An Obligate Peptidyl Brominase Underlies the Discovery of Highly Distributed Biosynthetic Gene Clusters in Marine Sponge Microbiomes

Article abstract of DOI:10.1021/jacs.1c03474

Marine sponges are prolific sources of bioactive natural products, several of which are produced by bacteria symbiotically associated with the sponge host. Bacteria-derived natural products, and the specialized bacterial symbionts that synthesize them, are not shared among phylogenetically distant sponge hosts. This is in contrast to nonsymbiotic culturable bacteria in which the conservation of natural products and natural product biosynthetic gene clusters (BGCs) is well established. Here, we demonstrate the widespread conservation of a BGC encoding a cryptic ribosomally synthesized and post-translationally modified peptide (RiPP) in microbiomes of phylogenetically and geographically dispersed sponges from the Pacific and Atlantic oceans. Detection of this BGC was enabled by mining for halogenating enzymes in sponge metagenomes, which, in turn, allowed for the description of a broad-spectrum regiospecific peptidyl tryptophan-6-brominase which possessed no chlorination activity. In addition, we demonstrate the cyclodehydrative installation of azoline heterocycles in proteusin RiPPs. This is the first demonstration of halogenation and cyclodehydration for proteusin RiPPs and the enzymes catalyzing these transformations were found to competently interact with other previously described proteusin substrate peptides. Within a sponge microbiome, many different generalized bacterial taxa harbored this BGC with often more than 50 copies of the BGC detected in individual sponge metagenomes. Moreover, the BGC was found in all sponges queried that possess high diversity microbiomes but it was not detected in other marine invertebrate microbiomes. These data shed light on conservation of cryptic natural product biosynthetic potential in marine sponges that was not detected by traditional natural product-to-BGC (meta)genome mining.

Full text of DOI:10.1021/jacs.1c03474

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Article
An Obligate Peptidyl Brominase Underlies the Discovery of Highly
Distributed Biosynthetic Gene Clusters in Marine Sponge
Microbiomes
Nguyet A. Nguyen, Zhenjian Lin, Ipsita Mohanty, Neha Garg, Eric W. Schmidt, and Vinayak Agarwal*
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ABSTRACT: Marine sponges are prolific sources of bioactive
natural products, several of which are produced by bacteria
symbiotically associated with the sponge host. Bacteria-derived
natural products, and the specialized bacterial symbionts that
synthesize them, are not shared among phylogenetically distant
sponge hosts. This is in contrast to nonsymbiotic culturable
bacteria in which the conservation of natural products and natural
product biosynthetic gene clusters (BGCs) is well established.
Here, we demonstrate the widespread conservation of a BGC
encoding a cryptic ribosomally synthesized and post-translationally
modified peptide (RiPP) in microbiomes of phylogenetically and
geographically dispersed sponges from the Pacific and Atlantic
oceans. Detection of this BGC was enabled by mining for
halogenating enzymes in sponge metagenomes, which, in turn, allowed for the description of a broad-spectrum regiospecific peptidyl
tryptophan-6-brominase which possessed no chlorination activity. In addition, we demonstrate the cyclodehydrative installation of
azoline heterocycles in proteusin RiPPs. This is the first demonstration of halogenation and cyclodehydration for proteusin RiPPs
and the enzymes catalyzing these transformations were found to competently interact with other previously described proteusin
substrate peptides. Within a sponge microbiome, many different generalized bacterial taxa harbored this BGC with often more than
50 copies of the BGC detected in individual sponge metagenomes. Moreover, the BGC was found in all sponges queried that possess
high diversity microbiomes but it was not detected in other marine invertebrate microbiomes. These data shed light on conservation
of cryptic natural product biosynthetic potential in marine sponges that was not detected by traditional natural product-to-BGC
(meta)genome mining.
by examining the chemical structures of the natural products
isolated from sponges. Subsequently, a potential biosynthetic
scheme to the desired natural product is postulated, with the
resulting hypothetical BGCs serving as templates to mine the
sponge metagenomic data. This natural product → BGC
approach has connected several sponge-derived natural
products to their respective BGCs, led to identification of
the physiological producers from among the microbial milieu
of the sponge holobiont, and illuminated microbiological and
enzymological ingenuities. However, as this approach does not
involve an untargeted mining of sponge microbiomes, an
appreciation for the diversity and conservation of natural
product BGCs present in marine sponge microbiomes has
INTRODUCTION
■
Highly specialized small organic molecules called natural
products are the universal language of symbiont-host
interactions. Often, a symbiont generates a natural product
that offers the host a survival and competitive advantage.1 This
leads to symbiont specialization, such as by drastic genome
reduction and/or by becoming a natural product superproducer
by accumulating the genetic potential to generate numerous
different bioactive natural products. The host, in turn, fulfills
primary metabolic demands of the symbiont, which is likely
why natural product producing symbionts resist laboratory
cultivation. Examples of such natural product mediated
symbioses are widespread in the nutrient poor benthic marine
environment.2 Benthic marine invertebrates that harbor rich
and diverse microbiomes, such as sponges,3,4 are engines for
marine natural product discovery.5 Several sponge-derived
natural products are produced by members of the bacterial
microbiome that is symbiotically associated with the eukaryotic
sponge host.2,6 Efforts to discover the biogenetic routes for
sponge holobiont-derived natural products have been initiated
Received: April 1, 2021
Published: July 2, 2021
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Figure 1. srp BGCs detected in the S. aurea metagenome. Of the 63 srp BGCs detected in S. aurea, ten representative examples are illustrated here.
