N-Phenylacetylation and Nonribosomal Peptide Synthetases with Substrate Promiscuity for Biosynthesis of Heptapeptide Variants, JBIR-78 and JBIR-95

Article abstract of DOI:10.1021/acschembio.7b00314

JBIR-78 (1) and JBIR-95 (2), both of which are heptapeptide derivatives isolated from Kibdelosporangium sp. AK-AA56, have the same amino acid sequences except for the second amino acid: phenylacetic acid (Paa)-l-Val-d-Asp (1)/d-cysteic acid (2)-l-Ala-(3S)-3-hydroxy-d-Leu-Gly-d-Ala-l-Phe. Heterologous expression of the biosynthetic gene cluster including genes encoding nonribosomal peptide synthetases (NRPS) and in vitro assays with recombinant Orf3, an l-cysteic acid synthase homologue, suggested the single A domain in module 2 activates both l-Asp and l-cysteic acid to yield 1 and 2, respectively, although the substrate specificities of the A domains of NRPSs are usually strict. Biosynthetic mechanism of introduction of N-terminal Paa was also investigated. Recombinant Orf1 and Orf2 similar to subunits of pyruvate dehydrogenase complex catalyzed the conversion of phenylpyruvate into phenylacetyl-CoA together with dihydrolipoyl dehydrogenase whose encoding gene is located outside of the gene cluster. Moreover, we showed that phenylacetyl-CoA was directly condensed with l-Val, which was tethered to a peptidyl carrier protein, at the first condensation domain in the NRPS.

Full text of DOI:10.1021/acschembio.7b00314

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Article
Unique N-phenylacetylation and NRPS with Substrate Promiscuity
for Biosynthesis of Heptapeptide Variants, JBIR-78 and JBIR-95
Kunpei Takeda, Kohei Kemmoku, Yasuharu Satoh, Yasushi Ogasawara, Kazuo Shin-ya, and Tohru Dairi
ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 15 May 2017
Downloaded from http://pubs.acs.org on May 15, 2017
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Unique N-phenylacetylation and NRPS with Substrate Promiscuity for Biosynthesis of
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Heptapeptide Variants, JBIR-78 and JBIR-95
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Kunpei Takeda†, Kohei Kemmoku†, Yasuharu Satoh†, Yasushi Ogasawara†, Kazuo
Shinꢀya‡, and Tohru Dairi†*
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†Graduate School of Engineering, Hokkaido University, N13ꢀW8, Kitaꢀku, Sapporo,
Hokkaido 060ꢀ8628, Japan
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‡Biomedicinal Information Research Center (BIRC), National Institute of Advanced
Industrial Science and Technology (AIST), 2ꢀ4ꢀ7 Aomi, Kotoꢀku, Tokyo 135ꢀ0064, Japan
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*Corresponding author. Eꢀmail: dairi@eng.hokudai.ac.jp, Tel: +81ꢀ11ꢀ706ꢀ7815
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ABSTRUCT
JBIRꢀ78 (1) and JBIRꢀ95 (2), both of which are heptapeptide derivatives isolated from
Kibdelosporangium sp. AKꢀAA56, have the same amino acid sequences except for the
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second amino acid; phenylacetic acid (Paa)–
(3S)ꢀ3ꢀhydroxyꢀ ꢀLeu–Gly– ꢀAla– ꢀPhe. Heterologous expression of the biosynthetic
gene cluster including genes encoding nonꢀribosomal peptide synthetases (NRPS) and in
LꢀVal–DꢀAsp (1)/Dꢀcysteic acid (2)–LꢀAla–
D
D
L
vitro assays with recombinant Orf3,
Aꢀdomain in module 2 activates both
L
ꢀcysteic acid synthase homolog, suggested the single
L
ꢀAsp and ꢀcysteic acid to yield 1 and 2, respectively,
L
although the substrate specificities of the Aꢀdomains of NRPSs are usually strict.
Biosynthetic mechanism of introduction of Nꢀterminal Paa was also investigated.
Recombinant Orf1 and Orf2 similar to subunits of pyruvate dehydrogenase complex
catalyzed the conversion of phenylpyruvate into phenylacetylꢀCoA together with
dihydrolipoyl dehydrogenase whose encoding gene is located at outside of the gene cluster.
Moreover, we showed that phenylacetylꢀCoA was directly condensed with LꢀVal, which
was tethered to a peptidyl carrier protein, at the first condensation domain in the NRPS.
