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New Metabolites from the Co-Culture of Marine-Derived Actinomycete Streptomyces Rochei MB037 and Fungus Rhinocladiella Similis 35

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New Metabolites from the Co-Culture of Marine-Derived Actinomycete Streptomyces Rochei MB037 and Fungus Rhinocladiella Similis 35 fmicb-10-00915 May 4, 2019 Time: 16:21 # 1 ORIGINAL RESEARCH published: 07 May 2019 doi: 10.3389/fmicb.2019.00915 New Metabolites From the Co-culture of Marine-Derived Actinomycete Streptomyces rochei MB037 and Fungus Rhinocladiella similis 35 Meilin Yu1,2,3, Yingxin Li1, Shivakumar P. Banakar1, Lu Liu2,3, Changlun Shao2,3, Zhiyong Li1* and Changyun Wang2,3,4* 1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China, 2 Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China, 3 Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China, 4 Institute of Evolution and Marine Biodiversity, Ocean University Edited by: of China, Qingdao, China Bey Hing Goh, Monash University Malaysia, Malaysia Co-culture of different microbes simulating the natural state of microbial community Reviewed by: Ayanabha Chakraborti, may produce potentially new compounds because of nutrition or space competition. University of Alabama at Birmingham, To mine its metabolic potential in depth, co-culture of Streptomyces rochei MB037 United States Phan Chia Wei, with a gorgonian-derived fungus Rhinocladiella similis 35 was carried out to stimulate University of Malaya, Malaysia the production of new metabolites in this study, using pure cultivation as control. Five Kai-Leng Tan, metabolites were isolated successfully from co-culture broth, including two new fatty Guangdong University of Technology, China acids with rare nitrile group, borrelidins J and K (1 and 2), one chromone derivative *Correspondence: as a new natural product, 7-methoxy-2,3-dimethylchromone-4-one (3), together with Zhiyong Li two known 18-membered macrolides, borrelidin (4) and borrelidin F (5). The structures [email protected] Changyun Wang of 1–3 were elucidated by using a combination of NMR and MS spectroscopy, [email protected] ester hydrolysis, and optical rotation methods. Interestingly, 1 and 2 were obtained only in co-culture. Though 3 was gained from either co-culture or single culture, its Specialty section: This article was submitted to production was increased significantly by co-culture. Compound 1 exhibited significant Antimicrobials, Resistance antibacterial activity against methicillin-resistant Staphylococcus aureus with a MIC and Chemotherapy, a section of the journal value of 0.195 mg/mL. Frontiers in Microbiology Keywords: co-culture, actinomycete, fungus, borrelidin, antibacterial activity Received: 15 November 2018 Accepted: 10 April 2019 Published: 07 May 2019 INTRODUCTION Citation: Yu M, Li Y, Banakar SP, Liu L, In nature, microbes generally exist in a community. One microbe may produce biological products Shao C, Li Z and Wang C (2019) New to inhibit other microbes for limited nutrition or space competition or against pathogenic microbes. Metabolites From the Co-culture Thus, co-culture of microorganisms which involves the cultivation of two or more microorganisms of Marine-Derived Actinomycete Streptomyces rochei MB037 in the same confined environment may produce potentially new compounds by stimulating and Fungus Rhinocladiella similis 35. the silent genes or gene clusters of one partner or increase the yields of previously described Front. Microbiol. 10:915. metabolites. For example, Sung et al.(2017) researched increased production of three antibiotics doi: 10.3389/fmicb.2019.00915 and enhanced biological activity against the Gram positive human pathogens via co-cultures of a Frontiers in Microbiology| www.frontiersin.org 1 May 2019| Volume 10| Article 915 fmicb-10-00915 May 4, 2019 Time: 16:21 # 2 Yu et al. New Metabolites From the Co-culture marine-derived Streptomyces sp. with human pathogens arrival to the Key Laboratory of Microbial Metabolism, Shanghai (Sung et al., 2017), and Zuck et al.(2011) gained a cytotoxic Jiao Tong University, China. N,N0-((1Z,3Z)-1,4-bis(4-methoxyphenyl)buta-1,3-diene-2,3- diyl)diformamide by co-culture of the fungus Aspergillus Fermentation, Extraction, and Isolation fumigatus with the actinomycete Streptomyces peucetius. The actinomycete S. rochei MB037 and fungal R. similis 35 Macrolide borrelidin has been reported to show broad- were cultivated in 25 L of ISP2 medium [malt extract 10 g, spectrum activities (Anderson et al., 1989; Ishiyama et al., 2011; anhydrous dextrose 4 g, yeast extract 4 g in 1 L of artificial Mirando et al., 2015). In our previous study, borrelidin was seawater (NaCl 132.6 g, MgCl ·6H O 55.86 g, CaCl 5.705 g, isolated and elucidated as a major product from a sponge- 2 2 2 KCl 3.625 g, NaHCO 1.01 g, NaBr 0.415 g), pH value 7.0] at derived actinomycete Streptomyces rochei MB037 (Li et al., 2018). 