N-Phenylacetylation and Nonribosomal Peptide Synthetases with

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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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ACS Chemical Biology

1

Unique N-phenylacetylation and NRPS with Substrate Promiscuity for Biosynthesis of

2

Heptapeptide Variants, JBIR-78 and JBIR-95

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Kunpei Takeda†, Kohei Kemmoku†, Yasuharu Satoh†, Yasushi Ogasawara†, Kazuo

5

Shin-ya‡, and Tohru Dairi†*

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†Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo,

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Hokkaido 060-8628, Japan

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‡Biomedicinal Information Research Center (BIRC), National Institute of Advanced

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Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan

11 12

*

Corresponding author. E-mail: [email protected], Tel: +81-11-706-7815

13 14 15 16 17 18 19

1 ACS Paragon Plus Environment


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ABSTRUCT

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JBIR-78 (1) and JBIR-95 (2), both of which are heptapeptide derivatives isolated from

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Kibdelosporangium sp. AK-AA56, have the same amino acid sequences except for the

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second amino acid; phenylacetic acid (Paa)–L-Val–D-Asp (1)/D-cysteic acid (2)–L-Ala–

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(3S)-3-hydroxy-D-Leu–Gly–D-Ala–L-Phe. Heterologous expression of the biosynthetic

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gene cluster including genes encoding non-ribosomal peptide synthetases (NRPS) and in

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vitro assays with recombinant Orf3, L-cysteic acid synthase homolog, suggested the single

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A-domain in module 2 activates both L-Asp and L-cysteic acid to yield 1 and 2, respectively,

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although the substrate specificities of the A-domains of NRPSs are usually strict.

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Biosynthetic mechanism of introduction of N-terminal Paa was also investigated.

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Recombinant Orf1 and Orf2 similar to subunits of pyruvate dehydrogenase complex

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catalyzed the conversion of phenylpyruvate into phenylacetyl-CoA together with

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dihydrolipoyl dehydrogenase whose encoding gene is located at outside of the gene cluster.

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Moreover, we showed that phenylacetyl-CoA was directly condensed with L-Val, which

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was tethered to a peptidyl carrier protein, at the first condensation domain in the NRPS.

35 36

INTRODUCTION

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Peptide antibiotics are a large family of natural products, including clinically important

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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

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can be categorized by their biosynthetic machineries as either ribosomally synthesized and

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post-translationally modified peptides (RiPPs) or nonribosomal peptides (NRPs).1,2 In the

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latter case, nonribosomal peptide synthetases (NRPSs) are representative biosynthetic

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enzymes. NRPSs are modular type large enzymes and a typical module consists of an

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adenylation (A) domain, a peptidyl carrier protein (PCP) domain and a condensation (C)

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domain. The A-domain activates the carboxylic acid of an amino acid with ATP and

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determines the amino acid to be selected and activated. Unlike ribosomes, NRPSs can

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utilize nonproteinogenic amino acids as building blocks. The C-domain catalyzes the

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peptide bond formation between the upstream peptidyl PCP and downstream amino acyl

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PCP. Besides NRPSs, amino acid ligases with ATP-grasp domains (ATP-grasp-ligases),

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tRNA-dependent aminoacyl transferase/cyclodipeptide synthases, stand-alone adenylation

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(A) domains and acyl-CoA synthetases (acyl-AMP-ligases) are also involved in NRP

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biosynthesis.3–6

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1 and 2, both of which are heptapeptide derivatives isolated from Kibdelosporangium

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sp. AK-AA56, have the same amino acid sequence except for the second amino acid

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(Figure 1).7 We are interested in their biosynthetic machineries from the following points of

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view. One is the difference of the second amino acid. Considering the presence of D-amino

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acids in 1 and 2, they could be biosynthesized by a NRPS. However, the substrate



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specificities of the A-domains of NRPSs are strict and one A-domain usually activates one

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amino acid. Therefore, we hypothesized three possibilities: (i) the producer possesses

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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) L-cysteic acid is independently biosynthesized

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and a single A-domain is exceptionally able to activate both L-Asp and L-cysteic acid. The

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other interesting feature is the presence of the N-terminal phenylacetate. To date, a dozen

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phenylacetylated natural polyketides and a few N-terminal phenylacetylated peptides such

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as penicillin G, microcystin,8 and JBIR-969 have been isolated. However, reports on the

