marine drugs Article

Biosynthetic Functional Gene Analysis of Bis-Indole Metabolites from 25D7, a Clone Derived from a Deep-Sea Sediment Metagenomic Library Xia Yan 1,† , Xi-Xiang Tang 2,3,† , Dan Qin 4 , Zhi-Wei Yi 2,3 , Mei-Juan Fang 1 , Zhen Wu 1, * and Ying-Kun Qiu 1, * 1 2 3 4

* †

School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen 361102, China; [email protected] (X.Y.); [email protected] (M.-J.F.) Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography State Oceanic Administration, Xiamen 361005, China; [email protected] (X.-X.T.); [email protected] (Z.-W.Y.) South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Xiamen 361005, China Key Laboratory of Urban Pollutant Conversion, Institute of Urbane Environment, Chinese Academy of Sciences, Xiamen 361021, China; [email protected] Correspondence: [email protected] (Z.W.); [email protected] (Y.-K.Q.); Tel./Fax: +86-592-218-9597 (Z.W.); +86-592-218-9868 (Y.-K.Q.) These authors contribute equally to this paper.

Academic Editor: Danielle Skropeta Received: 25 December 2015; Accepted: 13 May 2016; Published: 1 June 2016

Abstract: This work investigated the metabolites and their biosynthetic functional hydroxylase genes of the deep-sea sediment metagenomic clone 25D7. 5-Bromoindole was added to the 25D7 clone derived Escherichia coli fermentation broth. The new-generated metabolites and their biosynthetic byproducts were located through LC-MS, in which the isotope peaks of brominated products emerged. Two new brominated bis-indole metabolites, 5-bromometagenediindole B (1), and 5-bromometagenediindole C (2) were separated under the guidance of LC-MS. Their structures were elucidated on the basis of 1D and 2D NMR spectra (COSY, HSQC, and HMBC). The biosynthetic functional genes of the two new compounds were revealed through LC-MS and transposon mutagenesis analysis. 5-Bromometagenediindole B (1) also demonstrated moderately cytotoxic activity against MCF7, B16, CNE2, Bel7402, and HT1080 tumor cell lines in vitro. Keywords: deep-sea sediment; metagenomic clone; secondary metabolites; diindole derivatives; biosynthetic functional genes; cytotoxicity

1. Introduction Marine microorganisms exhibit unique metabolic properties because of the particularity of marine environments; as a result, many novel chemical structures are very complex and diverse. Many antitumor, antibacterial, and anti-inflammatory bioactive substances have been recognized and investigated. Deep marine subsurface sediments are extensive microbial habitats on Earth; these sediments contain numerous undeveloped functional gene clusters to encode the biosynthesis of natural products. However, numerous microorganisms cannot be cultivated [1,2]. Metagenomics, which involves culture-independent methods to access the collective genomes of natural bacterial populations, is applied to investigate secondary metabolites produced by large collections of bacteria that exist in the environment but remain recalcitrant to culturing [3,4]. In metagenomics, microbial DNA is directly extracted from environmental samples and cloned into appropriate educable host cells [5–7]; as such, metagenomics has been employed to utilize

Mar. Drugs 2016, 14, 107; doi:10.3390/md14060107

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Mar. Drugs 2016, 14, 107

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microbial genes from extreme environments, which are associated with the production of bioactive small molecules. We previously isolated a new compound and found a potent analgesic activity on fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) from the deep-sea sediment metagenomic clone 11F6 [8]. In further studies on deep-sea sediment eDNA libraries, two new indole alkaloids were isolated from a cytotoxic clone coded QD15 [9]. In addition to new metabolites from metagenomic libraries, their biosynthetic pathways should be elucidated because these pathways link genetic materials to biocatalytic enzyme functions and bioactive metabolites. The mechanisms by which metabolites are produced under ambient conditions can be determined by exploring biosynthesis-related relevant precursors and enzymes. On the basis of biosynthesis patterns, we can add the same or similar precursors or related enzyme inhibitors to obtain new structures or similar secondary metabolites. Our study investigated the metabolites of a deep-sea sediment metagenomic clone 25D7 with functional gene clusters of hydroxylase; this study also determined the proposed biosynthetic pathways of the metabolites. 5-Bromoindole was added to the culture medium to trace the secondary metabolites produced by the functional genes and to guide separation. As a result, two new brominated bis-indole metabolites, namely, 5-bromometagenediindole B (1) and 5-bromometagenediindole C (2), were isolated through LC-MS. The biosynthetic functional genes of the two new compounds were revealed through HPLC-MS and transposon mutagenesis analysis. 5-Bromometagenediindole B (1) demonstrated moderately cytotoxic activity against MCF7, B16, CNE2, Bel7402, and HT1080 tumor cell lines in vitro. By contrast, 5-bromometagenediindole C (2) did not elicit cytotoxic effects on the tumor cell lines. 2. Results and Discussion 2.1. Bromo-Substituted Substrate Fermentation and LC-MS-Guided Separation Our previous studies on the metabolites of metagenomic clones indicated that clones with exogenous genes can use indole generated by Escherichia coli as a material to synthesize new structures. In our present study, 5-bromoindole was added to the culture medium of 25D7 to obtain special metabolites produced by heterologously expressed functional genes and to verify their proposed biosynthetic pathways. The newly generated metabolites and their biosynthetic byproducts could be easily located through LC-MS. The isotope ion peak pair of [M]+ /[M + 2]+ (1:1) introduced by the bromo atom may emerge. The metabolites were also separated from metagenomic clone fermentation broth under the guidance of LC-MS. The total ion chromatogram of the crude extracts of 25D7 (Figure 1a) revealed the quasi-molecular ion peaks of brominated metabolites at a retention time (tR ) of 28.96 min: 340/342 [(M–OH + H)+ ], 359/361 [(M + H)+ ], and 380/382 [(M + Na)+ ]. The total extract was placed in a silica gel column and an ODS column for further separation. Each separated fraction was analyzed through LC-MS to specifically locate the target fraction with the brominated metabolites. Two new brominated bis-indole metabolites, namely, 5-bromometagenediindole B (1) and 5-bromometagenediindole C (2), were separated. The key by products of butanone addition, which involves nucleophilic addition when extraction is performed with butanone, were detected in the HPLC-MS spectra at tR of 24.09 min with m/z 298/300 [(M + H)+ ] and 319/321 [(M + Na)+ ] (Figure 1b). These by products were then isolated. The related biosynthetic products and intermediate hydroxylated indoles with or without 5-bromo substituent were obtained or detected through HPLC-MS (Supplementary Materials).

