Use of a phosphonate methyltransferase in the identification of the fosfazinomycin biosynthetic gene cluster

Article abstract of DOI:10.1002/anie.201308363

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for stable isotope labeling of phosphonates in extract (SILPE) that facilitates their purificatio

Full text of DOI:10.1002/anie.201308363

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Angewandte
Communications
DOI: 10.1002/anie.201308363
Natural Product Purification
Use of a Phosphonate Methyltransferase in the Identification of the
Fosfazinomycin Biosynthetic Gene Cluster**
Jiangtao Gao, Kou-San Ju, Xiaomin Yu, Juan E. Velꢀsquez, Subha Mukherjee, Jaeheon Lee,
Changming Zhao, Bradley S. Evans, James R. Doroghazi, William W. Metcalf,* and
Wilfred A. van der Donk*
Abstract: Natural product discovery has been boosted by
genome mining approaches, but compound purification is
often still challenging. We report an enzymatic strategy for
“stable isotope labeling of phosphonates in extract” (SILPE)
that facilitates their purification. We used the phosphonate
methyltransferase DhpI involved in dehydrophos biosynthesis
to methylate a variety of phosphonate natural products in
crude spent medium with a mixture of labeled and unlabeled S-
adenosyl methionine. Mass-guided fractionation then allowed
straightforward purification. We illustrate its utility by purify-
ing a phosphonate that led to the identification of the
fosfazinomycin biosynthetic gene cluster. This unusual natural
product contains a hydrazide linker between a carboxylic acid
and a phosphonic acid. Bioinformatic analysis of the gene
cluster provides insights into how such a structure might be
assembled.
been developed to aid in the purification process.[2] Herein
we illustrate the use of an enzymatic approach to rapidly
enrich a phosphonate from the spent medium of Streptomyces
sp. WM6372, a strain previously shown to encode an
uncharacterized phosphonate biosynthetic gene cluster. Iso-
lation and structure elucidation identified the molecule as
methyl-2-hydroxy-2-phosphonoacetic acid (Me-HPnA).
Realization that Me-HPnA is present in fosfazinomycins
prompted a targeted search after growth of the strain in
various media. We also examined Streptomyces XY332, which
was previously shown to encode the same phosphonate
biosynthetic genes. Both strains produced fosfazinomycins,
and heterologous expression of the genes in S. lividans led to
production of related phosphonic acids, providing strong
support for linking the gene cluster to fosfazinomycin
production. Analysis of the genes provides insights into the
biosynthetic pathway of hydrazine formation in nature.
Phosphonate natural products form a particular challenge
in regards to purification, as a consequence of their high
polarity and water solubility. These properties may explain
why only about 30 phosphonate natural products have been
isolated, despite the observation that about 5% of randomly
isolated actinomycetes contain the genetic capability to
produce phosphonates.[3] The phosphonate O-methyltransfer-
ase DhpI, from the dehydrophos biosynthetic pathway, is able
to non-specifically methylate other phosphonates such as
fosfomycin and fosmidomycin under defined reaction con-
ditions.[4] To determine whether DhpI could methylate these
compounds in a complex medium, fosfomycin and fosmido-
mycin were added to international streptomyces project 4
(ISP4) medium. Indeed, when supplied with S-adenosyl
methionine (SAM), DhpI methylated both compounds as
determined by 31P NMR spectroscopy (Figure 1a; see also the
Supporting Information, Figures S1 and S2, and Tables S1 and
N
atural products have gained renewed interest with the
realization that the genetic capacity of microorganisms to
produce these molecules is much larger than anticipated.
Indeed, genome mining exercises have resulted in the
discovery of a variety of new compounds.[1] Whereas the
sequenced bacterial and fungal genomes demonstrate the
untapped potential of natural products, connecting the
biosynthetic genes in genomes to novel compounds is still
a challenge. One aspect of this challenge is the difficulty of
purifying these molecules from complex spent media.
