molecules

Article Isolation and Biosynthetic Analysis of Haliamide, a New PKS-NRPS Hybrid Metabolite from the Marine Myxobacterium ochraceum

Yuwei Sun 1, Tomohiko Tomura 1, Junichi Sato 1, Takashi Iizuka 2, Ryosuke Fudou 3 and Makoto Ojika 1,*

Received: 11 November 2015 ; Accepted: 31 December 2015 ; Published: 6 January 2016 Academic Editor: Derek J. McPhee

1 Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; [email protected] (Y.S.); [email protected] (T.T.); [email protected] (J.S.) 2 Institute for Innovation, Ajinomoto Co., Inc., Kawasaki, Kanagawa 210-8681, Japan; [email protected] 3 R & D Planning Department, Ajinomoto Co., Inc., Chuo-ku, Tokyo 104-8315, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-52-789-4116; Fax: +81-52-789-4118

Abstract: of marine origin are rare and hard-to-culture microorganisms, but they genetically harbor high potential to produce novel antibiotics. An extensive investigation on the secondary metabolome of the unique marine myxobacterium SMP-2 led to the isolation of a new polyketide-nonribosomal peptide hybrid product, haliamide (1). Its structure was elucidated by spectroscopic analyses including NMR and HR-MS. Haliamide (1) showed cytotoxicity against HeLa-S3 cells with IC50 of 12 µM. Feeding experiments were performed to identify the biosynthetic building blocks of 1, revealing one benzoate, one alanine, two propionates, one acetate and one acetate-derived terminal methylene. The biosynthetic gene cluster of haliamide (hla, 21.7 kbp) was characterized through the genome mining of the producer, allowing us to establish a model for the haliamide biosynthesis. The sulfotransferase (ST)-thioesterase (TE) domains encoded in hlaB appears to be responsible for the terminal alkene formation via decarboxylation.

Keywords: marine myxobacterium; Haliangium ochraceum; haliamide; polyketide; biosynthesis

1. Introduction Myxobacteria are unique Gram-negative characterized by gliding on solid surfaces and formation of multicellular fruiting body [1–3]. Although long been regarded as terrestrial microorganisms, several halophilic myxobacterial strains have been noticed recently [4–8] and found to be excellent producers of novel secondary metabolites, such as polyketides (PKs), non-ribosomal peptides (NRPs) and their hybrids. A recent report suggested that the polyketide synthase (PKS) gene sequences in marine-derived myxobacterial isolates are highly novel (all of the sequences were less than 70% similar to the known ones) [9]. However, studies of the secondary metabolites of marine myxobacteria have been hampered by unfavorable factors such as difficulties in the isolation from environment, slow growth rates, strong tendency of cell aggregation and poor metabolite productivity. Up to now, there are a few groups of antibiotics that have been identified from the marine environment, including haliangicins [10–12], miuraenamides [13,14], salimabromides [15], salimyxins and enhygrolides [16]. Haliangium ochraceum SMP-2 is the first marine myxobacterium, from which we discovered a potent antifungal agent haliangicin [10,11]. During our ongoing investigation on the secondary

Molecules 2016, 21, 59; doi:10.3390/molecules21010059 www.mdpi.com/journal/molecules Molecules 2016, 21, 59 2 of 8

Molecules 2016, 21, 59 2 of 8 Haliangium ochraceum SMP-2 is the first marine myxobacterium, from which we discovered a potent antifungal agent haliangicin [10,11]. During our ongoing investigation on the secondary metabolome of H. ochraceum, a newnew PKS-NRPSPKS-NRPS hybridhybrid compound,compound, haliamide (1), was isolatedisolated and identifiedidentified (Figure1 ).1). InIn thisthis paper,paper, wewe reportreport thethe structuralstructural elucidation,elucidation, bioactivitybioactivity andand thethe decipherment of the biosynthetic system of haliamidehaliamide ((11).).

1 12 11 O 10 1' N H 3

Figure 1. Structure of haliamide (1).