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detailed phenylacetylation mechanism are limited. In the case of penicillin G, isopenicillin

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N acyltransferase was shown to convert isopenicillin N to penicillin G.10 In microcystin

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biosynthesis, phenylpropanoids were suggested to be loaded onto the PCP-domain rather

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than phenylacetate by ATP–PPi exchange assays and mass spectrometry, although the

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mechanism by which one carbon is excised from the starter substrate remains unknown.8

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Recently, Fu et al. reported that a pyruvate dehydrogenase-like protein complex catalyzed

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the decarboxylation of phenylpyruvate to form a phenylacetyl-S-acyl carrier protein, which

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would be utilized in a subsequent polyketide biosynthetic assembly line, in ripostatin

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biosynthesis.11 In this study, we investigated the phenylacetylation mechanism in JBIR-78

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(1) and JBIR-95 (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

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sequence for the producer and searched for orfs large enough to encode the seven

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A-domains essential for substrate activation in heptapeptide biosynthesis. From a BLAST

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search, we identified one candidate gene cluster (Figure 2, Table 1). The gene cluster

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consisted of eight orfs containing three orfs (orf4 to orf6) encoding NRPSs. Orf4 and Orf5

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had the same domain architectures; C-A-PCP-C-A-PCP-epimerization domain (E-domain).

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Orf6 was composed of C-A-PCP-C-A-PCP-E-C-A-PCP-thioesterase (TE) domains. The

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positions of the E-domains perfectly matched the locations of the D-amino acids in 1 and 2.

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Moreover, the substrate specificities of each of the A-domains predicted by

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NRPSpredictor2 (http://nrps.informatik.uni-tuebingen.de/Controller?cmd=SubmitJob) were

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roughly identical to the amino acid sequences of 1 (Supplementary Table 1, 2, and 3). Two

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orfs (orf1 and orf2), which encode two of the three component enzymes of pyruvate

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dehydrogenase complex, were found in the region upstream from the NRPS genes. Orf1

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was composed of pyruvate dehydrogenase (EC 1.2.4.1, E1β unit) and dihydrolipoyl

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transacetylase (EC 2.3.1.12, E2 unit). Orf2 was similar to the E1α unit of pyruvate

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dehydrogenase. Although no homologs of dihydrolipoyl dehydrogenase (EC 1.8.1.4, E3

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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,

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and the dihydrolipoyl dehydrogenase homolog perhaps catalyze the conversion of

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phenylpyruvate

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phenylacetylation.

into

phenylacetyl-CoA,

which

would

be

used

for

N-terminal

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Orf3 showed similarity to the MA3297 protein, which is a pyridoxal 5ʹ-phosphate

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dependent enzyme that catalyzes a β-replacement reaction converting L-phosphoserine and

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sulfite into

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responsible for the biosynthesis of 2.

L-cysteic

acid and inorganic phosphate.12 Therefore, Orf3 is plausibly

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Orf8 was similar to cytochrome P450 and presumably participates in the

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hydroxylation of Leu to form 3-hydroxy-D-Leu. Additionally, Orf7 was similar to MbtH, an

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integral component of NRPSs that is essential for amino acid activation.13 The identified

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gene cluster was the sole candidate and no paralogs of the identified genes existed in the

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draft genome database. Therefore, the production of both 1 and 2 was suggested to be

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governed by this gene cluster.

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Heterologous expression of the gene cluster

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Next, we performed a heterologous expression experiment to examine whether the

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gene cluster identified in this study contained all the genes responsible for 1 and 2

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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

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appropriate primers and the cosmid pC35 was introduced into Streptomyces lividans TK23.

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After cultivation of the transformants, the products were analyzed by LC-ESI-MS. As

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shown in Figure 3 and Supplementary Figure 1, the production of both 1 and 2 was

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suggested. Although the cosmid had one additional gene (Figure 2) downstream of orf8

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gene, this gene was suggested to have no relation to 1/2 biosynthesis. Taken together, our

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results suggested that both products were biosynthesized by this gene cluster alone and that

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the A-domain responsible for activation of the second substrate exceptionally activated both

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L-Asp

and L-cysteic acid.

