Guanidyl modification of the 1-azabicyclo[3.1.0]hexane ring in ficellomycin essential for its biological activity

Article abstract of DOI:10.1039/d0ob00339e

The 1-azabicyclo[3.1.0]hexane ring is a key moiety in natural products for biological activities against bacteria, fungi, and tumor through DNA alkylation. Ficellomycin is a dipeptide that consists of l-valine and a non-proteinogenic amino acid with the 1-azabicyclo[3.1.0]hexane ring structure. Although the biosynthetic gene cluster of ficellomycin has been identified, the biosynthetic pathway currently remains unclear. We herein report the final stage of ficellomycin biosynthesis involving ring modifications and successive dipeptide formation. After the ring is formed, the hydroxy group of the ring is converted into the guanidyl unit by three enzymes, which include an aminotransferase with a novel inter ω-ω amino-transferring activity. In the last step, the resulting 1-azabicyclo[3.1.0]hexane ring-containing amino acid is connected with l-valine by an amino acid ligase to yield ficellomycin. The present study revealed a new machinery that expands the structural and biological diversities of natural products. This journal is

Full text of DOI:10.1039/d0ob00339e

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DOI: 10.1039/D0OB00339E.
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DOI: 10.1039/D0OB00339E
Guanidyl modification of the 1-azabicyclo[3.1.0]hexane ring in
ficellomycin essential for its biological activity
Sumire Kurosawaa, Kenichi Matsudaa, Fumihito Hasebea, Taro Shiraishia,b,
Kazuo Shin-yac, Tomohisa Kuzuyamaa,b and Makoto Nishiyama*a,b
aBiotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8567, Japan.
bCollaborative Research Institute for Innovative Microbiology, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8567, Japan
cNational Institute of Advanced Industrial Science and Technology, 2-4-7
Aomi, Koto-ku, Tokyo 135-0064, Japan.
*Corresponding author: Makoto Nishiyama. Biotechnology Research Center,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8567, Japan.
E-mail: umanis@mail.ecc.u-tokyo.ac.jp; Fax: +81-3-5841-8030
Abstract
The 1-azabicyclo[3.1.0]hexane ring is a key moiety in the natural products for
biological activities against bacteria, fungi, and tumor through DNA
alkylation. Ficellomycin is a dipeptide that consists of L-valine and a non-
proteinogenic amino acid with the 1-azabicyclo[3.1.0]hexane ring structure.
Although the biosynthetic gene cluster of ficellomycin has been identified, the
biosynthetic pathway currently remains unclear. We herein report the final
stage of ficellomycin biosynthesis involving ring modifications and successive
dipeptide formation. After the ring is formed, the hydroxy group of the ring is
converted into the guanidyl unit by three enzymes, which includes an
aminotransferase with a novel inter ω-ω amino-transferring activity. In the
last step, the resulting 1-azabicyclo[3.1.0]hexane ring-containing amino acid
is connected with L-valine by an amino acid ligase to yield ficellomycin. The
present study revealed a new machinery that expands the structural and
biological diversities of natural products.
Introduction
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Ficellomycin isolated from Streptomyces ficellus1 is a dipeptide
consisting of -valine and the non-proteinogenic amino acid with a 1-
L
azabicyclo[3.1.0]hexane ring moiety (azabicyclo hexane ring)2.
Ficellomycin exhibits the antibacterial activity, for which the azabicyclo
hexane ring is a necessary skeleton3. We previously reported the novel
azabicyclo hexane ring-containing dipeptide, vazabitide A, from
Streptomyces sp. SANK 604044. Although the structure of vazabitide A
is similar to that of ficellomycin, vazabitide A did not exhibit any
antibacterial activity. The only structural difference between
ficellomycin and vazabitide A was the guanidyl group and hydroxy
group modifying the azabicyclo hexane ring (Fig. 1a). The difference in
the structure and activity of the related compounds indicates that the
modification of the azabicyclo hexane ring is important for the biological
activity. The guanidyl group is used in a pharmaceutical field because
it facilitates the membrane permeability of active coumpounds5 and
interaction with high molecular-weight proteins, such as protease6 and
ion channels7. Although the biosynthetic cluster of ficellomycin had
already been reported8, the biosynthetic pathway including the
guanidyl modification mechanism of the azabicyclo hexane ring
currently remains unclear.