2. Results Results and and Discussion Discussion

2.1. Isolation and Structural Elucidation The marinemarine myxobacteriummyxobacteriumH. H. ochraceum ochraceumSMP-2 SMP-2 showed showed an an optimal optimal NaCl NaCl demand demand of 2% of (2%w/ v(w) for/v) itsfor growthits growth and needsand needs approximately approximately two weeks two forwe theeks maximal for the productionmaximal production of secondary of metabolites.secondary Themetabolites. bacterial cellsThe bacterial from a total cells of 3.5from L cultures a total wereof 3.5 extracted L cultures with were methanol. extracted After with solvent methanol. partitioning After ofsolvent the methanol partitioning extract, of the a hexane–EtOAc methanol extract, (1:1) fractiona hexane–EtOAc was chromatographed (1:1) fraction on was silica chromatographed gel followed by reverse-phaseon silica gel followed HPLC toby give reverse-phase 1.9 mg of haliamide HPLC to give (1) as 1.9 well mg as of haliangicin. haliamide (1) as well as haliangicin. Haliamide (1) displayeddisplayed thethe IRIR absorptionabsorption bandsbands atat 3305,3305, 16351635 and and 1536 1536 cm cm´−11, which indicatedindicated the presence of an amide group. Its molecular formula was determined to be C19H25NO by the the presence of an amide group. Its molecular formula was determined to be C19H25NO by high-resolution ESI-MS. The 1H- and1 13C-NMR13 data of 1 in CDCl3 is summarized in Table 1. Three the high-resolution ESI-MS. The H- and C-NMR data of 1 in CDCl3 is summarized in partial structures, C6H5–, –NH–CH(CH3)–CH=CH– and –CH2–CH(CH3)–CH=CH2, were deduced by Table1. Three partial structures, C 6H5–, –NH–CH(CH3)–CH=CH– and –CH2–CH(CH3)–CH=CH2, wereDQF-COSY deduced correlations by DQF-COSY (Figure correlations 2), in which (Figure proton-carbon2), in which direct proton-carbon connectivity direct was connectivity determined was by determinedthe heteronuclear by the single heteronuclear quantum single coherence quantum (HSQC) coherence experiment. (HSQC) The experiment. remaining The three remaining quaternary three quaternarycarbons were carbons assigned were by assignedthe heteronuclear by the heteronuclear multiple bond multiple correlation bond (HMBC) correlation to the (HMBC) carbonyl to the at C-1′ (δC 166.5) 1and the two sp2 carbons 2at C-2′ (δC 134.8)1 and C-6 (δC 138.0) and connected to the carbonyl at C-1 (δC 166.5) and the two sp carbons at C-2 (δC 134.8) and C-6 (δC 138.0) and connected toabove-mentioned the above-mentioned partial partial structures structures (Figure (Figure 2). The2). Thegeometry geometry of the of thediene diene moiety moiety was was determined determined to be all-E based on the 3J3H3-H4 value of 15.2 Hz and the NOE correlations of H-4/H-12 and H-5/H-7 to be all-E based on the JH3´H4 value of 15.2 Hz and the NOE correlations of H-4/H-12 and H-5/H-7 (Figure2 2).). TheThe benzamidebenzamide structurestructure was was also also supported supported by by the the absorption absorption maximum maximum at at 242 242 nm. nm.

Table 1.1. NMRNMR datadata forfor haliamidehaliamide ((11)) inin CDClCDCl33.

Position δC δH mult. (J in Hz) Position δ δ mult. (J in Hz) 1 20.8C 1.38H d (6.8) 12 47.0 20.8 4.84 ddq 1.38(6.0, d 7.2, (6.8) 6.8) 23 131.9 47.0 5.63 4.84 dd ddq (6.0, (6.0, 15.2) 7.2, 6.8) 34 126.8 131.9 6.46 5.63 dd (10.8, dd (6.0, 15.2) 15.2) 4 126.8 6.46 dd (10.8, 15.2) 5 125.5 5.81 d (10.8) 5 125.5 5.81 d (10.8) 6 138.0 - 6 138.0 - 77 47.4 47.4 1.97 1.97 dd dd(7.6, (7.6, 13.6), 13.6), 2.10 2.10 dd dd(7.2, (7.2, 13.6) 13.6) 88 35.6 35.6 2.36 2.36 dddq dddq (6.8, (6.8, 7.2, 7.2, 7.6, 7.6, 6.4) 6.4) 99 144.2 144.2 5.73 5.73 ddd ddd (6.8, (6.8, 10.4, 10.4, 17.2) 17.2) 1010 112.4 112.4 4.91 4.91 d (10.4), d (10.4), 4.96 4.96 d (17.2) d (17.2) 1111 19.5 19.5 0.95 0.95 d (6.4) d (6.4) 1212 16.7 16.7 1.73 1.73 s s 1 11′ 166.5166.5 - - 1 22′ 134.8134.8 - - 31, 71 126.8 7.77 d (7.5) 3′, 7′ 126.8 7.77 d (7.5) 41,61 128.5 7.43 t (7.5) 4′,6′ 128.5 7.43 t (7.5) 51 131.4 7.49 t (7.5) NH5′ 131.4 - 7.49 6.04 t (7.5) d (7.2) NH - 6.04 d (7.2) Measured at 400 MHz for 1H and 100 MHz for 13C. Measured at 400 MHz for 1H and 100 MHz for 13C.

