Biosynthesis of the anti-infective marformycins featuring pre-nrps assembly line N -formylation and O -methylation and post-assembly line C -hydroxylation chemistries

Article abstract of DOI:10.1021/acs.orglett.5b00389

The biosynthetic gene cluster governing production of anti-infective marformycins was identified from deep sea-derived Streptomyces drozdowiczii SCSIO 10141. The putative mfn gene cluster (45 kb, 20 orfs) was found to encode six NRPSs and related proteins for cyclodepsipeptide core construction (mfnCDEFKL), a methionyl-tRNA formyltransferase (mfnA), a SAM-dependent methyltransferase (mfnG), and a cytochrome P450 monooxygenase for piperazic acid moiety hydroxylation (mfnN); notably, only MfnN uses an intact cyclodepsipeptide intermediate as its substrate.

Full text of DOI:10.1021/acs.orglett.5b00389

Letter
pubs.acs.org/OrgLett
Biosynthesis of the Anti-infective Marformycins Featuring Pre-NRPS
Assembly Line N‑Formylation and O‑Methylation and Post-Assembly
Line C‑Hydroxylation Chemistries
†
†
Jing Liu, Bo Wang, Hongzhi Li, Yunchang Xie, Qinglian Li, Xiangjing Qin, Xing Zhang, and Jianhua Ju*
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM
Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
Guangzhou 510301, China
*
S Supporting Information
ABSTRACT: The biosynthetic gene cluster governing production of anti-infective
marformycins was identified from deep sea-derived Streptomyces drozdowiczii SCSIO
1
0141. The putative mfn gene cluster (45 kb, 20 orfs) was found to encode six NRPSs
and related proteins for cyclodepsipeptide core construction (mfnCDEFKL), a
methionyl-tRNA formyltransferase (mfnA), a SAM-dependent methyltransferase
(
mfnG), and a cytochrome P450 monooxygenase for piperazic acid moiety
hydroxylation (mfnN); notably, only MfnN uses an intact cyclodepsipeptide
intermediate as its substrate.
yclic peptides have played an important role in natural
product drug discovery by virtue of their structural
SCSIO 10141; (ii) in vivo genetic and in vitro biochemical
C
characterization of mfnG as both an L-and D-tyrosine-O-
methyltransferase; and (iii) in vivo inactivation of mfnA, mfnK
mfnG, and mfnN, coupled with bioinformatics analysis and
metabolite identification revealing that the MFNs result from the
N-terminus preassembly line formylation and pre-NRPS
assembly line O-methylation of the Tyr residue as well as a
regio- and stereoselective post-NRPS hydroxylation of the
piperazic acid residue.
That the MFNs contain nonproteinogenic amino acid units
supported our initial hypothesis that MFN construction is
governed, in large part, by NRPSs. Accordingly, we sought to
identify the mfn cluster by application of whole genome scanning
of Streptomyces drozdowiczii SCSIO 10141 using 454 sequencing
technologies. Upon genome data annotation, we localized a 45 kb
DNA segment consisting of 20 opening reading frames (orfs)
likely to be involved in MFN biosynthesis. The putative mfn gene
cluster (Supporting Information, Table S1) was found to encode
diversity, well understood biosynthetic pathways, cell perme-
ability, and resistance to proteolytic digestion/inactivation
1
−4
pathways.
Many cyclopeptides such as the immunosuppres-
sive cyclosporine A, antibacterial agents tyrocidine A, colistin,
capreomycin and daptomycin, antifungal agent caspofungin, and
antitumor agent romidepsin are commonly used clinical drugs.
Two marine-derived cyclodepsipeptides, aplidine (plitidepsin or
dihydrodidemnin B), originally isolated from the Mediterranean
5
,6
tunicate Aplidium albicans, and kahalalide F (PM-92012),
originally isolated from the sacoglossan mollusk Elysia rufescens
and then subsequently from the algae Bryopsis pennata upon
7
,8
which it feeds, are currently being assessed as anticancer agents
9
,10
in phase II clinical trials by PharmaMar.
