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

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Isolation and Biosynthetic Analysis of Haliamide, a New PKS-NRPS Hybrid Metabolite from the Marine Myxobacterium Haliangium Ochraceum molecules Article Isolation and Biosynthetic Analysis of Haliamide, a New PKS-NRPS Hybrid Metabolite from the Marine Myxobacterium Haliangium 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: Myxobacteria 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 Haliangium ochraceum 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 bacteria 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. Molecules 2016, 21, 59 3 of 8 Molecules 2016, 21, 59 3 of 8 Molecules 2016, 21, 59 3 of 8 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).
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