The srpE genes are marked by arrows. Genes srpA−E and srpT1 and srpT2 were conserved in all srp BGCs. The halogenase is encoded by gene srpI.
Gene srpT1 also possesses the peptidase that cleaves at the leader/core boundary of the NHLP SrpE substrate.
been scarce. As was recently illustrated for natural product
geosmin, the BGC for which is present in nearly every
streptomycete genome, conserved BGCs furnish natural
products that fulfill primary ecological functions.7 While
natural products in sponges are broadly postulated to serve
as chemical defenses against herbivory and predation,8,9 it
remains unknown whether microbiomes of different sponges
share natural product BGCs, or whether certain class(es) of
natural products are prioritized for conservation in sponge
holobionts. Furthermore, access to cryptic natural products,
the biosynthetic potential for which remains locked away in
sponge microbiomes, has not been realized.
Sponges have been one of the most prolific sources of
natural products and sponge-derived natural products extend
across all known natural product chemical classes.5 However, it
is instructive to observe that the inventory of ribosomally
synthesized and post-translationally modified peptide (RiPP)
natural products from marine sponges is small. The biogenetic
basis of a solitary group of sponge-derived RiPPs has been
established, the polytheonamides.10−12 Enzymatic reactions
that furnish RiPPs are predicated upon substrate peptides that
are typically divided between an N-terminal leader and a C-
terminal core region.13 Amino acid residues in the core are
modified by biosynthetic enzymes, and the modified core is
proteolytically cleaved from the leader to furnish the mature
RiPP. Genes encoding the substrate peptide and the core-
modifying enzymes constitute the RiPP BGC. The poly-
theonamide BGC is harbored by bacterial symbionts associated
with the sponge host.11,12 All other sponge-derived peptidic
natural products for which BGCs have been deciphered fall
under the purview of nonribosomal peptide synthetase
enzymology.14
with exquisite pharmacological potential with the azol(in)e
heterocycles playing important roles in conferring bioactivity.17
Prominent examples include DNA gyrase inhibition by the
archetypical linear azol(ine) peptide microcin B17,18 the
ribosome-stalling activity of phazolicin,19 and the selective
antibiosis activity of plantazolicin against Bacillus anthracis- the
causative agent of anthrax.20 Curiously, from marine holobiont
sources, the only known azol(in)e containing RiPPs are the
cyanobactins that are produced by cyanobacteria symbiotically
associated with tunicates;21 no azol(in)e containing RiPPs
have been reported from sponges. Does this imply that the
biosynthetic potential for the production of azol(in)e
containing RiPPs is absent in sponge holobionts? Here, we
demonstrate that the genetic potential for the production of
azol(in)e containing RiPPs in marine sponge microbiomes is
quite extensive, but the natural products themselves remain
cryptic. RiPP BGCs in phylogenetically and geographically
disperse marine sponges harbor a highly conserved RiPP
substrate peptide and predicate the discovery of a broad-
spectrum peptide tryptophanyl brominase while opening up
new avenues for the combinatorial structural diversification of
RiPPs.
RESULTS
■
Detection of RiPP BGCs in a Sponge Metagenome. A
recent metabolome inventory of the Floridian marine sponge
Smenospongia aurea revealed the presence of brominated
indoles (Table S1).22 To query the enzymatic potential for
tryptophan bromination in S. aurea, we sequenced the S. aurea
metagenome (Table S2). We included the RiPP micro-
bisporicin flavin-dependent halogenase MibH23 as a query
sequence to mine the S. aurea metagenome for tryptophan
halogenases. MibH catalyzes the late-stage chlorination of the
microbisporicin tryptophan side chain indole (Figure S1).24,25
Several MibH homologues were detected in the S. aurea
metagenome. In the neighborhood of each of these MibH
homologues was a YcaO cyclodehydratase encoding gene.
RiPP substrate peptides with cysteine-rich C-termini were
A subclass of RiPPs contain azole/azoline heterocycles. The
azol(in)e heterocycles are derived from cysteine, serine, and
threonine residues and their formation is catalyzed by ATP-
dependent YcaO cyclodehydratases.15 The biosynthetic
potential for azol(in)e containing RiPPs is found widespread
in bacterial genomes.16 Several of these RiPPs are endowed
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Figure 2. Diversity and distribution of srp BGCs. (A) Top: Sponge microbiome α-diversity as denoted by Shannon indices. Bottom: Microbiome
architectures of sponge specimens used in this study. Bacterial phyla are color coded. Sponge specimens of identical genera collected from the same
site are distinguished by hyphenated numerals; this nomenclature is preserved in Tables S1 and S2. (B) Top: Cumulative fractional abundance of
bacteria harboring the srp BGCs. Bottom: Number of srp BGCs detected in metagenomes of sponge specimens used in this study. (C)
Metagenomic bins containing srp BGCs were organized into groups according to a genome wide average nucleotide identity network (Figure S2).