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(a) Total extract of 25D7

TIC of Fr. 6

(b) Total extract of 25D7

 

Figure 1. Brominated metabolites in the total extract and separated fractions determined through Figure  1. (a) Brominated  metabolites  in  found the  total  extract  fractions  determined  through  HPLC-MS. Brominated metabolites in the totaland  ion separated  chromatograms (TICs) of the total extract (a)  Brominated  found  in by the  total  ion  chromatograms  total  ofHPLC‐MS.  25D7 and subfraction Fr. 6;metabolites  (b) Key brominated products (butanone addition(TICs)  form)of  of the  the target extract of 25D7 and subfraction Fr. 6; (b) Key brominated by products (butanone addition form) of  metabolites found in the TIC of the total extract of 25D7 at tR 24.09 min. the target metabolites found in the TIC of the total extract of 25D7 at tR 24.09 min. 

2.2. Structural Identification of the Two New Bromodiindoles 2.2. Structural Identification of the Two New Bromodiindoles  5-Bromometagenediindole B (1) was isolated as a colorless crystal with a molecular formula of C16 H115‐Bromometagenediindole B (1) was isolated as a colorless crystal with a molecular formula of  N2 O3 Br on the basis of HRESIMS ([M + Na]+ at m/z 380.9841, calcd. for C16 H11 N2 O3 BrNa) C16H11N2O3Br  on  the  basis  of  HRESIMS  ([M  +  Na]+  at  m/z  380.9841,  calcd.  for  C16H11N2O3BrNa)  coupled with 1D NMR spectroscopic data. The IR spectrum of 1 exhibited absorption bands at coupled with 1D NMR spectroscopic data. The IR spectrum of 1 exhibited absorption bands at 3378  3378−1cm´1 (NH and/or OH) and 1693 (C=O) cm´1 . The UV spectrum of 1 in methanol exhibited cm  (NH and/or OH) and 1693 (C=O) cm−1. The UV spectrum of 1 in methanol exhibited maximum  maximum absorbance at 224 and 262 nm. 2In the sp2 region of 1 H NMR (Table 1), a set of three-spin absorbance  at  224  and  262  nm.  In  the  sp   region  of  1H  NMR  (Table  1),  a  set  of  three‐spin  AMX  AMX proton system signals at δH 7.07 (1H, overlapped, H-4), 7.40 (1H, dd, J = 8.3, 1.5 Hz, H-6), proton system signals at δH 7.07 (1H, overlapped, H‐4), 7.40 (1H, dd, J = 8.3, 1.5 Hz, H‐6), and 6.90  and 6.90 (1H, d, J = 8.5 Hz, H-7). A set of two-spin AX proton system signals at δH 7.06 (1H, d, (1H, d, J =  8.5 Hz,  H‐7). A  set  of  two‐spin AX  proton  system  signals at  δH  7.06  (1H,  d, J =  8.1  Hz,  J = 8.1 Hz, H-51 ), 6.51 (1H, d, J = 8.1 Hz, H-61 ) was also shown. Considering the biotransformation of H‐5′),  6.51  (1H,  d,  J  =  8.1  Hz,  H‐6′)  was  also  shown.  Considering  the  biotransformation  of  5-bromoindole substrates and the two labile proton at δH 10.95 (1H, s) and 10.58 (1H, br. s) in the low 5‐bromoindole substrates and the two labile proton at δ H 10.95 (1H, s) and 10.58 (1H, br. s) in the  field of the 1 H NMR of 1, we could propose the presence of two indole moieties. The 1 H–1 H COSY low  field  of  the  1H  NMR  of  1,  we  could  propose  the  presence  of  two  indole  moieties.  The  1H–1H  correlations of H-6/H-4,7 and H-51 /H-61 indicated that the AMX and AX spin systems were attributed COSY correlations of H‐6/H‐4,7 and H‐5′/H‐6′ indicated that the AMX and AX spin systems were  1 H-NMR spectrum of 1, toattributed  2,3,5-trisubstituted indole and 41indole  ,71 -dissubstituted indole moieties. In the to  2,3,5‐trisubstituted  and  4′,7′‐dissubstituted  indole  moieties.  In  the  1H‐NMR  1 -OH, as shown by the the aromatic hydroxyl signal at δ 9.65 (1H, br. s, –OH) could be attributed to 7 H spectrum of 1, the aromatic hydroxyl signal at δ H 9.65 (1H, br. s, –OH) could be attributed to 7′‐OH,  high field chemical shift of H-61 at δH 6.51. Another hydroxyl signal from alcohol was presented at δH as shown by the high field chemical shift of H‐6′ at δ H 6.51. Another hydroxyl signal from alcohol  6.58 (1H, br. s, –OH). This finding revealed the existence of sp3 carbon at one of the five-membered 3 carbon at one of  was presented at δ H 6.58 (1H, br. s, –OH). This finding revealed the existence of sp 13 C NMR spectra illustrated 13 rings in the two indole moieties. The the differences in the five-membered the five‐membered rings in the two indole moieties. The  C NMR spectra illustrated the differences  2 rings. In addition to benzene carbons, two additional sp carbon signals at δ2C carbon signals at δ 126.3 (s, C-71 a) and in the five‐membered rings. In addition to benzene carbons, two additional sp C  1 126.6 (s, C-3 a) were attributed to one of the indole moieties. A ketone carbon at δC 178.8 (s, C-2) and 126.3 (s, C‐7′a) and 126.6 (s, C‐3′a) were attributed to one of the indole moieties. A ketone carbon at  3 quaternary carbon at 3 quaternary carbon at δ anδCsp δC (s, C-3) belonged toC (s, C‐3) belonged to another indole moiety.  another indole moiety.  178.8 (s, C‐2) and an sp InIn  the HMBC 3, and  and 3a  3a explained  explainedthe  thereduction  reduction the  HMBC spectrum spectrum ofof 1,1, the the correlations correlations of of 3-OH/C-2, 3‐OH/C‐2,  3,  and hydroxyl H–11H  H COSY  COSY correlations  correlations and  hydroxyl substitution substitution ofof C-3 C‐3 inin the the indole indole moiety. moiety.  The The  observed observed  11H– between H‐5′ and H‐6′ and the HMBC correlation between H‐5′ and 6′/C‐7′ confirmed that C‐7′ of 

