Revised Structure of Anthelvencin A and Characterization of the Anthelvencin Biosynthetic Gene Cluster

Article abstract of DOI:10.1021/acschembio.9b00960

Anthelvencins A and B are pyrrolamide metabolites produced by Streptomyces venezuelae ATCC 14583 and 14585. Isolated in 1965, they were reported to exhibit anthelmintic and moderate antibacterial activities. In this study, we revise the structure of anthelvencin A and identify a third anthelvencin metabolite, bearing two N-methylated pyrrole groups, which we named anthelvencin C. We sequenced the genome of S. venezuelae ATCC 14583 and identified a gene cluster predicted to direct the biosynthesis of anthelvencins. Functional analysis of this gene cluster confirmed its involvement in anthelvencin biosynthesis and allowed us to propose a biosynthetic pathway for anthelvencins. In addition to a nonribosomal peptide synthetase (NRPS), the assembly of anthelvencins involves an enzyme from the ATP-grasp ligase family, Ant23. We propose that Ant23 uses a PCP-loaded 4-aminopyrrole-2-carboxylate as substrate. As observed for the biosynthesis of the other pyrrolamides congocidine (produced by Streptomyces ambofaciens ATCC 25877) and distamycin (produced by Streptomyces netropsis DSM 40846), the NRPS assembling anthelvencins is composed of stand-alone domains only. Such NRPSs, sometimes called type II NRPSs, are less studied than the classical multimodular NRPSs. Yet, they constitute an interesting model to study protein-protein interactions in NRPSs and are good candidates for combinatorial biosynthesis approaches.

Full text of DOI:10.1021/acschembio.9b00960

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Article
Revised structure of anthelvencin A and characterization
of the anthelvencin biosynthetic gene cluster
Céline Aubry, Paolo Clerici, Claude Gerbaud,
Laurent Micouin, Jean-Luc Pernodet, and Sylvie Lautru
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00960 • Publication Date (Web): 04 Mar 2020
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1 Revised structure of anthelvencin A and characterization
2 of the anthelvencin biosynthetic gene cluster
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Céline Aubrya, b, Paolo Clericic, Claude Gerbauda, Laurent Micouinc, Jean-Luc
Pernodeta, and Sylvie Lautrua*
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a
Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université
Paris-Saclay, 91198, Gif-sur-Yvette cedex, France
b
present address: Unité Molécules de Communication et Adaptation des Microorganismes
(MCAM, UMR 7245), Muséum National d’Histoire Naturelle (MNHN), Centre National de la
Recherche Scientifique (CNRS), CP 54 57 rue Cuvier, 75005 Paris, France
c Université de Paris, Laboratoire de Chimie et Biochimie pharmacologiques et toxicologiques,
LCBPT, UMR 8601 CNRS, F-75006 Paris, France
* Corresponding author: Sylvie LAUTRU, sylvie.lautru@i2bc.paris-saclay.fr
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ABSTRACT
Anthelvencins A and B are pyrrolamide metabolites produced by Streptomyces venezuelae
ATCC 14583 and 14585. Isolated in 1965, they were reported to exhibit anthelmintic and
moderate antibacterial activities. In this study, we revise the structure of anthelvencin A and
identify a third anthelvencin metabolite, bearing two N-methylated pyrrole groups, which we
named anthelvencin C. We sequenced the genome of S. venezuelae ATCC 14583 and
identified a gene cluster predicted to direct the biosynthesis of anthelvencins. Functional
analysis of this gene cluster confirmed its involvement in anthelvencin biosynthesis and
allowed us to propose a biosynthetic pathway for anthelvencins. In addition to a non-ribosomal
peptide synthetase (NRPS), the assembly of anthelvencins involves an enzyme from the ATP-
grasp ligase family, Ant23. We propose that Ant23 uses a PCP-loaded 4-aminopyrrole-2-
carboxylate as substrate. As observed for the biosynthesis of the other pyrrolamides
congocidine (produced by Streptomyces ambofaciens ATCC 25877) and distamycin
(produced by Streptomyces netropsis DSM 40846), the NRPS assembling anthelvencins is
composed of stand-alone domains only. Such NRPSs, sometimes called type II NRPSs, are
less studied than the classical multimodular NRPSs. Yet, they constitute an interesting model
to study protein-protein interactions in NRPSs and are good candidates for combinatorial
biosynthesis approaches.