Heat map showing the conservation of these groups in six different sponge genera, the values in the heat map is the number of bins that were
detected in each sponge genus. The taxonomic assignments for these groups are presented in Table S2. (D) The srp BGCs were mined from
sponge metagenomes using antiSMASH and parsed through BiG-SCAPE to generate a similarity network. Nodes are colored according to sponge
genera. The central core corresponds to the canonical srp BGCs as illustrated in Figure 1. (E) Representative srp BGCs from cluster_1, _2, and _3
as labeled in panel D. Putative activities of enzymes encoded by genes that were distinctly different from the canonical srp BGCs but highly
conserved within the respective clusters are highlighted. Note that the srpA and srpI genes are missing in cluster_3.
found adjacent to the cyclodehydratases. The RiPP substrate
peptides had large N-terminal leader peptides with similarity to
nitrile hydratases. The presence of nitrile hydratase like leader
peptides (NHLPs) has been recognized in bacterial genomes
and NHLP-derived RiPPs are named proteusins.26
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Surprisingly, not one, but multiple similar RiPP BGCs were
identified in the S. aurea metagenome (Figure 1, vide infra: 63
such RiPP BGCs were cataloged from S. aurea as illustrated in
Figure 2). Each BGC possessed the NHLP encoding gene in
close proximity to a gene encoding the YcaO cyclodehydratase.
Collectively, we have named these the sponge derived RiPP/
proteusin (srp) BGCs. In addition to the NHLP, YcaO
cyclodehydratase, and the halogenase, genes encoding trans-
porters, chaperones, and proteins with catalytic functions that
could not be discerned based on sequence similarity alone
could also be identified in the srp BGCs. Of these, genes srpA−
E and srpT1−T2, where srpE encodes the NHLP substrate,
srpC encodes the YcaO cyclodehydratase, and srpT1 and srpT2
encode ATP-dependent membrane transporters were highly
conserved in srp BGCs (Figure 1). The MibH-like halogenase
is encoded by srpI. While SrpI formed the basis for the
discovery of the srp BGCs, srpI was not present in all srp BGCs
(Figure 1). As determined by sequence similarity using
HHpred,27 srpA encodes a terpene cyclase with an N-terminal
nitrile hydratase like domain while srpD encodes a prenyl-
transferase. Gene srpB encodes a putative bifunctional enzyme
with an N-terminal metal-dependent oxidoreductase domain
and a C-terminal adenyltransferase domain.
collected in Puerto Rico also possessed srp BGCs. From the
Indo-Pacific, we detected srp BGCs in the microbiomes of
sponge samples of Ircinia spp. collected in the Solomon Islands
(Figure 2A,B). There is limited metabolomic overlap between
these sponge genera. Sponge S. aurea possesses brominated
indoles in high concentration.22 However, sponges of the
genera Aiolochroia, Aplysina, Verongula, and Pseudoceratina
harbor bromotyrosine alkaloids,31 and Ircinia meroditer-
penes.32 No RiPPs have been reported from any of these
sponge genera, and none were detected in the mass
spectrometry-based metabolomic data.
Characterized by the α-diversity Shannon indices, micro-
biomes of each of the above-mentioned sponges were highly
diverse (Figure 2A). We asked if srp BGCs are restricted to
sponges with highly diverse microbiomes only or do they
extend to low microbial diversity sponges as well. Low
microbial diversity sponge specimens belonging to genera
Ianthella and Aplysinella were collected in Guam (Figure 2A).
No srpE genes were detected in these low microbial diversity
sponges. This result suggests that srp BGCs may be associated
only with the high microbial diversity sponges; more
specimens of low microbial diversity sponges from more
locations are required to further query this observation.
Despite the abundance of srp BGCs in individual sponge
metagenomes, srp harboring bacteria collectively constitute a
minor fraction of the respective sponge microbiomes.
Illustratively, bacteria that possess the 63 srp BGCs detected
in the S. aurea metagenome make up 12.6% of the microbiome
fraction (Figure 2B). The cumulative fractional abundance of
srp BGC containing bacteria in sponge microbiomes ranges
from 7.5% to 17.4% for samples used in this study.
Only two proteusin compound families are currently known:
the polytheonamides11,28 and the landornamides.29 Neither of
these two compound families bear azol(in)e heterocycles or
halogenated residues (Figure S1). Amino acid epimerases
present in BGCs for both polytheonamides and landornamides
were absent in the srp BGCs. As such, none of the enzymes
that catalyze modifications that have been described previously
for polytheonamide and landornamide proteusins were found
encoded in the srp BGCs.
At this point, our data demonstrates that the srp BGCs are
not localized to a single specialized symbiont but distributed
throughout the sponge microbiome. Thus, we sought to
inventory which members of the microbiome harbored the srp
BGCs, and if the srp BGC harboring bacteria overlapped
among different sponge genera. For Aplysina, Aiolochroia,
Verongula, and Pseudoceratina spp. sponges used in this study,
we have reported that the microbiomes are conserved at the
host genus level.31 This observation also extends to the two
Ircinia spp. samples used in this study (Figure 2A). Hence,
subsequent analyses grouped sponge specimens used in this
study by genus. First, all metagenomic bins in which srp BGCs
were detected were grouped in a genome-wide average
nucleotide identity network, which was organized according
to sponge genus (Figure S2). Clusters and individual nodes
thus identified were ranked per the abundance of srp BGCs.
The srp BGC abundances for the top 45 phylogenetic groups
are illustrated in Figure 2C with taxonomic assignments of
these groups provided in Table S3. Bacterial groups 1−3 and 7,
which are present in all six sponge genera, belong to the
Latescibacterota, UBA8248, Nitrospirota, and Acidobacteriota
phyla, respectively. The next most widely distributed groups, 4
and 6, belong to Proteobacteria and Gemmatimonadota phyla.