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Figure 2.2. KeyKey 2-demensional 2-demensional NMR NMR correlations correlations in haliamidein haliamide (1). Bold(1). lines:Bold DQF-COSY,lines: DQF-COSY, curve arrows: curve

arrows:HMBC, HMBC, dotted arrows: dotted NOESY.arrows: NOESY. Figure 2. Key 2-demensional NMR correlations in haliamide (1). Bold lines: DQF-COSY, curve 2.2. Identificationarrows: HMBC, of the dotteBiosyntheticd arrows: PrecursorsNOESY. 2.2. Identification of the Biosynthetic Precursors To obtain supporting information for analyzing the haliamide biosynthetic machinery, we carried 2.2.To Identification obtain supporting of the Biosynthetic information Precursors for analyzing the haliamide biosynthetic machinery, we carried out feeding experiments with stable-isotope labeled compounds that were plausible as biosynthetic out feedingTo obtain experiments supporting with information stable-isotope for analyzing labeled the compounds haliamide biosyn that werethetic plausiblemachinery, as we biosynthetic carried building blocks (Supplementary Materials, Figure S1). The addition of sodium [1,2-13C2]acetate to the buildingout feeding blocks experiments (Supplementary with stable-isotope Materials, Figurelabeled S1).compounds The addition that were of sodiumplausible [1,2- as biosynthetic13C ]acetate to 13 2 culture medium of H. ochraceum led to the enhancement of C-NMR13 signals of C-4 (δ13C 126.8, d, J = 56 Hz), the culturebuilding mediumblocks (Supplementary of H. ochraceum Materials,led to theFigure enhancement S1). The addition of C-NMR of sodium signals [1,2- C of2]acetate C-4 (δC to126.8, the d, C-5 (δC 125.5, d, J = 56 Hz) and C-10 (δC 112.4, s), corresponding13 one intact acetate unit and one J = 56culture Hz), medium C-5 (δC of125.5, H. ochraceum d, J = 56led Hz) to the and enhancement C-10 (δC 112.4,of C-NMR s), corresponding signals of C-4 (δ oneC 126.8, intact d, J = acetate 56 Hz), unit acetate-derived terminal methylene in the haliamide structure (Figure 3). The second feeding andC-5 one ( acetate-derivedδC 125.5, d, J = 56 terminal Hz) and methylene C-10 (δC 112.4, in the s), haliamide corresponding structure one (Figureintact acetate3). The unit second and feedingone 13 experimentacetate-derived with sodium terminal [1- 13methyleneC]propionate in the gave halia twomide isotope-labeled structure (Figure carbons 3). Theof C-7 second (δC 47.4) feeding and C-9 experiment with sodium [1- C]propionate gave two isotope-labeled carbons of C-7 (δC 47.4) and C-9 (δC 144.2).experiment Therefore, with sodium the polyene [1-13C]propionate chain in haliamid gave twoe isotope-labeledis derived from carbons two acetate of C-7 and(δC 47.4) two andpropionate C-9 (δC 144.2). Therefore, the polyene chain in haliamide is derived from two acetate and two propionate units(δ C(Figure 144.2). Therefore,3). The third the polyene feeding chain experiment in haliamid withe is DLderived-[1-13C]alanine from two acetateenriched and 13 twoC nucleus propionate at C-3. units (Figure3). The third feeding experiment with DL-[1-13C]alanine enriched 13C nucleus at C-3. Finally,units the (Figure origin 3). of The the thirdbenzoyl feeding moiety experiment was clarified with toDL-[1- be 13benzoicC]alanine acid enriched because 13C the nucleus carbonyl at C-3. carbon Finally, the origin of the benzoyl moiety was clarified to be benzoic acid because the carbonyl carbon Finally,13 the origin of the benzoyl moiety was clarified to be benzoic acid because the carbonyl13 carbon of [1-13C]benzoic acid was incorporated into the C-11′ position in 1 (Figure 3). Since [1,2-13C2] acetate of [1- C]benzoic13 acid was incorporated into the C-1 position in 1 (Figure3). Since [1,2-13 C2] acetate wasof not [1- incorporatedC]benzoic acid into was theincorporated benzoyl intogroup, the C-1the′ positionbenzoate in unit1 (Figure may 3). be Since derived [1,2- Cvia2] acetateshikimate was not incorporated into the benzoyl group, the benzoate unit may be derived via shikimate pathway pathwaywas not but incorporatednot via polyketide into the one. benzoyl The benzoate group, thunite benzoate as a biosynthetic unit may precursor be derived is notvia veryshikimate common butpathway not via polyketide but not via one.polyketide The benzoate one. The benzoate unit as a unit biosynthetic as a biosynthetic precursor precursor is not is very not commonvery common and has and has been reported for a few natural products such as soraphen A [17] and enterocin [18]. beenand reported has been for reported a few natural for a few products natural products such as soraphensuch as soraphen A [17] andA [17] enterocin and enterocin [18]. [18].