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Characterization of Orf1 and Orf2

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To examine whether Orf1 and Orf2 catalyzed the conversion of phenylpyruvate to

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phenylacetyl-CoA, an in vitro assay with recombinant enzymes was carried out. A DNA

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fragment carrying both the orf1 and orf2 genes was inserted into the pET28a vector so that

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only Orf2 was expressed as a His-tagged recombinant enzyme because Orf1 was

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co-purified with Orf2 by Ni-NTA column chromatography (Supplementary Figure 2). A

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recombinant enzyme of the putative dihydrolipoyl dehydrogenase (E3 unit) was also

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prepared as a His-tagged recombinant enzyme (Supplementary Figure 2). After the

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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 co-factors in the pyruvate

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dehydrogenase reaction. A new product whose molecular mass was consistent with that of

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phenylacetyl-CoA was specifically detected by LC-ESI-MS analysis (Figure 4). Its high

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resolution molecular mass also agreed with that of phenylacetyl-CoA ([M+H]+ calcd. for

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C29H43N7O17P3S+, 886.16435: found 886.16534).

139 140 141

Characterization of Orf3 Orf3 showed similarity to the pyridoxal 5ʹ-phosphate dependent

L-cysteic

acid

142

synthase. To confirm its expected activity, we tried an in vitro assay with recombinant

143

enzymes. Although we could express Orf3 recombinant enzymes with several expression

144

vectors such as pET vectors, pMAL-c5X and pCold-TF, all recombinant enzymes formed

145

inclusion bodies. Therefore, we carried out heterologous expression of the gene cluster

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without the orf3 gene. We constructed a cosmid lacking orf3 gene (pC35∆orf3) from pC35.

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Unexpectedly, the transformant harboring pC35∆orf3 did not lose 2 productivity and still

148

produced a small amount of 2 concomitant with enhanced production of 1 (Figure 3 and

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Supplementary Figure 1). A possible reason for this is that L-cysteic acid might be supplied

150

by an alternative enzyme such as L-serine ammonia-lyase (EC 4.3.1.17) that catalyzes the

151

elimination of water from L-serine to form L-dehydroalanine, to which sulfite attacks.

152

Indeed, 2 productivity was dramatically recovered when L-cysteic acid was added into the


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medium (Figure 3 and Supplementary Figure 1), suggesting that the Orf3 product supplies

154

L-cysteic

155

L-cysteic acid, suggesting that L-cysteic acid competed with L-Asp in the incorporation into

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the peptide.

acid. Furthermore, the productivity of 1 was extremely reduced by the addition of

157

Previously, a few A-domains in NRPSs, which activated two different amino acids,

158

were reported. Pelgipeptin A/C and pelgipeptin B/D possess L-Val and L-Ile at the second

159

position, respectively. The substrate selectivity of A-domains (PlpE A1) responsible for

160

activation of the second amino acid was examined by in vitro experiments and PlpE A1 was

161

shown to activate both L-Val and L-Ile.

162

L-Leu) was also derived by the substrate selectivity of A-domains (LicC-A).

163

the A-domains recognized the structurally and chemically similar amino acids. For the

164

same reason, the A-domain in module 2 might accept L-Asp and L-cysteic acid, both of

165

which are acidic amino acids and have similar sizes.

15

C-terminal diversity of lichenysins (L-Ile or 16

In both cases,

166

We then tried denaturation and refolding of the inclusion body to obtain the active

167

form of the recombinant enzyme. After extensive trials to find the best conditions, we

168

successfully obtained active enzymes (Supplementary Figure 3). The refolded enzyme was

169

incubated with L-phosphoserine and sodium sulfite for 15 min at 30°C and the reaction

170

products were analyzed by LC-ESI-MS. As shown in Figure 5, a specific peak, which was

171

eluted at the same retention time and had the same molecular mass as authentic L-cysteic



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acid, was detected.

173 174

Characterization of Orf4

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Through detailed analysis of each of the domains in Orf4, we found that a

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PCP-domain to anchor phenylpyruvate was absent. Moreover, Orf1 and Orf2 catalyzed the

177

formation of phenylacetyl-CoA as mentioned above. These results suggested that

178

phenylacetyl-CoA is directly condensed with L-Val at the first C-domain in module 1 of

179

Orf4 in a similar manner to that of surfactin and amphi-enterobactin siderophore

180

biosynthesis.17,18 To examine this possibility, a truncated recombinant Orf4 (module 1) was

181

prepared and used for in vitro assay. We first tried to prepare a recombinant enzyme

182

possessing the first module with the C-, A-, and PCP-domains. However, we were unable to

183

obtain recombinant proteins. We then prepared recombinant enzymes divided into

184

C-A-domains and the PCP-domain. In this case, both recombinant enzymes were expressed

185

as soluble forms (Supplementary Figure 4).