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Anthelvencins A and B (Figure 1A) are specialized metabolites that were isolated in
1965 from cultures of Streptomyces venezuelae ATCC 14583 and 14585 and exhibit moderate
antibacterial and anthelmintic activities1. They belong to the family of pyrrolamide metabolites,
the best-characterized members of which are congocidine (also called netropsin, produced by
Streptomyces ambofaciens ATCC 25877) and distamycin (produced by Streptomyces
netropsis DSM 40846) (see Supplementary Figure S1). These metabolites are known to bind
to the DNA minor groove with some sequence specificity for regions of four (or more) A or T
bases2. During the last decade, the biosynthetic gene clusters of congocidine and distamycin
have been identified and the biosynthesis of these metabolites has been elucidated3–7. One
remarkable aspect of this biosynthesis is that it involves non-canonical non-ribosomal peptide
synthetases (NRPSs), solely constituted of stand-alone modules or domains. Such NRPSs are
sometimes called type II NRPSs. They participate in the biosynthesis of unusual precursors of
non-ribosomal peptides (NRP)8 and are involved in the assembly of some NRP such as
phosphinothricin9 and rhabdopeptide/xenortide peptides10.
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A structural analysis of anthelvencins shows that these metabolites most likely share
two precursors with congocidine and distamycin: 4-acetamidopyrrole-2-carboxylate (5) and 3-
aminopropionamidine (6). The remaining precursor is probably 5-amino-3,4-dihydro-2H-
pyrrole-2-carboxylate (4), a precursor shared with other pyrrolamides such as kikumycins11 or
TAN 868A12 (Figure S1). In fact, a limited number of precursors seems to be assembled in
various combinations to give birth to the different members of the pyrrolamide family.
Understanding how these precursors are assembled and combined may improve our
comprehension of type II NRPS enzymatic mechanisms and help to design functional synthetic
NRPSs using synthetic biology. For these reasons, we undertook to identify and characterize
the anthelvencin biosynthetic gene cluster of S. venezuelae ATCC 14583. In this study, we
revise the structure of anthelvencin A based on HR-MS2 and NMR data. We show that S.
venezuelae ATCC 14583 produces, in addition to the already known anthelvencins A and B, a
third anthelvencin, methylated on the two pyrrole groups. We named this new anthelvencin
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anthelvencin C. We also identify the gene cluster directing the biosynthesis of anthelvencins
in S. venezuelae ATCC 14583 genome and we functionally characterize this gene cluster.
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Figure 1. (A) Chemical structure of anthelvencins A, B and C; (B) Genetic organization
of the anthelvencin biosynthetic gene cluster in S. venezuelae ATCC 14583. Genes in
boldface are genes that were used as targets for genetic inactivation. The numbers in boldface
(1a-6) are used to refer to the corresponding chemical structures.
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RESULTS AND DISCUSSION
In silico identification of a gene cluster putatively involved in anthelvencin biosynthesis
in S. venezuelae ATCC 14583. To identify the gene cluster directing anthelvencin
biosynthesis, we sequenced the genome of the S. venezuelae ATCC 14583 strain by the
Illumina technology, using a paired-end genomic library. The 5.45 million reads of 301 bps
were assembled using Velvet v1.2.10, resulting in 63 contigs with a total length of 9.08 Mbps
(180-fold coverage).
