the “NH” group in the
glutarimide moiety). The fact that there is only one
methyltransferase (MT) domain in ChxE module-6 would
suggest that this MT most likely acts twice to introduce the
CH3 groups at both C-11 and C-13 of 1 and 2 (Figure 3).
While the latter prediction deviates from the collinear model
for type I PKS, the identical domain and module architecture
among the ChxE, SmdI, MgsF, and LtmF AT-less type I PKSs
would suggest a similar biosynthetic logic for the installation of
the analogous two CH3 groups in 4, 5, and 6 (Figure S3 in SI).
The chxE gene was subsequently inactivated (SI), and the
genotype (i.e., ΔchxE) of the resultant mutant strain SB19003
was confirmed by Southern analysis (Figure S4 and Table S2 in
SI). HPLC analysis of SB19003 fermentation showed the
abolishment of production of both 1 and 2 (Figure 2, panel
IV), confirming the essential role ChxE plays in 1 and 2
biosynthesis and establishing 1 and 2 production is governed by
single biosynthetic machinery (Figure 3).
We first cloned the gene cluster from Streptomyces sp.
YIM56141 taking advantage of the biosynthetic knowledge of
other glutarimide-containing polyketides. Thus, using degener-
ate primers designed according to the conserved genes
encoding biosynthesis of the glutarimide moiety in 5 and 6
(Table S1 in Supporting Information (SI)), we amplified a
fragment containing the amidotransferase gene (Supporting
Information and Table S2). Using this fragment as a probe, we
screened the genomic library of Streptomyces sp. YIM56141 and
identified two overlapping cosmids, pBS19001 and pBS19002,
that covered the chx cluster (Figure 1B). DNA sequencing of
the two cosmids revealed 18 open reading frames (Figure 1C),
and this sequence has been deposited in GenBank with the
accession number JX014302. To determine the chx cluster
boundaries, orf(-1) and orf1 were inactivated, affording mutant
strains SB19001 [Δorf(-1)] and SB19002 (Δorf1), the
genotypes of which were confirmed by Southern analysis
(Figures S1 and S2 in SI). HPLC analysis of SB19001 and
SB19002 fermentations confirmed that both strains still
produced 1 and 2 (Figure 2, panels II and III), hence
establishing boundaries of the chx cluster that spans ∼35 kb
and consists of 10 genes (Figure 1C).
Figure 2. Inactivation of selected genes within the chx cluster
supporting the proposed pathway for 1 and 2 biosynthesis. HPLC
analysis of fermentations from Streptomyces sp. YIM56141 wild-type
and recombinant strains: (I) wild-type, (II) SB19001 [Δorf(-1)], (III)
SB19002 (Δorf1), (IV) SB19003 (ΔchxE), (V) SB19004 (ΔchxJ),
(VI) SB19005 (ΔchxJ/chxJ), (VII) SB19006 (ΔchxI), (VIII) SB19007
(ΔchxI/chxI), (IX) SB19008 (ΔchxG), (X) SB19009 (ΔchxG/chxG),
(XI) SB19010 (ΔchxH), and (XII) SB19011 (ΔchxH/chxH).
ChxJ consists of three domains, an acyl-CoA ligase (AL), an
ACP, and a reductase (R) that specifically reduces a carboxylic
acid to an aldehyde via the intermediacy of an acyl-S-ACP and
shares 59% identity with CAR from Nocardia iowensis, which
reduces several carboxylic acids to their corresponding
aldehydes (Table S3 in SI).8 Inspired by the chemistry of
CAR, we propose that ChxJ catalyzes the reduction of the
carboxylic acid group in 7 to afford the aldehyde intermediate
(8). Thus, the AL domain of ChxJ activates 7 and loads it to the
ACP domain, and the resultant acyl-S-ACP intermediate is
⧫
◊
●
▽
Highlighted metabolites are 1 ( ), 2 ( ), 10 ( ), and 11 ( ).
Bioinformatics analysis of the 10 genes within the chx cluster
revealed (i) two genes encoding an acyl carrier protein (ACP)
(ChxC) and an amidotransferase (AMT) (ChxD) for
glutarimide moiety biosynthesis, (ii) two genes encoding a
discrete AT (ChxB) and a five-module AT-less type I PKS
(ChxE) for biosynthesis of the glutarimide-containing polyke-
tide backbone of both 1 and 2, (iii) four genes encoding an
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Figure 3. Proposed pathway for 1 biosynthesis featuring an AT-less type I PKS and proceeding via 2 as an intermediate. Abbreviations are ACP, acyl
carrier protein; AL, acyl-CoA ligase; AMT, amidotransferase; B, β-branching; DH, dehydratase; KR, ketoreductase; KS, ketosynthase; MT,
methyltransferase; R, acyl thioester reductase; SAM, S-adenosylmethionine; TE, thioesterase; and ?, unknown or nonenzymatic.
subsequently reduced by the R domain of ChxJ to afford 8.
ChxI belongs to the cytochrome P450 superfamily of
oxidoreductases (Table S3 in SI), serving as the candidate to
catalyze C-8 oxidation during the conversion from 7 to 8.
Oxidation at C-8 would set the stage for 8 to undergo an
intramolecular aldol condensation between C-9 and C-14 to
yield an intermediate such as preactiphenol (9), which features
the carbon scaffold of both 1 and 2 (Figure 3). While a cis-
double bond at C-12 and C-13 of 8 would be necessary to
facilitate C-9 and C-14 cyclization, it is not known when and
how this isomerization occurs from 7 to 8. We also failed to
identify any candidate responsible for the necessary aldol
condensation from 8 to 9, as well as the subsequent
dehydration of 9 to 4 within the chx cluster. While it is
tempting to speculate that both steps could be spontaneous, we
cannot exclude the possibility that they are catalyzed by enzyme
activities residing outside of the cloned chx cluster (Figure 3).