186

The purified recombinants were incubated with

L-valine

and phenylacetyl-CoA,

187

which was prepared by the enzyme reaction described above, in the presence of ATP and

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MgCl2. After the enzymes were precipitated by adding acetone, the thioester bonds were

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hydrolyzed with 0.1 M KOH. The resulting sample was analyzed by LC-ESI-MS and

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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

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authentic standard was clearly detected in the sample, showing that phenylacetyl-CoA was

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directly condensed with L-valine, which was activated at the A-domain and transferred to

194

PCP in module 1.

195

Recently, a similar mechanism for the formation of phenylacetate from phenylpyruvate

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by a pyruvate dehydrogenase-like protein complex in ripostatin biosynthesis was reported.11

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In this case, however, the phenylacetyl group of S-phenylacetyldihydrolipoamide was

198

directly transferred to an acyl carrier protein to form a phenylacetyl-S-acyl carrier protein

199

without the formation of phenylacetyl-CoA.

200

In summary, we studied the biosynthetic machinery of 1 and 2, which are both

201

heptapeptide derivatives and have the same amino acid sequence except for the second

202

amino acid. Through heterologous expression of the biosynthetic gene cluster and in vitro

203

assay with the recombinant Orf3, the single A-domain in module 2 was suggested to

204

activate both L-Asp and L-cysteic acid. We also showed that N-terminal phenylacetylation

205

was catalyzed by the first C-domain in module 1 with

206

phenylacetyl-CoA supplied by a phenylpyruvate dehydrogenase complex.

L-Val-tethered

PCP and

207 208

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(1998) Characterization of Sfp, a Bacillus subtilis Phosphopantetheinyl Transferase for

276

Peptidyl Carrier Protein Domains in Peptide Synthetases. Biochemistry 37, 1585–

277

1595.

278



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279

METHODS

280

General

281

All chemicals were purchased from Sigma-Aldrich Japan K.K. (Japan), Tokyo Chemical

282

Industry Co. Ltd. (Japan), or Wako Pure Chemical Industry (Japan). Oligonucleotides were

283

obtained from FASMAC Co. Ltd. (Japan). Enzymes, molecular weight standards and kits

284

for DNA manipulation were purchased from Takara Bio Inc. (Japan) or New England

285

Biolabs Japan Inc. (Japan). PCR reactions were carried out using a GeneAmp PCR System

286

9700 thermal cycler (Thermo Fisher Scientific Inc.) with Tks Gflex DNA polymerase

287

(Takara Bio). NMR spectra were obtained using a JEOL ECS-400 spectrometer (Japan).

288 289

Chemical synthesis of phenylacetyl valine

290

To a stirred solution of 1.03 g (8.8 mmol) of L-valine in 10 mL (20 mmol) of 2 M aq.

291

NaOH at 0°C was added dropwise 1.05 mL (8 mmol) of phenylacetyl chloride. The

292

reaction mixture was allowed to warm to room temperature and stirred. After 20 h of

293

reaction, water (30 mL) was added and the aqueous layer containing the product was

294

washed with Et2O (50 mL) twice. The water layer was then acidified with 1 M HCl to

295

obtain a white precipitate. The white crystalline product was washed thoroughly with water

296

and then with Et2O (606 mg, 29%). 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 8.6 Hz,

297

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


Page 16 of 34

Page 17 of 34

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298

= 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

299

(100 MHz, DMSO-d6) δ 173.1, 170.4, 136.6, 129.0, 128.1, 126.3, 57.1, 41.9, 29.9, 19.1,

300

18.0.

301 302

Draft genome sequences

303

Draft sequences of the Kibdelosporangium sp. AK-AA56 genome were determined by a

304

commercial company (Hokkaido System Science, Japan) using an Illumina HiSeq platform

305

(Illumina). A genomic DNA library (350 bp insert) was constructed with the TruSeq Nano

306

DNA LT Sample Prep Kit (Illumina). Paired end data (2 × 100 bp) were assembled using

307

Velvet (version 1.0.18). Gene prediction and annotation was carried out using Microbial

308

Genome Annotation Pipeline (MiGAP, http://www.migap.org/).