We recently demonstrated that the azabicyclo hexane ring in
vazabitide A is formed from (2S,6R)-diamino-(5R,7)-dihydroxy-
heptanoic acid (DADH)4. DADH is a non-proteinogenic amino acid that
is biosynthesized using amino-group carrier protein (AmCP)9. Since the
structures of ficellomycin and vazabitide A are similar to each other,
ficellomycin is probably biosynthesized from DADH. In fact, all DADH
biosynthetic gene homologs are conserved in the fic cluster (Fig. 1b). We
hypothesized that the four genes specifically present in the fic cluster
were the candidates for the enzyme catalyzing the last steps of the
ficellomycin biosynthesis. Through structural analyses of the
compounds accumulated in the knockout mutants of these candidate
genes coupled with in vitro enzymatic analyses, we revealed that three
out of the four enzymes are responsible for the formation of the
guanidylated azabicyclo hexane ring-containing amino acid and the
remaining enzyme plays a role in dipeptide formation by ligating L-
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valine with the guanidylated azabicyclo hexane ring-containing amino
acid to yield ficellomycin.
We herein report the final stage of ficellomycin biosynthesis (Fig.
1c).
Results & discussion
Involvement of fic22 and fic16 in ficellomycin biosynthesis
To verify that ficellomycin is biosynthesized using AmCP, we constructed an
in-frame knockout mutant of fic22 encoding AmCP. As expected, the fic22
mutant of S. ficellus JCM4946 did not produce ficellomycin (Fig. 2a),
suggesting that ficellomycin is synthesized using AmCP with DADH as an
intermediate. The fic cluster contains many genes similar to those in the
vazabitide A biosynthetic gene cluster. Among the genes in the fic cluster, the
genes present only in the fic cluster were the probable candidates for guanidyl
group modification because this group is unique to ficellomycin. Based on this
information, we listed four genes, fic13 (dehydrogenase), fic15 (amino acid
ligase), fic16 (ornithine aminotransferase) and fic36 (amidinotransferase),
and performed the in-frame deletion of fic16. When compounds accumulated
in its culture broth were analyzed by LC-MS, the fic16 mutation was found
to abolish the production of ficellomycin, but induce the accumulation of two
putative biosynthetic intermediates, 1 and 2, with m/z of 173.09 [M+H]+ and
171.08 [M+H]+, respectively, in the culture broth (Fig. 2b-d).
We purified 2 from the broth of fic16 and fed 2 into the culture of the
fic22 mutant that lacks the ability to produce ficellomycin. The feeding of 2
restored ficellomycin production by the mutant (Fig. 2e). This result indicated
that 2 is a biosynthetic intermediate of ficellomycin and produced via AmCP.
We then examined the chemical structure of 2 by NMR as the dinitrophenyl
(DNP) unit-conjugated derivative using 2,4-dinitrofluorobenzene (DNFB) to
increase its hydrophobicity10. 2 was elucidated as 2-amino-2-(4-oxo-1-
azabicyclo[3.1.0]hexan-2-yl)acetic acid (Figure S1-6). NOESY spectrum
revealed that the relative stereochemistry was in accordance with that of
ficellomycin11 (Figure S1 and S7). We attempted to isolate and purify 1;
however, it was unsuccessful because its DNP derivative still showed high
hydrophilicity. Nevertheless, MS/MS analysis supported information on the
proposal structure of 1 with the hydroxy group at the C4 position (Figure S8).
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In vitro reconstitution of the guanidyl unit formation
To clarify the functions of Fic13, Fic16, and Fic36 in the guanidyl group
modification, we prepared three enzymes, each as an N-terminal (His)8-
tagged protein, using recombinant Escherichia coli cells (Figure S9) and
performed an in vitro assay. Fic13 is annotated as a dehydrogenase12.