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the other indole ring was substituted by another hydroxyl. The HMBC signals between H‐5′/C‐2, 3  Mar. Drugs 2016, 14, 107 4 of 10 and 3‐OH/C‐4′ indicated that C‐4′ was attached to C‐3. The molecular framework was established  on  the  basis  of  the  HMBC  correlations  (Figure  2).  The  structure  of  1  is  between H-51 and H-61 and the HMBC correlation between H-51 and 61 /C-71 confirmed that C-71 of the 5‐bromo‐3,7′‐dihydroxy‐1,3‐dihydro‐1′H,2H‐3,4′‐biindol‐2‐one, named 5‐bromometagenediindole B.  1 /C-2, 3 and indoleisolated  ring wascompound  substitutedproduced  by anotherby  hydroxyl. The HMBC signals between H-5 It  other is  a  newly  the  deep‐sea  sediment  metagenomic  clone  25D7  1 indicated that C-41 was attached to C-3. The molecular framework was established on the 3-OH/C-4 through  bromo‐substrate  addition.  The  optical  rotation  of  1  is  close  to  zero,  and  this  finding  1 -dihydroxy-1,3-dihydrobasis ofthat  the HMBC correlations (Figure 2). The structure of 1 is 5-bromo-3,7 indicates  1  is  a  racemate.  Similar  indole‐based  compounds  have  also  been  found  in  different  1 H,2H-3,41 -biindol-2-one, named 5-bromometagenediindole B. It is a newly isolated compound 1 metagenomic  libraries.  For  example,  metagenetriindole  A  and  metagenediindole  A  with  produced by the deep-sea sediment metagenomic clone 25D7 through bromo-substrate addition. moderately cytotoxic activity against CNE2, Bel7402, and HT1080 tumor cell lines in vitro has been  The optical of 1 issediment  close to zero, and this finding indicates is aal. racemate. Similar isolated  from  rotation the  deep‐sea  metagenomic  clone  QD15  [9]. that Abe 1 et  constructed  a  indole-based compounds have also been found in different metagenomic libraries. For example, metagenomic  library  from  the  marine  sponge  Halichondria  okadai  and  then  isolated  a  novel  metagenetriindole A and metagenediindole A with moderately cytotoxic activity against CNE2, compound named halichrome A [10]. Yao Wang et al. obtained three new indole alkaloids, namely,  Bel7402, andA, HT1080 cell lines in vitro has been isolated from deep-sea sediment shewanellines  B,  and  tumor C,  from  Shewanella  piezotolerans  WP3  collected  in the deep‐sea  sediments;  metagenomic clone QD15 [9]. Abe et al. constructed a metagenomic library from the marine sponge shewanelline B can significantly inhibit the growth of human tumor cell line HL‐60 [11]. However,  Halichondria isolated a compound halichrome Aindole  [10]. Yao this  is  the  first okadai report and on then the  isolation  of novel bis‐indole  with  a named phenol‐substituted  ring Wang from eta al. obtained three new indole alkaloids, namely, shewanellines A, B, and C, from Shewanella piezotolerans metagenomic clone.  WP3 collected in deep-sea sediments; shewanelline B can significantly inhibit the growth of human tumor cell Table 1.  line HL-60 However, 1this is the first report on the isolation of bis-indole with a 1H, 13[11]. C NMR data and  H–1H COSY, HMBC correlations of compound 1.  phenol-substituted indole ring from a metagenomic clone. Position  δH (J in Hz) δC, Multiple 1H–1H COSY HMBC  1 13 1 1 1‐NH  10.58 br.s        n.o.  1. Table 1. H, C NMR data and H– H COSY, HMBC correlations of compound 2    178.8  C      1 H–1 H COSY Position δH (J in δC , Multiple HMBC 3    Hz) 77.8  C      3a  137.2  C     n.o. 1-NH 10.58  br.s 178.8 C 4  2 7.07 overlapped  127.7  CH  H‐6  C‐3, 5, 6, 7a  77.8 5  3   113.7  C C     3a 137.2 C 6  7.40 dd (8.3, 1.5)  132  CH  H‐4, 7  C‐4, 5, 7a  4 7.07 overlapped 127.7 CH H-6 C-3, 5, 6, 7a 7  5 6.90 d (8.2)  112.1  CH  H‐6  C‐3, 3a, 5  113.7 C 7a 6 141.8  C      5, 7a 7.40 dd  (8.3, 1.5) 132 CH H-4, 7 C-4, 7 6.90 d (8.2) 112.1 CH H-6 C-3, 3a, 5 1′‐NH  10.95 s      H‐2′, 3′  C‐2′, 3′, 3′a  7a 141.8 C 2′ 1 7.07 overlapped  124.8  CH  H‐1′, 3′  C‐3′, 3′a, 7′a  1 -NH 10.95 s H-21 , 31 C-21 , 31 , 31 a 3′ 21 5.91 br.s  101  CH  H‐2′  C‐2′, 3′a, 7′a  1 1 7.07 overlapped 124.8 CH H-1 , 3 C-31 , 31 a, 71 a 3′a 31 126.6  C    H-21 5.91  br.s 101 CH C-21 ,  31 a, 71 a 126.6 4′ 31 a   122.7  C C     1 4 122.7 C 5′  1 7.06 d (8.1)  117.4  CH  H‐6′  1 C‐2, 3, 3′a, 6′, 7′  5 7.06 d (8.1) 117.4 CH H-6 C-2, 3, 31 a, 61 , 71 6′  1 6.51 d (8.1)  104.8  CH  H‐5′  1 C‐4′, 7′, 7′a  6 6.51 d (8.1) 104.8 CH H-5 C-41 , 71 , 71 a 7′ 71   144  C C     144 1 7′a 7 a   126.3  C C     126.3 3-OH 6.58 br.s C-2, 3, 3a, 41 3‐OH  6.58 br.s        C‐2, 3, 3a, 4′  71 -OH 9.67 br.s n.o. 7′‐OH  9.67 br.s        n.o.  n.o. is not observed. n.o. is not observed. 

