Bacterial biosynthesis and maturation of the didemnin anti-cancer agents

Article abstract of DOI:10.1021/ja301735a

The anti-neoplastic agent didemnin B from the Caribbean tunicate Trididemnum solidum was the first marine drug to be clinically tested in humans. Because of its limited supply and its complex cyclic depsipeptide structure, considerable challenges were encountered during didemnin B's development that continue to limit aplidine (dehydrodidemnin B), which is currently being evaluated in numerous clinical trials. Herein we show that the didemnins are bacterial products produced by the marine α-proteobacteria Tistrella mobilis and Tistrella bauzanensis via a unique post-assembly line maturation process. Complete genome sequence analysis of the 6,513,401 bp T. mobilis strain KA081020-065 with its five circular replicons revealed the putative didemnin biosynthetic gene cluster (did) on the 1,126,962 bp megaplasmid pTM3. The did locus encodes a 13-module hybrid non-ribosomal peptide synthetase-polyketide synthase enzyme complex organized in a collinear arrangement for the synthesis of the fatty acylglutamine ester derivatives didemnins X and Y rather than didemnin B as first anticipated. Imaging mass spectrometry of T. mobilis bacterial colonies captured the time-dependent extracellular conversion of the didemnin X and Y precursors to didemnin B, in support of an unusual post-synthetase activation mechanism. Significantly, the discovery of the didemnin biosynthetic gene cluster may provide a long-term solution to the supply problem that presently hinders this group of marine natural products and pave the way for the genetic engineering of new didemnin congeners.

Full text of DOI:10.1021/ja301735a

Article
pubs.acs.org/JACS
Bacterial Biosynthesis and Maturation of the Didemnin Anti-cancer
Agents
Ying Xu,†,# Roland D. Kersten,‡,# Sang-Jip Nam,‡,¶ Liang Lu,† Abdulaziz M. Al-Suwailem,§
Huajun Zheng,∥ William Fenical,‡ Pieter C. Dorrestein,‡,⊥ Bradley S. Moore,*,‡,⊥ and Pei-Yuan Qian*,†
†KAUST Global Collaborative Research, Division of Life Science, School of Science, Hong Kong University of Science and
Technology, Clear Water Bay, Hong Kong, China
‡Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La
Jolla, California 92093, United States
§The Coastal and Marine Resources Core Lab, Red Sea Research Center, 4700 King Abdullah University of Science and Technology,
Thuwal, Makkah 23955-6900, Kingdom of Saudi Arabia
∥Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, 250 Bi
Bo Road, Shanghai 201203, China
⊥Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, United
States
S
* Supporting Information
ABSTRACT: The anti-neoplastic agent didemnin B from the
Caribbean tunicate Trididemnum solidum was the first marine
drug to be clinically tested in humans. Because of its limited
supply and its complex cyclic depsipeptide structure,
considerable challenges were encountered during didemnin
B’s development that continue to limit aplidine (dehydrodi-
demnin B), which is currently being evaluated in numerous
clinical trials. Herein we show that the didemnins are bacterial
products produced by the marine α-proteobacteria Tistrella
mobilis and Tistrella bauzanensis via a unique post-assembly
line maturation process. Complete genome sequence analysis of the 6,513,401 bp T. mobilis strain KA081020-065 with its five
circular replicons revealed the putative didemnin biosynthetic gene cluster (did) on the 1,126,962 bp megaplasmid pTM3. The
did locus encodes a 13-module hybrid non-ribosomal peptide synthetase−polyketide synthase enzyme complex organized in a
collinear arrangement for the synthesis of the fatty acylglutamine ester derivatives didemnins X and Y rather than didemnin B as
first anticipated. Imaging mass spectrometry of T. mobilis bacterial colonies captured the time-dependent extracellular conversion
of the didemnin X and Y precursors to didemnin B, in support of an unusual post-synthetase activation mechanism. Significantly,
the discovery of the didemnin biosynthetic gene cluster may provide a long-term solution to the supply problem that presently
hinders this group of marine natural products and pave the way for the genetic engineering of new didemnin congeners.
thus microbial fermentation offers the promise of a renewable
supply of important marine drug candidates.5 However, very
few examples exist in which clinically relevant compounds
originally extracted from marine invertebrates are produced by
cultured microbes. Herein we show that the didemnin family of
anti-cancer agents, including didemnin B as the first marine
natural product clinically tested in humans,6 is produced by
marine bacteria of the genus Tistrella through an unexpected
biosynthetic pathway.