INTRODUCTION
Peptide antibiotics are a large family of natural products, including clinically important
pharmaceuticals. In addition to isolated natural products, genome databases have revealed
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the presence of many biosynthetic genes for this category of compounds. These peptides
can be categorized by their biosynthetic machineries as either ribosomally synthesized and
postꢀtranslationally modified peptides (RiPPs) or nonribosomal peptides (NRPs).1,2 In the
latter case, nonribosomal peptide synthetases (NRPSs) are representative biosynthetic
enzymes. NRPSs are modular type large enzymes and a typical module consists of an
adenylation (A) domain, a peptidyl carrier protein (PCP) domain and a condensation (C)
domain. The Aꢀdomain activates the carboxylic acid of an amino acid with ATP and
determines the amino acid to be selected and activated. Unlike ribosomes, NRPSs can
utilize nonproteinogenic amino acids as building blocks. The Cꢀdomain catalyzes the
peptide bond formation between the upstream peptidyl PCP and downstream amino acyl
PCP. Besides NRPSs, amino acid ligases with ATPꢀgrasp domains (ATPꢀgraspꢀligases),
tRNAꢀdependent aminoacyl transferase/cyclodipeptide synthases, standꢀalone adenylation
(A) domains and acylꢀCoA synthetases (acylꢀAMPꢀligases) are also involved in NRP
biosynthesis.3–6
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1 and 2, both of which are heptapeptide derivatives isolated from Kibdelosporangium
sp. AKꢀAA56, have the same amino acid sequence except for the second amino acid
(Figure 1).7 We are interested in their biosynthetic machineries from the following points of
view. One is the difference of the second amino acid. Considering the presence of Dꢀamino
acids in 1 and 2, they could be biosynthesized by a NRPS. However, the substrate
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specificities of the Aꢀdomains of NRPSs are strict and one Aꢀdomain usually activates one
amino acid. Therefore, we hypothesized three possibilities: (i) the producer possesses
separate NRPSs for 1 and 2 biosynthesis; (ii) one NRPS biosynthesizes 1, which is then
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converted to 2 by tailoring reactions; (iii)
Lꢀcysteic acid is independently biosynthesized
and a single Aꢀdomain is exceptionally able to activate both
L
ꢀAsp and ꢀcysteic acid. The
L
other interesting feature is the presence of the Nꢀterminal phenylacetate. To date, a dozen
phenylacetylated natural polyketides and a few Nꢀterminal phenylacetylated peptides such
as penicillin G, microcystin,8 and JBIRꢀ969 have been isolated. However, reports on the
detailed phenylacetylation mechanism are limited. In the case of penicillin G, isopenicillin
N acyltransferase was shown to convert isopenicillin N to penicillin G.10 In microcystin
biosynthesis, phenylpropanoids were suggested to be loaded onto the PCPꢀdomain rather
than phenylacetate by ATP–PPi exchange assays and mass spectrometry, although the
mechanism by which one carbon is excised from the starter substrate remains unknown.8
Recently, Fu et al. reported that a pyruvate dehydrogenaseꢀlike protein complex catalyzed
the decarboxylation of phenylpyruvate to form a phenylacetylꢀSꢀacyl carrier protein, which
would be utilized in a subsequent polyketide biosynthetic assembly line, in ripostatin
biosynthesis.11 In this study, we investigated the phenylacetylation mechanism in JBIRꢀ78
(1) and JBIRꢀ95 (2) biosynthesis.
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RESULTS AND DISCUSSION
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Identification of 1 and 2 biosynthetic genes
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To identify the 1 and 2 biosynthetic gene cluster(s), we generated a draft genome
sequence for the producer and searched for orfs large enough to encode the seven
Aꢀdomains essential for substrate activation in heptapeptide biosynthesis. From a BLAST
search, we identified one candidate gene cluster (Figure 2, Table 1). The gene cluster
consisted of eight orfs containing three orfs (orf4 to orf6) encoding NRPSs. Orf4 and Orf5
had the same domain architectures; CꢀAꢀPCPꢀCꢀAꢀPCPꢀepimerization domain (Eꢀdomain).
Orf6 was composed of CꢀAꢀPCPꢀCꢀAꢀPCPꢀEꢀCꢀAꢀPCPꢀthioesterase (TE) domains. The
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positions of the Eꢀdomains perfectly matched the locations of the Dꢀamino acids in 1 and 2.
Moreover, the substrate specificities of each of the Aꢀdomains predicted by
NRPSpredictor2 (http://nrps.informatik.uniꢀtuebingen.de/Controller?cmd=SubmitJob) were
roughly identical to the amino acid sequences of 1 (Supplementary Table 1, 2, and 3). Two
orfs (orf1 and orf2), which encode two of the three component enzymes of pyruvate
dehydrogenase complex, were found in the region upstream from the NRPS genes. Orf1
was composed of pyruvate dehydrogenase (EC 1.2.4.1, E1β unit) and dihydrolipoyl
transacetylase (EC 2.3.1.12, E2 unit). Orf2 was similar to the E1α unit of pyruvate
dehydrogenase. Although no homologs of dihydrolipoyl dehydrogenase (EC 1.8.1.4, E3
unit) existed in the gene cluster, we identified one homolog outside of the gene cluster.
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Considering the reaction catalyzed by the pyruvate dehydrogenase complex, Orf1, Orf2,
and the dihydrolipoyl dehydrogenase homolog perhaps catalyze the conversion of
phenylpyruvate into phenylacetylꢀCoA, which would be used for Nꢀterminal
phenylacetylation.
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Orf3 showed similarity to the MA3297 protein, which is a pyridoxal 5ʹꢀphosphate
dependent enzyme that catalyzes a βꢀreplacement reaction converting
sulfite into
ꢀcysteic acid and inorganic phosphate.12 Therefore, Orf3 is plausibly
responsible for the biosynthesis of 2.
Orf8 was similar to cytochrome P450 and presumably participates in the
hydroxylation of Leu to form 3ꢀhydroxyꢀ ꢀLeu. Additionally, Orf7 was similar to MbtH, an
Lꢀphosphoserine and
L
D
integral component of NRPSs that is essential for amino acid activation.13 The identified
gene cluster was the sole candidate and no paralogs of the identified genes existed in the
draft genome database. Therefore, the production of both 1 and 2 was suggested to be
governed by this gene cluster.
Heterologous expression of the gene cluster
Next, we performed a heterologous expression experiment to examine whether the
gene cluster identified in this study contained all the genes responsible for 1 and 2
biosynthesis. A cosmid library of the producer was constructed with a shuttle cosmid vector,
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pOJ446.14 Positive cosmids carrying the gene cluster were obtained by PCR screening with
appropriate primers and the cosmid pC35 was introduced into Streptomyces lividans TK23.