We recently isolated a group of cyclodepsipetides, marformy-
cins A−F (MFN, 1−6), from fermentations of a deep sea
sediment-derived Streptomyces drozdowiczii SCSIO 10141. The
structures of 1−6, including their absolute stereochemistry, have
been determined by chiral-phase HPLC and single-crystal X-ray
diffraction data analyses by our group; all have been found to
consist of five nonproteinogenic amino acid residues implicating
the importance of nonribosomal peptide synthetases (NRPSs) in
their construction. These residues include a piperazic acid unit, an
O-methyl-D-Tyr, a D-allo-Ile/D-Val, an L-allo-Ile/L-Val, and an N-
(
i) six NRPSs and related proteins for cyclodepsipeptide core
construction (MfnCDEFKL), (ii) a methionyl-tRNA formyl-
transferase (MfnA), a SAM-dependent methyltransferase
(
MfnG), and a cytochrome P450 monooxygenase (MfnN) for
scaffold tailoring steps, (iii) a regulatory protein (MfnM) and a
transportation protein (MfnR), and (iv) seven proteins
associated with precursor supply or having unclear biosynthetic
function (MfnBHIJOPQ). The nucleotide sequences have been
deposited inthe GenBank with accession number KP715145, and
the genetic organization of the biosynthetic gene cluster is shown
in Figure 1.
1
1
methyl-Val. These compounds display no cytotoxicity,
appearing, instead, to exert selective anti-infective activity against
Micrococcus luteus and the bacteria Propionibacterium acnes and P.
granulosum, highlighting their potential as anti-infective drug
1
1,12
leads (e.g., for treating acne vulgaris).
To delineate the
biosynthetic machinery of this scaffold and to enable engineering
of new analogues, we report herein: (i) identification of the gene
cluster governing MFN biosynthesis in Streptomyces drozdowiczii
Received: February 6, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.5b00389
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Figure 1. (A) Organization of mfn biosynthetic gene cluster; (B) proposed biosynthetic pathway for the MFNs; (C) MfnG-catalyzed conversion of L(D)-
10
10
Tyr into O-methyl-L(D)-Tyr. N-Formylated intermediate 7 was identified from the engineered ΔmfnK mutant. N -fH F: N -formyltetrahydrofolate;
4
Met
fMet-tRNA : formylmethionine initiator tRNA.
We constructed a genomic library of S. drozdowiczii SCSIO
0141 using the SuperCos1 vector system and screened it with
paradigm agrees well with the pattern of amino acid residues
found in the MFN natural products. Inactivation of mfnD
abolished the MFN production (Figure 2, trace ii), demonstrat-
ing its involvement in the MFN core construction.
1
selected PCR primers to yield 16 positive clones. The boundary
of the mfn gene cluster was determined by bioinformatics analysis
and PCR-targeting mutagenesis methods that involved replacing
Besides the three basic domains that constitute a minimal
NRPS [i.e., the condensation (C), A, and PCP], within the
putative mfn cluster there are two epimerization (E) domains
(modules 3 and 4) and a methyltransferase domain (MT) in
module 7, consistent with the presence of D-Tyr, D-Val/D-allo-Ile,
and N-Me-L-Val units in the MFN scaffold. Additionally, the
mfnF gene encodes a putative MbtH-like protein composed of
only 76 amino acids. Recently, it was discovered that these MbtH-
like proteins are required for selected adenylation reactions in
antibiotic pathways; MbtH-like protein inactivation has been
1
3,14
each targeted gene with an spectinomycin resistance cassette.
On the basis of these efforts, it was found that orf(−1) encodes a
highly conserved DNA recombination−mediator protein and
that orf(−2) encodes a hypothetical protein; disruptions of these
two flanking genes orf(−1) and orf(−2) did not affect MFN
production, indicating their dispensability for MFN biosynthesis.
Inactivations of mfnB (encodes a LipE-like protein) and mfnP
and mfnQ (both encode hypothetical proteins) also did not affect
the MFN production, suggesting they are not necessary in MFN
biosynthesis (Supporting Information, Figure S1).