Invertebrate-derived natural products, especially those with
complex polyketide and non-ribosomal peptide structures, are
generally proposed to be microbial in origin since the molecular
basis governing their biosyntheses resides exclusively in
INTRODUCTION
■
Sessile marine invertebrates such as sponges and tunicates are
intimately linked with microbes that are consumed as food and
hosted for their protective chemistry. These marine animals are
rich sources of structurally diverse collections of unique
bioactive molecules that in some cases have been developed
into drugs for the benefit of human health.1,2 A major challenge
in the drug development of marine natural products, however,
has historically been the difficulty in supplying sufficient
material for pre-clinical and clinical evaluation due to natural
limitations of the source organism.3 Marine products are
frequently endowed with complex organic structures that
complicate their total syntheses in large scale to meet the
demands of a drug development program.4 Microbes are
commonly suggested as the actual producers of many
molecules originally isolated from invertebrate tissues, and
Received: February 22, 2012
Published: March 29, 2012
© 2012 American Chemical Society
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Figure 1. Structures of didemnin B and other representative didemnin analogues in which structural differences are colored red.
Figure 2. HPLC profiles of didemnin B and nordidemnin B in extracts of four different Tistrella strains.
microbes.7−9 Metagenomic studies have revealed the microbial
production of noteworthy bioactive natural products from
diverse marine invertebrates, including the pederin group of
polyketides from sponges,10 the patellamides and ecteinascidin-
743 (Yondelis) from tunicates,11,12 and the bryostatin macro-
lides from bryozoans.13 In each case, uncultivated bacterial
symbionts are thought to be the natural producers. Free-living
marine microbes are often not implicated in the synthesis of
marine invertebrate natural products, although a field-collected
marine cyanobacterium was shown to harbor the sponge
macrolide swinholide A.14 As a consequence, molecular
engineering approaches involving whole pathway expression
are envisaged to produce complex molecules from marine
invertebrate symbionts in laboratory microbial strains in order
to develop a renewable supply of the compound and allow for
the rational engineering of new chemical entities to probe
structure−function relationships.15 To date, only the cyano-
bactin group of ribosomal peptides, which includes the
patellamides, has been heterologously produced and engi-
neered.16 Complex polyketides, non-ribosomal peptides, and
hybrids thereof, on the other hand, present an unmet technical
challenge due to their large biosynthetic gene clusters that often
exceed 100 kb.
Didemnins (Figure 1) are cyclic depsipeptides with
remarkable anti-tumor, anti-viral, and immunosuppressive
properties6 that were first discovered in the Caribbean tunicate
Trididemnum solidum in 1981.17 The most potent analogue,
didemnin B, was the first marine natural product to enter
clinical trials as an anti-cancer agent, yet it ultimately failed to
demonstrate effective anti-tumor activity while displaying
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Figure 3. Genome maps of T. mobilis KA081020-065. From the outside inward, the first and second circles show predicted coding regions
transcribed on the forward and reverse strand, respectively, colored by functional categories according to COG classification. The third circle shows
RNAs (yellow for tRNA and red for rRNA). The fourth and fifth circles show G+C content and GC skew (G−C)/(G+C) calculated using a 5 kb
window (purple for values >1, orange for values <1), respectively.
cardiac and neuromuscular toxicities.18,19 A closely related
compound, dehydrodidemnin B (aplidine), was isolated from
the Mediterranean tunicate Aplidium albicans and shown to be
more potent and less toxic despite its minor structural
change.20,21 Aplidine has since replaced didemnin B in clinical
trials and is currently in multiple phase II and III trials for the
treatment of various cancers.6 Due to their cyclic depsipeptide
structures with highly modified amino acid residues, didemnins
have long been suspected as microbial products assembled by a
hybrid non-ribosomal peptide synthetase−polyketide synthase
(NRPS−PKS) enzymatic pathway.15 In this study, we report
that the didemnins are not only the products of the α-
proteobacterium Tistrella mobilis, which was also recently
reported by Tsukimoto and co-workers,22 but also of Tistrella
bauzanensis. More importantly, we show through the complete
genome sequencing of T. mobilis and MALDI-imaging MS
technique that didemnin B is biosynthesized by an unantici-
pated rare mechanism involving a post-assembly activation of
lipoglutamine congeners, didemnins X and Y.