After cultivation of the transformants, the products were analyzed by LCꢀESIꢀMS. As
shown in Figure 3 and Supplementary Figure 1, the production of both 1 and 2 was
suggested. Although the cosmid had one additional gene (Figure 2) downstream of orf8
gene, this gene was suggested to have no relation to 1/2 biosynthesis. Taken together, our
results suggested that both products were biosynthesized by this gene cluster alone and that
the Aꢀdomain responsible for activation of the second substrate exceptionally activated both
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LꢀAsp and Lꢀcysteic acid.
Characterization of Orf1 and Orf2
To examine whether Orf1 and Orf2 catalyzed the conversion of phenylpyruvate to
phenylacetylꢀCoA, an in vitro assay with recombinant enzymes was carried out. A DNA
fragment carrying both the orf1 and orf2 genes was inserted into the pET28a vector so that
only Orf2 was expressed as a Hisꢀtagged recombinant enzyme because Orf1 was
coꢀpurified with Orf2 by NiꢀNTA column chromatography (Supplementary Figure 2). A
recombinant enzyme of the putative dihydrolipoyl dehydrogenase (E3 unit) was also
prepared as a Hisꢀtagged recombinant enzyme (Supplementary Figure 2). After the
recombinant enzymes were expressed and purified, they were mixed and incubated with
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phenylpyruvate and CoA in the presence of known essential coꢀfactors in the pyruvate
dehydrogenase reaction. A new product whose molecular mass was consistent with that of
phenylacetylꢀCoA was specifically detected by LCꢀESIꢀMS analysis (Figure 4). Its high
resolution molecular mass also agreed with that of phenylacetylꢀCoA ([M+H]+ calcd. for
C29H43N7O17P3S+, 886.16435: found 886.16534).
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Characterization of Orf3
Orf3 showed similarity to the pyridoxal 5ʹꢀphosphate dependent
Lꢀcysteic acid
synthase. To confirm its expected activity, we tried an in vitro assay with recombinant
enzymes. Although we could express Orf3 recombinant enzymes with several expression
vectors such as pET vectors, pMALꢀc5X and pColdꢀTF, all recombinant enzymes formed
inclusion bodies. Therefore, we carried out heterologous expression of the gene cluster
without the orf3 gene. We constructed a cosmid lacking orf3 gene (pC35∆orf3) from pC35.
Unexpectedly, the transformant harboring pC35∆orf3 did not lose 2 productivity and still
produced a small amount of 2 concomitant with enhanced production of 1 (Figure 3 and
Supplementary Figure 1). A possible reason for this is that
by an alternative enzyme such as ꢀserine ammoniaꢀlyase (EC 4.3.1.17) that catalyzes the
elimination of water from ꢀserine to form ꢀdehydroalanine, to which sulfite attacks.
Lꢀcysteic acid might be supplied
L
L
L
Indeed, 2 productivity was dramatically recovered when Lꢀcysteic acid was added into the
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medium (Figure 3 and Supplementary Figure 1), suggesting that the Orf3 product supplies
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L
ꢀcysteic acid. Furthermore, the productivity of 1 was extremely reduced by the addition of
ꢀcysteic acid, suggesting that ꢀcysteic acid competed with ꢀAsp in the incorporation into
the peptide.
Previously, a few Aꢀdomains in NRPSs, which activated two different amino acids,
were reported. Pelgipeptin A/C and pelgipeptin B/D possess ꢀVal and ꢀIle at the second
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L
L
L
L
L
position, respectively. The substrate selectivity of Aꢀdomains (PlpE A1) responsible for
activation of the second amino acid was examined by in vitro experiments and PlpE A1 was
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shown to activate both
LꢀVal and
L
ꢀIle. Cꢀterminal diversity of lichenysins (
LꢀIle or
L
ꢀLeu) was also derived by the substrate selectivity of Aꢀdomains (LicCꢀA).16 In both cases,
the Aꢀdomains recognized the structurally and chemically similar amino acids. For the
same reason, the Aꢀdomain in module 2 might accept
which are acidic amino acids and have similar sizes.
LꢀAsp and Lꢀcysteic acid, both of
We then tried denaturation and refolding of the inclusion body to obtain the active
form of the recombinant enzyme. After extensive trials to find the best conditions, we
successfully obtained active enzymes (Supplementary Figure 3). The refolded enzyme was
incubated with
Lꢀphosphoserine and sodium sulfite for 15 min at 30°C and the reaction
products were analyzed by LCꢀESIꢀMS. As shown in Figure 5, a specific peak, which was
eluted at the same retention time and had the same molecular mass as authentic Lꢀcysteic
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acid, was detected.
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Characterization of Orf4
Through detailed analysis of each of the domains in Orf4, we found that a
PCPꢀdomain to anchor phenylpyruvate was absent. Moreover, Orf1 and Orf2 catalyzed the
formation of phenylacetylꢀCoA as mentioned above. These results suggested that
phenylacetylꢀCoA is directly condensed with LꢀVal at the first Cꢀdomain in module 1 of
Orf4 in a similar manner to that of surfactin and amphiꢀenterobactin siderophore
biosynthesis.17,18 To examine this possibility, a truncated recombinant Orf4 (module 1) was
prepared and used for in vitro assay. We first tried to prepare a recombinant enzyme
possessing the first module with the Cꢀ, Aꢀ, and PCPꢀdomains. However, we were unable to
obtain recombinant proteins. We then prepared recombinant enzymes divided into
CꢀAꢀdomains and the PCPꢀdomain. In this case, both recombinant enzymes were expressed
as soluble forms (Supplementary Figure 4).