We then analyzed genes anticipated to be critical to MFN core
construction. Three NRPS proteins MfnC, MfnD, MfnE, a free
adenylation (A) enzyme MfnK, and a free peptidyl carrier protein
PCP) MfnL constituting atotal of seven modules wereidentified
and envisioned to drive generation of the MFN core scaffold
Figure 1). The predicted specificity of A domains for amino acid
15
correlated to dramatically reduced antibiotic yields. Indeed, the
ΔmfnF mutant strain generated herein was found incapable of
MFN production (Figure 2, trace iii), consistent with previous
reports and suggesting an essential role in MFN NRPS assembly
line function.
(
Upstream of the MFN cluster resides mfnA encoding a
Met
(
methionyl-tRNA (Met-tRNA ) formyltransferase, containing a
10
10
activation (Supporting Information, Table S7) suggested that
MFN core peptide synthesis starts with L-Val/L-allo-Ile as
dictated by MfnC module 1 and terminates with the addition
of N-Me-L-Val carried out by MfnE module 7. This general
typical N -formyltetrahydrofolate (N -fH F) binding motif
4
(Figure S2, Supporting Information). In considering the
structure of MFNs, we envisioned mfnA to be involved in N-
formylation of the MFNs. We inactivated MfnA using PCR-
B
DOI: 10.1021/acs.orglett.5b00389
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to carry out Tyr O-methylation during MFN assembly. However,
mfnG inactivation completely ablated MFN production (Figure
2
, trace vi). This suggests that MfnG may methylate free Tyr; the
resulting O-methyl-Tyr may be the species recognized by the A3
domain and subsequently activated enabling introduction to the
NRPS assembly line. To verify this hypothesis, we added O-
methyl-D-Tyr and O-methyl-L-Tyr precursors, respectively, to
fermentations of the ΔmfnG mutant strain. As expected, the
ability of the ΔmfnG strain to produce MFNs was restored by the
addition of O-methyl-L-Tyr to the fermentation medium.
However, a substantial amount of MFN production was also
observed with the addition of O-methyl-D-Tyr. The ratio of
harvested MFNs obtained by providing ΔmfnG access to both O-
methyl-L-Tyr and O-methyl-D-Tyr was approximately 3:1 (Figure
2
, traces vii and viii). These data suggest that the A domain of
3
MfnC possesses relaxed substrate specificity but prefers O-
methyl-L-Tyr over O-methyl-D-Tyr.
Figure 2. HPLC profiles of S. drozdowiczii SCSIO 10141 wild-type and
The biochemical activity of MfnG was further explored in vitro.
The mfnG gene was cloned into the NdeI and EcoRI sites of the
pET28a (+) vector, overexpressed in Escherichia coli BL21
mutant strains, ΔmfnG mutant provided with O-Me-Tyr (L or
D
isomers) in the fermentation. See Figure 1 for structures.
(
DE3), and purified to homogeneity as an N-terminus His-tagged
protein. The enzymatic activity of MfnG in various buffer systems
i.e., HEPES, phosphate, and Tris buffers) and over a wide pH
targeting mutagenesis methodology directed at mfnA; fermenta-
tion and HPLC-based extract analysis for the ΔmfnA strain
revealed that MfnA inactivation abolished production of MFN
and corresponding intermediates (Figure 2, trace iv). These data
clearly demonstrate the indispensability of MfnA to MFN
production and suggest that it may direct incorporation of the
formyl group as a starter unit in the L-Val/L-allo-Val lateral chain
(
range (5.0−9.0) was evaluated. The optimal enzyme activity was
observed in a pH 8.0 phosphate buffer system at 30 °C. Typical in
vitro assays (50 μL) were performed in the presence of 50 mM
phosphate buffer (pH 8.0), 0.2 mM substrate L-Tyr or D-Tyr, 0.24
mM SAM, and 0.5 μM MfnG at 30 °C for 2 h, and were quenched
by addition of two volumes of MeOH. HPLC data were acquired
at different time points revealing MfnG-catalyzed generation of
O-methyl-L-Tyr and O-methyl-D-Tyr from L-Tyr and D-Tyr,
respectively, in a time-dependent manner (Figure 3). Detailed
(
Figure 1).