1H–11H COSY and HMBC correlations of 5‐bromometagenediindole B (1).  Figure 2. Structure and key  Figure 2. Structure and key H–1 H COSY and HMBC correlations of 5-bromometagenediindole B (1).

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5‐Bromometagenediindole C (2) was isolated as a colorless crystal with a molecular formula of  Mar. Drugs 2016, 14, 107 5 of 10 16H11N2O3Br  on  the  basis  of  HRESIMS  ([M  +  Na]+  at  m/z  380.9845,  calcd.  for  C16H11N2O3BrNa),  C which is the structural isomer of 1. The 5‐bromo‐substituted indole ring moiety is identical to that  13C  NMR  data  could  be  found  in  compound  2.  In  the  1H  in  1  5-Bromometagenediindole because  the  corresponding C1H  (2)and  was isolated as a colorless crystal with a molecular formula of NMR spectrum of compound 2 (Table 2), a set of three‐spin proton ABX system signals emerged at  C16 H11 N2 O3 Br on the basis of HRESIMS ([M + Na]+ at m/z 380.9845, calcd. for C16 H11 N2 O3 BrNa), δH 6.69 (1H, d, J = 7.5 Hz, H‐4′), 6.66 (1H, t, J = 7.5 Hz, H‐5′), and 6.44 (1H, d, J = 7.4 Hz, H‐6′). In the  which is the structural isomer of 1. The 5-bromo-substituted indole ring moiety is identical to that in 1 1H COSY spectra, the correlation between H‐4′/H‐5′/H‐6′ and H‐1′/H‐2′ indicated the presence of  1H‐ because the corresponding 1 H and 13 C NMR data could be found in compound 2. In the 1 H NMR 5′‐H  unsubstituted  7′‐OH  indole  which  differed  in  1.  The  linkage  two  spectrum of compound 2 (Table 2),moiety,  a set of three-spin protonfrom  ABXthat  system signals emergedof  atthe  δH 6.69 indole  by  HMBC  correlations  and  1 ), and 6.44 between  1 ).H‐4/C‐3.  1H (1H, d, Jmoieties  = 7.5 Hz,was  H-41revealed  ), 6.66 (1H, t, Jthe  = 7.5 Hz, H-5 (1H, d, J =H‐2′/C‐3  7.4 Hz, H-6 In the 1 H-The  structure  of  2  is  5‐bromo‐3,7′‐dihydroxy‐1,3‐dihydro‐1′H,2H‐3,3′‐biindol‐2‐one,  named  1 1 1 1 1 1 COSY spectra, the correlation between H-4 /H-5 /H-6 and H-1 /H-2 indicated the presence of 5 -H 1H–1H  COSY  and  HMBC  correlations  are  5‐bromometagenediindole  The  structure  and  key  unsubstituted 71 -OH indoleC.  moiety, which differed from that in 1. The linkage of the two indole shown  Figure  3.  Similar  bromo‐diindole  compounds  without  hydroxyl  at  The position  7′  were  1 /C-3 and moietiesin  was revealed by the HMBC correlations between H-2 H-4/C-3. structure of 2 13C  NMR  signal  of  the  synthesised  by  Chauhan  et  al.  with  5‐bromoistain  and  indole.  The  C‐7′  1 1 1 is 5-bromo-3,7 -dihydroxy-1,3-dihydro-1 H,2H-3,3 -biindol-2-one, named 5-bromometagenediindole reported compound was up‐field shifted compared with that of 2 [12].  C. The structure and key 1 H–1 H COSY and HMBC correlations are shown in Figure 3. Similar bromo-diindole compounds without hydroxyl at position 71 were synthesised by Chauhan et al. with Table 2. 1H, 13C NMR data, and 1H–1H COSY and HMBC correlations of compound 2.  5-bromoistain and indole. The C-71 13 C NMR signal of the reported compound was up-field shifted compared with that of 2 [12]. Position  δH (J in Hz)  δC, Multiple 1H–1H COSY HMBC  1‐NH  10.49 s        n.o.  1 H COSY and HMBC correlations of compound 2. Table 2  2. 1 H, 13 C NMR data, and 1 H–   178.4  C      3    75.4  C      1 H–1 H COSY Position δH (J in Hz) δC , Multiple HMBC 3a    136.5  C      1-NH 10.49 s n.o. 4  7.27 br.d (2.0)  127.8  CH  H‐6  C‐3, 5, 6, 7a  2 178.4 C 5    113.7  C      3 75.4 C 7.42 dd (8.3, 2.0)  136.5 132  CH  H‐4, 7  C‐4, 5, 7a  3a6  C 6.87 d (8.3)  112.2  CH  H‐6  C‐3, 3a, 5  4 7  7.27 br.d (2.0) 127.8 CH H-6 C-3, 5, 6, 7a 57a  113.7 C   141.4  C      6 7.42 dd (8.3, 2.0) 132 CH H-4, 7 C-4, 5, 7a 1′‐NH  10.88 br. S      H‐2′  C‐2′, 3′, 3′a  7 6.87 d (8.3) 112.2 CH H-6 C-3, 3a, 5 7.03 d (2.4)  123.4  CH  H‐1′  C‐3, 3′, 3′a, 7′a  7a2′  141.4 C 3′  115.5  C   H-21 11 -NH 10.88  br. S C-2  1 , 31 , 31 a 1 1 23′a  7.03 d (2.4) 123.4 CH C-3,  31 , 31 a, 71 a 126.9  C   H-1 1 3 4′  115.5 C 6.69 d (7.5)  111.3  CH  H‐5′, 6′  C‐3′, 6′, 7′a  31 a 126.9 C 5′  6.66 t (7.5)  119.9  CH  H‐4′, 6′  C‐3′, 3′a, 6′, 7′  41 6.69 d (7.5) 111.3 CH H-51 , 61 C-31 , 61 , 71 a 1 1 1 6′  6.45 d (7.3)  105.9  CH  H‐4′, 5′  C‐4′, 7′, 7′a  5 6.66 t (7.5) 119.9 CH H-4 , 6 C-31 , 31 a, 61 , 71 1 1 1 144.1  C    ,5 6 7′  6.45 d (7.3) 105.9 CH H-4 C-4  1 , 71 , 71 a 1 77′a  144.1 C   127.4  C      71 a 127.4 C 3‐OH  6.48 br.s        n.o.  3-OH 6.48 br.s n.o. 9.57 br.s        n.o.  717′‐OH  -OH 9.57 br.s n.o. n.o. is not observed.  n.o. is not observed.