Overall, Proteobacteria and Acidobacteriota were the most well
represented bacterial phyla that contain srp BGCs in
microbiomes of sponge specimens used in this study (Figure
S3). This finding is also supported by the %GC content of the
srpE genes detected in each sponge metagenome (Figure S4).
The inventory of over 600 srp BGCs detected here from six
sponge genera was organized using the Biosynthetic Genes
Similarity Clustering and Prospecting Engine (BiG-SCAPE)
(Figure 2D).33 Overall, srp BGCs clustered independently of
Of the constellation of srp BGCs detected in the S. aurea
metagenome, we chose one, which we term 1srp BGC, for
further characterization (Figure 1). The 1srp BGC was present
in the middle of a 117 kb metagenomic contig providing
confidence that open reading frames flanking the BGC termini
were not missed. The BGC boundary was determined so as to
exclude open reading frames with similarity to enzyme
sequences that could be rationalized to be involved in bacterial
primary metabolism.
Conservation, Abundance, and Diversity of srp BGCs.
Next, we queried if the detection of multiple srp BGCs in
marine sponge metagenomes was unique to S. aurea which was
collected in the Florida Keys. From the same location, we
collected and sequenced the metagenomes of Aiolochroia,
Aplysina, and Verongula spp. (Figure 2A,B, Table S1). Like S.
aurea, these sponge genera are ubiquitous on Floridian reefs. In
this study, multiple sponge specimens of the same genus
collected from the same geographical location represent
morphologically distinct species. In each of these sponge
specimens, we found srp BGCs to be present. These data
demonstrate that the presence of srp BGCs extends across
sponge hosts of different genera, families, and orders (Table
S1). The abundance of srp BGCs in each metagenome was
curated using antiSMASH,30 and the predicted BGCs that
contain the srpE gene were selected for inventory. In the
metagenome of an Aplysina specimen, at least 77 srp BGCs
could be identified; the S. aurea metagenome contained 63 srp
BGCs (Figure 2B).
The presence of srp BGCs is also not constrained
geographically. We found that the microbiomes of sponge
specimens Aiolochroia sp., Aplysina sp., and Pseudoceratina spp.
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sponge host genus. The central large network contained the
canonical srp BGCs that are illustrated in Figure 1. BGCs
distant from the central core revealed the presence of
additional open reading frames that were highly conserved
within the respective clusters. Three such clusters are
highlighted in Figure 2D with representative srp BGCs from
each of the three clusters illustrated in Figure 2E. The srp
BGCs in cluster_3 lacked the srpA and srpI genes and
possessed NHLP SrpE sequences with conspicuously different
C-termini (Figure S5).
Short Pentapeptide Core. At present, RiPPs encoded by
the srp BGCs are cryptic, meaning that we have not yet
detected the natural product in situ. To gain insight into the
possible natural products from srp, we focused our efforts on
the 1srp BGC (Figure 1). First, we sought to demarcate the
leader/core boundary for the 1SrpE substrate peptide. A Cys-
His-Asp catalytic triad containing C39 protease domain was
located at the 5′-terminus of the ATP-dependent trans-
membrane transporter encoding gene 1srpT1 (Figure 3A).
liquid chromatography/mass spectrometry (LC/MS) (Figure
3B). In addition, multiply charged ions corresponding to the
inferred 78-mer leader peptide with the appended (His)10 tag
were also detected with high accuracy (Figure 3C). While even
single amino acid excision natural products have been
described starting from peptidyl substrates,34,35 the SrpE
pentapeptide core is uncharacteristically short and is the
shortest among known proteusin substrate peptides. The SrpE
-LCCCW core was found to be highly conserved among
sponge microbiomes independent of sponge phylogeny and
geographical location with the highest variability observed at
the C-terminal tryptophan residue (Figure 3D).
Cleavage of 1SrpE was observed only when incubated with
equimolar or excess 1SrpT1protease. To circumvent the low
activity of 1SrpT1protease, we tested cleavage of 1SrpE by the
substrate promiscuous RiPP peptidase LahT150 (Figure S7).36
The leader peptide recognition determinants for LahT150,
which are the branched aliphatic amino acid side chains at the
−4, −7, and −12 positions,36 were found conserved in 1SrpE
(Figure 3D). Incubation of 1SrpE with purified LahT150
protease
offered identical cleavage products as 1SrpT1
but with
more favorable catalyst/substrate ratio and reduced reaction
times. Experiments described below utilize LahT150 in place
protease
of 1SrpT1
.
Cyclodehydrations in a Proteusin Peptide. While
widely represented in RiPP enzymology, ATP-dependent
YcaO catalyzed cyclodehydration of cysteine, serine, and
threonine residues to furnish azoline heterocycles has not been
observed for proteusin RiPPs (Figure 4A). Intransigence of
heterocyclase 1srpC expression in a soluble form in E. coli
precluded in vitro experiments. Five additional SrpC
homologues were tested each resulting in insoluble protein
expression. However, coexpression of 1srpC with 1srpE
provided an in vivo route to query the 1SrpC activity. After
coexpression of 1srpE and 1srpC, purified 1SrpE was treated
with the LysC protease to generate peptide fragments
amenable for high resolution and high accuracy mass
determination by MALDI-ToF mass spectrometry (Figure
4B). Three products, corresponding to mono-, di-, and
tridehydrated 1SrpE were observed (Figure 4C). The
monodehydrated product demonstrated two acetamide
additions upon treatment with the thiol alkylating reagent
iodoacetamide (Figure S8). These data imply that each of the
three cysteine residues in 1SrpE -LCCCW core could be
cyclodehydrated by 1SrpC. The inferred density of azoline
heterocycle installation by 1SrpC is reminiscent of plantazo-
licin biosynthesis in which two pairs of consecutive penta-
azol(in)es are installed by a YcaO cyclodehydratase.37
Further characterization of the cyclodehydrated 1SrpE
products was achieved by MS/MS fragmentation of the
modified 1SrpE core furnished by LahT150-catalyzed
proteolytic removal of the 1SrpE leader peptide. The
monodehydrated product demonstrated MS2 fragment ions
corresponding to cyclodehydration at Cys1 (1SrpE core:
-LC1C2C3W) (Figure S9). Subsequent di- and tricyclodehy-
drations were affected upon the Cys2 and Cys3 residues,
respectively (Figure S10, S11).
