Genome Mining of Amino Group Carrier Protein-Mediated Machinery

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Genome mining of amino group carrier protein-mediated machinery: discovery and biosynthetic characterization of a natural product with unique hydrazone unit Kenichi Matsuda, Fumihito Hasebe, Yuh Shiwa, Yu Kanesaki, Takeo Tomita, Hirofumi Yoshikawa, Kazuo Shin-ya, Tomohisa Kuzuyama, and Makoto Nishiyama ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00818 • Publication Date (Web): 16 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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ACS Chemical Biology

Genome mining of amino group carrier protein-mediated machinery: discovery and biosynthetic characterization of a natural product with unique hydrazone unit Kenichi Matsuda1, Fumihito Hasebe1, Yuh Shiwa2, Yu Kanesaki2, Takeo Tomita1, Hirofumi Yoshikawa2, Kazuo Shin-ya3, Tomohisa Kuzuyama1, Makoto Nishiyama1*

1

Biotechnology Research Center, The University of Tokyo, Tokyo 133-8657, Japan

2

Genome Research Center, NODAI Research Institute, Tokyo University of Agriculture, Tokyo 156-8502,

Japan 3

National Institute of Advanced Industrial Science and Technology (AIST), Tokyo 135-0064 Japan

ABSTRACT We recently revealed that a Streptomyces strain possesses the gene encoding amino group carrier protein (AmCP). AmCP is involved in the biosynthesis of a previously unidentified non-proteinogenic amino acid, (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH), which is a core compound for the synthesis of the dipeptide-containing novel natural product vazabitide A. We used polymerase chain reaction (PCR) screening to investigate the diversity of the biosynthetic machinery that uses AmCP; the results revealed that genes encoding AmCP are widely distributed among actinomycetes. The heterologous expression of the AmCP-containing gene cluster from Streptomyces sp. SoC090715LN-17 led to the discovery of s56-p1, a novel natural product. The structure of s56-p1 was determined by spectroscopic analysis; the results revealed that s56-p1 has the putative DADH-derived molecule as the core, and also possesses a unique hydrazone unit that is rarely observed in natural products. Our results pave the way for investigations of unexploited AmCP-mediated biosynthesis routes among actinomycetes and of the biosynthetic mechanism of the unique hydrazone unit.

AmCP was first identified as LysW in lysine biosynthesis of a thermophilic bacterium, Thermus 1

ACS Paragon Plus Environment


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thermophilus. In the lysine biosynthesis, LysW binds and protects the amino group of α-aminoadipate (AAA) and the tethered AAA portion is converted to lysine by several enzymes (Figure 1a).1 Recently, we showed that a hyperthermophilic archaeon, Sulfolobus acidocaldarius, uses a LysW ortholog in lysine and ornithine (arginine) biosynthesis for protection/binding of the α-amino groups of both AAA and glutamate (Figure 1b).2 In these biosynthesis processes, the protection of the α-amino group by LysW and the ortholog prevent undesirable intramolecular cyclization of the biosynthetic intermediates during conversion steps. In addition, these proteins also act as carrier proteins that guarantee efficient substrate recognition by biosynthetic enzymes through a series of reaction steps. Hereafter, we refer to this protein family as amino group carrier protein (AmCP). Streptomyces, which are mesophilic and terrestrial or marine bacteria, constitute a prolific source of bioactive compounds such as antibiotics and antioxidants.3,

4

We recently identified genes

encoding AmCP homologs in the genomes of several Streptomyces. These genes encoding AmCP homologs might not be involved in lysine or arginine biosynthesis in Streptomyces because Streptomyces synthesizes lysine via a diaminopimelate (DAP) pathway and arginine via a normal arginine biosynthetic pathway starting with acetylation of the α-amino group of glutamate.5 We recently found that one AmCP-containing Streptomyces strain, Streptomyces sp. SANK 60404, biosynthesizes vazabitide A using a novel non-proteinogenic amino acid, (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH), which is biosynthesized from glutamate by an AmCP-mediated system (Figure 1c).5 This system starts from the reaction catalyzed by LysX homolog (Vaz23), which activates the γ-carboxyl group of the C-terminal glutamate residue of AmCP (Vzb22) to form Vzb22-γ-Glu with an isopeptide bond linkage. Subsequently, LysZ homolog (Vzb25), LysY homolog (Vzb24), transketolase (Vzb27/Vzb28), and aminotransferase (Vzb9) catalyze phosphorylation, reduction, carbon-chain elongation, and transamination steps, respectively, to convert the glutamate adduct attached to Vzb22 into a DADH adduct. Finally, LysK homolog (Vzb26) hydrolyzes the isopeptide bond and releases DADH from the AmCP conjugate. DADH or its derivative then acts as a substrate of a non-ribosomal peptide synthetase (NRPS; Vzb7) and is incorporated into a novel dipeptide compound, vazabitide A, which contains a 1-azabicyclo[3.1.0]hexane 2