  key11 H–1 1 H

Figure 3. Structure and COSY and HMBC correlations of 5-bromometagenediindole C (2). Figure 3. Structure and key  H– H COSY and HMBC correlations of 5‐bromometagenediindole C (2). 

2.3. Biosynthetic Functional Gene Analysis of Bromo‐Diindoles  2.3. Biosynthetic Functional Gene Analysis of Bromo-Diindoles Gene expression-induced changes in metabolites should be elucidated to clarify the role of functional genes in metabolite production. Hence, biosynthetic pathways and associations between functional genes and metabolites should be investigated.

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Gene  expression‐induced  changes  in  metabolites  should  be  elucidated  to  clarify  the  role  of  6 of 10 functional genes in metabolite production. Hence, biosynthetic pathways and associations between  functional genes and metabolites should be investigated.  25D7  contains  a  36,627  bp  insertion  sequence  with  37  predicted  CDSs  (GenBank  accession  25D7 contains a 36,627 bp insertion sequence with 37 predicted CDSs (GenBank accession number: number:  KU883232). The  potential gene functional  clusters  with  six phenol hydroxylase  KU883232). The potential functional clustersgene  of 25D7 withof  six25D7  phenol hydroxylase subunit genes subunit  are 4a shown  in  Figure  and  Table  3.  The  proposed  pathways  of  are shown genes  in Figure and Table 3. The4a  proposed biosynthetic pathwaysbiosynthetic  of 5-bromodiindoles were 5‐bromodiindoles  were  illustrated  as  Figure  4b.  The  expression  of  hydroxylase  functional  genes  illustrated as Figure 4b. The expression of hydroxylase functional genes produced hydroxylase, which produced hydroxylase, which oxidizes C‐2 and C‐3 of the indole ring and produce 5‐bromoisatin,  oxidizes C-2 and C-3 of the indole ring and produce 5-bromoisatin, which is a key by product of this which  is  a  key The by  product  of  this  reaction  [13–15].  The  C‐3  carbonyl  group  of  electrophilic 5‐bromoisatin  is  a  reaction [13–15]. C-3 carbonyl group of 5-bromoisatin is a positively charged group, positively  charged  electrophilic  group,  which  can  be  easily  attacked  by  a  nucleophilic  group.  which can be easily attacked by a nucleophilic group. Hydroxylase also induces the hydroxylation ofHydroxylase also induces the hydroxylation of indole produced by E. coli at position 7. Position 3  indole produced by E. coli at position 7. Position 3 and 4 of 7-hydroxy indole is a negatively and 4 of 7‐hydroxy indole is a negatively charged nucleophilic group. 5‐Bromometagenediindole B  charged nucleophilic group. 5-Bromometagenediindole B (1) is produced when nucleophilic addition (1)  is  produced  when  nucleophilic  addition  occurs  at  C‐4  of  7‐hydroxy  indole.  occurs at C-4 of 7-hydroxy indole. 5-Bromometagenediindole C (2) is generated when nucleophilic 5‐Bromometagenediindole C (2) is generated when nucleophilic addition occurs at C‐3. The optical  addition occurs at C-3. The optical data of the new compounds are close to zero, indicating the data  of  the  new  compounds  are  close  to  zero,  indicating  the  non‐stereoselectivity  of  this  addition  non-stereoselectivity of this addition reaction step. reaction step.  Mar. Drugs 2016, 14, 107

(a)

(b) Figure  4.  Functional  gene  clusters  of  25D7  (a)  and  the  proposed  biosynthetic  pathways  of  Figure 4. Functional gene clusters of 25D7 (a) and the proposed biosynthetic pathways of 5‐bromodiindoles (b).  5-bromodiindoles (b). Table 3. Predicted hydroxylase genes of 25D7.  Table 3. Predicted hydroxylase genes of 25D7. CDS No.  CDS20  No. 21  2022  2123  2224  23 25  24 25