Figure 3. Biosynthetic building blocks of haliamide (1) deduced from feeding experiments. Figure 3. BiosyntheticBiosynthetic building building blocks blocks of haliamide ( 1)) deduced deduced from feeding experiments. 2.3. Proposed Biosynthetic Mechanism of Haliamide (1) 2.3. Proposed Proposed Biosynthet Biosyntheticic Mechanism of Haliamide (1) The chemical structure of haliamide (1) and its biosynthetic precursors described above suggest itThe to be chemical a hybrid structure compound of biosynthesizedhaliamide ( 1) and by PKS its biosynthetic and NRPS. The precursors in silico analysis described of the above genome suggest it to data be a of hybrid H. ochraceum compound using biosynthesized antiSMASH [19] by byrevealed PKS PKS and anda candidate NRPS. NRPS. The Thegene inin cluster, silico silico analysiswhich possesses of the genomeone dataNRPS of of H.H. module ochraceum ochraceum followed usingusing by antiSMASH antiSMASHfour PKS modules [19] [19 revealed] revealedand is ina candidatea a good candidate agreement gene gene cluster, with cluster, the which assembly which possesses possessesof the one NRPSonebiosynthetic NRPS module module followed units followed in 1 by(Figure four by 3). PKS four The modules PKSputative modules haliamideand is and in genea is good in cluster a agreement good (hla agreement) consists with ofthewith one assembly NRPS/PKS the assembly of the biosyntheticof thehybrid biosynthetic gene units (hlaA in units 1) (Figureand in one1 3).(Figure PKS The geneputative3). The(hlaB putativehaliamide), spanning haliamide gene a region cluster gene of ( hla21.7 cluster) consists kbp ( (Figurehla of) consistsone 4a). NRPS/PKS The of one hybridNRPS/PKSbiosynthesis gene hybrid (hlaA of )haliamide geneand (onehlaA ( 1)PKS) andwas gene oneproposed PKS(hlaB gene ),to spanningbegin (hlaB with), spanning a beregionnzoyl a CoA.of region 21.7 Although ofkbp 21.7 (Figure kbpthe (Figureloading 4a). The4a). mechanism of this starter remains unclear due to the absence of the loading module in the hla gene biosynthesisThe biosynthesis of haliamide of haliamide (1) (was1) was proposed proposed to tobegin begin with with be benzoylnzoyl CoA. CoA. Although Although the the loading cluster, the direct condensation of the benzoyl CoA starter and the forthcoming alanine unit on mechanism of of this starter starter rema remainsins unclear unclear due due to to the absence of the loading module in the hla gene cluster,module the 1direct could condensationbe possible as ofa similarthe benzoyl starter CoAloading starter mechanism and the was forthcoming recently reported alanine in unitthe on cluster,biosynthesis the direct of macyranones condensation [20]. of The the C benzoyl domain CoAin HlaA starter may andcatalyze the forthcomingthe formation of alanine the amide unit on module 1 could be possible as a similar starter loading mechanism was recently reported in the modulebond. 1 The could subsequent be possible chain as elongation a similar reactions starter loading take place mechanism by four PKS was modules recently (Module reported 2–5), in the biosynthesis of macyranones [20]. The C domain in HlaA may catalyze the formation of the amide biosynthesiswhich incorporate of macyranones two malonyl-CoAs [20]. The Cand domain two methylmalonyl-CoAs in HlaA may catalyze to theconstruct formation the polyketide of the amide bond.backbone The The subsequent subsequent of 1 (Figure chain chain4a). There elongation elongation are some reactions reactionsunusual featurestake take place place in the by by PKS four four modules. PKS PKS modules modules First, there (Module (Module are some 2–5), 2–5), whichmissing incorporate domains, two including malonyl-CoAs AT domains and in modules two methylmalonyl-CoAs 2 and 5, DH domain in to module construct 3 and theKR domain polyketide backbonein module of of 11 5(Figure(Figure (Supplementary 4a).4a). There There Materials, are are some some Figure unusual unusual S2 ).features The features function in the in of thePKS these PKS modules. missing modules. First,domains First, there may there are be some are missingsomecomplemented missing domains, domains, byincluding some including trans-acting AT doma AT enzymes.ins domains in modules For in the modules missing2 and 5, 2AT DH and doma domain 5, DHins, a domain instandalone module in moduleacyltransferase3 and KR 3 domain and KR in module 5 (Supplementary Materials, Figure S2). The function of these missing domains may be complemented by some trans-acting enzymes. For the missing AT domains, a standalone acyltransferase