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ring presumably derived from DADH. That study revealed that AmCP is involved in the generation of secondary metabolites in Streptomyces. As 1-azabicyclo[3.1.0]hexane ring-containing compounds, azinomycins A and B were isolated from Streptomyces sahachiroi and Streptomyces griseofuscus.6, 7 Among azinomycins, the 1-azabicyclo[3.1.0]hexane ring is important for its antitumor activity;8 however, the biosynthetic mechanism for the ring formation remains unknown. We previously reported that all homologs that biosynthesize DADH are conserved in the biosynthetic gene cluster of azinomycins.9 Therefore, we postulate that azinomycins are also biosynthesized using DADH as a biosynthetic intermediate. To date, a large number of natural products have been identified. In their biosynthesis, mega-synthases, such as polyketide synthase (PKS) and NRPS, play a crucial role in constructing the frameworks of compounds. In these systems, acyl carrier protein (ACP) and peptidyl carrier protein (PCP) are bound to the carboxyl group of a substrate through a phosphopantetheinyl group that is covalently bound to a conserved serine residue in these carrier proteins.10, 11 By contrast, AmCP binds directly to the α-amino group of a substrate and exhibits no sequence similarity to these carrier proteins, suggesting that it has a different evolutionary origin. We expect that this unique AmCP-mediated machinery may provide novel DADH-derived building blocks for expanding the structural diversity of natural products; however, no examples have been reported to date, except for vazabitide A, and azinomycins, which are biosynthesized using AmCP-mediated machinery. In this study, we used polymerase chain reactions (PCR) to screen 848 unidentified actinomycetes strains to investigate the diversity of AmCP-mediated machinery in actinomycetes. The results indicate that genes encoding AmCP are widely distributed among actinomycetes. The heterologous

expression

of

the

approximately 70-kb

gene

cluster

from

Streptomyces

sp.

SoC090715LN-17, which contains the gene encoding AmCP and functionally related genes, led to the discovery of a novel metabolite—s56-p1 (1)—with a unique hydrazone group.

3



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a) Thermus thermophilus

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b) Sulfolobus acidocaldarius

c) Streptomyces sp. SANK 60404

Lys biosynthesis

Lys biosynthesis

Arg biosynthesis

DADH biosynthesis

AAA

AAA

Glu

Glu

COOH

HOOC

COOH

HOOC

NH2

NH2

LysX

LysX

LysW

HOOC

COOH

HOOC

NH2

LysW

COOH

HOOC HN

HN

LysW

H2 O3 POOC HN

Vzb23

HOOC

COOH HN

LysW

LysZ COOH

Vzb22 ArgX

COOH

HOOC

HOOC

H2 O3 POOC HN

LysW

H2O3POOC

COOH HN

COOH HN

LysW

OHC

HN

OHC

COOH

OHC HN

LysW

COOH HN

LysW

LysJ

COOH HN

LysW

LysJ/ArgD

COOH HN

H2 N

COOH HN

LysW

OH COOH O

COOH

H2N HN

LysW

LysK H2 N

COOH

COOH

Vzb9

Lys

COOH

HO NH 2

COOH

H2N

NH2

NH2

Vzb22

OH

LysW H2 N

HN

LysW

LysK/ArgE

LysW

Vzb22

Vzb27/Vzb28 HO

H2 N

Vzb22

Vzb24

LysY/ArgC

COOH

OHC

Vzb22

Vzb25

H2O3POOC

LysW

LysY

COOH HN

LysW

LysZ/ArgB COOH

COOH NH2

NH2

Lys

HN

Vzb22

Vzb26

Orn

Vzb22 OH COOH

HO NH2

Arg

NH2

DADH

Vazabitide A

Figure 1. Relationship of lysine/arginine biosynthesis mediated by LysW in Thermus thermophilus and Sulfolobus acidocaldarius with DADH biosynthesis mediated by Vzb22 in Streptomyces sp. SANK 60404. AmCPs that play key roles in these pathways are highlighted by magenta circles.

H N

H2N O

O

O OH

O

O

O

N

O

HO

O

OH

O

O H N

N H N

HO

vazabitide A

azinomycin B

Figure 2. Chemical structures of vazabitide A and azinomycin B 4


O

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RESULTS AND DISCUSSION Gene-guided screening of putative AmCP-containing gene clusters. AmCP has two characteristic features in its amino acid sequence: a metal-binding motif with the sequence CXXC, which is responsible for zinc binding, and a strictly conserved EDWGE sequence at the C-terminus (Figure S1). PCR screening was conducted for genomes of 848 uncharacterized actinomycetes strains using a single set of primers corresponding to the conserved regions of the AmCP of Streptomyces sp. SANK 60404. By sequencing the amplified fragments, we identified 12 strains that were positive for carrying the gene encoding AmCP. The 16S rRNA sequence information of these strains indicated that among AmCP-positive strains, 9 strains (SpC080624G1-09, Sp080902JE-04, TB090715LN-01, TB090715LN-02, TB090715SC-02, TB090715SC-05, SoE090715HV-08, SoC090715LN-16, and SoC090715LN-17) belong to the Streptomyces genus, 2 strains (SpD080624G1-02, SS080624GE-03) belong to the Micromonospora genus, and 1 strain (SoC090715LN-10) belongs to the Actinomadura genus. The successive draft genome sequence analysis of AmCP-positive strains revealed that all the DADH biosynthetic genes (homologs of vzb23, vzb25, vab24, vzb27, vzb28, vzb9, and vzb26) were encoded in the vicinity of the gene encoding AmCP in most sequenced strains, suggesting that these strains may biosynthesize DADH. However, the other features of these gene clusters varied. For example, several NRPS genes were located in the flanking regions of genes encoding AmCP. Five strains (SpC080624G1-09, SS080624GE-03, Sp080902JE-04, SoC090715LN-10, and SoC090715LN-17) possessed adenylation (A) domains with the specificity signature12, 13 D V F D F G G V, identical to that of the A domain of Vzb7 (Vzb7-A), which is postulated to activate DADH or a putative biosynthetic intermediate derived from DADH. This finding suggests that these A domains activate a common intermediate (DADH or its derivative) in the same manner as Vzb7-A and utilize it as a building block for putative non-ribosomal peptides. By contrast, NRPSs found around genes encoding AmCPs of TB090715LN-01, TB090715LN-02, SoC090715LN-16, and SpD080624G1-02 lacked A domains with this signature but possessed A domains with different, uncharacterized signatures. Moreover, in 5