Therefore, we expected Fic13 to oxidize 1 to 2. When Fic13 was incubated
with 1 in the presence of 2 mM NAD(P)+ at 30˚C for 2 h, the generation of 2
along with consumption of 1 was observed in the LC-MS analysis of the
reaction mixture. This analysis also revealed that Fic13 exhibited a
preference for NAD+ to NADP+ as a coenzyme (Figure S9). Thus, Fic13 was
shown to function as a dehydrogenase that converts 1 into 2 in an NAD+-
dependent manner.
An in vitro assay of Fic16, annotated as an ornithine aminotransferase
(OAT), was then conducted. OAT transfers the α-amino group of glutamate to
glutamate-ϒ-semialdehyde and generates 2-oxoglutarate and ornithine13 (Fig.
3a). We firstly hypothesized that Fic16 transferred the α-amino group of
glutamate to 2 in order to produce 3. However, when Fic16 was incubated
with 2 and 0.5 mM glutamate at 30˚C for 2 h, no consumption of 2 was
observed. We examined several amino acids as a potential amino donor for
the reaction and found that Fic16 consumed 2 only in the presence of
ornithine (Fig. 3b). When Fic16 was incubated with asparagine, aspartate,
glutamine, and lysine, the consumption of 2 was not observed (Fig S11). We
also attempted to detect the putative intermediate 3 in the reaction mixture;
however, 3 was not detected, suggesting that 3 was unstable. Since ornithine
has two amino groups in its structure, we tried to identify which amino group
(α or δ) of ornithine Fic16 transferred to 2. When the ω-amino group of
ornithine is transferred, ornithine must be converted into glutamate-ϒ-
semialdehyde, which is spontaneously cyclized to 1-pyrroline-5-carboxylic
acid (P5C) (Fig. 3c). P5C can be converted to proline by reduction. We added
NaBH4 (final 20 mM) to the Fic16 reaction mixture and derivatized the
product with DNFB. LC-MS analysis revealed that proline-DNP was
produced in the presence of Fic16 and NaBH4 (Fig. 3d). Based on these results,
we concluded that Fic16 is the enzyme that converts 2 into 3 and the first ω-
ω specific aminotransferase that transfers the ω-amino group of ornithine to
the ω position of the substrate.
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Fic36, annotated as an amidinotransferase, shows 68% identity
with StrB1 in amino acid sequence14. StrB1 transfers the amidino group
of arginine to the amino group of the substrate. We hypothesized that
Fic36 catalyzes a similar reaction and converts 3 into 4, which has the
guanidyl group on the azabicyclo hexane ring. Since we were not able to
isolate 3, which may have been due to its instability, we examined the
function of Fic36 in a coupling assay with Fic16 using 2 as the substrate.
Fic16 and Fic36 was incubated with 2, 0.5 mM ornithine, and 5 mM
arginine at 30˚C for 2 h. After the reaction, we derivatized the reaction
product with DNFB at 60˚C for 1 h. An LC-MS analysis detected the
compound with of m/z 380.13 [M+H]+, which corresponded to that of 4-
DNP (Figure S12). MS/MS analysis supported information on the 4-
DNP structure with the guanidyl unit at the C4 position and DNP unit
at the -amino group (Figure S13). These results show that Fic36
converts 3 into 4, resulting in the guanidyl group modification of the
azabicyclo hexane ring.
In vitro reconstitution of ficellomycin production
Ficellomycin is a dipeptide composed of L-valine and 4. In the biosynthesis of
vazabitide A, a similar dipeptide composed of L-valine and the azabicyclo
hexane ring-containing amino acid with the hydroxy group, the two amino
acids are ligated by a non-ribosomal peptide synthase (NRPS)15, Vzb15.
However, a gene homologous to vzb15 was not found in the fic cluster,
suggesting that the bond is formed by a different mechanism without using
NRPS. As described above, four genes, fic13, fic15, fic16, and fic36, are
specifically present in the fic cluster. Among these, fic15 encodes a putative
ATP-grasp superfamily protein, some of which exhibit amino acid ligase
activity16. Therefore, we hypothesized that Fic15 ligates L-valine with 4 to
yield ficellomycin.