Tistrella strains to explore the extent of didemnin chemistry in
this poorly characterized genus. To our surprise, all strains
tested, including two Pacific Ocean T. mobilis isolates and a T.
bauzanensis strain, also produced didemnin B and nordidemnin
B at varying levels (Figure 2). During the preparation of this
manuscript, Tsukimoto and co-workers independently reported
their isolation from a T. mobilis strain isolated from marine
sediments in Japan.22 Based on these preliminary observations,
the didemnins appear to be commonly associated with this
recently described bacterial genus whose secondary metabolic
potential has not yet been reported.23 To further explore the
natural product biosynthetic capacity of Tistrella and to deduce
the molecular basis governing didemnin assembly, we
sequenced the genome of the Red Sea isolate T. mobilis
KA081020-065.
Genome Characteristics of T. moblis KA081020-065.
The complete genome sequence of T. mobilis strain KA081020-
065 totaling 6,513,401 bp was established by 454 pyrosequenc-
ing to reveal five replicons comprising a 3,919,492 bp circular
chromosome and four circular plasmids ranging in size from
83,885 bp (pTM4) to 1,126,962 bp (pTM3) (Figure 3). Its
genome organization with three megaplasmids greater than 600
kb each is reminiscent of several other α-proteobacteria
members such as the rice plant endophyte Azospirillum sp.
B510 that harbors six plasmids, five of which are in excess of
500 kb.24 General features of the T. mobilis genome, which has
a high average G+C content of 68%, are summarized in Table
S1.
RESULTS AND DISCUSSION
■
Isolation of Didemnins from Tistrella Species. We
isolated the α-proteobacterium T. mobilis KA081020-065 from
the Red Sea and measured a very potent cytotoxicity against
HeLa cells. Following a bioassay-guided fractionation of the
ethyl acetate extract, we isolated two metabolites that, based on
high-resolution MS and NMR characterization and comparison
with authentic standards, were identical to the cyclic
depsipeptides didemnin B (Figure S1) and nordidemnin B
(Figure S2) originally isolated from the Caribbean tunicate
Trididemnum solidum.17 Since this result showed that the
didemnins are bacterial products, we examined additional
Based on the chemical structure of the didemnin
depsipeptides that contains the two ketide-extended amino
acid residues isostatine (Ist) and α-(α-hydroxyisovaleryl)-
propionic acid (Hip),17 we hypothesized their biosynthesis by
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Figure 4. Proposed biosynthetic pathway of didemnins and domain organization of the biosynthetic did gene cluster in T. mobilis KA081020-065.
Domain notation: C, condensation; A, adenylation (substrate included); E, epimerase; KR, ketoreductase; KS, β-ketoacyl synthase; MT,
methyltransferase; T, thiolation; TE, thioesterase.
a modular hybrid non-ribosomal peptide synthetase−polyke-
tide synthase (NRPS-PKS) pathway reminiscent of nostopep-
tolide biosynthesis.25 Bioinformatic analysis of the Tistrella
genome revealed just one large NRPS-PKS biosynthetic gene
cluster (did) located on pTM3 (TMO_c0602−c0611) that was
consistent with the proposed biosynthesis of the didemnin
family of cyclic depsipeptides. We detected only one other
multimodular NRPS-based gene cluster in the genome, a
34,665 bp locus on pTM2 (TMO_b0323−b0325), which
rather encodes a putative eight-residue peptide product
inconsistent with didemnin assembly (Figure S3).