Numerous examples exist for RiPPs in which a single YcaO
enzyme catalyzes the cyclodehydration of cysteine, serine, and
threonine residues. To query whether serine and threonine
residues could be modified by 1SrpC, we generated 1srpE
mutants in which cysteines in the 1SrpE core were replaced by
serine and threonine residues. No mutations at Cys1 (1SrpE
Figure 3. Leader/core boundary for 1SrpE substrate peptide. (A)
Domain architecture for the 1SrpT1 transporter with the N-terminal
1SrpT1protease domain which possesses the Cys-His-Asp catalytic triad.
LC/MS data demonstrating cleavage products observed upon
treatment of 1SrpE with 1SrpT1protease. (B) Isotopic distribution for
the [M + H]1+ MS1 ion corresponding to the -LCCCW core. (C)
Isotopic distribution for the [M+12H]12+ MS1 ion corresponding the
1SrpE leader appended to the (His)10 tag. (D) Sequence conservation
for the SrpE NHLPs. 484 SrpE sequences were used to generate the
sequence logo. Residues that guide cleavage by LahT150 are marked
by arrows.
The corresponding nucleotide sequence was optimized for
expression in Escherichia coli and the recombinant protein,
henceforth referred to as 1SrpT1protease, purified (Figure S6).
The optimized 83-residue peptide encoding gene 1srpE was
also expressed in E. coli with an N-terminal (His)10-tag and the
recombinant peptide purified. Upon in vitro incubation of
1SrpE with 1SrpT1protease, we observed a product correspond-
ing to the 1SrpE C-terminal -LCCCW pentapeptide using
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this hypothesis, we introduced a nonmodifiable alanine residue
after the Cys1 and Cys2 to generate the -LCACCW and
-LCCACW 1SrpE core substrates. In accordance with the
hypothesis, a single heterocycle at Cys1 was installed by 1SrpC
for the -LCACCW core (Figures 4H, S14). Two heterocycles
at Cys1 and Cys2 could be installed by 1SrpC for the
-LCCACW core (Figure 4I). Mutations at Cys3 were not
tolerated: cyclodehydrated product formation was not
observed for the Cys3 → Ser and Cys3 → Thr mutants
(Figure 4J,K).
The 1SrpC specificity can be rationalized in view of the
narrow sequence space explored by the SrpE cores (Figure
3D). Contrary to inherently substrate tolerant marine RiPP
biosynthetic enzymes such as those that modify expanded
libraries of cyanobactin38 and prochlorosin39 substrate
peptides, the SrpC YcaO cyclodehydratase is a narrow
spectrum enzyme specifically tailored to modify the SrpE
-LCCCW core that is highly conserved in microbiomes of
diverse sponges around the world.
SrpI Is a Tryptophan-6-Brominase. To date, a single
RiPP halogenase has been described: the flavoenzyme MibH
that catalyzes tryptophan-5-chlorination during biosynthesis of
the lantibiotic NAI-107 (Figure S1).24 Akin to the activity of
MibH, we rationalized halogenation by SrpI to be a late-stage
biosynthetic modification. Activity of 1SrpI was thus evaluated
by coexpression of 1srpI with 1srpE and 1srpC. No
halogenation was observed with standard media conditions
(Figure 5A). Halogenation could be realized only when 1g/L
potassium bromide was added to the culture media (Figure
5B). MS/MS fragmentation demonstrated bromination to be
affected upon the tryptophanyl side chain indole (Figure S15).
Halogenases are named after the most electronegative halogen
that they incorporate,40 1SrpI was thus a physiological
tryptophan brominase. Mutating the catalytic lysine residue,
rationalized based on characterization of other flavoenzyme
halogenases, to alanine led to abolishment of activity (Figure
S16).41 Curiously, in addition to bromination of the
tricyclodehydrated 1SrpE, we observed bromination of the
mono- and dicyclodehydrated 1SrpE (Figure 5B). In addition
to the partially cyclodehydrated 1SrpE core, brominase 1SrpI
could also tolerate modifications adjacent to the tryptophan
residue. The modified 1SrpE-LCCCGW core was efficiently
processed by 1SrpC and 1SrpI to yield a brominated,
tricyclodehydrated product (Figure 5C). Similarly, brominated
products were detected for the 1SrpE-LCCCWG (Figure 5D)
and 1SrpE-LCCCWA (Figure S17) variants. To further
explore this unexpected substrate tolerance of 1SrpI, 1srpI
was coexpressed with 1srpE in the absence of 1srpC. Here, we
observed the production of the brominated -LCCCW 1SrpE
Figure 4. Cyclodehydration of the proteusin peptide 1SrpE. (A)
Azoline formation by YcaO cyclodehydratases. Each azoline formation
is accompanied by a mass decrease of 18 Da. (B) Coexpression assay
procedure to characterize 1SrpC activity. The protease LysC cleaved
at the C-terminus of the Lys30 residue in the 1SrpE leader. MALDI-
ToF mass spectra for (C) wild type and modified (D−K) 1SrpE
peptides obtained by coexpression of 1srpE and 1srpC genes in E. coli.