3 28◦C with shaking at 180 rpm, respectively. On day 3, 200 mL To mine its more metabolic potential, co-culture approach of actinomycete culture was inoculated into each 200 mL of the was applied on S. rochei MB037. A gorgonian-derived fungal fungal cultures (1:1 v/v) to initiate the co-culture experiment. The strain, Rhinocladiella similis 35, was selected as a partner against changes of secondary metabolite production between co-culture actinomycete S. rochei MB037. A literature survey revealed and single culture were analyzed by reverse-phased HPLC. that a few new and bioactive compounds were separated from After incubation for 11 additional days, the co-culture broth the fungus Rhinocladiella sp. (Wagenaar et al., 2000; Zhang was extracted with 50 L ethyl acetate using rotary evaporator to et al., 2014). The co-culture of S. rochei MB037 and R. similis yield 12 g of reddish brown oil material. The organic extract (12 g) 35 stimulated the production of new metabolites successfully. was subjected to Sephadex LH-20 eluting by MeOH to obtain Herein, we report the isolation, structural elucidation, and four fractions (Fr. 1–4). Fr. 2–4 was subjected to reversed phase evaluation of biological activities of the metabolites 1–5 chromatograph eluting with MeOH-H O (Cheetah Fs-9200t C , (Figure 1) produced by co-culture of S. rochei MB037 and 2 18 linear gradient 10, 30, 50, 70, 90, and 100% aqueous CH OH R. similis 35. A plausible biosynthesis pathway for the metabolites 3 for 30 min, respectively, at a flow rate of 20 mL/min) to give was also proposed and discussed. three fractions (Fr. 2–7, 3–18, and 4–8). Fr. 2–7 was further purified twice by preparative HPLC (Eclipse XDB C18 column, 4.6 mm × 150 mm, 5 mm, Agela) with 40 to 44% aqueous CH3CN MATERIALS AND METHODS to yield 5 (7 mg). Fr. 3–18 was further purified using Sephadex LH-20 with CH3OH and then followed by preparative HPLC with Instrumentation 50% aqueous CH3CN to give 2 (5 mg), 4 (20 mg) and 5 (4 mg), Optical rotations were determined on a JASCO P-2000 respectively. Fr. 4–8 was further purified by preparative HPLC polarimeter. 1D and 2D NMR spectra were recorded on an with 38% aqueous CH3CN to provide 3 (10 mg). Avance III 600 MHz NMR spectrometer. High-resolution electrospray ionization mass spectroscopy (HR-ESI-MS) was Borrelidin J (1) 25 1 13 recorded with an ACQUITYTM UPLC and Q-TOF mass A light yellow oil; [a] D – 25.5 (c 4.0, MeOH); H and C NMR spectrometer. High-performance liquid chromatography spectroscopic data, Tables 1, 2;(−)-HR-ESI-MS m/z 506.3117 (HPLC) analysis was performed with an Agilent 1200 [M-H]− (calcd for C28H44NO7, 506.3114). detector (G1322A), using a Durashell 150 Å C18 column × m (4.6 mm 250 mm, 5 m, Agela) and HPLC preparative-scale Borrelidin K (2) purification was performed with an Eclipse XDB C column 18 A light yellow oil; [a]25 – 14.0 (c 2.5, MeOH); 1H and 13C NMR (4.6 mm × 150 mm, 5 mm, Agela). The FT-IR spectra were D spectroscopic data, Tables 1, 2;(−)-HR-ESI-MS m/z 520.3264 recorded using a Nicolet 6700 spectrometer. − [M-H] (calcd for C29H46NO7, 520.3261). Microbial Strains 7-Methoxy-2,3-Dimethylchromone-4-One (3) The actinomycete S. rochei MB037 was derived from sponge 1 13 ◦ 0 ◦ A brown yellow solid; H and C NMR spectroscopic data, Dysidea arenaria collected from Yongxin Island (112 20 E; 16 C Table 3;(C)-HR-ESI-MS m/z 205.0885 [MCH] (calcd for 500 N) in the South China Sea (Karuppiah et al., 2015). It was C H O , 205.0888). identified as S. rochei on the basis of the 100% 16S rDNA sequence 12 13 3 identity with the type strain of this species, under the GenBank Accession No. AA2017041 (Li et al., 2018). The fungal strain Borrelidin (4) 25 1 R. similis 35 was isolated from a staghorn gorgonian collected A white amorphous powder; [a] D – 16.0 (c 2.5, MeOH); H and ◦ 0 ◦ 0 13C NMR spectroscopic data, Tables 1, 2;(−)-HR-ESI-MS m/z from Luhuitou fringing reefs (109 25 E; 18 15 N) in the South − China Sea in July 2014. The strain was identified as R. similis 520.3264 [M-H] (calcd for C29H46NO7, 520.3261). according to its morphologic traits and molecular identification. Its 616 base pair ITS sequence had 100% sequence identity to Borrelidin F (5) 25 1 that of R. similis (KY680425) isolate CMRP1259. The sequence A white amorphous powder; [a] D C 14.0 (c 2.5, MeOH); H data have been submitted to GenBank, under the Accession No. and 13C NMR spectroscopic data, Tables 1, 2;(−)-HR-ESI-MS ◦ − MH481284. Both of the strains were stored at −80 C after their m/z 520.3264 [M-H] (calcd for C29H46NO7, 520.3261).
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