The gene cluster directing the biosynthesis of anthelvencins was identified by mining
the genome of S. venezuelae ATCC 14583 for homologs of genes involved in the biosynthesis
of congocidine3. We identified a gene cluster (ant, GenBank accession number MK483114)
that spans 26 kb and contains 22 genes (Figure 1B). The deduced products of twenty of these
genes exhibit a high amino acid sequence identity with Cgc proteins involved in congocidine
biosynthesis (from 64 to 84 % sequence identity, Table 1) and they most likely have similar
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Table 1. Sequence identities between Ant and Cgc proteins
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Protein Putative protein function
Cgc ortholog
(Accession number)
%
sequence
identity
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Ant1
Ant2
Ant3
Transcriptional regulator
NRPS, C domain
Cgc1 (AKZ59702.1)
Cgc2 (AKZ59703.1)
Cgc3 (AKZ59704.1)
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4-Acetamidopyrrole-2-carboxaldehyde
dehydrogenase
Ant4
Ant5
Ant6
Ant8
Cytosine monophosphate hydrolase
Cytosine reductase
Cgc4 (AKZ59705.1)
Cgc5 (AKZ59706.1)
Cgc6 (AKZ59707.1)
Cgc8 (AKZ59709.1)
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Dihydrocytosine hydrolase
Nucleotidyl
N-acetylglucosamine
dehydrogenase
Ant9
Nucleotidyl-2-acetamido-2-
deoxyglucopyranuronate decarboxylase
Cgc9 (AKZ59710.1)
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Ant10
Ant11
Glycosyltransferase-like enzyme
Cgc10 (AKZ59711.1)
Cgc11 (AKZ59712.1)
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N-acetylglucosamine-1-phosphate
nucleotidyltransferase
Ant12
Nucleotidyl threo-2-acetamido-2-deoxy-
pentopyran-4-ulose aminotransferase
Cgc12 (AKZ59713.1)
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Ant13
Ant14
Glycoside hydrolase
Cgc13 (AKZ59714.1)
Cgc14 (AKZ59715.1)
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4-Acetamidopyrrole-2-carboxylate
deacetylase
Ant15
Ant16
Ant17
Methyltransferase
NRPS, C domain
Cgc15 (AKZ59716.1)
Cgc16 (AKZ59717.1)
Cgc17 (AKZ59718.1)
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83
4-Acetamidopyrrole-2-carboxaldehyde
dehydrogenase
Ant19
Ant20
Ant21
Ant22
Ant23
Ant24
NRPS, PCP domain
Cgc19 (AKZ59720.1)
Cgc20 (AKZ59701.1)
Cgc21 (AKZ59700.1)
Cgc22 (AKZ59699.1)
None
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ABC transporter
ABC transporter
Acyl co-A synthetase
ATP-grasp domain-containing protein
Ectoine synthase-like protein
None
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function to their Cgc homologs. Thus, the gene numbers attributed to the ant genes were
chosen to follow the cgc nomenclature whenever possible. The genetic organization of the ant
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cluster is remarkably similar to the one of the cgc cluster3 from S. ambofaciens ATCC23877
(Figure S2). Two cgc genes (cgc7 and cgc18) have no homologs in the ant gene cluster. These
two genes are involved in the biosynthesis of the guanidinoacetate precursor (absent in
anthelvencins) and its assembly. Based on the high level of sequence identity between Ant
and Cgc proteins and on the previously published biosynthetic pathways for congocidine and
distamycin3,4,6,7, we propose the following roles for the Ant proteins. Ant8 to Ant13, Ant3 and
Ant17 are involved in the biosynthesis of 4-acetamidopyrrole-2-carboxylate (5) and Ant14 is
involved in its deacetylation. Ant4 to Ant6 are involved in the biosynthesis of 3-
aminopropionamidine (6). Ant2, Ant16, Ant19, and Ant22 are involved in the assembly of
anthelvencins. In addition to these, proteins are necessary for the biosynthesis and assembly
of 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (4). The ant cluster contains two genes,
ant24 and ant23, which have no homolog in the cgc cluster. We hypothesized that they might
be involved in the biosynthesis of (4) and its assembly. Indeed, a protein blast13 and a
conserved domain searches14 on the Ant24 sequence suggested that Ant24 belongs to the L-
ectoine synthase (EC 4.2.1.108) family of enzymes. L-ectoine synthases catalyze the ring
closure of N-acetyl-L-2,4-diaminobutyric acid, yielding the osmolyte ectoine, a metabolite
structurally related to (4). In 2011, Witt and collaborators reported that the ectoine synthase
from Halomonas elongata can catalyze the intramolecular condensation of glutamine to form
(4) as a side reaction15. This enzyme has 34% sequence identity and 51% sequence similarity
with Ant24. Thus, it appears likely that Ant24 catalyzes the same reaction (Scheme 1).