Molecules 2016, 21, 59 4 of 8 domain in module 5 (Supplementary Materials, Figure S2). The function of these missing domains may be complemented by some trans-acting enzymes. For the missing AT domains, a standalone Molecules 2016, 21, 59 4 of 8 acyltransferase (Hoch_5652) was found in the genome of H. ochraceum, which harbors the APFH motif(Hoch_5652) that suggests was found this enzyme in the genome to be malonate-specific. of H. ochraceum, which Such harbors substrate the APFH specificity motif wellthat fitssuggests for the malonatethis enzyme incorporation to be malonate-specific. by modules 2Such and substrat 5, as demonstratede specificity well by thefits for feeding the malonate experiments incorporation (Figure3 ). Onby the modules other hand, 2 and no 5, candidatesas demonstrated of trans-acting by the feed DHing and experiments KR domains (Figure were 3). found. On the We other anticipated hand, no that somecandidates oxidoreductases of trans-acting downstream DH and KR to domains the hla cluster, were found. e.g., cytochromeWe anticipated P450 that (Hoch_0804, some oxidoreductases Hoch_0812, Hoch_0813downstream and to Hoch_0814) the hla cluster, and amine e.g., oxidasecytochrome (Hoch_0806), P450 (Hoch_0804, may complement Hoch_0812, these Hoch_0813 missing domains, and thoughHoch_0814) the real and roles amine of these oxidase proteins (Hoch_0806), remain unclear.may complement Second, the these AT missing domain domains, of module though 4 indicates the a malonatereal roles (acetateof these proteins unit)-specific remain conserved unclear. Second motif,HAFH the AT [domain21] (Supplementary of module 4 indicates Materials, a malonate Figure S3) despite(acetate the unit)-specific fact that this conserved module incorporated motif HAFH one[21] methylmalonate(Supplementary Materials, (propionate) Figure unit S3) as despite shown inthe the feedingfact that experiments. this module Such incorporated inconsistencies one methylmalonate are not common (propionate) in the typical unit bacterial as shown PKS in biosynthesis the feeding but sometimesexperiments. observed Such ininconsistencies myxobacteria. are We not noticed common that in the the AT typical domain bacteria of modulel PKS 4 isbiosynthesis phylogenetically but closesometimes to the AT observed domain in of myxobacteria EpoC (Supplementary. We noticed Materials, that the AT Figure domain S4), of a module promiscuous 4 is phylogenetically AT loading both malonateclose to andthe AT methylmalonate domain of EpoC in (S theupplementary epothilone biosyntheticMaterials, Figure pathway S4), a frompromiscuous terrestrial AT myxobacterium loading both Sorangiummalonate cellulosum and methylmalonate[22]. in the epothilone biosynthetic pathway from terrestrial myxobacterium Sorangium cellulosum[22]. After integrating the last biosynthetic block (acetate unit) by module 5, the biosynthesis undergoes After integrating the last biosynthetic block (acetate unit) by module 5, the biosynthesis a particular chain termination by decarboxylation, leading to the formation of a terminal olefin of the undergoes a particular chain termination by decarboxylation, leading to the formation of a terminal haliamide molecule, which may be mediated by the sulfotransferase (ST)-thioesterase (TE) domains olefin of the haliamide molecule, which may be mediated by the sulfotransferase (ST)-thioesterase encoded in the C-terminus of HlaB (Figure4b). These domains are homologous to CurM TE and CurM (TE) domains encoded in the C-terminus of HlaB (Figure 4b). These domains are homologous to β STCurM (identity TE and of 37% CurM and ST 41%, (identity respectively) of 37% and of curacin41%, respectively) A [23], in of which curacin-keto A [23], group in which is reduced β-keto by KRgroup domain is reduced and the by resulting KR domainβ-hydroxyl and the resulting intermediate β-hydroxyl is sulfated intermediate by ST, is followed sulfated by by ST, TE-catalyzed followed hydrolysisby TE-catalyzed coupled hydrolysis with decarboxylative coupled with eliminationdecarboxylative to give elimination a terminal to give olefin a terminal [24]. The olefin haliamide [24]. biosynthesisThe haliamide was biosynthesis predicted towas be predicted terminated to be in terminated a similar in way, a similar though way, a though KR domain a KR domain to generate to β-hydroxylgenerate β group-hydroxyl is absent group in is moduleabsent in 5 module (Figure 45b). (Figure 4b).