In considering MfnA and its precise role in MFN synthesis, we
noted that MfnC harbors a noncanonical C-A-PCP initiation
module (Figure 1); this unusual starter module has been
discovered in assorted N-acylated NRPS products such as the
1
6
xenematides. We thus proposed that MfnA may transfer a
Met
formyl group to Met-tRNA
from the primary metabolite
Met
pathway to generate formylmethionine tRNA (fMet-tRNA ).
The C-domain of module 1 (also containing the conserved
HHxxxD motif, Supporting Information, Figure S3) would
Met
condense the fMet-tRNA with L-Val/L-allo-Val that had been
loaded onto the module 1 PCP, thus forming N-formyl L-Val/L-
allo-Val of the MFN biosynthetic assembly line. Aminoacylated
tRNA-dependent peptide bond formation mediated by PacB, but
not catalyzed by the condensation domain, has been validated in
17
the nucleoside antibiotic pacidamycin pathway. Alternatively,
10
MfnA may directly transfer the formyl group from N -fH F to
4
the L-Val on the PCP domain of module 1, bypassing the C
domain of module 1. Notably, a formylation (F) domain
embedded within the NRPS module (F-A-PCP) has been
Figure 3. HPLC analyses MfnG-catalyzed reactions using substrates L-
Tyr and D-Tyr. Time-course experiments incubated with MfnG and
SAM for (i) 10 min, (ii) 30 min, and (iii) 60 min; panel (iv) in each set of
analyses represents a control reaction in which MfnG was heat denatured
prior to substrate presentation.
1
0
shown to transfer the N-formyl group from N -fH F to a PCP-
4
18
tethered L-Val in the gramicidin pathway.
A pre-NRPS assembly line N-formylation mechanism is
supported by LC−MS-based metabolite analysis of fermenta-
tions of a ΔmfnK mutant which revealed the accumulation of
small quantities of a biosynthetic intermediate 7 (Figure 2, trace
v). The compound 7 was structurally characterized following
kinetic analyses of MfnG were carried out using a 50 μL reaction
system containing 50 mM sodium phosphate buffer (pH 8.0).
The kinetic parameters of MfnG-catalyzed reactions with L- and
D-Tyr were determined and are summarized in Table 1; these
kinetic data reveal that MfnG utilizes L-Tyr as the preferred
substrate in accord with the previous feeding results.
1
large-scale fermentation, purification, and subsequent MS and H
13
and C NMR data analyses. It is notable that, despite its small
size, compound 7 bears terminal N-formylation indicative of
MfnA action at the start of MFN assembly.
Finally, we examined the role of putative cytochrome P450
enzyme MfnN. MfnN displays high similarity (77%) to and
identity (64%) with Ema5 from the abamectin biosynthetic
We then explored the role of mfnG encoding a putative
methyltransferase. MfnG shows high similarity (68%) to the S-
adenosylmethionine (SAM)-dependent O-methyltransferase
Sky37 from Streptomyces sp. acta 2897 and was thus proposed
19
pathway; Ema5 converts avermectin to 4″-oxoavermectin. The
mfnN gene was inactivated in a manner similar to that used to
inactivate mfnG. HPLC analyses of culture extracts revealed that
C
DOI: 10.1021/acs.orglett.5b00389
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Table 1. Kinetic Parameters of MfnG-Catalyzed Reactions
ASSOCIATED CONTENT
Supporting Information
■
kcat (min−1
k /K (μM−1 min−1
*
S
substrate
Km (μM)
)
)
cat
m
2.87 ± 10−1
Detailed experimental procedures, NMR data and spectra for
compound 7. This material is available free of charge via the
Internet at http://pubs.acs.org.