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TB090715SC-02, TB090715SC-05, and SoE090715HV-08, NRPS genes were not found in the flanking regions of genes coding for AmCP, suggesting the presence of responsible NRPSs encoded in other regions of the chromosome or the involvement of DADH modification machineries other than NRPSs. This characteristic diversity in the A domains around genes encoding AmCP may imply structural diversity of the compounds biosynthesized by these AmCP-containing gene clusters and suggests that AmCP-mediated machinery provides core molecules for a variety of non-proteinogenic amino acids that are utilized as the biosynthetic intermediate of different unprecedented non-ribosomal peptides. The specificity signatures of the A domains around the genes encoding AmCP identified in this study are listed in Table S4. Further investigations on these AmCP-containing gene clusters will clarify whether these putative NRPSs cooperate with AmCP-mediated machinery to biosynthesize secondary metabolites in these strains. Discovery of s56-p1 by heterologous expression of a putative AmCP-containing gene cluster. Among the 12 AmCP-containing gene clusters, we chose the cluster of Streptomyces sp. SoC090715LN-17 for heterologous expression. The draft genome sequence analysis of Streptomyces sp. SoC090715LN-17 revealed the presence of all the homologs (orf5, orf11, orf12, orf21, orf22, orf23, orf24, and orf28) necessary for DADH biosynthesis in the flanking regions of the genes encoding AmCP (Figure 3). In addition, the analysis also revealed that the cluster contained the putative gene cluster consisting of eight open-reading frames (orfs) (orf37 to orf44) that are conserved in the genomes of a large number of bacteria (Figure S2). Therefore, we postulated that this conserved gene cluster may encode biosynthetic machinery to synthesize a common but previously uncharacterized and peculiar partial structure in some secondary metabolites. Based on these observations, we expected that the machineries using the DADH biosynthetic genes and conserved gene cluster cooperate to produce an unknown natural product in Streptomyces sp. SoC090715LN-17.

6


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a) 1 kb -1

0

1

2

3

4

5

6

7

8

9

11 12 13 14

10

15

16 17 18 19 20 21 22 23

cosA 25 26 27

28

29

30

31

32 33

34

35

36

37

38

39

40

41 4243 44 45 46

47 48

cosB 49

50

24

51

b) ORF

AA

Proposed function

Homologous protein [Origin]

Id/Si

ORF0 ORF1

472 678

hypothetical protein aminotransferase

sporulation associated protein [Streptomyces pactum] asparagine synthase [Streptomyces flavogriseus ATCC 33331]

86/91 69/78

ORF2 ORF3

403 337

hypothetical protein hypothetical protein

DNA polymerase III subunit delta [Streptomyces bingchenggensis BCW-1] hypothetical protein SCAT_2854 [Streptomyces cattleya NRRL 8057 = DSM 46488]

80/87 39/54

ORF4 ORF5

522 278

hypothetical protein glutamate--AmCP ligase

probable exported protease [Streptomyces venezuelae ATCC 10712] Vzb23 [Streptomyces sp. SANK 60404]

62/75 65/76

ORF6 ORF7

590 412

NRPS (A-PCP) cytochrome P450

peptide synthetase [Renibacterium salmoninarum ATCC 33209] cytochrome P450 [Micromonospora aurantiaca ATCC 27029]

64/78 58/73

ORF8 ORF9

1030 256

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

peptide synthetase [Renibacterium salmoninarum ATCC 33209] thioesterase [Streptomyces tsukubaensis NRRL18488]

51/65 56/70

ORF10 ORF11

398 342

amide synthase AmCP-L-glutamyl-5-phosphate reducase

phosphoribosylglycinamide synthetase [Catenulispora acidiphila DSM 44928] Vzb24 [Streptomyces sp. SANK 60404]

79/90 63/74

ORF12 ORF13

290 187

AmCP-L-glutamate kinase adenylsulfate kinase

Vzb25 [Streptomyces sp. SANK 60404] Vzb20 [Streptomyces sp. SANK 60404]

62/70 70/81

ORF14 ORF15

315 451

sulfate adenyltransferase sulfate adenyltransferase


80/88 81/86

ORF16 ORF17

385 267

transporter transporter

sulfate ABC transporter substrate-binding protein [Streptomyces sp. CNT302] ABC transporter ATP-binding protein [Streptomyces pristinaespiralis ATCC 25486]