Predicted Gene phenol hydroxylase subunit  Predicted Gene phenol hydroxylase component phL  phenol hydroxylase subunit phenol hydroxylase component phM  phenol hydroxylase component phL phenol hydroxylase P3 protein  phenol hydroxylase component phM phenol hydroxylase conserved region  phenol hydroxylase P3 protein ferredoxin: oxidoreductase FAD/NAD(P)‐binding  phenol hydroxylase conserved region ferredoxin: oxidoreductase FAD/NAD(P)-binding

BLAST Result  83aa, 89% identity to Marinobacter algicola DG893  BLAST Result 333aa, 85% identity to Pseudomonas sp. OX1  83aa, 89% identity to Marinobacter algicola DG893 89aa, 96% identity to Pseudomonas sp. OX1  333aa, 85% identity to Pseudomonas sp. OX1 515aa, 94% identity to M. algicola DG893  89aa, 96% identity to Pseudomonas sp. OX1 119aa, 84% identity to M. algicola DG893  515aa, 94% identity to M. algicola DG893 353aa, 96% identity to M. algicola DG893  119aa, 84% identity to M. algicola DG893 353aa, 96% identity to M. algicola DG893

Considering the proposed biosynthetic scheme, debromo derivatives should exist in the extract.  Such derivatives were detected. For example, the debromo metagenediindole B/C were found in the  Considering the proposed biosynthetic scheme, debromo derivatives should exist in the extract. HPLC‐MS spectra at Rt 20.22 min with m/z 281 [(M + H)+] and 303 [(M + Na)+]. Indigo and indirubin,  Such derivatives were detected. For example, the debromo metagenediindole B/C were found in the including 5‐bromo substituted derivatives, were also produced by catalytic hydroxylation followed  HPLC-MS spectra at Rt 20.22 min with m/z 281as 5‐hydroxyl indole  [(M + H)+ ] and 303 [(M + Na)+ ]. Indigo and indirubin, by  auto‐oxidation.  Other  by‐products,  such  and 5‐bromo‐6‐hydroxyl indole,  including 5-bromo substituted derivatives, were also produced by catalytic hydroxylation followed by were isolated and their spectra data were given in Supplementary Materials.  auto-oxidation. Other by-products, such as 5-hydroxyl indole and 5-bromo-6-hydroxyl indole, were In vitro transposon mutagenesis was conducted and  HR‐ESI‐MS  was  performed  to locate the  isolated and their spectra data were given in Supplementary Materials. genes  responsible  for  bromo‐diindole  production.  25D7  appeared  brown  in  the  culture  medium,  Inhundreds  vitro transposon mutagenesis was conducted and HR-ESI-MS was performed to locate the and  of colorless mutants in  LB‐tyrosine  plates  were obtained. The sequencing of the 30  genes responsible for bromo-diindole production. 25D7 appeared brown in the culture medium, and hundreds of colorless mutants in LB-tyrosine plates were obtained. The sequencing of the 30 randomly selected mutants revealed that their sequences were disrupted; this result indicated the high identity

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randomly  selected  mutants  revealed that their sequences were disrupted; this result indicated the  of the genes to bacterial phenol hydroxylase subunit genes (CDS 20–25). The HR-ESI-MS data of high  identity  of  the  genes  to  bacterial  phenol  hydroxylase  subunit  genes  (CDS  20–25).  The  the HR‐ESI‐MS  mutants further that the two new bromo-diindoles and 5-bromoisatin were missing data  illustrated of  the  mutants  further  illustrated  that  the  two  new  bromo‐diindoles  and  (Figure 5). 5‐bromoisatin were missing (Figure 5).  RT: 0.00 - 45.00

NL: 1.44E9 TIC MS 25D7 transposon mutagenesi s

11.48

100 95 90 85 80 75 70

Relative Abundance

65 60

8.36

55 50

15.22

45 40

19.89

35 30

15.47

25

8.55

14.71

4.29

20 15

0.63 0.88 3.82

5

18.03 18.78

13.10

4.38 4.85 6.31

3.91

10

16.63

9.36

20.58 23.22 23.92 24.50

27.27 27.47

30.80 31.77

39.15

34.99

37.53

8.24

39.52 41.12 43.34

0 0

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4

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8

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16

18

20

22 24 Time (min)

25D7 transposon mutagenesis #10038-10484 RT: 23.47-24.51 AV: 447 NL: 5.45E7 T: FTMS + p ESI Full ms [100.00-1000.00]

m/z 298/300 [(M+H)+], 319/321 [(M+Na)+]: Disappeared.

28

15

14

14

13

13

12

12

11

11

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10

9 8 7 6

36

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144.98 173.08

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110.02

154.99

118.09 131.53

1 0 100

34

[(M+Na)+]: Disappeared.

315.16 4

2

32

6 161.08

5

3

30

m/z 340/342 [(M-OH+H)+], 359/361 [(M+H)+], and 380/382

16

15

Relative Abundance

Relative Abundance

16

26

25D7 transposon mutagenesis #12499-13285 RT: 29.22-31.05 AV: 787 NL: 5.04E7 T: FTMS + p ESI Full ms [100.00-1000.00]

264.23

171.15

3

140

217.11

175.03

160

171.15 146.98

2 317.16

224.13 239.09 259.02

120

112.09

219.17

173.08

180

200

220

240

260

267.04 280

309.20 295.14 300 m/z

391.28

321.24 342.24 351.21 320

340

360

383.20 380

395.26 400

413.27 417.19 420

138.08 118.09

1 443.24 454.29 440

460

475.23 480

499.29 500

154.99

413.27 217.10

182.14

163.13

196.02

215.13

315.16

237.07

227.13

239.09

271.19 282.28

301.07

391.28 352.34 355.27

317.16

0 100

120

140

160

180

200

220

240

260

280

300 m/z

320

340

360

381.28

395.26

380

400

419.20 443.24 420

440

454.29 461.25 460

485.29 496.34 480

500

Figure 5. HR‐ESI‐MS analysis of the transposon mutagenesis of 25D7.  Figure 5. HR-ESI-MS analysis of the transposon mutagenesis of 25D7.