Bioinformatic Analysis of the Didemnin Biosynthetic
Gene Cluster Reveals an Unexpected Post-assembly
Processing Mechanism. In silico analysis of the putative
didemnin biosynthetic gene cluster (did) revealed eight NRPS
and two PKS-encoding genes spanning from didA to didJ
(Figure S4), comprising 13 modules in total (Figure 4). Based
on the enzymatic logic of thiotemplate-mediated assembly line
biosynthesis, the projected product of the DidB−J mega-
synthetase perfectly correlates with the collinear synthesis of
didemnin B. The DidB adenylation (A) domain A3 is predicted
to load pyruvate and reduce it in cis via the adjacent
ketoreductase (KR) domain to give lactate bound to the
DidB thiolation (T) domain in a reaction reminiscent to that
proposed in valinomycin assembly.26 Further processing by the
monomodular DidC and the trimodular DidD NRPSs are next
proposed to assemble the Pro-DMeLeu-Thr-Ile tetrapeptide
fragment. The N-methylated leucine residue is the lone D-
amino acid in the didemnin B molecule, which correlates nicely
with the epimerase (E) and methyltransferase (MT) domains
that reside in the Leu-specific module 5 of DidD. Malonate
extension of the DidD product by the PKS DidE, which lacks
an integrated acyltransferase (AT) domain reminiscent of trans-
AT PKSs,27 yields the ketide-extended β-hydroxy-γ-amino acid
residue Ist. Analysis of the genes bordering didA−J and
elsewhere in the T. mobilis genome, however, did not reveal a
discrete AT from trans-AT systems, suggesting that the
didemnin polyketide synthase may co-opt the fatty acid
synthase AT FabD as reported recently for the biosynthesis
of FK228.28
Subsequent activation of 2-oxoisovaleric acid by the DidF A
domain, whose sequence is similar to the N-terminal HetE A
domain (43% identity at amino acid level) that is also
responsible for its selection in hectochlorin biosynthesis,29
sets up the incorporation of the α-hydroxy acid 2-
hydroxyisovaleric acid and a second round of malonate
extension by the DidG PKS. The DidG MT domain putatively
incorporates the α-methyl group in the Hip residue. Further
chain elongation of the DidG product by the sequential
addition of the residues Leu, Pro, and Me2Tyr via the
monomodular DidH−J NRPSs completes the assembly of the
linear didemnin B molecule. The C-terminal thioesterase (TE)
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Figure 5. Time-course production of didemnins by T. mobilis KA081020-065 detected by MALDI-imaging mass spectrometry.
domain of DidJ is hypothesized to offload didemnin B as the
cyclic depsipeptide product. In the case of co-produced
nordidemnin B, the DidD A7 domain is anticipated to exhibit
substrate flexibility toward aliphatic residues to also activate and
incorporate Val. [Hysp]didemnin B30 is an additionally
characterized didemnin B derivative from T. mobilis extract
(Figure S5) which further indicates substrate promiscuity in
DidF domain A9 for α-hydroxy acids 2-hydroxyisovaleric acid
(didemnin B) and 2-hydroxy-3-methylvaleric acid ([Hysp]-
didemnin B).
While the biosynthetic model described above is clearly
consistent with the observed didemnin B and nordidemnin B
chemistry of T. mobilis, this proposal ignores the DidA
bimodular NRPS that should function as the priming
synthetase prior to DidB catalysis. Inspection of both DidA A
domains revealed their preference for Gln. Furthermore,
sequence analysis of the DidA N-terminal condensation (C)
domain suggested its enzymatic role in the fatty acylation of the
first amino acid residue to yield a lipopeptide product (Figure
4) consistent with other important non-ribosomal peptides
such as surfactin.31 Among the natural didemnin congeners
previously identified from the tunicate T. solidum, only
didemnins X and Y have both Gln residues and a lipid
component (Figure 1).32 Interestingly, although these two
didemnin derivatives contain Gln residues, they each have more
than two residues as predicted by the bimodular DidA if each
module were to function just once in peptide elongation. We
thus explored the possibility that the immediate product of the
didemnin biosynthetic pathway is not (nor)didemnin B, but
rather an acylglutamine ester such as (nor)didemnin X or Y in
which the ester side chain facilitates the cellular export of the
cyclic depsipeptide from the Gram-negative bacterium for
extracellular unmasking of the didemnin B byproduct.