The purified recombinants were incubated with
Lꢀvaline and phenylacetylꢀCoA,
which was prepared by the enzyme reaction described above, in the presence of ATP and
MgCl2. After the enzymes were precipitated by adding acetone, the thioester bonds were
hydrolyzed with 0.1 M KOH. The resulting sample was analyzed by LCꢀESIꢀMS and
compared with authentic phenylacetyl valine prepared by chemical synthesis. As shown in
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Figure 6, a specific peak with the same retention time and molecular mass as those of the
authentic standard was clearly detected in the sample, showing that phenylacetylꢀCoA was
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directly condensed with
PCP in module 1.
Lꢀvaline, which was activated at the Aꢀdomain and transferred to
Recently, a similar mechanism for the formation of phenylacetate from phenylpyruvate
by a pyruvate dehydrogenaseꢀlike protein complex in ripostatin biosynthesis was reported.11
In this case, however, the phenylacetyl group of Sꢀphenylacetyldihydrolipoamide was
directly transferred to an acyl carrier protein to form a phenylacetylꢀSꢀacyl carrier protein
without the formation of phenylacetylꢀCoA.
In summary, we studied the biosynthetic machinery of 1 and 2, which are both
heptapeptide derivatives and have the same amino acid sequence except for the second
amino acid. Through heterologous expression of the biosynthetic gene cluster and in vitro
assay with the recombinant Orf3, the single Aꢀdomain in module 2 was suggested to
activate both
LꢀAsp and
Lꢀcysteic acid. We also showed that Nꢀterminal phenylacetylation
was catalyzed by the first Cꢀdomain in module 1 with
LꢀValꢀtethered PCP and
phenylacetylꢀCoA supplied by a phenylpyruvate dehydrogenase complex.
REFERENCES
(1) Fischbach, M. A., and Walsh, C. T. (2006) Assemblyꢀline Enzymology for Polyketide
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and Nonribosomal Peptide Antibiotics: Logic, Machinery, and Mechanisms. Chem.
Rev. 106, 3468–3496.
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(2) Ortega, M. A., and van der Donk, W. A. (2016) New Insights into the Biosynthetic
Logic of Ribosomally Synthesized and Postꢀtranslationally Modified Peptide Natural
Products. Cell. Chem. Biol. 23, 31–44.
(3) Giessen, T. W., and Marahiel, M. A. (2012) Ribosomeꢀindependent Biosynthesis of
Biologically Active Peptides: Application of Synthetic Biology to Generate Structural
Diversity. FEBS Lett. 586, 2065–2075.
(4) Kadi, N., OvesꢀCostales, D., BoronaꢀGomez, F., and Challis, G. L. (2007) A New
Family of ATPꢀdependent Oligomerizationꢀmacrocyclization Biocatalysts. Nat. Chem.
Biol. 3, 652–656.
(5) Maruyama, C., Toyoda, J., Kato, Y., Izumikawa, W., Takagi, M., Shinꢀya, K., Katano,
H., Utagawa, T., and Hamano, Y. (2012) A Standꢀalone Adenylation Domain Forms
Amide Bonds in Streptothricin Biosynthesis. Nat. Chem. Biol. 8, 791–797.
(6) Noike, M., Matsui, T., Ooya, K., Sasaki, I., Ohtaki, S., Hamano, Y., Maruyama, C.,
Ishikawa, J., Satoh, Y., Ito, H., Morita, H., and Dairi, T. (2015) A Peptide Ligase and
the Ribosome Cooperate to Synthesize the Peptide pheganomycin. Nat. Chem. Biol.
11, 71–76.
(7) Izumikawa, M., Takagi, M., and Shinꢀya, K. (2012) JBIRꢀ78 and JBIRꢀ95:
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Phenylacetylated Peptides Isolated from Kibdelosporangium sp. AKꢀAA56. J. Nat.
Prod. 75, 280–284.
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(8) Hicks, L. M., Moffitt, M. C., Beer, L. L., Moore, B. S., and Kelleher, N. L. (2006)
Structural Characterization of in vitro and in vivo Intermediates on the Loading
Module of Microcystin Synthetase. ACS Chem. Biol. 1, 93–102.
(9) Ueda, J., Izumilawa, M., Kozone, I., Yamamura, H., Hayakawa, M., Takagi, M., and
Shinꢀya, K. (2011) A Phenylacetylated Peptide, JBIRꢀ96, Isolated from Streptomyces
sp. RI051ꢀSDHV6. J. Nat. Prod. 74, 1344–1347.
(10) Tobin, M. B., Fleming. M. D., Skatrud, P. L., and Miller, J. R. (1990) Molecular
Characterization of the Acylꢀcoenzyme A: Isopenicillin N Acyltransferase Gene
(penDE) from Penicillium chrysogenum and Aspergillus nidulans and Activity of
Recombinant Enzyme in Escherichia coli. J. Bacteriol. 172, 5908–5914.
(11) Fu, C., Auerbach, D., Li,Y., Scheid, U., Luxenburger, E., Garcia, R., Irschik, H., and
Müller, R. (2017) Solving the Puzzle of Oneꢀcarbon Loss in Ripostatin Biosynthesis.
Angew. Chem., Int. Ed. 56, 2192–2197.
(12) Graham, D. E., Taylor, S. M., Wolf, R. Z., and Namboori, S. C. (2009) Convergent
Evolution of Coenzyme M Biosynthesis in the Methanosarcinales: Cysteate Synthase
Evolved from an Ancestral Threonine Synthase. Biochem. J. 424, 467–478.