L-Tyr
D-Tyr
78.74 ± 7.790
173.8 ± 13.90
22.73 ± 0.5665
22.07 ± 0.8226
1.27 ± 10−1
the ΔmfnN mutant failed to produce MFNs C (3) and D (4) but
generated MFNs A (1) and B (2) as the predominant products
AUTHOR INFORMATION
Corresponding Author
■
*
(Figure 2, trace ix). The structures of 1 and 2 produced by the
E-mail: jju@scsio.ac.cn.
Author Contributions
ΔmfnN mutant were found to be identical to MFNs A and B
isolated from the wild-type strain upon large scale (8-L)
1
13
†
fermentation, purification, and HRMS, H NMR, and
C
J.L. and B.W. contributed equally.
NMR data comparisons. These data clearly reveal that MfnN is a
cytochrome P450 monooxygenase responsible for the regio- and
stereospecific hydroxylation of the MFN piperazic acid moiety.
MFNs A−F (1−6) belong to a very small class of N-terminally
formylated nonribosomal peptide natural products. Members of
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by NSFC (41306146, 31290233,
81425022), and MOST (2012AA092104).
■
18
this family include the linear gramicidins, cyclic anabaenopep-
2
0
21
tilides, and the siderophores rhodochelin and coelichelin. In
the rhodochelin biosynthetic pathway, installation of the N-
formyl group is carried out by the formyltransferase Rft using N -
10
REFERENCES
■
(
1) Andavan, G. S.; Lemmens-Gruber, R. Mar. Drugs. 2010, 8, 810−
fH F and the small molecule δ-N-hydroxyornithine as sub-
4
2
1
834.
strates. In considering the MFN machinery, the noncanonical
initiation module with a C-A-PCP arrangement, the critical role
of the methionyl-tRNA formyltransferase MfnA and the captured
NRPS off-line N-formylated biosynthetic intermediate 7 strongly
suggests a different paradigm for N-terminus formylation of
NRPS natural products. Specifically, the mfn cluster indicates the
possibility that formylation may be accomplished via con-
(
2) Lemmens-Gruber, R.; Kamyar, M. R.; Dornetshuber, R. Curr. Med.
Chem. 2009, 16, 1122−1137.
3) Sarabia, F.; Chammaa, S.; Ruiz, A. S.; Ortiz, L. M.; Herrera, F. J.
Curr. Med. Chem. 2004, 11, 1309−1332.
4) Ballard, C. E.; Yu, H.; Wang, B. Curr. Med. Chem. 2002, 9, 471−498.
(5) Schmitz, F. J.; Yasumoto, T. J. Nat. Prod. 1991, 54, 1469−1490.
(6) Xu, Y.; Kersten, R. D.; Nam, S. J.; Lu, L.; Al-Suwailem, A. M.; Zheng,
H.; Fenical, W.; Dorrestin, P. C.; Moore, B. S.; Qian, P. Y. J. Am. Chem.
Soc. 2012, 134, 8625−8632.
7) Hamann, M. T.; Scheuer, P. J. J. Am. Chem. Soc. 1993, 115, 5825−
5826.
8) Ashour, M.; Edrada, R.; Ebel, R.; Wray, V.; Wat
Padmakumar, K.; Muller, W. E.; Lin, W. H.; Proksh, P. J. Nat. Prod.
006, 69, 1547−1553.
9) Mayer, A. M.; Glaser, K. B.; Cuevas, C.; Jacobs, R. S.; Kem, W.;
Little, R. D.; McIntosh, J. M.; Newman, D. J.; Potts, B. C.; Shuster, D. E.
Trends Pharmacol. Sci. 2010, 31, 255−265.
(10) Newman, D. J.; Cragg, G. M. Mar. Drugs 2014, 12, 255−278.
(11) Zhou, X.; Huang, H.; Li, J.; Song, Y.; Jiang, R.; Liu, J.; Zhang, S.;
Hua, Y.; Ju, J. Tetrehedron 2014, 70, 7795−7801.
12) Watanabe, M.; Kamigiri, K.; Tanaka, K.; Takeda, Y.; Yokoi, T.;
Shibazaki, K.; Suzumura, K.; Hashimoto, M.; Nishiwaki, S.; Takase, S.;
Abe, F. PCT Patent WO 2006073151, 2006.