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O
Ant24
N
-OOC
NH2
+H3N
NH2
COO-
Glutamine
4
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Scheme 1: Proposed biosynthesis of 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (4) by
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Ant24
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Ant23 contains an ATP-grasp domain. ATP-grasp enzymes usually catalyze the ATP-
dependent ligation of a carboxylate-containing molecule to an amino or thiol group-containing
molecule16. Some of these ATP-grasp enzymes play a role in the biosynthesis of specialized
metabolites17. They can function as an alternative to or in combination with non-ribosomal
peptide synthetases (NRPS), to elongate a peptide chain17,18. Thus, it appears plausible that
Ant23 catalyzes the amide bond formation between (4) and a PCP (Ant19)-bound 4-
aminopyrrole-2-carboxylate.
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Abolition of the production of four metabolites in a S. venezuelae ATCC 14583 mutant
deleted for ant8 (coding for
a
putative nucleotidyl N-acetylglucosamine
dehydrogenase). To verify that the ant gene cluster is involved in the biosynthesis of
anthelvencins, we inactivated ant8. This gene is the ortholog of cgc8, the product of which is
involved in the biosynthesis of the 4-acetamidopyrrole-2-carboxylate (5). The ant8 gene was
replaced by an aac(3)IV resistance cassette by homologous recombination using the pANT007
suicide plasmid, yielding the S. venezuelae ANT007 strain. This strain and the wild type
S. venezuelae strain were cultivated for three days in MP5 liquid medium. The culture
supernatants were then filtered and analyzed by HPLC. The chromatograms show that four
metabolites present in the wild type strain supernatant (peaks I to IV) are absent in the
supernatant of the ANT007 mutant strain (Figure 2). The first metabolite (peak I, retention time
of 11.5 min) corresponds to 4-aminopyrrole-2-carboxylate (5), identified by its UV spectrum
and by comparison with an authentic standard (Figure 2 and reference 7). The three peaks II
(retention time of 13.3 min), III (retention time of 14.3 min) and IV (retention time of 15.5 min)
have UV absorption spectra typical of pyrrolamides (Figure S3 and reference 4).
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Figure 2: HPLC analysis of culture supernatants of A) S. venezuelae ATCC 14583 wild
type and B) ANT007 (S. venezuelae ATCC 14583 ant8::aac(3)IV). C) Standard of 4-
acetamidopyrrole-2-carboxylate (5). Peaks I to IV are compounds the biosynthesis of which is
linked to the ant gene cluster. Peak I corresponds to (5).
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Chemical nature of metabolites II, III and IV. To determine the chemical nature of the
metabolites II, III and IV, we partially purified them. For that purpose, we used ANT012, a strain
that expressed a second copy of the genes ant23 and ant24 under the control of the promoter
rpsL(TP)19, as this strain produces compounds III and IV in slightly higher titers (data not
shown). The ANT012 culture supernatant was recovered after three days of culture in MP5
medium and the compounds of interest were partially purified on a XAD16 resin. The elution
fraction was concentrated to dryness solution, resuspended in water and analyzed by LC-HR-
MS².
The exact mass and fragmentation pattern of compound II (Figure S4) are consistent
with II being anthelvencin B (2) ([M+H]+ m/z = 414.1998; calculated 414.1997). The exact mass
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of compound III (Figure S5) is consistent with III being anthelvencin A ([M+H]+ m/z = 428.2151;
calculated 428.2153). The fragmentation pattern however (Figure S5, fragments 4 and 5),
indicates that the position of the methyl group is not on the B pyrrole ring, as previously
proposed (but never experimentally established1) but rather on the A pyrrole ring (Figure 1).
To confirm the structure of anthelvencin A, we purified compound III and carried out NMR
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experiments. H NMR of anthelvencin A hydrochloride proved to be very similar to the one
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reported for the authentic sample synthesized by Lee and coworkers17 assuming the position
of the methyl group on the B pyrrole ring. However, a combination of two-dimensional NMR
experiments confirmed the position of this methyl on the A ring. We therefore propose a revised
structure for anthelvencin A (Figure S6).
The exact mass and fragmentation pattern of compound IV (Figure S7) are consistent
with IV being an anthelvencin metabolite methylated on both pyrrole groups ([M+H]+ m/z =
442.2311; calculated 442.2310), a metabolite that we named anthelvencin C (Figure 1A, 3).