84/90 84/89

ORF18

296

transporter

transport system integral membrane protein [Streptomyces ambofaciens ATCC 23877]

76/85

ORF19 ORF20

110 234

hypothetical protein phosphopantetheinyl transferase

hypothetical protein [Kitasatospora setae KM-6054] phosphopantetheinyl transferase [Streptomyces pristinaespiralis ATCC 25486]

83/89 45/55

ORF21 ORF22

65 323

amino group carrier protein (AmCP) transketolase


59/78 58/71

ORF23 ORF24

280 431

transketolase aminotransferase


60/71 56-67

ORF25 ORF26

345 191



40/54 52/67

ORF27 ORF28

216 372

hypothetical protein AmCP-DADH carboxypeptidase


49/66 70/78

ORF29 ORF30

1350 383

NRPS (C-A-PCP-TE) dehydrogenase


38/51 40/56

Vzb5 [Streptomyces sp. SANK 60404] two component LuxR family transcriptional regulator [Streptomyces sviceus ATCC

40/48

ORF31

384

dehydrogenase

ORF32

270

regulator

ORF33

58

hypothetical protein

29083] predicted protein [Streptomyces sviceus ATCC 29083]

ORF34 ORF35

987 328

regulator amide synthase

transcriptional activator [Streptomyces sviceus ATCC 29083] phosphoribosylglycinamide synthetase [Streptomyces sviceus ATCC 29083]

ORF36

185


ORF37

383

dehydrogenase

conserved hypothetical protein [Streptomyces sviceus ATCC 29083] acyl-CoA dehydrogenase domain-containing protein [Catenulispora acidiphila

ORF38

437

oxygenase

DSM 44928] L-lysine 6-monooxygenase [Catenulispora acidiphila DSM 44928]

ORF39 ORF40

352 661

oxidoreductase ligase

FAD dependent oxidoreductase [Catenulispora acidiphila DSM 44928] methionine--tRNA ligase [Catenulispora acidiphila DSM 44928]

74/82 78/86

ORF41 ORF42

505 87

ligase hypothetical protein

AMP-dependent synthetase and ligase [Catenulispora acidiphila DSM 44928] hypothetical protein Caci_6316 [Catenulispora acidiphila DSM 44928]

78/86 83/86

ORF43

205

acetyltransferase

ORF44

342

acyltransferase

N-acetyltransferase GCN5 [Catenulispora acidiphila DSM 44928] peptidase C45 acyl-coenzyme A:6- aminopenicillanic acid acyl-transferase

ORF45

257

dehydrogenase

[Catenulispora acidiphila DSM 44928] short-chain dehydrogenase/reductase SDR [Catenulispora acidiphila DSM 44928]

ORF46 ORF47

393 294

esterase acyltransferase

acetyl esterase [Streptomyces sviceus ATCC 29083] 3-oxoacyl-ACP synthase [Catenulispora acidiphila DSM 44928]

69/82 73/83

ORF48 ORF49

87 661


hypothetical protein Caci_6311 [Catenulispora acidiphila DSM 44928] hypothetical protein [Streptomyces violaceusniger Tu 4113]

49/68 46/57

ORF50

652


hypothetical protein Caci_6300 [Catenulispora acidiphila DSM 44928]

62/75

Figure 3. 7


82/89 44/56 79/87 74/83 76/85 81/88 85/90

79/90 76/84 75/84


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a) Gene organization in cosA and cosB. The DADH biosynthetic gene homologs; “vzb23” (red), “vzb24” (yellow), “vzb25” (blue), “vzb27” (light gray), “vzb28” (light gray), “vzb9” (gold), and “vzb26” (green) are encoded in the vicinity region of the gene for AmCP (magenta) together with NRPS genes (dark gray) and the gene cluster distributed among bacteria (brown). b) Proposed functions of each ORF encoded in cosA and cosB from Streptomyces sp. SoC090715LN-17 and amino acid sequence identities/similarities with homologs found in a NCBI BLAST search.

To identify the natural product biosynthesized using these machineries, we planned to clone the approximately 50-kb genomic region containing putative DADH biosynthetic genes and conserved gene cluster (orf37 to orf44); however, due to the limited insert length possible for a single cosmid, it was impossible to clone the target region in a single cosmid. To overcome this limitation, we used two compatible cosmid vectors—the integrative vector pKU465cos14 and the replicative vector pOJ44615—to cover the full length of the cluster. Cosmid libraries of Streptomyces sp. SoC090715LN-17 were constructed using each vector, and the genomic libraries were screened by PCR using orf5 (vzb23 homolog) and orf45 (gene encoding a short-chain dehydrogenase/reductase (SDR) family protein) as probes. As a result, two different cosmids, cosA and cosB, were obtained from the pKU465cos-based and pOJ446-based libraries, respectively. Sequence analyses of cosA and cosB revealed that cosA covered orf0 to orf34 and that cosB covered orf22 to orf50 in a contiguous 67,922-bp region containing 51 ORFs. All of the DADH biosynthetic genes were contained in cosA and half were in cosB, whereas the entire conserved gene cluster (orf37-orf44), which is widely distributed among bacteria, was contained in cosB. Two cosmids were introduced one by one into the heterologous host Streptomyces lividans TK23, yielding the recombinant strain S. lividans TK23::cosA/cosB. When the culture broth of S. lividans TK23::cosA/cosB was analyzed by liquid chromatography mass spectrometry (LC-MS), the recombinant cells produced a new compound: s56-p1 (1) (Figure 4a). To isolate 1 for characterization, the culture broth of the recombinant cells was fractionated with an anion exchange column and successive preparative reverse-phase column chromatography. Since 1 was acid and base labile, the pH was adjusted to neutral during purification. The fraction containing 1 was then subjected to preparative LC and high-performance LC (HPLC) to obtain purified 1. The LC-MS data of 1 showed a molecular ion peak at m/z 479.1564 8