2.4. Cytotoxic Activity of 5‐Bromodiindoles  2.4. Cytotoxic Activity of 5-Bromodiindoles The  cytotoxic  activities  of  the  two  new  indole  alkaloids  were  evaluated  using  MCF7,  B16,  The cytotoxic of the alkaloids were evaluated using MCF7,that  B16, CNE2,  Bel7402,  activities and  HT1080  cell two lines new via  indole the  CCK‐8  method  [9].  The  results  revealed  CNE2, Bel7402, and HT1080 cell lines via the CCK-8 method [9]. The results revealed that 5‐bromometagenediindole B (1) demonstrated moderately cytotoxic activity against the five tumor  −1 respectively. By contrast,  5-bromometagenediindole B of 20.34, 16.60, 32.54, 27.48, and 15.26 μg∙mL (1) demonstrated moderately cytotoxic activity against the five tumor cell lines in vitro, with IC50 cell5‐bromometagenediindole C (2) did not elicit cytotoxic effects on the tumor cell lines.  lines in vitro, with IC50 of 20.34, 16.60, 32.54, 27.48, and 15.26 µg¨ mL´1 respectively. By contrast, 5-bromometagenediindole C (2) did not elicit cytotoxic effects on the tumor cell lines. 3. Experimental Section  3. Experimental Section 3.1. General Experimental Procedures  3.1. General Experimental Procedures LC‐MS and HR‐ESI‐MS analyses  were performed  on  a  Dionex  Ultimate  3000 UHPLC  system  LC-MS and HR-ESI-MS were performed onspectrometer  a Dionex Ultimate 3000 UHPLC system coupled  with  a  Thermo  analyses Q‐Exactive  Orbitrap  mass  (Thermo  Fisher  Scientific  coupled with a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific Corporation, Corporation,  Waltham,  MA,  USA)  equipped  with  an  electrospray  ionization  source  (ESI)  and  an  Waltham, MA, USA) equipped with an electrospray ionization source (ESI) and an analytical Cosmosil analytical Cosmosil ODS column (250 mm × 4.6 mm i.d., 5 μm; Cosmosil, Nakalai Tesque Co., Ltd.,  Kyoto, Japan). Preparative HPLC was conducted in a Varian binary gradient LC system (Varian Inc.,  ODS column (250 mm ˆ 4.6 mm i.d., 5 µm; Cosmosil, Nakalai Tesque Co., Ltd., Kyoto, Japan). Corporate,  Santa  Clara,  CA,  USA)  containing  solvent  modules  (PrepStar  218),  a  Preparative HPLC was conducted in a Varian binarytwo  gradient LCdelivery  system (Varian Inc., Corporate, Santa photodiode  detector  335),  and  a  fraction  collector  704)  array by  using  a  Clara, CA, USA)array  containing two(ProStar  solvent delivery modules (PrepStar 218), (ProStar  a photodiode detector preparative Cosmosil  ODS collector column  (250  mm  × 20.0 mm  i.d.,  5  Cosmosil,  Nakalai ODS Tesque  Co.,  (ProStar 335), and a fraction (ProStar 704) by using a μm,  preparative Cosmosil column

(250 mm ˆ 20.0 mm i.d., 5 µm, Cosmosil, Nakalai Tesque Co., Ltd., Kyoto, Japan). UV spectra were

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obtained using a Shimadzu UV-260 spectrometer (Shimadzu Corporation, Tokyo, Japan). IR spectra were determined by using a Perkin-Elmer 683 infrared spectrometer (PerkinElmer, Inc., Waltham, MA, USA) in KBr pellets. Optical rotations were performed by using a JASCO P-200 polarimeter (JASCO Corporation, Tokyo, Japan) with a 5 cm cell. NMR spectra were obtained using a Brucker Avance III 600 FT NMR spectrometer (Bruker Corporation, Billerica, MA, USA), with TMS as an internal standard. Column chromatography was performed on silica gel (Yantai Chemical Industry Research Institute, Yantai, China) and Cosmosil 75 C18 -OPN (75 µm, Nakalai Tesque Co., Ltd., Kyoto, Japan). 3.2. Fermentation Sediment samples for DNA extraction were collected from the subsurface sediments at water depths of 3006 m (102.612575˝ E, 2.022449˝ N) in Southwestern Indian Ocean by the Third Institute of Oceanography of China. The samples were maintained at 4 ˝ C before processing. The sediment samples were pre-cultured in 2216E medium for 3 days. Then, the DNA of the enrichment product was extracted and purified. The size-separated DNAs of 30–40 kb were pooled and end-repaired to blunt end and cloned into fosmid vector. The ligation mixture was packed; the packaged DNA was transformed into E. coli, and a library of 3500 clones was generated. Cytotoxic activity was then screened. The brown pigment-producing 25D7 was selected and characterized. 25D7 was incubated in a 60 L fermentation tank supplemented with 30 µg/mL 5-bromoindole and 0.01% inducer and fermented for 18 h at 37 ˝ C, 200 rpm, and pH 7.0. The supernatant from the fermentation broth was collected by using a continuous flow centrifuge at 60 L/min. A voucher specimen (25D7) has been deposited at the Third Institute of Oceanography, State Oceanic Administration of China. 3.3. Extraction and Isolation The fermentation broth (60 L) was extracted thrice with butanone (v/v 1:1). The butanone phase was evaporated at a reduced pressure to produce the total extract (9.4 g). The total crude extract was analyzed using an analytical Cosmosil ODS-MS column (250 mm ˆ 4.6 mm i.d., 5 µm, Nakalai Tesque Co., Ltd., Kyoto, Japan). The mobile phase used was acetonitrile (A) and water (B) in a linear gradient mode, as follows: A from 5% to 100% and B from 95% to 0% between 0 and 40 min. The flow rate of the mobile phase was 0.4 mL¨ min´1 and the effluents were monitored by a HR-ESI-MS. The total extract was subjected to liquid chromatography on silica gel by using CHCl3 -MeOH as an eluent at gradient elution ratios to produce seven fractions (Fr. 1 to 7). Each fraction was analyzed with HPLC-MS to fine metabolites containing a bromo atom. In Figure 1b, quasi-molecular ion peaks of brominated products appeared in Fr. 6 at a retention time of 27.76 min. Fr. 6 (0.86 g) was subjected to ODS chromatography eluted with MeOH-H2 O (10:90 to 100:0) to yield a subfraction Fr. 6.5. Fr. 6.5 (136.3 mg) was purified through preparative HPLC by using a C18 column (acetonitrile–H2 O, 30:70 to 35:65) to obtain 1 (24 mg) and 2 (2.5 mg). ˝ ´1 Metagenediindole B (1): colorless crystal; rαs29 D 0 (c = 0.1, MeOH); IR (KBr) (νmax ): 3378, 1693 cm . UV (MeOH) λmax (logε): 262 (4.03) and 224 (3.59) nm. 13 C NMR (125 MHz, DMSO-d6 ) and 1 H NMR (600 MHz, DMSO-d6 ) spectral data are listed in Table 1; ESIMS: m/z 380, 382 [M + Na]+ ; HR-ESI-MS: m/z 380.9841, 382.9822 (calcd. for C16 H11 N2 O3 BrNa, 380.9851, 382.9830). ˝ ´1 Metagenediindole C (2): colorless crystal; rαs29 D 0.001 (c = 0.1, MeOH); IR (KBr) (νmax ): 3392, 1652 cm . UV (MeOH) λmax (logε): 261 (4.03), 224 (3.59) nm. 13 C NMR (125 MHz, DMSO-d6 ) and 1 H NMR (600 MHz, DMSO-d6 ) spectral data were listed in Table 2; ESIMS: m/z 380, 382 [M + Na]+ ; HR-ESI-MS: m/z 380.9845, 382.9824 (calcd. for C16 H11 N2 O3 BrNa, 380.9851, 382.9830).