We tried to purify anthelvencin C to confirm its chemical structure with NMR analyses but this
metabolite turned out to be highly unstable, as already observed by Lee and coworkers20.
Involvement of ant24 in 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (4) biosynthesis.
To verify that ant24 is involved in the biosynthesis of anthelvencins, we replaced it by an
aac(3)IV resistance cassette by homologous recombination, following the same procedure as
described above. The culture supernatant of the resulting mutant strain, called ANT009, was
analyzed by HPLC. No production of anthelvencins was observed, confirming that ant24 is
necessary for the production of these metabolites (Figure 3A). To ensure that the observed
phenotype was due to the replacement of ant24 by the aac(3)IV cassette, we genetically
complemented the ANT009 strain using a plasmid expressing ant23 and ant24 under a
constitutive promoter. The production of anthelvencins was restored in the complemented
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strain, named ANT014 (Figure S8A), thus confirming that ant24 is involved in anthelvencin
biosynthesis.
To examine Ant24 putative function in the biosynthesis of (4), we undertook the chemical
complementation of ANT009 by adding to the culture medium (4), the putative product of
Ant24-catalyzed reaction. Thus, (4) was synthetized according to a previously described
synthetic procedure21 (Scheme 2).
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SOCl2 (2 equiv)
DMF (2 mol %)
(Et3O)BF4 (1.4 equiv)
O
O
O
O
O
EtO
N
N
N
MeOH
10 °C to room
temperature (rt), 24 h
DCM
rt, 24 h
H
H
OH
OMe
OMe
8
9
(98%)
(89%)
Pyroglutamic
7
acid ( )
NaH4Cl (1.05 equiv)
MeOH
reflux, 5 h
HClaq (10% v/v)
65 °C, 3 h
O
O
H2N
H2N
N
N
HCl
OH
HCl
OMe
4
10
(quant.)
(61%)
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Scheme 2: Synthesis of 5-amino-3,4-dihydro-2Hpyrrole-2-carboxylate (4)
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We next fed the ANT009 strain with (4). As shown in Figure 3B, this resulted in the
restoration of the production of anthelvencins A and C, hence confirming the involvement of
ant24 in the biosynthesis of the anthelvencin precursor (4).
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Figure 3: HPLC analysis of culture supernatants of (A) ANT009 (S. venezuelae ATCC 14583
ant24::aac(3)IV), (B) ANT009 (S. venezuelae ATCC 14583 ant24::aac(3)IV) cultivated in
presence of 1mM of (4), and (C) ANT008 (S. venezuelae ATCC 14583 ant23::aac(3)IV).
Numbers above peaks correspond to the metabolite numbers in the text.
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Involvement of ant23 in the biosynthesis of anthelvencins. To confirm that Ant23 is
involved in anthelvencin biosynthesis, we replaced ant23 by the aac(3)IV resistance cassette
following the previously described protocol. The resulting mutant strain was called ANT008. It
was cultivated for three days in MP5 medium at 28°C and the culture supernatant was
analyzed by HPLC. Figure 3C shows that no anthelvencin is produced by the ANT008 mutant.
To ensure that the observed phenotype was due to the replacement of ant23 by the aac(3)IV
cassette, we genetically complemented the ANT008 strain using the plasmid expressing ant23
and ant24 under a constitutive promoter. The production of anthelvencins was restored in the
complemented strain, named ANT013 (Figure S8B), thus confirming that ant23 is involved in
anthelvencin biosynthesis.