To clarify this hypothesis, we conducted a gene knockout experiment of
fic15. The knockout mutant lost the ability to produce ficellomycin (Figure
S14). This result demonstrated that fic15 is involved in the biosynthesis of
ficellomycin. We then performed an in vitro assay on Fic15 in a coupled
reaction with Fic13, Fic16, and Fic36 using 1 as the initial substrate. When
the reaction product was derivatized with DNFB and analyzed by LC-MS,
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ficellomycin-DNP was detected in the reaction mixture containing all the
necessary enzymes and compounds (Fig. 4).
Conclusions
We demonstrated that the amcp gene was necessary for the
biosynthesis of ficellomycin by gene deletion experiments. This has
become the second example that directly demonstrated the involvement
of AmCP in secondary metabolite production in Streptomyces4.
Ficellomycin is probably synthesized from DADH as a key intermediate,
which is biosynthesized using the AmCP-mediated machinery. DADH
must be converted into the azabicyclo hexane-ring containing amino
acid 1, and 1 is further converted into ficellomycin by the four enzymes,
Fic13, Fic16, Fic36, and Fic15. Among them, we assume that Fic16 and
Fic15 are key enzymes specifying the ficellomycin biosynthesis. Fic16 is
an aminotransferase that converts 2 to 3 using ornithine as the amino
donor. Aminotransferases can be divided into the following three types
according to the donor/acceptor relationship of the amino groups; (i) -
 type, (ii) -ω type, and (iii) amine-ketone type17. Fic16 transferred the
ω-amino group of ornithine to the ω position of the substrate. Thus,
Fic16 is the first specific ω-ω type aminotransferase that transfers the
ω-amino group of an amino donor to the keto group at the ω position of
an amino acceptor. Further studies are required to elucidate the
underlying mechanisms in more detail through crystal structure
analysis of Fic16.
Although ficellomycin and vazabitide A are both dipeptides
composed of an azabicyclo hexane ring-containing amino acid and L-
valine, their peptide bonds are formed via different mechanisms. While
the peptide bond in ficellomycin is formed by Fic15 annotated as an
amino acid ligase, the corresponding dipeptide formation is presumably
catalysed by Vzb7 (NRPS) in vazabitide A biosynthesis. We recently
provided another example of an amino acid ligase being involved in the
biosynthesis of azabicyclo hexane ring-containing natural product, s56-
p118, in which an amino acid ligase is proposed to ligate glycine with an
azabicyclo hexane ring-containing amino acid. An azabicyclo hexane
ring-containing amino acid appears to be a more versatile substrate of
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both amino acid ligase and NRPS than originally expected.
Interestingly, the relative stereochemistry of the azabicyclo ring in
ficellomycin is different from that of vazabitide A. In each biosynthesis,
the enzyme responsible for the peptide bond formation is different as
mentioned above. We speculate that C domain of the Vzb7-like protein
encoded in the ficellomycin biosynthetic gene cluster is not able to
recognize 1 as a substrate due to the different stereochemistry.
Structural similarity and resemblance of biosynthetic pathway between
ficellomycin and vazabitide A suggests that these natural products
share biosynthetic origin. It is intriguing how each stereochemistry had
been diverged from the common biosynthetic pathway. Bioinformatics
study is revealing that many gene clusters contain the DADH
biosynthetic gene homologs in Streptomyces4. Therefore, discovery of
unexploited azabicyclo hexane ring-containing natural products based
on their structures and functions will be expected, which will expand
the structural and biological diversities of these natural products to new
medicines.
Experimental
Bacterial strains and plasmids.
The bacterial strains and plasmids used in the present study are
listed in Table S1.
Biochemicals and enzymes for genetic manipulations were
purchased from TaKaRa Bio (Otsu, Japan), TOYOBO (Osaka, Japan), and
Bio-Rad Laboratories (Hercules, CA). Eluent additives in liquid
chromatography were purchased from Sigma-Aldrich (St. Louis, MO).