309 310

Cloning and heterologous expression of the gene cluster

311

To clone the gene cluster, a cosmid library of the producer was constructed with the shuttle

312

cosmid vector pOJ446.14 Genomic DNA of Kibdelosporangium sp. was partially digested

313

with Sau3AI and ligated with the pOJ446 vector digested with BamHI and HpaI and treated

314

bacterial alkaline phosphatase. This was packaged using Gigapack III XL Packaging

315

Extract (Agilent Technologies Inc.) and transfected to the E. coli XL1-Blue MRF′ strain

316

(∆(mcrA)183 ∆(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 34

317

[F′proAB lacIqZ∆M15 Tn10 (Tetr)]; Agilent Technologies). Positive cosmids carrying the

318

gene cluster were screened by PCR based on the amplification of both the orf1 and 8 genes

319

with

320

5ʹ-CGTTCCCGATCTCGGTGAAGGACTTGCTGAG-3ʹ

321

5ʹ-CATGGGCGAGTAGCGGAACGAGAGGAACAAG-3ʹ

322

5ʹ-GGGCCGTAGGCGAATGTCAGGTGTTTGTTC-3ʹ for orf8.

the

primers

5ʹ-GGCCGCGACGTGGGTGAGGAAGTTG-3ʹ for

orf1,

and and and

323

The obtained cosmid, pC35, was introduced into S. lividans TK23 and then the

324

transformants were cultured in 200 mL flasks with baffles containing 30 mL of SK#2

325

medium19 containing thiostrepton (10 µg mL−1) for 4 days at 30°C with agitation (200 rpm).

326

The whole culture broths were directly analyzed by LC-ESI-MS as follows: Waters

327

ACQUITY UPLC system equipped with an SQ Detector2 and ACQUITY PDA Detector

328

(Japan); Mightysil RP-18GP Aqua column (150 mm L × 2.1 mm ID, 3 µm, KANTO

329

CHEMICAL Co., Inc., Japan); flow rate, 0.2 mL min−1; temperature, 40°C; mobile phase A,

330

water containing 0.1% formic acid, mobile phase B, methanol containing 0.1% formic acid;

331

gradient conditions, 15% B, 0–2 min; 15–85% B, 2–40 min; detection, ESI-negative mode;

332

injection volume, 10 µL.

333

pC35∆orf3, in which the orf3 gene was in-frame deleted, was constructed using

334

Red/ET recombination.20 In brief, DNA fragments containing a Km-resistance gene cassette

335

(Gene Bridges GmbH, Germany) flanked with FRT sites and 50-bp homologous arms,


Page 19 of 34

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336

whose sequences were identical to the target regions, were amplified by PCR with the

337

primers

338

5ʹ-CCTGGTGTGGTGTGAATAGACCGCAGTCACGAAGGAGGTATGACCGGATGGA

339

ATTAACCCTCACTAAAGGGCGGC-3ʹ

340

5ʹ-CACCTGCCGCCTGTCCCGGGTGAGCTGTCATGTCACGCCCCACTCGGGGTAAT

341

ACGACTCACTATAGGGCTCG-3ʹ. The amplified DNA fragments were used to transform

342

E. coli XL1-Blue MRF′ harboring the pC35 cosmid and pRedET plasmid. Gene-disruption

343

in

344

5ʹ-GCGTAATGCGAGGTTGGGTCTATCGAAGG-3ʹ

345

5ʹ-CGCATCGACTGGGCCGTGGATCTC-3ʹ. The selection marker in the obtained cosmid

346

was removed with FLP-recombinase and the deletion was confirmed by PCR with the

347

primers used to check gene disruption as described above.