(13) Felnagle, E. A., Barkei, J. J., Park, H., Podevels, A. M., McMahon, M. D., Drott, D.
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W., and Thomas, M. G. (2010) MbtHꢀlike Proteins as Integral Components of
Bacterial Nonribosomal Peptide Synthetases. Biochemistry 49, 8815–8817.
(14) Vara, J., LewandowskaꢀSkarbek, M., Wang, Y. G., Donadio, S., and Hutchinson, C. R.
(1989) Cloning of Genes Governing the Deoxysugar Portion of the Erythromycin
Biosynthesis Pathway in Saccharopolyspora erythraea (Streptomyces erythreus). J.
Bacteriol. 171, 5872–5881.
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(15) Qian, C. D., Liu, T. Z., Zhou, S. L., Ding, R., Zhao, W. P., Li, O., and Wu, X. C.
(2012) Identification and Functional Analysis of Gene Cluster Involvement in
Biosynthesis of the Cyclic Lipopeptide Antibiotic Pelgipeptin Produced by
Paenibacillus elgii. BMC Microbiology, 12, 197.
(16) Konz, D., Doekel, S., and Marahiel, M. A. (1999) Molecular and Biochemical
Characterization of the Protein Template Controlling Biosynthesis of the Lipopeptide
Lichenysin. J. Bacteriol. 181, 133–140.
(17) Kraas, F. I., Helmetag, V., Wittmann, M., Strieker, M., and Marahiel, M. A. (2010)
Functional Dissection of Surfactin Synthetase Initiation Module Reveals Insights into
the Mechanism of Lipoinitiation. Chem. Biol. 17, 872–880.
(18) Zane, H. K., Naka, H., Rosconi, F., Snady, M., Haygoogd, M. G., and Butler, A.
(2014) Biosynthesis of Amphiꢀenterobactin Siderophores by Vibrio harveyi
BAAꢀ1116: Identification of a Bifunctional Nonribosomal Peptide Synthetase
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Condensation Domain. J. Am. Chem. Soc. 136, 5615–5618.
(19) Dairi, T., Nakano, T., Aisaka, K., Katsumata, R., and Hasegawa, M. (1995) Cloning
and Nucleotide Sequence of the Gene Responsible for Chlorination of Tetracycline.
Biosci. Biotech. Biochem. 59, 1099–1106.
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(20) Datsenko, K. A., and Wanner, B. L. (2000) Oneꢀstep Inactivation of Chromosomal
Genes in Escherichia coli Kꢀ12 Using PCR Products. Proc. Natl. Acad. Sci. U. S. A.
97, 6640–6645.
(21) Quadri, L. E., Weinreb, P. H., Lei, M., Nakano, M. M., Zuber, P., and Walsh, C. T.
(1998) Characterization of Sfp, a Bacillus subtilis Phosphopantetheinyl Transferase for
Peptidyl Carrier Protein Domains in Peptide Synthetases. Biochemistry 37, 1585–
1595.
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METHODS
General
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All chemicals were purchased from SigmaꢀAldrich Japan K.K. (Japan), Tokyo Chemical
Industry Co. Ltd. (Japan), or Wako Pure Chemical Industry (Japan). Oligonucleotides were
obtained from FASMAC Co. Ltd. (Japan). Enzymes, molecular weight standards and kits
for DNA manipulation were purchased from Takara Bio Inc. (Japan) or New England
Biolabs Japan Inc. (Japan). PCR reactions were carried out using a GeneAmp PCR System
9700 thermal cycler (Thermo Fisher Scientific Inc.) with Tks Gflex DNA polymerase
(Takara Bio). NMR spectra were obtained using a JEOL ECSꢀ400 spectrometer (Japan).
Chemical synthesis of phenylacetyl valine
To a stirred solution of 1.03 g (8.8 mmol) of Lꢀvaline in 10 mL (20 mmol) of 2 M aq.
NaOH at 0°C was added dropwise 1.05 mL (8 mmol) of phenylacetyl chloride. The
reaction mixture was allowed to warm to room temperature and stirred. After 20 h of
reaction, water (30 mL) was added and the aqueous layer containing the product was
washed with Et2O (50 mL) twice. The water layer was then acidified with 1 M HCl to
obtain a white precipitate. The white crystalline product was washed thoroughly with water
1
and then with Et2O (606 mg, 29%). H NMR (400 MHz, DMSOꢀd6) δ 8.24 (d, J = 8.6 Hz,
1H), 7.32–7.09 (m, 5H), 4.15 (dd, J = 8.6, 5.8 Hz, 1H), 3.56 (d, J = 13.8 Hz, 1H), 3.48 (d, J
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= 13.8 Hz, 1H), 2.05 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H). 13C NMR
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(100 MHz, DMSOꢀd6) δ 173.1, 170.4, 136.6, 129.0, 128.1, 126.3, 57.1, 41.9, 29.9, 19.1,
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18.0.
Draft genome sequences
Draft sequences of the Kibdelosporangium sp. AKꢀAA56 genome were determined by a
commercial company (Hokkaido System Science, Japan) using an Illumina HiSeq platform
(Illumina). A genomic DNA library (350 bp insert) was constructed with the TruSeq Nano
DNA LT Sample Prep Kit (Illumina). Paired end data (2 × 100 bp) were assembled using
Velvet (version 1.0.18). Gene prediction and annotation was carried out using Microbial
Genome Annotation Pipeline (MiGAP, http://www.migap.org/).