hlaA hlaB NRPS PKS PKS

2 kb

FigureFigure 4. 4.Proposed Proposed biosynthetic biosyntheticmachinery machinery ofof haliamidehaliamide ( (11).). ( (aa)) Genetic Genetic organization organization of ofthe the haliamide haliamide biosyntheticbiosynthetic gene gene clustercluster ( hla,, 21.7 21.7 kbp) kbp) and and a biosynthetic a biosynthetic pathway pathway for 1; for(b) Decarboxylative1;(b) Decarboxylative chain termination at the final step leading to the formation of the terminal olefin at C-9 and C-10. Abbreviations: chain termination at the final step leading to the formation of the terminal olefin at C-9 and C-10. A, adenylation domain; ACP, acyl carrier protein; AT, acyl transferase; C, condensation domain; DH, Abbreviations: A, adenylation domain; ACP, acyl carrier protein; AT, acyl transferase; C, condensation dehydratase; ER, enoyl reductase; KR, ketoreductase; KS, ketosynthase; PCP, peptide carrier protein; domain; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; KS, ketosynthase; PCP, peptide ST, sulfotransferase; TE, thioesterase. carrier protein; ST, sulfotransferase; TE, thioesterase.

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2.4. Bioactivities of Haliamide (1) The cytotoxic effect of the isolated compound haliamide (1) was evaluated by MTT assay using a tumor cell line (HeLa-S3). The IC50 was determined to be 12 µM (a positive control, paclitaxel: IC50 = 8.9 nM, Supplementary Materials, Figure S6). Haliamide (1) showed no inhibitory activity against the plant pathogen Phytophthora capsici (minimum inhibition dose: >30 µg/disk), whereas haliangicin isolated from the same myxobacterium showed an activity at 0.1 µg/disk or higher doses. In the other antimicrobial tests, 1 did not show inhibitory activities (MIC > 128 µg/mL) against a fungus (Candida rugosa), a gram negative (Escherichia coli) and a gram positive bacterium (Bacillus subtilis).