Both chxJ and chxI were subsequently inactivated (SI), and
the genotypes of the resultant mutant strains SB19004 (i.e.,
ΔchxJ) and SB19006 (i.e., ΔchxI) were confirmed by Southern
analysis (Figures S5, S6, and Table S2 in SI). Two additional
recombinant strains were also constructed (Table S2 in SI), in
which the ΔchxJ and ΔchxI mutations in SB19004 and
SB19006 were complemented by expressing a functional copy
of chxJ (SB19005) or chxI (SB19007) in trans. HPLC analysis
of their fermentations confirmed that 1 and 2 production was
completely abolished in SB19004 and SB19006 (Figure 2,
panels V and VII) and 1 and 2 production was restored, albeit
only partially, in SB19005 and SB19007 (Figure 2, panels V, VI,
VII, VIII), consistent with the essential roles proposed for ChxI
and ChxJ in 1 and 2 biosynthesis. Failure to accumulate any
discrete intermediate by SB19004 and SB19006, however,
prevented us from providing direct evidence supporting the
intermediacy of 7 or 8 in 1 and 2 biosynthesis or shedding light
into the timing of C-8 oxidation, which could occur before,
during, or after ChxJ catalysis (Figure 3).
We finally carried out in vivo and in vitro experiments on
chxG and chxH to delineate the biosynthetic relationship
between 1 and 2, unveiling 2 as a key intermediate to 1. ChxG
belongs to the Old Yellow Enzyme (OYE) family of
flavoprotein oxidoreductases that are capable of CC bond
reduction of a wide range of substrates,9 and ChxH is a member
of the short-chain dehydrogenase/reductase superfamily
consisting of a large number of NAD(P)H oxidoreductases
that provide varying enzymatic activities and act on a broad
spectrum of substrates10 (Table S3 in SI). Both chxG and chxH
were inactivated (SI), the genotypes of the resultant mutant
strains SB19008 (i.e., ΔchxG) and SB19010 (i.e., ΔchxH) were
confirmed by Southern analysis (Figures S7 and S8 in SI), and
the ΔchxG and ΔchxH mutations were also complemented by
expressing functional copies of chxG (SB19009) and chxH
(SB19011) in trans, respectively (Table S2 in SI). Remarkably,
HPLC analysis of their fermentations showed complete
abolishment of production of 1 but not 2 in both SB19008
and SB19010 (Figure 3, panels IX and XI) and partial
restoration of 1 production in SB19009 and SB19011 (Figure 3,
panels X and XII). These findings unambiguously established 2
as an intermediate for 1 biosynthesis, the transformation of
which to 1 requires minimally ChxG and ChxH (Figure 3).
Close examination of the HPLC profiles further revealed a
significantly increased production of 2, accompanied by the
accumulation of phenatic acid (11), a known metabolite of
nonenzymatic hydrolysis of 2,1c in SB19008 and accumulation
of dehydrocycloheximide (10), in addition to 2, in SB19010
(Figure 2, panels IX and XI). The identity of 10 was
unambiguously established by 1H and 13C NMR analysis
(Table S4 and Figure S9 in SI), which has been isolated
previously from 1 and 2 producers such as S. noursei.2d Taken
together, these results suggest that ChxG catalyzes reduction of
2 to 10, a provocative proposal for an enzymatic reduction of a
phenol to a cyclohexanone moiety in natural product
biosynthesis, and that ChxH catalyzes the final step of 1
biosynthesis, reducing 10 to 1 (Figure 3). Controlled reduction
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of a benzene ring has been difficult in both laboratories and
biological systems due to its high resonance energy. Although
members of the OYE family have been implicated in the
reduction of trinitrotoluene,9 structural characterization of the
partially reduced cyclic products remains elusive to date. ChxG,
which shows high sequence homology to known members of
OYE family (Figure S10 in SI), therefore could serve as an
excellent model to study how flavoproteins modulate redox
potential to catalyze the reduction of a benzene ring.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: shenb@scripps.edu.
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
Finally, we overexpressed chxH in E. coli (SI and Table S2)
and purified ChxH to near homogeneity (Figure S11 in SI).
ChxH catalyzed efficient reduction of 10 to 1, requiring
NADPH or NADH (Figure S12A in SI) and exhibiting an
optimal pH at 7.2 in 100 mM sodium phosphate (Figure S12B
in SI), and showed no activity toward 11 as an alternative
substrate. Pseudo-first-order kinetic studies under steady state
conditions (SI) showed that plots of initial velocity versus the
concentration of substrates or cofactors all displayed
Michaelis−Menten kinetics, allowing the determination of the
corresponding KM’s, and kcat’s (Figure S13 in SI). Thus, as
summarized in Table S5 in SI, ChxH exhibited apparent KM’s
for 10 at 44 4 μM and 139 23 μM upon saturation of
NADPH and NADH, apparent KM’s for NADPH and NADH
ACKNOWLEDGMENTS
■
We thank the John Innes Center, Norwich, U.K., for providing
the λ-Red-mediated PCR-targeting mutagenesis kit. This work
was supported in part by NIH grant CA106150 and the Natural
Products Library Initiatives at TSRI. M.Y. and Y.Y. were
supported in part by scholarships from the China Scholarship
Council.
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S
* Supporting Information
Complete description of materials and methods, supporting
tables (S1−S5), and supporting figures (S1−S13). This
material is available free of charge via the Internet at http://
pubs.acs.org.
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