The azoline heterocycles are denoted by yellow pentagons,
mutagenized residues in the SrpE core are highlighted in blue.
core without cyclodehydrations (Figure 5E). Incubation with
protease
1SrpT1
furnished the brominated -LCCCW 1SrpE core
(Figure S18). Mutating the 1SrpE C-terminal tryptophan
residue to tyrosine demonstrated the production of mono- and
dibrominated products (Figure 5F). Brominated product
formation was not observed for 1SrpE-LCCCF.
At this stage, the site for bromination upon the tryptophanyl
indole side chain catalyzed by 1SrpI was not known. Efforts to
spectroscopically characterize the brominated 1SrpE core were
impeded by the low expression level of 1srpE in E. coli. To
circumvent this challenge, brominated 1SrpE obtained after
coexpression of 1srpE and 1srpI in E. coli was digested with
carboxypeptidase to furnish the C-terminal bromotryptophan
residue (Figure 5G). Bromination by a flavin-dependent
core: -LC1C2C3W) were tolerated by 1SrpC; cyclodehydrated
products were not observed in either case (Figure 4D,E).
Mutations at Cys2 were moderately tolerated; singly
dehydrated products were observed for both the Cys2 → Ser
and Cys2 → Thr mutants (Figure 4F,G). MS/MS fragmenta-
tion established that the single heterocycle formation in the
Cys2 → Ser and Cys2 → Thr mutants was predicated upon
Cys1 (Figure S12, S13). These data lead to the hypothesis that
1SrpC cannot modify serine and threonine residues nor can it
skip over nonmodifiable residues in the 1SrpE core. To test
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Figure 5. Activity of RiPP brominase 1SrpI. (A) No halogenated products were observed upon coexpression of 1sprE with 1srpC and 1srpI when
culture media lacked bromide. (B) Bromination of the mono-, di-, and tricyclodehydrated 1SrpE was observed when culture media was
supplemented with bromide. Processing of the (C) 1SrpE-LCCCGW and (D) 1SrpE-LCCCWG by 1SrpC and 1SrpE to yield cyclodehydrated-
brominated products. (E) When gene 1srpC was omitted from the coexpression experiment, bromination of the unmodified 1SrpE peptide was
observed. (F) Mono- and dibromination was observed when the C-terminal tryptophan residue was modified to tyrosine. (G) To determine the
regiospecificity of tryptophan halogenation by 1SrpI, gene 1srpE was coexpressed with 1srpI and the purified 1SrpE peptide product was digested
with carboxypeptidase to excise the C-terminal monobrominated tryptophan residue. (H) Standards for 4-, 5-, 6-, and 7-bromotryptophan were
generated by condensation of L-serine with bromoindoles using tryptophan synthase Pf TrpB. (I) Scheme for synthesis of the 2-bromotryptophan
standard. NBS: N-bromosuccinimide. (J) The carboxypeptidase digestion reaction was coinjected with 2-, 4-, 5-, 6-, and 7-bromotryptophan
standards and the presence of monobrominated tryptophan was monitored by LCMS using extracted ion chromatograms for m/z 284.14 0.1 Da.
The 1SrpE-excised bromotryptophan residue from 1SrpE coeluted with the 6-bromotryptophan standard.
halogenase such as 1SrpI can be rationalized to be affected
upon either of the 2-, 4-, 5-, 6-, and 7-positions of the indole
ring. Standards for 4-, 5-, 6-, and 7-bromotryptophan were
synthesized by the enzymatic ligation of serine with
commercially available 4-, 5-, 6-, and 7-bromoindole by the
Pyrococcus furiosus tryptophan synthase (Pf TrpB, Figure
5H).42 2-Bromotryptophan was synthesized by bromination
of tryptophan methyl ester by N-bromosuccinimide followed
by mild hydrolysis of the methylester by α-chymotrypsin
(Figure 5I).43 Harsher saponification conditions led to product
degradation. Co-injection with the bromotryptophan standards
demonstrates that 6-bromotryptophan was produced by
carboxypeptidase catalyzed degradation of the 1SrpE peptide
obtained by coexpression of 1srpE and 1srpI (Figure 5J).
Hence, 1SrpI is rationalized to be a peptidyl tryptophan-6-
brominase.
Tolerance of Other Proteusin Leaders by Srp
Enzymes. By sequence similarity, SrpC is most closely aligned
with YcaO cyclodehydratases identified in marine cyanobacte-
rial symbionts that produce macrocylic cyanobactin natural
products containing azol(in)e heterocycles (Figure S19).