13) Gust, B.; Challis, G. L.; Fowler, K.; Kieser, T.; Chater, K. F. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 1541−1546.
14) Zhang, Y.; Huang, H.; Chen, Q.; Luo, M.; Sun, A.; Song, Y.; Ma, J.;
(
(
densation domain-mediated peptide bond formation using fMet-
Met
tRNA
and the PCP-tethered L-Val residue as substrates.
(
Alternatively, MfnA (not the F domain)-mediated peptide bond
1
0
formation may take place using N -fH Fand the PCP-tethered L-
4
(
̈
jen, W.;
Val residue as substrates.
̈
Among NRPS-based systems, it is a unique feature of the mfn
machinery that preassembly line methylation of Tyr takes place.
More surprising is that the agent of this change, MfnG, can use
both L- and D-Tyr as substrates yet the natural MFNs contain
exclusively the O-methyl-D-Tyr moiety. These observations are
especially intriguing in light of the putative epimerase domains
embedded within the NRPS machinery. Moreover, these findings
provide clues relevant to understanding the biosynthetic pathway
of the cyclic peptide aplidine. Importantly, aplidine, now in
clinical trials, also contains a methyl-O-Tyr moiety whose
2
(
(
(
5
generation remains unclear.
In summary, we have employed genome scanning, bio-
informatics analyses, gene inactivations, and in vitro biochemical
experiments to identify and validate the 45 kb mfn biosynthetic
gene cluster in Streptomyces drozdowiczii SCSIO 10141.
Significant attention has been paid to tailoring steps involved in
MFN production. These steps include two unusual pre-NRPS
assembly line transformations, (i) an N-terminal formylation step
and (ii) a pre-NRPS O-methylation step, to afford O-methyl-L-
Tyr and O-methyl-D-Tyr as MFN building blocks (although only
the D isomer appears within completed MFN scaffolds).
Following completion of NRPS-directed steps and liberation
from the NRPS machinery, a novel, regio- and stereospecific
hydroxylation of the MFN piperazic acid moiety was carried out
(
Ju, J. Org. Lett. 2013, 15, 3254−3257.
(15) Zhang, W.; Heemstra, J. R., Jr.; Walsh, C. T.; Imker, H. J.
Biochemistry 2010, 49, 9946−9947.
(16) Crawford, J. M.; Portmann, C.; Kontnik, R.; Walsh, C. T.; Clardy,
J. Org. Lett. 2011, 13, 5144−5147.
(
17) Zhang, W.; Ntai, I.; Kelleher, N. L.; Walsh, C. T. Proc. Natl. Acad.
Sci. U.S.A. 2011, 108, 12249−12253.
18) Schoenafinger, G.; Schracke, N.; Linne, U.; Marahiel, M. A. J. Am.
Soc. Chem. 2006, 128, 7406−7407.
19) Jungmann, V.; Molnar, I.; Hammer, P. E.; Hill, D. S.; Zirkle, R.;
(
(
́
Buckel, T. G.; Buckel, D.; Ligon, J. M.; Pachlatko, J. P. Appl. Environ.
Microbiol. 2005, 71, 6968−6976.
̈
(20) Rouhiainen, L.; Paulin, L.; Suomalainen, S.; Hyytiainen, H.;
(
by MfnN). These findings set the stage for full characterization
Buikema, W.; Haselkorn, R.; Sivonen, K. Mol. Microbiol. 2000, 37, 156−
of the biosynthetic mechanisms driving MFN production and
enable the engineering of new MFN analogues using
combinatorial or precursor-directed biosynthetic strategies for
antibacterial drug discovery.
167.
(
21) Bosello, M.; Mielcarek, A.; Giessen, T. W.; Marahiel, M. A.
Biochemistry 2012, 51, 3059−3066.
D
DOI: 10.1021/acs.orglett.5b00389
Org. Lett. XXXX, XXX, XXX−XXX