Cloning and heterologous expression of the gene cluster
To clone the gene cluster, a cosmid library of the producer was constructed with the shuttle
cosmid vector pOJ446.14 Genomic DNA of Kibdelosporangium sp. was partially digested
with Sau3AI and ligated with the pOJ446 vector digested with BamHI and HpaI and treated
bacterial alkaline phosphatase. This was packaged using Gigapack III XL Packaging
Extract (Agilent Technologies Inc.) and transfected to the E. coli XL1ꢀBlue MRF′ strain
(∆(mcrA)183 ∆(mcrCBꢀhsdSMRꢀmrr)173 endA1 supE44 thiꢀ1 recA1 gyrA96 relA1 lac
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[F′proAB lacIqZ∆M15 Tn10 (Tetr)]; Agilent Technologies). Positive cosmids carrying the
gene cluster were screened by PCR based on the amplification of both the orf1 and 8 genes
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with
the
primers
5ʹꢀGGCCGCGACGTGGGTGAGGAAGTTGꢀ3ʹ
and
and
and
5ʹꢀCGTTCCCGATCTCGGTGAAGGACTTGCTGAGꢀ3ʹ
5ʹꢀCATGGGCGAGTAGCGGAACGAGAGGAACAAGꢀ3ʹ
5ʹꢀGGGCCGTAGGCGAATGTCAGGTGTTTGTTCꢀ3ʹ for orf8.
for
orf1,
The obtained cosmid, pC35, was introduced into S. lividans TK23 and then the
transformants were cultured in 200 mL flasks with baffles containing 30 mL of SK#2
medium19 containing thiostrepton (10 ꢁg mL−1) for 4 days at 30°C with agitation (200 rpm).
The whole culture broths were directly analyzed by LCꢀESIꢀMS as follows: Waters
ACQUITY UPLC system equipped with an SQ Detector2 and ACQUITY PDA Detector
(Japan); Mightysil RPꢀ18GP Aqua column (150 mm L × 2.1 mm ID, 3 µm, KANTO
CHEMICAL Co., Inc., Japan); flow rate, 0.2 mL min−1; temperature, 40°C; mobile phase A,
water containing 0.1% formic acid, mobile phase B, methanol containing 0.1% formic acid;
gradient conditions, 15% B, 0–2 min; 15–85% B, 2–40 min; detection, ESIꢀnegative mode;
injection volume, 10 µL.
pC35∆orf3, in which the orf3 gene was inꢀframe deleted, was constructed using
Red/ET recombination.20 In brief, DNA fragments containing a Kmꢀresistance gene cassette
(Gene Bridges GmbH, Germany) flanked with FRT sites and 50ꢀbp homologous arms,
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whose sequences were identical to the target regions, were amplified by PCR with the
primers
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5ʹꢀCCTGGTGTGGTGTGAATAGACCGCAGTCACGAAGGAGGTATGACCGGATGGA
ATTAACCCTCACTAAAGGGCGGCꢀ3ʹ
and
5ʹꢀCACCTGCCGCCTGTCCCGGGTGAGCTGTCATGTCACGCCCCACTCGGGGTAAT
ACGACTCACTATAGGGCTCGꢀ3ʹ. The amplified DNA fragments were used to transform
E. coli XL1ꢀBlue MRF′ harboring the pC35 cosmid and pRedET plasmid. Geneꢀdisruption
in Kmꢀresistant colonies was checked by PCR using two primers,
5ʹꢀGCGTAATGCGAGGTTGGGTCTATCGAAGGꢀ3ʹ
and
5ʹꢀCGCATCGACTGGGCCGTGGATCTCꢀ3ʹ. The selection marker in the obtained cosmid
was removed with FLPꢀrecombinase and the deletion was confirmed by PCR with the
primers used to check gene disruption as described above.
Preparation and enzyme assay of recombinant enzymes of Orf1, Orf2, and
dihydrolipoyl dehydrogenase
A DNA fragment containing both the orf1 and 2 genes was amplified by PCR using
genomic DNA of Kibdelosporangium sp. as a template and the following primers;
5ʹꢀCCTATCGGCATATGGTGCTTGGCCGCCGATTCGACꢀ3ʹ
and
5ʹꢀATATAAGCTTCCGGCTACCCGACCATGATCAGACTGTCꢀ3ʹ. To amplify the
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dihydrolipoyl dehydrogenase gene of Kibdelosporangium sp., the primers
5ʹꢀAAGTGACCATATGGCCACAGTTGACGCGCGCGTAꢀ3ʹ
and
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5ʹꢀTATAAGCTTGGCTGACTTGGTGCCGACGGTCAGꢀ3ʹ were used. Restriction sites
(underlined) were introduced into the Nꢀ and Cꢀterminal regions. The PCR products were
cloned into the NdeIꢀHindIII sites of the pET28a vector (Merck KGaA, Germany). Each of
−
the obtained plasmids was introduced into E. coli BL21(DE3) (F−, dcm, ompT, hsdS(rB
−
mB ), gal, λ(DE3); NIPPON GENE CO., LTD, Japan). A liquid culture of each transformant
in LB supplied with kanamycin (30 µg mL−1) was induced by adding 0.5 mM IPTG when
the optical density at 600 nm reached about 0.6. The cultivation was continued for an
additional 16 h at 20°C. Purification of each recombinant protein was carried out as follows.