to analyze the bacterial culture extracts, whereupon we
successfully detected low-abundance masses consistent with
didemnins X and Y in addition to two novel derivatives,
nordidemnins X and Y (Figure S6). We confirmed their
structures by MSn analysis and high-resolution Fourier-
transform ion cyclotron resonance MS as reported in the
Supporting Information (Figure S7). The production of
didemnins X and Y not only suggests that they are the fully
elaborated biosynthetic product of the didemnin synthetase but
also indicates that the DidA bimodular NRPS functions
iteratively to install the three to four Gln residues in didemnins
X and Y, respectively (Figure 4).
In order to explore the biosynthetic connection between
these molecules, we next used MALDI-imaging MS33,34 to
study the temporal and spatial distribution of the didemnin
compounds. We interrogated T. mobilis colonies over a time
course ranging from 6 h to 6 d (Figure 5), which strikingly
revealed that didemnins X and Y are produced and excreted
from the colony during the early growth phase of the
bacterium, peaking at 24 h. Concomitant with their
disappearance by day 4 was the emergence of didemnin B as
the primary didemnin metabolite that steadily increased over
time. This time-lag difference between didemnins X and Y and
didemnin B was similarly observed in the nordidemnin series
(Figure 5) and is consistent with the proposed model in which
the lipoglutamine side chain facilitates the cellular export of the
mature didemnin molecule for extracellular processing.
We next set out to determine whether the maturation of
didemnins X and Y involved a secreted factor. To this end, we
performed a hydrolysis assay of purified didemnin precursors
and filtered growth media cultured with and without the
bacterium. The assay clearly showed that didemnin X/Y
turnover to didemnin B was dependent on the addition of a
protein extract from a 1 day T. mobilis culture supernatant
(Figure S8). While studies are ongoing to identify the secreted
hydrolytic factor involved in the peptide activation mechanism,
sequence analysis of the open reading frames adjacent to the
didA−J operon revealed a number of genes encoding
Conversion of Didemnins X and Y to Didemnin B
Captured by Imaging Mass Spectrometry. Repeated
attempts were unsuccessful to isolate additional didemnin
congeners by altering the culture medium in order to evaluate
the activation hypothesis. We thus turned to MALDI-TOF MS
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membrane-associated transport proteins (orf 3, orf6) and
hydrolytic enzymes (orf 2, orf14) (Figure S4), further
supporting the export-hydrolysis model. Recently an activation
mechanism was established in the non-ribosomal peptide
synthesis of the antibiotic xenocoumacin from the γ-
proteobacterium Xenorhabdus nematophila in which a precursor
molecule is processed by a membrane-bound and D-Asn-
specific peptidase.35 Although the characterized pathway in
xenocoumacin processing parallels the proposed pathway for
didemnin maturation, the did cluster does not harbor a
homologue of the xenocoumacin ABC transporter-trans-
peptidase XcnG. In addition, the maturation of xenocoumacin
takes place in the periplasmic space rather than the extracellular
space for didemnins, which may suggest an alternative
mechanism in T. mobilis.
lipopeptides didemnins X and Y may no longer be effective
in such a mutant for aplidine. We are actively pursuing answers
to the detailed mechanism of didemnin B biosynthesis and
whether we can re-engineer T. mobilis to directly produce
aplidine. A provisional patent has also been filed on the
didemnin biosynthetic gene cluster (U.S. provisional applica-
tion number 61/537,416).