Oligonucleotides used for genetic manipulations (Table S3) were purchased
from Operon Biotechnologies (Tokyo, Japan) or FASMAC (Kanagawa,
Japan). 2,4-Dinitrofluorobenzene (DNFB) was purchased from Tokyo
Chemical Industry (Tokyo, Japan). All other reagents were purchased from
Wako Pure Chemical Industries (Osaka, Japan), Kanto Chemicals (Tokyo,
Japan), and Nacalai Tesque (Kyoto, Japan) unless otherwise noted.
Gene deletion experiment of fic15, fic16, and fic22.
Streptomyces ficellus mutants each lacking the fic15, fic16, or fic22
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gene, were constructed. The plasmid for the in-frame knockout of each gene
was constructed as follows. The first polymerase chain reactions (PCR) were
performed with the primers fic16UpFw and fic16UpRv to amplify the
upstream region (2 kb) of the fic16 gene. The second PCR was performed
with the primers fic16DnFw and fic16DnRv to amplify the downstream
region (2 kb) of the fic16 gene. The first and second amplified fragments
were digested with XbaI/EcoRI and EcoRI/HindIII, respectively, and cloned
separately into pBlueScript II KS(+) (Stratagene, San Diego, CA) for
sequence verification. Two fragments with the correct sequences were
cloned into the HindIII site of the pUC118-aac(3)IV vector. The resulting
plasmid, pΔfic16, was introduced into S. ficellus to knockout the fic16 gene.
The polyethylene-glycol-assisted protoplast transformation of S. ficellus
JCM 4946 was performed as described previously19. Colonies that grew on
R2YE19 supplemented with 25 μg/ml apramycin were selected, and the
transformant was cultured in TSB19 medium for 2 d. Protoplasts were
prepared in YEME19 medium and regenerated on R2YE plates. Apramycin-
sensitive colonies from the regenerated colonies were selected. Knockout
was confirmed by PCR using fic16check-Fw and fic16check-Rv. The
resulting knockout mutant was named S. ficellus Δfic16. The S. ficellus
mutant lacking fic15 or fic22 was constructed in a similar manner using two
sets of primers.
Gene complementation of fic15, fic16, and fic22 in S. ficellus Δfic15, Δfic16,
and Δfic22.
The genetically complemented strains of S. ficellus fic15, fic16,
and S. ficellus fic22 were constructed as follows. Each gene was amplified
by PCR from the genome of S. ficellus using the primers listed in Table S1.
Primers were designed to amplify DNA regions, each containing the fic15,
fic16, or fic22 gene, with the upstream region including its ribosomal
binding site. The DNA fragment containing the target gene was cloned
downstream of the lacZ promoter in pSE10120 to give pSE101_fic15,
pSE101-fic16, and pSE101-fic22. S. ficellus fic15, Δfic16, and Δfic22 were
transformed with pSE101-fic15, pSE101-fic16, and pSE101-fic22,
respectively, according to a previously reported protocol19.
LC-MS analysis of the metabolites of the fic gene knockout mutants and
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genetically complemented strains.
The wild-type S. ficellus JCM 4946 and fic gene knockout mutants
were inoculated into 10 mL TSB medium (30 g L−1 tryptic soya broth) and
cultured with shaking (300 rpm) at 30°C for 3 d. The preculture (1 mL) was
transferred into 100 mL of ficellomycin production medium A (4.0% starch,
2.5% pharmamedia, 1.5% glucose, 2.0% molasses, and 0.8% CaCO3)1 with 1
g of XAD™7HP and further cultured with shaking (180 rpm) at 27°C for an
additional 2 or 3 d. The culture broth was centrifuged at 20,630 × g for 10
min and the resultant supernatant was diluted 10-fold with methanol. The
diluted sample was further centrifuged at 20,630 × g for 10 min. The
supernatant was applied to a high-resolution Triple TOF® 5600 system
(SCIEX, Tokyo, Japan) equipped with a UFLC Nexera system (Shimadzu,
Kyoto, Japan). Samples were separated by the ACQUITY UPLC® BEH
HILIC column, 130 Å, 1.7 µm, 2.1 × 50 mm (Waters). The LC conditions
were as follows: mobile phase A, H2O + 0.1% formic acid; mobile phase B,
acetonitrile + 0.1% formic acid; 95–40% B over 5 min, 40% B for 5 min, and
then 95% B for 5 min, at a flow rate of 0.4 ml min-1. MS analyses were
simultaneously performed using electrospray ionization in a positive mode.