After the cells were disrupted with an ultrasonic disruptor (TOMY, UDꢀ200), the Hisꢀtag
fused proteins were purified with NiꢀNTA column chromatography (QIAGEN K.K., Japan)
and their purities were analyzed by SDSꢀPAGE on 10% gels (Supplementary Figure 2). The
proteins were visualized by Coomassie brilliant blue staining and the protein concentrations
were determined by the Bradford method with bovine serum albumin as a standard.
Coꢀpurified recombinant Orf1 (approximately 0.42 µM) and Orf2 (approximately 2.3
µM), and purified recombinant dihydrolipoyl dehydrogenase (1.9 µM) were incubated with
CoA (1.0 mM), sodium phenylpyruvate (2.0 mM), MgCl2 (1.0 mM), thiamine
pyrophosphate (0.2 mM), NAD+ (2.5 mM), and dithiothreitol (0.6 mM) in potassium
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phosphate buffer (50 mM, pH 8.0) at 30°C for 16 h. The reaction product was directly
analyzed by LCꢀESIꢀMS as follows: Waters ACQUITY UPLC system equipped with an SQ
Detector2 and ACQUITY PDA Detector; XBridge BEH C8 column (150 mm L × 2.1 mm
ID, 2.5 µm, Waters); flow rate, 0.2 mL min−1; temperature, 35°C; mobile phase A, 100 mM
NH4COOH containing 5 vol% methanol, mobile phase B, 100 mM NH4COOH containing
50 vol% methanol; gradient conditions, 10% B, 0–5 min; 10–100% B, 5–40 min; detection,
ESIꢀnegative mode; injection volume, 10 µL. The fractionated product was also analyzed
by high resolutionꢀESIꢀFTꢀMS (Exactive, Thermo Fisher Scientific Inc.).
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Preparation and assay of recombinant
L
-cysteic acid synthase (Orf3) enzyme
ꢀcysteic acid synthase (MALꢀCS), the primer
To express maltose binding proteinꢀfused
L
pair pMal_CS_Fw: 5′ꢀGTATGACCATATGTGCGCTCGGCGGCATTACTCGATCꢀ3′ and
pMal_CS_Rv: 5′ꢀAGATAAGCTTCACGCCCCACTCGGGGTGGCTACGTAGꢀ3′ was
used. The PCR product was cloned into the NdeIꢀHindIII site of pMalꢀc5X (New England
Biolabs) to construct pMalꢀCS. Culture and purification conditions were the same as those
described above except for the use of amylose affinity column chromatography (New
England Biolabs) for purification. The purified recombinant Orf3 was treated with Factor
Xa protease (New England Biolabs) according to the manufacturer’s protocol to remove
MBP. To eliminate the MBP released, the reaction mixture was applied to an amylose
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affinity column again. Then, the flowꢀthrough fraction containing the enzyme was mixed
with 10 volumes of Tris·HCl buffer (20 mM, pH 8.0) containing 6 M urea and DTT (20
mM), and incubated at 37°C for 2 h. The solution was diluted ten times with refolding
buffer; Tris·HCl buffer (50 mM, pH 8.0) containing KCl (1 M), glycerol (20 %), MgCl2 (20
mM), and PLP (10 µM), and stirred at 4°C for 16 h. The enzyme solution was concentrated
to 2 mg mL−1 with Amicon Ultra filter units (Merck).
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The refolded recombinant Orf3 enzyme (8.8 µM) was incubated with PLP (10 µM),
sodium sulfite (5 mM), OꢀphosphoꢀLꢀserine (10 mM), and KCl (100 mM) in Tris·HCl
buffer (100 mM, pH 7.0) at 30°C for 15 min. The reaction product was directly analyzed by
LCꢀESIꢀMS as follows: Waters ACQUITY UPLC system equipped with an SQ Detector2
and ACQUITY PDA Detector; Scherzo SMꢀC18 column (150 mm L × 2 mm ID, 3 µm,
Imtakt Co., Japan); flow rate, 0.2 mL min−1; temperature, 40°C; mobile phase A, water
containing 0.1% formic acid, mobile phase B, acetonitrile containing 0.1% formic acid;
10% B isocratic conditions; detection, ESIꢀpositive mode; injection volume, 10 µL.
Preparation and assay of recombinant Orf4 enzyme
The first CꢀAꢀdomains and PCPꢀdomain in module 1 of Orf4 were expressed separately.
The
former
was
amplified
by
PCR
with
two
primers,
and
5ʹꢀAAAAAACATATGGACGCGGTCGCGGCCCAꢀ3ʹ
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5ʹꢀTGGCGTAAGCTTTCAGCGGGAAGGACTGGTꢀ3ʹ. The latter was amplified with the
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primers
5ʹꢀAAAAAACATATGCCCGTCACCAGTCCTTꢀ3ʹ
and
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5ʹꢀAAAAAGCTTGCCGACCAGCAGCGCGATꢀ3ʹ. Each amplified fragment was cloned
into the NdeIꢀHindIII site of pET28a to construct the plasmids pOrf4ꢀCA and pOrf4ꢀPCP, to
express recombinant enzymes fused with an Nꢀterminal Hisꢀtag. For activation of the
PCPꢀdomain, the plasmid (pACYCꢀSfp) expressing phosphopantetheinyl transferase of
Bacillus subtilis (Sfp) was constructed by digesting the pETꢀSfp plasmid21 with NdeI and
XhoI and then cloning the sfp fragment into the NdeIꢀXhoI site of pACYCDuetꢀ1 (Merck).
Each plasmid was used to transform E. coli BL21(DE3) for protein overexpression.
Purification procedures were the same as those described above.