Recently, chemoselective derivatization approaches have
[*] Dr. J. Gao, Dr. K. Ju, Dr. J. Lee, Dr. C. Zhao, Dr. B. S. Evans,
Dr. J. R. Doroghazi
Institute for Genomic Biology, University of Illinois at Urbana-
Champaign (USA)
X. Yu, Prof. W. W. Metcalf
University of Illinois at Urbana-Champaign
Department of Microbiology
601 South Goodwin Avenue, Urbana, IL 61801 (USA)
E-mail: metcalf@illinois.edu
Dr. J. E. Velꢀsquez, S. Mukherjee, Prof. W. A. van der Donk
University of Illinois at Urbana-Champaign
Howard Hughes Medical Institute and Department of Chemistry
600 South Mathews Avenue, Urbana, IL 61801 (USA)
E-mail: vddonk@illinois.edu
[**] This work was supported by the National Institutes of Health (GM
PO1 GM077596 to W.W.M. and W.A.V.) NMR spectra were recorded
on a 600 MHz instrument purchased with support from NIH Grant
S10 RR028833.
Figure 1. a) 31P NMR spectrum of fosfomycin (top) and after treatment
with DhpI and a 1:1 mixture of SAM and [D3]SAM (bottom). b) LC-MS
analysis of the sample in the bottom spectrum of (a).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201308363.
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S2). The broad substrate specificity and robust activity of
DhpI prompted the development of a method that relies on
“stable isotope labeling of phosphonates in extract” (SILPE).
The method uses the facilitated identification of pairs of
compounds of different stable isotope composition by mass
spectrometry, based on their well-defined mass difference,
which is similar to the SILAC method in proteomics.[5]
Accordingly, ISP4 media spiked with fosfomycin or fosmido-
mycin were treated with DhpI and a mixture of SAM and
CD3-SAM. The crude material was injected into an LCMS
system and the eluent analyzed for compounds giving rise to
two ions separated by 3.0188 Da, thus allowing the facile
detection of methylated fosfomycin and fosmidomycin (Fig-
ure 1b; see also Figures S1–S3).
We then turned our attention to unknown phosphonates
produced by Streptomyces sp. WM6372. This strain was
previously identified as a potential phosphonate producer by
screening for the presence of the phosphoenolpyruvate
mutase gene (pepM), a characteristic marker for phosphonate
biosynthesis.[3,6] Spent medium from this strain grown on ISP2
agar plates displayed two resonances in the phosphonate
window of the 31P NMR spectrum (Figure 2a). After several
unsuccessful attempts to isolate the compounds using tradi-
compounds before DhpI-treatment as methyl phosphonoa-
cetate (1) and methyl 2-hydroxy-2-phosphono-acetate (Me-
HPnA (2); Figure 2c); these assignments were confirmed by
chemical synthesis (Figure S5). A structure search revealed
that these molecules are new phosphonate natural products;
however, Me-HPnA is also a substructure in fosfazinomycins
(Figure 2c). We therefore cultivated Streptomyces sp.
WM6372 in a variety of media and monitored for fosfazino-
mycin production by LCMS analysis. In most media, produc-
tion of fosfazinomycins was not observed; however, the strain
produces small amounts of both fosfazinomycin A and B after
growth in R2AS medium containing phosphonoacetate.
Therefore, the organism has all the genes needed to produce
fosfazinomycin, although it appears that unknown regulatory
or metabolic limitations prevent its de novo synthesis under
the conditions examined.
We previously reported that Streptomyces sp WM6372
and Streptomyces sp. XY332 encode nearly identical phos-
phonate biosynthetic gene clusters (Figure S6).[6b] Therefore,
we tested whether Streptomyces sp. XY332 could produce
fosfazinomycins. Evaluation of various growth conditions and
analysis by LCMS demonstrated that two compounds were
produced with masses consistent with fosfazinomycin A and
B. A 31P NMR spectrum of the spent medium also revealed
two small new signals with close chemical shifts at 13.7 and
13.5 ppm in addition to the peaks for compounds 1 and 2
(Figure 3a). To provide confirmation that the new peaks
represented fosfazinomycin A and B, the compounds were
partially purified. A 1H-31P HMBC NMR spectrum displayed
correlations consistent with the fosfazinomycin core struc-
ture, and MS analysis showed molecular ions consistent with
fosfazinomycin A and B (Figure S7). Finally, Streptomyces
XY332 was grown on minimal medium containing
(15NH4)2SO4 as the sole nitrogen source, and the 31P NMR
spectrum was recorded. The two peaks in the 31P spectrum of
Figure 2. a) 31P NMR spectrum of spent medium of Streptomyces sp.
WM 6372. Only the chemical shift region of interest with respect to
phosphonates is depicted. b) 31P NMR spectrum after treatment with
DhpI and SAM. c) Structures of the phosphonates giving rise to the
signals in (a) and the structures of fosfazinomycin A and B.
tional purification methods, we attempted the SILPE method.