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[M+H]+, which corresponded to the molecular formula of C16H27N6O9S (calcd. as 479.1555). An LC-MS analysis of the metabolites of Streptomyces sp. SoC090715LN-17 revealed that the parental strain also produced 1, although to a markedly lesser degree than the S. lividans TK23 transformant. The one-dimensional (1D) nuclear magnetic resonance (NMR) spectra (1H and

13

C) and the

two-dimensional (2D) NMR spectra (1H-1H correlation spectroscopy (COSY), 1H-13C heteronuclear single quantum coherence (HSQC), 1H-13C heteronuclear multiple bond correlation (HMBC) and 1H-15N HMBC spectra) of 1 led to the assignment of the full structure of 1 (Figure 4a, Table S5, and Figures S3-S8). Briefly, 1 is a dipeptide compound with a glycine at the N terminus and a non-proteinogenic amino acid with an N-hydroxy pyrrolidine ring at the C terminus. N-acetyl cysteine is covalently bound to C16 via a thioether bond. The most interesting feature of 1 is the glyoxylate hydrazone unit bound to the N-hydroxy pyrrolidine ring with an ester bond, which is rare in natural products. 1 exhibits absorption at λmax 267 nm, similar to methylglyoxylate hydrazone (λmax at 269 nm).16 This structure was supported by 1H-15N HMBC, in which H13 is correlated with 2 nitrogen atoms (C=N (361.4 ppm) and -NH2 (121.0 ppm)) (Figure S7), confirming the presence of the hydrazone group. The treatment of 1 with acid in the presence of p-(dimethylamino)benzaldehyde yielded an azine product with λmax at 485 nm, indicating the generation of hydrazine upon the acid hydrolysis of 1 (Figure S9).17 This result clearly demonstrates the presence of a covalent linkage between nitrogen atoms and, thus, a unique hydrazone structure in 1. To investigate whether 1 is actually produced via AmCP, we constructed two altered cosmids: one cosmid was ∆orf5cosA, in which orf5, a homolog of vzb23 in cosA encoding a putative enzyme that catalyzes the formation of an iso-peptide bond between the α-amino moiety of the substrate glutamate and the γ-carboxyl moiety of the C-terminal glutamate residue of AmCP, was inactivated by replacement with the aac(3)IV gene, conferring apramycin resistance through the λ-Red recombination system.18 The other cosmid was cosBhph, which is a cosB modified by replacing the aac(3)IV gene in a vector region with the hph gene, thereby conferring hygromycin B resistance. The recombinant S. lividans TK23 cells harboring both ∆orf5cosA and cosBhph had no ability to produce 1 (Figure 4a), indicating that orf5 is actually responsible for the biosynthesis of 1. This result also implies that 1 is biosynthesized using 9



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AmCP. Notably, an LC-MS analysis of S. lividans TK23::cosA/pOJ446 revealed the accumulation of compound 2, which exhibited a molecular ion peak at m/z 336.1227 [M+H]+ corresponding to the molecular formula of C12H22N3O6S (calcd. as 336.1224) (Figure 4b). The production of 2 was abolished in S. lividans TK23:: ∆orf5cosA/cosBhph, indicating that 2 is also derived from AmCP-mediated machinery. This compound was isolated from the culture broth of S. lividans TK23::cosA using activated charcoal followed by anion exchange and successive reverse-phase column chromatography. The 1D NMR spectra (1H and

13

C) and 2D NMR spectra (1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC) led to the

assignment of the full structure of 2 (Figure 4b, Table S6, Figures S10-S14) as a core structure of 1 presumably derived from the DADH molecule. This result indicates that all the genes required for the biosynthesis of the core structure of 1 are encoded in cosA.

a

H N

H2 N O O H2 NN

1

O

O

b

OH

H2 N

N OH

HO

O S HO O

S

O

HO

N H

2

(2)

S. lividans TK23::cosA/cosB

S. lividans TK23::cosA/pOJ446

Streptomyces sp. SoC090715LN-17

S. lividans TK23::cosA/cosB

S. lividans TK23::∆orf5cosA/cosBhph

Streptomyces sp. SoC090715LN-17 S. lividans TK23::∆orf5cosA/cosBhph

S. lividans TK23::cosA/pOJ446 0.6

1.2 1.8 Minutes

2.4

O

N H

O

s56-p1 (1)