Km-resistant

colonies

was

and

checked

by

PCR

using

two

primers, and

348 349

Preparation and enzyme assay of recombinant enzymes of Orf1, Orf2, and

350

dihydrolipoyl dehydrogenase

351

A DNA fragment containing both the orf1 and 2 genes was amplified by PCR using

352

genomic DNA of Kibdelosporangium sp. as a template and the following primers;

353

5ʹ-CCTATCGGCATATGGTGCTTGGCCGCCGATTCGAC-3ʹ

354

5ʹ-ATATAAGCTTCCGGCTACCCGACCATGATCAGACTGTC-3ʹ.


and To

amplify

the


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dehydrogenase

gene

of

Kibdelosporangium

Page 20 of 34

355

dihydrolipoyl

sp.,

the

primers

356

5ʹ-AAGTGACCATATGGCCACAGTTGACGCGCGCGTA-3ʹ

357

5ʹ-TATAAGCTTGGCTGACTTGGTGCCGACGGTCAG-3ʹ were used. Restriction sites

358

(underlined) were introduced into the N- and C-terminal regions. The PCR products were

359

cloned into the NdeI-HindIII sites of the pET28a vector (Merck KGaA, Germany). Each of

360

the obtained plasmids was introduced into E. coli BL21(DE3) (F−, dcm, ompT, hsdS(rB−

361

mB−), gal, λ(DE3); NIPPON GENE CO., LTD, Japan). A liquid culture of each transformant

362

in LB supplied with kanamycin (30 µg mL−1) was induced by adding 0.5 mM IPTG when

363

the optical density at 600 nm reached about 0.6. The cultivation was continued for an

364

additional 16 h at 20°C. Purification of each recombinant protein was carried out as follows.

365

After the cells were disrupted with an ultrasonic disruptor (TOMY, UD-200), the His-tag

366

fused proteins were purified with Ni-NTA column chromatography (QIAGEN K.K., Japan)

367

and their purities were analyzed by SDS-PAGE on 10% gels (Supplementary Figure 2). The

368

proteins were visualized by Coomassie brilliant blue staining and the protein concentrations

369

were determined by the Bradford method with bovine serum albumin as a standard.

and

370

Co-purified recombinant Orf1 (approximately 0.42 µM) and Orf2 (approximately 2.3

371

µM), and purified recombinant dihydrolipoyl dehydrogenase (1.9 µM) were incubated with

372

CoA (1.0 mM), sodium phenylpyruvate (2.0 mM), MgCl2 (1.0 mM), thiamine

373

pyrophosphate (0.2 mM), NAD+ (2.5 mM), and dithiothreitol (0.6 mM) in potassium


Page 21 of 34

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374

phosphate buffer (50 mM, pH 8.0) at 30°C for 16 h. The reaction product was directly

375

analyzed by LC-ESI-MS as follows: Waters ACQUITY UPLC system equipped with an SQ

376

Detector2 and ACQUITY PDA Detector; XBridge BEH C8 column (150 mm L × 2.1 mm

377

ID, 2.5 µm, Waters); flow rate, 0.2 mL min−1; temperature, 35°C; mobile phase A, 100 mM

378

NH4COOH containing 5 vol% methanol, mobile phase B, 100 mM NH4COOH containing

379

50 vol% methanol; gradient conditions, 10% B, 0–5 min; 10–100% B, 5–40 min; detection,

380

ESI-negative mode; injection volume, 10 µL. The fractionated product was also analyzed

381

by high resolution-ESI-FT-MS (Exactive, Thermo Fisher Scientific Inc.).

382 383

Preparation and assay of recombinant L-cysteic acid synthase (Orf3) enzyme

384

To express maltose binding protein-fused L-cysteic acid synthase (MAL-CS), the primer

385

pair pMal_CS_Fw: 5′-GTATGACCATATGTGCGCTCGGCGGCATTACTCGATC-3′ and

386

pMal_CS_Rv:

387

used. The PCR product was cloned into the NdeI-HindIII site of pMal-c5X (New England

388

Biolabs) to construct pMal-CS. Culture and purification conditions were the same as those

389

described above except for the use of amylose affinity column chromatography (New

390

England Biolabs) for purification. The purified recombinant Orf3 was treated with Factor

391

Xa protease (New England Biolabs) according to the manufacturer’s protocol to remove

392

MBP. To eliminate the MBP released, the reaction mixture was applied to an amylose

5′-AGATAAGCTTCACGCCCCACTCGGGGTGGCTACGTAG-3′


was


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Page 22 of 34

393

affinity column again. Then, the flow-through fraction containing the enzyme was mixed

394

with 10 volumes of Tris·HCl buffer (20 mM, pH 8.0) containing 6 M urea and DTT (20

395

mM), and incubated at 37°C for 2 h. The solution was diluted ten times with refolding

396

buffer; Tris·HCl buffer (50 mM, pH 8.0) containing KCl (1 M), glycerol (20 %), MgCl2 (20

397

mM), and PLP (10 µM), and stirred at 4°C for 16 h. The enzyme solution was concentrated

398

to 2 mg mL−1 with Amicon Ultra filter units (Merck).