The solid medium was freeze-thawed and compressed to
release metabolites, evaporated to dryness, and taken up in
90% MeOH. Insoluble material was removed by centrifuga-
tion. The supernatant was concentrated and partially desalted
by Sephadex LH20. The resulting crude mixture was treated
with DhpI and a mixture of SAM and CD3-SAM. The two
original phosphonate peaks in the 31P NMR spectrum dis-
appeared and two new peaks were generated (Figure 2b; see
also Table S1). Analysis by LCMS using the mass difference
between compounds containing a CH3 or a CD3 group as
marker allowed the purification of both molecules and
Figure 3. a) 31P NMR spectrum of spent medium of Streptomyces sp.
XY332. b) 31P NMR spectrum of spent medium of the same strain
when grown on 15NH4SO4. The chemical shift variation is the result of
small changes in pH, as these compounds have pKa values near 7.
c) DNA fragment of the genome from Streptomyces sp. WM 6372 that
contains the pepM gene; Genes are color-coded according to their
putative function: formation of Me-HPnA (red), nitrogen methylation
1
determination of their masses. Characterization by H and
À
(dark green), amide ligation (pink), installation of the N N bond (light
1
13C NMR spectroscopy, as well as H-31P-HMBC and mass
green; the four genes shared with the clusters for the biosynthesis of
diazo-containing natural products are striped in light green).
spectrometry (Figure S4), allowed assignment of the parent
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the spent medium now displayed a doublet splitting pattern
with a coupling constant of 11.4 Hz (Figure 3b), and the high
Streptomyces sp. XY332 (Figure 3c and Table 1; see also
Figure S6), a putative biosynthetic pathway for fosfazinomy-
cin biosynthesis can be formulated (Figure 4). FzmC is the
resolution mass spectrum indicated the presence of seven 15
N
À
atoms, which is consistent with the assignment to fosfazino-
mycin A (Figure S8).
PEP mutase that installs the C P bond in phosphonopyruate
Two lines of evidence provide strong support to link the
gene cluster shown in Figure 3c with the biosynthesis of
fosfazinomycins. First, both organisms contain this gene
cluster, and both can produce the antibiotic. Moreover,
examination of draft genome sequences shows that these
clusters are the only PEP mutase-encoding loci in these
organisms; PEP mutase is involved in the biosyntheses of all
phosphonates that are currently understood. Furthermore, we
transferred the cluster to S. lividans, a non-phosphonate
producer, and showed that the recombinant strain produced
1 and 2. Thus, the transferred gene cluster clearly directs
synthesis of the phosphonate moiety of fosfazinomycin. We
suspect that, as with the parental strain, regulatory or
metabolic limitations prevent de novo synthesis of the final
product in S. lividans. Given that all characterized phospho-
nate biosynthetic pathways require PEP mutase, and that the
gene cluster in question directs the synthesis of the phospho-
nate moiety of fosfazinomycin, we believe that it is highly
likely, albeit not definitive in the absence of successful
heterologous production, that these loci are responsible for
fosfazinomycin synthesis.
Figure 4. Proposed biosynthetic pathway of fosfazinomycin A. Putative
steps are indicated with dashed arrows. FzmFIN are proposed to be
involved in formation of the three magenta-colored bonds, whereas
the FzmAMNOQR proteins are proposed to be involved in forming
and installing the hydrazine core.
Fosfazinomycin A and B were first isolated from Strepto-
myces lavendofoliae 630 as part of a screening program
focused on antifungal antibiotics.[7] The two peptide conge-
ners differ at their N termini (Figure 2c). Fosfazinomycins
contain a unique hydrazide linkage between the carboxylic
acid of Arg and the phosphonate of Me-HPnA. Nitrogen–
nitrogen bonds are relatively rare in nature[8] and the
mechanism of their formation is generally still poorly under-
stood.[9] Based on bioinformatics analysis of the putative
biosynthetic gene clusters of Streptomyces sp. WM6372 and
(PnPy) and FzmD decarboxylates PnPy to generate phos-
phonoacetaldehyde (PnAA). This intermediate is then pro-
posed to be oxidized to phosphonoacetate (PnA) and
methylated to generate 1, one of the observed metabolites.