0.0

OH NH

3.0

0.0

1.25

2.5 Minutes

3.75

5.0

Figure 4. a) LC-MS analyses of metabolites of S. lividans TK23 transformants and Streptomyces sp. SoC090715LN-17 and structure of 1. Extracted ion chromatograms for 1 (m/z 479.1555) from S. lividans TK23 harboring cosA/pOJ446, cosA/cosB, and ∆orf5cosA/cosBhph, respectively, and Streptomyces sp. SoC090715LN-17 are shown. b) LC-MS analyses of metabolites of S. lividans TK23 transformants and Streptomyces sp. SoC090715LN-17 and structure of 2. Extracted ion chromatograms for 2 (m/z 336.1224) from S. lividans 10


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TK23 harboring cosA/pOJ446, cosA/cosB, and ∆orf5cosA/cosBhph, respectively, and Streptomyces sp. SoC090715LN-17 are shown.

Gene knockout experiments and identification of putative biosynthetic intermediates produced in mutants. Three NRPS genes (orf6, orf8, and orf29) are present in the biosynthetic gene cluster of 1. Given that 1 is a dipeptide compound consisting of glycine and a DADH-derived amino acid, these NRPSs were first postulated to be involved in the biosynthesis of 1. A sequence analysis demonstrated that the domain architectures of ORF6, ORF8 and ORF29 were A-PCP, A-PCP-C and C-A-PCP-TE, respectively (PCP: peptidyl carrier protein domain, C: condensation domain, and TE: thioesterase domain). Further sequence analysis of the NRPS genes revealed that the ORF6-A and ORF8-A had specificity signatures: D A F L Q G L A and D A L W L G G I, respectively. Comparison of the signature of ORF6-A to all available models provided a negative result; therefore, whether this domain participates in the biosynthesis of 1 remains unclear. By contrast, ORF8-A was predicted to accept valine as a substrate. However, given that valine is not used as a building block of 1, the involvement of ORF8 in the biosynthesis of 1 is unlikely. The ORF29-A possesses the signature D V F D F G G V, the same as that of Vzb7-A in the vazabitide A biosynthetic gene cluster, which is postulated to activate DADH or a DADH-derived building block for loading the compound on the phosphopantetheinyl group of ORF29-PCP. Similar to the biosynthesis of vazabitide A, we expected that the dipeptide formation between the DADH-derived building block on ORF29-PCP and glycine building block that may be tethered to a PCP domain of another NRPS could occur in the biosynthesis of 1 via the function of ORF29-C. However, this scenario is unlikely because the catalytic motif “HHxxxDG”19, which is necessary for peptide bond formation, is replaced with 165PALVCDS in ORF29-C (Figure S15), indicating that this domain is probably inactive in condensing the building block tethered on ORF29-PCP and the glycine moiety. Therefore, the peptide bond between glycine and the DADH-derived molecule in 1 is presumably formed via a different mechanism. ORF10, which is encoded in a flanking region of the vzb24 homolog, belongs to the ATP grasp family. Because this protein family utilizes ATP to activate a carboxyl group, we hypothesized that 11



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Page 12 of 20

ORF10 was responsible for peptide bond formation in 1. To verify this hypothesis, a modified cosmid

∆orf10cosA was constructed and introduced together with cosBhph into S. lividans TK23 to yield S. lividans TK23:: ∆orf10cosA/cosBhph. An LC-MS analysis of this mutant revealed that the production of 1 was abolished, indicating the involvement of ORF10 in the biosynthesis of 1 (Figure S16a). Interestingly, a closer inspection of the metabolite profile of the knockout mutant revealed that accumulation of new compound 3, which exhibited a molecular ion peak at m/z 406.1390 [M+H]+ corresponding to the molecular formula of C14H24N5O7S (calcd. as 406.1391) (Figure S16b). This ion peak was also present in the fragmentation data of 1, suggesting that 3 has a partial structure of 1. The assignment of this fragment ion based on the structure of 1 suggests that it is a fragment of 1 lacking both the glycine residue and N-hydroxyl group of the pyrrolidine ring (Figure S17). Taken together, these results suggest that ORF10 plays a role in peptide bond formation in the biosynthesis of 1. Furthermore, the glycine residue may be ligated to the DADH-derived core molecule after the formation of the ester bond that connects the pyrrolidine ring with the glyoxylate hydrazone unit. We currently hypothesize that the loading of DADH or its derivative on ORF29-PCP is necessary for ring formation of DADH-derived core molecule; however, this remains to be verified. Next, to investigate the involvement of the conserved gene cluster (orf37–orf44) in the biosynthesis of 1, we constructed several modified cosmids. orf37, orf38, orf39, orf40, orf41, orf43, and orf44 in cosBhph were each individually substituted for the aac(3)IV gene using the λ Red recombination system

to

generate

the

modified

cosmids

∆orf37cosBhph,

∆orf38cosBhph,

∆orf39cosBhph,

∆orf40cosBhph, ∆orf41cosBhph, ∆orf43cosBhph, and ∆orf44cosBhph, respectively. Each of these modified cosmids was introduced into S. lividans TK23 harboring cosA to yield a knockout mutant. LC-MS analyses of the culture broths of these mutants revealed that all mutants abolished the production of 1, suggesting the necessity of these orfs in the biosynthesis of 1 (Figure S18). By contrast, all of the mutants retained the ability to produce 2, a DADH-derived core molecule that lacks the glyoxylate hydrazone unit (Figure S19), but failed to produce 3, which is a putative conjugate of a DADH-derived core molecule and the glyoxylate hydrazone unit (Figure S20). The amino acid sequence of ORF38, which is annotated as 12