399

The refolded recombinant Orf3 enzyme (8.8 µM) was incubated with PLP (10 µM),

400

sodium sulfite (5 mM), O-phospho-L-serine (10 mM), and KCl (100 mM) in Tris·HCl

401

buffer (100 mM, pH 7.0) at 30°C for 15 min. The reaction product was directly analyzed by

402

LC-ESI-MS as follows: Waters ACQUITY UPLC system equipped with an SQ Detector2

403

and ACQUITY PDA Detector; Scherzo SM-C18 column (150 mm L × 2 mm ID, 3 µm,

404

Imtakt Co., Japan); flow rate, 0.2 mL min−1; temperature, 40°C; mobile phase A, water

405

containing 0.1% formic acid, mobile phase B, acetonitrile containing 0.1% formic acid;

406

10% B isocratic conditions; detection, ESI-positive mode; injection volume, 10 µL.

407 408

Preparation and assay of recombinant Orf4 enzyme

409

The first C-A-domains and PCP-domain in module 1 of Orf4 were expressed separately.

410

The

411

5ʹ-AAAAAACATATGGACGCGGTCGCGGCCCA-3ʹ

former

was

amplified

by

PCR


with

two

primers, and

Page 23 of 34

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412

5ʹ-TGGCGTAAGCTTTCAGCGGGAAGGACTGGT-3ʹ. The latter was amplified with the

413

primers

414

5ʹ-AAAAAGCTTGCCGACCAGCAGCGCGAT-3ʹ. Each amplified fragment was cloned

415

into the NdeI-HindIII site of pET28a to construct the plasmids pOrf4-CA and pOrf4-PCP, to

416

express recombinant enzymes fused with an N-terminal His-tag. For activation of the

417

PCP-domain, the plasmid (pACYC-Sfp) expressing phosphopantetheinyl transferase of

418

Bacillus subtilis (Sfp) was constructed by digesting the pET-Sfp plasmid21 with NdeI and

419

XhoI and then cloning the sfp fragment into the NdeI-XhoI site of pACYCDuet-1 (Merck).

420

Each plasmid was used to transform E. coli BL21(DE3) for protein overexpression.

421

Purification procedures were the same as those described above.

5ʹ-AAAAAACATATGCCCGTCACCAGTCCTT-3ʹ

and

422

The recombinant C-A-domains (2.9 µM) and recombinant PCP-domain (82 µM)

423

were first incubated with L-valine (1 mM), ATP (1 mM), MgCl2 (10 mM), and DTT (2 mM)

424

in HEPES buffer (50 mM, pH 7.0) at 37°C for 30 min, before phenylacetyl-CoA

425

(approximately 0.1 mM) prepared from the in vitro enzyme assay and additional

426

recombinant C-A-domains (2.9 µM) were added and the reaction was incubated at 37°C for

427

30 min. The reaction product was precipitated by adding four volumes of acetone to the

428

reaction and keeping it at −80°C for 1 h. After centrifugation, the precipitates were

429

dissolved in 100 µL of 0.1 M KOH and incubated at 70°C for 10 min. After adding ethanol

430

into the solution (final concentration 40%) and centrifugation, the product was recovered



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

431

and analyzed with a Waters ACQUITY UPLC system equipped with an SQ Detector 2 and

432

ACQUITY PDA Detector. The analytical conditions were as follows; InertSustain C18

433

column (150 mm L × 2.1 mm ID, 3 µm GL Sciences Inc., Japan); flow rate, 0.2 mL min−1;

434

temperature, 40°C; mobile phase A, water containing 0.1% formic acid, mobile phase B,

435

acetonitrile containing 0.1% formic acid; gradient conditions, 20% B, 0–2 min; 20–80% B,

436

2–35 min; detection, ESI-negative mode; injection volume, 10 µL.

437 438

Accession Codes

439

The nucleotide sequences reported here have been submitted to the DDBJ/GenBank/EBI

440

Data Bank under accession nos. LC217607 (orf1 to orf8) and LC223607 (dihydrolipoyl

441

dehydrogenase gene).