EXPERIMENTAL SECTION
■
Strain, Fermentation, and Isolation of Didemnin Com-
pounds. Tistrella mobilis KA081020-065 was isolated from seawater
collected from the Red Sea during a 2009 research cruise. Its crude
extract showed remarkable cytotoxicity on HeLa cells in a bioactive
compound screening of Red Sea bacteria. T. mobilis KA081020-065
was grown in GYP medium [10 g glucose, 4 g yeast extract, 2 g
peptone, and 17 g sea salts per liter of deionized water] using 50-L
stirred fermenters at 25 °C. After 72 h, ethyl acetate was added to the
culture to extract the metabolites. The crude extract was fractionated
by reversed-phase (RP) C18 liquid chromatography and eluted with
increasing amounts of methanol in water. The active fraction was
further purified by semi-preparative RP HPLC with 63% MeCN in
water to give didemnin B and nordidemnin B at 0.2 and 0.1 mg L−1,
respectively. For MSn and FTMS analysis, crude extracts were
separated by RP-HPLC (10−100% MeCN in 0.1% trifluoroacetic
acid (TFA), 40 min gradient). For hydrolysis assay, didemnin X and Y
were purified from crude extract by Sephadex LH20 (GE Healthcare)
gel filtration with MeOH as a mobile phase and RP-HPLC (25−75%
MeCN in 0.1% TFA, 60 min gradient).
CONCLUSION
■
The discovery of the bacterial synthesis of the didemnins raises
the question about their origin in the Caribbean tunicate T.
solidum where they were first characterized over 30 years ago by
Rinehart and co-workers.17 Based on the findings reported
here, we suspect that the tunicate-derived didemnins are
similarly bacterial in origin. A recent metagenomic profile of the
tropical Pacific tunicate Lissoclinum patella revealed that the
distinctive patellamide ribosomal peptides and the anti-tumor
polyketide patellazole are biosynthetically linked to different
bacteria within the tunicate microbiome.36 Similarly, the
approved drug Yondelis (ET-743) from the tropical tunicate
Ecteinascidia turbinata was recently shown to be bacterial in
origin.12 With the T. mobilis genomic information now in hand,
molecular methods could be used to examine didemnin-
associated tunicates for the presence of Tistrella bacteria or
other microbes that carry the did genes. The finding that the
didemnin biosynthetic gene cluster resides on a plasmid is also
intriguing since other bacteria may acquire the metabolic
pathway by horizontal gene transfer. The did-containing
megaplasmid pTM3 contains several essential elements
required for conjugation, including a relaxase (TMO_c0026)
needed for DNA transfer. The bacterial chromosome addition-
ally contains a trb operon which encodes the components of a
bacterial mating apparatus, (TMO_2187−2198), another
important component in Gram-negative bacterial conjuga-
tion.37
MS Sample Preparation and MS Analysis of Didemnins. For
MALDI-TOF MS analysis, extract or peptide samples were dissolved
in MeOH, mixed 1:1 with saturated universal MALDI matrix, and
spotted on a Bruker MSP96 anchor plate. MALDI-TOF MS analysis
was performed on a Microflex Bruker Daltonics mass spectrometer
outfitted with Compass 1.3 software suite. For MSn and FTMS
analysis, HPLC-purified samples were redissolved in 50 μL of MeOH/
water/formic acid (50:49:1) and injected by a nanomate-electrospray
ionization robot (Advion) for consecutive electrospray into the MS
inlet of a LTQ 6.4T Fourier transform ion cyclotron resonance (FT-
ICR) mass spectrometer (Thermo Finnigan). MS and MSn data were
acquired in the positive ion mode. FTMS data were acquired in 400−
2000 m/z scans. Selected peptide mass signals were manually isolated
and fragmented by CID. MSn data were collected either in ion trap or
FT detection mode. All data were analyzed using QualBrowser, which
is part of the Xcalibur LTQ-FT software package (Thermo Fisher).
Genome Sequencing, Annotation, and Analysis. We used a
massively parallel pyrosequencing technology (Roche 454 GS FLX)40
to sequence the T. mobilis KA081020-065 genome. We obtained
315,496 reads with an average length of 334 bp, which provided a
16.2-fold coverage of the 6.4 Mb genome. From these reads, we
assembled 112 contigs (>500 bp) using the Newbler software of the
454 suite package. To enhance sequence quality and to construct
scaffolds, we sequenced a paired-end Illumina library of an additional
2,625,640 bp with average length of 115 bp, which were then mapped
to the genome sequence. All contig relationships within scaffolds were
then validated by PCR, while the relationships among scaffolds were
determined by multiplex PCR.41 Gaps were filled by sequencing PCR
products. The final sequence assembly was carried out using the
phred/phrap/consed package (http://www.genome.washington.edu)
in which we resequenced the low sequence quality regions. The final
error rate of the genome sequence was 0.28 per 100,000 bases. The
sequences of the five replicons were deposited in the GenBank
database as accession numbers CP003236−CP003240.