MS/MS analyses were performed with a collision energy of 35 eV.
Purification of 2 from the culture broth of S. ficellus Δfic16.
A single colony of S. ficellus Δfic16 was inoculated into 10 mL TSB
medium (30 g L−1 tryptic soya broth) and incubated with shaking (300 rpm)
at 30°C for 3 d. The preculture (1 mL) was inoculated into 100 mL of
ficellomycin production medium B, modified medium A lacking molasses,
with 1 g of XAD and the incubated with shaking (180 rpm) at 27°C for 3 d.
The culture broth was centrifuged at 2,500 × g for 10 min and the
supernatant was collected. The supernatant was subjected to an activated
charcoal column. The flow-through and water-washed fractions were
combined. After the pH of the fractions was adjusted to approximately 10 by
the addition of 1 N NaOH, they were subjected to the anion exchange
column AG 1-X8 (Bio-Rad) in bicarbonate forms. The adsorbed compounds
were eluted with 0, 0.05, and 0.1 M of NaCl and fractions containing 2 were
combined and concentrated.
Feeding experiment of 2 into the culture of S. ficellus Δfic22.
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S. ficellus Δfic22 was cultured in a same manner as described above.
The solution (0.1 mL) containing 2 or deionized water was added to 10 ml of
K media21 with 1 g of XAD and cells were cultured at 30˚C for 2 d. The
supernatant of the culture broth was diluted 10-fold with methanol and
centrifuged at 20,630 × g for 10 min. The supernatant of the culture broth
was diluted with appropriate volumes of methanol and subjected to LC-MS
using the same analytical conditions described above.
Structural elucidation of 2 isolated from S. ficellus Δfic16.
2, which was partially purified in the same manner described above,
was derivatized with DNFB. An equal volume of methanol with 5% DNFB,
0.5 × volumes of DMSO, and 0.2 × volumes of 1 N NaOH were mixed, and
the resultant mixture was incubated at 60˚C for 1 h. The mixture was
subjected to vacuum distillation to remove methanol, and then applied to
Purif-aios (SHOKO Science, Kanagawa, Japan). The evaporated sample was
separated by the ODS column (50 µm, 60 mL) (SHOKO Science). The LC
conditions used were as follows: mobile phase A, H2O; mobile phase B,
isopropanol; 2% B in 1 min, 2–50% B over 20 min, at a flow rate of 20 ml
min-1. Column elutes were monitored at 341 and 254 nm. Fractions
containing 2 modified with DNP molecules (2-DNP) were combined. The
resultant sample was subjected to separation by the JASCO Extrema HPLC
system (JASCO, Tokyo, Japan) equipped with CAPCELL PAK MGII (10 mm
× 250 mm) (Shiseido, Tokyo, Japan). The LC conditions for the preparation
of 2-DNP were as follows: mobile phase A, H2O; mobile phase B, methanol;
30% B, at a flow rate of 5 ml min-1. UV absorbance at 341 and 254 nm was
monitored. Fractions containing 2-DNP were combined. After the removal of
acetonitrile or methanol in vacuo, 2-DNP of 6.7 mg was purified as the
yellow powder from 7.5 L of the culture broth.
NMR and optical rotation measurement.
The NMR spectra of 2-DNP were obtained using the ECA-600
spectrometer (JEOL, Tokyo, Japan). 1H NMR spectra and all 2D spectra
were acquired in 600 MHz, and 13C NMR spectra were acquired in 150 MHz.
2-DNP were dissolved in d6-DMSO and filled into a 5-mm NMR tube. All
experiments were performed at an ambient temperature.
Optical rotation was measured on a JASCO P-2100 polarimeter
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(JASCO, Tokyo, Japan).
Preparation of Fic13, Fic16, Fic36, and Fic15 from recombinant E. coli.