Proposed biosynthetic pathway for anthelvencin biosynthesis. Based on the results
presented above and on previous characterizations of pyrrolamide biosyntheses3,4,6, we
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proposed that anthelvencins are assembled from 3-amidinopropionamidine, 4-aminopyrrole-
2-carboxylate and 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate following the biosynthetic
pathway presented in Figure 4. As already observed for the biosynthesis of other pyrrolamides
(congocidine, distamycin), the non-ribosomal peptide synthetase involved in anthelvencins is
constituted solely of stand-alone domains (C domains Ant2 and Ant16 and PCP domain
Ant19). No adenylation domain is involved in the activation of the carboxylate groups of the
precursors. Instead, activation of the carboxylate group of the pyrrole precursor (5) and the
covalent attachment of the activated precursor to the PCP domain Ant19 is catalyzed by Ant22,
which belongs to the family of acyl-CoA synthetases. The formation of the first amide bond
between (4) and Ant19-bound (5) is likely catalyzed by Ant23, an enzyme from the ATP-grasp
ligase family, which form acylphosphate intermediates. Two stand-alone condensation
domains, Ant16 and Ant2, catalyze the formation of the other amide bonds, adding respectively
a second pyrrole precursor and the 3-aminopropionamidine to Ant19-bound intermediates.
Thus, during anthelvencin assembly, the stand-alone PCP domain, Ant19, interacts with two
different families of enzymes (condensation domain and ATP-grasp ligase).
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O
Ant19
O
Ant19
NH2
Ant24
Ant19
N
H2N
COO-
S
+H3N
H
N
S
S
COO-
O
O
NH
Ac
Ant19
HN
O
O
Ant23
HN
HN
S
HN
Ant14
NH
Ant22
Ant19
O
Ac NH
NH
NH
Ant14, Ant16
COOH
N
N
C
H
HN
O
N
Ac
H2N
NH2
NH2
HN
HN
NH
H2N
NH2
HN
NH
NH2
Ant2
C
HN
NH2
O
O
O
NH
NH
N
CH3
NH
Ant15
HN
Ant15
H3C
HN
H3C
HN
N
H
O
N
N
O
O
O
N
NH
Anthelvencin C ( )
O
NH
O
NH
1
Anthelvencin A ( )
2
Anthelvencin B ( )
3
N
N
H2N
H2N
H2N
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Figure 4: Proposed biosynthetic pathway for anthelvencins A, B and C
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The blue ellipses represent PCP domains, and the red circles represent C domains
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In conclusion, we have identified and characterized the gene cluster directing the
biosynthesis of anthelvencins in Streptomyces venezuelae ATCC 14583. We showed that this
cluster directs the biosynthesis of two known metabolites, anthelvencin A, for which we
propose a revised structure, anthelvencin B, and new anthelvencin that we named
anthelvencin C. As already observed for the assembly of other pyrrolamides (congocidine,
distamycin), the assembly of anthelvencins involves a non-canonical NRPS constituted of
stand-alone domains only. Such structural organization, rarely encountered in NRPS, appears
to be characteristic of pyrrolamide NRPSs. Characterizing biosynthetic pathways involving
type II NRPSs is of interest for the production of novel specialized metabolites by synthetic
biology as the architecture of these NRPSs is particularly suited for combinatorial biosynthesis
approaches.
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MATERIALS AND METHODS
Bacterial strains, plasmids and growth conditions. Strains and plasmids used in this study
are listed in Table S1 and S2. Escherichia coli strains were grown at 37 °C in LB or SOB
complemented with MgSO4 (20 mM final), supplemented with appropriate antibiotics as
needed22. The Soya Flour Mannitol (SFM) medium23 was used for genetic manipulations of
Streptomyces strains and spore stocks preparations at 28°C. Streptomyces strains were grown
for three days at 28°C in MP524 for anthelvencins (1-3) production.
DNA preparation and manipulations. All oligonucleotides used in this study were purchased
from Eurofins and are listed in Table S3. The High fidelity DNA polymerase Phusion (Thermo
Fisher Scientific) was used to amplify the DNA fragments for the construction of the suicide
plasmids. DreamTaq polymerase (Thermo Fisher Scientific) was used for PCR verification of
plasmids and of the replacement of the targeted genes by the resistance cassette aac(3)IV.
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DNA fragments were purified from agarose gels using the Nucleospin Gel and PCR clean-up
kit from Macherey-Nagel. E. coli transformations and E. coli/Streptomyces conjugations were
performed according to standard procedures25,23
.
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S. venezuelae ATCC 14583 sequencing and assembly. Total DNA of S. venezuelae ATCC
14583 was extracted following the salting out procedure23 except that the mycelium was
ground in liquid nitrogen. A paired-end library of the whole genome was constructed and
sequenced at the high throughput sequencing core facility of I2BC with a MiSeq M01342
instrument (Illumina), generating 5.45 million 301 bp reads that were assembled using Velvet
v1.2.10. The GenBank accession number of the anthelvencin gene cluster is MK483114.