3. Materials and Methods

3.1. General Flash chromatography was carried out with a medium-pressure gradient system equipped with a Pump Module C-605 and a Pump Manager C-615 (BÜCHI, Flawil, Switzerland). Preparative HPLC was performed on a high-pressure gradient system equipped with PU-2087 plus pumps and a UV-2075 plus detector (Jasco, Tokyo, Japan). Specific rotation was measured by using a DIP-370 digital polarimeter (Jasco). FT-IR and UV spectra were recorded on FT/IR-4100 and V-530 spectrometers (Jasco), respectively. Mass spectra (MS) were recorded on a Mariner Biospectrometry Workstation (Applied Biosystems of Thermo Fisher Scientific, Waltham, MA, USA) in the positive-ESI mode. 1H- and 13C-NMR spectra were recorded at 27 ˝C on an Avance 400 (400 MHz for 1H) or Avance III HD 600 Cryo-Probe (600 MHz for 1H) spectrometer (Bruker, Rheinstetten, Germany). Chemical shifts (ppm) were referenced to the solvent (CDCl3) peaks at δH 7.26 ppm (residual CHCl3) and δC 77.0 ppm.

3.2. Isolation The marine myxobacterium Haliangium ochraceum SMP-2 was cultivated in 2 L flasks containing 500 mL of the production medium at 30 ˝C and 180 rpm for two weeks as previously reported [10]. The cells and resin were harvested from 3.5 L culture broth by filtration and extracted with methanol (300 mL, three times) at 30 ˝C for 60 min on a horizontal shaker (120 rpm).The combined methanolic extracts were concentrated in vacuo. The resulting crude extract (5.63 g) was further partitioned between water and hexane/EtOAc (1/1, v/v), and the organic layer were concentrated to yield an oily sample (435.7 mg). The crude oil was chromatographed on a HI-FLASH™ silica gel column (size L, 30 g, Yamazen Co., Osaka, Japan) with a linear gradient of 10%–50% EtOAc in hexane (60 min) at 10 mL/min. The fractions (32.5 mg) eluted with 21%–49% EtOAc in hexane were combined and subjected to preparative HPLC [Develosil ODS-HG-5 (20 ˆ 250 mm, Nomura Chemical Ltd., Seto, Japan), 75% MeOH, 6 mL/min, monitored at 205 nm] to obtain haliamide (1, 1.9 mg, tR = 56 min): 22 colorless oil, rαsD ´ 3 (c 0.12, MeOH); UV (MeOH) λmax 242 (ε 18,000) nm; IR (film) νmax 3305 (br), 3064, 3029, 1635, 1536, 964, 694 cm´1; ESI-MS m/z 284.20 [M + H]+, 306.19 [M + Na]+, 322.16 [M + K]+. + HR ESI-MS calcd. for C19H26NO 284.2009 [M + H] ; found 284.1989.

3.3. Feeding Experiments with Stable Isotope Labeled Precursors

13 The stable isotope-labeled compounds used were sodium [1,2- C2]acetate (Cambridge Isotope Laboratories, Tewksbury, MA, USA), sodium [1-13C]propionate (Sigma-Aldrich Co. St. Louis, MO, USA), DL-[1-13C]alanine (Isotec, Canton, GA, USA) and [1-13C]benzoic acid (Aldrich). H. ochraceum was cultured in the above-mentioned production medium (100 mL in six 500 mL flasks or 750 mL in one 2 L flask) in the presence of 2% (w/v) adsorbent resin (SEPABEADS SP207, Mistubishi Chemical Co., Tokyo, 13 Japan). A filter-sterile solution of an isotope-labeled compound (sodium [1,2- C2]acetate, sodium [1-13C]propionate, DL-[1-13C]alanine in water, or [1-13C]benzoic acid neutralized with 6 N NaOH and diluted with water, 200 mM) was added into 4-days bacterial culture to the final concentration of 4 mM. After two weeks cultivation in total, the cells and the resin were collected and extracted with methanol (300 mL, three times). The combined methanolic extracts were concentrated and then Molecules 2016, 21, 59 6 of 8 partitioned between water (200 mL) and EtOAc (200 mL). The ethyl acetate part was dissolved in 50% MeOH (50 mL) and extracted twice with hexane–EtOAc (4:1, 50 mL). The crude oil obtained from the upper layer was chromatographed on a HI-FLASH™ silica gel column (size S, flow rate: 3 mL/min) with a linear gradient using 10%–50% EtOAc in hexane (30 min). The haliamide-containing fractions were combined and then subjected to preparative HPLC [Develosil ODS-HG-5 (10 ˆ 250 mm, Nomura 13 Chemical Ltd.), 60% MeCN, 3 mL/min, monitored at 205 nm] to afford [1,2- C2]acetate-labeled haliamide (0.3 mg), [1-13C]propionate-labeled haliamide (0.5 mg), [1-13C]alanine-labeled haliamide (0.5 mg), and [1-13C]benzoic acid-labeled haliamide (0.2 mg), respectively.