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L-lysine 6-monooxygenase, has 32% identity with that of KtzI, an ornithine N-hydroxylase involved in the biosynthesis of the piperazic acid of kutzneride.20 KtzI catalyzes the hydroxylation of the primary amine in the ornithine side chain by nicotinamide adenine dinucleotide phosphate (NADPH) in a flavin-dependent manner.21 Although the transformation steps from N5-hydroxylated ornithine to piperazic acid have not yet been elucidated experimentally, the conversion of a primary amine to its corresponding hydroxylamine is required for the formation of the covalent N-N bond. Therefore, we postulate that ORF38 participates in the formation of the covalent N-N bond of 1. These genomic contexts also suggest that all members of the cluster—orf37, orf39, orf40, orf41, orf42, orf43, and orf44—functionally correlate with orf38. Based on these observations, we also hypothesize that the conserved gene cluster is involved in the biosynthesis of the glyoxylate hydrazone unit in 1. However, further investigations are necessary to verify this hypothesis. Proposed biosynthetic pathway of s56-p1. The observations obtained for gene knockout mutants prompted us to propose a convergent biosynthetic pathway of 1 as described in Figure 5. The DADH molecule synthesized via AmCP-mediated machinery is converted to 2, possibly through an unstable 1-azabicyclo[3.1.0]hexane-containing amino acid. The glyoxylate hydrazone unit biosynthesized via the conserved gene cluster encoded in cosB is connected to 2 to yield 3, which is then converted to 1 by the addition of the glycine residue and successive N-hydroxylation of the pyrrolidine ring (route a). ORF7 exhibits sequence similarity to cytochrome P450, which catalyzes a wide variety of oxidation reactions, including hydroxylation. Therefore, we hypothesize that ORF7 plays a role in the N-hydroxylation of the pyrrolidine ring in the biosynthesis of 1. All of the compounds produced by the heterologous host in this study were mercapturic acid derivatives, suggesting that these compounds are the products of a mycothiol (MSH)-dependent detoxification system.22, 23 MSH is a low-molecular-weight, thiol-containing compound consisting of a 1D-myo-inositol-2-amino-2-deoxylglucopyranoside residue (GlcN-Ins) and N-acetylcysteine attached to the amino group of GlcN, forming an amide bond.24, 25 In general, the thiol moiety of MSH attacks electrophilic toxins, such as alkylating agents, to form S-conjugates, which are hydrolyzed by amidase Mca to generate detoxified toxins with an N-acetyl cysteine adduct (mercapturic acid derivative) and GlcN-Ins.26 Subsequently, the modified toxins are 13



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Page 14 of 20

exported from the cell for complete detoxification. Therefore, it seems unlikely that all the mercapturic acid derivatives 1, 2, and 3 found in this study are products directed by the gene cluster on cosA and B. Instead, we postulate alternative intermediates in this biosynthesis that are not mercapturic acid derivatives. Because the biosynthetic gene cluster of 1 contains many biosynthetic gene homologs of vazabitide A, which contains the azabicyclo ring, we presume that compounds containing this moiety are “real” compounds synthesized by this cluster (route b). In azinomycins, the azabicyclo ring functions as a center of the alkylation activity of the purine base’s N7 atom through electrophilic attack.27 Although these compounds have not yet been detected in metabolites of the parental strain or S. lividans TK23 transformants, the structures of 1–3 are the same in that they have methylene group linked to N-acetylcysteine via thioether bonds, suggesting the nucleophilic attack of the thiol moiety of MSH to the azabicyclo ring. DADH

Glu O H 2N

OH

O

OH

AmCPmediated machinery

O H 2N

OH

NH 2

ORF5, ORF11, ORF12, HO ORF21, ORF22, ORF23, ORF24, ORF28

OH

route b O H2NN

H2 NN

H2N

OH ORF10

O O

H2 NN

O O H 2 NN

O H 2 NN ORF7

O

O

O OH

HO S

(N-hydroxylation)

N H

OH NH

O

ORF10

O

1

H2 N

NH

HO

HO

Mca

O

N OH

S

MSH

Mca

H2 N

route a

HO

MSH

OH

O

OH

N

ORF7 (N-hydroxylation)

O

H2 N N

O

MSH

H N

OH

O

N

O

Mca

H2 N

O

O

O

H N

H2N

OH

S

O

N H

3

HO

O

N H

O

2

Figure 5. Proposed biosynthetic pathway of 1 based on the observations of gene knockout experiments

Conclusions. We screened numerous actinomycetes strains to assess their potential to produce the recently discovered non-proteinogenic amino acid, DADH, which is biosynthesized using 14