A DNA fragment, carrying the fic13, fic16, fic36, or fic15 gene, was
amplified by PCR with a single set of the primers Fic_Fw/Fic_Rv using the
genome DNA of S. ficellus as the template. The amplified fragment was
inserted into pBluescript II KS(+) using the BamHI and HindIII sites. The
inserted fragment was further subcloned into pHIS822 using the same
restriction sites to yield pHis8_Fic for the production of Fic enzymes with
the (His)8 tag at the N terminus. The resulting plasmid was introduced into
E. coli BL21-CodonPlus(DE3)-RIL. Recombinant E. coli cells were grown in
200 ml of 2 × YT media (1.6% Bacto Tryptone, 1.0% Bacto Yeast Extract,
and 0.5% NaCl) supplemented with 50 µg/ml of kanamycin (Km) and 30
µg/ml of chloramphenicol (Cm) at 37 ˚C for 3 h. Isopropyl -D-1-
thiogalactopyranoside (IPTG) was then added to give a final concentration
of 0.1 mM for induction and cells were further cultured at 25˚C overnight.
Cells were harvested by centrifugation and washed with buffer A (20 mM
Tris-HCl, pH 8.0, 150 mM NaCl). The resultant cells were disrupted using
an ultrasonic homogenizer and debris was removed by centrifugation at 4˚C
(34,700 × g, 20 min). The supernatant was passed through a Minisart
syringe filter (Sartorius, Göttingen, Germany) and applied to a Ni-NTA His
Bind® Resin (EMD Millipore, Billerica, MA) column. The column was
washed with buffer B (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 20 mM
imidazole) and bound proteins were eluted by buffer C (20 mM Tris-HCl, pH
8.0, 150 mM NaCl, and 500 mM imidazole). The eluate was concentrated
using the VIVASPIN 10,000 MWCO PES membrane (Sartorius). The
concentration of the protein solution was measured using Bio-Rad Protein
Assay Dye Regent Concentrate (Bio-Rad).
Activity assay of Fic13.
The reaction mixture containing 25 mM Tris-HCl, pH 8.0, 0.2 mM
NAD(P)H, an appropriate amount of 1, and 20 µg of Fic13 was prepared in a
total volume of 100 µl. The reaction mixture was incubated at 30˚C for 2 h
and reactions were quenched by adding 400 µl of methanol. The resultant
mixtures were centrifuged at 20,630 × g for 10 min. The supernatant was
applied to a high-resolution Triple TOF® 5600 system equipped with a
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UFLC Nexera system. Samples were separated by the ACQUITY UPLC®
BEH HILIC column, 130 Å, 1.7 µm, 2.1 × 50 mm. LC conditions were the
same as those described above.
Activity assay of Fic16.
The reaction mixture containing 50 mM HEPES-NaOH, pH 8.0, 0.02
mM PLP, 0.5 mM amino acids (ornithine, glutamate, glutamine, arginine,
lysine, or asparagine), an appropriate amount of 2, and 50 µg of Fic16 was
prepared in a total volume of 100 µl. The reaction mixture was incubated at
30˚C for 2 h and reactions were quenched by adding 400 µl of methanol. The
resultant mixtures were centrifuged at 20,630 × g for 10 min. A decrease in
2 was analyzed by LC-MS as described in the previous section.
Conversion of glutamate--semialdehyde into proline to confirm the Fic16
reaction.
The reaction mixture of Fic16 incubated at 30˚C for 2 h as described
above was treated with 20 mM NaBH4 and incubated at 30 ˚C for 10 min. A
portion of the supernatant (50 µl) was treated with DNFB as described
above and proline-DNP formation was analyzed by LC-MS. Samples were
separated by the CAPCELL PAK C18 IF 2.0 × 50 mm (Shiseido). The LC
conditions used were as follows: mobile phase A, H2O + 0.1% formic acid;
mobile phase B, acetonitrile + 0.1% formic acid; 2–98% B over 5 min, 98% B
for 5 min, and then 2% B for 5 min, at a flow rate of 0.4 ml min-1. MS
analyses were simultaneously performed using electrospray ionization in a
positive mode.
Assay for Fic36 activity through coupled reactions with Fic16.