The cluster does not contain a canonical aldehyde dehydro-
genase, which suggests that a housekeeping enzyme may be
used for the conversion of PnAA into PnA, but methylation
of the carboxylate is likely catalyzed by the S-adenosylme-
Table 1: Summary of open reading frames (ORFs) in the genomic DNA of Streptomyces sp. WM 6372 that includes the fosfazinomycin biosynthetic
gene cluster.
ORF
No. of
Highest identity homologue (as of September 1, 2013)
Amino acid
residues
identity/similarity [%]
FzmA
FzmB
FzmC
FzmD
FzmE
FzmF
FzmG
FzmH
FzmI
727
233
284
378
253
465
317
349
426
411
231
492
655
519
500
575
134
435
341
Actinoplanes sp. SE50/110 asparagine synthase (YP_006269188.1)
Haliangium ochraceum DSM 14365 type 11 methyltransferase (YP_003269122.1)
Bacillus cereus VD102 PEP phosphomutase (WP_000571606.1)
Micromonospora sp. ATCC 39149 PnPy decarboxylase (WP_007071820.1)
Streptomyces ambofaciens ATCC 23877 phosphodiesterase (CAJ89340.1)
Methanococcoides burtonii DSM 6242 pyruvate carboxylase (YP_567027.1); ATP-GRASP family.
Streptomyces luridus dioxygenase (ACZ13452.1)
48/64
48/63
52/69
52/64
55/66
29/45
45/55
35/46
31/43
16/30
18/35
62/70
56/65
56/67
52/61
24/41
76/85
64/76
45/57
Nonomuraea sp. WU8817 N-methyl transferase (ACS83764.1)
Streptomyces rapamycinicus N-acyltransferase (CAA60475.1)
FzmJ
Corynebacterium matruchoti MFS transporter (WP_005523355.1)
Desulfovibrio hydrothermalis DSM 14728 thymidylate kinase (YP_007326724.1)
Streptomyces bingchenggensis BCW-1 lyase (YP_004961426.1)
Streptomyces davawensis JCM 4913 FAD(NAD)-dependent oxidoreductase (YP_007524522.1)
uncultured bacterium BAC AB649/1850 glutamine synthetase (AEE65491.1)
Salinispora tropica CNB-440 amidase (YP_001159034.1)
Staphylococcus epidermidis hypothetical protein (WP_002477807.1)
Streptomyces sp. N-acetyltransferase (WP_008740585.1)
Streptomyces ambofaciens ATCC 23877 adenylosuccinate lyase (CAI78075.1)
Streptomyces violaceusniger Tu 4113 transcriptional regulator (YP_004814876.1)
FzmK
FzmL
FzmM
FzmN
FzmO
FzmP
FzmQ
FzmR
FzmS
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thionine-dependent O-methyltransferase FzmB. FzmG is
a likely candidate for the hydroxylation of 1 to produce 2,
because its closest homologue is the a-ketoglutarate-depen-
dent oxygenase that hydroxylates 2-hydroxyethylphospho-
nate during dehydrophos biosynthesis.[10] To complete the
biosynthesis of fosfazinomycin A, three bonds need to be
made between nitrogen nucleophiles and acid electrophiles.
Interestingly, the cluster encodes proteins from three differ-
ent amidoligase families:[11] FzmF, a member of the ATP
GRASP enzyme family,[11–12] FzmI, a member of the GCN5-
related N-acetyltransferases (GNATs), which are enzymes
that function as aminoacyl tRNA-dependent peptidyl trans-
ferases,[11,13] and FzmN, a member of the glutamine synthase
family that is known to catalyze amide bond formation.[11,14]
Further studies are required to determine which of these
three enzymes makes which of the magenta-colored bonds in
Figure 4.
Keywords: antibiotics · biosynthesis · hydrazine ·
natural products
.
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construction of the hydrazide core. The methyl group on the
hydrazide is probably installed by the N-methyltransferase
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À
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À
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À
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In summary, we used the substrate tolerance of the
phosphonate methyltransferase DhpI as a chemoselective
tool to purify two phosphonate metabolites that are inter-
mediates in the biosynthesis of fosfazinomycin. Bioinformat-
ics analysis and heterologous production experiments provide
support for the involvement of two nearly identical gene
clusters in fosfazinomycin biosynthesis, thus providing
À
insights into how the N N bond might be fashioned and
showing the unusual combination of an ATP-GRASP ligase,
a tRNA-dependent peptidyl transferase, and a glutamine
synthetase in the biosynthesis of a natural product.
Received: September 24, 2013
Revised: November 16, 2013
Published online: December 27, 2013
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