Cyanobactin YcaO cyclodehydratases bind their substrates
using the L(S/T)EEXL sequence motif present in the substrate
leader peptide.44 Introducing this recognition sequence in
cyanobactin substrate peptides where it was otherwise absent
rendered them competent substrates for YcaO cyclodehy-
dratases.45 Sequence gazing identified the conservation of this
YcaO recognition sequence in the proteusin substrate peptide
SrpE (Figure 6A, here, one of the cyanobactin substrate
peptides TruE1 is illustrated). Curiously, even though the
proteusin RiPPs landornamides and polytheonamides do not
contain genes encoding YcaO cyclodehydratases in their BGCs
and azol(in)e heterocycles are not present in the mature
natural products, the respective substrate peptides, OspA and
PoyA also contain the YcaO cyclodehydratase recognition
sequence (Figure 6A).11,29 The leader/core boundaries for
TruE1, OspA, and PoyA have been described previously.11,29,46
The position of the YcaO recognition sequence relative to the
leader/core boundary was identical among the four peptides
(Figure 6A).
In light of this observation, we asked if the leader peptide
sequences from TruE1, OspA, and PoyA could support the
cyclodehydration activity of 1SrpC. Given the narrow tolerance
of 1SrpC for modifications of the 1SrpE core, the physiological
1SrpE core was preserved. We generated clones encoding the
TruE1-LCCCW, OspA-LCCCW, and PoyA-LCCCW chimeric
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1srpI (Figure 6B, 6D). Annotation of the MS2 spectra
identified the C-terminal tryptophan residue to be the site of
bromination (Figure 6F). Upon coexpression of 1srpC and
1srpI with ospA-LCCCW, bromination of unmodified and
singly cyclodehydrated core peptide was observed (Figure
S20).
DISCUSSION
■
First clues that bacterial symbionts could produce natural
products that had been isolated from sponges came from
physical separation of bacteria from sponge tissue wherein the
separated bacterial fractions were found enriched in the natural
products.47 Prominent examples include the colocalization of
cyanobacterial filaments from Dysideidae sponges with
polybrominated phenols and polychlorinated peptides and
that of various polyketide and peptidic natural products
isolated from sponge Theonella swinhoei with a filamentous
bacterial symbiont (reviewed in refs 2 and 6). These microbe-
molecule colocalization experiments benefited from the natural
product producing symbiont being highly abundant in the
sponge microbiome. In the post genomic age, rationalized
biosynthetic schemes guided the mining of sponge meta-
genomes, and the colocalization experiments were subse-
quently validated.48,49 Within this context, the work presented
here provides a glimpse into the cryptic biosynthetic potential
that is present in sponge microbiome members which are of
lesser abundance and traditionally not viewed as natural
product producing taxa.
We find the srp BGC to be distributed in high diversity
sponge microbiomes without constraints of sponge host
phylogeny or geographical location. While they were universal
in high microbial diversity sponges in our collection, we could
not detect srp BGCs in multiple other marine invertebrate
metagenomes that we have sequenced. Data presented in this
study likely underrepresent the distribution of srp BGCs in
sponge microbiomes with a greater spread of srp BGCs to be
realized as more sponge microbiomes are queried. Deeper
metagenomic sequencing can also likely uncover even more srp
BGCs within a sponge microbiome. Illustratively, over 200
NHLP encoding srpE genes are detected in the publicly
available metagenomic data for the marine sponge Ircinia
ramosa (collected in Australia).50
The presence of numerous highly similar srp BGCs within a
single sponge metagenome is without precedent. At present,
we do not know if some srp BGCs are plasmid encoded or not.
Biosynthesis of natural products by bacterial symbionts is
thought to provide the sponge host with chemical defense
against predation and a competitive growth advantage.9,51
Does then the conservation of srp BGCs in marine sponge
microbiomes point toward a conserved ecological role of the as
yet cryptic RiPP or represent the vestigial remnants of an
ancestral BGC that is being shuttled among microbial
symbionts? Within sponges, different symbionts share genetic
potential to interact with the host via exchange of primary
metabolites.50,52 Whether this redundancy extends to secon-
dary metabolites with specialized ecological roles remains a
tantalizing prospect.
Figure 6. Conservation of the YcaO recognition sequences in RiPP
substrates. (A) Sequence alignment of TruE1, SrpE, OspA, and PoyA
substrate peptides. The YcaO cyclodehydratase recognition sequence
is highlighted in yellow and the leader/core boundary is marked.
Extracted ion chromatograms (EICs) for m/z 609.2 (in blue) and m/
z 707.1 (in red) corresponding to the cyclodehydrated and
brominated -LCCCW core obtained when the gene encoding the
OspA-LCCCW chimeric substrate was expressed (B) without 1srpC
or 1srpI, (C) with 1srpC, and (D) with 1srpI. Structural annotation of
the (E) monocyclodehydrated product obtained after LahT150
digestion of modified OspA-LCCCW peptide by 1SrpC, and (F)
the brominated OspA-LCCCW peptide by 1SrpI.
peptides. Given the broad substrate scope of the brominase
1SrpI, we also queried if the activity of 1SrpI could be observed
for these chimeric substrates. The three chimeric peptide
encoding genes were coexpressed with 1srpC and 1srpI and the
resultant peptides purified, and after digestion with LahT150,
analyzed by LC/MS. For the OspA-LCCCW chimera, we
observed modification by both 1SrpC and 1SrpI (Figure 6B−
D). Modifications were not observed for the TruE1-LCCCW
and PoyA-LCCCW chimeras. Extracted ion chromatograms,
corresponding to the cyclodehydrated -LCCCW product, m/z
609.2, demonstrated product formation when ospA-LCCCW
was coexpressed with 1srpC (Figure 6B,C). Annotation of the
MS2 fragmentation spectra demonstrated that the single
cyclodehydration observed was localized to Cys1 (Figure
6E). No further cyclodehydrations could be observed.