Page 15 of 20

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AmCP-mediated machinery. We identified 12 actinomycetes strains possessing genes encoding AmCP. The combination of the heterologous expression of the targeted putative gene cluster and high-resolution LC-MS analysis of the transformant led to the discovery of novel natural product 1, which was produced in trace amounts in the parental strain under laboratory culture conditions. Although the characterization of a putative gene cluster is highly challenging, this bioassay-free strategy using a genome mining approach is a promising method to discover novel natural products. DADH is a key core molecule that expands the structural diversity of secondary metabolites. Therefore, the gene encoding AmCP is a good target for further exploration of structurally diverse novel natural products. Notably, 1 contains unique functional groups that are markedly different from those of vazabitide A and azinomycins. Indeed, one of the most interesting points of 1 is its glyoxylate hydrazone unit. More than 200 natural products containing covalent nitrogen-nitrogen bonds have been isolated to date;28 however, the biosynthetic mechanism of this bond remains poorly understood.29 Further investigations of the biosynthesis of 1 would provide insights into the mechanism underlying nitrogen-nitrogen bond formation.

METHODS Strains, plasmids, and oligonucleotides used in this study were listed in Tables S1, S2, and S3, respectively, in supporting information. PCR screening of the gene encoding AmCP was performed using Gotaq® green master mix (Promega) under the following condition: 1 cycle of 98.0 ˚C for 5.0 min; 34 cycles of 95.0 ˚C for 1.0 min, 56.4 ˚C for 30 sec, and 72.0 ˚C for 15 sec; and 15.0 ˚C hold. Bioinformatics analyses of AmCP-containing gene clusters were performed by a BLAST search of the NCBI non-redundant protein database, and NRPS/PKS analysis-Website. Streptomyces sp. SoC090715LN-17 and recombinant S. lividans TK23 were cultured in synthetic medium (6 % glucose, 0.2 % NaCl, 0.05 % K2HPO4 (pH7.0), 0.01 % MgSO47H2O, 0.2 % (NH4)2SO4, 0.2 % of yeast extract, 0.005 % of FeSO47H2O, 0.005 % MnSO44H2O, 0.005 % ZnSO47H2O, and 0.5 % CaCO3), and fermentation medium (2.5 % soluble starch, 1.5 % soybean meal, 0.2 % dry yeast, and 0.4 % CaCO3, and the pH was adjusted to 7.2) supplemented with 1.0 % Amberlite® XAD7HP (Sigma-Aldrich), respectively, and cultured at 27 ˚C with 15



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Page 16 of 20

shaking at 180 rpm for 4 days. Culture broths were analyzed by the Triple TOF® 5600 system (ABsciex) coupled to the Nexera UHPLC system (Shimadzu) equipped with an ACQUITY UPLC® BEH HILIC 1.7 µm 2.1 × 50 mm column (Waters). Compounds 1 and 2 were isolated from the culture broths of S. lividans TK23::cosA/cosB and S. lividans TK23::cosA, respectively, by following the procedure described in supporting information. Gene inactivation was accomplished by the λ Red recombination system. Detailed materials and methods are provided in supporting information.

ASSOCIATED CONTENT Supporting information Additional materials and methods, tables and figures.

Accession Codes The nucleotide sequence of the s56-p1 biosynthetic gene cluster has been deposited in DDBJ/EMBL/GenBank with accession no. LC177423. The accession numbers for the nucleotide sequences of NRPS genes around genes encoding AmCPs found in this study are as follows: SS080624GE-03_NRPS1,

(LC177424);

SS080624GE-03_NRPS2,

(LC177425);

Sp080902JE-04_NRPS1, (LC177426); Sp080902JE-04_NRPS2, (LC177427); Sp080902JE-04_NRPS3, (LC177428);

Sp080902JE-04_NRPS4,

(LC177429);


(LC177430);


(LC177431);


(LC177432);

SoC090715LN-10_NRPS2,

(LC177433);


(LC177434);


(LC177435);

TB090715LN-01_NRPS1,

(LC177436);


(LC177438);

SpD080624G1-02_NRPS2,

(LC177440);

TB090715LN-02_NRPS1,

(LC177437);

SpD080624G1-02_NRPS1, SpD080624G1-02_NRPS3,

(LC177439); (LC177441);

SpC080624G1-09_NRPS1,

SoC090715LN-10_NRPS1, (LC177443).

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] Present Addresses 16


(LC177442);

and

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Dr. F. Hasebe: Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan Dr. Y. Shiwa: Iwate Tohoku Medical Megabank Organization, Iwate Medical University Disaster Reconstruction Center, Iwate 028-3694, Japan

Notes The authors declare no competing financial interests.

Acknowledgments This work was supported in part by JSPS KAKENHI Grant No. 24228001 (M. Nishiyama) and the Japan Foundation for Applied Enzymology (M. Nishiyama). This study was also funded by the MEXT-supported Program for Strategic Research Foundation at Private Universities, 2013–2017 (S1311017), as well as a grant for “Project focused on developing key technology of discovering and manufacturing drug for next-generation treatment and diagnosis” from METI, Japan (to K. Shin-ya and T. Kuzuyama). We thank for Professor Haruo Ikeda (Kitasato University) for providing us with the λ Red recombination system, pKU487 and pKU493hph.

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Supporting Information Available: This material is available free of charge via the Internet.

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Graphical Table of Contents

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Genome Mining of Amino Group Carrier Protein-Mediated Machinery

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