442 443

Supporting Information

444

The Supporting Information is available free of charge on the ACS Publications website

445 446

Acknowledgments

447

This study was supported by Grants-in-Aid for Research on Innovative Areas from MEXT,

448

Japan (JSPS KAKENHI Grant Number 16H06452) and Grants-in-Aid for Scientific

449

Research from JSPS (15H03110) to T. Dairi.

450


Page 24 of 34

Page 25 of 34

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451


The authors declare no competing financial interest.

452 453

FIGURE LEGENDS

454

Figure 1. Chemical structures of JBIR-78 and -95.

455

JBIR-78 (1, upper) and -95 (2, lower).

456 457

Figure 2. JBIR-78 (1) and -95 (2) biosynthetic gene cluster.

458

The cloned DNA fragment (35,856 bp) contained 9 orfs. Orf4, 5, and 6 (yellow) were

459

NRPSs and their domain architectures are shown. A, adenylation domain (red); C,

460

condensation domain (blue); E, epimerization domain (green); PCP, peptidyl carrier protein

461

domain (gray); TE, thioesterase domain (black). They activated and condensed, in order,

462

L-Val, L-Asp

463

module 1 condensed L-Val-PCP and phenylacetyl-CoA, which was supplied by Orf1 and 2

464

from phenylpyruvate, CoA, and NAD+, to form phenylacetylated L-Val-PCP.

(or L-cysteic acid), L-Ala, L-Leu, Gly, L-Ala, and L-Phe. The C-domain in

465 466

Figure 3. Heterologous expression of the gene cluster.

467

The broths of the transformants and authentic standards were analyzed by LC-MS

468

monitoring at m/z 824 ([M−H]− of 1, traces (a)−(e)) or 860 ([M−H]− of 2, traces (f)−(j)). (a)

469

Standard of 1, (f) Standard of 2, (b) and (g) recombinant cells harboring pOJ446 (empty



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

470

vector), (c) and (h) recombinant cells harboring pC35, (d) and (i) recombinant cells

471

harboring pC35∆orf3 without

472

pC35∆orf3 with L-cysteic acid.

L-cysteic

acid, (e) and (j) recombinant cells harboring

473 474

Figure 4. LC-MS analysis Orf1, Orf2, and the dihydrolipoyl dehydrogenase reactions.

475

The LC-MS profile was monitored at m/z 884 ([M−H]− of phenylacetyl-CoA). (a) Reaction

476

products formed with Orf1 and Orf2. (b) Reaction products formed with dihydrolipoyl

477

dehydrogenase. (c) Reaction products formed with Orf1, Orf2, and dihydrolipoyl

478

dehydrogenase.

479 480

Figure 5. LC-MS analysis of the Orf3 reaction.

481

The LC-MS profile was monitored at m/z 170 ([M+H]+ of cysteic acid). (a) Standard of

482

L-cysteic

483

formed with refolded Orf3.

acid. (b) Reaction products formed with boiled Orf3. (c) Reaction products

484 485

Figure 6. LC-MS analysis of the Orf4 reaction. The LC-MS profile was monitored at m/z

486

234 ([M−H]− of phenylacetyl valine). (a) Standard of phenylacetyl valine. (b) Reaction

487

products formed with the recombinant PCP-domain. (c) Reaction products formed with

488

recombinant C-A- and PCP-domains.


Page 26 of 34

Page 27 of 34

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489

Table 1. Deduced functions of the Orfs.

490

Orf Amino acids

Proposed function

(no.)

491 492

1

677

pyruvate dehydrogenase complex, E1β and E2 units

493

2

312

pyruvate dehydrogenase complex, E1α unit

494

3

425

cysteic acid synthase

495

4

2565

NRPS (C-A-PCP-C-A-PCP-E)

496

5

2540

NRPS (C-A-PCP-C-A-PCP-E)

497

6

3825

NRPS (C-A-PCP-C-A-PCP-E-C-A-PCP-TE)

498

7

69

MbtH domain protein

499

8

406

cytochrome P450

500

9

223

hypothetical protein

501 502



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1 32x17mm (600 x 600 DPI)

ACS Paragon Plus Environment

Page 28 of 34

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3 98x161mm (600 x 600 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5 103x305mm (600 x 600 DPI)


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Figure 6 93x220mm (600 x 600 DPI)



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TOC 160x80mm (300 x 300 DPI)


Page 34 of 34

N-Phenylacetylation and Nonribosomal Peptide Synthetases with

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