Construction of the replacement mutants. The suicide plasmid pANT007 was constructed
to replace the ant8 gene by an aac(3)IV resistance cassette in S. venezuelae. This vector was
constructed by assembling the three following inserts in the backbone pOSV400 (bearing a
hygromycin resistance gene)26: a 1.8 kb fragment upstream of ant8, the resistance cassette
aac(3)IV and a 2.0 kb DNA fragment downstream of ant8. The 1.8 kb and 2.0 kb DNA
fragments from S. venezuelae ATCC 14583 were amplified by PCR with the primers
CEA001/CEA002 and CEA003/CEA004 respectively. The PCR products were purified and
ligated into pCR® Blunt, yielding pANT001 (containing the upstream DNA fragment) and
pANT002 (containing the downstream DNA fragment). Both plasmids were verified by
sequencing (Genewiz). The aac(3)IV resistance cassette was obtained by digestion of pW6027
by HindIII. The 1.8 kb HindIII-XhoI fragment from pANT001, the 1.0 kb HindIII aac(3)IV
fragment, and the 2.0 kb HindIII-SpeI fragment from pANT002 were then ligated into the XhoI-
SpeI-digested pOSV400, yielding pANT007. The pANT007 plasmid was verified by restriction
digestion and was introduced into S. venezuelae ATCC 14583 by intergeneric conjugation
from the E. coli ET12567/pUZ8002/pANT007 strain. Double-recombinant mutants were
selected on SFM plates with 50 µg/mL apramycin and screened for hygromycin sensitivity.
DNA from the resulting strain, ANT007, was isolated and analyzed by PCR using the primers
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A5, A6, and CEA013-CEA016 (See Figure S8). The same protocol was used for the
construction of the ANT008 mutant strain (replacement of ant23), using the plasmid pANT008,
and the ANT009 mutant strain (replacement of ant24), using the plasmid pANT009 (see Tables
S2 and S3 for plasmid names and for primer sequences).
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Construction of the ANT012 strain overexpressing Ant23 and Ant24. The DNA region
containing ant23-ant24 was amplified by PCR from S. venezuelae ATCC 14583 genomic DNA
using the primers CEA034/CEA035. The PCR product was purified and cloned into pCR®
Blunt, yielding pANT011. The sequence of the insert was confirmed by sequencing. The 2.0
kb NheI/AflII fragment from pANT011 was ligated into the SpeI/AflII-digested pCEA00528. The
obtained plasmid, containing ant23 and ant24 under the rpsL(TP) promoter, was named
pANT012 and confirmed by restriction digestion. This plasmid was introduced into S.
venezuelae ATCC 14583 by intergeneric conjugation. The correct integration of pANT012 was
verified on the extracted DNA by PCR using the primers CEA_vec_seq14 and
CEA_vec_seq15 and the strain was named ANT012.
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Genetic complementation of ANT008. As the ANT008 strain bears the aac(3)IV resistance
marker, the previously constructed pANT012 plasmid, bearing an aac(3)IV resistance gene
cassette, could not be used for the genetic complementation of the strain. Thus, the 2.4 kb
NsiI/AflII DNA fragment of pANT012 containing ant23 and ant24 under the control of the
rpsL(TP) promoter was ligated into the NsiI/AflII-digested pOSV80628. The resulting plasmid
was named pANT013 and was introduced into ANT008 by intergeneric conjugation. The strain
obtained was named ANT013.
Chemical synthesis of 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (4). Compound (4)
was prepared according to a previously described synthetic procedure29 (Scheme 2).
Commercially available DL-pyroglutamic acid (7) was converted into the corresponding methyl
ester (8) by treatment with thionyl chloride (2 equiv.), and N,N-dimethylformamide (DMF) (2
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mol %) in methanol. Derivative (8) was then submitted to a reaction with triethyloxonium
tetrafluoroborate (Meerwein's salt, 1.4 equiv.) in dichloromethane (DCM) to form carboximidate
(9) in quantitative yield. This compound subsequently reacted with ammonium chloride (1.05
equiv.) in refluxing methanol to provide product (10) in 61% yield. Hydrolysis of the ester moiety
of compound (10) finally afforded the desired acid (4) in a quantitative yield. A detailed
synthesis protocol is available in the Supporting Information.