3.4. Analysis of the Haliamide Biosynthetic Gene Cluster (hla) The genome data of H. ochraceum SMP-2T (Accession No. NC_013440.1 [25]) was analyzed by antiSMASH [19]. The sequence for the putative biosynthetic gene cluster hla was annotated by BLAST and CDD [26]. The multiple alignments of amino acid sequences were generated by Clustal Omega program provided by EMBL.

3.5. Bioassay

3.5.1. Cytotoxicity Assay HeLa-S3 (SC) cells were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. The cells were cultured in Eagle’s minimal essential medium (EMEM) (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA). A total of 10,000 cultured cells were seeded into each well of a 96-well plate containing 99 µL of the same ˝ medium. After preincubation for 24 h at 37 C in an atmosphere of 5% CO2, a compound (haliamide or paclitaxel as a positive control) in 1 µL of dimethyl sulfoxide (DMSO), or only 1 µL of DMSO as control, was added to each well, and the cells were incubated an additional 48 h. A solution (10 µL) of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffer saline (PBS) (5 mg/mL) was then added to each well, and the plate was incubated for an additional 3 h. Subsequently, the medium was removed by aspiration, any generated formazan was dissolved in 100 µL of DMSO, and the absorbance was measured at 595 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific). Values are means ˘ standard error (n = 4).

3.5.2. Anti-Oomycete Activity The phytopathogenic oomycete Phytophthora capsici NBRC 30696, purchased from NITE-Biological Resource Center (NBRC, Chiba, Japan), was cultured on a potato-agar medium (in 1 L, potato broth from 200 g of fresh potato, glucose (20 g), and agar (20 g)) in a 9-cm dish at 25 ˝C for 7 days in the dark. A piece of the colony was then inoculated on the center of a 5% V8-agar medium (V8 vegetable juice (5 mL), agar (1.5 g), and water (95 mL)) in a 9-cm dish and incubated at 25 ˝C for 48 h in the dark until the colony grew to approximately 4 cm in diameter. A paper disc (6 mm in diameter) impregnated with a sample was placed 1 cm away from the front of the colony. After incubating for 22–24 h, the distance between the edge of the colony and the paper disc (control: 0 mm) was measured. The activity was defined as a minimum dose that induced a definite distance (0.5 mm or wider) between the colony and the disk. The tested doses were 1, 3, 10, and 30 µg/disk.

3.5.3. MIC Assay The microorganisms, Candida rugosa AJ 14513, Bacillus subtilis AJ 12865, and Escherichia coli AJ 3837, were provided by the Institute for Innovation, Ajinomoto Co., Inc. (Kanagawa, Japan). The protocols used for the assay are in the followings. For B. subtilis and E. coli, see Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition. CLSI document M07-A9 (ISBN 1-56238-783-9), Clinical and Laboratory Standards Institute (Wayne, PA, USA), 2012. Molecules 2016, 21, 59 7 of 8

For C. rugosa, see Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Second Edition. NCCLS document M27-A2 (ISBN 1-56238-469-4), NCCLS (Wayne, PA, USA), 2002. The tested concentrations were 1, 2, 4, 8, 16, 32, 64, and 128 µg/mL.

4. Conclusions In the present study, we discovered a new PKS-NRPS hybrid type metabolite, haliamide (1), from the halophilic myxobacterium H. ochraceum and evaluated its biological activities. The identification of the biosynthetic units for 1 and the genome mining of its producer provided a putative biosynthetic gene cluster (hla). Further, genomic analysis suggests that at least five more PKS-NRPS gene clusters are harbored in the genome of H. ochraceum, which prompts us to discover additional unknown metabolites of this hard-to-culture marine myxobacterium.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/ 21/1/59/s1. Acknowledgments: Yuwei Sun is grateful for a scholarship provided by the China State-Funded Postgraduates Overseas Study Program from the Chinese Scholarship Council. Author Contributions: Y.S. deciphered the mechanism of the haliamide biosynthesis and prepared the manuscript. T.T. performed the bioassay. J.S. isolated the compound and completed the incorporation experiments. T.I. and R.F. cultured the myxobacteria. M.O. designed the study, analyzed the data and wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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