3.4. Transposon Mutagenesis and Sequence Analysis Random transposon mutations were generated with an EZ-Tn5™ insertion kit (Epicenter) in accordance with the manufacturer’s instructions. The 25D7 fosmid and transposon reaction mixture were incubated at 37 ˝ C for 4 h. The reaction was terminated by adding 1 µL of

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stop solution. Then, the mixture was incubated at 70 ˝ C for 10 min, electroporated into competent epi300 cells, and recovered in LAK (100 µg/mL ampicillin and 50 µg/mL kanamycin) plates. Colorless fosmid clones were selected randomly for sequencing (Sangon Inc., Shanghai, China). 3.5. Cytotoxic Activity The cytotoxic activities of these compounds were evaluated using MCF7, B16, CNE2, Bel7402, and HT1080 cell lines by the CCK8 method [9]. Human breast adenocarcinoma cell MCF7, melanoma cell B16, nasopharyngeal cell CNE2, hepatoma cell BEL7402, and osteosarcoma cell HT1080 (CCTCC, Wuhan, China) were grown in Dulbecco’s modified Eagle medium) supplemented with 10% fetal bovine serum and 1% (w/v) penicillin/streptomycin and were seeded as 100 µL aliquots into a sterile 96-well microtiter plate at a titer of approximately 1000 cells per plate and incubated at 37 ˝ C in 5% CO2 for 24 h. Compounds 1 and 2 resuspended in DMSO and a compound-free DMSO control were diluted in a fresh medium and added to the appropriate wells at final concentrations of 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, and 0.20 µg¨ mL´1 . These plates were then cultured for another 72 h. A CCK8 assay was conducted to assess the cytotoxic effects of compounds 1 and 2 on the cells. In brief, 10 µL of CCK8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well, and the 96-well plate was incubated at 37 ˝ C for 2 h. The OD of each well was read at a wavelength of 450 nm to determine the cell survival rate by using a microplate reader (Epoch; Biotek, Winooski, VT, USA). The assay was repeated thrice. IC50 was calculated using Origin 7.5 (OriginLab, Northampton, MA, USA). 4. Conclusions Metagenomics can be applied to investigate the secondary metabolites produced by large collections of bacteria present in extreme environments, such as deep sea, which remain recalcitrant to culturing. Metagenomics is also a major topic of international life science research. With modern molecular biology techniques, biosynthetic gene clusters with specific plasticity can be modified directly and expressed heterologously to create new structures called “artificial” compounds [16,17] with high activity and low toxicity. With these approaches, the production of microbial secondary metabolites can be improved to generate innovative drugs and to increase output. In our study, two new bis-indoles were obtained from 25D7 cultivated with 5-bromoindole, and their biosynthetic functional genes were determined. Our results revealed the heterologous expression and function of the 25D7 gene cluster. Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/14/6/107/s1, Figure S1: Isolated compounds related to the biosynthetic pathway of 5-bromometagenediindole B/C, Figure S2: Compounds related to the biosynthetic pathway of 5-bromometagenediindole B/C, detected by HPLC-MS spectum. Acknowledgments: The project was supported by the International S&T Cooperation Program of China (No. 2015DFA20500), National Basic Research Program of China (973 Program) (No. 2015CB755901). The authors would also like to acknowledge the financial supports from National Natural Science Foundation of China (No. 81273400), Fujian Key Science and Technology Program (No. 2013N0019), Xiamen Science and Technology Program (No. 3502Z20142009), and Xiamen South Marine Center project (13GZP002NF08). Author Contributions: X.X.T. and D.Q. conceived the deep-sea metagenomic strategies and performed the cytotoxic activities assay. X.X.T. and Z.W.Y conducted the tranposon mutagenesis analysis. Z.W. and Y.-K.Q. supervised the project. X.Y. isolated the two new compounds and analyzed the sample by HPLC-HRESIMS. M.J.F. was responsible for structural elucidation. Conflicts of Interest: The authors declare no conflict of interest.

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