Similarly, extracted ion chromatograms, corresponding to the
brominated -LCCCW product, m/z 707.1, demonstrated
product formation when ospA-LCCCW was coexpressed with
Cyclodehydration for the installation of azol(in)e hetero-
cycles and aromatic side chain halogenation are both activities
that were absent from previously known proteusin RiPPs. Our
data demonstrates that SrpC and SrpI can use their cognate
core ligated to other leader peptides (Figure 6). However,
leader peptide tolerance was not universal as the TruE and
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PoyA leaders did not support modification. Among the three
noncognate leader peptides tested here, sequence alignment
identifies OspA to be of closest similarity to SrpE (Figure S21).
Unlike the RiPP chlorinase MibH, which requires prior
modifications to be installed on the RiPP core region for
halogenation of the tryptophan indole, SrpI can brominate the
unmodified peptide. A winged helix-turn-helix RiPP precursor
peptide recognition element that is found in several RiPP
biosynthetic enzymes cannot be located at either the N- or the
C- termini of SrpI.53 Enzymatic bromination, without
contaminating chlorination in vivo, can be thought of as an
enabling platform for the cross-coupling of tryptophanyl
peptides with boronic acids, as has been achieved for other
classes of indolic natural products.54,55 Matching the
regiospecificity of SrpI, tryptophan-6-bromination is widely
detected in conotoxins,56 predicating further exploration of
SrpI as a general purpose peptide modification biocatalyst.
Sequence motifs that direct the C39 protease to cleave at the
leader/core boundary and the activity of the YcaO cyclo-
dehydratase were found conserved in SrpE.36,45 Both motifs
are placed toward the C-terminus of the SrpE leader. Proteusin
RiPPs possess unusually long leader peptide sequences. At the
N-terminus of the proteusin substrate OspA, a recognition
motif was identified which directs the activity of a radical SAM
epimerase which converts two L-amino acid residues in the
OspA core to D-amino acids.57 While radical SAM epimerases
have not been identified in our current inventory of srp BGCs,
the epimerase recognition sequence in the OspA leader is
partially conserved in the SrpE leader (Figure S21). The srp
BGCs contain many other genes encoding biosynthetic
enzymes that are not present in other proteusin BGCs,
activities and leader peptide dependencies for which remain to
be determined. Overall, in addition to beginning to construct
the widely distributed cryptic srp BGC encoded natural
product(s), we demonstrate that the Srp biosynthetic enzymes
can interact with other substrate peptides which now sets the
stage for combinatorial structural diversification of proteusin
RiPPs.
AUTHOR INFORMATION
■
Corresponding Author
Vinayak Agarwal − School of Chemistry and Biochemistry and
School of Biological Sciences, Georgia Institute of Technology,
Atlanta, Georgia 30332, United States; orcid.org/0000-
0002-2517-589X; Email: vagarwal@gatech.edu
Authors
Nguyet A. Nguyen − School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, Georgia 30332,
United States
Zhenjian Lin − Department of Medicinal Chemistry,
University of Utah, Salt Lake City, Utah 84112, United
States
Ipsita Mohanty − School of Chemistry and Biochemistry,
Georgia Institute of Technology, Atlanta, Georgia 30332,
United States
Neha Garg − School of Chemistry and Biochemistry, Georgia
Institute of Technology, Atlanta, Georgia 30332, United
States; orcid.org/0000-0002-2760-7123
Eric W. Schmidt − Department of Medicinal Chemistry,
University of Utah, Salt Lake City, Utah 84112, United
States; orcid.org/0000-0001-5839-694X
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c03474
Notes
The authors declare no competing financial interest.
The bacterial 16S rRNA, sponge 28S rRNA, and sponge ITS-2
amplicon sequences are deposited to GenBank with BioProject
ID PRJNA694437. Also included are the metagenomic reads
and annotated srp BGCs. Untargeted LC/MS data for sponge
extracts has been deposited to MassIVE database with ID
MSV000086758.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(GM142882 to V.A.; GM122521 to E.W.S.). The authors
thank V. J. Paul, J. S. Biggs, C. J. Freeman, and H. R. Thapa for
collection of sponge specimens. Sponge specimens from
Solomon Islands were acquired via export permit rp/2017/
003. Assistance from the Ministry of Environment, Climate
Change, Disaster Management, and Meteorology (Solomon
Islands) and Solomon Islands National University is gratefully
acknowledged. This work was supported by Georgia Institute
of Technology’s Systems Mass Spectrometry Core and the
Molecular Evolution Core facilities. The authors thank S. K.
Nair for insightful discussions.
CONCLUSION
■
Here, we describe the discovery of a RiPP BGC that is
conserved and exceptionally widely distributed in marine
sponge microbiomes. Encoded within this BGC was found an
obligate peptidyl tryptophan brominase, the activity of which
did not depend on prior modifications on the RiPP core
peptide. Sequence motifs that guide biosynthetic modifications
are shared within different RiPP compound classes which
provide avenues for combinatorial diversifications of the RiPP
chemical space. This study establishes marine sponges as
sources of as yet unknown cryptic natural products, in addition
to the already impressive catalog of bioactive natural products
that have been isolated from these benthic marine inverte-
brates.
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c03474.
Materials and methods used in this study describing
metagenomic sequencing, in vivo and in vitro bio-
chemical assays, and analytical data (PDF)
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