The reaction mixture containing 50 mM Tris-HCl, pH 8.0, 0.02 mM
PLP, 0.5 mM ornithine, 5 mM arginine, an appropriate amount of 2, 20 µg of
Fic16, and 150 µg of Fic36 was prepared in a total volume of 100 µl. The
reaction mixture was incubated at 30 ˚C for 2 h. The reaction mixture was
centrifuged at 20,630 × g for 10 min. A portion of the supernatant (50 µl)
was treated with DNFB and the formation of 4-DNP was analyzed by LC-
MS as described above. MS/MS analyses were performed with a collision
energy of 45 eV.
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Ficellomycin synthesis from 1 using Fic13, Fic16, Fic36, and Fic15.
The reaction mixture containing 50 mM Tris-HCl, pH 8.0, 0.2 mM
NAD+, 0.02 mM PLP, 5 mM ATP, 5 mM MgSO4, 0.5 mM ornithine, 5 mM
arginine, 0.5 mM valine, an appropriate amount of 1, 20 µg of Fic13, 40 µg of
Fic16, 40 µg of Fic36, and 70 µg of Fic15 was prepared in a total volume of
100 µl. The reaction mixture was incubated at 30˚C for 2 h and then
centrifuged at 20,630 × g for 10 min. A portion of the supernatant (50 µl)
was treated DNFB as described above and ficellomycin synthesis was
analyzed by LC-MS in the same manner.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by the JSPS KAKENHI (24228001 and
17H06168). We are grateful to Drs. Hirosato TAKIKAWA and Yusuke
OGURA, Department of Applied Biological Chemistry, Graduate School
of Agricultural and Life Sciences, The University of Tokyo, for their
assistance to measure the optical rotation.
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Fig. 1. (a) Chemical structures of ficellomycin and vazabitide A. The 1-azabicyclo[3.1.0]hexane rings are
circled by blue ring and the guanidyl unit in ficellomycin is shown in yellow. (b) The fic gene cluster. The
DADH biosynthetic gene homologs are colored in gray and the amcp gene is shown in red. Four candidate
genes responsible for the ring modification and dipeptide formation are highlighted in orange and green. (c)
The final four reactions of the ficellomycin biosynthetic pathway identified in the present study. The
proposed chemical structures of the intermediates are shown with the relative chemistry in brackets.
177x134mm (300 x 300 DPI)

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Fig. 2. Metabolite analysis of S. ficellus mutants and genetically complemented strains. (a), (b) Extracted
+
ion count (EIC) chromatograms for ficellomycin (m/z 313.20 [M + H] ) obtained via an LC-MS analysis of
the culture extracts from S. ficellus mutants and genetically complemented strains. (c) EIC chromatograms
+
+
for 1 and 2 (m/z 173.09 [M + H] and m/z 171.08 [M + H] ) obtained via an LC-MS analysis of the culture
extracts from Δfic16. (d) Feeding experiment of 2 into S. ficellus Δfic22_pSE101. EIC chromatograms for
+
ficellomycin (m/z 313.20 [M + H] ) are shown.
169x132mm (300 x 300 DPI)

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Fig. 3. In vitro assay of Fic16. (a) Reaction of ornithine aminotransferase. (b) LC-MS analysis of the in vitro
+
reaction mixture of Fic16. Extracted ion count (EIC) chromatograms for 2 (m/z 171.09 [M+H] ). (c) Fic16
reaction and a scheme to convert P5C into proline-DNP. (d) Detection of proline generated by the
spontaneous cyclization of glutamate-γ-semialdehyde followed by reduction with NaBH . EIC chromatogram
4
+
for proline-DNP (m/z 282.07 [M+H] ) in LC-MS.
165x151mm (300 x 300 DPI)

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Fig. 4. LC-MS analysis of the in vitro reaction mixture of Fic13, Fic16, Fic36, and Fic15. (a) EIC
+
chromatograms for 4-DNP (m/z 380.13 [M + H] ). (b) EIC chromatograms for ficellomycin-DNP (m/z
+
479.20 [M + H] ).
147x62mm (300 x 300 DPI)