The recombinant CꢀAꢀdomains (2.9 µM) and recombinant PCPꢀdomain (82 µM)
were first incubated with Lꢀvaline (1 mM), ATP (1 mM), MgCl2 (10 mM), and DTT (2 mM)
in HEPES buffer (50 mM, pH 7.0) at 37°C for 30 min, before phenylacetylꢀCoA
(approximately 0.1 mM) prepared from the in vitro enzyme assay and additional
recombinant CꢀAꢀdomains (2.9 µM) were added and the reaction was incubated at 37°C for
30 min. The reaction product was precipitated by adding four volumes of acetone to the
reaction and keeping it at −80°C for 1 h. After centrifugation, the precipitates were
dissolved in 100 µL of 0.1 M KOH and incubated at 70°C for 10 min. After adding ethanol
into the solution (final concentration 40%) and centrifugation, the product was recovered
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and analyzed with a Waters ACQUITY UPLC system equipped with an SQ Detector 2 and
ACQUITY PDA Detector. The analytical conditions were as follows; InertSustain C18
column (150 mm L × 2.1 mm ID, 3 µm GL Sciences Inc., Japan); flow rate, 0.2 mL min−1;
temperature, 40°C; mobile phase A, water containing 0.1% formic acid, mobile phase B,
acetonitrile containing 0.1% formic acid; gradient conditions, 20% B, 0–2 min; 20–80% B,
2–35 min; detection, ESIꢀnegative mode; injection volume, 10 µL.
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Accession Codes
The nucleotide sequences reported here have been submitted to the DDBJ/GenBank/EBI
Data Bank under accession nos. LC217607 (orf1 to orf8) and LC223607 (dihydrolipoyl
dehydrogenase gene).
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Supporting Information
The Supporting Information is available free of charge on the ACS Publications website
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Acknowledgments
This study was supported by GrantsꢀinꢀAid for Research on Innovative Areas from MEXT,
Japan (JSPS KAKENHI Grant Number 16H06452) and GrantsꢀinꢀAid for Scientific
Research from JSPS (15H03110) to T. Dairi.
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The authors declare no competing financial interest.
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FIGURE LEGENDS
Figure 1. Chemical structures of JBIR-78 and -95.
JBIRꢀ78 (1, upper) and ꢀ95 (2, lower).
Figure 2. JBIR-78 (1) and -95 (2) biosynthetic gene cluster.
The cloned DNA fragment (35,856 bp) contained 9 orfs. Orf4, 5, and 6 (yellow) were
NRPSs and their domain architectures are shown. A, adenylation domain (red); C,
condensation domain (blue); E, epimerization domain (green); PCP, peptidyl carrier protein
domain (gray); TE, thioesterase domain (black). They activated and condensed, in order,
LꢀVal,
LꢀAsp (or
Lꢀcysteic acid),
LꢀAla, LꢀLeu, Gly, LꢀAla, and LꢀPhe. The Cꢀdomain in
module 1 condensed
LꢀValꢀPCP and phenylacetylꢀCoA, which was supplied by Orf1 and 2
from phenylpyruvate, CoA, and NAD+, to form phenylacetylated
LꢀValꢀPCP.
Figure 3. Heterologous expression of the gene cluster.
The broths of the transformants and authentic standards were analyzed by LCꢀMS
monitoring at m/z 824 ([M−H]− of 1, traces (a)−(e)) or 860 ([M−H]− of 2, traces (f)−(j)). (a)
Standard of 1, (f) Standard of 2, (b) and (g) recombinant cells harboring pOJ446 (empty
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vector), (c) and (h) recombinant cells harboring pC35, (d) and (i) recombinant cells
harboring pC35∆orf3 without
pC35∆orf3 with ꢀcysteic acid.
Lꢀcysteic acid, (e) and (j) recombinant cells harboring
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L
Figure 4. LC-MS analysis Orf1, Orf2, and the dihydrolipoyl dehydrogenase reactions.
The LCꢀMS profile was monitored at m/z 884 ([M−H]− of phenylacetylꢀCoA). (a) Reaction
products formed with Orf1 and Orf2. (b) Reaction products formed with dihydrolipoyl
dehydrogenase. (c) Reaction products formed with Orf1, Orf2, and dihydrolipoyl
dehydrogenase.
Figure 5. LC-MS analysis of the Orf3 reaction.
The LCꢀMS profile was monitored at m/z 170 ([M+H]+ of cysteic acid). (a) Standard of
Lꢀcysteic acid. (b) Reaction products formed with boiled Orf3. (c) Reaction products
formed with refolded Orf3.
Figure 6. LC-MS analysis of the Orf4 reaction. The LCꢀMS profile was monitored at m/z
234 ([M−H]− of phenylacetyl valine). (a) Standard of phenylacetyl valine. (b) Reaction
products formed with the recombinant PCPꢀdomain. (c) Reaction products formed with
recombinant CꢀAꢀ and PCPꢀdomains.
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Table 1. Deduced functions of the Orfs.
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Orf Amino acids
(no.)
Proposed function
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pyruvate dehydrogenase complex, E1β and E2 units
pyruvate dehydrogenase complex, E1α unit
cysteic acid synthase
NRPS (CꢀAꢀPCPꢀCꢀAꢀPCPꢀE)
NRPS (CꢀAꢀPCPꢀCꢀAꢀPCPꢀE)
NRPS (CꢀAꢀPCPꢀCꢀAꢀPCPꢀEꢀCꢀAꢀPCPꢀTE)
MbtH domain protein
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cytochrome P450
hypothetical protein
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Figure 1
32x17mm (600 x 600 DPI)
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Figure 2
82x48mm (300 x 300 DPI)
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