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Chemical complementation of ANT009. S. venezuelae ANT009 strain was cultivated in
50 mL of MP5. After 24 h, the cultures were separated in two 25 mL cultures, and 1 mM of (4)
(final concentration) was added to one of the cultures. After a total of 72 h of cultivation, culture
supernatants were analyzed by HPLC as described below.
HPLC analysis of culture supernatants. S. venezuelae ATCC 14583 and its derivatives were
cultivated in MP5 medium for three days at 28°C. The supernatants were filtered using Mini-
UniPrep syringeless filter devices (0.2 µm, Whatman). The samples were analyzed on an
Atlantis C18 T3 column (250 mm x 4.6 mm, 5 µm, column temperature 28°C) using an Agilent
1200 HPLC instrument with a quaternary pump. Samples were eluted in isocratic conditions
with 0.1% v/v HCOOH in H20 (solvent A)/ 0.1% v/v HCOOH in CH3CN (solvent B) (95:5) at
1 ml.min-1 for 7 min, followed by a gradient to 40:60 A/B over 23 min. Anthelvencins were
detected by monitoring absorbance at 297 nm.
LC-HR-MS-MS analyses. The resuspended elution fraction obtained above was analyzed by
LC-HR-MS2.The analysis was performed using a Dionex Ultimate 3000 HPLC system coupled
with a Maxis II™ QTOF mass spectrometer (Bruker, MA, USA) fitted with an electrospray
ionization (ESI) source.
Chromatographic analysis was performed using a C18 AcclaimTM RSLC
PolarAdvantage II (2.1 x 100 mm, 2.2 µm pore size) column (Thermo Scientific, MA, USA).
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Column temperature was set at 40 °C and 2 μL of each sample was injected via an
autosampler cooled to 4 °C. A flow rate of 0.3 mL/min was used and the eluent was introduced
directly into the MS for ion detection. Elution was conducted with a mobile phase consisting of
0.1% v/v HCOOH in H20 (solvent A) and 0.1% v/v HCOOH in CH3CN (solvent B) following the
gradient elution profile: 0 min, 5% solvent B; 2 min, 5% solvent B; 9 min, 50% solvent B; 15
min 90% solvent B; 17 min 90% solvent B; 19 min 5% solvent B; 21 min 5% solvent B. In the
first half minute of each run, a sodium formate solution was injected directly as an internal
reference for calibration. The acquisition parameters of the ESI source were set up as follows:
electrospray voltage for the ESI source: 3500V, nebulising gas (N2) pressure: 35 psi, drying
gas (N2) flow: 8 L/min, and drying temperature: 200°C. Mass spectra were recorded over the
m/z range 100-1300 at a frequency of 2 Hz, in positive ion mode. For MS/MS analysis, the
cycle time was of 3 sec. Mass spectra were recorded over the m/z range 100-1300 at a
frequency of 2 Hz, in positive ion mode. Selected parent ion at m/z 442.23 was fragmented at
a fixed collision energy value of 40 eV and an isolation window of 0.5 amu.
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Accession codes. The GenBank accession number of the anthelvencin gene cluster is
MK483114. Its MiBig30 accession number is BGC0002042.
Supporting Information Available: The protocol for the synthesis of 5-amino-
3,4-dihydro-2H-pyrrole-2-carboxylate (4), the strains, plasmids and oligonucleotides used in
this study and seven supplementary figures are available in the Supporting Information. This
material is available free of charge via the internet at http://pubs.acs.org
Acknowledgements
We acknowledge the High-throughput sequencing facility of I2BC for its sequencing
and bioinformatics expertise (Centre de Recherche de Gif – http://www.i2bc.paris-saclay.fr/).
We thank Z. Hong, Y. Li, S. Prado and D. Touboul for their help with the LC-HRMS2 analysis.
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The research received funding from ANR-14-CE16-0003-01. The funders had no role in study
design, data collection and interpretation, or the decision to submit the work for publication.
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