With a bacterial production of the didemnins now in place,
the supply of these structurally complex and potent anti-cancer
agents may no longer be limiting. While clinical trials with
didemnin B have been suspended, phase II trials with aplidine
(dehydrodidemnin B; plitidepsin) from the Mediterranean
tunicate Aplidium albicans are actively underway.38 Presently,
multi-step total synthesis is employed to produce aplidine for
the clinic due to its limited natural supply.6,39 Biotechnology
now may offer an alternative solution. The T. mobilis DidB
tetradomain gene product is putatively responsible for the
selection and attachment of pyruvate, its ketoreduction to
lactate, and its ensuing condensation with the DidA fatty
acylglutamine residue (Figure 4). Gene inactivation of the didB
KR domain would maintain the oxidation state of the pyruvate
unit and thereby prevent the esterification reaction, effectively
switching the didemnin B biosynthetic pathway to directly
produce aplidine. While seemingly straightforward once a
genetics system is established in Tistrella, production levels may
be significantly impacted since the activation and export
mechanism that governs didemnin B synthesis from the
Putative protein-coding sequences (CDS) were determined by
combining the prediction results of the Glimmer 3.0242 and Z-Curve
programs.43 Functional annotations of CDS were performed by
searching the KEGG and the NCBI non-redundant protein databases.
tRNA genes were predicted with tRNAScan-SE (v1.23).44 Protein
domain predictions and COG assignments45 were performed by RPS-
BLAST using the NCBI CDD library.46
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In Silico Analysis of the Didemnin Biosynthetic Gene Cluster
and Other NRPSs in T. mobilis KA081020-065. The putative roles
of the proteins in the didemnin gene cluster were assigned using
protein−protein BLAST and Pfam analyses. NRPS A domain
specificities were predicted using the online program NRPSpredictor2
and antiSMASH.47−49
Time-Course Study of Didemnin Production in T. mobilis by
MALDI-Imaging MS. One microliter of fresh overnight T. mobilis
culture was spotted onto 1 mm thick GYP agar plates and allowed to
grow for 6 h to 6 d. Colonies with different growing times were
transferred to Bruker MSP 96 anchor plates and analyzed concurrently
using the IMS technique as previously described.33,50
Didemnin Precursor Hydrolysis Assay. Samples of 50 μg each
of HPLC-purified didemnin X and Y were dissolved in 5 μL of water,
mixed with (a) 45 μL of sterile GYP medium (negative control) or (b)
45 μL of protein filtrate (10 kDa cutoff, washed 2× with GYP
medium) of 1 mL of a 1 d T. mobilis KA081020-065 liquid culture (28
°C), and incubated for 24 h at 25 °C. Hydrolysis samples were taken
and spotted on a MALDI-TOF MS plate for subsequent MS analysis
at time points 5 min, 1 h, 12 h, and 24 h.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR spectra, annotated MSn spectra of the didemnins, and
hydrolysis experiment of didemnin X/Y. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
́
Joullie, M. M. Bioorg. Med. Chem. Lett. 2001, 11, 231.
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2007, 13, 3427.
Corresponding Author
boqianpy@ust.hk; bsmoore@ucsd.edu
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Present Address
¶College of Pharmacy, Sunchon National University, Suncheon
540-950, Republic of Korea.
Author Contributions
#Y.X. and R.D.K. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Z. Shao for providing the Pacific Ocean Tistrella
strains and N. Ziemert, J. Yang and P. Lai for assistance with
data analysis. Financial support was provided by King Abdullah
University of Science and Technology (SA-C0040/UK-C0016
to P.Y.Q.), China Ocean Mineral Resources Research and
Development Association (DY125-15-T-02 to P.Y.Q.), and the
National Institutes of Health (GM085770 to B.S.M.,
CA044848 to W.F., GM086283 and S10RR029121 to P.C.D.).
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